Role of Acetaldehyde in Metabolism: A Review 2. The Metabolism of Acetaldehyde in Cultured Dairy Products

Role of Acetaldehyde in Metabolism: A Review 2. The Metabolism of Acetaldehyde in Cultured Dairy Products G. J. LEES Russell Grimwade School of Biochemistry University of Melbourne Parkville, Victoria, 3052, Australia and G. R. JAGO Dairy Research Laboratory Division of Food Research, C.S.I.R.O. Highett, Victoria, 3190, Australia

ABSTRACT

Acetaldehyde, a product of the metabolism of microorganisms used in the manufacture of cultured dairy products, has attracted considerable interest because of its association with the development of desirable flavor and of flavor defects in these products. These microorganisms which form varying amounts of acetaldehyde and ethanol during growth contain enzymes which catalyze the formation of acetaldehyde from carbohydrate, protein, or nucleic acid sources. The enzyme activities of the lactic acid bacteria are reviewed in the light of their role in intermediary metabolism. INTRODUCTION

The ability to produce acetaldehyde is widespread among the lactic acid producing bacteria. Group N streptococci (3, 4, 6, 9, 12, 24, 28, 33, 35, 36, 41, 45, 46, 51, 54, 55, 60, 74, 76), Streptococcus thermopbilus (9, 10, 68), Streptococcus faecalis (4, 9), lactobacilli (4, 10, 11, 21, 34, 68), leuconostocs (6) and to a lesser extent pediococci (37) all produce acetaldehyde during growth in milk and other growth media. Propionibacteria, used in the manufacture of Swiss cheese, produce propionaldehyde in addition to acetaldehyde (30). The accumulation of acetaldehyde in the growth medium depends on whether the organism has enzymes which convert acetaldehyde to other metabolites, principally ethanol.

Received November 14, 1977. Department of Biochemistry, University of Auckland, Private Bag, Auckland, New Zealand. 1978 J Dairy Sci 61:1216-1224

The amount of acetaldehyde required for the development of a characteristic flavor varies widely between dairy products. In cheese, cultured butter, and butter-milk, only relatively small amounts are required for the development of a balanced flavor (31) while in yogurt relatively large amounts are required to give the characteristic flavor (21, 31, 58, 60, 62). That certain strains or species always have been associated with flavor defects arising from the accumulation of acetaldehyde or its further metabolites in certain cultured dairy products is not surprising. The yogurt-like or green flavor defect in cultured butter or butter-milk is due to an excess production of acetaldehyde by Group N streptococci, particularly strains of Streptococcus lactis subsp, diacetylactis (3, 33, 36, 45, 51). The defect can be prevented by the inclusion of Leuconostoc cremoris which reduces the acetaldehyde to ethanol (33, 35, 46). The fruity flavor defect of Cheddar cheese is characterized by large amounts of ethyl esters (7). The esterases catalyzing the reactions between short-chain fatty acids and ethanol are present in Group N streptococci and lactobacilli (52). High ethanol levels in cheese may increase the formation of these esters. Hence, the use of strains which have high alcohol dehydrogenase activity should be avoided. Strains of S. lactis subsp, diacetylactis and S. lactis in particular, appear to have high alcohol dehydrogenase activity (57, 74) and commonly cause a fruity flavor defect. The conditions under which Group N streptococci and other starter organisms produce acetaldehyde and ethanol during growth have not been investigated fully. Keenan et al. (36) found that the accumulation of acetaldehyde during the growth of Group N streptococci reached a peak and then declined (two

1216

ACETALDEHYDE 1N DAIRY PRODUCTS strains of S. lactis subsp, diacetylactis did not show this decline). The results (Fig. 1) of a study in this laboratory on the production of both acetaldehyde and ethanol by S. lactis subsp, diacetylactis DRC3 in growth media indicated that most of the acetaldehyde produced by this strain was reduced to ethanol. Until recently, the pathway of formation of acetaldehyde in Group N streptococci was not known, but it was assumed to be via the direct decarboxylation of pyruvate (6, 24, 59, 61) or from acetate (71). As shown in Table 1, enzymes are present in lactic acid bacteria which can form acetaldehyde from the end-products of carbohydrate, protein, or nucleic acid metabolism.

