瓦尼桑黄核苷类成分的液体发酵工艺优化及其细胞毒活性

Optimization of liquid fermentation and cytotoxic activity of nucleosides from Sanghuangporus vaninii

瓦尼桑黄(Sanghuangporus vaninii)是一种珍贵的药用真菌,其所含有的核苷类成分可开发为具有抗病毒、抗肿瘤、抗氧化、抗菌等活性的药品或保健产品等.该文通过单因素和正交试验对瓦尼桑黄液体发酵工艺进行优化,以核苷产量为指标,探究了最优发酵条件以及碳源、氮源、无机盐的最佳比例,并考察了添加不同前体物质以及调控因子对核苷产量的影响.试验筛选出影响核苷产量的关键因素之一为外源添加前体物质如次黄嘌呤、腺嘌呤,并确定了最优发酵条件:接种量10%、初始pH值为7、培养温度32℃、摇床转速140 r·min^(-1)、培养时间5 d;最佳培养基配比:葡萄糖15.00 g·L^(-1)、蔗糖20.00 g·L^(-1)、蛋白胨5.00 g·L^(-1)、牛肉膏2.50 g·L^(-1)、MgSO_(4)1.00 g·L^(-1)、CaCl_(2)1.50 g·L^(-1)、ZnSO_(4)1.00 g·L^(-1)、MnSO_(4)0.50 g·L^(-1);最优前体物质和调控因子及其添加量:次黄嘌呤1.50 g·L^(-1)、腺嘌呤2.50 g·L^(-1)、丙酮酸钠3.50 g·L^(-1)、槲皮素0.25 g·L^(-1).在最优工艺下瓦尼桑黄液体发酵核苷产量为291.16±6.21 mg·L^(-1),是优化前的32.3倍.此外,该研究还比较了优化前后以及子实体核苷富集产物的体外细胞毒活性,试验结果表明优化后的核苷富集产物对人肝癌细胞HepG2的抑制活性最好,抑制率高于优化前以及子实体的富集产物,为瓦尼桑黄进一步开发利用提供了理论基础.

Sanghuangporus vaninii is a valuable medicinal fungus,which contains nucleoside components that can be developed into pharmaceuticals and health products with antiviral,antitumor and antibacterial activities.In this paper,the fermentation conditions and ratio of carbon,nitrogen and inorganic salts for liquid fermentation of Sanghuangporus vaninii were optimized by single factor and orthogonal tests,using nucleoside yield as an indicator.And the effects of adding different precursors and regulatory factor on the yield of nucleoside were investigated.One of the key factors affecting nucleoside production was screened out as exogenous precursors such as hypoxanthine and adenine.The optimal fermentation conditions were determined as follows:10% inoculum,initial pH 7,incubation temperature 32℃,shaker speeds 140 r·min^(-1) and incubation time 5 d.The optimal medium ratios were as follows:glucose 15.00 g·L^(-1),sucrose 20.00 g·L^(-1),peptone 5.00 g·L^(-1),beef paste 2.50 g·L^(-1),MgSO_(4)1.00 g·L^(-1),CaCl_(2)1.50 g·L^(-1),ZnSO 41.00 g·L^(-1),MnSO_(4)0.50 g·L^(-1).Optimal amount of precursor and regulatory factor were hypoxanthine 1.50 g·L^(-1),adenine 2.50 g·L^(-1),sodium pyruvate 3.50 g·L^(-1) and quercetin 0.25 g·L^(-1).Under the optimum conditions,the nucleoside yield of liquid fermentation of Sanghuangporus vaninii was 291.16±6.21 mg·L^(-1),32.3 times higher than that before optimization.In addition,the cytotoxic activities of the nucleoside enriched products from the fruiting body and sample that before and after optimization were compared.The experimental results showed that the optimized liquid fermentation nucleoside products had the best inhibitory activity on human hepatoma cell line HepG2,with a higher inhibitory rate than the enriched products before optimization and the fruiting body products,This paper provided a theoretical basis for the further development and utilization of Sanghuangporus vaninii.