枯草芽孢杆菌沙漠亚种kc-16培养条件的优化

doi:10.3969/j.issn.1004-1524.20240710

浙江农业学报 ›› 2025, Vol. 37 ›› Issue (12): 2563-2573.DOI: 10.3969/j.issn.1004-1524.20240710

枯草芽孢杆菌沙漠亚种kc-16培养条件的优化

杨学宇¹^,²(), 牛丽¹, 谭琳³, 邓玉莲¹, 包强⁴^,^*(), 胡秋龙¹^,^*()

1.湖南农业大学园艺学院,湖南长沙 410128
2.湖南省烟草公司郴州市公司,湖南郴州 423000
3.湖南农业大学植物保护学院,湖南长沙 410128
4.湖南省农业科学院茶叶研究所,湖南长沙 410125

收稿日期:2024-08-06 出版日期:2025-12-25 发布日期:2026-01-09
作者简介:杨学宇(1998—),男,山西大同人,硕士,从事茶树病虫害生物防治研究。E-mail:1743501922@qq.com
通讯作者: *包强,E-mail:baoqiang0703@126.com;胡秋龙,E-mail:huqiulongnet@126.com
基金资助:
湖南省重点研发计划(2022NK2051);湖南省重点研发计划(2023NK2013);湖南省科技创新重大项目(2021NK1020);国家重点研发计划项目(2022YFD1600800)

Optimization of the culture conditions of Bacillus subtilis subsp. inaquosorum kc-16

YANG Xueyu¹^,²(), NIU Li¹, TAN Lin³, DENG Yulian¹, BAO Qiang⁴^,^*(), HU Qiulong¹^,^*()

1. College of Horticulture, Hunan Agricultural University, Changsha 410128, China
2. Chenzhou Branch of Hunan Tobacco Company, Chenzhou 423000, Hunan, China
3. College of Plant Protection, Hunan Agricultural University, Changsha 410128, China
4. Tea Research Institute, Hunan Academy of Agricultural Sciences, Changsha 410125, China

Received:2024-08-06 Online:2025-12-25 Published:2026-01-09

摘要/Abstract

摘要： 为优化枯草芽孢杆菌沙漠亚种(Bacillus subtilis subsp. inaquosorum)菌株kc-16的培养条件,提高其生物量与抑菌活性,本研究通过单因素试验初步筛选最适碳源、氮源、无机盐种类及其对应的质量分数范围,进一步采用Plackett-Burman试验确定影响菌液D₆₀₀(600 nm处的吸光度)的关键因子。在此基础上,以菌液D₆₀₀为响应值,通过Box-Behnken设计构建显著因子与生物量的非线性方程,预测并验证菌株kc-16的最佳培养条件。结果表明,最优培养基组分(质量分数)为0.5%乳糖、4.0%蛋白胨、0.1%硫酸镁、0.6%氯化钠、0.2%碳酸钙;培养条件为转速200 r·min^-1、初始pH值7.7、温度30 ℃、接种量0.6%(体积比)。在此条件下,模型预测的发酵液D₆₀₀为0.112,实测值为0.307,二者吻合较好。平板计数法结果显示,优化后的活菌数为6.7×10⁹ CFU·mL^-1,是优化前(4.0×10⁸ CFU·mL^-1)的16.75倍。本研究通过单因素试验结合响应面法实现了菌株kc-16培养条件的优化,为该菌株的规模化生产与应用提供支持。

关键词: 枯草芽孢杆菌沙漠亚种, 培养基, 响应面法, 发酵优化

Abstract:

