Abstract
In order to optimize the expression of antifreeze protein SF-P, using Nisin as the inducer to induced the expression of recombinant Lactococcus lactis. The expression conditions of induction time, induced pH, induced temperature and inducer Nisin concentration were optimized. The optimal expression conditions were determined by SDS-PAGE and Western blot. Furthermore, the physiological characteristics of recombinant bacteria under freezing stress were investigated by comparing the growth status, the acid production of bacteria and the changes of sodium and potassium contents before and after freezing stress. The results showed that the optimal expression conditions were determined: pH of 7.0, Nisin concentration of 15 ng/mL, temperature of 25 ℃, and incubation time of 6 h. The recombinant strain SF-P2 could significantly improved the growth of Lactococcus lactis in the logarithmic phase and the stable growth lag following freezing, and had a significant improvement in fermentation of L. lactis following freezing. It also could effectively reduced the effect of freezing stress on the permeability of cell membrane, which played a role in protecting the physiological function of cells. The results showed that the recombinant strain SF-P2 had significant protective effects against cold stress.
Publication Date
10-28-2017
First Page
123
Last Page
127,144
DOI
10.13652/j.issn.1003-5788.2017.10.028
Recommended Citation
Li, ZHANG; Xinyang, HUANG; Tianyi, MA; Jinhong, WU; Shaoyun, WANG; and Zhengwu, WANG
(2017)
"Expression of recombinant antifreeze peptide in Lactococcus lactis and its antifreeze activity,"
Food and Machinery: Vol. 33:
Iss.
10, Article 28.
DOI: 10.13652/j.issn.1003-5788.2017.10.028
Available at:
https://www.ifoodmm.cn/journal/vol33/iss10/28
References
[1] HASSAS-ROUDSARI M, GOFF H D. Ice structuring proteins from plants: mechanism of action and food application[J]. Food Research International, 2012, 46(1): 425-436.
[2] 赵金红, 胡锐, 刘冰, 等. 几种冷冻新技术对食品冻结过程中冰晶形成的影响[J]. 食品与机械, 2012, 28(6): 241-245.
[3] 张莉, 王晨笑, 陈姗姗, 等. 丝胶抗冻肽在马铃薯冷冻馒头中的应用研究[J]. 食品工业科技, 2017, 38(11): 49-54.
[4] 李向红, 刘忠祥, 邓海萍, 等. 鲢鱼酶解产物在冷冻鱼糜中的抗冻性能研究[J]. 食品与机械, 2015, 31(3): 125-130.
[5] 权国波, 吴国权, 吕春荣, 等. 抗冻蛋白对山羊精子冷冻保护效果的分析[J]. 中国畜牧杂志, 2017, 53(3): 53-57.
[6] DOXEY A C, YAISH M W, GRIFFITH M, et al. Ordered surface carbons distinguish antifreeze proteins and their ice-binding regions[J]. Nature Biotechnology, 2006, 24(7): 852-855.
[7] 张晖, 丁香丽. 抗冻蛋白在食品中应用研究进展及安全性分析[J]. 食品与生物技术学报, 2012, 31(5): 455-461.
[8] YEH C M, KAO B Y, PENG Hsuan-jung. Production of a recombinant type 1 antifreeze protein analogue by L. lactis and its applications on frozen meat and frozen dough[J]. Journal of Agricultural & Food Chemistry, 2009, 57(14): 6 216-6 223.
[9] LI Ling-ling, YANGSOO K, HUANG Wei-ning, et al. Effects of ice structuring proteins on freeze-thaw stability of corn and wheat starch gels[J]. Cereal Chemistry, 2010, 87(5): 497-503.
[10] AND V K, GOFF H D, KASAPIS S. Effect of Aging and Ice Structuring Proteins on the Morphology of Frozen Hydrated Gluten Networks[J]. Biomacromolecules, 2007, 8(4): 1 293-1 299.
