SOME FACTORS WHICH INFLUENCE THE MACE-TY L CONTENT IN COTTAGE CHEESE Thesis £0? the Degree of M. S. MICHIGAN STATE UNEVERSITY Chm-Tu 5. Wales 1956 THESlS SOME FACTORS WHICH INFLUENCE THE BIACETYL CONTENT' IN COTTAGE CHEESE By CHARLES S. WALES A THESIS \\ Submitted to the College of Agriculture of Michigan State University of Agriculture and Applied Science {Q in partial fulfillment of the requirements ‘~ f for the degree of l. 9p ‘ MAS TER OF SCIENCE Department of Dairy 1956 ‘.“3-50 ACKNOWLEDGMENTS The author wishes to express his sincere appreciation to Dr. L. G. Harmon for his valuable direction and encouragement during the course of this study and for his assistance in the preparation of this manuscript. The author is deeply grateful to the Dairy Industry Supplies Association for the fellowship which was awarded him, making this work possible. Gratitude is also expressed to Dr. G. M. Trout for his sug- gestions in preparing this manuscript. Sincere thanks is expressed to Mr. Milford D. Bonner. who supplied the cultures of microorganisms used in this experiment. ii EXPERIMENTAL PROCEDURE RESULTS TABLE OF CONT EN TS Addition of Various Organic Acids to Cottage Cheese Wash Water .................. Inoculation of the Cheese with Various Microorganisms ........................... Addition of Various Organic Acids and Starter to the Creaming Mixture ............... Effect of Various Organic Acids Used to Acidulate Wash Waters on the Biacetyl Content of Cottag e Che as e ......................... (8.) Influence of organic acids on the biacetyl content of cottage cheeSe stored at 40°F. (1)) Influence of organic acids on the biacetyl content of cottage cheeSe stored at 50°F. Effect of Various Organic Acids Used to Acidulate Wash Waters on the Acetylmethylcarbinol Content of Cottage Cheese ......................... (3.) Influence of organic acids on the acetyl- methylcarbinol content of cottage cheese stored at 40°F. .............................. iii --------------------------------- 12 l4 14 16 I7 17 17 19 27 27 Page (1)) Influence of organic acids on the acetyl- methylcarbinol content of cottage cheese stored at 50°F. ................................ 29 Destruction of Biacetyl by Microorganisms in Creamed Cottage Cheese ....................... 33 (a) Destruction of biacetyl in cottage cheeSe stored at 40°F. ........................... 33 (b) Destruction of biacetyl in cottage cheese stored at 50° F. ........................... 34 Destruction of Acetylmethylcarbinol by Microorganisms in Cottage Cheese ................ 35 (a) Destruction of acetylmethylcarbinol in cottage cheese stored at 40°F. ................ 35 (b) Destruction of acetylmethylcarbinol in cottage cheeSe stored at 50°F. ............... - . 40 The Biacetyl Content of Cottage Cheese as Affected by Adding Organic Acids and Starter to the Creaming Mixture ....................... 41 DISCUSSION .................................. 47 Effect of Various Organic Acids Used to Acidulate Cottage Cheese Wash Waters on the Biacetyl and Acetylmethylcarbinol Content of the Cottage CheeSe . . . . 47 Destruction of Biacetyl and Acetylmethylcarbinol by Microorganisms in Cottage Cheese ............. 52 Biacetyl Content as Affected by Adding Citric, Lactic, or Sorbic Acid or Commercial Starter to the Creaming Mixtur e .................................. 5 7 SUMMARY AND CONCLUSIONS .................... 5 8 LITERATURE CITED ........................... 6 l IN TR ODUC TION Biacetyl and acetylmethylcarbinol are recognized as chemical substances which contribute to the desirable flavor and aroma of dairy products. Biacetyl and acetylmethylcarbinol are produced by the decomposition of citric acid and lactose by Leuconostoc citro- vorous and Leuconostoc dextranicum. The mechanism of formation of these flavor components by Leuconostoc organisms and the fac- tors affecting their formation have been determined in previous studies primarily involving butter and butter cultures. Fine fla- vored butter contains from 0.0002 to 0.0004 percent biacetyl. The increasing consumer demand for cottage cheese has focused attention to the influence of biacetyl and acetylmethylcar- binol on desirable cottage cheese flavor. Moderate increases in amounts of these flavor components could result in a better fla- vored product which would be beneficial both to the producer and consumer of cottage cheese. One aim of this study was to deter- mine if additions of certain organic acids to wash waters used for cottage cheese would result in increases of biacetyl and acetyl- methylcarbinol. Biacetyl and acetylmethylcarbinol are produced and utilized by Leuconostoc organisms. The decrease in these constituents normally occurs slowly and in most instances does not result in a poor flavored cheese. Some organisms have been reported to de- stroy biacetyl and acetylmethylcarbinol rapidly and cause the cottage cheese to assume a flat flavor. However, very little information is available concerning destruction of these flavor compounds by or- ganisms which are naturally present in cottage cheese. Knowledge of the effect of organisms isolated from cottage cheese on the bi- acetyl and acetylmethylcarbinol content of cottage cheese could lead to retention of these flavor components. REVIEW OF LITERATURE The recognition of van Niel (1929) that biacetyl either is responsible for the aroma of butter or is the principal flavor com- ponent of butter led to studies of biacetyl and acetylmethylcarbinol content of dairy products. Tapernaux (1932) showed that addition of biacetyl improved the flavor of butter and margarine. Davies (1933) found that lactose fermentation yields acetylmethylcarbinol which is oxidized to biacetyl, giving the characteristic flavor and aroma of_ butter. According to Michaelian et a1. (1933). butter cultures with a satisfactory flavor and aroma contained considerable quantities of acetylmethylcarbinol and biacetyl, while cultures lacking in flavor showed little or none. Hammer (1934) reported that fine butter contained from 0.0002 to 0.0004 percent biacetyl. The relationship between acetylmethylcarbinol and biacetyl content, and a satisfactory flavor in butter and