| Food spoilage means the changed of food quality can not to eat. The caused of food spoilage are numerous, and the main factor is microorganisms. Microbial contamination can occur not only in the initial food source, but also in the process and transportation of finished or semi-finished products in the food industry. To avoid food spoilage caused by microorganisms, cold chain or freezing method usually is used to control microbial growth and reproduction. Due to the emergence of Psychrotrophys, it has been found that food quality can not be pretected by cold or frozen methods. Antimicrobial peptides as a natural preservative, has great potential in inhibiting psychrotrophs. The object of this dissertation is to the screen antimicrobial peptides from lactic acid bacteria to inhibit the psychrotrophs, establish appropriate separation and purification methods, investigated the antimicrobial mechanism and physico- chemical properties. The main results are as follows:Two strains T1 and Q7 with anti- psychrotrophs were selected from 325 LAB by 96-well agar diffusion method. The inhibitory substance is veriifed as peptides by neutralizing acid, excluding hydrogen peroxide, protease treatment and Tricine-SDS-PAGE. The results showed that antimicrobial peptides from Enterocin T1 Plantaricin Q7 present effective inhibition of the psychrotroph: Pseudomonas flurescens AS1.1802,Pseudomonas putida AS1.1819,Pseudomonas aeruginosa CICC21636, Listeria monocytogenes ATCC19111, and pathogenic bacteria: Escherichia coli ATCC25922, Salmonella typhimurium ATCC14028,Shigella flexneri ATCC12022, Shigella sonnei ATCC25931, Staphylococcus aureus. Antimicrobial peptides of Enterocin T1 and Plantaricin Q7 are stable at different p H values and temperatures, but are very sensitive to protease E, proteinase K, pepsin, trypsin and papain. By studying the effects of different chemical reagents for antimicrobial peptides, the results indicate that there were two kinds of antimicrobial peptides well tolerated. By 16 S r DNA gene sequence analysis and API identification profile, antimicrobial peptides producing lactic acid bacteria were identified as Enterococcus faecium and Lactobacillus plantarum.Fermentation broth of Enterococcus faecium T1 and Lactobacillus plantarum Q7 were loaded on SP-sepharose, CM-FF, Superdex Peptide 10/300 GL to obtain antimicrobial peptides Enterocin T1 and Plantaricin Q7. By LC-ESI/MS, molecular weight of Enteroicn T1 and Plantaricin Q7 were determined to 4629 Da and 4624 Da, respectively. Through fingerprint analysis, none amino acid sequence were correlated with these two kinds of antimicrobial peptides. For the composition of the amino acid antimicrobial peptide, Enterocin T1 contains 41 amino acids with partial amino acid sequence of M-A-E-T-F-A-P; And Plantarcin Q7 contains 40 amino acids with the partial amino acid sequence of V-H-A-K-I-K-F-D.The inhibitory mechanism on two kinds of antimicrobial peptides against Listeria monocytogenes and Pseudomonas fluorescens were explored by fluorescent probe method. By reflecting different positions numerals bacteria bacteriostatic antimicrobial peptides mode and mode of action. The results showed that two kinds of antimicrobial peptides can destroy these two kinds of indicator bacteria cell membrane and proton motive force destroys effectively, the indicator bacteria loss of physiological functions and death. Also it is learned that two kinds of antimicrobial peptides by Listeria cell surface by adsorbing single inserted inside the membrane acts on Listeria monocytogenes, Pseudomonas fluorescens for the mode of action is based on chelation way undermine Pseudomonas fluorescens bacterial outer membrane and cell membraneIn this study, milk, pork, and octopus were selected for research about the applications of Enteroicn T1 and Plantaricin Q7 in food preservation. The sensory evaluation, the total number of colonies, TVB-N and p H value were the main indicators for preservation effects of Enteroicn T1 and Plantaricin Q7. The results showed that two kinds of antimicrobial peptides for different types of foods can effectively inhibit the growth of Pseudomonas fluorescens, exerting very good preservation effect for different food, and this preservation effect of antimicrobial peptides were enhanced activty increased antimirobial activity... |
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