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Effect of Spoilage Bacteria on Biacetyl Content and Flavor of Cottage Cheese1
E F F E C T OF S P O I L A G E B A C T E R I A ON B I A C E T Y L C O N T E N T AND F L A V O R OF C O T T A G E C H E E S E 1 R. B . P A R K E R
AND P . R. E L L I K E I ¢
Oregon Agriwdtural Experiment Station, Corvallis
Observations on growth of certain cottage cheese spoilage bacteria indicated that their activity in the cheese usually was accompanied b y loss of desirable butter culture aroma in the product. The bacterial species involved and the nature of the gelatinous or slimy spoilage defect have been described in an earlier p a p e r (4). Elliker and Horrall (3) observed that samples of b u t t e r contaminated with Pseudomonas putrefaciens displayed a marked loss of aroma during keeping quality tests. The reduction of aroma preceded the typical cheesy or p u t r i d defect commonly associated with this organism. Chemical analyses correlated the loss of typical butter aroma with destruction of p a r t of the biacetyl present. Later, Elliker (2) was able to demonstrate the ability of a number of types of bacteria to destroy biacetyl. The importance of the Pseudomonas group was emphasized in studies on commercial and experimental butters. I t was pointed out that the loss of biacetyl due to microorganisms m a y be more i m p o r t a n t in some commercial foods than is generally realized. In the present study, as in those on butter, the loss of p r o p e r aroma often was observed to precede the pronounced physical or flavor manifestations of the spoilage. Thus it seemed obvious that at least some strains of the spoilage organisms were destroying a major aroma component (probably biacetyl) ; consequently, f u r t h e r studies were suggested. Purposes of the investigation were to study the relationship of hiacetyl to the desirable aromatic flavor of cottage cheese and to determine the effect of certain spoilage organisms on biacetyl content of cottage cheese, as well as on biacetyl added to milk. EXPERIMENTAL
Analytical method. Analyses for biacetyl were conducted with a modified Pien, Baisse, and Martin method (1, 5) more recently described b y Elliker (2). I n general, the analytical method represented a colorimetric analysis of a steamdistilled fraction of the product. A f u r t h e r modification included in the present study involved generation of steam for the distillation in a 2-1. balloon flask holding a 20 ohm nichrome wire coil immersed in water. Steam flow was regulated by adjusting the voltage applied to the coil by means of a Variac transformer. Fifty-g. portions of cottage cheese were acidified with 2 ml. of lactic acid which had been steamed previously to remove any traces of biacetyl. This mixture was carefully steam distilled and two 9-ml. portions of distillate collected Received for publication March 12, 1953. 1Approved for publication as Teehnical Paper No. 777 by the Director of the Oregon Agricultural Experiment Station. Contribution of the Department of Bacteriology. 843
844
R. B. P A R K E R
A N D P. R. E L L I K E R
in 15-ml. g r a d u a t e d centrifuge tubes containing 1 ml. of distilled water to cover the condenser tip. Steam flow was adjusted to allow about 10 minutes for each tube of distillate collected. 0ne-half ml. of concentrated tIC1 and 0.1 ml. of a 2.5 per cent freshly p r e p a r e d solution of orthodiaminobenzidine hydrochloride were added to each tube and the m i x t u r e allowed to stand for 15 minutes for color development. Color intensity was determined with a Beckman Model B spectrophotometer, using a wave length of 425 m~. P u r e biacetyl ( E a s t m a n ) was used to construct a s t a n d a r d transmittance curve and a 1 p.p.m, solution of biacetyl was used as a reference s t a n d a r d with each set of determinations. Using this method, highly reproducible results were obtained f r o m samples containing between 5 and 200 ~/of biacetyl. Total acetylmethylcarbinol plus biacetyl was determined by refluxing the