【作者】 张艳萍；

【导师】 戴志远；

【作者基本信息】 浙江工商大学 ， 食品科学， 2011， 博士

【摘要】 贻贝(Mytilus sp.),属软体动物门(Mollusca)瓣鳃纲(Lanellibranchia)异柱目(Anisomyaria)贻贝族(Mytilacea)贻贝科(Mytidea),是一种蛋白质含量较高的贝类水产。我国是目前世界上贻贝产量最大的国家,年产量超过60万吨。而由于加工技术落后,我国有90%的贻贝用于生鲜、冷冻制品的出口,导致贻贝的附加值低下,蛋白质资源浪费严重。本论文以紫贻贝为原料,对其蛋白质组成、营养价值、酶解特性进行系统分析,并对贻贝分离蛋白的提取及酶解制备ACE抑制肽进行了研究,旨在为贻贝的深度开发和利用提供理论依据和有效途径,实现贻贝资源的高效增值。论文首先就贻贝粉的化学组成、脂肪酸及矿物质元素含量进行分析。结果表明其化学组成为：水分5.02%,灰分13.09%,脂肪7.04%,蛋白质48.44%,碳水化合物23.75%；脂肪酸分析结果表明：贻贝中含有丰富的PUFA(31.09%),其中含量最高的为EPA和DHA,其含量分别为12.21%和7.18%,饱和脂肪酸中含量最高的为棕榈酸(29.95%),单不饱和脂肪酸中棕榈油酸最为丰富(10.07%)；而且,紫贻贝中含有丰富的Ca、Mg、A1、Fe、Zn和Se等元素。采用95%乙醇作为脱脂溶剂,将贻贝粉脱脂得到贻贝蛋白,其蛋白质含量高达60.23%。通过Osborne法分析其蛋白组成发现：贻贝蛋白中碱溶性蛋白含量最高(40.78%),其次是水溶性蛋白(24.59%)和醇溶性蛋白(6.88%),盐溶性蛋白含量最低(1.62%)。氨基酸分析表明,贻贝蛋白及其各个蛋白级分的氨基酸构成比较平衡,均含有丰富的谷氨酸、天冬氨酸、赖氨酸和丝氨酸,且必需氨基酸比例较高。营养特性分析表明,碱溶性蛋白的氨基酸评分(111.25)最高、蛋白质功效比最高,醇溶蛋白的生物价最高,碱溶性蛋白(65.44)次之。综合评价,贻贝蛋白中碱溶性蛋白的营养价值最高。考察不同蛋白酶对贻贝蛋白的酶解效果,结果表明：碱性蛋白酶Alcalase是生产贻贝蛋白ACE抑制肽的最适用酶,其水解贻贝蛋白4h后水解度(DH)为20.68%。较其它五种蛋白酶的作用,其酶解物的蛋白质回收率(PR)最高(85.77%),ACE抑制活性最强(IC50为66.34μg/mL)。且酶解产物主要分布在相对分子量为200-1000 Da的区间(54.36%)。比较不同蛋白级分对酶解物ACE抑制活性的贡献程度,确定碱溶性蛋白是赋予贻贝蛋白酶解物ACE抑制活性的优势蛋白级分。根据贻贝蛋白组成特点及其对ACE抑制活性的贡献度,确定采用碱溶酸沉工艺制备贻贝分离蛋白(MEPI)并优化了工艺条件,在优化的工艺条件下(料液比1：30,pH11.5,温度35。C,提取2.5小时,酸沉pH值为3.5),蛋白提取率为81.74%,蛋白质含量为74.32%。以DH和IC50-1为评价指标,通过单因素试验和响应面分析(RSM)确定Alcalase酶解MEPI制备ACE抑制肽的最佳酶解条件为：[E]/[S]=1.73%、pH9.2,温度56.8℃。在此条件下酶解所得产物的DH为25.4%,ACE抑制的IC50为35.21μg/mL。稳定性实验结果表明：MEPI酶解物在常规的食品加工条件下(20-100℃、pH2-10)具有良好的稳定性,且能在很大程度上抵抗消化酶的水解作用。并且,结果表明其是ACE的竞争性抑制剂。探讨了DH对酶解物的化学组成、相对分子量分布、氨基酸组成及ACE抑制活性的影响,结果表明：随着DH的增大,蛋白质含量缓慢增加；相对分子量范围在200-1000 Da的组分含量显著增加而后维持恒定；疏水性氨基酸、芳香族氨基酸及支链氨基酸的含量显著增加,酸性氨基酸的含量显著下降。这在一定程度上解释了DH对酶解物活性的影响趋势：随着DH的增加,酶解物的ACE抑制活性逐渐增强,但当酶解进行到一定程度,酶解物活性呈稳定或略微下降趋势。对水解过程进行动力学拟合表征,结果表明：MEPI的DH和水解产物的ACE抑制率的动力学回归方程为：DH=-4.24195+5.43497 1n(T-2.73073),R2=0.9934；而ACE抑制率随时间变化的回归方程为I(%)=47.68838+0.91943T-0.00559T2+1.30899×10-5 T3-1.06897×10-8T4,R2=0.8384。研究了料液浓度、操作压力、操作温度对膜通量的影响以及超滤膜对酶解物活性的影响,根据结果选择10 KD的超滤膜对MEPI酶解物进行超滤分离(料液浓度15 g/L、操作压力20 psi、操作温度25℃)；而后采用Sephadex G-10凝胶色谱、Sephadex LH-20凝胶色谱和半制备RP-HPLC等分离手段从MEPI酶解物中分离出VW、LGW和MVWT三种活性较强的ACE抑制肽,对应的IC50值分别为1.7μM、30.0μM、143.3μM;且均属于抑制剂类型。以VSHE描述子进行结构表征,比较了PLS、SVM和PCA-SVM对不同氨基酸残基数目的ACE抑制肽的建模结果。结果显示：对于ACE抑制二肽,三个模型结果相当；对于ACE抑制三肽,SVM模型和PCA-SVM模型的拟合能力明显优于PLS模型,而预测能力相当；对于ACE抑制四肽、五肽和六肽,SVM模型和PCA-SVM模型的拟合能力及预测能力尤其显著。同时发现,ACE二肽中,C末端氨基酸较N端氨基酸对其活性的影响更为明显；ACE抑制三肽的中间位置的氨基酸对其ACE抑制活性的影响最为显著；对于ACE抑制四肽,N端的三个氨基酸残基对其活性的影响较为明显；而对于ACE抑制五肽和六肽,C端的四个或五个氨基酸残基对其活性的影响较为显著。并且,采用贻贝中的ACE抑制肽对模型进行检验,结果表明：本论文所建ACE抑制肽的QSAR模型能够对已知结构的ACE抑制肽的活性进行准确预测,其中,SVM模型与PCA-SVM模型的预测结果最为准确。更多还原

【Abstract】 Mytilus edulis, a species of shellfish, is good resource of protein and favorite to Chinese. It belongs to phylum Mollusca, class Lanellibranchia, order Anisomyaria, tribe Mytilacea, family Mytidea. China has become the biggest producer of mussel in the world and its total annual yield is over 600000 tons. However, due to limitations in further processing technology,90% of them are traded in live, fresh, frozen, and dried forms. Therefore, mussel has a low commercial value and the waste of mussel protein is rather serious in China. In this thesis, protein composition, nutritional value and hydrolysis characteristic of mussel protein were evaluated. In addition, the preparation of mussel protein isolate (MEPI) and the production of angiotensinⅠ-converting enzyme (ACE) inhibitory peptides from MEPI by enzymatic hydrolysis were also studied. The purpose of this work was to provide some useful academic information and novel effective approach for deep processing of mussel protein, and to develop some high value-added products for human consumption.The chemical composition of the mussel powder was analyzed as follows:5.02% moisture, 