抗猕猴桃细菌性溃疡病蜡样芽孢杆菌MA23培养基及发酵条件优化

doi:10.11924/j.issn.1000-6850.casb2020-0108

摘要/Abstract

摘要：

蜡样芽孢杆菌MA23对猕猴桃溃疡病病原菌丁香假单胞菌猕猴桃致病变种(Pseudomonas syringae pv. actinidiae)有较好的抑制作用,优化其发酵培养基及发酵条件将为猕猴桃溃疡病的有效防治提供参考。本研究通过正交设计实验和单因素实验分别优化其发酵培养基和摇瓶发酵条件,提高摇瓶发酵的菌体量和抑菌活性。正交实验方差分析结果显示,蛋白胨、可溶性淀粉、K₂HPO₄和MgSO₄为高度显著因素,NaNO₃为显著因素,葡萄糖为不显著因素,它们对实验结果影响由大到小表现为可溶性淀粉>K₂HPO₄>蛋白胨>MgSO₄>NaNO₃>葡萄糖,综合考虑确定最优组合为A₁B₄C₄D₁E₂F₂。因此,蜡样芽孢杆菌MA23的最优培养基为葡萄糖0.8%,蛋白胨0.2%,可溶性淀粉3.5%,NaNO₃ 0.05%,K₂HPO₄ 0.2%,MgSO₄ 0.2%,CaCO₃ 0.12%,MnSO₄ 0.035%;培养基优化后,蜡样芽孢杆菌MA23发酵液的菌体量和抑菌活性分别提高了41%和37.2%。通过发酵条件优化,确定了蜡样芽孢杆菌MA23最佳发酵初始pH 6.5,最佳发酵温度为30℃,最佳接种量为8%,最佳装液量为70 mL,最佳摇床转速为180 r/min,菌体量和抑菌活性分别提高了28.2%和25.6%。通过培养基和发酵条件优化,蜡样芽孢杆菌的菌体量和抑菌活性都得到大幅提高,为其进一步应用提供了参考。

关键词: 蜡样芽孢杆菌MA23, 正交实验, 培养基优化, 发酵条件优化, 活菌数, 抑菌活性

Abstract:

As an important biocontrol bacterium, Bacillus cereus MA23 exhibits good antagonistic effect on the pathogen of kiwifruit canker, Pseudomonas syringae pv. actinidiae. To promote its application in the control of the diseases, it is necessary to improve the biomass and antimicrobial activity of strain MA23. Thus, orthogonal test and single factor experiment were selected for the optimization of culture medium and fermentation parameters. Through variance analysis of orthogonal experiment results, it was demonstrated that peptone, soluble starch, K₂HPO₄ and MgSO₄ were highly significant factors, and NaNO₃ was a significant factor while glucose was not a significant factor. These factors affected the test result in an order of soluble starch> K₂HPO₄> peptone> MgSO₄> NaNO₃> glucose, and A₁B₄C₄D₁E₂F₂was determined as the optimal combination. So the optimal culture medium for MA23 was 0.8% glucose, 0.2% peptone, 3.5% soluble starch, 0.05% NaNO₃, 0.2% K₂HPO₄, 0.2% MgSO₄, 0.12% CaCO₃, 0.035% MnSO₄. After optimization of the medium, the biomass and antimicrobial activity of strain MA23 fermentation broth increased by 41% and 37.2%, respectively. After the single factor experiments, the optimal fermentation condition was determined as fellows: the initial pH of the medium was 6.5, the fermentation temperature was 30℃, the inoculation size was 8%, the liquid medium volume was 70 mL, and the rotation speed was 180 r/min. After optimization of fermentation conditions, the biomass and antimicrobial activity of strain MA23 fermentation broth increased by 28.2% and 25.6%, respectively. In conclusion, the biomass and antimicrobial activity of Bacillus cereus MA23 are improved significantly through optimization of culture medium and fermentation parameters, and this will lay the foundation for the further application of strain MA23.

Key words: Bacillus cereus MA23, orthogonal test, optimization of fermentation medium, optimization of fermentation parameters, viable count, antagonistic activity

中图分类号:

Q939.96

朱海云, 马瑜, 柯杨, 李勃. 抗猕猴桃细菌性溃疡病蜡样芽孢杆菌MA23培养基及发酵条件优化[J]. 中国农学通报, 2021, 37(7): 112-118.

Zhu Haiyun, Ma Yu, Ke Yang, Li Bo. Optimization of Culture Medium and Fermentation Parameters of Bacillus cereus MA23 Antagonistic to Kiwifruit Canker[J]. Chinese Agricultural Science Bulletin, 2021, 37(7): 112-118.

