大豆孢囊线虫生防菌株Myrothecium verrucaria ZW-2发酵条件优化及活性物质分析

doi:10.13560/j.cnki.biotech.bull.1985.2021-0454

摘要/Abstract

摘要：

疣孢漆斑菌（Myrothecium verrucaria）ZW-2菌株对大豆孢囊线虫具有（Heterodera glycines）明显的杀线虫活性,为了确定该菌株的最优发酵条件以及代谢产物,采用4因素3水平的正交试验进行了发酵条件优化以及代谢组学分析。结果表明：菌株ZW-2以Czapek培养基为基础培养基,最佳培养温度为28℃,摇床转速为180 r/min,装瓶量为100 mL,培养基pH为8,最佳碳源和氮源为蔗糖和硝酸钠。经代谢组学分析,根据代谢物质荷比、精确分子量及一级碎片离子得分等信息共筛选出20种差异代谢物,包括酰胺类化合物和脂肪族化合物。菌株ZW-2上述试验结果为进一步开发利用该菌株提供重要的理论和应用价值。

关键词: 疣孢漆斑菌, 大豆孢囊线虫, 发酵条件优化, 代谢组学分析

Abstract:

Myrothecium verrucaria ZW-2 strain had obvious nematicidal activity against Heterodera glycines. In order to determine the optimal fermentation conditions and metabolites of the strain,the orthogonal experiment with four factors and three levels was used to optimize the fermentation conditions and analyze the metabolomics. The results showed that the strain ZW-2 was cultured on Czapek medium with the optimal culture temperature of 28℃,rotation speed of 180 r/min,bottle volume of 100 mL,medium pH of 8,and the optimal carbon and nitrogen sources were sucrose and sodium nitrate. After metabolomics analysis,20 differential metabolites,including amide compounds and aliphatic compounds,were screened according to the information of the charge ratio of metabolites,the accurate molecular weight and the score of first-order fragment ions. The above experiment results by strain ZW-2 further provide important theoretical basis and application value for the development and utilization of this strain.

Key words: Myrothecium verrucaria, Heterodera glycines, optimization of fermentation conditions, metabonomics analysis

陈倩, 张露源, 陈伯昌, 吴海燕. 大豆孢囊线虫生防菌株Myrothecium verrucaria ZW-2发酵条件优化及活性物质分析[J]. 生物技术通报, 2021, 37(7): 127-136.

CHEN Qian, ZHANG Lu-yuan, CHEN Bo-chang, WU Hai-yan. Optimization of Fermentation Conditions of Myrothecium verrucaria ZW-2,a Biocontrol Strain Against Heterodera glycines and Analysis of Active Substances[J]. Biotechnology Bulletin, 2021, 37(7): 127-136.

图/表 5

参考文献 41

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编号 Code	A发酵温度 Fermentation temperature/℃	B摇床转速Shaking speed/（r·min^-1）	C 培养基 pH Medium pH	D 装瓶量Bottle volume/ （mL·250 mL^-1）	72 h校正死亡率Adjusted mortality rate at 72 h/%
1	26	140	6	75	75.66
2	26	160	7	100	84.37
3	26	180	8	125	88.97
4	28	140	7	125	82.64
5	28	160	8	75	87.84
6	28	180	6	100	86.19
7	30	140	8	100	92.19
8	30	160	7	125	80.34
9	30	180	6	75	78.22
K1	249.00	250.49	240.07	241.72
K2	256.67	252.55	247.35	262.75
K3	250.75	253.38	269.00	251.95
k1	83	83.50	80.02	80.57
k2	85.56	84.18	82.45	87.58
k3	83.58	84.46	89.67	83.98
R	2.56	0.96	9.65	7.01
主次顺序 Primary and secondary order	B>A>D>C
最优水平 Optimal level	A2	B3	C3	D2
最优组合 Optimal combination	A2B3C3D2

编号 Code	A发酵温度 Fermentation temperature/℃	B摇床转速Shaking speed/（r·min^-1）	C 培养基 pH Medium pH	D 装瓶量Bottle volume/ （mL·250 mL^-1）	72 h校正死亡率Adjusted mortality rate at 72 h/%
1	26	140	6	75	75.66
2	26	160	7	100	84.37
3	26	180	8	125	88.97
4	28	140	7	125	82.64
5	28	160	8	75	87.84
6	28	180	6	100	86.19
7	30	140	8	100	92.19
8	30	160	7	125	80.34
9	30	180	6	75	78.22
K1	249.00	250.49	240.07	241.72
K2	256.67	252.55	247.35	262.75
K3	250.75	253.38	269.00	251.95
k1	83	83.50	80.02	80.57
k2	85.56	84.18	82.45	87.58
k3	83.58	84.46	89.67	83.98
R	2.56	0.96	9.65	7.01
主次顺序 Primary and secondary order	B>A>D>C
最优水平 Optimal level	A2	B3	C3	D2
最优组合 Optimal combination	A2B3C3D2

