基于响应面法的粘质沙雷氏菌BRC-CXG2发酵培养基优化

doi:10.13304/j.nykjdb.2022.0422

中国农业科技导报 ›› 2022, Vol. 24 ›› Issue (8): 133-143.DOI: 10.13304/j.nykjdb.2022.0422

• 动植物健康 • 上一篇

基于响应面法的粘质沙雷氏菌BRC-CXG2发酵培养基优化

李金荣¹(), 周彤², 林一琪¹, 黄祚骅¹, 盛亮菁¹, 张飞萍¹, 吴松青³()

^1.福建农林大学林学院, 福州 350002
^2.福建农林大学生命科学学院, 福州 350002
^3.福建农林大学生态公益林重大有害生物防控福建省高校重点实验室, 福州 350002

收稿日期:2022-05-19 接受日期:2022-06-20 出版日期:2022-08-15 发布日期:2022-08-22
通讯作者: 吴松青
作者简介:李金荣 E-mail：jinrongLi@fafu.edu.cn；
基金资助:
国家林业和草原局重大应急科技项目(2D202001);国家自然科学基金项目(U1905201);福建省林业科技重点项目(2021FKJ03);福建省自然科学基金项目(2019J05042)

Optimization of Serratia marcescens BRC-CXG2 Fermentation Medium Based on Response Surface Methodology

Jinrong LI¹(), Tong ZHOU², Yiqi LIN¹, Zuohua HUANG¹, Liangjing SHENG¹, Feiping ZHANG¹, Songqing WU³()

^1.College of Forestry，Fujian Agriculture and Forestry University，Fuzhou 350002，China
^2.College of Life Science，Fujian Agriculture and Forestry University，Fuzhou 350002，China
^3.Key Laboratory of Integrated Pest Management in Ecological Forests of Fujian Provincial University，Fujian Agriculture and Forestry University，Fuzhou 350002，China

Received:2022-05-19 Accepted:2022-06-20 Online:2022-08-15 Published:2022-08-22
Contact: Songqing WU

摘要/Abstract

摘要：

为了将粘质沙雷氏菌（Serratia marcescens）应用在松墨天牛（Monochamus alternatus）防治上，对粘质沙雷氏菌的培养基配方进行了优化。通过单因素试验筛选粘质沙雷氏菌发酵培养基的碳源、氮源和无机盐，利用Plackett-Burman试验设计确定3个主要影响因素，通过最陡爬坡试验，结合Box-Behnken Design试验设计及响应面分析法优化培养基组成成分，获得培养基最佳配方与发酵条件：葡萄糖23.27 g·L^-1、蚕蛹粉22.37 g·L^-1、酵母粉10 g·L^-1、硫酸镁2 g·L^-1、磷酸氢二钠3 g·L^-1，氯化钠2 g·L^-1；在28 ℃、150 r·min^-1条件下发酵36 h，装液量体积分数为50 mL/250 mL，接种量4.73 %，pH 7.1~7.3。最后，根据最优培养条件验证发现优化后培养基中生长的菌落数为6.0×10⁹cfu·mL^-1，是初始培养基的9.42倍，与模型预测值相符。研究结果为粘质沙雷氏菌的发酵生产提供了理论基础，为松材线虫病（pine wilt disease）的防治提供了新的途径。

关键词: 粘质沙雷氏菌, 单因素试验, 菌落数, 响应面分析, 松材线虫病

Abstract:

In order to develop the application of Serratia marcescens in the control of Monochamus alternatus， the medium formulation of Serratia marcescens was optimized in this study. The carbon source， nitrogen source and inorganic salt of S. marcescens fermentation medium were screened by single factor test， and 3 main influencing factors were determine by Plackett-burman design. The composition of the medium was optimized by the steepest climbing test combined with Box-Behnken Design and response surface analysis， and the optimal medium formula and fermentation conditions were obtained： glucose 23.27 g·L^-1， soybean cake meal 22.37 g·L^-1， yeast extract 10 g·L^-1， MgSO₄ 2 g·L^-1， Na₂HPO₄ 3 g·L^-1， NaCl 2 g·L^-1. At 28 ℃， 150 r·min^-1 for 36 h， the volume fraction of liquid was 50 mL/250 mL， the inoculation amount was 4.73 %， pH 7.1~7.3. Finally， according to the optimal culture conditions， the number of bacteria growing in the optimized medium was 6.0×10⁹cfu·mL^-1， 9.42 times of the initial medium， which was consistent with the predicted value of the model. These results provided theoretical basis for the fermentation production of S. marcescens and provide a new approach for the control of pine wilt disease.

