鲢鱼鱼鳞胶原蛋白肽的制备及其抗氧化活性的研究
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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( 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。
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.
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(
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。
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.
引文
[1]中华人民共和国农业部.中国农业发展报告[M].北京：中国农业出版社,2006.
[2]张俊杰,曾庆孝.鱼鳞的开发利用前景[J].中国水产,2004,5：74-75.
[3]钟朝辉,李春美,梁晋鄂,等.鱼鳞胶原蛋白提取工艺的优化[J].食品科学,2006,27(7)：162-165.
[4]Liu W T, Li G. Y, Miao Y Q, et al. Preparation and characterization of pepsin-solubilized type Ⅰ collagen from the scales of snakehead (Ophiocephalus argus) [J]. Journal of Food Biochemistry,2009,33(1):20-37.
[5]Shoulders M D, Raines R T. Collagen structure and stability [J]. Annual Review Biochemistry,2009,78(1):929-958.
[6]Liu Z, Oliveira A C M, Su Y C. Purification and characterization of pepsin-solubilized collagen from skin and connective tissue of giant red sea cucumber(Parastichopus californicus)[J]. Journal of agricultural and food chemistry,2009,58(2):1270-1274.
[7]刘刚.安全与功能性——关于胶原蛋白的探讨[J].明胶科学与技术,2010,30(2)：87-89.
[8]Gelse K, Poschl E, Aigner T. Collagens-structure, function, and biosynthesis [J]. Advanced Drug Delivery Reviews,2003,55(20):1531-1546.
[9]张楚福.生物化学原理[M].北京：高等教育出版社,2003：67-70.
[10]沈同,工镜岩,朱圣庚,等.生物化学[M].北京：高等教育出版社,2002.
[11]胡帅,王慧桂,但卫华,等.胶原蛋白结构的研究进展[J].西部皮革,2008,30(8)：16-19.
[12]陈胜军,曾名勇,董士远.水产胶原蛋白及其活性肽的研究进展[J].水产科学,2004,23(6)：44-46.
[13]吴波,陈运中.鱼鳞胶原蛋白的研究进展及应用[J].武汉工业学院学报,2007,26(4)：29-33.
[14]Ikoma T, Kobayashi H, Tanaka J, et al. Microstructure, mechanical, and biomimetic properties of fish scales from Pagrus major [J]. Journal of Structural Biology,2003, 142(3):327-333.
[15]Zylberberg L, Bereiter-Hahn J. The distribution of Tyr and Glu microtubules during fish scale regeneration [J]. European journal of cell biology,1991,54(1):132-139.
[16]Onozato H, Watabe N. Studies on fish scale formation and resorption [J]. Cell and Tissue Research,1979,201(3):409-422.
[17]段蕊,张俊杰,杜修桥.鱼鳞组成性质及其加工利用的研究进展[J].食品与机械,2006,22(5)：128-131.
[18]谢克难,周凤娇,袁锡东,等.鱼鳞胶滋阴养血作用初探[J].新中医,1996(1)：62-63.
[19]刘庆慧,刘从力,王采理.鱼鳞营养成分的分析及对高脂饲料大鼠血酯水平的影响[J].中国水产科学,2001,7(4)：56-59.
[20]赵连胜.鱼鳞杂谈(一,二)[J].内陆水产,1997(11)：28-30.
[21]郑本瑞,吕国桥,罗自文.龟甲胶药理作用研究进展[J].时珍国医国药,2001,12(5)：463-464.
[22]黄必胜.阿胶龟胶和鹿角胶中微量元素的测定与分析[J].时珍国医国药,2001,12(6)：487.
[23]Gomez-Guillen M C, Gimenez B, Lopez-Caballero M E, et al. Functional and bioactive properties of collagen and gelatin from alternative sources:A review [J]. Food Hydrocolloids,2011,25(8):1813-1827.
[24]曾江南,蒋柏泉,魏林生,等.鳙鱼鱼鳞脱钙工艺的优化[J].南昌大学学报(工科版),2011,33(4)：338-342.
[25]刘亚,陈孔才.微波辅助EDTA法对鱼鳞胶原多肽脱钙的影响[J].现代食品科技,2010,26(5)：512-515.
[26]Yan W, Joe M R. Effect of EDTA, HCl, and Citric Acid on Ca Salt Removal from Asian (Silver) Carp Scales Prior to Gelatin Extraction [J]. Journal of Food Science,2009,74(6): 426-431.
[27]朱秀灵,戴清源,蔡为荣,等.响应面法优化鲫鱼鱼鳞酸溶性胶原蛋白提取工艺[J].食品工业科技,2010,31(11)：247-251.
[28]Cheng F Y, Hsu F W, Chang H S, et al. Effect of different acids on the extraction of pepsin-solubilised collagen containing melanin from silky fowl feet[J]. Food chemistry, 2009,113(2):563-567.
[29]Skierka E, Sadowska M. The influence of different acids and pepsin on the extractability of collagen from the skin of Baltic cod (Gadus morhua) [J]. Food chemistry,2007, 105(3):1302-1306.
[30]张俊杰,段蕊,陈璐.鲤鱼鳞酸溶性胶原蛋白提取的研究[J].水产科学,2006, 25(12)：640-643.
[31]潘洪民,聂小宝,程丽林,等.武昌鱼鱼鳞胶原蛋白提取工艺的研究[J].安徽农业科学,2012,40(1)：‘111-112.
[32]曾少葵,蓝海明,章超桦,等.罗非鱼鳞胶原蛋白的提取及其酶解产物的抗氧化性[J].上海海洋大学学报,2009,18(5)：599-603.
[33]Wangtueai S, Noomhorm A. Processing optimization and characterization of gelatin from lizardfish (Sauriu spp.) scales [J]. LWT-Food Science and Technology,2009,42(4): 825-834.
[34]Woo J W, Yu S J, Cho S M, et al. Extraction optimization and properties of collagen from yellowfin tuna (Thunnus albacares) dorsal skin [J]. Food Hydrocolloids,2008,22(5): 879-887.
[35]Nalinanon S, Benjakul S, Visessanguan W, et al. Use of pepsin for collagen extraction from the skin of bigeye snapper (Priacanthus tayenus)[J]. Food Chemistry,2007,104(2): 593-601.
[36]王林.深海红鱼(Sebastes mentella)胶原蛋白的制备及其理化特性研究[D].南京：南京农业大学,2009.
[37]张俊杰,段蕊,潘秀楼.鲤鱼鱼鳞酶溶性胶原蛋白提取工艺的应用[J].淮海工学院学报(自然科学版),2006,4：55-58.
[38]刘艳,刘章武,唐婧苗,等.酶解淡水鱼鳞制备水解胶原蛋白的研究[J].武汉工业学院学报,2009,28(2)：11-14.
[39]董乐,林娈,王力.水提法提取鱼鳞胶原蛋白的研究[J].内蒙古民族大学学报：自然科学版,2010(005)：525-529.
[40]林家福,谢福美,易美华,等.热水提取罗非鱼鱼皮胶原蛋白的研究[J].中国热带医学,2008,8(8)：1317-1318.
[41]李敏.青鱼鱼鳞胶原蛋白和羟基磷灰石综合提取研究[D].长沙：湖南农业大学,2008.
[42]赵睿,王晓丹,祖国仁,等.热水法抽提鳕鱼皮胶原蛋白条件的响应面分析[J].大连工业大学学报,2011,30(2)：
[43]钱曼,武贤壮,邱承光,等.热力法和酶解法提取鱼鳞胶原蛋白的工艺及性质研究[J].食品工业科技,2007,28(10)：70-72.
[44]Kim H K, Kim Y H, Kim Y J, et al. Effects of ultrasonic treatment on collagen extraction from skins of the sea bass Lateolabrax japonicus [J]. Fisheries science,2012,78(2): 485-490.
[45]Li D, Mu C, Cai S, et al. Ultrasonic irradiation in the enzymatic extraction of collagen [J]. Ultrasonics sonochemistry,2009,16(5):605-609.
[46]Gomez-Guillen M C, Gimenez B, Montero P. Extraction of gelatin from fish skins by high pressure treatment [J]. Food hydrocolloids,2005,19(5):923-928.
[47]陈丽清,张宇昊,周梦柔,等.猪皮明胶提取过程中的超高压预处理工艺优化[J].农业工程学报,2012,28(19)：262-269.
[48]焦道龙,陆剑锋,张伟伟,等.水产动物胶原蛋白的研究现状及发展趋势[J].食品科学,2009(17)：334-338.
[49]曹俊.鱼鳞胶原蛋白的研究进展和应用前景[J].水产科技情报,2009,36(1)：41-44.