butter cultures has been confirmed repeatedly. HoweVer, a relatively high biacetyl content is no assurance of a good flavor because definite off-flavors due to a number of causes may be present. According to Wiley 3131. (1939) the assumption that the biacetyl content of a butter culture is an accurate guide to its value as a desirable flavor producer is fallacious. Farmer and Hammer (1931) and Ritter and Christen (1935) found that addition of citric acid greatly increased the amount of biacetyl in butter cultures. Ruehe (1937) obtained maximum biacetyl contents when citric acid was added to a twenty-four hour old culture and incubated for another twenty-four hours. A culture so treated could not be added to butter as a starter but was adapted to the preparation of starter distillate. Prill and Hammer (1939) reported that 0.15 percent citric acid proved to be the most prac- tical amount to add to butter cultures. Klubchandani (1939) added citric acid and sodium citrate to butter cultures as suggested by Prill and Hammer (1939) and reported improved butter flavor. When citric acid was added to butter cultures, a 70 to 90 percent increase in acetylmethylcarbinol and biacetyl content was observed by Gehrke and Weiser (1948). Nelson and Brence (1953) observed that the ad- dition of citric acid to cottage cheese increased the volatile acidity and improved the flavor. Hammer (1939) proved that biacetyl was the principal flavor component in cottage cheese. The biacetyl content of cottage cheese could be lowered by prolonged washing, or by high cooking tempera- tures which. destroyed flavor producing organisms. Ninety-two specimens of various kinds of cheese examined by Csiszar et a1. (1942) contained acetylmethylcarbinol in all samples and biacetyl in a large majority of the samples. Calvert and Price (1949) found biacetyl in samples of cheddar cheese but could not find a relation- ship between flavor and biacetyl content. Krishnaswamy and Babel (1951) found 1.06 to 2.25 parts per million of biacetyl in cottage Cheese curd obtained by the long set method. Parker and Elliker (1952) demonstrated the importance of biacetyl to cottage cheese flavor by classifying various samples of the product according to aromatic flavor and subjecting the same samples to a chemical analysis for biacetyl. Samples with high biacetyl contents received high flavor scores, with occasional samples criticized for high acid. Low scoring samples were low in biacetyl values. The Voges-Proskauer reaction (1898) was one of the earliest methods devised for the detection of acetylmethylcarbinol. It was a qualitative-colorimetric determination requiring considerable time. Barritt (1936) found that the Voges-Proskauer reaction (1898) could be intensified and made more delicate by addition of a small amount of alpha naphthol. Eggleston 3:33;. (1943) used a rapid col- orimetric method similar to that of Barritt (1936). A rapid qualita- tive method using creatine and sodium hydroxide was developed by O'Meara (1931). Pien 3:31.. (1936) mentioned a qualitative method reacting biacetyl with phenylhydrazine to yield biacetylphenylhydra- zone. Hammer (1935a) applied the rapid method of O'Meara (1931) to butter cultures. Small amounts of creatine and strong sodium hydroxide solution were added to culture in a test tube. The in- tensity of the resulting red color depended upon the amount of bi- acetyl and acetylmethylcarbinol present. A procedure for measuring 2,3-butylene glycol and acetylmethyl- carbinol was developed by Lemoigne (1920). Bromine was used to oxidize 2,3-butylene glycol to acetylmethylcarbinol. The acetyl- methylcarbinol was oxidized with ferric chloride to biacetyl and re- acted with nickel chloride to form nickel dimethyl glyoximate. The insoluble nickel salt precipitated and could be measured. Van Niel (1927) stated that the method of Lemoigne (1920) did not give quan- titative results and described a method, based on the Lemoigne reaction, which permitted a quantitative measurement of the biacetyl and acetylmethylcarbinol. This method required more careful dis— tillation of the biacetyl and a slightly modified procedure for col- lecting and weighing the precipitated nickel salt. Davies (1933) modified van Niel's method (1927) and obtained better results. Various factors which influence the determination of acetyl- methylcarbinol and biacetyl as nickel dimethyl gloximate were studied by Michaelian et a1. (1933). Biacetyl was distilled in a stream of carbon dioxide to prevent atmospheric oxidation. Conditions affecting the completeness of precipitation of acetylmethylcarbinol and biacetyl as nickel dimethyl glyoximate from butter were studied by Bairncoat (1935). This worker proposed a colorimetric method to determine traces of the nickel salt. The nickel dimethyl glyoximate was dis- solved in chloroform and compared with solutions of known amounts of the salt. Hammer (1935b) steam distilled cultures to which ferric chloride had been added for the purpose of oxidizing the acetyl— methylcarbinol to biacetyl. The distillates were treated with hydrox- ylamine hydrochloride, sodium acetate, and nickel chloride, and the resulting nickel salts determined quantitatively. Results indicated that the diketone produced was biacetyl rather than one of the homo- logs and if homologs were present, they were limited to relatively insignificant amounts. In the studies of Prill _e_t_a_l_. (1939), distil- lates from ordinary butter cultures gave no evidence of the higher homologs of biacetyl or acetylmethylcarbinol. Dehove and Desirrier (1938) were able to evaluate biacetyl with an accuracy of 0.5 milli- grams per kilogram of butter, by purification of the nickel dimethyl glyoximate. Other investigators including Pritzker and Jungkunz (1930), Vizern and Guillot (1932), Mohler and Helberg (1933), Stahly et a1. (1935), Mohler and Herzfeld (1935), Mohr and Wellm (1937), Schmalfuss and Werner (I937), Kniphorst and Kruisheer (1937), Parker and Shadwick (1937), Jungkunz (1940), and Wilson (1941) 8 have reported on the nickel dimethyl glyoximate method and modifica- tions of that method. A colorimetric method for determining biacetyl