【摘要】：皺紋盤鮑(Haliotis discus hannaiIno)是一種名貴海產(chǎn)品,富含蛋白質(zhì)、脂肪、人體必需的微量元素,營養(yǎng)價(jià)值極高,開發(fā)利用價(jià)值大。近年來,市面上作為添加輔料的抗氧化劑多為人工合成,因此制備天然抗氧化蛋白已成為研究熱點(diǎn),可為食品添加劑和化妝品產(chǎn)業(yè)帶來巨大的商業(yè)潛力。本研究以皺紋盤鮑為原料,通過構(gòu)建真核表達(dá)載體,體外表達(dá)、優(yōu)化其表達(dá)與純化工藝,對獲取的蛋白純品進(jìn)行活性評價(jià),為食品添加劑和化妝品的輔助原料研發(fā)提供參考。主要研究內(nèi)容和結(jié)果如下:1.對皺紋盤鮑Cu/Zn-SOD和TPX2進(jìn)行真核表達(dá)載體的構(gòu)建、重組表達(dá)、純化以及工藝條件優(yōu)化。結(jié)果表明:Cu/Zn-SOD在0.5%甲醇濃度,pH6條件下28 ℃培養(yǎng)24 h表達(dá)量最高;TPX2在2.0%甲醇濃度,pH7條件下28 ℃培養(yǎng)72 h表達(dá)量最高。2.比較皺紋盤鮑Cu/Zn-SOD在不同Cu2+濃度下的活性,對其熱穩(wěn)定性進(jìn)行評價(jià)以及測定抗氧化能力。同時(shí),也對TPX2進(jìn)行抗氧化能力的測定。結(jié)果表明:1.0%Cu2+濃度下的Cu/Zn-SOD活性最強(qiáng);溫度在80 ℃時(shí),Cu/Zn-SOD活性保持在50%以上;Cu/Zn-SOD和TPX2清除自由基的能力均高于Vc,清除羥自由基能力Cu/Zn-SOD是Vc的3倍,TPX2是Vc的9倍;清除超氧陰離子自由基能力 Cu/Zn-SOD 是 Vc 的 16 倍。3.采用MTS/PMS法進(jìn)行細(xì)胞水平安全性及活性評價(jià),另外研究重組蛋白是否引起馬血溶血。結(jié)果表明:100 μg/mL的Cu/Zn-SOD和TPX2均對L929細(xì)胞和L02細(xì)胞沒有毒害作用,不會引起馬血細(xì)胞溶血,并且對H202引起的細(xì)胞損傷均有一定的保護(hù)作用。由此可見,皺紋盤鮑Cu/Zn-SOD和TPX2是安全的抗氧化物,適合作為食品添加劑和化妝品的輔助原料。
[Abstract]:Haliotis discus hannai Ino is a rare seafood, rich in protein, fat, essential trace elements of human body, high nutritional value, great value of development and utilization. In recent years, the antioxidants in the market as supplementary materials are mostly synthetic, so the preparation of natural antioxidant proteins has become a research hotspot, which can bring great commercial potential for food additives and cosmetics industry. In this study, abalone was used as raw material, eukaryotic expression vector was constructed, expression and purification process was optimized in vitro, the activity of purified protein was evaluated, and reference was provided for the research and development of auxiliary raw materials of food additives and cosmetics. The main contents and results are as follows: 1. The eukaryotic expression vectors of Cu- / Zn-SOD and TPX2 were constructed, expressed, purified and optimized. The results showed that the highest expression of TPX2 was obtained at 0.5% methanol concentration and pH 6 at 28 鈩，

本文編號：2137991