Distribution of Diacetyl Reductase among Bacteria1

DISTRIBUTION

OF

DIACETYL

REDUCTASE

AMONG

BACTERIA 1

E. W. SEITZ, ~ W. E. SANDINE, P. R. ELLIKER, raced E. A. DAY Departments of Microbiology and Food Science and Technology, Oregon State University Corvallis SUMMARY

This work was undertaken to determine comparative amounts of the enzyme diacetyl reductase in several strains of lactic streptococci and Leuconostoc species. The level of enzyme in cultures of Pseudomonas, Alcaligenes, and coliform bacteria also was examined. Among the lactic streptococci, Streptococcus diacetilactis strains were the most active in reducing diaeetyl; one strain contained 100 units of diacetyl reductase per milligram of enzyme protein. Strains o f Streptococcus lactis~ Streptococcus cremoris~ Leuconostoc citrovorum, and L. dextranicum tested were comparatively inactive in diacetyl reduction; these bacteria contained from zero to eight units of enzyme per milligram of protein. F r o m this, the importance of selecting strains containing little or no diacetyl reductase for use in maximum flavor-producing mixed-strain lactic streptococcus starter cultures was indicated. With the exception of one strain each of Escherichia eoli and Pseudomonas viscosa, the coliform, Pseudomonas and Alcaligenes organisms showed considerable diacetyl reductase activity; Aerobacter aerogenes 8724 contained 345 units per milligram o f enzyme protein. These results emphasized the consequences of contamination of dairy products by diacetyl reductase-containing bacteria in terms of flavor loss. Possible uses of the enzyme for removal of diacetyl from fermented beverages and citrus juices are mentioned.

Diacetyl has long been regarded as one of the outstanding flavor compounds found in such cultured dairy products as Cottage cheese, cultured buttermilk, cultured sour cream, and ripened cream butter. Knowledge of factors contributing to the loss of diacetyl, therefore, becomes important to insure desirable flavor and aroma in these fermented foods. I n a preliminary study (11), cell-free extracts of Streptococcus diacetilactis 18-16 were shown to possess the enzyme diacetyl reductase which catalyzed the irreversible reduction of diacetyl to acetoin. The present investigation was made to determine the amount of this enzyme found among organisms used in the manufacture of cultured dairy products. The level of diacetyl reductase present in psychrophilic and coliform organisms that may contaminate dairy products also was examined. Diacetyl destruction was shown by Elliker (3) to be common to several species of bacReceived for publication October 13, 1962. 1 Technical paper no. 1605, Oregon Agricultural Experiment Station. Supported in part by a grant from the American Dairy Association. ~Present address: Dairy Technology Research Institute. Central Experimental Farm, Ontario, Canada.

teria. Organisms of the Bacillus, Pseudomonas, Escherichia~ Aerobacter~ Proteus, and Alcaligenes genera were active in this regard; only S. lactis revealed little ability to destroy diacetyl. Galesloot and Hassing (4) reported that Leuconostoe (Betacoccus cremoris) was capable of reducing diacetyl and acetoin to 2,3butanediol, whereas S. diacetilactis lacked this ability. The disappearance of diacetyl has, in some instances, been correlated with the appearance of nonaromatic compounds. P a r k e r and E1tiker (8) showed that Pseudomonas fragi and P. viscosa converted diacetyl to acetoin and possibly 2,3-butanediol, resulting in aroma loss in Cottage cheese. Green et al. (5) reported that pigeon-breast muscle contained a highly specific diacetyl mutase which converted two molecules of diacetyl to two molecules of acetate and one of acetoin. Aubert and Millet (1) obtained a purified extract from Neisseria winogradsky which attacked diacetyl but not acetoin. This enzyme also converted two molecules of diacetyl to two of acetate and one of aeetoin. Streeker and t I a r a r y (12) reported the presence of diacetyl reductase in S. aureus; the enzyme required reduced diphosphopyridine nucleotide ( D P N H ) for activity in cata186

DISTRIBUTION

OF DI•CETYL

lyzing the irreversible reduction of diacetyl to acetoin.

REDUCTASE

]87

4.8

EXPERIMENTAL PROCFA)URES

All the organisms used in this work were available from the stock culture collection in the Department of Microbiology, Oregon State University. The cultures were maintained by weekly transfer in sterile nonfat milk as well as a broth designed to support the growth of fastidious Leuconostoc species. The broth made with distilled water contained 1.0% yeast extract, 1.0% tryptone, 0.2% Whey-lae,~ 0.15% sodium aerate, 0.5% sodium citrate, 1.0% glucose, 0.07% ascorbic acid, 3.3% tap water, 0.2% sodium chloride, 0.2% monobasic potassium phosphate, and 0.2% anhydrous magnesium sulfate. The organisms were incubated for 18 h r at the optimum growth temperature for each species used. Diacetyl determinations on milk cultures of S. diacetilactis were made as described previously (9). Cell-free extracts were prepared as follows: Freshly washed cells from one liter of broth medium were suspended in 40 ml of 0.1 ~ potassium phosphate buffer, p i t 7.2, and disrupted in a Raytheon 10KC sonic oscillator for 15 rain. Cell debris was removed by eentrifugation at 15,000 r p m for 25 rain. The supernatant obtained was dialyzed for 24 hr against two changes (2,000 ml) of 0.1 ~ potassium phosphate buffer at p H 7.0. The resulting cell-free extracts were stored frozen at --20 C until used. Protein determinations were made by the method of Lowry et al. (6). Diacetyl reductase activity was measured with a Beckman Model DU spectrophotemeter by following the decrease in absorbancy at 340 m~ caused by the oxidation of D P N H . Conditions for the assay are indicated in the Results section. RESULTS

