合成绿原酸内生细菌的筛选、鉴定及发酵培养基的初步优化

Screening and Identification of Endophytic Bacteria for Chlorogenic Acid Synthesis and Preliminary Optimization of Fermentation Medium

目前绿原酸的主要来源为天然植物中提取,但天然植物中绿原酸含量较低且提取工艺复杂,导致绿原酸生产效率较低,因此有必要寻找新的、高效的制备绿原酸方法.本研究以金银花叶、红薯叶、薄荷叶、蒲公英叶和杜仲叶为材料,采用组织块法开展合成绿原酸的内生菌分离筛选,并利用高效液相色谱(HPLC)对筛选出内生菌的发酵产物进行分析,最终从植物样本中分离到具有合成绿原酸能力的5株内生菌,其中菌株XJ-1的绿原酸产量为9.9μg/mL.形态特征和16S rDNA序列分析表明,该菌株为枯草芽孢杆菌(Bacillus subtilis).对发酵培养基进行初步优化,优化后的发酵培养基主要成分为:果糖30 g/L、柠檬酸铵5 g/L、玉米浆6 g/L,所得绿原酸的产量达到24.02μg/mL,为优化前的2.43倍.本研究为开发微生物发酵法生产绿原酸提供了基础数据支持.

Nowadays,the main source of chlorogenic acid is extracted from natural plants.However,the content of chlorogenic acid in plants is low and the extraction process is complicated,thus causing low productivity of chlorogenic acid.Therefore,it is necessary to find a new and efficient method to prepare chlorogenic acid.In this study,the endophytic bacteria with synthesizing chlorogenic acid were isolated from honeysuckle leaves,sweet potato leaves,mint leaves,dandelion leaves and eucommia ulmoides leaves by tissue mass method.The fermentation products of endophytic bacteria were analyzed by HPLC.Finally,five endophytic bacteria with the ability to synthesize chlorogenic acid were isolated from the plant samples.Among the five endophytic bacteria,the yield of chlorogenic acid in strain XJ-1 was 9.9μg/mL.The endophytic strain was identified as Bacillus subtilis strain based on morphological characteristics and 16S rDNA sequence analysis.The fermentation medium was preliminarily optimized.The optimal fermentation medium was determined to be 30 g/L fructose,5 g/L ammonium citrate and 6 g/L corn starch.Under the optimized medium,the yield of chlorogenic acid reached 24.02μg/mL,and was 2.43 times higher than that before optimization.This study has laid a foundation for the development of microbial fermentation to produce chlorogenic acid.