Purification and characterization of a bacteriocin produced by Leuconostoc mesenteroides E131.

Leuconostoc mesenteroides E131, isolated from Greek traditional fermented sausage, prepared without the addition of starters, produces a bacteriocin which is active against the pathogen Listeria monocytogenes. The bacteriocin was purified by 50% ammonium sulphate precipitation, cation exchange, and reverse-phase chromatography. Bacteriocin is active at pH values between 4.0 and 9.0 and retains activity after incubation for 1h at 100°C. Proteolytic enzymes inactivated the bacteriocin after 1h of incubation, while renin resulted in full inactivation only after 24h. Lipase resulted in full inactivation after 4h. Applying molecular methods, it was determined that the bacteriocin produced, named as mesenterocin E131, was identical to mesenterocin Y105 and was expressed during the exponential growth phase.

Cloning and sequencing of the gene encoding X-prolyl-dipeptidyl aminopeptidase (PepX) from Streptococcus thermophilus strain ACA-DC 4.

AIMS: To clone and sequence the pepX gene from Streptococcus thermophilus. METHODS AND RESULTS: Three pairs of primers were used in polymerase chain reactions using as template the total DNA from Strep. thermophilus ACA-DC 4 in order to amplify, clone and sequence the pepX gene. Sequence analysis revealed an open reading frame of 2268 nucleotides encoding a protein of 755 amino acids. The calculated molecular mass of 85 632 Da agreed well with the apparent molecular mass of 80 000 Da previously determined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and gel filtration for the monomeric form of the purified enzyme. CONCLUSIONS: The pepX gene from Strep. thermophilus ACA-DC 4 was cloned and sequenced. The PepX protein showed significant sequence similarity with PepX enzymes from other lactic acid bacteria and contained a motif which was almost identical with the active site motif of the serine-dependent PepX family. SIGNIFICANCE AND IMPACT OF THE STUDY: There are economic and technological incentives for accelerating and controlling the process of cheese ripening. To achieve this, starters may be modified by introducing appropriate genes from other food-grade bacteria. New or additional peptidase activities may alter or improve the proteolytic properties of lactic acid bacteria.

Biochemical properties of Streptococcus macedonicus strains isolated from Greek Kasseri cheese.

A total of 32 Streptococcus macedonicus strains, isolated from Greek Kasseri cheese, were screened for biochemical properties of technological importance in milk fermentation processing, such as acid production, proteolytic and lipolytic activity, citrate metabolism, exopolysaccharide production, antimicrobial activity and biogenic amines production. All strains were found to be moderate acidifiers in milk. Only four strains could hydrolyse milk casein, while 11 strains showed lipolytic activity against tributyrin. Using amino acid derivatives of 4-nitroaniline as substrates, the highest peptidase activities were determined against phenylalanine- and glycine-proline-4-nitroanilide. Using fatty acid derivatives of 4-nitrophenol, it was shown that all strains exhibited esterase activities up to caprylate, with highest values against butyrate and caproate. Only one showed activity up to palmitate; this was also the most active strain against tributyrin. Five of the 32 strains could metabolize citrate but none of them produced exopolysaccharides. Nine strains displayed antimicrobial activity towards Clostridium tyrobutyricum, while no antimicrobial activity was detected against Listeria innocua and Propionibacterium freudenreichii subsp. shermanii. Finally, none was able to decarboxylize ornithine, histidine or lysine, and only four strains produced tyramine from tyrosine.