and acetyl- methylcarbinol was suggested by Testoni and Cuisa (1931). These investigators oxidized nickel dimethyl glyoximate and obtained a solu- ble red complex in which the nickel had a higher valence number. Kunze (1936) described a micromodification of the gravimetric method and recommended colorimetric methods for amounts less than 0.3 milligrams. Pien et a1. (1936) obtained a yellow color when biacetyl was reacted with m-p—toluenediamine and treated with strong sulfuric acid. These workers later (1937) obtained a stronger yellow color using diaminobenzine. This method was accurate for amounts of biacetyl as low as 0.5 milligrams per kilogram of butter. Pien (1948) modified the method of Pien et a1. (1937) by using a different pro- cedure for purifying the distillate and obtained more accurate re- sults for small amounts of biacetyl. Ritter and Nussbaumer (1939) used the method of Pien et a1. (1936) and suggested use of pure con- centrated sulfuric acid and a fresh solution of m-p-toluenediamine. Dehove and Dessirier (1938) noted that the method of Pien et a1. (1937) would not permit accuracy of four milligrams per kilogram when fifty grams of butter was used. Cox and Wiley (1939) extended 9 the method of Pien et a1. (1937) by standardizing the sample, the ap- paratus, and the method of distillation. A volumetric method for biacetyl determination, based on the oxidation of one molecule of biacetyl to two molecules of acetic acid with hydrogen peroxide was developed by Ruehe and Corbett (1937). Prill and Hammer (1938) developed a colorimetric method for the microdetermination of biacetyl based on the formation of the in- tensely colored ammo-ferrous dimethyl glyoximate. With this method, they were able to detect the difference between 0.001 milligrams of biacetyl and no biacetyl in five milliliters of water. Den Herder (1947) modified the method of Prill and Hammer (1938) using a special apparatus and distilling the biacetyl with a stream, of carbon dioxide. Folke Bange (1943a, 1943b, 1944a, 1944b, 1945) has shown that biacetyl and acetylmethylcarbinol are products of citric acid and sugar fermentation of Streptococcus citrovorous and Streptococcus paracitrovorous. These products are subject to utilization or de- struction by the above named flavor organisms during subsequent metabolism. Michaelian et a1. (1933) studied destruction of biacetyl and acetylmethylcarbinol using a skimmilk and butter culture medium that had been subjected to high heat treatment. When the medium was held twenty days at 6°C., the biacetyl and acetylmethylcarbinol 10 content remained constant, but when inoculated with butter culture and similarly held there was a pronounced decrease. The investigations of Williams and Morrow (1928) revealed that acetylmethylcarbinol is destroyed by certain strains of coli- aerogenes bacteria chiefly Aerobacter aerOgenes, by the green fluorescent bacteria and by all the aerobic spore formers tested. Acetylmethylcarbinol was not destroyed by certain representatives of the Salmonella, Eberthella, Proteus and Serratia groups. Vir- fi tanen and Kontio (1941) found that Bacillus punctatum and Bacillus vulgatus destroyed both biacetyl and acetylmethylcarbinol. A non- proteolytic coccus and Pseudomonas fluorescens destroyed very little acetylmethylcarbinol but up to half of the biacetyl content. Elliker and Horrall (1943) stated that Pseudomonas putrefaciens de- stroyed biacetyl in the aqueous phase of butter. A complete lack of aroma followed growth but one-third to one-fourth of the original biacetyl remained in the butter. Elliker (1945) found that Strepto- coccus lactis had no effect on biacetyl. However, Ps. fluorescens, "Ps. fluroscens var. liquefaciens,” Pseudomonas fragi, Ps. putre- faciens, Pseudomonas nigrificans, and some unidentified strains of Pseudodomonas markedly reduced the biacetyl content of butter stored at 60°F. Hietaranta and Gyllenberg (1950) reported that formation of biacetyl, but not of acetylmethylcarbinol, is suppressed 11 when the oxidation-reduction potential is lowered by the addition of strongly reducing bacteria such as A. aerogenes, Escherichia coli, and Serratia marcesens. The investigations of Parker and Elliker (1952, 1953) showed that Ps. fragi, Pseudomonas viscosa and Alcali- genes metalcaligenes destroyed biacetyl in cottage cheese. Biacetyl was destroyed prior to the appearance of the gelatinous or slimy curd defect associated with these organisms. Ps. fragi converted most of the biacetyl to acetylmethylcarbinol while slightly less was converted by Ps. viscosa. Alc. metalcaligenes reduced biacetyl more slowly to acetylmethylcarbinol and 2,3—butylene glycol than did Ps. fragi and Ps. viscosa. EXPERIMENTAL PROC EDUR E The cottage cheese curd used in this experiment was ob- tained from the Michigan State University creamery on the date of manufacture. The cheese was divided into the appropriate number of lots and placed in separate containers which had been rinsed with a two hundred parts per million hypochlorite solution. All creamed cottage cheese was packaged in twelve-ounce cartons and stored for twelve days. One-half of the cartons of each lot were stored at 40°F. and the other half were stored at 50°F. Biacetyl, acetylmethylcarbinol, pH, and flavor determinations were performed initially and at three-day intervals on each sample of-creamed cottage cheese. Biacetyl and acetylmethylcarbinol were determined by the method of Prill and Hammer (1938). Solutions were prepared con- taining pure biacetyl in increments of 0.05 milligram between the concentrations of 0 and 10 milligrams. These prepared solutions were develOped colorimetrically and the intensity measured on a Cenco-Sheard-Sanford photelometer. Six duplicate trials were per- formed and the average scale reading for each biacetyl concentration 12 13 was calculated. A standard curve was constructed, plotting photel- ometer scale readings versus milligrams of biacetyl. Sampling was done in an aseptic manner. Twenty-five grams of cheese were weighed directly into the distillation flasks. For acetylmethylcarbinol determinations, twenty milliliters of 40 percent ferric chloride solution were added to the weighed cheese sample and refluxed strongly for ten minutes and then distilled. All photelometer scale readings of the color intensity of all distilled samples were converted into biacetyl values using the standard curve. When it was impossible to perform biacetyl and acetylmethylcarbinol determinations on the sampling date, samples were frozen promptly and stored at 10°F. The storage time did not exceed two days. Flavor and pH were determined immediately after sampling. The pH was determined with a Beckman Model C pH meter. A l scorecard prOposed by the American Dairy Science Association was used in evaluating flavor. 