【作者】 尤娟；

【导师】 罗永康； 吴建平；

【作者基本信息】 中国农业大学 ， 水产品加工及贮藏工程， 2014， 博士

【摘要】 鲢鱼是我国主要的淡水鱼种,其生长快,产量大,但其特有腥味,骨刺多等造成鲢鱼低价值。近来,有关如何提高鲢鱼附加值的问题已成为国内水产加工研究者们研究和开发产品的热点,但仍然处在初级阶段。因此,本研究以鲢鱼鱼肉蛋白为原料,采用酶解的方法制备抗氧化活性肽,并通过糖基化作用对酶解产物和水溶性蛋白进行功能特性和抗氧化活性改性的研究,为鲢鱼蛋白综合加工和提高其附加值提供理论基础。本研究选择了五种蛋白酶酶解鲢鱼鱼肉蛋白,通过测定了鲢鱼鱼肉蛋白的水解度及酶解产物的抗氧化活性,并分析了水解度与抗氧化各指标间的相关性。结果表明,胃蛋白酶酶解产物的清除DPPH自由基能力和还原力较强：另外,酶解产物的水解度与亚铁离子螯合力具有显著的相关性(p<0.05)。通过超滤、凝胶过滤层析与反相高效液相色谱(RP-HPLC)相结合的方法对鲢鱼鱼肉蛋白多肽进行分离,并运用清除DPPH自由基、清除羟基自由基、亚铁离子螯合力、还原力及氧活性自由基吸收能力(ORAC)等方法测定其抗氧化活性。通过液质联用的方法鉴定了10个来源于鲢鱼肌球蛋白和肌动蛋白的肽,经过合成后测定了其抗氧化活性,其中有两个肽Tyr-Thr-Ile-Gln-Tyr(YTIQY)和Asn-Trp-Asp-Asp-Met (NWDDM)抗氧化活性较高,且高于相同浓度的茶多酚的ORAC值。鲢鱼鱼肉蛋白多肽和葡萄糖在不同温度和质量比的条件下经过糖基化改性制备鲢鱼鱼肉蛋白多肽-糖复合物。糖基化反应产物有较好的清除DPPH自由基能力和还原力,显著高于未经过糖基化反应的多肽(p<0.05)。当鲢鱼鱼肉蛋白多肽与葡萄糖在60℃,以2：1混合反应后其产物显现出较高的褐变程度和抗氧化活性,且294nm的吸光值与抗氧化活性之间具有显著的相关性(p<0.05)。因此糖基化改性技术可以在一定程度上提高鲢鱼鱼肉蛋白多肽的抗氧化活性。鲢鱼水溶性蛋白在鱼糜加工过程中被作为废水处理,为了更好的利用鲢鱼水溶性蛋白资源,研究了鲢鱼水溶性蛋白经过热加工、冷冻干燥以及不同pH条件处理下的鲢鱼水溶性蛋白的溶解性、乳化性、起泡性、热稳定性等功能特性和理化特性。结果表明,鲢鱼水溶性蛋白在40℃到55℃之间经历了变性和聚集,在50℃时变化速率最大。组成鲢鱼水溶性蛋白的蛋白质分子量主要集中在30～97kDa,大部分水溶性蛋白在55℃发生沉淀。经过冷冻干燥的水溶性蛋白溶解性降低至72%左右。在等电点时,鲢鱼水溶性蛋白的功能特性较差。鲢鱼水溶性蛋白在加热过程中容易发生变性聚集而降低其各方面功能特性,本研究将5种糖基供体(包括木糖、葡萄糖、半乳糖、低聚异麦芽糖、葡聚糖-10)引入到鲢鱼水溶性蛋白,经过糖基化反应后测定其产物的功能特性,包括溶解性、热稳定性、乳化活性、乳化稳定性,同时将其理化特性如表面疏水性、总巯基含量及SDS-PAGE电泳进行分析。结果表明：经过糖基化改性的鲢鱼水溶性蛋白在50℃条件下的溶解性、乳化活性、热稳定性得到提高。并且随着提供的糖基供体分子量的增大,溶解性、乳化活性及热稳定性有一定程度的增加。综合得出鲢鱼水溶性蛋白与葡聚糖-10的反应产物具有较好效果。更多还原

