The survival of food poisoning organisms in highly carbonated beverages

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THE SURVIVAL OP POOL POISONING- ORGANISMS IN HIGHLY CARBONATED BEVERAGES

A Thesis Presented to the Faculty of* the Department of Bacteriology University of

Southern California

In Partial Fulfillment of the Requirements for the Degree Master of

Science in Bacteriology

by James E. Stewart June

1950

UMI Number: EP55018

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T h is thesis, w r it t e n by

JAMES £• STEWART u n d e r the g u id a n c e 6 f Ais.... F a c u l t y C o m m itte e , a n d a p p r o v e d by a l l its m em b ers, has been p re se n te d to a n d a ccep ted b y the C o u n c i l on G ra d u a te S t u d y a n d Research in p a r t i a l f u l f i l l ­ m e n t o f the re q u ire m e n ts f o r the degree o f

MASTER OF SCIENCE

Dean D ate .

Faculty Committee

Chairman

..

ACKNOWLEDGMENT Thanks must go to Mr. Harry Lake of the Nehi Beverage Company who so kindly supplied the beverage used in this work.

To Dr. James W. Bartholomew go my thanks for sug­

gesting this problem and for his helpful advice both in this and in other problems*

TABLE OP CONTENTS CHAPTER

PAGE • • . . ..........................

1

II.

HISTORICAL R E V I E W .......... * .................

2

III.

EXPERIMENTAL M E T H O D S ..........................

6

IV.

EXPERIMENTAL R E S U L T S ..........................

8

I.

INTRODUCTION

V. DISCUSSION VI.

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

CONCLUSIONS . . ............................... .

BIBLIOGRAPHY

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

19 21

2

LIST OF TABLES TABLE 1#

PAGE Survival of .Test Organisms in Complete Carbonated Beverage

12

2* Effect of Size of Inoculation on the Survival of E. Coll and M. Aureus in 3.

cola type beverages

•

13

Cola Type Beverages •

14

Effect of Storage Temperatures on the Survival £• Coll and M. Aureus in

4* Survival of E* Coli in Cola

Type Beverage

Containing All IngredientsExcept Carbon Dioxide 5.

15

Survival of E. Coli Inoculated Into Bottles Con­ taining Only Carbonated Water ...............

6

15

. Survival of E* Coli in Complete Cola Type Beverage Adjusted to pH of 6*5-7»0 with KOH

16

7. Survival of E. Coll Inoculated into Distilled Water Adjusted to Final pH of 2 . 9 * . ......... 8

16

. Survival of E. Coli in Each of the Three Main Fractions Used In the Preparation of the Complete Cola Type Beverage

9*

17

Survival of E. Coli and M* Aureus Inoculated into ?/ater Containing *3 Grains of Caffeine per 350 ml

18

CHAPTER

I

INTRODUCTION It has long been noted that yeasts are the main cause of soft drink spoilage for the 6,500 soft drink bottling plants throughout the United States*

The overwhelming

presence of yeast as spoilage organisms has been attributed to the acid condition of the beverage which would favor yeast growth.

However the literature is not consistent con­

cerning those factors which retard bacterial growth.

Now,

the question naturally arises as to why, with such a sub­ strate as one finds in carbonated beverages, the growth is limited predominantly to yeasts.

We can assume that if

yeasts get Into the beverage then also bacteria gain entrance. What then happens to these bacteria?

With this thought in

mind the problem was attacked using various microorganisms in a commercial highly carbonated beverage.

Organisms which

might cause food poisoning were given particular attention since they have been generally ignored by previous workers.

CHAPTER II HISTORICAL

REVIEW

Cola type soft drinks are usually made up of five ingredients: and caffeine*

sugar, flavor, color, acid, carbon dioxide The specific items used will of course vary

with the kind or flavor of the beverage desired.

Most

authors readily agree that the bacterial population of a carbonated beverage will gradually decrease from the time of carbonation to the time of consumption.

Prom this point on

there is much debate as to just what agent is responsible for this diminution in numbers. Carbonic acid gas was first brought to this country from Germany in 1884.

Its use in beverages followed a few

years later, and, thus, it is quite natural that much work has been carried on with respect to the effect of carbon dioxide in beverages on bacteria.

Valley and Rettger (1927)

reported that whatever harmful action might be exerted by carbon dioxide in beverages was the result of Increased hydrogen Ion concentration and not due to the carbon dioxide as such.