[50]蒋挺大.胶原与胶原蛋白[M].北京：化学工业出版社,2006.
[51]Ogawa M, Portier R J, Moody M W, et al. Biochemical properties of bone and scale collagens isolated from the subtropical fish black drum (Pogonia cromis) and sheepshead seabream (Archosargus probatocephalus) [J]. Food Chemistry,2004,88(4):495-501.
[52]Muyonga J H, Cole C G B, Duodu K G. Fourier transform infrared (FTIR) spectroscopic study of acid soluble collagen and gelatin from skins and bones of young and adult Nile perch (Lates niloticus) [J]. Food Chemistry,2004,86(3):325-332.
[53]陆璐.Ⅰ型胶原的提取及其组装和表征[D].南京：东南大学,2006.
[54]Rigby B J. Amino-acid composition and thermal stability of the skin collagen of the Antarctic ice-fish [J].1968,219(150):166-167.
[55]Jongjareonrak A, Benjakul S, Visessanguan W, et al. Isolation and characterisation of acid and pepsin-solubilised collagens from the skin of Brownstripe red snapper (Lutjanus vitta)[J]. Food Chemistry,2005,93(3):475-484.
[56]Hattori S, Auchi E, Ebihara T, et al. Alkali-treated collagen retained the triple helical conformation and the ligand activity for the cell adhesion via α2 β1 integrin[J]. Journal of biochemistry,1999,125(4):676-684.
[57]段蕊,张俊杰,赵晓庆,等.鲤鱼鱼鳞胶原蛋白性质的研究[J].2001,32(2)：175-180.
[58]段蕊,张俊杰,今野久仁彦.鲤鱼鱼鳞胶原蛋白ASC与PSC的比较[J].水产科学, 2008,27(8)：397-400.
[59]汪海波,梁艳萍,汪海婴,等.草鱼鱼鳞胶原蛋白的提取及其部分生物学性能[J].水产学报,2012,36(4)：1-9.
[60]胡大林,廖建坤.自由基与DNA的氧化损伤[J].国外医学：卫生学分册,2002,29(5)：261-263.
[61]赵晶,白云.自由基相关疾病研究进展[J].生物学教学,2003,28(4)：6-8.
[62]刘兆红,岳炳辉.氧自由基对动物机体的毒性作用[J].上海畜牧兽医通讯,2005,3：57.
[63]胡柏成,黄龚.体内外自由基形成的研究及意义[J].中国疗养医学,2007,16(5)：317-319.
[64]Johansen J S, Harris A K, Rychly D J, et al. Oxidative stress and the use of antioxidants in diabetes:linking basic science to clinical practice [J]. Cardiovascular diabetology, 2005,4(1):5-15.
[65]Poli G, Leonarduzzi G, Biasi F, et al. Oxidative stress and cell signaling [J]. Current medicinal chemistry,2004,11(9):1163-1182.
[66]Wang J, Sun B, Cao Y, et al. Protection of wheat bran feruloyl oligosaccharides against free radical-induced oxidative umage in normal human erythrocytes [J]. Food and, Chemical Toxicology,2009,47(7):1591-1599.
[67]Ulle-Donne I, Rossi R, Colombo R, et al. Biomarkers of oxidative umage in human disease [J]. Clinical chemistry,2006,52(4):601-623.
[68]Dhalla N S, Temsah R M, Netticaun T. Role of oxidative stress in cardiovascular diseases [J]. Journal of hypertension,2000,18(6):655-673.
[69]Zhou C, Huang Y, Przedborski S. Oxidative stress in Parkinson's disease:A Mechanism of Pathogenic and Therapeutic Significance [J]. Annals of the New York Academy of Sciences,2008,1147(1):93-104.
[70]Sayre L M, Smith M A, Perry G. Chemistry and biochemistry of oxidative stress in neurodegenerative disease [J]. Current medicinal chemistry,2001,8(7):721-738.
[71]Di Bernardini R, Harnedy P, Bolton D, et al. Antioxidant and antimicrobial peptidic hydrolysates from muscle protein sources and by-products [J]. Food Chemistry,2011, 124(4):1296-1307.
[72]Gill I, Lopez-Fandino R, Jorba X, et al. Biologically active peptides and enzymatic approaches to their production [J]. Enzyme and Microbial Technology,1996,18(3): 162-183.
[73]Uwson P E, Muir T W, Clark-Lewis I, et al. Synthesis of proteins by native chemical ligation [J]. Science,1994,266(4):776-778.
[74]Seebach D, Albert M, Arvidsson P I, et al. From the biopolymer PHB to biological investigations of unnatural and peptides [J]. CHIMIA International Journal for Chemistry, 2001,55(4):345-353.
[75]Rao C, Tam J P. Synthesis of peptide dendrimer [J]. Journal of the American Chemical Society,1994,116(15):6975-6976.
[76]林琳.鱼皮胶原蛋白的制备及胶原蛋白多肽活性的研究[D].青岛：中国海洋大学,2006.
[77]Gimenez B, Aleman A, Montero P, et al. Antioxidant and functional properties of gelatin hydrolysates obtained from skin of sole and squid [J]. Food Chemistry,2009,114(3): 976-983.
[78]Mendis E, Rajapakse N, Kim S K. Antioxidant properties of a radical-scavenging peptide purified from enzymatically prepared fish skin gelatin hydrolysate [J]. Journal of agricultural and food chemistry,2005,53(3):581-587.
[79]Li B, Chen F, Wang X, et al. Isolation and identification of antioxidative peptides from porcine collagen hydrolysate by consecutive chromatography and electrospray ionization-mass spectrometry [J]. Food Chemistry,2007,102(4):1135-1143.
[80]韩凤杰,赵征,胡晓鹏.酶法制取鲮鱼皮胶原蛋白寡肽工艺的研究[J].现代食品科技,2006,22(2)：135-137.
[81]Aleman A, Gimenez B, Montero P, et al. Antioxidant activity of several marine skin gelatins[J]. LWT-Food Science and Technology,2011,44(2):407-413.
[82]Nagai T, Nagashima T, Abe A, et al. Antioxidative activities and angiotensin Ⅰ-converting enzyme inhibition of extracts prepared from chum salmon (Oncorhynchus keta) cartilage and skin [J]. International Journal of Food Properties,2006,9(4):813-822.
[83]任俊凤,任婷婷,朱蓓薇.河豚鱼皮胶原蛋白肽的提取及其抗氧化活性的研究[J].中国食品学报,2009,9(1)：77-83.
[84]庄永亮.海蜇胶原蛋白理化性质及其胶原肽的护肤活性研究[D].青岛：中国海洋大 学,2009.
[85]沈澎.鲤鱼鱼鳞胶原肽的制备及特性研究[D].长春：吉林农业大学,2011.
[86]胡娟.酶解鱼鳞明胶制备生物活性肽的研究[D].广州：华南理工大学,2010.
[87]申锋,杨莉莉,熊善柏,等.胃蛋白酶水解草鱼鱼鳞制备胶原肽的工艺优化[J].华中农业大学学报,2010,29(003)：387-391.
[88]Marcuse, R. Antioxidative effect of amino-acids [J].Nature,1960,186(11):886-887.
[89]Megias C, Pedroche J, Yust M M, et al. Production of copper-chelating peptides after hydrolysis of sunflower proteins with pepsin and pancreatin [J]. LWT-Food Science and Technology,2008,41(10):1973-1977.
[90]Xie Z, Huang J, Xu X, et al. Antioxidant activity of peptides isolated from alfalfa leaf protein hydrolysate [J]. Food Chemistry,2008,111(2):370-376.
[91]Wu H C, Chen H M, Shiau C Y. Free amino acids and peptides as related to antioxidant properties in protein hydrolysates of mackerel (Scomber austriasicus)[J]. Food research international,2003,36(9):949-957.
[92]Kim S K, Byun H G, Ito H. Articles:Purification and Characterization of Antioxidative Peptides from Bovine Skin [J]. Biochemistry and Molecurar Biology Reports,2001, 34(3):219-224.
[93]Je J Y, Qian Z J, Byun H G, et al. Purification and characterization of an antioxidant peptide obtained from tuna backbone protein by enzymatic hydrolysis [J]. Process Biochemistry,2007,42(5):840-846.
[94]Kim S K, Kim Y T, Byun H G, et al. Isolation and characterization of antioxidative peptides from gelatin hydrolysate of Alaska pollack skin [J]. Journal of Agricultural and Food Chemistry,2001,49(4):1984-1989.
