Citrus Nutrition and Quality 9780841205956, 9780841207592, 0-8412-0595-7

Table of contents :
Title Page......Page 1
Copyright......Page 2
ACS Symposium Series......Page 3
FOREWORD......Page 4
PdftkEmptyString......Page 0
PREFACE......Page 5
1 Nutrients and Nutrition of Citrus Fruits......Page 6
The Historical Role of Citrus Fruit in the Human Diet......Page 7
Carbohydrates......Page 8
Protein......Page 13
Fat-Soluble Vitamins in Citrus Fruit......Page 14
Water-Soluble Vitamins in Citrus Fruit......Page 15
Stability of Vitamin C in Citrus Fruits and Juices......Page 16
Mineral Nutrients in Citrus......Page 21
Bioflavonoids......Page 22
Conclusion......Page 23
Literature Cited......Page 24
Bioavailability of Water-soluble Vitamins......Page 28
Electrolyte Balance......Page 30
NUTRITIONAL ROLE OF DIETARY CITRUS PECTIN......Page 31
Literature Cited......Page 43
Variations in Citrus Extracts......Page 45
Trimodal Action of Bioflavonoids......Page 46
Blood Rheology and Capillary Fragility......Page 48
Therapeutic Effects in Disease......Page 49
Intact Organism, Physiological, Biochemical Levels......Page 50
Citrus Bioflavonoids and Disease......Page 51
Is a Theory Unifying Mode of Action Possible At Present?......Page 55
Summary......Page 57
Literature Cited......Page 59
Limonoid Structures and Relevant Chemistry......Page 62
Structural relationships and pathways......Page 66
Biosynthesis......Page 69
Metabolism......Page 70
Organoleptic Aspects......Page 73
Literature Cited......Page 79
5 Flavonoids and Citrus Quality......Page 82
Physical and Chemical Characteristics......Page 84
Methods of Analysis......Page 85
Taste and Structure of Citrus Flavonoids......Page 88
Flavonoid Bitterness Suppressors......Page 92
Taxonomic Significance of Flavonoids......Page 93
Effects of Fruit Maturity, Rootstocks and Horticultural Variables......Page 97
Effects of Processing on Juice Bitterness......Page 100
Literature Cited......Page 105
6 The Role of Pectin in Citrus Quality and Nutrition......Page 108
Quality......Page 110
Nutrition......Page 117
Literature Cited......Page 123
Chlorophyll in Peel......Page 128
Commercial Aspects of Degreening......Page 130
Carotenoids in Peel......Page 131
Isomer and Artifact Formation of Carotenoids......Page 133
Carotenoids and Peel Color......Page 134
Ethylene in Carotenoid Synthesis......Page 136
Color and Provitamin A......Page 140
Standards for Color in Citrus and Citrus Products......Page 142
Color Enhancement in Fresh Fruit......Page 144
Color Enhancement of Orange Juice......Page 145
Literature Cited......Page 146
8 Relationship of Citrus Enzymes to Juice Quality......Page 149
Pectinesterase (E.C. 3.1.1.11)......Page 150
Limonin D-Ring Lactonase (EC 3.1.1.36)......Page 157
Enzymes to Degrade Limonin Precursor......Page 158
Other Enzymes in Citrus Juices......Page 159
Literature Cited......Page 160
Components Important to Natural Orange Flavor......Page 165
Taste and Aroma Thresholds in Water of Individual Orange Juice Components......Page 166
Effect of Nonvolatile Juice Components on Individual Flavor Thresholds......Page 170
Influence of Selected Volatile Components on Flavor of a Bland Orange Juice Drink......Page 172
Components Important to Natural Grapefruit Flavor......Page 179
Components Important to Mandarin Flavor and Aroma......Page 182
Total Aldehydes As a Measure of Oil Quality......Page 185
Literature Cited......Page 186
Preharvest techniques which influence quality......Page 189
Handling techniques at harvest which influence quality......Page 192
Postharvest techniques and their influence upon quality......Page 195
Storage and Quality of Citrus Fruits......Page 203
Fruit Quality as Influenced by Packaging and Transportation......Page 208
Conclusions......Page 209
Literature Cited......Page 210
11 Citrus Juice Processing as Related to Quality and Nutrition......Page 221
1 Processing......Page 225
1.1 Juice Extraction......Page 229
1.3 Evaporation......Page 230
1.4 Ion-Exchange Processing.......Page 233
1.5 Pasteurization and Packaging......Page 234
2.1 Vitamin C......Page 236
2.2 Other Nutrients of Dietary Significance......Page 237
3 Effects of Processing on Nutritional Quality......Page 239
4 Processed Citrus Products......Page 243
4.1 Frozen Concentrated Juices......Page 245
4.2 Reduced-Acid Frozen Concentrated Orange Juice......Page 250
4.3 Chilled Citrus Juices......Page 253
4.4 Canned Juices......Page 257
4.5 Dehydrated Juices......Page 259
Literature Cited......Page 262
Dried Pulp and Pellets......Page 268
Peel Oils......Page 272
Pulp-Wash Solids......Page 277
Dried Juice Sacs......Page 280
Literature Cited......Page 281
Brix Determination......Page 284
Brix by Hydrometer.......Page 285
Acid Determination......Page 288
Calculation.......Page 290
Reagents.......Page 292
Pulp - Free and Suspended......Page 293
Pulp-Floating......Page 295
Equipment.......Page 297
Defects......Page 298
Procedure.......Page 299
Color Determination......Page 300
Procedure for Evaluating Color.......Page 302
Enumerating Microorganisms......Page 303
Literature Cited......Page 309
14 Problems in Sensory Evaluation of Citrus Products......Page 311
I. Panelists......Page 313
II. Sample Preparation......Page 315
III. Conducting Sensory Tests......Page 318
IV. Varietal Considerations......Page 323
VI. Specific Citrus Flavor Attributes......Page 324
VII. Problems in Citrus Sensory Evaluation Research......Page 328
Literature Cited......Page 331
Food Quality......Page 333
Problem Areas in Citrus Quality......Page 335
Current Analytical Methods......Page 336
Development of the Immunoassay in Plant Science......Page 337
ARIA......Page 343
Enzyme immunoassay......Page 345
Conclusions......Page 348
Acknowledgement......Page 349
Literature Cited......Page 350
16 Analysis of Trace Metals in Orange Juice......Page 352
Historical......Page 353
Sample Preparation......Page 358
End Measurement Techniques......Page 360
Experimental......Page 366
Results and Discussion......Page 370
Treatment of the Data......Page 373
Case II:......Page 376
Summary......Page 377
Abstract......Page 378
Literature Cited......Page 379
17 Methods for the Detection of Adulteration in Processed Citrus Products......Page 382
Physical Methods......Page 383
Chemical Methods......Page 386
Biological Assays......Page 399
Data Evaluations......Page 400
Conclusions......Page 402
Literature Cited......Page 403
18 Detection of Adulteration Visible and Ultraviolet Absorption and Fluorescence Excitation and Emission Characteristics of Florida Orange Juice and Orange Pulpwash......Page 409
Literature Cited......Page 427
Β......Page 429
C......Page 430
Ε......Page 432
F......Page 433
G......Page 434
J......Page 435
L......Page 436
M......Page 437
O......Page 438
Ρ......Page 439
R......Page 440
U......Page 441
W......Page 442

Citation preview

Citrus Nutrition and Quality Steven Nagy and John A. Attaway,

EDITORS

Florida Department of Citrus

Based on a symposium sponsored by the Division of Agricultural and Food Chemistry at the 179th Meeting of the American Chemical Society, Houston, Texas, March 26,

1980.

143

ACS SYMPOSIUM SERIES

AMERICAN

CHEMICAL

WASHINGTON, D. C.

SOCIETY 1980

In Citrus Nutrition and Quality; Nagy, S., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

Data Library of Congress Citrus nutrition and quality. (ACS symposium series; 143 ISSN 0097-6156) Includes bibliographies and index. 1. Citrus fruits—Congresses. 2. Citrus fruit indus­ try—Congresses. I. Nagy, Steven. II. Attaway, John Α., 1930III. American Chemical Society. Division of Agricul­ tural and Food Chemistry. IV. Series: American Chem­ ical Society. ACS symposium series; 143. [DNLM: 1. Citrus fruits—Congresses. 2. Nutrition—Congresses. WB430 C581 1980] TX558.C5C57 641.3'43 80-22562 ISBN 0-8412-0595-7 ASCMC8 143 1-456 1980

Copyright © 1980 American Chemical Society All Rights Reserved. The appearance of the code at the bottom of thefirstpage of each article in this volume indicates the copyright owner's consent that reprographic copies of the article may be made for personal or internal use or for the personal or internal use of specific clients. This consent is given on the condition, however, that the copier pay the stated per copy fee through the Copyright Clearance Center, Inc. for copying beyond that permitted by Sections 107 or 108 of the U.S. Copyright Law. This consent does not extend to copying or transmission by any means—graphic or electronic—for any other purpose, such as for general distribution, for advertising or promotional purposes, for creating new collective works, for resale, or for information storage and retrieval systems. The citation of trade names and/or names of manufacturers in this publication is not to be construed as an endorsement or as approval by ACS of the commercial products or services referenced herein; nor should the mere reference herein to any drawing, specification, chemical process, or other data be regarded as a license or as a conveyance of any right or permission, to the holder, reader, or any other person or corporation, to manufacture, repro­ duce, use, or sell any patented invention or copyrighted work that may in any way be related thereto. PRINTED IN THE UNITEDSTATESOFAMERICA

In Citrus Nutrition and Quality; Nagy, S., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

A C S Symposium Series M . Joa

Advisory Board David L. Allara

W . Jeffrey Howe

Kenneth B. Bischoff

James D . Idol, Jr.

Donald G . Crosby

James P. Lodge

Donald D . Dollberg

Leon Petrakis

Robert E. Feeney

F. Sherwood Rowland

Jack Halpern

Alan C. Sartorelli

Brian M . Harney

Raymond B. Seymour

Robert A . Hofstader

Gunter Zweig

In Citrus Nutrition and Quality; Nagy, S., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

FOREWOR The ACS SYMPOSIUM SERIES was founded in 1974 to provide

a medium for publishing symposia quickly in book form. The format of the Series parallels that of the continuing ADVANCES IN CHEMISTRY SERIES except that in order to save time the papers are not typeset but are reproduced as they are submitted by the authors in camera-ready form. Papers are reviewed under the supervision of the Editors with the assistance of the Series Advisory Board and are selected to maintain the integrity of the symposia; however, verbatim reproductions of previously published papers are not accepted. Both reviews and reports of research are acceptable since symposia may embrace both types of presentation.

In Citrus Nutrition and Quality; Nagy, S., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

PREFACE

T

here is a long romantic history of man's fascination, esteem, and food recognition of citrus fruits. According to one legend, it was not with an apple that Eve tempted Adam but with a citrus fruit (a primitive citron called the etrog or Adam's apple). Oranges were also once considered the fruit of the gods. The ancient dynasties of China regarded citrus fruits as prized tributes. During the reign of Ta Yu (about 2 2 0 5 - 2 1 9 7 B.C.), tributes of mandarins an sent to the imperial cour The importance of citrus fruits and their products as human food is underscored by the fact that more citrus is consumed than any other kind of fruit. To illustrate this point, a recent survey conducted by the Market Research Corporation of America showed that about 6 8 % of all juices (combined total of fruit and vegetable juices) consumed in the United States were citrus juices. The world's citrus crop for the 1 9 7 9 - 8 0 season was estimated at 39.5 million metric tons ( M M T ) , of which the United States contributed a total of 14.4 M M T (oranges 10.4 M M T , grapefruit 2.5 M M T , lemons 0.7 M M T , tangerines 0.5 M M T , other citrus cultivars 0.3 M M T ) . The citrus industry in Florida is the largest and encompasses 336,384 hectares (about 2 / 3 of the total U . S. hectarage). During the 1 9 7 8 - 7 9 season, Florida produced 10.1 M M T of citrus fruit with a fresh and processed sales value of $1.99 billion. World productionfiguresshow that the citrus crop is second only to the grape crop. However, most grapes are utilized primarily in fermented liquors rather than consumed as fresh fruit or juice products. As citrus fruits and their products contribute substantially to the American diet and are consumed in great abundance, we believe the time is appropriate to review in detail some important nutritional and quality properties of this important fruit. Twenty-eight scientists joined with us to cover extensively subjects in the following areas: nutrition and health; quality as related to specific biochemical components; effects of handling and processing; quality control and evaluation; regulatory implication; and adulteration. Florida Department of Citrus

STEVEN NAGY

Lakeland, Florida 3 3 8 0 2

JOHN A . A T T A W A Y

July 14, 1980 vii In Citrus Nutrition and Quality; Nagy, S., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

1 Nutrients and Nutrition of Citrus Fruits S. V. TING Florida Department of Citrus, University of Florida, Institute of Food and Agricultural Sciences, Agricultural Research and Education Center, P.O. Box 1088, Lake Alfred, FL 33850

A l l foods for human ments. These elements ar being metabolized in the body and those that are essential for the body to carry on this metabolism. Other qualifications for a food are i t s psychological and social effects. Many foods are eaten as a habit, custom, or t r a d i t i o n ; but all foods must possess acceptable physical attributes, i.e., color and texture, and desirable taste and p a l a t a b i l i t y . The acceptance of fruits as a staple i n human diet has only been practiced since the past century because of their perishability as fresh produce. In most instances, they are used as desserts because most of them are sweet in taste and because of their high economic values. With the advent of canning and other preservation industries and with the better knowledge of n u t r i t i o n , the use of fruits as staple foods has become more prevalent, especially in developed countries. The use o f c i t r u s f r u i t , e s p e c i a l l y oranges, d r a m a t i c a l l y increased i n the U.S. a f t e r World War II ( 1 ) because o f the i n t r o d u c t i o n o f f r o z e n concentrated orange j u i c e (FCOJ) t o the market. C i t r u s f r u i t s , being s u b t r o p i c a l products, d i d not enjoy the p o p u l a r i t y o f other f r u i t s , e . g . a p p l e s , because the l o c a l i t y of production were u s u a l l y not near the world population centers and because o f p e r i s h a b i l i t y o f c i t r u s f r u i t s during storage. T h e i r s u s c e p t i b i l i t y to p h y s i o l o g i c a l d i s o r d e r s and t o storage d i s e a s e s , e s p e c i a l l y molds and r o t s , made the cost a d e t e r r i n g f a c t o r t o consumers. These shortcomings were overcome w i t h the development o f FCOJ, which i s a t t r a c t i v e i n c o l o r , possesses f u l l f r e s h orange f l a v o r and g r e a t l y reduces the cost o f t r a n s p o r t a t i o n with n e a r l y no storage l o s s . Research i n the area o f storage disease c o n t r o l and i n t r a n s p o r t a t i o n o f f r e s h f r u i t have a l s o l e d t o increased consumption i n many developed c o u n t r i e s . C i t r u s f r u i t s and t h e i r products a r e important sources o f vitamin C i n the American d i e t , and are becoming i n c r e a s i n g l y more important t o other developed and developing c o u n t r i e s . Consumer awareness o f the h e a l t h f u l aspects o f c i t r u s , together 0-8412-0595-7/80/47-143-003$05.00/0 © 1980 A m e r i c a n Chemical Society

In Citrus Nutrition and Quality; Nagy, S., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

CITRUS NUTRITION AND QUALITY

4

w i t h i t s appealing c o l o r and d e l i g h t f u l aroma and t a s t e , makes c i t r u s products the most popular o f the processed f r u i t products. The improved technology of c i t r u s f r u i t p r o d u c t i o n , p r o c e s s i n g , storage and t r a n s p o r t a t i o n has placed the product w i t h i n economic reach of more people than ever. The c o s t of a s e r v i n g (6 f l o z , 177 ml) of orange j u i c e , g r a p e f r u i t j u i c e , and other j u i c e s were 9 . 7 , 9.0 and 10.7 c e n t s , r e s p e c t i v e l y , as reported i n September 1979 (2:). Table I shows the gallonage and expenditures f o r c i t r u s f r u i t j u i c e s and f r u i t - f l a v o r e d d r i n k s , i n c l u d i n g orange f l a v o r e d , i n the U.S. during September. Table I. Consumption of F r u i t J u i c e s and Other F r u i t Beverages i n the United States and T h e i r Total Values-September, 1979 M i l l i o n s of gallons

M i l l i o n s of dollars

Orange j u i c e Grapefruit juice A l l other f r u i t j u i c e s

26.1

70.0

Orange f l a v o r d r i n k s A l l other f l a v o r d r i n k s

11.6 18.4

16.2 26.2

Source:

{2)

The H i s t o r i c a l Role of C i t r u s F r u i t i n the Human D i e t C i t r u s i s g e n e r a l l y regarded as one of the most important sources of a s c o r b i c a c i d . The r e l a t i o n between c i t r u s f r u i t and a n t i s c o r b u t i c a c t i v i t y a c t u a l l y was f i r s t reported by a Hungarian p h y s i c i a n , Kramer, i n 1732 (3J. The dreadful disease of scurvy was found to be completely prevented w i t h the i n g e s t i o n of green vegetable or pulp of c i t r u s f r u i t . The use of lime and orange i n the d i e t of seamen i n the Royal B r i t i s h A d m i r a l i t y was the o r i g i n of the name "Limey" f o r B r i t i s h s a i l o r s . Harden and Z i l v a (4) found a concentrated f r a c t i o n from the lemon f r u i t t h a t possessed strong reducing p r o p e r t i e s . L a t e r Szent Gyorgyi (5) i s o l a t e d the a n t i s c o r b u t i c substance i n c r y s t a l l i n e form from pepper and c i t r u s fruit. Many vegetables and f r u i t s , other than c i t r u s , c o n t a i n a s c o r b i c a c i d . I t was estimated t h a t c i t r u s f r u i t s and tomatoes p r o vided only 18% of the t o t a l v i t a m i n C i n t a k e i n the American d i e t during the decade of 1910. These two f r u i t s s u p p l i e d 41% of v i t a m i n C i n 1956-58 ( 6 ) . Today orange j u i c e alone provides n e a r l y 60% of the U.S. Recommended D a i l y Allowance (U.S. RDA) of v i t a m i n C i n the American d i e t ( 7 ) . C i t r u s f r u i t s and t h e i r products are now recognized as an important food i n the human d i e t , not only because of t h e i r v i t a m i n C c o n t e n t s , but a l s o because of t h e i r other food a t t r i butes, such as t h e i r pleasant aroma, appealing c o l o r , and p l e a s ant t a s t e of a p p r o p r i a t e r a t i o s of sweetness and t a r t n e s s

In Citrus Nutrition and Quality; Nagy, S., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

1.

TING

Nutrition

of Citrus

5

Fruits

and because of the awareness by the p u b l i c of t h e i r n u t r i t i v e values and the other n u t r i e n t s they c o n t a i n . Macronutrients i n C i t r u s The energy-supplying n u t r i e n t s are g e n e r a l l y carbohydrates, p r o t e i n and f a t . While c i t r u s products provide l i t t l e p r o t e i n and f a t , t h e i r c o n t r i b u t i o n of carbohydrate i s an e s s e n t i a l p a r t of the n u t r i t i v e value of c i t r u s . The proximate composition of several kinds of c i t r u s f r u i t s (8) are shown i n Table II. Because Table II.

Proximate Composition of C i t r u s F r u i t s (g/100 g)

Fruits

Moisture

Protein

Orange Whole f r u i t Juice

86. 88.3

.6

.2

10.5

.1

.4

Grapefruit Whole f r u i t Juice Segment

88.9 90.2 91.3

.5 .5 .6

.1 .1 .1

10.1 9.0 7.6

.2 — .2

.4 .2 .4

Tangerine Whole f r u i t Juice

87.0 88,9

.8 J>

.2 .2

11.6 10.1

.5 .1

.4 .3

Source:

Fat

Carbohydrate Sol. Insol.

Ash

(8)

the compositions these values can main p a r t of the hydrate and most amino a c i d s .

vary g r e a t l y due to f r u i t m a t u r i t y and v a r i e t y , f l u c t u a t e c o n s i d e r a b l y from a c t u a l samples. The c a l o r i c values s u p p l i e d by c i t r u s i s from c a r b o of the p r o t e i n value i s a c t u a l l y from f r e e

Carbohydrates Simple sugars. The main p o r t i o n of carbohydrates i n c i t r u s f r u i t are the three simple sugars: sucrose, glucose and f r u c t o s e (9). Together they represent about 80% of the t o t a l s o l u b l e s o l i d s of orange j u i c e (jLO), and the r a t i o s of sucrose: glucose: f r u c t o s e are g e n e r a l l y about 2:1:1 (11_). In over-mature e a r l y and mid-season F l o r i d a oranges, and i n t a n g e r i n e s , the r a t i o s of sucrose to reducing sugars have been found to increase but not i n the l a t e season F l o r i d a oranges (12). In g r a p e f r u i t , the sucrose to non-reducing sugar r a t i o s are l e s s than 1. Most of the f r e e sugars i n lemon and lime j u i c e s are reducing sugars (Table I I I ) and the main s o l u b l e s o l i d i n these f r u i t j u i c e s i s c i t r i c a c i d . In an a c i d i c medium such as c i t r u s j u i c e s , sucrose can be e a s i l y hydrolyzed; t h i s f a c t may account f o r the low sucrose values sometimes found i n canned j u i c e s s u b j e c t to long term storage.

In Citrus Nutrition and Quality; Nagy, S., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

CITRUS NUTRITION AND QUALITY

6

Table I I I .

Average Sugar Composition of C i t r u s J u i c e s (g/100 g) Total Total Sugars Reducing Sucrose Glucose Fructose

Fruit Orange Grapefruit Tangerine Lemon Lime Source:

2.03 1.66 1.13 1.40

2.48 1.75 1.54 1.35

4.51 3.41 2.67 2.75 3.48

4.81 2.56 6.53 0.41 0

9.32 5.97 9.20 3.16 3.48

(12)

The main sugars i n the peel of c i t r u s f r u i t s are a l s o sucr o s e , glucose and f r u c t o s e , although a t r a c e of f r e e xylose was reported (13J. The r e l a t i v e amounts of these sugars are shown i n Table IV. These f r e e sugars are a l s o the major i n g r e d i e n t s of c a t t l e feed manufactured fro c i t r u l d processing r e s i d u e e s p e c i a l l y when c i t r u s molasse Table IV.

Average Sugar Composition i n C i t r u s Peel Sucrose Fructose ( Ï dry weight)

Total sugar

F r u i t and variety

Time of harvest

Hamlin oranges Pineapple oranges V a l e n c i a oranges Marsh seedless grapefruit

December January April

10.3 10.8 10.9

16.8 21.2 10.9

11.8 13.9 16.5

38.8 45.8 37.4

December

11.6

12.8

14.3

38.7

Source:

Glucose

(13)

Polysaccharides and Polyuronides. These compounds are found i n the a l c o h o l - i n s o l u b l e f r a c t i o n and c o n s i s t of p e c t i c subs t a n c e s , h e m i c e l l u l o s e , c e l l u l o s e and l i g n i n . The recent i n t e r e s t of d i e t a r y f i b e r i n human n u t r i t i o n has placed s p e c i a l emphasis upon these substances i n foods. Between 45 and 75 percent of the t o t a l s o l i d s i n c i t r u s peel and membrane i s not s o l u b l e i n a l c o hol (13), and most of these a l c o h o l i n s o l u b l e s o l i d s c o n s i s t e d of polysaccharides or polyuronides (15). In orange f r u i t , the peel i s not g e n e r a l l y eaten except i n such s p e c i a l i t y products as candied peel or marmalade. The b i t t e r n e s s i n the peel and the segment membrane of g r a p e f r u i t i s due to n a r i n g i n and l i m o n i n (12) and makes t h a t p o r t i o n of the f r u i t u n p a l a t a b l e . Roe and Bruemmer (16) developed a method to d e b i t t e r the albedo by vacuum i n f u s i o n of t h i s t i s s u e w i t h n a r i n g i n a s e , thus, rendering the e n t i r e f r u i t , except the f l a v e d o , e d i b l e . A d i s t r i b u t i o n of the v a r i o u s component parts of d i f f e r e n t c i t r u s f r u i t i s shown i n Table V (17, 18). Approximately 20 percent of the weight of orange and 30 percent of t h a t of g r a p e f r u i t i s peel ( i n c l u d e s both

In Citrus Nutrition and Quality; Nagy, S., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

1.

TING

Nutrition

of

Citrus

7

Fruits

the albedo and f l a v e d o ) , and about 10 percent of the weight of the f r u i t i s segment membrane. Table V. D i s t r i b u t i o n of the Components of Oranae and Grapef r u i t (% Fresh Weight) Fruit

Peel

Segment Membrane

Juice vesicles

Seeds

Oranges Pineapple Valencia

19.9 19.2

13.7 9.0

62.6 71.0

3.8 0.8

Grapefruit Seedy

27.2

10.0

59.4

3.4

Source:

(17,

18)

About 30 percent of the polysaccharides i n the peel and pulp may be c l a s s i f i e d as c e l l u l o s e (Table V I ) . No separate c e l l u l o s e f r a c t i o n can be d i s t i n g u i s h e d from the j u i c e p o l y s a c c h a r i d e . Over 50 percent of the t o t a l polysaccharide i n the p e e l , 60 p e r cent or more of t h a t of the pulp and over 90 percent of t h a t of the j u i c e are e x t r a c t e d w i t h the p e c t i c substance, the balance being h e m i c e l l u l o s e . Separate h y d r o l y s i s of these f r a c t i o n s , i n d i c a t e d t h a t some monosaccharides, such as arabinose and g a l a c t o s e , were found i n a l l f r a c t i o n s . Xylose occurred mostly i n the h e m i c e l l u l o s e f r a c t i o n whereas g a l a c t u r o n i c a c i d and glucose were the main monosaccharides i n the p e c t i c substances and the c e l l u l o s e f r a c t i o n s , r e s p e c t i v e l y (Table V I I ) . D i e t a r y F i b e r . The d e f i n i t i o n of d i e t a r y f i b e r i s not very s p e c i f i c and i t g e n e r a l l y includes t h a t group of substances found i n the a l c o h o l - i n s o l u b l e f r a c t i o n s of c i t r u s f r u i t s . The crude f i b e r value as c o n v e n t i o n a l l y reported i s only t h a t p o r t i o n of the d i e t a r y f i b e r c o n s i s t i n g of p a r t i a l l y p u r i f i e d c e l l u l o s e and l i g n i n . Although these compounds are not attacked by the human d i g e s t i v e system as they t r a v e l through the alimentary t r a c t , they are s u b j e c t to p a r t i a l h y d r o l y s i s by the m i c r o f l o r a i n the lower p a r t of the d i g e s t i v e system. The b e n e f i t of d i e t a r y f i b e r has been a t t r i b u t e d to i t s a b i l i t y to decrease the t r a n s i t time of food through the g a s t r o i n t e s t i n a l t r a c t (19). Some f r a c t i o n of the d i e t a r y f i b e r such as p e c t i n has Been a s s o c i a t e d with the property of lowering the c h o l e s t e r o l i n mammals (20), and the methyl content of the polymer has been reported to be c o r r e l a t e d to t h i s c a p a b i l i t y . C i t r u s p e c t i n has a methyl content of 7-10 percent as compared to 6-9 percent f o r apple p e c t i n . P e c t i n from f l e s h y f r u i t s such as strawberry has only 0.2 percent (21). The polysaccharides of the peel and pulp of c i t r u s f r u i t s

In Citrus Nutrition and Quality; Nagy, S., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

CITRUS NUTRITION AND QUALITY

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In Citrus Nutrition and Quality; Nagy, S., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

In Citrus Nutrition and Quality; Nagy, S., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

(13, 17)

+ ++ +++ t --

Juice

Source:

t

+

P e c t i c substances Hemicellulose and Cellulose

Pulp t

t

t

t

Rhamnose

= 50% = trace = not found

+

t ++ +

+ +

P e c t i c substances Hemicellulose Cellulose

Peel

++

t ++ +

+ + +

Fractions

Xylose

Arabinose

P e c t i c substances Hemicellulose Cellulose

Component part + +++ t + +++ ——

+ + t + + + + ++

Glucose

Galactose

+

+++

+++ +

+++ t

t +

t +

Other uronic acids

Polysaccharide

Galacturonic acid

Table VII. R e l a t i v e Amounts of Various Monosaccharides Found i n the Hydrolysate of F r a c t i o r ι of the A l c o h o l - I n s o l u b l e S o l i d s of C i t r u s P e e l , Pulp and J u i c e

H ι—ι

VO

ο

10

CITRUS NUTRITION AND QUALITY

provide a good source of d i e t a r y f i b e r . Church and Church (22) reported t h a t an average s i z e orange could supply about 0.8 g of d i e t a r y f i b e r , whereas 236 ml (8 f l oz) s e r v i n g of j u i c e contains only o n e - h a l f t h a t amount. Organic A c i d s . The most predominate s o l u b l e c o n s t i t u e n t s of c i t r u s j u i c e , f o l l o w i n g the sugars, are the organic acids and t h e i r s a l t s . They represent about 10 percent of the t o t a l s o l u b l e s o l i d s i n c i t r u s j u i c e s . The proper r a t i o s of sugar and the acids and t h e i r b u f f e r s give the c i t r u s j u i c e s t h e i r d e l i g h t f u l t a s t e . The organic acids of c i t r u s f r u i t i n c l u d e a group of c a r b o x y l i c acids (23) w i t h d i f f e r e n t a c i d s predominant i n various component parts of the f r u i t . C i t r i c a c i d i s the main a c i d i n the j u i c e , r e p r e s e n t i n g from 80 percent of the t o t a l a c i d i t y i n j u i c e from ripened oranges, about 90 percent of t h a t of g r a p e f r u i t and n e a r l y a l l of t h a t o a f f e c t s the sourness o by the c a t i o n s , e s p e c i a l l y potassium. The major a c i d s i n c i t r u s peel are m a l i c , o x a l i c (25), malonic (26), and qui n i c (27). Organic acids are metabolized i n the body and should be considered as a source of energy. In the case of the s a l t s of these a c i d s , the organic p o r t i o n i s metabolized l e a v i n g the f r e e c a t i o n s to be combined with other anions. Thus, c i t r u s j u i c e i s c l a s s e d as an a l k a l i n e food (28). Protein The amount of p r o t e i n i n c i t r u s f r u i t i s r e l a t i v e l y low (Table I I ) , and the j u i c e and peel have about the same amount (29). Much of the value t h a t i s considered as p r o t e i n i s e i t h e r f r e e amino a c i d s or non-protein c o n s t i t u e n t s which c o n t a i n n i t r o g e n . The t o t a l n i t r o g e n of orange j u i c e s was found to increase w i t h the m a t u r i t y of the f r u i t and ranged between .068 to .120 g per 100 ml (30). The a c t u a l p r o t e i n values obtained by Clements (31) were about 20 percent of the acetone powder. Nearly 30 percent of the a l c o h o l - i n s o l u b l e s o l i d s of j u i c e and about 20 percent of t h a t of v e s i c u l a r pulp were found to be p r o t e i n as determined by the K j e l d a h l procedure (32). These values are the actual p r o t e i n that was p r e c i p i t a t e d by a l c o h o l and are only a f r a c t i o n of the t o t a l p r o t e i n values u s u a l l y reported f o r orange j u i c e ( 8 ) . The main source of p r o t e i n s i n c i t r u s j u i c e i s probably i n the form of enzymes and the p l a s t i d s . At l e a s t 47 d i f f e r e n t enzymes have been reported to occur i n c i t r u s f r u i t s (33). C i t r u s f r u i t s a l s o contain several phenolic amines (34), some of which such as synepherine, may have p h y s i o l o g i c a l importance (35). Among the v a r i o u s f r e e amino acids reported i n c i t r u s j u i c e s (32), a r g i n i n e i s the only semi-indispensable amino a c i d t h a t occurs i n moderate amounts. The m a j o r i t y of amino acids i n c i t r u s are considered to be nonessential according to the c l a s s i f i c a t i o n

In Citrus Nutrition and Quality; Nagy, S., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

1.

TING

Nutrition

of Citrus

11

Fruits

by Block and B o i l i n g (36). At most, the c o n t r i b u t i o n of f r e e amino acids i n c i t r u s j u i c e s to human n u t r i t i o n i s minimal. Lipids From a d i e t a r y s t a n d p o i n t , the c o n t r i b u t i o n of c i t r u s l i p i d s i s i n s i g n i f i c a n t ; and only between .06 and .09 percent has been found i n oranges (37). They a r e , however, of importance because of t h e i r e f f e c t s on the development of o f f - f l a v o r s (12), thus lowering the p a l a t a b i l i t y of these products. The near absence of l i p i d s i n c i t r u s makes i t a d e s i r a b l e food f o r those on a limited fat diet. Micronutrients in Citrus The term micro i s used purely to i n d i c a t e the p h y s i c a l t i t i e s required in n u t r i t i o These n u t r i e n t s represen stances t h a t have d i e t a r y s i g n i f i c a n c e .

quan­

F a t - S o l u b l e Vitamins i n C i t r u s F r u i t Among the several vitamins i n t h i s c l a s s i f i c a t i o n , only v i t a m i n A i s present i n a p p r e c i a b l e q u a n t i t y as c a r o t e n o i d p r o ­ v i t a m i n A i n c i t r u s (38). No v i t a m i n D has ever been reported i n c i t r u s nor any p l a n t v i t a m i n D p r e c u r s o r s , such as e r g o s t e r o l . Several of the s t e r o l s present i n c i t r u s f r u i t s are reported (39, 40, 4 1 ) , but they are not r e l a t e d to v i t a m i n D. Vitamin Ε. The amount of vitamin Ε i n c i t r u s i s n u t r i t i o n ­ a l l y i n s i g n i f i c a n t . Braddock (42) reported only 0.1 mg i n 100 ml of orange j u i c e . The U.S. RDA f o r t h i s v i t a m i n i s 30 mg. Newhall and Ting (43) found as much as 1 mg i n 100 grams of f l a v e d o on a f r e s h weight b a s i s . Its a n t i o x i d a n t property plays an impor­ t a n t r o l e i n the keeping q u a l i t y of c i t r u s o i l s . Vitamin A. The v i t a m i n A of c i t r u s f r u i t i s e n t i r e l y i n the form of provitamin A c a r o t e n o i d s . The carotenes (both the alpha and beta form) and the cryptoxanthins i n c i t r u s are considered as the main p r e c u r s o r s . The carotenes are only a minor component of the t o t a l carotenoids of oranges ranging from about 5 to 10 p e r ­ cent (44-46). In Dancy tangerine and V a l e n c i a oranges, c r y p t o xanthin i s the main v i t a m i n A precursor (47, 48). High perform­ ance l i q u i d chromatography (HPLC) has been usëcT to separate cryptoxanthin from other oxygenated carotenoids (413, 50). Only the b e t a - c r y p t o x a n t h i n has provitamin a c t i v i t y . P r i o r use of open column chromatography (51) could not separate the d i f f e r e n t monooxygenated c a r o t e n o i d s , thus g i v i n g higher v a l u e s . Using the HPLC method, Stewart (52) analyzed the carotenoids of several v a r i e t i e s of oranges and mandarins, and found t h a t b e t a - c r y p t o x a n t h i n i s the

In Citrus Nutrition and Quality; Nagy, S., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

12

CITRUS NUTRITION AND QUALITY

main provitamin A c i t r u s c a r o t e n o i d . The mandarin-type c i t r u s f r u i t i s a good source o f provitamin A, whereas oranges are s i g n i f i c a n t l y poorer (Table V I I I ) . With g r a p e f r u i t , the white v a r i e t i e s have no v i t a m i n A, but the pink and red v a r i e t i e s have been found to c o n t a i n a p p r e c i a b l e amounts o f beta-carotene (53, 54). Table V I I I .

Vitamin A Content o f C i t r u s J u i c e s % U. S. RDA 177 ml (6 f 1 . oz)

Fruit E a r l y and mid-season Late season Tangerines Dancy Honey Robinson Tangélos Orlando Grapefruit White Ruby Source:

1.6-2.7 1.7 19 6 2 4.7 None 30

(44, 52-54)

Wàter-Sôluble Vitamins i n C i t r u s F r u i t Vitamin C. Perhaps the most important c o n t r i b u t i o n of c i t rus f r u i t s to human n u t r i t i o n i s a t t r i b u t e d to t h e i r high ascorbic acid. Although c i t r u s products are not the only source f o r high contents of v i t a m i n C among f r u i t s and v e g e t a b l e s , t h e i r p o p u l a r i t y are l a r g e l y due to t h e i r d e s i r a b l e f l a v o r , t a s t e and c o l o r . In Table IX (55) are l i s t e d some of the more common f r u i t s and vegetables and t h e i r v i t a m i n C content. An average 177 ml (6 f l oz) s e r v i n g o f e i t h e r orange or g r a p e f r u i t j u i c e could provide 100 percent U.S. RDA of v i t a m i n C (60 mg). Tangerine j u i c e , a l though c o n t a i n i n g l e s s v i t a m i n C than other c i t r u s j u i c e s , would provide a s u b s t a n t i a l amount toward the recommended d a i l y a l l o w ance. Nearly 3/4 of a l l v i t a m i n C i n an orange and 5/6 i n a grapef r u i t i s found i n the peel ( 5 6 ) , however, c i t r u s j u i c e s and t h e i r products provide a major p o r t i o n of the v i t a m i n C i n the American d i e t . Considerable v a r i a t i o n s i n v i t a m i n C content can be found i n d i f f e r e n t c i t r u s products due to such f a c t o r s as v a r i e t y s , m a t u r i t y and c u l t u r a l p r a c t i c e s of the f r u i t (57) from which the products o r i g i n a t e and to the processing p r a c t i c e s and storage c o n d i t i o n s of these products before they reach the consumer. The decrease of a s c o r b i c a c i d w i t h f r u i t m a t u r i t y i s i l l u s -

In Citrus Nutrition and Quality; Nagy, S., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

1.

TING

Nutrition

of

Citrus

13

Fruits

Table IX. Average Vitamin C Content of Freshly Harvested F r u i t s and Vegetables Product Guava Broccoli Green pepper Turnip greens Cabbage Orange Lemon Grapefruit Tangerine Potato Tomato Pineapple Banan Appl Peaeh Source:

mg/100 g 300 120 120 120 60 50 50 40 25 30 25 25 4

(55)

t r a t e d i n Figure 1. Vitamin C content of the e a r l y and midseason oranges are higher than t h a t of the l a t e season f r u i t . These f a c t s i n d i c a t e t h a t high q u a l i t y does not c o r r e l a t e w i t h higher n u t r i t i o n a l q u a l i t y . The more mature f r u i t and those of the l a t e season v a r i e t y are g e n e r a l l y regarded as of b e t t e r q u a l i t y . S t a b i l i t y of Vitamin C i n C i t r u s F r u i t s and J u i c e s The ease of o x i d a t i o n of reduced a s c o r b i c a c i d i s the b a s i s f o r a simple method of a n a l y s i s by dye t i t r a t i o n (58). Ascorbic a c i d as i t occurs i n c i t r u s j u i c e i s i n the reduced form. When subjected to o x i d a t i o n , a s c o r b i c a c i d changes to the dehydro form. Dehydroascorbic a c i d has nearly the same p h y s i o l o g i c a l a c t i v i t y as the reduced form and i s e a s i l y converted to the l a t t e r . Further o x i d a t i o n of the dehydroascorbic a c i d converts i t to 2 , 3 - d i k e t o gulonic a c i d . This r e a c t i o n i s i r r e v e r s i b l e , and the o x i d i z e d product i s devoid of b i o l o g i c a l a c t i v i t y . These r e a c t i o n s are shown i n Figure 2. Nearly 90 percent or more of the v i t a m i n C found i n c i t r u s j u i c e and c i t r u s products i s i n the reduced form (Table X) (59). Vitamin C i n c i t r u s j u i c e i s remarkably s t a b l e during the short period i t i s g e n e r a l l y kept a f t e r e x t r a c t e d from the f r u i t . F r e s h l y e x t r a c t e d orange and g r a p e f r u i t j u i c e s r e t a i n e d about 98 percent of t h e i r o r i g i n a l v i t a m i n C at 21,1 f o r 3 days. At 4.4 orange and g r a p e f r u i t j u i c e s r e t a i n e d 96 and 99 percent, respect i v e l y , of the o r i g i n a l amount a f t e r one-week's storage (60). Orange j u i c e t h a t has been heated to b o i l i n g f o r 15 min s t i l l r e t a i n e d about 96 percent of the v i t a m i n C (59). Atmospheric oxygen Q

In Citrus Nutrition and Quality; Nagy, S., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

14

CITRUS NUTRITION AND QUALITY

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In Citrus Nutrition and Quality; Nagy, S., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

PC

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s

In Citrus Nutrition and Quality; Nagy, S., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

46 15 12 410 305 73 34

27 + 1.0 8 + 0.0 t 175 + 5.0 90 + 0.0 26 + 2.0 10 + 1.0

Hamlin Pineapple Valencia Robinson Dancy Orlando Murcott

+ + + + + + +

+ + + + + + +

42 87 127 84 130 55 80

0.0 9.0 9.0 5.0 4.0 1.0 3.0

+ + + + + + +

61 110 68 65 120 16 150

Hamlin Pineapple Valencia Robinson Dancy Orlando Murcott

0.0 1.0 0.0 20 15.0 0.0 2.0

0.0 0.0 0.0 0.0 1.0 0.0 1.0

t t t 300 + 10.0 54 + 2.0 9 + 1.0 t

t t t 85 + 5.0 t t t

Hamlin Pineapple Valencia Robinson Dancy Orlando Murcott

10-27

10-7

(Continued)

Cultivar

Table I.

15.0 10.0 0.0

2.0

115 59 8 660 465 210 74

+ + + + + + + 5.0 2.0 0.0 0.0 25.0 0.0 1.0

20 + 0.0 36 + 0.0 51 + 0.0 t 101 + 0.0 27 + 1.0 75 + 9.0

17 + t t 465 + 140 + 64 + t

11-19

2.0

2.0

1.0 9.0

Violaxanthin 260 + 10.0 160 + 0.0 120 + 0.0 735 + 15.0 1005 + 15.0 460 + 20.0 195 + 5.0

Lutein t 25 + 38 + t 150 + t 56 +

3-Citraurin 47 + 2.0 65 + 0.0 10 + 0.0 665 + 45.0 545 + 50.0 205 + 5.0 57 + 2.0

305 405 265 1115 1275 885 245

+ 5.0 + 1.0 + 5.0 + 5.0 + 5.0 +_ 5.0 + 5.0

1090 + 20.0 1235 + 5.0 1040 + 0.0 2705 + 55.0 1160 + 10.0 1645 + 5.0

2000 + 20.0 625 + 5.0 1065 + 35.0

t t

t t t t

485 + 5.0 285 + 5.0

210 + 0.0 175 + 5.0 54 + 2.0

2-23

945 + 15.0 855 + 35.0 540 + 10.0

47 + 0.0 t 170 + 27.0 t t

320 + 0.0 215 + 5.0

255 + 5.0 245 + 5.0 58 + 0.0

43 + 0.0 t 130 + 5.0 t 29 + 1.0

5.0 5.0 1.0 25.0 10.0 5.0 0.0

t t

+ + + + + + +

1-28

t t

105 145 43 755 700 445 80

Sampling dates 12-19 1_7

t

t t t

215 + 15.0

185 + 5.0 160 + 0.0 190 + 0.0

3-30

β-CAROTENE

Figure 4.

Comparison of vitamin A and β-carotene. The bond between C C ' is broken as an initial step in forming vitamin A.

15

15

In Citrus Nutrition and Quality; Nagy, S., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

and

7.

STEWART

Color

As

Related

to

Citrus

Quality

141

p e r i o d , the synthesis of carotenoids could be v i r t u a l l y con­ t r o l l e d by changing temperature and ethylene concentrations Carotenoids

(9).

i n Juice

The carotenoids i n c i t r u s j u i c e i n comparison with those i n the p e e l d i f f e r i n two important respects. In F l o r i d a , we have found no C-30 carotenoids i n j u i c e . This i s based on observa­ tions made on s e v e r a l hundred samples, using a v a r i e t y of tech­ niques. However, Gross et aJL. (32) reported f i n d i n g c i t r a u r i n i n Shamouti orange j u i c e and i n V a l e n c i a orange j u i c e (48). Secondly, our studies (49) i n d i c a t e that the main carotenoids i n orange and tangerine j u i c e are α-carotene, |3-carotene, zeta carotene, α-cryptoxanthin, {3-cryptoxanthin, l u t e i n zeaxanthin, antheraxanthin and v i o l a x a n t h i n (Figure 3). Although anther­ axanthin and v i o l a x a n t h i n occur i n the l a r g e s t amounts, they are yellow pigments, g-cryptoxanthin main c o n t r i b u t o r to the In pink and red g r a p e f r u i t j u i c e s the main pigment i s lycopene. Ting and Deszyck (50) found 1.3 mg and 2.3 mg of lycopene i n 100 g of pink and red g r a p e f r u i t , r e s p e c t i v e l y . They a l s o reported the f r u i t to have 0.5 mg to 1.4 mg β-carotene per 100 g. P u r c e l l (5_1, 52) found up to 1.8 mg lycopene and 0.49 mg carotene/100 g i n j u i c e sacs i n Texas Ruby Red g r a p e f r u i t . Maximum lycopene content was reached i n August and September and dropped o f f a f t e r that time. Very l i t t l e i s known regarding f a c t o r s that a f f e c t the c o l o r of c i t r u s j u i c e . The author has used many combinations of temperature and ethylene with f r u i t of s e v e r a l c u l t i v a r s . Although the peel c o l o r was a f f e c t e d , no changes were observed i n the j u i c e c o l o r . Recently, Houck et a_l. (_53) found storage atmospheres c o n t a i n i n g 407 and 807 O2 caused navel but not V a l e n c i a endocarp to turn a darker orange a f t e r 2 weeks of storage. This i s the only work that we are aware of i n which postharvest treatment i n f l u e n c e d the j u i c e c o l o r . o

o

Color and Provitamin A I t i s well-known that plants do not synthesize vitamin A. A l s o , animals can only synthesize vitamin A from β-carotene or carotenoids i n which one-half of the molecule i s l i k e β-carotene. In nature, the v i t a m i n A precursor comes e i t h e r from plants or microorganisms. The most common sources of vitamin A i n c i t r u s are a- and β-carotenes and β-cryptoxanthin. In a d d i t i o n to the above name c a r o t e n o i d s , β-apo-S -carotenal i n c i t r u s peel could be a source of provitamin A. However, the peel i s not u s u a l l y consumed. Provitamin A compounds are cleaved to form vitamin A aldehyde i n the i n t e s t i n e by β-carotene 15,15 -oxygenase (Figure 4) (54). Aldehyde reductase reduces the aldehyde to the a l l t r a n s - v i t a m i n A. β-Carotene i s cleaved between the 15,15' carbon 1

1

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Provitamin A Content of C i t r u s

Juice

A N D QUALITY

(49)

b Daily recommended

Vitamin A va lue , Cultivar 80

1.6

133

2.7

Valencia

83

1.7

Robinson

1142

23

Dancy

965

19

Orlando

236

Murcott

3195

Hamlin Pineapple

4.7 64

C a l c u l a t i o n s based o n : β - c a r o t e n e e q u a l s 1.667 I n t e r n a t i o n a l u n i t s v i t a m i n A ^ g , e q u a l s 100%; α - c a r o t e n e , 52.7%; βc r y p t o x a n t h i n , 577 . C o n v e r s i o n f a c t o r f r o m 100 mL o f j u i c e t o 6 o z , 1.77 ( 7 0 ) . 0

Values daily

based on 6 oz o f j u i c e allowance

and c a l c u l a t e d

on the

dietary

o f 5000 I U .

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atoms g i v i n g two molecules of r e t i n a l . α-Carotene, which con­ s i s t s of a β r i n g and an α r i n g , i s s p l i t but only the β r i n g p o r t i o n i s a c t i v e . With β-cryptoxanthin, which i s the l a r g e s t source of provitamin A i n c i t r u s j u i c e , the 3-OH r i n g h a l f i s not a c t i v e , l e a v i n g only the β-ring s i d e . In case of acryptoxanthin, which l i k e α-carotene contains an α r i n g i n a c t i v e and a β r i n g which would be a c t i v e provided the OH group was on the α r i n g . However, i n case of c i t r u s , the OH group i n α-cryptoxanthin i s on the 3 carbon of the β - r i n g (40). V i o l a x a n t h i n , the carotenoid that occurs i n the l a r g e s t amounts i n c i t r u s j u i c e , does not have any provitamin A a c t i v i t y because of s u b s t i t u t i o n s on both r i n g s . The v i t a m i n A content of orange j u i c e as given by Adams (55) i s about 200 I n t e r n a t i o n a l u n i t s of provitamin A i n 100 g. In more recent studies (Table II) (49), orange j u i c e was found to have the l e a s t amount of provitamin A whereas Murcott (a reputed tangor) had the highest lowest found was i n the units/6 oz of j u i c e . The highest was Murcott with 3195 I n t e r n a t i o n a l u n i t s / 6 oz of j u i c e . A l l samples were taken from mature f r u i t except V a l e n c i a . When mature, V a l e n c i a j u i c e normally contains more provitamin A than Hamlin or Pineapple juice. In g r a p e f r u i t j u i c e there was a wide d i f f e r e n c e between provitamin A i n white and red or pink. White g r a p e f r u i t j u i c e contained approximately 8 I n t e r n a t i o n a l u n i t s of provitamin A per 100 g of j u i c e , whereas pink and red contained 440 (55). This d i f f e r e n c e i n provitamin A i s due to β-carotene. Ting and Deszyck (50) showed red and pink g r a p e f r u i t j u i c e to c o n t a i n 1.0 to 1.4 mg of β-carotene per 100 g of j u i c e when the f r u i t was f u l l y mature. When c a l c u l a t e d on the value of 1.667 I n t e r n a t i o n a l u n i t s of vitamin A = 1 μg of β-carotene, the F l o r i d a j u i c e would have contained the equivalent of 1667 to 2334 u n i t s of v i t a m i n A/100 g. P u r c e l l (5_1, 52) reported approximately one-half t h i s value f o r red g r a p e f r u i t j u i c e from Texas f r u i t . Standards f o r Color i n C i t r u s and C i t r u s Products The United States Department of A g r i c u l t u r e (56), the F l o r i d a Department of C i t r u s (_57), and the C a l i f o r n i a Department of Food and A g r i c u l t u r e (58) have set standards f o r grades of f r e s h f r u i t , concentrated orange j u i c e and other c i t r u s products. Processed products are given one of two grades by government i n s p e c t o r s : U. S. Grade A and U. S. Grade B. Any product graded below t h i s i s c l a s s e d as substandard. In grading c i t r u s pro­ ducts, a s c o r i n g system based on 100 points i s used. In case of orange j u i c e the system i s as f o l l o w s :

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Points 40

defects

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flavor

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t o t a l score

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100

Orange j u i c e with good c o l o r , having a c o l o r score of 36 to 40 p o i n t s , may be c l a s s i f i e d as Grade A. J u i c e with good c o l o r but with a c o l o r score of 32 to 35 cannot be graded above U. S. Grade Β regardless of other c h a r a c t e r i s t i c s . Orange j u i c e with a c o l o r score of 31 or below must be graded substandard. Two methods are used to measure c o l o r o f orange j u i c e . The o l d e s t of these procedures i s made by comparing the c o l o r of r e c o n s t i t u t e d products i f i t i s concentrated with the USDA orange j u i c e c o l o r standards yellow to yellow orange pared with the standard tubes using s p e c i f i e d l i g h t i n g conditions. The second method f o r e v a l u a t i n g c o l o r i s by the use o f c o l o r ­ imeters approved by the USDA. In F l o r i d a , the Department of C i t r u s has r u l e d that the Hunterlab Model D-45 C i t r u s Colorimeter s h a l l be used e x c l u s i v e l y to determine the c o l o r scores f o r orange j u i c e . The Hunterlab C i t r u s Colorimeter was developed i n 1963 f o r the F l o r i d a C i t r u s Commission. This i s a r e f l e c t a n c e instrument that measures c i t r u s redness (CR) and c i t r u s yellow­ ness (CY). By means of a formula, the c o l o r score i s c a l c u l a t e d : Color score = 22.51 + (CR χ 0.165) + (CY χ 0.11). Color scores obtained with the c i t r u s c o l o r i m e t e r have been c o r r e l a t e d with those using the USDA c o l o r standards (_59, 60). In F l o r i d a , there are r e g u l a t i o n s on the d i l u t i o n of con­ centrated orange j u i c e that can s u b s t a n t i a l l y a f f e c t the c o l o r score. For example, j u i c e concentrated to 45°Brix must be r e c o n s t i t u t e d to 12.8°Brix before a c o l o r determination i s made. That concentrated to 42°Brix must be r e c o n s t i t u t e d to 11.8°Brix. Concentrated orange j u i c e f o r manufacturing i s r e c o n s t i t u t e d to 12.3°Brix. G r a p e f r u i t j u i c e standards l i k e those f o r orange j u i c e a l l o c a t e 40 points out of 100 f o r c o l o r . However, the s i m i l a r i t y of s c o r i n g the two j u i c e s ends at that point. There are no instrumental methods i n use f o r determining the c o l o r of grape­ f r u i t j u i c e . Rather ambiguous terms are used to d e s c r i b e c o l o r of g r a p e f r u i t j u i c e such as U. S. Grade A or U. S. Fancy which requires that the j u i c e have "very good c o l o r , " meaning that i t i s b r i g h t and t y p i c a l o f f r e s h e x t r a c t e d g r a p e f r u i t j u i c e . I t may be pale yellow to very s l i g h t l y amber, t y p i c a l of white g r a p e f r u i t j u i c e or s l i g h t l y red t y p i c a l of red or pink grape­ f r u i t juice. Obviously, i t i s d i f f i c u l t to enforce these regulations. Standards f o r c o l o r of a l l other processed c i t r u s products are e q u a l l y as s u b j e c t i v e . This does not i n f e r that

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there are not s a t i s f a c t o r y o b j e c t i v e techniques f o r determining the c o l o r of processed c i t r u s other than orange j u i c e . Huggart and Petrus (61) and Huggart et a l . (62_) found the v i s u a l c o l o r changes i n white and pink g r a p e f r u i t j u i c e were c l o s e l y a s s o c i a t e d with Hunterlab C i t r u s Colorimeter measurements. P r e v i o u s l y , Ting and Deszyck (50) had shown a c l o s e c o r r e l a t i o n between Hunter a/b values and lycopene content i n pink and red grapefruit. Color Standards

f o r Fresh F r u i t

In F l o r i d a , c o l o r standards f o r f r e s h f r u i t are based e n t i r e l y on the c o l o r of the p e e l (57). S u b j e c t i v e means are used to d e s c r i b e the c o l o r . For example, some of the USDA c o l o r grade standards f o r F l o r i d a oranges and tangelos are: U. S. Fancy - W e l l - c o l o r e d meaning that the f r u i t i s yellow or orange i n c o l o r wit U. S. No. 1 B r i g h t - E a r l f a i r l y w e l l - c o l o r e d . This means the yellow and orange c o l o r s predominate over the green c o l o r . An aggregate area of green c o l o r may not exceed the area of a c i r c l e 1 inch i n diameter. For V a l e n c i a s and other l a t e v a r i e t i e s not l e s s than 50? by count s h a l l be f a i r l y w e l l - c o l o r e d and the remainder reasonably w e l l - c o l o r e d . Reasonably w e l l - c o l o r e d means the yellow or orange c o l o r predominates on at l e a s t two-thirds of the f r u i t surfaces i n the aggregate. There are s t i l l other grades with d e s c r i p t i v e c o l o r requirements. In C a l i f o r n i a , the c o l o r requirements are much d i f f e r e n t (58). The c o l o r of the peel i s t i e d i n with the j u i c e q u a l i t y , namely, the r a t i o which i s the s o l u b l e s o l i d s d i v i d e d by the a c i d content. F r u i t having a b r i g h t p e e l c o l o r can be marketed with poorer j u i c e q u a l i t y (lower r a t i o ) than that with poor peel color. This i s a case where the best looking f r u i t may not be the best t a s t i n g . o

Color Enhancement i n Fresh F r u i t In F l o r i d a , i t i s not uncommon i n the f a l l f o r the i n t e r n a l part of the f r u i t to be p a l a t a b l e at maturity but the p e e l to be mostly green with a c o l o r break. A f t e r the f r u i t has passed through the degreening process, i t has a yellow appearance. The pigments are mainly l u t e i n , v i o l a x a n t h i n , and neoxanthin. This f r u i t may then be passed through a color-add tank c o n t a i n i n g C i t r u s Red No. 2 (6_3). The f r u i t i s then thoroughly r i n s e d and a maximum of 2 ppm residue i s permitted. The o p e r a t i o n i s time and temperature dependent and takes place under the s u p e r v i s i o n of government i n s p e c t i o n (_57). Color-add i s permitted only on oranges, Temples and tangelos and the f r u i t must have higher i n t e r n a l q u a l i t y than n a t u r a l c o l o r e d f r u i t .

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Color Enhancement of Orange J u i c e No a r t i f i c i a l c o l o r i n g m a t e r i a l i s permitted to be added to orange j u i c e . Almost one-half of the orange j u i c e processed i n F l o r i d a comes from e a r l y and midseason v a r i e t i e s . In many cases t h i s j u i c e does not have a c o l o r score of 35, the minimum r e quired f o r Grade A. Color standards are met by blending. This i s done i n s e v e r a l ways: (a) E a r l y and midseason orange j u i c e i s stored and blended with f r u i t from the late-maturing V a l e n c i a f r u i t which has more than s u f f i c i e n t c o l o r to meet Grade A standards; (b) V a l e n c i a j u i c e may be stored u n t i l the f o l l o w i n g season when i t i s blended; (c) Tangerine and h y b r i d j u i c e which has a b r i g h t c o l o r may be blended but only to a maximum of 10? , according to FDA standards of i d e n t i t y f o r orange j u i c e ; (d) F i n a l l y , another source of c o l o r f o r orange j u i c e i n F l o r i d a i s the use of imported j u i c e s e l e c t e d f o r b r i g h t c o l o r . In the l a b o r a t o r y orange p e e l f o r the purpos Ting and Hendrickson (64) e x t r a c t e d orange peel with acetone and hexane. The peel e x t r a c t from 1226 kg of pineapple oranges was s u f f i c i e n t to increase the c o l o r score i n 3785 & of j u i c e from 37 to 38. In other studies (65), i t was pointed out that 1 g of c o l o r concentrate from e i t h e r Pineapple or V a l e n c i a peel i n 2 l i t e r s of j u i c e increased the c o l o r score from 35 to 40. In s i m i l a r s t u d i e s , Berry et a_l. (66) reported on the e x t r a c t i o n of carotenoids from c i t r u s p e e l with hexane. Another approach to the same problem was to concentrate the chromoplasts of deeply c o l o r e d tangerine j u i c e . Barron e_t a l . (67) and Barron and M e t c a l f (68) d i d t h i s by means of c e n t r i fuging j u i c e and c o l l e c t i n g the c o l o r e d p e l l e t s . I t has been suggested that the r e g u l a r run V a l e n c i a orange j u i c e be s p l i t and h a l f be used f o r c o l o r e x t r a c t i o n . The chromoplasts would be used to improve the c o l o r of j u i c e from e a r l y and midseason varieties. The j u i c e l e f t from the chromoplast e x t r a c t i o n would be mixed with the remaining h a l f of the V a l e n c i a j u i c e , g i v i n g a c o l o r score s u f f i c i e n t l y high to meet Grade A standards. N a t u r a l l y o c c u r r i n g c o l o r i n g m a t e r i a l obtained by t h i s means could be added to orange j u i c e without any a d u l t e r a t i o n being considered a c o l o r a d d i t i o n (69). None of the c o l o r concentrates are p r e s e n t l y being used i n orange j u i c e . o

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Literature Cited 1. Campbell, C. W. Fla. Agr. Exp. Sta. Bull. 187, Gainesville, FL, 1979. 2. Sites, J. W.; Deszyck, E. J. Proc. Fla. State Hort. Soc., 1952, 65, 92. 3. Reese, R. L.; Koo, R. C. J. Proc. Fla. State Hort. Soc., 1974, 87, 1. 4. Lewis, L. N.; Coggins, C. W. Plant and Cell Physiol., 1964, 5, 457. 5. Eilati, S. K.; Goldschmidt, Ε. E.; Monselise, S. P. Experientia, 1969, 25, 209. 6. Stearns, C. R. Jr.; Young, G. T. Proc. Fla. State Hort. Soc., 1942, 55, 59. 7. Young, L. B.; Erickson, L. C. Proc. Am. Soc. Hort. Sci., 1961, 78, 197. 8. Sonnen, H. D.; Lenz 1979, 44 (2), 49. 9. Wheaton, Τ. Α.; Stewart, I. J. Am. Soc. Hort, Sci., 1973, 98, 337. 10. Vines, H. M.; Grierson, W.; Edwards, G. J. Proc. Am. Soc. Hort. Sci., 1968, 92, 227. 11. Apelbaum, Α.; Goldschmidt, Ε. E.; Ben-Yehoshua, S. Plant Physiol., 1976, 57, 836. 12. Shimokawa, K. Scientia Hort., 1979, 11, 253. 13. Jahn, O. L.; Young, R. J. Am. Soc. Hort. Sci., 1976, 101, 416. 14. Barmore, C. R. HortScience, 1975, 10, 595. 15. Shimokawa, K.; Shimada, S.; Yaco, K. Scientia Hort., 1978, 8, 129. 16. Shimokawa, K.; Sakanoshita, Α.; Horiba, K. Plant and Cell Physiol., 1978, 19, 229. 17. Grierson, W.; Newhall, W. F. Fla. Agr. Exp. Sta. Bull. 620, Gainesville, FL, 1979. 18. Denny, F. E. J. Agr. Res., 1924, 27, 757. 19. Wardowski, W. F.; McCornack, A. A. Fla. Agr. Exp. Sta. Circ. 389, Gainesville, FL, 1973. 20. McCornack, Α. Α.; Wardowski, W. F. In "Proc. Int. Soc. Citriculture", Grierson, W. ed., P. O. Box 1088, Lake Alfred, FL, 1977, 1, p. 211. 21. Cohen, E. In "Proc. Int. Soc. Citriculture", Grierson, W. ed., P. O. Box 1088, Lake Alfred, FL, 1977, 1, p. 215. 22. Kitagawa, H.; Kawada, K.; Tarutani, T. In "Proc. Int. Soc. Citriculture", Grierson, W. ed., P. O. Box 1088, Lake Alfred, FL, 1977, 1, p. 219. 23. Eaks, I. L. In "Proc. Int. Soc. Citriculture", Grierson, W. ed., P. O. Box 1088, Lake Alfred, FL, 1977, 1, p. 223. 24. Brown, G. E.; Barmore, C. R. Proc. Fla. State Hort. Soc., 1976, 89, 198.

In Citrus Nutrition and Quality; Nagy, S., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

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25. Barmore, C. R.; Brown, G. E. Plant Pis. Reptr., 1978, 62, 541. 26. Zechmeister, L.; Tuzson, P. Naturwissenschaften, 1931, 19, 307. 27. Zechmeister, L.; Tuzson, P. Hoppe Seylers Zeitschriet Fir Physiol. Chem., 1931, 221, 278. 28. Zechmeister, L.; Tuzson, P. Ges. Deut. Chem. Ber., 1936, 69, 1878. 29. Natarajan, C. P.; MacKinney, G. Indian J. Sci. and Indus. Res., 1952, 11B, 416. 30. Stewart, I.; Wheaton, T. A. In "I Congreso Mundial de Citricultura", Carpena, O. ed.; Ministerio de Agricultura, Murcia, España, 1973, 2, p. 325. 31. Yokoyama, H.; Vandercook, C. E. J. Food Sci., 1967, 32, 42. 32. Gross, J.; Gabai, M.; Lifshitz, A. J. Food Sci., 1971, 36, 466. 33. Subbarayan, C.; Cama 34. Stewart, I. In "Proc , , W., ed., P. O. Box 1088, Lake Alfred, FL, 1977, 1, p. 308. 35. Straub, O. In "Carotenoids" and "Supplement." Isler, O., ed., Birkhauser Verlag, Basel, 1971, p. 771. 36. Weeden, B. C. L. In "Chemistry of the Carotenoids", Goodwin, T. W., ed., Academic Press, New York, 1965, p. 112. 37. Stewart, I.; Wheaton, T. A. J. Chromatography, 1971, 55, 325. 38. Rodriquez, D. B.; Tanaka, Y.; Katayama, T.; Simpson, K. L.; Lee, T.; Chichester, C. O. J. Agr. Food Chem., 1976, 24, 819. 39. Stewart, I.; Wheaton, T. A. Phytochemistry, 1973, 12, 2947. 40. Stewart, I. J. Assoc. Off. Anal. Chem., 1977, 60, 132. 41. Sonnen, H. P. In "Proc. Int. Soc. Citriculture", Grierson, W., ed., P. O. Box 1088, Lake Alfred, FL, 1977, 3, p. 1089. 42. Stewart, I.; Wheaton, T. A. Proc. Fla. State Hort. Soc., 1971, 84, 264. 43. Weeden, B. C. L. In "Carotenoids", Isler, O., ed., Berkhäuser Verlag, Basel, 1971, p. 48. 44. Leuenberger, U.; Stewart, I. Phytochemistry, 1976, 15, 227. 45. Leuenberger, U.; Stewart, I. J. Org. Chem., 1976, 41, 891. 46. Taylor, R.; Davies, B. Biochem. J . , 1974, 139, 75. 47. Stewart, I.; Wheaton, T. A. J. Agr. Food Chem., 1972, 20, 448. 48. Gross, J . ; Carmon, M.; Lipshitz, A. J. Food Sci., 1971, 36, 466. 49. Stewart, I. J. Agr. Food Chem., 1977, 25, 1132. 50. Ting, S. V.; Deszyck, E. J. Proc. Am. Soc. Hort. Sci., 1958, 71, 271. 51. Purcell, A. E. J. Rio Grande Valley Hort. Soc., 1959, 13, 39. 52. Purcell, A. E. J. Rio Grande Valley Hort. Soc., 1959, 13, 45. 53. Houck, L. G.; Aharoni, Y.; Fouse, P. C. Proc. Fla. State Hort. Soc., 1978, 91, 136.

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54. Pitt, G. A. J. In "Carotenoids", Isler, O., ed., Birkhäuser Verlag, Basel, 1971, p. 771. 55. Adams, C. F. Agriculture Handbook No. 456, Agric. Res. Serv., U. S. Dept. of Agric, Washington, D. C. (1975). 56. U. S. Standards for Grades of Florida Oranges and Tangelos 1975; Florida Grapefruit 1975. Florida Tangerines 1975; Grades of Lemons 1964; Orange Juice 1976; Canned Tangerine Juice 1969; Concentrated Tangerine Juice for Manufacturing 1955; Grapefruit Juice 1968; Frozen Concentrated Grapefruit Juice 1970; Concentrated Grapefruit Juice for Manufacturing 1957; Grapefruit Juice and Orange Juice 1972; Concentrated Lemon Juice for Manufacturing 1959; Canned Lemon Juice 1962; Frozen Concentrate for Lemonade 1968; Frozen Concentrate for Limeade 1968. Frozen Concentrated Blended Grapefruit Juice and Orange Juice 1968; Canned Grapefruit 1973; Frozen Grapefruit 1948; Canned Grapefruit and Orange for Salad 1975. U Service, Washington 57. Official Rules Affecting the Florida Citrus Industry. State of Florida Dept. of Citrus, Lakeland, FL (1975). 58. Food and Agriculture Code, California Administrative Register 75, No. 4-B Dept. of General Services (1975). 59. Hunter, R. S. Food Technol., 1967, 21, 100. 60. Huggart, R. L.; Wenzel, F. W.; Martin, F. G. Proc. Fla. State Hort. Soc., 1969, 82, 200. 61. Huggart, R. L.; Petrus, D. R. Proc. Fla. State Hort. Soc., 1976, 89, 194 62. Huggart, R. L.; Petrus, D. R.; Buslig, B. S. Proc. Fla. State Hort. Soc., 1977, 90, 173. 63. Grierson, W.; Miller, W. M.; Wardowski, W. F. Fla. Agr. Exp. Sta. Bull. 803, Gainesville, FL, 1978. 64. Ting, S. V.; Hendrickson, R. Food Technol., 1969, 23, 87. 65. Ting, S. V.; Hendrickson, R. Proc. Fla. State Hort. Soc., 1968, 81, 264. 66. Berry, R. E.; Wilson, C. W. III; Bissett, O. W. J. Food Sci., 1972, 37, 809. 67. Barron, R. W.; Fellers, P. J.; Huggart, R. L. U. S. Patent 3,725,083, 1973. 68. Barron, R. W.; Metcalf, J. F. Proc. Fla. State Hort. Soc., 1969, 82, 197. 69. Attaway, J. Α.; Atkins, C. D.; Maraulja, M. D. In "Citrus Science and Technology", Nagy, S., Shaw, P. and Veldhuis, M. (eds.), AVI Publishing Co., Westport, Conn., 1977, II, p. 397. 70. Bauernfeind, J. C.; Brubacher, G. B.; Kläui, Η. M.; Marusich, W. L. In "Carotenoids", Isler, O., ed., Birkhäuser Verlag, Basel, 1971, p. 744. "Florida Agricultural Experiment Stations Journal Series, No. 2365" RECEIVED May 20, 1980.

In Citrus Nutrition and Quality; Nagy, S., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

8 Relationship of Citrus Enzymes to Juice Quality JOSEPH H. BRUEMMER U.S. Citrus and Subtropical Products Laboratory, U.S. Department of Agriculture, Science and Education Administration, Southern Region, P.O. Box 1909, Winter Haven, FL 33880

The quality of citru development--fruit set, ripening on the tree. During this development period compounds are formed that are responsible for the color and flavor of the ripe fruit. Many of these compounds have been identified, and research is now directed toward identifying the enzymic reactions that regulate their biosynthesis through metabolic pathways. Stewart (1) recently reviewed the carotenoid pigments identified as present in citrus and assessed their contribution to citrus color. Reported with that review in the same journal was the mode of action of bioregulators in controlling carotenoid biosynthesis through enzyme inhibition (2). Many terpenoids, aliphatic esters and aliphatic aldehydes in citrus fruit have been identified, but the pathways for their biosynthesis have not yet been determined (3). Bruemmer et al. (4) recently reviewed the working hypotheses on the mechanism for regulating the biosynthesis of citrus acids and concluded that supportive data were inadequate to identify the enzyme reactions that regulate acid metabolism in citrus. The q u a l i t y of extracted c i t r u s j u i c e s depends on enzyme r e a c t i o n s that occur not only i n the f r u i t during the development p e r i o d , but a l s o i n the j u i c e during p r o c e s s i n g . When j u i c e i s e x t r a c t e d from c i t r u s f r u i t , enzymes are released from t h e i r normal r e s t r a i n t i n the c e l l . Several of these enzymes c a t a l y z e r e a c t i o n s that adversely a f f e c t t a s t e and appearance of the j u i c e . Unless the r e a c t i o n s are c o n t r o l l e d , the j u i c e products w i l l not meet the standards of q u a l i t y set up by the USDA Food Safety and Quality Service. The two r e a c t i o n s of commercial importance are the h y d r o l y s i s of p e c t i n to p e c t i c a c i d , which c l a r i f i e s j u i c e , and the l a c t o n i z a t i o n of limonoic a c i d Α - r i n g lactone to the b i t t e r compound, l i m o n i n . Research e f f o r t s to i d e n t i f y and c h a r a c t e r i z e the r e a c t i o n s , to i s o l a t e and p u r i f y the enzymes, and to develop methods to c o n t r o l the r e a c t i o n s are described i n t h i s review.

