表达牛乳铁蛋白肽的重组乳酸乳球菌微胶囊制备工艺的优化

doi:10.16431/j.cnki.1671-7236.2021.12.029

中国畜牧兽医 ›› 2021, Vol. 48 ›› Issue (12): 4607-4617.doi: 10.16431/j.cnki.1671-7236.2021.12.029

表达牛乳铁蛋白肽的重组乳酸乳球菌微胶囊制备工艺的优化

张珺妍¹, 谭宏超¹, 陈佩佩², 王晓娜^1,3, 姜艳平^1,3, 崔文^1,3, 乔薪瑗^1,3, 王丽^1,3, 周晗^1,3, 徐义刚^1,3, 李一经^1,3, 唐丽杰^1,3

1. 东北农业大学动物医学学院, 哈尔滨 150030;
2. 江苏省宿迁市宿城区畜牧兽医站, 宿迁 223800;
3. 农业农村部动物疫病病原生物学东北科学观测实验站重点实验室, 哈尔滨 150030

修回日期:2021-05-19 出版日期:2021-12-20 发布日期:2021-12-02
通讯作者: 唐丽杰 E-mail:tanglijie@163.com
作者简介:张珺妍(1997-),女,内蒙古包头人,硕士生,研究方向:动物微生物学与免疫学,E-mail:1169043166@qq.com;谭宏超(1993-),男,山东青岛人,硕士生,研究方向:动物微生物学与免疫学,E-mail:996929204@qq.com
基金资助:
十三五重点研发计划（2017YFD0501000）

Optimization of Microcapsule Preparation Technology of Recombinant Lactococcus lactis Expressing Bovine Lactoferrin Peptide

ZHANG Junyan¹, TAN Hongchao¹, CHEN Peipei², WANG Xiaona^1,3, JIANG Yanping^1,3, CUI Wen^1,3, QIAO Xinyuan^1,3, WANG Li^1,3, ZHOU Han^1,3, XU Yigang^1,3, LI Yijing^1,3, TANG Lijie^1,3

1. College of Veterinary Medicine, Northeast Agricultural University, Harbin 150030, China;
2. Animal Husbandry and Veterinary Station, Sucheng District, Suqian City, Jiangsu Province, Suqian 223800, China;
3. Key Laboratory of Animal Disease Pathogen Biology, Northeast Scientific Observation and Experiment Station, Ministry of Agriculture and Rural Affairs, Harbin 150030, China

Revised:2021-05-19 Online:2021-12-20 Published:2021-12-02

摘要/Abstract

摘要： 为使重组乳酸乳球菌在发酵生产后便于存储、运输,本试验利用可表达牛乳铁蛋白肽的重组乳酸乳球菌pAMJ399-LFBA/MG1363制备微胶囊,优化微胶囊的制备工艺条件,并对胶囊化后重组菌的相关生物学特性进行检测。通过对不同壁材及壁材浓度进行控制单一变量试验,测定其包埋量,以筛选出最佳壁材及浓度。通过模拟胃肠液环境试验,比较微胶囊释放率,并对不同壁材进行保存期试验,比较最低活菌数,利用响应面试验确定最佳工艺条件,使用最佳工艺条件制备微胶囊后进行验证。优化后的结果为:选取海藻酸钠和壳聚糖作为壁材,在海藻酸钠浓度为2.49%,壳聚糖浓度为0.96%,CaCl₂浓度为6.67%,凝固时间为57 min的条件下微胶囊效果最佳,预测包埋量为7.85×10⁸ CFU/g。微胶囊在模拟胃液中稳定存在,在模拟肠液中可完全破裂,能释放出微胶囊内95%以上的乳酸乳球菌。海藻酸钠-壳聚糖微胶囊在4 ℃保存3周后,微胶囊中活菌数为1.41×10⁷ CFU/g。对微胶囊的最佳工艺条件验证结果为微胶囊的包埋量为8.08×10⁸ CFU/g;常温保存2周后,活菌数仍可达到3.89×10⁶ CFU/g;ELISA方法检测微胶囊内重组乳酸乳球菌可见表达的牛乳铁蛋白肽,抑菌试验可见微胶囊内重组乳酸乳球菌表达的牛乳铁蛋白肽对沙门氏菌和金黄色葡萄球菌仍具有明显的抑制作用。以上结果表明,表达牛乳铁蛋白肽的重组乳酸乳球菌可制备成为低成本、保存期长、耐胃液的微胶囊,为重组乳酸菌在生产中的进一步应用奠定基础。