Key words: Serratia marcescens, single factor test, colony number, response surface analysis, pine wilt disease

中图分类号:

S476+.11

李金荣, 周彤, 林一琪, 黄祚骅, 盛亮菁, 张飞萍, 吴松青. 基于响应面法的粘质沙雷氏菌BRC-CXG2发酵培养基优化[J]. 中国农业科技导报, 2022, 24(8): 133-143.

Jinrong LI, Tong ZHOU, Yiqi LIN, Zuohua HUANG, Liangjing SHENG, Feiping ZHANG, Songqing WU. Optimization of Serratia marcescens BRC-CXG2 Fermentation Medium Based on Response Surface Methodology[J]. Journal of Agricultural Science and Technology, 2022, 24(8): 133-143.

图/表 11

参考文献 21

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因素 Factor	水平 Level
因素 Factor	低 Low（-1）	高 High（+1）
X₁∶油酸 Oleic acid/（g·L^-1）	10	15
X₂∶葡萄糖 Glucose/（g·L^-1）	10	12
X₃∶虚拟项 Virtual items	/	/
X₄∶蚕蛹粉 Soybean cake meal/（g·L^-1）	8	12
X₅∶酵母粉 Yeast extract/（g·L^-1）	8	12
X₆∶虚拟项 Virtual items	/	/
X₇∶硫酸镁 MgSO₄/（g·L^-1）	1	2
X₈∶磷酸氢二钠 Na₂HPO₄/（g·L^-1）	2	3
X₉∶虚拟项 Virtual items	/	/
X₁₀∶接种量 Inoculatin amount/ %	1	2
X₁₁∶装液量 Liquid volume /（mL/250 mL）	50	100

因素 Factor	水平 Level
因素 Factor	低 Low（-1）	高 High（+1）
X₁∶油酸 Oleic acid/（g·L^-1）	10	15
X₂∶葡萄糖 Glucose/（g·L^-1）	10	12
X₃∶虚拟项 Virtual items	/	/
X₄∶蚕蛹粉 Soybean cake meal/（g·L^-1）	8	12
X₅∶酵母粉 Yeast extract/（g·L^-1）	8	12
X₆∶虚拟项 Virtual items	/	/
X₇∶硫酸镁 MgSO₄/（g·L^-1）	1	2
X₈∶磷酸氢二钠 Na₂HPO₄/（g·L^-1）	2	3
X₉∶虚拟项 Virtual items	/	/
X₁₀∶接种量 Inoculatin amount/ %	1	2
X₁₁∶装液量 Liquid volume /（mL/250 mL）	50	100

方差来源 Source	平方和 Sum of squares	自由度 Degree of freedom	均方 Mean square	F值 F value	P值 P value
模型 Model	40 471.24	8	5 058.91	22.87	0.013 1
油酸 Oleic acid	2 691.61	1	2 691.61	12.17	0.039 8
葡萄糖 Glucose	10 783.21	1	10 783.21	48.75	0.006 0
蚕蛹粉 Soybean cake meal	17 414.54	1	17 414.54	77.50	0.003 1
酵母粉 Yeast extract	1 384.17	1	1 384.17	6.26	0.087 6
硫酸镁 MgSO₄	176.95	1	176.95	0.80	0.437 0
磷酸氢二钠 Na₂HPO₄	2 072.97	1	2 072.97	9.37	0.054 9
接种量 Inoculatin amount	5 837.96	1	5 837.96	26.39	0.014 3
装液量 Liquid volume	382.84	1	382.84	1.73	0.279 8
残差Residual	663.55	3	221.18	—	—
总和Cor Total	41 134.80	11	—	—	—