This chapter not subject to U.S. copyright. Published 1980 American Chemical Society In Citrus Nutrition and Quality; Nagy, S., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

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E a r l y i n the development of the c i t r u s j u i c e p r o c e s s i n g i n d u s t r y , c l a r i f i c a t i o n of b o t t l e d and canned c i t r u s j u i c e s and c i t r u s beverages was recognized as r e s u l t i n g from the a c t i o n of p e c t i c enzyme(s) that had not been destroyed by heat p a s t e u r i z a t i o n (5). I n v e s t i g a t i o n s of cause and prevention of c l a r i f i c a t i o n and g e l a t i o n i n c i t r u s j u i c e s and t h e i r concentrates were stimulated by the r a p i d expansion of the frozen-orange conc e n t r a t e i n d u s t r y i n the l a t e f o r t i e s . These i n v e s t i g a t i o n s were thoroughly reviewed by J o s l y n and P i l n i k (6). During the past twenty years some progress was made on p u r i f i c a t i o n and mode of a c t i o n of p e c t i c enzymes (7)· More r e c e n t l y , m u l t i p l e forms of p e c t i n e s t e r a s e (PE) that d i f f e r i n k i n e t i c p r o p e r t i e s and temperat u r e s t a b i l i t y have been i s o l a t e d (8). Some of the i r r e g u l a r i t i e s i n heat i n a c t i v a t i o n of PE and j u i c e cloud s t a b i l i t y can be a t t r i b u t e d to the a c t i o n recent research on PE an z a t i o n are reviewed to r e l a t e PE to the problem of j u i c e q u a l i t y . J u i c e C l a r i f y i n g Enzyme. One of the e a r l i e s t reports on a c l a r i f y i n g and c l o t t i n g enzyme i n orange j u i c e was by Cruess (9). He reported that f r e s h orange j u i c e formed a j e l l y - l i k e suspension a few hours a f t e r expression, and that a f t e r a few days the suspended matter coalesced and s e t t l e d , l e a v i n g a c l e a r supernatant l i q u i d . He speculated that heating the j u i c e to 85°C (185°F) destroyed enzyme(s) and prevented the j u i c e from c l e a r i n g . J o s l y n and Sedky (10) showed that c l a r i f i c a t i o n of c i t r u s j u i c e s was always accompanied by decomposition of p e c t i c substances and used the rate of c l a r i f i c a t i o n as a measure of the a c t i v i t y of the p e c t i c enzymes. They (11) found that the rate at which heat p a s t e u r i z e d c i t r u s j u i c e s cleared v a r i e d i n v e r s e l y with both temperature and d u r a t i o n of the heat treatment. They showed that h e a t i n g orange j u i c e to 80°C (176°F) at pH 4 f o r 1 min i n a c t i v a t e d the c l e a r i n g enzyme and that i n a c t i v a t i o n was more r a p i d at pH 2.5 than at pH 4.0. Stevens (12) elaborated on the pH-temperature r e l a t i o n s h i p i n h i s patent f o r s t a b i l i z i n g c i t r u s j u i c e products by s e t t i n g f o r t h four s p e c i f i c ranges of minimum temperatures for short time (0.1 to 3 min) heating of j u i c e s at pH 2.2 to 3.79. A l s o he speculated that c i t r u s j u i c e s contain two cloud-coagulating enzymes of d i f f e r e n t t h e r m o s t a b i l i t i e s . One enzyme, most a c t i v e at low pH and temperature, appeared to be destroyed by h e a t i n g the j u i c e at 65 to 70°C (149 to 158°F). The second enzyme, most a c t i v e at pH 3.0 to 3.3 and about 35°C (95°F), appeared to r e q u i r e h e a t i n g to 88°C (191°F) f o r i n a c t i v a t i o n (12). Stevens (13) d e s c r i b e d a r a p i d t e s t f o r p e c t i c enzymes i n c i t r u s j u i c e . It i n v o l v e d adding p e c t i n under c o n t r o l l e d c o n d i t i o n s of temperature and sample p r e p a r a t i o n , and measuring the time r e q u i r e d f o r flocculation. Stevens, and coworkers (14) f u r t h e r elaborated on the patent work (12, 13). They produced a trend curve of the

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minimum temperatures required to s a t i s f a c t o r i l y i n a c t i v a t e p e c t i c enzymes i n n a t u r a l strength c i t r u s j u i c e s of d i f f e r e n t pHs. Their data i n d i c a t e d that the pulp content of j u i c e was the most important f a c t o r determining the rate and completeness of f l o c c u l a t i o n . Pulpy j u i c e required higher temperature f o r cloud s t a b i l i z a t i o n . They a l s o found that the p a s t e u r i z a t i o n c o n d i t i o n s necessary to s t a b i l i z e n a t u r a l strength or 42°Brix concentrated orange j u i c e f o r frozen storage (-23.3°C; -10°F) was about 65°C (149°F) f o r 1 min. They reported that these c o n d i t i o n s c o i n c i d e d with the minimum c o n d i t i o n s necessary to destroy spoilage organisms. A q u a n t i t a t i v e o b j e c t i v e measurement of c i t r u s j u i c e t u r b i d i t y was used by L o e f f l e r (15, 16) to show that p e c t i c enzyme changes occurred so r a p i d l y a f t e r the j u i c e was reamed from the f r u i t that at l e a s t a p a r t i a l coagulation of the cloud occurred before the j u i c e could be screened, deaerated and heated to a p a s t e u r i z a t i o n temperature increased by f l a s h - p a s t e u r i z a t i o the j u i c e before p a s t e u r i z a t i o n . L o e f f l e r (15, 16) presented data on t u r b i d i t y of f l a s h - p a s t e u r i z e d c i t r u s j u i c e s (heat exposure f o r 16 to 18 sec) a f t e r storage at s e v e r a l temperatures. He found that "samples p a s t e u r i z e d at 91°C (196°F) l o s t t h e i r cloud when stored at 35°F (95°F) but others p a s t e u r i z e d at 93-95°C (199-203°F) r e t a i n e d t h e i r cloud almost i n d e f i n i t e l y " . E x t r a c t i o n and I d e n t i f i c a t i o n . I d e n t i f i c a t i o n of PE as the c l e a r i n g enzyme i n c i t r u s j u i c e s progressed r a p i d l y a f t e r MacDonnell et a l . (17) reported on c a t i o n requirement f o r e x t r a c t i o n and s o l u b i l i z a t i o n of the enzyme from various p o r t i o n s of the f r u i t . PE was assayed by the method introduced by Kertesz (18) and modified by Lineweaver and B a l l o u (19). The method involved measuring the rate at which the methyl e s t e r groups i n the p e c t i n molecule are hydrolyzed by t i t r a t i n g the f r e e carboxyl groups with 0.1N NaOH as they are formed. One u n i t of PE was defined as the amount of enzyme which w i l l hydrolyze 1 meq carboxyl groups per min from a 0.5% s o l u t i o n of p e c t i n i n 0.15M NaCl at pH 7.5, 30°C (86°F). McDonnell et a l . (17) showed that the enzyme e x t r a c t e d from orange flavedo had a rather broad pH optimum at about 7.5 i n 0.15M NaCl, but that the enzyme required high i o n i c s t r e n g t h s o l u t i o n s at lower pH to be o p t i m a l l y a c t i v e . For 0.15M CaCl^ the plateau of optimum a c t i v i t y extended from pH 4.0 to 8.5 but a c t i v i t y was only about 50% of the l e v e l of a c t i v i t y found f o r 0.05M CaCl^ at pH 7.5. The increased a c t i v i t y caused by c a t i o n was greater at pH 5 than at pH 7.5 with 0.05M C a C l . 2

Occurrence and D i s t r i b u t i o n . PE was found a s s o c i a t e d with s t r u c t u r a l elements of the orange. McDonnell et a l . (17) reported no a c t i v i t y i n f i l t e r e d orange j u i c e , but found 58, 44 and 28 PE u n i t s per kg wet t i s s u e i n flavedo, albedo and c e l l sacs r e s p e c t i v e l y . Working with four v a r i e t i e s of F l o r i d a oranges and Dancy tangerine, Rouse (20) showed that j u i c e sacs had the highest

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a c t i v i t y and c e n t r i f u g e d (not c l a r i f i e d ) j u i c e the l e a s t . The r a g (segment w a l l t i s s u e e n c l o s i n g the j u i c e sacs) had the next highest a c t i v i t y followed by the peel (flavedo and albedo). In g r a p e f r u i t he found that j u i c e sacs had the highest a c t i v i t y but that albedo and flavedo had more a c t i v i t y than the rag. The d i s t r i b u t i o n of PE i n lemon and lime t i s s u e s was somewhat d i f f e r e n t from that i n orange or g r a p e f r u i t . Rouse and Atkins (21) showed that lemon and lime p e e l (flavedo plus albedo) had the highest a c t i v i t y followed by j u i c e sacs and r a g i n that order. P r e v i o u s l y , Rouse (22) found that j u i c e samples prepared to contain i n c r e a s i n g l y higher amounts of pulp ( j u i c e s a c s ) , a l s o had correspondingly higher PE a c t i v i t i e s . Rouse, Atkins and Huggart (23) showed that PE a c t i v i t y i n orange j u i c e was d i r e c t l y p r o p o r t i o n a l to pulp content. Orange PE was shown to be bound to c e l l w a l l s as an enzymesubstrate complex with p e c t i n (24). The s o l u b i l i z a t i o n of PE (pH 7.5 and 0.15N NaCl) and d e - e s t e r i f i c a t i o n of c e l l w a l l p e c t i n were s i m i l a r l y temperatur d e - e s t e r i f i c a t i o n of th e s t a b l i s h e d between bound and f r e e PE i n the e x t r a c t i o n medium. At the pH o f j u i c e (4.5 and below) the enzyme bound to c e l l w a l l s was not s o l u b i l i z e d unless s o l u b l e p e c t i n was added. The bound enzyme was i n a c t i v e at pH 4, whereas the f r e e s o l u b l e enzyme was about 20% more a c t i v e at pH 4 than at the optimum pH (7.5). Assays f o r PE. Because PE hydrolyzes p e c t i n t o p e c t i c a c i d and methanol, the enzyme concentration can be assayed by measuring the r a t e at which f r e e carboxyl groups or methanol i s released from the substrate. Kertesz (18) t i t r a t e d the f r e e carboxyl groups as they were formed by the a c t i o n o f the enzyme on p e c t i n . He used methyl red to i n d i c a t e the pH (6.2) and added 0.1N NaOH at frequent i n t e r v a l s to maintain the pH r e l a t i v e l y constant f o r 30 min. The pH meter replaced the use of i n d i c a t o r s i n subsequent m o d i f i c a t i o n s (17, 19). Current procedures use automatic pH t i t r a t o r s to t i t r a t e a l k a l i at constant pH (25). A blank i s used to c o r r e c t f o r the consumption of a l k a l i due to i t s r e a c t i o n with atmospheric C 0 , or the r e a c t i o n s o l u t i o n i s protected from CO- with a blanket o f N^. The broad pH optimum f o r PE was used By Somogyi and Romani (26) t o devise a r a p i d assay based on the r a t e at which the pH of unbuffered 1% p e c t i n i n 0.2M NaCl changed from 7.0 to 7.5 a f t e r the a d d i t i o n o f the enzyme. At low enzyme concentration the r e a c t i o n r a t e was f i r s t order f o r the_ f i r s t 1-min i n t e r v a l , and r e p r o d u c i b i l i t y was w i t h i n - 5%. Methanol can be determined c o l o r i m e t r i c a l l y but must u s u a l l y be d i s t i l l e d from the r e a c t i o n mixture before being analyzed. Wood and S i d d i q u i (27) described a simple and p r e c i s e s p e c t r o p h o t o m e t r y method f o r measuring methanol i n an assay f o r PE. The method involved permanganate o x i d a t i o n of methanol to formaldehyde and r e a c t i o n o f the formaldehyde with pentane-2,4-dione. The c o l o r r e a c t i o n was developed d i r e c t l y i n the PE r e a c t i o n mixture without i n t e r f e r e n c e from the p e c t i n . However, Termote e t a l . (28) 2

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increased the s e n s i t i v i t y and r e p r o d u c i b i l i t y of the method by developing the c o l o r with methanol d i s t i l l e d from the PE r e a c t i o n mixture. Gas chromatographic (GC) analyses are more s e n s i t i v e and r e p r o d u c i b l e than c o l o r i m e t r i c analyses. Gessner (29) developed a method f o r r e a c t i n g n i t r o u s a c i d with the a l c o h o l s i n e x t r a c t s of t i s s u e homogenates and then analyzing the head space by GC. The d e r i v a t i v e s formed have higher vapor pressures than the a l c o h o l s so that s e n s i t i v i t y of head space analyses i s increased according­ ly. Gessner claimed good r e p r o d u c i b i l i t y of values with concen­ t r a t i o n s of methanol at 1 mg/1. The d e r i v a t i z a t i o n method was used to assay PE i n plant t i s s u e (30) . A probable e r r o r of 2% was claimed, and s e n s i t i v i t y was 3 mg/1. The most s e n s i t i v e assay method f o r PE a c t i v i t y i s to use, as s u b s t r a t e , p e c t i n e s t e r i f i e d b i o l o g i c a l l y (31) or chemically (32) with C methanol. In s t u d i e s by Gessner (29) and Bartolome and Hoff (30) the substrate reacj^on mixture with a c i d i f i e the C methanol i n the supernate was determined. The r a d i o a c t i v e assay method was about 100 times as s e n s i t i v e as the t i t r i m e t r i c method f o r PE a c t i v i t y (32). The r a d i o a c t i v e method may be used when the t i t r a t i o n method i s not a p p l i c a b l e , e.g., when the pH of the r e a c t i o n i s near the pK of p e c t i n (about 4.0), or when the r e a c t i o n r a t e i s low because of l i m i t e d amount of enzyme or substrate. However, the automatic t i t r a t i o n method when a p p l i c a b l e , i s advantageous because the r e a c t i o n can be monitored continuously and any d e v i a t i o n s from l i n e a r i t y r e a d i l y recognized. Purification. Orange PE was i s o l a t e d i n i t i a l l y from Navel orange flavedo by e x t r a c t i o n with a borate-acetate b u f f e r at pH 8.2 and then p r e c i p i t a t i o n from the e x t r a c t with (NH,) SO^ (17). L a t e r MacDonnell et a l . (33) used (NH,) S0^ to f r a c t i o n a t e the flavedo e x t r a c t (pH 7) and adsorbed the 40 to 80% s a t u r a t i o n f r a c t i o n on f i l t e r paper pulp (1-2 g/1). PE was extracted from the paper pulp with 0.1M NaCl and t r a n s f e r r e d to a C e l i t e 505 column (0.4U PE/g). The column was f i r s t washed with 0.025M Na HP0^, and the PE was then eluted from the column with 1.0M NaCl c o n t a i n i n g 0.025M Na HP0,. The enzyme was p r e c i p i t a t e d from the e x t r a c t with ( N H ^ S O ^ (90% s a t . , pH 7), s o l u b i l i z e d , d i a l y z e d and then t r a n s f e r r e d to a D u c i l column (0.75U PE/g). The column was washed w i t h 0.025M Na^HPO,, and PE was e l u t e d from the column with 1.0M NaCl c o n t a i n i n g 0.025M Na^PO^. The eluate was d i a l y z e d and f r e e z e - d r i e d ; and i t s powder r e f r i g e r a t e d . This procedure p u r i f i e d orange PE over 100-fold on a t o t a l Ν b a s i s . More r e c e n t l y , Manabe (34) p u r i f i e d PE from C i t r u s n a t s u d a i d a i f r u i t by chromatography on a DEAE-cellulose column followed by Sephadex G-100 column adsorption of the a c t i v e f r a c t i o n . The f i n a l preparation was homogeneous as determined by d i s c e l e c t r o p h o r e s i s and was 460 times as a c t i v e as the o r i g i n a l e x t r a c t on a p r o t e i n b a s i s . 2

2

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Specificity. Orange PE hydrolyzed methyl and e t h y l e s t e r s of polygalacturonans, but at optimal pH, the r a t e on e t h y l e s t e r s was about 10% f a s t e r than that on the methyl e s t e r s (33, 35). At pH 4 the r a t e s were about equal (35). G l y c y l and g l y c e r y l e s t e r s of polygalacturonans were not hydrolyzed by orange PE (35, 36). Nongalacturonate e s t e r s (over 50 tested) were not hydrolyzed by the p u r i f i e d PE p r e p a r a t i o n of MacDonnell et a l . (33). P u r i f i e d c i t r u s PE d i d not hydrolyze methyl e s t e r s of d i g a l a c t u r o n i c or t r i g a l a c t u r o n a c i d but r e a d i l y hydrolyzed polymeric u n i t s of 10 or more (37). So f a r , the minimum chain length of the o l i g o galacturonan methyl e s t e r s required f o r PE a c t i o n has not been determined. P a r t i a l reduction of the galactopyranosiduronate s t r u c t u r e by NaBH^ decreased PE a c t i o n markedly, i n d i c a t i n g s p e c i f i c i t y of the carboxyl group f o r enzyme r e a c t i o n (38). A c t i v a t i o n - I n h i b i t i o n and Function In Vivo. When 0.15M NaCl was added to an orange PE-pecti a c t i v i t y was increased 5 - f o l d f o l d (17). As explained by Lineweaver and B a l l o u (19), NaCl caused the apparent a c t i v a t i o n by f r e e i n g the enzyme from the i n a c t i v e i o n i c complex ( p e c t i n - c a r b o x y l ) . They showed that at pH 5.7 p e c t i c a c i d i n h i b i t e d a l f a l f a PE a c t i v i t y 55% i n 0.015M NaCl but only 17% i n 0.2M NaCl. At pH 8.5 p e c t i c a c i d i n h i b i t e d PE a c t i v i t y only 9% i n 0.015 NaCl. They concluded that the s t i m u l a t i o n of a c t i v i t y by c a t i o n s at low pH (17) d i d not show that c a t i o n s were e s s e n t i a l f o r a c t i v i t y , but, r a t h e r , that c a t i o n s f u n c t i o n by preventing product i n h i b i t i o n , which i s greater at low pH. Termote et a l . (28) studied product i n h i b i t i o n of orange PE as an approach to s t a b i l i z i n g cloud of orange j u i c e . Both chemically and enzymically prepared hydrolysates of p e c t i c a c i d with an average degree of polymerization of 8 or higher i n h i b i t e d PE a c t i v i t y at pH 5 i n 0.1M NaCl. Unhydrolyzed p e c t i c a c i d showed the greatest i n h i b i t i o n . No i n h i b i t i o n was observed when the i o n i c s t r e n g t h of the r e a c t i o n mixture was increased to 0.5M NaCl. P e c t i c a c i d hydrolysates with degree of polymerization of 8 to 15 were e f f e c t i v e i n extending the period of cloud s t a b i l i t y i n PE a c t i v e j u i c e s by a f a c t o r of 3 to 5 but d i d not prevent the j u i c e s from c l a r i f y i n g e v e n t u a l l y . Versteeg (8) speculated on the f u n c t i o n o f PE i n v i v o . He noted the high a c t i v i t y of PE i n c i t r u s f r u i t compared to the amount of a v a i l a b l e p e c t i n . The f r u i t contain s u f f i c i e n t a c t i v i t y to d e e s t e r i f y the p e c t i n to low methoxy p e c t i n i n 10 min at optimum pH. He suggested that the methyl t r a n s f e r a s e found by Kauss and Hassid (39) to e s t e r i f y p e c t i c a c i d to p e c t i n i n mung bean shoots and to be l o c a t e d i n a lipid-membrane complex (31) functioned as p e c t i n e s t e r a s e a f t e r the l i p i d membranes were destroyed and the environment changed. However, no d e f i n i t i v e experiments to e s t a b l i s h the r o l e of PE i n f r u i t s were reported.

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Isoenzymes. M u l t i p l e forms of c i t r u s PE were reported by Evans and McHale (40) and Versteeg et a l . (41). PE was p u r i f i e d from West Indian limes and Navel oranges by f r a c t i o n a t i o n of the whole f r u i t e x t r a c t s with (NH.^SO^ (40-65%), adsorption and e l u t i o n from Sephadex G-75 columns (40). The PE a c t i v e f r a c t i o n s were combined and concentrated before separation i n t o two a c t i v e PEs on the b a s i s of t h e i r e l u t i o n volume from a DEAE Sephadex A-50 column. Orange PEI (OPEI) and lime PEI (LPEI) had the same e l u t i o n volume; a l s o OPEII and LPEII had the same e l u t i o n volume. A higher c o n c e n t r a t i o n of NaCl was required at a l l pH values f o r optimum a c t i v i t y of OPEI and LPEI than of OPEII and LPEII. When the component parts of oranges were s e p a r a t e l y analyzed chromâtοgraphi­ c a l l y with DEAE-Sephadex A-50, OPEI was detected only i n the p e e l , whereas OPEII was i d e n t i f i e d i n j u i c e sacs and s e c t i o n w a l l s (40). Versteeg et a l . (41) p u r i f i e d e x t r a c t s of Navel orange pulp and p e e l by (NH^^SO^ f r a c t i o n a t i o n (30 to 75% s a t u r a t i o n ) and chromatography on Bio-Ge from Bio-Gel P-100 was separate graphy on c r o s s - l i n k e d pectate (42) . Each PE was f u r t h e r p u r i f i e d by chromatography on CM Bio-Gel. PEI d i d not bind as s t r o n g l y as PEII to e i t h e r the c r o s s - l i n k e d pectate or CM B i o - G e l . The a c t i v i t y of the p u r i f i e d PEI and PEII combined was about 26% of the a c t i v i t y of the crude e x t r a c t ; and PEI had about twice the a c t i v i t y of PEII. Both enzymes had molecular weight of 36,200 as determined from t h e i r m o b i l i t y based on dodecyl s u l f a t e e l e c t r o p h o r e s i s . PEI had optimum a c t i v i t y at pH 7.6 and PEII at pH 8.0. A l s o , PEI was r e l a t i v e l y more a c t i v e at low pH v a l u e s . PEI had low a f f i n i t y f o r p e c t i n and was weakly i n h i b i t e d by polygalacturonate. PEII had high a f f i n i t y and was s t r o n g l y i n h i b i t e d . Rombouts et a l . (42) synthesized c r o s s - l i n k e d pectate with 0.46 degree of c r o s s - l i n k i n g and was able to get strong b i n d i n g of both PEI and PEII to the pectate. Through PE a c t i v i t y measurements on m e t h y l - e s t e r i f i e d c r o s s - l i n k e d pectate they concluded that b i n d i n g of the enzymes to the pectate matrix involved b i o s p e c i f i c a f f i n i t y as w e l l as i o n exchange e f f e c t s . Versteeg (8) i s o l a t e d a t h i r d isoenzyme of Navel oranges and found that i t was more heat s t a b l e than e i t h e r PEI or PEII and had an i s o e l e c t r i c point s i m i l a r to that of PEI (pH 10.05). The s p e c i f i c a c t i v i t y of the heat s t a b l e f r a c t i o n was low compared to that of PEI or PEII and was not improved much by B i o Gel P-100 chromatography. The molecular weight of t h i s t h i r d PE isoenzyme was estimated to be 54,000 and was c a l l e d high molecular weight (HM-PE) by the author (8). C r o s s - l i n k e d pectate chromato­ graphy separated HM-PE i n t o three f r a c t i o n s . HM-PEII contained about 85% of the a c t i v i t y of the o r i g i n a l HM-PE and was p u r i f i e d about 20-fold by c r o s s - l i n k e d pectate chromatography (8). Thinl a y e r pH gradient e l e c t r o p h o r e s i s of crude PE e x t r a c t s from d i f f e r e n t c i t r u s f r u i t (oranges, lemon, g r a p e f r u i t and tangerine) gave good separation of PEI, PEII and HM-PEII and revealed a c t i v i t y peaks corresponding to a t o t a l of 12 forms of PE. However PEI and PEII accounted f o r more than 80% of the a c t i v i t y i n oranges (8).

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Mode of A c t i o n of PEI and PEII. Versteeg (8) measured r e a c t i o n rates of PEI and PEII with p e c t i n s p r e s a p o n i f i e d so that degree of e s t e r i f i c a t i o n (DE) ranged from 95.6 to 32%. He found that maximum v e l o c i t i e s (Vmax) f o r the r e a c t i o n s with a l l the substrates were about the same. However, the a f f i n i t y of PEI and PEII to the substrates increased d r a m a t i c a l l y as the DE of the p e c t i n s decreased. A f f i n i t y of PEII f o r p e c t i n was almost 150-fold greater f o r p e c t i n with DE of 32% than of 95.6%. The l o g of a f f i n i t y c o r r e l a t e d with the l o g of percentage free carboxyl groups i n the p e c t i n f o r both PEI and PEII even though a f f i n i t y of PEII was much l a r g e r than that of PEI f o r the p e c t i n s . The slopes of the c o r r e l a t i o n equation were 2.08 f o r PEI and 2.03 f o r PEII, i n d i c a t i n g that doubling the number of f r e e carboxyl groups gave a f o u r - f o l d increase i n a f f i n i t y . Versteeg (8) c a l c u l a t e d the percentage of p o s s i b l e u n i t s that had two carboxyl groups f o r p e c t i n s with DEs of 95 to 32%. He found that l o g % u n i t s w i t h two carboxyl groups i n some 2) w i t h the l o g % f r e e carboxy PE i s i n h i b i t e d by g a l a c t u r o n i c a c i d oligomers with degree of polymerization of 8 or more (28) he concluded that a substrate with two f r e e carboxyl groups separated by s i x monomers was required f o r the optimal enzyme-substrate complex. Versteeg (8) found that PEI and PEII were s i m i l a r i n the products they cleaved from p e c t i n with DE of 95.6%. Separation of r e a c t i o n products a f t e r t h i s substrate was d e - e s t e r i f i e d to DE of 60% showed that about 30% of the substrate was not acted on by the enzyme. In contrast a l l the e s t e r bonds i n p e c t i n with DE of 95.6% could be hydrolyzed by a l k a l i . PEI and PEII d i d not completely d e - e s t e r i f y p e c t i n s with DE ranging from 95.6 to 32%. The lower the i n i t i a l DE of the s u b s t r a t e , the lower was the DE of the f i n a l product of the r e a c t i o n . However, none of the products from PEI and PEII r e a c t i o n s with p e c t i n s (DE 95.6 to 32%) had DEs of l e s s than 11%. Solms and Deuel (38) e a r l i e r noted that 11% DE was the lower l i m i t f o r d e - e s t e r i f i c a t i o n of p e c t i n by PE. S t a b i l i t y of PE Isozymes. The p u r i f i e d forms of PE l o s t only 15% of t h e i r a c t i v i t i e s during 2 years at 4°C (40°F) with 0.1M NaCl i n phosphate b u f f e r (pH 7.5) (8). At 30°C (86°F) PEI was s t a b l e f o r 24 hr at pH 4 and 7, but PEII l o s t a l l a c t i v i t y w i t h i n 6 hr at pH 4. Tested at 30°C (86°F) i n s i n g l e - s t r e n g t h j u i c e r e c o n s t i t u t e d from concentrate, a c t i v i t y of PEI and crude orange PE d e c l i n e d but HM-PE r e t a i n e d i t s a c t i v i t y over the 15 days storage. Versteeg (8) t e s t e d the heat s t a b i l i t y of the p u r i f i e d PEs i n orange j u i c e , pH 4.0, and found that PEII was the l e a s t s t a b l e , being completely i n a c t i v a t e d at 55°C (131 F); PEI was i n a c t i v a t e d at 65°C (149°F), and HM-PE at 85°C (185°F). Heat i n a c t i v a t i o n curves of a crude preparation of orange PE i n orange j u i c e i n d i c a t e d that about 5% of the t o t a l PE a c t i v i t y was due to HM-PE, 60% to PEI and 30% to PEII. e

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Cloud S t a b i l i t y and PE A c t i v i t y . The three forms o f PE were t e s t e d f o r d e s t a b i l i z a t i o n o f orange j u i c e cloud (8). Only PEI and HM-PE were a c t i v e at 5°C (41°F) and 30°C (86°F). PEI was much l e s s a c t i v e than HM-PE a t 5°C (41°F). Versteeg (8) concluded that HM-PE a c t i v i t y was r e s p o n s i b l e f o r d e s t a b i l i z i n g orange j u i c e during storage and that cloud s t a b i l i z a t i o n required heating j u i c e t o 90°C (194°F) because HM-PE r e t a i n e d a c t i v i t y i n j u i c e heated to l e s s than that temperature. The heat s t a b i l i t y of the a c t i v e HM-PE a l s o accounted f o r the observations on i r r e g u l a r heat i n a c t i v a t i o n and cloud s t a b i l i t y patterns (43, 44·, 45^, 46). B i s s e t t e t a l . (45) reported that heating orange j u i c e to 82°C (180°F) i n a c t i v a t e d 94 to 95% o f the PE a c t i v i t y but d i d not g r e a t l y improve cloud s t a b i l i t y o f s i n g l e - s t r e n g t h or concentrated orange j u i c e . A s i m i l a r report by Atkins et a l . (46) showed that 90% i n a c t i v a t i o n of PE a c t i v i t y of g r a p e f r u i t j u i c e heated to 85°C (185°F) d i d not prevent g e l a t i o n and c l a r i f i c a t i o n i n the concentrate. I d e n t i f i c a t i o n and t o l e r a n t enzyme r e s p o n s i b l c i t r u s j u i c e s represent a major breakthrough i n understanding the r e l a t i o n s h i p between PE and j u i c e q u a l i t y . Progress i n e s t a b l i s h i n g the r o l e of HM-PE i n v i v o , i t s o r i g i n and r e l a t i o n s h i p to PEI and PEII could lead to procedures to c o n t r o l HM-PE formation d u r i n g f r u i t development and maturation. Knowing the d i s t r i b u t i o n of t h i s a c t i v e form of PE i n the s t r u c t u r a l l y defined f r u i t p a r t s could a s s i s t the f r u i t j u i c e t e c h n o l o g i s t to adopt processing c o n d i t i o n s to minimize HM-PE presence i n the product. Limonin D-Ring Lactonase (EC 3.1.1.36) The need to u t i l i z e packinghouse r e j e c t s o f substandardq u a l i t y Navel oranges r e s u l t e d i n i n v e s t i g a t i o n s on the cause and prevention of b i t t e r n e s s i n p a s t e u r i z e d Navel orange j u i c e . Juice from the Navel orange was not b i t t e r when f r e s h l y expressed but became b i t t e r on standing (47). Higby (47) i s o l a t e d s e v e r a l b i t t e r substances from V a l e n c i a and Navel orange pulps, and p u r i f i e d and c h a r a c t e r i z e d one of them as limonin, a substance p r e v i o u s l y i s o l a t e d from seeds of s e v e r a l v a r i e t i e s o f c i t r u s (48). Higby suggested that the precursor of limonin i s limonoic a c i d and that i t i s s t a b l e i n the i n t a c t f r u i t but i s l a c t o n i z e d a t the pH of j u i c e . Emerson (49, 50) compared the r a t e at which b i t t e r n e s s developed i n early-season Navel oranges with the r a t e at which limonin formed from limonoic a c i d i n V a l e n c i a orange j u i c e and i n water at the pH o f the j u i c e and concluded that the precursor i n Navel oranges was probably one of the lactone a c i d s rather than the d i a c i d . He a l s o suggested that i f the d i a c i d were the precursor of limonin, the r e a c t i o n would probably be enzyme catalyzed because a c i d c a t a l y s i s o f the d i a c i d was too slow.

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Maier and Beverly (51) used high voltage e l e c t r o p h o r e s i s to separate limonin, limonoic a c i d and limonoate monolactone i n e x t r a c t s from early-season and l a t e r - s e a s o n Navel oranges and Marsh g r a p e f r u i t . They were able to show that limonoate monolactone was the primary limonoid i n the endocarp and albedo t i s s u e s o f the early-season f r u i t and that f r e e limonin and limonoic a c i d were not n a t u r a l c o n s t i t u e n t s of healthy i n t a c t navel oranges or g r a p e f r u i t . They a l s o showed that the monolactone was not present i n l a t e r season f r u i t . Maier and M a r g i l e t h (52) i d e n t i f i e d the monolactone as limonoic a c i d Α-ring lactone a f t e r separating the A- and D-ring lactones by paper e l e c t r o p h o r e s i s and TLC. They a l s o obtained i n d i c a t i o n s of an enzyme i n c a r p e l l a r y membrane t i s s u e that converted limonoic a c i d Α-ring lactone to limonin. The enzyme was i s o l a t e d and p u r i f i e d from peeled seeds (53) and the r e a c t i o n shown to be s p e c i f i c f o r the D-ring lactone group. The enzyme c a t a l y z e d the l a c t o n i z a t i o n r e a c t i o n at pH 6 and the h y d r o l y t i c r e a c t i o n at pH 8.0. Th shown to be e s s e n t i a l l y e l e c t r o p h o r e s i s (53). Hasegawa (54) reported on p r o p e r t i e s of the lactonase. The enzyme p u r i f i e d from g r a p e f r u i t seeds hydrolyzed limonoids that have the D-ring i n t a c t but d i f f e r from limonin i n the v i c i n i t y of the A and A - r i n g s , namely, obacunone, nomilin and ichangin. Limonin D-ring lactonase was shown to be markedly heat r e s i s t a n t , r e t a i n i n g about 30% of i t s a c t i v i t y a f t e r 5 min at 100°C (54). C i t r u s leaves were shown to be the s i t e of limonoic a c i d Ar i n g lactone b i o s y n t h e s i s i n c i t r u s (55). The lactone accumulated to the l e v e l of 2000 ppm i n very small leaves but as the l e a f grew, the lactone content d e c l i n e d . The lactone content o f the f r u i t increased as the l e v e l i n the leaves d e c l i n e d . Hasegawa and Hoagland (55) a l s o showed that limonoic a c i d Α-ring lactone was not synthesized i n the f r u i t but i n the leaves. The r a d i o a c t i v e l a b e l e d lactone was i s o l a t e d from a f r u i t adjacent to a l e a f a c t i v e l y s y n t h e s i z i n g i t from l a b e l e d acetate, i n d i c a t i n g that the lactone was synthesized i n the leaves and transported to the f r u i t (55). Although limonoic a c i d Α-ring lactone has been shown to be a substrate of a lactonase that c a t a l y z e s the formation of limonin i n c i t r u s f r u i t , s e v e r a l other enzymes i n c i t r u s can a l s o use the Ar i n g lactone as substrate, however, the products are not b i t t e r products. These enzymes and t h e i r r e a c t i o n s are reviewed i n the next s e c t i o n . f

Enzymes to Degrade Limonin Precursor Hasegawa et a l . (56) defected the enzymic conversion of 19deoxylimonoic a c i d 3-methyl C e s t e r to the 17-dehydro d e r i v a t i v e by albedo t i s s u e s l i c e s of Navel oranges. They i s o l a t e d the product and i d e n t i f i e d i t by TLC as the r e a c t i o n product formed when the substrate was dehydrogenated by limonoate dehydrogenase (EC 1.1.1)

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i s o l a t e d from c e l l - f r e e e x t r a c t s of Arthrobacter g l o b i f o r m i s (57). They a l s o showed that the 17-dehydro d e r i v a t i v e i s o l a t e d from the r e a c t i o n mixture with the albedo t i s s u e could £g converted back to the substrate, 19-deoxy-limonoic a c i d 3-methyl C e s t e r by b a c t e r i a l limonoate dehydrogenase (56). The c i t r u s and b a c t e r i a l limonoate dehydrogenases both required NAD f o r the dehydrogenase r e a c t i o n (56, 57). Although the c i t r u s enzyme was not i s o l a t e d , i d e n t i f i c a t i o n of the product of the r e a c t i o n as the 17-dehydro d e r i v a t i v e and i s o l a t i o n of 17-dehydrolimonoate Α-ring lactone from Navel orange j u i c e and Navel orange peel (58) i n d i c a t e d that one of the limonoid metabolic pathways i n c i t r u s was the dehydrogenation of carbon 17 i n the l a c t o n e . The 17-dehydrolimonoate Α-ring lactone i s not b i t t e r , so the enzymic d e b i t t e r i n g of f r e s h Navel orange j u i c e with limonoate dehydrogenase from A. g l o b i f o r m i s was suggested (57) and then demonstrated (59). A patent was issued f o r using limonoate dehydrogenase to d e b i t t e the b a c t e r i a l enzyme a c t i v i t pH (3.5-4.5), so that the r a t e of d e b i t t e r i n g was very slow (59) i n j u i c e not adjusted to higher pH. Limonoate dehydrogenase i s o l a t e d from a Pseudomonas sp. had a lower pH optimum (pH 8.0) and showed considerable a c t i v i t y even at pH 4 (61). The Pseudomonas enzyme used NADP twice as e f f e c t i v e l y as NAD, and i t s a c t i v i t y was stimulated by ZnCl^. Compared to the A. g l o b i f o r m i s dehydrogenase, the Pseudomonas enzyme was much more s t a b l e at pH 3.5 (62). As l i t t l e as 200 u n i t s of the enzyme decreased the limonin precursor l e v e l i n one l i t e r of f r e s h orange j u i c e to 4 ppm from 21 ppm i n 2 hr at 24°C (75°F). Prospects look good f o r use of the Pseudomonas dehydrogenase to d e b i t t e r orange j u i c e commercially (62). Recently, a n o n s p e c i f i c enzyme capable of degrading the precursor was extracted from Navel orange albedo (63). N i c o l and Chandler (63) used a proximate assay f o r the degrading enzyme; that i s , the substrate was not i d e n t i f i e d except as a limonin precursor. The crude e x t r a c t s from albedo contained degradation a c t i v i t y that was concentrated by 40 to 60% s a t u r a t i o n with (NH^)^SO^. I n s t a b i l i t y of the enzyme prevented f u r t h e r purification. Other Enzymes i n C i t r u s J u i c e s P e c t i n e s t e r a s e and limonin D-ring lactonase are the only enzymes known to c a t a l y z e r e a c t i o n s that adversely a f f e c t the q u a l i t y of c i t r u s j u i c e s . Bruemmer et a l . (64) l i s t e d other enzymes that have been detected i n c i t r u s j u i c e s and described some of the r e a c t i o n s that can occur i n the j u i c e s . None of the r e a c t i o n s appear to n o t i c e a b l y a f f e c t the q u a l i t y of commercial juices. F r e s h l y extracted c i t r u s j u i c e s contain esterase (EC 3.1.1.1) (65^, 66) and phosphatase (EC 3.1.32) (66, 67) a c t i v i t i e s . Native substrates i n orange j u i c e f o r peroxidase

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(EC 1.11.1.7) (68) and diphenol oxidase (EC 1.10.3.1) (69) have been i d e n t i f i e d . The p o t e n t i a l r o l e of p y r u v i c decarboxylase (EC 4 . 1 . 1 . 1 ) c a t a l y z e d r e a c t i o n as a source of acetaldehyde and other aldehydes i n j u i c e was discussed (70). Raymond et a l . (71) i s o l a t e d the decarboxylase from orange j u i c e s e c t i o n s and demonstrated that only 10 to 15% of the enzyme was i n an a c t i v e form. Since the p u r i f i e d enzyme was only a c t i v e w i t h p y r u v i c a c i d and 2 - k e t o b u t y r i c a c i d of the s e r i e s of 2-ketoacids examined, they (71) concluded that the d i r e c t c o n t r i b u t i o n of orange p y r u v i c decarboxylase to the orange v o l a t i l e p r o f i l e was l i m i t e d to acetaldehyde and p o s s i b l y propionaldehyde.

Literature Cited 1. Stewart, I. Citrus color-a review. Proc. Int. Soc. Citriculture, 1977, 1, 308-311. 2. Yokoyama, H.; Hsu Debenedict, C. Bioregulator Int. Soc. Citriculture, 1977, 3, 717-722. 3. Bruemmer, J. H. Aroma substances of citrus fruits and their biogenesis. In "Geruch-und Geschmackstoffe". F. Drawert, ed. Verlag Hans Carl, Nurnberg, Germany, 1975, p 167-176. 4. Bruemmer, J. H.; Buslig, B. S.; Roe, B. Citrus Enzyme Systems: Opportunities for Control of fruit quality. Proc. Int. Soc. Citriculture, 1977, 3, 712-717. 5. Joslyn, Μ. Α.; Marsh, G. L. Some factors involved in the preservation of orange juice by canning. Fruit Prod. J., 1934, 14, 45-50. 6. Joslyn, Μ. Α.; Pilnik, W. Enzymes and enzyme activity. In "The Orange, Its Biochemistry and Physiology". W. B. Sinclair, ed. Univ. of Calif. Press, Berkeley, Calif. 1961, p. 373-435. 7. Rexova-Benkova, L.; Markovic, O. Pectic enzymes. In "Advances in Carbohydrate Chemistry and Biochemistry. R. S. Tipson and D. Horton, eds. Acad. Press, New York, 1976, 33, 323-385. 8. Versteeg, C. Pectinesterases from the orange fruit - their purification general characteristics and juice cloud destabilizing properties. Agric. Res. Rep. 892. 1979, Purdoc, Wageningen, Netherlands. 9. Cruess, W. V. Utilization of waste oranges. Calif. Agr. Exp. Sta. Bull., 1914, 244, 157-170. 10. Joslyn, Μ. Α.; Sedky, A. The relative rates of destruction of pectin in macerates of various citrus fruits. Plant Physiol., 1940, 15, 675-687. 11. Joslyn, Μ. Α.; Sedky, A. Effect of heating on the clearing of citrus juices. Food Res., 1940, 5., 223-232. 12. Stevens, J. W. Method of conserving fruit juices. 1940, U.S. Patent 2,217,261. 13. Stevens, J. W. Method of testing fruit juices. 1941, U.S. Patent 2,267,050.

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8.

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Enzymes

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163

14. Stevens, J. W.; Pritchett, D. E.; Baier, W. E. Control of enzymatic flocculation of cloud in citrus juices. Food Tech., 1950, 4, 469-473. 15. Loeffler, H. J. Processing of orange juice: effect of storage temperature on quality factors of bottled juice. Ind. & Eng. Chem., 1941, 33, 1308-1314. 16. Loeffler, H. J. Maintenance of cloud in citrus juice. Inst. Food Tech. Proc., 1941, 29-36. 17. MacDonnell, L. R.; Jansen, E. F.; Lineweaver, H. The properties of orange pectinesterase. Arch. Biochem., 1945, 16, 389-401. 18. Kertesz, Ζ. I. Pectic enzymes. I. The determination of pectin-methoxylase activity. J. Biol. Chem., 1937, 121, 589-598. 19. Lineweaver, H.; Ballou, G. A. The effect of cations on the activity of alfalfa pectinesterase (pectase) Arch Biochem. 1945, 6, 373-387. 20. Rouse, A. H. Distributio in component parts of citrus fruits. Food Tech., 1953, 7, 360-362. 21. Rouse, A. H.; Atkins, C. D. Lemon and lime pectinesterase and pectin. Proc. Fla. State Hort. Soc., 1954, 67, 203-206. 22. Rouse, A. H. Effect of insoluble solids and particle size of pulp on the pectinesterase activity in orange juice. Proc. Fla. State Hort. Soc., 1951, 64, 162-166. 23. Rouse, A. H.; Atkins, C. D.; Huggart, R. L. Effect of pulp quantity on chemical and physical properties of citrus juices and concentrates Food Tech., 1954, 8, 431-435. 24. Jansen, E. F.; Jang, R.; Bonner, J. Orange pectinesterase binding and activity. Food Res., 1960, 25, 64-72. 25. Rouse, A. H.; Atkins, C. D. Pectinesterase and pectin in commercial citrus juices as determined by methods used at the Citrus Experiment Station. Fla. Agr. Exp. Sta. Bull., 1955, 570, 1-19. 26. Somogyi, L. P.; Romani, R. J. A simplified technique for the determination of pectin methylesterase activity. Anal. Biochem., 1964, 8, 498-501. 27. Wood, J. R.; Siddiqui, I. R. Determination of methanol and its application to measurement of pectinester content and pectin methylesterase activity. Anal. Biochem., 1971, 39, 418-428. 28. Termote, F.; Rombouts, F. M.; Pilnik, W. Stabilization of cloud in pectinesterase active orange juice by pectic acid hydrolysates. J. Food Biochem., 1977, 1, 15-34. 29. Gessner, P. K. Method for the assay of ethanol and other aliphatic alcohols applicable to tissue homogenates and possessing a sensitivity of 1 μg/ml. Anal. Biochem., 1970, 38, 499-505. 30. Bartolome, L. G.; Hoff, J. H. Gas chromatographic methods for the assay of pectin methylesterase, free methanol and

In Citrus Nutrition and Quality; Nagy, S., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

164

31.

32. 33.

34. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46.

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methoxyl groups in plant tissues. J. Agric. Food Chem., 1972, 20, 262-266. Kauss, H.; Swanson, A. L.; Arnold, R.; Odzuck, W. Biosynthesis of pectic substances. Localization of enzymes and products in a lipid-membrane complex. Biochim. Biophys. Acta, 1969, 192, 55-61. Milner, Y.; Avigard, G. A sensitive radioisotope assay for pectin methylesterase activity. Anal. Biochem., 1973, 51, 116-120. MacDonnell, L. R.; Jang, R.; Jansen, E. F.; Lineweaver, H. Specificity of pectinesterases from several sources with some notes on purification of orange pectinesterase. Arch. Biochem., 1950, 28, 260-273. Manabe, M. Purification and properties of Citrus natsudaidai pectinesterase. Agric. Biol. Chem., 1973, 37, 1487-1491. Deuel, H. Über Glykoleste de Pectinsäure Helvetic Chim Acta, 1947, 30, 1523-1534 McCready, R. M.; Seegmiller, C. G. Action of pectic enzymes on oligogalacturonic acids and some of their derivatives. Arch. Biochem. Biophys., 1954, 50, 440-450. Solms, J.; Deuel, H. Uber den Mechanismus der enzymatischen Verseifung von Pektinstoffen. Helvetica. Chim. Acta, 1955, 38, 321-329. Kauss, H.; Hassid, W. Z. Enzymic introduction of the methyl ester groups of pectin. J. Biol. Chem., 1967, 242, 3449-3453. Evans, R.; McHale, D. Multiple forms of pectinesterase in limes and oranges. Phytochem., 1978, 17, 1073-1075. Versteeg, C.; Rombouts, F. M.; Pilnik, W. Purification and some characteristics of two pectinesterase isoenzymes from orange. Lebensm. Wiss. Technol., 1978, 11, 267-274. Rombouts, F. M.; Wissenburg, A. K.; Pilnik, W. A. Chromatographic separation of orange pectinesterase isoenzymes on pectates with different degrees of cross-linking. J. Chromatogr., 1979, 168, 151-161. Rouse, A. H.; Atkins, C. D. Further results from a study on heat inactivation of pectinesterase in citrus juices. Food Technol., 1953, 7, 221-223. Rouse, A. H.; Atkins, C. D. Heat inactivation of pectinesterase in citrus juices. Food Technol., 1952, 6, 291-294. Bissett, O. W.; Veldhuis, M. K.; Rushing, Ν. B. Effect of heat treatment temperature on the storage life of Valencia orange concentrates. Food Tech., 1953, 7, 258-260. Atkins, C. D.; Rouse, A. H.; Huggart, R. L.; Moore, E. L.; Wenzel, F. W. Gelation and clarification in concentrated juices. III. Effect of heat treatment of Valencia oranges and Duncan grapefruit juices prior to concentration. Food. Tech., 1953, 7, 62-66.

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47. Higby, R. H. The bitter constituents of Navel and Valencia oranges. J. Amer. Chem. Soc., 1938, 60, 3013-3018. 48. Bernay, S. Limonin. Annalen, 1841, 40, 317. 49. Emerson, O. H. The bitter principles of citrus fruit. I. Isolation of nomilin, a new bitter principle from the seeds of oranges and lemons. J. Am. Chem. Soc., 1948, 70, 545-549. 50. Emerson, O. H. The bitter principle in Navel oranges. Food Tech., 1949, 3, 248-250. 51. Maier, V. P.; Beverly, G. D. Limonin monolactone, the non­ -bitter precursor responsible for delayed bitterness in certain citrus juices. J. Food Sci., 1968, 33, 488-492. 52. Maier, V. P.; Margileth, D. A. Limonoic acid Α-ring lactone, a new limonin derivative in citrus. Phytochem., 1969, 8, 243-248. 53. Maier, V. P.; Hasegawa, S., Hera, E. Limonin D-ring lactone hydrolase. A new enzyme from citrus seeds. Phytochem., 1969, 8, 405-407. 54. Hasegawa, S. Metabolis hydrolase activity in Pseudomonas. J. Agri. Food Chem., 1976, 24, 24-26. 55. Hasegawa, S.; Hoagland, J. E. Biosynthesis of limonoids in citrus. Phytochem., 1977, 16, 469-471. 56. Hasegawa, S.; Maier, V. P.; Bennett, R. D. Detection of limonoate dehydrogenase activity in albedo tissues of Citrus sinensis. Phytochem., 1974, 13, 103-105. 57. Hasegawa, S.; Bennett, R. D.; Maier, V. P.; King, A. D., Jr. Limonoate dehydrogenase from Arthrobacter globiformis. J. Agric. Food Chem., 1972, 20, 1031-1034. 58. Hsu, A. C.; Hasegawa, S.; Maier, V. P.; Bennett, R. D. 17-Dehydrolimonoate Α-ring lactone, a possible metabolite of limonoate Α-ring lactone in citrus fruits. Phytochem., 1973, 12, 563-567. 59. Hasegawa, S.; Brewster, L. C.; Maier, V. P. Use of limonoate dehydrogenase of Arthrobacter globiformis for the prevention or removal of limonin bitterness in citrus products. J. Food Sci., 1973, 38, 1153-1155. 60. Hasegawa, S.; Brewster, L. C. Limonoate dehydrogenase and debittering of citrus juice therewith. U. S. Patent 3,911,103. 61. Hasegawa, S.; Maier, V. P.; King, A. D. Jr. Isolation of new limonoate dehydrogenase from Pseudomonas. J. Agric. Food Chem., 1974, 22, 523-526. 62. Brewster, L. C.; Hasegawa, S.; Maier, V. P. Bitterness prevention in citrus juices. Comparative activities and stabilities of the limonoate dehydrogenase from Pseudomonas and Arthrobacter. J. Agric. Food Chem., 1976, 24, 21-24. 63. Nicol, K. J . ; Chandler, Β. V. The extraction of the enzyme degrading the limonin precursor in citrus albedo. J. Sci. Food Agric., 1978, 29, 795-802.

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64. Bruemmer, J. H.; Baker, R. Α.; Roe, B. Enzymes affecting flavor and appearance of citrus products. "Enzymes in Food and Beverage Processing". R. L. Ory and A. J. St. Angelo, eds. ACS Symposium Series #47, 1977, American Chemical Society, Washington, D. C. p. 1-11. 65. Jansen, E. F.; Jang, R.; MacDonnell, L. R. Citrus acetylesterase. Arch. Biochem. Biophys., 1947, 15, 415-431. 66. Bruemmer, J. H.; Roe, B. Esterase activity and orange juice quality. Proc. Fla. State Hort. Soc., 1975, 88, 300-303. 67. Axelrod, B. Citrus fruit phosphatase. J. Biol. Chem., 1947, 167, 57-72. 68. Bruemmer, J. H.; Roe, B.; Bowen, E. R. Peroxidase reactions and orange juice quality. J. Food Sci., 1976, 41, 186-189. 69. Bruemmer, J. H.; Roe, B. Enzymic oxidation of simple diphenols and flavonoids by orange juice extracts. J. Food Sci., 1970, 35, 116-119. 70. Roe, B.; Bruemmer orange juice. J. 71. Raymond, W. R.; Hostettler, J. B.; Assar, K.; Varsel, C. Orange pyruvic decarboxylase: isolation and mechanistic studies. J. Food Sci., 1979, 44, 777-781. RECEIVED May 23, 1980.

In Citrus Nutrition and Quality; Nagy, S., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

9 Importance of Selected Volatile Components to Natural Orange, Grapefruit, Tangerine, and Mandarin Flavors PHILIP E. SHAW and CHARLES W. WILSON III U.S. Citrus and Subtropical Products Laboratory, U.S. Department of Agriculture, Sciences and Education Administration, Southern Region, P.O. Box 1909, Winter Haven, FL 33880 During the past tw information on compound citrus cultivars, orange, grapefruit, mandarin (tangerine), lemon, and lime. Widespread use of gas chromatography has enabled researchers to identify and quantitate many of the flavor constituents in these citrus cultivars (1, 2, 3). In a limited way, we are able to describe the mixture of components necessary to create the unique flavor of each of the major citrus fruits. However, the contributions to citrus flavor of many of the volatile and nonvolatile citrus components that have been identified are undetermined or only partly characterized. The interrelationship of citrus components and their effect on citrus flavor, particularly orange, is widely known, but few definitive studies have been carried out. Several compounds have been implicated as important to the flavor of certain types of citrus, especially mandarin, but evidence supporting these relationships is not definitive. In t h i s chapter, we present some s p e c i f i c evidence on c e r t a i n components important to c i t r u s f l a v o r . The i n t e r r e l a t i o n s h i p of c e r t a i n v o l a t i l e components to orange f l a v o r i s described and the f l a v o r of g r a p e f r u i t and the importance of s p e c i f i c compounds to the f l a v o r of mandarin and tangerine are r e l a t e d to recent t a s t e panel s t u d i e s at our l a b o r a t o r y . Components Important to N a t u r a l Orange Flavor Orange f l a v o r i s one of the most popular and g e n e r a l l y accepted f r u i t f l a v o r s ; i t i s widely used i n f l a v o r i n g beverages, c a n d i e s , and other foods i n which f r u i t f l a v o r notes are desirable. E a r l y research workers f e l t that the unique f l a v o r of orange could be a s c r i b e d to one or a few components of the j u i c e and o i l , such as s p e c i f i c aldehydes (4), but extensive a n a l y t i c a l s t u d i e s during the past 20 years have led to the conclusion that a mixture of s e v e r a l compounds i n the proper p r o p o r t i o n s i s necessary for a good orange f l a v o r (1_) .

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In t e s t s to b e t t e r define t h i s mixture of components (and t h e i r proper proportions) necessary f o r good orange f l a v o r , v o l a t i l e components b e l i e v e d from p r i o r a n a l y t i c a l studies to be important to orange f l a v o r were examined (5_) . I n d i v i d u a l t a s t e and aroma thresholds i n water were determined on the compounds s e l e c t e d . Then, the i n f l u e n c e of n o n v o l a t i l e j u i c e c o n s t i t u e n t s on the t a s t e threshold of c e r t a i n of the v o l a t i l e components was studied. F i n a l l y , s e l e c t e d i n d i v i d u a l compounds and mixtures c o n t a i n i n g from two to s i x components were evaluated i n a bland j u i c e medium f o r t h e i r c o n t r i b u t i o n to orange f l a v o r . Taste and Aroma Thresholds i n Water of I n d i v i d u a l Orange J u i c e Components Table I l i s t s the aroma and t a s t e thresholds i n water f o r 29 orange j u i c e components b e l i e v e d to c o n t r i b u t e to orange f l a v o r (6^ 7) and the estimate of these compounds base 15). For some components, no q u a n t i t a t i v e data i s a v a i l a b l e f o r even an estimation of t h e i r concentrations i n orange j u i c e . Several d i f f i c u l t i e s are encountered i n t r y i n g to estimate q u a n t i t i e s of i n d i v i d u a l components i n orange j u i c e . I f d i s t i l l a t i o n , e x t r a c t i o n with an organic s o l v e n t , and d e r i v a t i v e formation are used to separate i n d i v i d u a l components q u a n t i t a t i v e l y , as i n the e a r l y work of Kirchner and M i l l e r (6), q u a n t i t a t i v e values w i l l be low because of i n e v i t a b l e l o s s e s . J u i c e free from peel o i l (9) w i l l have abnormally low l e v e l s of j u i c e components that are a l s o present i n the peel o i l , because most commercial j u i c e s contain 0.015-0.025% o i l . Reasonably accurate estimates f o r most commercial j u i c e s prepared from frozen concentrated orange j u i c e can be c a l c u l a t e d with q u a n t i t a t i v e values i n p e e l o i l i f the o i l l e v e l i s w i t h i n the above range (e.g., 0.0175% o i l ) . However, s i n g l e - s t r e n g t h orange j u i c e processed d i r e c t l y from the e x t r a c t o r s contains about 0.005% j u i c e o i l (16) , and thus i t s o i l content would c o n s i s t of about 75% p e e l o i l and 25% j u i c e o i l . Most of the estimates i n Table I are c a l c u l a t e d on the assumption that a l l o i l i n the j u i c e i s p e e l o i l . Few accurate q u a n t i t a t i v e values e x i s t on i n d i v i d u a l c o n s t i t u e n t s of j u i c e o i l because of the d i f f i c u l t y i n o b t a i n i n g a sample uncontaminated with p e e l o i l . Thus, the estimates i n v o l v i n g o i l c o n s t i t u e n t s must be based on p e e l o i l values only. S t a t i s t i c a l a n a l y s i s of the aroma and t a s t e threshold values i n Table I showed that w i t h i n 95% confidence l i m i t s there was no d i f f e r e n c e between aroma and t a s t e threshold values f o r a l l compounds but four. Octanal and c i t r a l had aroma threshold values s i g n i f i c a n t l y higher than the corresponding t a s t e threshold values. Nonanal and trans-2-hexenal had higher t a s t e threshold than aroma t h r e s h o l d values.

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169

Comparison of the t a s t e t h r e s h o l d w i t h e s t i m a t e d c o n c e n t r a t i o n i n o r a n g e j u i c e (where a v a i l a b l e ) i n T a b l e I r e v e a l s t h a t i n a l l c a s e s e x c e p t o c t y l a c e t a t e and α - p i n e n e , t h e c o n c e n ­ t r a t i o n i n orange j u i c e exceeds the t a s t e t h r e s h o l d i n water f o r most v a l u e s r e p o r t e d . P a t t o n and J o s e p h s o n (17) postulated that components p r e s e n t i n a f o o d a t above t h r e s h o l d l e v e l make a p o s i t i v e c o n t r i b u t i o n to the f l a v o r , w h i l e those p r e s e n t at below t h r e s h o l d l e v e l make l i t t l e o r no c o n t r i b u t i o n t o f l a v o r . This g e n e r a l i z a t i o n i s now c o n s i d e r e d an o v e r s i m p l i f i c a t i o n , f o r s y n e r g i s t i c e f f e c t s among f o o d c o n s t i t u e n t s h a v e b e e n shown t o d e c r e a s e t h e t h r e s h o l d l e v e l o f some compounds, and n o n v o l a t i l e c o n s t i t u e n t s a r e known t o e i t h e r i n c r e a s e o r d e c r e a s e t h e t a s t e t h r e s h o l d o f c e r t a i n v o l a t i l e and n o n v o l a t i l e c o n s t i t u e n t s . I n e a r l y s t u d i e s , t h e f l a v o r o f o r a n g e was a s c r i b e d t o t h e p r e s e n c e o f a l d e h y d e s , e s p e c i a l l y o c t a n a l and d e c a n a l (18). One t a s t e p a n e l j u d g e d 1 ppm a q u e o u s s o l u t i o n s o f o c t a n a l , n o n a n a l , d e c a n a l , or dodecanal t t a s t e Ç7). The a l d e h y d e s i g n i f i c a n t l y to f l a v o r of orange j u i c e are those w i t h concentrat i o n s i n j u i c e t h a t g r e a t l y exceed the i n d i v i d u a l t a s t e t h r e s h o l d levels. The e s t i m a t e d c o n c e n t r a t i o n s o f e t h a n a l (acetaldehyde), o c t a n a l , and d o d e c a n a l i n j u i c e a r e more t h a n 100 times higher than t h e i r taste thresholds. O c t a n a l and d o d e c a n a l h a v e l o w e r t a s t e t h r e s h o l d s t h a n w o u l d be p r e d i c t e d f r o m v a l u e s f o r o t h e r s t r a i g h t c h a i n a l d e h y d e s i n the homologous s e r i e s . This phenomenon has b e e n o b s e r v e d b e f o r e (19) for aldehydes with chain l e n g t h s i n m u l t i p l e s o f f o u r c a r b o n atoms. Of t h e compounds l i s t e d i n T a b l e I , e t h y l b u t y r a t e was p r e s e n t i n orange j u i c e at the g r e a t e s t l e v e l above i t s t a s t e t h r e s h o l d v a l u e (2200 t i m e s i t s t h r e s h o l d l e v e l ) . Kefford (18) s t a t e d t h a t a l d e h y d e s and e s t e r s were i m p o r t a n t t o o r a n g e f l a v o r . E t h y l b u t y r a t e i s one o f t h e m a i n e s t e r s i n a q u e o u s o r a n g e e s s e n c e (2^). I t s low t a s t e t h r e s h o l d r e l a t i v e t o i t s e s t i m a t e d concentrat i o n i n j u i c e and i t s d e f i n i t e c o n t r i b u t i o n t o " f r u i t y " f l a v o r n o t e s s u g g e s t t h a t e t h y l b u t y r a t e i s a m a j o r c o n t r i b u t o r t o good o r a n g e f l a v o r . The t a s t e t h r e s h o l d f o r e t h y l b u t y r a t e l i s t e d i n T a b l e I (7_) i s s u b s t a n t i a l l y l o w e r t h a n two e a r l i e r r e p o r t e d v a l u e s f o r t h i s compound o f 450 ppb (20) and 150 ppb (21). The e a r l i e r s t u d i e s involved s t a r t i n g at a high c o n c e n t r a t i o n and d e c r e a s i n g t h e c o n c e n t r a t i o n u n t i l t h e compound c o u l d n o t be d e t e c t e d so t h a t f a t i g u e c o u l d h a v e b e e n a f a c t o r i n t h e r e l a t i v e l y high values obtained. The v a l u e i n T a b l e I was d e t e r m i n e d by a p r o c e d u r e i n w h i c h f a t i g u e was n o t a p o s s i b l e factor (7_). L i m o n e n e , a t e r p e n e h y d r o c a r b o n , i s t h e m a j o r component o f o r a n g e o i l (3), and i t i s p r e s e n t i n o r a n g e j u i c e a t a l e v e l a l m o s t 800 times i t s taste threshold i n water. Limonene p o s s e s s e s a weak, c i t r u s - l i k e aroma b u t does n o t by i t s e l f i m p a r t an o r a n g e l i k e f l a v o r n o t e t o a b l a n d o r a n g e j u i c e b a s e (5^, 6). Limonene and t h e o t h e r t e r p e n e h y d r o c a r b o n s p r o b a b l y make a s i g n i f i c a n t

In Citrus Nutrition and Quality; Nagy, S., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

170

CITRUS

NUTRITION

AND

QUALITY

Table I. Aroma and taste thresholds i n water o f s e l e c t e d orange j u i c e components [from Ahmed et a l . ( 7 ) ] .

J u i c e component

Concn. (ppb) i n orange j u i c e

Thresholds (ppb) Aroma Taste

Aldehydes Ethanal Butanal Hexanal Octanal Nonanal Decanal Dodecanal trans-2-Hexenal Citral Geranial Citronellal Perillaldehyde 3-Sinensal

17. 15.9 9.18 1.41 2.53 1.97 0.53 24.2 85.3 — 66 30.1 3.8

5.26 3.66 0.52 4.25 3.02 1, 07 49. 3 41. 4 40 35 25. 3.

C

4θΐ\ 260 60 . 510 , 950 160 14 , 735 . 1,110 105 , 138 e

u

C

e

u

~"f 14 2 6

45 , 280 , 385

fi

53

d

Hydrocarbons Limonene Myrcene a-Pinene p-Cymene

60 36 9.5 11.4

210 42 1013.8 13.3

4100°, 166,000 , 6 8 , 2200 , 3500 2 2 , 175 , 875 12 e

e

e

e

d

e

Esters E t h y l butyrate E t h y l propionate Methyl butyrate Nonyl acetate O c t y l acetate

0. 13 9, 9 43 57 47

0.13 4.9 59 270 210

290"

175*

In Citrus Nutrition and Quality; Nagy, S., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

d,e

9.

SHAW

A N D WILSON

Volatile

Components

and

Citrus

Flavor

171

Alcohols Octanol Decanol Dodecanol Linalool a-Terpineol

C

b

190 47 73 5.3 28

54 23 66 3.8

10 - 210 100 — , , 150°, 525 , 930

2.7 0.9

86.0 1.2

3 5 , 175

b

Ketones d-Carvone l-Penten-3-one

determined

b

with a l a r g e group o f 55-73 untrained

h

panelists.

^ K i r c h n e r and M i l l e r ( 8 ) . S c h r e i e r et a l . (9^, f o r j u i c e with no p e e l o i l present. ^Estimated from data of Shaw and Coleman (10), assuming a l e v e l of 0.0175% p e e l o i l i n j u i c e . Estimated from the data o f L i f s h i t z et a l . (11) , f o r F l o r i d a orange o i l , assuming a l e v e l o f 0.0175% p e e l o i l i n j u i c e . f

Naves (12).

^Estimated from data of Z i e g l e r peel o i l i n juice.

(13), assuming a l e v e l o f 0.0175%

^ S t a n l e y et a l . (14), u s i n g reported percent aldehyde value (15), and assuming 0.0175% p e e l o i l i n j u i c e . Journal of Agricultural and Food Chemistry

In Citrus Nutrition and Quality; Nagy, S., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

172

CITRUS

NUTRITION

AND

QUALITY

c o n t r i b u t i o n t o o r a n g e j u i c e f l a v o r , p e r h a p s as c a r r i e r s f o r t h e o x y g e n a t e d c o n s t i t u e n t s i n c o l d - p r e s s e d o i l s (22) , f o r c o n c e n t r a t e d ( f o l d e d ) o i l s i n w h i c h much o f t h e t e r p e n e h y d r o c a r b o n s h a v e b e e n removed by d i s t i l l a t i o n h a v e somewhat d i f f e r e n t c h a r a c t e r i s t i c s from the c o r r e s p o n d i n g c o l d - p r e s s e d oils. C o n c e n t r a t e d o i l s a r e u s e d w i d e l y i n b a k e d goods b e c a u s e t h e y r e t a i n t h e i r f l a v o r s w e l l i n the f i n i s h e d p r o d u c t s . They a r e a l s o used i n the p r e p a r a t i o n of c e r t a i n foods because of t h e i r i n c r e a s e d s o l u b i l i t y due t o t h e d e c r e a s e d amounts o f h y d r o c a r b o n s present. E f f e c t of Nonvolatile Thresholds

J u i c e Components on

Individual

Flavor

I n t e r a c t i o n o f v o l a t i l e and n o n v o l a t i l e c o n s t i t u e n t s i n r e s u l t s i n f l a v o r modifications of varying i n t e n s i t i e s . The e f f e c t s of 5 -nucleotide and t h e e f f e c t s o f a c i d o f l i m o n e n e (24) h a v e b e e n s t u d i e d i n o r a n g e j u i c e .

foods

1

Some 5 ' - n u c l e o t i d e s h a v e b e e n i m p l i c a t e d as f l a v o r m o d i f i e r s , and o r a n g e j u i c e i s known t o c o n t a i n r e l a t i v e l y l a r g e q u a n t i t i e s o f c e r t a i n 5 - n u c l e o t i d e s , w i t h a t o t a l l e v e l o f a b o u t 40 ppm (25, 26). I n a s t u d y o f t h e i n f l u e n c e o f s i x 5 - n u c l e o t i d e s a t t h e 10 ppm l e v e l on t h e t a s t e t h r e s h o l d o f o c t a n a l i n w a t e r ( T a b l e I I ) , two o f t h e 5 - n u c l e o t i d e s (GMP and ADP) s i g n i f i c a n t l y lowered the t h r e s h o l d o f o c t a n a l (23). GMP i s known t o e n h a n c e f l a v o r i n f o o d s , b u t ADP had b e e n r e p o r t e d t o h a v e l i t t l e o r no modifying e f f e c t on f o o d f l a v o r s (23). A n a l y s i s o f v a r i a n c e showed t h a t GMP, ADP and GDP e n h a n c e d t h e f l a v o r o f o c t a n a l i n a q u e o u s 1

T

?

Table I I . Taste thresholds of o c t a n a l s o l u t i o n s of s e l e c t e d 5 -nucleotides f

Threshold c o n c n . (ppb)

5'-Nucleotide C o n t r o l (none) ATP ( a d e n o s i n e GTP ( g u a n o s i n e AMP (adenosine GDP (guanosine GMP (guanosine ADP

(adenosine

5 5 5 5 5

Significantly by

t

test.

f

-triphosphate) -triphosphate) -monophosphate) -diphosphate) -monophosphate) 5 -diphosphate) f

1

f

1

1

Twelve screened

and

determined i n aqueous a t 10 ppm (23)

1.3S 1.22 1.21 1.20 0.99 0.86" 0.85

from c o n t r o l at

1.20 0.91 1.00 0.87 0.51 0.57 0.76

b

t r a i n e d p a n e l i s t s were

different

95% C o n f i d e n c e limits (ppb)

95%

-

1.59 1.65 1.47 1.64 1.68 1.29 0.95

employed. level

as

determined Journal of Food Science

In Citrus Nutrition and Quality; Nagy, S., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

9.

SHAW A N D WILSON

Volatile

Components

and

Citrus

173

Flavor

s o l u t i o n . Thus, t h i s study seems to answer the question r a i s e d by Woskow (27) whether 5'-nucleotides enhance the f l a v o r of the food rather than suppress undesirable o f f - f l a v o r s , f o r no other f l a v o r s were present to be suppressed i n t h i s study. E f f e c t s of organic a c i d s , sugars, and p e c t i n at the concent r a t i o n s normally present i n orange j u i c e on the f l a v o r threshold of d-limonene i n water were a l s o studied by Ahmed et a l . (24). Included i n that study were f l a v o r e f f e c t s i n v o l v i n g i n t e r a c t i o n s between the n o n v o l a t i l e c o n s t i t u e n t s of orange j u i c e and e f f e c t s of the n o n v o l a t i l e c o n s t i t u e n t s on p r e c i s i o n among the t a s t e panel members (Table III). These data show that n e i t h e r i n d i v i d u a l nonv o l a t i l e components nor combinations of them v a r i e d s i g n i f i c a n t l y i n t h e i r e f f e c t on the threshold of d-limonene. Because p e c t i n i s g e n e r a l l y regarded as a t h i c k e n i n g agent, i t s e f f e c t on the t h r e s h o l d of d-limonene may be due to t e x t u r a l p r o p e r t i e s (24). Table I I I . Taste threshold containing pectin, acid

Solutions

Thresholds (ppm)

95% Confidence limits (ppm)

Water only Acid Pectin Sugar P e c t i n and a c i d P e c t i n and sugar Acid and sugar P e c t i n , a c i d , and sugar

0.21 0.41 0.22 0.35 0.31 0.23 0.38 0.36

0.05-0.79 0.17-0.99 0.07-0.61 0.12-0.96 0.08-1.20 0.08-0.64 0.14-1.10 0.13-1.00

^Twelve screened and b

All

Correlation^ c o e f f i c i e n t (r) 0.82 0.91 0.88 0.89 0.83 0.89 0.88 0.89

t r a i n e d p a n e l i s t s were employed.

c o r r e l a t i o n c o e f f i c i e n t s were s i g n i f i c a n t at the 0.01

level.

