Eleven strains of homofermentative and heterofermentative lactic acid bacteria were screened for acetoin (A) and diacetyl (D) production from pyruvate and citrate in a peptone-yeast extract-glucose broth. The homofermenters, except Streptococcus faecalis subsp. liquefaciens, produced much more AD from pyruvate than from citrate; the opposite was true for the heterofermenters. Acetoin and diacetyl were produced from pyruvate as soon as growth was initiated. The production was exponential up to 24 h. Destruction of the accumulated AD coincided with entry into the stationary phase. Production of AD from citrate did not begin until 6 h of the logarithmic phase of growth. Formation of gas from citrate by Lactobacillus plantarum did not implicate greater ability to form AD from citrate than from pyruvate. Fifty μmoles ml−1 citrate caused about 50% inhibition of growth of Streptococcus lactis subsp. diacetylactis. All strains examined for ability to use pyruvate as a sole source of carbon were able to do so. Acetate (50 μmoles ml−1 generally stimulated AD formation from pyruvate. With the exception of a Pediococcus sp. and S. faecalis subsp. liquefaciens, acetaldehyde (100 μg ml−1) enhanced AD production but not growth. Concentrations higher than 100 μg ml−1 had different effects.

One hundred and fourty five lactobacilli, leuconostocs and pediococci were isolated from salted raw milks incubated at 30°C for 4 to 21 d. Of 126 lactobacilli isolated, mostly from 9 to 12% salted milk, 115 were identified as homofermentative, nonthermophilic lactobacilli-73 were classified as Lactobacillus plantarum, 31 Lactobacillus casei, 8 strains were motile and 3 Lactobacillus xylosus. The remaining 11 isolates were heterofermentative lactobacilli-8 were Lactobacillus cellobiosus and 3 Lactobacillus brevis-buchneri. Strains of L. plantarum fermented many oligosaccharides, produced DL lactate and gas from L(+) but not from D(−) tartrate and their cell wall peptidoglycan was of the mesodiaminopimelic acid type. Eight strains of L. casei proved to be subsp. pseudoplantarum on the basis of inactive lactic acid production; 8 were subsp. rhamnosus and 2 subsp. alactosus, according to their pattern of sugar fermentation. L. xylosus simulated L. casei morphologically but differed from it in fermentation of xylose. The motile strains fermented arabinose and mostly sucrose but not lactose and produced 73.2 to 94 μmoles ml−1 inactive lactate from 1 % glucose. None of 10 Leuconostoc isolates produced dextran from sucrose but they fermented trehalose and were identified with Leuconostoc paramesenteroides. Three strains belonged to Pediococcus and produced 62 μmoles ml−1 of inactive lactate, whereas other six strains were atypical pediococci. Nineteen strains representing L. plantarum, L. casei, motile strains and Pediococcus gave, on examination for isomers of lactic acid, 32.8 to 171 μmoles ml−1 inactive lactate; the L(+) enanthiomorph generally predominated.

Whole-cell suspensions of Lactobacillus plantarum grown on lactose in the presence of citrate did not produce acetoin and diacetyl (AD) (D) from citrate in succinate buffer, pH 4.4 unless both a source of energy and nitrogen was present, but did from pyruvate. The total AD and the amount of D, produced by citrate-grown cells, from citrate were about two times the amounts formed from pyruvate, calculated on a molar basis. It appears, that AD are formed not only from pyruvate resulting from cleavage of citrate but also from acetyl-coenzyme A arising during a probable breakdown of citrate in a reversible reaction of citrate synthetase. Neither acetate nor acetaldehyde had any effect on the total AD or the amount of D produced from pyruvate by pyruvate-grown cells. The rates of AD production from pyruvate by whole-cell suspensions of pyruvate-grown L. plantarum and Streptococcus subsp. diacetylactis represented only 69.7 and 6.6%, respectively, of that produced by Lactobacillus casei. These were 0.075 μmoles/mg dry wt−1 ml−1 min−1 for L. casei, 0.053 for L. plantarum and 0.005 for S. lactis subsp. diacetylactis.

The ability of Lactobacillus casei subsp. pseudoplantarum to produce acetoin and diacetyl (AD) was evaluated in succinate buffer (initial pH 4.4) containing sodium pyruvate, at 30°C. Cells grown in MRS broth containing pyruvate produced AD more rapidly than did an equal number of cells either grown in broth without pyruvate or even stored, after harvesting during logarithmic growth, in MRS broth in the presence of pyruvate for 120 minutes. One or more of the enzymes catalyzing formation of AD appears to be formed originally during growth in the presence of pyruvate. The rate of AD production by pyruvate-grown cells was exponential, being 0.08 μmoles mg dry wt−1 ml−1 min−1 during the first 30 min of the reaction. Storage of pyruvate grown cells at 7°C for 6 h in 0.3 mmol/L KH2PO4 buffer, pH 7.0 resulted in 77% loss of activity. Inclusion of 0.5 ml of MRS broth in the assay mixture led to a considerable increase in AD production by both cells grown in the absence and presence of pyruvate. Lactose slightly stimulated AD production in cells grown on lactose, whereas glucose had practically no effect on glucose-grown cells. Acetate and acetaldehyde reduced AD production. The effect varied with the compound used and strain studied. Of 7 concentrations of cetylpyridinium chloride tested for their effect on AD production, the least and most inhibitory concentration were 1 and 10 μg ml−1 of assay mixture.

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