The attempted correlation of dye reduction time and the organoleptic qualities of ground meat

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THE ATTEMPTED CORRELATION OF DYE REDUCTION TIME AND THE ORGANOLEPTIC QUALITIES OF GROUND MEAT

A thesis Presented to the Faculty of the Department of Bacteriology The University of Southern California

In Partial Fulfillment of the Requirements for the Degree Master of Science

by Eugene P* Hess June 1950

UMI Number: EP55011

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TABLE OF COHTENTS CHAPTER

PAGE

I* Introduction . . II.

................

Experimental ........

• « « • • • •

1 7

III.

Discussion

• • • • • ............... 17

IV.

Conclusions

• • • • • • • • • • • •

BIBLIOGRAPHY

.........

. i .

20 21

LIST OP TABLES TABLE I*

PAGE Methylene blue and resazurin reduction times of various meat samples • • • • • • • » • •

II*

0

Correlation of dye reduction time, plate count and organoleptic qualities

III*

« • » • • • • • • • •

11

Further studies on dye reduction time and organoleptic qualities

15

LIST OF FIGURES FIGURE 1.

PAGE Correlation of plate count and dye reduction time

12

THE ATTEMPTED CORRELATION OP DYE REDUCTION TIME AND THE ORGANOLEPTIC QUALITIES OF GROUND MEAT CHAPTER I INTRODUCTION Today, even though the beef is examined both ante and post mortem at the time of slaughter, there is little sanitary control before it is purchased by the consumer* As a result most ground beef sold has an excessively high bacterial count* leptically sound*

This is true even of meats organo­

These high bacterial counts do not

necessarily mean that disease producing organisms are present, but show that ample opportunities for their introduction are available during the handling of the meat*

It is known that meat satisfies the food require­

ments for most micro-organisms as is shown by the use of meat Infusion media for the cultivation of the more fastidious organisms; and pathogens, if introduced, would multiply rather than die off*

The potentiality of patho­

genic and toxinogenic bacteria in meat is also emphasized by reports in the literature of food infections and poisonings caused by organisms in meat products (Savage, 1913)*

To safeguard public health it would seem desirable

to have standards of evaluation based on microbiological data viz* numbers rather than on organoleptic tests* This principle is applied in many allied fields but has not as yet been used in the meat industry*

2 Weinzirl and Newton (1914) proposed a standard of ten million organisms per gram of sample and Lefevre (1917) set as an acceptable maximum one million organisms per gram of sample.

These standards have not been widely

adopted, however, for several reasons*

One reason is

that either of the proposed standards would prohibit the sale of most ground beef.

In the majority of cases not

only as reported in this paper but also by other workers, (Brewer, 1925; Geer et al.,1935). the count of ground beef purchased on the open market far exceeded the maxi­ mum of ten million organisms per gram of sample. Another reason is that there are no commonly accepted standard methods of estimating the number of organisms in any given sample, although numerous procedures of determining bacterial numbers have been reported in the literature.

Non-acceptance of the proposed procedures

Is due mainly to the fact that the methods either take too long to be of value or they involve the use of extensive equipment.

These proposed methods may be

divided into two general groups: 1) those based on plate counts made using a saline or aqueous extract of the meat; and 2) those based on special properties of partly putrefied samples. The methods in the first group are all dependant on using some means of dislodging the bacteria from the

5 meat particles*

Bacterial numbers are then determined

by means of a plate count.

There have been numerous

procedures used to accomplish the dislodging.

Weinzirl

and Newton (1914) found that by grinding the meat in a sterile mortar containing sterile sand and water a much higher count was obtained than when the meat was shaken with glass beads#

Geer et al* (1933) obtained

counts comparable to the mortar method by shaking the sample with sterile sand* Methods utilizing special properties of putrefying meats have been reported*

Strohecker (1920) proposed

a test based on oxygen consumption#

Gorowitz and

Wlassawa (1928) investigated pH, the refractive index, and the biuret reaction but found that these tests did not indicate the freshness of the meat*

The total N,

the N of the tannins and the iodine value were also investigated by these workers; however their results were negative*

They proposed a rapid test carried out

by heating a meat sample at 50°G in ammonia free water containing magnesium oxide for a period of five minutes# This solution can then be tested with litmus paper and is found sensitive to 0#02$ ammonia* not give a positive test*

Fresh meat does

Ottolenghi (1913) found that

the amino nitrogen in a meat extract as determined by a formol titration increases as the bacterial population