Journal of Agricultural and Food Chemistry

When the average e f f e c t of each component was evaluated with a t_ t e s t , some d i f f e r e n c e s were found (Table IV). The t a s t e threshold f o r limonene i n s o l u t i o n s c o n t a i n i n g a c i d was s i g n i f i c a n t l y higher than that i n s o l u t i o n s not containing a c i d . These r e s u l t s a l s o show a tendency f o r threshold values to be lower i n s o l u t i o n s c o n t a i n i n g p e c t i n and higher i n s o l u t i o n s c o n t a i n i n g sugar than those without. Although d-limonene may not be r e p r e s e n t a t i v e of a l l v o l a t i l e f l a v o r components i n orange j u i c e , these r e s u l t s i n d i c a t e that a c i d probably plays an important r o l e i n masking the e f f e c t of some v o l a t i l e f l a v o r components. Pangborn (28) reported that i n f r u i t nectar, the greater the a c i d i t y , the greater the depressing e f f e c t on the i n t e n s i t y of the compound added. The observation that sugar tended to r a i s e the threshold of d-limonene agrees with the reported observation (29) that sweetness beyond that imparted by 15% sugar i n t e r f e r e s with f l a v o r perception.

In Citrus Nutrition and Quality; Nagy, S., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

174

CITRUS

NUTRITION A N D QUALITY

C e r t a i n o f the n o n v o l a t i l e compounds a f f e c t e d the a b i l i t y of p a n e l i s t s to determine the t h r e s h o l d o f d-limonene (Table V ) . Thus, p r e c i s i o n appeared high i n s o l u t i o n s o f water and i n the aqueous s o l u t i o n s of sugar alone, but was l e s s i n s o l u t i o n s con­ t a i n i n g p e c t i n and l e a s t i n s o l u t i o n s c o n t a i n i n g a c i d . Table IV. Average t a s t e t h r e s h o l d o f d-limonene i n aqueous s o l u t i o n s with and without p e c t i n , a c i d , and sugar.(24) Average thresholds (ppm)

Solution

With a c i d Without a c i d With suga Without suga With p e c t i n Without p e c t i n a

0.36 0.25

a

a

0.28 0.34

S i g n i f i c a n t l y d i f f e r e n t at Ρ 6.2 6.5 6.5 6.6 6.7 7.1 7.5 7.7 7.7 8.1 8.1 8.2

n

6.7 6.7 6.7 6.7 6.7 7.0 7.1 7.3 7.3 7.3 7.^ 7.3 7.5 7.5 7.7 7.7 7.9 8.3

4.3 ± 0.4 4.7 ± 0.4 4 . 7 ±±0 . 4 0.5 4.9 4.9±0.5 5.0 ± 0.3 5.1 ± 0.5 5.4 ± 0.4 5.7 ± 0.4 5.8 ± 0.4 5 . 8 ±±0 0.2 .4 6.1 6.1 ± 0.2 6.3 ± 0.3

1

5.1 + 0.3 5.1 + 0.3 ± 0.4 5.1 ± 5.1 ± ± 0.5 ± 0.5 5.1 ± 5.3 5 . 3 ±±0 0.3 .3 5.4 5 . 4 ±±0 0.4 .4 5.5 5 . 5 ±±0 0.3 .3 5.5 5 . 5 ±±0 0.4 .4 ± η 0.3^ 5.5 s ς + 5.5 ± 0.3 5.7 5 . 7 ±±0 0.3 .3 5.7 5 . 7 ±±0 0.4 .4 5.8 ° ± 0.3 ° 5.8 ± 0.4 ± 00.2 6.0 6.0± .2 6.3 ' ~ ± 0.5 " 7 7

66 70 70

57 r62 -> b2 65 65 66 67 71 75 77 77 81 81 82

Λ Ο

67 67 67 67 67 70 71 73 73 73 73 75 73 75 75 77 77 79 83 *

^

§

a

«s

§

£

^

~ g 2

g

>

a ^

In Citrus Nutrition and Quality; Nagy, S., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

15 45 55 75

4.5 5.5 7.5

3.4 ± 0.4 4.2 ± 0.4 5.7 ± 0.3

81

1.5

8.1

1.1 ± 0.1

6.1 ± 0.2

Based on r e f e r e n c e orange j u i c e score as 100% of maximum. Journal of Agricultural and Food Chemistry

Sensory s c o r e s : 1 s i m i l a r to pumpout j u i c e , 10 s i m i l a r to r e f e r e n c e j u i c e . A c t u a l data presented as mean ± standard d e v i a t i o n . A l l mixtures were prepared by adding components to bland orange j u i c e d r i n k at l e v e l s reported by Ahmed e t a l . (30).

Acetaldehyde, c i t r a l , c i t r o n e l l a l , decanal, e t h y l b u t y r a t e , d-limonene, l i n a l o o l , myrcene, nonanal, o c t a n a l , a-pinene, trans-2-hexenal Acetaldehyde, c i t r a l , c i t r o n e l l a l , decanal, e t h y l butyrate, d-limonene, l i n a l o o l , o c t a n a l , a-pinene Acetaldehyde, c i t r a l , c i t r o n e l l a l , e t h y l b u t y r a t e , d-limonene, o c t a n a l Acetaldehyde, c i t r a l , c i t r o n e l l a l , e t h y l b u t y r a t e , d-limonene, l i n a l o o l , o c t a n a l , a-pinene

Six-twelve components

Acetaldehyde, e t h y l b u t y r a t e , c i t r a l , d-limonene, o c t a n a l

9.

SHAW AND WILSON

Volatile

Components

and

Citrus

Flavor

181

These s t u d i e s suggest that mixtures of d-limonene, e t h y l b u t y r a t e , and c e r t a i n aldehydes seem to be best at p r o v i d i n g good orange f l a v o r when mixtures of only a few f l a v o r components are used. Mixtures of more than four components seemed to have l e s s d e s i r a b l e f l a v o r i n g q u a l i t i e s than simpler t h r e e - and f o u r component mixtures. P r e c i s e q u a n t i t a t i v e values f o r i n d i v i d u a l f l a v o r components i n c i t r u s j u i c e s are s t i l l l a c k i n g (3). Perhaps i n the'mixtures c o n t a i n i n g f i v e or more f l a v o r components, where i n t e r a c t i o n s between components are accentuated, a blend of components more n e a r l y i d e n t i c a l to that n a t u r a l l y o c c u r r i n g i n orange j u i c e i s c r i t i c a l to avoid c r e a t i o n of u n d e s i r a b l e f l a v o r e f f e c t s due to these i n t e r a c t i o n s . Components Important to N a t u r a l G r a p e f r u i t

Flavor

G r a p e f r u i t f l a v o r has become i n c r e a s i n g l y popular i n recent years, p a r t l y because f r e s worldwide i n i n c r e a s i n g nootkatone, the primary f l a v o r impact component of g r a p e f r u i t , i s a v a i l a b l e f o r use i n s y n t h e t i c g r a p e f r u i t - f l a v o r e d beverages and other foods. Since the d i s c o v e r y and s y n t h e s i s of nootkatone (31, 32), food f l a v o r i s t s have discussed i t s r e l a t i v e importance i n g r a p e f r u i t f l a v o r , and whether i t i s even necessary f o r good g r a p e f r u i t f l a v o r (33). Nootkatone. The t a s t e threshold of nootkatone was f i r s t reported to be 20-40 ppm i n sucrose s o l u t i o n , and the odor was d e t e c t a b l e at l e s s than 10 ppm (31). MacLeod and Buigues estimated the l e v e l of nootkatone i n j u i c e s f r e e from p e e l o i l at about the t a s t e threshold l e v e l found i n sucrose s o l u t i o n (31). T h e i r data of 77 mg of crude nootkatone i s o l a t e d from 1 g a l of p e e l - o i l - f r e e j u i c e and rag represent a l e v e l of about 20 ppm nootkatone i n the j u i c e . Taste thresholds of 1 ppm nootkatone i n water and 5-6 ppm i n g r a p e f r u i t j u i c e were determined by Berry et a l . (34), who s t a t e d that g r a p e f r u i t j u i c e c o n t a i n i n g 0.005% o i l contains an average l e v e l of l e s s than 0.5 ppm nootkatone, which i s c o n s i d e r a b l y lower than the t h r e s h o l d l e v e l i n j u i c e . They considered only the nootkatone present i n the o i l , and any nootkatone present i n the o i l - f r e e j u i c e would have added to t h i s v a l u e . Some reported odor t h r e s h o l d values f o r nootkatone were c o n s i d e r a b l y lower than the t a s t e t h r e s h o l d v a l u e s . Odor t h r e s h o l d s of 0.8 ppm i n water and 30 ppm i n a i r were reported f o r (+)-nootkatone, the enantiomer of t h i s sesquiterpene ketone present i n g r a p e f r u i t (35) . An odor t h r e s h o l d of 0.15 ppm i n water was reported f o r c r y s t a l l i n e nootkatone i s o l a t e d from grapef r u i t o i l (36). In that study, mother l i q u o r from c r y s t a l l i z a t i o n of nootkatone was 30 times more potent (odor t h r e s h o l d of mother l i q u o r 5 ppb) than nootkatone alone and the panel f e l t that the aroma of the mother l i q u o r more c l o s e l y resembled g r a p e f r u i t aroma

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than d i d that of c r y s t a l l i n e nootkatone. When s y n t h e t i c nootkatone i s used to f l a v o r g r a p e f r u i t - f l a v o r e d products, the t e c h n i c a l grade, which contains more i m p u r i t i e s , i s g e n e r a l l y p r e f e r r e d over the pure grade (37). Thus, i t i s c l e a r that other g r a p e f r u i t o i l components, probably s i m i l a r to nootkatone i n chemical s t r u c t u r e , c o n t r i b u t e to the f l a v o r and aroma of grapefruit o i l . We c a r r i e d out a recent study to b e t t e r d e f i n e the r o l e of nootkatone i n the aroma and f l a v o r of cold-pressed g r a p e f r u i t o i l (33). We obtained good q u a l i t y commercial cold-pressed g r a p e f r u i t o i l s w i t h r e l a t i v e l y h i g h and low l e v e l s of nootkatone and compared t h e i r aromas with those of limonene (the major component of g r a p e f r u i t o i l ) and nootkatone i n limonene at the l e v e l corresponding to that i n the high-nootkatone g r a p e f r u i t o i l sample. Then we added these o i l s to s i n g l e - s t r e n g t h j u i c e from commercially prepared frozen concentrated g r a p e f r u i t j u i c e that contained no added p e e l o i l or f l a v o and compared the t a s t e o a l l cases an experienced 12-member panel was used. Table V I I I . E f f e c t of nootkatone content on aroma and t a s t e of g r a p e f r u i t o i l and j u i c e . Confidence l i m i t (%) Aroma pangl Taste panel with o i l s with j u i c e s

O i l samples compared

Limonene v s . high-nootkatone o i l Limonene v s . nootkatone i n limonene Nootkatone i n limonene v s . h i g h nootkatone o i l Low-nootkatone o i l v s . high-nootkatone oil Nootkatone + low-nootkatone o i l v s . high-nootkatone o i l

99

99.9 N.S.

99

95

N.S.

N.S.

95

N.S.

High-nootkatone o i l , nootkatone i n limonene, and nootkatone + lownootkatone o i l a l l c o n t a i n 0.83% nootkatone; low-nootkatone o i l contains 0.02% nootkatone. P a i r e d comparison d i f f e r e n c e

test.

T r i a n g l e comparison d i f f e r e n c e

test.

The t a s t e panel could r e a d i l y d i s t i n g u i s h a sample of j u i c e c o n t a i n i n g high-nootkatone (0.83%) g r a p e f r u i t o i l from a sample of j u i c e c o n t a i n i n g limonene at the same e f f e c t i v e o i l l e v e l , thus showing that f l a v o r of the g r a p e f r u i t j u i c e d i d not mask the e f f e c t of added g r a p e f r u i t o i l . In aroma t e s t s w i t h limonene samples, the panel d i s t i n g u i s h e d 0.83% nootkatone i n limonene from limonene and 0.83% nootkatone i n limonene from high-nootkatone o i l

In Citrus Nutrition and Quality; Nagy, S., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

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at the 99% confidence l e v e l . However, the t a s t e o f j u i c e c o n t a i n i n g these samples was l e s s a f f e c t e d . The panel was unable to d i s t i n g u i s h j u i c e c o n t a i n i n g only added limonene from j u i c e c o n t a i n i n g 0.83% nootkatone i n limonene, but i t could d i s t i n g u i s h j u i c e c o n t a i n i n g high-nootkatone o i l from that c o n t a i n i n g nootkatone i n limonene at the 95% confidence l e v e l . These r e s u l t s i n d i c a t e the importance o f c o n s t i t u e n t s other than nootkatone t o the f l a v o r of g r a p e f r u i t j u i c e c o n t a i n i n g added cold-pressed o i l . Comparison of low- and high-nootkatone o i l s g e n e r a l l y showed i n s i g n i f i c a n t d i f f e r e n c e s i n aroma or t a s t e . Low-nootkatone o i l was not s i g n i f i c a n t l y d i f f e r e n t from high-nootkatone o i l e i t h e r by aroma o f the neat o i l s or by t a s t e imparted to s i n g l e - s t r e n g t h g r a p e f r u i t j u i c e . When c r y s t a l l i n e nootkatone was added to lownootkatone o i l t o r a i s e the l e v e l of t h i s compound t o that i n the high-nootkatone o i l , the aromas of the two o i l s were s i g n i f i c a n t l y d i f f e r e n t , but j u i c e s f l a v o r e d with the two o i l s could not be d i s t i n g u i s h e d by t a s t e the f l a v o r o f the low-nootkaton e f f e c t s with other o i l components. However, the nootkatone l e v e l i n the o i l i s i n s u f f i c i e n t to cause dramatic changes i n the t a s t e of g r a p e f r u i t j u i c e f l a v o r e d with these o i l s . One c r i t i c a l f a c t o r i n the e f f e c t o f cold-pressed g r a p e f r u i t o i l c o n t a i n i n g nootkatone on the f l a v o r of g r a p e f r u i t j u i c e i s the l e v e l o f nootkatone present i n the j u i c e p r i o r t o a d d i t i o n o f the oil. We estimated the l e v e l of nootkatone i n our g r a p e f r u i t j u i c e sample t o be 7 ppm by t h i n - l a y e r chromatography (33, 38). This i s w i t h i n the range of 6-7 ppm nootkatone reported (34) f o r optimum flavor. Thus, a d d i t i o n of even high-nootkatone o i l adds only about 0.8 ppm t o t h i s value, which i s already above the t h r e s h o l d level. I t i s perhaps not s u r p r i s i n g that the t a s t e panel had d i f f i c u l t y d i s t i n g u i s h i n g high-nootkatone o i l from low-nootkatone o i l i n s i n g l e - s t r e n g t h g r a p e f r u i t j u i c e . This s i t u a t i o n i s comparable to that found f o r commercial samples o f g r a p e f r u i t j u i c e f l a v o r e d with added cold-pressed o i l , s i n c e both frozen concentrated g r a p e f r u i t j u i c e and s i n g l e - s t r e n g t h j u i c e would be expected t o cont a in nootkatone p r i o r to a d d i t i o n of cold-pressed o i l (31). Nootkatone i s one of the l e s s - v o l a t i l e o i l components so that frozen concentrated and d e o i l e d s i n g l e - s t r e n g t h j u i c e might be expected t o have higher nootkatone l e v e l s than the o i l content as determined by standard methods might i n d i c a t e . Aldehydes and E s t e r s . Aldehydes have long been considered important t o the f l a v o r of g r a p e f r u i t and other cold-pressed o i l s from c i t r u s . The t o t a l l e v e l of aldehydes i n F l o r i d a g r a p e f r u i t o i l reaches a maximum i n f r u i t harvested during mid-season (39). Kesterson e t a l . (39) s t a t e d that optimum q u a l i t y g r a p e f r u i t o i l s c o n t a i n the highest l e v e l of t o t a l aldehydes (about 1.8%) and a moderately high l e v e l o f nootkatone (0.5-0.7%). Thus, the aldehydes are c l e a r l y important to g r a p e f r u i t o i l q u a l i t y . Esters have a l s o been i m p l i c a t e d as important c o n t r i b u t o r s to f l a v o r of

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g r a p e f r u i t o i l (40). Q u a n t i t i e s of s e v e r a l of these e s t e r s have r e c e n t l y been determined i n g r a p e f r u i t o i l (41), but t h e i r p r e c i s e c o n t r i b u t i o n to g r a p e f r u i t f l a v o r has not been determined. Although nootkatone i s a f l a v o r impact compound i n grape­ f r u i t , i t does not by i t s e l f provide the f u l l - b o d i e d f l a v o r provided by good q u a l i t y cold-pressed g r a p e f r u i t o i l . The proper blend of nootkatone, aldehydes, other as yet u n i d e n t i f i e d components, and perhaps e s t e r s i s necessary f o r f u l l - b o d i e d grape­ fruit flavor. Components Important to Mandarin F l a v o r and Aroma The f l a v o r of most c i t r u s c u l t i v a r s i s complex, and compound­ i n g c i t r u s f l a v o r s r e q u i r e s the blending of s e v e r a l components i n s p e c i f i c p r o p o r t i o n s to o b t a i n the unique f l a v o r of each c i t r u s c u l t i v a r (3). Studies on S i c i l i a n mandarin o i l suggest the d i s t i n c t f l a v o r and arom compounds, thymol and methyl-N-methy a n t h r a n i l a t e ) but no evidence to support t h i s c l a i m has been presented (42). Thymol has been i d e n t i f i e d i n Dancy tangerine p e e l o i l , and both thymol and dimethyl a n t h r a n i l a t e have been i d e n t i f i e d i n S i c i l i a n mandarin o i l (3). The reported q u a n t i t i e s of thymol i n mandarin o i l v a r i e d from 0.04-0.2% of the o i l , where­ as o n l y one value (0.9%) was reported f o r dimethyl a n t h r a n i l a t e . In a recent study (44), we determined the q u a n t i t i e s of thymol and dimethyl a n t h r a n i l a t e i n Argentine ( S i c i l i a n ) mandarin o i l and i n cold-pressed tangerine o i l . We assessed the f l a v o r and aroma e f f e c t s of these two compounds on cold-pressed o i l s from mandarins and tangerines and determined the t a s t e t h r e s h o l d of dimethyl a n t h r a n i l a t e i n water, and of thymol and dimethyl a n t h r a n i l a t e i n s i n g l e - s t r e n g t h tangerine j u i c e . The t a s t e t h r e s h o l d of thymol i n water was reported e a r l i e r (43). We prepared o i l samples f o r aroma and t a s t e e v a l u a t i o n by adding mixtures of thymol and dimethyl a n t h r a n i l a t e or thymol, dimethyl a n t h r a n i l a t e , α-pinene and γ-terpinene, to e i t h e r cold-pressed tangerine o i l or d-limonene (the major component of both tangerine and mandarin o i l s ) at the l e v e l s found i n mandarin o i l . a-Pinene, γ-terpinene, thymol, and dimethyl a n t h r a n i l a t e were q u a n t i t a t e d i n tangerine and mandarin o i l by gas chromatography on a methyl s i l i c o n e , fused s i l i c a , g l a s s c a p i l l a r y column (Table IX) (44). Aroma samples were evaluated by a p a i r e d comparison d i f f e r e n c e t e s t using a t r a i n e d twelve-member aroma panel. Then, the o i l samples were added to s i n g l e - s t r e n g t h tangerine j u i c e and evaluated by a t r i a n g l e d i f f e r e n c e t e s t with the same twelvet r a i n e d panel members (Table X). We determined t a s t e thresholds i n water by adding an appropriate amount of t e s t m a t e r i a l i n e t h a n o l to water, and i n s i n g l e - s t r e n g t h j u i c e by adding dimethyl a n t h r a n i l a t e or a s o l u t i o n of thymol i n ethanol to the concentrated tangerine j u i c e and d i l u t i n g to s i n g l e - s t r e n g t h j u i c e with water (Table X I ) .

In Citrus Nutrition and Quality; Nagy, S., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

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The aroma a n d t a s t e p a n e l r e s u l t s ( T a b l e X) p o i n t t o some s i g n i f i c a n t c o n c l u s i o n s about t h e e f f e c t o f thymol and d i m e t h y l a n t h r a n i l a t e on t h e aroma and t a s t e o f m a n d a r i n o i l . T h e aroma p a n e l s r e a d i l y d e t e c t e d a d i f f e r e n c e i n samples o f t a n g e r i n e o i l c o n t a i n i n g added t h y m o l a n d d i m e t h y l a n t h r a n i l a t e t h a t were c o m ­ p a r e d t o m a n d a r i n o i l b u t d i d n o t d e t e c t a d i f f e r e n c e as e a s i l y when t h y m o l a n d d i m e t h y l a n t h r a n i l a t e i n l i m o n e n e were compared t o m a n d a r i n o r t a n g e r i n e o i l s , o r when t h y m o l a n d d i m e t h y l a n t h r a n i ­ l a t e i n t a n g e r i n e o i l were compared t o t a n g e r i n e o i l . T a s t e p a n e l r e s u l t s show t h a t p a n e l members r e a d i l y d e t e c t e d a d i f f e r e n c e i n a l l samples t e s t e d . These r e s u l t s suggest that a combination o f t h y m o l and d i m e t h y l a n t h r a n i l a t e i s n o t a s i m p o r t a n t t o t h e f l a v o r and aroma o f m a n d a r i n o i l as o r i g i n a l l y p r o p o s e d . T h e y a l s o show that tangerine o i l contains other constituents that modify the f l a v o r a n d aroma o f t h e o i l e v e n when t h y m o l and d i m e t h y l a n t h r a n i l a t e a r e a d j u s t e d t o t h e same l e v e l s f o u n d i n m a n d a r i n oil. I n one aroma t e s t were added t o t a n g e r i n e a n t h r a n i l a t e , t h e p a n e l c o u l d n o t d i s t i n g u i s h t h e compounded o i l from mandarin o i l . These r e s u l t s i n d i c a t e the importance o f m o n o t e r p e n e h y d r o c a r b o n s t o t h e aroma o f m a n d a r i n o i l . T h e t a s t e p a n e l r e a d i l y d e t e c t e d a t a s t e d i f f e r e n c e i n t h i s same s a m p l e o f o i l i n single-strength juice. Thus, i t i s apparent that other c i t r u s o i l components m o d i f y t h e t a s t e o f m a n d a r i n o i l . Table IX. Q u a n t i t i e s of important mandarin and t a n g e r i n e o i l s .

Component Thymol Dimethyl anthranilate •y^Terpinene α-Pinene

f l a v o r components i n

Weight Tangerine o i l 0.022 0.072 1.74 0.17

percent Mandarin o i l 0.182 0.652 14.0 1.8

The t a s t e t h r e s h o l d s f o r d i m e t h y l a n t h r a n i l a t e and t h y m o l ( T a b l e X I ) i n d i c a t e t h e e f f e c t o f t h e s e two components on t h e aroma o f m a n d a r i n o i l a n d t h e f l a v o r o f t a n g e r i n e j u i c e c o n t a i n i n g m a n d a r i n o i l . D i m e t h y l a n t h r a n i l a t e i s b y f a r t h e more p o t e n t f l a v o r component, f o r i t i s d e t e c t a b l e a t a l e v e l t h a t i s o n e h u n d r e d t h t h a t o f t h y m o l i n w a t e r and o n e - t h o u s a n d t h t h a t o f t h y m o l i n b l a n d t a n g e r i n e j u i c e ( T a b l e X I ) . We e s t i m a t e d t h e l e v e l s o f t h y m o l and d i m e t h y l a n t h r a n i l a t e i n t a n g e r i n e j u i c e f l a v o r e d w i t h m a n d a r i n o i l t o b e 220 ppb o f t h y m o l and 782 ppb o f d i m e t h y l a n t h r a n i l a t e b a s e d on a l e v e l o f 0.012% o i l i n t h e j u i c e . The l e v e l o f d i m e t h y l a n t h r a n i l a t e p r e s e n t i s a b o u t f i v e t i m e s i t s t a s t e t h r e s h o l d i n t a n g e r i n e j u i c e , but the l e v e l o f thymol i s a l m o s t one s e v e n - h u n d r e d t h ( 1 / 7 0 0 ) i t s t a s t e t h r e s h o l d i n t a n g e r i n e juice. T h u s , d i m e t h y l a n t h r a n i l a t e i s much more l i k e l y t o

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Table X. C o n t r i b u t i o n of thymol, dimethyl a n t h r a n i l a t e , β-pinene and γ-terpinene to the aroma and t a s t e of coldpressed tangerine and mandarin o i l s . Confidence Aroma panel with o i l s

O i l samples compared

level Taste panel with j u i c e s

Tangerine vs mandarin

99.9

99.9

Tangerine, DMA vs mandarin

99.9

99.9

99.9

99.9

95.0

99.9

Tangerine, thymol, vs mandarin

DMA

Limonene, DMA vs mandarin Limonene, thymol, vs mandarin

DMA

Tangerine, thymol, γ-terpinene vs mandarin

DMA, 95.0

Tangerine, thymol, DMA, γ-terpinene, 3-pinene vs mandarin Limonene, thymol, vs tangerine

NS

99.0

95.0

99.9

95.0

99.0

DMA

Tangerine, thymol, vs tangerine

DMA

DMA = dimethyl a n t h r a n i l a t e . NS = not s i g n i f i c a n t at the 95% confidence l e v e l . a„ D i f f e r e n c e between the two samples evaluated. ^Paired comparison

difference

" T r i a n g l e comparison

test.

difference

test.

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i n f l u e n c e the f l a v o r o f j u i c e s c o n t a i n i n g Argentine mandarin o i l than i s thymol. Table XI. Taste t h r e s h o l d values f o r thymol and dimethyl a n t h r a n i l a t e i n water and i n tangerine j u i c e .

— Compound water Thymol Dimethyl a n t h r a n i l a t e In tangerine j u i c e Thymol Dimethyl a n t h r a n i l a t e

Threshold (ppb)

r

In

a

1,700° 20.3 147,000 157

Reported e a r l i e r by Moshonas et a l determined (ND),

ND 0.98 0.93 0.96

2 (1972); r not

Taste panel members f e l t that dimethyl a n t h r a n i l a t e was d e f i n i t e l y a f l a v o r impact compound i n mandarin o i l . These s t u d i e s suggest however, that a mixture of components i n c l u d i n g thymol, dimethyl a n t h r a n i l a t e , and s e v e r a l monoterpene hydrocarbons i n the proper p r o p o r t i o n s i s necessary f o r acceptable f l a v o r i n mandarins. T o t a l Aldehydes As a Measure o f O i l Q u a l i t y One of the standards of p u r i t y f o r c i t r u s o i l s i s the t o t a l aldehyde content determined by a procedure described i n the U. S. Pharmacopoeia (USP) (45). Some buyers or processors o f c i t r u s e s s e n t i a l o i l s have been unable to d u p l i c a t e t o t a l aldehyde v a l u e s on a given o i l because of s l i g h t v a r i a t i o n s i n c a r r y i n g out the standard procedure. S p e c i f i c a l l y , Dennis and Hendrix (46) have found that i f the s o l u t i o n of p e e l o i l and hydroxylamine h y d r o c h l o r i d e i s not s t i r r e d (or allowed to stand w i t h o c c a s i o n a l shaking) f o r a f u l l 30 min before t i t r a t i o n , a low t o t a l aldehyde value may r e s u l t . Varying t h i s time i n t e r v a l can cause v a r i a t i o n s of as much as 0.25% i n the aldehyde v a l u e f o r a given o i l . Samples of pure decanal, o c t a n a l , and c i t r a l d i l u t e d to about 1.5% s o l u t i o n s were q u a n t i t a t i v e l y accounted f o r , showing that the major aldehydes i n c i t r u s o i l s react completely i n the USP procedure. A maximum t o t a l aldehyde value i s h i g h l y d e s i r a b l e when an o i l i s being s o l d on the b a s i s o f i t s aldehyde content, as i s o f t e n the case. Recent q u a n t i t a t i v e s t u d i e s on i n d i v i d u a l aldehydes i n c i t r u s o i l s i n v o l v i n g gas chromatography have suggested that t o t a l aldehydes i n some o i l s may be higher than t o t a l aldehydes obtained by the USP procedure (3, 41). Although the reasons f o r t h i s d i f f e r e n c e a r e unclear, the c a l c u l a t i o n o f t o t a l aldehydes i n orange, g r a p e f r u i t , and tangerine o i l s as decanal may cause some

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e r r o r , p a r t i c u l a r l y s i n c e o c t a n a l i s sometimes the major aldehyde i n these o i l s . Another reason t h i s d i f f e r e n c e may e x i s t i s the p o s s i b i l i t y that gas chromatographic peaks assigned to some i n d i v i d u a l aldehydes may c o n t a i n i m p u r i t i e s , thus c r e a t i n g abnormally high t o t a l aldehyde v a l u e s . This p o s s i b i l i t y i s less l i k e l y when analyses are being c a r r i e d out on g l a s s c a p i l l a r y gas chromatographic columns where contamination of peaks by t r a c e i m p u r i t i e s i s minimized.

Literature Cited 1. Shaw, P. E. In "Citrus Science and Technology", Nagy, S., Shaw, P. E. and Veldhuis, M. Κ., Ed., Avi Publishing Co.: Westport, Conn., 1977; Vol. 1, Chapter 11. 2. Shaw, P. E. In "Citrus Science and Technology", Nagy, S., Shaw, P. E. and Veldhuis M Κ. Ed. Avi Publishing Co.: Westport, Conn., 1977 3. Shaw, P. E. Revie essential oils. J. Agric. Food Chem., 1979, 27, 246-257. 4. Crocker, E.C. "Flavor". McGraw-Hill Book Co.: New York, 1945. 5. Ahmed, Ε. M. Flavor enhancing potential of selected orange oil and essence components and their relationship to product quality. U. S. Dept. of Agric., Agric. Res. Serv., Contract No. 12-14-100-10337 (72), 1975. 6. Shaw, P. E.; Ahmed, E. M.; Dennison, R. A. Orange juice flavor: contribution of certain volatile components as evaluated by sensory panels. Proc. Int. Soc. Citriculture, 1977, 3, 804-807. 7. Ahmed, Ε. M.; Dennison, R. Α.; Dougherty, R. H.; Shaw, P. E. Flavor and odor thresholds in water of selected orange juice components. J. Agric. Food Chem., 1978, 26, 187-191. 8. Kirchner, J. G.; Miller, J. M. Volatile water-soluble and oil constituents of Valencia orange juice. J. Agric. Food Chem., 1957, 4, 283-291. 9. Schreier, P.; Drawert, F.; Junker, Α.; Mick, W. The quantitative composition of technologically changed aromas of plants. II. Aroma compounds in oranges and their changes during juice processing. Z. Lebensm. Unters.-Forsch., 1977, 164, 188-193. 10. Shaw, P. E.; Coleman, R. L. Quantitative composition of cold-pressed orange oils. J. Agric. Food Chem., 1974, 22, 785-787. 11. Lifshitz, Α.; Stanley, W. L.; Stepak, Y. Comparison of Valencia essential oil from California, Florida and Israel. J. Food Sci., 1970, 35, 547-548. 12. Naves, Y. R. The aldehyde fraction of essential oil of sweet orange. Perfum. Essent. Oil. Rec., 1947, 38, 295-298, 320. 13. Ziegler, E. The examination of citrus oils. Flavour Ind., 1971, 647-653.

In Citrus Nutrition and Quality; Nagy, S., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

9.

SHAW AND WILSON

Volatile

Co mponents and Citrus

Flavor

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14. Stanley, W. L.; Ikeda, R. M.; Vannier, S. H.; Rolle, L. Α. Determination of the relative concentrations of the major aldehydes in lemon, orange and grapefruit oils by gas chromatography. J. Food Sci., 1961, 26, 43-48. 15. Braddock, R. J.; Kesterson, J. W. Quantitative analysis of aldehydes, esters, alcohols and acids from citrus oils. J. Food Sci., 1976, 41, 1007-1010. 16. Blair, J. S.; Godar, Ε. M.; Masters, J. E.; Riester, D. W. Exploratory experiments to identify chemical reactions causing flavor deterioration during storage of canned orange juice. I. Incompatibility of peel oil constituents with the acid juice. Food Res., 1952, 17, 235-260. 17. Patton, S.; Josephson, D. V. A method for determining significance of volatile flavor compounds in foods. Food Res., 1957, 22, 316-318. 18. Kefford, J. F. The chemical constituents of citrus fruits. Adv. Food Res., 1959 19. Amoore, J. E.; Johnston stereochemical theory of odor. Sci. Am., 1964, 210, 42-49. 20. Keith, E. S.; Powers, J. J. Determination of flavor threshold levels and subthreshold, additive and concentration effects. J. Food Sci., 1968, 33, 213-218. 21. Siek, T. J.; Albin, I. Α.; Suther, L. Α.; Lindsay, R. C. Taste thresholds of butter volatiles in deodorized butteroil medium. J. Food Sci., 1969, 34, 265-267. 22. Shaw, P. E. Citrus essential oils. Perfum. Flav., 1979, 3, 35-39. 23. Schinneller, D. J.; Dougherty, R. H.; Biggs, R. H. Influence of selected 5'-nucleotides on flavor threshold of octanal. J. Food Sci., 1972, 37, 935-937. 24. Ahmed, Ε. M.; Dennison, R. Α.; Dougherty, R. H.; Shaw, P. E. Effect of nonvolatile orange juice components, acid, sugar and pectin on the flavor threshold of d-limonene in water. J. Agric. Food Chem., 1978, 26, 192-194. 25. Barmore, C. R.; Biggs, R. H. Acid-soluble nucleotides of juice vesicles of citrus fruit. J. Food Sci., 1972, 37, 712-714. 26. Bennett, M. C. Extraction, separation and quantitation of acid-soluble nucleotides in citrus. J. Agric. Food Chem., 1977, 25, 219-221. 27. Woskow, M. H. Selectivity in flavor modification by 5'-ribonucleotides. Food Technol., 1969, 23, 1364, 1369. 28. Pangborn, R. M. Taste interrelationships. Food Res., 1960, 25, 245-256. 29. Valdes, R. M.; Hinreiner, E.; Simone, M. J. Effect of sucrose and organic acids on apparent flavor intensity. I. Aqueous Solutions. Food Technol., 1956, 10, 282-285. 30. Ahmed, Ε. M.; Dennison, R. Α.; Shaw, P. E. Effect of selected oil and essence volatile components on flavor quality of pumpout orange juice. J. Agric. Food Chem., 1978, 26, 368-372.

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31. MacLeod, W. D., Jr.; Buigues, Ν. M. Sesquiterpenes. 1. Nootkatone, a new grapefruit flavor constituent. J. Food Sci., 1964, 29, 565-568. 32. Hunter, G. L. K.; Brogden, W. Β., Jr. Conversion of valencene to nootkatone. J. Food Sci., 1965, 30, 876-878. 33. Shaw, P. E.; Wilson, C. W., III. Importance of nootkatone to aroma and flavor of cold-pressed grapefruit oil. J. Food Sci., 1980, 45, in press. 34. Berry, R. E.; Wagner, C. J. Jr.; Moshonas, M. G. Flavor studies of nootkatone in grapefruit juice. J. Food Sci., 1967, 32, 75-78. 35. Haring, H. G.; Rijkens, F.; Boelens, H.; van der Gen, A. Olfactory studies on enantiomeric eremophilane sesquiterpenoids. J. Agric. Food Chem., 1972, 20, 1017-1020. 36. Stevens, K. L.; Guadagni, D. G., Stern, D. J. Odour character and threshold values of nootkatone and related compounds. J. Sci 37. W. A. Bucek, Coca communication. 38. Tatum, J. Η., Berry, R. E. Proc. 1975 Citrus Chemistry & Technology Conference, Winter Haven, Fla., October 8, 1975, p. 19. 39. Kesterson, J. W.; Hendrickson, R.; Braddock, R. J. Florida citrus oils. Univ. Fla. Inst. Food Agric. Bull., 749, 1971, 67. 40. Moshonas, M. G. Analysis of carbonyl flavor constituents from grapefruit oil. J. Agric. Food Chem., 1971, 19, 769-770. 41. Wilson, C. W. III; Shaw, P. E. Glass capillary gas chromatography for quantitative determination of volatile constituents in cold-pressed grapefruit oil. J. Agric. Food Chem., 1980, 28, in press. 42. Kugler, E.; Kovats, E. Information on Mandarin peel oil. Helv. Chim. Acta, 1963, 46, 1480-1513. 43. Moshonas, M. G.; Shaw, P. E.; Veldhuis, M. K. Analysis of volatile constituents from Meyer lemon oil. J. Agric. Food Chem., 1972, 20, 751-572. 44. Wilson, C. W., III; Shaw, P. E. Importance of thymol, methyl N-methyl anthranilate, and monoterpene hydrocarbons to the aroma and flavor of cold-pressed mandarin oils. J. Agric. Food Chem., 1980, in press. 45. "United States Pharmacopoeia"; 17th Rev.; Mack Printing, Easting, Pa., 1965. 46. Dennis, S.; Hendrix, C. Μ., Jr. 1980. Personal communication. RECEIVED May 28, 1980.

In Citrus Nutrition and Quality; Nagy, S., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

10 Fruit Handling and Decay Control Techniques Affecting Quality G. ELDON BROWN Florida Department of Citrus, University of Florida, Institute of Food and Agricultural Sciences, Agricultural Research and Education Center, P.O. Box 1088, Lake Alfred, FL 33850

Information concernin niques used i n the handlin which affect quality has been compiled and summarized within the scope of this chapter. Major cultural and genetic influences on quality are not considered. Quality i s influenced by physiological and pathological factors and is measured i n both specific and general terms. Quality parameters involved with internal composition are usually defined, but i n many instances causes related to off-flavors have not yet been i d e n t i f i e d . Factors which detract from the external appearance and affect the saleability of citrus f r u i t d e f i n i t e l y have to be considered when discussing quality. These factors associated with the rind include color, softness, and various forms of rind breakdown. Keeping quality i s another important aspect, and various handling and decay control techniques influence the occurrence of decay caused by several different fungal pathogens. Quality can be adversely affected by many of the techniques, but several specific methods have been developed to improve quality at harvest and/or maintain it during storage or t r a n s i t . Preharvest techniques which i n f l u e n c e q u a l i t y Lead arsenate s p r a y s . I n t e r n a l f r u i t q u a l i t y can be i n fluenced by spraying t r e e s w i t h lead arsenate a t post-bloom Oranges ( C i t r u s s i n e n s i s ( L . ) Osbeck) are more a f f e c t e d than g r a p e f r u i t ( C i t r u s p a r a d i s i M a c f . ) . The use o f lead arsenate to reduce t i t r a t a b l e a c i d i t y o f F l o r i d a g r a p e f r u i t has been a standard procedure f o r many y e a r s . Red g r a p e f r u i t sprayed w i t h lead arsenate contained s i g n i f i c a n t l y l e s s a c i d than d i d the unsprayed f r u i t ( 2 ) . A c i d i n f r u i t sprayed w i t h the highest r a t e (1.9 g/1) was only h a l f t h a t w i t h the lowest r a t e (O.5 g / 1 ) . In a d d i t i o n to a c i d i t y r e d u c t i o n , arsenated g r a p e f r u i t contained s l i g h t l y l e s s reducing sugar, s i g n i f i c a n t l y more non-reducing

0-8412-0595-7/80/47-143-193$08.00/0 © 1980 American Chemical Society

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sugar, and more t o t a l sugar than nonarsenated f r u i t . The b i o f l a v o n o i d content and pH were a l s o s i g n i f i c a n t l y higher i n the sprayed f r u i t (3J. A p p l i c a t i o n s of lead arsenate to Temple oranges lowered the t i t r a t a b l e a c i d content but not the s o l u b l e s o l i d s or percentage j u i c e ( 4 ) . Decay, peel i n j u r y , or c r e a s i n g (a r i n d malformation) were not i n f l u e n c e d but l e g a l m a t u r i t y was advanced by 15 to 20 days. F l o r i d a r e g u l a t i o n s (5) r e s t r i c t the use of lead arsenate to g r a p e f r u i t . C o l o r . Color of c i t r u s f r u i t at m a t u r i t y i s a major c r i t e r i o n of consumer appeal. In humid, s u b t r o p i c a l c l i m a t e s , c i t r u s f r u i t s w i l l reach m a t u r i t y w i t h a considerable amount of c h l o r o p h y l l s t i l l remaining i n the r i n d . T h e r e f o r e , c o n s i d e r a b l e i n t e r e s t has been shown i n the area of c o l o r enhancement through the use of m a t e r i a l s a p p l i e d to mature f r u i t before harvest. The f i n a l c o l o r of most orange c u l t i v a r s i s produced by a dec l i n e i n c h l o r o p h y l l pigment enoids. The f i n a l c o l o lime ( C i t r u s a u r a n t i f o l i a (Christm.) Swing) and g r a p e f r u i t c u l t i v a r s i s produced by a d e c l i n e i n c h l o r o p h y l l pigments and l i t t l e or no net increase i n carotenoids (6_). Removal of c h l o r o p h y l l from c i t r u s f r u i t s w i t h preharvest a p p l i c a t i o n s of ethephon (2-chloroethylphosphonic a c i d ) , which decomposes to produce ethylene ( 7 ) , has been s u c c e s s f u l l y achieved (8, 9, 10^, 11_, 12). Concentrations between 50 to 200 mg/1 as a preharvest spray 5 to 20 days before harvest to Robinson and Lee tangerines ( C i t r u s r e t i c u l a t a Blanco) r e s u l t e d i n p a r t i a l c h l o r o p h y l l l o s s (3egreening). Less postharvest degreening was r e q u i r e d to achieve acceptable c o l o r , and keeping q u a l i t y of the f r u i t during storage was improved (10^, 11). Concentrations of 200 to 500 mg/1 induced varying degrees of f r u i t loosening and, o f t e n , f r u i t drop of Robinson, Lee, Nova, and Dancy tangerines and Hamlin oranges (10). Leaf a b s c i s s i o n a l s o o c c u r r e d , p a r t i c u l a r l y a t the higher c o n c e n t r a t i o n . Damage to f r u i t by •'plugging", removal of a p o r t i o n of the peel at the stem-end at harvest, was reduced by the ethephon sprays. Preharvest a p p l i c a t i o n s of ethephon to Robinson tangerines s i g n i f i c a n t l y reduced the i n c i dence of anthracnose caused by C o l l e t o t r i c h u m g l o e o s p o r i o i d e s (Penz.) Sacc. during storage (13JT Control was a t t r i b u t e d to p h y s i o l o g i c a l changes w i t h i n the r i n d a s s o c i a t e d w i t h c a r t e n o i d accumulation which occurred due to the presence of low l e v e l s of e t h y l e n e . A l l c u l t i v a r s of c i t r u s , however, do not respond to ethephon treatment. Preharvest a p p l i c a t i o n s to Bearss lemons were r e l a t i v e l y i n e f f e c t i v e f o r inducing degreening even though the ethephon was absorbed and ethylene was produced (9). Simil a r r a t e s of a p p l i c a t i o n as a postharvest dip induced degreening, suggesting t h a t a f a c t o r such as a u x i n , g i b b e r e l l i n , or c y t o k i nin from the t r e e i n h i b i t e d the response to e t h y l e n e . E l Zeftawi and G a r r e t t (8) observed t h a t ethephon markedly reduced a c i d i t y of j u i c e from green-colored V a l e n c i a oranges. In f r u i t

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excluded from l i g h t a f t e r ethephon a p p l i c a t i o n , f l a v o n o i d s and polyphenols were increased i n j u i c e from green-colored f r u i t or f r u i t undergoing regreening. Carotenoids were a l s o increased at the orange-colored and regreened ( a d d i t i o n a l s y n t h e s i s of c h l o r o ­ p h y l l ) stages, but decreased at the green stage. Ethephon d i d not cause e a r l i e r maturation of Shamouti oranges (14). Growth r e g u l a t o r s . G i b b e r e l l i c a c i d (GA), 2 , 4 - d i c h l o r o phenoxyacetic a c i d (2,4-D), and 2 , 4 , 5 - t r i c h l o r o p h e n o x y a c e t i c a c i d (2,4,5-T) are growth r e g u l a t o r s evaluated on c i t r u s f r u i t s to improve q u a l i t y a t / o r a f t e r harvest. In g e n e r a l , s i g n i f i c a n t changes i n i n t e r n a l q u a l i t y have not been observed w i t h these growth r e g u l a t o r s {6, 15, 16, 17, 18, 19, 20), t h e i r e f f e c t s being r e s t r i c t e d p r i m a r i l y to the r i n d . The use of GA has been p a r t i c u l a r l y s u c c e s s f u l on Washington Navel oranges grown i n most c i t r u s r e g i o n s . A p p l i c a t i o n s of GA r e t a r d senescence of the r i n d due to high enzymati the season (21_, 22) whic l a t i o n of c a r o t e n o i d pigments ((5, 1!5, 16_, 17, 23, 2£, 25, 26, 27_, 28). A p p l i c a t i o n s of 2,4-D or c l o s e l y r e l a t e d compounds" have been s u c c e s s f u l l y combined w i t h GA. The e f f e c t of 2,4-D has been p r i n c i p a l l y to reduce f r u i t drop or increase f r u i t s i z e due to a t h i n n i n g e f f e c t (6_, _15, Γ7, 28, 29, 30, 31, 32). A p p l i c a t i o n s of GA and 2,4-D have been shown to extend the harvesting season of g r a p e f r u i t (30, 31_, 33, 34), producing a f i r m e r f r u i t and, i n some i n s t a n c e s , i n c r e a s i n g f r u i t s i z e (33). F r u i t t r e a t e d w i t h these growth r e g u l a t o r s withstood deformity due to handling and packing s i g n i f i c a n t l y b e t t e r than untreated f r u i t (34). G i b b e r ­ e l l i c a c i d and 2,4-D alone and i n combination reducicT germination of seed i n mature g r a p e f r u i t (30). Darkened blemishes, some d i s c o l o r a t i o n o f the r i n d and increased r i n d t h i c k n e s s were some of the u n d e s i r a b l e e f f e c t s of GA on g r a p e f r u i t (33). Regreening due to the a p p l i c a t i o n of GA can be a problem on V a l e n c i a oranges and g r a p e f r u i t (15_, 30, 35, 36, 3 7 ) , and f r u i t deformity has o c c a s i o n a l l y been observed w i t h V a l e n c i a oranges (35). Postharvest decay of f r u i t sprayed w i t h growth r e g u l a t o r s has been reported to be reduced (15, 16, Γ7, 23, 31). This has been a t t r i b u t e d to fewer i n j u r i e s being formed i n f i r m f r u i t r e ­ c e i v i n g growth r e g u l a t o r s as w e l l as to growth of the decay organisms being decreased i n l e s s senescent peel (16). Anatomi­ cal changes a s s o c i a t e d w i t h r i n d senescence were retarded by GA sprays (_6, 16). G i b b e r e l l i c a c i d caused an i n c r e a s e i n v a l e n cene, an e s s e n t i a l o i l component (27). V a l e n c i a oranges sprayed with 2,4-D and GA were f i r m e r , evolved l e s s e t h y l e n e , and showed slower malonic a c i d accumulation i n the j u i c e (20). G i b b e r e l l i c a c i d reduced B r i x ( p r i m a r i l y sugars) and a c i d i t y i n green-colored V a l e n c i a oranges ( 8 ) . Chlorophenoxyacetic a c i d (CLPA) and 2,4,5-T a p p l i e d to mandarins increased j u i c e content and peel moisture. Treated f r u i t s u f f e r e d l e s s l o s s of weight, v i t a m i n C, and sugar during storage (38). G i b b e r e l l i c a c i d

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a p p l i e d to lemon t r e e s caused the f r u i t to remain greener and to develop the y e l l o w lemon c o l o r l e s s r a p i d l y i n storage (39). The b e n e f i t s of GA a p p l i c a t i o n s to lemons are a more d e s i r a b l e seasonal harvest p a t t e r n i n r e l a t i o n to market demands, a l a r g e r percentage of f r u i t w i t h a long storage l i f e , and a decrease i n the number of small f r u i t ( 6 ) . A p p l i c a t i o n s of GA to Clementine mandarins s i g n i f i c a n t l y delayed f r u i t c o l o r i n g , l o s s of a c i d , and an i n c r e a s e i n s o l u b l e s o l i d s (40). Growth r e g u l a t o r s have been s u c c e s s f u l l y used to c o n t r o l p h y s i o l o g i c a l r i n d d i s o r d e r s . Rind s t a i n i n g of navel oranges was reduced w i t h GA a p p l i c a t i o n s (41, 42, 4 3 ) , as was the accumulation of r i n d exudate on mature f r u i t (44). Water spot of navel oranges, caused by surface deposits of water e n t e r i n g the r i n d , can be reduced by GA sprays (45, 46). Rough and t h i c k peel of l a r g e f r u i t s i z e s of Shamouti oranges was reduced s i g n i f i c a n t l y by applying 2 , 2 r d i m e t h y l h y d r a z i d e (SADH) and 2chloroethyltrimethylammoniu duced p u f f i n g and c r e a s i n u l a t i o n i n V a l e n c i a oranges was reduced w i t h 2,4-D sprays (29). Preharvest sprays of benzyl adenine, a c y t o k i n i n , and 2,4-D tended to decrease s u s c e p t i b i l i t y of g r a p e f r u i t to c h i l l i n g i n j u r y , a c o l d storage d i s o r d e r of the r i n d (50). Additional i n f o r m a t i o n on the e f f e c t of growth r e g u l a t o r s on c i t r u s has been compiled by Coggins and Hi e l d ( 6 ) . Aging, a w r i n k l i n g of the peel at the sten-end of V a l e n c i a oranges caused from l o s s of m o i s t u r e , was reduced by applying Pinolene ( p o l y - l - p - m e n t h e n - 8 , 9 - d i y l ) , an a n t i t r a n s p i r a n t p l a s t i c , 1-2 months before harvest. At both 1 and 3%, Pinolene reduced stem-end aging and moisture l o s s of V a l e n c i a oranges during storage (51). S o l u b l e s o l i d s and a c i d i t y were reduced, but i n t e r n a l q u a l i t y was s t i l l acceptable (52). Handling techniques at harvest which i n f l u e n c e q u a l i t y Harvest methods. C i t r u s f r u i t s are harvested almost e n ­ t i r e l y by hand. I n i t i a l l y , c i t r u s f r u i t s were c l i p p e d when harvested because the stem does not separate e a s i l y from the f r u i t at p i c k i n g . However, as t h i s procedure was slow and a c e r t a i n amount of c l i p p e r i n j u r y o c c u r r e d , h a r v e s t i n g by p u l l i n g became the predominant method (53). P u l l i n g or snapping, a breaking-by-bending process, causes the stem to break c l o s e to the f r u i t . In many i n s t a n c e s , a l l or p o r t i o n s of the button ( c a l y x and d i s k ) may be removed from the f r u i t . The button harbors fungi which may l a t e r cause decay during storage and thus reduce keeping q u a l i t y . A comparison of decay i n p u l l e d versus c l i p p e d f r u i t showed t h a t stem-end r o t , caused by D i p l o d i a n a t a l e n s i s P. Evans and Phomopsis c i t r i F a w c , was reduced by p u l l i n g (53, 54, 55). Levels of decay caused by A l t e r n a r i a c i t r i E l l . and P i e r c e , however, were increased (55). Spot p i c k i n g , p r i n c i p a l l y on the b a s i s of c o l o r , can be used to

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aid i n the harvest of high q u a l i t y f r u i t . S i t e s and R e i t z (56, 57, 58) showed t h a t the best c o l o r e d f r u i t contained the highest s o l u b l e s o l i d s and v i t a m i n C c o n t e n t , and u s u a l l y had a higher s o l u b l e s o l i d s / t i t r a t a b l e a c i d r a t i o . Spot p i c k i n g f o r j u i c e content had no p r a c t i c a l a p p l i c a t i o n (58). Unnecessary rough h a n d l i n g , no matter whether f r u i t are harvested by c l i p p i n g or p u l l i n g , causes i n j u r i e s to the r i n d which are r e a d i l y i n f e c t e d by decay f u n g i . The simple p r a c t i c e of c a r e f u l handling during harvesting has an i n d u b i t a b l y s i g n i f ­ i c a n t i n f l u e n c e upon q u a l i t y . I t has been shown i n numerous studies t h a t rough handling s i g n i f i c a n t l y reduces keeping q u a l i t y of c i t r u s f r u i t s (59^, 6Ό, 6^, 62) and q u a l i t y decreases with increased s e v e r i t y o f the i n j u r i e s (61). Gently handled g r a p e f r u i t held under humid, shaded c o n d i t i o n s u n t i l packing were more r e s i s t a n t to deformation than f r u i t roughly-handled and exposed to the sun S u s c e p t i b i l i t y t o permanent deform a t i o n increased w i t h advancin Physiological disorders. P h y s i o l o g i c a l d i s o r d e r s are a l s o i n f l u e n c e d by handling a t harvest. Blossom-end c l e a r i n g i s a symptom of b r u i s i n g which i s common on f u l l y mature t h i n skinned, seedless g r a p e f r u i t t h a t have been subjected to rough handling (63). This d i s o r d e r i s u s u a l l y v i s i b l e w i t h i n 24 hours a f t e r b r u i s i n g and i s manifested by water soaking of the blossom-end caused by exudation of j u i c e from broken v e s i c l e s w i t h i n the pulp. The d i s o r d e r i s more p r e v a l e n t i n rough handled mature f r u i t (62). O l e o c e l l o s i s or o i l s p o t t i n g occurs when f r u i t are i n j u r e d during handling causing a r e l e a s e of o i l from damaged glands. The o i l i s t o x i c to surrounding healthy c e l l s of the r i n d causing a c o l l a p s e and d i s c o l o r a t i o n o f the area (65). Turgid f r u i t are more s u b j e c t to the d i s o r d e r (42, 66, (D7, 68, 69, 70) and harvesting should p r e f e r a b l y be delayed u n t i l the r a t e o f water l o s s from the t r e e exceeds the r a t e o f water uptake. F r u i t harvested w h i l e wet or damp from r a i n , overhead i r r i g a t i o n , dew or fog are very s u s c e p t i b l e to o l e o c e l l o s i s . Damaged areas c o l l a p s e and darken, causing the o i l glands to remain prominent. Such areas w i l l not degreen with ethylene but w i l l darken unless f r u i t are held i n very high ( c a , 95% RH) humidity a f t e r harvest p r i o r to washing and waxing (68). Use o f ethephon to enhance r i n d c o l o r break caused an increase i n s u s c e p t i b i l i t y t o o l e o c e l l o s i s i n Washington Navel oranges (71). I t was suggested t h a t an increase i n f r u i t r i n d hydration brought about by stomatal c l o s u r e induced i n ­ creased s u s c e p t i b i l i t y to o l e o c e l l o s i s . Z e b r a - s k i n , a p h y s i o ­ l o g i c a l d i s o r d e r of t a n g e r i n e s , i s i n f l u e n c e d a l s o by water c o n d i t i o n s of the t r e e . Losses can be d i s a s t r o u s i f f r u i t are harvested from t r e e s subjected to heavy r a i n s or i r r i g a t i o n f o l l o w i n g a period of severe drought (72, 73). S u s c e p t i b l e f r u i t are so f r a g i l e t h a t the normal packing processes o f washing, d r y i n g , and waxing cause the peel to darken over the

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segments, p a r t i c u l a r l y i n f r u i t p r e v i o u s l y degreened w i t h ethylene. Once f r u i t are separated from the t r e e , they are no longer provided w i t h a source of moisture. Loss of water from the f r u i t a f t e r harvest before waxing can occur during delays i n handling. This can o f t e n lead to a p h y s i o l o g i c a l d e t e r i o r a t i o n of the r i n d near the stem-end c a l l e d stem-end r i n d breakdown (68, 74, 75, 76). The d i s o r d e r i s p a r t i c u l a r l y troublesome i f f r u i t are helcFtwo or more days at low r e l a t i v e h u m i d i t i e s and high r a t e s of a i r f l o w between harvesting and waxing. R e l a t i v e h u m i d i t i e s l e s s than 85% during degreening aggravate t h i s d i s order (74). The c o n d i t i o n i s c h a r a c t e r i z e d by a c o l l a p s e of the r i n d t i s s u e around the button which may extend several c e n t i meters towards the f r u i t equator. A r i n g of t i s s u e of approximately 3 mm immediately surrounding the button which i s immune to breakdown was observed to l a c k stomata and to be covered w i t h a heavy d e p o s i t of e p i c u t i c u l a S t y l a r - e n d breakdown, physiologica l i m e , i s a l s o enhanced by rough handling a t harvest or during packing (78, 79, 80, 81). Rough h a n d l i n g , however, i s not the primary cause. Davenport and h i s co-workers (82, § 2 * 84, 85) found t h a t rupture of the v e s i c l e s i n the p u l p T i b e r a t i r l j u i c e which invaded the r i n d . The j u i c e passed more e a s i l y from the c e n t r a l a x i s to the r i n d at the s t y l a r - e n d than a t the stem-end. F r u i t turgor a s s o c i a t e d w i t h thermal expansion of f l u i d w i t h i n the j u i c e v e s i c l e s increased i n t e r n a l pressure to a magnitude s u f f i c i e n t to rupture some j u i c e v e s i c l e s , e s p e c i a l l y those i n l a r g e r s i z e d f r u i t . S t y l a r - e n d breakdown i n limes can be cont r o l l e d by m a i n t a i n i n g a s t r i c t p i c k i n g schedule so t h a t f r u i t i s harvested before i t becomes too l a r g e , by c o n t r o l l i n g p o s t harvest f r u i t temperature by hydrocooling i n the f i e l d and/or assuring t h a t harvested f r u i t i s kept shaded throughout the day, p i c k i n g f r u i t w i t h r i n d o i l r e l e a s e pressure of 4*55 kg and g r e a t e r , or reducing t u r g o r by f o r c e d evaporation (84, 85). Central C a l i f o r n i a Washington Navel oranges are s u s c e p t i b l e to a r i n d s t a i n i n g d i s o r d e r which i s a l s o p h y s i o l o g i c a l i n nature. The d i s o r d e r becomes apparent soon a f t e r processing and v a r i e s from brownish d i s c o l o r a t i o n s on the r i n d to severe cases of surface breakdown. E a r l y i n the season w e l l - c o l o r e d f r u i t showed more s t a i n i n g than f r u i t picked green and degreened with e t h y l e n e . F r u i t harvested l a t e i n the season s t a i n e d more than f r u i t harvested i n e a r l y or mid-season. Rough p i c k i n g p r a c t i c e s and prolonged brushing increased r i n d s t a i n i n g (42, 86). Mechanical h a r v e s t i n g . E f f o r t s to develop systems to mechanically harvest c i t r u s f r u i t s , p a r t i c u l a r l y oranges, have been i n progress f o r s e v e r a l y e a r s . U s u a l l y , these systems have been developed to handle f r u i t destined f o r processed as opposed to f r e s h u t i l i z a t i o n . Some e f f o r t s , however, have been made to

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u t i l i z e mechanically harvested f r u i t f o r f r e s h f r u i t marketing. Due to the rough handling procedures i n v o l v e d , f r u i t harvested mechanically are s u b j e c t t o more i n j u r i e s than are f r u i t harvested by hand. Increased i n j u r i e s lead t o higher l e v e l s o f decay (87, 88, 89) and increased moisture l o s s (90), In a d d i t i o n t o i n j u r i e s , mechanically harvested f r u i t often have a d hering long stems (89, 9 1 ) , which r e q u i r e removal before f r e s h u t i l i z a t i o n . Use o f e f f e c t i v e f u n g i c i d e s , a p p l i e d before or immediately a f t e r h a r v e s t , suppressed decay so t h a t l e v e l s were comparable to t h a t i n hand-harvested f r u i t (91, 9 2 ) . F r u i t harvested mechanically f o r processed purposes r e q u i r e the use o f c e r t a i n grading techniques a t the processing plants (93, 94, 95) t o remove c u l l s and t r a s h . Such techniques are g e n e r a l l y not needed to such an extent f o r hand-harvested f r u i t . A b s c i s s i o n chemicals, which reduce the attachment f o r c e of the f r u i t t o the stem hav bee s t u d i e d a i d t mechan i c a l h a r v e s t i n g (96). Th act by damaging the r i n d , g ethylen weakens c e l l s w i t h i n the a b s c i s s i o n l a y e r , r e s u l t i n g i n a b s c i s s i o n . The technique o f loosening c i t r u s f r u i t s w i t h a b s c i s s i o n chemicals can i n f l u e n c e e s s e n t i a l o i l composition and degrade the f l a v o r o f the orange j u i c e (97, 9 8 ) . Q u a n t i t a t i v e a n a l y s i s of the major v o l a t i l e compounfls o f cold-pressed orange o i l showed t h a t o i l s from a b s c i s s i o n chemical t r e a t e d , b a r e l y mature, oranges contained l e s s l i n a l o o l and more * < - s i n e n s a l , c i t r o n e l l a l , dodecanal and valencene than o i l s from the unsprayed f r u i t . In o i l s from mature f r u i t ( 9 9 ) , d i f f e r e n c e s could not be det e c t e d and the composition was s i m i l a r t o t h a t o f the b a r e l y mature c h e m i c a l l y t r e a t e d oranges. I t was concluded t h a t abs c i s s i o n chemicals which i n j u r e the f r u i t a c c e l e r a t e maturation (98). Postharvest techniques and t h e i r i n f l u e n c e upon q u a l i t y Degreening, washing, and grading. The use o f ethylene t o remove c h l o r o p h y l l from the r i n d o f c i t r u s f r u i t s i n order t o expose the orange, orange-red, r e d , or y e l l o w pigments i s p r a c t i c e d e x t e n s i v e l y . This i s p a r t i c u l a r l y t r u e i n s u b t r o p i c a l c i t r u s regions where f r u i t maturation u s u a l l y precedes the low temperatures necessary t o remove c h l o r o p h y l l from the p e e l . Q u a l i t y based on eye appeal i s enhanced e x t e n s i v e l y by such a treatment. Understandably but i l l o g i c a l l y , green c o l o r w i t h i n the r i n d represents immaturity t o consumers and, t h e r e f o r e , they are r e l u c t a n t t o purchase the product. I t has been reported t h a t green c o l o r i s the major reason f o r low pack-outs i n F l o r i d a c i t r u s (100). The degreening treatment does not improve e a t i n g q u a l i t y b u t , on the c o n t r a r y , i t c o n t r i b u t e s to p h y s i o l o g i c a l d i s o r d e r s , senescence, and losses from decay (101). However, because o f the consumer's expectations t h a t oranges and mandarins are o r a n g e - c o l o r e d , lemons and g r a p e f r u i t are

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y e l l o w and limes are green, research e f f o r t s continue i n an e f f o r t to improve the c o l o r q u a l i t y c h a r a c t e r i s t i c o f each c u l t i v a r and type. I f performed c o r r e c t l y , degreening does not harm the i n t e r n a l q u a l i t y of c i t r u s f r u i t (98, 102). U n f o r t u n a t e l y , c r i t i c a l a t t e n t i o n to the c o n d i t i o n s necessary f o r proper degreening i s not always g i v e n . Since degreening never improves i n t e r n a l q u a l i t y but only the e x t e r n a l appearance o f the r i n d , only f r u i t of proper m a t u r i t y and i n t e r n a l q u a l i t y should be degreened. C i t r u s f r u i t s as they mature on the t r e e do not produce n a t u r a l c o l o r u n i f o r m l y , t h e r e f o r e , e a r l y i n the season, much v a r i a b i l i t y in color exists. In such i n s t a n c e s , a l l f r u i t , regardless of c o l o r , are held i n the degreening room u n t i l f r u i t of the greenest c o l o r has degreened. Spot p i c k i n g of f r u i t w i t h the best c o l o r break e l i m i n a t e s the need f o r excessive degreening time which occurs i f f r u i t of mixed c o l o r are harvested. Losses during degreening of tangerine f o r c o l o r (103). S o r t i n harvest i s a l s o another technique a v a i l a b l e to reduce degreening time (104, 105). F r u i t which may regreen on the t r e e , such as V a l e n c i a oranges, are d i f f i c u l t to degreen (101). There are numerous reviews d e s c r i b i n g degreening (101, 106, 107, 108, 109, 110, 111, 112, 113). The most common procedure i n v o l v e s continuous metering of ethylene i n t o a room exposing f r u i t t o a c o n c e n t r a t i o n o f 1 to 20 ul ethylene/1 of a i r f o r 1 to as many as 3 days. Increased concentrations of ethylene w i l l not shorten the degreening t i m e . Humidities are maintained at l e v e l s from about 85 to 95% and temperatures range from 21.1 to 30°C, depending upon geographical areas (114, 115, 116). One exception to t h i s procedure i s p r a c t i c e d i n Japan where f r u i t are exposed to a high i n i t i a l c o n c e n t r a t i o n of 1000 ul ethylene/ 1 of a i r f o r 15 hours and then removed t o ambient c o n d i t i o n s (117, 118). Degreening continues f o r the f o l l o w i n g 2 or 3 days depending on c u l t i v a r . V a r i a t i o n s from these procedures during degreening which cause delays i n removal of c h l o r o p h y l l or excessive moisture l o s s can reduce f r u i t q u a l i t y . The most r a p i d r a t e of removal o f c h l o r o p h y l l occurs at the higher tempe r a t u r e s , but w i t h r e l a t i v e l y l i t t l e synthesis of c a r o t e n o i d s . A d i f f e r e n t degreening procedure has been adopted r e c e n t l y i n I s r a e l (106, 119). I n t e r m i t t e n t exposure of f r u i t to ethylene and heat i n 12 hour c y c l e s a t low rates o f v e n t i l a t i o n caused decreased r e s p i r a t i o n r a t e s , l e s s peel i n j u r y and r o t and maint a i n e d the f r u i t at a higher r e l a t i v e humidity. Under these c o n d i t i o n s of low v e n t i l a t i o n , carbon d i o x i d e concentrations reached l e v e l s of 2.5 to 5.0%, which d i d not i n t e r f e r e w i t h c h l o r o p h y l l removal as long as oxygen l e v e l s were adequate (120). Oxygen concentrations below 10% caused a reduction i n the degreening r a t e (121). Other workers have reported t h a t inadequate v e n t i l a t i o n rates a l l o w i n g an excess of 1% carbon d i o x i d e accumulation w i l l i n t e r f e r e w i t h the degreening process (108, 109).

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Q u a l i t y o f lemons r e p o r t e d l y was improved by degreening which increased the j u i c e q u a n t i t y (122). Ethylene degreening p r i o r to c o l d storage reduced c h i l l i n g i n j u r y of green-colored g r a p e f r u i t . However, ethylene tended t o i n c r e a s e c h i l l i n g i n j u r y o f f u l l y c o l o r e d y e l l o w g r a p e f r u i t (123). Exposure o f c i t r u s f r u i t s t o ethylene has been shown t o increase the i n c i d e n c e of stem-end r o t caused p r i m a r i l y by D_. n a t a l e n s i s (113, 124, 125, 126, 127). Higher than recommended ethylene concentrât!oni"T75, 106, 116, 128, 129) and temperature (116) and an increase i n degreening d u r a t i o n (116, 125) caused s i g n i f i c a n t increases i n stem-end r o t . Anthracnose, caused by £. g l o e o s p o r i o i d e s , i s a s e r i o u s disease o f s p e c i a l t y c i t r u s hybrids such as Robinson, Lee, Nova, and Page (130, 131), when f r u i t are exposed t o e t h y l e n e . Incidence was r e l a t e d d i r e c t l y to length o f degreening (130), ethylene c o n c e n t r a t i o n (132, 133), and f r u i t c o l o r (133 134) The occurrence o f mold caused by P é n i c i l l i u m d i g i t a t u greening under F l o r i d a held a t 30°C. Under these c o n d i t i o n s , growth o f the organism i s s i g n i f i c a n t l y reduced and l i g n i f i c a t i o n o f the i n j u r e d e p i c a r p at the high temperatures and r e l a t i v e h u m i d i t i e s i s encouraged (136). Once l i g n i f i c a t i o n i s i n i t i a t e d , entry o f the fungus i s impeded. Normally, f r u i t are not washed before degreening because washing r e t a r d s the loss o f c h l o r o p h y l l (108). Jahn (137) r e ported t h a t washing had l i t t l e e f f e c t on c h l o r o p h y l l l o s s during degreening but c a r o t e n o i d s y n t h e s i s was s i g n i f i c a n t l y reduced by washing before degreening. Carotenoid s y n t h e s i s was reduced by l e s s brushing than was r e q u i r e d t o clean the f r u i t , but "over b r u s h i n g " had l i t t l e f u r t h e r e f f e c t . Washing before degreening i n t e r f e r r e d w i t h c o l o r development i n orange and g r a p e f r u i t more so than lemons and mandarins (138). Several b e n e f i t s from washing before degreening would be gained i f i t were not f o r the degreening problem. Processes which f o l l o w washing, such as grading o r c o l o r s o r t i n g , would be more e f f e c t i v e i f done before degreening (137). Washing before degreening has been shown t o c o n t r i b u t e s u b s t a n t i a l l y t o decay c o n t r o l (131, 138) by removing propagules o f JJ. n a t a l e n s i s and £. g l o e o s p o r i o i d e s from the f r u i t surfaces and, thereby, p r e venting i n f e c t i o n during o r f o l l o w i n g degreening (131, 132). Damage t o c i t r u s peel due to the a c t i o n o f ethylene has been r e p o r t e d , p a r t i c u l a r l y on mandarin types (109, 117), and c e r t a i n d i s o r d e r s such as o l e o c e l l o s i s (106) and r i n d s t a i n i n g (42) have been i n c r e a s e d . A burn o f Temples during degreening (125) has been shown t o be suppressed by a s u f f i c i e n t l y high r e l a t i v e humidity ( 7 5 ) . Under extremely high ethylene concent r a t i o n s (200 ul/1) and exposure f o r long periods o f time (4 weeks), s t i c k i n e s s o f the r i n d o f Marsh g r a p e f r u i t has been r e ported (139). Synthesis o f n a t u r a l wax a f t e r harvest i s s t i m u l a t e d by ethylene (140).

