【作者】 刘恩岐；

【导师】 李华；

【作者基本信息】 西北农林科技大学 ， 食品科学， 2013， 博士

【摘要】 酶法水解开发生物活性肽是大豆蛋白精深加工研究的一个重要方向，国内外大豆肽的研究均以普通大豆蛋白为主，而有关黑豆蛋白肽的研究文献报道较少且不够系统和深入。黑豆蛋白质亚基结构和氨基酸组成与普通大豆蛋白基本一致，且蛋白质含量较高，氨基酸组成总体优于黄豆，是一种优质蛋白资源。本研究在黑豆蛋白单酶水解试验的基础上，探讨多酶分阶段水解黑豆蛋白的最佳酶解组合，通过超滤与大孔树脂吸附分离初步筛选出体外抗氧化与降胆固醇效果各自最优的黑豆肽组分，进行抗氧化、缓解体力疲劳和辅助降血脂动物实验。采用高速逆流、凝胶过滤和反相高效液相等色谱分离技术，获得高纯度的黑豆功能肽。采用Edman降解N-端测序的方法，对黑豆肽的氨基酸序列进行鉴定，旨在为酶法制备功能肽的分离纯化与结构表征提供理论与技术依据。通过SDS-PAGE凝胶电泳图谱和光密度分析表明，两个供试黑豆品种分离蛋白和两种商品大豆分离蛋白的各亚基电泳条带数量一致，但黑豆蛋白的β亚基和Basic亚基带较宽且染色深，黑豆分离蛋白的11S组分（Acid+Basic）与7S（++β）组分的比值明显高于商品大豆分离蛋白，且晚熟黑豆蛋白的11S球蛋白含量高于早熟黑豆蛋白。氨基酸组成分析表明，黑豆蛋白的氨基酸组成与普通大豆蛋白基本一致，且总必需氨基酸和疏水性氨基酸含量均明显高于普通大豆分离蛋白。不同蛋白酶有不同的水解特异性，Alcalase酶水解黑豆蛋白产物的DPPH·与O－2·清除率和胆固醇胶束溶解度抑制率最高，Neutrase酶次之，Flavourzyme酶较差。多酶组合分阶段水解采用以Alcalase和Neutrase酶为主、辅以Flavourzyme酶短时间水解的黑豆蛋白水解物的DPPH·与O－2·的清除率和胆固醇胶束溶解度抑制率均高于单酶水解物，且水解时间从单酶水解的3h缩短为2h，说明通过合理的多酶组合水解方式可以提高酶解速度并改善水解液的抗氧化与降胆固醇活性。由于晚熟黑豆蛋白疏水性氨基酸含量较高，水解后释放出的疏水性氨基酸侧链活性基团较多，晚熟黑豆蛋白酶水解产物的的抗氧化和降胆固醇活性均高于相同酶解方式的早熟黑豆。采用超滤与大孔树脂吸附分离技术，对酶法水解试验确定的抗氧化和降胆固醇活性最强的晚熟黑豆蛋白多酶组合水解产物分别进行分离纯化，相对分子质量＜3000、75%乙醇洗脱的OABSP-MR-Ⅲ抗氧化组分和DCBSP-MR-Ⅲ降胆固醇组分的活性最强。对OABSP-MR-Ⅲ组分进行抗氧化与缓解体力疲劳动物实验，灌胃黑豆肽500mg·kg-1·d-1中剂量组和1000mg·kg-1·d-1高剂量组小鼠血清和肝脏组织中MDA含量降低实验结果阳性，且小鼠血清GSH-Px和小鼠肝脏组织中SOD、GSH-Px活性提高实验结果阳性，提示黑豆蛋白肽能够抑制细胞脂质过氧化损伤，提高机体内源性抗氧化酶活性，具有抗氧化的生物学功能；黑豆蛋白肽中、高剂量组小鼠负重游泳、血乳酸和肝糖原二项生化指标实验结果皆为阳性，判定黑豆蛋白肽具有缓解体力疲劳功能的作用。对DCBSP-MR-Ⅲ组分进行辅助降血脂动物实验，黑豆蛋白肽高剂量组小鼠血清总胆固醇TC和低密度脂蛋白胆固醇LDL-C水平与高脂饲料模型组具有极显著差异(P＜0.01)，同时血清高密度脂蛋白胆固醇HDL-C不显著低于模型对照组，判定该受试样品具有辅助降低胆固醇的作用；且小鼠血清甘油三酯TG与模型对照组差异显著，提示黑豆蛋白肽具有辅助降血脂作用。以氧自由基清除能力ORAC值为评价指标，对抗氧化AOBSP-MR-Ⅲ黑豆肽组分通过HSCCC和Sephadex G-25凝胶色谱分离得到1个抗氧化较强的G-25-P4峰组分，再经RP-HPLC分离得到2个抗氧化较强的RP-HPLC-P2和P4峰组分，最终采用SEC排阻色谱分离获得RP-HPLC-P4组分SEC-P3肽段和RP-HPLC-P2组分SEC-P2肽段2个高纯度的抗氧化黑豆肽，通过Edman降解后蛋白测序仪测定，其氨基酸序列结构分别为Trp－Asn－Pro和Tyr－Asn－Ile，均是肽链长度为3个氨基酸的小肽。以胆固醇胶束溶解度抑制率为评价指标，对降胆固醇DCBSP-MR-Ⅲ黑豆肽组分进行HSCCC、Sephadex G-25、RP-HPLC和SEC色谱分离纯化，获得RP-HPLC-P5组分SEC-P2肽段和RP-HPLC-P7组分SEC-P5肽段2个高纯度的降胆固醇黑豆肽，其序列结构分别为Ala－Phe－Pro－Lys－Asp和Ile－Leu－Ser－Tyr－Ala－Met－Asp－Gly，是肽链长度分别为5个和8个氨基酸的短肽。更多还原

【Abstract】 Preparation of bioactive peptides from soybean protein by enzymatic hydrolysis plays animportant role in deep processing of soybean. Up to now, numerous researches about soybeanpeptides at home and abroad were reported, which were used ordinary soy protein as rawmaterials, but the papers about black soybean peptides (BSP) were rare and not systematicand thorough. Black soybean protein is a good plant protein resource, the subunit structureand amino acid compositions were basically similar to ordinary soy protein. In this paper,black soybean protein were hydrolyzed with different combinations and steps of three kindsof commercial proteases (the Alcalase