sample with 50 ml. of 40 p e r cent ferric chloride for 20 minutes in order to convert acetylmethylearbinol to biacetyl a n d then proceeding as with biacetyl determinations. Acetylmethylcarbinol was determined b y difference in the values. Because of high levels of acetylmethylcarbinol in cottage cheese, it usually was found necessary to make a 1 to 10 dilution of the distillate before addition of the HC1 and color reagent in order to provide a final biaeetyl concentration in the range of 5 to 200 v. B i a c e t y l c o n t e n t of commercial cottage cheeses. Thirty-eight commercial cottage cheese samples were obtained in as fresh a condition as possible f r o m various sources. All cheeses were graded according to aromatic flavor as flat, moderate aroma, or high aroma, respectively. Also, included in the s u r v e y were six cheeses submitted to the Oregon D a i r y M a n u f a c t u r e r s ' Association cottage cheese contest. I n addition to the aromatic flavor grade, the six contest cheeses also received an average numerical flavor score f r o m four experienced judges. A f t e r grading for flavor, representative portions of each of the 44 cheeses were analyzed for biacetyl and the results correlated. Average flavor grades and biacetyl values of all samples are shown in Table 1. Simple inspection of the data in Table 1 indicates t h a t there is a close correlation of desirable aromatic flavor with high biacetyl concentrations in cottage cheese. I t is of interest that the numerical flavor score of the six contest cheeses ranged in almost direct relationship to the biacetyl content. I n order of decreasing flavor score the cheeses contained 3.2, 2.4, 2.4, 1.8, 1.9, a n d 1.2 p.p.m, biacetyl, respectively. Cheeses with high acid or yeasty defects were not included in the TABLE 1 Relationship of biavetyl content to flavor of 63 commercial cottage cheese samples
Flavor :Flat aromu
Minimum
Biacetyl content Maximum
Average
p.p.m,
p.p.m,
p.p.m,
0.15
0.62
0.41
:Moderate aroma
0.62
1.40
1.05
lCIigharoma
1.60
3.25
2.26
BIACETYL CONTENT OF COTTAGE CHEESE
845
flavor data, although most cheese with acid defect had relatively high biacetyl concentrations. Loss of biacetyl during keeping quality tests. About one-fourth of the graded samples were held at 10 ° C. for a keeping quality test. Biacetyl analyses were made at 0, 24, and 48 hours, and physical condition of the cheese was noted for an additional 5-day period. Representative results of biacetyl determinations during keeping quality tests are included in Figure 1. Although all samples included in Figure I were in satisfactory physical condition at the end of the initial 48-hour period, sample D displayed a typical gelatinous defect at the end of 96 hours. Samples A and C remained normal in physical appearance and aroma for the full 7-day period, whereas sample B developed a yeasty odor during the latter part of the keeping quality test. By culturing sample D on tryptone glucose milk agar and veal infusion agar containing 40 units of penicillin per milliliter, it was possible to isolate species resembling Pseudomonas viscosa. When these organisms were inoculated onto fresh uncontaminated cottage cheese curd, a typical gelatinous defect appeared within 64 hours.
Effect of pure cultures of three different species of spoilage bacteria on biacetyl content of cottage cheese. A fresh sample of uncontaminated cottage cheese was analyzed for biacetyl and then divided into 300-g. portions, three of which were inoculated respectively with 0.1 ml. of P. viscosa, Pseudomonas fragi, or Alcaligenes metalcaligenes. A fourth uninoculated portion was used as a con-
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~I(]. 1. Change in biacetyl content of four representative commercia] cottage cheeses during keeping quality test. Samples A, B, and C are examples of cheeses with satisfactory keeping quality. Sample D developed a gelatinous defect within 96 hours.
R. B. P A R K E R
~46
A N D P . R. E L L I K E I ~
2.5
2.0
i to5
A.METALCALIGSN~
I,O
o:
P"~4OI
Z4
48
77.
