D. S. CLARK, R. H. WALLACE, AND J. J. DAVID

BoISSEVAIN, C. H. 1928 Replacement of potassium by otherelements in culture mediums. J. Infectious Diseases,43, 194-199.

BREWER, C. R., MCCULLOUGH, W. G., MILLS, R. C., ROESSLER,W. G., HERBST, E. J., AND HOWE, A. F. 1946 Studieson the nutritional requirements of Bacillus anthracis.Arch. Biochem., 10, 65-75.

BURTON, M. O., CAMPBELL, J. J. R., AND EAGLES, B. A. 1948The mineral requirements for pyocyanin production.Can. J. Research, C, 26, 15-22.

EDDY, A. A., AND HINSHELWOOD, C. 1949-1950 The utiliza-tion of potassium by Bacterium lactis aerogenes. Proc.Roy. Soc. (London) B, 136, 544-562.

FEENEY, R. E., AND GARIBALDI, J. A. 1948 Studies on themineral nutrition of the subtilin-producing strain ofBacillus subtilis. Arch. Biochem., 17, 447-458.

KATZNELSON, H. 1947 Studies with Bacillus polymyxa.V. Potassium as a factor in the production of 2, 3 bu-tanediol from starch. Can. J. Research, C, 25, 129-136.

LOCKEMANN, G. 1919 What substances are absolutely neces-sary for the growth of the tubercle bacillus? Centr.Bakteriol. Parasitenk., Abt. I, 83, 420-425 (thru C. A.,14, 754, [19201).

MAcLEOD, R. A., AND SNELL, E. E. 1947 Some mineralrequirements of the lactic acid bacteria. J. Biol. Chem.,170, 351-365.

MUELLER, J. H. 1935 Studies on cultural requirements ofbacteria. VI. The diphtheria bacillus. J. Bacteriol.,30, 513-524.

PORTER, J. R. 1946 Bacterial Chemistry and Physiology.John Wiley and Sons, Inc., New York, page 622.

RAHN, 0. 1936 Substitutes for potassium in metabolism ofthe lowest fungi. J. Bacteriol., 32, 393-399.

ROBINSON, G. L. 1932 The growth of Bacillus pyocyaneusin synthetic media. Brit. J. Exptl. Pathol., 13, 310-317.

ROGOSA, M. 1944 Microbiological method for the quantita-tive determination of small quantities of potassium.J. Biol. Chem., 154, 307-308.

SAUTON, B. 1912 Sur la nutrition minerale du bacille tuber-culeux. Compt. rend., (Acad. Sci.) 155, 860-861.

SNELL, E. E., AND STRONG, F. M. 1939 A microbiologicalassay for riboflavin. Ind. Eng. Chem., Anal. Ed., 11,346-350.

SPEAKMAN, H. B. 1923 Molecular configuration in the sugarsand acid production by Bacillus granulobacter pectino-vorum. J. Biol. Chem., 58, 395-413.

WAKSMAN, S. A. 1932 Principles of Soil Microbiology.Ed. 2. The Williams & Wilkins Co., Baltimore, page 506.

ZUNZ, E., AND GYORGY, P. 1916 Observations sur l'influencechimique des milieux de culture sur le d6veloppement etla production de l'indol par les colibacilles et par lesbacilles typhiques. J. Bacteriol., 1, 627-661.

Factors Affecting the Fermentation of Apple JuiceI

D. -S. CLARK2, R. H. WALLACE3, AND J. J. DAVID4

Macdonald College, Quebec, Canada

Received for publication June 10, 1954

The production of apple cider has been establishedas a domestic enterprise for centuries and as an in-dustrial enterprise for decades. Beginning with thework of Pasteur, much has been done to investigatethe conditions necessary for the production of alcoholin beer and wine. It is questionable if the factors af-fecting the fermentation of wort and grape juice wouldalso apply to that of apple juice; nothing in the litera-ture has indicated that any comparisons of this naturehave been made. It appears also that relatively littlehas been published about the numerous factors thataffect the quality of apple cider. Tressler et al. (1941)have reported that blends of ciders made from two or

1 Contribution from the Faculty of Agriculture, McGillUniversity, Macdonald College, Quebec, Canada; MacdonaldCollege Journal Series No. 355.