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1217

F o rm ation of Acetaldehyde from Carbohydrate

Group N streptococci form acetaldehyde and ethanol from glucose (42, 53), but only trace amounts are formed by L. bulgaricus (42). Aldehyde dehydrogenase activity (E.C. 1.2.1.10) in Group N streptococci was correlated with their ability to form acetaldehyde from glucose. These results suggest that aldehyde dehydrogenases may be involved in the pathway forming acetaldehyde from glucose. The dependence of the aldehyde dehydrogenase in S. lactis subsp, diacetylactis DRC3 on coenzyme A (CoA) and NAD for activity has been established tentatively (42). The enzyme in S. lactis UN is specific for NAD (71). Pyruvate appears to be the major source of acetate, which either as the free acid (42, 71) or as acetyl-CoA is the substrate for aldehyde dehydrogenase activity in Group N streptococci, Apart from its synthesis from glucose, pyruvate (and acetate) can be formed from citrate via citrate lyase (in S. lactis subsp. diacetylactis only (25, 26, 75) and from alanine via an alanine:t~-ketoglutarate aminotransferase (E.C.2.6.1.2) (40). Pyruvate is used as a source of acetaldehyde and ethanol by whole cell suspensions of strain DRC3 (Table 2). Acidic conditions, such as exists in cheese, promote synthesis of acetaldehyde and ethanol from pyruvate in whole cells. This is probably due to an increased uptake of pyruvate at the lower pH.

08 0

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Formation of Acetaldehyde from Protein

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FIG. 1. Rate of formation of acetaldehyde and ethanol from glucose during growth of S. lactis subsp. diacetylactis DRC3. a) o, glucose utilized; A, acetaldehyde formed; *, ethanol formed; a, total deoxyribose formed, b) =, pH of growth medium; n, cell mass. Assays according to Lees and Jago (42).

The possible formatio'n of acetaldehyde and other volatile flavor components from protein sources has been neglected. Pyruvate is an end-product of the catabolism of many amino acids such as serine, cysteine, and alanine. An analine :a-ketoglutarate aminotransferase in Group N streptococci (40) demonstrates that alanine is a potential source of acetaldehyde in dairy products. Threonine aldolase (E.C.4.1.2.5) (43) is another acetaldehyde-forming enzyme which has been found in many lactic acid bacteria. This enzyme appears to be the major enzyme forming acetaldehyde in cultures of E. bulgaricus. However, the threonine aldolase of Group N streptococci may have a different function from the threonine aldolase of L. bulgaricus. In Group N streptococci, the threonine aldolase activity is inhibited strongly by glycine, whereJournal of Dairy Science Vol. 61, No. 9

t~

TABLE 1. Specific activities of e n z y m e s metabolizing acetaldehyde in lactic acid bacteria. ga

Specific activities a

Organism

Aldehyde dehydrogenase

Threonine aldolase

Deoxyriboaldolase

Alcohol dehydrogenase

S. lactis subsp, diacetylactis DRC1 S. lactis subsp, diacetylactis DRC~ S. lactis subsp, diacetylactis 1 8 - 1 6

.38 5.58 6.00

1.29 1.56 .98

2.45 2.66 3.36

48.8 33.8 (.3) 21.9

S. S. S. S.

1.86 .28 .71 1.25

.37 .40 1.36 .57

1.31 1.24 3.68 3.74

13,4 16.5 21.2 33.0

S, cremoris Z8 S. cremoris HP S. cremoris ML1

1.52 2.87 4.55

.00 1.28 .35

.72 .91 1.47

12,3 8.8 16.9

S. faecalis SF1

5.29

.04

2.99

.0

.15 1.05 .17 .04

.30 .10 .26 .33

.00 2.32 .00 .00

.0 5.0 .7 19.0

.28 .53 .10 .26

1.07 .69 .62 .70

.00 .00 .00 .00

.0 (.2) .2 (.0) .0 (.1) .0

[39 .53

.09 1.03 .01

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5913 (104.8) .0 (.0)

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L. L. L. L.

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~7

CSIRO TW1 ST7 NIZO

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L. plantarum 8041 Leuc. cremoris 9 1 4 0 4 Ped. cerevisiae 8042

.

(.1) (.0) (.1) (.0)

a~moles × 10 -2 of product formed or substrate utilized/min/mg protein. The values shown for aldehyde dehydrogenase have been based on per m g dry wt o f cells. Values in parentheses give the alcohol dehydrogenase activities with NADPH 2 as cofactor. Compiled from data of Lees and Jago (42, 43, 44).

