Intracellular Expression of Antifreeze Peptides in Food Grade Lactococcus lactis and Evaluation of Their Cryoprotective Activity

Antifreeze peptides can 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 also in food production. In this study, a recombinant SF-P gene was constructed and inserted into pNZ8149 to construct a food grade expression vector, which was then electroporated into Lactococcus lactis NZ3900. The expression of the target protein was induced using Nisin, and the optimal expression condition was determined to be a pH of 6.0, Nisin concentration of 25 ng/mL, temperature of 37 degrees C, and incubation time of 6 hr. Compared to the strain NZ3900 and the recombinant strain SF-P-1 without addition of Nisin, the recombinant strain SF-P-2 showed the highest cell survival and thermal hysteresis activity, and had a reduction in the changes of activitiesof extracellular and intracellular lactate dehydrogenase and -galactosidase after freezing. Moreover, analysis by SEM showed that SF-P-2 cells were more completely and regularly shaped than other strains, displayed no obvious leakage of cell contents, and had an intact boundary between cells after freezing. These results indicate that the recombinant strain SF-P-2 has a protective effect against freezing. This paper presents a food grade expression system for an antifreeze peptide SF-P using L. lactis as a host, and shows that the intracellular expression of antifreeze peptide could protect the cellular integrity and physiological functions of L. lactis.