柔毛淫羊藿黄酮类成分转化菌株的筛选及其对朝藿定C的生物转化作用

Screening of a strain for biotransformation of flavonoids in Epimedium pubescens and evaluation of its biotransformation effect on epimedin C

摘要:

【背景】 柔毛淫羊藿(Epimedium pubescens Maxim.)是传统药用植物，其药用成分主要为黄酮类化合物。部分黄酮类成分因糖基化程度较高导致生物利用度受限。生物转化技术可定向制备低糖基黄酮，是提升活性成分成药性的潜在策略。【目的】 筛选高效转化朝藿定C生成淫羊藿苷的功能菌株，解析其关键酶系特性，并通过微生物驯化提升底物耐受性及转化效率。【方法】 采用生境特异性采样策略，从淫羊藿根际土壤和叶际微生物群落中筛选菌株。通过薄层色谱法(thin layer chromatography, TLC)-HPLC联用技术建立转化产物监测体系；结合16S rRNA基因系统发育分析和糖苷酶活性解析，鉴定功能菌株分类地位及其功能酶类。采用单因素试验优化粗酶反应体系(温度、pH、底物浓度、酶活力浓度)；以梯度胁迫法(0.01-0.12 g/mL水提液)进行六轮定向驯化，评估菌株适应性进化效果。【结果】 分离获得芽孢杆菌(Bacillus sp.) MY202402-3，其粗酶液中α-L-鼠李糖苷酶(18.7 U/mL)与β-葡萄糖苷酶(3.3 U/mL)活性显著；在含0.01 g/mL水提液的培养基中，朝藿定C平均转化率与宝藿苷Ⅰ平均生成率分别为88.10%与21.13%。粗酶最适反应条件为50 ℃、pH 7.5、底物浓度1.2 mg/mL、酶活10 U/mL，2 h内朝藿定C转化率达95.20%，淫羊藿苷纯度96.78%。经浓度梯度驯化后，菌株在0.10 g/mL水提液中朝藿定C转化率提升至90.93%，0.08 g/mL体系中宝藿苷Ⅰ生成率达26.96%。【结论】 本研究采用的微生物筛选与驯化相结合的策略显著提高了朝藿定C的生物转化效率，为提升中药提取物中活性成分的生物利用度提供了参考；浓度依赖性优化进一步展示了其在工业应用中的可扩展潜力；菌株MY202402-3是芽孢杆菌属被报道用于淫羊藿黄酮类成分转化的菌株，扩大了芽孢杆菌属的应用。

Abstract:

[Background] Epimedium pubescens Maxim., a traditional medicinal plant, contains flavonoids as its primary bioactive constituents. The bioavailability of certain highly glycosylated flavonoids is limited due to high glycosylation. Biotransformation enables directional preparation of low-glycosylated flavonoids, representing a potential strategy to enhance the druggability of active components. [Objective] To screen functional strains capable of efficiently converting epimedin C to icariin, characterize their key enzyme systems, and improve substrate tolerance/conversion efficiency through microbial domestication. [Methods] We isolated strains from the rhizosphere microbiota and the phyllosphere microbiota of Epimedium by habitat-specific sampling. A thin layer chromatography (TLC)-HPLC system was established for the monitoring of transformation products. The phylogenetic analysis (16S rRNA gene) and glycosidase activity profiling were employed to identify the strain and functional enzymes, respectively. Single-factor experiments were conducted to optimize the crude enzyme reaction parameters (temperature, pH, substrate concentration, and enzyme activity). Six rounds of gradient stress domestication (0.01-0.12 g/mL aqueous extract of Epimedium) were implemented to evaluate the adaptive evolution of the strain. [Results] Bacillus sp. MY202402-3 was isolated, with its crude enzymes demonstrating notable α-L-rhamnosidase (18.7 U/mL) and β-glucosidase (3.3 U/mL) activities. In the medium containing 0.01 g/mL aqueous extract, the average epimedin C conversion rate and the average baohuoside Ⅰ yield reached 88.10% and 21.13%, respectively. The optimal reaction conditions of the crude enzyme were 50 ℃, pH 7.5, 1.2 mg/mL substrate, and 10 U/mL enzyme, under which the epimedin C conversion rate and the icariin purity reached 95.20% and 96.78%, respectively, within 2 h. Post-domestication, the conversion rate improved to 90.93% in the medium containing 0.10 g/mL aqueous extract, and the baohuoside Ⅰ yield reached 26.96% in the system containing 0.08 g/mL aqueous extract of Epimedium. [Conclusion] The integrated microbial screening-domestication strategy significantly enhances epimedin C biotransformation efficiency, providing insights for improving the bioavailability of herbal extracts. Concentration-dependent optimization demonstrates scalable industrial potential. As the Bacillus strain reported for Epimedium flavonoid transformation, MY202402-3 expands the application scope of Bacillus.