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Improved c o l o r of degreened f r u i t was obtained when i t was discovered t h a t ethylene i n the form of the gas or ethephon s t i m u l a t e d c a r o t e n o i d s y n t h e s i s (141, 142, 143, 144) but at temperatures w e l l below 30°C. This high temperature caused r a p i d c h l o r o p h y l l removal but d i d not s t i m u l a t e continued c a r o t ­ enoid synthesis and a c t u a l l y i n h i b i t e d accumulation of B - c i t r a u r i n (142, 143). C r y p t o x a n t h i n , Β - c i t r a u r i n , and to a l e s s e r e x t e n t , v i o l a x a n t h i n , accumulated i n the e p i c a r p at temperatures of 15-25°C. The y e l l o w pigments, trans-and c i s - v i o l a x a n t h i n i n ­ creased l e s s than orange or reddish-orange pigments. Levels of ethylene r e q u i r e d f o r optimum carotenoid synthesis decreased be­ low 10 ul/1 o f a i r as temperatures were lowered from 30°C to 15°C at 5°C increments (143). Ethylene caused some increase i n r e s p i r a t i o n r a t e , a b s c i s s i o n of the c a l y x , and an e f f e c t on aroma of the f r u i t , but no e f f e c t on percentage s o l u b l e s o l i d s , a c i d , or c o l o r of the j u i c e (143). s t i m u l a t i o n of carotenoid synthesis could a l s o b ethephon (142, 144). T s u l t i n g from c a r o t e n o i d s y n t h e s i s , f r u i t had to be held longer than the 1 to 3 days normally needed to remove c h l o r o p h y l l at higher temperatures (142, 143, 144). U n f o r t u n a t e l y , wastage due to decay can be excessive during periods of 8-10 days or more r e q u i r e d f o r c o l o r development under commercial c o n d i t i o n s . In an e f f o r t to reduce degreening time w i t h ethylene or to replace i t s use, postharvest a p p l i c a t i o n s of ethephon e i t h e r alone or i n combination w i t h ethylene have been evaluated (14, 145, 146, 147, 148, 149, 150). In c e r t a i n i n s t a n c e s , p o s t harvest a p p l i c a t i o n s of ethephon to green-colored f r u i t to achieve degreening during t r a n s i t would be d e s i r a b l e . Results w i t h t h i s procedure may be inadequate since waxing i n some s t u d i e s and w i t h c e r t a i n c u l t i v a r s has reduced the degreening a c t i o n of ethephon (146, 149, 151). Ethephon has had no appre­ c i a b l e e f f e c t on changes i n percentage j u i c e , c i t r i c a c i d , t o t a l s o l u b l e s o l i d s , vitamin C or pH (152). A c o n c e n t r a t i o n of 5000 ul ethephon/1 caused r i n d i n j u r i e s to lemons and delayed degreening of Clementine mandarins and Shamouti oranges (145). In g e n e r a l , postharvest a p p l i c a t i o n s of ethephon have not proved more e f f e c t i v e or p r a c t i c a l under commercial c o n d i t i o n s than the standard ethylene degreening p r a c t i c e . To remove the green c o l o r from lemons, the standard F l o r i d a p r a c t i c e i s to hold unwashed f r u i t (washing r e t a r d s c o l o r de­ velopment) at 15.6°C and high r e l a t i v e humidity u n t i l the c h l o ­ r o p h y l l i s removed and the y e l l o w c o l o r predominates (126). This procedure requires 2 to 3 weeks during which time j u i c e y i e l d and c i t r i c a c i d increase (153). In an e f f o r t to speed up the process, ethephon or standard degreening p r a c t i c e s have been used but these u s u a l l y enhance decay, p a r t i c u l a r l y stem-end r o t (126, 148, 150, 153). Degreening was achieved w i t h standard degreening p r a c t i c e s i f mature f r u i t were t r e a t e d w i t h an e f f e c -

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t i v e f u n g i c i d e before exposure to ethylene to c o n t r o l decay (154, 155). Chemicals, p a r t i c u l a r l y 2 - ( 4 - c h l o r o p h e n y l t h i o ) - t r i e t h y l a m i n e (CPTA) and some r e l a t e d compounds have been a p p l i e d postharvest to c i t r u s f r u i t t o enhance c a r o t e n o i d s y n t h e s i s (22, 2£, 151, 152, 156, 157, 158, 159). The m a t e r i a l CPTA, which has been evaluated most e x t e n s i v e l y , p r i n c i p a l l y s t i m u l a t e s the red l y c o ­ pene c a r o t e n o i d which i s not a normal pigment f o r c i t r u s . There­ f o r e , i t s p r a c t i c a l use appears l i m i t e d (157). The compound 2 , 4 ' - d i c h l o r o - l - c y a n o e t h a n e s u l p h o n a n i l i d e a p p l i e d p r e - or p o s t harvest s t i m u l a t e s r a p i d and uniform removal of c h l o r o p h y l l (26, 160) i f f r u i t are subjected to l i g h t a f t e r treatment. In dark­ ness, i t does not serve as a degreening agent, but i t does i n ­ t e r f e r e w i t h ethylene degreening. Since i t s mode of a c t i v i t y appears somewhat d i f f e r e n t to t h a t of e t h y l e n e , i t i s speculated that a degreening system might be developed without the d e t r i mental senescence a c t i o One o f the i n i t i a l f r u i t on the packing l i n e i s to remove f r u i t u n s u i t a b l e f o r f r e s h consumption d i v e r t i n g i t to p r o c e s s i n g . G e n e r a l l y , those f r u i t removed because of s i z e , c o l o r , or blemishes have l i t t l e i n f l u e n c e on the q u a l i t y of processed products. However, i t has been suggested t h a t e a r l y season small s i z e d f r u i t of l e s s matu­ r i t y or l a t e season o v e r s i z e d f r u i t of poor q u a l i t y could a f f e c t the processed product (161). 1

Decay c o n t r o l techniques. Fungicides are commonly a p p l i e d to c i t r u s f r u i t s to p r o t e c t them from decay caused by numerous f u n g i . U n l i k e many f r u i t s , healthy p o r t i o n s of decaying c i t r u s f r u i t s are normally not salvaged, and any decay of a f r u i t means t h a t the e n t i r e f r u i t i s d i s c a r d e d . Postharvest f u n g i c i d e s are u t i l i z e d t o improve keeping q u a l i t y . These chemicals appear t o have l i t t l e i n f l u e n c e on i n t e r n a l q u a l i t y or n u t r i t i o n . At l e a s t i f they do, the e f f e c t i s so s u b t l e t h a t research w i t h i n t h i s area has not been j u s t i f i e d . Several e x t e n s i v e reviews on the s u b j e c t of postharvest f u n g i c i d e s have been published (162, 163, 164, 165), as have s p e c i f i c f u n g i c i d e recommendations to c o n t r o l c i t r u s decay (166, 167, 168). A p p l i c a t i o n techniques can i n f l u e n c e the e f f i c a c y of p o s t harvest f u n g i c i d e s and, t h u s , a l t e r the keeping q u a l i t y of t r e a t e d f r u i t . A p p l i c a t i o n of sodium ortho-phenylphenate (SOPP) i n a foam washer w i t h an exposure time of only 15 to 20 sec i s not as an e f f e c t i v e method as a soak or drench treatment r e ­ q u i r i n g 2-4 min (167). However, i f proper pH c o n t r o l i s not maintained, f r u i t may be burned w i t h the soak or drench t r e a t ­ ment (169, 170, 171). A p p l i c a t i o n s of SOPP i n wax were l e s s p h y t o t o x i c ΤΊ72). Within the l a s t 14 y e a r s , development of the benzimidazoles, t h i a b e n d a z o l e £ 2 - ( 4 ' - t h i a z o l y l ) b e n z i m i d a z o l e ^ and .benomyl {methyl 1-(butyl carbamoyl)-2-benzimidazole-carbam a t e j , has l e d to the a v a i l a b i l i t y of f u n g i c i d e s w i t h high

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l e v e l s of a c t i v i t y against the major decay pathogens. Litera­ t u r e p e r t a i n i n g to the a p p l i c a t i o n of the benzimidazoles and techniques which a l t e r t h e i r e f f i c a c y has r e c e n t l y b e e n reviewed (173). A p p l i c a t i o n of the benzimidazoles by suspension i n water emulsion wax or by d i s s o l v i n g i n a hydrocarbon-soluble ( s o l v e n t ) wax has not u s u a l l y provided as good decay c o n t r o l as when the m a t e r i a l s are a p p l i e d i n water. N o n a v a i l a b i l i t y o f the f u n g i c i d e at the i n f e c t i o n s i t e because of i n c o r p o r a t i o n w i t h i n the wax and v a r i a b l e coverage with the wax a p p l i c a t i o n , p a r t i c u l a r l y under the c a l y x f o r stem-end r o t c o n t r o l , have been suggested as reasons f o r reduced decay c o n t r o l . Less f u n g i c i d a l a c t i v i t y by benomyl may occur i n a l k a l i n e water waxes where the m a t e r i a l de­ grades to methyl 2-benzimidazole carbamate (MBC) and 1,2,3,4t e t r a h y d r o - 3 - b u t y l - 2 , 4 - d i o x o - S - t r i a z i n o l a j benzimidazole (STB) (174, 175), which has very weak, i f any, f u n g i c i d a l a c t i v i t y (164). A p p l i c a t i o n s of TBZ, benomyl and MBC i n wax f o r sporu­ l a t i o n c o n t r o l of P. d i g i t a t u tamination of healthy f r u i benomyl i s the most e f f e c t i v e of the three m a t e r i a l s (167, 175, 176). Benomyl penetrated the peel more e f f i c i e n t l y than MBC, forming a f u n g i s t a t i c b a r r i e r i n the e p i c a r p t h a t prevented e r u p t i o n of the hyphae of F\ d i g i t a t u m through the epidermis (175). Preharvest a p p l i c a t i o n s of benomyl to V a l e n c i a oranges f o r the c o n t r o l of post-harvest decay s i g n i f i c a n t l y reduced aging ( s h r i v e l i n g of the r i n d a t the stem-end) (52). Vapor-phase fumigants are used f o r c i t r u s decay c o n t r o l , diphenyl being the most e f f e c t i v e and commonly used m a t e r i a l . The undesirable odor imparted to t r e a t e d f r u i t d i s s i p a t e s w i t h i n a few days a f t e r removal of the f r u i t from the diphenyl atmo­ sphere. Control of stem-end r o t s caused by A. c i t r i , D. n a t a l e n s i s , and J P . c i t r i w i t h postharvest a p p l i c a t i o n s of the growth r e g u l a t o r s 2, 4-D and, o c c a s i o n a l l y , 2,4,5-T i s a t t r i b u t e d to p r e s e r ­ v a t i o n of the green button and prevention of a b s c i s s i o n (106, 177, 178, 179, 180, 181, 182). Freshness and r e t a r d a t i o n of c o l o r changes a s s o c i a t e d w i t h senescence are b e n e f i t s of the treatment (183, 184). G i b b e r e l l i c a c i d a p p l i e d a f t e r harvest was a l s o reported to reduce decay of lemons (183), but i t s p r i ­ mary e f f e c t i s to r e t a i n c h l o r o p h y l l (183, 185ΤΓ Total s o l u b l e s o l i d s , a c i d i t y and a s c o r b i c a c i d were not s i g n i f i c a n t l y i n ­ fluenced by 2,4-D (184) or GA (183, 184). Treatments of c i t r u s f r u i t s to c o n t r o l decay fungi which leave no chemical residues would be d e s i r a b l e . E r a d i c a t i o n of i n c i p i e n t i n f e c t i o n s of decay fungi w i t h heat treatments and gamma r a d i a t i o n have been t r i e d . Immersion of c i t r u s f r u i t s i n water at 48 C f o r 2-4 min to c o n t r o l species of Phytophthora has been shown to p h y s i o l o g i c a l l y weaken lemons, i n some i n ­ stances, and increase t h e i r s u s c e p t i b i l i t y to other p a t h o l o g i c a l diseases during storage (186). Decay caused by I P . d i g i t a t u m , P. c i t r i , and D. n a t a l e n s i s was reduced by t r e a t i n g oranges a t

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53°C f o r 5 min (187, 188), but p h y s i o l o g i c a l breakdown of the r i n d was i n c r e a s e d . Cold t u r g i d lemons r e q u i r e d s l i g h t dehydration before immersion to reduce o l e o c e l l o s i s (189). T r e a t ment a t elevated temperatures to enhance decay c o n t r o l w i t h SOPP increased the i n c i d e n c e of o l e o c e l l o s i s and darkened i n j u r i e s of the r i n d (169). Use of gamma rays to destroy pathogens i n e s t a b l i s h e d l e s i o n s w i t h i n the f r u i t has g e n e r a l l y been unsuccessful. Dosages r e q u i r e d to e r a d i c a t e or r e t a r d c e r t a i n pathogens, p a r t i c u l a r l y the stem-end r o t fungi D i p l o d i a and A l t e r n a r i a , g e n e r a l l y caused adverse e f f e c t s to the f r u i t . These were mani f e s t e d as damage to the r i n d (190, 191, 192), o f f - f l a v o r s (191, 193, 194) and l o s s i n i n t e r n a l q u a l i t y and weight (191, 195). R e s p i r a t i o n r a t e s (191, 195) and ethylene evolution~TT%T"â"re increased by gamma r a d i a t i o n treatments. There i s a marked l o s s i n a s c o r b i c a c i d i n lemons t r e a t e d w i t h 200 Krad or above (195), but not i n Washington Navel oranges t r e a t e d w i t h 200 Krad or l e s s (191). A thorough revie citrus fruits is availabl Waxing. S o - c a l l e d wax coatings are normally a p p l i e d to c i t r u s f r u i t s to r e p l a c e n a t u r a l wax removed i n washing, to impart gloss to increase consumer a p p e a l , and to reduce s h r i n k age from l o s s of moisture. Both n a t u r a l waxes and s y n t h e t i c r e s i n s are used and are a p p l i e d as aqueous emulsions or ( f o r the r e s i n s ) as s o l u t i o n s i n an organic s o l v e n t . Some a d d i t i o n a l e f f e c t s from wax a p p l i c a t i o n s have been observed. Wax coatings lowered i n t e r n a l oxygen and r a i s e d carbon d i o x i d e l e v e l s (197, 198, 199, 200, 201) and t h i c k e r than normal a p p l i c a t i o n s may cause ethanol accumulation (197, 202, 203) and o f f - f l a v o r s . A thickness of c o a t i n g which reduced the weight l o s s to 50% of t h a t of untreated f r u i t produced i n t e r n a l anaerobic c o n d i t i o n s and o f f - f l a v o r s (197, 204). Ethanol b u i l d - u p i n j u i c e and o f f f l a v o r s occurred a f t e r m u l t i p l e coatings of water wax or s o l v e n t type wax; l e s s ethanol accumulation and no o f f - f l a v o r s were noted i n polyethylene-coated f r u i t s (203). Reductions i n c h i l l ing i n j u r y of g r a p e f r u i t (205, 206), p i t t i n g of V a l e n c i a oranges (207), black spot (Guignardia c i t r i c a r p a K i e l y ) of V a l e n c i a oranges (208), decay of lemons"Tl83), and an i n c r e a s e i n c h i l l ing i n j u r y of limes (209) has been obtained w i t h wax a p p l i c a t i o n s . Waxing w i t h aqueous emulsion waxes has been shown to r e t a r d degreening (210). Fumigation. Many c o u n t r i e s importing c i t r u s f r u i t s r e q u i r e t h a t the f r u i t be t r e a t e d to e r a d i c a t e eggs, l a r v a e , and pupae of f r u i t f l i e s so as to prevent t h e i r i n t r o d u c t i o n i n t o u n i n f e s t e d areas. Treatment f o r the e r a d i c a t i o n of a t l e a s t 5 species of 3 genera of f r u i t f l i e s has been r e p o r t e d . Ethylene dibromide (EDB) and o c c a s i o n a l l y methyl bromide, g e n e r a l l y a p p l i e d as fumigants, have been u t i l i z e d most e x t e n s i v e l y f o r t h i s purpose. I f c a r e f u l handling and proper treatment are not

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f o l l o w e d , r i n d of c i t r u s f r u i t s can be d i s c o l o r e d or p i t t e d by the fumigation treatment (211, 212, 213, 214, 215, 216, 217, 218). Fumigation damage r e s u l t i n g i n r i n d i n j u r y may be assoc i a t e d with p r i o r p r e c o o l i n g before treatment (217) or w i t h i n adequate v e n t i l a t i o n a f t e r fumigation (212, 214, 217, 219). R e f r i g e r a t i o n should be delayed f o r at l e a s t 24 hours a f t e r treatment (217). Injury to Marsh g r a p e f r u i t and Shamouti and V a l e n c i a oranges was due to the p e r s i s t e n c e of residues of the fumigant i n the f r u i t peel (212). The desorption r a t e during a e r a t i o n increased w i t h temperature. Incidence of peel i n j u r y was highest i n f r u i t s t o r e d at low temperature or wrapped i n polyethylene bags, probably due to prolonged a c t i o n of the EDB residues on the p e e l . Storage of fumigated f r u i t i n an atmosphere c o n t a i n i n g an increased c o n c e n t r a t i o n of carbon d i o x i d e delayed the appearance o f damage. Fumigation i n j u r y was e n hanced by the presence of diphenyl impregnated i n pads (217). Ethylene degreened g r a p e f r u i EDB fumigation than non-degreene lemon and orange f r u i t s was s t i m u l a t e d by EDB exposure and was p r o p o r t i o n a l to the dosage l e v e l (221). Treated f r u i t s a l s o evolved more e t h y l e n e , p a r t i c u l a r l y those e x h i b i t i n g i n j u r y (211). The e f f e c t of chamber load on EDB absorption and r i n d i n j u r y showed t h a t reducing the l o a d i n g r a t e from 30 to 15% i n creased EDB absorption considerably and often caused development of severe r i n d i n j u r y (215). Increasing loading rates from 45 to 60% had l i t t l e e f f e c t on absorption of EDB by the f r u i t . N u t r i t i o n a l value of lemons, as determined by percentage j u i c e and s o l u b l e s o l i d s , c i t r i c a c i d , pH, and a s c o r b i c a c i d , was not a f f e c t e d by fumigation (221). The a p p l i c a t i o n of TBZ to c i t r u s f r u i t i n the wax coating e i t h e r before or a f t e r fumigation markedly reduced the i n c i d e n c e of EDB peel i n j u r y (222). A p p l i c a t i o n s of TBZ i n a water suspension or i n a water suspension followed by the wax c o a t i n g which d i d not c o n t a i n the f u n g i c i d e d i d not e f f e c t i v e l y reduce the incidence of the i n j u r y . EDB r e p o r t e d l y was s t a b l e and nonreactive with the c o n s t i t u e n t s of the t i s s u e s of V a l e n c i a and navel oranges, and lemons (219). Handling P r a c t i c e s . Methods used i n the packinghouse to prepare f r u i t f o r the f r e s h market may a l s o i n f l u e n c e the s h e l f l i f e and i n t e r n a l q u a l i t y . Processing Dancy tangerines on the packinghouse l i n e c u r t a i l e d the subsequent l i f e of the f r u i t , p a r t i c u l a r l y when degreened (103), Rind breakdown i n oranges was shown to increase w i t h processing (223). Washing, c o l o r adding, and waxing treatments had l i t t l e e f f e c t upon the t o t a l s o l u b l e s o l i d s , t o t a l a c i d s , or v i t a m i n C content of the j u i c e . Taste t e s t s , however, i n d i c a t e d t h a t f l a v o r was never improved, and i n some l o t s r e c e i v i n g the f u l l packinghouse treatment, t a s t e was unmistakably impaired (223). Dropping of F l o r i d a g r a p e f r u i t during h a r v e s t i n g (64) or of Satsuma mandarins during the packing process (224) was shown to decrease firmness during

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storage. The r e s p i r a t i o n r a t e o f c i t r u s f r u i t i s increased by rough handling (81, 201, 224, 225), and handling a l s o hastened y e l l o w i n g o f Persian~Umes~T8l7T~ In the case o f dropped Satsuma mandarins held i n storage (224), the r a t i o of dehydroascorbic a c i d to t o t a l a s c o r b i c a c i d i n the pulp increased i n p r o p o r t i o n to the number o f times the f r u i t were dropped. Most types of mechanical i n j u r y t o mandarins were produced upon dropping impact, compression, and abrasion (226). Two major f a c t o r s c o n t r o l l i n g breakdown of j u i c e sacs were dropping height and compression (227). Several postharvest treatments to c i t r u s f r u i t s have been t e s t e d i n an e f f o r t to improve the q u a l i t y o f the e x t r a c t e d j u i c e . Bruemmer and Roe subjected c i t r u s f r u i t s to anaerobic c o n d i t i o n s f o r periods of 20 t o 32 hours a t 32.2 to 43°C (228, 229). This treatment reduced the t i t r a t a b l e a c i d i t y and i n creased the B r i x - a c i d r a t i o by about 10% The decrease i n a c i d i t y was accompanied ethanol (229). Since th c r i t e r i o n of c i t r u s j u i c e q u a l i t y , t h i s procedure, i f p e r f e c t e d , could a l l o w e a r l i e r harvesting of f r u i t and a more c o n s i s t e n t supply of f r u i t during the processing season. Bitterness of products from navel oranges, lemons, and g r a p e f r u i t i s r e l a t e d to l i m o n i n content. A 3-hour treatment o f f r u i t w i t h 20 ul ethylene/1 of a i r lowered the limonin content, reduced b i t t e r ness, and the j u i c e was judged more p a l a t a b l e than j u i c e from untreated f r u i t (230). Storage and Q u a l i t y of C i t r u s

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Cold Storage. C i t r u s f r u i t s may be stored to provide a source o f f r u i t a t times other than during the normal harvesting season, to hold f r u i t u n t i l a market i s a v a i l a b l e , or w h i l e f r u i t i s i n t r a n s i t to a d i s t a n t market. U n l i k e deciduous f r u i t s , c i t r u s does not undergo r a p i d chemical or p h y s i c a l changes a f t e r harvest. C i t r u s f r u i t s are n o n - c l i m a c t e r i c , hence, they do not r i p e n o r improve i n q u a l i t y a f t e r harvest. The only exception may be the lemon, which i s picked hard and green, and which i n creases i n j u i c e and becomes y e l l o w during normal storage periods. I f c i t r u s f r u i t s are stored under proper temperatures, h u m i d i t i e s , and atmospheres, and are marketed a f t e r a proper d u r a t i o n of s t o r a g e , the q u a l i t y of the f r u i t does not d i f f e r g r e a t l y from t h a t which e x i s t e d a t harvest. Storage of f r u i t at lowered temperatures reduces f r u i t r e s p i r a t i o n and r a t e o f growth o f decay pathogens w i t h a r e s u l t i n g p r o p o r t i o n a t e i n crease i n f r u i t storage l i f e . Since numerous reviews on the subject o f changes i n c i t r u s f r u i t s during c o l d storage have been w r i t t e n ( 1 , 231, 232, 233, 234, 235, 236, 237), d i s c u s s i o n of the t o p i c w i l l be l i m i t e d p r i m a r i l y to some o f the more recent studies. Some of the changes c h a r a c t e r i s t i c of m a t u r a t i o n , notably

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decreasing a c i d i t y , continue a f t e r storage. The f r u i t begins to l o s e weight as soon as i t leaves the t r e e and there i s g e n e r a l l y a l o s s of s o l u b l e s o l i d s and a s c o r b i c a c i d , on the basis of the o r i g i n a l f r e s h weight of the f r u i t . I f the weight l o s s i s exc e s s i v e , these c o n s t i t u e n t s may a c t u a l l y increase i n c o n c e n t r a t i o n (233). However, some q u i t e s i g n i f i c a n t losses i n a s c o r b i c a c i d have been r e p o r t e d , p a r t i c u l a r l y w i t h lemons (238) and t a n gerines (239). Q u a l i t y of V a l e n c i a oranges stored f o r 12 and 18 weeks at 15 and 5 C, r e s p e c t i v e l y , was compared to s i m i l a r f r u i t l e f t on the t r e e (240). Storage f r u i t was d e f i n i t e l y of b e t t e r q u a l i t y . Color was b e t t e r , s i n c e regreening which occurred i n f r u i t on the t r e e d i d not occur i n storage. G r a n u l a t i o n and stem-end browning a l s o developed i n f r u i t held on the t r e e but not i n storage f r u i t . C o o l - s t o r e d f r u i t was a l s o higher i n B r i x , B r i x / a c i d r a t i o , and a s c o r b i c a c i d . Loss of f l a v o r i n storage a f t e r 24 weeks a t 15 C was a s s o c i a t e d w i t h a r a p i a c i d , polyphenols, and f l a v o n o i d stored a t 3.3 C f o r 6 months decreased i n a c i d i t y , increased i n t o t a l s o l u b l e s o l i d s , and v a r i e d i n a s c o r b i c a c i d content (241). C i t r i c a c i d was the main a c i d i n j u i c e , malonic and m a l i c a c i d s predominated i n the r i n d of Shamouti oranges stored 3 months at 17 C (242). With a g i n g , m a l o n i c , s u c c i n i c and a d i p i c a c i d s i n creased i n r i n d and j u i c e , but c i t r i c and m a l i c a c i d s decreased i n the j u i c e . The authors (242) suggest t h a t malonic a c i d could serve as an i n d i c a t o r of f r u i t t i s s u e senescence i n s t o r a g e , because accumulation of the a c i d w i t h i n the r i n d begins a t f r u i t m a t u r i t y , and increased t h r e e - f o l d during three months of p o s t harvest storage. I t has been r e c e n t l y suggested (243) t h a t ethanol may a l s o be an i n d i c a t o r of f r u i t c o n d i t i o n s s i n c e i t showed g r e a t e r changes during storage and subsequent holding than d i d acetaldehyde, t o t a l s o l u b l e s o l i d s , t i t r a t a b l e a c i d , or pH. Ethanol increased more i n g r a p e f r u i t stored f o r 3 months a t 1.0 C than at 4.5 or 10°C. In oranges, ethanol increased more a t 10°C than a t 4.5 or 1.0 C. Ethanol content i n j u i c e of Hamlin, P i n e apple and V a l e n c i a oranges and Marsh g r a p e f r u i t increased w i t h decreasing oxygen ( 0 ) c o n c e n t r a t i o n i n CA storage and w i t h wax a p p l i c a t i o n s (202). The ethanol content of j u i c e of Marsh grapef r u i t increased w i t h i n c r e a s i n g carbon d i o x i d e (C0 ) c o n c e n t r a t i o n s from 10 to 30%, w i t h length of time of exposure to C 0 , and w i t h t o t a l time of storage. The acetaldehyde content o f the j u i c e , although much lower than the ethanol c o n t e n t , increased i n a s i m i l a r manner (244). Under these various storage c o n d i t i o n s , the ethanol content was apparently i n s u f f i c i e n t to a f f e c t f l a v o r s i g n i f i c a n t l y . The c o n d i t i o n of f r u i t during c o l d storage (245) was estimated by continuously monitoring the C 0 produced hourly/ kg of f r u i t and c a l c u l a t i n g the r e l a t i v e increase i n the r e s p i r a t o r y i n t e n s i t y . Increases i n r e s p i r a t i o n were due to i n f e c t e d f r u i t plus the e f f e c t of ethylene emanation on metabolism of healthy f r u i t . 2

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C h i l l i n g i n j u r y . A major problem w i t h c o l d storage of c i t r u s f r u i t s i s t h a t o f c h i l l i n g i n j u r y o r p i t t i n g , which occurs i n c h i l l - s e n s i t i v e f r u i t s such as g r a p e f r u i t , lemon, and lime a t temperatures of 5-10 C. Eaks (42) reported t h a t limes had to be stored a t 5 C to r e t a i n green c o l o r , but c h i l l i n g i n j u r y d e v e l oped i n about 4-5 weeks. Storage a t 10°C e l i m i n a t e d the c h i l l i n g i n j u r y problem, but f r u i t became y e l l o w - c o l o r e d a f t e r 2-3 weeks. Several techniques i n handling c i t r u s f r u i t s , p r i n c i p a l l y grapef r u i t , have been u t i l i z e d to m i t i g a t e the incidence of c h i l l i n g i n j u r y . Exposure of f r u i t to high r e l a t i v e humidity (95-100%) f o r several days before storage (123, 246) or a t lower moisture l e v e l s (curing) f o r 1 to 2 weeks T247, 248, 249) have reduced incidence of the d i s o r d e r . Waxing has a l s o been shown to have a f a v o r a b l e e f f e c t (250, 251). I n t e r m i t t e n t warming during the storage period has"âTso~Bêen s u c c e s s f u l (252, 253). C h i l l i n g i n j u r y was reduced by removing f r u i t p e r i o d i c a l l y from storage a t 4.4°C to 21.1°C f o r 8 hour Treatment w i t h ethylen technique f o r c o n t r o l , however, i t was more e f f e c t i v e on greenc o l o r e d f r u i t (123). C h i l l i n g i n j u r y of limes was markedly r e duced by storage i n a p a r t i a l vacuum a t 220 mm of Hg (123), and some i n f l u e n c e from growth r e g u l a t o r s (benzyladenine, GA, 2,4-D) a p p l i e d p r e - or postharvest has been observed (50). Treatments w i t h high l e v e l s of 0 0 (40%) f o r up to seven days a t 21.1°C s i g n i f i c a n t l y reduced c h i l l i n g i n j u r y (247). Success using C 0 treatments i n a d d i t i o n a l studies g e n e r a l l y a t lower C 0 l e v e l s has been obtained (254, 255, 256, 257, 258). Fungicides a p p l i e d f o r the c o n t r o l o f decay fungi unexpectedly reduced c h i l l i n g i n j u r y (251, 258, 259, 260). Control w i t h TBZ and benomyl g e r s i s t e d during 16 weeks storage of g r a p e f r u i t a t 2, 5, and 8 C (251}. TBZ was more e f f e c t i v e than benomyl and treatments were most e f f e c t i v e when the m a t e r i a l s were incorporated i n t o the wax. The e f f e c t of TBZ was enhanced by i n c r e a s i n g c o n c e n t r a t i o n and r e s i d u e s , but the e f f e c t i v e n e s s of benomyl was not improved w i t h increased c o n c e n t r a t i o n s . Reduced c h i l l i n g i n j u r y of g r a p e f r u i t w i t h the use o f benomyl was a l s o observed by McCornack (261), but use of the f u n g i c i d e diphenyl s i g n i f i c a n t l y increased c h i l l i n g i n j u r y . F o r t u n a t e l y , the benzimidazoles, TBZ and benomyl, are used e x t e n s i v e l y on c i t r u s f r u i t s f o r decay c o n t r o l and the m i t i g a t i o n of c h i l l i n g i n j u r y from t h e i r a p p l i c a t i o n provides an a d d i t i o n a l b e n e f i t . U n f o r t u n a t e l y , however, the cause of c h i l l ing i n j u r y has y e t to be d e f i n e d , and none o f these handling techniques provide complete c o n t r o l of t h i s d i s o r d e r . 2

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P r e - c o o l i n g . P r e - c o o l i n g , a technique to remove f i e l d heat from c i t r u s f r u i t s , i s advised s i n c e r e f r i g e r a t i o n f a c i l i t i e s w i t h i n most t r a n s i t containers do not have s u f f i c i e n t c a p a c i t y to cool f r u i t s r a p i d l y . F r u i t i s o f t e n d e l i v e r e d from the f i e l d a t r e l a t i v e l y high temperatures. Processes w i t h i n the packinghouse such as degreening, c o l o r - a d d i n g , and drying c o n t r i b u t e f u r t h e r

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heat to the f r u i t . P r e - c o o l i n g can be accomplished by a i r c o o l i n g or h y d r o - c o o l i n g . Forced a i r a t -2 C f o r 6 to 24 hours p r i o r to v e n t i l a t e d shipment o f Shamouti, V a l e n c i a and navel oranges and Marsh g r a p e f r u i t (262) reduced weight l o s s during simulated shipment and storage. P r e - c o o l i n g of f r u i t to temperatures below 0 C was not advised because of p o s s i b l e e f f e c t s on q u a l i t y . P r e - c o o l i n g of F l o r i d a oranges (263) i n i t i a l l y reduced decay l e v e l s but then decay l e v e l s increased during simulated s h e l f - l i f e . Water damage from hydro-cooling caused increased decay of tangerines and g r a p e f r u i t (264) and oranges (265). Decay a f t e r hydro-cooled f r u i t are allowed to come to ambient temperature, as i n markets or homes, f r e q u e n t l y exceeded t h a t of non-hydro-cooled f r u i t (266). Peel i n j u r y , p a r t i c u l a r l y of Pineapple oranges, was o f t e n increased by h y d r o - c o o l i n g . C o n t r o l l e d Atmosphere Storage Use of c o n t r o l l e d atmosphere (CA), an atmosphere c o n t a i n i n maintain the q u a l i t y o no more e f f e c t i v e than c o l d storage i n a i r . Several reviews of the s u b j e c t published p r e v i o u s l y cover r e s u l t s o f some o f the e a r l i e r work (1 207, 236, 237, 267, 268, 269). Various r e s u l t s have been obtained and i n d i c a t e t h a t f a c t o r s other than 0 and C 0 c o n c e n t r a t i o n s , such as v a r i e t y , m a t u r i t y and humidity may be i n v o l v e d . Decay i s o f t e n enhanced w i t h c e r t a i n CA storage c o n d i t i o n s (268, 270, 271, 272), d i s o r d e r s of the r i n d may be i n creased (209, 270, 27T, 273) and o f f - f l a v o r s may develop (271, 274). However, some improvement i n keeping q u a l i t y w i t h CA storage has been r e p o r t e d . V a l e n c i a oranges held i n 15% 0 -0% C 0 f o r 12 weeks a t 1 C plus 1 week a t 21°C had higher f l a v o r r a t i n g s than s i m i l a r f r u i t held i n other c o n t r o l l e d atmospheres or i n a i r (267) A d d i t i o n o f e i t h e r 2.5 o r 5% C 0 c o n t r o l l e d aging. No marked d i f f e r e n c e s were found i n t o t a l s o l u b l e s o l i d s , t o t a l a c i d , pH, a s c o r b i c a c i d , or h e s p e r i d i n between V a l e n c i a oranges from CA storage and those from a i r (207). Less decay developed f o l l o w i n g storage of V a l e n c i a oranges a t 15 or 10% 0 w i t h 0% C 0 than when s t o r e d i n a i r (275). P i t t i n g ( c h i l l i n g i n j u r y ) o f g r a p e f r u i t was e l i m i n a t e d by CA storage at 10 C but not lower temperatures (267). C o n t r o l l e d atmosphere storage of Satsuma mandarin increased the p r o p o r t i o n of marketable f r u i t , f r u i t d e n s i t y , and c i t r i c a c i d c o n c e n t r a t i o n . Optimum c o n d i t i o n s were 1% C 0 w i t h 6-9% 0 (276). Storage of navel oranges at 15 C a t high 0 l e v e l s (40-80%) improved the orange c o l o r of the endocarp and of e x t r a c t e d j u i c e (277). Some of the most o p t i m i s t i c r e s u l t s w i t h CA storage have been reported from work w i t h lemons. B i a l e (278) i n d i c a t e d t h a t lemons may b e n e f i t from CA storage. Storage l i f e was prolonged markedly by atmosphere of 5 and 10% 0 . R e s p i r a t i o n r a t e s were reduced, c o l o r changes were r e t a r d e d , and decay, caused p r i m a r i l y by A. c i t r i , was reduced. An atmosphere of .6% 0 without C 0 was the best treatment f o r lemons a t 15.6°C (272, 279). Levels of 5 to 9

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8% 0 reduced decay to l e s s than t h a t i n a i r storage (279). The importance of removing e t h y l e n e , which may accumulate i n storage f a c i l i t i e s , to enhance keeping q u a l i t y was emphasized by some recent s t u d i e s (280). V a l e n c i a oranges were stored a t 10°C i n 5% C0 and 3% 0^ without and w i t h an ethylene absorbant P u r a f i l (porous alumina beads impregnated w i t h potassium permanganate). By keeping ethylene concentrations to l e s s than 0.8 ul/1 of a i r , stem-end decay caused by a species o f Fusarium, green mold, l o s s of orange f l a v o r , and development o f o f f - f l a v o r s were minimized. Other v o l a t i l e s present w i t h and without the ethylene absorber apparently had no e f f e c t on q u a l i t y . E t h y l e n e , produced i n copious q u a n t i t i e s by r o t s , enhances r e s p i r a t i o n and senescence of healthy f r u i t w i t h i n the storage f a c i l i t y (281). The importance o f ethylene removal has been i l l u s t r a t e d a l s o i n recent storage s t u d i e s of lemons i n A u s t r a l i a (282, 283, 284, 285). Lemons remained i n good c o n d i t i o n f o r up to 6 months under an atmosphere of 6-10% 0 an tinuous removal of the c e n t r a t i o n s s i g n i f i c a n t l y reduced the development o f green mold (284). Oxygen c o n c e n t r a t i o n i n the atmosphere was a l s o shown to r e g u l a t e the e f f e c t of ethylene on the r a t e of green c o l o r l o s s from lemon r i n d . An atmosphere o f 11% 0 and 6 ul/1 o f accumul a t e d ethylene caused a more r a p i d l o s s o f green c o l o r than d i d 5% 0 and 300 ul ethylene/1 of a i r . Removal of ethylene was usef u l i n m a i n t a i n i n g the green c o l o r but i t was more e f f e c t i v e when combined w i t h CA storage. By t r e a t i n g f r u i t w i t h 2,4-D and GA, both a t a c o n c e n t r a t i o n of 500 mg/1, optimum r e s u l t s were obtained i n an e t h y l e n e - f r e e CA. The 2,4-D e f f e c t i v e l y prevented senescence o f the button w h i l e the GA retarded the development of the deep y e l l o w c o l o r a s s o c i a t e d w i t h o v e r - m a t u r i t y . Attempts t o s t o r e limes i n CA o f t e n r e s u l t e d i n s c a l d - l i k e i n j u r y t o the r i n d and an i n c r e a s e i n the i n c i d e n c e o f decay (209, 272). Limes stored i n a CA of 5% 0 w i t h 7% C 0 maint a i n e d acceptable green c o l o r , but had low j u i c e c o n t e n t , t h i c k r i n d s , and a high i n c i d e n c e o f decay 286). However, limes were s u c c e s s f u l l y stored when held under low pressure (hypobaric) c o n d i t i o n s . Berg and Berg (287) reported limes were s t i l l green a f t e r 8 weeks a t 150 mm Hg ancT~15 C, whereas, 50% of c o n t r o l f r u i t s turned y e l l o w i n 10 days. Spalding (286, 288) observed t h a t T a h i t i (Persian) limes r e t a i n e d green c o l o r , j u i c e c o n t e n t , and f l a v o r acceptable f o r marketing and had a low i n c i d e n c e o f decay during storage a t a low atmospheric pressure o f 170 mm Hg f o r up to 6 weeks a t 10°C or 15.6 C and a r e l a t i v e humidity of 98-100%. Check f r u i t s turned y e l l o w w i t h i n 3 weeks a t normal atmospheric pressure. Low pressure storage d i d not prevent c h i l l i n g i n j u r y of f r u i t stored a t 2.2°C. At 21.1°C, f r u i t stored a t 170 mm Hg remained green and s u i t a b l e f o r marketing a f t e r 3-4 weeks i f t r e a t e d w i t h TBZ or benomyl f o r decay c o n t r o l . Removal o f ethylene a t low pressure was suggested to be involved i n r e t a r d a t i o n o f r i p e n i n g (287), and appears to be c r i t i c a l to 2

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the success of storage of both lemons and l i m e s . Low pressures l e s s than 50 mm Hg were r e q u i r e d to r e t a r d growth of common postharvest decay fungi (289). A c t i o n was f u n g i s t a t i c r a t h e r than f u n g i c i d a l . F r u i t Q u a l i t y as Influenced by Packaging and T r a n s p o r t a t i o n Packaging. Techniques used i n packaging and t r a n s p o r t i n g c i t r u s f r u i t s i n f l u e n c e f r u i t q u a l i t y . The package and/or cont a i n e r must p r o t e c t the f r u i t from mechanical damage and provide a f a v o r a b l e m i c r o c l i m a t e f o r the f r u i t i f q u a l i t y i s to be maintained from harvest to consumption. Various reviews on the subj e c t of packaging and t r a n s p o r t i n g c i t r u s have been published p r e v i o u s l y (250, 268, 290, 291, 292, 293, 294). T h e r e f o r e , only the major aspects as w e l l as recent developments not p r e v i o u s l y reviewed w i l l be discussed here R e l a t i v e humidity i of q u a l i t y , both i n i t s pathogens (295). Humidity w i t h i n the package or c o n t a i n e r can vary e x t e n s i v e l y . M i c r o c l i m a t e changes occur due to t r a n s p i r a t i o n , r e s p i r a t i o n , and to changes i n ambient c o n d i t i o n s (291, 296, 297). I n t e r a c t i o n s between f r u i t and a i r temperatures when f r u i t are t r a n s f e r r e d i n t o and out of r e f r i g e r a t e d cond i t i o n s can have d r a s t i c e f f e c t s on humidity (292). High humidi t y and poor v e n t i l a t i o n i n polyethylene bags or s h r i n k - f i l m wrapped paper t r a y s have led to increased amounts of decay (290, 291, 298, 299, 300). T h e r e f o r e , p e r f o r a t i o n s w i t h i n the f i l m are required to reduce humidity and C 0 l e v e l s (301). On the other hand, f r u i t packed i n mesh bags can l o s e moisture e x c e s s i v e l y and become f l a c c i d and s h r i v e l e d (300). Decay of only one f r u i t w i t h i n a package makes i t unacceptable to the consumer, and v o l a t i l e s from decaying f r u i t w i t h i n a polyethylene f i l m bag may produce o f f - f l a v o r s i n the j u i c e of sound f r u i t s w i t h i n the same bag (302). Films possessing d i f f e r e n t p e r m e a b i l i t y to 0 and C 0 have been used to develop atmospheres and/or h u m i d i t i e s which improve q u a l i t y . C h i l l i n g - i n j u r y was reduced i n g r a p e f r u i t by packaging f r u i t under various types of f i l m s (206, 303, 304). Some recent success i n improving keeping q u a l i t y of c i t r u s f r u i t s has been obtained by wrapping i n d i v i d u a l f r u i t w i t h i n f i l m of high dens i t y p o l y e t h y l e n e . Ben-Yehoshua and co-workers (305, 306) markedly delayed d e t e r i o r a t i o n of i n d i v i d u a l l y wrapped Shamouti and V a l e n c i a oranges, Marsh g r a p e f r u i t , and Eureka lemons. Peel shrinkage, s o f t e n i n g , deformation, and l o s s of f l a v o r was delayed. Sealed f r u i t maintained the f r e s h appearance more than twice as long as c o n v e n t i o n a l l y handled f r u i t . Weight l o s s was reduced about f i v e - f o l d . R e s p i r a t o r y a c t i v i t y and ethylene production was a l s o reduced i n wrapped and sealed f r u i t . In a separate study (307), g r a p e f r u i t wrapped i n polyethylene bags l o s t l e s s weight, maintained optimum c o l o r and g l o s s , and developed l e s s stem-end r o t than unwrapped f r u i t . 2

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T r a n s p o r t a t i o n . Deformation of c i t r u s f r u i t , due to i n adequate p r o t e c t i o n by the c o n t a i n e r , d e t r a c t s g r e a t l y from e x t e r n a l appearance. O v e r - f i l l i n g o f c a r t o n s , so t h a t weight o f stacked cartons i s supported by the f r u i t r a t h e r than the cont a i n e r , c o n t r i b u t e s s i g n i f i c a n t l y to deformation (294). Problems w i t h malformation occur more f r e q u e n t l y i n s i t u a t i o n s where f r u i t are transported over long d i s t a n c e s , such as during export. Hale (308) reported t h a t 33 to 60% of the F l o r i d a g r a p e f r u i t shipped to Japan a r r i v e d s e r i o u s l y deformed. Deformed g r a p e f r u i t were more p r e v a l e n t i n the bottom l a y e r of the carton regardless of the type of carton used. U s u a l l y , the l a r g e r the f r u i t , the more s e r i o u s l y the f r u i t were deformed. By packing f r u i t i n e x perimental (309) or i n t e r n a t i o n a l standard shipping cartons (310), which were somewhat deeper than the standard 4/5 bushel c a r t o n , deformity was reduced s i g n i f i c a n t l y s i n c e more of the weight was supported by the c a r t o n . C o n d i t i o n and appearance o f l a r g e s i z e d g r a p e f r u i t were improve pack containers (311). Seriou was reduced to an average of 2.7%, compared to 27.9% f o r f r u i t shipped i n the standard export box. During handling and shipping g r a p e f r u i t from South A f r i c a to Europe, percentage j u i c e and r i n d thickness decreased (34). The percentage of t o t a l s o l u b l e s o l i d s increased and the a c i d content remained undamaged. Conclusions Q u a l i t y o f c i t r u s f r u i t s i s a l t e r e d q u i t e s i g n i f i c a n t l y by numerous techniques a p p l i e d before and a f t e r harvest. Since appearance of the f r u i t i s so v i t a l to consumer a c c e p t a b i l i t y , c o n s i d e r a b l e e f f o r t s towards improving and preserving the q u a l i t y of the r i n d can be expected i n f u t u r e s t u d i e s . Damage r e s u l t i n g from unnecessary rough handling during harvesting and processing predisposes f r u i t to increased decay caused by several wound pathogens. Even though e f f e c t i v e f u n g i c i d e s such as the b e n z i m i dazoles have been developed, these m a t e r i a l s can not be expected to cure the damage caused by improper handling. Rather, proper f u n g i c i d e a p p l i c a t i o n s combined w i t h good handling and s a n i t a r y techniques w i l l insure c i t r u s f r u i t s o f e x c e l l e n t keeping q u a l i t i e s . Problems w i t h fungal r e s i s t a n c e and lack of c o n t r o l of minor decays are s u f f i c i e n t to warrant f u r t h e r i n v e s t i g a t i o n s w i t h postharvest chemicals f o r decay c o n t r o l . Cold storage s t i l l appears to be the most e f f e c t i v e method f o r preserving q u a l i t y i n c i t r u s f r u i t s except f o r lemons and l i m e s . Recent s t u d i e s w i t h lemons have shown b e n e f i c i a l e f f e c t s of CA storage where ethylene i s e f f e c t i v e l y removed. S i m i l a r r e s u l t s have been obtained w i t h limes stored under low pressure (hypobaric) storage. Recent r e s u l t s i n improving keeping q u a l i t y o f c i t r u s f r u i t by i n d i v i d u a l l y wrapping them i n high d e n s i t y polyethylene await a d d i t i o n a l c o n f i r m a t i o n and a p p l i c a t i o n to commercial p r a c t i c e s .

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Literature Cited 1. Ting, S. V.; Attaway, J. A. In "The Biochemistry of Fruits and Their Products", Hulme, A. C., Ed. Academic Press: London and New York, 1971; 2, p. 107-169. 2. Deszyck, E. J.; Ting, S. V. Proc. Am. Soc. Hortic. Sci., 1958, 72, 304. 3. Deszyck, E. J.; Ting, S. V. Proc. Am. Soc. Hortic. Sci., 1960, 75, 266. 4. Reese, R. L.; Brown, G. E. HortScience, 1964, 4, 96. 5. Anonymous. State of Florida Department of Citrus, Citrus Fruit Laws, Lakeland, FL 1974. 6. Coggins, C. W., Jr.; Hield, H. Z. In "The Citrus Industry"; Reuther, W.; Batchelor, L. D.; Webber, J. H., Eds.; Univ. Calif. Div. Agric. Sci.: Riverside, CA, 1968; 2, p. 373-389. 7. Warner, H. L.; Leopold, A. C. Plant Physiol., 1969, 44, 156. 8. El-Zeftawi, Β. M. 215. 9. Jahn, O. L.; Young, R. J. Am. Soc. Hortic. Sci., 1972, 97, 544. 10. Young, R.; Jahn, O. J. Am. Soc. Hortic. Sci., 1972, 97, 237. 11. Young, R.; Jahn, O.; Cooper, W. C.; Smoot, J. J. HortScience, 1970, 5, 268. 12. Young, R.; Jahn, O.; Smoot, J. J. Proc. Fla. State Hortic. Soc., 1974, 87, 24. 13. Barmore, C. R.; Brown, G. E. Plant Dis. Rep., 1978, 62, 541. 14. Fishler, M.; Monselise, S. P. Israel J. Agric. Res., 1971, 21, 67. 15. Coggins, C. W., Jr. Acta Hortic., 1969, 34, 469. 16. Coggins, C. W., Jr. Proc. First Int. Citrus Symp., 1968, 3, 1177. 17. El-Zeftawi, Β. M.; Peggie, I. D. Food Technol. Aust., 1973, 25, 359. 18. Monselise, S. P. Acta Hortic., 1973, 34, 457. 19. Monselise, S. P. Proc. Int. Soc. Citriculture, 1977, 2, 664. 20. Monselise, S. P.; Sasson, A. Proc. Int. Soc. Citriculture, 1977, 1, 232. 21. Lewis, L. N.; Coggins, C. W., Jr.; Labananskas, C. K.; Dugger, W. M., Jr. Plant Cell Physiol., 1967, 8, 151. 22. Monselise, S. P.; Goren, R. Phyton, 1965, 22, 61. 23. Bevington, Κ. B. Aust. JL Exp. Agric. Anim. Husb., 1973, 13, 196. 24. Coggins, C. W., Jr.; Eaks, I. L. Calif. Citrogr., 1967, 52, 475. 25. Coggins, C. W., Jr.; Hield, H. Z.; Eaks, I. L.; Lewis, L. N.; Burns, R. M. Calif. Citrogr., 1965, 50, 457. 26. Coggins, C. W., Jr.; Jones, W. W. Proc. Int. Soc. Citri­ culture, 1977, 2, 686. 27. Coggins, C. W., Jr.; Scora, R. W.; Lewis, L. N.; Knapp, J. C. F. J. Agric. Food Chem., 1969, 17, 807.

In Citrus Nutrition and Quality; Nagy, S., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

10.

BROWN

Fruit

Handling

and

Decay

Control

215

28. El-Zeftawi, Β. M. J. Aust. Inst. Agric. Sci., 1971, 37, 151. 29. Erickson, L. C.; Richards, S. J. Proc. Am. Soc. Hortic. Sci. 1955, 65, 109. 30. Krezdorn, A. H.; Ali Dinar, H. M.; Rose, A. J. Proc. Fla. State Hortic. Soc., 1976, 89, 4. 31. Monselise, S. P. First World Congr. of Citriculture (Spain), 1973, 2, 393. 32. Stewart, W. S.; Hield, H. Z.; Brannaman, B. L. Hilgardia, 1952, 21, 301. 33. Gilfillan, I. M.; Koekemoer, W.; Stevenson, J. First World Congr. of Citriculture (Spain), 1973, 3, 355. 34. Gilfillan, I. M.; Stevenson, J. A. Citrus Subtrop. Fruit J., 1976, 507, 5. 35. Coggins, C. W., Jr.; Hield, H. Z.; Garber, M. J. Proc. Am. Soc. Hortic. Sci.., 1960, 76, 193. 36. Coggins, C. W., Jr.; Lewis L N Plant Physiol. 1962 37 625. 37. Embleton, Τ. Α.; Jones, ; Coggins, , Soc. Hortic. Sci., 1973, 98, 281. 38. Rodrigues, J.; Subramanyam, H. J. Sci. Food Agric., 1966, 17, 425. 39. Coggins, C. W., Jr.; Hield, H. Z.; Boswell, S. B. Proc. Am. Soc. Hortic. Sci., 1960, 76, 199. 40. Soost, R. K.; Burnett, P. H. Proc. Am. Soc. Hortic. Sci., 1961, 77, 194. 41. Coggins, C. W., Jr.; Eaks, I. L.; Hield, Η. Z.; Jones, W. W. Proc. Am. Soc. Hortic. Sci., 1963, 82, 154, 42. Eaks, I. L. Proc. First Int. Citrus Symp., 1968, 3, 1343. 43. Eaks, I. L.; Jones, W. W. Calif. Citrogr., 1959, 44, 390. 44. Coggins, C. W., Jr.; Lewis, L. N. Proc. Am. Soc. Hortic. Sci., 1965, 86, 272. 45. Riehl, L. Α.; Coggins, C. W., Jr.; Carman, G. E. Calif. Citrogr., 1965, 51, 2. 46. Riehl, L. Α.; Coggins, C. W., Jr.; Carman, G. E. J. Econ. Entomol., 1966, 59, 615. 47. Erner, Y.; Goren, R.; Monselise, S. P. J. Am. Soc. Hortic. Sci., 1976, 101, 513. 48. Leggo, D. Agric Gaz. N.S.W., 1968, 79, 112. 49. Monselise, S. P.; Weiser, M.; Shafir, N.; Goren, R.; Goldschmidt, Ε. E. J. Hortic. Sci., 1976, 51, 341. 50. Ismail, Μ. Α.; Grierson, W. HortScience, 1977, 12, 118. 51. Albrigo, L. G.; Brown, G. E.; Fellers, P. J. Proc. Fla. State Hortic. Soc., 1970, 83, 263. 52. Albrigo, L. G.; Brown, G. E. First World Congr. Citriculture (Spain), 1973, 3, 361. 53. Hopkins, E. F.; Loucks, K. W. Proc. Fla. State Hortic. Soc., 1944, 57, 80. 54. Pelser, P. Du T. S. Afr. Citrus Subtrop. Fruit J., 1973, 475, 5. 55. PFTseri P. Du T. S. Afr. Citrus Subtrop. Fruit J., 1977,519, 5.

In Citrus Nutrition and Quality; Nagy, S., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

216

CITRUS NUTRITION AND QUALITY

56. Sites, J. W.; Reitz, H. J. Proc. Am. Soc. Hortic. Sci., 1949, 54, 1. 57. Sites, J. W.; Reitz, H. J. Proc. Am. Soc. Hortic. Sci., 1950, 55, 73. 58. Sites, J. W.; Reitz, H. J. Proc. Am. Soc. Hortic. Sci., 1951, 56, 103. 59. Christ, R. A. S. Afr. Citrus J., 1966, 387, 7. 60. McCornack, A. A. Proc. Fla. State Hortic. Soc., 1973, 86, 284. 61. Smoot, J. J.; Melvin, C. F. Proc. Fla. State Hortic. Soc., 1961, 74, 285. 62. Smoot, J. J.; Melvin, C. F. Proc. Fla. State Hortic. Soc., 1975, 88, 276. 63. McCornack, A. A. Proc. Fla. State Hortic. Soc., 1966, 79, 258. 64. Rivero, L. G.; Grierson W.; Soule J J Am Soc Hortic Sci., 1979, 104, 551 65. Fawcett, H. S. Calif Agric Exp , , 66. Cahoon, G. Α.; Grove, B. L.; Eaks, I. L. Proc. Am. Soc. Hortic. Sci., 1964, 84, 188. 67. Eaks, I. L. Proc. Am. Soc. Hortic. Sci., 1955, 66, 141. 68. McCornack, A. A. Proc. Fla. State Hortic. Soc., 1970, 83, 267. 69. Miller, Ε. V.; Winston, J. R. Citrus Ind., 1943, 24, 7. 70. Wardowski, W. F.; McCornack, Α. Α.; Grierson, W. Fla. Coop. Ext. Serv. Circ. 410, 1976, p. 6. 71. Levy, Y.; Greenberg, J . ; Ben-Anat, S. Sci. Hortic., 1979, 11, 61. 72. Grierson, W.; Brown, G. E. Citrus Ind., 1966, 47, 8. 73. Grierson, W.; Koo, R. C. J. Citrus Veg. Mag., 1958, 21, 8. 74. Hopkins, E. F.; McCornack, A. A. Proc. Fla. State Hortic. Soc., 1960, 73, 263. 75. McCornack, A. A. Proc. Fla. State Hortic. Soc., 1972, 85, 232. 76. McCornack, Α. Α.; Grierson, W. Fla. Coop. Ext. Serv. Circ. 286, 1965, 3 p. 77. Albrigo, L. G. J. Am. Soc. Hortic. Sci., 1972, 97, 220. 78. Grierson, W.; Pantastico, Ε. B. Proc. Trop. Reg., Am. Soc. Hortic. Sci., 1968, 11, 1. 79. Hatton, T. T., Hr.; Reeder, W. F. Citrus Ind., 1967, 48, 23. 80. Hatton, T. T., Jr.; Reeder, W. F. Proc. Fla. State Hortic. Soc., 1968, 81, 344. 81. Pantastico, Ε. B.; Grierson, W.; Soule, J. Proc. Fla. State Hortic. Soc., 1966, 79, 388. 82. Cunha, G. A.; Davenport, T. L.; Soule, J.; Campbell, C. W. J. Am. Soc. Hortic. Sci., 1978, 103, 622. 83. Davenport, T. L.; Campbell, C. W. HortScience, 1977,12,246. 84. Davenport, T. L.; Campbell, C. W. J. Am. Soc. Hortic. Sci., 1977, 102, 484.

In Citrus Nutrition and Quality; Nagy, S., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

10.

BROWN

Fruit

Handling

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Decay

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85. Davenport, T, L.; Campbell, C. W.; Orth, P. G. Proc. Fla. State Hortic. Soc., 1976, 89, 245. 86. Eaks, I. L. Proc. Am. Soc. Hortic Sci., 1964, 85, 245. 87. Brown, G. K.; Schertz, C. E. Trans. ASAE., 1967, 10, 577. 88. El-Zeftawi, Β. M.; Thornton, I. R.; Gould, I. V. HortScience, 1977, 52, 461. 89. Grierson, W. Proc. Fla. State Hortic. Soc., 1968, 81, 53. 90. Chesson, J. H. Trans. ASAE., 1972, 15, 31. 91. Rackham, R. L.; Grierson, W. HortScience, 1971, 6, 163. 92. Ben-Yehoshua, Α.; Sarig, Y.; Golcomb, A. First World Congr. Citriculture (Spain), 1973, 3, 647. 93. Bryan, W. L. Trans. Citrus. Eng. Conf. ASME, 1974, 20, 24. 94. Bryan, W. L. Proc. Int. Soc. Citriculture, 1977, 3, 763. 95. Coppock, G. E.; Grierson, W. Citrus Ind., 1970, 51, 6. 96. Wilson, W. C.; Holm, R. E.; Clark, R. K. Proc. Int. Soc. Citriculture, 1977 2, 404 97. Moshonas, M. G.; Shaw 1976, 41, 809. 98. Moshonas, M. G.; Shaw, P. E.; Sims, D. A. Proc. Int. Soc. Citriculture, 1977, 3, 802. 99. Moshonas, M. G.; Shaw, P. E. J. Agric. Food Chem., 1977, 25, 1151. 100. Grierson, W. Proc. Fla. State Hortic. Soc., 1958, 71, 166. 101. Eaks, I. L. Proc. Int. Soc. Citriculture, 1977, 1, 223. 102. Moshonas, M. G.; Shaw, P. E. Int. Flav. Food AddTt., 1977, July-Aug., 149. 103. Grierson, W.; Newhall, W. F. Proc. Am. Soc. Hortic. Sci., 1956, 67, 236. 104. Gaffney, J. J.; Jahn, O. L Proc. Fla. State Hortic. Soc., 1967, 80, 296. 105. Jahn, O. L.; Yost, G. E.; Soule, J. U.S. Dep. Agric., Agric. Res. Ser. Rep. 51-14, 1967, p. 14. 106. Cohen, E. Spec. Pub. 128, Div. Scientific Pub. Volcanic Ctr.: Bet Dagan, Israel, 1979; p. 72. 107. Gillespie, K.; Tugwell, B. L. Dep. Agric. S. Aust. Spec. Bull. No. 4.75, 1975, p.7. 108. Grierson, W.; Newhall, W. F. Fla. Agric. Exp. Stn. Bull. 620, 1960, p. 80. 109. Hall, E. G.; Leggo, D.; Seberry, J. A. Agric. Gaz. J. N.S.W., 1968, 79, 721. 110. Jahn, O. L. First World Congr. of Citriculture (Spain), 1973 3 291 111. Jorgensen, K. R. Queensl. Agric. J., 1977, 103, 85. 112. Wardowski, W. F.; McCornack, A. A. Fla. Coop. Ext. Serv. Circ. 389, 1973, p. 3. 113. Winston, J. R. U.S. Dep. Agric. Circ. 961, 1955, p. 13. 114. Cohen, E. J. Hortic. Sci., 1978, 53, 143. 115. Grierson, W.; Newhall, W. F. Proc. Fla. State Hortic. Soc., 1953, 66, 42.

In Citrus Nutrition and Quality; Nagy, S., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

218

CITRUS NUTRITION AND QUALITY

116. Jahn, O. L.; Chace, W. G., Jr.; Cubbedge, R. H. J. Am. Soc. Hortic. Sci., 1973, 98, 177. 117. Kitagawa, H.; Kawada, K.; Tarutani, T. Proc. Int. Soc. Citriculture, 1977, 1, 219. 118. Kitagawa, H.; Kawada, K.; Tarutani, T. J. Am. Soc. Hortic. Sci., 1978, 103, 113. 119. Cohen, E. Proc. Int. Soc. Citriculture, 1977, 1, 215. 120. Cohen, E. First Int. Congr. Citriculture (Spain), 1973, 3, 297. 121. Jahn, O. L.; Chace, W. G., Jr.; Cubbedge, R. H. J. Am. Soc. Hortic. Sci., 1969, 94, 123. 122. Zamorani, Α.; Russo, C.; Monaco, M. First World Congr. of Citriculture (Spain), 1973, 3, 303. 123. Grierson, W. Proc. Trop. Reg., Am. Soc. Hortic. Sci., 1974, 18, 66. 124. Fulton, H. R.; Stevens, H. E.; Wooten, J. F. Proc. Fla. State Hortic. Soc., 125. Grierson, W.; Newhall 1955, 65, 244. 126. Grierson, W.; Ting, S. V. Proc. Fla. State Hortic. Soc., 1960, 73, 284. 127. Loucks, K. W.; Hopkins, E. F. Phytopathol., 1946, 36, 750. 128. Cohen, E. J. Hortic. Sci., 1978, 53, 139. 129. McCornack, A. A. Proc. Fla. State Hortic. Soc., 1971, 84, 270. 130. Smoot, J. J.; Melvin, C. F. Proc. Fla. State Hortic. Soc., 1967, 80, 246. 131. Smoot, J. J.; Melvin, C. F.; Jahn, O. L. Plant Dis. Rep., 1971, 55, 149. 132. Brown, G. E. Phytopathol., 1975, 65, 404. 133. Brown, G. E.; Barmore, C. R. Phytopathol., 1977, 67, 120. 134. Brown, G. E. Phytopathol., 1978, 68, 700. 135. Hopkins, E. F.; Loucks, K. W. Fla. Agric. Exp. Stn. Bull. 450, 1948, p. 26. 136. Brown, G. E. Phytopathol., 1973, 63, 1104. 137. Jahn, O. L. J. Am. Soc. Hortic. Sci., 1975, 100, 586. 138. Smoot, J. J.; Melvin, C. F. Proc. Fla. State Hortic. Soc., 1972, 85, 235. 139. Hatton, T. T., Jr.; Cubbedge, R. H. HortScience, 1973, 8, 101. 140. Albrigo, L. G. First World Congr. of Citriculture (Spain), 1973, 3, 107. 141. Stewart, I.; Wheaton, T. A. Proc. Fla. State Hortic. Soc., 1971, 84, 264. 142. Stewart, I.; Wheaton, T. A. J. Agric. Food Chem., 1972, 20, 448. 143. Wheaton, Τ. Α.; Stewart, I. J. Am. Soc. Hortic. Sci., 1973, 98, 337 144. Young, R.; Jahn, O. J. Am. Soc. Hortic. Sci., 1972, 97, 258.

In Citrus Nutrition and Quality; Nagy, S., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

10.

BROWN

Fruit Handling

and

Decay

Control

219

145. Fuchs, Y.; Cohen, A. J. Am. Soc. Hortic. Sci., 1969, 94, 617. 146. Grierson, W.; Ismail, F. H.; Oberbacher, M. F. J. Am. Soc. Hortic. Sci., 1972, 97, 541. 147. Jahn, O. L. Am. Soc. Hortic. Sci., 1973, 98, 230. 148. Jahn, O. L. Proc. Fla. State Hortic. Soc., 1974, 87, 218. 149. Jahn, O. L. J. Am. Soc. Hortic. Sci., 1976, 101, 597. 150. Wardowski, W. F.; Barmore, C. R.; Smith, T. S.; Dubois, C.W. Proc. Fla. State Hortic. Soc., 1974, 87, 216. 151. Gertman, E.; Fuchs, Y. HortScience, 1975, 10, 231. 152. Gertman, E.; Fuchs, Y. Israel J. Agric. Res., 1973, 23, 25. 153. Oberbacher, M. F.; Grierson, W. Proc. Fla. State Hortic. Soc., 1960, 73, 236. 154. Barmore, C. R.; Wheaton, Τ. Α.; McCornack, A. A. Hort­ Science, 1976, 11, 588. 155. Barmore, C. R.; Wheaton, Τ. Α.; McCornack, A. A. Citrus Ind., 1976, 57, 38 156. Hayman, E. P.; Yokoyama Chem., 1977, 25, 1251. 157. Jahn, O. L.; Young, R. J. Am. Soc. Hortic. Sci., 1975, 100, 244. 158. Valadon, L. R. G.; Mummery, R. S. Phytochemistry, 1978, 17, 818. 159. Yokoyama, H.; Debenedict, C.; Coggins, C. W., Jr.; Henning, G. L. Phytochemistry, 1972, 11, 1721. 160. Coggins, C. W., Jr.; Hall, A. E. J. Am. Soc. Hortic. Sci., 1975, 100, 484. 161. Grierson, W.; Wardowski, W. F. In "Citrus Science and Technology"; Nagy, S.; Shaw, P. E.; Veldhuis, M. K. Eds.; Avi Publishing Co., Inc.: Westport, CT, 1977; 2, p.128-140. 162. Eckert, J. W. In "Fungicides: An Advanced Treatise"; Torgeson, D. C., Ed.; Academic Press: New York and London, 1967; 1, p. 287-378. 163. Eckert, J. W. World Rev. Pest Control, 1969, 8, 116. 164. Eckert, J. W. In "Antifungal Compounds"; Siegel, M. R.; Sisler, H. D., Eds.; Marcel Dekker, Inc.: New York, NY, 1977; 1, p. 269-352. 165. Eckert, J. W.; Sommer, N. F. Ann. Rev. Phytopathol., 1967, 5, 391. 166. Eckert, J. W. Outlook Agric., 1978, 9, 225. 167. Eckert, J. S.; Kolbezen, M. J. Proc. 6th British Insec­ ticide and Fungicide Conf., 1971, 3 683. 168. McCornack, Α. Α.; Wardowski, W. F.; Brown, G. E. Fla. Coop. Ext. Serv. Circ. 359A, 1976, 6 p. 169. Cresswell, G. A. S. Afr. Citrus J . , 1964, 361, 19. 170. Eckert, J. W.; Kolbezen, M. J.; Krainer, B. A. Proc. First Int. Citrus Symp., 1968, 2, 1097. 171. Hopkins, E. F.; Loucks, K. W. Science, 1950, 112, 720. 172. Razzman, Α.; Apelbaum, Α.; Heller, H. Pest. Sci., 1972, 3, 585. 9

In Citrus Nutrition and Quality; Nagy, S., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

220

CITRUS NUTRITION AND QUALITY

173. Brown, G. E. Proc. Int. Soc. Citriculture, 1977, 1, 283. 174. Eckert, J. W.; Kolbezen, M. J. Neth. J. Plant Pathol., Suppl., 1, 1977, 83, 343. 175. Eckert, J. W.; Kolbezen, M. J . ; Rahm, M. L.; Eckard, K. J. Phytopathol., 1979, 69, 934. 176. Eckert, J. W.; Kolbezen, M. J. First World Congr. of Citri­ culture (Spain), 1973, 3 559. 177. DeWolfe, T. Α.; Erickson, L. C.; Brannaman, B. L. Proc. Am. Soc. Hortic. Sci., 1968, 74, 367. 178. Schiffmann-Nadel, M. Proc. First Int. Citrus Symp., 1968, 3, 1295. 179. Schiffmann-Nadel, M.; Lattar, F. S.; Waks, J. HortScience, 1972, 7, 120. 180. Stewart, W. S. Proc. Am. Soc. Hortic. Sci., 1949, 54, 109. 181. Stewart, W. S.; Palmer, J. E.; Hield, Η. Z. Proc. Am. Soc. Hortic. Sci., 1952 59 327 182. Weyer, F. P. S. Afr 183. El-Nabawy, S.; El-Hammady Gazyawy, A. Proc. Int. Soc. Citriculture, 1977, 3, 1135. 184. Fahmy, Β. Α.; Rizk, S. S.; Khalil, R. I. Agric. Res. Rev., 1972, 50, 83. 185. Blunden, G.; Jones, E. M.; Passam, H. C.; Metcalf, E. Trop. Agric., 1979, 56, 311. 186. Harding, P. R., Jr.; Savage, D. C. Plant Dis. Rep., 1964, 48, 808. 187. Melvin, C. F.; Smoot, J. J. Proc. Fla. State Hortic. Soc., 1963, 76, 322. 188. Smoot, J. J . ; Melvin, C. F. Plant Dis. Rep., 1965, 49, 463. 189. Klotz, L. J . ; De Wolfe, T. A. Plant Dis. Rep., 1961, 45, 264. 190. Barkai-Golan, R.; Kahan, R. S. Plant Dis. Rep., 1966, 50, 874. 191. Guerrero, F. P.; Maxie, E. C.; Johnson, C. F.; Eaks, I. L.; Sommer, N. F. Proc. Am. Soc. Hortic. Sci., 1967, 90, 515. 192. Grierson, W.; Dennison, R. A. Proc. Fla. State Hortic, Soc., 1965, 78, 233. 193. Mahmood, T. Plant Dis. Rep., 1972, 56, 582. 194. Maxie, E. C.; Sommer, N. F.; Eaks, I. L. First Int. Citrus Symp., 1968, 3, 1375. 195. Maxie, E. C.; Eaks, I. L.; Sommer, N. F. Rad. Bot., 1964, 4, 405. 196. Maxie, E. C.; Eaks, I. L.; Sommer, N. F.; Rae, H. L.; El-Batel, S. Plant Physiol., 1965, 40, 407. 197. Ben-Yehoshua, S. Israel J. Agric. Res., 1967, 17, 17. 198. Ben-Yehoshua, S.; Garber, M. J . ; Huszar, C. K. Trop. Agric., 1970, 47, 151. 199. Oavis, P. L.; Chace, W. G., Jr.; Cubbedge, R. H. Hort­ Science, 1967, 2, 168. 200. Eaks, I. L.; Ludi, W, A. Proc Am. Soc. Hortic. Sci., 1960, 76, 220.

In Citrus Nutrition and Quality; Nagy, S., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

10.

BROWN

Fruit

Handling

and

Decay Control

221

201. Vines, H. M.; Grierson, W.; Edwards, G. J. Proc. Am. Soc. Hortic. Sci., 1968, 92, 227. 202. Davis, P. L. Proc. Fla. State Hortic. Soc., 1970, 83, 294. 203. Davis, P. L.; Hofmann, R. C. J. Agric. Food Chem., 1973, 21, 455. 204. Long, W. G. Fla. Agric. Exp. Stn. Bull. 681, 1964, p. 38. 205. Davis, P. L.; Harding, P. L. J. Am. Soc. Hortic. Sci., 1960, 75, 271. 206. Grierson, W. Proc. Trop. Reg., Am. Soc. Hortic. Sci., 1971, 15, 76. 207. Chace, W. G., Jr.; Davis, P. L.; Smoot, J. J. XII Int. Cong. Refrig. (Madrid), 1967, 3, 383. 208. Seberry, J. Α.; Leggo, D.; Keily, T. B. Aust. J. Exp. Agric. Anim. Husb., 1967, 7, 593. 209. Hatton, T. T., Jr.; Reeder, W. F. Proc. Trop. Reg., Am. Soc. Hortic. Sci. 1968 11, 23 210. Aharoni, Y.; (Littauer Science, 1973, 8, 211. Alumot, E.; Chalutz, E. Pest Sci., 1972, 3, 539. 212. Chalutz, E.; Schiffmann-Nadel, M.; Waks, J . ; Alumot, E.; Carmi, Y.; Bussel, J. J. Am. Soc. Hortic. Sci. 1971, 96, 782. 213. Grierson, W.; Hayward, F. W. Proc. Fla. State Hortic. Soc., 1959, 73, 267. 214. Grierson, W.; Miller, W. M.; Wardowski, W. F.; Ismail, M. A. Proc. Fla. State Hortic. Soc., 1976, 89, 172. 215. Leggo, D.; Gellatley, J. G.; Seberry, J. Α.; Peggie, I. D.; Long, J. K.; Hall, E. G. Agric. Gaz. N.S.W., 1965, 76, 274. 216. Lindgren, D. L.; Sinclair, W. B. J. Econ. Entomol., 1951, 44, 980. 217. Norman, G. Α.; Grierson, W.; Wheaton, Τ. Α.; Dennis, J. D. Proc. Fla. State Hortic. Soc., 1975, 88, 323. 218. Swaine, G., Corcoran, R. J.; Cranny, A. E.; Davey, M. A. Pest Sci., 1978, 9, 22. 219. Sinclair, W. B.; Lindgreen, D. L.; Forbes, R. J. Econ. Entomol., 1962, 55, 236. 220. Bussel, J . ; Kamburov, S. S. J. Am. Soc. Hortic. Sci., 1976, 101, 11. 221. Eaks, I. L.; Ludi, W. A. Proc. Am. Soc. Hortic. Sci., 1958, 72, 297. 222. Chalutz, E.; Biron, S.; Alumot, E. Int. Inst. Refrig. Commission C2, (Jersualem) 1973, 205. 223. Winston, J. R.; Roberts, C. L. Proc. Fla. State Hortic. Soc., 1944, 57, 140. 224. Iwamato, M.; Hayakawa, Α.; Kawano, S.; Manago, M. J. Soc. Agric. Mach (Japan), 1977, 38, 539. 225. Eaks, I. L. Proc. Am. Soc. Hortic. Sci., 1960, 78, 190. 226. Yamashita, Y.; Kitano, Y.; Hatta, S.; Wada, T.; Uda, H. J. Jpn. Soc. Hortic. Sci., 1979, 48, 231.

