罗伊氏粘液乳杆菌HCS02-001培养方法优化及改善睡眠功能研究

doi:10.19586/j.2095-2341.2023.0103

生物技术进展 ›› 2024, Vol. 14 ›› Issue (1): 133-140.DOI: 10.19586/j.2095-2341.2023.0103

罗伊氏粘液乳杆菌HCS02-001培养方法优化及改善睡眠功能研究

宋佳¹^,²(), 李雪龙³, 林欣梅¹^,², 余萍¹^,², 杨思程¹^,², 彭永振¹, 矫艳平¹^,²(), 陈雪娇¹^,²

^1.江西仁仁健康微生态科技有限公司，江西樟树 331200
^2.仁仁微生物科技研究（沈阳）有限公司，沈阳 110170
^3.杭州民生健康药业股份有限公司，杭州 310000

收稿日期:2023-09-18 接受日期:2023-09-12 出版日期:2024-01-25 发布日期:2024-02-05
通讯作者: 矫艳平
作者简介:宋佳 E-mail: 573147127@qq.com；

Study on Optimization of Culture Method and Improvement of Sleep Function of Limosilactobacillus reuteri HCS02-001

Jia SONG¹^,²(), Xuelong LI³, Xinmei LIN¹^,², Ping YU¹^,², Sicheng YANG¹^,², Yongzhen PENG¹, Yanping JIAO¹^,²(), Xuejiao CHEN¹^,²

^1.Jiangxi Renren Health Microecological Technology Co. ，Ltd. ，Jiangxi Zhangshu 331200，China
^2.Renren Microbial Technology Research （Shenyang） Co. ，Ltd. ，Shenyang 110170，China
^3.Hangzhou Minsheng Health Pharmaceutical Co. ，Ltd. ，Hangzhou 310000，China

Received:2023-09-18 Accepted:2023-09-12 Online:2024-01-25 Published:2024-02-05
Contact: Yanping JIAO

摘要/Abstract

摘要：

为了优化罗伊氏粘液乳杆菌(Limosilactobacillus reuteri)HCS02-001的培养方法，并探究其助睡眠功能，以γ-氨基丁酸（γ-aminobutyric acid，GABA）的产量为考察指标，通过单因素实验、响应面实验和小鼠实验进行条件筛选和功能评价。在培养温度37 ℃，培养时间24 h，初始pH 6.5时，罗伊氏粘液乳杆菌的GABA产量为5.58 g·L^-1。直接睡眠实验中小鼠均未出现直接睡眠现象，延长戊巴比妥钠催眠小鼠的睡眠时间实验中，高剂量组的小鼠睡眠时间有效延长，戊巴比妥钠阈下剂量催眠实验结果显示高剂量组的小鼠入睡率高达40%，巴比妥那睡眠潜伏期实验显示高剂量组的小鼠减少了睡眠潜伏期，可与巴比妥钠协同发挥作用。结果表明罗伊氏粘液乳杆菌HCS02-001具备助睡眠的作用。

关键词: 罗伊氏粘液乳杆菌, 单因素, 响应面, 助睡眠

Abstract:

In order to optimize the cultivation method of Limosilactobacillus reuteri HCS02001 and explore its sleep promoting function， the optimal cultivation was screened through single factor experiment and response surface experiment based on the γ-aminobutyric acid （GABA） yield，and its sleep promoting function was evaluated through mouse experiment. Under the condition of that culture temperature was 37 ℃， the culture time was 24 h and the initial pH was 6.5， the GABA yield of L. reuteri HCS02-001 was 5.58 g·L^-1. No direct sleep phenomenon occurred in mice in the direct sleep experiment. The experimental results of prolonging the sleep time of mice hypnotized by pentobarbital sodium showed that the mice in the high-dose group effectively prolonged the sleep time， and the results of pentobarbital sodium subthreshold dose hypnosis experiment showed that the sleep rate of mice in the high-dose group was as high as 40%. Barbiturate sleep latency experiments showed that mice in the high-dose group had reduced sleep latency and could act synergistically with barbiturate sodium. The results showed that L. reuteri HCS02-001 had the function of sleep aid.

Key words: Limosilactobacillus reuteri, single factor, response surface, sleep aid

中图分类号:

Q935

宋佳, 李雪龙, 林欣梅, 余萍, 杨思程, 彭永振, 矫艳平, 陈雪娇. 罗伊氏粘液乳杆菌HCS02-001培养方法优化及改善睡眠功能研究[J]. 生物技术进展, 2024, 14(1): 133-140.

