JOURNAL OF BACTERIOLOGY, Jan. 1968, p. 174-180Copyright © 1968 American Society for Microbiology

Diacetyl Biosynthesis in Streptococcus diacetilactisand Leuconostoc citrovorum

R. A. SPECKMAN A.ND E. B. COLLINSDepartment ofFood Science and Technology, University of California, Davis, California 95616

Received for publication 30 August 1967

Pyruvate was shown to be the precursor of diacetyl and acetoin in Streptococcusdiacetilactis, but dialyzed cell-free extracts of S. diacetilactis and Leuconostoccitrovorum that had been treated with anion-exchange resin to remove coenzyme A(CoA) formed only acetoin from pyruvate in the presence of thiamine pyrophos-phate (TPP) and Mg++ or Mn++ ions. The ability to produce diacetyl was restoredby the addition of acetyl-CoA. Acetyl-phosphate did not replace the acetyl-CoA.Neither diacetyl nor acetoin was formed when the otherwise complete reaction sys-tem was modified by using boiled extract or by omitting the extract, pyruvate, TPP,or the metal ions. Free acetaldehyde was not involved in the biosynthesis of diacetylor acetoin from pyruvate, dialyzed cell-free extracts of the bacteria produced onlyacetoin (besides C02) from a-acetolactate, and acetoin was not involved in the bio-synthesis of diacetyl. Only one of the optical isomers present in racemic a-acetolac-tate was attacked by the extracts, and there was no appreciable spontaneous de-carboxylation of the a-acetolactate at the pH (4.5) used in experiments.

The mechanism by which citrate-fermentingbacteria form diacetyl has not been established,although this volatile compound has been recog-nized since 1929 as an important food-flavor com-ponent (19). Historically, it has been assumed thatacetoin (3-hydroxy-2-butanone) is the immediateprecursor of diacetyl (2,3-butanedione). Sebekand Randles (22) reported that Pseudomonasfluorescens formed diacetyl when 2,3-butanediolwas the only source of carbon, and they supporteda pathway involving successive oxidations from2, 3-butanediol to acetoin to diacetyl. This oxida-tion of acetoin to diacetyl was considered appro-priate for cultured dairy products as recently as1966 (12), though Michaelian and Hammer (17),Cox (1), and Pette (20) had found lactic culturesunable to convert acetoin to diacetyl under avariety of experimental conditions, and Streckerand Harary (26) had found Aerobacter aerogenesand Staphylococcus aureus unable to form diacetylfrom acetoin.The biosynthesis of acetoin in bacteria has been

shown by Juni (8) to proceed by enzymatic con-densation of two molecules of pyruvate to yieldone of CO2 and one of a-acetolactate. The reac-tion requires thiamine pyrophosphate (TPP) andmanganous or magnesium ions. In a subsequentreaction, the a-acetolactate is decarboxylated togive acetoin and CO2. This a-acetolactate is op-tically active, which distinguishes it from the

racemic a-acetolactate that is formed by pyruvateoxidase preparations (10).De Ley (13) reported that some species of

Acetobacter can form acetoin from pyruvate andfree acetaldehyde in reactions that are analogousto the mechanism that occurs in yeasts and mam-malian tissues, but studies with other bacteriaindicate that free acetaldehyde is not involved intheir synthesis of acetoin (9, 11, 14).Some bacteria can form acetoin by reducing

diacetyl. This conversion of diacetyl to acetoin bydiacetyl reductase has been reported to be bothreversible (22) and irreversible (1, 17, 23, 27).

After a-acetolactate was established as anintermediate in the formation of acetoin (8), therewere conflicting reports as to the possibility thatdiacetyl can be formed in addition to acetoin inthe decarboxylation of a-acetolactate. Dolin andGunsalus (3) found that Streptococcusfaecalis didnot yield diacetyl, acetaldehyde, or lactate froma-acetolactate, but de Man (16) reported thatferric, cupric, or aluminum ions catalyze a non-enzymatic liberation of diacetyl from ca-acetolac-tate. Mizuno and Jezeski (18) and Seitz et al.(E. W. Seitz, Ph.D. Thesis, Oregon State Univ.,Corvallis, 1962; and 23) have reported that cell-free extracts of starter bacteria can decarboxylatea-acetolactate.