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ACETALDEHYDE IN DAIRY PRODUCTS

1219

TABLE 2. The production of acetaldehyde and ethanol from pyruvate by S. lactis subsp, diacetylactis DRC3. Fermentation productsa Pyruvate (mM)

pH of assay

Acetaldehyde

Ethanol

Acetaldehyde + ethanol

50 50

7.0 4.5

.0 3.1

.25 1.46

.25 4.56

attmoles product X 10-4/min/mg (dry wt) cells. Assays according to Lees and Jago (42).

as the threonine aldolase activity in L. bulgaricus is much less sensitive to inhibition by glycine, especially at physiological concentrations (Fig. 2). Moreover, the amounts of threonine aldolase in S. lactis subsp, diacetylactis and S. tbermopbilus, but not in L. bulgaricus are decreased markedly by growing the organisms at 37 C instead of at 30 C (43). L. bulgaricus is the species largely responsible for the formation of acetaldehyde in yogurt (21, 58, 63, 68), and if threonine aldolase is the enzyme responsible, the glycine formed as a by-product would stimulate growth of S. tbermopbilus (5). Veringa (personal communication) has shown that the addition of free threonine to yogurt increased acetaldehyde in that product. It appears that L. bulgaricus is also able to contribute to the acetic acid and ethanol in cheese. Czulak, Hammond, and Horwood (15) were able to restore the flavor and acetic acid and ethanol in cheese manufactured from

cow's milk containing approximately 20% linoleic acid content in the fat, by adding L. bulgaricus with the normal starter. In the absence of the lactobacillus, acetate and ethanol were depressed markedly and the cheese developed little flavor. Anders and Jago (3) have shown that unsaturated fatty acids inhibit the pyruvate dehydrogenase system in Group N streptococci. This enzyme system catalyzes the initial step in the pathways forming diacetyl, acetic acid, acetaldehyde, and ethanol from pyruvate. L. bulgaricus appears able to bypass this inhibition of the pyruvate dehydrogenase system by forming acetaldehyde from threonine (43), which then could be converted in cheese to acetate and ethanol by the following mechanism:

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FIG. 2. Inhibition by glycine of the threonine aldolase activities in e, S. lactis subsp, diactylactis DRC3; o, S. cremoris HP; • Leuc. cremoris 91404; zx, L. bulgaricus LB1. Assays according to Lees and Jago (43).

This dismutation of acetaldehyde to ethanol and acetic acid can be catalyzed by resting ceils of S. lactis subsp, diacetylactis and Leuc. cremoris (Fig. 3). By contrast, Collins and Speckman (14) have shown that acetaldehyde is converted only to ethanol in growing cells of Leuc. cremoris and that no acetate is formed. The discrepancy is probably due to the necessity of using acetaldehyde as a hydrogen acceptor in growing cells to regenerate the oxidized pyridine nucleotides required for the catabolism of glucose. Unlike the threonine aldolase in L. bulgaricus, the activity of this enzyme in the Group N Journal of Dairy Science Vol. 61, No. 9

1220

LEES AND JAGO

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derivatives have been found in Cheddar cheese (69). The breakdown of thymidine derivatives involved in the catabolism of glucose also could serve as a source of acetaldehyde. Other enzymes synthesizing acetaldehyde, such as pyruvate decarboxylase (41) (E.C.4.1.1.1), dioldehydrase (E.C.4.2.1.28), and ethanolamine deaminase (E.C.4.3.1.7), were absent from Group N streptococci, even after growth on glucose, ethanediol, and ethanolamine, respectively, as inducers (40). There may, however, be some nonenzymic synthesis of acetaldehyde via the Strecker degradation involving 0l-keto acids and amino acids such as alanine (38, 47). U tilization of Acetaldehyde

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ATTENUATION

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FIG. 3. Formation of acetic acid and ethanol from acetaldehyde by resting whole cells of S. lactis subsp. diacetflactis DRC3 and Leuc. cremoris 91404. Leuc. cremoris a) acetaldehyde added; b) no acetaldehyde added. S. lactis subsp, diacetylactis DRC3; c) acetaldehyde added; d ) n o acetaldehyde added. Peak numbers 1) acetaldehyde; 2) ethanol; 3) unknown; 4) acetic acid. Assays according to Lees and Jago (42).

streptococci is reduced markedly in cells grown at 37 to 38 C (the normal cooking temperature of Cheddar cheese) (43) and in the presence of glycine (Fig. 2). Therefore, Group N streptococci would be less able to use threonine as an alternate substrate to pyruvate for the production of ethanol and acetate. Formation of Acetaldehyde from Nucleic Acid

Deoxyriboaldolase appears to be the only other enzyme forming acetaldehyde in lactic acid bacteria (Table 1) (44). However, the synthesis of acetaldehyde via this enzyme is probably limited although the gradual breakdown of DNA over a long time, as occurs during the ripening of cheese, could provide the necessary precursors for the synthesis of acetaldehyde by this pathway. 2-Deoxyribose Journal of Dairy Science Vol. 61, No. 9

