Optimization Of Fermentation Conditions For Bacteriocin-like Substance Production By Lactococcus Lactis NFL

Compared with bacteriocin, bacteriocin-like substances?BLS? have a broader antimicrobial spectrum and higher stability, which indicates BLS have a wider application prospect. Our team isolated a strain NFL of Lactococcus lactis, which produces BLS with obvious antibacterial activities against common G+and G-bacteria, strong thermal stability and sensitivity to proteases, suggesting the BLS has the potential to become a food preservative. Nowadays, the agar diffusion method was the most widespread method for determining the potency of BLS. In order to accurately measure the production of the BLS produced by L. lactis NFL, we optimized the factors which influenced the results of double agar plate diffusion experiment; In order to make L.lactis NFL capable of producing stable and high yields of bacteriocin-like substance, we optimized the seed medium and synthetic fermentation medium; In order to get the best culture conditions of the optimal synthetic fermentation medium, we optimized the culture conditions. The main research results are as follows:The optimal conditions for determination the antibacterial activity of the BLS produced by L.lactis NFL using double layer agar diffusion method were as follows: the indicator Escherchia coli CMCC44102, the concentration of agar in the NA medium 0.75%, the inoculum volume 0.1%, temperature 30?and incubation time 12h.Optimization of the seed medium and fermentation medium of L.lactis NFL: Among the tested carbon sources?fructose, glucose, sucrose, maltose, lactose?, the highest biomass of L.lactis NFL was obtained with glucose as the carbon source, 3.1% higher than fructose as the carbon source. The highest bacteriocin-like substance production of L.lactis NFL was obtained with the fructose as the carbon source, but was only 1.6% higher than glucose as the carbon source. For the aim of low cost, glucose was chosen as the optimum carbon source of the seed and fermentation medium; Among the tested organic nitrogen source?peptone, beef extract, yeast powder, urea? and inorganic nitrogen source??NH₄?₂SO₄?NH₄NO₃?KNO₃?, the highest biomass of L.lactis NFL was obtained with peptone as the nitrogen source, the lower biomass was obtained with the NH₄NO₃ as the nitrogen source.but the unit cell production of bacteriocin-like substance was significantly higher when the nitrogen source was NH₄NO₃ than organic nitrogen source, thus peptone was chosen as the optimum nitrogen source of the seed medium, and NH₄NO₃ was chosen as the optimum nitrogen source of the fermentation medium; 20 kinds of basic amino acids were optimized by single factor experiments, results showed that no significant effect was observed on thebiomass and production, so it was determined that amino acid was not added into the medium; the original buffer salt system of MRS medium was optimized by orthogonal experiments, results showed that the optimal composition of buffer salt system of the seed medium was 2g/L K₂HPO₄, 3g/L di-ammonium hydrogen citrate and 3g/L sodium acetate, and the optimal buffer salt system of the fermentation medium was composed of 1g/L K₂HPO₄, 2g/L di-ammonium hydrogen citrate and 7g/L sodium acetate; Initial pH value of the medium was optimize within the scope of 5.2 to 7.7, the results showed that the biomass increased significantly with the increase of pH, and was maintained stable after reaching to the maximum at pH 7.2, the production peaked at pH 5.7. Therefore, the optimal initial pH of the seed medium was 7.2, and the optimal initial pH of the fermentation medium was 5.7; Among the tested inorganic salts?MgSO₄?MnSO₄?K₂SO₄?ZnSO₄?CuSO₄??Fe?₂?SO₄?₃?FeSO₄?FeCl₂?CaCl₂?, the highest biomass and production were obtained when supplemented with MnSO₄ alone, and slightly higher than the control?Mg SO4 and MnSO₄'s combination?, so adding MnSO₄ alone was chosen as the optimal inorganic salt of the seed and fermentation medium; No significantly increase of the biomass was observed when supplemented with the emulgator?Tween- 20, Tween 40,Tween- 80, Triton- 100?, but the production was increased by 5.5% with the presence of Tween – 80, so Tween- 80 was chosen as the optimal emulgator of the fermentation culture; Decrease of the biomass and production was observed when supplemented with the tested stimulating factor?Na Cl, ethanol?, it was determined that these compounds were not added into the medium; Among the tested B group vitamins?VitB1, VitB2, VitB3,VitB5, VitB6, VitB7, VitB11, Vit12, Vit C?, 6 kinds of vitamin significantly increased the biomass, 5 kinds of vitamins?VitB3, VitB5, VitB6, VitB7, VitB11? significantly increased the production. Vitamins combination was further optimized by orthogonal experimental design, results showed that the optimal vitamin combination of the seed medium was composed of VitB3 20mg/L, VitB7 5mg/L,VitB11 20mg/L, VitB12 5mg/L and Vit C 20mg/L, the optimal vitamin combination of the fermentation medium was composed of VitB3 20mg/L, 20mg/L VitB5 and VitB7 20mg/L; Among the tested nitrogen bases?thymine, cytosine, uracil, adenine?, the biomass was increased by 46.6% when supplemented with uracil, but the four types of nitrogen base had almost no influence on the production, so uracil was chosen as the optimal nitrogen baseof the seed medium.Compared to MRS medium, in optimized seed medium, the biomass of L. lactis NFL was increased by 13.8%; in optimized synthetic fermentation medium, the production of bacteriocin-like substance was increased by 22.2%.The culture conditions for L.lactis NFL producing bacteriocin-like substance was optimized by single factor experiments. Results showed that the optimal culture conditions were as follows: the post-exponential phase culture?8h? was inoculated into fermentation medium with an inoculum size of 5%, then incubated statically at 30? for 12h.