96
HOURS STORED AT IO*C (50°F)
FIG. 2. Effect of growth of pure cultures of -P. viscosa, P. fragi and A. metalcaZigenes, on Joiaeetyl content of eottage cheese. ~rol. The samples were incubated at ] 0 ° C. and biacetyl analyses made at 24, 48, and 96 hours. Results of the s t u d y appear in F i g u r e 2. At 24 and 48 hours all samples were in normal physical condition, although the portions inoculated with Pseudomonas species displayed an appreciable drop in biaeetyl level at 48 hours. A t 96 hours all samples inoculated with spoilage organisms displayed slight evidence of the typical gelatinous or slimy defect. At this time the uninoculated control remained in satisfactory physical condition and retained 1.7 p.p.m, of the 2.6 p.p.m, biacetyl originally present in the cheese. The sample inoculated with A. metalcaligenes showed slightly higher loss in that only 1.2 p.p.m, biaeetyl was retained. This quantity, however, is appreciably larger than the 0.2 p.p.m. remaining in samples inoculated with P. fragi and P. viscosa. Depletion of biacetyl by pure cultures in milk. In order to investigate the chemical path of the destruction of biacetyl, it was necessary to transfer studies to milk with added pure biacetyl because of the large quantities of acetyhnethylcarbinol normally present in cottage cheese. Reconstituted n o n f a t milk made up to 10 per cent total solids was sterilized and adjusted to p H 5.2 to simulate p i t conditions in cottage cheese, and sufficient biacetyl was added to provide 6 p.p.m. Analyses of the sterilized milk before addition of biacetyl indicated 0.8 p.p.m. aeetyhnethylcarT,~inoI but biaeetyl was not detected. Liter quantities of milk with the added 6 p.p.m, biacetyI were inoculated with 0.1 per cent of a 24-hour culture of P. viscosa, P. fragi, and A. metalcaligenes, respectively, and one uninoeulated portion was retained as a control. Duplicate flasks were used for each organism. W i t h d r a w a l for analyses was made after 0-, 24-, and 54-hour incubation at 15 ° C. As shown in F i g u r e 3, the control demonstrated no loss or gain of either biacetyl or acetylmethylcarbinol at 24 hours. All inoculated flasks showed loss of biacetyl and corresponding gain of acetylmethylcarbinol indicating reduction of biacetyl to acetylmethylcarbinol. The milk inoculated with P. fragi showed a
BIACETYL
71
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CONTROL
CONTENT
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OF
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I
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:FIa. 3. Effect of growth of P. viscosa, P. fragi and A. ~netalcaligenes, respectively, on reduction of added biacetyl, (AC)~, and consequent formation of acetylmethylcarbinol, AMC, in milk at p i t 5.2.
1.3 p.p.m, loss of biaeetyl and a gain of 1.2 p.p.m, of aeetylmethylcarbinol, with 0.1 p.p.m, unaccounted for. The milk inoculated with P. viscosa showed a 1.9 p.p.m, loss of biacetyl and a 1.1 p.p.m, gain in acety]nlethylearbinol, leaving 0.8 p.p.m, unaccounted for. Milk inoculated with A. metalcaligenes showed a loss of 1.45 p.p.m, biacetyl and a gain of I).5 p.p.m, acetylmethylcarbinol, leaving 1.0 p.p.m, not accounted for. A t the end of 54 hours of incubation, the control sample displayed slight loss of biacetyl. Milks inoculated with either Pseudomonas species showed a loss of almost all biaeetyl present, and in the ease of P. frag" almost all biacetyl lost was accounted for in the gain in acetylmethylcarbinol value. The milk inoculated with P. viscosa contained 0.15 p.p.m, biaeetyl and 5.1 p.p.m, aeetylmethylcarbinol, which left 1.5 p.p.m, unaccounted for. A. metalcaligenes did not a p p e a r to be as active as the Pseudomonas species in the reduction 02 biaeetyl; the portion lost was not accounted for as aeetylmethylearbinol and therefore m a y have been reduced to 2-3 butylene glycol or other eompoun.ds. DISCUSSION
Undoubtedly a number of substances contribute to the proper flavor and aroma of cottage cheese. However, the intensity of the aroma contributed by biacetyl suggests t h a t it is a p r i m a r y agent. Also the lability of biacetyl would indicate one reason for wide differences in flavor of various cottage cheeses. The