13.09% ash,7.04% fat,48.44% protein and 23.75% polysaccharide. The results of fatty acids determination with GC and minerals analysis with ICP-MS showed that PUFA accounted for 31.09% of total fatty acids amounts and the contents of EPA and DHA were highest (12.21% and 7.18%, respectively). Palmitic acid was the major saturated fatty, and palmitoleic acid was the major monounsaturated fatty acid. Their contents were determined as 29.95% and 10.07%, respectively. Also, the mussel was an excellent source of minerals, such as Ca, Mg, Al, Fe, Zn and Se.The effect of four solvents on the defatting of mussel powder was studied. The results showed that 95% ethanol was the most suitable solvent. Four protein fractions (albumin, globulin, prolamine and glutelin) from defatted mussel powder (mussel protein) were fractionated by using the method of Osborne and then characterized by amino acid analysis and SDS-PAGE. Glutelin was the major fraction (40.78%) extracted, followed by albumin (24.59%), prolamine(6.88%), and globulin(1.62%). These protein fractions showed an excellent balance of amino acid, with a relatively high level of Glu, Asp, Lys and Ser. All the estimated mutritional quality parameters based on amino acid composition showed that mussel protein had good nutritional quality (E/T> 36%). Glutelin possessed the highest AAS and PER value. However, prolamine possessed the highest BV, followed by glutelin. Thus, considering both the yield and the quality parameters, glutelin was thought to possess the highest nutritional values in mussel protein.Hydrolysis of the mussel protein was investigated in the presence of various proteolysis enzymes. Alcalase was found to be the optimal protease with the highest hydrolysis degree (DH) (20.68% for 4h). The protein recovery (PR) and the ACE inhibitory activity of various hydrolysates of mussel protein were also compared, the hydrolysates obtained by the treatment with alcalse possessed the highest PR (85.77%) and ACE inhibitory activity, having a IC50 value of 66.34μg/mL. Moreover, the relative molecular weights of these hydrolysates were rather low, especially the hydrolysates obtained by alcalse were mainly distributed in the range of 200-1000 Da (54.36%).The relative molecular weight distribution and ACE inhibitory activity of four protein fractions hydrolysates with alcalse were studied. The result showed that glutelin was major fraction, which contributed more on ACE inhibitory activity of mussel protein. According to the composition feature of protein fractions and their contribution to the ACE inhibitory activity, the alkaline extraction-isoelectric precipitation technology was employed to prepare MEPI, and the parameters were optimized. Under the optimum conditions, i.e. solid-to-liquid ratio,1:30; pH,11.5; temperature,35℃; time,2.5h; precipation pH,3.5, the protein extraction ratio was 81.74%, and the protein content of the product was 74.32%.Taking the DH and the inverse of IC50 as responses, the hydrolysis parameters using alcalse were optimized with RSM as follows:enzyme to substrate ratio 1.73%, pH 9.2 and temperature 56.8℃. Under these conditions, MEPI hydrolysate was prepared with a DH of 25.4% and an IC50 value of 35.21μg/mL. Also, the stability of MEPI hydrolysate was examined. The results showed that the influence of food processing, such as heat and adjustment of pH on its ACE inhibitory activity is not significant, and MEPI hydrolysate showed resistance to in vitro digestion by gastrointestinal proteases. Moreover, MEPI hydrolysate was found to be competitive ACE inhibitors.Effects of DH on chemical composition, relative molecular weight distribution, amino