1 American Dairy Science Association Committee on Cottage Cheese Scorecard. H. C. Olson, Dairy Department, Oklahoma A. and M. College, Stillwater, chairman. 14 Addition of Various Organic Acids to Cottage Cheese Wash Water Citric, lactic, and sorbic acid were added to cottage cheese wash waters to determine if they would increase the biacetyl and acetylmethylcarbinol content of the cottage cheese. The cheese curd, which had been washed once, was divided into ten equal lots. Each lot of cheese was washed separately with wash water which con- tained lactic, citric, or sorbic acid. Sufficient citric and lactic acids were used to produce pH levels of 6.0, 5.5, and 5.0 in the wash water. pH levels of 6.2, 5.8, and 5.2 were obtained by adding 0.05, 0.1, and 0.25 percent sorbic acid, respectively. The cottage cheese curd was allowed to stand one hour in the treated wash water before draining. One lot of cottage cheese was washed with untreated tap water and used as a control. A creaming mixture containing 14 percent butterfat and 4 percent salt was added to the drained curd to standardize the final product to 4 percent fat and 1.3 percent salt. Inoculation of the Cheese with Various Microorganisms Various microorganisms were inoculated into cottage cheese to determine if they reduced the biacetyl and acetylmethylcarbinol 15 content. Cottage cheese curd was divided into twenty-one equal lots preparatory to creaming. An appropriate amount of creaming mixture of the composition previously described was prepared and also di- vided into twenty-one equal portions. One lot of cheese was creamed and used as a control. Each of the remaining lots of creaming mixture was inoculated with 0.1 milliliter of a twenty-four hour trypticase soy broth culture of the following organisms: Micrococcus flavus, Micrococcus conglomeratus, Micrococcus candidus, Pseudom- onas fragi, Pseudomonas fluorescens, Pseudomonas desmolyticum, Pseudodomonas tralucida, Achromobacter butyri, Achromobacter eurydice, Escherichia coli, Escherichi freundii, Alcaligenes metal- caligenes, Bacillus firmus, Bacterium erythrcgenes, Geotrichum candidum, Mucor plumbeus, Penicillium frequentans, Rhodotorula flava, and Torulopsis candida. These organisms were obtained from stock cultures possessed by the Michigan State University Dairy De- partment. Not all of these organisms are responsible for cottage cheese spoilage, but all have been isolated from spoiled cottage cheese and were included to determine their effect upon biacetyl and acetylmethylcarbinolcontent. Samples of each creaming mixture were taken before and after inoculation. The total microbiological p0pulation of each sample was enumerated according to procedures described by Standard Methods (1953). The counts of organisms 16 added ranged from 300 to 450,000 per milliliter of cream. Mold and yeast numbers were the lowest, while counts for Micrococci, Achromobacter, and Escherichia tended to be high. After sampling. the inoculated creaming mixture was added to the cottage) cheese curd. Addition of Various Organic Acids and Starter to the Creaming Mixture Citric, lactic, and sorbic acids and commercial starter were added to creaming mixtures in an effort to increase the biacetyl content of the creamed cottage cheese. A creaming mixture of previously described composition was divided into five equal lots. One-tenth percent lactic acid was added to one portion, 0.1 percent citric acid to a second portion, 0.1 percent sorbic acid to a third portion, and 1.0 percent commercial starter to a fourth portion. The fifth portion had no addition and was used as a control. These fiVe lots of cream were then added to uncreamed cottage cheese. RESULTS Effect of Various Organic Acids Used to Acidulate Wash Waters on the Biacetyl Content of Cottage Cheese The initial biacetyl values of fresh cottage cheese analyzed in this experiment usually ranged from 0.3 to 0.8 milligrams per 100- gram sample (3 to 8 parts per million), and most samples were within the range of 0.4 to 0.6 milligrams (4 to 6 parts per million). Variations were noted in the biacetyl content of individual samples. Generally, however, biacetyl increased to a maximum on the sixth or ninth day of storage and decreased on the twelfth day. (a) Influence of organic acids on the biacetyl content of cot- tage cheese stored at 40°F. The effect of washing curd with wash waters acidified to pH 6.0, 5.5, and 5.0 with lactic or citric acid, and to pH 6.2, 5.8, and 5.2 with sorbic acid on the biacetyl content of creamed cottage cheese stored twelve days at 40°F. is shown in Figures 1 to 6, inclusive. A comparison of data in Figures 1, 2, and 3 shows that the samples washed with waters acidulated with lactic acid contained more biacetyl than the other samples. The data indicate that lactic acid was the most effective in permitting the biacetyl content to increase above that of the control. The 17 18 higher amount of biacetyl is evident especially in the data of Figures 2 and 3. The effect of various levels of lactic acid in the wash water on the biacetyl content of the cheese is shown in Figure 5. The biacetyl values of each sample are significantly higher than those of the control. Unlike the control in which a decrease in biacetyl was noted at three days, the biacetyl content of all samples washed with waters containing lactic acid increased similarly, reaching a maximum at nine days. Beyond the nine-day storage period, the biacetyl value varied according to the pH of the wash water. The biacetyl