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.

Crude cell-free extracts of some strains of each L. casei and L. plantarum were assayed for their amino-, imino- and endopeptidase as well as the caseinolytic activity. L-alanine-, L-phenylalanine- and L-leucine-p-nitroanilide but not L-glutamic acid-p-nitroanilide, were hydrolyzed by all the strains indicating an amino-peptidase activity. The activity of proline iminopeptidase was very low compared to that of the aminopeptidase. L. casei could be distinguished from L. plantarum by its high endopeptidase activity against succinyl-phenylalanine- and glutaryl-phenylalanine-p-nitroanilide. The caseinolytic activity of cell-free extract of L. casei ATCC 393 was about one seventh the caseinolytic activity of intact cells, suggesting that the bulk of the cellular proteinase activity is located in the cell wall. It appears that a metallo aminopeptidase and a probable cysteine one are involved in the hydrolysis of amino acid-p-nitroanilide, whereas the endopeptidase activity appears to be related to a cysteine enzyme. Incubation of gels with L-leucine-p-nitroanilide after electrophoresis allowed the revealing of 2 zones of aminopeptidase activity in a strain of L. casei and only one in two others, but in L. plantarum it did not allow the revealing of any. The high proteolytic activities of L. casei compared to those of L. plantarum may explain its more frequent occurrence in cheese and its probable role in the ripening of many cheese varieties.

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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.

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