High concentrations of acetaldehyde in yogurt must be due, in part, to a low rate of utilization of acetaldehyde. Thus, the main enzymes responsible for the utilization of acetaldehyde, alcohol dehydrogenases (E.C.1.1.1.1), are absent in L. bulgaricus and in most strains of S. tbermopbilus (Table 1) (42). Moreover, the lactose concentration of milk, and the higher temperatures used in the manufacture of yogurt, may decrease further alcohol dehydrogenase, as these factors decreased alcohol dehydrogenase in S. lactis subsp. diacetylactis (42). Lactose could be involved in catabolite repression of this enzyme. Utilization of free acetaldehyde for the formation of acetoin or diacetyl appears unlikely as no stimulation of acetoin and diacetyl production by acetaldehyde (10 mM)

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and threonine aldolase (o) of S. lactis subsp, diacetylactis DRC3. Assays according to Lees and Jago (43, 44).

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1221

nine dehydrogenase (NAD-dependent) (43); 39) aminoacetone synthetase (40); 40) glycine oxidase (40); 41) glycine:a-ketoglutarate transminase (40); 42) thymidine phosphorylase (44); 43) deoxyribomutase (44); 44) deoxyriboaldolase (44); 45) leucine: a-ketoglutarate transaminase (48, 49); 46) a-keto acid decarboxylase (specific for branched chain keto acids) (48, 49, 70); 47) arginine deaminase (50); 48) ornithine transcarbamylase (39.50); 49) phosphoglucoisomerase (56); 50) phosphofructokinase (56); 51)triose phosphate isomerase (53); 52) glyceraldehyde-3-phosphate dehydrogenase (56); 53) D-galactose-6-phosphate isomerase (8); 54) D-tagatose-6-phosphate kinase (8); 55) D-tagatose-l,6-diphosphate aldolase (8).

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FIG. 5. Intermediary metabolism of Group N streptococci. -~, enzymes present; ...*, enzymes tentatively identified; - - -% enzymes assumed present; ~, enzymes not found. 1)phosphotransferase system (8); 2) fl-D-phosphogalactosidase (8); 3) hexokinase (23, 56, 66, 71); 4) galactokinase (76); 5)galactowaldenase (71); 6) phosphoglucomutase (66); 7) glucose6-phosphate dehydrogenase (53, 56, 61, 66); 8) 6phosphogluconate dehydrogenase (53, 56, 61, 66); 9) phosphoribose isomerase (40, 72); 10) transketolase (40, 72); 11) aldolase (53, 56, 71, 72); 12) phosphoketolase (induced after growth on xylose) (42); 13) citrate lyase (citritase) (25, 26, 64, 75); 14) malate dehydrogenase (40); 15) oxaloacetate decarboxylase (25, 64); 16: alanine:c~-ketoglutarate transaminase (40); 17) lactate dehydrogenase (1, 29, 56, 66, 71, 72); 18) acetohydroxy acid synthetase (64); 19) c~acetolacetate decarboxylase (64); 20) pyruvate decarboxylase (42); 21) pyruvate dehydrogenase (53, 67); 22) diacetyl synthetase (13, 67); 23) carboxyligase (acetoin synthetase) (40, 64, 67); 24) diacetyl reductase (1, 64, 65, 67); 25) butanediol dehydrogenase (13, 40, 64); 26) butanediol dehydrase (39); 27)aldehyde dehydrogenase (CoA-dependent) (42); 28) aldehyde dehydrogenase (71, 72); 29) acetate kinase (23, 42, 56); 30) phosphotransacetylase (42); 31)acetoacetate decarboxylase (32, 40); 32) alcohol dehydrogenase (42, 53, 71, 72); 33) ethanolamine deaminase (40); 34) dioldehydrase (40); 35) threonine aldolase (43); 36) threonine dehydrogenase (NADP-dependent) (43); 37) threonine dehydratase (43); 38) threo-

