【作者】 陈日春；

【导师】 郑宝东；

【作者基本信息】 福建农林大学 ， 农产品加工及贮藏工程， 2013， 博士

【摘要】 鲢鱼由于其生长速度快,易养殖,营养价值高,已成为我国一种主要的淡水鱼,其加工过程中产生的大量副产物(如鱼头、鱼皮、鱼鳞、鱼骨及内脏等)通常都被当做废弃物而丢弃。而鱼鳞中蛋白质占其总重的50%-70%,且主要为胶原蛋白和角蛋白,因而鱼鳞是非常好的胶原蛋白生产原料。利用鱼鳞生产胶原蛋白,既可以保护环境,又显著提高水产品加工附加值。因此本文以鲢鱼鱼鳞为原料,从中提取胶原蛋白。以自由基清除率为指标,筛选出制备抗氧化活性肽的最佳用酶,并优化制备条件。采用体外和体内抗氧化试验,系统地研究了鲢鱼鱼鳞胶原蛋白肽的抗氧化作用及其理化特性和稳定性,运用现代生化分离技术对鲢鱼鱼鳞胶原蛋白肽进行抗氧化肽的分离纯化和鉴定。研究成果为鲢鱼鱼鳞胶原蛋白抗氧化肽的开发利用提供理论基础。主要研究成果如下：1.为获得较高的鲢鱼鱼鳞胶原蛋白提取率,首先对鱼鳞进行脱钙处理,通过单因素试验摸索和正交试验设计优化得到最优的脱钙工艺条件为：料液比1：30,EDTA浓度0.17mol/L,浸泡时间30h；在此最优条件下,鲢鱼鱼鳞的脱钙率为94.11%。采用响应面试验设计对胃蛋白酶提取鲢鱼鱼鳞胶原蛋白的工艺条件进行优化,得到最优提取条件为：液料比(v/w)18.0,胃蛋白酶添加量25.51U·mg-1,提取时间61.9h；在该最优条件下提取率为4.02±0.11%。采用SDS-PAGE凝胶电泳对鲢鱼鱼鳞胶原蛋白进行分析,并通过与标准Ⅰ型胶原蛋白比较,确定鲢鱼鱼鳞胶原蛋白属于Ⅰ型胶原蛋白；紫外扫描显示鲢鱼鱼鳞胶原蛋白的特征吸收波长位于235nm处；氨基酸组成进行分析结果显示甘氨酸是鲢鱼鱼鳞胶原蛋白中含量最多的氨基酸,其次是谷氨酸、丙氨酸、脯氨酸和羟脯氨酸；傅里叶红外光谱扫描结果表明所提取的胶原蛋白较好地保持了螺旋结构。鲢鱼鱼鳞胶原蛋白的等电点在7.0左右,变性温度为24.1℃,其溶解性最高与最低的pH值分别为3.0与7.0(P<0.05),氯化钠浓度对胶原蛋白溶解性影响较大,吸水性与持水性均优于甘油,乳化性与乳化稳定性、起泡性与泡沫稳定性均较好。2.以水解度(DH)、DPPH·、O2-·和·OH自由基清除率为指标,比较碱性蛋白酶、中性蛋白酶和木瓜蛋白酶对鲢鱼鱼鳞胶原蛋白的酶解效果及产物抗氧化活性,结果显示碱性蛋白酶酶解产物的抗氧化活性最强,其对DPPH·、O2-·和·OH自由基清除率分别为76.93±0.64%、43.11±0.48%和58.21±0.80%。采用响应面试验设计优化碱性蛋白酶制备鲢鱼鱼鳞胶原蛋白抗氧化活性肽的条件,得到最优的制备条件为底物浓度5.47%,酶解时间4.24h,酶添加量4200U·g-1：在该条件下,所得抗氧化肽对DPPH·、O2-·和·OH自由基清除率分别为79.62±0.1.04%、46.28±0.96%和63.47±0.88%,与优化前比较,抗氧化肽对各自由基的清除率得到显著提高(P<0.05)。3.采用截留分子量分别为5、3、1ku的超滤膜对鲢鱼鱼鳞胶原蛋白肽(SCSCP)进行分离分级,得到四个不同超滤组分：SCSCP-Ⅰ (>5ku)、SCSCP-Ⅱ (3-5ku)、SCSCP-Ⅲ (1-3ku)、SCSCP-Ⅳ(<lku)。采用六种体外抗氧化指标评价各超滤组分抗氧化活性的强弱；结果表明,分子量小于1ku的SCSCP-Ⅳ体外抗氧化活性最强(P<0.05);氨基酸组成分析结果显示,SCSCP-Ⅳ中总疏水性氨基酸和精氨酸含量均较高,推测可能与其较高的抗氧化活性有关；动物实验结果表明,SCSCP-Ⅳ能显著提高大鼠血清中抗氧化酶SOD与GSH-Px活力(P<0.05),降低MDA含量(P<0.05)。4.对SCSCP-Ⅳ理化特性的研究结果表明,与鲢鱼鱼鳞胶原蛋白比较,SCSCP-Ⅳ溶解度受pH变化的影响较小,其溶解度都在90%以上；SCSCP-Ⅳ的乳化性和乳化稳定性均优于胶原蛋白(P<0.05); SCSCP-Ⅳ的吸水性和持水性均优于胶原蛋白和甘油(P<0.05)。对SCSCP-Ⅳ的稳定性研究结果表明,避光、日光和自然光对SCSCP-Ⅳ活性均有一定的破坏作用,其中避光保存影响较小,日光和自然光影响较大。SCSCP-Ⅳ具有良好的耐热性。SCSCP-Ⅳ在酸性环境中活性保持较好,在碱性环境中活性很快丧失。氯化钠、蔗糖和葡萄糖虽然对SCSCP-Ⅳ的自由基清除活性均有一定的破坏作用,但SCSCP-Ⅳ对自由基的清除活性仍保持在95%以上。金属离子均会在不同程度上降低对SCSCP-Ⅳ的自由基清除活性。苯甲酸钠和山梨酸钾均会在不同程度上降低SCSCP-Ⅳ活性,其中山梨酸钾的负作用较苯甲酸钠更强。与喷雾干燥相比,采用冷冻干燥所得产品色泽较深、颗粒较大、溶解速度较快,对超O2-·和·OH自由基清除活性优于喷雾干燥产品(P<0.05)。5.连续采用DEAE-52阴离子交换层析、Sephadex G-15葡聚糖凝胶层析和RP-HPLC对相对分子量小于1kU的抗氧化肽(SCSCP-Ⅳ)进行分离纯化,收集到1个对自由基清除活性较强的组分E3-4,对DPPH·、O2-·和·OH的清除率分别为91.80±1.08%、79.23±4.10%和82.17±2.35%。组分E3-4经分析型RP-HPLC鉴定为纯度较高的单一组分。氨基酸组成分析表明,组分E3-4由Glu、His、Pro和Tyr组成,4种氨基酸的摩尔比为n(Glu):n(His):n(Pro):n(Tyr)=1:1:1:1;组分E3-4经MALDI-TOF/TOF MS和TOF-MS/MS分析,其相对分子量为576.9,氨基酸序列为HPEY (His-Pro-Glu-Tyr);经检索,为一种新型抗氧化肽；初步分析该抗氧化四肽HPEY ((His-Pro-Glu-Tyr)的抗氧化活性是由Tyr提供质子能力、His螯合金属离子能力、强疏水性能以及其自身结构的稳定性决定的。更多还原

【Abstract】 Silver carp (Hypophthalmichthys molitrix) is one of the main freshwater species in China because of its fast growth, easy to farming, high nutritional value. Large byproducts (eg, head, skin, scales, bones and internal organs, etc.) generated during processing silver carp that are usually treated as waste and discarded. The total weight of protein in the scale about50%-70%, and mainly collagen and keratin, which is a very good fish scale collagen raw materials. Use scale production collagen, which can protect environment, but also significantly improve the added value of aquatic products processing. In this paper, silver carp scale as the raw material and extracted collagen. Free radical scavenging rate as the main indicators, selected the best enzyme