Young and Sherwood (1911) using cultures of

£• typhosus, B. coli and B. prodigiosus and applying carbon dioxide at 18 pounds pressure, found that there was not a complete killing of the organisms during the entire experi­ ment.

Carbonic acid gas was noted by Pinnell (1915) to be

3 antiseptic to a decided extent but did not kill or inhibit all bacteria, therefore could not be counted on as a sanitary safe-guard*

Levine (1935) found that low concentrations of

carbon dioxide were stimulating to bacteria, but that concen­ trations of

3

to

5

volumes of carbon dioxide gas prevented

the survival of many bacteria and that none of them grew under these high carbon dioxide pressures*

Koser and Skinner

(1922) inoculated Escherichia coli, Salmonella paratyphi B, Bberthella typhi * Bacillus mesentericus and Clostridium sporogenes into a carbonated beverage and found that E. coli lived from 4 to 14 days*

SI* paratyphiB survived 24

hours and E* typhi was still viable up to 48 hours.

to 48 B*

mesentericus and Cl* sporogenes showed no diminution in count up to one month* Many others have thought that the acidity developed in the beverage was responsible for its bactericidal action* Koser and Skinner (1922) believed that the acidity of the dissociated carbonic acid was the main factor responsible for the decrease in bacterial counts.

Moore and Buchanan

(1930) on the other hand have shown that the lower limits reached by high carbonation is between pH 3*4 and pH 3*3, and that these limits were usually no greater than that due to the citric or phosphoric acid in carbonated beverages, thus the inhibiting power of carbon dioxide must result from some other factor beside that of Increased hydrogen ion

4 concentration. To some the answer is the pressure obtained by the addition of carbon dioxide under pressure.

Donald, et al

(19S4) reported that it was possible to obtain complete sterilization with carbon dioxide and a sufficiently high pressure.

They used nitrogen instead of carbon dioxide and

obtained good growth, and consequently claimed that pressure alone had no effect on the bacterial count.

Hon-spore

forming bacteria were killed by carbon dioxide under 50 atmospheres pressure in about one and one half hours in *

these experiments.

- -

•

• • , . .

. ■-

. . .

They noted also that pressure was a

minor factor in increasing the acidity of carbon dioxide solutions beyond a certain point. Levine (1934) stated that bacteria are very sensitive to citric acid and that

20

to 30 grains of citric acid per

gallon of beverage will prevent spoilage due to bacteria. Koser and Skinner (1922) reported that if citric acid, tartaric acid, phosphoric acid or lactic acid was added to carbonated beverages, then these were the chief causative agents of bacterial destruction, irrespective of the effect of carbon dioxide. Caffeine possibly is another antibacterial agent present.

Other products which have high caffeine content

such as tea, coffee and cocoa have been noted to display bacteriostatic action, although the agent responsible for

5 this action has not been identified as yet probably because of the limited work on these substances*

Caffeine appears

also in pepsi cola and coca cola, in the latter case as much as *112 grains per fluid ounce.

The above mentioned colas

have been investigated by Tobey (1941)*

Bates in 1958 found

that strychnine sulphate acted to increase growth of Rhizopus nigricans and Aspergillus niger but that growth of both were decreased by caffeine citrate. that the addition of

1%

Zanda (1927) found

caffeine prevented putrification of

protein solutions and that pathogenic organisms, e.g. anthrax, typhoid, cholera, staphylococcus and colon bacteria were destroyed in varying periods of time according to their individual power of resistance.

The observation that

a Is1 0 0 solution of caffeine retarded the growth of a number of pathogenic and non-pathogenic microorganisms was confirmed by Sechi (1930).

In dilutions of Is1000 or

Is1 0 , 0 0 0 caffeine was found to stimulate bacterial growth. Mezzadrali and Amati (1932) noted the action of strychnine, quinine and caffeine on the development of Aspergillus niger.

The first favors development with doses of

.5

to

3cc per 1000 cc*s, the action increasing with larger doses. Quinine also favored development while caffeine, even at .5% concentration, inhibited growth.