This study aimed to optimize the culture conditions of Bacillus subtilis subsp. inaquosorum kc-16 to enhance its biomass and antimicrobial activity. The optimal carbon source, nitrogen source, types of inorganic salts, and their corresponding mass fraction ranges were preliminarily screened by single-factor experiments. Subsequently, the Plackett-Burman experiment was performed based on the D₆₀₀ (absorbance at 600 nm) as an important factor from the single-factor screening results, a non-linear equation between significant factors and biomass was fitted and solved with Box-Behnken Design to predict the optimal culture conditions for strain kc-16, and the predicted results were validated. Results showed that the optimized culture medium components (mass fraction) were determined to be 0.5% lactose, 4.0% peptone, 0.1% magnesium sulfate, 0.6% sodium chloride, and 0.2% calcium carbonate. The optimal culture conditions were identified as 200 r·min^-1 agitation speed, initial pH value of 7.7, temperature of 30 ℃, and inoculation amount of 0.6%. Under these conditions, the model predicted an D₆₀₀ value of 0.112, which closely matched with the validated value of 0.307. Plate count results indicated that the viable cell count after optimization reached 6.7×10⁹ CFU·mL^-1, which was 16.75 times that the initial count (4.0×10⁸ CFU·mL^-1). This study achieved optimized culture conditions for strain kc-16 through a combination of single-factor experiments and response surface methodology, providing support for its large-scale production and application.

Key words: Bacillus subtilis subsp. inaquosorum, culture medium, response surface methodology, fermentation optimization

中图分类号:

S435.711

杨学宇, 牛丽, 谭琳, 邓玉莲, 包强, 胡秋龙. 枯草芽孢杆菌沙漠亚种kc-16培养条件的优化[J]. 浙江农业学报, 2025, 37(12): 2563-2573.

YANG Xueyu, NIU Li, TAN Lin, DENG Yulian, BAO Qiang, HU Qiulong. Optimization of the culture conditions of Bacillus subtilis subsp. inaquosorum kc-16[J]. Acta Agriculturae Zhejiangensis, 2025, 37(12): 2563-2573.

图/表 10

参考文献 29

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水平Level	A/%	B/%	C/%	D/%	E/%	F	G/(r·min^-1)	H/℃	I/%
高水平	1.0	5	0.2	0.8	0.4	8	220	32	1.0
低水平	0.5	3	0.1	0.4	0.1	7	180	28	0.5

水平Level	A/%	B/%	C/%	D/%	E/%	F	G/(r·min^-1)	H/℃	I/%
高水平	1.0	5	0.2	0.8	0.4	8	220	32	1.0
低水平	0.5	3	0.1	0.4	0.1	7	180	28	0.5

组别Group	A/%	B/%	C/%	D/%	E/%	F	G/(r·min^-1)	H/℃	I/%	D₆₀₀
1	1.0	5	0.1	0.8	0.4	8	180	28	0.5	1.68
2	0.5	5	0.2	0.4	0.4	8	220	28	0.5	0.32
3	1.0	3	0.2	0.8	0.1	8	220	32	0.5	0.10
4	0.5	5	0.1	0.8	0.4	7	220	32	1.0	0.16
5	0.5	3	0.2	0.4	0.4	8	180	32	1.0	0.80
6	0.5	3	0.1	0.8	0.1	8	220	28	1.0	0.18
7	1.0	3	0.1	0.4	0.4	7	220	32	0.5	1.90
8	1.0	5	0.1	0.4	0.1	8	180	32	1.0	0.33
9	1.0	5	0.2	0.4	0.1	7	220	28	1.0	1.98
10	0.5	5	0.2	0.8	0.1	7	180	32	0.5	1.97
11	1.0	3	0.2	0.8	0.4	7	180	28	1.0	1.89
12	0.5	3	0.1	0.4	0.1	7	180	28	0.5	1.77

组别Group	A/%	B/%	C/%	D/%	E/%	F	G/(r·min^-1)	H/℃	I/%	D₆₀₀
1	1.0	5	0.1	0.8	0.4	8	180	28	0.5	1.68
2	0.5	5	0.2	0.4	0.4	8	220	28	0.5	0.32
3	1.0	3	0.2	0.8	0.1	8	220	32	0.5	0.10
4	0.5	5	0.1	0.8	0.4	7	220	32	1.0	0.16
5	0.5	3	0.2	0.4	0.4	8	180	32	1.0	0.80
6	0.5	3	0.1	0.8	0.1	8	220	28	1.0	0.18
7	1.0	3	0.1	0.4	0.4	7	220	32	0.5	1.90
8	1.0	5	0.1	0.4	0.1	8	180	32	1.0	0.33
9	1.0	5	0.2	0.4	0.1	7	220	28	1.0	1.98
10	0.5	5	0.2	0.8	0.1	7	180	32	0.5	1.97
11	1.0	3	0.2	0.8	0.4	7	180	28	1.0	1.89
12	0.5	3	0.1	0.4	0.1	7	180	28	0.5	1.77