[95]Yang J I, Ho H Y, Chu Y J, et al. Characteristic and antioxidant activity of retorted gelatin hydrolysates from cobia (Rachycentron canadum) skin [J]. Food Chemistry,2008, 110(1):128-136.
[96]Mendis E, Rajapakse N, Byun H G, et al. Investigation of jumbo squid (Dosidicus gigas) skin gelatin peptides for their in vitro antioxidant effects [J]. Life Sciences,2005, 77(17):2166-2178.
[97]Aleman A, Gimenez B, Perez-Santin E, et al. Contribution of Leu and Hyp residues to antioxidant and ACE-inhibitory activities of peptide sequences isolated from squid gelatin hydrolysate [J]. Food Chemistry,2011,125(2):334-341.
[98]Kim S K, Byun H G, Park P J, et al. Angiotensin I converting enzyme inhibitory peptides purified from bovine skin gelatin hydrolysate[J]. Journal of Agricultural and Food Chemistry,2001,49(6):2992-2997.
[99]Davalos A, Miguel M, Bartolome B, et al. Antioxidant activity of peptides derived from egg white proteins by enzymatic hydrolysis[J]. Journal of Food Protection,2004,67(9): 1939-1944.
[100]Hernandez-Ledesma B, Davalos A, Bartolome B, et al. Preparation of antioxidant enzymatic hydrolysates from a-lactalbumin and β-lactoglobulin. Identification of active peptides by HPLC-MS/MS [J]. Journal of Agricultural and Food Chemistry,2005, 53(3):588-593.
[101]Qian Z J, Jung W K, Kim S K. Free radical scavenging activity of a novel antioxidative peptide purified from hydrolysate of bullfrog skin, Rana catesbeiana Shaw [J]. Bioresource Technology,2008,99(6):1690-1698.
[102]Guillen G, Lopez Caballero M E, Aleman A, et al. Antioxidant and antimicrobial peptide fractions from squid and tuna skin gelatin[M].2010. In E. Le Bihan,& N. Koueta (Eds.), Sea by-products as a real material:New ways of application (pp.89-115). Kerala, India:Transworld Research Network Signpost, Chapter 7.
[103]Yang H, Wang Y, Zhou P, et al. Effects of alkaline and acid pretreatment on the physical properties and nanostructures of the gelatin from channel catfish skins [J]. Food Hydrocolloids,2008,22(8):1541-1550.
[104]田亚平.生化分离技术[M].化学工业出版社,2006.
[105]耿莉,安丽娟.分子排阻色谱法检测胰岛素中的大分子蛋白质[J].徐州工程学院学报.2007,22(4)：50
[106]Un N, Ganesan R, Flood K G, et al. Determination of enantiomers in a synthetic argininal peptide using capillary zone electrophoresis and high-performance liquid chromatography [J]. Journal of Chromatography A,2000,891(1):115-127.
[107]Sanz-Nebot V, Benavente F, Toro I, et al. Optimization of HPLC conditions for the separation of complex crude mixtures produced in the synthesis of therapeutic peptide hormones[J]. Chromatographia,2001,53(1):S167-S173.
[108]吴熙然,胡红华,张相年,等.亲和层析法分离纯化蒜氨酸酶[J].食品科学,2009(19)：244-247.
[109]张智武,许建香.种电泳方法定量分析牛奶蛋白的比较[J].中国乳品工业,2005,33(8)：51-57.
[110]象丽丽,季旼,任宇鹏,等.毛细管区带电泳法研究新型喜树碱衍生物的水解反应.分析化学[J].2009,37(11)：1657-1661.
[111]黄莉莉,蔡怀鸿,洪爱华,等.毛细管区带电泳法分离四种蛋白质的研究[J].现代仪器.2008,14(6)：68-69.
[112]尹开吉,谢荣继,谢瑶,等.压力驱动的毛细管等电聚焦分离牛血清白蛋白和血红蛋白[J].生命科学仪器.2006,4(4)：34-35.
[113]李艳红.鹰嘴豆蛋白酶解物的制备及其抗氧化肽的研究[D].无锡：江南大学博士学位论文,2008.
[114]贺玖明,再帕尔,小川亮,等.快原子轰击质谱(FAB-MS)法与抗氧化作用的相关性[J].质谱学报,2003,24(2)：346-350.
[115]Stapels M D, Barofsky D F. Complementary use of MALDI and ESI for the HPLC-MS/MS analysis of DNA-binding proteins [J]. Analytical chemistry,2004, 76(18):5423-5430.
[116]Kim S E, Kim H H, Kim J Y, et al. Anticancer activity of hydrophobic peptides from soy proteins[J]. Biofactors,2000,12(1-4):151-155.
[117]Rejtar T, Chen H, Andreev V, et al. Increased identification of peptides by enhanced uta processing of high-resolution MALDI TOF/TOF mass spectra prior to utabase searching[J]. Analytical chemistry,2004,76(20):6017-6028.
[118]张玉奎,张维冰.分析化学手册-液相色谱分析[M].北京：化学工业出版社.2000：19-45.
[119]Kittiphattanabawon P, Benjakul S, Visessanguan W, et al. Characterisation of acid-soluble collagen from skin and bone of bigeye snapper (Priacanthus tayenus)[J]. Food chemistry,2005,89(3):363-372.
[120]Zhang J, Duan R, Tian Y, et al. Characterisation of acid-soluble collagen from skin of silver carp (Hypophthalmichthys molitrix)[J]. Food Chemistry,2009,116(1):318-322.
[121]Ahmad M, Benjakul S. Extraction and characterisation of pepsin-solubilised collagen from the skin of unicorn leatherjacket(Aluterus monocerous) [J]. Food Chemistry,2010, 120(3):817-824.
[122]张帅,罗非鱼皮胶原蛋白提取及性质研究[D],上海：上海海洋大学,2010.
[123]Matmaroh K, Benjakul S, Prodpran T, et al. Characteristics of acid soluble collagen and pepsin soluble collagen from scale of spotted golden goatfish (Parupeneus heptacanthus)[J]. Food Chemistry,2011,129(3):1179-1186.
[124]Ikoma T, Kobayashi H, Tanaka J, et al. Physical properties of type I collagen extracted from fish scales of Pagrus major and Oreochromis niloticas[J]. International Journal of Biological Macromolecules,2003,32(3):199-204.
[125]赵振国,吴佩强,羌笛.胶体与界面化学实验[M].北京：北京大学出版社,1993,300-304.
[126]Duan R, Zhang J, Du X, et al. Properties of collagen from skin, scale and bone of carp (Cyprinus carpio)[J]. Food chemistry,2009,112(3):702-706.
[127]Li F, Jia D, Yao K. Amino acid composition and functional properties of collagen polypeptide from Yak (Bos grunniens) bone[J]. LWT-Food Science and Technology, 2009,42(5):945-949.
[128]Kimura S, Zhu X P, Matsui R, et al. Characterization of fish muscle type I collagen[J]. Journal of Food Science,1988,53(5):1315-1318.
[129]刘海英.斑点叉尾鮰鱼皮胶原蛋白及胶原蛋白多肽的研究[D].江南大学,2007.
[130]Liu D, Liang L, Regenstein J M, et al. Extraction and characterisation of pepsin-solubilised collagen from fins, scales, skins, bones and swim bladders of bighead carp (Hypophthalmichthys nobilis)[J]. Food Chemistry,2012,133(4): 1441-1448.
[131]金成.草鱼鱼鳞胶原蛋白的提取及特性研究[D].武汉：华中农业大学,2005.
[132]Kristinsson H G, Rasco B A. Fish protein hydrolysates:production, biochemical, and functional properties [J]. Critical reviews in food science and nutrition,2000,40(1): 43-81.
[133]Liu R H. Potential synergy of phytochemicals in cancer prevention:mechanism of action [J]. The Journal of nutrition,2004,134(12):3479S-3485S.
[134]Finkel T, Holbrook N J. Oxidants, oxidative stress and the biology of ageing [J]. Nature, 2000,408(6809):239-247.
[135]Shimau K, Fujikawa K, Yahara K, et al. Antioxidative properties of xanthan on the autoxidation of soybean oil in cyclodextrin emulsion[J]. Journal of Agricultural and Food Chemistry,1992,40(6):945-948.
[136]Wang B, Li Z R, Chi C F, et al. Preparation and evaluation of antioxidant peptides from ethanol-soluble proteins hydrolysate of Sphyrna lewini muscle [J]. Peptides,2012.36(2): 240-250.
[137]荣建华,李小定,谢笔钧.大豆肽体外抗氧化效果的研究[J].食品科学,2002,23(11)：1]8-120.
[138]崔剑,李兆陇,洪啸吟.自由基生物抗氧化与疾病[J].清华大学学报(自然科学版).2000,40(6)：9-12.
