利用根黴菌之深部培養法開發機能性醱酵黑豆食品之研究

Abstract

本研究利用省產黑豆為原料以根黴菌之液態醱酵法生產機能性醱酵黑豆食品，並對液態醱酵黑豆產品進行功能性檢測及功能性成分分析，以期確立機能性醱酵黑豆產品之醱酵生產體系，開拓省產黑豆在健康食品上之用途為目的。 。 以高效能液相層析法可有效分離四種指標性異黃酮。探討液態醱酵黑豆最佳萃取條件，得知以80% 甲醇萃取可得到最高異黃酮含量及最佳DPPH自由基之清除效應。以黑豆台南三號為基質篩選適合液態醱酵之根黴菌，並檢討液態醱酵期間代謝產物之變化，得知三株檢測菌皆可將帶醣基之異黃酮（daidzin及genistin）經由 β-葡萄醣苷酶之作用轉換成不具醣基之異黃酮（daidzein及genistein）。依據異黃酮轉換效率、自由基清除之效應及醱酵時間等因子，選擇以Rhizopus oligosporus NTU-5為最適液態醱酵菌株。 在擴大生產試驗中，檢討攪拌速率及通氣量對R. oligosporus NTU-5於五公升醱酵槽中液態醱酵黑豆之影響，並分析醱酵期間代謝產物之變化，得知最適液態培養條件為培養溫度，30℃；通氣量，0.1 vvm；攪拌速率，100 rpm；接種量，10% 及醱酵槽裝液量， 3 公升/ 5 公升。 。 以矽膠管柱層析法進行液態醱酵黑豆粗萃液之溶劑沖提，得知以乙酸乙酯：甲醇（2：1; v/v）之區分5具有最大抗氧化活性。以薄層層析法及半製備式高效能液相層析法分離出不具醣基之異黃酮 daidzein、genistein 及具抗氧化活性未知化合物 FBE5-PA。Daidzein 及 genistein 之回收率分別為 90.6% 及 70.5%。未知化合物FBE5-PA對DPPH自由基之有效半抑制濃度（IC50）為 7.5 μg/mL。 液態醱酵黑豆粗萃液之乾燥產物在濃度為400μg/mL時對人類肝癌細胞 Hep 3B 及子宮頸癌細胞 Hela 具有抑制增生之能力，抑制率分別為 61.5% 及 64.4%，對於人類正常肺纖維母細胞 MRC-5 則無顯著抑制效果。

The purpose of this study is to develop a submerged fermentation process to produce functional fermented black soybean foods from domestic black soybean by Rhizopus spp. .The functional components of fermented black soybean and its physiological function will also be investigated. These results can provide information to establish a submerged fermentation process to manufacture functional fermented black soybean foods. . A fast and precise method of high performance liquid chromatography can separate four indicator isoflavones efficiently. Using 80% methanol to extract the fermented black soybean, the content of isoflavones and 1, 1-diphynel-2-dipicrylhydrazyl (DPPH) radical scavenging effects can reach to the best. Black soybean Tainan NO.3 was used as substrate for submerged fermentation by Rhizupus spp. . The changes of metabolites during the submerged fermentation were analyzed. It was found that all Rhizupus spp. tested exhibited the ability to convert the isoflavones glycosides （daidzin and genistin）in black soybean to isoflavones aglycones （daidzein and genistein）by β-glucosidase. According to the content of isoflavone aglycones formation, scavenging effect and cultivation time, Rhizupus oligosporous NTU-5 was selected as potent strain for submerged fermentation of black soybean. . The effects of agitation speed and aeration rate on isoflavones content and scavenging effect of fermented black soybean were carried out in a 5L fermentor, and the changes of metabolites during the submerged fermentation were analyzed. It was found that the optimal cultivation condition were as follows: temperature, 30℃; aeration rate, 0.1 vvm; agitation speed, 100 rpm; inoculum size, 10% and working volume, 3 L/ 5 L. . A fraction 5 had the highest scavenging effect was isolated by silica gel column chromatography, eluting with ethyl acetate/methanol （2: 1, v/v）. Then, the fraction 5 was analyzed and purified by TLC and semi-preparative HPLC. Daizein, genistein and an unknown antioxidant compound, FBE5-PA were isolated. The recovery of daidzein and genistein were 90.6% and 70.5%, respectively. The IC50 value of FBE5-PA for DPPH free radicals was 7.5 μg/mL. . The fermented black soybean extracts exhibited inhibition effect on the viability of human cancer cell line, Hep 3B and Hela when the concentration was up to 400μg/mL. The inhibition rate was 61.5% and 64.4%, respectively. However, it showed no inhibition effect on normal human embryonal lung cell MRC-5.