[11] UHLIG C, KABISCH J, PALM G J, et al. Heterologous expression, refolding and functional characterization of two antifreeze proteins from Fragilariopsis cylindrus (Bacillariophyceae)[J]. Cryobiology, 2011, 63(3): 220-228.
[12] MOK Y F, LIN F H, GRAHAM L A, et al. Structural basis for the superior activity of the large isoform of snow flea antifreeze protein[J]. Biochemistry, 2010, 49(11): 2 593-2 603.
[13] 吕绘倩, 蒋洁兰, 姜志强, 等. 太平洋鳕抗冻基因AFP4的原核表达及多克隆抗体的制备[J]. 大连海洋大学学报, 2017, 32(2): 127-133.
[14] 苏松坤, 晏励民, 刘芳. 乳酸菌食品级表达系统的研究进展[J]. 食品与生物技术学报, 2012, 31(12): 1 233-1 238.
[15] 崔月倩, 王菁蕊, 王艳萍. 乳酸菌基因表达载体及其应用研究进展[J]. 食品科学, 2015, 36(9): 224-229.
[16] 谭嘉圣. 人源及杂合抗菌肽在乳酸乳球菌中重组分泌表达[D]. 广州: 南方医科大学, 2015: 217-230.
[17] 朱东升. 乳酸菌冻干保活关键技术研究[D]. 杭州: 浙江大学, 2010: 52-69.
[18] CARVALHO A L, CARDOSO F S, BOHN A, et al. Engineering trehalose synthesis in Lactococcus lactis for improved stress tolerance[J]. Applied and Environmental Microbiology, 2011, 77(12): 4 189-4 199.
[19] PENTELUTE B L, GATES Z P, TERESHKO V, et al. X-ray structure of snow flea antifreeze protein determined by racemic crystallization of synthetic protein enantiomers[J]. Journal of the American Chemical Society, 2008, 130: 9 695-9 701.
[20] 陈帅印. 幽门螺杆菌基因在乳球菌中食品级表达及免疫反应性[D]. 郑州: 郑州大学, 2010: 45-52.
[21] CAVANAGH D, FITZGERALD G F, MCAULIFFE O. From field to fermentation: the origins of Lactococcus lactis and its domestication to the dairy environment[J]. Food Microbiology, 2015, 47: 45-61.
[22] 周方方, 吴正钧, 艾连中, 等. 蛋白组学技术在乳酸菌环境胁迫应激研究中的应用[J]. 食品与发酵工业, 2012, 38(8): 101-106.
[23] 贺松, 龚芳红, 张德纯, 等. 乳酸链球菌素对乳酸菌抑菌作用的研究[J]. 食品科学, 2009, 30(23): 352-355.
[24] 蔡鲁峰, 杜莎, 谭雅, 等. 乳酸菌肉品发酵剂的发酵特性研究[J]. 食品工业科技, 2015, 36(17): 150-156.
[25] 杨俊俊. 西藏牦牛奶渣中微生物的分离鉴定及优良乳酸菌的筛选[D]. 咸阳: 西北农林科技大学, 2014: 62-70.
[26] HANSEN G, JOHANSEN C L, MARTEN G, et al. Influence of extracellular pH on growth, viability, cell size, acidification activity, and intracellular pH of Lactococcus lactis, in batch fermentations[J]. Applied Microbiology & Biotechnology, 2016, 100(13): 1-12.
[27] BISCHOF J C, WOLKERS W F, TSVETKOVA N M, et al. Lipid and protein changes due to freezing in Dunning AT-1 cells[J]. Cryobiology, 2002, 45(1): 22-32.
[28] 吴文茹, 汪政煜, 范梦茹, 等. 乳酸菌的抗冷冻性及冻干保护[J]. 食品工业, 2017, 38(5): 246-249.
[29] 王学良, 韩雪, 王海娟, 等. 乳酸菌在各种胁迫下的应激反应研究进展[J]. 食品工业科技, 2015, 36(6): 365-369.