[139]Halliwell B, Whiteman M. Measuring reactive species and oxidative umage in vivo and in cell culture:how should you do it and what do the results mean? [J]. British journal of pharmacology,2004,142(2):231-255.
[140]Halliwell B. Role of free radicals in the neurodegenerative diseases [J]. Drugs & aging, 2001,18(9):685-716.
[141]Pihlanto A. Antioxidative peptides derived from milk proteins [J]. International Uiry Journal,2006,16(11):1306-1314.
[142]Halliwell B. Free radicals, antioxidants, and human disease:curiosity, cause, or consequence [J]. The lancet,1994,344(8924):721-724.
[143]Maritim A C, Sanders R A, Watkins J B. Diabetes, oxidative stress, and antioxidants:a review [J]. Journal of biochemical and molecular toxicology,2003,17(1):24-38.
[144]Pappenheimer J R, Volpp K. Transmucosal impeunce of small intestine:correlation with transport of sugars and amino acids [J]. American Journal of Physiology-Cell Physiology,1992,263(2):480-493.
[145]Kaur R, Arora S, Singh B. Antioxidant activity of the phenol rich fractions of leaves of Chukrasia tabularis A. Juss [J]. Bioresource Technology,2008,99(16):7692-7698.
[146]Chung Y C, Chang C T, Chao W W, et al. Antioxidative activity and safety of the 50 ethanolic extract from red bean fermented by Bacillus subtilis IMR-NK1 [J]. Journal of Agricultural and Food Chemistry,2002,50(8):2454-2458.
[147]Yang J I, Liang W S, Chow C J, et al. Process for the production of tilapia retorted skin gelatin hydrolysates with optimized antioxidative properties [J]. Process Biochemistry, 2009,44(10):1152-1157.
[148]国家质量监督检验检疫总局.实验动物小鼠大鼠配合饲料[S].北京：中国标准出版社,2001,485-487.
[149]Chen H M, Muramoto K, Yamauchi F, et al. Antioxidant activity of designed peptides based on the antioxidative peptide isolated from digests of a soybean protein [J]. Journal of Agricultural and Food Chemistry,1996,44(9):2619-2623.
[150]Rajapakse N, Mendis E, Byun H G, et al. Purification and in vitro antioxidative effects of giant squid muscle peptides on free radical-mediated oxidative systems [J]. The Journal of nutritional biochemistry,2005,16(9):562-569.
[151]Wang J, Zhao M, Yang X, et al. Improvement on functional properties of wheat gluten by enzymatic hydrolysis and ultrafiltration [J]. Journal of Cereal Science,2006,44(1): 93-100.
[152]Chen H M, Muramoto K, Yamauchi F, et al. Antioxidative properties of histidine-containing peptides designed from peptide fragments found in the digests of a soybean protein [J]. Journal of Agricultural and Food Chemistry,1998,46(1):49-53.
[153]Saiga A I, Tanabe S, Nishimura T. Antioxidant activity of peptides obtained from porcine myofibrillar proteins by protease treatment [J]. Journal of Agricultural and Food Chemistry,2003,51(12):3661-3667.
[154]Megias C, Pedroche J, Yust M M, et al. Affinity purification of copper-chelating peptides from sunflower protein hydrolysates [J]. Journal of agricultural and food chemistry,2007,55(16):6509-6514.
[155]刘静娜.壳聚糖降脂作用机理研究[D].中国无锡：江南大学,2008.
[156]丁进锋,苏秀榕,李妍妍,等.海蜇胶原蛋白肽的降血脂及抗氧化作用的研究[J].天然产物研究与开发,2012,24(3)：362-365.
[157]董士远,孟艳丽,赵元晖,等.鲢鱼寡肽对高血脂大鼠脂质代谢的调节与抗氧化作用研究[J].营养学报,2011,33(2)：121-125.
[158]裴新荣,杨睿悦,张召锋,等.海洋胶原肽的分子组成及其降血脂和抗氧化作用研 究[J].中华预防医学杂志,2008,42(4)：235-238.
[159]游丽君.泥鳅蛋白抗氧化肽的分离纯化及抗疲劳,抗癌功效研究[D].广州：华南理工大学,2010.
[160]牛慧慧,马美湖,杨昆.蛋清肽的抗氧化稳定性与功能特性[J].食品科学,2011,32(15)：139-143.
[161]赵谋明,何婷,赵强忠,等.蓝园鲹抗氧化肽抗氧化稳定性研究[J].食品科学,2009(1).128-130.
[162]朱淑云,董英,周越.水飞蓟粕蛋白酶解物抗氧化稳定性研究[J].林产化学与工业,2012,32(2).153-157.
[163]姚翔.紫菜可控酶解产抗氧化活性肽的研究[D].无锡：江南大学,2012.
[164]李景军,陈正行,毛健.抑制羟自由基的粟水溶性蛋白胁迫培养基试验[J].江苏大学学报：自然科学版,2008,29(1)：9-12.
[165]李高阳,丁霄霖.双液相萃取对亚麻籽分离蛋白功能特性的影响[J].江苏大学学报：自然科学版,2006,27(5)：383-387.
[166]Guerard F, Sumaya-Martinez M T. Antioxidant effects of protein hydrolysates in the reaction with glucose [J]. Journal of the American Oil Chemists' Society,2003,80(5): 467-470.
[167]汪家政,范明.蛋白质技术手册[M].北京：科学出版社.2001.
[168]陈怀侠,韩凤梅,杜鹏,等.苦参生物碱电喷雾质谱分析[J].分析化学,2006,34(2)：205-208.
[169]越皓,皮子凤,赵宇峰,等.电喷雾串联质谱分析附子炮制中的化学成分变化[J].分析化学.2007,35(7)：959-963
[170]赵玉芬.生物有机质谱[M].郑州大学出版社,2005.
[171]Jeon Y J, Byun H G, Kim S K. Improvement of functional properties of cod frame protein hydrolysates using ultrafiltration membranes [J]. Process Biochemistry,1999, 35(5):471-478.
[172]Suetsuna K, Ukeu H, Ochi H. Isolation and characterization of free radical scavenging activities peptides derived from casein [J]. The Journal of nutritional biochemistry, 2000,11(3):128-131.
[173]Ma Y, Xiong Y L, Zhai J, et al. Fractionation and evaluation of radical scavenging peptides from in vitro digests of buckwheat protein[J]. Food chemistry,2010,118(3): 582-588.
[174]Guo H, Kouzuma Y, Yonekura M. Structures and properties of antioxidative peptides derived from royal jelly protein [J]. Food Chemistry,2009,113(1):238-245.
[175]Hsu K C, Lu G H, Jao C L. Antioxidative properties of peptides prepared from tuna cooking juice hydrolysates with orientase(Bacillus subtilis) [J]. Food Research International,2009,42(5):647-652.
[176]You L, Zhao M, Regenstein J M, et al. Purification and identification of antioxidative peptides from loach (Misgurnus anguillicauutus) protein hydrolysate by consecutive chromatography and electrospray ionization-mass spectrometry [J]. Food Research International,2010,43(4):1167-1173.
[177]Cheng Y, Chen J, Xiong Y L. Chromatographic separation and tandem MS identification of active peptides in potato protein hydrolysate that inhibit autoxidation of soybean oil-in-water emulsions [J]. Journal of agricultural and food chemistry,2010, 58(15):8825-8832.
[178]张苗.甘薯蛋白酶解肽的抗氧化及结肠癌活性研究[D].北京：中国农业科学院,2012.
[179]Saito K, Jin D H, Ogawa T, et al. Antioxidative properties of tripeptide libraries prepared by the combinatorial chemistry [J]. Journal of Agricultural and Food Chemistry,2003,51(12):3668-3674.
[180]Zhang H Y. Structure-activity relationships and rational design strategies for radical-scavenging antioxidants [J]. Current Computer-Aided Drug Design,2005,1(3): 257-273.
[181]Park P J, Jung W K, Nam K S, et al. Purification and characterization of antioxidative peptides from protein hydrolysate of lecithin-free egg yolk [J]. Journal of the American Oil Chemists' Society,2001,78(6):651-656.
[182]Pena-Ramos E A, Xiong Y L, Arteaga G E. Fractionation and characterisation for antioxidant activity of hydrolysed whey protein [J]. Journal of the Science of Food and Agriculture,2004,84(14):1908-1918.
[183]孙月梅.大豆抗氧化肽酶法制备及其活性保护技术研究[D].哈尔滨：东北农业大 学,2008.