This study was undertaken to evaluate themechanisms indicated in the literature and to de-

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termine their relationships to diacetyl biosynthesisin Streptococcus diacetilactis and Leuconostoccitrovorum. Until recently, research on the bio-synthesis of diacetyl has been hampered by theuse of complex test systems and methods inade-quate for quantitative separation and determina-tion of the microgram quantities of diacetyl andacetoin found in cell-free test systems. This prob-lem is resolved by the column salting-out chro-matographic method we developed (25) for sepa-rating diacetyl, acetoin, and 2,3-butylene glycol.The present study has disclosed a new mecha-

nism that accounts for the biosynthesis of di-acetyl. A possible role of free acetaldehyde in thesynthesis of diacetyl and acetoin by S. diacetilactiswas eliminated. The results show the two mecha-nisms by which these bacteria form acetoin, andreveal that neither a-acetolactate nor acetoin is aprecursor of diacetyl in these bacteria.

MATERIALS AND METHODS

S. diacetilactis 18-16 and L. citrovorum CAF,were propagated routinely in sterile litmus milk.Cell-free extracts were prepared as follows. Cells ofS. diacetilactis were grown for 16 hr (L. citrovorum,36 hr) at 22 C, without shaking, in citrate broth atpH 6.6 (5), harvested by centrifugation, washed twicewith cold 1 N tris(hydroxymethyl) aminomethane-cysteine (Tris-cysteine) buffer atpH 7.3 (0.001 M withrespect to L-cysteine), and resuspended in the samebuffer at a cell density of ca. 50 mg (dry weight) perml. Preparative operations with the cell-free extractswere performed in a refrigerated room maintained at4 C except where indicated otherwise. Cells weredisrupted by ballistic disintegration with a Mickletissue disintegrator operated at maximal amplitudefor 45 min at 0 C. After centrifugation for 30 min at2 C in a refrigerated centrifuge at 15,000 X g, thecell-free supernatant fluid was dialyzed for 24 hragainst 100 volumes of 1 N Tris-cysteine buffer, atpH 7.3. For certain indicated experiments, coenzymeA was removed from dialyzed extracts by treatingthem with an anion-exchange resin (acid-washedDowex 1-X2). One volume of the extract was stirredslowly for 5 min with one-quarter volume of the resin.The pH was checked periodically and maintainedbetween 7 and 7.3 by dropwise addition of 1 N Tris-cysteine buffer. The resin was sedimented by centri-fugation for 3 min at 5,000 X g, and the supernatantfluid was removed with a Pasteur pipette. One jAmoleof ethylenediaminetetraacetate (EDTA) per mg ofextract protein was added to the supernatant fluid toremove metal ions.

Protein was determined by the biuret method (4)or the method of Lowry et al. (15). Acetoin and diac-etyl were separated by the method of Speckman andCollins (25) and determined quantitatively by theWesterfeld method (28).

Sodium pyruvate, thiamine pyrophosphate (TPP),oxidized and reduced nicotinamide adenine dinucleo-tide (NAD, NADH2), flavin adenine dinucleotide(FAD), flavin mononucleotide (FMN), reduced

nicotinamide adenine dinucleotide phosphate(NADPH2), acetyl-phosphate, acetyl-coenzyme A(acetyl-CoA), and sodium pyruvate-3-14C (specificactivity, 4.6 X 104 counts per min per ,umole) wereobtained from Calbiochem (Los Angeles, Calif.).Acetyl-CoA-1-14C (specific activity, 25 mc/mmole)was obtained from New England Nuclear Corp.(Boston, Mass.). Isotopically labeled acetoin (spe-cific activity, 5.9 X 104 counts per min per ;&mole)was prepared by the action of cell-free extracts of S.diacetilactis 18-16 on sodium pyruvate-3-14C. Thelabeled acetoin was recovered and purified by salting-out chromatography. Diacetyl, acetaldehyde, andacetoin were obtained from Eastman Organic Chem-icals Co. Acetoin, obtained as the crystalline dimer,was washed with ether until free from diacetyl asshown by both column and gas chromatography.Esterified a-acetolactate was obtained from K & KLaboratories (Jamaica, N.Y.). An infrared spectrumdetermined for the compound received from K & K(Fig. 1) matched the published spectrum for the ace-toxy ethyl ester of ca-acetolactate (21). a-Acetolactatewas prepared from the ester by saponifying with twoequivalents of cold 0.025 N NaOH added slowly tothe system held in a water bath at 5 C and continu-ously purged with a stream of nitrogen.

