目的 青黛Indigo Naturalis浸泡发酵环节存在工艺稳定性差、有效成分波动大的问题，为提高青黛的品质，系统解析马蓝叶的浸泡发酵机制是关键。方法 采用溶氧仪等连续监测6、12、24、36、48、60、72 h发酵液中的pH值、氧化还原电位（oxidation-reduction potential，ORP）、溶氧量等物理指标。利用HPLC法测定不同发酵时间下样品中靛蓝、靛玉红、色胺酮、靛红等关键化学成分含量变化，同时采用高通量测序对样品中微生物组成进行鉴定和考察。对微生物和理化参数进行相关性分析，微生物群落动态演替与理化参数的关联变化。结果 随着发酵时间的不断增加，pH值、ORP、溶氧量等物理指标均呈现先下降后相对平稳的趋势，靛蓝、靛玉红等靛类成分均呈现先上升后缓慢下降的趋势。发酵6 h样本细菌和真菌的多样性最丰富，且群落结构与后期发酵样品的差异显著。主要的优势菌群包括细菌中的梭状芽孢杆菌属Clostridium_sensu_stricto_1、乳酸球菌属Lactococcus、肠杆菌属Enterobacter、肠球菌属Enterococcus，以及真菌中的丝孢酵母属Trichosporon、unclassified_f_Didymellaceae、季也蒙迈耶氏酵母属Meyerozyma、帚枝霉属Sarocladium、Cutaneotrichosporon。相关性分析揭示，门水平上，细菌中的厚壁菌门（Firmicutes）与pH值和ORP呈显著负相关（P＜0.01），而变形菌门（Proteobacteria）则相反；真菌中的担子菌门（Basidiomycota）与之呈显著负相关（P＜0.01），而子囊菌门（Ascomycota）则相反；属水平上，梭状芽孢杆菌属、乳酸球菌属等细菌属与关键成分的含量呈显著正相关（P＜0.01），与丝孢酵母属、皮肤孢菌属Cutaneotrichosporon等真菌属呈显著性正相关（P＜0.01）。结论 初步阐明了在青黛浸泡发酵过程中，微生物群落动态演替与理化参数的显著互作，构建了二者的关联网络，为进一步通过理化参数对发酵过程靛类成分含量与发酵程度进行快速预测，从而实现青黛工业生产的可观、可控及可调提供理论支撑。

Objective Qingdai (Indigo Naturalis, IN) soaking and fermentation faces challenges such as poor process stability and significant fluctuations in active ingredients. To improve its quality, systematically elucidating the soaking and fermentation mechanism of Baphicacanthus cusia leaves is crucial. Methods In this study, dissolved oxygen meters and other devices were used to continuously monitor physical parameters such as pH value, oxidation-reduction potential (ORP) and dissolved oxygen content in the fermentation broth for 6, 12, 24, 36, 48, 60 and 72 h. The content changes of key chemical components such as indigo, indirubin, tryptanthrin and isatin in the samples under different fermentation times were determined by HPLC. Meanwhile, high-throughput sequencing was used to identify and investigate the microbial composition in the samples. Correlation analysis was conducted on microorganisms and physicochemical parameters, as well as the correlation changes between the dynamic succession of microbial communities and physicochemical parameters. Results With the continuous increase of fermentation time, physical indicators such as pH value, ORP and dissolved oxygen content all showed a trend of first decreasing and then relatively stable, while indigoid compounds such as indigo and indirubin all showed a trend of first increasing and then slowly decreasing. The microbial diversity of bacterial and fungal was highest in the 6 h fermentation samples, and their community structures were significantly different from those in later fermentation stages. The dominant microbial taxa identified were the bacteria Clostridium_sensu_stricto_1, Lactococcus, Enterobacter, Enterococcus, and the fungal Trichosporon, unclassified_f__Didymellaceae, Meyerozyma, Sarocladium, Cutaneotrichosporon. Notably, correlation analysis showed at the phylum level, the bacterial phylum Firmicutes were significantly negatively correlated with pH value and ORP (P < 0.01), whereas Proteobacteria exhibited the opposite trend. The fungal phyla Basidiomycota was significantly negatively correlated with pH and ORP (P < 0.01), whereas Ascomycota exhibited the opposite trend. At the genus level, the bacterial genera Clostridium_sensu_stricto_1 and Lactococcus were significantly positively correlated with the content of key components (P < 0.01), as were the fungal genera Trichosporon and Cutaneotrichosporon (P < 0.01). Conclusion This study preliminarily elucidated the significant interactions between microbial community succession and physicochemical parameters during the soaking and fermentation of IN and constructed an association network between them. These findings provide a theoretical basis for the rapid prediction of indigo-related compound contents and degree of fermentation based on physicochemical parameters, thereby enabling observable, controllable, and tunable industrial production of IN.

R283.6

国家重点研发计划（2018YFC1707205）；国家自然科学基金面上项目（82173976）