Isolation Of Lactic Acid Bacteria From Xixiang Jiangshui And Purification Of Its Bacteriocin

Bacteriocin is natural antimicrobial protein or peptide which can be used aspreservatives in food industry and has potential therapeutic applications as antibioticsubstitution. In this paper,31lactic acid bacteria （LAB） strains were isolated fromXixiang jiangshui. Standard Escherichia coli ATCC25922and standardStaphylococcus aureus ATCC25923were used as indicators,20LAB strains withdifferent antimicrobial activity were screened by overlay assay method.4optimalstrains were selected according to their inhibitory activity, and MXJ32strain wasscreened to study further for its most significant inhibitory activity by well diffusionassay. The16S rDNA gene was amplified via PCR and sequenced, and MXJ32strainwas identified as Lactobacillus coryniformis on NCBI.Crude bacteriocin from fermentation broth of MXJ32strain was obtained byammonium sulphate precipitation, it was purified by dialysis, ion exchangechromatography, cation-exchange chromatography and anioin-exchangechromatography respectively, and was named lactocin MXJ32A. Molecular mass isabout3520Da by LC-ESI/MS. N-terminal12amino acid residues sequence of thebacteriocin as following: NH2-GEPGPMGPAGAD, which was used to search in theNCBI database and no similarity to any other known bacteriocins was found. It’s totalhydrophobic residue is about52%among amino acid compositions.Bacteriocin lactocin MXJ32A has thermostability, after treated at60,80,100and121℃for5,10and20min repectively, it still maintained activity without anydecrease. The bacteriocin revealed good stability in broad pH range（pH2-11）, andthe lower pH is, the stronger inhibitory activity will be. Nature of the bacteriocin isprotein without any carbohydrate chain or aliphatic chain. Hemolysis activity ofbacteriocin lactocin MXJ32A is close to ampicillin, and it is safe under the range ofapplication.Bacteriocin lactocin MXJ32A is broad spectrum antibacterial substancewhich had different antimicrobial ability to both gram-positive and gram-negative strains including foodborne drug-resistant bacteria and pathogenic bacterium. MICs ofthe bacteriocin against both Saureus and Ecoli was10mg/mL. Scanning electronmicroscopy （SEM） and Transmission electron microscopy （TEM） were used toobserve the morphological changes of Ecoli and Saureus after treated by bacteriocin.Compared with blank, treated indicators exhibited shrinkage, rupture, leakage, cavity,and so on, which leaded the death of target bacteria.