CHAPTER

III

EXPERIMENTAL METHODS The organisms used in this experiment were Mierococcus aureus, Escherichia coli, Micrococcus albus, Streptococcus fecalls, Streptococcus llquefaclens, Salmonella paratyphi A, Salmonella newport, Shigella flexner* Shigella sonnei» Proteus vulgaris and a culture of pseudomonas* These were chosen as representative organisms of food poisoning and food infection, and it was our object to find out if these organisms could survive If by chance they gained entrance Into the beverage*

The beverage itself was

a commercial product of the cola type obtained direct from the bottling line of the plant* The organisms were transferred several times into nutrient broth to obtain healthy normal cultures*

At the

proper time a suspension of cells of known numbers were added to an aseptically opened bottle of beverage which was then recapped with a sterile crown* the bottle was opened and

1

At the end of 48 hours

ml of the liquid plated out

according to Standard methods to determine the number of organisms present*

All of the above organisms were tested

for survival* From this point on instead of using all the stock cultures studied in the first experiment it was decided that a typical Gram negative and Gram positive organism

7 would be used*

Escherichia coli and Micrococcus aureus

were the organisms decided upon*

CHAPTER IV EXPERIMENTAL RESULTS The results of the first experiment are shown in Table 1*

It can be seen that all the organisms were

killed within 48 h o w s after inoculation. It is well known that the size of inoculation influences the survival time of microorganisms under various conditions.

The Influence of inoculation size

on survival in cola beverage was studied by using E. coli and M# aureus as test organisms. Table 2. 1000

The results are shown in

Even though the size of inoculation varied between

ml to

10,000

per ml all of the organisms were killed

within three days time. The above results indicated that the cola beverage stored at 30° C was very lethal to the organisms tested. However, beverage is usually stored at a cold temperature. The above experiment was repeated using a storage tempera­ ture of 30° C and 5° C.

Table 3 shows that the death rate

£• coli and M. aureus at 5° C closely followed that which was obtained at 30° C.

Therefore cold storage would not

greatly Increase the chance of survival of microorganisms.* We were next lead to the problem of effect of con­ stituents, that is, just which factor or factors contributed to the bactericidal properties of the beverage displayed in

9 the first three experiments. The first ingredient studied was carbon dioxide. A series of bottles containing all of the constituents of the finished beverage except carbon dioxide were inoculated with E. coli.

One bottle was opened each day and plated

out to determine total number of E. coll present per ml of the beverage.

Table 4 shows complete killing of all or­

ganisms within three days time. In the next experiment a series of bottles were pre­ pared containing Just the water used in the beverage manu­ facture and carbon dioxide under the same pressure as found in the highly carbonated beverage.

These bottles were

inoculated with E. coli and the results shown In Table 5. It Is obvious that the carbon dioxide was not the bacteri­ cidal agent in this cola. The next experiment was designed to determine if the pH of the beverage was the lethal agent.

To determine the

effect of the hydrogen ion concentration on E. coli we ad­ justed the pH of the complete beverage to pH potassium hydroxide.

It ean be seen in Table

6

.5-7.0 with 6

that the

organisms suffered an Initial shock and then recovered and started growing rapidly.

Thus, the acid properties of the

beverage were necessary for the activities of the bacteri­ cidal agent. Since pH seemed to be an important factor it was

thought advisable to next conduct an experiment to deter­ mine if pH alone was the bactericidal agent.

Since both

phosphates and citrates are present in this beverage, both were used in obtaining low pH* a.

Distilled water was ad­

justed to pH of 2.9 with H3 PO4 and inoculated with E. coli. Table 7 shows that H 3 PO4 at this pH had little effect on this organism.

Distilled water adjusted to a pH of 2.9 with

citric acid not only supported growth but also enhanced it. This is shown in Table 7. The above results show that while an acid reaction is necessary for the bactericidal action of the cola* the action is not due to the acidity alone* therefore there must be some bactericidal agent in the ingredients used.

The

three main fractions of the ingredients were obtained from the Bottling Plant and these separate samples were prepared in proper concentrations using distilled water as a base. The three fractions contained: Fraction 1 - Sucrose syrup* gum acacia, citrus oils* spice oils* vanilla, extraction from cola nuts. Fraction 2 - Phosphoric acid* caffeine. Fraction 3 - Caramel color produced from corn sugar. £• ooll was inoculated into the proper concentrations of each of the three fractions and by looking at Table

8

it is obvious that the portion containing the phosphoric acid and the caffeine was bacteriostatic to E. coli.

Each

of the other portions was non-toxic* Prom the above tests we narrowed the field down to the solution of phosphoric acid and caffeine.