was observed in S. lactis subsp, d i a c e t y l a c t i s DRC3 (40), thus supporting the results of Collins and Speckman (14). However, when hydroxyethylthiamine pyrophosphate (active acetaldehyde) is formed from pyruvate in the pyruvate dehydrogenase system in Group N streptococci, the hydroxyethylthiamine pyrophosphate can react with either pyruvate or acetyl CoA to form acetoin or diacetyl, respectively (13). When hydroxyethylthiamine pyrophosphate is formed from pyruvate by pyruvate decarboxylase (in yeast) it immediately is converted to acetaldehyde. Utilization of acetaldehyde by L e u c . crem o r i s was accompanied by a stimulation of growth (46). Lindsay et al. (46) proposed that with this organism, the addition of acetaldehyde to the growth medium supplied a hydrogen acceptor for the oxidation of reduced pyridine nucleotides. In the absence of an alternative hydrogen acceptor, such as acetaldehyde, the acetylphosphate produced from glucose must be reduced to acetaldehyde and ethanol, a step which does not produce ATP. In the presence of acetaldehyde, acetyl phosphate could be converted to acetate and ATP. The mechanism appears to operate in L e u c . m e s e n t e r o i d e s , an organism closely related to L e u c . cremoris. L e u c . m e s e n t e r o i d e s ferments glucose via the heterofermentative hexose monophosphate shunt (20, 22). It contains an NAD(P)-dependent glucose-6-phosphate dehydrogenase (17, 18) and an NAD-dependent 6-phosphogluconate dehydrogenase (18), as well as NAD- and NADP-dependent alcohol dehydrogenases (16, 19). The rate-limiting step for the fermentation of glucose by L e u c m e s e n t e r o i d e s was a hydrogen acceptor such as acetaldehyde (20). Journal of Dairy Science Vol. 61. No. 9

1222

LEES AND JAGO

S o m e e x p e r i m e n t a l evidence in favor of the h y d r o g e n a c c e p t o r hypothesis of Lindsay et al. (46) was a highly active N A D ( P ) - d e p e n d e n t alcohol dehydrogenase(s) (Table 1) and an acetate kinase in Leuc. cremoris (40). This strain rapidly converted glucose to ethanol, and acetaldehyde to acetate, or a derivative of acetate, and ethanol (42) (Fig. 3). N o n e n z y m a t i c utilization of acetaldehyde by condensation reactions is also possible. Such well k n o w n reactions include condensation with a m i n o acids and o t h e r amines, with itself to f o r m aldol, and with ethanol to f o r m acetal. In addition to knowing which e n z y m e s are involved in the synthesis of acetaldehyde it is also i m p o r t a n t to understand the factors which m a y affect their activity. Thus, t h r e o n i n e aldolase, and d e o x y r i b o a l d o l a s e are irreversibly inactivated at 37 C when the pH is b e l o w 5 (Fig. 4). Hence, a c o m b i n a t i o n of high acidity and high t e m p e r a t u r e during growth of the organism during m a n u f a c t u r e m a y decrease the rate at which acetaldehyde and o t h e r flavor c o m p o u n d s are p r o d u c e d during cheese ripening. A l t h o u g h the presence of the e n z y m e s responsible for the synthesis and utilization o f acetaldehyde in lactic acid bacteria n o w appear to be established, the pathways by which the precursors of acetaldehyde are f o r m e d have n o t been w o r k e d o u t in great detail. Even less is k n o w n of the properties of the enzymes catalyzing the reactions in these pathways. A summ a r y of s o m e of the k n o w n e n z y m e s in G r o u p N streptococci and of the reactions t h e y catalyze, is in Fig. 5. The properties and relative activities of the e n z y m e s forming acetaldehyde in lactic acid bacteria suggest that threonine may be the major precursor of the acetaldehyde and ethanol f o r m e d in y o g u r t while c a r b o h y d r a t e m a y be the m a j o r precursor of the acetaldehyde and ethanol f o r m e d in cheese and c u l t u r e d butter. F u r t h e r studies with radioactive tracers could provide additional i n f o r m a t i o n concerning the relative i m p o r t a n c e of protein and carbohydrate as precursors o f the acetaldehyde f o r m e d in cultured dairy products.

REFERENCES

1 Anders, R. F. 1967. Aspects of the growth and metabolism of Group N streptococci. Ph.D. Thesis, Journal of Dairy Science Vol. 61, No. 9