[184]Rajapakse N, Mendis E, Jung W K, et al. Purification of a radical scavenging peptide from fermented mussel sauce and its antioxidant properties [J]. Food Research International,2005,38(2):175-182.
[2]张俊杰,曾庆孝.鱼鳞的开发利用前景[J].中国水产,2004,5：74-75.
[3]钟朝辉,李春美,梁晋鄂,等.鱼鳞胶原蛋白提取工艺的优化[J].食品科学,2006,27(7)：162-165.
[4]Liu W T, Li G. Y, Miao Y Q, et al. Preparation and characterization of pepsin-solubilized type Ⅰ collagen from the scales of snakehead (Ophiocephalus argus) [J]. Journal of Food Biochemistry,2009,33(1):20-37.
[5]Shoulders M D, Raines R T. Collagen structure and stability [J]. Annual Review Biochemistry,2009,78(1):929-958.
[6]Liu Z, Oliveira A C M, Su Y C. Purification and characterization of pepsin-solubilized collagen from skin and connective tissue of giant red sea cucumber(Parastichopus californicus)[J]. Journal of agricultural and food chemistry,2009,58(2):1270-1274.
[7]刘刚.安全与功能性——关于胶原蛋白的探讨[J].明胶科学与技术,2010,30(2)：87-89.
[8]Gelse K, Poschl E, Aigner T. Collagens-structure, function, and biosynthesis [J]. Advanced Drug Delivery Reviews,2003,55(20):1531-1546.
[9]张楚福.生物化学原理[M].北京：高等教育出版社,2003：67-70.
[10]沈同,工镜岩,朱圣庚,等.生物化学[M].北京：高等教育出版社,2002.
[11]胡帅,王慧桂,但卫华,等.胶原蛋白结构的研究进展[J].西部皮革,2008,30(8)：16-19.
[12]陈胜军,曾名勇,董士远.水产胶原蛋白及其活性肽的研究进展[J].水产科学,2004,23(6)：44-46.
[13]吴波,陈运中.鱼鳞胶原蛋白的研究进展及应用[J].武汉工业学院学报,2007,26(4)：29-33.
[14]Ikoma T, Kobayashi H, Tanaka J, et al. Microstructure, mechanical, and biomimetic properties of fish scales from Pagrus major [J]. Journal of Structural Biology,2003, 142(3):327-333.
[15]Zylberberg L, Bereiter-Hahn J. The distribution of Tyr and Glu microtubules during fish scale regeneration [J]. European journal of cell biology,1991,54(1):132-139.
[16]Onozato H, Watabe N. Studies on fish scale formation and resorption [J]. Cell and Tissue Research,1979,201(3):409-422.
[17]段蕊,张俊杰,杜修桥.鱼鳞组成性质及其加工利用的研究进展[J].食品与机械,2006,22(5)：128-131.
[18]谢克难,周凤娇,袁锡东,等.鱼鳞胶滋阴养血作用初探[J].新中医,1996(1)：62-63.
[19]刘庆慧,刘从力,王采理.鱼鳞营养成分的分析及对高脂饲料大鼠血酯水平的影响[J].中国水产科学,2001,7(4)：56-59.
[20]赵连胜.鱼鳞杂谈(一,二)[J].内陆水产,1997(11)：28-30.
[21]郑本瑞,吕国桥,罗自文.龟甲胶药理作用研究进展[J].时珍国医国药,2001,12(5)：463-464.
[22]黄必胜.阿胶龟胶和鹿角胶中微量元素的测定与分析[J].时珍国医国药,2001,12(6)：487.
[23]Gomez-Guillen M C, Gimenez B, Lopez-Caballero M E, et al. Functional and bioactive properties of collagen and gelatin from alternative sources:A review [J]. Food Hydrocolloids,2011,25(8):1813-1827.
[24]曾江南,蒋柏泉,魏林生,等.鳙鱼鱼鳞脱钙工艺的优化[J].南昌大学学报(工科版),2011,33(4)：338-342.
[25]刘亚,陈孔才.微波辅助EDTA法对鱼鳞胶原多肽脱钙的影响[J].现代食品科技,2010,26(5)：512-515.
[26]Yan W, Joe M R. Effect of EDTA, HCl, and Citric Acid on Ca Salt Removal from Asian (Silver) Carp Scales Prior to Gelatin Extraction [J]. Journal of Food Science,2009,74(6): 426-431.
[27]朱秀灵,戴清源,蔡为荣,等.响应面法优化鲫鱼鱼鳞酸溶性胶原蛋白提取工艺[J].食品工业科技,2010,31(11)：247-251.
[28]Cheng F Y, Hsu F W, Chang H S, et al. Effect of different acids on the extraction of pepsin-solubilised collagen containing melanin from silky fowl feet[J]. Food chemistry, 2009,113(2):563-567.
[29]Skierka E, Sadowska M. The influence of different acids and pepsin on the extractability of collagen from the skin of Baltic cod (Gadus morhua) [J]. Food chemistry,2007, 105(3):1302-1306.
[30]张俊杰,段蕊,陈璐.鲤鱼鳞酸溶性胶原蛋白提取的研究[J].水产科学,2006, 25(12)：640-643.
[31]潘洪民,聂小宝,程丽林,等.武昌鱼鱼鳞胶原蛋白提取工艺的研究[J].安徽农业科学,2012,40(1)：‘111-112.
[32]曾少葵,蓝海明,章超桦,等.罗非鱼鳞胶原蛋白的提取及其酶解产物的抗氧化性[J].上海海洋大学学报,2009,18(5)：599-603.
[33]Wangtueai S, Noomhorm A. Processing optimization and characterization of gelatin from lizardfish (Sauriu spp.) scales [J]. LWT-Food Science and Technology,2009,42(4): 825-834.
[34]Woo J W, Yu S J, Cho S M, et al. Extraction optimization and properties of collagen from yellowfin tuna (Thunnus albacares) dorsal skin [J]. Food Hydrocolloids,2008,22(5): 879-887.
[35]Nalinanon S, Benjakul S, Visessanguan W, et al. Use of pepsin for collagen extraction from the skin of bigeye snapper (Priacanthus tayenus)[J]. Food Chemistry,2007,104(2): 593-601.
[36]王林.深海红鱼(Sebastes mentella)胶原蛋白的制备及其理化特性研究[D].南京：南京农业大学,2009.
[37]张俊杰,段蕊,潘秀楼.鲤鱼鱼鳞酶溶性胶原蛋白提取工艺的应用[J].淮海工学院学报(自然科学版),2006,4：55-58.
[38]刘艳,刘章武,唐婧苗,等.酶解淡水鱼鳞制备水解胶原蛋白的研究[J].武汉工业学院学报,2009,28(2)：11-14.
[39]董乐,林娈,王力.水提法提取鱼鳞胶原蛋白的研究[J].内蒙古民族大学学报：自然科学版,2010(005)：525-529.
[40]林家福,谢福美,易美华,等.热水提取罗非鱼鱼皮胶原蛋白的研究[J].中国热带医学,2008,8(8)：1317-1318.
[41]李敏.青鱼鱼鳞胶原蛋白和羟基磷灰石综合提取研究[D].长沙：湖南农业大学,2008.
[42]赵睿,王晓丹,祖国仁,等.热水法抽提鳕鱼皮胶原蛋白条件的响应面分析[J].大连工业大学学报,2011,30(2)：
[43]钱曼,武贤壮,邱承光,等.热力法和酶解法提取鱼鳞胶原蛋白的工艺及性质研究[J].食品工业科技,2007,28(10)：70-72.
[44]Kim H K, Kim Y H, Kim Y J, et al. Effects of ultrasonic treatment on collagen extraction from skins of the sea bass Lateolabrax japonicus [J]. Fisheries science,2012,78(2): 485-490.
[45]Li D, Mu C, Cai S, et al. Ultrasonic irradiation in the enzymatic extraction of collagen [J]. Ultrasonics sonochemistry,2009,16(5):605-609.
[46]Gomez-Guillen M C, Gimenez B, Montero P. Extraction of gelatin from fish skins by high pressure treatment [J]. Food hydrocolloids,2005,19(5):923-928.
[47]陈丽清,张宇昊,周梦柔,等.猪皮明胶提取过程中的超高压预处理工艺优化[J].农业工程学报,2012,28(19)：262-269.
[48]焦道龙,陆剑锋,张伟伟,等.水产动物胶原蛋白的研究现状及发展趋势[J].食品科学,2009(17)：334-338.
[49]曹俊.鱼鳞胶原蛋白的研究进展和应用前景[J].水产科技情报,2009,36(1)：41-44.
[50]蒋挺大.胶原与胶原蛋白[M].北京：化学工业出版社,2006.
