Purification And Partial Characteriaztiong Of Bacteriocin Produced By Lactobacillus Alimentarius FM-MM₄

A large number of drug-resistant strains have emerged owing to the abuse of antibiotics and chemical preservatives,which are harmful to human health.Such situation aroused people's extensive concern,so more and more people have a desire to develop a safe and efficient biopreservative to replace the chemical preservatives.Lactobacillin is a peptide or protein synthesized by lactic acid bacteria in ribosome,having a inhibitory effect on homologous microorganisms.Lactobacillin has become a research focus due to its character of efficiency,non-toxicity,no residue,and non-resistance.In this paper,a bacteriocin-producing lactic acid bacteria having broad spectrum antimicrobial abilities was screened from a traditional Chinese fermented food.In order to provide a theoretical basis for the development and application of the new lactobacillin in food,the bacteriocin was purified and its characteristics such as the primary structure,fermentation conditions were investigated.The main results are given as follows:1.Total of 63 Gram-positive and catalase-negative LAB strains were isolated from the traditional fermented meat products by gradient dilution method,and the cell-free supernatants of seven strains exhibited inhibitory activity against Escherichia coli,Staphylococcus aureus,and Salmonella by agar diffusion method.After eliminating the contribution of organic acid and hydrogen peroxide to the antimicrobial activity though adjusting the pH of the supernatant to 5.0 and adding catalase to supernatant,three strains(FM-MM4,SS2-6,SR4-1)still possessed antimicrobial activity against indicator strains.Proteases,such as trypsin,protease K,papain and pepsin,could completely inactivate the antimicrobial activity,indicating the chemical nature of protein or polypeptide.Among them,the strain FM-MM4 was chosen for further study because of the highest antimicrobial activity.The strain FM-MM4 was identified as Lactobacillus alimentarius based on its physiobiochemical characteristics and 16S rDNA sequencing.2.The bacteriocin produced by Lactobacillus alimentarius FM-MM4 was purified using three steps including ethyl acetate extraction,cation exchange and semi-preparative RP-HPLC,with an activity of 27242.40 AU/mg,a purification fold of 89.9 and the final recovery of 0.51%.The molecular mass of the bacteriocin was 1104.58 Da with N-terminal sequences of QGVGPLGQGHR according to the LC-MS/MS and N-terminal sequence analysis.The results indicated that it is a novel Class ? bacteriocin.3.Lactocin MM4 had a broad antimicrobial spectrum against not only genetically related Lactobacillus strains,but also various Gram-positive and Gram-negative foodbome pathogenic bacteria.In addition,Lactocin MM4 had slight inhibitory activity against three tested yeasts,Saccharomyces cerevisiae,Pichia sp.and Candida sp.It was considerably thermo-stable with 84.7%residual antimicrobial activity after exposure to121 ? for 15 min and remained above 60%residual antimicrobial activity exposing in acidic or neutral environment.Different chemicals have no effect on the antibacterial activity of the bacteriocin,but EDTA has a synergistic effect with it.Lactocin MM4 is sensitive to different metal,especially divalent metal ions.The minimal inhibitory concentration of the Lactocin MM4 agaist Staphylococcus aureus and Escherichia coli are 0.02mg/ml and 0.04 mg/ml,respectively.The action of the bacteriocin on Staphylococcus aureus and Escherichia coli is bacteriostatic,instead of sterilizable.4.The optimum fermentation conditions of bacteriocin-producing of the Lactobacillus alimentarius FM-MM4 was obtained though single factor test and orthogonal test.The optimum fermentation conditions were:fermentation temperature 30°C,fermentation time 24 h,inoculation quantity 4%,and initial medium pH 7.According to the results of Plackett-Burman Design,the key factors which affect the antimicrobial activity of Lactocin MM4 were glucose,yeast powder and sodium acetate.Response surface method was used to optimize the medium components,and a reasonable and reliable quadratic polynomial regression model of the inhibitory diameters with glucose,yeast powder and sodium acetate was established.The best optimum medium formula were glucose 24.40 g/L,yeast powder 4.34 g/L,sodium acetate 5.05 and other ingredients remained unchanged.Under these conditions,the maximum antimicrobial activity was obtained(18.54 mm)which was increased by 40.13%,in comparison with the initial activity.