「靈芝-豆科」發酵產物之抗氧化活性及其對倉鼠脂質之影響

Antioxidative and lipid-modulating effects of the fermentation products of Lingzhi (Ganoderma lucidum) cultivated in a medium containing leguminous plants

簡永亮(Yung-Liang Chien)

指導教授 ： 孫璐西

國立臺灣大學/生物資源暨農學院/食品科技研究所/博士(2010年)

https://doi.org/10.6342/NTU.2010.00292

摘要

動脈粥狀硬化所造成的腦血管疾病，心臟病與高血壓疾病分別高居國人十大死因的第二、三及十名。過高的血脂濃度與體內的氧化壓力（導致低密度脂蛋白（LDL）的氧化）是動脈粥狀硬化的肇因。人體天然存在的內源性抗氧化物質會與自由基反應而降低其傷害性，許多體外（in vitro）及離體（ex vivo）實驗亦證實飲食中的抗氧化物質可增強LDL對氧化的抵抗力。靈芝中的多醣體、三萜類等活性成分被證實具有抗氧化及降膽固醇等效果。從豆科植物如黑豆及黃耆中萃取出的異黃酮素，更具有抑制脂質過氧化及延緩低密度脂蛋白氧化的能力。以黑豆及黃耆為部分發酵基質，在發酵過程中微生物的作用可能使抗氧化活性成分如異黃酮或其他類黃酮素的醣苷鍵被分解，成為醣苷配基（aglycone）形式，多醣體亦可能經由發酵所產生酵素的作用，使其降解為較小的分子，增加對水的溶解度與生物活性，而更易為人體所吸收及利用。
本論文研究探討的主題為「靈芝-豆科」發酵產物抗氧化活性及其對血脂之影響，分成三個部分進行探討：(1)探討不同發酵條件之「靈芝-豆科」發酵產物之體外抗氧化活性；(2) 以動物模式探討樣品對倉鼠血脂及抗氧化之影響：(a)動物試驗I：探討靈芝子實體對倉鼠血脂之影響；(b) 動物試驗II：探討「靈芝-豆科」發酵濾液對倉鼠血脂之影響；(c) 動物試驗III：探討探討「靈芝-豆科」發酵濾液乙醇萃出物對倉鼠血脂之影響；(d) 動物試驗IV：探討不同飼料及油脂配方對倉鼠血脂之影響；(3) 探討「靈芝-豆科」發酵濾液中具抗氧化活性之成分。
不同發酵條件之「靈芝-豆科」發酵產物抗氧化活性結果顯示：靈芝發酵濾液去除多醣者，其抑制LDL過氧化能力較佳；發酵溫度對靈芝發酵濾液抑制銅離子誘導人類LDL氧化能力為30℃＞18℃＞24℃；通氣量在發酵溫度為18℃時為0.75 vvm效果最佳，發酵溫度為24℃則以0.25 vvm最佳；接菌濃度比較上可發現在發酵溫度30℃時抑制LDL氧化能力為10%＞1%＞0.1%。整體而言對於抑制LDL氧化能力最佳者為GL-D3去除多醣物(發酵條件為30℃、0.75 vvm)，且抗氧化能力隨著發酵時間而增加。在異黃酮素含量方面，總異黃酮素隨著發酵時間而遞減，不含醣基的異黃酮素於發酵初期略有增加，但在第3天後亦呈遞減狀態，至發酵第11天則檢測不到異黃酮素。在抗氧化活性方面，抗氧化能力與總多酚含量有良好的相關性。
動物試驗I結果顯示，餵食添加10％黑豆或黃耆之飼料皆會增加倉鼠血中HDL-C濃度及肝臟中膽固醇濃度，此外黃耆亦會減少倉鼠肝臟中脂質過氧化物含量；餵食添加8％靈芝子實體之飼料對倉鼠血清及肝臟之膽固醇與三酸甘油酯均無降低之效果，但在血清中對過氧化物含量影響則是與黑豆組、黃耆組及正控制組(probucol)相同，均具有降低倉鼠血清MDA之效果。動物試驗II結果顯示，餵食添加10％靈芝發酵濾液之飼料能降低倉鼠肝臟中膽固醇及血清中MDA含量，但倉鼠之每日攝食量、體重及肝臟中之MDA卻顯著增加。動物試驗III結果顯示，餵食倉鼠含1％靈芝發酵濾液乙醇萃出物之飼料對於誘導高血脂之倉鼠血清及肝臟中膽固醇、三酸甘油酯、氧化遲滯期及過氧化物(MDA)均無降低之效果。
動物試驗IV結果顯示，餵食Purina 5001 rodent chow diet之血清總膽固醇、VLDL、LDL及LDL-C/HDL-C均顯著高於餵食AIN-93M，肝臟中膽固醇含量亦為餵食 Purina 5001 rodent chow diet較高。在以AIN-93M為基礎飼料比較不同油脂飽和程度之影響方面，結果顯示除了餵食添加6％豬油降低倉鼠血中膽固醇外，其餘均無顯著差異；本次研究顯示不同基礎飼料對倉鼠本身的血脂所造成的影響遠大於油脂不飽和程度所造成的影響。
探討靈芝發酵濾液中有效抗氧化成分方面，研究結果顯示pyrogallol及protocatechuic acid為靈芝發酵濾液有效抗氧化成分。Protocatechuic acid在靈芝發酵過程中隨著發酵天數延長而增加，總多酚含量及抗LDL氧化活性亦隨發酵天數延長而增加，顯示protocatechuic acid為靈芝發酵液中主要影響其抗氧化活性成分。