content of the cheese washed with waters acidified to pH 5.5 with lactic acid increased on the twelfth day, whereas the bi- acetyl of the cheese washed with waters acidified to pH 5.0 and 6.0 with lactic acid decreased on the twelfth day. No significant differences between the biacetyl contents of the control and the cheese washed with waters containing citric and sorbic acids were noted (Figures 4 and 6). However, slight deviae tions were evident. The biacetyl of the control decreased at three days, while that in the samples washed with waters adjusted to pH 5.0 with citric acid and to pH 5.2 with sorbic acid increased the. third day. Also, the biacetyl content of the control increased on the twelfth day, whereas the biacetyl content of samples washed with 19 waters acidified to pH 6.2 with sorbic acid, as well as that of all samples washed with citric acid, decreased on the twelfth day. The pH values of all samples stored at 40°F. varied from pH 5.48 to 4.88 regardless of sampling interval (Table 1). The pH of most of the samples decreased slightly to a minimum on the ninth day and were perceptibly higher the twelfth day. (b) Influence of organic acids on the biacetyl content of cot- tage cheese stored at 50°F. The changes occurring in the biacetyl content of cottage cheese washed with acidulated waters and stored twelve days at 50°F. are shown in Figures 7 to 12, inclusive, and in Table l. The higher storage temperature had a great effect upon the biacetyl content of the control. The biacetyl content of the control sample stored at 40°F. (Figure 1) decreased slightly the third day before increasing continuously and uniformly to a maximum of 1.28 milligrams per lOO-gram sample on the twelfth day. The biacetyl content of the control stored at 50°F. (Figure 7) increased gradually to a maximum of 1.0 Inilligrams per lOO-gram sample on the sixth day and uniformly decreased to a minimum of 0.5 milligrams on the twelfth day. With the exception of the cheese sample washed with water acidified to pH 5.2 with sorbic acid (Fig- ure 9), similar differences due to temperature were noted between 20 the biacetyl content of cheese stored at 40° and that of the corres- ponding sample stored at 50°F. The biacetyl contents resulting from acidifying wash waters to pH 6.0, 5.5, and 5.0 are shown in Figures 7, 8, and 9, respectively. The biacetyl contents of the cheese washed with water acidulated with citric acid (Figure 7) and- lactic acid (Figure 8) were higher on the third day than the control, but decreased and remained below the control on the sixth, ninth, and twelfth day. The remaining cheese samples in Figures 7 and 8 and those washed with citric and lactic acid in Figure 9 had biacetyl contents that were lower than the control for the duration of the storage period. The biacetyl content of the cheese washed with water contain- ing sorbic acid (Figure 9) increased markedly and continuously above the biacetyl level of the control. This increase may be due to the continued production of biacetyl and inhibition of the fermentation which destroys biacetyl. Leuconostoc citrovorous and/or Leucon- ostoc dextranicum which produce biacetyl, were quite tolerant to sorbic acid. These organisms grew well in broth containing all combinations of sorbic acid and pH used in this experiment (Table 2). Good growth was observed in trypticase soy broth containing 0.05 to 0.25 percent sorbic acid and adjusted to a pH range of 4.0 21 to 6.6. Growth was slightly heavier in broth of pH 5.0 and below, than in broth above pH 5.0. Data representing the biacetyl values of the cheese washed with the three pH levels of each acid have been grouped (Figures 10, 11, and 12). Analysis of these graphs indicates that the sample washed with water acidified to pH 5.2 with sorbic acid (Figure 12) was the only sample with a biacetyl content significantly higher than the control. This sample contained 1.1 milligrams per lOO-gram sample more biacetyl than the control on the twelfth day. The pH values of all cheese samples stored at 50°F. are shown in Table 1. The pH of all samples decreased to a minimum on the sixth and ninth day and increased slightly on the Welfth day. At the same sampling interval, the pH of the cheese stored at 50°F. was lower in all cases than the corresponding sample stored at 40°F. Attention should be focused on the pH values of the cheese washed with waters containing sorbic acid. These pH values did not decrease as rapidly or extend as low as the other samples. A cor- relation is evident between the amount of sorbic acid and the de- crease in pH. The larger the amount of sorbic acid, the higher' the pH of the sample, which indicates that acid production by organisms is inhibited by sorbic acid. The sample washed with water adjusted /./. 'Iz...- . :X "‘/.a..'-'.'.."'~, 2' :rra'm'i 22:12.2: Lees: 1:41-- a. 1'; -‘ .214“. 22'22132 :-°..':: .3:’..:. c: s: 4’ ’33 a"; it’d-“i for :. ---.-: days a: 4.7." and 53‘? Was: 'o‘faters :Se’l 0 ( [Ip’p1’ll‘ , . 545 1’;'f “I: It.‘ I .‘.r.'. sad ’9” 6.0) , 5.35 r Arm, anxi- (pii 5 5) , 5.25 I ,‘.r.r. at?! (pH 5 O; , 5.22 35:! 9.2!. acid (pH 6 0) . . . , 5. 18’1”; 811’1 (I111 5.5) 5. 1m 1.2!. acid (pH 5 0) , . 5- sorbic Atld ([111 (12) . . sorbic mid (p11 5 8) . . 5. ”orbit; acid (pH 5.2) . . . . . 23 Table 1 (Continued) pH of Cottage Cheese at Various Storage Conditions When Washed with Acidified Water v—v iv Viva f- 3 da. 6 da. 9 da. 12 da. 40°F. 50°F. 40°F. 50°F. 40°F. 50°F. 40°F. 50°F. 5.25 4.65 5.18 4.30 5.00 4.32 5.08 4.40 5.00 4.68 5.08 4.40 4.98 4.30 5.05 4.40 5.00 4.65 4.95 4.35 4.88 4.35 5.00 4.45 5.05 4.78 4.90 4.35 4.90 4.38 5.00 4.43 5.12 4.70 5.00 4.35 4.95 4.35 5.02 4.45 5.10 4.65 5.00 4.32 4.98 4.32 5.00 4.40 5.05 4.60 4.95 4.28 4.98 4.28 5.00 4.35 5.00 4.90 4.88 4.45 4.80 4.35 5.00 4.48 5.00 4.95 4.90 4.65 4.82. 4.50 5.00 4.60 4.90 4.95 4.90 4.75 4.85 4.75 4.90 4.80 24 Table 2. Sorbic acid tolerance of Leuconostoc citrovorous and/or Leuconostoc dextranicum in trypticase soy broth at vari- ous levels of pH (avg. of 4 trials). ’1 1 f vi v f fv—f Viv vv va ff v v f v v Sorbic Acid Added to Broth Before Adjusting Final Reacti‘m Turbidity 9f . . , of Broth Culture Indi- pH w1th Lactic Ac1d . (PH) cating Growth (‘70) _ 0.05 6.65 + 0.05 6.00 + 0.05 5.00 + 0.05 4.50 + 0.05 4.00 + 0.10 6.60 + 0.10 6.00 + 0.10 5.00 + 0.10 4.50 + 0.10 4.00 + 0.25 6.00 + 0.25 5.90 + 0.25 5.00 + 0.25 4.50 + 0.25 4.00 + _ I Control I Lactic acld * o Citric acid a Sorbic acid Days “.3. 