848
R. B. P A R K E R
A N D P. R. E L L I K E R
importance of this compound to proper aromatic flavor is emphasized by the presence of high levels of biacetyl in cottage cheese with pleasing aroma and, conversely, lack of biacetyl in flat cheese. Also the destruction of biacetyl in cottage cheese results in loss of desirable aroma and a distinctly flat flavor. Observations ill these and other studies also have indicated that another important factor responsible for flat, unpalatable commercial cottage cheese is insufficient biaeetyl production by the starter culture employed in its manufacture. Several samples of commercial cottage cheeses were found to be extremely flat when first received. When incubated at 10 ° C. for 3 to 7 days, most of these samples developed gelatinous defects. Other samples were satisfactory when first tested but displayed marked loss of biacetyl during keeping quality tests. Invariably, such samples developed a typical gelatinous defect within 2 to 4 days after loss of the aroma compound. It should be pointed out that these conditions are more rigorous than would be found in most trade and household channels. However, there would appear to be ample time for the depletion of some, if not most, of the biacetyl during retail and household storage, provided the cheese was contaminated with Pseudomonas types. Such a cheese would be flat and unpalatable although succeeding stages of the defect were not apparent. In some instances it has been possible to predict subsequent spoilage by gelatinous or slime-producing organisms on the basis of abrupt loss of biacetyl during keeping quality tests. Studies of the chemical nature of the depletion of biacetyl indicate that most of the compound is converted to acetylmethylcarbinol by P. fragi and slightly less by P. viscosa. Although A. metalcaligenes does not deplete biacetyl as rapidly as the Pseudomonas species, the portion that is affected is extensively reduced. This is emphasized by data which show that only about half of the reduced biacetyl is accounted for in the acetylmethylcarbinol fraction of the distillate. Presumably, a portion of the remaining biacetyl has been reduced to 2,3-butylene glycol. SUMMARY
A simple, sensitive, and accurate method for determination of microquantities of biacetyl was applied in studies on the relationship of biacetyl to aromatic flavor of cottage cheese and the effect of gelatinous or slimy spoilage bacteria on aroma of cottage cheese. Results indicated that the desirable aromatic flavor of cottage cheese is directly related to biacetyl content, and samples low in biacetyl were typically flat and less palatable. Cultures of P. viscosa and P. fragi isolated from gelatinous cottage cheese were capable of rapidly reducing almost all biacetyl present in cottage cheese or in milk whereas a culture of A. metalcaligenes was much less active in such destruction. When P. fragi or P. viscosa were present, the complete loss of aroma preceded development of any apparent physical manifestation of spoilage. Results demonstrated that the biacetyl involved is converted to acetyhuethylcarbinol and some possibly to 2,3-butylene glycol.
B I A C E T Y L C O N T E N T OF C O T T A G E C H E E S E
849
I~EFERENCES (i) Cox, G. A., AND WILEY, W. J. The Colorimetric Estimation of Diacetyl and Aeetoin in Dairy l)roclncts. J. Australian Council Sci. and Ind. Research, 12: 227-231. 1939. (2) ELLIKEa, P. I~. Effect of Various Bacteria on Diacetyl Content and Flavor of Butter. J. Dairy Sci., 28: 93-102. 1945. (3) ELLIKEI~, P. 1~., AND ]:~01~RALL, B . E . Effect of Growth of Pseudo~nonas putrefaciens on Diacetyl and Flavor of Butter. J. Dairy Sci., 26: 943-949. 1943. (4) PARKER, l~. B., S:~ITlff, V° ~r, AND ELLIKER, P. ]~. Bacteria Associated with a Gelatinous or Slimy Curd Defect of Cottage Cheese. J. Dairy Sci., 34: 887-893. 1951. (5) PIEI~, J., BAISSE, S., AND MA~TIhr, R. Le Dosage du Diacetyl dans les Beurres. Lait., 17: 675-698. 1937.