acids composition and ACE inhibitory activity of MEPI hydrolysate were also studied. With the increase of DH, protein content increased slowly, the contents of hydrophobic amino acids, aromatic amino acids and branched-chain amino acids increased significantly, while the content of acidic amino acids decreased markedly. Also, the contents of fraction in the range of 200-1000 Da increased obviously at first, and then become a stable level. All above results can explain the effects of DH on ACE inhibitory activity of MEPI hydrolysate to some extent. The ACE inhibitory activity of MEPI hydrolysate significantly increased with the increase of DH, then increased slowly, and finally decreased slowly. Dynamic characteristics results of hydrolysis process showed that the kinetics fitting equations for DH and ACE inhibitory rate were as follows:DH=-4.24195+5.43497 In (T-2.73073) and I(%)-47.68838+0.91943T-0.00559T2 +1.30899×10-5 T3-1.06897X 10-8T4.Using ACE inhibitory activity as an index, MEPI hydrolysate was purified firstly by ultrafiltration (UF). The influence of UF conditions on flux and the molecular weight cutoff (MWCO) of membrane on ACE inhibitory activity were studied. The results showed that the ACE inhibitory peptides in MEPI hydrolysates could be effectively separated and enriched by membrane (MWCO:10 KDa) under suitable conditions (sample concentration of 15 g/L, operation pressure of 20 psi and temperature of 25℃). Three kinds of ACE inhibitory peptides with strong activities were isolated from MEPI hydrolysates by sequential Sephadex G-10, LH-20 and two-step RP-HPLC, and their amino acid sequences were identified by UPLC-MS as VW, LGW and MVWT with IC50 values of 1.7μM 30.0μM and 143.3μM respectively. Stability tests showed that these peptides could not be hydrolyzed in the presence of ACE, indicating that three ACE inhibitory peptides could be regarded as the competitive inhibitors, which means that three peptides could have antihypertensive effect in vivo.In order to study the quantitative structure-activity relationships of angiotensin I-converting enzyme (ACE) inhibitory peptides, a database including 182 dipeptides,237 tripeptides,94 tetrapeptides,94 pentapeptides and 88 hexapeptides was constructed on the basis of published literatures.18 models were computed using partial least squares regression (PLS), support vector machine (SVM) and principal component analysis (PCA)-SVM based on the VHSE descriptors of 20 coded amino acids and further validated by cross-validation. The results showed that SVM and PCA-SVM achieved better performance in QSAR than PLS for ACE inhibitory tetrapeptides, pentapeptides and hexapeptides. While SVM and PCA-SVM achieved equivalent predication results with PLS and better fitness than PLS for tripeptides. Also, equivalent results were obtained in comparision with three QSAR models for ACE inhibitory dipeptides. In addition, variable importance in the projection (VIP) analysis of individual amino acid residues at each position revealed that the C-terminal amino acid residues of dipeptides, the middle amino acid residues of tripeptides, the N-terminal tripeptide residues in tetrapeptides, the C-terminal tripeptide residues in pentapeptides and the C-terminal tetrapeptide residues in hexapeptides were more important to their ACE-inhibitory activity than the other residues in the peptide sequences.The models were validated with ACE inhibitory peptides from MEPI hydrolysates. The results showed that the QSAR models constructed in this paper could predicate activity of ACE inhibitory peptides exactly. Especially, the predicated results of SVM and PCA-SVM models were very well.更多还原