[51]Ogawa M, Portier R J, Moody M W, et al. Biochemical properties of bone and scale collagens isolated from the subtropical fish black drum (Pogonia cromis) and sheepshead seabream (Archosargus probatocephalus) [J]. Food Chemistry,2004,88(4):495-501.
[52]Muyonga J H, Cole C G B, Duodu K G. Fourier transform infrared (FTIR) spectroscopic study of acid soluble collagen and gelatin from skins and bones of young and adult Nile perch (Lates niloticus) [J]. Food Chemistry,2004,86(3):325-332.
[53]陆璐.Ⅰ型胶原的提取及其组装和表征[D].南京：东南大学,2006.
[54]Rigby B J. Amino-acid composition and thermal stability of the skin collagen of the Antarctic ice-fish [J].1968,219(150):166-167.
[55]Jongjareonrak A, Benjakul S, Visessanguan W, et al. Isolation and characterisation of acid and pepsin-solubilised collagens from the skin of Brownstripe red snapper (Lutjanus vitta)[J]. Food Chemistry,2005,93(3):475-484.
[56]Hattori S, Auchi E, Ebihara T, et al. Alkali-treated collagen retained the triple helical conformation and the ligand activity for the cell adhesion via α2 β1 integrin[J]. Journal of biochemistry,1999,125(4):676-684.
[57]段蕊,张俊杰,赵晓庆,等.鲤鱼鱼鳞胶原蛋白性质的研究[J].2001,32(2)：175-180.
[58]段蕊,张俊杰,今野久仁彦.鲤鱼鱼鳞胶原蛋白ASC与PSC的比较[J].水产科学, 2008,27(8)：397-400.
[59]汪海波,梁艳萍,汪海婴,等.草鱼鱼鳞胶原蛋白的提取及其部分生物学性能[J].水产学报,2012,36(4)：1-9.
[60]胡大林,廖建坤.自由基与DNA的氧化损伤[J].国外医学：卫生学分册,2002,29(5)：261-263.
[61]赵晶,白云.自由基相关疾病研究进展[J].生物学教学,2003,28(4)：6-8.
[62]刘兆红,岳炳辉.氧自由基对动物机体的毒性作用[J].上海畜牧兽医通讯,2005,3：57.
[63]胡柏成,黄龚.体内外自由基形成的研究及意义[J].中国疗养医学,2007,16(5)：317-319.
[64]Johansen J S, Harris A K, Rychly D J, et al. Oxidative stress and the use of antioxidants in diabetes:linking basic science to clinical practice [J]. Cardiovascular diabetology, 2005,4(1):5-15.
[65]Poli G, Leonarduzzi G, Biasi F, et al. Oxidative stress and cell signaling [J]. Current medicinal chemistry,2004,11(9):1163-1182.
[66]Wang J, Sun B, Cao Y, et al. Protection of wheat bran feruloyl oligosaccharides against free radical-induced oxidative umage in normal human erythrocytes [J]. Food and, Chemical Toxicology,2009,47(7):1591-1599.
[67]Ulle-Donne I, Rossi R, Colombo R, et al. Biomarkers of oxidative umage in human disease [J]. Clinical chemistry,2006,52(4):601-623.
[68]Dhalla N S, Temsah R M, Netticaun T. Role of oxidative stress in cardiovascular diseases [J]. Journal of hypertension,2000,18(6):655-673.
[69]Zhou C, Huang Y, Przedborski S. Oxidative stress in Parkinson's disease:A Mechanism of Pathogenic and Therapeutic Significance [J]. Annals of the New York Academy of Sciences,2008,1147(1):93-104.
[70]Sayre L M, Smith M A, Perry G. Chemistry and biochemistry of oxidative stress in neurodegenerative disease [J]. Current medicinal chemistry,2001,8(7):721-738.
[71]Di Bernardini R, Harnedy P, Bolton D, et al. Antioxidant and antimicrobial peptidic hydrolysates from muscle protein sources and by-products [J]. Food Chemistry,2011, 124(4):1296-1307.
[72]Gill I, Lopez-Fandino R, Jorba X, et al. Biologically active peptides and enzymatic approaches to their production [J]. Enzyme and Microbial Technology,1996,18(3): 162-183.
[73]Uwson P E, Muir T W, Clark-Lewis I, et al. Synthesis of proteins by native chemical ligation [J]. Science,1994,266(4):776-778.
[74]Seebach D, Albert M, Arvidsson P I, et al. From the biopolymer PHB to biological investigations of unnatural and peptides [J]. CHIMIA International Journal for Chemistry, 2001,55(4):345-353.
[75]Rao C, Tam J P. Synthesis of peptide dendrimer [J]. Journal of the American Chemical Society,1994,116(15):6975-6976.
[76]林琳.鱼皮胶原蛋白的制备及胶原蛋白多肽活性的研究[D].青岛：中国海洋大学,2006.
[77]Gimenez B, Aleman A, Montero P, et al. Antioxidant and functional properties of gelatin hydrolysates obtained from skin of sole and squid [J]. Food Chemistry,2009,114(3): 976-983.
[78]Mendis E, Rajapakse N, Kim S K. Antioxidant properties of a radical-scavenging peptide purified from enzymatically prepared fish skin gelatin hydrolysate [J]. Journal of agricultural and food chemistry,2005,53(3):581-587.
[79]Li B, Chen F, Wang X, et al. Isolation and identification of antioxidative peptides from porcine collagen hydrolysate by consecutive chromatography and electrospray ionization-mass spectrometry [J]. Food Chemistry,2007,102(4):1135-1143.
[80]韩凤杰,赵征,胡晓鹏.酶法制取鲮鱼皮胶原蛋白寡肽工艺的研究[J].现代食品科技,2006,22(2)：135-137.
[81]Aleman A, Gimenez B, Montero P, et al. Antioxidant activity of several marine skin gelatins[J]. LWT-Food Science and Technology,2011,44(2):407-413.
[82]Nagai T, Nagashima T, Abe A, et al. Antioxidative activities and angiotensin Ⅰ-converting enzyme inhibition of extracts prepared from chum salmon (Oncorhynchus keta) cartilage and skin [J]. International Journal of Food Properties,2006,9(4):813-822.
[83]任俊凤,任婷婷,朱蓓薇.河豚鱼皮胶原蛋白肽的提取及其抗氧化活性的研究[J].中国食品学报,2009,9(1)：77-83.
[84]庄永亮.海蜇胶原蛋白理化性质及其胶原肽的护肤活性研究[D].青岛：中国海洋大 学,2009.
[85]沈澎.鲤鱼鱼鳞胶原肽的制备及特性研究[D].长春：吉林农业大学,2011.
[86]胡娟.酶解鱼鳞明胶制备生物活性肽的研究[D].广州：华南理工大学,2010.
[87]申锋,杨莉莉,熊善柏,等.胃蛋白酶水解草鱼鱼鳞制备胶原肽的工艺优化[J].华中农业大学学报,2010,29(003)：387-391.
[88]Marcuse, R. Antioxidative effect of amino-acids [J].Nature,1960,186(11):886-887.
[89]Megias C, Pedroche J, Yust M M, et al. Production of copper-chelating peptides after hydrolysis of sunflower proteins with pepsin and pancreatin [J]. LWT-Food Science and Technology,2008,41(10):1973-1977.
[90]Xie Z, Huang J, Xu X, et al. Antioxidant activity of peptides isolated from alfalfa leaf protein hydrolysate [J]. Food Chemistry,2008,111(2):370-376.
[91]Wu H C, Chen H M, Shiau C Y. Free amino acids and peptides as related to antioxidant properties in protein hydrolysates of mackerel (Scomber austriasicus)[J]. Food research international,2003,36(9):949-957.
[92]Kim S K, Byun H G, Ito H. Articles:Purification and Characterization of Antioxidative Peptides from Bovine Skin [J]. Biochemistry and Molecurar Biology Reports,2001, 34(3):219-224.
[93]Je J Y, Qian Z J, Byun H G, et al. Purification and characterization of an antioxidant peptide obtained from tuna backbone protein by enzymatic hydrolysis [J]. Process Biochemistry,2007,42(5):840-846.
[94]Kim S K, Kim Y T, Byun H G, et al. Isolation and characterization of antioxidative peptides from gelatin hydrolysate of Alaska pollack skin [J]. Journal of Agricultural and Food Chemistry,2001,49(4):1984-1989.
[95]Yang J I, Ho H Y, Chu Y J, et al. Characteristic and antioxidant activity of retorted gelatin hydrolysates from cobia (Rachycentron canadum) skin [J]. Food Chemistry,2008, 110(1):128-136.