4 increases*

Tillmans et al* (1921) found that if 5 grams

of well mixed meat is incubated at 45°G with one milli­ liter of a methylene blue solution incipient deeay is indicated when the dye reduction time is less than one hour*

Arbenz (1925) also utilized dye reduction for

determining incipient putrefaction*

He obtained satis­

factory results in testing beef, pork, veal, mutton, horse and several kinds of fish*

Proctor and Greenlie

(1959) using a similar technique concluded that dye reduction time might be used as a reasonable accurate means for determining abnormally large numbers of bacteria* Their method was found to be applicable to a number of foods including hamburg steak and other comminuted meats, broken eggs and other food products*

Besuits could be

obtained within an hour when the dye, resazurin, was used* The technique used by these investigators apparently was sufficiently sensitive to satisfy the requirements for a practical, standardized test* The dye reduction method would be an ideal one for the inspector in that the apparatus is simple, the time of incubation is short, and the procedure could be easily standardized*

The present investigation was

undertaken to determine if the method could be made sensitive enough to detect incipient spoilage*

5 A test based on dye reduction should have, the following properties if it is to be of value in the sanitary control of ground beef*

First, the reduction

time should decrease as the microbiological population increases*

Second, the test should indicate gross

contamination when the meat is still organoleptically sound*

By the terms organoleptically sound we mean the

meat appears fresh, and possesses no ttofftt odors or Mofffl color; in other words, the meat would be saleable*

As

stated before the results must be obtained in a short time, the equipment should be simple, and the method easily standardized* On this basis resazurin would be the dye of choice for several reasons*

Ramsdell et al. (1935) describes

a one hour test for use in grading milks that utilizes resazurin*

Golding (1943) found a good correlation

between the standard plate count and the resazurin test on milk when the results were read in one hour. Barrett et al* (1937) found that this dye is extremely sensitive to physiological abnormal and pathological milks*

Johns (1941) found that there are marked color

changes in the dye when the milk has a high leucocyte content even though there is no change in the redox potential*

This would be especially valuable in meat

6 control because the presence of an abundance of leuco­ cytes might indicate a possible infection in the animal before slaughter# Methylene blue on the other hand has long been used in the rapid grading of milks#

Elleriberger et al# (1927)

state that the methylene blue reduction test is a more reliable index of milk quality than is the agar plate count.

Hastings (1919) found that the reduction time

corresponds with the number of organisms present in milk.

Methylene blue dye reduction is one of the methods

outlined in Standard Methods for The Examination of Dairy Products (1949).

Standard Methods divides milks

in four different grades on the basis of reduction times: 1) Excellent, not decolorized in eight hours; 2) Good, decolorized in less than eight hours but not less than six hours; 3) Pair, decolorized in less than six hours but not less than two hours; 4) Poor, decolorized in less than two hours#

Prom these data it can be noted

that the reduction time is longer than that of resazurin# However, because both dyes have been used in dye reduction tests, both dyes were tested in the present experiments#

CHAPTER II EXPERIMENTAL Stock solutions of the dyes were made fresh each week#

These solutions were stored in the dark since

Prayer (1934) found that light caused a deterioration of the dyes#

The resazurin stock solution consisted of

a 0.05$ aqueous solution of the dye#

Methylene blue

stock solution was a 1:200,000 aqueous dilution of methylene blue thiocyanate# In general the procedure for each experiment was the same#

The bacteria were dislodged from the meat particles

either by shaking with glass beads or by comminuting in a Waring Blendor#

The dye was added to the tubes

or bottles containing the meat extract and these were incubated in a covered 37°G water bath# Methylene blue reduction time was based on the time required for the dye to be reduced to the colorless or leucobase end point*

Resazurin reduction time was

considered to be the amount of time it took the dye to reach the definite pink stage#

The tubes were examined

every five minutes as the end point was approached# Duplicate samples of the appropiate dilutions were plated out on Difco nutrient agar except in certain experiments when an enriched medium was used#

All

plates were incubated at 37°C for 48 hours before counting#

8 The first set of experiments were of a preliminary nature to determine whether or not dye reduction corre­ lated at all with organoleptic qualities of the meat sample.

They were also designed to show the approxi­

mate meat and dye concentration to use in order to obtain results in less than a four hour period. One to ten grams of the meat samples were placed in sterile six ounce bottles that contained 100 ml. of sterile tap water plus forty glass beads.

The

bottles were stoppered and were shaken vigorously for three minutes. added.