Results on the synthesis and degradation of diacetyl at 30 C by S. diacetilactis 18-16 in nonfat milk supplemented with 2.0% citrate are shown in Figure 1. Rapid destruction of diacetyl occurred after 24 hr. I n unsupplemented nonfat milk found to contain 0.2% citrate, diacetyt destruction was a p p a r e n t after about 12 hr of incubation. The cause for this dramatic loss of accumulated diacetyl in milk cultures led to a search for diacetyl reduetase in S. diacetilactis; spectrophotometrie studies 8 Deionized, spray-dried whey, Consolidated Dairy Products, 635 Elliott Avenue, Seattle 99, Washington.

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Fie. 1. Diacetyl produced in nonfat milk at 21 C. by S. diacetilavtis 18-16. using cell-free extracts showed that diacetyl was enzymatically reduced, presumably to AMC. The reaction was not reversible and D P N H was required as the coenzyme. Divalent metal activators were not required by diacetyl reductase; E D T A and dialysis did not inactivate the enzyme. Figure 2 shows typical spectrophotometric data obtained in testing cell-free extracts for diacetyl reductase. I

1

I

oE.500

I

PNH

rO i

>--.375

I'-03 Z

c~.250

~--ENZYME +LDPNH +

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4 6 MINUTES

I

8

FIG. 2. Oxidation of DPNIt by diacetyl reductase in crude enzyme preparation from S. d/aeetilactis 18-16. The complete system contained 10 ~moles of diacetyl, 2.0 mg of DPNH, 2.0 nag of enzyme protein, and sufficient 0.1 ~ potassium phosphate at pH 7.2 to provide a volume of 3.0 ml.

188

E. w . SEITZ, W. E. SANDINE, P. R. E L L I K E R , AND E. A. DAY

The amount of this enzyme found in S. diacetilactis~ S. lactis~ and S. cremoris is shown in Table 1. I t may be seen that the specific

by Leuconostoc amy be rapidly destroyed, especially since Figure I suggests diacety] reduetase may be active outside the cell.

TABLE 1 Diacetyl reduetase found in lactic streptococci, Le~tconostoc, psyehrophilc, and coliform bacteria Specific activity ~

Organism S. diacetilaetis 18-16 26-2 DRC-1 DRC-2 DRC-3 gM-1 S. laetis E 27 C2 CI0 7962 11454 S. cremoris 1 W C3 C13 E8

Organism

Specific ~etivity ~

L. citrovor~m Lcl~ 6 LcF8 0 91404 6 8081 0 82 6 L. dextranic~lm LdF 8 4 688 7 0 83,58 0 8 62-9 7 2 2-5 0 0 8359 6 4 Aerobacter aerogenes 8724 345 Escherichia eoli OSU 5 8 Pseudomonas putrefac@ns OSU 64 7 Alcaligenes metalcaligenes OSU 55 5 Pseudomonas fragi OSU 44 3 Pse~tdomonas fluoreseens OSU 19 0 Pseudomonas viscosa OSU 3 Expressed as units per milligram of enzyme protein, where one unit was defined as the amount of enzyme necessary to cause a 0.0'01 change in absorbancy at 340 m~ during the second 30-see time interval after beginning the reaction by adding enzyme.

100 3 16 8 20 20

activity varied from three to 100 for S. diacetiZactis and zero to eight for both S. lactis and S. cremeris. The levels found in Leuconostoc (Table 1) were uniformly low, varying from zero to eight units per milligram enzyme protein. Examination of psyehrophiles and coliform organisms for diacetyl reductase provided the findings also recorded in Table 1. Among the psychrophiles, only P: viscosa wss relatively inactive; A . aerogen~s was about 70 times as active as E. coll. DISCUSSION

The exclusive use of S. diacetilactis as aroma bacteria in mixed-strain starter cultures has recently been criticized by Badings and Galesloot (2); such cultures were high in acetaldehyde and consequently imparted a yogurt-like flavor to %rmented products. F r o m Table 1, still another reason may be seen for exercise of caution when compounding starters containing only S. diacetilactis as aroma bacteria; some strains of this orgomism, such as 18-16, may produce sufficient diacetyl reduetase to prevent the accumulation of diacetyl. Even in mixed-strain cultures containing both Leuconostoc and S. diacetilactis, diacetyl produced