In Citrus Nutrition and Quality; Nagy, S., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

222

CITRUS NUTRITION AND QUALITY

227. Yamashita, S.; Wada, T.; Kitano, Y.; Hatta, S.; Uda. H. J. Jpn. Soc. Hortic. Sci., 1979, 48, 242. 228. Bruemmer, J. H. Proc. Fla. State Hortic. Soc., 1970, 83, 290. 229. Bruemmer, J. H.; Roe, B. Proc. Fla. State Hortic. Soc., 1969, 82, 212. 230. Maier, V. P.; Brewster, L. C.; Hsu, A. C. J. Agric. Food Chem., 1973, 21, 490. 231. Bartholomew, E. T.; Sinclair, W. B. "The Lemon Fruit". Its Composition, Physiology and Products"; Univ. Calif. Press: Berkeley, CA, 1951; p. 163. 232. Biale, J. B. In "The Orange, Its Biochemistry and Physi­ ology"; Sinclair, W. B. Ed; Univ. of Calif., Div. Agric. Sci.: Riverside, CA, 1961; p. 96-113. 233. Kefford, J. F.; Chandler, Β. V. "The Chemical Constituents of Citrus Fruits"; Advances in Food Research Suppl 2; Academic Press: Ne 234. Miller, Ε. V. Bot , , , 235. Miller, Ε. V. Bot. Rev., 1958, 24, 43. 236. Rose, D. H.; Cook, H. T.; Redit, W. H. USDA Bibliogr. Bull. No. 13, 1951, p. 178. 237. Sinclair, W. B. In "The Grapefruit, its Composition, Physi­ ology, and Products"; Sinclair, W. B., Ed.; Univ. of Calif., Div. of Agric. Sci.: Riverside, CA, 1972; p. 426-501. 238. Eaks, I. L. J. Food Sci., 1961, 26, 593. 239. Bratley, C. O. Proc.Am.Soc. Hortic. Sci., 1939, 37, 526. 240. El-Zeftawi, B. M. J. Hortic. Sci., 1976, 51, 411. 241. Isshak, Y. M.; Rizk, S. S.; Khalil, R. I.; Fahmi, B. A. Agric. Res. Rev. Mar., 1974, 52, 80. 242. Sasson, Α.; Monselise, S. P. J. Am. Soc. Hortic. Sci., 1977, 102, 331. 243. Davis, P. L.; Hofmann, R. C.; Hatton, T. T., Jr. HortScience, 1974, 9, 376. 244. Davis, P. L. Proc. Fla. State Hortic. Soc., 1971, 84, 217. 245. Caro, J.; Prestamo, G.; Calvo, J. M. Proc. Int. Soc. Citri­ culture, 1977, 3, 1120. 246. Pantastico, Ε. B.; Soule, J.; Grierson, W. Proc. Trop. Reg., Am. Soc. Hortic. Sci., 1968, 12, 171. 247. Hatton, T. T., Jr.; Cubbedge, R. H.; Grierson, W. Proc. Fla, State Hortic. Soc., 1975, 88, 335. 248. Hawkins, L. A. J. Agric. Res., 1921, 22, 263. 249. Hawkins, L. Α.; Barger, W. R. U.S. Dep. Agric. Tech. Bull. 1368, 1926, p. 7. 250. Chace, W. G., Jr.; Harding, P. L.; Smoot, J. J . ; Cubbedge, R. H. U.S. Dep. Agric. Mark. Res. Rep. 739, 1966, p. 21. 251. Schiffmann-Nadel, M.; Chalutz, E.; Waks, J . ; Dagan, M. J. Am. Soc. Hortic. Sci., 1975, 100, 270. 252. Brooks, C.; McColloch, L. P. J. Agric. Res., 1936, 52, 319. 253. Davis, P. L.; Hofmann, R. C. J. Food Sci., 1973, 38, 871.

In Citrus Nutrition and Quality; Nagy, S., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

10.

BROWN

Fruit

Handling

and

Decay

Control

223

254. Brooks, C.; Bratley, C. O.; McColloch, L. P. U.S. Dep. Agric. Tech. Bull. No. 519, 1936, p. 24. 255. Brooks, C.; McColloch, L. P. J. Agric. Res., 1937, 55, 795. 256. Hatton, T. T., Jr.; Smoot, J. J . ; Cubbedge, R. H. Proc. Trop. Reg., Am. Soc. Hortic. Sci., 1972, 16, 49. 257. Vakis, N.; Grierson, W.; Soule, J. Proc. Trop. Reg., Am. Soc. Hortic. Sci., 1970, 14, 89. 258. Wardowski, W. F.; Albrigo, L. G.; Grierson, W.; Barmore, C. R.; Wheaton, T. A. HortScience, 1975, 10, 381. 259. Kokkalos, T. I. HortScience, 1974, 9, 456. 260. Schiffmann-Nadel, M.; Chalutz, E.; Waks, J . ; Lattar, F. S. HortScience, 1972, 7 394. 261. McCornack, A. A. Proc. Fla. State Hortic. Soc.,1976,89,200. 262. Chalutz, E.; Schiffmann-Nadel, M.; Waks, J.; Lattar, F. S. J. Am. Soc. Hortic. Sci., 1974, 99, 368. 263. Grierson, W.;HaywardF W Proc Am Soc Hortic Sci. 1960, 76, 229. 264. Hayward, F. W. Proc , , , 302. 265. Eaks, I. L. Calif. Citrogr., 1955, 41, 68. 266. Grierson, W.; Hayward, F. W. Proc. Fla. State Hortic. Soc., 1958, 71, 205. 267. Chace, W. G., Jr. Proc. First Int. Citrus Symp., 1968, 3, 1365. 268. Chace, W. G., Jr.; Smoot, J. J.; Cubbedge, R. H. Citrus Ind., 1970, 51, 16. 269. Seberry, J. A.; Hall, E. G. Agric. Gaz. N.S.W., 1970, 81, 564. 270. Aharoni, Y.; Lattar, F. S. Phytopathol. Z., 1972, 73, 371. 271. Harding, P. R., Jr. Plant Dis. Rep., 1969, 53, 585. 272. Salama, S. B.; Grierson, W.; Oberbacher, M. F. Proc. Fla. State Hortic. Soc., 1965, 78, 353. 273. Carreres, R.; Tuset, J. J . ; Lloret, J. L. Proc. Int. Soc. Citriculture, 1977, 3, 1133. 274. Davis, P. L.; Roe, B.; Bruemmer, J. H. J. Food Sci., 1973, 38, 225. 275. Smoot, J. J. Proc. First Int. Citrus Symp., 1968, 3, 1285. 276. Oogaki, C.; Manago, M. Proc. Int. Soc. Citriculture, 1977, 3, 1127. 277. Houck, L. G.; Aharoni, Y.; Fouse, D. C. Proc. Fla. State Hortic. Soc., 1978, 91, 136. 278. Biale, J. B. Calif. Citrog., 1953, 38, 427. 279. Grierson, W.; Vines, H. M.; Oberbacher, M. F.; Ting, S. V.; Edwards, G. J. Proc. Am. Soc. Hortic. Sci., 1966, 88, 311. 280. McGlasson, W. B.; Eaks, I. L. HortScience, 1972, 7, 80. 281. Schiffmann-Nadel, M. Proc. Int. Soc. Citriculture, 1977, 1, 311. 282. Hall, E. G.; Scott, K. J.; Wild, B. L.; Rippon, L. E. First 9

World Congr. of Citriculture (Spain) 1973, 3, 347.

In Citrus Nutrition and Quality; Nagy, S., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

224

CITRUS NUTRITION AND QUALITY

283. Wild, B. L.; McGlasson, W. B.; Lee, T. H. HortScience, 1976, 11, 114. 284. Wild, B. L.; McGlasson, W. B.; Lee, T. H. Proc. Int. Soc. Citriculture, 1977, 1, 259. 285. Wild, B. L.; McGlasson, W. B.; Lee, T. H. Food Technol. Aust., 1977, 29, 351. 286. Spalding, D. H.; Reeder, W. F. Proc. Trop. Reg., Am. Soc. Hortic. Sci., 1974, 18, 128. 287. Burg, S. P.; Burg, E. A. Science, 1966, 153, 314. 288. Spalding, D. H.; Reeder, W. F. J. Am. Soc. Hortic. Sci., 1976, 101, 367. 289. Apelbaum, Α.; Barkai-Golan, R. Phytopathol., 1977, 67, 400. 290. Grierson, W. Proc. Fla. State Hortic. Soc., 1966, 79, 274. 291. Grierson, W. Proc. First Int. Citrus Symp., 1968, 3, 1389. 292. Grierson, W. Int. Inst. Refrig., Commission C2 (Jerusalem), 1973, 51. 293. Risse, L. Α.; Hatton 294. Smith, R. J. Proc 295. Grierson, W.; Wardowski, W. F. HortScience, 1978, 13, 570. 296. Hardenburg, R. E. HortScience, 1971, 6, 198. 297. Harding, P. R., Jr. Plant Dis. Rep., 1959, 43, 893. 298. Hayward, F. W.; Grierson, W.; Edwards, G. J. Proc. Fla. State Hortic. Soc., 1965, 78, 244. 299. Hayward, F. W.; Oberbacher, M. F.; Grierson, W. Proc. Fla. State Hortic. Soc., 1961, 74, 237. 300. Kaufman, J . ; Hardenburg, R. E.; Lutz, J. M. Proc. Am. Soc. Hortic. Sci., 1956, 67, 244. 301. Vines, H. M.; Oberbacher, M. F. Proc. Fla. State Hortic. Soc., 1961, 74, 243. 302. Rygg, G. L. Citrus Leaves, 1951, 32, 12. 303. Stahl, A. L.; Fifield, W. M. Fla. Agric Exp. Stn. Bull. 304, 1936, p. 78. 304. Wardowski, W. F.; Grierson, W.; Edwards, G. J. HortScience, 1973, 8, 173. 305. Ben-Yehoshua, S. Proc. Int. Soc. Citriculture, 1978, (In Press). 306. Ben-Yehoshua, S.; Kobiler, I.; Shapiro, B. J. Am. Soc. Hortic. Sci., 1979, 104, 868. 307. Kawada, K.; Albrigo, L. G. Proc. Fla. State Hortic. Soc., 1979, 92, (In Press). 308. Hale, P. W.; Smoot, J. J. Citrus Veg. Mag., 1973, 37, 20. 309. Smoot, J. J . ; Hale, P. W. Proc. Fla. State Hortic. Soc., 1977, 90, 152. 310. Hale, P. W.; Smoot, J. J . ; Miller, W. R. Proc. Fla. State Hortic. Soc., 1978, 91, 133. 311. Hale, P. W. Citrus Veg. Mag., 1976, 40, 12. RECEIVED May 13,

1980.

In Citrus Nutrition and Quality; Nagy, S., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

11 Citrus Juice Processing as Related to Quality and Nutrition CHARLES VARSEL The Coca-Cola Company Foods Division, 7105 Katy Road, Houston, TX 77024

There is much tha of fresh fruits and vegetable the world, citrus is consumed primarily as the fresh fruit, but in the United States processed products are consumed as the major source of citrus in the diet. The main staple of processed citrus juices in the U.S. is frozen concentrated orange juice (FCOJ). Were it not for the processing of citrus fruits, this rich source of nutritious food, in the forms of juices and drinks, would be available to us for only limited periods of time throughout the course of any year. Processing techniques practiced today in the citrus industry ensure the availability of a continuous supply of citrus juices and their allied products to people in a l l regions of the United States and, indeed, in many parts of the world. Our increased knowledge of nutrients in the food supply and how they are affected by processing has led to an increased awareness on the p a r t of processors about the n u t r i t i o n a l aspects and q u a l i t i e s of t h e i r products, and f o r a greater d e s i r e to improve p r o c e s s i n g techniques so that the consumer can derive maximum b e n e f i t s from those n u t r i e n t s . There has been an increased r e c o g n i t i o n i n the food i n d u s t r y that we have some r e s p o n s i b i l i t y for the n u t r i t i o n a l q u a l i t y of our food supply. This awareness of r e s p o n s i b i l i t y has led to increased safeguards i n processing so that not only the n u t r i t i o n a l q u a l i t y , but also the f l a v o r a c c e p t a b i l i t y i s b e t t e r r e t a i n e d i n the processing of n a t u r a l foods. The increased awareness on the part of consumers about nut r i t i o n has l e d to an increased demand f o r c i t r u s j u i c e s and p r o d u c t s , a demand that i s greater today than i t has ever been. This has l e d to a tremendous growth w i t h i n the c i t r u s i n d u s t r y , and developing nations of the world that have climates s u i t a b l e f o r the production of c i t r u s f r u i t s have b e n e f i t t e d tremendously from t h i s consumer demand. B r a z i l i s a prime example. The growth of the c i t r u s i n d u s t r y i n B r a z i l has been a great economic f a c t o r i n

0-8412-0595-7/80/47-143-225$l 1.75/0 © 1980 American Chemical Society In Citrus Nutrition and Quality; Nagy, S., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

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the w e l f a r e of i t s people. B r a z i l today i s a major f a c t o r i n the worldwide supply of c i t r u s products and i s second only to the U.S. i n the p r o d u c t i o n of c i t r u s . T h i s i s evidenced i n Table I, which presents data on c i t r u s f r u i t production by s p e c i f i e d c o u n t r i e s (j.) . The o v e r a l l growth of the c i t r u s i n d u s t r y i s , o f course, f o s t e r e d by improved economic c o n d i t i o n s i n many count r i e s and e v o l v i n g technologies that permit the production, storage, and shipment of c i t r u s products over long d i s t a n c e s . R e f r i g e r a t i o n , new d i s t r i b u t i o n methods, and new packaging techniques represent developments without which many people i n the world could not enjoy the f u l l f l a v o r and n u t r i t i o n of c i t r u s products. The unique and d i s t i n c t i v e f l a v o r s o f the c i t r u s f r u i t s and the general a c c e p t a b i l i t y of these f l a v o r s by peoples throughout the world have a l s o been f a c t o r s c o n t r i b u t i n g to the growth of the c i t r u s i n d u s t r y . Orange f l a v o r i s probably the most widely recognized an erage i n d u s t r y worldwide and aroma i t i s used to f l a v o r many foods and beverages and to aromatize many household products. G r a p e f r u i t i s l e s s popular than the orange but perhaps more popular than the lemon r e l a t i v e t o consumption of the j u i c e . P o p u l a r i t y of g r a p e f r u i t j u i c e i s i n c r e a s i n g i n many p a r t s of the world, p a r t i c u l a r l y i n the United States and i n Japan. How much i t s c h i m e r i c a l image as a d i e t e t i c food has c o n t r i b u t e d tc t h i s i n c r e a s i n g p o p u l a r i t y i s not known, but i t undoubtedly has been a f a c t o r . C h i l l e d and b o t t l e d g r a p e f r u i t j u i c e s are growing i n p o p u l a r i t y whereas the usage of frozen concentrated g r a p e f r u i t j u i c e continues to grow, but at a s l i g h t l y slower r a t e . Lemon j u i c e has many uses i n the food i n d u s t r y that other j u i c e s do not have because of i t s uniquely d i f f e r e n t composition i n r e l a t i o n to other j u i c e s , except perhaps lime. Large q u a n t i t i e s of lemon j u i c e are used to enhance food f l a v o r s and to develop and balance the f l a v o r s of many food items, seafood being an outstanding example. P o s s i b l y only sugar and s a l t are used more e x t e n s i v e l y i n the development and enhancement of food f l a v o r s . Lemon j u i c e has a l s o gained i n p o p u l a r i t y because o f t e c h n o l o g i c a l advances that now permit the manufacture of concentrated j u i c e s and the p r o d u c t i o n of a frozen concentrate f o r lemonade. The f l a v o r of lemon, c o n t r i b u t e d by the p e e l o i l , i s probably second only to orange f l a v o r i n o v e r a l l p o p u l a r i t y . The growth i n market f c r the powdered s o f t d r i n k mixes and the f r u i t d r i n k mixes, p a r t i c u l a r l y f o r lemon-flavored products, has i n creased the demand f o r lemon o i l . Added to t h i s i s the i n c r e a s ing demand f o r lemon o i l s f o r use i n the carbonated and noncarbonated s o f t d r i n k s that are i n c r e a s i n g i n p o p u l a r i t y worldwide.

In Citrus Nutrition and Quality; Nagy, S., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

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TABLE I Citrus Fruit: P r o d u c t i o n , by Selected C o u n t r i e s , of P r i n c i p a l Types, Crop Years 1976-77 through 1918-19(1)

Country

i

1 1976-77

j

Crop Years 1977-78

1978-79 2J

1,000 M e t r i c Tons 11 Orange s and Tangerines United S t a t e s Brazil Japan Spain Italy Morocco Israel Argentina Mexico Egypt Turkey Greece South A f r i c a Australia Cyprus Chile TOTAL

10,144 6,087 3,575

9,256 8,205 4,119

8,725 7,256 3,663

784 968 990 1,283 840 671 533 463 384 100 45

1,055 949 925 750 747 735 455 591 393 109 47

992 975 926 783 780 780 629 583 395 113 48

31,591

32,792

30,820

Lemons United States Italy Brazil Argentina Turkey Spain Greece TOTAL

1/ 2J

896 792 371 320 278 220 190

900 800 363 280 280 313 194

758 600 367 267 250 239 175

3,067

3,130

2,656

One m e t r i c ton i s equivalent to 2204.6 pounds P r e l i m i n ary. The Florida Crop and Livestock Reporting Service

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TABLE I I

U.S.

Crop Y e a r

P r o d u c t i o n o f Oranges Y e a r s 1 9 7 3 - 7 8 ÇL)

Utilizatio 1000 M e t r i c Tons

Fresh

Processed

Processed

Total

1973-74

1613

6900

8514

81.0

1974-75

1951

7339

9290

79.0

1975-76

1803

7717

9519

81.1

1976-77

1680

7886

9567

82.4

1977-78

1598

7059

8657

81.5

1978-79*

1451

6854

8306

82.5

1683

7292

8976

81.2

AVERAGE

^Estimate The Florida Crop and Livestock Reporting Service

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Processing

Processing

T e c h n o l o g i c a l developments i n high vacuum evaporation techniques have been r e s p o n s i b l e f o r the r a p i d growth o f the domestic c i t r u s i n d u s t r y . These techniques were developed and r e f i n e d , f o r the most p a r t , during World War I I and they made p o s s i b l e the manufacture and production o f many p e r i s h a b l e foods and medicines. Most n o t a b l e f o r the domestic c i t r u s i n d u s t r y was the development of f r o z e n concentrated c i t r u s j u i c e s which was made p o s s i b l e by the development of these high vacuum evaporators Frozen concentrated orange j u i c e began to capture a r e a l segment of the c i t r u s market i n 1948, and s i n c e then, i t s presence has been a dominant c o n t r i b u t i n g f a c t o r to the i n c r e a s i n g per c a p i t a consumption of c i t r u s j u i c e s worldwide. Processed orange products accounted f o r the usage of about 81% of the domestic orange crop between the years 1973 and 1978 as can be seen i n Table I I p e r i o d was by f a r the majo which i s concentrated i n 4 s t a t e s ; F l o r i d a , C a l i f o r n i a , Texas, and A r i z o n a , w i t h F l o r i d a being the dominant f a c t o r i n the i n d u s t r y . About 94% of the F l o r i d a orange crop went i n t o the product i o n o f orange j u i c e products during the 6-year p e r i o d , 1973-1978, and f r o z e n concentrated orange j u i c e accounted f o r approximately 81% of that usage. About h a l f o f the orange crcp of Texas and about 40% o f the A r i z o n a crop were u t i l i z e d i n processed products, but only about o n e - t h i r d of the C a l i f o r n i a crop was so u t i l i z e d . The major p o r t i o n o f the l a t t e r crop went to the f r e s h f r u i t market. These data are summarized i n Table I I I . TABLE I I I U.S.

Production of Oranges by Region 6-Year Average 1973-1978 (1)

1000 State Florida California Texas Arizona TOTAL

M e t r i c Tons

Processed

T o t a l Production

6614 500 129 49 7292

7069 1550 235 122 8976

% Processed 93.6 32.3 54.9 40.2 81.2

The Florida Crop and Livestock Reporting Service

S i m i l a r trends to those noted above e x i s t i n the domestic

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230

TABLE IV

U.S. Production of G r a p e f r u i t Years 1973-1978 (1)

Crop Year

U t i l i z a t i o n of Production 100

Fresh

Processed

% Tons

Total

1973-74

1023

1416

2439

58.1

1974-75

1038

1231

2270

54.2

1975-76

1193

1390

2583

53.8

1976-77

1034

1716

2750

62.4

1977-78

1101

1646

2747

59.9

1978-79*

1038

1452

2490

58.3

1071

1475

2547

57.9

AVERAGE

*Estimate The Florida Crop and Livestock Reporting Service

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g r a p e f r u i t market, but that t o t a l market i s only about 30% as l a r g e as that f o r oranges as seen i n Table IV, Almost two-thirds of the F l o r i d a g r a p e f r u i t crop goes to the production of processed products with frozen concentrated g r a p e f r u i t j u i c e accounting f o r about 35% of the processed j u i c e . C h i l l e d g r a p e f r u i t j u i c e accounts f o r about 12% of the processed j u i c e , and that market segment i s growing along w i t h the b o t t l e d g r a p e f r u i t j u i c e market. Of the g r a p e f r u i t produced i n the other U.S. growing r e g i o n s , C a l i f o r n i a , Texas, and A r i z o n a , more than h a l f go to the f r e s h f r u i t markets. About 46% i s processed. These data are shown i n Table V. TABLE V U.S. Production of G r a p e f r u i t by Region 6-Year Average 1973-1978 (1)

1000 M e t r i c Tons State Florida California Texas Arizona TOTAL

Processed

Total

% Processed

1173 93 167 42

1894 200 375 78

61.9 46.5 44.5 53.8

1475

2547

57.9

The Florida Crop and Livestock Reporting Service

Table VI shows the average amounts of the F l o r i d a orange and g r a p e f r u i t crops that went i n t o processed products during the f i v e - y e a r p e r i o d from 1973-1977. Frozen concentrates accounted f o r the major p o r t i o n of the processed orange j u i c e and about one-third of the processed g r a p e f r u i t j u i c e . C h i l l e d orange j u i c e i n b o t t l e s and i n d a i r y cartons accounted f o r a s i g n i f i c a n t p o r t i o n of the processed F l o r i d a orange crop and t h i s i s presently the f a s t e s t growing segment of the market. C h i l l e d g r a p e f r u i t j u i c e i s a growing market, but b o t t l e d , s h e l f - s t a b l e g r a p e f r u i t j u i c e i s a l s o e x p e r i e n c i n g major growth at the present time. C h i l l e d g r a p e f r u i t j u i c e accounted f o r about one-eighth of the processed j u i c e ; but, canned and b o t t l e d g r a p e f r u i t j u i c e s accounted f o r a major p o r t i o n of the processed g r a p e f r u i t j u i c e as can be seen i n Table VI.

In Citrus Nutrition and Quality; Nagy, S., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

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232 TABLE VI

Processed F l o r i d a C i t r u s Products 6-Year Average 1973-1978 (JL)

Product Category

Gallons Produced (000 s)

Fruit Utilization (1000 M e t r i c Tons)

% Of Processed Oranges

5644 405

81.0

978 144

14.0

40,393 66,328 68 5,861

278 588

4.0

64 7,639 465

21 38

0.3

T

% Of Processed Grapefruit

Frozen Concentrated Juice (Reconst. B a s i s ) Orange Grapefruit Tangerine Blended

Chilled

684,419 40,912 4,700 28

34.5

Juice

Orange* Grapefruit

125,517 16,244 (est.)

12.3

Canned J u i c e Orange* Grapefruit Tangerine Blended

50.0

F r u i t Sections Orange Grapefruit Blended

3.2

*Includes Temples, Tangelos, and Honey Tangerines The Florida Crop and Livestock Reporting Service

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Juice Extraction

Citrus f r u i t i s delivered to a processing plant i n truckload q u a n t i t i e s of 20,410 t o 21,430kg or 20.4 to 21.4 m e t r i c tons. The f r u i t i s unloaded, inspected f o r maturity and graded to remove unwholesome and damaged f r u i t , a f t e r which i t i s conveyed to f r u i t b i n s f o r storage. F r u i t from the b i n s i s washed w i t h a detergent i n a r o t a r y brush washer, r i n s e d , then inspected and graded a second time to remove unwholesome f r u i t . J u i c e e x t r a c t o r s d i f f e r i n design, but a l l a r e f a s t , rugged, easy t o c l e a n , and a d j u s t a b l e to accomodate f r u i t o f d i f f e r e n t sizes. P r i o r to the i n v e n t i o n of automatic e x t r a c t o r s , the r o t a r y j u i c e press was i n common use, and i s s t i l l used commercially i n many p a r t s of the world, p r i n c i p a l l y I t a l y , Spain, & South America. The FMC In-Line E x t r a c t o r i s widely used i n the domestic i n dustry, most p a r t i c u l a r l i Florida becaus i t effect s i multaneous recovery o f tor can process from 32 s i s t s of a bottom cup, i n t o which the f r u i t i s f e d , and an upper cup that meshes w i t h the bottom as c i r c u l a r plugs are cut from the top and bottom o f the f r u i t . The f r u i t i n the bottom cup i s compressed as the upper cup descends and j u i c e and other f r u i t components are f o r c e d through the bottom plug i n t o a s t r a i n e r tube. The contents o f the s t r a i n e r tube, rag, seeds, and c e l l sacs, are squeezed between the top and bottom plugs r e s u l t i n g i n almost complete e x t r a c t i o n o f j u i c e and, i n essence, a f i r s t - f i n i s h i n g o p e r a t i o n s i n c e t h e plug (seeds, pulp, and peel) i s separated from t h e j u i c e . As the f r u i t i s squeezed i n the cup, p e e l o i l expressed from the flavedo and s m a l l p i e c e s of p e e l are washed i n t o a conveyer by a water spray that surrounds the e x t r a c t o r cup. The v a l u a b l e o i l i s recovered from the o i l / w a t e r slurry. S e v e r a l types o f Brown e x t r a c t o r s a r e used i n the c i t r u s i n dustry throughout the world. The Model 400 produces a j u i c e that i s low i n p e e l o i l content and h i g h i n j u i c e q u a l i t y . The f r u i t i s halved and the j u i c e removed by a r o t a t i n g reamer that exerts pressure t o e f f e c t e x t r a c t i o n . The Brown Model 700 E x t r a c t o r operates i n a manner s i m i l a r to the Model 400 and produces j u i c e o f the same h i g h q u a l i t y with low o i l content. I t expresses the j u i c e from about 700 f r u i t / m i n . compared t o the 350 f r u i t / m i n . that can be processed by the Model 400. A more s o p h i s t i c a t e d e x t r a c t o r , the Brown Model 1100, accepts three p a r a l l e l l i n e s of s i n g l e - f i l e f r u i t , and has a p r o c e s s i n g c a p a c i t y of almost 11 metric tons of f r u i t per hour. I t produces maximum j u i c e y i e l d s . F r u i t e n t e r i n g the e x t r a c t o r i s halved by a s t a i n l e s s - s t e e l k n i f e and each h a l f passes between a s t a i n l e s s s t e e l g r i d and a r o t a t i n g d i s c . The j u i c e i s expressed i n two stages e q u i v a l e n t to a l i g h t e x t r a c t i o n (low pulp/low o i l ) and a hard e x t r a c t i o n (higher o i l / h i g h e r pulp), t h e n flows t o the bottom of the c o l l e c t o r where i t can be d i v i d e d i n t o two f r a c t i o n s .

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The s t a i n l e s s - s t e e l g r i d provides a coarse f i r s t - s t a g e f i n i s h i n g o p e r a t i o n simultaneously w i t h the e x t r a c t i o n . The j u i c e flows through o u t l e t s at the bottom of the c o l l e c t o r s and i s conveyed to f i n i s h e r s . 1.2

Finishing

Operations

In the f i n i s h i n g o p e r a t i o n , seeds are removed from the j u i c e and the pulp content i s lowered. As w i t h e x t r a c t o r s , f i n i s h e r s vary i n design. The two types most commonly used are the screwtype and the paddle-type. In both designs, s e p a r a t i o n i s accomp l i s h e d by a c y l i n d r i c a l p e r f o r a t e d screen. J u i c e and a cont r o l l e d amount of i n s o l u b l e s o l i d s or f i n e pulp pass through the screen w h i l e the remainder of the s o l i d s i s discharged at the end of the f i n i s h e r . The s i z e of the screen p e r f o r a t i o n s d e t e r mines the s i z e of the s o l i d p a r t i c l e s that remain with the j u i c e The j u i c e e x t r a c t o r and the c h a r a c t e r i s t i c s of th the j u i c e y i e l d and q u a l i t y . The j u i c e c h a r a c t e r i s t i c s controlled by these operations i n c l u d e the pulp content and s i z e , and o i l content. The f i n i s h e d j u i c e i s conveyed to blend tanks at which time the a c i d i t y and s o l u b l e s o l i d s l e v e l may be determined. I f necessary, the j u i c e can be deaerated and d e o i l e d , dependent upon the product to be produced. 1.3

Evaporation

According to Cook ( 2 ) , the f i r s t commercial orange concent r a t e was produced i n F l o r i d a i n 1938 on a low-temperature (20°25°C) evaporator w i t h 13 stages that operated under h i g h vacuum. Most of the evaporators used i n the s t a t e of F l o r i d a p r i o r to 1947 u t i l i z e d h i g h temperatures and long residence times. These evaporators operated somewhere between 48.9° and 82.2°C (120°-180°F). F r u i t s o l i d s remained i n these evaporators f o r a minimum of 30 minutes; hence, the products produced on such evaporators were of poor q u a l i t y and e x h i b i t e d a s t r o n g heat processed f l a v o r . In 1946, the f i r s t commercial f r o z e n concentrated orange j u i c e was produced i n a f a l l i n g - f i l m type evaporator operated at low temperature and high vacuum. T h i s evaporator, i n s t a l l e d by the Minute Maid Company (now The Coca-Cola Company Foods Division) employed a l a r g e steam j e t pumping system to remove water vapor at h i g h enough vacuum to maintain an o p e r a t i n g temperature of about 18.3°C (65°F). The j u i c e stayed i n the evaporator f o r a long p e r i o d of time, but the concentrate produced was f a r superior to t h a t produced i n high-temperature evaporators. Low-temperature evaporators of the f a l l i n g - f i l m type were h e a v i l y u t i l i z e d i n the c i t r u s i n d u s t r y through the 1950 s and f

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and e a r l y 6 0 s . The f i r s t high-temperature short-time evaporator was i n s t a l l e d i n F l o r i d a i n 1959. T h i s was a d o u b l e - e f f e c t twostage u n i t that employed some r e c i r c u l a t i o n of steam. T h i s r e search i n t o evaporation p r i n c i p l e s l e d to the development of the present u n i t s that are used almost e x c l u s i v e l y today i n the domestic c i t r u s i n d u s t r y . These u n i t s are known as TASTE (thermallya c c e l e r a t e d short-time evaporation) evaporators. Most of these u n i t s employ a s i n g l e pass of j u i c e , and they are composed of four to s i x e f f e c t s w i t h s i x or seven s t a g e s . The water removal c a p a c i t y i s normally 18,000 to 23,000 l i t e r s per hour, but some of the new u n i t s being c o n s t r u c t e d have water removal c a p a c i t i e s of n e a r l y 41,000 l i t e r s / h o u r . F i g u r e 1 i l l u s t r a t e s the o p e r a t i n g p r i n c i p l e s of a TASTEtype evaporator (2). J u i c e passes through the preheaters and i s heated to the temperature of s t a b i l i z a t i o n , about 205°-210°F (96.l°-98.9°C), to destroy enzyme a c t i v i t y . After stabilization, the j u i c e passes throug stage where i t f l a s h e s t sponds to the pressure at that p o i n t . The r e s u l t i n g mixture of j u i c e and vapor i s p r o j e c t e d i n t o the tube bundle i n the f i r s t stage where f u r t h e r evaporation of water occurs as the j u i c e passes down through the tubes. T h i s i s not a f a l l i n g - f i l m evaporator because the l i q u i d ( j u i c e ) i s mostly suspended i n the vapor, and heat t r a n s f e r i s to the turbulent mixture. The e x i t v e l o c i t y of the j u i c e from the tubes i s on the order of 20 to 100 meters per second (2). As i t e x i t s from the tubes, the l i q u i d and vapor mixture t r a v e l s i n t o a c e n t r i f u g a l - t y p e vapor s e p a r a t o r , and the l i q u i d then flows down the s u c t i o n l i n e to a heavy-duty pump. On b e i n g pumped to the next stage, the l i q u i d f l a s h e s through the n o z z l e and the mixture t r a v e l s down the tube bundle as i t d i d i n the f i r s t stage. Vapor from the f i r s t - e f f e c t s e p a r a t o r prov i d e s the heat f o r evaporation i n the second stage. The j u i c e passes through a d d i t i o n a l stages, normally about seven i n a l l , and a f t e r the l a s t stage, the j u i c e enters a chamber where i t i s f l a s h cooled to about 10°C (50°F). A f t e r f l a s h c o o l ing, the concentrate, which w i l l be at 65°-68°Brix, i s pumped i n to drums or to a h o l d i n g tank i n one of the newly constructed tank farms that are becoming more p r e v a l e n t i n the domestic citrus industry. In a f o u r - e f f e c t evaporator steam i s put i n t o the f i r s t e f f e c t , where an e f f e c t r e l a t e s to vapor flow, and the heat from t h a t steam i s used four times b e f o r e condensation occurs i n a barometric condenser. In theory, a f o u r - e f f e c t evaporator w i l l remove four l i t e r s of water per k i l o g r a m of steam usage, but i n a c t u a l p r a c t i c e that water removal i s about 3.4 1 per kg of steam. Heat losses and the change i n the heat of v a p o r i z a t i o n w i t h tempe r a t u r e account f o r the d i f f e r e n c e . A t y p i c a l TASTE evaporator i n use i n a c i t r u s p l a n t i s shown i n F i g u r e 2. The Coca-Cola Company Foods D i v i s i o n operates three

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Technologist

Figure 1.

Figure 2.

Flow diagram for TASTE

TASTE

evaporator (2)

evaporator (courtesy of George Craddock)

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c i t r u s p r o c e s s i n g p l a n t s i n the s t a t e of F l o r i d a , and i n these p l a n t s are e i g h t TASTE evaporators dedicated to the p r o d u c t i o n of orange j u i c e concentrate. A d d i t i o n a l evaporators of the same type are a l s o used f o r by-product p r o d u c t i o n , e.g., c i t r u s molasses and washed pulp s o l i d s . The e i g h t evaporators used f o r the p r o d u c t i o n of orange j u i c e concentrate have a t o t a l r a t e d c a p a c i t y f o r water removal of 170,000 1 per hour. The l a r g e s t of these evaporators i s r a t e d at 36,400 1 per hour of water removal. Berry and Veldhuis (3) r e c e n t l y presented a comprehensive treatment of evaporators, and the reader i s r e f e r r e d to that art i c l e f o r a more in-depth review. 1.4

Ion-Exchange Processing-

A c i d removal from c i t r u s j u i c e s was f i r s t reported by K i l burn and Drager (4) i n the e a r l y 1960 s. They employed e l e c t r o d i a l y s i s to remove c i t r a t and Wagner (5) used calciu in citrus juices. A more recent process, i . e . , a c i d r e d u c t i o n by a n i o n i c ion exchange, was developed at the Research and Development Laborat o r i e s of The Coca-Cola Company Foods D i v i s i o n i n Plymouth, F l o r ida. Removal of c i t r a t e i o n by an a n i o n i c ion-exchange process can be accomplished by exchange w i t h hydroxy1 i o n and the subsequent formation of water, which i s a component of j u i c e and which can be removed by evaporation; hence, i t should be pref e r a b l e to a method that r e l i e s on the a d d i t i o n of a n e u t r a l i z i n g substance to a c i t r u s j u i c e . The ion-exchange process i s i l l u s t r a t e d i n the f o l l o w i n g equation: T

3

+

3

3 R+ ' OH" + C H 5 0 7 ~ ^ ( R ) · C H 0 - + 30H", where R equates to a p o s i t i v e l y charged u n i t of the r e s i n s t r u c t u r e . 6

3

6

5

7

+

The ion-exchange r e s i n employed i n the a c i d r e d u c t i o n process i s weakly b a s i c and i s approved f o r food use as p r e s c r i b e d i n the food a d d i t i v e r e g u l a t i o n 173.25(a)(14) i n T i t l e 21, Code of Fede r a l Regulations (21CFR) (6). Because the a n i o n i c r e s i n i s weakly b a s i c , the r e t e n t i o n of stronger acids i s favored. As a r e s u l t , when p r o c e s s i n g orange j u i c e , the r e t e n t i o n of c i t r i c a c i d i s favored with r e s p e c t to the weaker organic a c i d s , a s c o r b i c and f o l i c , which a r e w e l l recognized n u t r i e n t s i n orange j u i c e . A l s o , mass a c t i o n favors the removal of c i t r i c a c i d . The process permits the treatment of e i t h e r b u l k concentrate or f r e s h l y e x t r a c t e d j u i c e . Freshly extracted j u i c e i s f i r s t s t a b i l i z e d at 175°-180°F (79.4°-82.2°C) then c e n t r i f u g e d i n a h i g h speed c e n t r i f u g e to e f f e c t a pulp r e d u c t i o n to 2% to i n h i b i t development of e x c e s s i v e back pressures i n the column due to plug-

In Citrus Nutrition and Quality; Nagy, S., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

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ging. T h i s pulp can be re-added to the j u i c e stream f o l l o w i n g a c i d r e d u c t i o n i n the ion-exchange column. Bulk concentrate must f i r s t be d i l u t e d to about 15°Brix a f t e r which pulp r e d u c t i o n i s accomplished by c e n t r i f u g a t i o n . S t a b i l i z a t i o n of the d i l u t e concentrate i s not necessary because of i t s p r i o r s t a b i l i z a t i o n during concentration. A c i d r e d u c t i o n of orange j u i c e i s e f f e c t e d by downflow passage through the r e s i n . J u i c e i s passed through the column u n t i l the e l u a t e (reduced-acid j u i c e ) drops te a pH below 4.6 as monitored by a pH meter. T h i s method assures minimal l o s s of ascorbic acid. The column e l u a t e i s discharged i n t o an evaporator feed tank where i t s pH i s adjusted to a maximum of 4.6 through the a d d i t i o n of f r e s h l y e x t r a c t e d , but u n t r e a t e d , j u i c e or concentrated orange juice. At t h i s time the pulp removed i n c e n t r i f u g a t i o n can a l s o be re-added. T h i s adjustment of pH ensures that no growth of pathogenic organisms ca a t o r i e s have confirmed F o l l o w i n g pH adjustment of the acid-reduced j u i c e , i t i s concentrated to 65°Brix i n a TASTE evaporator. 1.5

P a s t e u r i z a t i o n and Packaging

The purpose of p a s t e u r i z a t i o n , as i t i s p r a c t i c e d i n the domestic i n d u s t r y today, i s to destroy s p o i l a g e organisms, i n a c t i vate enzymes, or both. Heating to temperatures of only 150°F (65.6°C) w i l l destroy most s p o i l a g e organisms but some heat r e s i s t a n t molds may r e q u i r e p a s t e u r i z a t i o n temperatures as h i g h as 210°F (98.9°C) f o r c o n t r o l . C i t r u s j u i c e s that are p a s t e u r i z e d at the lower temperatures, 65-66°C, can undergo c l a r i f i c a t i o n , i . e . , a process of s e p a r a t i o n that r e s u l t s i n a lower l a y e r of l i q u i d and sediment and an upper l a y e r of c l e a r l i q u i d . T h i s process i s brought about by the n a t u r a l enzyme, p e c t i n e s t e r a s e , that occurs i n c i t r u s f r u i t s . Studies have shown that p r o c e s s i n g of the j u i c e at temperatures of 170-210°F (76.7-99°C) f o r a f r a c t i o n of a second to 40 seconds w i l l destroy the p e c t i n e s t e r a s e a c t i v i t y i n c i t r u s j u i c e s (7-10). The temperature necessary to s t a b i l i z e the j u i c e i s pH dependent. J u i c e s at higher pH r e q u i r e h i g h e r temperatures f o r s t a b i l i z a t i o n . With the new high-temperature short-time techniques and equipment, s t a b i l i z a t i o n can u s u a l l y be e f f e c t e d i n a f r a c t i o n of a second. F l a s h p a s t e u r i z a t i o n can be accomplished i n e i t h e r a p l a t e - t y p e or a tube-type heat exchanger. C h i l l e d j u i c e s , both orange and g r a p e f r u i t , are i n c r e a s i n g i n p o p u l a r i t y and, indeed, t h i s market segment i s p r e s e n t l y the f a s t e s t growing i n the i n d u s t r y . These products are p a s t e u r i z e d , cooled, and f i l l e d i n t o paper cartons l i n e d w i t h a p l a s t i c or a l u minum f o i l laminated w i t h a p l a s t i c . The c h i l l e d j u i c e market experienced much of i t s i n i t i a l growth through d a i r y p r o c e s s i n g and d e l i v e r y systems, but today

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much o f the product i s processed i n p l a n t s owned by companies i n v o l v e d i n the c i t r u s i n d u s t r y , even though the technology employed i s based on that developed f o r the d a i r y i n d u s t r y . The products are p a s t e u r i z e d by the HTST (high-temperature s h o r t time) process i n which the j u i c e i s heated to a high temperature, on the order of 175°-180°F (79.4°-82.2°C) f o r a very short time, about 0.5 second. I t i s then cooled and f i l l e d i n t o cartons a t about 32°-40°F (0°-4.4°C). Such a process i s not damaging to the f l a v o r and t e x t u r e of the j u i c e and the r e s u l t i n g product has a very acceptable f l a v o r and aroma. C h i l l e d products, which have s h e l f l i v e s i n the order of f i v e to s i x weeks, a r e s o l d a t r e f r i g e r a t e d temperatures (4.4°7.2°C)in r e t a i l o u t l e t s . Open code d a t i n g of these products en-*sures a supply o f f r e s h product f o r the consumer at r e t a i l . Some c h i l l e d products a r e packaged i n g l a s s c o n t a i n e r s , 32f l . oz. and 6 4 - f l . oz., but the d a i r y - t y p e cartons account f o r the major segment o f t h i that t h e q u a l i t y of thes of time i f maintained a t 50°F (10°C) o r lower, and products i n g l a s s e x h i b i t e d b e t t e r a s c o r b i c a c i d and f l a v o r s t a b i l i t y than those i n paper o r p l a s t i c cartons. Higher temperatures l e d t o more,rapid d e t e r i o r a t i o n o f a s c o r b i c a c i d and f l a v o r . Canned and b o t t l e d j u i c e s a r e p a s t e u r i z e d at r e l a t i v e l y h i g h temperatures (76. 7°-90.6° C) and the containers a r e f i l l e d hot. The hot f i l l serves to s t e r i l i z e the c o n t a i n e r and, i n the case o f a can, i t i s i n v e r t e d f o r 60-90 seconds a f t e r seaming to s t e r i l i z e the l i d . The cans a r e then cooled to about 105°F (40.6°C) i n a s p i n c o o l e r o r a spray c o o l e r before b e i n g l a b e l e d and cased. B o t t l e s are f i l l e d i n the same manner, but the caps are s t e r i l i z e d w i t h steam or a chemical s t e r i l a n t b e f o r e being app l i e d t o the b o t t l e . The f i l l e d containers are then cooled gradu a l l y i n a spray c o o l e r . When f r e s h l y e x t r a c t e d j u i c e i s b e i n g f i l l e d i n t o cans o r b o t t l e s , the temperature of p a s t e u r i z a t i o n must be s u f f i c i e n t t o i n a c t i v a t e the n a t u r a l enzymes, p a r t i c u l a r l y p e c t i n e s t e r a s e . F o r orange j u i c e , t h i s temperature i s somewhere between 185° and 195°F (85°-90.6°C) and i s , to a degree, dependent upon the pH of the juice. G r a p e f r u i t j u i c e , g e n e r a l l y , need not be subjected to temperatures as high as a r e necessary to orange j u i c e i n order to achieve s t a b i l i z a t i o n . The temperatures r e q u i r e d f o r g r a p e f r u i t j u i c e are between 170°-189°F (76.7°-87.2°C). J o s l y n and Sedky (7) showed that the heat i n a c t i v a t i o n o f enzymes r e s p o n s i b l e f o r cloud i n s t a b i l i t y i n g r a p e f r u i t j u i c e was more r a p i d a t pH 2.5 than a t pH 4.0. Rouse and A t k i n s (9) and P r a t t and Powers (12) corroborated these f i n d i n g s . Of course, w i t h any j u i c e the i n a c t i v a t i o n of enzymes i s dependent upon both time and temperature. As the temperature of p a s t e u r i z a t i o n i s i n c r e a s e d , the length of time i n the p a s t e u r i z e r can be decreased.

In Citrus Nutrition and Quality; Nagy, S., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

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240 1.5.1

A s e p t i c Packaging

Canned and b o t t l e d c i t r u s j u i c e s are examples of products that are packed a s e p t i c a l l y , and these processes have been used i n the i n d u s t r y f o r many years. One of the newer processes f o r a s e p t i c packaging employs a paperboard package that i s s t e r i l i z e d with hydrogen peroxide p r i o r to the form, f i l l , and s e a l operation. This process, developed by T e t r a Pak Ab of Lund, Sweden, i s i n use i n many p a r t s of the world, but i t has not yet been approved by the U.S. Food and Drug A d m i n i s t r a t i o n f o r domestic use. The new packaging system developed by T e t r a Pak i s known as the T e t r a B r i k ® , and i s g e n e r a l l y a v a i l a b l e i n 1-1, 200-ml, and 250-ml s i z e s . The system i s considered to be an a l t e r n a t i v e to metal and g l a s s c o n t a i n e r s . The packaging m a t e r i a l comes i n r o l l stock form and i s a 6-or 7-layer laminate. Polyethylene and aluminum f o i l o f f e r the major b a r r i e r p r o p e r t i e s to the package. The system enable with minimal heat i n p u t are s i m i l a r to those employed f o r the dairy-type paper cartons. The package i n t e g r i t y , when p r o p e r l y s t e r i l i z e d and maintained, i s such that m i c r o b i a l r e i n f e c t i o n i s i n h i b i t e d . Because oxygen can permeate the package along the l o n g i t u d i n a l and l a t e r a l seams, i t i s not t r u l y hermetic even though i t does provide a s e p s i s . One major advantage of the package i s that i t contains no headspace because the top s e a l i s a c t u a l l y formed through a column of s t e r i l e product. T h i s l a c k of headspace o f f e r s some p r o t e c t i o n , at l e a s t i n i t i a l l y , to oxygen-sensitive products. In many of the developing c o u n t r i e s of the world, the T e t r a B r i k ® system o f f e r s the only economical and p r a c t i c a l package f o r j u i c e s and j u i c e products ( a l s o m i l k ) . The major disadvantages of the T e t r a B r i k ® process are the slow l i n e speeds (70 u n i t s per minute) and the l i m i t e d mechanical and p h y s i c a l s t r e n g t h of the package. The l a t t e r makes c a r e f u l handling and adequate secondary packaging q u i t e e s s e n t i a l . 2

N u t r i t i o n a l Q u a l i t y of C i t r u s J u i c e s

2.1

Vitamin

C

C i t r u s f r u i t s have long been noted as e x c e l l e n t sources of a s c o r b i c a c i d (Vitamin C), which i s the most abundant v i t a m i n i n the c i t r u s f r u i t s . C i t r u s f r u i t s are a l s o q u i t e r i c h i n the mine r a l element, potassium, and are o f t e n recommended f o r p a t i e n t s who must use d i u r e t i c drugs. Healthy a d u l t s r e q u i r e 60mg per day of Vitamin C and about 2.5g per day of potassium (13) . A t k i n s et a l . (14) reported that most of the a s c o r b i c a c i d that occurs i n the orange i s present as a c o n s t i t u e n t of the peel. Based on the weight of whole f r u i t , the j u i c e contains about 25% of the t o t a l a s c o r b i c a c i d content. The j u i c e of the g r a p e f r u i t contains only about 17% of the t o t a l a s c o r b i c a c i d content on the same b a s i s .

In Citrus Nutrition and Quality; Nagy, S., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

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The a s c o r b i c a c i d content of the j u i c e of d i f f e r e n t c i t r u s f r u i t s v a r i e s c o n s i d e r a b l y , and the content w i l l vary with stage of f r u i t maturity, f r u i t v a r i e t y , and climate. Soil conditions and f e r t i l i z i n g p r a c t i c e s have only minimal e f f e c t s i f any at a l l (15). According to Ting and Attaway (16), oranges g e n e r a l l y cont a i n from 40-70mg/100ml of j u i c e , whereas g r a p e f r u i t , tangerine, and lemon j u i c e contain between 20 and 50mg/100ml of j u i c e . The a s c o r b i c a c i d c o n c e n t r a t i o n i s high i n immature f r u i t , and i t decreases as the f r u i t r i p e n and increase i n s i z e , according to Harding e t a l . (17). As w i t h oranges, g r a p e f r u i t e x h i b i t an i n v e r s e r e l a t i o n s h i p between a s c o r b i c a c i d content and m a t u r i t y . M e t c a l f e e t a l . (1_8) examined f i v e v a r i e t i e s of g r a p e f r u i t grown i n s i x l o c a t i o n s i n the Rio Grand V a l l e y of Texas and concluded that, although there were only small d i f f e r e n c e s among the v a r i e t i e s , there were great v a r i a t i o n s i n a s c o r b i c a c i d content of the f r u i t from any given tree. These workers a l s c o r b i c a c i d content du t i o n s i n a s c o r b i c a c i d content of g r a p e f r u i t from trees i n d i f f e r e n t areas, as w e l l as from trees i n the same grove. He c o r r e l a t e d the a s c o r b i c a c i d content with a c i d i t y and reported that i t increased w i t h a c i d i t y . A number of workers examined the e f f e c t of l i g h t exposure on a s c o r b i c a c i d content and the general conclusion i s that d i r e c t s u n l i g h t has a p o s i t i v e e f f e c t on i t s content; i . e . , exposure to d i r e c t s u n l i g h t tends to i n c r e a s e the a s c o r b i c a c i d content of fruit. Long et a l . (20) found that the a s c o r b i c a c i d content of g r a p e f r u i t was i n v e r s e l y r e l a t e d to t h e i r s i z e . In V a l e n c i a oranges, S i t e s and R e i t z (21) found a p o s i t i v e c o r r e l a t i o n between a s c o r b i c a c i d and the s o l u b l e s o l i d s of f r u i t from the same tree. As might w e l l be expected, other c i t r u s f r u i t s e x h i b i t the same type of seasonal d e c l i n e i n a s c o r b i c a c i d content of the j u i c e w i t h maturity. Harding and Sunday (22) reported that the a s c o r b i c a c i d content of tangerines may be 35mg/100ml of j u i c e i n the e a r l y season and as low as 10-15mg per 100ml i f the f r u i t i s allowed to overmature. 2.2

Other N u t r i e n t s of D i e t a r y

Significance

Other n u t r i e n t s i n orange j u i c e that are of d i e t a r y s i g n i f i cance, according to standards s e t by the U.S. Food and Drug Adm i n i s t r a t i o n , i . e . , they are present at a l e v e l of 10% or more of the U.S. RDA (Recommended D a i l y Allowance) per s e r v i n g , are f o l i c a c i d and thiamine (Vitamin B-^). The f a c t o r of s i g n i f i c a n c e (10% of the U.S. RDA per serving) i s s e t f o r t h i n 21CFR 101.9(c) (7)(v)(6). A s e r v i n g s i z e f o r orange j u i c e i s g e n e r a l l y regarded as s i x f l u i d ounces or 177ml.

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Ting (23) reported values f o r thiamine i n orange j u i c e between 0.75 and 0.85mcg per gram of j u i c e , and a survey of the l i t e r a t u r e i n d i c a t e s that other c i t r u s j u i c e s c o n t a i n l e s s e r amounts. Analyses of orange concentrate by an i n d u s t r i a l l a b o r a tory f o r our C i t r u s R&D L a b o r a t o r i e s r e s u l t e d i n a value of 75mcg per 100g of r e c o n s t i t u t e d orange j u i c e , i n good agreement with the values reported by T i n g (23). Adams (24) r e p o r t s 91mcg/100g f o r r e c o n s t i t u t e d orange j u i c e . In the compilation of Adams (24) , r e c o n s t i t u t e d g r a p e f r u i t j u i c e i s reported to d e l i v e r about 38mcg of thiamine per 100g and tangerine j u i c e ( r e c o n s t i t u t e d ) about 59mcg/100g. Per s e r v i n g of 177ml, these values would be 170, 70, and lOOmcg, respect i v e l y , f o r orange, g r a p e f r u i t , and tangerine. With a U.S. RDA of 1.5mg, the percentage of the U.S. RDA of thiamine d e l i v e r e d by these j u i c e s would be 11%, 5%, and 7%, r e s p e c t i v e l y . F o l i c a c i d , g e n e r i c a l l y d e s c r i b e d as f o l a c i n i s chemically known as pteroylmonoglutami that e x h i b i t f o l i c a c i ber of glutamic a c i d residues they c o n t a i n . These polyglutamates, as they are known, must be acted upon by the enzyme, conjugase, to r e l e a s e the f o l i c a c i d f o r metabolic a c t i v i t y . A deficiency of t h i s v i t a m i n leads to m e g a l o b l a s t i c anemia (25) . Most e v i dence p l a c e s the d a i l y requirement f o r f o l i c a c i d at about 50mcg per day of c r y s t a l l i n e f o l i c a c i d (26,27) ; however, the U.S. RDA f o r t o t a l food f o l a c i n i s set at 400mcg f o r the adolescent, f o r a d u l t males, and f o r non-pregnant, n o n - l a c t a t i n g females. The higher RDA i s s p e c i f i e d to allow f o r a b s o r p t i o n of only 25% of f o l i c a c i d a c t i v i t y i n a manner comparable to the c r y s t a l l i n e f o l i c a c i d and to allow f o r a wide range of a v a i l a b i l i t y of the polyglutamate form (13). E a r l y work p l a c e d the f o l a c i n content of orange j u i c e at between three and s i x micrograms/100ml (28). L a t e r , Hurdle et a l . (29) r e v i s e d t h i s to 20-45mcg/100g f o r orange products, and they a l s o reported that canned g r a p e f r u i t products contained about llmcg/lOOg. More recent work by S t r e i f f (30) i n d i c a t e d a f o l a c i n value o f from 50-100mcg with i n g e s t i o n of 100-125ml of orange juice. G r a p e f r u i t j u i c e and tangerine j u i c e were reported to have lower l e v e l s . Dong and Oace (31) reported a f o l a c i n value of 50mcg/100ml f o r orange j u i c e , and a somewhat lower l e v e r f o r grapefruit juice. Ting et a l . (32) reported an average f o l a c i n value f o r recons t i t u t e d F l o r i d a orange j u i c e of about 45mcg/100ml. In our own s t u d i e s , with analyses conducted by independent a n a l y t i c a l l a b o r a t o r i e s , we have not seen values of that magnitude, but r a t h e r have observed values on the order of 26-33mcg/100ml of j u i c e . These values would be on the order of 12%-15% of the U.S. RDA. T i n g (23) has a l s o reported that wide v a r i a t i o n i n the f o l i c a c i d content occurs throughout the growing season and that the concent r a t i o n i n c r e a s e s as the season progresses.

In Citrus Nutrition and Quality; Nagy, S., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

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Other N u t r i e n t s

A number of n u t r i e n t s of l e s s e r d i e t a r y s i g n i f i c a n c e are present i n orange j u i c e and other c i t r u s j u i c e s . Measurable l e v e l s of Vitamin A, r i b o f l a v i n (Vitamin I ^ ) , n i a c i n , p y r i d o x i n e ( V i t a min B 5 ) and pantothenic a c i d have been reported i n orange j u i c e . The l e v e l s of these n u t r i e n t s are g e n e r a l l y i n the range of 2-3% of t h e i r r e s p e c t i v e U.S. RDA's. For a more extensive review of these n u t r i e n t s , one should consult T i n g (23) and Araujo (33)· In a d d i t i o n to the vitamins mentioned above, c i t r u s j u i c e s are a r i c h source of potassium. Even though potassium i s an e s s e n t i a l m i n e r a l i n human n u t r i t i o n , the U.S. Food and Drug Adm i n i s t r a t i o n does not i n c l u d e i t i n i t s n u t r i t i o n a l l a b e l i n g program because i t i s w i d e l y d i s t r i b u t e d i n foods. The Food and N u t r i t i o n Board of the N a t i o n a l Academy of Sciences (13) has determined that healthy a d u l t s r e q u i r e about 2.5g of potassium per day. Based on the (177ml) of orange j u i c Values produced i n our own l a b o r a t o r i e s would approximate a potassium content of about 0.4g per 177ml of orange j u i c e . T i n g (23) r e p o r t s a potassium content of 0.30-0.4g per 177ml f o r orange j u i c e . Other m i n e r a l elements are present i n c i t r u s j u i c e s i n measurable q u a n t i t i e s . McHard et a l . (34) reported on the t r a c e element contents of F l o r i d a and B r a z i l i a n orange j u i c e . They c i t e d c o n c e n t r a t i o n ranges f o r 25 elements. T i n g (23) reported that calcium, i r o n , phosphorus, magnesium, z i n c , and copper are present i n r e c o n s t i t u t e d FCOJ at l e v e l s e q u i v a l e n t to about 1% to 5% of t h e i r r e s p e c t i v e U.S. RDA's. Phosphorus r e p o r t e d l y occurs i n orange j u i c e at l e v e l s of about 10-30mg/100g of j u i c e (24,34,35), e q u i v a l e n t to 1.9-5.6% of the U.S. RDA; magnesium was reported at l e v e l s between 8-15mg/100 ml of j u i c e by B i r d s a l l et a l . (36), whereas Ingwalson et a l . (37) reported l e v e l s i n r e c o n s t i t u t e d orange j u i c e of 12-14mg/100g. McHard et a l . (34) reported s i m i l a r v a l u e s . The maxiumum would be about 6.5% of the U.S. RDA per 177ml of orange j u i c e . Orange j u i c e was reported tc c o n t a i n from 50 to 160mcg of copper per 100ml by B i r d s a l l e t a l . (36) , whereas others reported values i n the range of 30-50mcg/100g (34,37), a maximum of 1.7% of the U.S. RDA, but p o s s i b l y as low as 0.3% of the U.S. RDA. Calcium has been reported at 6.5-15.4mg/100g of r e c o n s t i t u ted orange j u i c e (34,35,37). T h i s l e v e l , which i s 1.2% to 2.7% of the U.S. RDA, i s not of any great s i g n i f i c a n c e . Likewise, i r o n , which has been reported at l e v e l s of 0.08 to 0.7mg/100g of orange j u i c e (34,37) i s not of any great n u t r i t i o n a l s i g n i f i cance because the l e v e l i s only 0.8% to 7% of the U.S. RDA. 3

E f f e c t s of P r o c e s s i n g on N u t r i t i o n a l Q u a l i t y

P r o c e s s i n g as i t i s p r a c t i c e d i n the i n d u s t r y r e q u i r e s the input of heat to e f f e c t p a s t e u r i z a t i o n , enzyme s t a b i l i z a t i o n , and/

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or c o n c e n t r a t i o n . Heat p r o c e s s i n g to achieve one of these three r e s u l t s would not be expected to have any d e t r i m e n t a l e f f e c t on the mineral composition of c i t r u s j u i c e s . These m i c r o n u t r i e n t s should not be l o s t during p r o c e s s i n g ; n e i t h e r should they be degraded. The same cannot be s a i d f o r the organic m i c r o n u t r i e n t s , the v i t a m i n s , and the s o - c a l l e d macronutrients, carbohydrates, p r o t e i n s , and f a t s , which supply energy as w e l l as n u t r i t i o n to the human body. C i t r u s f r u i t s are not regarded as good n u t r i t i o n a l sources of f a t based on 21CFR 101.9(c)(6) that the d e l i v e r y of l e s s than one gram of f a t per s e r v i n g i s not of d i e t a r y s i g n i f i c a n c e (6). According to Adams (24) , r e c o n s t i t u t e d FCOJ d e l i v e r s about 0.2g of f a t per 177ml; r e c o n s t i t u t e d f r o z e n concentrated g r a p e f r u i t j u i c e about 0.2g/177ml. Tangerine j u i c e may be s l i g h t l y higher i n f a t content. According to Nagy (38), the l i p i d s that occur i n c i t r u s j u i c e c o n t a i n high unsaturated/saturated f a t t y a c i d r a t i o s (>4). The c o n t r i b u t i o n o development has been s t u d i e d by many workers, and a review of these s t u d i e s has been presented by Nagy (38). I t i s g e n e r a l l y agreed that the c o n t r i b u t i o n of the l i p i d o x i d a t i v e products to the f l a v o r d e t e r i o r a t i o n of processed c i t r u s products i s r e l a t i v e l y minor when compared to the c o n t r i b u t i o n s by the products formed by the a c i d - c a t a l y z e d h y d r o l y s i s of f l a v o r i n g o i l s and the products of M a i l l a r d browning (39,40). C i t r u s j u i c e s and t h e i r products cannot be considered s i g n i f i c a n t d i e t a r y sources of p r o t e i n because the p r o t e i n e f f i c i e n c y r a t i o (PER) of c i t r u s p r o t e i n i s l e s s than 20% that of c a s e i n (23, 41). According to the r e g u l a t i o n s set f o r t h i n 21CFR 101.9(c) ( 7 ) ( i i ) ( b ) , p r o t e i n w i t h a PER l e s s than 20% that of c a s e i n i s not of d i e t a r y s i g n i f i c a n c e (6). The p r o t e i n i n c i t r u s is"~generally a s s o c i a t e d w i t h the s o l i d p o r t i o n s of the f r u i t , i . e . , the seeds, f l a v e d o , albedo, chromatophores, and pulp. Some of these components f i n d t h e i r way i n t o the j u i c e along with the a v a i l a b l e f r e e amino acids during extract i o n and p r o c e s s i n g and storage. Studies conducted i n our l a b o r a t o r i e s (42,43,44) and by others (45) have shown that reductions i n the pulp content of j u i c e slow the rate of browning. According to Ting (23,41), a s e r v i n g (177ml) of reconstituted FCOJ d e l i v e r s about 19g of carbohydrate and 84 c a l o r i e s c o n t r i b u ted p r i m a r i l y by the sugars, sucrose, glucose, and f r u c t o s e . Adams (24) , i n d i c a t e s that a s e r v i n g of r e c o n s t i t u t e d FCOJ d e l i v e r s 92 c a l o r i e s , whereas g r a p e f r u i t and tangerine, j u i c e del i v e r 76 and 68 c a l o r i e s per 177ml, r e s p e c t i v e l y . C i t r u s j u i c e s c o n t a i n both nonreducing (sucrose) and reduc i n g ( f r u c t o s e and glucose) sugars. Mature oranges c o n t a i n almost equal amounts of the two types and the reducing sugar content i s composed of almost equal amounts of f r u c t o s e and glucose. Grapef r u i t tend to have n e a r l y e q u i v a l e n t amounts of reducing and non-

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reducing sugars, but at times, the reducing sugars tend to be s l i g h t l y more dominant (16). In tangerines, the nonreducing sugar dominates except i n immature f r u i t ; i n j u i c e from mature f r u i t , sucrose may account f o r 60-65% of the t o t a l sugar content. In lemon j u i c e , the reducing sugars dominate (46) and may account for about 90% of the t o t a l sugars. The sugars, which c o n t r i b u t e much to the a c c e p t a b i l i t y of c i t r u s j u i c e s , under adverse c o n d i t i o n s can play a major r o l e i n the formation of o f f f l a v o r s that reduce the a c c e p t a b i l i t y of the c i t r u s j u i c e s and t h e i r products. The sugars, p r i m a r i l y the hexoses, can p a r t i c i p a t e i n "browning" r e a c t i o n s that cause darkening of the j u i c e and these r e a c t i o n s g i v e r i s e to components that are d e s c r i b e d g e n e r a l l y as a p r i c o t - l i k e or p i n e a p p l e - l i k e i n flavor. In general, the more processed f l a v o r that a c i t r u s product e x h i b i t s , the l e s s acceptable i t becomes to the consumer. Some authors have i n d i c a t e d that the sugar-amino a c i d react i o n s of the M a i l l a r d typ j u i c e s because of the hig l a b o r a t o r i e s (42-44) would tend to i n d i c a t e that, to the contrary, the amino acids and sugars are of more than j u s t minor importance i n the darkening of c i t r u s j u i c e s . Huffman (42) t r e a t e d c i t r u s j u i c e s w i t h c a t i o n i c ion-exchange r e s i n s to remove amino a c i d s , p r o t e i n s , and the mineral c a t i o n s , then r e s t o r e d the c a t i o n s . The j u i c e s from which the f r e e amino a c i d s were removed were l e s s s u b j e c t to darkening and o f f - f l a v o r development than were t h e i r r e s p e c t i v e c o n t r o l s a f t e r heating f o r long periods of time to temperatures near 100°C, then s t o r i n g at room temperature. J u i c e s were a l s o dehydrated i n a vacuum s h e l f dryer and on a chain b e l t dryer with l e s s v i s i b l e darkening than c o n t r o l samples. The ion-exchange t r e a t e d j u i c e s were judged by sensory panels to be much more acceptable than untreated c o n t r o l s when presented as p a s t e u r i z e d j u i c e or as dehydrated j u i c e . Addit i o n a l s t u d i e s conducted i n our l a b o r a t o r i e s (43) corroborated the f i n d i n g s of Huffman. In a d d i t i o n , lowering the pulp content of j u i c e p r i o r to dehydration decreased the tendency f o r j u i c e to darken during the d r y i n g process. Seaver and Kertesz (47) reported that D-galacturonic and D-glucuronic a c i d s , when heated alone or i n the presence of amino a c i d s , formed c o l o r e d compounds at a r a t e exceeding that found with common sugars. They f u r t h e r r e ported that L - a s c o r b i c a c i d formed c o l o r e d compounds more r a p i d l y than the sugars, but s t i l l at a slower r a t e than the u r o n i c acids. C u r l (48), i n a study conducted with a s y n t h e t i c orange j u i c e , reported that the l o s s of a s c o r b i c a c i d occurred i n the presence of c i t r i c a c i d and potassium c i t r a t e b u f f e r alone, but that the l o s s e s were i n c r e a s e d by the a d d i t i o n of the sugars, lévulose, sucrose, and dextrose, i n that order. He found that darkening of the s y n t h e t i c j u i c e occurred p r i n c i p a l l y when both amino a c i d s and sugars were present; and, the e f f e c t was even more pronounced by the presence of a s c o r b i c a c i d . P r u t h i and L a i (49), i n a study of d i f f e r e n t methods f o r

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p r e s e r v i n g and s t o r i n g c i t r u s j u i c e s , reported that the a d d i t i o n of 5% cane sugar to the j u i c e s a c c e l e r a t e d the darkening and d i d not a i d i n a s c o r b i c a c i d r e t e n t i o n . Kato and Sakurai (50) studi e d the e f f e c t s of a s c o r b i c a c i d , organic a c i d s , amino a c i d s , and i n o r g a n i c ions on browning i n a model system. They determined that 3-deoxyglucosone and 5-hydroxymethylfurfural were formed by the a c t i o n o f organic a c i d on f r u c t o s e (formed by i n v e r s i o n of sucrose). Browning, they reported, was a f f e c t e d by organic acids, amino a c i d s , o x i d i z e d a s c o r b i c a c i d , and the i n o r g a n i c i o n s , S n \ F e 3 , S n , and A l . These i n v e s t i g a t o r s reported that 3-deoxyglucosone and 5-hydroxymethylfurfural were intermediates i n the browning of concentrated lemon j u i c e that occurred when the concentrate was d i l u t e d to s i n g l e - s t r e n g t h j u i c e with a sucrose s o l u t i o n . They concluded that amino acids had a r o l e i n the browning (51). +

+

+ 2

+ 3

Wolfrom e t a l . (52) s t u d i e d the nonenzymic browning of dehydrated orange j u i c e an of p a r t i c u l a r s i g n i f i c a n c The i n i t i a l phase of the browning r e a c t i o n l e d to a l o s s o f D-glucose and 4-aminobutyric a c i d . Kampen (53) s t o r e d f r e e z e d r i e d orange j u i c e c r y s t a l s and a s y n t h e t i c mixture f o r 40 days at 50°C and monitored l o s s e s of t o t a l amino acids (73%), a s c o r b i c a c i d (100%), c i t r i c a c i d (5.1%), and sucrose (4.4%). The orange j u i c e c r y s t a l s were d i s c o l o r e d from M a i l l a r d browning, and severa l carbonyl compounds and f u r f u r a l d e r i v a t i v e s were i d e n t i f i e d as products o f the r e a c t i o n s . Berry e t a l . (54) s t u d i e d foammat d r i e d i n s t a n t orange j u i c e s t o r e d a t 70°F and a t 85°F and reported that the s t a b i l i t y of the product was improved by the use o f more a c i d i c j u i c e s , but adding a c i d , or removing sugar. They reported an i n v e r s e r e l a t i o n s h i p between s t a b i l i t y of the i n s t a n t orange j u i c e and the pH of the orange concentrate dried. Although the importance o f the sugars i n the browning of c i t r u s j u i c e s has been debated by many i n v e s t i g a t o r s , v i r t u a l l y a l l agree on the importance of a s c o r b i c a c i d i n t h i s r e a c t i o n . J o s l y n (55) reported that a s c o r b i c a c i d was the most r e a c t i v e of the system, a s c o r b i c acid-amino acid-sugar, that occurs i n orange juice. Sugars, he reported, e x e r c i s e a p r o t e c t i v e e f f e c t on the enzymic and nonenzymic o x i d a t i o n of a s c o r b i c a c i d ; hence, both glucose and f r u c t o s e are i n h i b i t o r y to browning. The amino acids were reported t o have an i n h i b i t o r y e f f e c t i n the e a r l y stages o f the browning r e a c t i o n , but i n l a t e r stages, these components increased the r a t e o f browning. Moore et a l . (56) reported that the decomposition of ascorb i c a c i d i n orange j u i c e was d i r e c t l y a s s o c i a t e d w i t h darkening. According to C u r l (57), the development of o f f f l a v o r s i n orange j u i c e s a t 13-71% s o l u b l e s o l i d s was c l o s e l y p a r a l l e l e d by the l o s s of a s c o r b i c a c i d and by darkening. In mandarin j u i c e , Aiba et a l . (58) found t h a t the r a t e of browning was r e l a t e d to the decomposition of a s c o r b i c a c i d . Studies with the j u i c e of natsu-

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d a i d a i (a Chinese c i t r o n ) by Imai et a l . (59) and others a l s o i m p l i c a t e d a s c o r b i c a c i d as a major reactant r e s p o n s i b l e f o r browning. Imai e t a l . (59) reported that the f r e e amino a c i d s played an important r o l e i n the browning o f the j u i c e . Clegg (60) s t u d i e d the nonenzymic browning of lemon j u i c e and reported that the phenomenon was a t t r i b u t a b l e to a s c o r b i c a c i d r a t h e r than sugar-amino a c i d condensations. She reported that f u r f u r a l was produced during the. development of browning, but d i d not consider that i t played an important r o l e i n the a e r o b i c a l l y - p r o d u c e d browning. In model systems that simulated lemon j u i c e , she reported that amino a c i d s i n a s c o r b i c systems were major c o n t r i b u t o r s to browning (61). I t has been reported and i s g e n e r a l l y agreed that a s c o r b i c a c i d i n c i t r u s j u i c e s can be degraded through a e r o b i c and anaerob i c pathways (53,62). The a c i d i s q u i t e s e n s i t i v e t o o x i d a t i o n and dehydroascorbic a c i d i s the primary o x i d a t i o n product, though r e l a t i v e l y unstable. I i c a c i d . In a d d i t i o n t h y d r o x y f u r f u r a l have been i d e n t i f i e d as products of the degradat i o n of a s c o r b i c a c i d (60,63,64,65). Bauernfeind and P i n k e r t (66) proposed pathways f o r both the aerobic and anaerobic pathways i n aqueous media. In these pathways, shown i n F i g u r e 3, f u r f u r a l can r e s u l t from e i t h e r mode o f a s c o r b i c a c i d d e s t r u c t i o n while h y d r o x y f u r f u r a l i s a product of the o x i d a t i v e system. In processed c i t r u s products, a s c o r b i c a c i d l o s s can occur through aerobic or anaerobic mechanisms. The o x i d a t i o n of ascorb i c a c i d i n orange j u i c e was s t u d i e d by Evenden and Marsh (67) and was reported to be a f i r s t order r e a c t i o n whose r a t e was a f u n c t i o n o f temperature. In a very recent review, Nagy (65) reported that the degradation of a s c o r b i c a c i d was best explained by a f i r s t - o r d e r r e a c t i o n and that f o r g r a p e f r u i t j u i c e , the A r r henius p l o t showed a l i n e a r p r o f i l e f o r the temperature region 10-50°C. With orange j u i c e , h i s data suggested that two d i f f e r ent r e a c t i o n mechanisms were operative w i t h the k i n e t i c change o c c u r r i n g a t about 28°C. Between 10°and 27°C the r a t e of ascorb i c a c i d l o s s doubled f o r each 10°C r i s e ; from 27°to 37°C the r a t e quadrupled. The data of Nagy (65) a l s o confirmed e a r l i e r data by Ross (68) and Lamb (69) that i n d i c a t e d f o r s i m i l a r s t o r age temperatures the l o s s o f a s c o r b i c a c i d was greater f o r orange j u i c e than f o r g r a p e f r u i t j u i c e . 4

Processed C i t r u s Products

Of the 1978-79 domestic c i t r u s crop, some 6,855,000 metric tons o f oranges from a t o t a l o f 8,340,000 metric tons went to the production o f processed products. Of the 2,490,000 metric tons of g r a p e f r u i t that were harvested, 1,510,000 metric tons were u t i l i z e d i n processed products. A s i m i l a r p i c t u r e can be p a i n t e d f o r the other domestic c i t r u s crops. I t i s easy to see that the market f o r processed c i t r u s f r u i t i n the U.S. i s ex-

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248

ASCORBIC

ACID

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Figure 3.

