目的 以中药固体双向发酵方法，建立至真方的新型发酵工艺。方法 ①通过文献研究拟定候选发酵菌 种。对候选发酵菌种进行平板混合实验，考察各菌种间生长兼容性。采用 CCK-8 细胞增殖抑制实验，比较不 同发酵条件下所得产物对人结直肠癌细胞（HCT-8）的半数抑制浓度（IC50），评判发酵体系的优劣，从而确定发 酵菌种、发酵时长等关键发酵参数，建立发酵工艺。②通过 HCT-8 细胞增殖抑制率，以及巨噬细胞 （RAW264.7）NO 释放率和吞噬活性测定，比较至真方发酵前后的体外药效差异。结果 黑曲霉和酵母菌为最 优发酵菌种组合，4 d 为最优发酵时长。在此发酵条件下，发酵组对 HCT-8 细胞的 IC50为 67.41 µg·mL-1，生 药组对 HCT-8 细胞的 IC50为 1 032 µg·mL-1；与生药组比较，发酵组能显著抑制 RAW264.7 释放炎症因子 NO， 并显著提升其吞噬活性。结论 该研究建立了至真方固体双向发酵新工艺。在该发酵体系下，至真方的体外抗 结直肠癌活性、抗炎活性和促免疫活性得到显著提升。

Objective To establish a new fermentation process for Zhizhen Recipe by using bi-directional solid fermentation method of traditional Chinese medicine. Methods ① Candidate strains for fermentation were primarily recruited through literature research. Then plate incorporation assay of candidate strains were conducted to investigate the growth compatibility between the strains. CCK-8 cell proliferation assay was used to compare the half proliferation inhibitory concentration of the products obtained under different fermentation conditions against human colorectal cancer cell line HCT-8. The advantages of different fermentation systems were evaluated and the key fermentation parameters including strains and incubation periods were determined， which gave rise to the establishment of the optimal fermentation process for Zhizhen Recipe. ② In vitro pharmacological effects were compared between the fermented and unfermented Zhizhen Recipe by measuring proliferation inhibition of HCT-8 cells，as well as NO release and phagocytic activity of RAW264.7 cells. Results The results showed that Aspergillus niger/Saccharomyces cerevisiae was the optimal combination of strains，and the optimal fermentation time is 4 days. Under optimized conditions，the half inhibitory concentration （IC50） of the fermentation group on HCT-8 cells was 67.41 µg·mL-1， while IC50 value of the unfermented Zhizhen Recipe was 1 032 µg·mL-1. Compared with unfermented preparation， the fermented preparation exhibited significant effects on inhibiting the release of inflammatory factor NO of macrophages RAW264.7 cells and obviously enhanced their phagocytic activity. Conclusion In this present study，we established a new bi-directional solid fermentation process for Zhizhen Recipe. Under this fermentation system， the anti-colorectal cancer activity， anti-inflammatory activity and pro-immune activity of Zhizhen Recipe in vitro were significantly improved.

R283.1；285.5

上海交通大学“医工交叉基金”项目（YG2019ZDA28）。