| Bacteriocins are a kind of ribosomal synthesized antimicrobial peptides or proteins produced by bacteria, which can kill or inhibit bacterial strains closely-related, but don’t harm the bacteria themselves. Bacteriocins are very popular due to the advantages of their safety, efficiency, no drug resistance and digestibility in body. Therefore, it has become the trend that the bacteriocins would be used as food preservatives or additives in the development of food industry in the future. China is a country rich in the resource of lactic acid bacteria and has a comprehensive study on lactic acid bacteria. However, the studies on bacteriocins of lactic acid bacteria were started relatively later and research content was not very deeply, compared to abroad. So this study is aimed to develop an novel bacteriocin, improve the production of bacteriocins and make a preliminary study on the mechanism of bacteriocins.Firstly, the strain that is capable of producing a high yield of bacteriocin, having a higher antimicrobial ability against Escherichia coli ATCC 25922, was screened from seven lactic acid bacteria strains by using double layer tablets. Then the fermentation medium composition and condition of target strains were optimized for bacteriocin production by the response surface and single factor experiment. Further bacteriocin was purified by saturated ammonium sulfate precipitation and SP Sepharose Fast Flow cation exchange chromatography and molecular mass of purified bacteriocin was estimated by tricine-SDS-PAGE. Finally, biological characteristics of bacteriocin and action mode of CFS were studied. Bacteriostatic spectrum was further drawn.The following results were obtained through experimental investigation.The strain KLDS 4.0325 that is capable of producing a high yield of bacteriocin, was screened from seven lactic acid bacteria strains. We confirmed the antimicrobial role of this bacteriocin by eliminating the interference of organic acid and hydrogen peroxide and by conducting protease sensitivity tests. The strain was also identified as lactococcus lactis on the basis of morphological, physiological, biochemical characteristics and 16 S r RNA sequence analysis.In order to obtain the optimum condition, the fermentation medium composition and condition were optimized. And the optimum fermentation medium composition was finally determined as: 128.99 g/L sucrose, 17.59 g/L peptone, 10 g/L casein peptone, 10 g/L K2HPO4, 60 g/L Ca CO3, 1.5 g/L Mg SO4.7H2 O, 1.49 g/L sodium ascorbate, 1.5 g/L β-glycerophosphate, 1.0 g/L CH3 COONa.7H2O, 1.0 g/L NaCl and 1.0 g/L Mn SO4. Meanwhile, bacteriocin production reached the maximal value under the condition of 33 ℃, original p H 7.0 and 120 r/min for 20 h.Bacteriocin from L.Lactis KLDS 4.0325 was purified. The cell-free supernatant of L.Lactis KLDS 4.0325 was precipitated by 60% ammonium sulphate. Then, the precipitates were subjected to cation exchange chromatography with COLUMN XK 16/20 chromatographic column filled with SP Sepharose for further purification and the bacteriocin was washed with acetate buffer solution(p H 4.0) at flow rate of 0.4 mL/min, sampling amount of 0.4 m L and wavelength of 215 nm. The molecular mass of purified bacteriocin was approximately 3.4 k Da by means of tricine-SDS-PAGE.Biological characteristics of bacteriocin were studied. The results showed bacteriocin exhibited a wide range of antimicrobial activity, strong heat stability( 30 min at 121 ℃) and p H stability(p H 2.0-10.0). Following treatment by pepsin and trypsin, the antibacterial activity was disappear. The results indicated that this antagonistic substance was a kind of protein. The action mode of the CFS was confirmed as bactericidal. This bacteriocin exhibited broad-spectrum antimicrobial activity against various species of gram-positive and gram-negative bacteria.The strain KLDS 4.0325 was screened from seven lactic acid bacteria strains and identified as lactococcus lactis. Further, under the optimal culture condition, the bacteriocin inhibitory activity was increased by 3.31 times and reached up to 5000 IU/m L. The specific activity of purified bacteriocin was 51.02 IU/mg and increased by 18 times. As well as, bacteriocin exhibited a wide range of antimicrobial activity, strong heat stability(30 min at 121 C) and p H stability(p H 2.0-10.0). This bacteriocin also exhibited broad-spectrum antimicrobial activity against common species of gram-positive and gram-negative bacteria. It also laid the foundation for the industrial production and application. |
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