which prepared the antioxidant activity peptides, and optimized preparation conditions. Using In vitro and in vivo anti-oxidation test, systematic research of the silver carp scale collagen peptide (SCSCP) antioxidant activity, physicochemical properties and stability. Silver carp scale collagen antioxidant activity peptide were purified and identification by the modern biochemical separation techniques. Research results for the development and utilization of the silver carp scale collagen proteolysis antioxidant peptides provide a theoretical basis. The main results are as follows:1. In order to obtain a higher extraction rate of collagen from silver carp scale, first of all decalcification for scales. Explored by single factor experiment and orthogonal experiment design optimization to obtain the optimal process conditions of decalcification:liquid ratio1:30, EDTA concentration0.17mol/L, soaking time30h. In optimal conditions, the silver carp scales decalcification was94.11%. Optimization of pepsin extracts collagen conditions from silver carp scale by using response surface experiment design. Optimal extraction conditions:liquid ratio (v/w)18.0, the amount of pepsin added25.51U·mg-1, extraction time61.9h. In optimal conditions, collagen extraction rate was4.02±0.11%.Using SDS-PAGE gel electrophoresis to analysis the silver carp scale collagen, And compared with the stanurd type I collagen to determine the silver carp scale collagen is type Ⅰ collagen. UV scan shows silver carp scale collagen characteristic absorption at the wavelength of235nm. Amino acid composition analysis showed that glycine is the most abununt amino acids in silver carp scale collagen, followed by glutamic, alanine, proline and hydroxyproline. FTIR scan results show that the collagen better keep a helical structure.Silver carp scale collagen isoelectric point was about7.0, denaturation temperature24.1 ℃. Its solubility highest and lowest pH values were3.0and7.0(P<0.05), respectively, sodium chloride concentration greater impact on collagen solubility, water absorption and water retention are better than glycerin, emulsifying and emulsion stability, foaming and foam stability are better.2. The degree of hydrolysis (DH), DPPH·,O2-·and·OH free radical scavenging rate as the indicators. Compare the effects and the antioxidant activity of product of alkaline protease, neutral protease and papain hydrolysis on the silver carp scale collagen. The results showed strongest antioxidant activity that alkaline protease hydrolyzate. Its DPPH·,O2-·and·OH free radical scavenging rate were76.93±0.64%,43.11±0.48%and58.21±0.80%, respectively.Optimization the preparation antioxidant activity peptides conditions of alkaline protease hydrolysis silver carp scale collagen by using response surface experimental design. Optimal preparing conditions:the substrate concentration5.47%, hydrolysis time4.24h, enzyme dosage4200U·g-1. In optimal conditions, Antioxidant peptides on DPPH·,O2-·and·OH and·OH free radical scavenging rate was79.62±0.1.04%,46.28±0.96%and63.47±0.88%, respectively. Compared with before optimization, antioxidant peptide on the free radical scavenging rate has been significantly increased (P<0.05).3. Silver carp scale collagen peptide (SCSCP) was separation