新种苦豆子无毛毛壳菌Achaetomium sophora HY17产槐定碱发酵条件优化

doi:10.11686/cyxb2024444

摘要/Abstract

摘要：

本研究旨在从苦豆子内生真菌中筛选获得高产槐定碱的菌株，并对其进行分类鉴定和产碱发酵条件优化，提高碱产率，为发酵过程提供优良的菌种资源。以前期从苦豆子健康种子中分离的50株内生真菌为材料，采用生物碱沉淀法和酸性染料比色法初筛，利用高效液相色谱法复筛得到高产槐定碱菌株，通过形态学和分子生物学确定其分类地位；通过单因素试验、Plackett-Burman试验、最陡爬坡试验和Box-Behnken design试验考察培养基成分（培养基种类、碳源和氮源）、发酵条件（培养天数和pH）、前体物质和诱导子对该菌株碱产率的影响，确定最佳产碱培养基、发酵条件和前体物质。结果表明，筛选获得一株高产槐定碱的菌株HY17，经鉴定为新种，命名为苦豆子无毛毛壳菌。Achaetomium sophora HY17菌株产槐定碱的最佳发酵参数为：在初始pH=6，碳源和氮源分别为玉米粉和干酪素的SDY液体培养基上培养8 d，添加L-赖氨酸、L-哌啶酸和苯丙氨酸浓度分别为1.044 g·L^-1，0.081 g·L^-1，1.995 g·L^-1时，碱产率达到最大，为1.369 mg·g^-1。与对照相比，优化后碱产率提高了61.95%。A. sophora HY17菌株能够稳定高产槐定碱，这为通过微生物发酵生产槐定碱提供了一种新方法。

关键词: 苦豆子无毛毛壳菌Achaetomium sophora HY17, 筛选鉴定, 槐定碱, 发酵条件优化, 响应面法分析

Abstract:

The aim of this study was to screen for endophytic fungal strains of Achaetomium sophora infecting Sophora alopecuroides that produce sophoridine alkaloid， classify and identify them， and then identify in-vitro culture conditions that optimize the alkaloid production of the endophyte fungus. The overall goal was to improve alkaloid yield and obtain excellent strains for culture. Fifty strains of A. sophora isolated from healthy S. alopecuroides seeds were screened using alkaloid precipitation， acid dye colorimetry， and high-performance liquid chromatography analyses. The strains were identified through morphological and molecular analyses. Subsequently， a series of methods including the single factor test， Plackett-Burman （PB） test， steepest climb test， and response surface test （Box-Behnken test） were used to investigate the effects of medium composition （medium type， carbon and nitrogen sources）， culture conditions （incubation time and pH）， and precursors and inducers on the alkali yield of selected fungal strains. From the results of these analyses， the optimal alkali-producing medium， culture conditions， and precursors for sophoridine alkaloid production were determined. The fungal strain HY17 producing sophoridine at high levels was identified as a new species， Achaetomium sophora. The optimal culture conditions for A. sophora HY17 were determined to be SDY liquid medium with an initial pH of 6 and a culture period of 8 days， with maize flour as the carbon source and casein as the nitrogen source. Tyrosine， L-lysine， L-piperidinic acid， and phenylalanine had significant effects on sophoridine production by strain HY17 during culture. The addition of L-lysine （1.044 g·L^-1）， L-piperidinic acid （0.081 g·L^-1）， and phenylalanine （1.995 g·L^-1） resulted in a maximum alkaloid yield of 1.369 mg·g^-1 mycelium under these optimized conditions， representing a remarkable increase of 61.95% compared with that obtained under the original culture conditions. A. sophora HY17 produced sophoridine at a high and stable rate. Optimization of the culture conditions resulted in a notable increase in alkaloid production. The results of this study offer a novel approach for sophoridine production through in-vitro culture of the fungal endophyte， A. sophora strain HY17 isolated from S. alopecuroides.

Key words: Sophora alopecuroides HY17, screening and identification, sophoridine, optimization of fermentation conditions, response surface methodology （RSM）

唐致云, 王文凯, 刘冠兰, 顾沛雯. 新种苦豆子无毛毛壳菌Achaetomium sophora HY17产槐定碱发酵条件优化[J]. 草业学报, 2025, 34(10): 151-163.

Zhi-yun TANG, Wen-kai WANG, Guan-lan LIU, Pei-wen GU. Optimization of culture conditions for the sophoridine-producing new fungal species Achaetomium sophor strain HY17 isolated from seeds of Sophora alopecuroides[J]. Acta Prataculturae Sinica, 2025, 34(10): 151-163.

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目标基因Target gene	引物名称Primer	引物序列Primer sequences （5′-3′）	退火温度Annealing temperature （℃）	参考文献Reference
ITS	ITS4	TCCTCCGCTTATTGATATGC	55	［23］
ITS	ITS5	GGAAGTAAAAGTCGTAACAAGG	55	［23］
LSU	LSU1Fd	GRATCAGGTAGGRATACCCG	55	［24］
LSU	LR5	TCCTGAGGGAAACTTCG	55	［24］
TUB2	Bt2a	GGTAACCAAATCGGTGCTGCTTTC	58	［25］
TUB2	Bt2b	ACCCTCAGTGTAGTGACCCTTGGC	58	［25］

目标基因Target gene	引物名称Primer	引物序列Primer sequences （5′-3′）	退火温度Annealing temperature （℃）	参考文献Reference
ITS	ITS4	TCCTCCGCTTATTGATATGC	55	［23］
ITS	ITS5	GGAAGTAAAAGTCGTAACAAGG	55	［23］
LSU	LSU1Fd	GRATCAGGTAGGRATACCCG	55	［24］
LSU	LR5	TCCTGAGGGAAACTTCG	55	［24］
TUB2	Bt2a	GGTAACCAAATCGGTGCTGCTTTC	58	［25］
TUB2	Bt2b	ACCCTCAGTGTAGTGACCCTTGGC	58	［25］

变量 Codes	因素 Factors	水平Levels
变量 Codes	因素 Factors	-1	1
X₁	L-赖氨酸 L-lysine	1.00	2.00
X₂	L-哌啶酸 L-piperidinic acids	0.08	0.16
X₃	α-酮戊二酸 α-ketoglutaric acid	0.01	0.02
X₄	苯丙氨酸 Phenylalanine	2.00	5.00
X₅	丙酮酸 Pyruvate	1.00	4.00
X₆	水杨酸 Salicylate	1.00	2.00
X₇	茉莉酸甲酯Methyl jasmonate	0.10	0.50
X₈	种子提取物 Seed extract	2.00	5.00

变量 Codes	因素 Factors	水平Levels
变量 Codes	因素 Factors	-1	1
X₁	L-赖氨酸 L-lysine	1.00	2.00
X₂	L-哌啶酸 L-piperidinic acids	0.08	0.16
X₃	α-酮戊二酸 α-ketoglutaric acid	0.01	0.02
X₄	苯丙氨酸 Phenylalanine	2.00	5.00
X₅	丙酮酸 Pyruvate	1.00	4.00
X₆	水杨酸 Salicylate	1.00	2.00
X₇	茉莉酸甲酯Methyl jasmonate	0.10	0.50
X₈	种子提取物 Seed extract	2.00	5.00

项目 Item	因子 Factors	水平 Levels
项目 Item	因子 Factors	-1	0	1
X₁	L-赖氨酸 L-lysine	0.50	1.00	1.50
X₂	L-哌啶酸 L-piperidinic acids	0.04	0.08	0.12
X₄	苯丙氨酸 Phenylalanine	0.50	2.00	3.50