All spectrophotometric measurements were madewith a Beckman spectrophotometer, model DB. AKlett-Summerson colorimeter, model 800-3 withfilter no. 54, was used for Westerfeld analyses. ThepH was measured with a Radiometer pH meter,model 22 (Copenhagen, Denmark). Effluent wascollected and distributed during chromatographicseparations with a Radi-Rak fraction collector(Stockholm, Sweden). Radioactivity was determinedin a Packard Tri-Carb liquid scintillation spectrome-ter with 0.3% 2,5-diphenyloxazole (PPO) and 0.01%1,4-bis-2-(5 phenyl-oxazolyl)-benzene (POPOP) in atotal volume of 20 ml of toluene.

RESULTSConversion of pyruvate to acetoin and diacetyl.

A quantity of cell-free extract of S. diacetilactiswas held in a water bath at 25 C with sodiumpyruvate-3-_4C and cofactors as indicated inFig. 2. After 30 min, a sample of the reactionmixture was removed and the acetoin and diacetyl

100E

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WAVENUMBER (cffr)

FIG. 1. Thin-film infrared absorption spectrum ofcommercial esterified a-acetolactate.

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400

0'300

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ACE TOIN* 1

.10\;i sm

2 3 4 5 6 7 8 9 10I1 12 13 1415156FRACTION NUMBER

FiG. 2. Conversion ofpyruvate to acetoin and diacetylby a cell-free extract of Streptococcus diacetilactis.The reaction mixture contained: Na-pyruvate-3-14C,100 ,umoles (specific activity 4.6 X 10 counts permin per pmole); TPP, 0.02 ,umole; MgSO4, 4.5 .moles;extract protein, 3 mg; 0.1 m phosphate buffer, pH 4.5;total volume, 3.0 ml; temperature, 25 C.

were separated, determined quantitatively, andassayed for radioactivity. Each was radioactive,indicating that pyruvate had served as its pre-cursor. Specific activity of the acetoin was 5.9 X104 counts per min per ,umole; specific activity ofthe diacetyl was 6.2 X 104 counts per min per,umole.

Free acetaldehyde not involved in the formationof acetoin or diacetyl. A dialyzed cell-free extractof S. diacetilactis 18-16 was used to determinewhether this organism can use free acetaldehydein the biosynthesis of acetoin or diacetyl. Reactionmixtures containing 3 mg of extract protein,100 ,umoles of pyruvate, 0.025 ,umole of TPP,and 4.5 ,umoles of Mg- (with and without100 ,Amoles of acetaldehyde) in 0.1 M KH2PO4buffer, pH 4.5, were held 2 hr in a water bath at25 C. Determination of the reaction productsrevealed that 42.1 ,umoles of acetoin and 4.8,umoles of diacetyl were formed in the presence ofacetaldehyde, and that 41.7 ,umoles of acetoin and5.9 ,umoles of diacetyl were formed when noacetaldehyde was added.

Additional evidence that free acetaldehyde isnot involved in the formation of acetoin or di-acetyl from pyruvate was obtained in the aboveexperiment by adding 500 ,umoles of Na2SO3 to areaction mixture (without added acetaldehyde) tobind any acetaldehyde formed from the pyruvate.The cell-free extract produced 41.5 inmoles ofacetoin and 5.6 ,umoles of diacetyl in the presenceof the Na2SO3, though it is known that excessNa2SO3, by binding free acetaldehyde, completelyinhibits the formation of acetoin in yeasts (27).We confirmed the reported influences of acet-

aldehyde and Na2SO3 on acetoin formation inyeasts by using a cell-free extract of Saccharo-myces cerevisiae. Reaction mixtures contained3 mg of extract protein, 25 ,umoles of pyruvate,0.025 Amole of TPP, and 4.5 ,umoles of Mg++,with and without 125 Amoles of Na2SO3 and withand without 25 ,umoles of acetaldehyde. Reactionmixtures, in 0.1 M KH2PO4 at pH 4.5, were held2 hr in a water bath at 25 C. The extract produced180 jig of acetoin without Na2SO3 and only 9 ,ugwith Na2SO3 added. The addition of acetalde-hyde stimulated the production of acetoin; withacetaldehyde added, there was 227 Mug of acetoin.

