| Nowadays, consumers are aware of the health concerns regarding food additives;the health benefits of “natural” and “traditional” foods, processed without anyaddition of chemical preservatives, are becoming more attractive. One of thealternatives to satisfy this request are bacteriocins, which are antimicrobial peptidesproduced by a large number bacteria, including lactic acid bacteria, normally actingagainst closely related and some spoilage and disease-causing Gram-positive pathogens.For this reason they are used in several applications, among which are biopreservation,self-life extension, clinical antimicrobial action and control of fermentation microflora.This study showed that a strain4121that produced broad-spectrum, efficient andspecific antibacterial peptide was screened from the91isolates lactic acid bacteriawhich come from infant guts. The antibacterial peptide was purified with the optimizedpurification parameters. We also studied that the effect of different acid, heat, proteaseand chemical treatment on the bacteriostatic activity of antimicrobial peptide producedby strain4121. Our research laid a theoretical foundation for the application of theantibacterial peptides in the aspect of food antiseptic.This study used Escherichia coli, staphylococcus aureus, shiga bacillus,salmonella and Listeria monocytogenes as indicator bacteria, and5strains screenedfrom91strains of lactic acid bacteria, respectively strains3123,3222,3325,3521and4121. Through the16SrDNA molecular biology identification, results showed that3123is Weissella hellenica NCFB2973;3521is Weissella viridescens NRIC1536T,3222and3521are Weissella minor NRIC1625,4121is Lactobacillus rhamnosusJCM1136. And its relatives similarity were above99%. In addition, the genome of5strains of lactobacillus were extracted, which can be specific amplified by a kind ofClass Ⅱ a antibacterial peptide gene conserved sequence primers(p1)BCgr2NSf:5’-ATGAAAAAGAAAKTAKTWAA-3’ and primer (p2)BCgr2R1K:5’-TTACAATAMASACCATTTCC-3’. Thus antibacterial peptides produced by the5strains wereidentified for ClassⅡa antibacterial peptides by preliminary judgment.Combined with antibacterial spectrum, antibacterial strength and the PCR test,antibacterial peptide produced by strain4121was purified with cell adsorption orSP-Sepharose Fast Flow cation exchange chromatography respectively. During the purification, the cation exchange chromatography parameter was determined for theoptimum condition. Research has shown that in liquid chromatography elution with0.1~1mol/L NaCl phosphate buffer (pH6.5) linear gradient elution in step3, and elutionflow rate of1ml/min was the best condition. Under the condition, with antibacterialpeptide yield29.10%, and purified ratio78.38, Specific activity3695.15AU/mg. bycontrast, the cell adsorption method has a higher productivity with peptide yield40.80%and purified ratio78.38, Specific activity10543.65AU/mg. in order to further improvethe antibacterial peptide activity, design the cell adsorption-cation exchange adsorptionchromatography two-step purification method, eventually make the antibacterial peptidepurification multiples of91.99times. Make that be a effective means of purification.Purified antibacterial peptide to Tricine-SDS-PAGE electrophoresis and found that theantimicrobial peptide molecular weight size for around10KDa, belongs to the ClassⅡaantimicrobial peptide molecular weight size category, a further proof that antimicrobialpeptide produced by strain4121is ClassⅡa antibacterial peptide.Through research of antibiotic activity of antimicrobial peptide produced by4121,results shown that antimicrobial activity at pH2.0-8.0, and thermal stability at20-120℃. Different treatments of protease showed that this antimicrobial peptide is notsensitive to pepsin or trypsin, but protease K and α-chymotrypsin can both deactive thisantimicrobial peptide. Moreover, different chemical agents’ treatment lay different impa-cts on antimicrobial activity. Tween80and Tween20have no remarkable antimicrobialactivity, whereas, NaCl, protease inhibitor EDTA and abluent SDS can de-crease antimi-crobial activity under different concentration, and have certain time-dos-age relationship... |
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