University of Melbourne. 2 Anders, R. F., and G. R. Jago. 1970. The effect of fatty acids on the metabolism of pyruvate in lactic acid bacteria. J. Dairy Res. 37:445. 3 Badings, H. T., and Th. E. Galesloot. 1962. Studies on the flavour of different types of butter starters with reference to the defect "yoghurt flavour" in butter. 16th Int. Dairy Congr. B:199. 4 Bassette, R., R. E. Bawdon, and T. J. Claydon. 1967. Production of volatile materials in milk by some species of bacteria. J. Dairy Sci. 50:167. 5 Bautista, E. S., R. S. Dayiya, and M. L. Speck. 1966. Identification of compounds causing symbiotic growth of Streptococcus tbermopbilus and Lactobacillus bulgaricus in milk. J. Dairy Res. 33:299. 6 Bills, D. D., and E. A. Day. 1966. Dehydrogenase activity of lactic streptococci. J. Dairy Sci. 49:1473. 7 Bills, D. D., M. E. Morgan, L. M. Libbey, and E. A. Day. 1965. Identification of compounds responsible for fruity flavor defect of experimental cheddar cheeses. J. Dairy Sci. 48:1168. 8 Bissett, D. L., and R. L. Anderson. 1974. Lactose and D-galactose metabolism in Group N streptococci: Presence of enzymes for both the Dgalactose-l-P and D-tagatose-6-P pathways. J. Bacteriol. 117 : 318. 9 Bottazzi, V., and F. Dellaglio. 1967. Acetaldehyde and diacetyl production by Streptococcus tbermopbilus and other lactic streptococci. J. Dairy Res. 34:109. 10 Botazzi, V., and M. Vescovo. 1969. Carbonyl compounds produced by yoghurt bacteria. Netherlands Milk Dairy J. 23:71. 11 Cantoni, C., and M. R. Molnar. 1967. Investigations on the glycerol metabolism of lactobacilli. J. Appl. Bacteriol. 30:197. 12 Chou, T. C. 1963. The chemical nature of the characteristic flavour of cultured buttermilk. L~issertation Abstr. 23 : 3589. 13 Chuang, L. F., and E. B. Collins. 1968. Biosynthesis of diacetyl in bacteria and yeast. J. Bacteriol. 95:2083. 14 Collins, E. B., and R. A. Speckman. 1974. influence of acetaldehyde and growth and acetoin production by Leuconostoc citrovorum. J. Dairy Sci. 57:1428. 15 Czulak, J., L. A. Hammond, and J. F. Horwood. 1974. Cheese and cultured dairy products from milk with high linoleic acid contents: 1. Manufacture and physical and flavour characteristics. Australian J. Dairy Technol. 29:124. 16 De Moss, R. D. 1954. A triphosphopyridine nucleotide-dependent alcohol dehydrogenase from Leuconostoc mesenteroides. J. Bacteriol. 68:252. 17 De Moss, R. D. 1953. Routes of ethanol formation in bacteria. J. Cellular Comp. Physiol. 41, Suppl. 1:207. 18 De Moss, R. D. 1955. Glucose-6-phosphate and 6-phosphogluconic dehydrogenases from Leuconostoc mesenteroides. Page 328 in Methods in enzymology. Vol. 1, S. P. Colowick and N. O. Kaplan, ed. Academic Press, New York.

ACETALDEHYDE IN DAIRY PRODUCTS 19 De Moss, R. D. 1955. TPN alcohol dehydrogenase from Leuconostoc mesenteroides. Page 504 in Methods in enzymology. Vol. 1, S. P. Colowick and N. O. Kaplan, ed. Academic Press, New York. 20 De Moss, R. D., R. C. Bard, and I. C. Gunsalus. 1951. The mechanism o f the heterolactic fermentation: a new route o f ethanol formation. J. Bacteriol. 62:499. 21 Goerner, V. F., V. Palo, and M. Bertan. 1968. Veranderungen des Gehaltes der fluchtigen Stoffe wakrend der Joghurtreifung. Milchwissenshaft 23:94. 22 Gunsalus, I. C., and M. Gibbs. 1952. The heterolactic fermentation. II. Position of C 14 in the products of glucose dissimilation by Leuconostoc mesenteroides. J. Biol. Chem. 194:871. 23 Harvey,.R.J. 1965. Damage to Streptococcus tactis resulting from growth at low pH. J. Bacteriol. 90:1330. 24 Harvey, R. J. 1960. Production of acetone and acetaldehyde by lactic streptococci. J. Dairy Res. 27:41. 25 Harvey, R. J., and E. B. Collins. 1961. Role o f citritase in acetoin formation by Streptococcus diacetilactis and Leoconostoc citrovorum. J. Bacteriol. 82:954. 26 Harvey, R. J., and E. B. Collins. 1963. The citrase o f Streptococcus diacetilactis. Substrate products and equilibrium. J. Biol. Chem. 238:2648. 27 Holmes, B., W. E. Sandine, and P. R. Elliker. 1968. Some factors influencing carbon dioxide production by Leuconstoc citrovorum. Appl. Microbiol. 16:56. 28 Jackson, H. W., and M. E. Morgan. 1954. Identity and origin o f the malty aroma substance from milk cultures o f Streptococcus lactis var. maltigenes. J. Dairy Sci. 37:1316. 29 Jonas, H. A. 1968. Mammalian and bacterial lactate dehydrogenases. M.S. Thesis, University of Melbourne. 30 Keenan, T. W,, and D. D. Bills. 1968. Volatile compounds produced by Propionibacterium sbermanii, J. Dairy Sci. 51:797. 31 Keenan, T. W., and D. D. Bills. 1968. Metabolism of volatile compounds by lactic starter culture microorganisms. A review. J. Dairy Sci. 51:1561. 32 Keenan, T. W., D. D. Bills, and R. C. Lindsay. 1967. Acetone in milk cultures o f lactic streptococci and Leuconostoc citrovorum. Can. J. Microbiol. 13:1118. 33 Keenan, T. W., and R. C. Lindsay. 1966. Removal of green flavor from ripened b u t t e r cultures. J. Dairy Sci. 49:1563. 34 Keenan, T. W., and R. C. Lindsay. 1967. Dehydrogenase activity of Lactobacillus species. J. Dairy Sci. 50:1585. 35 Keenan, T. W., R. C. Lindsay, and E. A. Day. 1966. Acetaldehyde utilization by Leuconostoc species. Appl. Microbiol. 14:802. 36 Keenan, T. W., R. C. Lindsay, M. E. Morgan, and E. A. Day. 1966. Acetaldehyde production by single-strain lactic streptococci. J. Dairy Sci. 49:10. 37 Keenan, T. W., C. E. Parmelee, and A. L. Branen. 1968. Metabolism o f voltaile compounds of