Acetoin from decarboxylation of a-acetolactate.Dialyzed cell-free extracts of S. diacetilactis andL. citrovorum were held in a water bath at 25 Cwith a-acetolactate as indicated in Fig. 3, sampleswere removed, and the products were separatedand determined quantitatively. The pH for theexperiments was 4.5 because these bacteria nor-

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FIG. 3. Decarboxylation of a-acetolactate by dia-lyzed cell-free extracts of Streptococcus diacetilactisand Leuconostoc citrovorum. Reaction mixtures con-tained: a-acetolactate, 10 ,umoles; MgSO4, 4.5 ,moles;extract protein, 5 mg; 0.1 M phosphate buffer, pH 4.5;total volume, 3 ml. The reaction time was 15 min at25 C. Acetoin and diacetyl in the reaction mixtureswere separated by salting-out chromatography andwere determined quantitatively. STD is a standardelution graph ofa mixture of acetoin and diacetyl (100,ug of each). EXP is an elution graph of the experimen-tal reaction mixture.

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DIACETYL BIOSYNTHESIS

mally produce much larger amounts of diacetylat low pH.

Decarboxylation of a-acetolactate by the ex-tracts resulted in the formation of acetoin, sub-stantiating the finding of Juni (8) that a-acetolac-tate is an important precursor of acetoin in bac-teria. Although we found a-acetolactate to de-carboxylate spontaneously under prolongedacidic conditions (about 5% per hr at 25 C),results showed no significant nonenzymatic de-carboxylation in our experiments, in which thereaction times were usually 15 min and not longerthan 30 min. From 10 Amoles of a-acetolactate,only 4.8 ,moles of acetoin was formed. Additionof 3 N H2SO4 resulted in decarboxylation of theremaining a-acetolactate, indicating that theenzyme can attack only one of the optical isomerspresent in racemic a-acetolactate.Only acetoin was formed by S. diacetilactis or

L. citrovorum in the enzymatic decarboxylation ofa-acetolactate. There was no detectable diacetyl.This finding of acetoin as the only end product(other than C02) was not altered by adding thefollowing cofactors to the test system given inFig. 3: FAD, NAD, FAD plus NAD, NADPH2,FMN, or FMN plus NAD. The substitution offerric ions for the magnesium ions in equimolaramounts did not change the products of a-aceto-lactate decarboxylation.

Acetoin not involved in theformation of diacetyl.Dialyzed cell-free extracts of S. diacetilactis andL. citrovorum were held in a water bath at 25 Cwith diacetyl and cofactors as indicated in Fig. 4,samples of the reaction mixtures were removed,and the products were separated and determinedquantitatively. The conversion of diacetyl toacetoin was enzymatic and dependent on thepresence of NADH2. Results for each of theorganisms showed that acetoin is the product ofthis diacetyl reductase reaction and that diacetylis in fact reduced. The results for S. diacetilactisare in Fig. 4. It was important to show that di-acetyl is reduced, because we have found bothS. diacetilactis and L. citrovorum to have NADH2oxidase activity similar to that reported by Dolin(2) for S. faecalis. This NADH2 oxidase activitywas found to interfere with spectrophotometricanalysis of NADH2 oxidation as a measure ofdiacetyl reductase activity. Its presence accountsfor the fact that only about 85% of the diacetylwas converted to acetoin (Fig. 4).