2 Lecturer, Department of Agricultural Bacteriology,McGill University, Montreal, Quebec, Canada.

3Assistant Professor, Department of Agricultural Bacteriol-ogy, McGill University, Montreal, Quebec, Canada.

4Associate Professor of Horticulture, McGill University,Montreal, Quebec, Canada, and Food Technologist, QuebecDepartment of Agriculture.

more varieties of apples produce a beverage which hasa better quality than that of an unblended cider; theyalso noted that the juices from different varieties ofapples fermented at approximately the same rate.Ball (1946) has reported that the juice of a mixture ofwild apples is better raw material for the production ofcider than that of any one cultivated variety. It seemsthat only a few papers have been published since 1900about conditions that affect the efficiency of the yeastin the fermentation of apple juice, especially in rela-tion to the rate of alcohol formation. Barker (1908)investigated such factors as aeration, temperature, thechemical composition of the apple juice, and the kindsof yeast; he noted that the main factor in determiningthe rate of fermentation appears to be the nitrogenousmatter present in the juice, and that the quantity ofthis material is generally insufficient for satisfactorynutrition of the yeast. Other investigators have con-firmed Barker's findings (Charley, 1938; Dauthy andAbadie, 1936; Grove, 1919; and Steuart, 1947). Thepurpose of the work reported in this paper was to ex-amine the effects of the age of the inoculum, the con-

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FACTORS AFFECTING THE FERMENTATION OF APPLE JUICE

centration of yeast extract, and the presence of non-fermenting yeasts on the rate of alcohol formation inapple juice.

EXPERIMENTAL

Methods

Apple juice that had been pressed from mixed varie-ties of wild apples was filtered and adjusted to 150Balling with glucose. It was sterilized by Seitz filtra-tion and then transferred in 500 ml quantities to sterile500 ml Erlenmeyer flasks. The flasks were stopperedwith cotton and kept at 5 C until required.The three yeasts used in the experiments had been

isolated from apple cider and identified as strains ofSaccharomyces cerevisiae, Saccharomyces oviformis, andSaccharomyces steineri (Clark, 1953). Each was grownin apple juice and subcultured at least four times at24-hour intervals before it was used for inoculatingthe experimental media. One milliliter of the fourthsubculture was pipetted into 12.5 ml of the inoculationmedium, which, in each instance, was a portion of thetest medium. When this culture had reached the agerequired for the experiment it was poured into theflask of test medium. A rubber stopper fitted with anair trap was placed tightly in the mouth of each flask;the air trap was filled with paraffinol which allowed gasto escape but prevented air from entering. The cultureswere incubated at 28-30 C.The rate of alcohol formation was ascertained by

determining the amount of alcohol at regular intervalsby means of the dichromate-sulphuric acid method ofSemichon and Flanzy (1929) modified by David(1949). All the tests were made in duplicate.

Age of the Inoculum

Cultures of S. cerevisiae 20, 30, 40 and 50 hours old,and of S. oviformis 25, 35, 45, 60 and 75 hours old wereused as inocula. The total numbers of cells and thenumbers of viable cells in 1 ml were determined at thetime the various inocula were transferred to the flasksof apple juice; the former were calculated by means ofan haemocytometer and the latter by plating suitabledilutions with apple juice agar (Marshall and Walkley,1951). The results are shown in figures 1 and 2, and intables 1 and 2. The 20-hour-old inoculum of S. cere-visiae produced the slowest rate of alcohol formation,and the 50-hour-old inoculum the most rapid. The 30-and 40-hour inocula produced rates that were inter-mediate between those of the youngest and oldest, butthe 30-hour inoculum caused the greatest final concen-tration of alcohol. It will be noted that the age of theinoculum for S. oviformis did not appreciably affect itsrate of alcohol formation.The rate of alcohol production by S. cerevisiae

varied inversely with the number of viable cells in the

1

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FIG. 1. Saccharomyces cerevisiae. Effect of age of inoculumon the rate of alcohol formation.

