Intracellular Expression Of An Antifreeze Peptide By Lactococcus Lactis And Evaluation Of Cryoprotective Effect In Recombinant Bacteria

Antifreeze peptides protect living organisms from low temperatures by preventing damage or killing due to ice crystal formation between cells.Therefore,antifreeze peptides can be used as a low temperature protectant for cryopreservation of cells and tissues,and in food production.In this study,an antifreeze peptide sequence was constructed based on codon bias of Lactococcus lactis,and then the food grade gene engineering bacteria were constructed by molecular regulation method,so as to obtain recombinant bacteria which could express antifreeze protein successfully.Elliker agar plates,colony polymerase chain reaction and sequencing analysis were used to verify the recombinant plasmids were successfully constructed.The expression of target genes was induced by Nisin,SDS-PAGE and Western-Blot identified the expression of antifreeze protein successfully.The expression of antifreeze proteins was improved by optimizing several key factors.The optimum single factor condition of recombinant Lactococcus lactis expressing antifreeze protein for the induction time of 6 h,p H of 7.0,temperature of 25 ? and Nisin concentration of 15 ng/m L.The optimal expression conditions of orthogonal combinatorial optimization were found to be a p H of 6.0,Nisin concentration of 25 ng/m L,temperature of 37 ?,and incubation period of6 h.Finally,the effects of antifreeze protein expression on freeze stress and freeze drying stress of Lactococcus lactis were investigated.After freezing stress,compared with the control group,recombinant strain SF-P2 did notshow obvious fluctuated and mitigated at the later stage of log growth and stable growth lag phase,the fermentation activity and the key enzyme activity of intracellular is stable.The survival rate of cells increased from21.00 % and 22.03 % to 49.28 %,the proportion of living cells increased from 3.40 % to 56.30 % and the proportion of damaged cells dropped from91.00 % to 41.80 %.The TH and ?Hr values of SF-P2 were significant higher than those of SF-P1 and NZ3900.After freeze drying stress,compared with the control group,the damage of cell membrane permeability was mild and SF-P2 cells displayed a normal ultrastructural morphology with integrated cell walls,round cell profiles and observable boundaries between the cells.The results of this study show that,compared to non-induced recombinant strain SF-P1 and airborne strain NZ3900,the recombinant strain SF-P2 showed the highest cell survival and thermal hysteresis activity,and decreased the changes of activities of extracellular and intracellular lactate dehydrogenase and ?-galactosidase before and after freezing.Moreover,it was found that SF-P2 cells were more completely and regularly shaped than other strains,displayed no obvious leakage of cell contents,and had an intact boundary between cells.These results indicated that the recombinant strain SF-P2 had a protective effect against freezing.