基于液体发酵的高三萜灵芝菌生物转化大豆异黄酮及其抑制肿瘤活性研究

发布时间：2018-09-09 17:17

【摘要】：灵芝Ganoderma lucidum [(Leyss.ex.Fr.) Karst]是一种珍贵的药用真菌,其作为药物在我国已有2000多年的历史,本文以高产三萜灵芝菌株为研究对象,研究内容包括：灵芝酸成分的初步鉴定,灵芝转化大豆异黄酮及其转化产物的抗癌活性和机理。对5株供试灵芝菌株进行菌株筛选得到最优菌株为美国灵芝,其菌丝生物量、胞内三萜、胞内多糖、胞外多糖、和胞外三萜产量分别可达1.22±0.03g/100ml, 39.29±0.25mg/100ml,23.44±0.76mg/100ml,11.50±0.74mg/ml,14.24±0.55 mg/ml;对其进行菌种鉴定,基于ITS序列分析,该菌株鉴定为赤芝(G. lucidum),是可以用于保健食品的灵芝菌种,具备食用安全性；进而对美国灵芝液体发酵培养基及培养条件进行优化,结果显示,灵芝菌丝接种到种子液中生长8d后,按接种量10%(v/v)接种于优化后的发酵培养基(麦芽汁4.10%,酵母浸出粉1.8%,KH2PO4 0.3%, MgSO4 0.15%, VB1 0.005%, pH 5.40),于180rpm,28℃,培养7d,可使灵芝菌丝生物量和胞内三萜产量达到最高。此外,本文按照European Brewery Cenvention (EBC)方法制备麦芽汁,将其应用于灵芝液体发酵以提高灵芝三萜产量,至今未见报道。经HPLC-ESI-MS初步鉴定,结果显示灵芝菌丝中含有的10种灵芝酸为：Ganolucidic acid A, Ganoderic acid A, Ganoderenic acid B, Elfvingic acid A, Ganoderic acid F,7,15-dihydroxy-4,4,14-trimethyl-3,11-dioxochol-8-en-24-oic acid, Lucidenic acid C,3β-hydroxy-4,4,14-trimethyl-7,11,15-trioxochol-8-en-24-oic acid, Ganoderic acid H,3,7,15-trihydroxy-4,4,14-trimethyl-11-oxo-chol-8-en-24-oic acid。此外,灵芝在液体发酵过程中可以有效的富集氨基酸,尤其是人体必需氨基酸,同时还可富集微量元素,但对各元素的富集能力各有不同。将含有菌丝体的灵芝发酵液匀浆,以β-葡萄糖苷酶酶活为1.0U/ml的灵芝匀浆液100ml(pH=5)作为转化反应液,加入5g大豆异黄酮粗提物,于60℃下转化48h,得到大豆苷元及染料木素转化率分别为96.63%,87.82%。利用富集了生物活性物质的灵芝匀浆液转化大豆异黄酮至今尚未见报道。测定转化产物抗氧化能力,随着底物浓度(0.5-10mg/ml)增加,转化产物(TSI)、转化前(SI)溶液各抗氧化指标均随之上升,并且在相同底物浓度下,TSI溶液抑制羟自由基能力(底物浓度6mg/ml)、DPPH清除能力、SOD酶活及T-AOC均高于SI溶液。分别经30%、50%、75%(v/v)乙醇浸提灵芝转化大豆异黄酮产物得到TSI-1、 TSI-2、TSI-3提取液。经MTT法检测,TSI-1 (120μg/ml), TSI-2 (100μg/ml), TSI-3 (60μg/ml)对细胞HTL9, MCF-7, HepG2的增殖均有显著的抑制作用,且都可以促进细胞凋亡,其中,TSI-2 (100μg/ml)对细胞HTL9凋亡影响最为显著,晚凋细胞由8.27%增至40.13%,早凋细胞由0.95%增至9.05%。此外,经TSI-2 (100μg/ml)处理后,细胞HTL9、MCF-7被阻滞于G1期,HepG2被阻滞在S期,无法进行分裂而诱导凋亡。细胞HTL9经TSI(100μg/ml)处理后,细胞中Bax相对含量增加,Bcl-2/Bax比值下降,同时Cyto-C蛋白表达含量显著增加,激活Caspase-3, Caspase-8使其含量增加,由此可说明TSI主要是通过线粒体途径诱导细胞凋亡的。此外,抗凋亡因子Survivin相对表达量、NF-κB蛋白含量显著降低,而促凋亡p53的相对表达量增加。结果表明,TSI(100μg/ml)可通过对多个与凋亡相关基因的调控,来诱导细胞HTL9凋亡。
[Abstract]:Ganoderma lucidum [(Leyss.ex.Fr.) Karst] is a kind of valuable medicinal fungi. It has been used as a drug for more than 2000 years in China. In this paper, Ganoderma lucidum strains with high triterpene yield were selected as the research object. The primary identification of Ganoderma lucidum components, the anticancer activity and mechanism of Ganoderma lucidum transformed soybean isoflavones and their transformed products were studied. The optimum strains of Ganoderma lucidum were obtained by screening five strains of Ganoderma lucidum. The mycelial biomass, intracellular triterpenoids, intracellular polysaccharides, extracellular polysaccharides, and extracellular triterpenoids yields were 1.22 (+ 0.03g/100ml), 39.29 (+ 0.25mg/100ml), 23.44 (+ 0.76mg/100ml), 11.50 (+ 0.74mg/ml) and 14.24 (+ 0.55 mg/ml), respectively. Column analysis showed that the strain was identified as G. lucidum, which could be used in health food and had food safety. Then the liquid fermentation medium and culture conditions of Ganoderma lucidum were optimized. The results showed that the mycelium of Ganoderma lucidum was inoculated into the optimized