方差来源 Source	平方和 Sum of squares	自由度 Degree of freedom	均方 Mean square	F值 F value	P值 P value
模型 Model	40 471.24	8	5 058.91	22.87	0.013 1
油酸 Oleic acid	2 691.61	1	2 691.61	12.17	0.039 8
葡萄糖 Glucose	10 783.21	1	10 783.21	48.75	0.006 0
蚕蛹粉 Soybean cake meal	17 414.54	1	17 414.54	77.50	0.003 1
酵母粉 Yeast extract	1 384.17	1	1 384.17	6.26	0.087 6
硫酸镁 MgSO₄	176.95	1	176.95	0.80	0.437 0
磷酸氢二钠 Na₂HPO₄	2 072.97	1	2 072.97	9.37	0.054 9
接种量 Inoculatin amount	5 837.96	1	5 837.96	26.39	0.014 3
装液量 Liquid volume	382.84	1	382.84	1.73	0.279 8
残差Residual	663.55	3	221.18	—	—
总和Cor Total	41 134.80	11	—	—	—

编号 Number	葡萄糖 Glucose/（g·L^-1）	蚕蛹粉 Soybean cake meal/（g·L^-1）	接种量 Inoculatin amount/%	菌落数 Number of bacterial colonies/（10⁸cfu·mL^-1）
1	16	20	3.0	13.73
2	18	22	3.5	21.00
3	20	24	4.0	31.77
4	22	26	4.5	34.37
5	24	28	5.0	20.93
6	26	30	5.5	13.13

基于响应面法的粘质沙雷氏菌BRC-CXG2发酵培养基优化

Optimization of Serratia marcescens BRC-CXG2 Fermentation Medium Based on Response Surface Methodology

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图/表 11

参考文献 21

相关文章 5

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Metrics

编号 Number	X₁	X₂	X₃	X₄	X₅	X₆	X₇	X₈	X₉	X₁₀	X₁₁	菌落数 Number of bacterial colonies/（10⁸ cfu·mL^-1）
1	1	1	-1	1	1	1	-1	-1	-1	1	-1	15.133
2	-1	1	1	-1	1	1	1	-1	-1	-1	1	7.022
3	1	-1	1	1	-1	1	1	1	-1	-1	-1	3.980
4	-1	1	-1	1	1	-1	1	1	1	-1	-1	13.367
5	-1	-1	1	-1	1	1	-1	1	1	1	-1	1.633
6	-1	-1	-1	1	-1	1	1	-1	1	1	1	16.900
7	1	-1	-1	-1	1	-1	1	1	-1	1	1	1.637
8	1	1	-1	-1	-1	1	-1	1	1	-1	1	4.533
9	1	1	1	-1	-1	-1	1	-1	1	1	-1	10.967
10	-1	1	1	1	-1	-1	-1	1	-1	1	1	18.533
11	1	-1	1	1	1	-1	-1	-1	1	-1	1	6.333
12	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	3.100

编号 Number	葡萄糖 Glucose/（g·L^-1）	蚕蛹粉 Soybean cake meal/（g·L^-1）	接种量 Inoculatin amount/%	Y：菌落数 Number of bacterial colonies/（10⁸cfu·mL^-1）
1	15	20	5	10.5
2	25	20	5	49.2
3	15	30	5	24.9
4	25	30	5	29.7
5	15	25	2.5	3.0
6	25	25	2.5	32.0
7	15	25	7.5	8.1
8	25	25	7.5	34.7
9	20	20	2.5	37.8
10	20	30	2.5	5.4
11	20	20	7.5	20.0
12	20	30	7.5	26.5
13	20	25	5	44.3
14	20	25	5	47.6
15	20	25	5	50.4
16	20	25	5	59.5
17	20	25	5	55.5

方差来源 Source	平方和 Sum of squares	自由度 Degree of freedom	均方 Mean square	F值 F value	P值 P value	显著性 Significance
模型 Model	4 965.87	9	551.76	17.31	0.000 5	**
X₁	1 227.60	1	1 227.60	38.51	0.000 4	**
X₄	120.13	1	120.13	3.77	0.093 4
X₁₀	15.40	1	15.40	0.48	0.509 4
X₁X₄	287.30	1	287.30	9.01	0.019 9	*
X₁X₁₀	1.44	1	1.44	0.045	0.837 8
X₄X₁₀	378.30	1	378.30	11.87	0.010 8	*
X₁²	703.94	1	703.94	22.08	0.002 2	**
X₄²	417.27	1	417.27	13.09	0.008 5	**
X₁₀²	1 532.83	1	1 532.83	48.08	0.000 2	**
残差 Residual	223.16	7	31.88
失拟项 Lack of fit	74.91	3	24.97	0.67	0.611 7
纯误差 Pure error	148.25	4	37.06
总和Cor total	5 189.04	16
R²	0.957 0
R_adj²	0.901 7

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