Lactobacillus Plantarum Genome Shuffling And Application Of Bacteriocin On Meatball Preservation

Bacteriocin is a group of antimicrobial peptide produced by lactic acid bacteria(LAB)which have many advantages,such as broad inhibitory bacterial spectrum,high security,stable quality and so on.However,there is a difficulty in industrial application due to its generally low yield of wild-type strain.Genome shuffling technology has been widely used to promote the yield of bacteriocin for its simple protocol and high efficiency.People are confronted with serious problems of meat product deterioration in the food industry.The ineradicable chemical preservatives remaining in the meat products could bring about threats to human health.Thus,it is of great significance to develop novel,safe and effective preservatives.Plus,the used of combined preservatives help to solve the problem of low yield,high cost in the process of separation and purification of bacteriocins,which has been studied extensively.In this study,a bacteriocin-producing Lactobacillus plantarum JL-A65 isolated from sauerkraut was employed.On the basis of the high yield strains(M2-58?M7-111?A1-40,and A2-23),which were obtained from pre-mutagenesis,the higher yield strains were screened with the help of genome shuffling technology.Further,the combination of crude extraction of bacteriocin and sodium diacetate was used to extend the shelf life of meat products.The main findings are shown as follows:1.To obtain high-yield bacteriocin producing strains through genome shufflingThe L.plantarum JL-A65 was used in the protoplast preparation,inactivation and fusion in this study.By measuring the protoplast formation rate and regeneration rate,the optimal conditions for protoplast preparation were determined as follows:the cell growth time was 8 h,the concentration of lysozyme was 25 mg/mL,and the enzyme was digested at 37?for 30 min.By measuring the protoplast fatality rate,the optimal conditions for protoplast inactivation were determined as follows:the distance of UV lamp and cell was 19.5 cm,irradiation time was 35 min,water bathing for 40 min.By measuring the protoplast fusion rate,the optimal conditions for protoplast fusion were determined as follows:the concentration of PEG was 40%,and the fusion time was 20 min at room temperature.Using the high-yield strains obtained from mutagenesis breeding as the parent strains for fusion,a strain named F4-23 was screened after four rounds of genome shuffling.The yield reached 6.48 ?g/mL,which was 103.48%higher than the original strain.And the strain F4-23 was stable heredity.2.The application of combined preservatives in meat products.The sodium diacetate and bacteriocin crude extract were used in this study.The ratio of the two preservatives were determined with checkerboard method.L0 is control group without preservatives.Experiment groups with 0.5 g/kg sodium diacetate+8 mg/kg bacteriocin crude extract,1.0 g/kg sodium diacetate+16 mg/kg bacteriocin crude extract,1.5 g/kg sodium diacetate+24 mg/kg bacteriocin crude extract and 2.0 g/kg sodium diacetate+32 mg/kg bacteriocin crude extract were corresponding to groups LI,L2,L3,and L4,respectively.The results showed that:while on the 6th day,the total bacterial counts exceeded 1×106 CFU/g and it began to spoil.The group L1 started to deteriorate on the 8th day while the groups L2,L3,and L4 showed no signs of spoilage until the 11th day because of the preservatives added.The addition of combined preservatives have a mitigation effect on TVB-N,TBARS,pH and a*.The TVB-N value increased fast in the control group.It reached 20 mg/ml on the 8th day and started to spoil.While the in groups L2,L3,and L4,the TVB-N value exceeded 15 mg/ml until 11th day,whuch was basically consistent with the tendency of changes in the total bacterial counts.The TBARS values in the experiment groups(L1,L2,L3,L4)lower than that of the control group(L0)in this study.The combined preservatives effectively mitigated the upward trend of pH in the meat products during storage.The pH in the control group reached 6.4 on the 6th day and the meat products started to spoil.But it showed no significant changes in the experiment groups until the 11th day.The groups of L2,L3,and L4 can keep the color of fresh meat during storage.Overall,the combination ratio in group L2 showed the best effect,and the shelf life of meat products was extended for about 5 days compared to the control group L0.