方差来源 Soruce	平方和 Sum of squares	自由度 Degree of freedom	均方差 Mean square	F值 F value	p	标准化效应 Standardization effect	系数 Coefficient	贡献率 Contribution rate/%	重要性排序 Importance rank
A	0.145 200	1	0.145 200	256.235 300	0.003 880	-0.220 000	-0.110 000	14.508 390	4
B	0.003 333	1	0.003 333	5.882 353	0.136 132	-0.033 330	-0.016 670	0.333 067	8
C	0.090 133	1	0.090 133	159.058 800	0.006 228	0.173 333	0.086 667	9.006 128	6
D	0.104 533	1	0.104 533	184.470 600	0.005 377	-0.186 670	-0.093 330	10.444 980	5
E	0.014 700	1	0.014 700	25.941 180	0.036 454	0.070 000	0.035 000	1.468 825	7
F	0.252 300	1	0.252 300	445.235 300	0.002 238	0.290 000	0.145 000	25.209 830	1
G	0.003 333	1	0.003 333	5.882 353	0.136 132	0.033 333	0.016 667	0.333 067	9
H	0.172 800	1	0.172 800	304.941 200	0.003 263	0.240 000	0.120 000	17.266 190	3
I	0.213 333	1	0.213 333	376.470 600	0.002 646	0.266 667	0.133 333	21.316 280	2
残差Residual	0.001 133	2	0.000 567	—	—	—	—	—	—
总离差平方和	1.000 798	11	—	—	—	—	—	—	—
Cor Total
模型Model	0.999 665	9	0.111 074	196.013 100	0.005 086	Significant	—	—	—

枯草芽孢杆菌沙漠亚种kc-16培养条件的优化

Optimization of the culture conditions of Bacillus subtilis subsp. inaquosorum kc-16

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组别Group	F	I/%	H/℃	D₆₀₀
1	7.0	0.10	30	1.441
2	8.0	0.10	30	1.868
3	7.0	1.00	30	1.913
4	8.0	1.00	30	1.916
5	7.0	0.55	28	1.929
6	8.0	0.55	28	1.994
7	7.0	0.55	32	1.929
8	8.0	0.55	32	1.994
9	7.5	0.10	28	1.741
10	7.5	1.00	28	1.864
11	7.5	0.10	32	1.761
12	7.5	1.00	32	1.864
13	7.5	0.55	30	0.200
14	7.5	0.55	30	0.005
15	7.5	0.55	30	0.095
16	7.5	0.55	30	0.136
17	7.5	0.55	30	0.027

方差来源 Source	平方和 Sum of squares	自由度 Degree of freedom	均方差 Mean square	F值 F value	p
模型Model	0.430	9	0.048	7.100	0.008 5
F	0.039	1	0.039	5.780	0.047 2
I	0.070	1	0.070	10.250	0.015 0
H	5.000×10^-5	1	5.000×10^-5	7.368×10^-3	0.934 0
FI	0.045	1	0.045	6.620	0.036 8
FH	5.551×10^-17	1	5.551×10^-17	8.180×10^-15	1.000 0
IH	1.000×10^-4	1	1.000×10^-4	0.015	0.906 8
F²	0.025	1	0.025	0.680	0.096 4
I²	0.220	1	0.220	0.120	0.000 7
H²	0.012	1	0.012	1.810	0.220 2
残差Residual	0.048	7	6.787×10^-3	—	—
失拟项Lack of fit	0.022	3	7.368×10^-3	1.160	0.428 0
纯误差Pure error	0.025	4	6.350×10^-3	—	—
总和Cor total	0.936	32	—	—	—

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