Optimization Of Bacteriocin-producing Lactic Acid Bacteria And Its Application In Mutton Preservation

In this thesis,199 lactic acid bacteria strains isolated and purified from cheese in Yili,Hami and Tachheng regions of Xinjiang were selected as research objects.Through the antibacterial test against Escherichia coli,Enterococcus faecalis,Megalococcus tyrolysoides and Bacillus subtilis,the interference of acid and hydrogen peroxide and the protease sensitivity test,the bacteriocin-producing lactic acid bacteria strains were screened.The bacteriocin produced by the selected strains were separated and purified by ammonium sulfate precipitation,ion exchange chromatography,gel filtration chromatography and High Performance Liquid Chromatography.The fresh-keeping effects of different doses of crude bacteriocin and Nisin on cold fresh mutton were studied.The optimal treatment group was combined with three different lock fresh bags to explore the fresh-keeping effects on mutton.The test results are as follows:(1)Screening of bacteriocin-producing lactic acid bacteriaThrough the Oxford Cup method,97 strains of lactic acid bacteria with antibacterial activity were preliminarily screened out from 199 strains of lactic acid bacteria,but only 22 strains of lactic acid bacteria had antibacterial effect on the four indicator bacteria.After excluding the influence of organic acid and hydrogen peroxide,13 strains of lactic acid bacteria were retained.After the protease sensitivity test,The13 strains of lactobacillus were identified as bacteriocin-producing lactobacillus.Strain F2-1 with the best bacteriostatic effect was selected for 16 S r DNA analysis and identification.The results showed that strain F2-1 was Lactobacillus fermentans..(2)Isolation and purification of lactobacillus bacteriocin and its bacteriostatic effectThe bacteriocin produced by Lactobacillus fermentum F1-1 was purified by using 80% ammonium sulfate saturated solution for crude extraction,cellulose DEAE-52 for ion exchange chromatography,Sephadex G-50 for gel filtration chromatography,and then highly purified by high performance liquid chromatography.Finally,the purification ratio of bacteriocin was 30.54.The molecular weight of bacteriocin measured by Tricine-SDS-PAGE gel electrophoresis was in the range of 10 ～ 15 k Da.Crude bacteriocin has good thermal stability,active in the p H range of 2 ～ 6,sensitive to trypsin,pepsin and protease K,and has no significant change in antibacterial activity after surfactant treatment.The minimum inhibitory concentration against Escherichia coli is 12.5 μg/m L,which can effectively inhibit the growth of Escherichia coli and the formation of biofilm.(3)Application of bacteriocin-producing lactic acid bacteria in mutton preservation6%,8%,10% crude bacteriocin and 0.5%,1.5%,2.5% Nisin had different effects on sensory indexes,color,juice loss rate,p H,TVB-N and total number of colonies of mutton.All indexes of mutton treated with crude bacteriocin were better than those treated with Nisin.Moreover,10% crude bacteriocin can prolong the storage period of mutton for 6 days compared with the control group.Compared with the control group,the storage period of mutton could be prolonged for 3 d,6 d and 9 d by using 10% crude bacteriocin combined with 3 different kinds of fresh bags.