Dept. of Food Science and Engineering, School of Agriculture and Biology, Shanghai Jiao Tong Univ., Shanghai, 200240, China.
College of Biological Science and Technology, Fuzhou Univ., Fuzhou, 350108, China.
J Food Sci. 2018 May;83(5):1311-1320. doi: 10.1111/1750-3841.14117. Epub 2018 Apr 16.
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 °C, and incubation time of 6 hr. Compared to the strain NZ3900 and the recombinant strain SF-P without addition of Nisin, the recombinant strain SF-P showed the highest cell survival and thermal hysteresis activity, and had a reduction in the changes of activities of extracellular and intracellular lactate dehydrogenase and β-galactosidase after freezing. Moreover, analysis by SEM showed that SF-P 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 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.
The recombinant Lactococcus lactis with intracellular expression of antifreeze peptides SF-P could reduce the damage of bacteria cells induced by freezing or freeze drying, so, it could be applied in the process of freezing food without separation, such as the manufacture of yoghurt ice cream, frozen dough, and so on.
抗冻肽可以通过防止冰晶在细胞之间形成而导致的细胞损伤或死亡来保护生物体免受低温影响。因此，抗冻肽可用作细胞和组织低温保护剂，也可用于食品生产。本研究构建了重组 SF-P 基因并插入 pNZ8149 中，构建了食品级表达载体，然后电转化入 Lactococcus lactis NZ3900。使用乳链菌肽诱导目标蛋白表达，确定了最佳表达条件为 pH6.0、乳链菌肽浓度 25ng/mL、温度 37°C、孵育时间 6 小时。与 NZ3900 菌株和未添加乳链菌肽的重组菌 SF-P 相比，重组菌 SF-P 表现出最高的细胞存活率和热滞活性，并且在冷冻后细胞外和细胞内乳酸脱氢酶和β-半乳糖苷酶活性的变化减少。此外，SEM 分析表明，SF-P 细胞比其他菌株更完整和规则，细胞内容物无明显泄漏，细胞间边界完整。这些结果表明重组菌 SF-P 具有抗冻作用。本文介绍了一种使用 Lactococcus lactis 作为宿主的抗冻肽 SF-P 的食品级表达系统，并表明抗冻肽的细胞内表达可以保护 Lactococcus lactis 的细胞完整性和生理功能。
具有细胞内表达抗冻肽 SF-P 的重组乳球菌可以减少细菌细胞因冷冻或冷冻干燥而受到的损伤，因此可应用于无需分离的冷冻食品过程中，如酸奶冰淇淋、冷冻面团等的制造。