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Pediococcus cerevisiae and their occurrence in cheddar cheese. J. Dairy Sci. 51:1737. 38 Keeney, M., and E. A. Day. 1957. Probable role o f the Strecker degradation of amino acids in development o f cheese flavor. J. Dairy Sci. 40:874. 39 Korzenovsky, M., and C. H. Werkman. 1952. Bacterial metabolism o f arginine. Arch. Biochem. Biophys. 41:233. 40 Lees, G. J. 1969. The role o f acetaldehyde in the metabolism o f Group N streptococci. Ph.D. Thesis, University of Melbourne. 41 Lees, G. J., and G. R. Jago. 1969. Methods for the estimation of acetaldehyde in cultured dairy products. Australian J. Dairy Technol. 24 : 181. 42 Lees, G. J., and G. R. Jago. 1976. Acetaldehyde: An intermediate in the formation o f ethanol from glucose by lactic acid bacteria. J. Dairy Res. 43:63. 43 Lees, G. J., and G. R. Jago. 1976. Formation o f acetaldehyde from threonine by lactic acid bacteria. J. Dairy Res. 43:75. 44 Lees, G. J., and G. R. Jago. 1976. Formation o f acetaldehyde from 2-deoxy-D-ribose-5-phosphate in lactic acid bacteria. J. Dairy Res. 44:139. 45 Lindsay, R. C., and E. A. Day. 1965. Rapid quantitative method for determination o f acetaldehyde in lactic starter cultures. J. Dairy Sci. 48:665. 46 Lindsay, R. C., E. A. Day, and W. E. Sandine. 1965. Green flavor defect in lactic starter cultures. J. Dairy Sci. 48:863. 47 Mabbitt, L. A. 1961. Reviews of the progress of dairy science. Section B. Bacteriology. The flavour of cheddar cheese. J. Dairy Res. 28:303. 48 MacLeod, P., and M. E. Morgan. 1955. Leucine metabolism o f Streptococcus lactis vat. rnaltigenes. I. Conversion o f alpha-ketoisocaproic acid to leucine and 3-methylbutanal. J. Dairy Sci. 38:1208. 49 MacLeod, P., and M. E. Morgan. 1956. Leucine metabolism of Streptococcus lactis vat. rnaltigenes. II. Transaminase and decarboxylase activity of acetone powders. J. Dairy Sci. 39:1125. 50 Mikolajcik, E. M. 1964. Arginine metabolism by an oxytetracycline-resistant strain o f Streptococcus lactis. J. Dairy Sci. 47:667. 51 Morgan, M. E., R. C. Lindsay, L. M. Libbey, and R. L. Pereira. 1966. Identity o f additional aroma constituents in milk cultures of Streptococcus lactis var. maltigenes, J. Dairy Sci. 49:15. 52 Morichi, T., M. E. Sharpe, and B. Reiter. 1968. Esterases and other soluble proteins o f some lactic acid bacteria. J. Gen. Microbiol. 53:405. 53 Nandan, R. 1967. Pathways and enzymes involved in gloazose catabolism by lactic streptococci. Dissertation Abstr. B, 28:2052. 54 Nikkila, O. E. 1947. Page 521 in Malty flavour in milk and milk products. 4th Int. Congr. Microbiol. 55 Nikkila, O. E. 1948. Malty flavour in milk products. Meijeritier Arkakausk. 10:101. Dairy Sci. Abstr. 1950, 12:70. 56 Oram, J. D., and B. Reiter. 1966. The inhibition o f streptococci by lactoperoxidase, thiocyanate and hydrogen peroxide. The effect of the inhibitory system on susceptible and resistant strains of Group N streptococci. Biocbem. J. 100:373. 57 Perry, K. D. 1961. A comparison of the influence o f Streptococcus lactis and Str. cremoris starters Journal of Dairy Science Vol. 61, No. 9