关键词: 重组乳酸乳球菌; 乳铁蛋白肽; 微胶囊; 工艺优化

Abstract: In order to facilitate the storage and transportation of recombinant Lactococcus lactis after fermentation, microcapsules were prepared by recombinant Lactococcus lactis pAMJ399-LFBA/MG1363, which could express lactoferrin peptide.The preparation conditions of microcapsules were optimized, and the related biological characteristics of the encapsulated recombinant bacteria were detected.In order to select the best wall material and its concentration, the embedding amount was measured by controlling the single variable experiment of different wall materials and wall material concentrations.The release rate of microcapsules was compared through simulated gastric and intestinal fluid environment experiment, and the shelf life experiment of different wall materials was carried out to compare the lowest viable bacteria number.The response surface experiment was used to determine the optimal process conditions, and the microcapsules were prepared under the optimal process conditions for verification.The optimized results were as follows:Sodium alginate and chitosan were finally selected as wall materials.Under the conditions of sodium alginate was 2.49%, chitosan was 0.96%, CaCl₂ was 6.67% and solidification time was 57 min, the microcapsule effect was the best, and the prediction embedding amount was 7.85×10⁸ CFU/g.Microcapsules existed stably in simulated gastric juice, and could completely break in simulated intestinal juice, and could release more than 95% of lactic acid bacteria in microcapsules.After the sodium alginate-chitosan microcapsules were stored at 4 ℃ for 3 weeks, the number of viable bacteria in microcapsules was 1.41×10⁷ CFU/g.The results showed that the embedding amount of microcapsules was 8.08×10⁸ CFU/g.After being stored at room temperature for 2 weeks, the viable bacteria count could still reach 3.89×10⁶ CFU/g.The lactoferrin peptide expressed by recombinant Lactococcus lactis in microcapsules was detected by ELISA, and the antibacterial assay showed that the lactoferrin peptide expressed by recombinant Lactobacillus lactis in microcapsules still had significant inhibitory effect on Salmonella and Staphylococcus aureus.These results indicated that the recombinant Lactococcus lactis expressing lactoferrin peptide could be prepared into microcapsules with low cost, long shelf life and gastric juice resistance, which laid a foundation for the further application of recombinant lactic acid bacteria in production.

Key words: recombinant Lactococcus lactis; lactoferrin peptide; microcapsules; optimization of process

中图分类号:

S852.61

张珺妍, 谭宏超, 陈佩佩, 王晓娜, 姜艳平, 崔文, 乔薪瑗, 王丽, 周晗, 徐义刚, 李一经, 唐丽杰. 表达牛乳铁蛋白肽的重组乳酸乳球菌微胶囊制备工艺的优化[J]. 中国畜牧兽医, 2021, 48(12): 4607-4617.

ZHANG Junyan, TAN Hongchao, CHEN Peipei, WANG Xiaona, JIANG Yanping, CUI Wen, QIAO Xinyuan, WANG Li, ZHOU Han, XU Yigang, LI Yijing, TANG Lijie. Optimization of Microcapsule Preparation Technology of Recombinant Lactococcus lactis Expressing Bovine Lactoferrin Peptide[J]. China Animal Husbandry and Veterinary Medicine, 2021, 48(12): 4607-4617.

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表达牛乳铁蛋白肽的重组乳酸乳球菌微胶囊制备工艺的优化

Optimization of Microcapsule Preparation Technology of Recombinant Lactococcus lactis Expressing Bovine Lactoferrin Peptide

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