fermentation medium (10% (v / v) after 8 days of growth in the seed liquid. The biomass of Ganoderma lucidum mycelium and the production of intracellular triterpenoids reached the highest level after 7 days of culture at 180 rpm, 28 C for 7 days. In addition, the wort was prepared by the method of European Brewery Cenvention (EBC) and applied to the liquid fermentation of Ganoderma lucidum to improve the production of triterpenoids. The results of preliminary identification by HPLC-ESI-MS showed that Ganoderic acid A, Ganoderic acid A, Ganoderenic acid B, Elfvingic acid A, Ganoderic acid F, 7,15-dihydroxy-4,4,14-trimethyl-3,11-xochol-8-en-24-oic acid, Lucidenic acid C, 3 beta-hydroxy-4,4,14-dioxylic acid C, Lucidenic acid C. - trimethyl-7,11,15-trioxochol-8-en-24-oic acid, Ganoderic acid H, 3,7,15-trihydroxy-4,4,14-trimethyl-11-oxo-chol-8-en-24-oic acid. In addition, Ganoderma lucidum can effectively enrich amino acids, especially essential amino acids of human body, and trace elements, but the enrichment ability of each element is different. Similarly, the conversion rates of daidzein and genistein were 96.63% and 87.82% respectively by adding 5g soybean isoflavone crude extract to the homogenate of Ganoderma lucidum fermentation broth containing mycelium, 100ml of Ganoderma lucidum homogenate with activity of beta-glucosidase 1.0U/ml (pH=5) as the conversion reaction solution and 48 hours at 60 C. The antioxidant capacity of the transformed product was determined. With the increase of substrate concentration (0.5-10mg/ml), the antioxidant indexes of the transformed product (TSI) and the solution before transformation (SI) increased. At the same substrate concentration, the TSI solution inhibited hydroxyl radical (substrate concentration 6mg/ml), DPPH scavenging capacity, SOD and so on. TSI-1, TSI-2 and TSI-3 were obtained from Ganoderma lucidum by ethanol extraction with 30%, 50% and 75% (v/v) respectively. The results of MTT assay showed that TSI-1 (120 ug/ml), TSI-2 (100 ug/ml) and TSI-3 (60 ug/ml) significantly inhibited the proliferation of HTL-9, MCF-7 and HepG2 cells, and could promote cell apoptosis. Among them, TSI-2 (100 ug/ml) had the most significant effect on the apoptosis of HTL9 cells, the late apoptotic cells increased from 8.27% to 40.13%, the early apoptotic cells increased from 0.95% to 9.05%. In addition, after TSI-2 (100 ug/ml) treatment, HTL9 and MCF-7 were blocked in G1 phase, HepG2 was blocked in S phase and could not divide and induce apoptosis. The relative content of Bax increased, the ratio of Bcl-2 to Bax decreased, and the expression of Cyto-C protein increased significantly. Caspase-3 and Caspase-8 were activated, which indicated that TSI induced apoptosis mainly through mitochondrial pathway. The results showed that TSI (100 ug/ml) could induce apoptosis of HTL9 cells by regulating several apoptosis-related genes.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：TQ929;TQ460.1
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本文编号：2233056