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on the flavour o f Cheddar cheese. J. Dairy Res. 28:221. Pette, J. W., and H. Lolkema. 1950. Yogurt. lII. Zuurvormingen aromavorming in yoghurt. Netherlands Milk Dairy J. 4:261. Platt, T. B., and E. M. Foster. 1958. Products of glucose metabolism by h o m o f e r m e n t i v e streptococci under anaerobic conditions. J. Bacteriol. 75:453. Rasic, J., and Z. Milanovic. 1966. Influence of Str. diaeetilactis culture on the flavour of yoghurt. 17th Int. Dairy Congr. E/F:637. Reiter, B., and A. Moller-Madsen. 1963. Reviews o f the progress o f dairy science. Section B. Cheese and butter starters. J. Dairy Res. 30:419. Schulz, M. E., and G. Hingst. 1954. Beitrage zue Chemie des Joghurts. I. Acetaldehyd Farbreaktionen zur Joghurt Untersuchung. Milchwissenschaft 9:330. Schulz, M. E., E. Voss, and W. Kley. 1954. Beitrage zue Chemie des Joghurts. II. U n t e r s u c h u n g uber die Anwendbarkeitder Acetaldehyd Farbreaktionen zue Beurteilung yon Joghurt. Milchwissehschaft 9:361. Seitz, E. W., W. E. Sandine, P. R. Elliker, and E. A. Day. 1963. Diacetyl biosynthesis b y Streptococcus diacetilactis. Can. J. Microbiol. 9:431. Seitz, E. W., W. E. Sandine, P. R. Elliker, and E. A. Day. 1963. Distribution of diacetyl reductase a m o n g bacteria. J. Dairy Sci. 46:186. Shahani, K. M., and J. R. Vakil. 1962. Certain e n z y m e s of glycolytic and h e x o s e m o n o p h o s p h a s e s h u n t pathways o f Streptococcus lactis. J. Dairy Sci. 45:655. Speckman, R. A., and E. B. Collins. 1968. Diacetyt biosynthesis in Streptococcus diacetilactis and Leucon ostoc citrovorum. J. Bacteriol. 95 : 174.

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68 Stoyanov, I., and Ya. Yankov. 1966. Metabolic products o f L. bulgaricus and Str. tbermopbilus. lzv. Nauchnoizsled. Inst. Mlechn. Prom., Vidin. 1:99. Dairy Sci. Abstr. 1967, 29:212. Abstr. no. 1502. 69 Sullivan, R. A., and D. G. lnfantino. 1969. Carbohydrate metabolism in Cheddar cheese. I. Nacetalneuraminic acid, 2-deoxy-D-ribose and phosphorylated sugars. J. Dairy Sci. 52:761. 70 Tucker, J. S., and M. E. Morgan. 1967. Decarboxylation o f c~-keto acids by Streptococcus lactis var. maltigenes. Appl. Microbiol. 15:694. 71 Vakil, J. R., and K. M. Shahani. 1969. Carbohydrate metabolism of lactic acid cultures. IIl. Glycolytic e n z y m e s of Streptococcus lactis and their sensitivity to antibiotics. Can. J. Microbiol. 15:753. 72 Vakil, J. R., and K. M. Shahani. 1964. Sensitivity o f certain e n z y m e s of glycolysis and hexose p h o s p h a t e s h u n t pathways of Streptococcus lactis to antibiotics. J. Dairy Sci. 47:675. 73 Vakil, J. R., and K. M. Shahani. 1969. Carbohydrate metabolism of lactic acid cultures. II. Differe n t pathways of lactose metabolism o f Streptococcus laetis and their sensitivity to antibiotics. J. Dairy Sci. 52:162. 74 V e d a m u t h u , E. R., W. E. Sandine, and P. R. Elliker. 1966. Flavor and texture in cheddar cheese. 1I. Carbonyl c o m p o u n d s produced b y m i x e d strain lactic starter cultures. J. Dairy Sci. 49:151. 75 Ward, R. L., and P. A. Srere. 1965. A magnetic resonance s t u d y on the citric lyase of Streptococcus diacetilactis. Biochim. et Biophys. Acta. 99:270. 76 Zuraw, E., and M. E. Morgan. 1952. Acetaldehyde production b y Streptococcus lactis and Streptococcus lactis var. maltigenes. J. Dairy Sci. 35:483.