Identification And Screening Of Bacteriocin-producing Lactic Acid Bacteria Isolated From Fermented Sausages And Fermentation Condition

The aim of this paper was to isolate lactic acid bacteria strain of high yield bacteriafrom natural fermented sausage. According to the morphological features，physiologicaland biochemical features，the strain was identified as Lactobacillus plantarum. Thefactors affecting the growth and bacteriocin production of this Lactobacillus plantarumwas studied. It was conducted to investigate the significance of different factors throughthe Placket-Burman experiment, and the interaction of the significant factors using theBox-Behnken experiment. Fermentation conditions of the strain producing bacteriocinwere optimized, we determined the best fermentation technology.The biological featuresof coarse baeteriocin were also determined.The main results of this research were asfollows:（1） One hundred and twenty-seven strains were isolated from natural fermentedsausage using repeated separation and purification. Microscopy were gram-positivebacterias, eighty-five isolated belonged to bacillus, forty-two strains belonged tostreptococcus. The preliminary screening results showed that strain ratio was11.8%thathas inhibitory effect on e. coli and strain rate accounted for40.9%that has inhibitoryeffect on staphylococcus aureus. There were7strains have inhibitory effect on bothindicator bacterias, strain rate accounted for5.5%. Strain T102had strong inhibitoryeffect on e. coli and staphylococcus aureus. After screening results showed that strainT102had antibacterial activity after excluded inhibitive effect of H2O2and organic acid.The inhibitive activity decreased after the treatment with proteases.The resultesconfirmed that the anti-bacteria product produced by T102was protein, which could beclassed as bacteriocin. According to the morphological features，physiological andbiochemical features，the strain T102was identified as Lactobacillus plantarum.（2） In cultures without pH control, the bacteriocin activity of L. plantarum T102was higher at30oC. However, the cell growth rate was higher at37oC. In MRS brothwith an initial pH6.0, the cell growth was lower, but high bacteriocin levels wererecorded than at pH7.0. Lactose was the best carbon sources and yeast extract was thebest nitrogen sources. The bacteriocin activity was maximal in medium containing2.5%lactose or2.0%yeast extract or2.0%KH₂PO₄or1.0%NaCl or2.0mmol/LMnSO₄or1.0g/L Tween80. Tween80didn’t promote strain growth, but had great influence onbacteriocin production. （3） The Plackett-Burman experiment indicated that temperature,lactoseconcentration and yeast extract concentration were the significant factors that effect theproduction of bacteriocin.Lactose and yeast extract have negative influence onbacteriocin production and temperature, pH, NaCl, KH₂PO₄and Tween80have positiveeffect.The Box-Behnken experiment showed that the interaction between temperatureand yeast extract concentration was significant, so was lactose concentration and yeastextract concentration（P<0.05）.The condition optimized by Design-Expert8.07was that:temperature34.06℃, lactose concentration2.23%and yeast extract concentration2.08%,bacteriocin production1077.44IU/mL.Considering the actual situation, the resultswas:temperature34℃, lactose concentration2.2%and yeastextract concentration2.1%.（4） The biomass of Lactobacillus plantarum T102was the largest at24h and theantibacterial activity of was the strongest at36h.The pH of Fermentation liquid has beena declining trend,the lowest pH was3.91.The biological features of coarse bacteriocinshowed that the inhibition spectrums of the bacteriocin were very wide. it could inhibitboth Gram-bacteria and some Gram+bacteria，but not effected on beer yeast. It had goodthermal stability and its optimum pH was5.0. This kind of bacteriocin produced by8strainT102was sensitive to proteinaseK and trypsase,it indicated that this bacteriocinbelonged to the protein.