AND P . R. E L L I K E I ¢
Oregon Agriwdtural Experiment Station, Corvallis
Observations on growth of certain cottage cheese spoilage bacteria indicated that their activity in the cheese usually was accompanied b y loss of desirable butter culture aroma in the product. The bacterial species involved and the nature of the gelatinous or slimy spoilage defect have been described in an earlier p a p e r (4). Elliker and Horrall (3) observed that samples of b u t t e r contaminated with Pseudomonas putrefaciens displayed a marked loss of aroma during keeping quality tests. The reduction of aroma preceded the typical cheesy or p u t r i d defect commonly associated with this organism. Chemical analyses correlated the loss of typical butter aroma with destruction of p a r t of the biacetyl present. Later, Elliker (2) was able to demonstrate the ability of a number of types of bacteria to destroy biacetyl. The importance of the Pseudomonas group was emphasized in studies on commercial and experimental butters. I t was pointed out that the loss of biacetyl due to microorganisms m a y be more i m p o r t a n t in some commercial foods than is generally realized. In the present study, as in those on butter, the loss of p r o p e r aroma often was observed to precede the pronounced physical or flavor manifestations of the spoilage. Thus it seemed obvious that at least some strains of the spoilage organisms were destroying a major aroma component (probably biacetyl) ; consequently, f u r t h e r studies were suggested. Purposes of the investigation were to study the relationship of hiacetyl to the desirable aromatic flavor of cottage cheese and to determine the effect of certain spoilage organisms on biacetyl content of cottage cheese, as well as on biacetyl added to milk. EXPERIMENTAL
Analytical method. Analyses for biacetyl were conducted with a modified Pien, Baisse, and Martin method (1, 5) more recently described b y Elliker (2). I n general, the analytical method represented a colorimetric analysis of a steamdistilled fraction of the product. A f u r t h e r modification included in the present study involved generation of steam for the distillation in a 2-1. balloon flask holding a 20 ohm nichrome wire coil immersed in water. Steam flow was regulated by adjusting the voltage applied to the coil by means of a Variac transformer. Fifty-g. portions of cottage cheese were acidified with 2 ml. of lactic acid which had been steamed previously to remove any traces of biacetyl. This mixture was carefully steam distilled and two 9-ml. portions of distillate collected Received for publication March 12, 1953. 1Approved for publication as Teehnical Paper No. 777 by the Director of the Oregon Agricultural Experiment Station. Contribution of the Department of Bacteriology. 843
844
R. B. P A R K E R
A N D P. R. E L L I K E R
in 15-ml. g r a d u a t e d centrifuge tubes containing 1 ml. of distilled water to cover the condenser tip. Steam flow was adjusted to allow about 10 minutes for each tube of distillate collected. 0ne-half ml. of concentrated tIC1 and 0.1 ml. of a 2.5 per cent freshly p r e p a r e d solution of orthodiaminobenzidine hydrochloride were added to each tube and the m i x t u r e allowed to stand for 15 minutes for color development. Color intensity was determined with a Beckman Model B spectrophotometer, using a wave length of 425 m~. P u r e biacetyl ( E a s t m a n ) was used to construct a s t a n d a r d transmittance curve and a 1 p.p.m, solution of biacetyl was used as a reference s t a n d a r d with each set of determinations. Using this method, highly reproducible results were obtained f r o m samples containing between 5 and 200 ~/of biacetyl. Total acetylmethylcarbinol plus biacetyl was determined by refluxing the sample with 50 ml. of 40 p e r cent ferric chloride for 20 minutes in order to convert acetylmethylearbinol to biacetyl a n d then proceeding as with