Immediately after shaking the dye was

The bottles were inverted twice and then were

placed in the covered waterbath. Meat samples were purchased at a local market and were sampled within one-half an hour from the purchase time.

The results of these preliminary experiments

can be found in Table I. The results of these preliminary experiments show that there is a great difference in the reduction time of both dyes with samples of different organoleptic qualities.

The sound meats do not decolorize the dyes

within a relatively long period, whereas, only a few minutes were required for meats with only a slight off odor.

These wide variations of reduction time indicate

that with the proper technique, it might be possible

Table I. METHYLENE BLUE AND RESAZURIN REDUCTION TIMES OF VARIOUS MEAT SAMPLES * Sample number

Description

Organoleptic qualities

Reduction time in minutes Methylene Blue Resazurin

1.

ground beef 1 gm/lOO ml. sterile water

sound

480

240

2.

ground beef 10 gny^lOO ml. sterile water

sound

220

120

3.

ground beef 10 gm/lOO ml. sterile water

slight off odor

20

15

4.

ground beef 10 gm/lOO ml. sterile water

off odor & color

10

10

# 10 ml. Methylene blue stock and 2 ml. Resazurin stock used per 100 ml. suspension. Incubation temperature 57°C.

10 to detect the presence of a large microbiological pop­ ulation in meat previous to organoleptically detectable spoilage with a rapid decolorization test.

These results

also show that the meat and dye concentration used on samples 2,3, & 4, are adequate to give results in a relatively short time. Further experiments were undertaken to determine if any correlation exists between reduction time, plate count and organoleptic qualities.

This was done by

checking the above factors periodically on a large sample of meat that was in the process of spoiling.

The

methods used were the same as those previously described with one exception.

Plate counts were made on a meat

extract obtained by shaking a one gram sample suspended in sterile water that contained approximately forty glass beads.

This bottle was shaken vigorously for

a period of three minutes and then samples were taken for plating.

The results of these experiments can be

found in Table II. From the results in Table II we can see an apparent correlation between plate count and reduction time. This is shown even more clearly in Fig. 1 in which the plate count is plotted against reduction time.

The

data from both samples (6 & 7) were used for the curve. In general an Increase in reduction time with decreasing

Table II CORRELATION OF DYE REDUCTION TIME, PLATE COUNT AND ORGANOLEPTIC QUALITIES Sample number

6

Time of sampling in hours!

Description

ground beef 10 gm/100 ml. sterile water

Reduction time in minutes Methylene blue Resaizurin Plate count

sound

120

40

3x10®

sound

120

60

1.3x10

5

sound

45

40

1.4x10®

7

sound

120

40

3x10®

9

slight off odor

45

30

3.5x10®

definite off color & odorlO

5

0 3

23 7

Organoleptic qualities

*

Q

1x10®

sound

180

180

sound

180

180

29

sound

180

60

4xl07

31

slight off odor

180

60

4xl07

slight off odor & < color 180

60

7.6xl07

0 5

33

Round steak in sterile grinder 10 gm/100 ml. sterile water

* 10 ml. Methylene blue stock and 2 ml. resazurin stock per 100 ml. Incubation temperature 37°C.

1.5x10® C* 1.5x10

b a c ftria of Log. no.

10

20

30

4o

SO

60

r» Fig. 1.

Correlation

op plate count

tc and

dye

reduction ti me.

15 numbers of bacteria is observed. It can also be seen that there is some correlation between the length of the reduction time and organoleptic qualities.

It is very definitely shown that a distinct

drop in reduction time does occur when organoleptic qualities are off. Further experiments were run in an attempt to make the test more sensitive by varting meat and dye concen­ trations.

A change in procedure was also instigated at

this point.

This was done for two reasons.

First, it

was felt that by using such a large sample there were enough substances present in the meat to poise the potential so that the significant drop in reduction time did not occur until after definite decomposition had begun.

Second, the one hundred ml. of meat sus­

pension did not completely fill the bottle.

This

may have allowed enough of an air space to delay the reduction time due to the presence of oxygen (Thornton, 1929). In the modified procedure one gram of meat was homogenized with 100 ml. of sterile water in a sterile / Waring Blendor cup for 2 minutes. Then 10 ml. of meat extract and meat fibers was pipetted into sterile 15 ml. tubes.

Essentially this gives approximately

0.1 gram meat sample per tube.

The dye was added and

14 the tubes were incubated at 37°C.