grading by using MWCO of ultrafiltration membrane were5kU,3kU and1kU, respectively. Get four different ultrafiltration components:SCSCP-Ⅰ (>5kU), SCSCP-Ⅱ (3-5kU), SCSCP-Ⅲ (1-3kU), SCSCP-Ⅳ(<1kU). Evaluate the antioxidant activity strength of ultrafiltration components by six indicators of the in vitro antioxidant. The results showed that the molecular weight less than1kU (SCSCP-Ⅳ) have strongest antioxidant activity in vitro(P<0.05). Amino acid composition analysis showed. SCSCP-Ⅳ total content of hydrophobic amino acids and arginine were higher, presumably with a higher antioxidant activity are closely related. Animal experiments show that SCSCP-Ⅳ can significantly increase serum antioxidant enzymes SOD and GSH-Px activity (P<0.05), decreased MU content (P<0.05).4. Physicochemical properties for SCSCP-Ⅳ findings suggest that, compared with the silver carp scale collagen, SCSCP-Ⅳ solubility less affected by pH changes, its solubility more than90%. SCSCP-Ⅳ emulsifying and emulsion stability are better than collagen (P <0.05). SCSCP-Ⅳ water absorption and water holding capacity superior to collagen and glycerol (P<0.05).The results of research on stability of SCSCP-Ⅳ showed that urk, sunlight and natural light had some umaging effects on SCSCP-Ⅳ antioxidant activity. Compared with sunlight and natural light, urk stored less impact. SCSCP-Ⅳ has good heat resistance. SCSCP-Ⅳ antioxidant activity in an acidic environment to maintain good activity, but in an alkaline environment lost quickly. Sodium chloride, sucrose and glucose although for SCSCP-Ⅳ radical scavenging activity had some umaging effects, but SCSCP-Ⅳ on free radical scavenging activity remained at95%. Metal ions in varying degrees reduce the SCSCP-Ⅳ radical scavenging activity. Sodium benzoate and potassium sorbate at different levels reduce the activity SCSCP-Ⅳ and potassium sorbate more negative effect than sodium benzoate. Compared with the spray-drying, the product has urker color, larger particles and dissolves faster by freeze-drying. In addition, it’s for O2-·and·OH free radical scavenging activity better than spray-dried product (P<0.05).5. The relative molecular weight less than1kU antioxidant peptide (SCSCP-Ⅳ) were isolated and purified by continuous use of DEAE-52anion exchange chromatography, Sephadex G-15Sephadex chromatography and RP-HPLC. One pair of free radicals scavenging activity stronger component (E3-4) was collected. E3-4on DPPH·,O2-·and·OH free radical scavenging rate was91.80±1.08%,79.23±4.10%and82.17±2.35%, respectively. Component E3-4was identified as a single component of high purity by the analytical RP-HPLC. Amino acid composition analysis showed that the component E3-4was composed by Glu, His, Pro, and Tyr, and the four amino acids in a molar ratio was n(Glu): n(His):n(Pro):n(Tyr)=1:1:1:1. Component E3-4was analyzed by TOF-MS/MS and MALDI-TOF/TOF MS, the relative molecular weight was576.9, the amino acid sequence HPEY (His-Pro-Glu-Tyr). Through the retrieval, it’s novel antioxidant peptides. Preliminary analysis of the antioxidant tetrapeptide HPEY (His-Pro-Glu-Tyr) antioxidant activity is provided by the proton ability Tyr, His ability to chelate metal ions, strong hydrophobic properties and structural stability of its own.更多还原