Optimization of Pretreatment Methods for Metabolites of Rhizobia Isolated from Lespedeza davurica Using Box-Behnken Response Surface Methodology

doi:10.11733/j.issn.1007-0435.2026.08.007

Acta Agrestia Sinica ›› 2026, Vol. 34 ›› Issue (8): 2826-2836.DOI: 10.11733/j.issn.1007-0435.2026.08.007

Optimization of Pretreatment Methods for Metabolites of Rhizobia Isolated from Lespedeza davurica Using Box-Behnken Response Surface Methodology

LI Yi-juan^1,2,3, BAI Yu-fang^1,2,3, CHEN Yu-chong^1,2,3, MA Jie⁴, YANG Kai-yuan^1,2,3, CUI Qing-liang⁴, ZHAO Xiang^1,2,3

1. College of Grassland Science, Shanxi Agricultural University, Taigu, Shanxi Province 030800, China;
2. Shanxi Key Laboratory of Grassland Ecological Protection and Native Grass Germplasm Innovation, Taigu, Shanxi Province 030801, China;
3. Youyu Loess Plateau Grassland Ecosystem National Research Station, Youyu, Shanxi Province 037200, China;
4. College of Agricultural Engineering, Shanxi Agricultural University, Taigu, Shanxi Province 030800, China

Received:2025-09-29 Revised:2025-11-21 Published:2026-08-04

Box-Behnken响应面法优化达乌里胡枝子根瘤菌代谢产物的前处理方法

李宜娟^1,2,3, 白玉芳^1,2,3, 陈禹翀^1,2,3, 马洁⁴, 杨凯元^1,2,3, 崔清亮⁴, 赵祥^1,2,3

1. 山西农业大学草业学院, 山西太谷 030800;
2. 草地生态保护与乡土草种质创新山西省重点实验室, 山西太谷 030801;
3. 山西右玉黄土高原草地生态系统国家定位观测研究站, 山西右玉 037200;
4. 山西农业大学农业工程学院, 山西太谷 030800

通讯作者: 赵祥,E-mail:sxndzhaox@126.com
作者简介:李宜娟(2000-),女,汉族,甘肃白银人,硕士研究生主要从事草地微生物学研究,E-mail:1328636321@qq.com
基金资助:
山西省重点研发计划项目（2402140601011）；中央引导地方科技发展资金项目（YDZJSX2024B008）资助

Abstract

Abstract: The symbiont formed by legumes and rhizobia plays a crucial role in the nitrogen cycle， and root exudates significantly affect the nodulation process. The pretreatment and detection methods for metabolites of rhizobia from Lespedeza daurica have not been systematically optimized. In this study， the extraction efficiency of rhizobial metabolites was improved by optimizing pretreatment conditions， including ultrasonic temperature （A）， ultrasonic time （B）， derivatization temperature （C）， and derivatization time （D）. Single-factor experiments were used to determine the ranges of detection conditions， followed by a four-factor， three-level Box-Behnken response surface methodology （ultrasonic temperature： 30-40℃ ultrasonic time： 7-11 min derivatization time： 20-100 min derivatization temperature： 50-90℃） for optimization design. A quadratic regression model was established to determine the optimal conditions and verify their reliability. The optimal conditions determined by single-factor experiments were ultrasonic temperature 35℃， ultrasonic time 9 min， derivatization temperature 70℃， and derivatization time 60 min. The response surface model （R²=0.9035） showed that the linear terms A and C， the interaction term CD， and the quadratic terms A²， C²， and D² had extremely significant effects on the results （P<0.01）， while the interaction term AD had a significant effect （P<0.05）. After optimization， 145 metabolites were detected， representing an increase of 198.60% compared with the pre-optimization level， with an error of 3.5%. Methodological validation showed that the peak area RSD of the internal standard L-2-chloro-phenylalanine was 8.2% （n=6）， and the signal-to-noise ratio （S∶N） was 9.9∶1， which met the requirements for metabolomic detection. This study established a pretreatment method for the detection of metabolites from rhizobia of Lespedeza daurica， providing a technical basis for their extraction and analysis.

Key words: Lespedeza daurica, Metabolites of Rhizobia, Optimization of pretreatment methods

摘要： 豆科植物-根瘤菌共生体在氮循环中至关重要，其根系代谢产物显著影响结瘤过程。达乌里胡枝子（Lespedeza daurica）根瘤菌代谢产物前处理检测方法尚未进行系统优化。通过优化前处理（超声温度A及时间B、衍生温度C及时间D），提高其代谢产物提取效率。采用单因素试验筛选检测条件范围，再采用四因素三水平Box-Behnken响应面法优化设计（超声温度30~40℃、超声时间7~11 min、衍生时间20~100 min、衍生温度50~90℃），建立二次回归模型确定最优条件并验证其可靠性。单因素试验确定检测条件：超声温度35℃、超声时间9 min、衍生温度70℃、衍生时间60 min；响应面模型（R²=0.9035）显示一次项A、C，交互项CD和二次项A²、C²、D²对结果影响极显著（P<0.01），交互项AD影响显著（P<0.05）。优化后共检测到代谢产物145个，较优化前提高了198.60%，误差3.5%；方法学验证：内标物质L-2-氯-苯丙氨酸的峰面积RSD为8.2%（n=6），信噪比（S∶N）=9.9∶1，符合代谢组学检测要求。确定达乌里胡枝子根瘤菌代谢产物检测前处理方法，为其提取与检测提供技术依据。

关键词: 达乌里胡枝子, 根瘤菌代谢产物, 前处理方法优化

CLC Number:

S541.5

LI Yi-juan, BAI Yu-fang, CHEN Yu-chong, MA Jie, YANG Kai-yuan, CUI Qing-liang, ZHAO Xiang. Optimization of Pretreatment Methods for Metabolites of Rhizobia Isolated from Lespedeza davurica Using Box-Behnken Response Surface Methodology[J]. Acta Agrestia Sinica, 2026, 34(8): 2826-2836.

李宜娟, 白玉芳, 陈禹翀, 马洁, 杨凯元, 崔清亮, 赵祥. Box-Behnken响应面法优化达乌里胡枝子根瘤菌代谢产物的前处理方法[J]. 草地学报, 2026, 34(8): 2826-2836.

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Optimization of Pretreatment Methods for Metabolites of Rhizobia Isolated from Lespedeza davurica Using Box-Behnken Response Surface Methodology

Box-Behnken响应面法优化达乌里胡枝子根瘤菌代谢产物的前处理方法

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