Possible ascorbic acid degradation pathways

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tremely important to the c i t r u s i n d u s t r y . Almost 95% of the F l o r i d a orange crop i s u t i l i z e d by processors i n the production of j u i c e products. Since the d i e t s of domestic consumers contain processed products as t h e i r major source of c i t r u s , i t i s r e a sonable to look at these products and how they are a f f e c t e d nut r i t i o n a l l y by the p r o c e s s i n g techniques i n p r a c t i c e i n the industry. 4.1

Frozen Concentrated J u i c e s

Frozen concentrated orange j u i c e (FCOJ) i s by f a r the most widely d i s t r i b u t e d of the processed c i t r u s products. First marketed i n the mid-1940 s, i t has grown i n consumer acceptance u n t i l the present day, and to the p o i n t where i t s volume consumption exceeds the combined t o t a l f o r a l l other processed c i t r u s products. FCOJ and other froze duced by the process o u t l i n e the process i n c l u d e s e x t r a c t i o n , f i n i s h i n g , and b l e n d i n g . In the evaporator, the j u i c e may be concentrated to 45°Brix (% s o l u b l e s o l i d s ) or higher; and, as a matter of r o u t i n e p r a c t i c e , most of the evaporator pumpout (concentrate) i s at 65-68°Brix. The concentrate can go to low-temperature s t c r a g e or d i r e c t l y to p r o c e s s i n g f o r FCOJ. During the f r u i t p r o c e s s i n g season, cutback j u i c e may be used to d i l u t e the concentrate to 45°Brix. At other times, essence and water are used to prepare FCOJ. Berry and Veldhuis (3) reviewed t h i s process i n great d e t a i l . The l o s s of a s c o r b i c a c i d during e x t r a c t i o n , f i n i s h i n g , and b l e n d i n g i s minimal. Based on the data of Sale (70) and Hayes et a l . (71), the l o s s should be no g r e a t e r than 2%. Hayes et a l . (71) prepared orange j u i c e concentrates having 50-60% s o l i d s and reported a mean r e t e n t i o n of 96.6% of a s c o r b i c a c i d . T i n g et a l . (32) c o l l e c t e d samples of FCOJ from 23 manufact u r i n g p l a n t s i n F l o r i d a during 1973 and 1974 and analyzed them f o r s e l e c t e d n u t r i e n t s , those s p e c i f i e d by the U.S. Food and Drug A d m i n i s t r a t i o n as being e s s e n t i a l to human n u t r i t i o n . The average n u t r i e n t content of FCOJ r e c o n s t i t u t e d to 12.8°Brix expressed as percent of the U.S. RDA i s shown i n Table VII along with the U.S. RDA s as s p e c i f i e d by the Food and Drug A d m i n i s t r a t i o n (6). Based on these data, i t can be seen that FCOJ i s of d i e t a r y s i g n i f i c a n c e with respect to Vitamin C ( a s c o r b i c a c i d ) , f o l i c a c i d , and thiamine, i . e . , i t provides 10% or more of the r e s p e c t i v e U.S. RDA per 177ml s e r v i n g . Much has been w r i t t e n about the e f f e c t s of p r o c e s s i n g , tempe r a t u r e , and storage c o n d i t i o n s on the s t a b i l i t y of a s c o r b i c acid i n citrus juices. On the other hand, l i t t l e i s known about the s t a b i l i t y of f o l i c a c i d under s i m i l a r c o n d i t i o n s . Chen and Cooper (72) s t u d i e d the e f f e c t s of temperature and oxygen and a s c o r b i c a c i d on the thermal degradation of f o l i c a c i d , and they reported that a s c o r b i c a c i d i n c r e a s e d the s t a b i l i t y of the tetraf

T

In Citrus Nutrition and Quality; Nagy, S., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

250

CITRUS NUTRITION AND QUALITY

A * IT corns OUT next Figure 4.

^

L

Flow diagram for frozen concentrated orange juice production (courtesy Adney Reed)

In Citrus Nutrition and Quality; Nagy, S., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

11.

VARSEL

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Processing

TABLE V I I Average N u t r i e n t D e l i v e r y p e r Serving o f R e c o n s t i t u t e d FCOJ (12.8°Brix) i n R e l a t i o n to U.S. RDA 77^ U.S. KDk K±J

Nutrient Vitamin A

5000

IU

Vitamin C

60

mg

Average % U.S. RDA/ j(2) 1

7

7

m

l

F C O

1.4 131

Thiamine

1.5mg

9.8

Riboflavin

1.7mg

2.4

1.0g

1.8

Niacin Calcium

18

mg

1.1

2

mg

4.9

Folic Acid

0.4mg

20.3

Phosphorus

1.0g

3.3

Iron Vitamin B

6

400

mg

4.9

15

mg

0.7

2

mg

4.4

10

mg

3.3

Vitamin D

400

IU

Vitamin Ε

30

IU

Magnesium Zinc Copper Pantothenic A c i d

Vitamin

B^

Iodine

meg

150

meg

0. 3 mg

Biotin

Source:

6

1) 2)

U.S. Food and Drug A d m i n i s t r a t i o n ( 6 ) . Ting et a l . (32).

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hydro- and 5 - m e t h y l f o l i c a c i d at 1 0 0 ° C T h e i r data i n d i c a t e d t h a t the degradation of these f o l a t e s at high temperature was due to an o x i d a t i v e process that r e q u i r e d the presence of molecular oxygen. Floyd and Rogers (73) analyzed authentic samples of F l o r i d a orange j u i c e s and concentrates to determine the e f f e c t s of conc e n t r a t i o n on chemical composition. They reported no s i g n i f i c a n t e f f e c t s of concentration on the chemical composition of orange j u i c e concentrate as compared to s i n g l e - s t r e n g t h j u i c e . S e t t y et a l . (74) reported that the n u t r i e n t content of mandarin orange j u i c e when concentrated to 62°Brix and higher was very l i t t l e affected. Horton and Dickman (75) reported that the p h y s i o l o g i c a l l y a v a i l a b l e a s c o r b i c a c i d ( a s c o r b i c a c i d and dehydroascorbic acid) i n r e c o n s t i t u t e d orange j u i c e was s t a b l e over a two-week p e r i o d , both at 4°C and at room temperature. A e r a t i o n caused by blendori z i n g at high speed f o acid s t a b i l i t y . B i s s e t t and Berry (76) reported on the a s c o r b i c a c i d r e t e n t i o n i n orange j u i c e as a f u n c t i o n of container type. They stored FCOJ i n f o i l - l i n e d cardboard, r e c t a n g u l a r cartons and i n polyethylene ( P E ) - l i n e d f i b e r c y l i n d r i c a l cans f o r a year at -20.5°, -6.7°, and 1.1°C. At -20.5°C, the a s c o r b i c a c i d r e t e n t i o n was 93.5% i n the f o i l - l i n e d cartons and 91.5% i n the PEl i n e d cans. Neither c o n t a i n e r proved e f f e c t i v e above f r e e z i n g due to m i c r o b i a l s p o i l a g e . The f o i l - l i n e d carton was s u p e r i o r at 1.1°C, i n that 89% of the a s c o r b i c a c i d was r e t a i n e d a f t e r three months. In the P E - l i n e d can, the r e t e n t i o n was 44% a f t e r three months at 1.1°C. G a r c i a (77) s t u d i e d the e f f e c t of storage on 45°Brix and 54°Brix orange j u i c e concentrates packaged i n 6-oz. f o i l - l i n e d , spiral-wound cans and i n 2 0 0 - m l f o i l - l i n e d , r e c t a n g u l a r cartons ( T e t r a B r i k ® ). The l a t t e r were both c o l d f i l l e d and a s e p t i c a l l y f i l l e d v i a high-temperature short-time p a s t e u r i z a t i o n . These products were stored f o r one year at -17.8°, 7.2°, and 23.9°C. The a s e p t i c a l l y - p r o c e s s e d concentrates r e t a i n e d s t e r i l i t y throughout the course of the study and were s t o r e d at 7.2° and 23.9°C. The samples i n 6-oz. f o i l - l i n e d composite cans and those c o l d f i l l e d i n t o the 200-ml r e c t a n g u l a r packages were stored only at -17.8°C because they were not a s e p t i c a l l y packed and were subject to m i c r o b i a l s p o i l a g e . Figures 5 and 6 show the a s c o r b i c a c i d r e t e n t i o n i n the 200-ml packages as a f u n c t i o n of time. The data f o r the 6-oz. composite can are not shown because the a s c o r b i c a c i d r e t e n t i o n i n these packages was s i m i l a r to but j u s t m a r g i n a l l y poorer than was the r e t e n t i o n i n the 200-ml packages at -17.8°C. This marg i n a l d i f f e r e n c e was a t t r i b u t e d to the presence of some headspace oxygen i n the 6-oz. cans which r e s u l t e d i n the l o s s of s l i g h t l y more a s c o r b i c a c i d . At -17.8°C, the r e t e n t i o n of a s c o r b i c a c i d over 12 months

In Citrus Nutrition and Quality; Nagy, S., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

VARSEL

Citrus Juice

100 0,,

'±

g

η

ο

1.5

253

Processing

°-

4.5

3.C

6.0

7.5

12 0

9.0

MONTHS Figure 5. Ascorbic acid retention in 54° Brix concentrated orange juice as a function of storage temperature ((O) -17.8°C; Ο 7.2°C; (A) 23.9°C)

Τ

ΔΔ

—"—α......

Δ"- .

1.5

3.0

a..

^

4.5

6.0

7.5

9.0

10.5

12.0

MONTHS

Figure 6. Ascorbic acid retention in 45° Brix concentrated orange juice as a function of storage temperature ((O) -17.8°C; Ο 7.2°C; (A) 23.9°C)

In Citrus Nutrition and Quality; Nagy, S., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

254

CITRUS NUTRITION AND QUALITY

exceeded 97% i n the 200-ml packages and 94% i n the 6-oz. compos i t e cans. The a s c o r b i c a c i d r e t e n t i o n i n both the 45°Brix and 54°Brix concentrates s t o r e d at 7.2°C were very s i m i l a r over 12 months. The l o s s of a s c o r b i c a c i d amounted to about 8% to 10% over the storage p e r i o d . At 23.9°C, the 45°Brix concentrate i n i t i a l l y e x h i b i t e d a more r a p i d l o s s of a s c o r b i c a c i d than d i d the 54°Brix concentrate, but at the end of e i g h t months both had l o s t about 25% of t h e i r s t a r t i n g ascorbic acid. The more r a p i d l o s s of a s c o r b i c a c i d may have been caused by the amount of d i s s o l v e d oxygen i n the 45°Brix concentrate i n i t i a l l y . I t may have been the r e s u l t of more r a p i d d i f f u s i o n of oxygen i n t o the lower °Brix concentrate once i t entered the package along the l o n g i t u d i n a l seam and at the corners. N e i t h e r f a c t o r was measured. As might have been expected, f l a v o r d e t e r i o r a t i o n followed the same p a t t e r n as d i the concentrates at 23.9° more r a p i d l y than d i d those at 7.2°C; those at 7.2°C d e t e r i o r a t e d at a f a s t e r rate than d i d those s t o r e d f r o z e n . The products stored at 23.9°C remained acceptable i n f l a v o r f o r about s i x months; those at 7.2°C remained acceptable f o r e i g h t to ten months. At -17.8°C, the products were s t i l l acceptable a f t e r a year of storage. Packages of the type used i n t h i s study by G a r c i a are pres e n t l y used i n many p a r t s of the world f o r the packaging of m i l k , j u i c e s , and j u i c e products. B r i k Pak, Inc., a s u b s i d i a r y of T e t r a Pak Ab has p e t i t i o n e d the £ood and Drug A d m i n i s t r a t i o n for approval to use the T e t r a B r i k © package i n the U.S., but a f i n a l d e c i s i o n regarding t h e i r p e t i t i o n i s s t i l l pending. Frozen concentrated g r a p e f r u i t j u i c e i s produced i n essent i a l l y the same manner as FCOJ. P r i o r to evaporation, the j u i c e i s s t a b i l i z e d at 66° to 88°C to prevent g e l a t i o n and clarificat i o n during storage. As i s done i n the manufacture of FCOJ, coldpressed g r a p e f r u i t p e e l o i l i s added to the g r a p e f r u i t conc e n t r a t e to enhance i t s f l a v o r . Essence, the v o l a t i l e water s o l uble f l a v o r from the f r u i t , may be added during the manufacture of a g r a p e f r u i t concentrate, but t h i s system of f l a v o r s does not seem e s s e n t i a l to h i g h f l a v o r q u a l i t y . T h i s i s contrary to what i s g e n e r a l l y found w i t h FCOJ where essences do enhance f l a v o r q u a l i t y (78). 4.2

Reduced-Acid Frozen Concentrated

Orange J u i c e

I n t e r e s t i n reduced-acid c i t r u s j u i c e s o r i g i n a t e d i n the e a r l y I960's when K i l b u r n and Drager (4) employed e l e c t o d i a l y s i s tc remove c i t r a t e ions from j u i c e . The F l o r i d a Department of C i t r u s t e s t e d the reduced-acid concept w i t h consumers at the New York World's F a i r i n 1965, and followed t h i s t e s t with a n a t i o n a l consumer survey i n 1972. The Coca-Cola Company Foods D i v i s i o n

In Citrus Nutrition and Quality; Nagy, S., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

11.

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Processing

255

conducted an independent consumer survey i n 1973, and a l l of the s t u d i e s i n d i c a t e d s u b s t a n t i a l consumer i n t e r e s t i n reduceda c i d c i t r u s j u i c e s . A f t e r f u r t h e r t e s t i n g with consumers i n which products were t e s t e d i n homes, the Foods D i v i s i o n was granted a permit from the U.S. Food and Drug A d m i n i s t r a t i o n and the F l o r i d a Department of C i t r u s to manufacture the product and to d i s t r i b u t e i t i n i n t e r s t a t e commerce. L a t e r the FDA was p e t i t i o n e d to e s t a b l i s h a new standard of i d e n t i t y f o r reduceda c i d f r o z e n concentrated orange j u i c e . The issuance of that standard i s s t i l l pending. A reduced-acid frozen concentrated orange j u i c e i s presently being t e s t marketed by The Coca-Cola Company Foods D i v i s i o n . This product i s produced by blending r e g u l a r concentrated orange j u i c e with acid-reduced concentrate i n p r o p o r t i o n s that w i l l r e s u l t i n a f i n a l f r o z e n concentrated orange j u i c e with a B r i x / a c i d r a t i o between 21 and 26 to 1; the p r e c i s e blend being dependent on the B r i x / a c i A f t e r b l e n d i n g , the concentrat the a d d i t i o n of water and essence. Coldpressed orange o i l i s added f o r good f l a v o r q u a l i t y . The product i s then canned and s t o r e d at -17.8°C. The f a t e s of the n u t r i e n t s of orange j u i c e were n a t u r a l l y of concern i n the process of producing an acid-reduced orange conc e n t r a t e , so many s t u d i e s were conducted to a s c e r t a i n the l e v e l s of the major n u t r i e n t s before and a f t e r p r o c e s s i n g . The major concerns were i n regard to a s c o r b i c and f o l i c a c i d s , s i n c e these components might w e l l be removed during the ion-exchange process to remove c i t r a t e i o n . Since the a n i o n i c r e s i n employed i s weakly b a s i c , the retent i o n of stronger a c i d s i s favored with respect to the weaker a c i d s , a s c o r b i c and f o l i c . A l s o , because of the law of mass a c t i o n , the removal of c i t r a t e ion i s favored over ascorbate and f o l a t e . The change i n the a s c o r b i c a c i d c o n c e n t r a t i o n of j u i c e during a c i d r e d u c t i o n p r o c e s s i n g i s i l l u s t r a t e d i n F i g u r e 7. Some a s c o r b i c a c i d i s i n i t i a l l y r e t a i n e d by the r e s i n but i t i s l a t e r r e p l a c e d by the stronger a c i d , c i t r i c , as the exchange c a p a c i t y of the r e s i n i s depleted. T h i s i n i t i a l r e d u c t i o n i n a s c o r b i c a c i d i s on the order of 15%, but as the column i s exhausted t h i s a c i d i s r e p l a c e d by c i t r i c and i t i s e l u t e d i n the j u i c e stream. Near the end of treatment, the a s c o r b i c a c i d l e v e l r i s e s tc i t s i n i t i a l l e v e l and even exceeds i t as that which was i n i t i a l l y h e l d by the column i s r e p l a c e d by c i t r a t e . I t can be seen i n F i g u r e 7 that some a s c o r b i c a c i d can be l o s t i f the ion-exchange r e s i n i s not completely exhausted during p r o c e s s i n g . When the column i s exhausted t h i s l o s s of a s c o r b i c a c i d i s minimized. A s c o r b i c a c i d l o s s i n acid-reduced j u i c e s never exceeded 10% except i n cases where the ion-exchange r e s i n was not comp l e t e l y exhausted and g e n e r a l l y i t was i n the range of 3% to

In Citrus Nutrition and Quality; Nagy, S., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

256

CITRUS NUTRITION AND QUALITY

50

pH4.2

A 3.9

40

si

r

312

CITRUS

NUTRITION

AND

QUALITY

but yeast count w i l l be r e l a t i v e l y the same even a f t e r eight months storage at below 0°F (22). Even though there are v a r i o u s types of organisms that s u r v i v e the manufacture of c i t r u s j u i c e s , most of them w i l l not grow or reproduce. The microorganisms i n the j u i c e that are capable of growing, even at r e f r i g e r a t o r tempe r a t u r e s , are Leuconostoc, L a c t o b a c i l l u s and v a r i o u s genera of yeasts (23). These organisms, given the opportunity, w i l l develop o f f - f l a v o r s and o f f - o d o r s i n j u i c e . The Leuconostoc and L a c t o b a c i l l u s produce d i a c e t y l which gives the j u i c e a strong " b u t t e r m i l k " l i k e f l a v o r and odor. Yeasts w i l l cause a sour, a l c o h o l i c f l a v o r and odor with gas production. Several species of the L a c t o b a c i l l i w i l l grow at r e l a t i v e l y high temperatures. One s t r a i n has been i s o l a t e d from the regenerative s e c t i o n of a heat exchanger which grew very r a p i d l y i n s i n g l e strength j u i c e at 49°C (24). The enumeration of b a c t e r i a before and a f t e r heat exchangers i s e s p e c i a l l y important when reprocessing bulk concentrate i n t o c i t r u s j u i c Standard P l a t e Count. This i s a means of estimating the number of v i a b l e b a c t e r i a l c e l l s per given q u a n t i t y of j u i c e and i s g e n e r a l l y reported i n terms of the number of microorganisms per ml of concentrated j u i c e , s i n g l e strength j u i c e or the packing medium of c h i l l e d s e c t i o n s . I t i s of the utmost importance to insure that a s e p t i c technique i s used throughout these procedures. The work place must be s u i t a b l e f o r b a c t e r i o l o g i c a l work. I t i s very d i f f i c u l t to produce meaningful work where there are a i r currents and laboratory t r a f f i c . Normal a i r contains b a c t e r i a and mold spores which can e a s i l y contaminate samples and p l a t e s . Medium P r e p a r a t i o n and S t e r i l i z a t i o n . Orange Serum Agar has been developed s p e c i f i c a l l y f o r the b a c t e r i o l o g i c a l examination of c i t r u s products. I t gives a high t o t a l count of microorganisms and i s e s p e c i a l l y recommended f o r the enumeration and c u l t i v a t i o n of organisms causing s p o i l a g e i n c i t r u s products. Dehydrated medium i s a v a i l a b l e commercially and should be prepared according to the d i r e c t i o n s given by the manufacturer. The prepared medium i s dispensed i n t o screw cap b o t t l e s ( p r e s c r i p t i o n ovals 125 ml to 250 ml c a p a c i t y ) , which should never be f i l l e d more than two-thirds f u l l . B o t t l e s of the f r e s h l y prepared medium are capped l o o s e l y and s t e r i l i z e d i n an autoclave or pressure cooker f o r 15 minutes at 15 pounds pressure (120°C). When the medium has been s t e r i l i z e d and cooled the caps should be tightened upon removal from the autoc l a v e or pressure cooker, and stored i n a c o o l p l a c e . Sampling Containers. P r e - s t e r i l i z e d disposable sampling containers are p r e f e r r e d f o r the c o l l e c t i o n of samples. Screw capped b o t t l e s may be used i f they are f i r s t s t e r i l i z e d under 15 pounds pressure f o r 15 minutes. I f g l a s s b o t t l e s are

In Citrus Nutrition and Quality; Nagy, S., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

13.

MCALLISTER

Quality

of

Citrus

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s t e r i l i z e d they should c o n t a i n about 1 ml of water per b o t t l e . B o t t l e s which are dry i n s i d e cannot be considered s t e r i l e at t h i s recommended time and pressure. Samples should be taken d i r e c t l y i n the s t e r i l i z e d c o n t a i n e r . S t e r i l e metal sampling dippers may be used to remove samples from vats or tanks. Samples should not be stored but should be p l a t e d immediately a f t e r sampling, as storage might i n c r e a s e the counts. Samples which cannot be p l a t e d immediately must be c h i l l e d r a p i d l y and thoroughly. P i p e t t e s and P e t r i Dishes. I t i s p r e f e r a b l e to use pres t e r i l i z e d d i s p o s a b l e p i p e t t e s and p e t r i d i s h e s . They are not only more economical to use, but the method of s t e r i l i z i n g and s e a l i n g i s g e n e r a l l y b e t t e r than that accomplished i n most l a b o r a t o r i e s . P i p e t t e s should be 1 ml s e r o l o g i c a l type graduat­ ed i n 0.1 ml. I f concentrates are to be sampled the l a r g e bore pipette for viscous l i q u i d should be standard 100 χ S t e r i l i z a t i o n of D i l u t i o n B o t t l e s . Pyrex m i l k d i l u t i o n b o t t l e s with screw caps are p r e f e r r e d . The b o t t l e s are f i l l e d with 99 ml of water and s t e r i l i z e d under 15 pounds pressure (121°C) f o r twenty minutes. B o t t l e s should not be opened except before and a f t e r d e l i v e r y of the sample. D i l u t i o n of Samples. Samples are g e n e r a l l y p l a t e d i n d i l u t i o n s of 1-100 or 1-1000. The d i l u t i o n used w i l l depend upon the number of c o l o n i e s present on the p l a t e s a f t e r a 48-hour i n c u b a t i o n p e r i o d , or what previous experience with the product has shown. D i l u t i o n s should be adjusted so that the p l a t e counts f a l l between 30 and 300 c o l o n i e s per p l a t e . I f the count f a l l s outside t h i s range, higher or lower d i l u t i o n s are necessary. Upon i n i t i a l p l a t i n g , when the d i l u t i o n range i s unknown, samples should be p l a t e d i n d i l u t i o n s of 1-100, 1-1000, 1-10,000 and made i n d u p l i c a t e . I f s p o i l a g e i s suspected the lower d i l u t i o n s are not necessary and even higher d i l u t i o n s made. To prepare d i l u t i o n s , 1 ml of concentrate, s i n g l e s t r e n g t h j u i c e , or packing medium from c h i l l e d s e c t i o n s i s removed from the sample and t r a n s f e r r e d to a s t e r i l e 99 ml water blank. This gives a 1-100 d i l u t i o n . The 1-100 d i l u t i o n i s shaken v i g o r o u s l y at l e a s t 25 times over a 7 second p e r i o d . T h i s i s to break any b a c t e r i a l clumps that might be present. Using a s t e r i l e p i p e t t e 0.1 i s t r a n s f e r r e d to a p e t r i d i s h f o r the 1-1000 p l a t e . 1 ml i s t r a n s f e r r e d to another p e t r i d i s h f o r the 1-100 p l a t e . To prepare a 1-10,000 d i l u t i o n , 1 ml of the 1-100 d i l u t i o n i s t r a n s f e r r e d to a s t e r i l e 99 ml water blank and shaken as d e s c r i b e d . 1 ml of t h i s d i l u t i o n i s d e l i v e r e d to a p e t r i d i s h f o r the 1-10,000 d i l u t i o n and 0.1 ml would be a 1-100,000 i f t h i s high a d i l u t i o n i s needed. When p l a t i n g packing media from c h i l l e d s e c t i o n s

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that c o n t a i n p r e s e r v a t i v e s lower d i l u t i o n s may be needed. 1 ml of the packing medium t r a n s f e r r e d to a p e t r i d i s h w i l l give a 1-1 d i l u t i o n . B a c t e r i o l o g i c a l Procedures. Microorganisms m u l t i p l y r a p i d l y under i d e a l c o n d i t i o n s . Consequently, i t i s necessary that samples be p l a t e d as soon as p o s s i b l e a f t e r they are drawn. I f the samples are i n a frozen s t a t e , they should be thawed i n t e p i d water. I f there are a l a r g e number of samples, they should be thawed i n workable groups. I t i s not advisable to allow a sample to stand over an hour. I f the samples are i n t w i s t top s t e r i l e bags or screw cap b o t t l e s they should never be immersed i n water. I f the sample i s i n a can i t should be d r i e d thoroughly, the top swabed with 70 percent a l c o h o l and opened with a beer opener that i s s t i l l warm from flaming. I t i s e s s e n t i a l that from the puncturing of the can or opening the sample throug to be exercised i n a s e p t i b o t t l e must be flamed p r i o r to and a f t e r d e l i v e r y of the sample. Caps on the b o t t l e s must be c a r e f u l l y removed and held between the f i n g e r s i n such a p o s i t i o n that the bottom or i n s i d e of the cap does not touch the f i n g e r s . I f t h i s happens i n any way the b o t t l e must not be used. This a p p l i e s to p i p e t t e s as w e l l . P i p e t t e s can only be used once. P r i o r to p l a t i n g a s u f f i c i e n t amount of agar must be melted. P l a c e the r e q u i r e d number of b o t t l e s i n a sauce pan and f i l l with water two-thirds the height of the b o t t l e s , b o i l the water u n t i l the agar i s completely melted. A f t e r m e l t i n g the agar i s cooled and maintained at a temperature of 45°C u n t i l used. I t i s very important that the agar i s not hot when poured since microorganisms are very s u s c e p t i b l e to high temperature. The procedure i s as f o l l o w s : A. Using a s t e r i l e p i p e t t e a s c e p t i c a l l y t r a n s f e r the sample to a d i l u t i o n b o t t l e , the mouth of which has been flamed. Flame the mouth again and replace cap. B. Shae the d i l u t i o n b o t t l e at l e a s t 25 times. C. Remove the d i l u t i o n b o t t l e cap, flame and remove a sample with s t e r i l e p i p e t t e . Transfer t h i s to a flamed d i l u t i o n b o t t l e or a s t e r i l e p e t r i d i s h . The p e t r i d i s h cover should not be f u l l y removed, j u s t t i l t e d up enough that the p i p e t t e can be i n s e r t e d . D. The cap i s removed from the agar b o t t l e , the b o t t l e flamed and approximately 15 ml i s poured i n t o the p e t r i d i s h with the sample. The sample should be mixed c a r e f u l l y with the agar by a slow r o t a t i o n of the p l a t e , i n a f i g u r e 8 p a t t e r n . The agar should not be allowed to s l o s h up on the rim or top of the d i s h . E. The agar p l a t e s are allowed to harden p r i o r to incubation.

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When the agar i s hardened the p l a t e s are i n v e r t e d and placed i n an incubator f o r 48 hours at 30°C. A c o n t r o l p l a t e should be run with each batch. This c o n s i s t s of pouring agar i n t o a p e t r i d i s h c o n t a i n i n g 1 ml of the s t e r i l e d i l u t i o n water. This gives a check on the s t e r i l i t y o f glassware, medium, and d i l u t i o n water.

Counting the P l a t e s . A f t e r the p l a t e s are incubated f o r 48 hours the counts may be made. A Qubec Colony Counter f a c i l i t a t e s counting the p l a t e s . I t s u p p l i e s the r i g h t l i g h t ing and m a g n i f i c a t i o n to detect and count p i n p o i n t c o l o n i e s which otherwise might be overlooked. The t o t a l p l a t e count i s a r r i v e d a t by counting the number of c o l o n i e s and m u l t i p l y i n g that number by the d i l u t i o n f a c t o r . For example, i f there are 30 c o l o n i e s on the p l a t e the d i l u t i o n of which i s 1-100 the t o t a l count f o r that p l a t milliliter. I f duplicat both are counted and the average count i s reported. It i s not very l i k e l y that p l a t e s made i n d u p l i c a t e w i l l show the same counts. Some p l a t e s may vary as much as 10 c o l o n i e s per d i l u t i o n . However, i f there i s a wider v a r i a t i o n i n the t o t a l counts on d u p l i c a t e p l a t e s , i t i s more l i k e l y that the technique i s at f a u l t and should be thoroughly checked from beginning to end. P l a t e s containing l e s s than 30 c o l o n i e s or more than 300 do not give a v a l i d count. When t h i s i s encountered other d i l u t i o n s should be made. S i g n i f i c a n c e of Counts. The t o t a l p l a t e counts w i l l vary throughout the season depending on the c o n d i t i o n of f r u i t . Generally speaking, the higher the r a t i o of the f r u i t the more l i k e l y the higher the count. Normal counts on 45° B r i x orange concentrate w i l l vary from 25,000 to 75,000 microorganisms. A count of 100,000 i s considered e x c e s s i v e . C h i l l e d or p a s t e u r i z e d j u i c e s packed i n cartons with a t o t a l p l a t e count of 300 to 500 organisms per m i l l i l i t e r , i f handled p r o p e r l y , can expect a s h e l f l i f e to three weeks. C h i l l e d j u i c e s with a count of 5,000 per m i l l i l i t e r probably w i l l not have a s h e l f l i f e of more than a week. Use of the permitted p r e s e r v a t i v e s sorbates and benzoates w i l l i n h i b i t the growth of microorganisms but w i l l not apprec i a b l y extend the s h e l f l i f e of c h i l l e d c i t r u s j u i c e s or c h i l l e d s e c t i o n s that have a heavy b a c t e r i a l l o a d .

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Literature Cited 1. McAllister, J. W. Sanitation Workshop for Food Industry; Food Processors Institute and Management Association Food Division, Orlando, Florida, September 10-12, 1974. 2. Hendrix, C. J. Jr. In "Citrus Science and Technology"; Nagy, S.; Shaw, P.; Veldhuis, M.; Eds.; AVI Publishing Co.: Westport, CT, 1977; Vol. 2, Chapter 12. 3. Stevens, J. W.; Baier, W. E. Ind. Eng. Chem., Anal. Ed., 1939 11, 447. 4. Scott, W. C.; Morgan, D. Α.; Veldhuis, M. Food Technol. 1960, 14, 423-428. 5. Scott, W. C.; Morgan 1961, 15, 180-186. 6. Scott, W. C.; Morgan, D. Α.; Veldhuis, M. Food Technol. 1961, 15, 388-391. 7. "Official Methods of Analysis of the Analytical Chemists"; Horowitz, W., Ed.; 13th edition, 1980. 8.

Scott, W. C. J. Assoc. Off. Anal. Chem., 1968, 51, 928-931.

9. United States Standards for Grades of Grapefruit Juice. U. S. Dept. of Agric. Food Safety and Quality Service, Washington, D. C., 1968. 10. Davis, W. B. Anal. Chem., 1947, 19, 476-478. 11. Kesterson, J. W.; Hendrickson, R. Agric. Exp. Stn. Tech. Bull. 511; University of Florida: Gainesville, 1953. 12. Maurer, R. H.; Burdick, E. M.; Waihel, C. W. Lower Rio Grande Valley Citrus and Vegetable Institute Fourth Annual Proceedings. 1950. Weslaco, TX. 13. Judd, D. B. U. S. Dept. Commerce, Nat. Bureau Standards circ. 478, 1950. 14. Kramer, Α.; Twigg, B. A. "Fundamentals of Quality Control for the Food Industry"; AVI Publishing Co.: Westport, CT. 1962. 15. Hunter, R. S. U. S. Dept. Commerce, Nat. Bureau Standards Circ. C429, 1952.

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16. Hunter, R. S. "The Measurement of Appearance"; Hunter Associates Laboratory, Inc.: Fairfax, VA. 1973. 17. Huggart, R. L.; Wenzel, F. W.; Martin, F. C. Proc. Fla. State Hortic. Soc. 1969, 82, 200-205. 18. "Official Rules Affecting the Florida Citrus Industry"; State of Florida Department of Citrus; Lakeland, FL Chapters 20-65. 19. "United States Standards for Grades of Frozen Concentrated Orange Juice." U. S. Dept. of Agric. Food Safety and Quality Service, Washington, D. C., 1976. 20. McAllister, J. W. Masters Thesis; University of Florida: Gainesville, 1951 21. Faville, L. W.; Hill, E. C. Food Technol., 1951, 10, 423-425. 22. Faville, L. W.; Hill, E. C. Food Technol., 1951, 1, 33-36. 23. Patrick, R.; Hill, E. C. Agric. Exp. Tech. Bull. 618; University of Florida: Gainesville, 1959. 24. Murdock, D. I. In "Citrus Science and Technology"; Nagy, S.; Shaw, P.; Veldhuis, M.; Eds.; AVI Publishing Co.: Westport, CT, 1977; Vol. 2, Chapter 11. RECEIVED June

26, 1980.

In Citrus Nutrition and Quality; Nagy, S., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

14 Problems in Sensory Evaluation of Citrus Products P. J. F E L L E R S Florida Department of Citrus, University of Florida, Institute of Food and Agricultural Sciences, Agricultural Research and Education Center, P.O. Box 1088, Lake Alfred, FL 33850

As i n most food industries is interested at all time quality of its products. To obtain this information, use of some sort of sensory evaluation i s often employed. This may be by an experienced quality control person or group, a government food inspector, a small experienced group of judges gleaned from the technical plant personnel, a larger consumer-type sensory panel made up of diverse plant personnel, or perhaps a combination of the above. Most plants are also vitally interested i n flavor quality of product manufactured elsewhere, including states other than Florida and foreign countries, including B r a z i l , already a citrus giant. Of course, to control q u a l i t y , raw product coming into the plant requires at least a cursory flavor evaluation by plant personnel in addition to objective tests which normally are run such as degrees B r i x , % a c i d , color, etc. In F l o r i d a , all f r u i t delivered to a fresh f r u i t packer or processor i s inspected by personnel from the Florida Department of Agriculture, Division of F r u i t and Vegetable I n s p e c t i o n . J u i c e content and degrees B r i x to % a c i d r a t i o are used i n determining m a t u r i t y according to s p e c i f i c standards during fresh f r u i t i n s p e c t i o n U). F r u i t passing m a t u r i t y t e s t s are inspected to determine grade c o n s i d e r i n g i n t e r n a l f a c t o r s such as j u i c i n e s s , dryness, f i r m n e s s , f l a b b i n e s s , sponginess, absence or presence o f decay, freeze damage, i n j u r y , d i s e a s e , e t c . A l l f r u i t f a i l i n g to pass e i t h e r minimum j u i c e content requirements or B r i x to a c i d r a t i o m a t u r i t y t e s t s w i l l be destroyed o r l e g a l l y d i v e r t e d to some other s u i t a b l e use. Matur i t y t e s t s are a l s o made on f r u i t f o r p r o c e s s i n g . These t e s t s i n c l u d e j u i c e content, a c i d , s o l u b l e s o l i d s , and s o l u b l e s o l i d s to a c i d r a t i o . In a d d i t i o n , wholesomeness i s determined wholesome f r u i t c o n s i s t i n g o f " f r u i t free from r o t , decay, sponginess, soundness, leakage, s t a l e n e s s , o r other c o n d i t i o n s showing p h y s i c a l defects o f the f r u i t " . I f a f t e r any r e q u i r e d r e g r a d i n g , f r u i t i s s t i l l found to be unwholesome or decomposed so as to be u n f i t f o r processing purposes, i t w i l l be destroyed 0-8412-0595-7/80/47-143-319$05.50/0 © 1980 American Chemical Society In Citrus Nutrition and Quality; Nagy, S., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

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at the expense o f the owner, under the personal s u p e r v i s i o n o f the i n s p e c t o r . For the f r e s h c i t r u s f r u i t packer, no f u r t h e r q u a l i t y t e s t s are g e n e r a l l y necessary. However, f o r the c i t r u s processor, f u r t h e r f l a v o r q u a l i t y t e s t s may be undertaken by q u a l i t y c o n t r o l personnel a t any of several points during p r o duction should a p o s s i b l e problem a r i s e wherein f l a v o r q u a l i t y may have been adversely a f f e c t e d . The main emphasis, however, i s made on the f i n a l product where g e n e r a l l y a c r o s s - s e c t i o n of each d a y ' s pack i s checked f o r f l a v o r q u a l i t y e i t h e r during the day of production or the next day ( 2 ) . In F l o r i d a , USDA i n s p e c t o r s determine t h a t f l a v o r q u a l i t y on a l l product meet e x p l i c i t U.S. Standards and State of F l o r i d a Department of C i t r u s Statutes f o r Grades o f the several types o f c i t r u s product packed. To maint a i n c o n s i s t e n c y i n f l a v o r g r a d i n g , weekly panels are convened a t the USDA Processed Products Branch Area O f f i c e i n Winter Haven, FL to cover a l l c i t r u s products ( 3 ) The primary c i t r u s products evaluated are f r o z e n concentrate t r a t e d orange j u i c e f o t r a t e , p a s t e u r i z e d orange j u i c e and g r a p e f r u i t j u i c e , the l a t t e r d e s i g n a t i o n covering both " c h i l l e d " g r a p e f r u i t j u i c e and canned s i n g l e - s t r e n g t h g r a p e f r u i t j u i c e . To o b t a i n a U.S. Grade A c l a s s i f i c a t i o n f o r f l a v o r of FCOJ, f o r example, standards s t a t e : "Frozen concentrated orange j u i c e of which the r e c o n s t i t u t e d j u i c e possesses a very good f l a v o r may be given a score o f 36 to 40 p o i n t s . 'Very good f l a v o r ' means t h a t the f l a v o r i s f i n e , d i s t i n c t , and s i m i l a r to t h a t of f r e s h orange j u i c e . . . " ( 4 ) . There are a l s o c e r t a i n other f a c t o r s a s s o c i a t e d w i t h f l a v o r grade, namely minimum and maximum degrees B r i x to % a c i d r a t i o and o i l content requirements. Should there be a question as to the f l a v o r by e i t h e r the i n s p e c t o r or the p l a n t p e r s o n n e l , the i n s p e c t o r w i l l submit a sample to the USDA Processed Products Branch area o f f i c e f o r another e v a l u a t i o n . In order to monitor the f l a v o r of t h e i r products more c l o s e l y , the F l o r i d a C i t r u s Processors A s s o c i a t i o n has c o n t r a c t e d w i t h the USDA Processed Products Branch to operate a t a s t e panel i n which c u r r e n t FCOJ production i s t e s t e d on a weekly b a s i s . The panel c o n s i s t s of l o c a l consumers ( g e n e r a l l y housewives) and p e r sonnel from each o f the p a r t i c i p a t i n g p l a n t s (other types o f c i t rus products have been evaluated i n the p a s t ) . P a n e l i s t s r a t e samples on a 9 - p o i n t s c a l e from a low of "extremely poor" to a high of " e x c e l l e n t e J u i c e s are ranked according to f l a v o r score and a copy of the rankings are sent t o each processor w i t h o n l y t h a t p a r t i c u l a r p r o c e s s o r ' s product i d e n t i f i e d ( 3 ) . A s i m i l a r f l a v o r survey i s c a r r i e d out a t the U n i v e r s i t y of F l o r i d a , T n s t i t u t e of Food and A g r i c u l t u r a l S c i e n c e s , A g r i c u l t u r a l Research and Education Center, Lake A l f r e d , FL (AREC). FCOJ, p a s t e u r i z e d orange j u i c e , and orange j u i c e from concentrate are examined on a monthly b a s i s . Canned and b o t t l e d p a s t e u r i z e d s i n g l e - s t r e n g t h g r a p e f r u i t j u i c e s are checked biweekly. Sensory panels comprising 10-12 experienced t a s t e p a n e l i s t s grade samples

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on a 9 - p o i n t hedonic s c a l e . Unusual sample r e s u l t s are v e r i f i e d during f u r t h e r t e s t i n g . Results o f a l l samples examined each period are t a b u l a t e d and sent to the p a r t i c i p a t i n g p l a n t s , w i t h only the hedonic score of the p l a n t r e c e i v i n g the l e t t e r being i d e n t i f i e d , however. In a d d i t i o n , a comprehensive sensory e v a l u a t i o n program i n v o l v i n g c i t r u s and c i t r u s - b a s e d products i s being conducted by the author. Many f a c e t s o f c i t r u s f l a v o r research may be ongoing at any p a r t i c u l a r time u t i l i z i n g an assortment o f sensory e v a l u a t i o n methods. In the United States and other c o u n t r i e s , companies ( p r i m a r i l y d a i r i e s ) t h a t r e c o n s t i t u t e frozen concentrate f o r manuf a c t u r e i n t o s i n g l e - s t r e n g t h products o r manufacture FCOJ from bulk concentrate a r e a l s o i n t e r e s t e d i n monitoring f l a v o r q u a l i t y of t h e i r products. Minimal a t t e n t i o n w i l l be devoted i n t h i s chapter to a r e view o f the d i f f e r e n t type on operating and a n a l y z i n a number o f e x c e l l e n t p u b l i c a t i o n s a v a i l a b l e d e s c r i b i n g and e x p l a i n i n g n e a r l y every f a c e t of the aforementioned f a c t o r s i n d e t a i l (6-11). This paper i s an attempt to document c e r t a i n problems pecul i a r t o sensory e v a l u a t i o n o f c i t r u s f r u i t or products p r i m a r i l y as they p e r t a i n to F l o r i d a and t o perhaps o f f e r some s o l u t i o n s o r a l t e r n a t i v e s t o these problems. I.

Panelists

Special C r i t e r i a f o r Selecting P a n e l i s t s . In a d d i t i o n to many o f the usual c r i t e r i a used i n choosing t a s t e p a n e l i s t s f o r sensory e v a l u a t i o n such as a b i l i t y to d i f f e r e n t i a t e b i t t e r , sour, sweet, s a l t y and bland basic f l a v o r f a c t o r s , determination of t h r e s h o l d values f o r these f a c t o r s , proven a b i l i t y to t a s t e c e r t a i n c i t r u s j u i c e s , w i l l i n g n e s s to p a r t i c i p a t e i n t a s t e panels on a r e g u l a r b a s i s , e t c . , there are a s i g n i f i c a n t number o f p o t e n t i a l judges who cannot p a r t i c i p a t e because o f the chemical nature o f c i t r u s . For c e r t a i n persons, f o r example, the a c i d present i n c i t r u s may a f f e c t them a d v e r s e l y , e s p e c i a l l y when the j u i c e i s not taken w i t h a meal. Another p o s s i b l e problem i s persons s u f f e r i n g from a l l e r g i e s t o c e r t a i n foods such as c i t r u s . F o r t u n a t e l y , most consumers are very fond o f oranges and orange products and there i s not much of a problem e n l i s t i n g i n d i v i d u a l s to p a r t i c i p a t e i n e v a l u a t i o n t e s t s . However, g r a p e f r u i t and g r a p e f r u i t products are not nearly as u n i v e r s a l l y accepted f l a v o r w i s e , thus a s p e c i a l e f f o r t must be made t o generate a g r a p e f r u i t t a s t e panel t h a t w i l l give c o n s i s t e n t r e l i a b l e r e s u l t s . E n l i s t i n g an i n d i v i d u a l who does not p a r t i c u l a r l y l i k e g r a p e f r u i t on a g r a p e f r u i t t a s t e panel (perhaps even though the i n d i v i d u a l d o e s n ' t mind s e r v i n g on the panel) can r e s u l t i n u n r e l i a b l e d a t a . The i n d i v i d u a l w i l l most l i k e l y expectorate the j u i c e i n t o an empty cup a f t e r only a cursory

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examination, thus not a l l o w i n g f o r a complete sensory e v a l u a t i o n of the product. Furthermore, s i n c e the product i s d i s t a s t e f u l to the p a n e l i s t , proper m o t i v a t i o n to do a good c o n s c i e n t i o u s e v a l uation w i l l be l a c k i n g , again r e s u l t i n g i n u n r e l i a b l e d a t a . I n a b i l i t y of some p o t e n t i a l p a n e l i s t s to t a s t e c e r t a i n f l a vor c h a r a c t e r i s t i c s t h a t may occur i n c i t r u s products a t low or moderately low c o n c e n t r a t i o n l e v e l s may exempt these i n d i v i d u a l s from p a r t i c i p a t i n g i n r o u t i n e survey, q u a l i t y c o n t r o l , and perhaps c e r t a i n other f l a v o r e v a l u a t i o n work. For example, d i a c e t y l i n a c i t r u s product imparts a b u t t e r m i l k - l i k e o f f - f l a v o r . I t may be detected i n very small amounts by c e r t a i n i n d i v i d u a l s , whereas the t h r e s h o l d f o r others may be s i g n i f i c a n t l y higher which would a l l o w the presence of the d i a c e t y l to go undetected. About two out of three otherwise q u a l i f i e d t a s t e p a n e l i s t s cannot d e t e c t d i a c e t y l i n amounts t h a t may be found i n c i t r u s products. Another compound, n a r i n g i n , c o n t r i b u t e s s i g n i f i c a n t l y to the b i t t e r sensation r e a l i z e d i n g r a p e f r u i not by a l l . B i t t e r n e s able spread i n the a b i l i t y of i n d i v i d u a l s to d e t e c t b i t t e r n e s s due to n a r i n g i n . Two i n d i v i d u a l s were found who could not d e t e c t n a r i n g i n concentrations a t 2,000 ppm (or about f i v e times the amount normally found i n F l o r i d a g r a p e f r u i t j u i c e ) i n e i t h e r model g r a p e f r u i t j u i c e systems or as an a d d i t i v e to otherwise pure g r a p e f r u i t j u i c e . Number of P a n e l i s t s . Very o f t e n p r a c t i c a l c o n s i d e r a t i o n s o v e r r i d e the t h e o r e t i c a l when i t comes to the s e l e c t i o n of the numbers of i n d i v i d u a l s to c o n s t i t u t e an e f f e c t i v e c i t r u s t a s t e panel. For several d i v e r s e reasons, many p o t e n t i a l t a s t e p a n e l i s t s e i t h e r can n o t , w i l l n o t , or would r a t h e r not p a r t i c i p a t e i n sensory e v a l u a t i o n panels. These i n d i v i d u a l s , along w i t h those w i l l i n g but found to l a c k s u f f i c i e n t t a s t e a c u i t y , can o f t e n r e s u l t i n only a few w i l l i n g and able p a n e l i s t s a t a p a r t i c u l a r l o c a t i o n . Should t h i s be the case, however, e s p e c i a l l y as p e r t a i n s to d i s c r i m i n a t o r y t e s t i n g , numbers of i n d i v i d u a l e v a l u a t i o n s may be increased through r e p l i c a t i o n using the same p a n e l i s t s . Larmond (6j i n d i c a t e s a minimum number of p a n e l i s t s to be 4 or 5 to c o n s t i t u t e a sensory p a n e l , w i t h l a b o r a t o r y panels u s u a l l y being composed of 10 to 20 persons w i t h 3 or 4 r e p l i c a t i o n s per judge per treatment. At AREC, out of a pool of about 35 i n d i v i d u a l s a v a i l a b l e f o r everyday panel work, about h a l f p a r t i c i p a t e i n orange product e v a l u a t i o n whereas s l i g h t l y l e s s than h a l f p a r t i c i p a t e w i t h g r a p e f r u i t . Panels c o n s i s t i n g of about 12 semitrained and t r a i n e d i n d i v i d u a l s are used. A 5 to 6 member e f f i c i e n t t r a i n e d panel i s g e n e r a l l y a v a i l a b l e f o r c e r t a i n sensory e v a l u a t i o n work. In a d d i t i o n , between 30 to 70 i n d i v i d u a l s can be u t i l i z e d l o c a l l y f o r small consumer-type panels. Large consumer t e s t s o f t e n i n volve as many as 600 responses w i t h data being obtained from several d i f f e r e n t demographic l o c a t i o n s .

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Products

Sample P r e p a r a t i o n

Fresh C i t r u s J u i c e . F r e s h l y e x t r a c t e d commercial orange j u i c e may c o n t a i n a high peel o i l content making sensory e v a l u a t i o n of t h i s product very d i f f i c u l t because o f the o v e r r i d i n g , b i t i n g and even b i t t e r f l a v o r e f f e c t o f the peel o i l . This i s one o f the reasons why i n c e r t a i n p l a n t s , minimal a t t e n t i o n i s paid to the f l a v o r q u a l i t y o f the j u i c e u n t i l the product i s standardized e i t h e r i n r e t a i l o r bulk form (2). Excessive pressure used i n hand-reaming o f c i t r u s f r u i t can a l s o r e s u l t i n j u i c e c o n t a i n i n g high peel o i l content and perhaps s i g n i f i c a n t amounts o f undesirable f l a v o r c o n s t i t u e n t s ( p r i m a r i l y of a b i t t e r nature) as w e l l . Since high peel o i l and u n d e s i r a b l e f l a v o r c o n s t i t u e n t s would s e r i o u s l y a f f e c t sensory e v a l u a t i o n r e s u l t s i n a negative way, care should be taken i n hand-reaming c i t r u s f r u i t t o apply only as much pressure as necessary to e x t r a c t most but not a l peel o i l and u n d e s i r a b l levels. The number o f f r u i t necessary to comprise a v a l i d sample f o r f l a v o r e v a l u a t i o n purposes may very w e l l be i n the range r e q u i r e d by the F l o r i d a Department of A g r i c u l t u r e i n determining c i t r u s f r u i t m a t u r i t y , namely 20 oranges, t a n g e r i n e s , t a n g e l o s , Temples or M u r c o t t s , or 10 g r a p e f r u i t ( 1 ) . To t e s t the v a l i d i t y of using a 1 0 - g r a p e f r u i t sample, f r u i t were u t i l i z e d from an e a r l y season storage study i n v o l v i n g 3 r e p l i c a t e s f o r each of 3 treatments. Each r e p l i c a t e sample contained several boxes of f r u i t from the original l o t of f r u i t . In a l l , 9 separate samples o f 10 f r u i t each were taken a t random from each of the r e p l i c a t e samples. The m a t u r i t y i n d i c a t o r s , degrees B r i x , % a c i d , and the r a t i o between these two f a c t o r s were determined on the j u i c e o f each sample. J u i c e was obtained by using a small e l e c t r i c handreamer t h a t provided about 1,500 ml of j u i c e per sample. The mean values f o r degrees B r i x was 9.40 ± 0.125 (as determined by a t a b l e r e f r a c t o m e t e r ) , 1.47 ± 0.03 percent a c i d (as determined by t i t r a t i o n w i t h NaOH), and 6.36 +.0.125 degrees B r i x to percent a c i d r a t i o ( c a l c u l a t e d ) . Since p r i o r work by the author (13>) has shown t h a t a degrees B r i x d i f f e r e n t i a l o f about 1 i n s i n g l e strength g r a p e f r u i t o r orange j u i c e was r e q u i r e d before d i f f e r ences could be detected by experienced judges, a d i f f e r e n c e of 0.25 degrees B r i x between the highest and lowest samples, as found i n the above study, would presumably not s i g n i f i c a n t l y a f f e c t f l a v o r . In l i k e manner, a r a t i o o f degrees B r i x to % a c i d of about 0.8 had been p r e v i o u s l y found to be d i f f e r e n t i a t e d i n s i n g l e - s t r e n g t h g r a p e f r u i t j u i c e using experienced judges. Again t h i s amount was s i g n i f i c a n t l y higher than the 0.25 r a t i o found between the highest and lowest r a t i o samples i n the above study, thus having an apparent n e g l i g i b l e e f f e c t on f l a v o r . C i t r u s Sections or Segments.

Sensory e v a l u a t i o n o f t h i s

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type of product presents the problem of o f t e n s i g n i f i c a n t l y d i f f e r e n t f l a v o r e d s e c t i o n s w i t h i n and/or between f r u i t thus making i t d i f f i c u l t f o r p a n e l i s t s to evaluate the o v e r a l l f l a v o r q u a l i t y . Since the p a n e l i s t w i l l most l i k e l y sample only 1 or 2 i n d i v i d u a l s e c t i o n s or segments before passing judgment, no easy way e x i s t s to reduce the odds of maverick s e c t i o n s being t e s t e d w i t h ambiguous judgments. An a l t e r n a t i v e method i s to homogenize the p r o duct i n a blender f o r as b r i e f a period of time as p o s s i b l e to achieve a homogeneous mixture which may then be f i l t e r e d through cheesecloth to produce a j u i c e . Using t h i s method, compensation can be made f o r any can-to-can v a r i a t i o n by using several cans. Of course, t e x t u r e cannot be taken i n t o c o n s i d e r a t i o n using t h i s procedure and, t h e r e f o r e , a p o s s i b l e s i g n i f i c a n t p a r t of the o v e r a l l f l a v o r may be compromised. I f s e c t i o n s or segments are to be e v a l u a t e d , i t i s a good idea to present the e n t i r e contents of a can, i f p r a c t i c a l , f o r each judge to make an o v e r a l l e v a l u a t i o n . Seeds, i f present would only serve to confus a t t r i b u t e s are being evaluated such as f i r m n e s s , sweetness, e t c . F a i l u r e to remove seeds i n t h i s case could lead to a type of stimulus e r r o r , the seeds being an i r r e l e v a n t c h a r a c t e r i s t i c . For c i t r u s s e c t i o n s or segments packed as c h i l l e d product e i t h e r a l o n e , as mixed types of c i t r u s , or w i t h other f r u i t such as c h e r r i e s or pineapple chunks, there may be a problem f o r c e r t a i n i n d i v i d u a l s t a s t i n g these products due to the presence of small amounts o f sodium benzoate (0.05-0.06% w/w) which i s added as a p r e s e r v a t i v e . .Sodium benzoate can impart a sharp f l a v o r sensation to c e r t a i n i n d i v i d u a l s even at the r e l a t i v e l y low l e v e l s found i n c h i l l e d c i t r u s products. The sharpness of the benzoate f l a v o r thus acts as an o v e r r i d i n g f a c t o r , and the p r o duct w i l l be graded down a c c o r d i n g l y . Since i n d i v i d u a l s who have low t o l e r a n c e to sodium benzoate w i l l most l i k e l y not consume t h i s p a r t i c u l a r product, or grade i t down s e v e r e l y because of sodium benzoate, i n d i v i d u a l s chosen to evaluate c h i l l e d c i t r u s products should have a r e l a t i v e l y high t o l e r a n c e to sodium benzoate. FCOJ. Proper r e c o n s t i t u t i o n of FCOJ i s e s s e n t i a l f o r v a l i d t a s t e panel r e s u l t s . Since orange j u i c e f l a v o r i s of a r a t h e r d e l i c a t e , m i l d , or l i g h t t y p e , the water source used to r e c o n s t i t u t e the concentrate must be r e l a t i v e l y f r e e of o f f - f l a v o r s , such as s u l f u r o u s . D i s t i l l e d water should be used whenever p r a c t i c a l . In a d d i t i o n , i f the water used f o r r e c o n s t i t u t i o n i s placed i n a r e f r i g e r a t o r , w a l k - i n c o o l e r , e t c . f o r c h i l l i n g , care must be taken to have the water c o n t a i n e r covered s e c u r e l y to e l i m i n a t e p o s s i b l e absorption of f o r e i g n f l a v o r s . And, of course, the r e c o n s t i t u t e d product i t s e l f , i f being c h i l l e d p r i o r to t a s t i n g , should be p r o t e c t e d . There are several methods a v a i l a b l e f o r r e c o n s t i t u t i n g FCOJ so t h a t the f i n a l d e s i r e d d i l u t i o n i s c o r r e c t . R e c o n s t i t u t i o n of concentrate r e q u i r e s use of a p r e c i s e s c a l e or balance, sugar

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t a b l e s , and a p r e c i s e r e f r a c t o m e t e r . At times t h i s p r e c i s i o n may not be o b t a i n a b l e f o r sensory e v a l u a t i o n work when a good s c a l e or balance or refractometer i s u n a v a i l a b l e . For r e c o n s t i t u t i o n of r e t a i l products on a volume/volume b a s i s , the f o l l o w i n g p r o cedures or parts thereof may be f o l l o w e d : thaw f r o z e n cans i n running tap water (about 30 min f o r 6-oz c a n s ) ; open can and place on a l e v e l surface and w i t h a small k n i f e or sharp i n s t r u ment mark the i n s i d e of the can by making a short l i n e a t the surface of the c o n c e n t r a t e ; pour the contents of the can i n t o a vessel l a r g e enough to c o n t a i n a l l of the r e c o n s t i t u t e d j u i c e ; c a r e f u l l y f i l l the can to the mark w i t h d i s t i l l e d water ( c h i l l e d or room temperature, whichever i s p r e f e r r e d ) the p r e s c r i b e d number of times and add to the v e s s e l ; and s t i r very w e l l u n t i l the concentrate and water are thoroughly mixed. Another p o s s i b i l i t y i s to pour a c e r t a i n amount of thoroughly thawed concentrate i n t o a v o l u m e t r i c c y l i n d e r , c a r e f u l l y add the necessary volume of water and mix thoroughly of FCOJ i n 3 U.S. c i t i e B r i x e s of 12.8 and 11.8 degrees were r e q u i r e d f o r about 1.5 1 of each j u i c e per session the f o l l o w i n g method was d e v i s e d : make up cans of FCOJ c o n t a i n i n g e x a c t l y 6 f 1 . oz of product having a c e r t a i n degrees B r i x f o r the s p e c i f i c use i n the t e s t ; a t the p o i n t of t e s t i n g place f r o z e n cans i n running tap water f o r 30 min; pour c h i l l e d d i s t i l l e d water i n t o a 2,000 ml p l a s t i c v o l u m e t r i c c y l i n d e r u n t i l the water i s e x a c t l y l e v e l w i t h the top of the black mark (placed there i n the l a b o r a t o r y p r e v i o u s l y marking e x a c t l y the number of ml or f 1 . oz r e q u i r e d f o r e x a c t l y 12 f 1 . oz of FCOJ to make the d e s i r e d f i n a l r e c o n s t i t u t e d B r i x p r o d u c t ) ; from the graduated c y l i n d e r , pour about o n e - t h i r d of the water i n t o a clean mixing v e s s e l ; open two cans of FCOJ t a k i n g care not to i n c o r p o r a t e water i n the contents and not to s p i l l the p r o d u c t add the contents of the 2 cans to the c h i l l e d water i n the mixing v e s s e l ; from the graduated c y l i n d e r pour water i n t o each of the emptied cans to r i n s e the remaining product t a k i n g care not to o v e r f l o w ; add the r i n s i n g s c a r e f u l l y to the mixing v e s s e l ; pour a l l the remaining water from the graduated c y l i n d e r i n t o the mixing v e s s e l ; and f i n a l l y , s t i r or mix the contents of the mixing vessel very w e l l making c e r t a i n there i s no unmixed concent r a t e on the bottom. This method i n p r a c t i c e worked extremely w e l l using premarked v o l u m e t r i c c y l i n d e r s , two of which were a v a i l a b l e at each t e s t i n g l o c a t i o n (glass c y l i n d e r s should not be considered i n t h i s s i t u a t i o n because of the ease of breakage). Of course, i d e n t i c a l r e c o n s t i t u t i o n methodology could be a p p l i e d to f r o z e n concentrated g r a p e f r u i t or other c i t r u s j u i c e s as w e l l as f o r FCOJ as discussed above. The presence of surface or f l o a t i n g j u i c e sacs may s i g n i f i c a n t l y a f f e c t sensory e v a l u a t i o n of c i t r u s j u i c e s , Amount of f l o a t i n g pulp i n any p a r t i c u l a r r e t a i l product i s p r i m a r i l y dependent on corporate s p e c i f i c a t i o n s as per t h e i r b e l i e f s . When t e s t i n g frozen concentrated c i t r u s products c o n t a i n i n g s i g n i f i -

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cant amounts of f l o a t i n g p u l p , care must be e x e r c i s e d i n the r e c o n s t i t u t i o n process and i n dissémination of j u i c e s f o r sensory e v a l u a t i o n work. I f the e n t i r e can contents are to be used, there i s no problem i n r e c o n s t i t u t i o n . However, i f a h a l f can i s to used f o r one treatment, f o r example, and the other h a l f f o r another treatment, a thorough mixing of the thawed concentrate j u s t p r i o r to pouring w i l l a i d i n o b t a i n i n g equal d i s t r i b u t i o n of the s u r f a c e pulp i n both samples. Again a t the time of dispensing of j u i c e s to the i n d i v i d u a l cups, another thorough mixing w i l l insure reasonably equal d i s t r i b u t i o n of top pulp among the s e v e r a l cups. Of course, mixing w i l l a l s o i n s u r e good bottom pulp d i s t r i b u t i o n as w e l l . The above holds t r u e f o r r e t a i l s i n g l e strength c i t r u s j u i c e s as w e l l . Holding of C i t r u s J u i c e s P r i o r to Sensory E v a l u a t i o n . A l l types of c i t r u s j u i c e s should undergo sensory e v a l u a t i o n i n as s h o r t a p e r i o d o f time because o f the p o s s i b i l i t room temperature, time between p r e p a r a t i o n and a c t u a l t e s t i n g can be e s p e c i a l l y c r i t i c a l . C e r t a i n l y , j u i c e prepared one day f o r next day t e s t i n g should not be considered even w i t h overnight r e f r i g e r a t i o n . G e n e r a l l y , the most p r a c t i c a l s i t u a t i o n i s morning p r e p a r a t i o n of j u i c e s f o r a morning sensory e v a l u a t i o n s e s s i o n and an afternoon p r e p a r a t i o n f o r an afternoon s e s s i o n . Can-to-Can V a r i a t i o n . Experience has shown the v a r i a t i o n i n commercial products w i t h i d e n t i c a l codes f o r F l o r i d a product to be minimal. The small amount of v a r i a t i o n t h a t does occur i s due to the nature of the manufacturing process which g e n e r a l l y i s not of the batch type. Production runs of from one to several hours d u r a t i o n , and i n c l u d i n g tremendous amounts of product, c a r r y the same code w i t h a l l of the v a r i a t i o n s of product e x i s t i n g , w i t h i n c e r t a i n p l a n t s p e c i f i c a t i o n s of course. However, p l a n t s having modern concentrate bulk-tank storage c a p a c i t y i n the 50,000150,000 gal range (tank farms) and which u t i l i z e computer systems to meter out product f o r r e t a i l pack are i n a p o s i t i o n to be able to produce a more uniform product, perhaps, than a p l a n t without such a c a p a b i l i t y . I t has been the p r a c t i c e i n r e t a i l FCOJ and c h i l l e d j u i c e seasonal surveys a t AREC to r e - e v a l u a t e samples exh i b i t i n g any f l a v o r q u a l i t y problems. O c c a s i o n a l l y the second or t h i r d cans t e s t e d w i l l vary s i g n i f i c a n t l y i n f l a v o r q u a l i t y from the o r i g i n a l i n d i c a t i n g r e a l can-to-can v a r i a t i o n . For a l l p r a c t i c a l purposes, however, s i n g l e cans of s i m i l a r l y coded product can be used i n sensory e v a l u a t i o n work w i t h a minimum amount of can-to-can v a r i a t i o n to be expected. III.

Conducting Sensory Tests

Choosing a Sensory T e s t . The types o f sensory methods a v a i l able f o r c i t r u s e v a l u a t i o n are numerous. Two e x c e l l e n t r e f e r -

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ences covering these methods i n d e t a i l are the "Manual on Sensory Testing Methods" {5) and "Laboratory Methods f o r Sensory E v a l u a t i o n of Food" (6). S e l e c t i n g the r i g h t t e s t f o r e v a l u a t i n g a c i t r u s product as f o r other products depends p r i m a r i l y on what i n formation i s d e s i r e d . Often several types of t e s t may be a v a i l able f o r answering the same q u e s t i o n ( s ) . I t then may be a matter of t r i a l and e r r o r to f i n d the one t e s t best s u i t e d f o r a p a r t i c u l a r s i t u a t i o n . O c c a s i o n a l l y , m o d i f i c a t i o n s of e x i s t i n g t e s t s or newly designed t e s t s are r e q u i r e d i n order to o b t a i n c e r t a i n i n f o r m a t i o n . Whatever t e s t i s chosen, s t a t i s t i c a l e v a l u a t i o n of the data should be made. Experience has shown t h a t d i f f e r e n c e s between c i t r u s samples can perhaps best be detected using the t r i a n g l e t e s t f o r orange, Temple, tangerine and a l l i e d products and p a i r e d comparison f o r g r a p e f r u i t , K-early and s i m i l a r products. Because of the i n herent b i t t e r n e s s and r e l a t i v e l y high a c i d i n g r a p e f r u i t the amount of back and f o r t can r e s u l t i n s i g n i f i c a n U t i l i z a t i o n o f a standard c i t r u s j u i c e product i n a sensory method i s not a d v i s a b l e i n c e r t a i n instances because of the t r e mendous amount of v a r i a b i l i t y i n the j u i c e s due to c u r r e n t commercial production p r a c t i c e s . The primary v a r i a b l e s t h a t e x i s t i n the processing i n d u s t r y i n c l u d e : v a r i e t a l d i f f e r e n c e s , e x t r a c t o r and f i n i s h e r v a r i a b l e s , and v a r i a b l e usages of essence and/or essence o i l , peel o i l , and cutback j u i c e . C e r t a i n l y , a seasonal survey-type f l a v o r study would be a poor place to employ a standard j u i c e . However, a standard j u i c e could perhaps be used to good advantage i n a p l a n t by q u a l i t y - c o n t r o l personnel where v a r i a b l e s would conceiveably be reduced to a minimum. As more p l a n t s convert to tank-farm operations equipped w i t h s o p h i s t i c a t e d computers, an end r e s u l t w i l l most s u r e l y be a more u n i form or standardized product. Of course there are some b a s i c problems i n making s t a n d a r d - f l a v o r e d c i t r u s j u i c e s . However, using c a r e , volumes of j u i c e s to be used as standard j u i c e s and having s a t i s f a c t o r y f l a v o r a t t r i b u t e s could be produced each year and u t i l i z e d from f r o z e n storage as necessary i n p l a n t s producing r e l a t i v e l y standardized products. Serving V e s s e l s . For c i t r u s j u i c e s both g l a s s and paper s e r v i n g c o n t a i n e r s may be s u c c e s s f u l l y used. I f g l a s s c o n t a i n e r s are used, c e r t a i n precautions should be taken to i n s u r e best r e s u l t s : (a) make sure t h a t the g l a s s e s are thoroughly r i n s e d f o l l o w i n g washing to e l i m i n a t e any r e s i d u a l soap or detergent o f f - f l a v o r s which might enter the c i t r u s j u i c e served next i n the g l a s s e s ; (b) f o r obvious reasons make sure t h a t high standards of hygiene are p r a c t i c e d i n washing the g l a s s e s by using c l e a n , h o t , soapy w a t e r , no washing of ash t r a y s i n the d i s h w a t e r , e t c . ; (c) use of ruby-red g l a s s e s aids i n reducing any c o l o r bias arid; (d) any g l a s s e s developing cracks or having chipped rims should be discarded immediately.