1 Wth IatorI acidulatod to ii 6.0 with citric and lactic acida and to pH 6.2 Iith sorbic acid. 2' I Control I Lactic cold 0 Cltrlc cold A Sorblc acld - a O U '5 9 l 6 8 10 I2 Days ' 11¢. 2 flash Iatcra acidulated to pH 5.5 with citric all! lactic act“ and to fl 5o8 with Iorbia ”no 0 '0 Q fig. biacetyl par lOOa. choc“ .N o I Control I Lactlc acld ’ o Citric acid A Sorblc acld - O O T 3a 9 Ito. blocatyl par IOOg.ctIaaao o 2 2 4 6 0 IO 12 m. 3 Waah vatcra actdulatod to pH 5.0 with citric and lactic actda and to pH 5.2 with aarbtc acid. mummMmummm. mmmumnu 21m 024501012 Dan Fig. 16 Huh natal-a actdulatod to m 6.0. 5.5 ad 5.0 with citric acid. of oControI “biacetyl permanence“ o 2 4 c a toll! Days Fig. 5 Wash IatcrI actdulatad to pH 6.0. 505 I“ 500 “th lactic ”mo tofo Control val-t 5.8 vatt6.2 09:10.2 1.6 a. .. 3L4- §oo 3‘ C 804 :5 go‘é‘i'i‘o I0 I! Days 1’13. 6 Wash IatorI actdulatod to IE 6.2. 5.8 m 502 “th IMO “I olggttg gr. sortie 191.9. 20 5112 mm}. mm 2.0 3 _ IControl I Lactic acid ' g oCutrlc acld ASorbic acid 0 LS ~ 4 O. l- d 3 '.2 t- It 36. :5 o. 1: Ci a 7 1 1 1 Mk 1 1 1 1 1 1 O 2 4 6 8 1C) l2 Days Elf. 7 "ash waters acidulated to 0H 3.0 with citric and lactic acids and to OH 6.2 with sorbic acid. 2.0 3 I Control I Lactic acid E r oCitric acid aSorbic acid ‘ " 1.6 r ‘ o. - fl 8 L4 4 . 8 Q E '0 11 .9 A: 6 2 7 m 1 1 4 1 1 1 1 L 1 O 2 4 6 8 IO 12 Days Fig. 8 flash waters acidulated to pH 5.5 with citric and lactic acids and to pH 5.8 with sorbic acid. 3 2'0 IControl I Lactic acid g ’ D Citric acid A Sorbic acid " o L6h . 1 d» E «I (a .2 .0 g l l L l l l l 1 I l l O 2 4 6 8 IO 12 Days Fig. 9 hash Haters acidulated to pH 5.0 with citric and lactic acids and to 0H 5.2 with sorbic acid. 2'0); I Control I pH 5.5 v pH 6.0 0 pH 5.0 ° LG) . '02 P ‘ .0 a Ma biacetyl par I00 a. chant. ,0 1 1 1 m1 1 l 1 14 - 8 IO 12 Fig. 10 Lash waters acidulated to pH 6.0, 5.5 and 5.0 with citric acid. (3 00 It a! :20 IControl val-l 5.5 g 3 ' va 6.0 9115.0 5|.6- ‘ d‘ ' 4 81.4- « g. 2‘ 80.. .5 D g 1 1 1 1 1 1 1 1 1 L 1 , O 2 4 6 8 10 12 Days Fig. ll «ash waters acidulated to 0H 8.0, 5.5 and 5.0 with lactic acid. .N o I Control " v pH 6.2 'cn L a a 10 :2 Days Mg. biacetyl par I00 9. chaos. o a C) ”1- Jh Fig. 12 Wash waters acidulated to pH 6.2. 5.8 and 5.2 with sorbic acid. mammmmm.§1finmmm§mmmmnmtot ummmmumuflr. 27 to pH 5.2 with sorbic acid had a pH value of 4.80 on the twelfth day (Table l) as compared to 4.40 for the control on the twelfth day. Effect of Various Organic Acids Used to Acidulate Wash Waters on the Acetylmethylcarbinol Content of Cottage Cheese The initial acetylmethylcarbinol values for fresh cottage cheese ranged from 1.5 to 5.0 milligrams per IOO-gram sample with the average ranging from 3.2 to 3.8 milligrams. Acetylmethylcar- binol values tended to increase considerably during the storage period and usually attained a maximum on the sixth or ninth days. The values tended to be lower on the twelfth day. (a) Influence of organic acids on the acetylmethylcarbinol con- tent of cottage cheese stored at 40°F. The acetylmethylcarbinol contents of cottage cheese, washed with waters acidified to pH 6.0, 5.5, and 5.0 with citric or lactic acid and to pH 6.2. 5.8, and 5.2 with sorbic acid and stored twelve days at 40° F., are shown in Figures 13 through 18. The acetylmethylcarbinol content of the control increased from the initial value of 3.6 milligrams to a maximum of 5.95 milligrams per IOO-gram sample on the sixth day and on the twelfth day decreased to approximately the original value. The acetylmethylcarbinol contents of the cheese washed with waters adjusted to pH 6.0 are illustrated in Figure 18. The 28 acetylmethylcarbinol was highest on the sixth day in the cheese washed with water containing lactic acid, but decreased more abruptly on the ninth day than did the control. When washed with waters acidulated with citric acid, the cheese reached a maximum acetylmethylcarbinol content on the ninth day and remained higher than the control on the twelfth day. The use of lactic acid in the wash waters, as shown in Figures 14 and. 15, enormously increased the acetylmethylcarbinol content of the cheese. Maximum values of 8.6 and 7.7 milligrams per lOO-gram sample, respectively, were attained on the third day and the acetylmethylcarbinol content remained above the control until the twelfth day, at which time it was slightly lower. Cheese washed with waters containing citric acid reached a maximum acetyl- methylcarbinol content later than the corre5ponding control but these acetylmethylcarbinol values did not decrease as rapidly as the other samples, including the control. The acetylmethylcarbinol content of the samples which are grouped according to the acid used in the wash waters (Figures 16. 17, and 18) show a conformity between the acetylmethylcarbinol contents at the three pH values produced by each acid. The data in Figure 16 show that the acetylmethylcarbinol content of all samples washed with waters containing citric acid reached a 29 maximum on the ninth day and decreased slightly on the twelfth day. The acetylmethylcarbinol content of cheese washed with waters con- taining lactic acid (Figure .17) increased significantly above that of the control. The acetylmethylcarbinol contents of cheese washed with waters acidified with sorbic acid (Figure 18) were below the level of the corresponding control. A comparison of data in Figures 4, 5, and 6 with data in Figures 16, 17, and 18, respectively, indicates a general relationship between biacetyl and acetylmethylcarbinol in the cheese samples. There was a tendency for high biacetyl values to be associated with high acetylmethylcarbinol values and for low biacetyl values to be associated with low acetylmethylcarbinol values. (b) Influence of organic acids on the acetylmethylcarbinol gontent of cottage cheese stored at 50°F. The data for