Results indicated that the reaction catalyzed bydiacetyl reductase is not reversible and thatacetoin is not involved in the biosynthesis of di-acetyl. No diacetyl was formed when acetoin wassubstituted for diacetyl in the test system de-scribed for Fig. 4, with or without the substitu-tion of NAD for NADH2. Results were the same

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FiG. 4. Conversion of diacetyl to acetoin by the dia-cetyl reductase of Streptococcus diacetilactis. Com-plete reaction mixture contained: diacetyl, I umole;NADH2, 1 ,umole; 0.05 mt phosphate buffer, pH 6.2;extract protein, 3 mg; total volume, 3 ml; temperature,25 C. (A) Standardelution graph ofa mixture ofacetoinanddiacetyl (100 ugof each). (B toF) Elution graphs ofthe complete reaction mixture and of the completemixture modified as indicated on the graphs. Reactiontimes (in minutes) are indicated in parentheses.

for both organisms over the pH range of 4.5 to8.1.

Additional evidence was obtained for S. di-acetalactis in two different experiments involvingradioactive compounds. The test system for oneexperiment contained 0.12 ,umole of "4C-labeledacetoin, 100 ,moles of unlabeled sodium pyru-vate, 0.025 ,mole of TPP, 4.5 ,umoles of Mg++ions, 10 ,umoles of NAD, and 3 mg of extractprotein, all in 0.1 M KH2PO4 buffer atpH 4.5. Themixture was held for 30 min at 25 C, and a samplewas removed for separation of acetoin and di-acetyl and examination of the diacetyl for radio-activity. The resulting diacetyl was not radio-active, although it had been formed in the pres-ence of radioactive acetoin.

In a different experiment, 50 ;umoles of un-labeled acetoin was added to a mixture of sodiumpyruvate-3-'4C, extract, and cofactors (similar tothose indicated in Fig. 2) to determine whether ornot the unlabeled acetoin would result in dilutionof the radioactivity of the diacetyl formed fromthe pyruvate. It did not. The specific activity ofthe diacetyl formed in the presence of the addedunlabeled acetoin was 7.4 X 104 counts per minper ,umole compared to 6.2 X 104 counts per minper inmole for diacetyl that was formed in theabsence of added acetoin.Mechanism of diacetyl biosynthesis. The organ-

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SPECKMAN AND COLLINS

isms obviously formed diacetyl by a mechanismdifferent from that used in the formation ofacetoin. Consideration was given to mechanismsinvolving TPP in a-keto acid decarboxylationsand to the apparent mechanism by which acetoinis formed via a-acetolactate. It seemed that con-densation of the acetaldehyde-TPP complex witha compound having an acetyl-ester linkage mightallow the necessary migration of electrons toyield diacetyl rather than acetoin. Consequently,we decided to determine whether acetyl-CoA,with its thiol-ester linkage, is involved in the bio-synthesis of diacetyl.Coenzyme A was removed from dialyzed cell-

free extracts of S. diacetilactis and L. citrovorumwith anion-exchange resin (acid-washed Dowex1-X2). In the presence ofTPP and Mg+ or Mnlions, the resin-treated extracts formed onlyacetoin from pyruvatey-not diacetyl and acetoin,as did untreated extracts-and the ability to formacetoin was retained only if the pH during theresin treatment was maintained between 7.0 and7.3. Subsequently, it was found possible to restorethe diacetyl-producing property of resin-treatedextracts by adding acetyl-CoA to the reactionmixtures (Fig. 5). Acetyl-phosphate did not re-place the acetyl-CoA. Neither diacetyl nor acetoinwas formed when the otherwise complete reaction

S. diocetilactis250f

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FIG. 5. Influence of acetyl-CoA on diacetyl biosyn-thesis by Dowex-l-X2-treated cell-free extracts ofStreptococcus diacetilactis and Leuconostoc citro-vorum. Reaction mixtures contained: Na-pyruvate, 25,umoles; TPP, 0.02 ,umole; MgSO4, 4.5 ,umoles; resin-treated extract protein, 5 mg; 0.05 M phosphate buffer,pH 5.3; total volume, 3.0 ml; temperature, 25 C; withor without acetyl-CoA, 10 Amoles.

system was modified by using boiled extract or byomitting the extract, pyruvate, TPP, or the metalions.The role of acetyl-CoA in diacetyl biosynthesis

was confirmed for S. diacetilactis in an experi-ment in which 1 ,uc of acetyl-CoA-1-_4C was addedto a test system containing 100 ,umoles of un-labeled sodium pyruvate, 0.025 ,umole of TPP,4.5 j,moles of Mg+ ions, and 3 mg of extractprotein, in 0.1 M KH2PO4 buffer at pH 4.5. Themixture was held for 30 min at 25 C, and a sam-ple was removed for separation of acetoin and di-acetyl and examination of each for radioactivity.The resulting diacetyl was found to be highlylabeled, with a specific activity of 4.77 X 105counts per min per ,umole. The acetoin, however,was not radioactive, showing that its biosynthesishad not involved acetyl-CoA.

