药用真菌戴氏虫草中透明质酸的定性定量分析

目的: 建立定性、定量分析真菌来源透明质酸的方法。 方法: 采用十六烷基三甲基溴化铵(CTAB)比浊法定性检测真菌发酵液中透明质酸,采用改良Bitter-Muir(BM)法、共振瑞利散射(RRS)法定量分析真菌发酵液中透明质酸,并比较两者的优劣。 结果: CTAB能够与真菌发酵液中透明质酸形成沉淀,导致溶液浑浊。改良BM法测定透明质酸在10~50 μg·mL^-1范围内具有良好的线性关系,平均加样回收率(n=9)为97.52%;RRS法测定透明质酸在0.5~2.5 μg·mL^-1范围内具有良好的线性关系,平均加样回收率(n=9)为100.1%。发酵基质中果糖、蔗糖、乳糖、葡萄糖等碳源可严重影响BM法的测定结果,而对RRS法测定干扰小。 结论: CTAB法可以作为快速鉴别真菌发酵生产透明质酸的检测方法,RRS法可作为真菌发酵生产透明质酸过程中大量样品的快速定量分析方法。

Objective: To establish the qualitative and quantitative assays for fungus-derived hyaluronic acid (HA). Methods: A cetyltrimethylammonium bromide (CTAB) turbidity method was employed to detect HA in fungal fermented broth.The quantitative analysis of HA concentration in fermentation broth was performed using methods of modified Bitter-Muir (BM),and resonance Rayleigh scattering (RRS) method. Results: CTAB could generate a complexation reaction with HA in fermented broth,which resulted in a turbid CTAB-HA precipitate.For the modified BM quantitative assay of HA,whereas,there was a good linearity in the range of 10 to 50 μg·mL^-1,the average recovery was 97.52%.For the RRS quantitative assay of HA,the calibration curve showed a good correlation in the range of 0.5 to 2.5 μg·mL^-1,the average recovery (n=9) was 100.1%.More importantly,the constituents in fermentation broth,especially the carbon source such as fructose,sucrose,glucose,and lactose,could lead to a considerable interference with modified BM method,but only very slight interference with RRS method. Conclusion: Owing to its simplicity and rapidity,CTAB method can be directly used to detect HA in the fermentation broth of Cordyceps taii.The findings also indicate that RRS method can be applied as a rapid quantitative determination of HA in fungal fermentation broth.