[96]Mendis E, Rajapakse N, Byun H G, et al. Investigation of jumbo squid (Dosidicus gigas) skin gelatin peptides for their in vitro antioxidant effects [J]. Life Sciences,2005, 77(17):2166-2178.
[97]Aleman A, Gimenez B, Perez-Santin E, et al. Contribution of Leu and Hyp residues to antioxidant and ACE-inhibitory activities of peptide sequences isolated from squid gelatin hydrolysate [J]. Food Chemistry,2011,125(2):334-341.
[98]Kim S K, Byun H G, Park P J, et al. Angiotensin I converting enzyme inhibitory peptides purified from bovine skin gelatin hydrolysate[J]. Journal of Agricultural and Food Chemistry,2001,49(6):2992-2997.
[99]Davalos A, Miguel M, Bartolome B, et al. Antioxidant activity of peptides derived from egg white proteins by enzymatic hydrolysis[J]. Journal of Food Protection,2004,67(9): 1939-1944.
[100]Hernandez-Ledesma B, Davalos A, Bartolome B, et al. Preparation of antioxidant enzymatic hydrolysates from a-lactalbumin and β-lactoglobulin. Identification of active peptides by HPLC-MS/MS [J]. Journal of Agricultural and Food Chemistry,2005, 53(3):588-593.
[101]Qian Z J, Jung W K, Kim S K. Free radical scavenging activity of a novel antioxidative peptide purified from hydrolysate of bullfrog skin, Rana catesbeiana Shaw [J]. Bioresource Technology,2008,99(6):1690-1698.
[102]Guillen G, Lopez Caballero M E, Aleman A, et al. Antioxidant and antimicrobial peptide fractions from squid and tuna skin gelatin[M].2010. In E. Le Bihan,& N. Koueta (Eds.), Sea by-products as a real material:New ways of application (pp.89-115). Kerala, India:Transworld Research Network Signpost, Chapter 7.
[103]Yang H, Wang Y, Zhou P, et al. Effects of alkaline and acid pretreatment on the physical properties and nanostructures of the gelatin from channel catfish skins [J]. Food Hydrocolloids,2008,22(8):1541-1550.
[104]田亚平.生化分离技术[M].化学工业出版社,2006.
[105]耿莉,安丽娟.分子排阻色谱法检测胰岛素中的大分子蛋白质[J].徐州工程学院学报.2007,22(4)：50
[106]Un N, Ganesan R, Flood K G, et al. Determination of enantiomers in a synthetic argininal peptide using capillary zone electrophoresis and high-performance liquid chromatography [J]. Journal of Chromatography A,2000,891(1):115-127.
[107]Sanz-Nebot V, Benavente F, Toro I, et al. Optimization of HPLC conditions for the separation of complex crude mixtures produced in the synthesis of therapeutic peptide hormones[J]. Chromatographia,2001,53(1):S167-S173.
[108]吴熙然,胡红华,张相年,等.亲和层析法分离纯化蒜氨酸酶[J].食品科学,2009(19)：244-247.
[109]张智武,许建香.种电泳方法定量分析牛奶蛋白的比较[J].中国乳品工业,2005,33(8)：51-57.
[110]象丽丽,季旼,任宇鹏,等.毛细管区带电泳法研究新型喜树碱衍生物的水解反应.分析化学[J].2009,37(11)：1657-1661.
[111]黄莉莉,蔡怀鸿,洪爱华,等.毛细管区带电泳法分离四种蛋白质的研究[J].现代仪器.2008,14(6)：68-69.
[112]尹开吉,谢荣继,谢瑶,等.压力驱动的毛细管等电聚焦分离牛血清白蛋白和血红蛋白[J].生命科学仪器.2006,4(4)：34-35.
[113]李艳红.鹰嘴豆蛋白酶解物的制备及其抗氧化肽的研究[D].无锡：江南大学博士学位论文,2008.
[114]贺玖明,再帕尔,小川亮,等.快原子轰击质谱(FAB-MS)法与抗氧化作用的相关性[J].质谱学报,2003,24(2)：346-350.
[115]Stapels M D, Barofsky D F. Complementary use of MALDI and ESI for the HPLC-MS/MS analysis of DNA-binding proteins [J]. Analytical chemistry,2004, 76(18):5423-5430.
[116]Kim S E, Kim H H, Kim J Y, et al. Anticancer activity of hydrophobic peptides from soy proteins[J]. Biofactors,2000,12(1-4):151-155.
[117]Rejtar T, Chen H, Andreev V, et al. Increased identification of peptides by enhanced uta processing of high-resolution MALDI TOF/TOF mass spectra prior to utabase searching[J]. Analytical chemistry,2004,76(20):6017-6028.
[118]张玉奎,张维冰.分析化学手册-液相色谱分析[M].北京：化学工业出版社.2000：19-45.
[119]Kittiphattanabawon P, Benjakul S, Visessanguan W, et al. Characterisation of acid-soluble collagen from skin and bone of bigeye snapper (Priacanthus tayenus)[J]. Food chemistry,2005,89(3):363-372.
[120]Zhang J, Duan R, Tian Y, et al. Characterisation of acid-soluble collagen from skin of silver carp (Hypophthalmichthys molitrix)[J]. Food Chemistry,2009,116(1):318-322.
[121]Ahmad M, Benjakul S. Extraction and characterisation of pepsin-solubilised collagen from the skin of unicorn leatherjacket(Aluterus monocerous) [J]. Food Chemistry,2010, 120(3):817-824.
[122]张帅,罗非鱼皮胶原蛋白提取及性质研究[D],上海：上海海洋大学,2010.
[123]Matmaroh K, Benjakul S, Prodpran T, et al. Characteristics of acid soluble collagen and pepsin soluble collagen from scale of spotted golden goatfish (Parupeneus heptacanthus)[J]. Food Chemistry,2011,129(3):1179-1186.
[124]Ikoma T, Kobayashi H, Tanaka J, et al. Physical properties of type I collagen extracted from fish scales of Pagrus major and Oreochromis niloticas[J]. International Journal of Biological Macromolecules,2003,32(3):199-204.
[125]赵振国,吴佩强,羌笛.胶体与界面化学实验[M].北京：北京大学出版社,1993,300-304.
[126]Duan R, Zhang J, Du X, et al. Properties of collagen from skin, scale and bone of carp (Cyprinus carpio)[J]. Food chemistry,2009,112(3):702-706.
[127]Li F, Jia D, Yao K. Amino acid composition and functional properties of collagen polypeptide from Yak (Bos grunniens) bone[J]. LWT-Food Science and Technology, 2009,42(5):945-949.
[128]Kimura S, Zhu X P, Matsui R, et al. Characterization of fish muscle type I collagen[J]. Journal of Food Science,1988,53(5):1315-1318.
[129]刘海英.斑点叉尾鮰鱼皮胶原蛋白及胶原蛋白多肽的研究[D].江南大学,2007.
[130]Liu D, Liang L, Regenstein J M, et al. Extraction and characterisation of pepsin-solubilised collagen from fins, scales, skins, bones and swim bladders of bighead carp (Hypophthalmichthys nobilis)[J]. Food Chemistry,2012,133(4): 1441-1448.
[131]金成.草鱼鱼鳞胶原蛋白的提取及特性研究[D].武汉：华中农业大学,2005.
[132]Kristinsson H G, Rasco B A. Fish protein hydrolysates:production, biochemical, and functional properties [J]. Critical reviews in food science and nutrition,2000,40(1): 43-81.
[133]Liu R H. Potential synergy of phytochemicals in cancer prevention:mechanism of action [J]. The Journal of nutrition,2004,134(12):3479S-3485S.
[134]Finkel T, Holbrook N J. Oxidants, oxidative stress and the biology of ageing [J]. Nature, 2000,408(6809):239-247.
[135]Shimau K, Fujikawa K, Yahara K, et al. Antioxidative properties of xanthan on the autoxidation of soybean oil in cyclodextrin emulsion[J]. Journal of Agricultural and Food Chemistry,1992,40(6):945-948.
[136]Wang B, Li Z R, Chi C F, et al. Preparation and evaluation of antioxidant peptides from ethanol-soluble proteins hydrolysate of Sphyrna lewini muscle [J]. Peptides,2012.36(2): 240-250.
[137]荣建华,李小定,谢笔钧.大豆肽体外抗氧化效果的研究[J].食品科学,2002,23(11)：1]8-120.
[138]崔剑,李兆陇,洪啸吟.自由基生物抗氧化与疾病[J].清华大学学报(自然科学版).2000,40(6)：9-12.