A sample of

each of these was obtained from the manufacturer of the carbonated beverage and then made up to the concentrations of the finished beverage using distilled water as a base* In the case of the caffeine .3 grains per 350 ml of dis­ tilled water was used.

Since the phosphorie acid results

have already been reported in Table 7 no further words are necessary.

E. coli was Inoculated into the water containing

the caffeine and Table 9 shows that it is definitely inhi­ bitory.

M. aureus when Inoculated into the same material

displayed marked Inhibition but not to the extent shown by E. coli in Table

8

.

12

TABLE

I

SURVIVAL OF TEST ORGANISMS IN CARBONATED COLA TYPE BEVERAGE

count after 2 days incubation at 25° C Micrococcus aureus Micrococcus albus

negative w

Streptococcus fecalis

H

Streptococcus liquefaciens

tt

Salmonella paratyphi A

H

Salmonella newport

tt

Shigella flexner

ft

Shigella sonnei

tt

Proteus vulgaris

M

Escherichia coli

If

Pseudomonas s pecies

tt

1 ee of actively growing culture cultivated in enriched nutrient broth transferred to each bottle of beverage

13 TABLE

2

EFFECT OF SIZE OF INOCULATION ON THE SURVIVAL OF E. COLI a n d M. AUREUS IN COLA T3TPE BEVERAGES

Incubation time lOM/ml

M/ml

Inoculation HT/ml lOT/ml

T/rnl

E. coli 800

500

200

75

50

days

10

15

neg

neg

neg

3 days

neg

neg

neg

neg

neg

1

day

2

M. aureus 1

day

500

500

120

100

70

2

days

100

50

20

neg

neg

3 days

neg

neg

neg

neg

neg

Incubation temperature 25° C figures indicate count per ml

14

TABLE

3

EFFECT OF STORAGE TEMPERATURES OH THE SURVIVAL OF E. COLI AND M. AUREUS IH COLA TIPE BEVERAGES

M. aureus

I* coli 5°G

30°C

5°C

30 °C

1

day

600

550

500

100

2

days

100

25

neg

neg

3 days

neg

neg

neg

neg

original Inoculation -

1

,0 0 0 , 0 0 0 per ml

15 TABLE

4

SURVIVAL OP E. COLI IN COLA TYPE BEVERAGE CONTAINING ALL iIGREd H N T S EXCEPT CARBON DIOXIDE

1 st

1

day

2 nd

test

test

500

200

2 days

50

50

3 days

neg

neg

original inoculation - lOM/ml temperature - 25° C

TABLE

5

SURVIVAL OP E. COLI INOCULATED INTO BOTTLES CONTAINING ONLY CARBONATED WATER

1

day

4,000,000

2

days

5,000,000

3 days

1 0 ,0 0 0 , 0 0 0

4 days

original Inoculation - lOM/ml temperature - 25° C

2

,0 0 0 , 0 0 0

16 TABLE

6

SURVIVAL OP E. COLI IN COMPLETE COLA TYPE BEVERAGE ADJUSTED TO pH OF 6.5-7iG WITH KOH

2nd Test

1st Test 7,000

100,000

days

248,000

600,000

5 days

700,000

4 days

5,000,000

1

day

2

3

,0 0 0 , 0 0 0

3,000,000

original inoculation - 10M/ml

TABLE

7

SURVIVAL OF E. COLI INOCULATED INTO DISTILLED WATER IB JUSTS© TO FINAL pH QP 2.9

H3 PO4

Citric acid

9,000,000

19,000,000

1

day

2

days

1 0 ,0 0 0 , 0 0 0

22

,0 0 0 , 0 0 0

3 days

1 1 ,0 0 0 , 0 0 0

21

,0 0 0 , 0 0 0

4 days

6

,0 0 0 , 0 0 0

original inoculation - lOM/ml

24,000,000

17 TABLE

8

SURVIVAL GP E. COLI IN EACH OF THE THREE MAIN FRACTIONS USED IN THE PREPARATION OF THE COMPLETE COLA TYPE BEVERAGE

Fraction 1 - Sucrose syrup, gum acacia, citrus oils, spice oils, vanilla, extraction from, cola nuts. 1