DISCUSSIONS. diacetilactis and L. citrovorum, the impor-

tant diacetyl-producing bacteria used in makingcultured products, were found unable to producediacetyl from a-acetolactate, acetoin, or acetalde-hyde. Apparently their only mechanism for pro-ducing diacetyl is the mechanism newly indicatedby the results reported here, wherein an acetalde-hyde-TPP complex formed from pyruvate at-tacks the carbonyl carbon of acetyl-CoA. By thisproposed mechanism (Fig. 6), the thiol-esterlinkage provided by the acetyl-CoA moietyallows a migration of electrons to yield diacetyl asthe product of rearrangement. Whether othermicroorganisms use this mechanism, and its rela-tions to other biochemical processes, are beinginvestigated.These bacteria have two mechanisms for pro-

ducing acetoin, each involving the intermediateformation of an acetaldehyde-TPP complex frompyruvate. One mechanism is by decarboxylationof a-acetolactate following its formation by con-densation of the complex with another moleculeof pyruvate. This mechanism, first indicated byJuni (8) to be important in bacteria, is substanti-ated by the results reported here. Rapid conver-sion of only about 50% of commercial a-aceto-lactate to acetoin by cell-free extracts of S. di-acetilactis and L. citrovorum indicates that thisenzymatic conversion involves only one of theoptically active forms, since the commerciala-acetolactate employed as substrate was racemic.This result distinguishes the acetoin formed bythis enzyme from that produced by pyruvic oxi-dase preparations, which yielded racemic acetoin(10).The second mechanism used by these bacteria

for producing acetoin is by reducing part or all ofthe diacetyl that is formed from condensation of

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DIACETYL BIOSYNTHESIS

-N/ -N|

CH C-CO2 CH C-CO-H

-N / _ -N/=NS \S

CH C-OH CH C-OH

CH43 SCoA +CQ2

-N -N+N

s

N

CH3C-OH CH CCH, SCoA CH3C=O

+ CoASH

00_N + CH C-CCH3

FIG. 6. Proposed mechanism for diacetyl biosyn-thesis by Streptococcus diacetilactis and Leuconostoccitrovorum.

the acetaldehyde-TPP complex with acetyl-CoA.Harvey and Collins (6, 7) pointed out that theproduction of acetoin in S. diacetilactis serves asa detoxification mechanism for removing intra-cellular pyruvate not required for the synthesis ofcell material. Successive reductions of diacetyl toacetoin to 2, 3-butylene glycol also appear usefulto the bacterial cell. They provide a path for re-generating the cell's NAD supply with the simul-taneous formation of only neutral molecules. Incontrast, NAD regeneration by glycolysis istightly coupled to the formation of toxic lacticacid.

In addition to providing evidence for a newmechanism for the biosynthesis of diacetyl, thepresent results have an important bearing on pre-vious interpretations of results. Failure of thedecarboxylation of a-acetolactate to yield detect-able diacetyl, failure of acetoin to be involved inthe biosynthesis of diacetyl, and the considerablevariations that have been observed among bac-terial strains and species in ability to reducediacetyl (24) leave little reason to suppose thatthere is correlation between the acetoin and di-acetyl contents of lactic cultures. These findingsenhance the importance of using methods thatseparate or distinguish between diacetyl and ace-toin, and place in question the results of a large

number of published papers in which the sum ofacetoin and diacetyl has been considered to indi-cate the production of diacetyl by bacteria.

ACKNOWLEDGMENTThis investigation was supported by Public Health

Service research grant Ul-00101 from the NationalCenter for Urban and Industrial Health.