图/表 9

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编号	因素/%						活菌数/(×10⁸ CFU/mL)
编号	A	B	C	D	E	F	Ⅰ	Ⅱ	Ⅲ
1	3(2.4)	3(0.15)	3(2.5)	1(0.05)	1(0.1)	2(0.2)	5.7	5.5	6.7
2	3	2(0.1)	1(0.5)	4(0.2)	2(0.2)	1(0.1)	3	4.5	4.2
3	1(0.8)	1(0.05)	1	1	1	1	5.1	5.8	6.9
4	2(1.6)	3	4(3.5)	4	1	1	7.9	9	11.6
5	1	3	2(1.5)	3(0.15)	2	1	9	5	8.7
6	4(3.2)	4(0.2)	4	1	2	1	13.4	15.3	15.2
7	4	2	3	3	1	1	7.0	5.9	5.4
8	1	2	4	2(0.1)	1	2	8.3	9.5	10.1
9	3	1	4	3	2	2	13	13.7	12.9
10	3	4	2	2	1	1	7.8	9.1	7.5
11	4	1	2	4	1	2	10.1	7.5	7.2
12	2	1	3	2	2	1	6.1	6.6	6.6
13	2	4	1	3	1	2	6.8	6.6	7.2
14	1	4	3	4	2	2	9.5	8.6	10.3
15	4	3	1	2	2	2	7.9	5.9	7.0
16	2	2	2	1	2	2	11	8.7	9.8
K₁_j	96.8	101.5	70.9	109.1	180.2	186.6
K₂_j	97.9	87.4	101.4	92.4	215.9	209.5
K₃_j	93.6	89.9	83.9	101.2
K₄_j	107.8	117.3	139.9	93.4

编号	因素/%						活菌数/(×10⁸ CFU/mL)
编号	A	B	C	D	E	F	Ⅰ	Ⅱ	Ⅲ
1	3(2.4)	3(0.15)	3(2.5)	1(0.05)	1(0.1)	2(0.2)	5.7	5.5	6.7
2	3	2(0.1)	1(0.5)	4(0.2)	2(0.2)	1(0.1)	3	4.5	4.2
3	1(0.8)	1(0.05)	1	1	1	1	5.1	5.8	6.9
4	2(1.6)	3	4(3.5)	4	1	1	7.9	9	11.6
5	1	3	2(1.5)	3(0.15)	2	1	9	5	8.7
6	4(3.2)	4(0.2)	4	1	2	1	13.4	15.3	15.2
7	4	2	3	3	1	1	7.0	5.9	5.4
8	1	2	4	2(0.1)	1	2	8.3	9.5	10.1
9	3	1	4	3	2	2	13	13.7	12.9
10	3	4	2	2	1	1	7.8	9.1	7.5
11	4	1	2	4	1	2	10.1	7.5	7.2
12	2	1	3	2	2	1	6.1	6.6	6.6
13	2	4	1	3	1	2	6.8	6.6	7.2
14	1	4	3	4	2	2	9.5	8.6	10.3
15	4	3	1	2	2	2	7.9	5.9	7.0
16	2	2	2	1	2	2	11	8.7	9.8
K₁_j	96.8	101.5	70.9	109.1	180.2	186.6
K₂_j	97.9	87.4	101.4	92.4	215.9	209.5
K₃_j	93.6	89.9	83.9	101.2
K₄_j	107.8	117.3	139.9	93.4

方差来源	Ⅲ型平方和	自由度	均方	F	P
矫正模型	3.332E+18^*	14	2.380E+17	16.826	0.000
截距	3.269E+19	1	3.269E+19	2310.567	0.000
A	9.387E+16	3	3.129E+16	2.212	0.105
B	4.654E+17	3	1.551E+17	10.967	0.000
C	2.247E+18	3	7.489E+17	52.942	0.000
D	1.515E+17	3	5.049E+16	3.569	0.024
E	2.655E+17	1	2.655E+17	18.769	0.000
F	1.093E+17	1	1.093E+17	7.723	0.009
误差	4.668E+17	33	1.415E+16
总计	3.649E+19	48
校正的总计	3.799E+18	47

方差来源	Ⅲ型平方和	自由度	均方	F	P
矫正模型	3.332E+18^*	14	2.380E+17	16.826	0.000
截距	3.269E+19	1	3.269E+19	2310.567	0.000
A	9.387E+16	3	3.129E+16	2.212	0.105
B	4.654E+17	3	1.551E+17	10.967	0.000
C	2.247E+18	3	7.489E+17	52.942	0.000
D	1.515E+17	3	5.049E+16	3.569	0.024
E	2.655E+17	1	2.655E+17	18.769	0.000
F	1.093E+17	1	1.093E+17	7.723	0.009
误差	4.668E+17	33	1.415E+16
总计	3.649E+19	48
校正的总计	3.799E+18	47

优化阶段	活菌数		抑菌圈半径
优化阶段	数据/(CFU/mL)	优化效率/%	数据/cm	优化效率/%
A	13.9±0.89	/	9.84±0.87	/
B	19.6±1.20	41.0^*	13.50±0.76	37.2^*
C	25.12±0.60	28.2^**	16.96±1.24	25.6^**