protease, Neutrase and Flavourzyme protease) on thebasis of the single enzyme hydrolysis experiments, and the BSP fractions, which displayedbest antioxidant activity and hypocholestermic effect were separated and purified byultrafiltration (UF) and macroporous adsorption resin (MAR), and their antioxidant activity,relieviing physical fatigue and hypolipidemic effect in mice were investigated. In order topreparated highly purified targeted BSP fractons, high-speed countercurrent chromatography(HSCCC), gel filtration and reverse-phase HPLC were used, and the amino acid sequences ofBSPs were analyzed by N-terminal amino acid sequence analysis as well.The SDS-PAGE and photodnsitometry were used to analyzed black soybean proteinisolate and commercial soybean protein isolate, the results showed that all samples had thesame subunit electrophoresis band number, compared with the commercial soybean proteinisolate, the bands of-subunit and Basic-subunit of black soybean protein were wider and haddeeper color, the value of11S/7S of black soybean protein was higher. Moreover, the contentof11S globulin of late-maturing black soybean protein was higher than early-maturing cultivar.Amino acid composition analyed showed that the amino acid compositions of black soybeanprotein were basically similar to ordinary soy protein, but the contents of total essential aminoacids and hydrophobic amino acids were higher than ordinary soy protein.To clear the hydrolysis properties of different protease, black soybean protein werehydrolyzed by using single enzyme. The results showed that the Alcalase protease enzymatic hydrolysates possessed the highest DPPH·, O2－· scavenging capability and inhibitory rates ofcholesterol micelle solubility (CMSIR), that by Neutrase took the second place, and that ofFlavourzyme was the lowest. On the basis of single enzyme hydrolysis expriments, thedifferent combinations and steps of multienzyme hydrolysis processing were investigated, theresults showed that the BSPs hydrolyzed by multienzyme (the Alcalase protease, Neutraseand Flavourzyme protease) for2h had better DPPH·, O2－·scavenging capability and CMSIRthan BSPs hydrolyzed by single enzyme for3h, it indicated that the hydrolysates produced byreasonable combinations with different enzymes had better antioxidant activity andhypocholesterolemic effect, and it can also save hydrolysis time. Compared with theearly-maturing cultivar, the late-maturing BSPs had better antioxidant activity andhypocholesterolemic effect using the same hydrolysis processing, which may be that thelate-maturing black soybean protein had greater contents of hydrophobic amino