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FIG. 2. Saccharomyces oviformis. Effect of age of inoculumon the rate of alcohol formation.

TABLE 1. The number of cells of Saccharomyces cerevisiae ininocula of different ages

Age Total Viable Dead

hr.

20 31,680,000 16,000,000 15,680,00030 37,200,000 21,000,000 16,200,00040 42,520,000 24,000,000 18,520,00050 46,560,000 26,000,000 20,560,000

inoculum. Between the 20th and 30th hours the totalnumbers in 1 ml had increased by about 5.6 millioncells; between the 30th and 40th hours by 5.3 million;and between the 40th and 50th by 4.0 million. Duringthose intervals, however, the numbers of viable cells

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D. S. CLARK, R. H. WALLACE, AND J. J. DAVID

TABLE 2. The number of cells of Saccharomyces oviformis ininocula of different aaes- -".u "' -Xv"" "

Age Total Viable Dead

hr.

25 20,440,000 12,000,000 8,440,00035 33,200,000 22,000,000 11,200,00045 37,720,000 23,000,000 14,720,00060 40,280,000 25,000,000 15,280,00075 42,600,000 24,000,000 18,600,000

had increased in diminishing increments of 5.0, 3.0, and2.0 millions, respectively.

In contrast to S. cerevisiae, the viable cells of S.oviformis increased by 10 million between the 25th and35th hours, but only by 1, 2, and 1 millions at the 45th,60th, and 75th hours, respectively. It seems that therewas relatively little increase in the viable cells after35 hours, and that the large increase between the 25thand 35th hours had little or no effect on the rate ofalcohol formation.

Concentration of Yeast ExtractIt has been established that the nitrogen and vita-

min-B contents of apple juice are low (Smock andNeubert, 1950). Nevertheless they appear to be inhigh enough concentration to support the growth andmetabolism of yeasts. It seemed possible that the addi-tion of an organic nutrient, such as yeast extract, tothe apple juice would considerably increase the rate offermentation. Indeed, Barker (1908) emphasized thefact that the rate of fermentation of apple juice de-pends largely upon the assimilable nitrogenous matterpresent in the juice.Yeast extract (Bacto) in concentrations of 0.01, 0.10,

0.50, and 1.00 per cent, respectively, was added toflasks of apple juice. S. cerevisiae, S. oviformis and S.steineri were used for the tests, and alcohol formationwas determined at intervals during 390 hours. Dupli-cate flasks of apple juice without yeast extract werealso inoculated to serve as controls.The results for S. cerevisiae are shown in figure 3. It

can be seen that the rate of alcohol formation becamemore rapid with increasing concentrations of yeastextract. This was especially evident between concen-trations of 0.01 and 0.1 per cent. Five-fold and ten-fold increases above 0.1 per cent caused relativelysmall increases in the rate of fermentation. This yeastconverted 45.73, 46.13, and 48.00 per cent of the sugarinto alcohol in flasks of apple juice containing 0.10,0.50, and 1.00 per cent yeast extract, respectively.The results for S. oviformis are given in figure 4, in

which it may be seen that the effect of yeast extract onthe rates of alcohol formation are similar to those of S.cerevisiae. S. oviformis, however, produced such a rapidrate of fermentation in the flasks containing 0.10, 0.50,and 1.00 per cent yeast extract that the fermentation

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FIG. 3. Saccharomyces cerevisiae. Effect of various concen-trations of yeast extract on the rate of alcohol formation.

