發(fā)布時(shí)間：2018-09-09 17:17

【摘要】：靈芝Ganoderma lucidum [(Leyss.ex.Fr.) Karst]是一種珍貴的藥用真菌,其作為藥物在我國(guó)已有2000多年的歷史,本文以高產(chǎn)三萜靈芝菌株為研究對(duì)象,研究?jī)?nèi)容包括：靈芝酸成分的初步鑒定,靈芝轉(zhuǎn)化大豆異黃酮及其轉(zhuǎn)化產(chǎn)物的抗癌活性和機(jī)理。對(duì)5株供試靈芝菌株進(jìn)行菌株篩選得到最優(yōu)菌株為美國(guó)靈芝,其菌絲生物量、胞內(nèi)三萜、胞內(nèi)多糖、胞外多糖、和胞外三萜產(chǎn)量分別可達(dá)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;對(duì)其進(jìn)行菌種鑒定,基于ITS序列分析,該菌株鑒定為赤芝(G. lucidum),是可以用于保健食品的靈芝菌種,具備食用安全性；進(jìn)而對(duì)美國(guó)靈芝液體發(fā)酵培養(yǎng)基及培養(yǎng)條件進(jìn)行優(yōu)化,結(jié)果顯示,靈芝菌絲接種到種子液中生長(zhǎng)8d后,按接種量10%(v/v)接種于優(yōu)化后的發(fā)酵培養(yǎng)基(麥芽汁4.10%,酵母浸出粉1.8%,KH2PO4 0.3%, MgSO4 0.15%, VB1 0.005%, pH 5.40),于180rpm,28℃,培養(yǎng)7d,可使靈芝菌絲生物量和胞內(nèi)三萜產(chǎn)量達(dá)到最高。此外,本文按照European Brewery Cenvention (EBC)方法制備麥芽汁,將其應(yīng)用于靈芝液體發(fā)酵以提高靈芝三萜產(chǎn)量,至今未見報(bào)道。經(jīng)HPLC-ESI-MS初步鑒定,結(jié)果顯示靈芝菌絲中含有的10種靈芝酸為：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,靈芝在液體發(fā)酵過程中可以有效的富集氨基酸,尤其是人體必需氨基酸,同時(shí)還可富集微量元素,但對(duì)各元素的富集能力各有不同。將含有菌絲體的靈芝發(fā)酵液勻漿,以β-葡萄糖苷酶酶活為1.0U/ml的靈芝勻漿液100ml(pH=5)作為轉(zhuǎn)化反應(yīng)液,加入5g大豆異黃酮粗提物,于60℃下轉(zhuǎn)化48h,得到大豆苷元及染料木素轉(zhuǎn)化率分別為96.63%,87.82%。利用富集了生物活性物質(zhì)的靈芝勻漿液轉(zhuǎn)化大豆異黃酮至今尚未見報(bào)道。測(cè)定轉(zhuǎn)化產(chǎn)物抗氧化能力,隨著底物濃度(0.5-10mg/ml)增加,轉(zhuǎn)化產(chǎn)物(TSI)、轉(zhuǎn)化前(SI)溶液各抗氧化指標(biāo)均隨之上升,并且在相同底物濃度下,TSI溶液抑制羥自由基能力(底物濃度6mg/ml)、DPPH清除能力、SOD酶活及T-AOC均高于SI溶液。分別經(jīng)30%、50%、75%(v/v)乙醇浸提靈芝轉(zhuǎn)化大豆異黃酮產(chǎn)物得到TSI-1、 TSI-2、TSI-3提取液。經(jīng)MTT法檢測(cè),TSI-1 (120μg/ml), TSI-2 (100μg/ml), TSI-3 (60μg/ml)對(duì)細(xì)胞HTL9, MCF-7, HepG2的增殖均有顯著的抑制作用,且都可以促進(jìn)細(xì)胞凋亡,其中,TSI-2 (100μg/ml)對(duì)細(xì)胞HTL9凋亡影響最為顯著,晚凋細(xì)胞由8.27%增至40.13%,早凋細(xì)胞由0.95%增至9.05%。此外,經(jīng)TSI-2 (100μg/ml)處理后,細(xì)胞HTL9、MCF-7被阻滯于G1期,HepG2被阻滯在S期,無法進(jìn)行分裂而誘導(dǎo)凋亡。細(xì)胞HTL9經(jīng)TSI(100μg/ml)處理后,細(xì)胞中Bax相對(duì)含量增加,Bcl-2/Bax比值下降,同時(shí)Cyto-C蛋白表達(dá)含量顯著增加,激活Caspase-3, Caspase-8使其含量增加,由此可說明TSI主要是通過線粒體途徑誘導(dǎo)細(xì)胞凋亡的。此外,抗凋亡因子Survivin相對(duì)表達(dá)量、NF-κB蛋白含量顯著降低,而促凋亡p53的相對(duì)表達(dá)量增加。結(jié)果表明,TSI(100μg/ml)可通過對(duì)多個(gè)與凋亡相關(guān)基因的調(diào)控,來誘導(dǎo)細(xì)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.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2015
【分類號(hào)】：TQ929;TQ460.1
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本文編號(hào)：2233056