Characterization of two bacteriocins produced by Leuconostoc mesenteroides L124 and Lactobacillus curvatus L442, isolated from dry fermented sausages

Leuconostoc mesenteroides L124 and Lactobacillus curvatus L442, isolated from dry fermented sausages, produce bacteriocins antagonistic towards closely related species and pathogens, such as Listeria monocytogenes. The bacteriocins were inactivated by proteolytic enzymes and lipase but not by catalase and lysozyme. They were also heat stable, retaining activity after heating at 100 °C for 60 min. The bacteriocins were stable at pH values ranging from 2.0 to 8.0. Bacteriocin production was observed at low temperatures (10 and 4 °C) and in meat juice. The maximum bacteriocin activity was observed at the end of the exponential growth phase. The bacteriocins were produced in media with initial pH values ranging from 5.0 to 7.5, but not in media with a pH lower than 5.0 (weak bacteriocin activity of the antibacterial compound produced by Ln. mesenteroides L124 was observed at pH 4.5). Both bacteriocins exhibited strong bactericidal activity following cell/bacteriocin contact.     

Taxonomic studies on some leuconostoc-like organisms from fermented sausages: description of a new genus Weissella for the Leuconostoc paramesenteroides group of species

Taxonomic studies were performed on some unknown Leuconostoc -like organisms from fermented Greek sausage. Comparative 16S rRNA sequence analysis showed the unidentified organisms represent a new line within the Leuconostoc paramesenteroides group of species. On the basis of the results of this and earlier phylogenetic investigations, it is proposed that Leuconostoc paramesenteroides and related species be reclassified in a new genus Weissella. In addition a new species, Weissella hellenica , is proposed for the isolates from fermented sausage.     

Microbial interaction in cooked cured meat products under vacuum or modified atmosphere at 4 degrees C

AIMS: To investigate the antagonistic activity of two lactic acid strains against the spoilage microflora in cooked cured meat products, vacuum or modified atmosphere packed at 4 degrees C and to determine the inhibitory capacity of their bacteriocins. METHODS AND RESULTS: Frankfurter-type sausages and sliced cooked cured pork shoulder were inoculated with Leuconostoc mesenteroides L124 and Lactobacillus curvatus L442 or with their bacteriocins. The microbial, physico-chemical (pH, L- and D-lactate, acetate and ammonia) and colour changes were studied. Results under vacuum packaging showed that in the uninoculated samples of the pork product the spoilage microflora grew but in the inoculated ones the spoilage microorganisms (e.g. Brochothrix thermosphacta and enterococci) reduced during the storage. This observation was more pronounced in the samples with the addition of bacteriocins. In the frankfurter-type sausages the spoilage microflora did not grow in the uninoculated and inoculated samples. In the modified atmosphere enriched in CO2 the population of spoilage microflora remained at low levels in both products, indicating that CO2 has an effect on the spoilage microorganisms' growth. In the pork product the concentrations of acetate and d-lactate increased while L-lactate decreased, but in the frankfurter-type sausages increase of acetate and D-lactate was not observed. CONCLUSIONS: Lactic acid strains had an effect on the spoilage microflora growth but did not affect, negatively, the organoleptic properties of the products. These strains may be used as biopreservative cultures or their bacteriocins could be an important contribution to microbiological quality of meat products. SIGNIFICANCE AND IMPACT OF STUDY: Establishment of biopreservation as a method for extension of shelf life of meat products.     

Purification and characterization of curvaticin L442, a bacteriocin produced by Lactobacillus curvatus L442

Lactobacillus curvatus L442, isolated from Greek traditional fermented sausage prepared without the addition of starters, produces a bacteriocin, curvaticin L442, which is active against the pathogen Listeria monocytogenes. The bacteriocin was purified by 50% ammonium sulphate precipitation, cation exchange, reverse phase and gel filtration chromatography. Partial N-terminal sequence analysis using Edman degradation revealed 30 amino acid residues, revealing high homology with the amino acid sequence of sakacin P. Curvaticin L442 is active at pH values between 4.0 and 9.0 and it retains activity even after incubation for 5 min at 121 °C with 1 atm of overpressure. Proteolytic enzymes and α-amylase inactivated this curvaticin, while the effect of lipase was not severe.     

