【作者】 杨卓；

【导师】 王艳辉； 董金皋；

【作者基本信息】 河北农业大学 ， 植物学， 2018， 硕士

【摘要】 香菇(Lentinus edodes)是一种药食两用的真菌。香菇多糖具有提高机体免疫功能、降血脂、抗肿瘤、抗衰老、抗病毒等生物活性,且没有任何毒副作用。香菇柄作为香菇生产中的下脚料被丢弃,造成了极大的资源浪费。为了充分开发香菇柄的利用价值,并且为香菇柄多糖的工业生产提供数据参考。本文的主要研究结果和创新点如下:1.对比了香菇柄多糖与香菇盖多糖的体外抑癌活性。香菇柄也有很好的提取率,说明香菇柄具有良好的多糖提取加工价值;香菇柄与香菇盖粗多糖对8个离体癌细胞系HCT116、HT29、KM12L4、SW620、SW480、RKO、H4、HepG2的增殖均有明显抑制作用,表现出较强的抑癌活性;利用流式细胞仪研究表明,香菇柄与香菇盖粗多糖均可抑制HCT116细胞有丝分裂,使停留在G0/G1期的细胞比例明显增加,这可能与其抑癌功能相关;利用实时定量PCR技术,发现香菇柄、香菇盖粗多糖对与抗癌机制有关的基因ATF4的表达有显著促进作用,对C-ymc的表达有抑制作用。在以上试验中,说明香菇柄多糖同香菇盖多糖一样具有较好的开发价值。2.对比了粗多糖和纯化多糖的体外抗氧化和抑癌效果。为了研究香菇柄中的蛋白成分是否具有生物活性,用Sevage法将香菇柄样品进行脱蛋白处理,得到纯化多糖。粗多糖对自由基的清除率高于纯化多糖;结果表明,粗多糖具有更好的体外抗氧化和抑癌效果,开发粗多糖产品具有更好的应用前景。3.通过响应面法优化了香菇柄多糖的提取工艺。利用响应面软件进行设计得到17个试验点。进行响应面分析后得到参数为液料比(mL/g)43:1、浸提温度79℃、浸提时间124 min,在此条件下多糖的含量为18.66%,所得优化条件为大规模提取香菇柄多糖提供了试验和理论参考。4.研究了不同提取方法对香菇柄多糖的体外抗氧化活性影响。冷冻干燥得到的多糖比喷雾干燥得到的多糖具有更强的清除自由基的能力,说明冷冻干燥法更有利于多糖抗氧化活性的保持;浸提液要比醇沉物多糖具有更强的清除自由基的能力,说明浸提液有更好的体外抗氧化活性。在考虑药效和经济成本平衡的前提下,应选择醇沉冷冻干燥法进行多糖提取。5.研究了储藏时间长短对香菇柄粗多糖抗氧化和抑癌活性的影响。将新提取的香菇柄粗多糖样品在-20℃保存40 d,香菇柄粗多糖仍具有良好的抗氧化活性。因加工简单,生产成本低,抗氧化效果良好,使香菇菇柄粗多糖具有良好的开发前景。将新提取的香菇柄粗多糖液保存于-20℃,检测了40 d内香菇柄粗多糖对人肝癌SMMC-7721细胞增殖的抑制作用。如以液体状态储存香菇柄粗多糖,储藏温度应为低于-20℃,储藏时间应小于30 d。更多还原

【Abstract】 Lentinus edodes are medicinal and edible fungi.Lentinan has biological activities such as improving immune function,reducing blood fat,antitumor,anti-aging,and antivirus,without any toxic side effects.Shitake mushroom stems were discarded as waste in the production of shiitake mushrooms,resulting in a great waste of resources.In order to fully exploit the potential value of shitake mushroom stems,and provided a reference for the industrial production of shitake mushroom stems polysaccharides.The main findings and innovations of this paper are as follows:1.Comparison of the anticancer activity of the crude polysaccharides extracted from shitake mushroom stems and shitake mushroom caps in vitro.Shitake mushroom stems also have a good extraction rate,indicating that the shitake mushroom stems have good extraction and processing value of polysaccharides.Polysaccharides extracted from shitake mushroom stems and shitake mushroom caps both can significantly inhibited the proliferation of eight isolated cancer cell lines HCT116,HT29,KM12L4,SW620,SW480,RKO,H4,and HepG2,showing strong anticancer activity.Flow cytometry assays showed that both shitake mushroom stems and shitake mushroom caps polysaccharide could impede mitosis in HCT116 cells,and the proportion of cells staying in G0/G1 phase increased significantly,which may be due to its tumor suppressor function.Through real-time quantitative PCR assay,it was found that the crude polysaccharides extracted from shitake mushroom stems and shitake mushroom caps both can significantly promote the expression of the anticancer related gene ATF4 and inhibit the expression of C-ymc.In the above experiments,indicating that shitake mushroom stems polysaccharides have at least the same exploitation value to the shitake mushroom caps.2.Comparison of the antioxidant and anticancer effects of crude polysaccharides and purified polysaccharides in vitro.In order to examine whether the proteins in the stems of shitake mushroom have biological activity,the Sevage method was used to remove the protein from the shitake mushroom stems sample to obtain the purified polysaccharides.It suggested that higher free radical scavenging rate of crude polysaccharides than the purified polysaccharides.The results indicate that the crude polysaccharides have better antioxidant and anticancer effects in vitro,and the development of crude polysaccharides products have better application prospects.3.Optimization the extraction technology of polysaccharides of shitake mushroom stems by response surface methodology.The response surface software was used to design and obtain 17 test points.The parameters obtained from the response surface analysis were liquid/material ratio(mL/g)43:1,extraction temperature 79°C,and extraction time 124 min.Using these parameters,the obtained polysaccharides content was 18.66%.The optimized parameters provide experimental and theoretical references for the large-scale extraction of shitake mushroom stems polysaccharides.4.The effects of different extraction methods on the antioxidant activity of shitake mushroom stems polysaccharides in vitro were studied.It indicates that the polysaccharides obtained by freeze-drying have stronger ability to scavenge free radicals than spray-dried polysaccharides,the freeze-drying method is more conducive to the preservation of the antioxidant activity.It indicates that the without alcohol-precipitated polysaccharides have a stronger ability to scavenge free radicals than the alcohols-precipitated,the without alcohol-precipitated method is more conducive to the preservation of the antioxidant activity.Under the premise of considering the balance of efficacy and economic cost,alcohol freeze-drying should be used to extract polysaccharides.5.The effects of storage time on antioxidant activity and anticancer activity of crude polysaccharides of shitake mushroom stems.The freshly extracted shitake mushroom stems crude polysaccharide samples were stored at-20°C for 40 days.It indicates that the crude polysaccharide still has good antioxidant activity.Due to the simple processing,low cost and good antioxidant effect,the crude polysaccharides from shitake mushroom stems have superior prospects.The freshly extracted shitake mushroom stems crude polysaccharides solution was stored at-20°C.The inhibition effects of polysaccharides on the proliferation of human hepatocellular carcinoma SMMC-7721 cells were detected within 40 days.The results showed that if storing the crude polysaccharides of shitake mushroom stems in liquid state,the storage temperature should be lower than-20°C,and the storage time should be less than 30 days.更多还原