The ability of S. diacetilactis to reduce diacetyl as found in the present work appears contrary to the report of Galesloot and Hassing (4). However, these latter authors measured the disappearance of diacetyl plus acetoin as an index of diacetyl reduction. Also, it is now known that N. diacetilactis possesses a reversible 2,3-butanediol dehydrogcuase (11), so that some aeetoin would always be present in cultures of this organism. Selection of strains low in diaeetyl reductase for designing" mixed-strain starter cultures may have some merit. Strain 26-2, Yor example, has been used in the manufacture of superior-flavored ripened cream butter (10). The success of the recently patented aroma process of Lundstedt (7), using S. diacetilactis 26-2, i s perhaps partly accounted for by the low level of diaeetyl reductase which this strain possesses. ]~Yxtracts of S. diacetilactis possessing 100 units of diacetyl rednctase per milligram of enzyme protein have been found capable of completely reducing at least 9.0 p p m of diacetyl within 10 min (11). Since the level of diacetyl in cultured dairy products seldom exceeds this amount, even the presence of six to eight units of enzyme, which some S. lactis,

DISTRIBUTION OF DIACETYL I~EDUCTASE S. cremoris, and Leuconostoc possess (Table 1), may be significant. However, since optim u m t e m p e r a t u r e and p H conditions f o r diacetyl reduetase are as yet undetermined, such reasoning is only speculative. Nevertheless, because this study revealed that all lactic streptococci and Leuco~ostoc exhibit strains that show little or no evidence of diacetyl reductase, use of such strains in mixed-strain starter cultures would seem warranted. Results in Table 1 re-emphasize the hazards of product contamination by coliform and psychrophilic organisms. E v e n slight contamination by an organism as high in diacetyl reductase as A . ae~ogenes would undoubtedly yield a final p r o d u c t lacking flavor and aroma. The irreversible nature of diacetyl reductase seems unfortunate, since this would even prevent equilibrium stabilization of low amounts of diacetyl. However, since the enzyme is irreversible, it m a y be used to advantage in removal of diaeetyl f r o m fermented alcoholic beverages, such as beer, and also f r o m citrus juices, or, as will be reported in a f u t u r e paper, in the measurement of diacetyl. I t m a y be presumptive to assume that all diacetyl which is enzymatically destroyed in starter cultures is due to diaeetyl reductase. Diacetyl mutase may also be involved. REFERENCES ( ] ) AUtCEI~T, J. P., AND MILLET, ,]-. Degradation of Diacetyl by a Bacterial Enzymatic Extract. Academie des Sciences, Paris Comptes R.endus, 236: ]512. 1953. (2) BADINGS, H. T., AND GALE.sLeeT, TH. E. Studies on the Flavor of Different Types of Butter Starters with Reference to the " Y o g h u r t F l a v o r " in Butter. 16th In-

189

tern. Dairy Congr., B ( I I I ) : 1 9 9 . ]962. (3) ELLIKEJ¢, P. R. Effect of Various Bacteria on Diaeetyl Content and Flavor of Butter. J. Dairy Sci., 28: 93. 194.5. (4) GALESLOOT,TH. E., AND HASSING, ~. Some Differences in Behavior Betweel~ Starters Containing as Aroma Bacterium Either Streptococcus diacetilaetis or Betacocc~s cremoris. Netherlands Milk Dairy J., 15." 225. 1961. (5) GRF~I~L% r, D. E., STUI~[PI~, P. g., AN]) ZARUNDNAYA, K. J. Diacetyl Mutase. J. Biol. Chem., 167: 811. 194.5. (6) LowRY, O. H., ROS]~BROUG~t, N. J., 1OARS~AND, A. L., ANn RANDALL, R. J. Protein Measurement with the Fotin Phenol Reagent. J. Biol. Chem., 193: 265. 195.1. (7) LUNDS~Z])T, E]UK. Aroma Process for Dairy Products and the Resulting Products. U. S. Pat. No. 3048490. August 7, ]962. (8) PAKK~, R. B., AR~) ELLIKEK, P. R. Effect of Spoilage Bacteria on Biacetyl Content and Flavor of Cottage Cheese. J. Dairy Sci., 36: 843. 1953. (9) SANDIN]~, W. E., ELLIKFA~, P. R., A N n HAYS, HELE~IV. Bacteriophagc-lysis of Streptococcue diacetilactis an(! Its Effect on Biacetyl Production in Mixed Strain Starter Cultures. J. Dairy Sci., ~e3: 755. 1960. (10) S~I~z, E. W., SANmNE, W. E., DAY, E. A., AN]) ELLIKEt¢, P. R. Studies on Factors Affecting Diacetyl Production by Streptococcus diacetilaeti~'. J. Dairy Sci., 44: 1159. ]961. (11) SEI~Z, E. W., S±N])INn, W. E., E~IKE~, P. R , AND DAY, E. A. Enzymatic Destruction of Diacetyl by Bacteria. Bacteriol. Prec., p. 23. 1962. (12) STRE'CKER,H. J., A~TDHARA]~Y, I. :Bacterial Butyiene Glycol Dehydrogenase and Diacetyl Reductase. J. :Biol. Chem., 211: 263. 1954.