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I f paper cups are used, only the t r e a t e d type should be employed as c e r t a i n untreated cups can impart a "paper" or " c a r d board" f l a v o r to the j u i c e . Treated cups w i l l a l s o maintain t h e i r i n t e g r i t y longer. Use of c e r t a i n marking pens to mark the cups should be avoided i f i t i s noted t h a t the ink imparts a l i n g e r i n g s o l v e n t aroma. Volume of J u i c e to Serve. Experience has shown t h a t about 1 1/2 to 2 oz of c i t r u s j u i c e i s s u f f i c i e n t f o r a s u c c e s s f u l sensory judgment. Anything l e s s than about 1 1/2 oz w i l l very l i k e l y r e s u l t i n 1 or 2 i n d i v i d u a l s asking f o r more j u i c e , e s p e c i a l l y i f a t e s t such as the t r i a n g l e t e s t i s being used. However, i t i s important t h a t however much j u i c e i s presented to the p a n e l i s t , enough empty space i s l e f t i n the g l a s s or cup to a l l o w a t l e a s t a g e n t l e s w i r l i n g of the j u i c e by the p a n e l i s t i n order f o r t h a t i n d i v i d u a l to b e t t e r d e t e c t aroma c h a r a c t e r i s t i c s of the j u i c e . Of course 1 1/2 or 2 oz of j u i c e as f o r c e r t a i n consumer-type t a s t e panels or when a new product i s being looked a t ; i t then may be d e s i r a b l e f o r consumption o f a f u l l 4 o r 6 oz s e r v i n g , an amount such as an o r d i n a r y consumer might i n g e s t a t one time. G e n e r a l l y , only a s i n g l e j u i c e or perhaps two a t the most should be considered per session w i t h the l a r g e r volume of j u i c e . Number of J u i c e s to Serve. S u b s t a n t i a l l y more orange, t a n g e r i n e , Temple, M u r c o t t , e t c . j u i c e samples can e f f e c t i v e l y be served f o r sensory e v a l u a t i o n a t one session than can g r a p e f r u i t j u i c e s . The primary reasons f o r t h i s are probably because of the notable amount of b i t t e r n e s s found i n g r a p e f r u i t j u i c e and the r e l a t i v e l y l e s s a c i d a s s o c i a t e d w i t h the orange and s i m i l a r j u i c e s than g r a p e f r u i t j u i c e . B i t t e r n e s s does not normally occur i n F l o r i d a orange j u i c e . However, b i t t e r n e s s can e x i s t i n orange j u i c e under c e r t a i n circumstances as discussed i n the s e c t i o n i n t h i s chapter on b i t t e r n e s s . As to a c t u a l numbers of j u i c e s t h a t may be sampled a t one s e s s i o n , a maximum of about 4-6 orange j u i c e s or 3-4 g r a p e f r u i t j u i c e s are the p r a c t i c a l l i m i t f o r judges. We have found t h a t anything much beyond these maximums s e v e r e l y tax the p h y s i c a l and mental c a p a b i l i t i e s of the average p a n e l i s t r e s u l t i n g i n a d e t e r i o r a t i o n of r e l i a b i l i t y of r e s u l t s . In a consumer-type p a n e l , probably, 1 or 2 j u i c e s per session would y i e l d the best r e s u l t s . However, f o r a q u a l i t y c o n t r o l person i n a p l a n t , USDA i n s p e c t o r , o r other person i n v o l v e d w i t h many e v a l u a t i o n s , numerous orange and perhaps somewhat fewer g r a p e f r u i t j u i c e samples could be t e s t e d s u c c e s s f u l l y w i t h short breaks i n between samples over several hours time. At AREC the author and two other e x perienced c i t r u s sensory evaluators once s u c c e s s f u l l y ranked by preference a t one session as many as 16 d i f f e r e n t f o r m u l a t i o n s of a new c i t r u s j u i c e product c a l l e d "Sunshine-?" i n which 7 types of

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c i t r u s were blended. Taste f a t i g u e was evident i n t h i s arduous session but not so much as to s i g n i f i c a n t l y compromise the o v e r all results. Serving Temperature. For consumer or acceptance/preference t e s t i n g , c i t r u s j u i c e s should be served a t a temperature i n the range o f 7-13°C or the approximate temperature a t which j u i c e s are presumably consumed. However, f o r d i s c r i m i n a t i o n and perhaps c e r t a i n other t e s t s , t e s t i n g a t warmer temperatures i n the approximate range 19-24°C may be s u p e r i o r to the lower temperature range. To t e s t t h i s hypothesis derived through e x p e r i ence, the f i r s t o f several t e s t s along these l i n e s was r e c e n t l y completed (15). In t h i s p a r t i c u l a r study, three d i f f e r e n t comp o s i t e commercial FCOJs were evaluated over a 2-month period by an experienced 12-member t a s t e p a n e l , 3 times a t 10°C and 3 times at 24°C, without g i v i n g the p a n e l i s t s any knowledge o f the purpose o f the t e s t i n g e i t h e the samples, mean f l a v o higher a t the 95% confidence l e v e l f o r the samples served a t 10°C than those a t 24°C. No s i g n i f i c a n t d i f f e r e n c e was found i n the remaining sample. E x a c t l y why a p a r t i c u l a r c i t r u s j u i c e may be p r e f e r r e d c h i l l e d t o u n c h i l l e d by experienced p a n e l i s t s i s not y e t known. The c o o l e r s e r v i n g temperature could be the o v e r r i d i n g f a c t o r . Another p o s s i b i l i t y might be i n a suppression or masking o f c e r t a i n undesirable f l a v o r notes by the c h i l l e d j u i c e . At whatever temperature c i t r u s products are served, care must be e x e r c i s e d to make c e r t a i n a l l samples have l i k e s e r v i n g temperatures. The l e a s t d i f f e r e n c e i f detected by p a n e l i s t s w i l l confuse them and r e s u l t i n questionable d a t a . Number o f Types o f C i t r u s Products Per Session. Should ever the occasion a r i s e when more than one type o f c i t r u s product r e q u i r e s sensory e v a l u a t i o n , i t i s best to schedule a separate s e s s i o n f o r each product or group o f l i k e products. The human mind has a great tendency f o r automatic comparison and carryover of one experience to the next which i n the case o f sensory e v a l u a t i o n i s not good should two or more d i s s i m i l a r products be compared a t one s e s s i o n . In l a b o r a t o r y t e s t s a t AREC when a few f r e s h orange j u i c e samples were included g e n e r a l l y one a t a time several times among many r e c o n s t i t u t e d FCOJ samples i n a l a r g e survey study, the f r e s h j u i c e samples graded out lower i n f l a v o r q u a l i t y than the r e c o n s t i t u t e d FCOJ samples. However, when these same f r e s h j u i c e samples were graded together as a group the scores i n c r e a s e d . What to T e l l the P a n e l i s t . P a n e l i s t s should be given as l i t t l e information as p o s s i b l e about the t e s t a t hand so as not to i n f l u e n c e r e s u l t s . P a r t o f the same study as was described i n the s e c t i o n d e a l i n g w i t h sensory e v a l u a t i o n of more than one type of c i t r u s product a t one s e s s i o n a l s o d e a l t w i t h the problem a t

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hand. Results showed the f r e s h j u i c e samples grading out w e l l below the r e c o n s t i t u t e d FCOJ samples when no mention of the f r e s h j u i c e sample was made. When several o f the f r e s h j u i c e s were run together and the p a n e l i s t s were s t i l l not t o l d what the product was, scores improved s i g n i f i c a n t l y . However, when the p a n e l i s t s were f i n a l l y t o l d t h a t f r e s h orange j u i c e was to be t e s t e d , the scores again improved measurably showing e x i s t e n c e of a preconceived impression o r e x p e c t a t i o n e r r o r . A s i t u a t i o n where p a n e l i s t s should be informed of the p r o duct they are e v a l u a t i n g i s p o s s i b l e when c e r t a i n new and/or q u i t e d i f f e r e n t products are being t e s t e d . Two prime examples of t h i s s i t u a t i o n o c c u r r i n g a t AREC involved f l a v o r s t u d i e s of an i s o t o n i c orange j u i c e t h i r s t quencher type o f new product d e v e l oped a t the Lake A l f r e d Center, and b i t t e r n e s s s t u d i e s u t i l i z i n g model g r a p e f r u i t j u i c e f o r m u l a t i o n s . Some p a n e l i s t s were q u i t e u n f o r g i v i n g when not t o l d beforehand what i t was they were about to e v a l u a t e . For d i s c r i m i n a t i o from product i d e n t i t y bein Another f a c t o r to consider i s whether or not to have a l i s t of f l a v o r a t t r i b u t e s a v a i l a b l e f o r the t a s t e p a n e l i s t to peruse w h i l e judging the product. Perhaps i n c e r t a i n s p e c i a l i n s t a n c e s , such as when s p e c i f i c f l a v o r a t t r i b u t e s or i n t e n s i t y of a t t r i b u t e s are being c o n s i d e r e d , a l i s t i n g may be used. However, normally no such l i s t i n g should be given a l l o w i n g each p a n e l i s t to judge the product "from s c r a t c h " w i t h a minimum of preconceived impressions e n t e r i n g i n . Also,no matter how many items might appear on a l i s t i n g of f l a v o r a t t r i b u t e s (and there could e a s i l y be as many as 20 f o r c i t r u s p r o d u c t s ) , the occasional odd f l a v o r w i l l appear and the p a n e l i s t should f e e l a b s o l u t e l y f r e e to d e s c r i b e i t without the presence o f d i s t r a c t i n g d a t a . Value of S o l i c i t i n g Comments. Very o f t e n number data t h a t i s c o l l e c t e d through sensory e v a l u a t i o n , whether found s i g n i f i cant or not, can be explained f u l l y or a t l e a s t p a r t i a l l y through examination of p a n e l i s t s ' comments which were a l s o obtained a t the time of t e s t i n g . Every sensory e v a l u a t i o n t e s t should have space f o r comments. This i s both f o r the p s y c h o l o g i c a l advantage i t gives the p a n e l i s t s to express t h e i r f e e l i n g s on some f a c t o r t h a t perhaps d i d not or could not be included as p a r t o f the t e s t and, a l s o , to b e t t e r understand how/why the p a n e l i s t s answered the way they d i d . I t i s p o s s i b l e a t times to u t i l i z e frequency of i n d i v i d u a l comments and/or t o t a l comments f o r samples i n s t a t i s t i c a l e v a l u a t i o n o f d a t a , thus p o s s i b l y g a i n i n g a d d i t i o n a l i n formation from the t e s t . Value of Using a Regular,Trained or Experienced Sensory Panel. An advantage i n having t h i s type of panel i s the f a c i l i t a t i o n of i n t e r p r e t a t i o n and understanding o f each p a n e l i s t ' s e v a l u a t i o n i t makes f o r the s u p e r v i s o r . In time an a l e r t panel s u p e r v i s o r w i l l know and understand i n d i v i d u a l p r e f e r e n c e s , d i s -

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l i k e s , and i d i o s y n c r a s i e s and thus, make a b e t t e r e v a l u a t i o n of the data gleaned from the sensory t e s t . For example, one capable p a n e l i s t a t AREC possesses e x c e l l e n t aroma and t a s t e a c u i t y f o r the presence o f d i a c e t y l i n c i t r u s products; however, u n l i k e the other p a n e l i s t s , does not p a r t i c u l a r l y o b j e c t to the presence of t h i s " o f f - f l a v o r " i n c i t r u s products. A s i m i l a r s i t u a t i o n can e x i s t i f b i t t e r n e s s i s detected i n orange j u i c e by c e r t a i n capable p a n e l i s t s . B i t t e r n e s s normally i s considered h i g h l y u n d e s i r able i n orange j u i c e , however, the occasional i n d i v i d u a l able to d e t e c t b i t t e r n e s s does not consider i t e s p e c i a l l y undesirable and w i l l f a i l to r a t e the j u i c e down s i g n i f i c a n t l y . Each i n d i v i d u a l has h i s or her own ranking f o r r e l a t i v e importance of f l a v o r a t t r i b u t e s c o n s t i t u t i n g good j u i c e and bad j u i c e . The a l e r t panel s u p e r v i s o r w i l l a l s o come to know about each r e g u l a r p a n e l i s t ' s a b i l i t y to d e t e c t the many o f f - f l a v o r s t h a t could p o s s i b l y occur i n c i t r u s products Of primary concern other than the common swee p a n e l i s t s ' a b i l i t i e s to ( n a r i n g i n and/or l i m o n i n induced), (b) heated, processed or pumpout o f f - f l a v o r , (c) excess peel o i l y , (d) excess and/or poor essency f l a v o r , and (e) the f o l l o w i n g o f f - f l a v o r s : cardboard, t a l l o w y , c a s t o r o i l , d i a c e t y l or b u t t e r m i l k , green or immature, overmature or s t a l e f r u i t , and s p o i l e d f r u i t . IV.

Varietal

Considerations

In F l o r i d a the range of f l a v o r s i n orange j u i c e products due to v a r i e t a l d i f f e r e n c e s can be very wide. Each mature major F l o r i d a v a r i e t y possesses a general f l a v o r p r o f i l e as f o l l o w s : Hamlin ( e a r l y season) - moderate orange f l a v o r w i t h no top notes; Pineapple (midseason) - moderate to f u l l orange f l a v o r w i t h a f r u i t y top note; V a l e n c i a ( l a t e season) - f u l l orange f l a v o r w i t h good body; C i t r u s r e t i c u l a t a , (or hybrids t h e r e o f ) , such as t a n g e r i n e s , Temples and tangelos possess d i s t i n c t f l a v o r top notes q u i t e u n l i k e the 3 major orange v a r i e t i e s . For t h i s reason i n the manufacture of FCOJ, f o r example, only up to 10% by volume of the concentrated blend of j u i c e can be of the C i t r u s r e t i c u l a t a type (4J. The same standards l i m i t C i t r u s aurantiurn (sour orange) to 5% by volume. The major F l o r i d a g r a p e f r u i t v a r i e t i e s are the n e a r l y seedl e s s Marsh white and a l l i e d pigmented forms, such as Marsh P i n k , Thompson and Ruby Red, and the seedy Duncan. D i f f e r e n c e s i n f l a v o r between the n e a r l y seedless and seedy v a r i e t i e s are not l a r g e , however, the consensus of the author and others f a m i l i a r w i t h both types i s t h a t seedy f r u i t possess a somewhat f u l l e r , more balanced g r a p e f r u i t f l a v o r than seedless. Extreme care must be e x e r c i s e d when applying general f l a v o r terminology to orange, mandarin or g r a p e f r u i t types of c i t r u s when there are so many v a r i e t a l f l a v o r d i f f e r e n c e s w i t h i n the basic types.

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Of course there are many other f a c t o r s involved i n f l a v o r of i n d i v i d u a l v a r i e t i e s o f c i t r u s such as seasonal v a r i a t i o n , time of harvest or m a t u r i t y , s o i l t y p e , r o o t s t o c k , geographical considerations, etc. V.

Color E f f e c t

A l a r g e consumer sensory t e s t conducted a t the 1965 World's F a i r i n New York (16) i n v o l v i n g several d i f f e r e n t orange j u i c e s of v a r y i n g q u a l i t y a t t r i b u t e s , i n c l u d i n g c o l o r , showed t h a t c o l or was one of the most i n f l u e n t i a l f a c t o r s i n j u i c e preference. In a recent study by Huggart et^ a l . (17), g r a p e f r u i t j u i c e s were prepared having varying l e v e l s o f v i s u a l c o l o r from white through p i n k , w i t h a l l other f a c t o r s being equal. A l a r g e consumer t e s t of the 5 products showed c o l o r to have a s i g n i f i c a n t e f f e c t i n acceptance of p a i r e d j u i c e s In g e n e r a l the y e l l o w i s h - w h i t e to brownish-yellow (chamois or pink j u i c e s . With there being c o l o r biases/preferences i n both orange and g r a p e f r u i t j u i c e s , care must be e x e r c i s e d i n sensory e v a l u a t i o n work to i n s u r e against these c o l o r b i a s e s . An e f f e c t i v e means of n e u t r a l i z i n g any c o l o r bias i n c i t r u s sensory e v a l u a t i o n work has been found to be the u t i l i z a t i o n of red l i g h t s i n the booths used i n making the e v a l u a t i o n . A s p e c i a l s i t u a t i o n can e x i s t i n F l o r i d a and other c i t r u s producing areas r e l a t i v e to c o l o r bias i n orange j u i c e . Indiv i d u a l s working w i t h c i t r u s are f a m i l i a r w i t h the d i f f e r e n t v a r i e t i e s and have developed t h e i r opinions as to r e l a t i v e q u a l i t y . Hamlin orange j u i c e s , f o r example, are considered by many to be the l e a s t p r e f e r r e d of the major v a r i e t i e s whereas V a l e n c i a g e n e r a l l y passes as the most p r e f e r r e d . Color of Hamlin j u i c e i s l i g h t orange whereas V a l e n c i a j u i c e i s dark orange, thus should p a n e l i s t s who are f a m i l i a r w i t h the f l a v o r of d i f f e r e n t orange v a r i e t i e s be able to see the c o l o r of the orange j u i c e s being e v a l u a t e d , a p o s s i b l e bias due to c o l o r might e x i s t . A g a i n , the use of red l i g h t s i n the panel area to n e u t r a l i z e product c o l o r should be u t i l i z e d . VI.

S p e c i f i c Citrus Flavor Attributes

Bitterness Considerations. B i t t e r n e s s i s not normally found i n commercial F l o r i d a orange j u i c e , but when i t does o c c u r , the source of the b i t t e r n e s s i s d i f f i c u l t to determine from a sensory e v a l u a t i o n standpoint. B i t t e r n e s s , p r i m a r i l y from l i m onin (18), may be due to one or more of the f o l l o w i n g : too severe j u i c e e x t r a c t i o n , use of c e r t a i n B r a z i l i a n j u i c e (19), use of Navel oranges, excessive peel o i l probably g r e a t e r than 0.020% by volume, and perhaps other f a c t o r s as w e l l . Orange pulpwash (water e x t r a c t i o n of s o l u b l e f r u i t s o l i d s from orange pulp) can c o n t r i b u t e s i g n i f i c a n t l y to the b i t t e r n e s s

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and harshness (roughness) of orange j u i c e to which i t has been added (20). U.S. standards f o r grades of FCOJ (4) s t a t e "In i t s p r e p a r a t i o n , seeds, (except f o r embryonic seeds and small f r a g ments of seeds t h a t cannot be separated by good manufacturing p r a c t i c e ) and excess pulp are removed, and a properly prepared water e x t r a c t of the excess pulp so removed may be added." F l o r i d a , however, does not a l l o w pulpwash i n FCOJ ( 1 ) . Orange pulpwash i s allowed i n concentrated orange j u i c e f o r manufacturing " i f product i s not to be used i n the production of FCOJ i n r e t a i l or i n s t i t u t i o n a l s i z e d c o n t a i n e r s , i t may c o n t a i n s o l u b l e orange j u i c e s o l i d s recovered by aqueous e x t r a c t i o n or washing of pulp removed from the j u i c e contained i n the product, but not from pulp removed from other j u i c e " (21). And concentrated orange j u i c e f o r manufacturing can be used i n the manufacture of orange j u i c e from concentrate (22) and as 25% of the t o t a l o r ange j u i c e s o l i d s i n f i n i s h e d p a s t e u r i z e d orange j u i c e (23) A study i s c u r r e n t l y underwa use of orange pulpwash o u t s i d from concentrate and how much pulpwash has been used i n each of the products (20). Sensory e v a l u a t i o n of each product i s a l s o being c a r r i e d out along w i t h the other analyses. G r a p e f r u i t contain the b i t t e r p r i n c i p l e s l i m o n i n and n a r i n gin and these give g r a p e f r u i t products the c h a r a c t e r i s t i c " b i t e " f o r which the f r u i t i s noted. In an e f f o r t to understand more about consumer l i k e s and d i s l i k e s of g r a p e f r u i t and g r a p e f r u i t products, a p a r t of the f l a v o r research a t AREC has focused on the b i t t e r n e s s a t t r i b u t e . In conducting t h i s r e s e a r c h , and some o t h e r , a few r a t h e r unique problems arose t h a t w i l l be considered here. From a t e c h n i c a l p o i n t of view, there are extreme d i f f i c u l t i e s i n g e t t i n g w a t e r - i n s o l u b l e l i m o n i n i n t o aqueous m i x t u r e s , such as c i t r u s j u i c e s or model c i t r u s j u i c e s , without a d d i t i o n of an unwanted s o l v e n t such as a l c o h o l . Naringin may be added to c i t r u s j u i c e o r model c i t r u s j u i c e s without heating the products using the f o l l o w i n g method: s t a r t i n g w i t h high B r i x c i t r u s conc e n t r a t e , c a l c u l a t e the amount of water necessary to achieve the d e s i r e d f i n a l lower degrees B r i x concentrate or r e c o n s t i t u t e d j u i c e . Determine the amount of n a r i n g i n to add to achieve des i r e d ppm i n the f i n a l product. Heat a p o r t i o n of the water to about 65 C and add the n a r i n g i n w h i l e s t i r r i n g . Bring the water/ n a r i n g i n s o l u t i o n to room temperature and add to the concentrate or r e c o n s t i t u t e d j u i c e . Add any remaining r e q u i r e d water. This method w i l l work up to a t l e a s t 2,000 ppm n a r i n g i n i n the f i n a l r e c o n s t i t u t e d j u i c e . Concentrate t r e a t e d t h u s l y may be frozen f o r l a t e r use. Care must be taken, however, not to c h i l l any s i n g l e - s t r e n g t h j u i c e having added n a r i n g i n as p r e c i p i t a t i o n w i l l occur. One method used by F e l l e r s and C a r t e r (24) to i n c o r p o r a t e natural b i t t e r n e s s i n t o g r a p e f r u i t j u i c e was to add c e r t a i n p e r centages o f a frozen concentrated g r a p e f r u i t j u i c e pulpwash obt a i n e d from use of "hard-squeeze" f r u i t . Reference has already been made i n the s e c t i o n d e a l i n g with

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" S p e c i a l C r i t e r i a f o r S e l e c t i n g P a n e l i s t s " on the i n a b i l i t y of c e r t a i n i n d i v i d u a l s to d e t e c t n a r i n g i n a t concentrations as high as 2,000 ppm i n aqueous model g r a p e f r u i t j u i c e systems or grapef r u i t j u i c e . Obviously those p a n e l i s t s would be o f no value i n d i s c r i m i n a t o r y sensory work i n v o l v i n g n a r i n g i n . In a recent l a r g e consumer t e s t (25) i n which n a r i n g i n was added to f i v e l i k e p o r t i o n s o f a good f r o z e n concentrated grapef r u i t j u i c e i n i n c r e a s i n g q u a n t i t i e s up to about 2,000 ppm i n the r e c o n s t i t u t e d j u i c e , a key f i n d i n g was t h a t g e n e r a l l y , as the l e v e l o f n a r i n g i n i n c r e a s e d , perceived b i t t e r n e s s and t a r t n e s s o r sourness increased and perceived sweetness decreased. The primary r a m i f i c a t i o n of t h i s f i n d i n g i s t h a t extreme care must be used when i n t e r p r e t i n g sensory e v a l u a t i o n data on f r e s h and processed g r a p e f r u i t j u i c e products as regards b i t t e r n e s s , sweetness and sourness. O c c a s i o n a l l y g r a p e f r u i t j u i c e samples can be e x c e s s i v e l y b i t t e r , and can cause problem u a t i n g j u i c e s having thes lem i s t h a t the intense b i t t e r n e s s can render one s sensory a c u i t y n e a r l y useless f o r a s i g n i f i c a n t p e r i o d of time r e s u l t i n g i n p o s s i b l e erroneous data f o r succeeding samples, even w i t h i n g e s t i o n of a c r a c k e r and/or water. According to Dougherty and Barros (26) i t i s p o s s i b l e to have g r e a t e r than 1,000 ppm n a r i n g i n and/ or more than 10 ppm l i m o n i n i n c e r t a i n commercial g r a p e f r u i t samp l e s . However, to have both maxima occur together would be indeed r a r e . Of course,randomization of samples should work to e q u a l i z e any c o n t r a s t e f f e c t caused by the b i t t e r sample. 1

Heated, Processed or Pumpout F l a v o r . P r i o r to the advent of the t e m p e r a t u r e - a c c e l e r a t e d , s h o r t - t i m e evaporator (TASTE), commercial FCOJ and f r o z e n concentrated g r a p e f r u i t j u i c e samples o f t e n possessed an u n d e s i r a b l e heated, processed or pumpout f l a v o r t h a t was not masked e n t i r e l y by o i l , cutback, or essence a d d i t i o n . Current manufacturing p r a c t i c e s have e f f e c t i v e l y e l i m i n a t e d t h i s problem i n the products. However, p a s t e u r i z e d orange j u i c e , orange j u i c e from c o n c e n t r a t e , canned orange j u i c e and a l l t h e i r g r a p e f r u i t j u i c e counterparts possess a wide range o f heated or processed f l a v o r , from a n e g l i g i b l e amount to extreme. As to how t h i s heated f l a v o r i s evaluated apparently d i f f e r s from person to person and from group to group. According to U. S. standards f o r grades o f orange j u i c e from c o n c e n t r a t e (21), f o r example, f o r t h i s product to make U.S. Grade A i t "possesses a very good f l a vor" when "very good f l a v o r means t h a t the f l a v o r i s f i n e , d i s t i n c t , and s u b s t a n t i a l l y t y p i c a l o f orange j u i c e e x t r a c t e d from f r e s h , mature sweet oranges; i s f r e e from o f f - f l a v o r s of any k i n d ; e t c " . Since a heated or processed f l a v o r i s considered to be an o f f - f l a v o r and e x i s t s i n most commercial orange j u i c e from conc e n t r a t e , apparently the U.S. Standards do not take t h i s f l a v o r i n t o account. I t i s i n t e r e s t i n g to note t h a t f o r proposed U.S. standards f o r grades o f g r a p e f r u i t j u i c e ( 2 7 ) , t h a t i n the p r o -

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posed f l a v o r d e f i n i t i o n s f o r both g r a p e f r u i t j u i c e and grapef r u i t j u i c e from c o n c e n t r a t e , t h a t "The g r a p e f r u i t j u i c e may be s l i g h t l y a f f e c t e d by p r o c e s s i n g , packaging or storage c o n d i t i o n s . " Apparently l i k e so many other f l a v o r a t t r i b u t e s o f c i t r u s products, heated, processed or pumpout type o f o f f - f l a v o r s vary c o n s i d e r a b l y i n i n d i v i d u a l t h r e s h o l d and t o l e r a n c e l e v e l s . The author, f o r example, possesses both a low t h r e s h o l d and t o l e r a n c e f o r t h i s p a r t i c u l a r o f f - f l a v o r i n any type o f c i t r u s product. Cardboard, C i t r u s O x i d i z e d , Castor O i l , Tallowy and CQF O f f - F l a v o r . For t h i s group o f o f f - f l a v o r s there e x i s t s much conf u s i o n . Olsen e t al_. (28) b e l i e v e d these o f f - f l a v o r s were the r e s u l t o f o x i d a t i o n o f substances i n orange concentrate s i n c e they were able to produce these o f f - f l a v o r s i n concentrate by whipping a i r i n t o the product. B l a i r e t al_. (29) i n t h e i r s t u d i e s r e f e r r e d to these o f f - f l a v o r s as the "COF e f f e c t " COF being an abbreviation f o r eithe or c a s t o r o i l f l a v o r . A c t u a l l deed be a l l i e d as being products o f o x i d a t i o n , however, i n most instances j u i c e s so a f f e c t e d may be c h a r a c t e r i z e d by the e x p e r i enced i n d i v i d u a l as possessing e i t h e r cardboard, c a s t o r o i l , or t a l l o w y o f f - f l a v o r q u i t e d i s t i n c t l y . Should none o f the above apply then usage o f the more general term c i t r u s o x i d i z e d f l a v o r could be used. Vague terminology such as COF should not be used and can only serve to confuse t h i s p a r t i c u l a r f l a v o r s i t u a t i o n . Essence C o n s i d e r a t i o n s . P a r t i c i p a t i o n i n survey-type sensory e v a l u a t i o n panels of F l o r i d a FCOJ, orange j u i c e from conc e n t r a t e , p a s t e u r i z e d orange j u i c e , and g r a p e f r u i t from concent r a t e a t AREC f o r the past 15 years has r e s u l t e d i n many observations by the author as to the dynamic f l a v o r p i c t u r e presented to the consumer by the c i t r u s i n d u s t r y . Perhaps one o f the major changes noted has been the marked increase i n the use o f essence and essence o i l i n recent y e a r s , notably i n the orange j u i c e products. No f i g u r e s are a v a i l a b l e on amount o f FCOJ packed i n F l o r i d a c o n t a i n i n g essence/essence o i l but a f i g u r e between 50 and 75% may not be u n r e a l i s t i c . P r i o r t o essence usage as a f l a v o r - e n h a n c i n g m a t e r i a l , cutback ( f r e s h l y e x t r a c t e d orange j u i c e ) , cold-pressed orange o i l , f o l d e d o i l s or combinations o f the above were the prime f l a v o r enhancement m a t e r i a l s . The a d d i t i o n of cutback j u i c e to the concentrated j u i c e from the evaporator was the key t o the i n v e n t i o n o f FCOJ (30) i n 1948 and i s s t i l l being used today by a s i g n i f i c a n t number o f F l o r i d a processors. The major problem i n sensory e v a l u a t i o n of c i t r u s products c o n t a i n i n g essence appears to l i e mainly i n i n d i v i d u a l p r e f e r ences as regards essence-add products and i n d i v i d u a l thresholds f o r the essence f r a c t i o n i n these products. A s i g n i f i c a n t number of r e g u l a r , t r a i n e d p a n e l i s t s a t AREC, f o r example, upon d e t e c t i o n o f c e r t a i n essences i n c i t r u s products might grade the p r o -

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duct down and comment t h a t the product i s "perfumey", " a r t i f i c i a l " or " s y n t h e t i c " f l a v o r e d . Others, a l s o d e t e c t i n g the presence of essence, might grade the product up and comment " t a s t e s l i k e f r e s h j u i c e " or something s i m i l a r . And then there are those i n d i v i d u a l s who apparently are not a f f e c t e d one way or the other by essence a d d i t i o n . Dougherty et afL (31) found t h a t orange essences produced by d i f f e r e n t recovery systems were d i s t i n c t and d i f f e r e d c o n s i d e r ably i n t h e i r chemical composition and s t r e n g t h ; i n some cases there was an optimum l e v e l of essence t h a t could be added to FCOJ t h a t would produce a b e t t e r f l a v o r e d j u i c e ; t h a t higher l e v e l s of essence could r e s u l t i n a lowering of f l a v o r q u a l i t y ; and t h a t essence-enhanced FCOJs d i d not l o s e s i g n i f i c a n t amounts of essence f l a v o r over a 30-month f r o z e n storage p e r i o d . Results of t h i s study showed some of the reasons why there were o f t e n such a d i s p a r i t y of grades and comments when essence-bearing FCOJs were being evaluate s t u d i e s ) . Not only wer due to numerous f a c t o r s (many of which have been discussed e l s e where i n t h i s c h a p t e r ) , but the essences used i n these products a l s o proved to be anything but standardized r e s u l t i n g i n a mixed bag o f f l a v o r sensations. Serving temperature has not been shown to a f f e c t f l a v o r i n essence-bearing c i t r u s products. However, i f temperature was found to be or suspected of being a s i g n i f i c a n t f a c t o r , s e r v i n g temperature o f samples destined f o r preference-type sensory e v a l u a t i o n should be made a t temperatures a t which the product would normally be consumed (presumably about 7-13 C). A f i n a l c o n s i d e r a t i o n concerning essence i n c i t r u s products i s t h a t depending on the type and/or q u a n t i t y of essence used, d e t e c t i o n may be by aroma, t a s t e or by both. Proper sensory e v a l u a t i o n of a l l c i t r u s products should i n c o r p o r a t e both aroma and t a s t e judgments - a t a s t e judgment alone j u s t not being enough i n many i n s t a n c e s . Should, f o r example, a sample c o n t a i n ing poor essence, "wet dog" essence, " b u t t e r m i l k " essence, e t c . occur p r i m a r i l y i n the aroma f r a c t i o n , a judgment based on t a s t e alone might miss the off-aroma. VII.

Problems i n C i t r u s Sensory E v a l u a t i o n

Research

Avoiding F l a v o r H i g h l i g h t s i n Control Samples. Every e f f o r t should be made i n u t i l i z i n g a c h a r a c t e r i s t i c type of c i t r u s p r o duct when conducting c e r t a i n sensory e v a l u a t i o n s t u d i e s , espec i a l l y i f studying one or more s p e c i f i c q u a l i t y a t t r i b u t e s . The f o l l o w i n g are the major f l a v o r h i g h l i g h t s t h a t should be avoided: excessive cold-pressed o i l (greater than about 0.017% (v/v) i n orange; 0.010% (v/v) i n g r a p e f r u i t ) , moderate or high essence product, orange product having any d e t e c t a b l e b i t t e r n e s s or grapef r u i t having excessive b i t t e r n e s s ( g r e a t e r than about 4 ppm l i m o n i n or 500 ppm n a r i n g i n ) , degrees B r i x to % a c i d r a t i o s n e i -

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ther too low nor too high thus keeping product n e i t h e r too a c i d nor too sweet, too t h i n or watery,excessive top p u l p , and no h i n t of any o f the f o l l o w i n g o f f - f l a v o r s : cardboard, c a s t o r o i l , t a l lowy, d i a c e t y l ( b u t t e r m i l k ) , green o r immature, overmature o r s t a l e , o r s p o i l e d o r r o t t e n f r u i t ; i n short nothing unusual about the aroma o r t a s t e t h a t might confuse or d i s t r a c t the p a n e l i s t . Use o f Model Systems. From time to time i t may be d e s i r a b l e to u t i l i z e a model o r a r t i f i c i a l c i t r u s j u i c e system as an a i d i n studying c e r t a i n f l a v o r a t t r i b u t e s . A b i g advantage, of course, i s the r e s u l t a n t standard " j u i c e " being completely reproducible at any time. However, the b i g disadvantage i s t h a t no matter how well a c i t r u s product i s simulated i n a model j u i c e system, many t a s t e p a n e l i s t s apparently cannot f e e l r e a l l y comfortable when e v a l u a t i n g a purely a r t i f i c i a l product. A basic f o r m u l a t i o n f o r making a model o r a r t i f i c i a l grapef r u i t j u i c e , f o r example r i c a c i d (sourness), potassiu nate (body), n a r i n g i n ( b i t t e r n e s s ) , and cold-pressed g r a p e f r u i t o i l emulsion ( g r a p e f r u i t f l a v o r ) . Of course, use o f a d d i t i o n a l i n g r e d i e n t s to f u r t h e r r e f i n e the product could be added. To achieve an o i l emulsion s u i t a b l e to add t o a product such as d e s c r i b e d above, the f o l l o w i n g f o r m u l a t i o n may be used ( f o r an a p proximate 10% emulsion, f o r example, to produce about 200 m l ) : mash5ggum a c a c i a and 50 g c e r e l o s e to a f i n e powder and mix; add 20 ml o f good cold-pressed g r a p e f r u i t o i l to the mix and s t i r w e l l ; measure out 125 ml water and add j u s t enough t o the mix t o form a s l u r r y ; pour i n t o a blender using some more o f the measured water to r i n s e the c o n t a i n e r ; mix the s l u r r y using the blender f o r a short period o f time; pour the s l u r r y i n t o a cont a i n e r using the remainder o f the water to r i n s e the blender; c l o s e the c o n t a i n e r t i g h t l y and keep r e f r i g e r a t e d u n t i l ready to use. Approximate usage l e v e l s f o r such a f l a v o r emulsion might be 4 drops/100 ml of model or a r t i f i c i a l j u i c e . I s o t o n i c or High Energy Thirst-Quencher Type C i t r u s P r o ducts" Several years ago many formulations o f t h i s type orange j u i c e product were s u c c e s s f u l l y made a t AREC (32). F l a v o r s t u d i e s a s s o c i a t e d w i t h t h i s work by the author were complicated by the f a c t t h a t the energy d r i n k product was designed f o r a c t i v e people to quench t h i r s t and r e s t o r e energy. So t h a t even though b a s i c sensory e v a l u a t i o n work could be accomplished by p a n e l i s t s i n a booth, the products had to be thoroughly f i e l d t e s t e d , which they were, using l o c a l high school and c o l l e g e f o o t b a l l teams p r i m a r i l y . The a c t i v e f i e l d t r i a l s , f o r example, showed c o n c l u s i v e l y t h a t pulp l e v e l s had to be lowered s i g n i f i c a n t l y - a f a c t o r not considered important during the s t a t i c e v a l u a t i o n s . The importance o f t e s t i n g new o r improved c i t r u s products and where and how they w i l l u l t i m a t e l y be consumed cannot be o v e r l y s t r e s s e d . The success r a t e f o r new products i s poor

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enough without a product being introduced without having had a thorough research and development program c a r r i e d out on i t , i n c l u d i n g t r i a l consumer t e s t s . During the t e s t i n g o f the many f o r m u l a t i o n s of the t h i r s t quencher product, another problem arose - t h a t o f excessive time being r e q u i r e d f o r storage t e s t s of the products packed as s i n g l e strength j u i c e s i n g l a s s and cans. The answer here was a c c e l e r ated storage t e s t s u t i l i z i n g a l a r g e 43 C i n s u l a t e d box. Using t h i s system months o f storage a t room temperature (20-22 C) were condensed t o d a y s or a t most weeks w i t h sampling periods running every 2-3 days f o r sensory e v a l u a t i o n . Geographic C o n s i d e r a t i o n s . O c c a s i o n a l l y r e s u l t s o f d e c i s i o n s made a t AREC r e l a t i v e to new products or c e r t a i n f l a v o r a t t r i butes are found to d i f f e r when checked elsewhere, e x h i b i t i n g c e r t a i n geographic d i f f e r e n c e s i n t a s t e s or preferences. For e x ample, i n two d i f f e r e n (33)), minimum b i t t e r n e s both small t r a i n e d panel t e s t s and l a r g e r consumer t e s t s to d e f i n i t e l y i n c l u d e l e v e l s found to r e s u l t i n unacceptable o v e r a l l f l a v o r i n g r a p e f r u i t j u i c e products. The same samples, however, were r a t e d s i g n i f i c a n t l y higher o u t s i d e F l o r i d a . Perhaps Cent r a l F l o r i d i a n s are more aware of the tremendous range i n f l a v o r of g r a p e f r u i t harvested over the season and n a t u r a l l y tend to r a t e down a product e x h i b i t i n g what they f e e l and know to be an i n f e r i o r flavored j u i c e . A p o i n t to c o n s i d e r w i t h f r e s h f r u i t f l a v o r i s t h a t a l o t of f r e s h f r u i t , e s p e c i a l l y oranges, found to possess f r e s h , c l e a n f l a v o r i n a c i t r u s producing area may be anything but t h a t f o l lowing long periods of t r a n s i t and storage o u t s i d e of t h a t a r e a . U n l i k e many other f r u i t s and v e g e t a b l e s , c i t r u s f r u i t never i n crease i n f l a v o r q u a l i t y f o l l o w i n g harvest - only decrease, the r a t e depending on many v a r i a b l e s . Any sensory e v a l u a t i o n work c a r r i e d on w i t h o l d f r u i t , whether i n or out of the c i t r u s p r o ducing area most l i k e l y would possess a s i g n i f i c a n t amount of s t a l e f l a v o r r e s u l t i n g i n p o s s i b l e erroneous or v a r i a b l e d a t a , e s p e c i a l l y i f the supposed " f r e s h " j u i c e i s being used i n r e search work. T e s t i n g of P o t e n t i a l l y Hazardous Compounds. A p o s s i b l e danger e x i s t s i n t e s t i n g the e f f e c t on f l a v o r of f e d e r a l l y nonc l e a r e d substances t h a t may enter i n t o c i t r u s products. A sensory e v a l u a t i o n s u p e r v i s o r has a c l e a r o b l i g a t i o n to r e f r a i n from a l l o w i n g unknown p o t e n t i a l l y hazardous substances i n h i s or her research.

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Literature Cited 1. "Official Rules Affecting the Florida Citrus Industry". Published by the State of Florida, Dept. of Citrus: Lake­ land, FL, 1975. 2. McDuff, O., Adams Packing Assoc., Inc.: Auburndale, FL, personal communication, 1980. 3. Pinner, A. USDA Processed Products Branch Area Office, Winter Haven, FL, personal communication, 1980. 4. "U. S. Standards for Grades of Frozen Concentrated Orange Juice". USDA Consumer and Marketing Service: Washington, D.C., 1968. 5. "Manual on Sensory Testing Methods". ASTM Tech. Publ. 434, 1968. 6. Larmond, E. "Laboratory Methods for Sensory Evaluation of Food", Publication 1637; Food Res.Inst.: Ottawa Ont. Canada, 1977. 7. Jelllnek, G. J. Nutr 8. Jellinek, G. "Modern Scientific Sensory Methodology and Its Application to the Sensory Evaluation of Fruit Juices", International Federation of Fruit Juice Producers Meeting: Madrid, Spain, 1967. 9. Hepburn, W. J. "Taste Testing in the Food Industry"; Rollins College: Winter Park, FL, 1966. 10. Spencer, H. W. The Flavour Ind., 1971, 2 (5), 293-302. 11. Amerine, Μ. Α.; Pangborn, R. M.; Roessler, Ε. B. "Principles of Sensory Evaluation of Foods"; Academic Press: New York, 1965. 12. Fellers, P. J., unpublished data, 1978-79. 13. Fellers, P. J., unpublished data, 1976. 14. Carter, R. D.; Savant, W.; Fellers, P. J., unpublished data, 1980. 15. Fellers, P. J., unpublished data, 1979. 16. "World's Fair Consumer Taste Test". Ceco Marketing, Con­ sulting and Research, Inc.; prepared for the State of FL, Dept. of Citrus, Lakeland, FL, 1965. 17. Huggart, R. L.; Fellers, P. J.; DeJager, G.; Brady, J. Proc. Fla. State Hortic. Soc., 1979, 92, 148-150. 18. Higby, R. H. J. Am. Chem. Soc. 1938, 60, 3013-3018. 19. Fellers, P. J., unpublished data, 1979. 20. Petrus, D. R.; Fellers, P. J.; Ting, S. V., unpublished data, 1979-80. 21. "U. S. Standards for Grades of Concentrated Orange Juice for Manufacturing". USDA Consumer and Marketing Service: Washington, D.C., 1964. 22. "U. S. Standards for Grades of Orange Juice from Concen­ trate". USDA Consumer and Marketing Service: Washington, D.C., 1969. 23. "U. S. Standards for Grades of Pasteurized Orange Juice". USDA Consumer and Marketing Service: Washington, D.C., 1969.

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24. Fellers, P. J.; Carter, R. D., unpublished data, 1972. 25. Fellers, P. J.; Huggart, R. L . ; DeJager, G.; Brady, J., un­ published data, 1979. 26. Dougherty, M. H.; Barros, S. M., unpublished data, 1975-80. 27. Federal Register 1980, 45 (33), 10356-10359. 28. Olsen, R. W.; Moore, E. L . ; Wenzel, F. W.; Huggart, R. L. Citrus Station Mimeo Report 56-1. Univ. of FL, AREC: Lake Alfred, 1955. 29. Blair, J. S.; Godar, E. M.; Reinke, H. G.; Marshall, J. R. Food Technol. 1957, 11, 61. 30. MacDowell, L. G.; Moore, E. L . ; Atkins, C. D. U. S. Patent 2,453,109, 1948. 31. Dougherty, M. H.; Petrus, D. R.; Fellers, P. J. J. Food Sci. 1974, 39, 855-856. 32. Atkins, C. D.; Attaway, J. A. U. S. Patent 3,657,424, 1972. 33. Ting, S. V.; Attaway J Α.; Blair J G.; Carter R D.; Fellers, P. J.; Fisher Meeting Report 72-23 p. 4. RECEIVED

July 1, 1980.

In Citrus Nutrition and Quality; Nagy, S., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

15 Immunological Tests for the Evaluation of Citrus Quality RICHARD L. MANSELL Department of Biology, University of South Florida, Tampa, FL 33620 ELMAR W. WEILER Institute fur Pflanzenphysiologie, Ruhr Universität, 463 Bochum, West Germany

Quality of food product values, such as color, physiological thresholds and sensitivities to taste, flavor and aroma. Ultimately the acceptance of foods and their products is dependent upon the physiological or biochemical state of the product, however, the subjective evaluations cannot be ignored except in times of hunger and famine. Each food and food product is, then, a veritable collection of chemical constituents which is neither static nor stable. It might be best to describe the endogenous chemistry as dynamic and, as such, the quality characteristics must be a function of the composition of the chemical constituents at any given time. Since the basis of chemical composition is, in fact, controlled by the inherent genetics of any given organism, we can at least focus in on the fundamental site responsible for each chemical compound. In addition, however, we must also be aware that the interactions of these same genes with a multitude of complex regulatory mechanisms also plays a major role in determining the ultimate chemical composition at any given period of development. I n v e s t i g a t i o n i n t o the chemical composition of c i t r u s has led to the i s o l a t i o n and i d e n t i f i c a t i o n of hundreds of i n d i v i d u a l compounds. As work has continued, b i o c h e m i c a l , n u t r i t i o n a l and o r g a n o l e p t i c s t u d i e s have made great s t r i d e s i n determining which compounds are paramount i n the f i n a l determination of f r u i t and f r u i t product q u a l i t y . For the purpose of t h i s d i s c u s s i o n , we w i l l concern ourselves with only those q u a l i t y aspects a f f e c t e d by endogenous chemical composition and c o n s i d e r a t i o n of p h y s i c a l or d e s c r i p t i v e parameters w i l l be omitted. Food Q u a l i t y Q u a l i t y i s defined as "the nature, excellence or i n t r i n s i c l e v e l of a thing" and thus e v a l u a t i o n i s a complicated and d i f f i c u l t task. The major problem r e l a t e d to q u a l i t y e v a l u a t i o n i s inherent i n the methodology used to make the determination.

0-8412-0595-7/80/47-143-341$05.00/0 © 1980 American Chemical Society In Citrus Nutrition and Quality; Nagy, S., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

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The most obvious method i s s u b j e c t i v e and here the e v a l u a t i o n i s based on the o p i n i o n of the t e s t e r and the response of the sensory organs. Obviously t h i s technique presents many problems and there i s wide v a r i a t i o n i n the range of r e s u l t s obtained. O b j e c t i v e methods are based on s p e c i f i c analyses and, thereby, exclude t e s t e r o p i n i o n . Thus, the i n t r i n s i c aspects of q u a l i t y are reduced to chemical, p h y s i c a l , or b i o l o g i c a l measurements and only the i n t e r p r e t a t i o n of the r e s u l t i n g data i s r e l a t e d back to the s u b j e c t i v e o p i n i o n . Reduction of the s u b j e c t i v e values or c r i t e r i a to a s p e c i f i e d compound, group of compounds or p h y s i c a l f a c t o r i s o f t e n a very d i f f i c u l t endeavor, but one by one each of the s o - c a l l e d " q u a l i t a t i v e c r i t e r i a " i s being l i n k e d to the presence or absence of s p e c i f i c c o n s t i t u e n t s . Sometimes only a s i n g l e compound i s i n v o l v e d , however, o f t e n the q u a l i t a t i v e character i s the r e s u l t of a d d i t i v e or even s y n e r g i s t i c i n t e r a c t i o n s of two or more constituents. Thus, sinc t e r i s t i c s that can c o n t r i b u t food product, i t i s paramount that p r e c i s e o b j e c t i v e methodology be developed f o r each parameter. In c i t r u s f r u i t s and products, most f l a v o r s and aromas are produced by p o l y p h e n o l i c compounds or e s s e n t i a l o i l s plus a v a r i e t y o f n o n - v o l a t i l e organic compounds. For d e t a i l e d d i s c u s s i o n s of c i t r u s f l a v o r s and chemical composition, the reader i s r e f e r r e d to the e x c e l l e n t and comprehensive reviews which have been published (j.,.2,_3,4) . During the past h a l f - c e n t u r y , the c i t r u s i n d u s t r y has grown world-wide and q u a l i t y standards, both l o c a l and i n t e r n a t i o n a l , have c o n t i n u a l l y become more comprehensive and r i g o r o u s . Numerous d e f i n i t i o n s and c r i t e r i a of q u a l i t y have been p u b l i s h e d (_5,6^_7) and i n some areas, e.g. F l o r i d a , wide ranging g u i d e l i n e s have been adopted and laws* enacted f o r the s o l e purpose of maintaining q u a l i t y l e v e l s . Extensive advances have been made i n q u a l i t y c o n t r o l or q u a l i t y assurance programs (8) y e t many d i f f i c u l t and complex problems remain as i n t r i n s i c components of the i n d u s t r y . Thus as the impetus f o r q u a l i t y improvement has evolved, i t has become a widely recognized f a c t that c e r t a i n t e c h n o l o g i c a l advances were required before q u a l i t a t i v e progress could be r e a l i z e d . The purpose o f q u a l i t y c o n t r o l or q u a l i t y assurance i n the c i t r u s i n d u s t r y i s to help provide f o r the p r o d u c t i o n of a uniform, h i g h q u a l i t y and commercially acceptable product. For most of the major q u a l i t a t i v e c h a r a c t e r i s t i c s , standardized, accurate and simple methods have been developed, and high and low l i m i t s have been c a r e f u l l y been e s t a b l i s h e d . Some of these include: B r i x , a c i d , c o l o r , r e c o v e r a b l e o i l , and f r e e and suspended pulp. For each of these c r i t e r i a , a simple and r a p i d assay has been developed and good c o r r e l a t i o n e x i s t s between the o b j e c t i v e assay r e s u l t s and the s u b j e c t i v e q u a l i t y e v a l u a t i o n s .

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Problem Areas i n C i t r u s Q u a l i t y In s p i t e of the progress which has already been made, there s t i l l e x i s t , i n c i t r u s f r u i t and products, numerous q u a l i t a t i v e aspects which are e i t h e r very d i f f i c u l t to measure or whose c o n t r i b u t i o n to q u a l i t y i s a f u n c t i o n of the o r g a n o l e p t i c s e n s i t i v i t i e s of the consumer. For the purpose of t h i s paper we would l i k e to concentrate on two groups of n a t u r a l l y o c c u r r i n g compounds which have a major impact on the t a s t e of c i t r u s f r u i t and t h e i r products. As p r e v i o u s l y discussed by Maier and co-workers (Chapter 4 ) , i n Navel, Shamouti and c e r t a i n other orange c u l t i v a r s , the presence of l i m o n i n , a b i t t e r t r i t e r p e n o i d , causes many economic and o r g a n o l e p t i c problems and g r e a t l y a f f e c t s the t a s t e q u a l i t y of processed f r u i t . Limonin i s a l s o prevalent i n the g r a p e f r u i t but the i n t r i n s i c q u a l i t y of t h i s f r u i t i s f u r t h e r complicated by the presence of n a r i n g i n (Chapter 5 ) . Numerous o r g a n o l e p t i c s t u d i e s have been done on b i t t e r n e s s as a f u n c t i o n of l i m o n i n and/or n a r i n g i n , and a myriad of a d d i t i v e and perhaps s y n e r g i s t i c r e s u l t s have been obtained ( 9 ) . Table I presents a g e n e r a l i z e d response as measured i n a v a r i e t y of t a s t e s t u d i e s . Table I Organoleptic Responses to Limonin and N a r i n g i n

Non-bitter Slightly bitter Bitter Very b i t t e r

Threshold

However:

Limonin ppm

Naringin ppm

6 7-9 10-16 18

20 20-300 500-1300 1500

ave.=1

ave.=20

a s o l u t i o n or j u i c e with 0.75 ppm limonin and 5 ppm n a r i n g i n i s b i t t e r (99% confidence l e v e l ) (9)

During the past two decades i n t e r e s t and concern about the b i t t e r p r i n c i p l e s of c i t r u s products have increased g r e a t l y and although some e x c e l l e n t chemical s t u d i e s have been done, there

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has been l i t t l e i n f o r m a t i o n generated which f r u i t quality. The major problem which has to overcome i n these s t u d i e s i s that u n l i k e f a c t o r s , e.g. sugar, a c i d , e t c . , the b i t t e r r a p i d l y assayed and q u a n t i f i e d .

has had an e f f e c t on proved most d i f f i c u l t other q u a l i t y p r i n c i p l e s cannot be

Current A n a l y t i c a l Methods When one examines the a n a l y t i c a l methods which are p r e s e n t l y a v a i l a b l e f o r these b i t t e r compounds, i t i s c l e a r why the study and c o n t r o l of b i t t e r n e s s i n c i t r u s has been so hampered and quality control v i r t u a l l y lacking. For the flavanone, n a r i n g i n , only an approximate t e s t i s a v a i l a b l e (Davis Test) (see Chapter 5 f o r a d i s c u s s i o n of t h i s method) and f o r limonin there i s no method a v a i l a b l e f o r monitoring i n p r o c e s s i n g p l a n t s . In the past ten years t h i n l a y e r chromatograph (TLC) has proven u s e f u l f o r s e m i - q u a n t i t a t i v j u i c e samples, however t h i rather i n s e n s i t i v e (yg range); 2) slow (1 hour or more f o r sample a p p l i c a t i o n and solvent development); 3) r e q u i r e s p r e - p u r i f i c a t i o n steps ( l i q u i d - l i q u i d e x t r a c t i o n or p r e c i p i t a t i o n ) ; 4) d e t e c t i o n i s d i f f i c u l t ( e s p e c i a l l y true f o r limonoids) and 5) sample throughput i s normally l e s s than 50 per person per day. Within the past s e v e r a l years good s e p a r a t i o n and q u a n t i f i c a t i o n have been achieved with high-performance l i q u i d chromatography (HPLC) and t h i s procedure has proven very u s e f u l . However, HPLC i s not without i t s drawbacks: 1) equipment cost i s very h i g h (thus small p r o c e s s i n g operations would probably be unable to purchase and maintain such an instrument); 2) only one compound can be measured at a time s i n c e d i f f e r e n t columns and s o l v e n t s are used f o r each c l a s s of compounds ( t h i s a l s o means that time must be spent i n changing the system from one a n a l y s i s to the n e x t ) ; 3) p r e - p u r i f i c a t i o n i s r e q u i r e d and f o r good r e s o l u t i o n repeated l i q u i d - l i q u i d and evaporation steps are i n v o l v e d ; 4) the procedure i s slow (only 10-15 samples can be processed per person per day); 5) i t i s s e n s i t i v e only to the p a r t s per m i l l i o n (ppm) range (ug/gm). Thus, i n summing the current s t a t u s of l i m o n i n and n a r i n g i n q u a n t i f i c a t i o n , a quotat i o n i s most a p p r o p r i a t e . "None of the methods developed thus f a r can be considered i d e a l , e s p e c i a l l y f o r r o u t i n e q u a l i t y c o n t r o l purposes. Among t h e i r disadvantages are s u b j e c t i v e readout methods, time-consuming separat i o n s , questionable s p e c i f i c i t y , and l i m i t e d applicability. Recent work showing that t a s t e thresholds f o r l i m o n i n are lower than had p r e v i o u s l y been assumed f o r s i g n i f i c a n t p r o p o r t i o n s of the p o p u l a t i o n emphasizes the need f o r a b e t t e r assay method. The use of a p r o t e i n s p e c i f i c f o r l i m o n i n , such as an enzyme or antibody, could provide the b a s i s f o r an improved assay." (9)

In Citrus Nutrition and Quality; Nagy, S., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

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Development of the Immunoassay i n Plant

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Science

As we began to develop i n t e r e s t i n the b i t t e r n e s s problem i n c i t r u s f r u i t , a major advance or breakthrough i n plant science was achieved when i t was shown that an immunological assay method could be developed f o r the measurement of n a t u r a l l y o c c u r r i n g plant products (10). In t h i s study i t was demonstrated that i t was p o s s i b l e to develop antibodies which were s p e c i f i c f o r the compound under i n v e s t i g a t i o n , and the authors furthermore suggested that t h i s immunological method should render i t s e l f exceedingly u s e f u l i n plant screening and breeding programs; i n c e l l c u l t u r e screening; and f o r studies of b i o s y n t h e s i s , transport and metabolism. Extension of such v e r s a t i l i t y would l o g i c a l l y include q u a l i t y c o n t r o l s t u d i e s and monitoring. The r o l e of the immunoassay, e s p e c i a l l y the radioimmunoassay (RIA), i n c l i n i c a l biochemistry has been the major f a c t o r i n the tremendous advances mad 1959 (11). At present th t o o l a v a i l a b l e f o r q u a n t i t a t i v e d e t e c t i o n of molecules of d i v e r s e s t r u c t u r e and f u n c t i o n i n b i o l o g i c a l f l u i d s of human, animal and now p l a n t o r i g i n . The immunoassay comprises a unique combination of s e n s i t i v i t y and s p e c i f i c i t y as w e l l as p r e c i s i o n and a p p l i c a bility. With t h i s assay technique, i t i s now p o s s i b l e to detect and very a c c u r a t e l y measure compounds at endogenous p h y s i o l o g i c a l concentrations which f r e q u e n t l y are i n the range of 10 M or lower. In Table I I the major c h a r a c t e r i s t i c s of the immunoassay are l i s t e d . This method i s v e r s a t i l e , s p e c i f i c , can be u t i l i z e d f o r almost an u n l i m i t e d number of compounds and has a high throughput p o t e n t i a l . Table II C h a r a c t e r i s t i c s of the Immunoassay System 1)

Most s e n s i t i v e a n a l y t i c a l technique f o r both r o u t i n e and s o p h i s t i c a t e d analyses. picogram (femtomole) range = parts per b i l l i o n range = c e l l u l a r l e v e l

2)

S p e c i f i c i t y - n e a r l y absolute f o r each compound p e r m i t t i n g d i r e c t measurement i n crude e x t r a c t s , n u t r i e n t s o l u t i o n s or processed samples

3)

S u i t a b l e f o r automation or semi-automation. a n a l y s i s p o s s i b l e , 20-30 minutes.

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4)

Can be used f o r s i n g l e sample a n a l y s i s or f o r mass screening. Screening p o t e n t i a l i s as high as 3000 samples per day per person.

5)

U s e f u l as a q u a n t i t a t i v e or s e m i - q u a n t i t a t i v e assay

The r o l e of the immunoassay i n food science has developed more slowly and, o v e r a l l , r a t h e r few immunological t e s t s are c u r r e n t l y being performed on a r o u t i n e b a s i s . Table I I I provides a l i s t of some of the major analyses which are a v a i l a b l e (12). Table I I I Immunoassay i n Food Science A.

B.

C.

D. E.

Food P r o t e i n s 1) meat 2) soybean Toxic Plant & Animal Constituents 1) a g g l u t i n i n s 2) proteinase i n h i b i t o r s B a c t e r i a l and V i r a l Toxins 1) enterotoxins from Staphylococcus, IS. c o l i , Clostridium 2) f i s h and s h e l l f i s h poisons 3) foodborne v i r u s e s Food A l l e r g y 1) a l l e r g e n s C i t r u s (discussed below) 1) E s t i m a t i o n of orange j u i c e content i n s o f t d r i n k s 2) B i t t e r p r i n c i p l e a n a l y s i s (limonin and n a r i n g i n )

Within the l a s t s e v e r a l years there has been a very r a p i d expansion of the RIA i n t o p l a n t science, and assays are p r e s e n t l y i n use with such d i v e r s e compounds as n i c o t i n e (13), i n d o l a c e t i c a c i d (14), a b s c i s s i c a c i d (15), serpentine (16), d i g o x i n (10,17) and many others. The unique f e a t u r e s of the RIA have shown that i t i s an e x c e l l e n t and very necessary technique f o r mass screening programs i n v o l v i n g genetic breeding of p l a n t characters (18,19) screening of c e l l c u l t u r e s f o r primary or secondary metabolites (20), f o r i n v e s t i g a t i o n s of b i o - s y n t h e s i s (21) and i n p r a c t i c a l s i t u a t i o n s such as q u a l i t y c o n t r o l monitoring of p l a n t e x t r a c t s (20). The immunoassay i t s e l f can be performed i n a wide array of procedural v a r i a t i o n s , but the RIA i s p r e s e n t l y the most widely used. In p r i n c i p l e , the immunoassay i s based on the h i g h l y s p e c i f i c r e a c t i o n of a n t i b o d i e s with antigens against which the a n t i b o d i e s have been d i r e c t e d . The m a j o r i t y of antigens known to b i o l o g y are p r o t e i n s or other molecules of high molecular weight. Compounds with a molecular weight below 1000 are u s u a l l y not

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immunogenic when introduced i n t o the bloodstream of an animal. However, i f these low molecular weight compounds are bound c o v a l e n t l y to p r o t e i n c a r r i e r s (such as human serum albumin or p o l y - l y s i n e ) as haptens they become immunogenic and s p e c i f i c a n t i b o d i e s a g a i n s t these haptens are produced. The production of a n t i b o d i e s i s normally done i n r a b b i t s and i t t y p i c a l l y takes a minimum of three months, with booster i n j e c t i o n s , to produce a f i n a l antiserum with a high t i t r e . Characteri z a t i o n of the antiserum i s done by the f o l l o w i n g : a) determinat i o n of the t i t r e , ( t h i s i s defined as that d i l u t i o n of the a n t i serum which binds 50% of a known amount of r a d i o a c t i v e or l a b e l l e d hapje^i undejj constant assay c o n d i t i o n s ) . In most cases the l a b e l is I or H but n o n - r a d i o a c t i v e immunoassays are a l s o a v a i l a b l e i n which an enzyme o r f l u o r e s c e n t compound f u n c t i o n s as l a b e l , b) determination of the e f f e c t s o f pH, s o l v e n t s and chemicals, e.g. ethanol, a z i d e , e t c . ; c) c o r r e l a t i o n of the immunoassay with standard methods; d) determinatio characterization factor other than the hapten might bind with the antibody. This phenomenon i s known as cross r e a c t i v i t y and a wide v a r i e t y of known r e l a t e d compounds must be t e s t e d . A f t e r production of the a n t i serum and i t s c h a r a c t e r i z a t i o n , a s i n g l e animal should produce enough m a t e r i a l to be used i n tens o f thousands of s i n g l e assays. The p r i n c i p l e o f the RIA i s diagrammed below and the assay i s done as f o l l o w s : Ag + Ag* + Ab p=f where:

AgAb + Ag*Ab + Ag + Ag*

Ab = antibody Ag = antigen Ag*= antigen l a b e l l e d with

1

2

5

3

I , H, enzyme, e t c .

To a s o l u t i o n of known t i t r e antiserum i s added a known c o n c e n t r a t i o n of r a d i o a c t i v e or t r a c e r hapten (antigen) and an a l i q u o t of the p l a n t e x t r a c t . There w i l l be a competition of the l a b e l l e d hapten (added) and u n l a b e l l e d (plant e x t r a c t ) hapten f o r the f i x e d number o f antibody s i t e s (antiserum of known t i t r e ) and t h i s r e s u l t s i n some of the l a b e l l e d hapten being bound while the remainder remains f r e e . The d i s t r i b u t i o n of the r a d i o a c t i v e hapten between the f r e e and bound s t a t e w i l l be a f u n c t i o n of the amount o f u n l a b e l l e d hapten present i n the assay tube. A f t e r e q u i l i b r i u m has been reached, the bound and unbound hapten can be separated ( F i g . 1) ( s e v e r a l methods are a v a i l a b l e ) and determination of the r a d i o a c t i v i t y i n e i t h e r f r a c t i o n gives an exact measure of the unknown hapten (plant e x t r a c t ) as computed from a standard curve ( F i g . 2 ) . Since the immunoassay should prove to have great p o t e n t i a l i n a l l areas o f p l a n t s c i e n c e , we would l i k e to d e s c r i b e i n some

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leaf unit

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Figure 1.

Performance of the semiautomated RIA technique (36)

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Figure 2. Limonin standard curve shown in two different plots (\-0—\ Mean s.d. of triplicate determinations; tracer is Η limonin (Sp. Act. 22 Ci/mmol)) (23) 3

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d e t a i l r e s u l t s of s t u d i e s which we have done using limonin as the antigen. During the past two years we have developed and charac­ t e r i z e d two^cj^fferent RIA s f o r l i m o n i n (22,23) . One RIA was done using I-limonin as t r a c e r and the antibody d e v e l o p e d i s c h a r a c t e r i z e d by having a high a f f i n i t y (k = 1.1 χ 10 1/mol) f o r l i m o n i n . The d e t e c t i o n l i m i t of the assay i s 0.07 ng per 0.1 ml o r 0.7 p a r t s per b i l l i o n (ppb) and the standard curves are l i n e a r over a range of 0.5-100 ng l i m o n i n . Assays can be performed i n crude e x t r a c t s and 400-800 samples can be processed per day by one person. C o e f f i c i e n t s o f v a r i a t i o n of t r i p l i c a t e B/Bo values throughout the measuring range were 2.5 + 1.8% and recovery values were found to be 93%. The antibody produced against l i m o n i n has a very high t i t r e and 30,000 samples can be assayed per ml of serum. The s e n s i t i v i t y of t h i s RIA i s more than 10,000 times greater than that of any of the present a n a l y t i c a l methods (24,25,26,27). Data c a l c u l a t i o n i s done on a programmable computer an minutes. In the s p e c i f i c i t y t e s t s , i t was observed that only deoxylimonin (27%) and d e a c e t y l n o m i l i n (66%) cross reacted with the antibody. However, previous s t u d i e s (28,29) on the r e l a t i v e concentration of these two compounds i n c i t r u s t i s s u e s showed that deoxylimonin i s present a t only 0.47% that of limonin and, t h e r e f o r e , w i l l not be detected i n d i l u t e d samples. D e a c e t y l ­ n o m i l i n i s a l s o one of the minor c o n s t i t u e n t s of the t o t a l limonoid f r a c t i o n and w i l l not c o n t r i b u t e to l i m o n i n v a l u e s . In a study of the d i s t r i b u t i o n of limonin w i t h i n v a r i o u s g r a p e f r u i t t i s s u e s ( F i g . 3), the values obtained were i n good agreement with values a v a i l a b l e i n the l i t e r a t u r e (27,29,30) and the c o n c e n t r a t i o n gradient from growing leaves i n t o the f r u i t conductive t i s s u e and u l t i m a t e l y the seed supports the recent r e s u l t s obtained by Hasegawa and Hoagland (31). Although an extensive q u a n t i t a t i v e a n a l y s i s of both f r u i t and v e g e t a t i v e t i s s u e and the k i n e t i c s of accumulation have not yet been done, with the development of an RIA f o r l i m o n i n these s t u d i e s are now possible. ^ The second RIA system was developed using H-limonin as a tracer. Establishment of a second system was important s i n c e many l a b o r a t o r i e s possess s c i n t i l l a t i o n equipment f o r counting beta e m i t t i n g i s o t o p e s , but gamma counting equipment i s l e s s readily available. In a d d i t i o n , the stabij.j^y and h a l f - l i f e of a t r i t i a t e d t r a c e r i s very long compared to I and, thus, a s i n g l e r a d i o s y n t h e s i s can produce enough t r a c e r f o r s e v e r a l years or more. The c h a r a c t e r i s t i c s of the o r i g i n a l assay are: 1) a d e t e c t i o n l i m i t of 0.22 ng o r 2.2 ppb, 2) the standard curve i s l i n e a r over a range of 0.5-100 ng ( F i g . 2 ) , 3) no p u r i f i c a t i o n of the e x t r a c t s i s necessary, 4) c o e f f i c i e n t s of v a r i a t i o n f o r t r i p l i c a t e determinations (B/Bo) throughout the measuring range are 3.0 + 1.8% and 5) recovery i s 97%. The t i t r e of the antibody 1

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Figure 3.

Distribution of limonin in leaves, stems, and fruit parts of grapefruit (Citrus paradisi; (22)

2 1 2 5

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Figure 4.

Scatter diagram for various plant extracts assayed by both the I and H RIA (n = 67; r = 0.95; y = 0.96x + 0.07 (23) 125

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was 400, which means that 4000 samples could be assayed per ml of serum. Since the f i n a l t i t r e of the serum i s a f u n c t i o n of the s p e c i f i c a c t i v i t y of the t r a c e r used, the t i t r e value could be increased by using higher s p e c i f i c a c t i v i t y t r a c e r . Thus, when a second H-limonin was synthesized (108 Ci/mmol vs 22 Ci/mmol f o r the f i r s t ) the t i t r e of the antj.tjç)dy was found to i n c r e a s e to 1,500. In comparison with the I assay there a l s o was a high degree of c o r r e l a t i o n when e x t r a c t s from v a r i o u s t i s s u e s were assayed by both t r a c e r s ( F i g . 4 ) . HPLC/RIA C o r r e l a t i o n . We compared the l i m o n i n content of 180 samples of canned, s i n g l e - s t r e n g t h g r a p e f r u i t j u i c e which had been^analyzed by HPLC at the Lake A l f r e d Experiment S t a t i o n with our H RIA system ( F i g . 5 ) . A p a i r e d t t e s t showed no d i f f e r e n c e between the two methods and a l i n e a r r e g r e s s i o n gave r = 0.794. We were able to analyze these samples i n d u p l i c a t e i n a s i n g l e working day, whereas th using the HPLC. I t was were higher than the corresponding HPLC, but s i n c e the HPLC r e q u i r e s extensive e x t r a c t i o n and p r e p u r i f i c a t i o n p r i o r to a n a l y s i s whereas the RIA i s done with crude e x t r a c t s , t h i s r e s u l t i s perhaps p r e d i c t a b l e . Due to the s e n s i t i v i t y of the RIA, i t i s now p o s s i b l e to measure l i m o n i n c o n c e n t r a t i o n i n any p a r t of the p l a n t from embryos to s m a l l l e a f d i s c s , seed coats or any other d e s i r a b l e s i t e or sample. In t h i s type of a n a l y s i s or screening study i t i s p o s s i b l e f o r a s i n g l e person to analyze 2000 or more samples per week arid i t i s i r o n i c that "sample t a k i n g " i s now l i k e l y to become the l i m i t i n g step i n any l a r g e s c a l e study. In the recent s t u d i e s of Hasegawa and Hoagland (31) i t was found that limonoids ( i n the form of the Α-ring lactone) are synthesized i n the leaves of t r e e s and are t r a n s l o c a t e d to the fruit. T h i s o b s e r v a t i o n makes the RIA very a p p l i c a b l e i n a screening program. From very recent work i n our l a b o r a t o r y , we have found that i t i s a c t u a l l y p o s s i b l e to measure both the Ar i n g l a c t o n e and l i m o n i n w i t h i n a s i n g l e t i s s u e sample using j u s t the l i m o n i n antibody. We have found that the Α-ring l a c t o n e has a very low cross r e a c t i v i t y with the antibody, t h e r e f o r e i f we measure the l i m o n i n i n n e u t r a l i z e d e x t r a c t s and then a c i d i f y these same s o l u t i o n s and again measure the l i m o n i n content, the d i f f e r e n c e w i l l r e f l e c t the c o n c e n t r a t i o n of Α-ring lactone which was present (32). Thus the s t u d i e s of both compounds can be expanded and the k i n e t i c s of each followed as a f u n c t i o n of both l e a f development and growth. In a d d i t i o n , the e f f e c t of e n v i r o n ­ mental and p h y s i o l o g i c a l f a c t o r s can a l s o be analyzed. ARIA To the standard RIA d e s c r i b e d above, another dimension has r e c e n t l y been added with the development of an autoradiographic

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2

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6 HPLC

Figure 5. 3

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Scatter diagram for canned single-strength grapefruit juice assayed by H-RIA and HPLC (n = 180; r = 0.794; y = 0.844x + 1.381)

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immunoassay (ARIA) (33)• In t h i s modified RIA, the purpose i s not to measure with extreme accuracy the content of substances, but r a t h e r to merely i d e n t i f y those p l a n t s , c e l l c u l t u r e s or j u i c e samples w i t h i n l a r g e populations which c o n t a i n high or low amounts of the compound under study. In t h i s procedure the RIA p r i n c i p l e i s employed but, r a t h e r than doing r a d i o a c t i v e counting on each sample, the amount of r a d i o a c t i v i t y i s determined on Xray f i l m . By a d j u s t i n g the d i l u t i o n of the antibody the procedure can be made s e l e c t i v e f o r amju^its of substances below or above a particular level. By using I as the r a d i o a c t i v e t r a c e r as l i t t l e as 3,000 CPM can be detected a f t e r a 20 hr f i l m exposure. The procedure's r e a l usefulness comes from the f a c t that whereas the RIA can process 400-800 samples per day the ARIA can e a s i l y screen 2,000-3,000 or even more per day. Thus by s a c r i f i c i n g the s e n s i t i v i t y by a f a c t o r of about 10, the number of samples which can be measured per day can be increased by the same value. The s e n s i t i v i t y of t h i s assa s e n s i t i v e enough f o r i n t a c analysis. Immunoassay f o r N a r i n g i n At the present time we can only i n c l u d e p r e l i m i n a r y r e s u l t s on t h i s assay as i t s c h a r a c t e r i z a t i o n i s s t i l l i n progress. In the determination of b i t t e r flavanone g l y c o s i d e s the b a s i c importance i s that the assay i s able to d i s t i n g u i s h between the b i t t e r d i s a c c h a r i d e , neohesperidose, l i n k a g e (glucose 1-2 rhamnose) and the n o n - b i t t e r r u t i n o s e l i n k a g e (glucose 1-6 rhamnose). From the serum pools which are j u s t being harvested we have been able to a s c e r t a i n that the antibody being produced i s completely s p e c i f i c f o r the neohesperidoside and no cross r e a c t i v i t y has been observed f o r the r u t i n o s i d e at concentrations up to >5,000 ng/0.1 ml. Synthesis of an immunogenic n a r i n g i n molecule was a l s o done using a hapten-protein conjugate and complete d e t a i l s of the antibody production procedures and c h a r a c t e r i z a t i o n s t u d i e s w i l l be published i n depth at a l a t e r date. However, i t i s both important and encouraging to r e p o r t that through use of the immunoassay, major progress i n q u a l i t y c o n t r o l improvement i n c i t r u s now becomes a d i s t i n c t p o s s i b i l i t y . As a cautionary note, however, i t should a l s o be r e a l i z e d that much work, o p t i m i z a t i o n and t e s t i n g remains to be done before the f u l l p o t e n t i a l of these assays can be r e a l i z e d . Enzyme immunoassay In the previous pages we have d e s c r i b e d , i n p r i n c i p l e , an immunological assay f o r the q u a n t i f i c a t i o n of l i m o n i n and n a r i n g i n i n citrus^gamples.^ In these assays we have employed r a d i o a c t i v e tracers ( I and H) and have found that both are useable i n a

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research l a b o r a t o r y s i t u a t i o n . As with any assay method, however, the RIA a l s o has s e v e r a l major l i m i t a t i o n s e s p e c i a l l y when t r y i n g to employ such a technique i n commercial c i t r u s processing operations. F i r s t , and most importantly, i t i s undesirable and p o t e n t i a l l y unsafe to use r a d i o i s o t o p e s i n the v i c i n i t y of foods; secondly, the personnel using these compounds must be r i g o r o u s l y t r a i n e d i n t h e i r use and r o u t i n e h e a l t h and contamination surveys must be done. T h i r d l y , the equipment needed to measure r a d i o a c t i v i t y i s expensive to purchase and maintain. Thus, although the RIA method i s undoubtedly the p r e f e r r e d system i n an i s o l a t e d research l a b o r a t o r y s i t u a t i o n , an a l t e r n a t i v e method would be most acceptable i n p r a c t i c a l , " i n house" a p p l i c a t i o n s . As mentioned e a r l i e r , the immunoassay can be done with a number of procedural m o d i f i c a t i o n s and i n t h i s instance one must s u b s t i t u t e the isotope with another molecule ( f l u o r e s c e n t dye, magnetic p a r t i c l e , enzyme) which can be measured and t h e r e f o r e serve as the source of t r a c e r chosen to use the enzym present time the EIA i s s t i l l i n i t s infancy and although a number of s u c c e s s f u l EIA's have been developed the method cannot be considered a panacea (34). The f u t u r e of t h i s assay appears to be very b r i g h t and e x c i t i n g , and there i s considerable i n t e r e s t i n the a p p l i c a t i o n of the EIA to problems i n both microbiology and c l i n i c a l medicine (34). Many of the procedures and p r o t o c o l s are derived from RIA procedures and the EIA, l i k e the RIA, has the p o t e n t i a l to be performed i n a multitude of procedural v a r i a t i o n s ; but, f o r the purpose of t h i s manuscript we w i l l d e s c r i b e only the system we have chosen f o r our use. Consider, f o r example, a s e n s i t i v e competitive assay where the antibody i s immobilized on the s u r f a c e of a polystyrene tube or cuvette with l a b e l l e d and u n l a b e l l e d antigen competing f o r the antibody s i t e s . In t h i s instance the l a b e l l e d antigen i s limonin c o v a l e n t l y bonded to an enzyme molecule and u n l a b e l l e d antigen i s the l i m o n i n present i n j u i c e sample. We have done a number of p r e l i m i n a r y limonin-enzyme coupling analyses and of the enzymes we have used (3-glucosidase, a l k a l i n e phosphatase and h o r s e r a d i s h peroxidase) we have chosen the peroxidase (HPR) as our t e s t system. At present our data are s t i l l i n the p r e l i m i n a r y stage and thus i t would be somewhat premature to attempt to present many f i n d i n g s at t h i s time. We have, however, been able to demonstrate that the assay does f u n c t i o n and moreover we e n v i s i o n being able to develop t h i s system to a p o i n t where i t could be used i n both a research l a b o r a t o r y as w e l l as i n q u a l i t y c o n t r o l monitoring centers. In Figure 6 the procedures or steps involved i n an EIA are diagrammed. For t h i s assay i t i s f i r s t necessary to bind the antibody to the w a l l s of a polystyrene tube. (These tubes could be made a v a i l a b l e through s e v e r a l commercial l a b o r a t o r i e s or could be prepared by l a b o r a t o r y t e c h n i c i a n s . ) For the assay, a sample of j u i c e i s incubated together with the t r a c e r antigen.