LITERATURE CITED1. Cox, G. A. 1945. The effect of acidity on the

production of diacetyl by betacocci in milk.J. Dairy Res. 14:28-35.

2. DOLIN, M. 1955. The DPNH-oxidizing enzymesof Streptococcus faecalis. II. Arch. Biochem.Biophys. 55:415-435.

3. DOLIN, M. I., AND I. C. GUNSALUS. 1951. Pyruvicacid metabolism. II. An acetoin-forming en-zyme system in Streptococcus faecalis. J.Bacteriol. 62:199-214.

4. GORNALL, A. G., C. J. BARDSWILL, AND M. M.DAVIS. 1949. Determination of serum proteinsby means of the biuret reaction. J. Biol. Chem.177:751-766.

5. HARVEY, R. J., AND E. B. COLLINS. 1961. Role ofcitritase in acetoin formation by Streptococcusdiacetilactis and Leuconostoc citrovorum. J.Bacteriol. 82:954-959.

6. HARVEY, R. J., AND E. B. COLLINS. 1962. Factorsresponsible for the relationship between pHand acetoin formation by Streptococcus diace-tilactis. Intern. Congr. Microbiol., 8th, Mon-treal, p. 46.

7. HARVEY, R. J., AND E. B. COLLINS. 1963. Roles ofcitrate and acetoin in the metabolism of Strep-tococcus diacetilactis. J. Bacteriol. 86:1301-1307.

8. JUNI, E. 1952. Mechanisms of formation of acet-oin by bacteria. J. Biol. Chem. 195:715-726.

9. JUNI, E. 1952. Mechanisms of the formation ofacetoin by yeast and mammalian tissue. J.Biol. Chem. 195:727-734.

10. JUM, E., AND G. A. HEYM. 1956. Acyloin con-densation reactions of pyruvic oxidase. J.Biol. Chem. 218:365-378.

11. KEENAN, T. W., R. C. LINDSAY, M. E. MORGAN,AND E. A. DAY. 1966. Acetaldehyde productionby single-strain lactic streptococci. J. DairySci. 49:10-14.

12. KoSncoWSKl, F. 1966. Cheese and fermentedmilk foods. Edwards Bros., Ann Arbor, Mich.

13. DE LEY, J. 1959. On the formation of acetoin byAcetobacter. J. Gen. Microbiol. 21:352-365.

14. LINDSAY, R. C., E. A. DAY, AND W. E. SANDINE.1965. Green flavor defect in lactic starter cul-tures. J. Dairy Sci. 48:863-869.

15. LOWRY, 0. H., N. J. ROSEBROUGH, A. L. FARR,AND R. J. RANDALL. 1951. Protein measure-ment with the Folin phenol reagent. J. Biol.Chem. 193:265-275.

16. DE MAN, J. C. 1959. The formation of diacetyland acetoin from a-acetolactic acid. Rec.Trav. Chim. 78:480-486.

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17. MICHAELIAN, M. B., AND B. W. HAMMER. 1936.The oxidation of acetylmethylcarbinol to diace-tyl in butter cultures. Iowa Agr. Expt. Sta. Res.Bull. 205.

18. MIZUNO, W. G., AM) J. J. JEZESKI. 1959. Startermetabolism. IV. Effects of various substrateson the formation of acetoin by a mixed strainstarter culture. J. Dairy Sci. 42:251-263.

19. VAN NIEL, C. B., A. J. KLUYVER, AND H. G. DERX.1929. Uber das Butteraroma. Biochem. Z.210:234-251.

20. PEruE, J. W. 1949. Some aspects of the butteraroma problem. 12th Intern. Dairy Congr.2:572-579.

21. Sadtler Research Laboratories. 1959-65. TheSadtler standard spectra. Midget ed., Phila-delphia.

22. SEBEK, 0. K., AND C. I. RANLES. 1952. The oxi-dation of the stereoisomeric 2,3-butanediolsby Pseudomonas. Arch. Biochem. Biophys.40:373-379.

23. SErTZ, E. W., W. E. SANDINE, P. R. ELLIKER,

AND E. A. DAY. 1963. Studies on diacetylbiosynthesis by Streptococcus diacetilactis.Can. J. Microbiol. 9:431-441.

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