acids, and theactive groups on side chains of hydrophobic amino acids would expose when hydrolysed byenzymes.The UF and MARs were used to separation and purification of targeted enzymatichydrolysates, the results showed that the OABSP-MR-Ⅲ and DCBSP-MR-Ⅲ fractions withMr＜3000eluted by75%ethanol displayed strongest antioxidant activity andhypocholesterolemic effect, respectively. The antioxidant activity and relieving physicalfatigue effect were carried out with OABSP-MR-Ⅲ fractions in mice, the results showed thatthe contents of MDA,GSH-Px in serum and hepar in mice were positive, and the content ofSOD in hepar in mice were positive as well, when oral administrated500mg·kg-1·d-1(middledosage) and1000mg·kg-1·d-1(high dosage) BSPs, this impled that the BSPs can inhibitlipid peroxidative injury and impove the activity of endogenous antioxidant enzymes in vivo.Compared with the control group, the middle and high dose group can significantly (P＜0.01)prolong loaded-swimming time, blood lactic acid (BLA) can be reduced notable (P＜0.01),and improving hepatic glycogen (HG) storage status greatly (P＜0.01), it indicated that oraladministration of the BSP can relieve physical fatigue in mice. Furthermore, Mice withhyperlipidemia were made by feeding high fat feed in order to evaluate the effect of theDCBSP-MR-Ⅲ fraction in regulating serum cholesterol. Results showed that compared withthe model control group, high dosage group of DCBSP-MR-Ⅲ could notable（P＜0.01）reduce the levels of TC,TG and LDL-C in serum, it hinted that the DCBSP-MR-Ⅲ hadhypolipidemic effect in mice.The antioxidant capacities of BSPs were evaluated by oxyen radical absorbance capacity(ORAC), the fraction, AOBSP-MR-Ⅲ, exhibited the highest antioxidative activity in vitio andin vivo was further purified using consecutive methods on HSCCC, Sephadex G-25column, reversed phase high-performance liquid chromatography (RH-HPLC) and size exclusionchromatography (SEC), then the highly purified antioxidant peptides named SEC-P3andSEC-P2were obtained, and the sequences were Trp－Asn－Pro and Tyr－Asn－Ile,respectively. In addition, the CMSIR was chosen as an index to isolate highly purifitedhypocholesterolemic peptides from the DCBSP-MR-III fraction, which demonstrated goodhyocholesterolemic effect in mice, the methods of HSCCC, Sephadex G-25column,RH-HPLC and SEC were applied, and then the highly purifited hypocholesterolemic peptidesnamed SEC-P2and SEC-P5were got, which isolated from RP-HPLC-P5and RP-HPLC-P7fractions, respectively. Moreover, the sequences of SEC-P2and SEC-P5were Ala－Phe－Pro－Lys－Asp and Ile－Leu－Ser－Tyr－Ala－Met－Asp－Gly, respectively.更多还原