關鍵字

靈芝 ； 黑豆 ； 黃耆 ； 抗氧化 ； 液態發酵

並列摘要

Atherosclerosis related diseases, such as cerebral hemorrhage, coronary heart disease and high blood pressure, are the 2nd, 3rd and 10th major causes of death in Taiwan. High blood lipids and oxidative stress, which cause the oxidation of low density lipoprotein (LDL), play important roles in atherosclerosis. Intrinsic antioxidants are known to scavenge free radicals in the body and thus lower the oxidative damage. Many in vitro and ex vivo studies have shown that dietary antioxidants could enhance the resistance of LDL toward oxidation. Recent studies suggested that the polysaccharides and triterpenes present in Ganoderma lucidum have antioxidative activities and are also capable of reducing cholesterol. Leguminous plants such as black soybean and Astragalus membranaceus contain isoflavones which are shown to inhibit lipid peroxidation and delay the oxidation of low density lipoprotein. Using black soybean and A. membranaceus as part of the fermentation medium, isoflavones might be transformed into the more bioavailable form (aglycone) by microbial enzymatic hydrolysis of the glycosidic linkage. The immunomodulatory polysaccharides might also undergo enzymatic degradation into smaller molecules with higher hydrophilicity and bioavailability.
The objectives of this study were to investigate the antioxidative effect and lipid modulating ability of the fermentation broth produced by using leguminous plants as part of the liquid fermentation medium of G. lucidum. This study was, therefore, divided into three parts: (1) in vitro antioxidative effect of the fermentation products of G. lucidum under different fermentation conditions; (2)Antioxidative and lipid-modulating effect of fermentation products with animal model：(a) animal study I：effect of black bean, A. membranaceus and G. lucidum on serum lipids of hamsters; (b) animal study II：effect of fermentation broth filtrate of G. lucidum on serum lipids of hamsters；(c) animal study III：effect of ethanolic extract of G. lucidum fermentation broth filtrate on serum lipids of hamsters；(d) animal study IV：effect of different diet and fat composition on serum lipids of hamsters. (3) To investigate the antioxidant compounds of G. lucidum broth filtrate.
Results of antioxidative effect of fermentation products of G. lucidum under different fermentation conditions showed: G. lucidum broth filtrate after removal of polysaccharide had better antioxidative effect. Comparing the effect of fermentation condition, broth filtrate from fermentation at 30℃ had better effect than 18 and 24℃. The better effects on inhibition of LDL oxidation at 18℃ and 24 C was aeration at 0.75 and 0.25 vvm, respectively. Also, 10% inoculum ratio had better antioxidative effect then 1% and 0.1% at 30℃. Overall, fermentation product with the best antioxidative effect was shown by GL-D after removal of polysaccharide and the antioxidative ability increased with fermentation time. Content of total isoflavone decreased with fermentation time. Isoflavone aglycone increased in the first three days, but then decreased and became undetectable at the 11th day. Total polyphenol contents of the fermentation broth filtrate increased with fermentation time and correlated well with the antioxidative effect, therefore polypheonl may play an important role on antioxitative ability of G. lucidum broth filtrate.
Results of animal study I showed that after feeding 10% black bean or A. membranaceus hamsters had higher serum HDL-C and hepatic cholesterol. Hamsters fed with A. membranaceus also reduced hepatic TBAR value. Hamsters fed with 10% black bean, 10% A. membranaceus and 8% G. lucidum all showed lowered levels of serum MDA than the high fat high cholesterol control group. Results of animal study II showed that hamsters fed with 10% G. lucidum broth filtrate had lower hepatic cholesterol and serum MDA, but the daily feed intake, body weight and hepatic TBAR value were higher than high fat high cholesterol group. Result of animal study III showed that after feeding hamsters 1% ethanolic extract of G. lucidum broth filtrate there were no significant differences in either blood or liver lipids with high fat high cholesterol control group.
Results of animal study IV showed that after feeding Purina 5001 rodent chow diet hamsters had higher serum cholesterol, VLDL, LDL, LDL-C/HDL-C ratio and hepatic cholesterol than feeding AIN-93M diet. Based on AIN-93M diet, hamster feed containing 6% lard resulted in lower serum cholesterol than feed containing 6% corn oil. Our data indicated that hamster fed on Purina 5001 rodent chow diet was a more suitable model for evaluating the effect of test sample on blood lipids than AIN-93M diet
To investigate antioxidant compounds, we found 2 compounds, pyrogallol and protocatechuic acid, were the main active compounds of G. lucidum broth filtrate. We also found that protocatechuic acid, polyphenol content and inhibiting Cu2＋-induced LDL oxidation depended on days of fermentation. The results indicate protocatechuic acid was the main antioxidant compound during fermentation of G. lucidum broth filtrate.

並列關鍵字

Ganoderma lucidum ； black soybean ； A. membranaceus ； antioxidative

參考文獻

楊棋明、楊智旭、趙壁欲。1999。市售黑豆與黃豆抗氧化力之灰預測比較。中華民國營養學會雜誌。24: 201-214。

楊舒秦。2005。靈芝發酵液之免疫活性分析及以靈芝多醣處理人類單核細胞之蛋白質體學探討。國立台灣大學食品科技研究所碩士論文。

謝孟志、黃士懿、錢信、楊蕙婷、流配姎、謝明哲。2000。高膽固醇飲食添加紅麴粉末對倉鼠體內脂質代謝之影響。中華營養學會誌。15:11-22。

謝孟潔。2006。龍眼花抗氧化成分之研究。國立台灣大學食品科技研究所碩士論文。

李儀儂。2003。培養條件對靈芝菌絲多醣與其(1, 3；1, 6)-β-D-聚葡萄醣生成量及性質之影響。國立台灣大學食品科技研究所碩士論文。