Plate counts were

made on the meat extract remaining in the blendor cup. Tbe medium used for plate counts was an enriched one consisting of Tryptose 20 gm., dextrose 1 gm., yeast extract 7 gm., NaCl 5 gpu, Agar 15 gm., distilled water 1,000 ml.

The pH was adjusted to pH 7.

The

results of these experiments can be found in Table III. Prom the results found in Table III it can be seen that there is no significant drop in dye reduction time before organoleptic qualities are off. In fact even with obvious spoilage the reduction time remained less than previous reduction times of sound samples.

This may be due to the presence of a

different type of flora in the meat samples. Also using the enriched medium there is little correlation of plate counts and dye reduction time. Apparently this medium promotes the growth of micro­ organisms that contribute only slightly to the red­ uction of the dye. Many further experiments were carried out in an attempt to increase the sensitivity of the test.

Both

the dye concentration and the amount of sample were varied but all combinations used were found unsatis­ factory for numerous reasons.

If the dye content

was decreased, the end point was obscured by the color

Table III FURTHER STUDIES OH DYE REDUCTIOH TIME AID ORGAIOLEPTIC QUALITIES * Sample number 9

Time of sampling in hours 0 15

10

ground beef 1 gm/100 ml. sterile water

Organoleptic qualities

Reduction time in minutes Methylene blue Resazurin Plate count

sound

180

80

4x10 5

sound

165

75

lSxlO6

21

slight odor

150

40

15xl07

41

off odor & color

150

30

19xl07

sound

210

210

sound

320

210

9.4x10® ry 1.2x10'

sound

150

180

5.9x10®

0 14 18

12

Description

round steak ground in sterile grinder 1 gm/100 ml. sterile water

42

slight odor

180

150

2.7x10®

45

off odor & color

120

60

1.3xl09

slight odor

60

30

1.2x10®

slight odor

60

30

1.7x10®

0 2

ground beef 1 gm/100 ml. sterile water

24

definite off odor

40

25

3x10®

28

definite off odor

17

17

3.3X10®

* 10 ml. meat extract in 15 ml. tube; 1 ml. resazurin stock, 5 ml. methylene blue stock Plate counts were made on the enriched medium

16 of the meat extract and if the dye concentration was increased, the test took too long to be of value.

CHAPTER III DISCUSSION Weinzirl (1924) states that meat spoilage is a complex process*

In the first place there is a souring of the

meat due to fermentation of the carbohydrates present by aerobic, facultative, and anaerobic bacteria. Secondly, there is a digestion of proteins by bacteria without the abundant evolution of sulfides.

Thirdly, there is a

production of indole, skatol, and other vile smelling compounds*

Fourthly, there is a putrefaction with the

production of sulfides by anaerobes. Boffstadt (1924) forrd there was no correlation between the plate count and organoleptic qualities of ground beef.

Thornton (1929, 1950) stated that the

reduction of methylene blue is dependant on two factors: 1) the using up of the oxygen dissolved in the milk by microorganisms; 2) the reduction of the dye by substances present in the milk.

From the results of these two

workers it can be seen that in all probability, a test utilizing a correlation of dye reduction time and organoleptic qualities of ground beef could never be successful. However, Ellenberger (1927) has found that methy­ lene blue reduction time is a more accurate means of determining the keeping quality of milk than is the

18 agar plat© method.

The results of present experiments

would tend to show the dye reduction time is not a satisfactory method of judging the sanitary condition of ground meat. Fred (1912) early in the history of dye reduction pointed out that most organisms have the power to reduce dyes but that some organisms reduce the dye more rapidly than others.

This would tend to show that the results

of reduction times would be extremely variable depending on the predominating flora at the time the sample was taken. One of the great difficulties encountered in this work was the determination of what constitutes organo­ leptically sound meat and conversly when can we say the meat has an "off” odor or color.

Fellers (1929)

defines fresh meat as meat that appeals to our aesthetic senses.

This is probably as good a definition as any.

On the other hand there is wide variation in opinion as to what constitutes an off odor.

During the course

of this work numerous samples were checked by different people and quite often there was disagreement as to whether the sample should be considered unsatisfactory or still saleable.

This factor accounts for some of

the experimental results that do not correlate with the rest of the data.

19 Another difficulty encountered was the determination of the end point when resazurin was used*

During the

course of reduction the color change is from a purple through several shades of purple-pink and finally to pink#

Other investigators using the one hour test

compared the tubes to a specific color chart#

But even

by the use of a color chart the change is so gradual that a great deal of personal error is introduced. This factor may also account for some of the non­ correlation of experimental results.