the acetyl- methylcarbinol content in cottage cheese samples stored at 50°F. are presented in Figures 19 to 24, inclusive. These graphs show that the sample washed with water acidified to pH 5.2 with sorbic acid (Figure 21) had a much higher acetylmethylcarbinol content than the control. This is the only sample in which the acetylmeth- ylcarbinol content exceeded the control. Generally, the acetylmeth- ylcarbinol content of cheese samples stored at 50° tended to be lower than that of the corresponding sample stored at 40°F. 30 An examination of data in Figures 22, 23, and 24 shows a consistent relationship between the acetylmethylcarbinol content of the cheese samples washed with waters containing three different amounts of each acid. The acetylmethylcarbinol contents of all samples washed with waters containing citric acid (Figure 22) were lower than the control; these values were maximum the sixth day and de- creased thereafter. An extremely close relationship is apparent among the acetylmethylcarbinol contents of cheese washed with waters acidulated with lactic acid (Figure 23). These acetylmethyl- carbinol values decreased on the third day, increased sharply on the sixth day, decreased abruptly on the ninth day, and increased on the twelfth day. The acetylmethylcarbinol contents of the cheese washed with waters acidified to pH 6.2 and 5.8 with sorbic acid (Figure 24) decreased on the third day and remained well below the control. The acetylmethylcarbinol content of the sample washed with water adjusted to pH 5.2 with sorbic acid remained above the control for the duration of the storage period and was 4.3 milligrams per 100— gram sample higher than the control on the twelfth day. A comparison of data in Figures 10, 11, and 12 with data in Figures 22, 23, and 24, respectively, shows a general relationship between biacetyl and acetylmethylcarbinol contents of corresponding samples stored at 50°F. High acetylmethylcarbinol values seemed IG . . IO 3 _ I Control I Lactic acid 3 I Control v pH 5.5 3 a Citric acid A Sorbic acid . v pH 6.0 0 pH 5.0 ‘ 1: 5" ‘ i! ‘f . . 11 5’ ci 8 o - 2 I- 8 i 11 s E d» ' . a so 1 1 1 1 J E1 L J . 0 2 4 6 8 I0 I2 0 2 4 6 3 l3 l2 Days Days Fig. 13 Wash waters acidulated to 0H Fig. 16 waan waters acidulated to pH 6.0 with citric and lactic acid and 6.0, 5.5 and 5.0 with citric acid. to pH 6.2 with sorbic acid. I . . IC) IControl o Citric acid 4 : IControl v pH 6 0 3 I Lactic acid 3 ' v pH 5 5 3 A Sorbic acid 5 0 pH 5 O 8 6i 6» C) 9 8 ‘g a o 0 S 5 :5 :5 s 1 1 1 1 1 1 1 L 1 1 1 3 1 J 1 1 1 1 1 J 1 l 1 (J 2i 4' 6 8 "3 I2 C) 2 4 (5 6 IO’ I2 Days Days Fig. in «ash waters acidulated to pH 5.5 Fig. 17 hash waters acidulated to pH with citric and lactic acids and to 6.0. 5.5 and 5.0 with lactic acid. pH 5.8 with sorbic acid. '0 IControl I Lactic acid '0 IControl vol-l 5.8 L i oCitric acid ASdrbic acid g * va-I 6.2 opt-I 5.2 ‘ 13- . 0 5 )- . :5 ah I- b a a 11 11 S S o 6 2 11111111141 2 11111111111” Cl 2! ,4 i6 13 IC) I2. () 2i ‘4 £5 8! ICi I2 Day: Day: . Fig. 15 Wash waters acidulated to OH Fig. 18 hash waters acidulgted to pH 5.0 with citric and lactic acids and 6.2, 5.8 and 5.2 with sorbic acid. to pH 5.2 with sorbic acid. mammmmm.mdrmmaaMmmmmmm- 91mm 12mm 22 amused sauna L.__heese 1___tcred n 9.11; iii L02- 0 Control a Citric acid I Lactic acid A Sorbic acid Mo. anic par loo 9. chasss Days Fig. 19 “ash waters acidulated to on 6.0 with citric and lactic acids and to pH 0.2 with sorbic acid. I Control I Lactic acid a Citric acid A Sorbic acid ‘ Mg. ainc par loo 9. chasss O 2 4 6 8 IO I2 Days Fig. 20 dash waters acidulated to pH 5.5 with citric and lactic acids and to pH 5.8 with sorbic acid. I Control I Lactic acid a Citric acid A Sorbic acid Ila anic par loo 9. chasss ‘ 9 :6 l2 0 2 40031 Fig. 21 Wish waters acidulated to pH 5. with citric and lactic acids and to pH 5.2 with sorbic acid. mummmmm.mmm .mnmnmn Mg. arnc par IOO g. chsIss Days Fig. 22 «ash waters acidulated to 9H 0.0, 5.5 and 5.0 with citric acid. l0 I Control pH 5.5 8 v an 5.0 0 pH 5.0 ‘ 8 S 0 6 a 0 E O 6 2 1 1 1 7 O 2 4 6 8 IO l2 Days big. 23 hash waters acidulated to pH 6.0. 5.5 and 5.0 with lactic acid. '0 I Control Mg. amc par loo 9. chasss l l l l L J_ l I. l o' 2466IOI2 Days Fig. 24 "ash waters acidulated to pH 6.2. 5.8 and 5.2 with sorbic acid. Slamm- M 9; “sinned gottgge gheesg staged 3 5131; Q1 5g _. .- a - O‘5¥-" . A pov'fi'v‘ I .4....... at d r ~v\~ Giana . 33 to occur in samples with high biacetyl contents and low acetylmethyl- carbinol values seemed to occur in samples with low biacetyl con- te nts . Destruction of Biacetyl by Microorganisms in Creamed Cottage Cheese (3) Destruction of biacetyl in cottage cheese stored at 40° F. The p0pulation of microorganisms inoculated into cottage cheese creaming mixtures. is shown in Table 3. Counts ranged from 300 per milliliter in cream inoculated with Mucor plumbeus to 450,000 per milliliter in cream inoculated with E. coli. The effect of microorganisms on the biacetyl content of cot- tage cheese stored twelve days at 40°F. is shown by data in Figures 25 through 30. The cheese sample inoculated with Ach. butyri (Fig- ure 29) is the only one in which the maximum biacetyl content of an inoculated sample exceeds that of the control. An examination of all data presented in Figures 25 through 30 indicates that the cheese samples inoculated with G. candidum and Pen. frequentans (Figure 25), Ps. desmolyticum, Ps. fluorescens, and Pa. tralucida (Figure 26), T. candida (Figure 28), E. freundii (Figure 29), and Alc. metalcaligenes (Figure 30) achieved a maximum biacetyl content on the sixth day, were much lower on the ninth day and increased on the twelfth day. 34 The biacetyl content of cheese inoculated with P5. fragi (Figure 26) and R. flava (Figure 28) reached a maximum on the third day and decreased continuously thereafter. The biacetyl content of cheese inoculated with M. flavus, M. conglomeratus, and M. candidus (Fig- ure 26), Mucor plumbeus (Figure 25), Bact. erythrogenes, C. filamentosum, and B. firmus (Figure 30) did not vary significantly from the biacetyl content of the control. Increases or decreases in pH values (Table 4) could not be