利用裏氏木黴固態發酵玉米胚芽與麩皮之產物對降低類澱粉β-胜肽毒性之功效

Abstract

阿茲海默症主要由類澱粉胜肽(Amyloid β-peptide, Aβ)堆積在腦中形成斑塊，使神經細胞發生氧化與發炎反應，最後導致神經細胞凋亡。近年來許多研究顯示，自然界中的抗氧化劑，例如維生素E、多酚類和類胡蘿蔔素，具有清除自由基和抑制ROS形成之功能，並可修復Aβ產生的發炎與氧化反應，使腦神經細胞受到保護。玉米為世界上重要的穀類作物，研究指出玉米的總酚含量和抗氧化活性皆高於小麥、燕麥和稻米，其富含具抗氧化活性的植化素，主要分布於胚芽與麩皮。玉米胚芽與麩皮為具高活性的抗氧化物，但過去並無文獻探討其對抑制Aβ毒性之功效。另外，日常生活中攝取玉米胚芽主要來自於玉米油，而傳統的製油方式在乾燥胚芽的過程中，經常會使油類發生高度的氧化作用，使胚芽內部的營養物質變質，有研究指出，利用裏氏木黴生產的纖維素酶可將細胞壁分解而釋放油脂，可保有玉米胚芽中的抗氧化物。麩皮除了具有抗氧化物質外，也因富含纖維素可提供微生物良好的生長碳源。因此，本研究目的以裏氏木黴進行固態發酵，產生纖維素酶分解玉米胚芽與麩皮的細胞壁，進而釋放內部的有效物質，接著以細胞平台(IMR-32 cell line)測定發酵液的細胞毒性與抑制Aβ毒性之功效，並用不同溶劑進行萃取以分析有效物質。實驗組別將胚芽與麩皮混和成不同比例，共有五組，分別有胚芽與麩皮比例1：2和2：1，以及水分含量較少的組別1：3、3：1和2：2。結果顯示，各組發酵產物之萃取液皆於濃度100μg/mL以下，細胞存活率達80%以上。在抑制Aβ毒性的能力試驗上，以發酵五天之發酵萃取物的功效最佳，各組在濃度100μg/mL時皆有90%以上之細胞存活率，其中，以胚芽與麩皮的比例3：1細胞存活率達110%，其抑制Aβ毒性之功效最顯著，因此，選此組別進行萃取分析。在不同溶劑萃取發酵液的試驗中，以正己烷、乙酸乙酯、正丁醇和水萃取，結果發現，正丁醇和水層之萃取發酵液，其抑制Aβ毒性效果最好，細胞存活率分別達95%和104%，推測在正丁醇和水層中可萃取出具神經保護功效之物質。將各層萃取液做成分分析，結果顯示正丁醇之總酚含量有507.8 mg/g，而水層萃取液其總酚含量則達324.6 mg/g，推測總酚為功效物質。水層萃取液的總酚含量雖然為次高，但其抑制Aβ毒性之功效為最好，參考前人研究，推測水層中可能含有小分子的碳水化合物，具有清除羥自由基(OH•)的能力。因此，經由裏氏木黴固態發酵玉米胚芽與麩皮之發酵產物，具有預防阿茲海默症的潛力。

The major hallmarks of Alzheimer’s disease (AD) is the accumulation of amyloid-β peptides (Aβ). Aβ would promote pro-inflammatory responses and activate neurotoxic pathways, leading to dysfunction and death of brain cells. Recently studies showed that the nature antioxidants, such as vitamin E, polyphenols and carotenoids, could protect neurons from inflammation and oxidation triggered by Aβ. Corn is the important cereal in the world. Studies indicate that the content of polyphenols and antioxidant activity in corn are higher than wheat, oats, and rice, and mainly distribute in the corn germ and bran. However, there were very few studies about the corn germ and bran on reducing cytotoxicity of amyloid β peptide. In addition, the dietary source of corn germ is mainly from corn oil. In the traditional way for making oil, the drying process would destroy the nutrient in corn germ. Recently, some studies indicate that enzymes producing form Trichoderma reesei not only could decompose the cell wall to release oil, but also have much more effective compounds in the milder process. And corn brans are rich in cellulose to provide T. reesei grow. In this study, we used Trichoderma reesei to decompose the corn germ and bran’s cell wall, and then released the effective compounds during the solid fermentation state. The human neuroblastoma (IMR-32) and amyloid-β peptides was used as in vitro model to evaluate the bioactivity of the fermentation product. The effective compounds were extracted from the fermentation product by different solvents. We mixed corn germ and bran in different ratio and there were six sample, such as 1:2, 1:3, 2:1, 3:1 and 2:2. Results showed that the dose below 100μg/mL would not exert cytotoxic effect on the human neuroblastoma. The ethanol extracts of 5 day fermented products could reduce the cytotoxic of Aβ best. Because 3:1 sample had the significant inhibit effect on cytotoxic of Aβ, we used it for further extraction. We use n-hexane, ethyl acetate, n-butanol and water for extraction. Results showed that the layer of n-butanol and water had the significant effect on reducing the cytotoxic of Aβ. We thought the layer of n-butanol and water had the protective compounds for neurons. Results of the extraction analysis showed that the layer of n-butanol had highest total phenolic contents about 507.8 mg/g, but the layer of water had lower total phenolic contents about 324.6 mg/g. Maybe the bioactivity of the fermentation product are phenolic compound in corn. In addition, previous studies show that the layer of water may have small molecule carbohydrates, and they could scavenge the hydroxyl radicals. To sum up, corn germ and bran fermented by T. reesei have potential neuroprotective effect on AD.