Jia SONG, Xuelong LI, Xinmei LIN, Ping YU, Sicheng YANG, Yongzhen PENG, Yanping JIAO, Xuejiao CHEN. Study on Optimization of Culture Method and Improvement of Sleep Function of Limosilactobacillus reuteri HCS02-001[J]. Current Biotechnology, 2024, 14(1): 133-140.

图/表 10

表1 响应面各因素水平

Table 1 The levels of response surface factors

因素	水平
因素	-1	0	1
A：培养温度/℃	36	37	38
B：培养时间/h	22	24	26
C：初始pH	6.3	6.5	6.7

图1 培养温度对罗伊氏粘液乳杆菌HCS02-001中GABA产量的影响注：不同字母间代表组间在P<0.05水平上具有统计学意义（n=3)。

Fig. 1 Effect of culture temperature on GABA production by Limosilactobacillus reuteri HCS02-001

图2 培养时间对罗伊氏粘液乳杆菌HCS02-001中GABA产量的影响注：不同小写字母代表组间在P<0.05水平上具有统计学意义（n=3)。

Fig. 2 Effect of culture time on GABA production byLimosilactobacillus reuteri HCS02-001

图3 初始pH对罗伊氏粘液乳杆菌HCS02-001中GABA产量的影响注：不同小写字母代表组间在P<0.05水平上具有统计学意义（n=3)。

Fig. 3 Effect of initial pH on GABA production by Limosilactobacillus reuteri HCS02-001

表2 响应面设计方案和结果

Table 2 Response surface design scenarios and results

试验号	A：培养温度	B：培养时间	C：初始pH	GABA产量/（g·L^-1）
1	0	0	0	5.66
2	-1	-1	0	3.56
3	0	-1	1	4.22
4	1	1	0	3.61
5	1	-1	0	3.65
6	0	0	0	5.62
7	1	0	-1	3.92
8	0	0	0	5.59
9	-1	0	1	3.82
10	0	0	0	5.58
11	-1	0	-1	3.94
12	1	0	1	3.91
13	0	1	1	4.35
14	0	0	0	5.51
15	0	1	-1	4.52
16	-1	1	0	3.55
17	0	-1	-1	4.62

表3 回归模型方差分析

Table 3 Regression model ANOVA

项目	平方和	自由度	均方	F值	P
模型	10.77	9	1.20	219.95	<0.000 1
A	6.050×10^-3	1	6.050×10^-3	1.11	0.326 7
B	5.000×10^-5	1	5.000×10^-5	9.191×10^-3	0.926 3
C	0.061	1	0.061	11.26	0.012 2
AB	2.250×10^-4	1	2.250×10^-4	0.041	0.844 6
AC	3.025×10^-3	1	3.025×10^-3	0.56	0.480 1
BC	0.013	1	0.013	2.43	0.169 2
A²	6.74	1	6.74	1 238.07	<0.000 1
B²	2.27	1	2.27	417.85	<0.000 1
C²	0.78	1	0.78	142.95	<0.000 1
残差	0.038	7	5.440×10^-3
失拟项	0.026	3	8.600×10^-3	2.80	0.172 6
纯误差	0.012	4	3.070×10^-3
总离差	10.81	16

图4 培养时间、培养温度和初始pH对GABA产量的影响交互作用

Fig. 4 Interaction of culture time, culture temperature and initial pH on GABA production

图5 罗伊氏粘液乳杆菌HCS02-001对小鼠睡眠持续时间的影响（n=10）

Fig. 5 Effects of Limosilactobacillus reuteri HCS02-001 on sleep duration in mice（n=10）

表4 罗伊氏粘液乳杆菌HCS02-001对小鼠睡眠率的影响

Table 4 Effects of Limosilactobacillus reuteri HCS02-001 on sleep duration in mice

分组	小鼠数量/只	睡眠数量/只	入睡率/%
空白对照组	10	0	0b
GABA水溶液组	10	1	10b
低剂量组	10	1	10b
中剂量组	10	2	20b
高剂量组	10	4	40a

图6 巴比妥那小鼠睡眠潜伏实验注：不同字母表示组间在P<0.05水平上有统计学意义(n=10)

Fig. 6 Sleep latency experiment of barbital sodium mice

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罗伊氏粘液乳杆菌HCS02-001培养方法优化及改善睡眠功能研究

Study on Optimization of Culture Method and Improvement of Sleep Function of Limosilactobacillus reuteri HCS02-001

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