In Citrus Nutrition and Quality; Nagy, S., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

MANSELL

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antibody

WEILER

coated

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tube

incubate

decant

& wash

add

enzyme

follow

color

assay

solution

development

Figure 6. Diagrammatic scheme for pe formance of a solid-phase enzyme immunoassay ((%) juice sample (limonin); ( \-E) tracer (limonin-enzyme conjugate))

In Citrus Nutrition and Quality; Nagy, S., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

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The tube i s then decanted, washed twice or more with b u f f e r and then an enzyme assay s o l u t i o n i s added and the c o l o r development or change i n absorbance i s measured. The amount of enzyme remaining bound to the antibody w i l l be a f u n c t i o n of the amount of antigen present i n the j u i c e sample and, thus, the c o l o r development can be used to c a l c u l a t e the amount of j u i c e antigen. In theory t h i s assay would be quite easy to use, i t would be r a p i d , safe and the only equipment necessary would be a s a t i s f a c t o r y spectrophotometer or c o l o r i m e t e r . Work i s c u r r e n t l y progressing toward a complete c h a r a c t e r i z a t i o n and o p t i m i z a t i o n of t h i s assay. Immunoassay f o r Orange J u i c e While our work on the RIA and EIA f o r limonin was i n progress, a research group i n I s r a e l (35) reported on an immunoassay f o r estimating the orange j u i c reconstituted juice. Thi i n d u s t r y s i n c e a d u l t e r a t e d or improperly r e c o n s t i t u t e d products g r e a t l y a f f e c t the q u a l i t y of the product and thus u l t i m a t e l y r e f l e c t upon c i t r u s q u a l i t y c o n t r o l c r e d i b i l i t y . A great d e a l of research e f f o r t has already been expended i n t h i s area of product r e c o n s t i t u t i o n and the problem seems to be never ending as new methods of a d u l t e r a t i o n always seem to be emerging. In t h i s new report the authors report that they were able to e l i c i t antibody production by i n j e c t i o n of pure orange juice. The t e s t f o r determination of orange j u i c e content was that of g e l d i f f u s i o n (35) and the d e t e c t i o n l i m i t s were s t a t i s t i c a l l y v a l i d f o r concentrations as low as 2.5%. From the data presented i n t h i s paper, i t i s evident that t h i s immunological method i s more accurate and s e n s i t i v e than any method p r e s e n t l y a v a i l a b l e . More importantly the assay i s cheap, simple, r a p i d and can be performed i n any o r d i n a r i l y equipped l a b o r a t o r y . P r e s e r v a t i v e s , c o l o r a n t s , o i l s and j u i c e s of other f r u i t s do not a f f e c t the assay. I n t e r e s t i n g l y , the p r e l i m i n a r y s t u d i e s of the antigen-antibody i n t e r a c t i o n suggest that the antigen i s not a protein. Conclusions I t i s both encouraging and e x c i t i n g to observe that the f i e l d of immunology has f i n a l l y been d i r e c t e d toward p l a n t s and molecules of p l a n t o r i g i n . For a l l of plant science, the future developments of the RIA, EIA and other immunoassays should a i d g r e a t l y i n s o l v i n g some of our most troublesome problems. We are of the o p i n i o n that the immunoassay has the p o t e n t i a l to become a major a n a l y t i c a l t o o l i n the c i t r u s industry and i n Table IV are l i s t e d j u s t some of the areas where t h i s procedure might be employed.

In Citrus Nutrition and Quality; Nagy, S., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

15.

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AND WEILER

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IV

Uses of Immunoassay i n Q u a l i t y Related

Programs

A)

Basic Research: 1) D i s t r i b u t i o n analyses i n v e g e t a t i v e parts 2) D i s t r i b u t i o n and l o c a l i z a t i o n w i t h i n f r u i t parts 3) I n t e r - and i n t r a - p l a n t and f r u i t v a r i a t i o n 4) C u l t i v a r v a r i a t i o n analyses 5) Stock/Scion r e l a t i o n s h i p s 6) Geographic & n u t r i e n t e f f e c t s on q u a l i t y 7) Crop improvement s t u d i e s ( s e l e c t i o n of i n d i v i d u a l p l a n t s possessing improved q u a l i t y t r a i t (s)) 8) Tissue c u l t u r 9) Biochemica metabolism 10) E a r l y d e t e c t i o n of b a c t e r i a l and v i r a l i n f e c t i o n s 11) Seasonality s t u d i e s

B)

Applied Research: 1) Use i n t e s t houses to monitor incoming f r u i t 2) Measurement of compound l e v e l s i n the f i n i s h e d s i n g l e strength and concentrate products 3) Use i n blending (e.g. navel j u i c e with n o n - b i t t e r orange) 4) B a c t e r i a l contamination-detection of organisms or compounds 5) Pulp wash s t u d i e s - a d u l t e r a t e d j u i c e

Since an immunoassay can be developed f o r each compound or antigen, i t i s conceivable that w i t h i n the not too d i s t a n t future we w i l l have many such assays a v a i l a b l e f o r both b a s i c research and a p p l i e d uses. Acknowledgement A p o r t i o n of t h i s research was supported by a grant from the F l o r i d a C i t r u s Commission, Lakeland, F l o r i d a and by a grant of the Bundesminister f u r Forschung und Technologie, Bonn (to Prof. Zenk, Bochum).

In Citrus Nutrition and Quality; Nagy, S., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

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Literature Cited 1. Nagy, S.; Shaw, P.E.; Veldhuis, M.K. "Citrus Science and Technology"; Avi: Westport, Ct., 1977; Vol. 1. 2. Maier, V.P.; Brewster, L.C. Proc. Int. Soc. Citriculture, 1977, 3, 709. 3. Ting, S.V.; Attaway, J.A. In "The Biochemistry of Fruits and Their Products"; Ed. by A.C. Hulme, Academic Press: New York, 1971; Vol. 2. 4. Blandstone, H.A.W.; Woodman, J.S.; Adams, J.B. In "The Biochemistry of Fruits and Their Products"; Ed. by A.C. Hulme, Academic Press: New York, 1971; Vol. 2. 5. Wood, J.F.; Reed, H.M 1935, 48, 246. 6. Wood, J.F.; Reed, H.M. Texas Agric. Exp. Stn. Bull., 1938, 563, 1. 7. Kebby, R.G.; Kepper, A.H. Agric. Gaz. N.S.W., 1948, 59, 357. 8. Hendrix, C.M. Jr.; Viale, H.E.; Johnson, J.D.; Vilece, R.J. In "Citrus Science and Technology"; Ed. by S. Nagy, P.E. Shaw, and M.K. Veldhuis; Avi: Westport Ct., 1977; Vol. 2, 482. 9. Maier, V.P.; Bennett, R.D.; Hasegawa, S. In "Citrus Science and Technology"; Ed. by S. Nagy, P.E. Shaw, and M.K. Veldhuis, Avi: Westport, Ct., 1977; Vol. 1, 355. 10. Weiler, E.W.; Zenk, M.H. Phytochemistry, 1976, 15, 1537. 11. Yalow, R.S.; Berson, S.A. Nature, 1959, 184, 1648. 12. Catsimpoolas, N. "Immunological Aspects of Foods"; Avi: Westport, Ct., 1977. 13. Langone, J.J.; Gjika, H.B.; van Vunakis, H. Biochemistry, 1973, 2, 5025. 14. Pengelly, W.; Meins, F. Jr. Planta, 1977, 135, 173. 15. Weiler, E. Planta, 1979, 144, 255. 16. Zenk, M.H.; El-Shagi, H.; Ahrens, H.; Stockigt, J.; Weiler, E.W.; Deus, B. In "Plant tissue culture and its biotechnological application"; Ed. by W. Barz, E. Reinhard, M.H. Zenk; Springer: New York, 1977; 27.

In Citrus Nutrition and Quality; Nagy, S., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

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17. Nickel, S.; Staba, E.J. In "Plant tissue culture and its Bio-technological application"; Ed. by W. Barz, E. Reinhard and M.H. Zenk, Springer: New York, 1977; 278. 18. Ahrens, H. In "Production of Natural Compounds by Cell Culture Methods"; Ed. by A.W. Alfermann and E. Reinhard, Gesell. Strahlen-und Umweltfor.: Munich, 1978. 19. Weiler, E.W.; Wostekemper, P. Planta Medica, 1979, 35, 316. 20. Zenk, M.H. In "Frontiers of Plant Tissue Culture 1978"; Ed. by T.A. Thorpe, U. Calgary: Calgary, 1978; 1. 21. Treimer, J.F.; Zenk, M.H. Phytochemistry, 1978, 17, 227. 22. Mansell, R.L.; Weiler E.W Phytochemistry in press 23. Weiler, E.W.; Mansell

Agric

press

24. Fisher, J.F. J. Agric. Food Chem., 1973, 21, 1109. 25. Fisher, J.F. J. Agric. Food Chem., 1975, 23, 1199. 26. Fisher, J.F. J. Agric. Food Chem., 1978, 26, 497. 27. Maier, V.P.; Grant, E.R. J. Agric. Food Chem., 1970, 18, 250. 28. Dreyer, D.L. J. Org. Chem., 1965, 30, 749. 29. Barton, D.H.R.; Pradhan, S.K.; Sternhell, S.; Templeton, J.F. J. Chem. Soc., 1961, 255. 30. Scott, W.C. Proc. Fla. State Hortic. Soc., 1970, 83, 270. 31. Hasegawa, S.; Hoagland, J. Phytochemistry, 1977, 16, 469. 32. Mansell, R.L.; Tisdale, R. Unpublished results. 33. Weiler, E.W.; Zenk, M.H. Anal. Biochem., 1979, 92, 147. 34. Engvall, E.; Pesce, A.J. "Quantitative Enzyme Immunoassay"; Blackwell Scientific: Oxford, 1978. 35· Firon, N.; Lifshitz, Α.; Rimon, Α.; Hochbert, Y. Lebensm.Wiss. U-Technol., 1979, 12, 143. 36. Weiler, E.W. In "Plant tissue culture and its biotechnological application"; Ed. by W. Barz, E. Reinhard, M.H. Zenk; Springer: New York, 1977; 266. RECEIVED June

27, 1980.

In Citrus Nutrition and Quality; Nagy, S., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

16 Analysis of Trace Metals in Orange Juice JAMES A. McHARD, SUSAN J. FOULK, JEANETTE L. JORGENSEN, SAM BAYER, and JAMES D. WINEFORDNER Department of Chemistry, University of Florida, Gainesville, FL 32611

Orange j u i c e h a s becom accepted n a t u r a l source i n v i g o r a t i n g f l a v o r , i t s source o f quick energy i n t h e form o f n a t u r a l sugars, i t s other n u t r i e n t q u a l i t i e s , e s p e c i a l l y as a s o u r c e f o r v i t a m i n C, and i t s h i g h p o t a s s i u m c o n t e n t r e l a t i v e t o o t h e r i n o r g a n i c m e t a l s , p a r t i c u l a r l y sodium. T h i s l a t t e r a t t r i b u t e ( h i g h p o t a s s i u m l o w sodium) makes o r a n g e j u i c e a h i g h l y d e s i r e d f o o d s o u r c e f o r t h o s e who must r e s t r i c t t h e i n t a k e o f s o d i um i n t h e i r d i e t . The a u t h o r s ' i n t e r e s t i n o r a n g e j u i c e i s , however, not i n t h e n u t r i t i v e area but i n f o r m u l a t i n g a complete des c r i p t i o n o f the elemental composition, e s p e c i a l l y the t r a c e i n o r g a n i c e l e m e n t a l c o m p o s i t i o n , and how t h i s c o m p o s i t i o n r e l a t e s t o geographical o r i g i n . Orange j u i c e i s m a r k e t e d i n t h e f o r m s o f s i n g l e s t r e n g t h ( u s u a l l y r e c o n s t i t u t e d from c o n c e n t r a t e ) and as f r o z e n concentrate. In F l o r i d a t h e s e forms a r e d e f i n e d i n degrees B r i x ( a v a l u e w h i c h r e l a t e s t o t h e s u g a r o r t o t a l s o l i d s c o n t e n t ) . I n most i n s t a n c e s , i n t h i s paper, c o n c e n t r a t i o n s o f c o n s t i t u e n t s w i l l be g i v e n based on " s i n g l e s t r e n g t h " j u i c e , a t e r m w h i c h d e n o t e s t h e c o n c e n t r a t i o n at w h i c h t h e j u i c e i s u s u a l l y consumed r e g a r d l e s s o f t h e f o r m i n w h i c h i t was p u r c h a s e d . There a r e v a r i o u s e x p r e s s i o n s r e l a t i n g t o e l e m e n t a l composit i o n t h a t d e s c r i b e t h e magnitude o r c o n c e n t r a t i o n range o f t h e p a r t i c u l a r e l e m e n t u n d e r c o n s i d e r a t i o n . I n t h e l i t e r a t u r e , one f i n d s e x p r e s s i o n s "major", " p r i n c i p a l " , "minor", " t r a c e " , " u l t r a t r a c e " and o t h e r s r e l a t i n g t o c o n c e n t r a t i o n l e v e l s . Inthis disc u s s i o n we w i l l adopt t e r m i n o l o g y f o r t h r e e c a t e g o r i e s ; macro ( t h o s e e l e m e n t s above 1%) m a j o r t r a c e ( e l e m e n t s f r o m 1 0 ppm t o 1%) and m i n o r t r a c e ( e l e m e n t s l e s s t h a n 1 0 ppm). These r a n g e t e r m s a r e p u r e l y a r b i t r a r y a n d a r e n o t meant t o b e o f f e r e d f o r a d o p t i o n as a g e n e r a l l y a c c e p t e d t e r m i n o l o g y s e t . T h e r e p o s s i b l y s h o u l d be one more c a t e g o r y s u c h a s u l t r a t r a c e f o r t h o s e e l e m e n t s w h i c h may b e p r e s e n t b u t a r e a t c o n c e n t r a t i o n s t o o l o w t o b e d e t e c t e d b y modern d a y i n s t r u m e n t a t i o n . F o r t h e p r e s e n t d i s c u s s i o n , h o w e v e r , t h i s range has l i t t l e v a l u e . 9

0-8412-0595-7/80/47-143-363$07.50/0 © 1980 American Chemical Society In Citrus Nutrition and Quality; Nagy, S., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

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W a t e r , s u g a r s , o r g a n i c a c i d s a n d amino a c i d s make up a l l b u t a few t e n t h s o f 1 ? o f " s i n g l e s t r e n g t h " orange j u i c e . A breakdown o f t h e c o m p o s i t i o n o f o r a n g e j u i c e i s p r e s e n t e d i n T a b l e I . Table Principal

I . Components o f S i n g l e S t r e n g t h Orange J u i c e Constituents

P r i n c i p a l C o n s t i t u e n t s o f Major Elements (Column I ) a n d O t h e r E l e m e n t s (Column I I )

8 6 ?

Water

I 1 1 . 5 ?

Sugars Amino a c i d s Organic

Oxygen

8 3 . 8 5 ?

Hydrogen

1 0 . 6 5 ?

1 ?

0 . 2 ?

Nitrogen

0 . 1?

Phosophorous

acids 1 ?

Inorganic Ash 0 . 5 ?

TOTALS

Potassium

1 0 0 ?

0 . 0 2 ?

Magnesium

0 . 0 1 ?

A l l Other Elements

0 . 0 1 ?

9 9 . 6 5 ?

0 . 3 5 ?

The v a l u e s i n T a b l e I a r e a p p r o x i m a t e a n d w i l l depend on g e o g r a p h i c a l l o c a l e , s o i l c o n d i t i o n s , f r u i t v a r i e t y and f e r t i l i z a t i o n practices. I t i s t h e group o f elements c o m p r i s i n g l e s s t h a n 0 . 0 1 ? o f s i n g l e s t r e n g t h o r a n g e j u i c e t h a t w i l l be t h e p r i n c i p a l s u b j e c t of t h i s d i s c u s s i o n . Historical The r e c o g n i t i o n i n t h e e a r l y p a r t o f t h e t w e n t i e t h c e n t u r y t h a t c e r t a i n m i n e r a l elements i n s m a l l c o n c e n t r a t i o n s had observa b l e e f f e c t s o n p l a n t , a n i m a l a n d human w e l l - b e i n g h a s l e d a h o s t o f i n v e s t i g a t o r s t o t r y t o measure t h e s e e f f e c t s q u a n t i t a t i v e l y . A r e m a r k a b l e and n o t e w o r t h y b e g i n n i n g l e a d i n g t o t h e c o n t i n u e d modern d a y i n t e r e s t i n a p p l y i n g a n a l y t i c a l s p e c t r o s c o p i c methods t o t h e a n a l y s i s o f a g r i c u l t u r a l p r o d u c t s was made b y H e n r i k L u n d e gardh i n t h e l a t e 1 9 3 0 s and e a r l y 1 9 U 0 s . L u n d e g a r d h was t h e f i r s t t o e s t a b l i s h t h e u t i l i t y o f f l a m e and spark s p e c t r o g r a p h i c methods f o r s o i l a n d l e a f a n a l y s i s ( l ^ f ^ ^ M Th results of t h i s work i n d i c a t e d a r e l a t i o n s h i p o f t h e s o i l c o n t e n t o f m a j o r t r a c e n u t r i e n t s l i k e c a l c i u m , phosphorus and n i t r o g e n and minor t r a c e n u t r i e n t s l i k e manganese, c o p p e r a n d i r o n t o optimum p l a n t and f r u i t d e v e l o p m e n t . I t h a s now b e e n w e l l e s t a b l i s h e d b y t h e C i t r u s Experiment S t a t i o n , Lake A l f r e d , F l o r i d a , ( 5 . ) , t h a t these e l e m e n t s a l o n g w i t h b o r o n , magnesium,molybdenum, p o t a s s i u m a n d z i n c a r e e s s e n t i a l elements i n t h e promotion o f s a t i s f a c t o r y growth o f o r a n g e t r e e s a n d t h e p r o d u c t i o n o f f r u i t . !

!

e

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Juice

365

Because o f t h e important n u t r i t i v e a t t r i b u t e s o f t h e e l e v e n e l e m e n t s s p e c i f i c a l l y l i s t e d a b o v e , most o f t h e a n a l y t i c a l e f f o r t i n t h e p a s t few decades h a s been d i r e c t e d toward t h e d e t e r m i n a t i o n o f t h e c o n c e n t r a t i o n s o f some o r a l l o f t h e s e e l e m e n t s i n f o o d s and p l a n t s o f i n t e r e s t . However, t h e r e c a n a l s o b e j u s t i f i a b l e i n t e r e s t i n what o t h e r e l e m e n t s a r e p r e s e n t . Some e l e m e n t s a r e , o f c o u r s e , t o x i c e v e n a t l o w c o n c e n t r a t i o n s . Some e l e m e n t s may h a v e a s y e t u n d i s c o v e r e d n u t r i t i v e v a l u e . Beyond t h e s e c o n s i d e r a t i o n s i t may b e i m p o r t a n t t o know i f t h e r e a r e e l e m e n t s whose c o n c e n t r a t i o n depends o n , a n d t h u s may b e i n d i c a t i v e o f , t h e g e o g r a p h i c a l l o c a l e i n w h i c h t h e p l a n t s were grown. I f s u c h e l e m e n t s a r e p r e s e n t a n d i f t h e i r c o n c e n t r a t i o n s c a n be m e a s u r e d , t h i s i n f o r m a t i o n , perhaps, c o u l d be used f o r " f i n g e r p r i n t i n g " o r charact e r i z i n g t h e j u i c e as t o i t s o r i g i n . The l i t e r a t u r e i s r e p l e t e w i t h s t u d i e s on t h e measurement o f a l i m i t e d number o f e l e m e n t s p r e s e n t i n s o i l , p l a n t , l e a v e s a n d f r u i t ; and e s t i m a t e s hav content t o growth, m a t u r i t 1 3 y e a r s ( t a k e n f r o m C h e m i c a l A b s t r a c t s ) , t h e a u t h o r s were a b l e to account f o r n e a r l y 1 0 0 r e f e r e n c e s r e l a t i n g t o t h i s s u b j e c t . Many o f t h e s e r e f e r e n c e s , h o w e v e r , were f r o m o b s c u r e f o r e i g n publ i c a t i o n s and o n l y a few p r e s e n t e d m e a n i n g f u l comparisons o f j u i c e s from d i f f e r e n t sources. I n a f a i r l y recent review, Kefford and C h a n d l e r {6) gave a c o m p r e h e n s i v e a c c o u n t o f g e o g r a p h i c a l c o m p a r i s o n s w h i c h h a d b e e n made t o t h a t d a t e ( S e e T a b l e I I ) . An e x a m i n a t i o n o f t h e s e c o m p a r i s o n s r e v e a l s t h a t t h e y d e a l m o s t l y w i t h t h e more common n u t r i e n t e l e m e n t s l i k e c a l c i u m , p h o s phorus, p o t a s s i u m a n d n i t r o g e n . An i n t e r e s t i n g a s p e c t o f t h i s c o m p i l a t i o n i s t h e wide range i n c o n c e n t r a t i o n v a l u e s f o r elements i n j u i c e s from d i f f e r e n t l o c a t i o n s throughout t h e w o r l d . Of t h e g e o g r a p h i c a l areas s t u d i e d b y u s , t h e s e wide v a r i a t i o n s have n o t been observed. A s e l e c t i o n o f r e f e r e n c e s from t h e e x t e n s i v e l i t e r a t u r e i s g i v e n a t t h e end o f t h i s c h a p t e r (j-22). T h e r e have b e e n o n l y t w o c o m p r e h e n s i v e s t u d i e s made o n t h e elemental content o f orange j u i c e s produced i n t h e U n i t e d S t a t e s . The f i r s t o f t h e s e was r e p o r t e d b y R o b e r t s a n d Gaddum ( 2 3 ) a l i t t l e o v e r hO y e a r s a g o . That s t u d y was a s p e c t r o g r a p h i c a n a l y s i s of t h e elemental contents o f s e v e r a l v a r i e t i e s o f F l o r i d a oranges and g r a p e f r u i t w h i c h were grown d u r i n g t h a t p e r i o d o f t i m e . E x c e r p t s f r o m t h e i r r e s u l t s a r e shown i n somewhat m o d i f i e d f o r m i n Table I I I . I t i s n o t e w o r t h y t h a t t h e r e s u l t s o f t h e R o b e r t s a n d Gaddum s t u d y compare r e m a r k a b l y w e l l f o r most e l e m e n t s w i t h modern d a y r e s u l t s o b t a i n e d b y u s u s i n g t h e more s o p h i s t i c a t e d i n s t r u m e n t a t i o n (see T a b l e V I I ) . T h i s c a n be observed b y comparing t h e v a l u e f o r V a l e n c i a o r a n g e s w i t h t h e modern v a l u e s s i n c e t h e B l o o d , S e e d l i n g , a n d L u e Gim Gong v a r i e t i e s a r e no l o n g e r c o m m e r c i a l l y p r o duced i n F l o r i d a . The o n l y o t h e r c o m p r e h e n s i v e s t u d y was r e p o r t e d i n t h e e a r l y s i x t i e s b y B i r d s a l l e t a l . (2k). B i r d s a l l and coworkers, l i k e

In Citrus Nutrition and Quality; Nagy, S., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

In Citrus Nutrition and Quality; Nagy, S., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

Extremes o f ranges o f f r u i t

5 0 - 1 U 7

. 5 - 6

U 2 - 2 U 0

5 7 0 - 1 8 0 0

1 2 0 0 - 2 U 6 0

Ν

Locales

c

2 0 - 5 5

Cl

and A r i z o n a .

from I s r a e l , S p a i n , S o u t h A f r i c a , U.S.A., West I n d i e s , a n d S o u t h A m e r i c a .

U 9 - 2 0 6

1 3 0 - 2 8 0

C o n s t i t u e n t s o f Orange J u i c e f r o m V a r i o u s (mg/kg) Ca Mg Fe Ρ

'Extremes o f r a n g e s f r o m I s r a e l , C a l i f o r n i a

D

2 - 3 0

Na

Inorganic

From K e f f o r d and C h a n d l e r ( 6 )

5 2 0 - 2 8 U 0

Juice

1 8 0 0 - U 6 0 0

9 2 0 - 2 7 8 0

Κ

Table I I .

Whole

Orange

Total Ash

>

c

> σ ο

δ

H

2

G H

C

Η

ο

as

In Citrus Nutrition and Quality; Nagy, S., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

Elemental

-

h

1 .h

3

-

N.D.

1 0 0

N.D.

2

-

-

1 .

0.13 1 . 3

1

0 . 3 9

2 . 8

-

70

-

2.3

N.D.

0 . 0 3

0 . 1 U -

-

.IU 0.

1 . 3

TRACE

-

0 . 3 3

0 .. 3 8

-

0 ., 3 8

N.D.

-

0 . 1 U

0 . 0 0 U

TRACE O.OOU - O.Oh N.D.

1 .,h

0 .,lh

g i v e n i n p a r t s p e r m i l l i o n based on s i n g l e s t r e n g t h j u i c e .

0 . U 5

N.D. N.D.

-

0 . 3 7

-

1 . 2

-

See r e f .

(23).

0 . 3 1

N.D.

-

TRACE

0 . 1 1

0 . 3 0

N.D.

0,

-

0 . 1 2

0 . 0 3

0 .12

-

-

N.D. 0 . 1 2

0 . 3 2

. 0 0 U - . 0 U

0 ,. 0 3

.03 -

.lh

0 . 0 3

0 . 0 0 U - 0 . 0 U 0

0

1 3.6

20

ho

50

30

0.03 -

60 60

70 70

90

0 . 0 U

TRACE TRACE TRACE

TRACE

80

N.D.

1 5 0 1 7 7 0

300 1 5 7 0

320 1 7 2 0

0 . 1 2

3 . 2

N.D. TRACE

1 ,. 2

0 . 1 2

0 . 3 0

1 . 2

1 2 . 1

TRACE

3 .. 2

-

N.D.

0 ,, 0 3

TRACE N.D. TRACE

50

TRACE

0 . 1 9

o.ooU-o.oUo N.D.

2 8 0

- O.Ul N.D. N.D.

50

N.D.

1

-

N.D.

-

I 8 5 O

0 . 2 0

0 . 3 3

0 . 0 3

300

h

0 . lh

N.D. TRACE

0 . 3 8

60

1 .

h

0 .1 5

N.D.

-

0.03 -

0 . 3 8

o.oh

I 8 7 O

N.D. N.D.

.h

Λ

.lh

30

0

30

-

N.D.

N.D.

30

N.D.

-

30

0 ,. 1 2

N.D.

- 0 . 1 2

3

N.D.

0 ,. 3 2

30

0.03

N.D.

0 . 0 0 U - 0 . 0 H 0

N.D.

0 . 3 6 - 1

3 . 6

0 . 1 2

1 U 0

0 .U8

6 -

N.D. O.OUl - 0 . 2 1

0 . 0 3

Tangerine

1 1 0

-

N.D.

1 5 . 0

L u e Gim Gong

90

0 . 3 6 - 1

0 . 0 3

8 -

V a l e n c i a Orange

( 1 9 3 7 )

1 1 0

0 . 2 3

3 . 8

B l o o d Orange

C o n t e n t o f S e v e r a l V a r i e t i e s o f F l o r i d a Orange J u i c e s (PPM)

l 6 0

N.D. 0.13 - 0 . 3 7

0 . 3 6

S e e d l i n g Orange

Concentrations

aluminum barium bismuth boron calcium cadmium chlorine chromium cobalt copper iron lead magnesium manganese molybdenum nickel phosphorus potassium silver sodium sulfur strontium tin titanium vanadium zinc zirconium

Element

Table I I I .

368

CITRUS NUTRITION AND

QUALITY

R o b e r t s and Gaddum, r e p o r t e d on e l e m e n t s m e a s u r e d s p e c t r o g r a p h i c a l l y ; e x c e p t t h e i r measurements were done on o r a n g e s and lemons grown i n C a l i f o r n i a . T h i r t y - o n e e l e m e n t s , a l l o f w h i c h were meta l s , e x c e p t b o r o n and p h o s p h o r u s , were m e a s u r e d . The r e s u l t s were r e p o r t e d i n c a t e g o r i e s as p e r c e n t o f t h e a s h o f t h e j u i c e . Select i n g o n l y t h e j u i c e d a t a , r e a r r a n g i n g i t s l i g h t l y , and e x p r e s s i n g i t i n t e r m s o f p a r t s p e r m i l l i o n b a s e d on t h e o r i g i n a l j u i c e i n s t e a d o f as a p e r c e n t a g e o f t h e a s h , t h e v a l u e s a p p e a r i n T a b l e IV. Table IV. Elemental Content o f C a l i f o r n i a V a l e n c i a and N a v e l Orange J u i c e s (PPM) ( l 9 6 l ) a

J u i c e Type/PPM o f J u i c e Sample

Greater t h a n hO PPM

Valencia

Ca, Κ

Navel

a

Data

Ca, Κ

Concentrât i o n s between 0 . h PPM and hO PPM

Trace Less t h a n O.h PPM

Not Detected

Mg

Mg,

from B i r d s a l l et a l .

P, S i , Na, A l , Fe

Mo, S n , Z r , Co, B a , Zn, Sr

Sb, Cb,

Ti, Cr, Zn, Sr,

B i , Ba, Co, Sb, A s , Cb, W, P b , Mo

Cu, N i , V, Mn, L i , Zr, Ag, Β

As, W

(2h).

As one c a n r e a d i l y s e e , t h e q u a n t i t a t i v e s c a l e o f t h e r e s u l t s o f T a b l e I V i s l e s s d e f i n i t i v e t h a n t h e s c a l e i n T a b l e I I I . However, o r d e r o f m a g n i t u d e agreement i s a p p a r e n t f r o m a c o m p a r i s o n o f t h e two s e t s o f d a t a s h o w i n g t h a t t h e r e a r e p r o b a b l y no g r o s s d i f f e r ­ e n c e s i n t h e m e t a l c o n t e n t s o f j u i c e s f r o m t h e two s o u r c e s - F l o r ­ i d a and C a l i f o r n i a . I t w i l l be n o t e d t h a t t h e c o n c e n t r a t i o n r a n g e f o r p h o s p h o r u s i s an o r d e r o f m a g n i t u d e l e s s i n t h i s d a t a t h a n t h e R o b e r t s and Gaddum d a t a a n d , j u d g e d by t h a t and t h e v a l ­ ues we o b t a i n , we c o n c l u d e t h e r a n g e f o r p h o s p h o r u s i n T a b l e I V t o be i n e r r o r . I t c o u l d be s t a t e d h e r e t h a t Lundegârdh d i d most o f h i s s t u d i e s on s o i l and l e a f a n a l y s i s c o n t e n d i n g t h a t t h e f r u i t ( i n t h i s case t h e orange i t s e l f ) v a r i e d l i t t l e i n composition. I n r e c e n t p u b l i c a t i o n s , we ( 2 5 , 2 6 , 2 7 , 2 8 ) h a v e d i s c u s s e d some modern a p p r o a c h e s t o t h e a n a l y s i s o f o r a n g e j u i c e , and t h e r e s u l t s o f t h e s e s t u d i e s w i l l be p r e s e n t e d i n t h e f o l l o w i n g s e c tions of t h i s chapter.

In Citrus Nutrition and Quality; Nagy, S., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

16.

MCHARD E T AL.

Trace Metals

in Orange

Juice

369

Methodology I n a n y a n a l y t i c a l p r o b l e m t h e a n a l y s t must o u t l i n e t h e g o a l s t o be a c h i e v e d b y t h e a n a l y s i s b e f o r e d e c i d i n g on m e t h o d o l o g y . O f t e n i t i s d e s i r e d t o measure t h e c o n c e n t r a t i o n o f p e r h a p s , o n e , o r a t m o s t , j u s t a few e l e m e n t s . I n t h e s e c i r c u m s t a n c e s , t h e i n v e s t i g a t o r may, f o r e x a m p l e , b e i n t e r e s t e d i n a p p l y i n g a n e l e m e n t l i k e manganese o r z i n c t o t h e s o i l o r f e r t i l i z e r and m o n i t o r i n g t h e r e s u l t i n g i n f l u e n c e on orange t r e e development o r f r u i t q u a l i ty. A l t e r n a t i v e l y , the o b j e c t i v e behind a p a r t i c u l a r a n a l y s i s m i g h t be t o measure t h e c o n t a m i n a t i o n l e v e l s o f e l e m e n t s l i k e cadmium and l e a d w h i c h r e s u l t f r o m t h e i n v a s i o n o f t h e g r o w i n g area by f o r e i g n environmental agents. I n s i t u a t i o n s l i k e t h e s e t h e method o f measurement o f t h e element ( o r elements) o f i n t e r e s t can be t a i l o r e d s p e c i f i c a l l y t o s u i t the p a r t i c u l a r needs A matte ffact fo fe metal l i k e m e r c u r y and t i n an mony, and t e l l u r i u m s p e c i a u s u a l l y r e q u i r e d ( 2 9 _ , 3 0 _ , 3 1 _ , 3 2 , 3 3 _ ) . The r e a s o n s p e c i a l p r e c a u t i o n s and s p e c i a l methods a r e r e q u i r e d f o r t h e e l e m e n t s l i s t e d a b o v e , and f o r c e r t a i n o t h e r s , i s t h a t t h e y e i t h e r a r e r e a d i l y r e d u c e d t o v o l a t i l e f o r m s , r e a c t c h e m i c a l l y t o f o r m v o l a t i l e compounds, or are i n f l u e n c e d i n t h e i r a n a l y s i s by the matrices u s u a l l y present . Sample

Preparation

Orange j u i c e i n c o n c e n t r a t e f o r m ( t h e f o r m u s u a l l y s e l e c t e d for a n a l y s i s ) i s g e n e r a l l y about f o u r t i m e s the c o n c e n t r a t i o n o f s i n g l e s t r e n g t h j u i c e and t h e r e f o r e i s p r i m a r i l y s u g a r and w a t e r ( r o u g h l y 5 0 / 5 0 ; see T a b l e I ) . U s u a l l y , f o r a n a l y t i c a l p u r p o s e s , t h e o r g a n i c m a t t e r must b e d e s t r o y e d o r b r o k e n down i n s u c h a way t h a t i t does n o t i n t e r f e r e w i t h t h e a n a l y s i s . C o n s i d e r a b l e emphas i s has b e e n g i v e n t o t h i s a s p e c t i n t h e l i t e r a t u r e on f o o d a n d b i o l o g i c a l a n a l y s i s . No c l e a r c o n s e n s u s o f o p i n i o n seems t o p r e v a i l o n what t h e b e s t a p p r o a c h e s a r e . P r o b a b l y t h e s i m p l e s t method i s t o d r y a s u i t a b l e sample u n d e r a lamp o r i n a n open d r a f t o v e n u n t i l t h e w a t e r i s d r i v e n o f f and t h e n a s h a t t e m p e r a t u r e s up t o 5 5 0 ° C A l t h o u g h t h e t o t a l t i m e i n v o l v e d i n t h i s a s h i n g proc e d u r e i s u s u a l l y t h e b e t t e r p a r t o f one day, t h i s method r e q u i r e s l i t t l e o p e r a t o r a t t e n t i o n a n d a number o f s a m p l e s can b e c a r r i e d t h r o u g h t h e p r o c e s s a t one t i m e . An a d d i t i o n a l a d v a n t a g e t o a s h i n g i s t h a t r e l a t i v e l y l a r g e s a m p l e s can b e u s e d (up t o 2 0 g i n most i n s t a n c e s ) . D i s a d v a n t a g e s a r e t h a t v o l a t i l e e l e m e n t s ( o r e l e m e n t s f o r m i n g v o l a t i l e compounds) c a n b e l o s t t h r o u g h v o l a t i l ization. D e p e n d i n g o n what o t h e r i o n s a r e p r e s e n t , t h e l i s t o f elements g e n e r a l l y l o s t by ashing c e r t a i n l y w i l l i n c l u d e elements l i k e m e r c u r y , a r s e n i c , cadmium, s e l e n i u m , a n t i m o n y and t e l l u r i u m . Some l o s s e s , a l s o d e p e n d i n g o n t h e c i r c u m s t a n c e s , may be o b s e r v e d w i t h m e t a l s l i k e s i l v e r , l e a d and p o s s i b l y a l s o c o p p e r a n d z i n c

In Citrus Nutrition and Quality; Nagy, S., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

370

CITRUS

NUTRITION

AND

QUALITY

a l t h o u g h t h e a u t h o r s h a v e n o t o b s e r v e d s i g n i f i c a n t p r o b l e m s by d r y a s h i n g when d e a l i n g w i t h t h e s m a l l c o n c e n t r a t i o n s o f s i l v e r , c o p p e r and z i n c p r e s e n t i n o r a n g e j u i c e . T h i s i s p r o b a b l y due t o the r e t e n t i v e e f f e c t o f phosphate s i n c e phosphorus i s present i n o r a n g e j u i c e i n r e l a t i v e l y m a s s i v e amounts compared t o t h e c o n ­ c e n t r a t i o n s of the l a t t e r t h r e e elements. A l s o , the high a l k a l i m e t a l content o f orange j u i c e , e s p e c i a l l y the h i g h potassium con­ c e n t r a t i o n r e n d e r s t h e f i n a l a s h a l k a l i n e , p r i n c i p a l l y as t h e c a r ­ bonate. This m a t r i x i s a l s o conducive t o r e t e n t i o n of metals whi­ ch m i g h t o t h e r w i s e be v o l a t i l e o r be s u b s t a n t i v e on t h e a s h i n g crucible. S i l i c a c r u c i b l e s are l e s s d e s i r a b l e f o r ashing than p l a t i n u m o n e s . T h i s r e s u l t s f r o m two c o n s i d e r a t i o n s : l ) t h e s i l i c a s u r f a c e has open r e a c t i v e s i t e s f o r i o n exchange a n d / o r i o n a b s o r p t i o n and 2 ) s i l i c a t e n d s t o p i t and e r o d e i n t o t h e h i g ­ h l y a l k a l i n e m a t r i x of the f i n a l ash. A l o w t e m p e r a t u r e a s h i n g t e c h n i q u e has r e c e n t l y begun t o r e ­ c e i v e a t t e n t i o n ( 3^_, 3 5 . , 3 6 , 3 χ , 3 8 _ ) vacuum ( a f t e r r e m o v i n g t h o r a m i x t u r e o f o x y g e n and f r e o n . The l a t t e r gas seems t o s p e e d up t h e a s h i n g p r o c e s s . H e a t i n g i s p r o v i d e d by h i g h f r e q u e n c y c o i l s s u r r o u n d i n g t h e a s h i n g chamber. The chamber and a s h i n g con­ t a i n e r s must be made o f q u a r t z i f f r e o n i s t o be u s e d as a comp­ onent o f t h e gas a t m o s p h e r e . The a u t h o r s h a v e v e r i f i e d t h a t up t o 1 0 g o f f r o z e n c o n c e n t r a t e d o r a n g e j u i c e i n m u l t i p l e u n i t s may be a s h e d i n a s u i t a b l y s i z e d chamber. The t e m p e r a t u r e o f t h e ash­ i n g p r o c e s s c a n be v a r i e d up t o 2 0 0 ° C d e p e n d i n g on t h e f r e q u e n c y power. I t i s c l a i m e d t h a t many o f t h e m e t a l s c o n s i d e r e d t o be v o l a t i l e at h i g h e r temperatures are c o m p l e t e l y o r n e a r l y complete­ l y r e t a i n e d by t h i s a s h i n g method ( 3 8 ) . A number o f d i s c u s s i o n s ( 3 2 , 3 9 7 ^ 0 ^ 1 , U 2 ) can be founo. i n t h e l i t e r a t u r e r e l a t i n g t o t h e m e r i t s o f d r y v e r s u s wet a s h i n g . Wet a s h i n g e n t a i l s d e c o m p o s i n g t h e o r g a n i c components o f t h e sample by means o f m i x t u r e s o f o x i d i z i n g a c i d s . The u s u a l m i x t u r e s a r e n i t r i c and s u l f u r i c a c i d s ; n i t r i c and p e r c h l o r i c a c i d s ; n i t r i c , s u l f u r i c and p e r c h l o r i c a c i d s ; and s u l f u r i c a c i d and h y d r o g e n p e r ­ oxide. T h e r e a r e two p r i n c i p a l a d v a n t a g e s t o a c i d a s h i n g ( s o c a l l e d wet a s h i n g ) : l ) t h e r e a r e l i t t l e i f any l o s s e s by v o l a t i l i ­ z a t i o n ; and 2 ) t h e e l e m e n t s a r e k e p t i n an o x i d i z e d s t a t e and i n a s t r o n g l y a c i d medium; t h u s , d i m i n i s h i n g , i f n o t e l i m i n a t i n g , t h e chance of s u b s t a n t i v e r e a c t i o n s o r r e a c t i o n s w i t h the s u r f a c e o f t h e c o n t a i n e r . On t h e o t h e r h a n d , t h e r e a r e some v e r y s e r i o u s c o n s i d e r a t i o n s w h i c h t e n d t o r u l e a g a i n s t wet a s h i n g . The v o l u m e o f a c i d t o w e i g h t o f sample i s h i g h , u s u a l l y on t h e o r d e r o f 1 5 1 0 m l ( o r more) o f a c i d p e r gram o f sample e s p e c i a l l y when d e a l i n g w i t h orange j u i c e . T h i s means t h a t i f one w i s h e s t o decompose s a y 1 0 - 2 0 g o f sample f o r a s u r v e y o f many e l e m e n t s , t h e v o l u m e o f a c i d n e e d e d i s l a r g e , and b e c a u s e o f t h e h a z a r d o u s n a t u r e o f m i x t u r e s c o n t a i n i n g p e r c h l o r i c a c i d , c o n s i d e r a b l e t i m e and c o n ­ s t a n t a t t e n t i o n i s demanded o f t h e o p e r a t o r . A l s o , w i t h o r a n g e j u i c e i n the presence o f s t r o n g a c i d s , c o n s i d e r a b l e foaming

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o c c u r s a t t h e b e g i n n i n g s t a g e s o f t h e o x i d a t i o n , a n d i t becomes n e c e s s a r y t o a l l o w t h e samples t o r e a c t s l o w l y a t l o w t e m p e r a t u r e s u n t i l t h e f o a m i n g t e n d e n c y i s s u b d u e d , a p e r i o d o f t i m e t h a t may t a k e s e v e r a l h o u r s . The c o p i o u s amounts o f a c i d n e e d e d i n c r e a s e the l i k e l i h o o d o f m e t a l c o n t a m i n a t i o n from t h e a c i d s themselves s i n c e i t i s n e a r l y i m p o s s i b l e t o remove a l l t r a c e s o f i m p u r i t i e s during t h e i r manufacture. F u r t h e r m o r e , one r u n s t h e r i s k o f t h e a c i d anions r e a c t i n g w i t h t h e metals t o form r e l a t i v e l y s t a b l e but i n s o l u b l e p r o d u c t s , e.g., b a r i u m , c a l c i u m a n d o t h e r a l k a l i n e e a r ths form s u l f a t e s o f v a r y i n g degrees o f i n s o l u b i l i t y . I f one o r j u s t a few elements a r e t o be measured and i f i t i s determined t h a t v o l a t i l e l o s s e s o c c u r s u c h a s w i t h m e r c u r y , a r s e n i c , antimony and g r o u p V I e l e m e n t s , t h e n a c i d a s h i n g o f f e r s a n a c c e p t a b l e way f o r p r e p a r i n g t h e sample. I f a b r o a d r a n g e o f e l e m e n t s a r e t o be m e a s u r e d , a n d i n t e r e s t i n t h e above l i s t o f v o l a t i l e ones i s m i n o r t h e n we c o n s i d e r d r y a s h i n g t o be t h e method o f c h o i c e , e s p e c i a l l y f o r p r e p a r i n g orang t i o n s bear out t h e though be t a i l o r e d t o t h e s p e c i f i c a n a l y t i c a l p r o b l e m . For a s e l e c t e d l i s t o f elements ( 2 6 ) t h e r e i s another a c i d treatment procedure which i s r e a d i l y a p p l i c a b l e t o the a n a l y s i s of orange j u i c e ; t h i s procedure i n v o l v e s h y d r o l y s i s w i t h moderatel y s t r o n g n i t r i c a c i d t o b r e a k d o w n most o f t h e s u g a r s a n d t o d e crease the s i z e o f the pulpy constituents. The s o l u t i o n i s t h e n f i l t e r e d , d i l u t e d , and measured by atomic a b s o r p t i o n . For elements t h a t c a n b e d e t e r m i n e d w i t h a n a i r - a c e t y l e n e f l a m e u s i n g a high s o l i d s (three s l o t ) burner, t h i s procedure o f f e r s a u s e f u l alternative. F r i c k e e t a l . ( 3 3 ) a l s o mentions the u t i l i t y o f t h i s method a n d g i v e s c o m p a r a t i v e r e s u l t s on t h e u s e o f t h i s method i n sample p r e p a r a t i o n . I f d r y a s h i n g h a s b e e n u s e d , o r i f t h e samples f o l l o w i n g a c i d a s h i n g have b e e n e v a p o r a t e d t o d r y n e s s , t h e f i n a l s t e p s i n t h e p r e p a r a t i o n o f t h e sample f o r a n a l y s i s w i l l depend on t h e t y p e o f end measurement t o b e u s e d ; t h e s e e n d measurements w i l l b e d e a l t w i t h i n some d e t a i l i n t h e d i s c u s s i o n t o f o l l o w . End Measurement

Techniques

The f i n a l measurement s t e p can b e done i n a number o f ways. A f e w o f t h e e l e m e n t s i n o r a n g e j u i c e a r e p r e s e n t i n l a r g e enough c o n c e n t r a t i o n s and/or have c h e m i c a l c h a r a c t e r i s t i c s which p e r m i t the u s e o f c l a s s i c a l a n a l y s i s . P h o s p h a t e a n d some h e a v y m e t a l s l i k e z i n c a n d i r o n may b e m e a s u r e d c o l o r i m e t r i c a l l y . C a l c i u m has b e e n m e a s u r e d a s t h e o x a l a t e , b u t s u c h methods a r e t i m e consuming and may r e q u i r e p r i o r s e p a r a t i o n f r o m t h e i n e v i t a b l e m a t r i x b e f o r e analysis. A t o m i c A b s o r p t i o n S p e c t r o m e t r y . The most w i d e l y a c c e p t e d measurement t e c h n i q u e s i n modern d a y a n a l y s i s a r e f o u n d i n t h e f i e l d o f spectroscopy. A search o f the l i t e r a t u r e reveals that

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atomic a b s o r p t i o n spectroscopy i s the most popular modern t e c h n i que f o r the measurement of t r a c e metals i n foods (26,30,32,33,^1, k2 k3 kk k5 k6). Crosby (k3) i n a comprehensive review a r t i c l e surveyed the l i t e r a t u r e over a f i v e - y e a r p e r i o d using two appropr i a t e j o u r n a l s (one B r i t i s h and one American) and found t h a t there was a t o t a l of 8 3 a r t i c l e s d e a l i n g with atomic a b s o r p t i o n as compared t o a combined number of 6 2 d e a l i n g with molecular absorption spectrometry, flame photometry, neutron a c t i v a t i o n , x-ray f l u o r e s cence, chemical t i t r a t i o n , e t c . Atomic a b s o r p t i o n c e r t a i n l y has advantages because of the extensive and a u t h o r i t a t i v e s t u d i e s that have been made to improve instrumentation and work out the d e t a i l s f o r the a n a l y s i s o f metals i n many types of chemical m a t r i c e s . Methods have been worked out f o r some 6 0 t o TO elements c o v e r i n g c e r t a i n l y the most important ones i n the p e r i o d i c t a b l e . Modern atomic a b s o r p t i o n instrumentation has been developed which almost completely e l i m i n a t e s the p o s s i b i l i t y of operator e r r o r , and with multi-element lamps a v a i l a b l e has been i n c o r p o r a t e d . lamps and r e o p t i m i z i n g instrumental c o n d i t i o n s f o r each new metal to be analyzed. Atomic a b s o r p t i o n can a l s o be made very s e n s i t i v e by the use o f the g r a p h i t e furnace i n s t e a d o f a flame as a sample c e l l (30,^7,^8). Furnaces have been used i n a number o f d i f f e r e n t forms and c o n f i g u r a t i o n s ranging from c e l l s with open tops t o f i l a m e n t s and tube designs. Samples can be i n the form o f s o l i d s , p a s t e s , or l i q u i d s , and by programming the h e a t i n g o f the furnace, p r e l i m i n a r y ashing can u s u a l l y be avoided. 9

9

9

9

Atomic Emission Spectrometry. Emission spectroscopy was the e a r l i e s t developed multielement measurement technique (1+9,50,51, 52). I t s widest acceptance was by the metal i n d u s t r y where i t was p a r t i c u l a r l y u s e f u l i n determining a few elements r e p e t i t i v e l y i n a metal ( u s u a l l y some form of s t e e l ) matrix which was w e l l defined. I t was a l s o used i n the food and a g r i c u l t u r a l f i e l d and was r e s p o n s i b l e f o r much o f the e a r l y knowledge of the concentrations o f a number of t r a c e elements i n orange j u i c e (23, 2k). Recently, a very important development has been made t o enhance the ease o f manipulation and the range of a p p l i c a b i l i t y o f emission spectroscopy t o t r a c e metal a n a l y s i s . T h i s development i s the plasma source which can be employed as an. accessory source i n most d i r e c t reading emission spectrometers i n p l a c e o f the arc or spark or may be i n c o r p o r a t e d d i r e c t l y i n the design o f the spectrometer by the manufacturer. This development has been d i s cussed i n d e t a i l i n the recent l i t e r a t u r e ( U 9 , 5 3 - 5 8 ) . There are two popular types of plasma sources: l ) the d i r e c t current plasma (DCP), and 2) the i n d u c t i v e l y coupled plasma (ICP). In the commercial v e r s i o n of the former plasma source (marketed by Spectrometries, I n c . ) , the sample i s a s p i r a t e d with argon through a small o r i f i c e i n t o a chamber where the l a r g e d r o p l e t s s e t t l e out and the f i n e mist i s conveyed by the argon stream t h r ough a chimney to the v e r t e x of a plasma which i s i n the form of

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an i n v e r t e d Y c o n f i g u r a t i o n . The v a p o r s a r e h e a t e d and a t o m i c emission takes p l a c e i n t h i s area ( v e r t e x of the Y ) . T h i s emiss i o n f l u x i s f o c u s e d on an e n t r a n c e s l i t t o t h e m o n o c h r o m a t o r and p r o g r e s s e s through t h e o p t i c a l path which c o n s i s t s o f a system o f m i r r o r s , an échelle g r a t i n g and a p r i s m t o s e p a r a t e t h e w a v e l e n g ths i n t o a two-dimensional p a t t e r n c o n s i s t i n g o f s e v e r a l wavelengt h o r d e r s . Measurement o f i n t e n s i t i e s c a n be s i m u l t a n e o u s ( i . e . , m u l t i e l e m e n t ) o r s e q u e n t i a l d e p e n d i n g how t h e i n s t r u m e n t has b e e n designed t o operate. The o t h e r t y p e o f p l a s m a s o u r c e , t h e i n d u c t i v e l y c o u p l e d p l a sma, has b e e n e n g i n e e r e d i n t o s p e c i f i c a l l y d e s i g n e d i n s t r u m e n t a t i o n m o d u l e s by A p p l i e d R e s e a r c h L a b o r a t o r i e s , J a r r e l l A s h ( a d i v i s i o n of F i s h e r S c i e n t i f i c Corp.), P e r k i n Elmer, Instrumentation L a b o r a t o r i e s , and B a i r d A t o m i c i n t h e U n i t e d S t a t e s ; f o r e i g n models are a l s o a v a i l a b l e . When t h e I C P p l a s m a i s i n o p e r a t i o n , t h e s a m p l e i s d i s p e r s e d as l i q u i d d r o p l e t s i n t o an a r g o n s t r e a m and d i r e c t e d t o t h e c e n t e r zon mode i s d i f f e r e n t f r o m t h i t means t h a t t h e e x c i t a t i o n r e g i o n i s p r o b a b l y c o n s i d e r a b l y h i g h er i n t e m p e r a t u r e . T h e r e a r e some a d v a n t a g e s and some d i s a d v a n t ages t o t h e I C P s y s t e m compared t o t h e DCP s y s t e m . Elements, l i k e v a n a d i u m , molybdenum and t u n g s t e n w h i c h t e n d t o f o r m h i g h l y r e f r a c t o r y compounds a r e p r o b a b l y more c o m p l e t e l y a t o m i z e d . Theref o r e , t h e r e i s l e s s i n t e r f e r e n c e from t h e m a t r i x , a t y p e o f i n t e r f e r e n c e which i s n e a r l y always a problem w i t h s p e c t r o s c o p i c analysis. The p r i n c i p a l d r a w b a c k s a r e : l ) n e b u l i z e r d e s i g n i s c r i t i c a l i n the o p e r a t i o n o f i n d u c t i v e l y coupled plasmas; the r e ason i s t h a t c l o s e c o n t r o l o f t h e r a t e o f f l o w o f sample t o t h e p l a s m a must be a c h i e v e d t o a v o i d p l a s m a i n s t a b i l i t y , 2 ) t h e v i e w ing ( o b s e r v a t i o n ) a r e a t o which t h e o p t i c s are exposed i s d i r e c t l y i n t h e c e n t e r o f t h e p l a s m a and b a c k g r o u n d e m i s s i o n i s h i g h comp a r e d t o t h e s i t u a t i o n w i t h t h e d i r e c t c u r r e n t p l a s m a where v e r y l i t t l e of the plasma r e g i o n i s observed d u r i n g a n a l y s i s , 3 ) mult i e l e m e n t f a c i l i t i e s , t h e t y p e o f i n s t r u m e n t a t i o n u s u a l l y accompa n y i n g t h e i n d u c t i v e l y c o u p l e d p l a s m a s , a r e e x p e n s i v e and may be o u t o f t h e f i n a n c i a l r e s o u r c e s o f many s m a l l l a b o r a t o r i e s , and k) when a m u l t i e l e m e n t f a c i l i t y i s c h o s e n t h e b u y e r must make a dec i s i o n as t o w h i c h e l e m e n t s he i s i n t e r e s t e d i n d e t e r m i n i n g ; i n d o i n g s u r v e y w o r k t h i s d e c i s i o n i s h a r d t o make b e c a u s e one s e l dom knows i n a d v a n c e t h e e l e m e n t s t h a t may be i m p o r t a n t . Because o f t h e d i s a d v a n t a g e s o f t h e ICP systems l i s t e d above, we c h o s e t o do o u r s t u d y on t h e s i m p l e r l e s s e x p e n s i v e s e q u e n t i a l d i r e c t c u r r e n t plasma system ( 2 7 , 5 9 - 6 5 ) · It w e l l to recognize t h a t i n any c h o i c e o f t h i s k i n d t r a d e - o f f s may become n e c e s s a r y . F o r e x a m p l e , t h e DC p l a s m a i s s u b j e c t t o more o r l e s s s e v e r e matr i x e f f e c t s , and t h e s e must be a c c o u n t e d f o r i n s e t t i n g up t h e m e t h o d o l o g y ( 2 8 ) . T h e s e e f f e c t s a r e i l l u s t r a t e d i n F i g u r e s 1 and 2 w h i c h show t h e i n f l u e n c e o f t h e l a r g e p o t a s s i u m c o n c e n t r a t i o n s on b o t h t h e atom and i o n l i n e s o f b a r i u m . i s

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Ba

Concentration (ng/rnl) Analytical Chemistry

Figure /. Effect of analytical curve for ppm) in 0.1 M HNO ; K/Ba ratio (4000/L) A

potassium on barium calibration curves using DC P A ES: (a) barium in matrix containing constant Κ concentration (800 (b) analytical curve for barium in matrix containing constant in 0.1M H NO(c) analytical curve for barium in 0.1M HNOj (2S)

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Analytical Chemistry

Figure 2. Matrix effects on the calibration curve of barium using DCPAES at the atom line (553.55 nm) and the ion line (455.40 nm): (a) barium in 0.1M HNO , atom line; (b) barium in 0.1 M HNO containing 100 ppm K, atom line; (c) barium in 0.1M HN0 , ion line; (d) barium in 0.1M HN0 containing 1000 ppm K, ion line (28j s

s

3

3

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CITRUS NUTRITION AND

QUALITY

Atomic F l u o r e s c e n c e Spectrometry. A spectroscopic technique r e l a t e d t o some o f t h e t y p e s m e n t i o n e d above i s a t o m i c f l u o r e s cence spectrometry (AFS). L i k e atomic a b s o r p t i o n s p e c t r o m e t r y ( A A S ) , AFS r e q u i r e s a l i g h t s o u r c e s e p a r a t e f r o m t h a t o f t h e h e a t ed f l a m e c e l l . T h i s c a n be p r o v i d e d , as i n AAS, by i n d i v i d u a l ( o r m u l t i e l e m e n t l a m p s ) , o r by a c o n t i n u u m s o u r c e s u c h as x e n o n a r c or by s u i t a b l e l a s e r s o r c o m b i n a t i o n o f l a s e r s and d y e s . The l a s er i s s t i l l p r e t t y much i n i t s i n f a n c y b u t i t i s l i k e l y t h a t f u t u r e d e v e l o p m e n t w i l l c a u s e t h e l a s e r , and c o n s e q u e n t l y t h e many s p e c t r o s c o p i c i n s t r u m e n t s t o w h i c h i t c a n be a d a p t e d t o , t o b e come i n c r e a s i n g l y p o p u l a r . C o m p l e t e f r e e d o m o f w a v e l e n g t h s e l e c t i o n s t i l l remains a problem. U n l i k e AAS t h e l i g h t s o u r c e i n AFS i s n o t i n d i r e c t l i n e w i t h t h e o p t i c a l p a t h , and t h e r e f o r e , t h e r a d i a t i o n e m i t t e d i s a r e s u l t o f e x c i t a t i o n by t h e lamp o r l a s e r source. Atomic f l u o r e s c e n c e spectrometry p r o v i d e s h i g h s e n s i t i v i t i e s f o r e l e m e n t s whose a n a l y t i c a l l the u l t r a - v i o l e t region i s h e s as one a p p r o a c h e s t h e u p p e r end o f t h e v i s i b l e r e g i o n (i+00700 nm). We made a c o m p a r a t i v e s t u d y (28) d e t e r m i n i n g s e v e r a l e l ements i n s e v e n b r a n d s o f F l o r i d a o r a n g e j u i c e b y i n d u c t i v e l y coupled plasma atomic emission spectrometry (ICPAES), d i r e c t c u r r e n t p l a s m a a t o m i c e m i s s i o n s p e c t r o m e t r y (DCPAES), f l a m e a t o m i c f l u o r e s c e n c e s p e c t r o m e t r y ( F A F S ) , and f l a m e a t o m i c a b o s r p t i o n s p e c t r o m e t r y (FAAS). T h e s e c o m p a r i s o n s a r e shown i n T a b l e V. The s a m p l e s f o r t h i s s t u d y were d r y a s h e d and d i s s o l v e d i n e i t h e r n i t r i c o r h y d r o c h l o r i c a c i d s , i . e . , t h e s e were s e p a r a t e s e t s d i s s o l v e d i n e a c h a c i d ( t h e a c i d c o n c e n t r a t i o n d u r i n g a n a l y s i s was 0.1 M). I t i s o b s e r v e d t h a t t h e n i t r i c and h y d r o c h l o r i c a c i d m e d i a show some d i f f e r e n c e s , b u t t h e d i f f e r e n c e s do n o t seem t o be particularly consistent. The p r e c i s i o n o f r e p l i c a t i o n u s i n g t h e i n s t r u m e n t t y p e s l i s t e d was g e n e r a l l y t h e b e s t w i t h FAAS. I n s p i t e o f t h e v a r i a b i l i t y n o t e d between some o f t h e m e t h o d s , t h e r e s u l t s seem w e l l w i t h i n s i m i l a r r a n g e s o f c o n c e n t r a t i o n s and t h e degree of c o n f o r m i t y g i v e s c o n f i d e n c e t o the g e n e r a l v a l i d i t y of the r e s u l t s . Neutron A c t i v a t i o n Spectrometry. Another i n s t r u m e n t a l t e c h n i q u e w h i c h has a p p l i c a b i l i t y t o a w i d e r a n g e o f e l e m e n t s i s n e u tron activation analysis. I n t h i s method t h e sample ( w h i c h c o u l d be o r a n g e j u i c e w i t h o u t any p r i o r sample t r e a t m e n t ) i s i r r a d i a t e d with a strong neutron f l u x . The e l e m e n t s o f a n a l y t i c a l i n t e r e s t are thus c o n v e r t e d t o u n s t a b l e i s o t o p e s which decay w i t h c h a r a c t e r i s t i c e n e r g i e s and t h u s measurement o f t h e i n t e n s i t i e s r e s u l t s i n a n a l y t i c a l v a l u e s f o r t h e elements o f i n t e r e s t . T h e r e a r e some s e r i o u s d r a w b a c k s t o t h i s m e t h o d , however. The m a t r i x c a n c a u s e s e v e r e b a c k g r o u n d e f f e c t s e s p e c i a l l y when t h e sample c o n t a i n s l a r g e amounts o f an e l e m e n t , l i k e p o t a s s i u m , w h i c h i s t h e s i t u a t i o n w i t h orange j u i c e . I n t h i s event t e d i o u s c h e m i c a l s e p a r a t i o n s must be c a r r i e d out t o a c h i e v e a d e q u a t e s e l e c t i v i t y , a c c u r a c y

In Citrus Nutrition and Quality; Nagy, S., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

16.

MCHARD

ET AL.

Trace Metals

in Orange

Juice

377

and p r e c i s i o n . F a c i l i t i e s f o r neutron a c t i v a t i o n are v e r y expens i v e a n d , t h u s , most o f t e n t h e a n a l y s t w o u l d g e n e r a l l y h e o b l i g e d t o send samples t o a s e r v i c e l a b o r a t o r y equipped t o p e r f o r m such analysis. A sample o f F l o r i d a o r a n g e j u i c e h a s b e e n a n a l y z e d b y n e u t r o n a c t i v a t i o n ( 6 6 ) a n d t h e r e s u l t s a r e g i v e n i n T a b l e V I . F o r some elements, a c t u a l c o n c e n t r a t i o n s a r e shown a l o n g w i t h t h e p r e c i s i o n o f t h e d a t a , b u t f o r most e l e m e n t s o n l y m a x i m a l c o n c e n t r a t i o n s are l i s t e d . T h i s means t h a t v a l u e s c a n n o t b e h i g h e r t h a n t h o s e g i v e n b u t c o u l d be much l o w e r . A g a i n , t h e s e d a t a a r e i n c l u d e d i n t h i s d i s c u s s i o n o n l y f o r c o m p a r i s o n w i t h t h a t t o be p r e sented i n subsequent paragraphs and i t should be emphasized t h a t t h e v a l u e s l i s t e d as maxima a r e r e l a t i v e l y m e a n i n g l e s s . F o r many o f t h e e l e m e n t s , t h e d a t a a g r e e w e l l w i t h i n an o r d e r of magnitude w i t h the d a t a gathered by us and w i t h t h e d a t a o f R o b e r t s a n d Gaddum. T h e r e a r e some o b v i o u s d i s c r e p e n c i e s The upper l i m i t s g i v e n f o r s u l f u s i l i c o n (UOOO ppm), a n A l s o , t h e r e seems t o be no r e l i a b l e e v i d e n c e t h a t y t t r i u m , t i t a n ium a n d n i c k e l a n d z i r c o n i u m a r e above 1 ppm i n s i n g l e s t r e n g t h orange j u i c e . O t h e r w i s e , t h e d a t a a r e c r e d i b l e , a n d i t s h o u l d be n o t e d t h a t t h e s e v a l u e s w e r e o b t a i n e d f r o m o n l y one sample a n d no a t t e m p t was made t o i m p r o v e t h e v a l u e s b y r a d i o c h e m i c a l s e p a r a t ions . S p a r k S o u r c e Mass S p e c t r o m e t r y . A n o t h e r method f o r t r a c e a n a l y s i s p r o b a b l y s h o u l d be mentioned and t h a t i s spark source mass s p e c t r o m e t r y . I n t h i s t e c h n i q u e , t h e sample i n t h e f o r m o f a s o l i d s e r v e s as an e l e c t r o d e and v a p o r s , formed by s p a r k i n g , a r e a t o m i z e d a n d i o n i z e d t o m e t a l i o n s w h i c h a r e s e p a r a t e d b y a mass s p e c t r o m e t e r and m e a s u r e d . The equipment i s e x p e n s i v e a n d good r e s u l t s r e q u i r e t h e a t t e n t i o n o f a s k i l l e d o p e r a t o r . Even under t h e b e s t c o n d i t i o n s o r d e r o f m a g n i t u d e agreement o f r e s u l t s i s about t h e b e s t t h a t can be a c h i e v e d . C o m p a r i s o n o f F l o r i d a Orange J u i c e w i t h J u i c e s f r o m O t h e r phic Locales

Geogra-

I n an attempt t o update the e a r l i e r s t u d i e s on orange j u i c e and t o t r y t o a r r i v e a t some q u a n t i t a t i v e v a l u e s f o r a b r o a d r a n ge o f e l e m e n t s i n F l o r i d a a n d o t h e r s o u r c e j u i c e s a m p l e s , we u n dertook the a n a l y t i c a l p r o j e c t described i n the f o l l o w i n g d i s c u s sion. Orange j u i c e s o u r c e s o f p r i m a r y i n t e r e s t t o u s were f r o m F l o r i d a a n d B r a z i l b u t some v a l u e s were o b t a i n e d o n j u i c e s f r o m o t h e r l o c a l e s , i . e . , M e x i c o a n d C a l i f o r n i a a r e i n c l u d e d f o r comparison. Experimental Twenty-four

F l o r i d a j u i c e s , s e v e n t y - f o u r B r a z i l i a n a n d some

In Citrus Nutrition and Quality; Nagy, S., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

In Citrus Nutrition and Quality; Nagy, S., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

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Comparative

Analytical Chemistry

C = A v e r a g e c o n c e n t r a t i o n , ppm; ?RSD = | R e l a t i v e S t a n d a r d D e v i a t i o n ; LOD = L i m i t o f D e t e c t i o n , ppm; %ΑΌ = A v e r a g e D e v i a t i o n o f M e a s u r e m e n t . A l l c o n c e n t r a t i o n s a r e c a l c u l a t e d o n t h e b a s i s o f s i n g l e ICPAES = I n d u c t i v e l y C o u p l e d P l a s m a s t r e n g t h orange j u i c e (SSOJ). °Values n o t m e a s u r e d , s e e t e x t . E m i s s i o n S p e c t r o m e t r y ; DCPAES = D i r e c t C u r r e n t P l a s m a E m i s s i o n S p e c t r o m e t r y ; FAFS = F l a m e A t o m i c F l u o r e s c e n c e S p e c t r o m e t r y ; FAAS = F l a m e A t o m i c A b s o r p t i o n S p e c t r o m e t r y .

Zinc

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Rubidium

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1

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In Citrus Nutrition and Quality; Nagy, S., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

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