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21(1 *-o ~0.01 ItPcENT YEAsr XTrmAcTIih~~~ 9~0020 PEARCIfT YEAST 4EXTrAwcTa.- 5 5Q RJNT Year ExTNcr0 ir //0_4 N Y(A$~tr EXRA

0 s0 20 250 too 4IO "1 IO "7/AX IAd P011*5

FIG. 4. Saccharomyces oviformis. Effect of various concen-trations of yeast extract on the rate of alcohol formation.

was almost completed in 140 hours. However, therewas still a detectable difference after 390 hours in theamounts of alcohol produced in flasks having thehigher concentrations of yeast extract compared withthose produced in apple juice containing the lower con-centrations of yeast extract and the controls. In applejuice containing both 0.50 and 1.00 per cent yeast ex-tract, S. oviformis converted 46.86 and 48.53 per centof the sugar into alcohol after 225 and 390 hours, re-spectively.The results for S. steineri are shown in figure 5. The

response to yeast extract in the apple juice was some-what similar qualitatively but not quantitatively tothat of the other yeasts used in this study. The rate of

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FACTORS AFFECTING THE FERMENTATION Or APPLE JUICE

6

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I_ Iju I CE

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FIG. 5. Saccharomyces steineri. Effect of various concentra-tions of yeast extract on the rate of alcohol formation.

*fermentation in flasks containing 0.01 per cent yeastextract, however, was not different from that of thecontrol. This yeast fermented at the greatest rate andproduced the largest amount of alcohol in the presenceof 1.0 per cent yeast extract. Appreciable amounts ofalcohol were formed only after a lag of almost fourdays. After approximately 17 days this yeast convertedonly 19.93 per cent of the sugar into alcohol in flaskscontaining 1.00 per cent yeast extract.

Nonfermenting YeastsA large number of different strains of yeasts could

be effective in the fermentation of apple juice undernatural conditions. Indeed the presence of nonferment-ing yeasts may contribute to the activity of the fer-menters. An experiment was done, therefore, to testthis hypothesis, using S. cerevisiae, S. oviformis, and S.

1.

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FIG. 6. Effect of the presence of nonfermenting yeasts on

the rate of alcohol production by the fermenting yeasts.

steineri, respectively, and a mixture of 13 nonferment-ing yeasts that had been isolated during another phaseof the work (Clark, 1953). The inocula of the non-fermenting yeasts were prepared by growing each yeastseparately in 5 ml of apple juice; after 4 subsequenttransfers to 5 ml of apple juice, 1 ml of each final cul-ture was pipetted into a sterile culture tube. The yeastswere mixed and then added aseptically to a flask ofapple juice. Fermentations without the addition of thenonfermenting yeasts served as controls. The resultsshown in figure 6 indicate that the addition of the non-fermenting yeasts did not influence the rates of alcoholproduction by the fermenting yeasts.

DISCUSSION

The metabolic activity of microorganisms in a givenmedium is dependent on the species used and, further-more, is related in some considerable measure to theage of the cells that are used for the inoculum. It wasnoted in this investigation that between a minimum of20 hours and a maximum of 50 hours ten-hour differ-ences in the age of S. cerevisiae resulted in increasedrates of alcohol formation. The numbers of viablecells, however, appeared to increase at a slower rate.It seems that the cells were physiologically more activeat or near the end of their logarithmic phase of growth.Possibly the increased number of dead cells may havecontributed nutritionally to the metabolism of theliving cells. In similar circumstances the age of S.oviformis, however, did not have any appreciable effecton its rate of alcohol formation.The results show that S. cerevisiae and S. oviformis

ferment apple juice efficiently, since they convertedapproximately 46 per cent of the sugar at 150 Ballinginto alcohol. Niehaus (1937) has stated that the bestwine yeasts seldom ferment over 48 per cent of invertsugar, glucose or fructose into alcohol.The rates of alcohol formation were greatly increased

when yeast extract was added to the apple juice.Furthermore, the quantity of alcohol formed afterapproximately 16 days was greater in the presence ofyeast extract than in its absence. S. cerevisiae and S.oviformis both converted 48 per cent of the sugar intoalcohol when the concentration of yeast extract wasraised to 1.00 per cent. These findings confirm those ofprevious workers with regard to the value of enrichingapple juice with assimilable nitrogenous material.Further work is necessary to determine the effects ofvarious concentrations of organic and inorganic nitro-gen compounds along with various concentrations ofsugar on the rate of alcohol formation. It may be pos-sible also that the addition of small quantities of oneor more of the intermediate compounds of fermentationmay enhance the activity of the yeasts.Barker (1908) concluded that the kind of yeast