day

40,000,000

2

days

33,000,000

3 days

30,000,000

4 days

35,000,000

Fraction 2 - Phosphoric acid and caffeine. 16,000

1

day

2

days

360

3 days

1

4 days

neg

Fraction 3 - Caramel color produced from corn sugar. 1

day

21

,0 0 0 , 0 0 0

2

days

20

,0 0 0 , 0 0 0

3 days

21

,0 0 0 , 0 0 0

4 days

2 0 ,0 0 0 , 0 0 0

original inoculation - lOM/ml temperature - 25° C

18

TABLE

9

SURVIVAL OF E. COLI AND M. AUREUS INOCULATED INTO WATER 'CONTAINING .3 GRAINS OF CAFFEINE PER 350 ML

E* coli

M. aureus

200,000

400,000

90,000

250,000

3 days

500

200,000

4 days

200

25,000

1 2

day days

original E* coli inoculation 10M/ml original M* aureus inoculation M/ml Incubated at 25° C pH of solution

CHAPTER

V

Discussion The results have shown that even If* food poisoning organisms would gain entrance into highly carbonated cola type beverages containing caffeine, they would be killed during the normal storage time of the beverage.

This of

course is a very important fact to the beverage manufacturer as well as to the consumer*

In our work the inoculations

used were many times larger than would occur in even a very poorly kept plant*

Since all the organisms were killed

within three days regardless of the size of inoculation used, It Is obvious that there is a powerful bacteriostatic agent present in the beverage. Our results have shown that of the three main fractions used in the preparation of the beverage the one containing caffeine and phosphoric acid displayed the bacteriostatic action noted in the complete beverage.

When the caffeine was

tested alone It was found to have a definite inhibitory effect although not to the extent that was noted when used in combination with phosphoric acid.

In going over the

literature one is struck with the complete lack of work on this agent.

As was pointed out In the review of the litera­

ture just about every Ingredient present in cola beverages have been given credit for bactericidal action, but in only

one or two cases, e.g. Bates, Zanda, Sechi and Mezzadrali, is caffeine even mentioned* It is now necessary to explain the fact that growth takes place when the pH of the complete beverage is ad­ justed to 6*5 to 7.0.

It is possible that in the presence

of an adequate food supply the caffeine was not able to produce such noticable results as shown when it was used alone or in combination with the acid.

Another possibility

was that perhaps the potassium hydroxide used in adjusting the pH of the beverage reacted with the caffeine producing a substance which was no longer bactericidal.

CHAPTER VI COHCLUSIOHS From this work it scorns that bottlers of carbonated beverages have depended too much on carbon dioxide as a bacteriostatic agent in the preservation of their produet. It becomes apparent from our results that the bacteriostatic agent found in this particular highly car­ bonated beverage is caffeine, whose action Is enhanced by phosphoric acid.

Since these agents are found in most cola

beverages it Is probable that the same conditions result. This particular phase of preservation has not been studied in this country to any extent, thus the exact limits are not understood.

It is hoped that this bit of experimental

work will interest others to investigate the exact effect of caffeine on bacterial growth. It was shown that the chance of bacteriological food poisoning resulting from a cola type beverage is almost nil, since the causative organisms are so rapidly killed.

BIBLIOGRAPHY

22 Allen, R. M* 1915. Hon-alcoholic carbonated beverages, sanitary condition and composition. Kentucky Agri­ cultural Experiment Station Bulletin. 192:59. Bates, J. Cm 1938. Effects of certain alkaloids on the growth of Aspergillus niger and Rhizopus nigricans. University of Kansas Scientific Bulletin, 25:85. Cannizzaro, J. 1923. Carbon dioxide content of beverages. Journal of Industrial and Engineering Chemistry. 16:1074. Corper, H. J., H. Oauss, and 0. B. Rensch. 1921. Studies on the influence of carbon dioxide on resistance to tuberculosis. American Review of Tuberculosis, 5:562. Donald, J. R., C. L. Jones and A. R. M. MacLean. 1924. effect of carbonation on bacteria in beverages. American Journal of Public Health, 14:122.