Production of Italian Dry Salami: Effect of Starter Culture and Chemical Acidulation on Staphylococcal Growth in Salami Under Commercial Manufacturing Conditions

The effect of starter culture and chemical acidulation on the growth and enterotoxigenesis of Staphylococcus aureus strain S-6 in Italian dry salami under commercial manufacturing conditions was studied. The experimental design included two levels of S. aureus (10⁴ and 10⁵/g), three levels of starter culture (0, 10⁵, and 10⁶/g), three levels of initial pH (pH₀) (6.1, 5.5, and 4.8), two manufacturing plants, and three replications. S. aureus growth in the salami was affected significantly (P < 0.005) by pH₀, initial levels of S. aureus (staph₀) and lactic acid bacteria (LAB₀), day of fermentation, and by the interactions of pH₀ × day, pH₀ × LAB₀, LAB₀ × staph₀, pH₀ × staph₀, and pH₀ × location of fermentation. In general, the lower the pH₀ and the higher the LAB₀, the greater the inhibition of S. aureus. The LAB levels during the fermentation were affected significantly (P < 0.005) by pH₀, LAB₀, day of fermentation, location, LAB₀ × pH₀, and LAB₀ × day. Derived regression equations related level of S. aureus and LAB at any day of fermentation to a number of microbiological and chemical variables. Close similarity of observed and predicted levels of S. aureus and LAB growth demonstrated the usefulness of the experimental approach in evaluating the safety of a process. No detectable enterotoxin or thermonuclease was found at any stage of processing even when S. aureus reached levels of 10⁷/g of salami.     

Differentiation of Lactobacillus sake and Lact. curvatus isolated from naturally fermented Greek dry salami by SDS-PAGE of whole-cell proteins

J. SAMELIS, E. TSAKALIDOU, J. METAXOPOULOS AND G. KALANTZOPOULOS. 1995. To confirm the phenotypic characterization of 169 predominant sausage isolates of the Lactobacillus sake/curvatus group, SDS-polyacrylamide gel electrophoresis (SDS-PAGE) of whole-cell proteins of 29 representative strains was undertaken. The results of the electrophoretic analysis indicated that each species yielded a specific protein profile, identical to the respective type strain. It was shown that all melibiose-positive isolates belonged to Lact. sake , whether or not they were arginine- and maltose-positive, whereas all melibiose- and arginine-negative isolates were assigned to Lact. curvatus. A group which phenotypically appeared to lie between Lact. sake and Lact. curvatus , on the basis of arginine (+), melibiose (-) and maltose (-) reactions, was shown to be more closely related to Lact. sake. The SDS-PAGE method was valuable in distinguishing between species, but did not allow further differentiation of Lact. curvatus or Lact. sake strains displaying high heterogeneity in secondary physiological and biochemical properties.     

Evaluation of the extent and type of bacterial contamination at different stages of processing of cooked ham

In an attempt to determine the composition and origin of the spoilage flora of refrigerated vacuum-packed cooked ham, the changes in microbial numbers and types were followed along the processing line. Results revealed Lactobacillus sake and Leuconostoc mesenteroides ssp. mesenteroides as the major causative agents of spoilage of sliced ham stored at 4 °C and 12 °C, due to recontamination in the cutting room. On the contrary, the progressive deterioration of whole ham under the same storage conditions was associated with a non-identifiable group of leuconostoc-like bacteria. Except for lactic acid bacteria, no other organism grew in vacuum packs of either sliced or whole ham. Although atypical leuconostocs could not be detected among isolates recovered from freshly produced whole ham, they appeared to survive cooking and proliferate during storage. Neither these organisms however, nor Lact. sake and Leuc. mesenteroides were important in curing and tumbling as carnobacteria, mainly Carnobacterium divergens, and Brochothrix thermosphacta dominated at this stage. A progressive inversion of the ham microflora from mostly Gram-negative at the beginning of processing to highly Gram-positive prior to cooking was noted. Listeria monocytogenes cross-contaminated ham during tumbling. However, the pathogen was always absent from the vacuum-packed product provided that heating to a core temperature of 70 °C occurred and recontamination during slicing and packing was prevented. The percentage distribution of different species of lactic acid bacteria as well as the uncommon phenotypic characteristics of some strains were discussed.     

Influence of nutrients on growth and bacteriocin production by Leuconostoc mesenteroides L124 and Lactobacillus curvatus L442

The aim of this study was to investigate the effect of complex nutrients on microbial growth and bacteriocin production, in order to improve bacteriocin synthesis during the growth cycle of Leuconostoc mesenteroides L124 and Lactobacillus curvatus L442. The fermentations were conducted at the optimum pH and temperature for bacteriocin production (pH 5.5+/-0.1 and temperature 25+/-0.1 degrees C). Because of their association with the final biomass, conditions favouring the increase of the produced biomass resulted in the increase of bacteriocin activity in the growth medium. Since the produced final biomass and the final concentration of the bacteriocins were associated with the amount of the carbon (glucose) and nitrogen source, better growth of the lactic acid bacterial strains favoured the increase of the specific bacteriocin production. Additionally, the bacteriocin production was influenced by carbon/nitrogen ratio.