correlated with increases or decreases in biacetyl content. pH values of all samples stored at 40°F. varied between 5.4 and 4.5 during the sampling period. (b) Destruction of biacetyl in cottage cheese stored at 50°F. The samples illustrated in Figures 31 to 35, inclusive, were stored at 50°F. Biacetyl determinations were performed on all inoculated samples up to and including the sixth day but were discontinued thereafter as spoilage was evident. Studies of the graphs (Figures 31 to 35) indicate that biacetyl destruction is more rapid and com- plete at 50° than at 40°F. The biacetyl contents of samples inoculated with G. candidum and Pen. frequentans (Figure 31), Ps. v—vfi desmalyticum, Ps. fluorescens, and Ps. tralucida (Figure 32), T. candida (Figure 34), E. freundii (Figure 35), and Ale. metalcaligenes 35 (Figure 36) and stored at 50°F. were substantially below the control throughout the storage period. Attention should be focused on the cheese samples inoculated with the organisms which appeared to re- duce the biacetyl content at 40°F. These organisms also appeared to reduce the biacetyl content in samples stored at 50°F. Compari- son with the control shows a significantly lower biacetyl content at three and six days in samples inoculated with Mucor plumbeus (Fig- ure 31), Ps. fragi (Figure 32), M. conglomeratus (Figure 33), R. flava (Figure 34), Ach. eurydice, and E. coli (Figure 35). The bi- aCetyl content of cheese inoculated with M. flavus and M. candidus (Figure 33), C. filamentosum, Bact. erythrogenes, and B. firmus (Figure 36) did not vary significantly from that of the control. All organisms which did not appear to reduce biacetyl in samples stored at 50°F., did not appear to reduce biacetyl in samples stored at 40°F. The pH values of all samples stored at 50°F. varied between 5.2 and 4.5 regardless of sampling interval (Table 4). High or low pH values could not. be correlated with high or low biacetyl contents. Destruction of Acetylmethylcarbinol by Microorganisms in Cottage Cheese (a) Destruction of acetylmethylcarbinol in cottage cheese v—V—v stored at 40°F. Data showing the acetylmethylcarbinol content of Table 3. 36 Microorganism population inoculated into the cottage cheese creaming mixture. V—f f vvfiv v V v vv v7 7 Organism Inoculated into the Creaming Mixture fv'f fivfif 7V v—vv—v Plate Count Per M1. When Added to Cottage Cheese Bacteria: Ach. butyri Ach . eurydic e Alc. metalcalig ene s B. firmus Bact. er ythrog enes .......... Vv—v—vfiv C . filame ntos um E. coli M. candidus M. conglomeratus ........... M. flavus Ps . desmolyticum ........... Ps. fragi ................. Ps. fluorescens Ps. tralucida Molds : Ci . candidum Muc or plumbeus ............ Pen. fr equentans Yeasts: ................ oooooooooooo ----------- 50,000 33,000 150,000 77,000 78,000 107,000 450,000 400,000 200,000 140,000 340,000 186,000 270,000 246,000 400,000 2,500 300 6,000 400 2,000 vvvvvvv 37 Table 4. pH of cottage cheese inoculated with twenty microorgan- isms associated with cottage cheese spoilage under dif- fer ent s torag e conditions. v v V v vfi v f vf v V f v Viv v v v w v f r a The pH of Inoculated Cottage Cheese at Various Storage Times and Temperatures Organisms Inocu- lated into _ . 3 da. 6 da. 9 da. 12 da. Creaming Mixture 0 da. fl 40°F. 50°F. 40°F. 50°F. 40°F. 40°F. Control ......... 4.85 5.00 5.10 4.70 4.68 4.70 4.65 Bacteria: Ach. butLri ..... 4.85 4.85 4.75 4.90 4.70 5.30 5.02 Ach. eurydice 4.85 5.10 5.20 4.73 4.75 4.62 4.65 Alc. metalcaligenes. 4.85 4.82 4.73 5.05 5.00 ' 5.35 4.95 B. firmus .. 4.85 5.00 5.05 4.78 4.70 4.55 4.60 Bact. erythrogenes . 4.85 4.75 4.85 5.04 4.70 5.25 4.85 C. filamentosum 4.85 4.80 4.82 5.08 4.73 5.28 4.90 E. coli ........ 4.85 5.10 5.10 4.70 4.80 4.75 4.60 E. freundii ...... 4.85 4.78 4.80 5.02 4.75 5.24 4.88 M. candidus ..... 4.85 4.85 4.80 5.05 4.75 5.30 5.00 M. conglomeratus 4.85 5.05 5.00 4.75 4.50 4.72 4.65 M. flavus ....... 4.85 5.00 5.05 4.70 4.72 4.60 4.58 Ps. desmolyticum 4.85 4.74 4.80 5.02 4.80 5.42 5.02 Ps. fragi ....... 4.85 5.00 5.05 4.70 4.80 4.65 4.60 Ps. fluorescens 4.85 4.98 5.15 4.85 4.80 4.72 4.65 Ps. tralucida 4.85 4.80 4.78 5.08 4.85 5.28 4.90 Molds: Ci. candidum ..... 4.85 5.05 5.00 4.80 4.52 4.50 4.60 Mucor plumbeus. . . 4.85 5.00 5.10 4.75 4.68 4.60 4.80 Pen. frequentans . . 4.85 4.80 4.68 5.10 4.68 5.25 4.78 Yeasts: R. flava ........ 4.85 5.10 5.10 4.80 4.80 4.60 4.58 T. candida . . . . . . 4.85 4.75 4.82 5.05 4.70 5.05 4.85 rfvvf' rij’vvf' vav 00!! . Fig. 25 Variations in tho bisaotyl con- tast at crossed aottago choose inoculstod with 1. am. m m or in. W- O‘ 2 4 O I Fig. 26 Variations in the biacetyl con- tent. at creased cottage choose inoa ulster! with h- 9.12- 21- {imma- Wm . mm. "El. IControl Inductor-ntos ‘ outlaws oucsndldos 00!! m. 2? Variations in the usastyl son taut of croaissd aottsgo chosss inoculated with 5- mm. '.; min-r1111.” ii. mm. Fig. 26 Variations in the bisaotyl son- tant or eras-Id aattags obsess inoaalstsd Huh 2- am or 1- 11111.11. Fig. 29 Variations in tho biacetyl soa- tant at arosnsd aattago choose inoculatod with m. m. m. m. l- 191.1 ms ori. ”Minimachoosa Fig. 30 Variation: in the hisz can- toat or aresnisd cottage choose inasalstod With as I 0 a . 2!!! II C'_s o mammmmmxmmmnmnnm Mg. biacotyl par loo 9. chooso Ma. hiacotyl par I00 9. chooso ‘ IControl IMacor plunbous ascendiduin oPon. troauontans‘ oizsegée Days Fig. 31 Variations in the biacetv] bsn‘=nt of creamed cottage cheese inOCi1«‘ed with Q. C‘ndidun. .ucor olumbeus or Pgn. {:qugntags. ' 2‘ COMM” 0Ps. desmolyticum A Po. fragi A Ps. tralucida ‘ Fig. 3? Variatiens in thc biacety‘ content of creamed crttsze cheese inoculzted *ith fig. fragi fig. fiuoresgggg. g3. tra ucida or £5. desnolvticun. ‘ IControl InconaIainoratus . "2 outlaws 0M.candidus — I . l i1 . # <3 9 7 1 1 1 1 O I 2 5 4 5 6 Days Fig. 33 Variation: in the biacetyl content of creased cottage cheese inoculated with 5. (Layig. :- ggngloneratus or ;. ganiid_§. M9. biacetyl par IOO a. chooso Ma. hiacotyl par loo 9. chooso ‘2 Mg. biacetyl par I00 9. chooso l2 IOantral I Total - ORhodatarula and do 1 flows 0 l. < Q «i O. O. 0.2 7 M L 1 1 I O I 2 3 4 5 6 Days Pig. 3w .ari.