biacetyl determinations. Acetylmethylcarbinol was determined b y difference in the values. Because of high levels of acetylmethylcarbinol in cottage cheese, it usually was found necessary to make a 1 to 10 dilution of the distillate before addition of the HC1 and color reagent in order to provide a final biaeetyl concentration in the range of 5 to 200 v. B i a c e t y l c o n t e n t of commercial cottage cheeses. Thirty-eight commercial cottage cheese samples were obtained in as fresh a condition as possible f r o m various sources. All cheeses were graded according to aromatic flavor as flat, moderate aroma, or high aroma, respectively. Also, included in the s u r v e y were six cheeses submitted to the Oregon D a i r y M a n u f a c t u r e r s ' Association cottage cheese contest. I n addition to the aromatic flavor grade, the six contest cheeses also received an average numerical flavor score f r o m four experienced judges. A f t e r grading for flavor, representative portions of each of the 44 cheeses were analyzed for biacetyl and the results correlated. Average flavor grades and biacetyl values of all samples are shown in Table 1. Simple inspection of the data in Table 1 indicates t h a t there is a close correlation of desirable aromatic flavor with high biacetyl concentrations in cottage cheese. I t is of interest that the numerical flavor score of the six contest cheeses ranged in almost direct relationship to the biacetyl content. I n order of decreasing flavor score the cheeses contained 3.2, 2.4, 2.4, 1.8, 1.9, a n d 1.2 p.p.m, biacetyl, respectively. Cheeses with high acid or yeasty defects were not included in the TABLE 1 Relationship of biavetyl content to flavor of 63 commercial cottage cheese samples
Flavor :Flat aromu
Minimum
Biacetyl content Maximum
Average
p.p.m,
p.p.m,
p.p.m,
0.15
0.62
0.41
:Moderate aroma
0.62
1.40
1.05
lCIigharoma
1.60
3.25
2.26
BIACETYL CONTENT OF COTTAGE CHEESE
845
flavor data, although most cheese with acid defect had relatively high biacetyl concentrations. Loss of biacetyl during keeping quality tests. About one-fourth of the graded samples were held at 10 ° C. for a keeping quality test. Biacetyl analyses were made at 0, 24, and 48 hours, and physical condition of the cheese was noted for an additional 5-day period. Representative results of biacetyl determinations during keeping quality tests are included in Figure 1. Although all samples included in Figure I were in satisfactory physical condition at the end of the initial 48-hour period, sample D displayed a typical gelatinous defect at the end of 96 hours. Samples A and C remained normal in physical appearance and aroma for the full 7-day period, whereas sample B developed a yeasty odor during the latter part of the keeping quality test. By culturing sample D on tryptone glucose milk agar and veal infusion agar containing 40 units of penicillin per milliliter, it was possible to isolate species resembling Pseudomonas viscosa. When these organisms were inoculated onto fresh uncontaminated cottage cheese curd, a typical gelatinous defect appeared within 64 hours.
Effect of pure cultures of three different species of spoilage bacteria on biacetyl content of cottage cheese. A fresh sample of uncontaminated cottage cheese was analyzed for biacetyl and then divided into 300-g. portions, three of which were inoculated respectively with 0.1 ml. of P. viscosa, Pseudomonas fragi, or Alcaligenes metalcaligenes. A fourth uninoculated portion was used as a con-
Z
.
0
~ "--O
"°i
0.5
o
0
13 I
I
I
24
I
I
--o I
48
HOURS STORED AT IO°C (50°F}
~I(]. 1. Change in biacetyl content of four representative commercia] cottage cheeses during keeping quality test. Samples A, B, and C are examples of cheeses with satisfactory keeping quality. Sample D developed a gelatinous defect within 96 hours.
R. B. P A R K E R
~46
A N D P . R. E L L I K E I ~
2.5
2.0
i to5
A.METALCALIGSN~
I,O
o:
P"~4OI
Z4
48
77.