活性物质英文名 Name of an active substance in English	中文名 Name in Chinese	分子式 Formula	质荷比 M/z	得分 Score	保留时间 Retaining time/min
Trimethylsilyl N,N-bis（trimethylsilyl）glycylglycinate	-	C₁₃H₃₂N₂O₃Si₃	348.1724	36.9	9.6076
Glycol palmitate	乙二醇棕榈酸酯	C₁₈H₃₆O₃	323.2568	49	8.01246
Stearyl glycol	硬脂二醇	C₁₈H₃₈O₂	309.2784	48.5	10.27076
Tris（butoxyethyl）Phosphate	磷酸三（丁氧基乙基）酯	C₁₈H₃₉O₇P	399.2522	35.5	6.4744
2-[4-（2,4-Dichlorobenzoyl）-1,3-dimethylpyrazol-5-yloxy]acetophenone	2-（4-（2,4-二氯苯甲酰基）-1,3-二甲基吡唑-5-基氧）乙酰苯	C₂₀H₁₆Cl₂N₂O₃	385.0529	35.6	2.7755
2-Nonadecanone,O-methyloxime	-	C₂₀H₄₁NO	311.3186	39.3	10.6652
p-（6-Chloro-4-phenyl-2-quinolyl）aniline	-	C₂₁H₁₅ClN₂	330.0915	37.1	0.7172
Isoscoparin	异金雀花素	C₂₂H₂₂O₁₁	445.1125	44.2	11.5114
Octylphenoxy Polyethoxyethanol	NP-40乙基苯基聚乙二醇	C₂₂H₃₈O₅	365.2676	49.9	7.2005
8,11-Dihydroxypimar-15-en-12-yl acetate	-	C₂₂H₃₆O₄	365.2676	49.4	7.2005
（2-Methyl-3-biphenylyl）methyl 3-[（1Z）-2-Chloro-3,3,3-trifluoro-1-propenyl]-2,2-dimethylcyclopropanecarboxylic acid（2-methylbiphenyl-3-yl）methyl ester	氟氯菊酯	C₂₃H₂₂ClF₃O₂	445.1125	42.9	11.5114
3,6,9,12,15,18-Hexaoxatriacontan-1-ol	六聚乙二醇单十二醚	C₂₄H₅₀O₇	450.3528	49.7	9.4589
Pyrinoline	吡诺林	C₂₇H₂₀N_4O	416.1608	39.1	10.1850
Verrucarin A	疣孢菌素A	C₂₇H₃₄O₉	541.1870	36.5	5.61113
Verruculogen	疣孢青霉原	C₂₇H₃₃N₃O₇	512.2436	28.2	6.4058
3-Hydroxy-17,23-epoxyveratraman-11-one	-	C₂₇H₃₉NO₃	426.2990	51.9	7.0519
Epidihydrocholesterin	胆甾烷醇	C₂₇H₄₈O	427.3301	42.1	10.1049
Roridin A	漆斑菌素A	C₂₉H₄₀O₉	571.2342	43.6	5.2110
4,4'-Furfurylidenebis（2,6-di-tert-butylphenol）	-	C₃₃H₄₆O₃	513.3318	37.3	8.0467
N,N-Didodecyl-1-dodecanamine	三月桂胺	C₃₆H₇₅N	522.5961	39.1	10.5508

活性物质英文名 Name of an active substance in English	中文名 Name in Chinese	分子式 Formula	质荷比 M/z	得分 Score	保留时间 Retaining time/min
Trimethylsilyl N,N-bis（trimethylsilyl）glycylglycinate	-	C₁₃H₃₂N₂O₃Si₃	348.1724	36.9	9.6076
Glycol palmitate	乙二醇棕榈酸酯	C₁₈H₃₆O₃	323.2568	49	8.01246
Stearyl glycol	硬脂二醇	C₁₈H₃₈O₂	309.2784	48.5	10.27076
Tris（butoxyethyl）Phosphate	磷酸三（丁氧基乙基）酯	C₁₈H₃₉O₇P	399.2522	35.5	6.4744
2-[4-（2,4-Dichlorobenzoyl）-1,3-dimethylpyrazol-5-yloxy]acetophenone	2-（4-（2,4-二氯苯甲酰基）-1,3-二甲基吡唑-5-基氧）乙酰苯	C₂₀H₁₆Cl₂N₂O₃	385.0529	35.6	2.7755
2-Nonadecanone,O-methyloxime	-	C₂₀H₄₁NO	311.3186	39.3	10.6652
p-（6-Chloro-4-phenyl-2-quinolyl）aniline	-	C₂₁H₁₅ClN₂	330.0915	37.1	0.7172
Isoscoparin	异金雀花素	C₂₂H₂₂O₁₁	445.1125	44.2	11.5114
Octylphenoxy Polyethoxyethanol	NP-40乙基苯基聚乙二醇	C₂₂H₃₈O₅	365.2676	49.9	7.2005
8,11-Dihydroxypimar-15-en-12-yl acetate	-	C₂₂H₃₆O₄	365.2676	49.4	7.2005
（2-Methyl-3-biphenylyl）methyl 3-[（1Z）-2-Chloro-3,3,3-trifluoro-1-propenyl]-2,2-dimethylcyclopropanecarboxylic acid（2-methylbiphenyl-3-yl）methyl ester	氟氯菊酯	C₂₃H₂₂ClF₃O₂	445.1125	42.9	11.5114
3,6,9,12,15,18-Hexaoxatriacontan-1-ol	六聚乙二醇单十二醚	C₂₄H₅₀O₇	450.3528	49.7	9.4589
Pyrinoline	吡诺林	C₂₇H₂₀N_4O	416.1608	39.1	10.1850
Verrucarin A	疣孢菌素A	C₂₇H₃₄O₉	541.1870	36.5	5.61113
Verruculogen	疣孢青霉原	C₂₇H₃₃N₃O₇	512.2436	28.2	6.4058
3-Hydroxy-17,23-epoxyveratraman-11-one	-	C₂₇H₃₉NO₃	426.2990	51.9	7.0519
Epidihydrocholesterin	胆甾烷醇	C₂₇H₄₈O	427.3301	42.1	10.1049
Roridin A	漆斑菌素A	C₂₉H₄₀O₉	571.2342	43.6	5.2110
4,4'-Furfurylidenebis（2,6-di-tert-butylphenol）	-	C₃₃H₄₆O₃	513.3318	37.3	8.0467
N,N-Didodecyl-1-dodecanamine	三月桂胺	C₃₆H₇₅N	522.5961	39.1	10.5508