The

Epstein, S. S*, and M. Kirsch. 1941. Quality control of operations in the beverageplant. Part I - Control of ingredients. Food Industries, 13:54. Epstein, S. S., and M. Kirsch. 1941. Quality control of operations in the beverageplant. Part II - Carbonation, bottle cleaning and control coordination. Food Industries, 13:44. Gershenfeld, L. 1921. Bacteria in (so called) soft drinks. Journal of the American Pharmacological Association, 1571887-------------------- : ---- -----------------Kilcourse, J. P., 1923. The sanitation of bottled beverages and soda fountain drinks. American Journal Public Health, 13:550. “ Knapp, H. J., and F. B. Buchanan. 1925. Sanitary control of beverages. American Journal Public Health, 15:1053. Koser, S. A. and W. W. Skinner. 1922. Viability of the colon-typhoid group in carbonated water and carbonated beverages. Journal of Bacteriology, 7:111. La Bach, J. L. 1915. Hon-aleoholic carbonated beverages, chemical examination of pops. Kentucky Agricultural Experiment Station Bulletin, 192:82. Larson, W. P., T. B. Hartzell, and H. S. Diehl. 1918. effect of high pressure on bacteria. Journal of Infectious Diseases. 22:271.

The

23 Lee, W. H. 1942. The study of pH. Bottler, 74. no. 450*44.

National Carbonator

lievine, M. 1925. The relation of bottling practice to spoilage of carbonate beverages. Fruit Products Journal and American Vinegar Industry, 4*20. Levine, M., and J. H. Toulouse. 1934. elimination of spoilage. Part I. Gazette, ^53*51.

Suggestions for the National Bottlers

Levine, M., and J. H. Toulouse. 1934. Suggestions for the elimination of spoilage. Part II. National Bottlers Oazette, 53*60. Levine, M. 1935. Bacteriology for the bottler. National Bottlers Oazette, 54*67.

Part I.

Levine, M. 1935. Bacteriology for the bottler. National Bottlers Oazette, 54*73.

Part II*

Levine, M. 1937. Applying scientific control to beverage bottling. Food Industries, 9*568. McKelvey, C. E., 1926. Notes on yeasta in carbonated beverages. Journal of Bacteriology, 11*98. Medbery, H. E. 1948. beverage plants.

Modern practices in carbonated Food Industry, 20*83.

Mezzadrali, 0 ., and A. Amati. 1932. The action of certain alkaloids on the development of Aspergillus niger. Atti Accad Lincel, 16*366. Moore, M. B., and J. H. Buchanan. 1930. The variation of Hydrogen-ion concentration with carbon dioxide pressure above one atmosphere. Iowa State College Journal of Science, 4*431. Patten, H. E., and 0. H. Mains. 1918. Carbonation Studies. Part II - The carbonation of distilled water. Journal of Industrial and Engineering Chemistry, 10*279. Pinnell, W. R. 1915. Non-alcoholic carbonated beverages, Bacteriological examination of pops. Kentucky Agri­ cultural Experiment Station Bulletin, 192*85. Sechi, E. 1930. The action of caffeine. pharma cadynamle, 37;181.

Arch intern

24 Sharf, J. M. 1939* What is satisfactory carbonation. Food Industries. 11?261. Sharf, J. M. 1943. Factors associated with the retention of carbon dioxide in carbonated beverages. Iowa State College Journal of Science. 18:84• Slater, G. 1893. A bacteriological investigation of artificial mineral waters. Journal of Pathology. 1:468. Stokes, W. R. 1920. Bacteriological examination of soft drinks. American Journal of Public Health. 10:308. Swearingen, J. S. and I. M. Lewis. 1933. Hie nature of the effect of COo under pressure upon bacteria. Journal of Bacteriology. 26:201. Tobey, E. R. 1941. Beverages containing caffeine and theobromine. Maine Agricultural Experiment Station Official Inspection Bulletin. 179:133. Toulouse, J. H., J. H. Buchanan, and M. Levine. \1931. The action of citric acid and Its salts In sugar solutions. Iowa State College Journal of Science. 5:89. Turner, W. R. 1925. Yeasts of carbonated beverages. Proceedings of the Iowa Academy of Science. 52:95. Valley, G., and L. F. Bettger. 1927. The Influence of carbon dioxide on bacteria. Journal of Bacteriology. Wickenden, L. 1940. Clearing tap the confusion about sugar sirups. Food Industries. 12:41* Wiley, H. W., Beverages and Their Adulteration, 1919. P. Blakiston*s Son and Company, Philadelphia, Pennsyl­ vania. Young, C. G., and N. P. Sherwood. 1911. The effect of the environment of carbonated beverages on bacteria. Journal of Industrial and Engineering Chemistry. 3:495. Zanda, G. B. 1927. Action of caffeine on microorganisms* Arch Farmacal Sper, 43:277.

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