96
HOURS STORED AT IO*C (50°F)
FIG. 2. Effect of growth of pure cultures of -P. viscosa, P. fragi and A. metalcaZigenes, on Joiaeetyl content of eottage cheese. ~rol. The samples were incubated at ] 0 ° C. and biacetyl analyses made at 24, 48, and 96 hours. Results of the s t u d y appear in F i g u r e 2. At 24 and 48 hours all samples were in normal physical condition, although the portions inoculated with Pseudomonas species displayed an appreciable drop in biaeetyl level at 48 hours. A t 96 hours all samples inoculated with spoilage organisms displayed slight evidence of the typical gelatinous or slimy defect. At this time the uninoculated control remained in satisfactory physical condition and retained 1.7 p.p.m, of the 2.6 p.p.m, biacetyl originally present in the cheese. The sample inoculated with A. metalcaligenes showed slightly higher loss in that only 1.2 p.p.m, biaeetyl was retained. This quantity, however, is appreciably larger than the 0.2 p.p.m. remaining in samples inoculated with P. fragi and P. viscosa. Depletion of biacetyl by pure cultures in milk. In order to investigate the chemical path of the destruction of biacetyl, it was necessary to transfer studies to milk with added pure biacetyl because of the large quantities of acetyhnethylcarbinol normally present in cottage cheese. Reconstituted n o n f a t milk made up to 10 per cent total solids was sterilized and adjusted to p H 5.2 to simulate p i t conditions in cottage cheese, and sufficient biacetyl was added to provide 6 p.p.m. Analyses of the sterilized milk before addition of biacetyl indicated 0.8 p.p.m. aeetyhnethylcarT,~inoI but biaeetyl was not detected. Liter quantities of milk with the added 6 p.p.m, biacetyI were inoculated with 0.1 per cent of a 24-hour culture of P. viscosa, P. fragi, and A. metalcaligenes, respectively, and one uninoeulated portion was retained as a control. Duplicate flasks were used for each organism. W i t h d r a w a l for analyses was made after 0-, 24-, and 54-hour incubation at 15 ° C. As shown in F i g u r e 3, the control demonstrated no loss or gain of either biacetyl or acetylmethylcarbinol at 24 hours. All inoculated flasks showed loss of biacetyl and corresponding gain of acetylmethylcarbinol indicating reduction of biacetyl to acetylmethylcarbinol. The milk inoculated with P. fragi showed a
BIACETYL
71
I
6
CONTROL
CONTENT
I
OF
COTTAGE
I
C~EESE
I
I
847 I
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HOURSINCUBATEDAT 15"C(59"F)
:FIa. 3. Effect of growth of P. viscosa, P. fragi and A. ~netalcaligenes, respectively, on reduction of added biacetyl, (AC)~, and consequent formation of acetylmethylcarbinol, AMC, in milk at p i t 5.2.
1.3 p.p.m, loss of biaeetyl and a gain of 1.2 p.p.m, of aeetylmethylcarbinol, with 0.1 p.p.m, unaccounted for. The milk inoculated with P. viscosa showed a 1.9 p.p.m, loss of biacetyl and a 1.1 p.p.m, gain in acety]nlethylearbinol, leaving 0.8 p.p.m, unaccounted for. Milk inoculated with A. metalcaligenes showed a loss of 1.45 p.p.m, biacetyl and a gain of I).5 p.p.m, acetylmethylcarbinol, leaving 1.0 p.p.m, not accounted for. A t the end of 54 hours of incubation, the control sample displayed slight loss of biacetyl. Milks inoculated with either Pseudomonas species showed a loss of almost all biaeetyl present, and in the ease of P. frag" almost all biacetyl lost was accounted for in the gain in acetylmethylcarbinol value. The milk inoculated with P. viscosa contained 0.15 p.p.m, biaeetyl and 5.1 p.p.m, aeetylmethylcarbinol, which left 1.5 p.p.m, unaccounted for. A. metalcaligenes did not a p p e a r to be as active as the Pseudomonas species in the reduction 02 biaeetyl; the portion lost was not accounted for as aeetylmethylearbinol and therefore m a y have been reduced to 2-3 butylene glycol or other eompoun.ds. DISCUSSION
Undoubtedly a number of substances contribute to the proper flavor and aroma of cottage cheese. However, the intensity of the aroma contributed by biacetyl suggests t h a t it is a p r i m a r y agent. Also the lability of biacetyl would indicate one reason for wide differences in flavor of various cottage cheeses. The