present in apple juice does not materially affect the

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D. S. CLARK, R. H. WALLACE, AND J. J. DAVID

rate of fermentation. He based his conclusion on theprobability that the mixture of yeasts normally presentin apple juice would have at least one species thatwould complete the fermentation at a maximum rate.It is conceivable that the occasion may arise in whichsuch a species would not be present. Our findings showthat a yeast such as S. steineri could be present andwould ferment apple juice slowly and inefficiently onlywhen an adequate supply of nitrogen is available.

ACKNOWLEDGMENT

The authors wish to thank the Scientific ResearchBureau, Quebec Department of Trade and Commerce,for a grant to defray the expenses of this investigation.

SUMMARY

A study was made on the effects of the age of theinoculum, the concentration of yeast extract, and thepresence of nonfermenting yeasts on the rate of alcoholformation in apple juice adjusted to 150 Balling. Ten-hour differences between the 20th and 50th hour of theage of Saccharomyces cerevisiae caused irregular in-creases in the rate of alcohol formation. It was foundalso that the rate of fermentation appeared to be cor-related inversely with the rate of increase of viablecells in the inoculum. Similar effects did not occur withSaccharomyces oviformis. The yeasts fermented applejuice at increasing rates when it was enriched withincreasing concentrations of yeast extract. The pres--ence of nonfermenting yeasts did not influence therates of alcohol production by the fermenting yeasts.It is considered that further studies should be made ofthe effects of varying concentrations of nitrogenouscompounds and glucose on the rate of fermentation ofapple juice.

REFERENCES

BALL, E. 1946 The raw materials for cider making. J. Soc.Chem. Ind., 65, 38-39.

BARKER, B. T. P. 1908 The rate of fermentation of cidersand perries. J. Agr. Sci., 3, 1-21.

CHARLEY, V. L. S. 1938 Fermentation control of ciders bythe centrifuge method. Relationship between changes innitrogen content in juices and ciders and the subsequentrates of fermentation. Long Ashton Ann. Report 1938,174-187.

CLARK, D. S. 1953 Taxonomy of yeasts isolated from apples.M.Sc. Thesis, McGill University, Montreal.

DAUTHY, E., AND ABADIE, G. 1936 Retardation of ciderfermentation by lack of nitrogen. Bull. Assoc. Chini.,53, 523-532. Chem. Abstr. 30, 5718.

DAVID, J. J. 1949 Acetaldehyde and related carbonyl com-pounds in frozen peas. Ph.D. Thesis, University of Cali-fornia, Berkeley.

GROVE, 0. 1919 The relation between the rate of fermenta-tion and the content of nitrogenous matter in apple juice.Long Ashton Ann. Report 1919, 20-22.

MARSHALL, C. R., AND WALKLEY, V. T. 1951 Some aspectsof microbiology applied to commercial apple juice pro-duction. 1. Distribution of microorganisms on the fruit.Food Research, 16, 448-456.

NEIHAUS, C. J. G. 1937 Sugar alcohol ratios in South Afri-can musts and wines. South Africa Dept. Agr. For. Sci.Bull. 161.

SEMICHON, L., AND FLANZY, M. 1929 Determination of alco-hol in wines and spirits by use of the sulphuric-chroinicmixture. Ann. Fals et fraudes 22, 139-152. Chem. Abstr.,23, 3537.

SMOCK, R. M., AND NEUBERT, A. M. 1950 Apples and appleproducts, Interscience Publishers, Inc., New York.

STEUART, D. W. 1947 The nitrogen content of apple juiceand its importance in cider making. Chemistry & In-dustry, 66, 55-56.

TRESSLER, D. K., CELMER, R. F., AND BEAVENS, E. A. 1941Bulk fermentation process for sparkling cider. Ind.Eng. Chem., 33, 1027-1031.

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