Optimization of Culture Medium and Fermentation Parameters of Bacillus cereus MA23 Antagonistic to Kiwifruit Canker

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

Abstract

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

CLC Number:

Q939.96

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.

Figures/Tables 9

References 32

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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^**