848
R. B. P A R K E R
A N D P. R. E L L I K E R
importance of this compound to proper aromatic flavor is emphasized by the presence of high levels of biacetyl in cottage cheese with pleasing aroma and, conversely, lack of biacetyl in flat cheese. Also the destruction of biacetyl in cottage cheese results in loss of desirable aroma and a distinctly flat flavor. Observations ill these and other studies also have indicated that another important factor responsible for flat, unpalatable commercial cottage cheese is insufficient biaeetyl production by the starter culture employed in its manufacture. Several samples of commercial cottage cheeses were found to be extremely flat when first received. When incubated at 10 ° C. for 3 to 7 days, most of these samples developed gelatinous defects. Other samples were satisfactory when first tested but displayed marked loss of biacetyl during keeping quality tests. Invariably, such samples developed a typical gelatinous defect within 2 to 4 days after loss of the aroma compound. It should be pointed out that these conditions are more rigorous than would be found in most trade and household channels. However, there would appear to be ample time for the depletion of some, if not most, of the biacetyl during retail and household storage, provided the cheese was contaminated with Pseudomonas types. Such a cheese would be flat and unpalatable although succeeding stages of the defect were not apparent. In some instances it has been possible to predict subsequent spoilage by gelatinous or slime-producing organisms on the basis of abrupt loss of biacetyl during keeping quality tests. Studies of the chemical nature of the depletion of biacetyl indicate that most of the compound is converted to acetylmethylcarbinol by P. fragi and slightly less by P. viscosa. Although A. metalcaligenes does not deplete biacetyl as rapidly as the Pseudomonas species, the portion that is affected is extensively reduced. This is emphasized by data which show that only about half of the reduced biacetyl is accounted for in the acetylmethylcarbinol fraction of the distillate. Presumably, a portion of the remaining biacetyl has been reduced to 2,3-butylene glycol. SUMMARY
A simple, sensitive, and accurate method for determination of microquantities of biacetyl was applied in studies on the relationship of biacetyl to aromatic flavor of cottage cheese and the effect of gelatinous or slimy spoilage bacteria on aroma of cottage cheese. Results indicated that the desirable aromatic flavor of cottage cheese is directly related to biacetyl content, and samples low in biacetyl were typically flat and less palatable. Cultures of P. viscosa and P. fragi isolated from gelatinous cottage cheese were capable of rapidly reducing almost all biacetyl present in cottage cheese or in milk whereas a culture of A. metalcaligenes was much less active in such destruction. When P. fragi or P. viscosa were present, the complete loss of aroma preceded development of any apparent physical manifestation of spoilage. Results demonstrated that the biacetyl involved is converted to acetyhuethylcarbinol and some possibly to 2,3-butylene glycol.
B I A C E T Y L C O N T E N T OF C O T T A G E C H E E S E
849
I~EFERENCES (i) Cox, G. A., AND WILEY, W. J. The Colorimetric Estimation of Diacetyl and Aeetoin in Dairy l)roclncts. J. Australian Council Sci. and Ind. Research, 12: 227-231. 1939. (2) ELLIKEa, P. I~. Effect of Various Bacteria on Diacetyl Content and Flavor of Butter. J. Dairy Sci., 28: 93-102. 1945. (3) ELLIKEI~, P. 1~., AND ]:~01~RALL, B . E . Effect of Growth of Pseudo~nonas putrefaciens on Diacetyl and Flavor of Butter. J. Dairy Sci., 26: 943-949. 1943. (4) PARKER, l~. B., S:~ITlff, V° ~r, AND ELLIKER, P. ]~. Bacteria Associated with a Gelatinous or Slimy Curd Defect of Cottage Cheese. J. Dairy Sci., 34: 887-893. 1951. (5) PIEI~, J., BAISSE, S., AND MA~TIhr, R. Le Dosage du Diacetyl dans les Beurres. Lait., 17: 675-698. 1937.