CHAPTER IV CONCLUSIONS From the experimental work described it can be concluded that there is not a significant correlation between dye reduction time and organoleptic qualities of ground beef.

BIBLIOGRAPHY American Public Health Association# 1948 Standard Methods for the Examination of Dairy Products# pp# 36-39, 9th ed#, Amer# Pub# Health Assoc#, New York Arbenz, E* 1925 Deteoting the beginning of purefaction in meat and meat products# Mitt# Lebensm# Hyg# 16:84# C# A* 19*2988 Barrett, W# D* , Rutan, H#, and Keenan, J# A# 1937 The resazurin test, its use and practicability as applied to quality control of raw milk* J# Dairy Sci* 20* 705-709 Brewer, C* M# 1925 The bacteriological content of market meats# J# Bact# 10*545*560# Ellenberger, H# B#, Bond, M# C#, Robertson, A# H#, and Moody, R* I# 1927 A comparison of the methylene blue reduction test and the agar plate count for determining quality of milk# Vermont Agr# Exp# Sta. Bull* 264 Fellers, C# R# 1929 When are meats or fish spoiled# Amer# J# Pub# Health 19 *389*592 Frayer, J# M# 1934 Light and temperature as factors in the methylene blue reduction test# Vermont Agr* Exp# Sta# Bull# 374 Fred, E* B# 1912 A study of the quantitative reduction of methylene blue by bacteria found in milk and the use of this stain in determining the keeping quality of milk# Cent# Bakt# Parasltenk# II Abt# 35*391-428 Geer, L# F#, Murray, W. T#, and Smith, E* 1933 Bacterial content of frosted hamhurg steak* Am# J# Pub# Health 23:673-676 Goulding, N# S# and Jorgenson, S# I* 1943 A correlation of the resazurin grade with the standard plate count ofraw milk* J# Milk Tech# 8*(4) 189-195*

22 Hoffstadt, R* E* 1924 Bacterial Examination of ground beef with reference to standard analysis* X* Relation of bacterial count and aerobic species present to spoilage* Amer* J* Hyg* 4:33-42; 43-51 Horowitz-Wlassowa 1928 The sanitary valuation of meat* Etschr* f* Rntersuch. d* Lebensmitt* 55:239 C* A* 22:3465 Hastings, E* G* 1919 The comparative value of quantita­ tive and qualitative bacteriological methods with especial consideration of the methylene blue test* J. Dairy Sci. 2:295-311 Johns, C. K* 1941 Some aspects of the resazurin test* Fifteenth Annual Report of the Hew York State Association of Dairy and Milk Inspectors* Le Fevre, E* 1917 Sanitary control of ground meats* Am* Food J* 12:140 Ottolenghi 1913 Studies on the maturation and putre­ faction of meat* Genussmittel 26:728-58* C. A* 8:1175 Proctor, B* E* and Greenlie, D. G* 1939 Reductionoxidation potential indicators in quality control of foods* I* Correlation of resazurin reduction rates and bacterial plate counts as indexes of the bacterial condition of fresh and frozen foods* Food Research 4:441-6 Ramsdell, G. A*, Johnson, W* T*, and Evans, F* R* 1935 Investigation of resazurin as an indicator of the sanitary condition of milk* J* Dairy Sci* 18:705717 ~ Savage

1913 Rept* of Loca). Gov*t* Bd. Series Ho* 77, Cited by Tanner, F. W* 1933 Food borne infections and intoxications* The Twin City Printing Co*, Champaign, 111*

23 Strohecker, R* 1920 Detection of incipient putrefaction in meat* Chem* 2tg* 44:744* C. A* 15:2678 Thornton, H* R* and Hastings, E* G* 1929 Studies on the oxidation-reduction potential in milk* J* Bact. 18:295-352 Thornton, H* R* and Hastings, E* G* 1930 Studies on the oxidation-reduction of milk - The methylene blue test* J* Dairy Sci* 15:221-245 Tillmans, J*, Strohecker, R*, and Schults, W* 1921 The detection of incipient decay in meat* Z* Nahr*Genussm* 42:65-75* G* A* 16:444 Weinzlrl, J* and Newton, E* B* 1914 Bacteriological methods for meat analysis* Amer* J* Pub* Health 4:408-413 Weinzirl, J* 1924 Concerning the relation of the bacterial count to the putrefaction of meat* Amer* J* Pub* Health 14:946-949