【Abstract】 In China, silver carp with fast growing and large production is one of the main freshwater fish species. However it is a low market-value resource because of its muddy flavor and many fish bones. Nowerday, the development of silver carp products became to be most popular research area for aquatic researchers, but the researches are still in the early stages. The research objective of this study was to utilize silver carp fish protein and to impove the additional value for this freshwater fish. Antioxidant peptides from silver carp protein by enzymes hydrolysation were prepared. And functional properties and antioxidant acitivities of glycosylated hydrolysates and water-soluble proteins from silver carp were studied.Five common enzymes were used for hydrolysation at the same enzyme activity. The hydrolysis degree and antioxidant activities of hydrolysates were determined. The relativity between the degree of hydrolysis and antioxidant activities were also dicussed. The results showed that the DPPH radical scavenging capacity and reducing power of pepsin hydrolysates were higher than other hydrolysates, and the values were61%and1.6, respectively. There were marked relativity between the degree of hydrolysis and metal-chelating activity (p<0.05)Silver carp peptides were separated by ultrafiltration, gel filtration and reverse phrase high pressure liquid chromatogram. The DPPH radical scavenging capacity, hydroxyl radical scavenging capacity, iron chelating, reducing power and oxygen radical absorbance capacity of fractions were determined. Ten peptides from silver carp myosin and actin were identified by LC-MS/MS, and their antioxidant activities were determined using synthetic peptides. Two pentapeptides (Tyr-Thr-Ile-Gln-Tyr and Asn-Trp-Asp-Asp-Met) showed higher antioxidant activity, and the ORAC values of them were higher than catechin.The Maillard reaction products (MRPs) were prepared at different temperatures and ratios between silver carp protein hydrolysates (SPH) and glucose by Maillard reaction in powdered state, respectively. MRPs possessed a strong2,2-diphenyl-l-picrylhydrazyl (DPPH) radical scavenging activity and reducing power (p<0.05). The hydrolysate and glucose heated with the ratio of2:1at60℃showed high browning intensity and good antioxidant properties (p<0.05). According to the correlation coefficients of variables included in the hydrolysate-glucose system, good correlations were observed among the antioxidant activities and the absorbance at294nm. The results suggested that Maillard reaction has a good potential to improve the antioxidant activity of SPH.Water-soluble proteins (WSP) from silver carp muscle are currently underutilized in China and are discarded into waste streams in surimi processing. In order to utilize WSP resources efficiently, this work investigated the effect of three processing conditions on the physicochemical properties and fuctional properties such as solubility, heat stability, emulsifying activity and foamability of water-soluble proteins from silver carp. The results showed that silver carp water-soluble proteins would suffer huge denaturation and aggregation from40to55℃. Most of silver carp water-soluble proteins precipitated at55℃. Lyophilization could increase surface hydrophobicity of water-soluble proteins, but decrease its solubility and sulfhydryl groups. The functional properties of WSP were greatly affected by pH value, the lowest solubility was observed at pH4.0-5.0.WSP denature easily during thermal processing, leading to the reduction of functional properties. Five different kinds of carbohydrates (i.e., xylose, glucose, galactose, oligoisomaltose, and dextran-10) were used to conjugate with WSP by Maillard reaction. Results suggested that the use of the Maillard reaction improved the solubility, heat stability, and emulsifying properties of WSP.With the increase of molecular weight (MW) of the sugars, the solubility, emulsifying properties, and total sulfhydryl (SH) group of WSP increased, while the surface hydrophobicity decreased. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of the five Maillard reaction products (MRPs) showed that xylose reacted easily with WSP than the other four kinds of sugar did. Dextran-10conjugated to WSP could efficiently improve the functional properties of WSP during heat processing.更多还原