[139]Halliwell B, Whiteman M. Measuring reactive species and oxidative umage in vivo and in cell culture:how should you do it and what do the results mean? [J]. British journal of pharmacology,2004,142(2):231-255.
[140]Halliwell B. Role of free radicals in the neurodegenerative diseases [J]. Drugs & aging, 2001,18(9):685-716.
[141]Pihlanto A. Antioxidative peptides derived from milk proteins [J]. International Uiry Journal,2006,16(11):1306-1314.
[142]Halliwell B. Free radicals, antioxidants, and human disease:curiosity, cause, or consequence [J]. The lancet,1994,344(8924):721-724.
[143]Maritim A C, Sanders R A, Watkins J B. Diabetes, oxidative stress, and antioxidants:a review [J]. Journal of biochemical and molecular toxicology,2003,17(1):24-38.
[144]Pappenheimer J R, Volpp K. Transmucosal impeunce of small intestine:correlation with transport of sugars and amino acids [J]. American Journal of Physiology-Cell Physiology,1992,263(2):480-493.
[145]Kaur R, Arora S, Singh B. Antioxidant activity of the phenol rich fractions of leaves of Chukrasia tabularis A. Juss [J]. Bioresource Technology,2008,99(16):7692-7698.
[146]Chung Y C, Chang C T, Chao W W, et al. Antioxidative activity and safety of the 50 ethanolic extract from red bean fermented by Bacillus subtilis IMR-NK1 [J]. Journal of Agricultural and Food Chemistry,2002,50(8):2454-2458.
[147]Yang J I, Liang W S, Chow C J, et al. Process for the production of tilapia retorted skin gelatin hydrolysates with optimized antioxidative properties [J]. Process Biochemistry, 2009,44(10):1152-1157.
[148]国家质量监督检验检疫总局.实验动物小鼠大鼠配合饲料[S].北京：中国标准出版社,2001,485-487.
[149]Chen H M, Muramoto K, Yamauchi F, et al. Antioxidant activity of designed peptides based on the antioxidative peptide isolated from digests of a soybean protein [J]. Journal of Agricultural and Food Chemistry,1996,44(9):2619-2623.
[150]Rajapakse N, Mendis E, Byun H G, et al. Purification and in vitro antioxidative effects of giant squid muscle peptides on free radical-mediated oxidative systems [J]. The Journal of nutritional biochemistry,2005,16(9):562-569.
[151]Wang J, Zhao M, Yang X, et al. Improvement on functional properties of wheat gluten by enzymatic hydrolysis and ultrafiltration [J]. Journal of Cereal Science,2006,44(1): 93-100.
[152]Chen H M, Muramoto K, Yamauchi F, et al. Antioxidative properties of histidine-containing peptides designed from peptide fragments found in the digests of a soybean protein [J]. Journal of Agricultural and Food Chemistry,1998,46(1):49-53.
[153]Saiga A I, Tanabe S, Nishimura T. Antioxidant activity of peptides obtained from porcine myofibrillar proteins by protease treatment [J]. Journal of Agricultural and Food Chemistry,2003,51(12):3661-3667.
[154]Megias C, Pedroche J, Yust M M, et al. Affinity purification of copper-chelating peptides from sunflower protein hydrolysates [J]. Journal of agricultural and food chemistry,2007,55(16):6509-6514.
[155]刘静娜.壳聚糖降脂作用机理研究[D].中国无锡：江南大学,2008.
[156]丁进锋,苏秀榕,李妍妍,等.海蜇胶原蛋白肽的降血脂及抗氧化作用的研究[J].天然产物研究与开发,2012,24(3)：362-365.
[157]董士远,孟艳丽,赵元晖,等.鲢鱼寡肽对高血脂大鼠脂质代谢的调节与抗氧化作用研究[J].营养学报,2011,33(2)：121-125.
[158]裴新荣,杨睿悦,张召锋,等.海洋胶原肽的分子组成及其降血脂和抗氧化作用研 究[J].中华预防医学杂志,2008,42(4)：235-238.
[159]游丽君.泥鳅蛋白抗氧化肽的分离纯化及抗疲劳,抗癌功效研究[D].广州：华南理工大学,2010.
[160]牛慧慧,马美湖,杨昆.蛋清肽的抗氧化稳定性与功能特性[J].食品科学,2011,32(15)：139-143.
[161]赵谋明,何婷,赵强忠,等.蓝园鲹抗氧化肽抗氧化稳定性研究[J].食品科学,2009(1).128-130.
[162]朱淑云,董英,周越.水飞蓟粕蛋白酶解物抗氧化稳定性研究[J].林产化学与工业,2012,32(2).153-157.
[163]姚翔.紫菜可控酶解产抗氧化活性肽的研究[D].无锡：江南大学,2012.
[164]李景军,陈正行,毛健.抑制羟自由基的粟水溶性蛋白胁迫培养基试验[J].江苏大学学报：自然科学版,2008,29(1)：9-12.
[165]李高阳,丁霄霖.双液相萃取对亚麻籽分离蛋白功能特性的影响[J].江苏大学学报：自然科学版,2006,27(5)：383-387.
[166]Guerard F, Sumaya-Martinez M T. Antioxidant effects of protein hydrolysates in the reaction with glucose [J]. Journal of the American Oil Chemists' Society,2003,80(5): 467-470.
[167]汪家政,范明.蛋白质技术手册[M].北京：科学出版社.2001.
[168]陈怀侠,韩凤梅,杜鹏,等.苦参生物碱电喷雾质谱分析[J].分析化学,2006,34(2)：205-208.
[169]越皓,皮子凤,赵宇峰,等.电喷雾串联质谱分析附子炮制中的化学成分变化[J].分析化学.2007,35(7)：959-963
[170]赵玉芬.生物有机质谱[M].郑州大学出版社,2005.
[171]Jeon Y J, Byun H G, Kim S K. Improvement of functional properties of cod frame protein hydrolysates using ultrafiltration membranes [J]. Process Biochemistry,1999, 35(5):471-478.
[172]Suetsuna K, Ukeu H, Ochi H. Isolation and characterization of free radical scavenging activities peptides derived from casein [J]. The Journal of nutritional biochemistry, 2000,11(3):128-131.
[173]Ma Y, Xiong Y L, Zhai J, et al. Fractionation and evaluation of radical scavenging peptides from in vitro digests of buckwheat protein[J]. Food chemistry,2010,118(3): 582-588.
[174]Guo H, Kouzuma Y, Yonekura M. Structures and properties of antioxidative peptides derived from royal jelly protein [J]. Food Chemistry,2009,113(1):238-245.
[175]Hsu K C, Lu G H, Jao C L. Antioxidative properties of peptides prepared from tuna cooking juice hydrolysates with orientase(Bacillus subtilis) [J]. Food Research International,2009,42(5):647-652.
[176]You L, Zhao M, Regenstein J M, et al. Purification and identification of antioxidative peptides from loach (Misgurnus anguillicauutus) protein hydrolysate by consecutive chromatography and electrospray ionization-mass spectrometry [J]. Food Research International,2010,43(4):1167-1173.
[177]Cheng Y, Chen J, Xiong Y L. Chromatographic separation and tandem MS identification of active peptides in potato protein hydrolysate that inhibit autoxidation of soybean oil-in-water emulsions [J]. Journal of agricultural and food chemistry,2010, 58(15):8825-8832.
[178]张苗.甘薯蛋白酶解肽的抗氧化及结肠癌活性研究[D].北京：中国农业科学院,2012.
[179]Saito K, Jin D H, Ogawa T, et al. Antioxidative properties of tripeptide libraries prepared by the combinatorial chemistry [J]. Journal of Agricultural and Food Chemistry,2003,51(12):3668-3674.
[180]Zhang H Y. Structure-activity relationships and rational design strategies for radical-scavenging antioxidants [J]. Current Computer-Aided Drug Design,2005,1(3): 257-273.
[181]Park P J, Jung W K, Nam K S, et al. Purification and characterization of antioxidative peptides from protein hydrolysate of lecithin-free egg yolk [J]. Journal of the American Oil Chemists' Society,2001,78(6):651-656.
[182]Pena-Ramos E A, Xiong Y L, Arteaga G E. Fractionation and characterisation for antioxidant activity of hydrolysed whey protein [J]. Journal of the Science of Food and Agriculture,2004,84(14):1908-1918.
[183]孙月梅.大豆抗氧化肽酶法制备及其活性保护技术研究[D].哈尔滨：东北农业大 学,2008.
[184]Rajapakse N, Mendis E, Jung W K, et al. Purification of a radical scavenging peptide from fermented mussel sauce and its antioxidant properties [J]. Food Research International,2005,38(2):175-182.