【作者】 张建忠；

【导师】 安辛欣；

【作者基本信息】 南京农业大学 ， 食品科学， 2007， 硕士

【摘要】 本文研究了草鱼皮的预处理工艺条件，采用氢氧化钠中添加双氧水方法可以同时起到脱色、脱杂蛋白、脱脂肪的作用，以及其对胶原蛋白的溶解性的影响。提取关键参数—温度对胶原蛋白提取率及性质的影响；酸溶性胶原蛋白和酶溶性胶原蛋白的性质区别；草鱼皮和猪皮中Ⅰ型胶原蛋白和Ⅲ型胶原蛋白纯化及电泳鉴定。1．不同浓度双氧水和氢氧化钠溶液处理草鱼皮，可以同时起到脱色、脱杂蛋白以及脱脂作用，氢氧化钠浓度越高，脱色速度越快，反应也越剧烈，双氧水浓度对胶原蛋白脱色无明显影响，在0．1 mol／L的氢氧化钠溶液含1％双氧水中只需6小时就可以完全脱去色素，在0．01 mol／L的氢氧化钠含1％双氧水溶液中，鱼皮浮在溶液表面色素渐退，无法看到黑色素体转变为淡黄色；0．01 mol／L-0．2 mol／L氢氧化钠能够有效的去除鱼皮中的非胶原蛋白，但是氢氧化钠浓度越高，可能导致胶原蛋白的降解在预处理溶液中；在20℃条件下，含1％H₂O₂ 0．01mol／L NaOH溶液可以有效脱去脂肪，脱脂率约为70％。2．在含1％双氧水不同浓度氢氧化钠处理鱼皮以后，对鱼皮胶原蛋白性质产生了影响。在20℃，1％过氧化氢，0．1 mol／L的氢氧化钠溶液中处理草鱼皮24小时后，即使在加酶的醋酸中，提取48小时，提取率也只有8％。表明胶原蛋白之间发生了交联，而在0．01 mol／L氢氧化钠或4℃条件下处理胶原蛋白，对胶原蛋白的提取率影响较小，所以只能选择浓度较低的氢氧化钠或在低温处理鱼皮。3．温度和是否加酶对胶原蛋白提取率影响很大。在醋酸溶液或醋酸加酶溶液中，温度越高，提取率越高。相同温度条件下，醋酸加酶的溶液胶原蛋白提取率高于醋酸溶液。但是SDS-PAGE电泳显示，在30℃条件下，不论醋酸溶液或醋酸加酶溶液提取的胶原蛋白都发生了变性，胶原蛋白肽链发生了降解，在4℃和20℃提取的胶原蛋白三螺旋结构保持完整，具有完整生物学活性。4．从草鱼皮中提取的酸溶性胶原蛋白和酶溶性胶原蛋白，紫外光谱分析，最大吸收峰都接近223 nm处；SDS-PAGE电泳图谱显示：酸溶性胶原蛋白含有较多β、γ和多聚体，平均分子量高于酶溶性胶原蛋白，草鱼皮ASC和PSC热变性温度分别为33．8℃、34．5℃，只比猪皮的热变性温度（37℃）低3℃左右。表明草鱼皮胶原蛋白在功能食品、医药、化妆品、制药等方面有潜在的应用。5．在pH 7．4条件下，在不同盐浓度下分别沉淀草鱼皮和猪皮胶原蛋白，Ⅲ型胶原蛋白在1．8 mol／L盐浓度（pH 7．4）完全沉淀，此时绝大多数Ⅰ型胶原蛋白溶解在溶液中，最终在2．5 mol／L盐浓度（pH 7．4）的条件下沉淀。纯化得到不同盐浓度的沉淀样品，再用含尿素的SDS-PAGE电泳分析，可以将电泳迁移率相同的a1（Ⅰ）和a1（Ⅲ）区分开，电泳显示，草鱼皮样品中不含a1（Ⅲ），猪皮中含有a1（Ⅲ），表明草鱼皮仅含有Ⅰ型胶原蛋白，不含Ⅲ型胶原蛋白，猪皮主要含有Ⅰ型胶原蛋白同时也含少量的Ⅲ型胶原蛋白。更多还原

【Abstract】 In this paper, we study grass carp skin pretreatment conditions, using the method of sodium hydroxide adding hydrogen peroxide can bleaching and removing non-collagen and defatted to grass carp skin, the role of its collagen solubility effects. Effects of the key parameter extraction—temperature to the collagen extraction rate and characterization, Characterization different between Acid-soluble collagen and enzyme-soluble collagen; Purifying typeⅠand typeⅢcollagens from Grass carp skin and pig skin and electrophoresis identification.1. Different concentrations of hydrogen peroxide and sodium hydroxide treatment grass carp skin, can also play a role on bleaching, removing non-collagen and defatted, the higher the concentration of sodium hydroxide, the faster shedding pigment. Concentration of hydrogen peroxide had no significant effect on bleaching. In 0.01 mol/L the sodium hydroxide solution containing 1% hydrogen peroxide, skin floating in surface of the solution, pigment gradual withdrawal, unable to see melanin turn to yellow; 0.01 mol/L-0.2 mol/L sodium hydroxide can effectively remove the skin of non-collagenous proteins, However, the higher concentration of sodium hydroxide, may lead to the degradation of collagen protein in the pretreatment solution; At 20℃conditions, 0.01 mol/L NaOH solution containing 1% H₂O₂ can effectively remove fat, defatted rate about 70%.2. The treatment of different concentrations of hydrogen peroxide and sodium hydroxide, have an impact on the Characterizations of collagen. At 20℃, 1% hydrogen peroxide, sodium hydroxide concentration solution, leaching treatment 24 hours later, have great impact on the Characterizations of collagen, even in the acid+enzyme xtracted 48 hours, Extraction rate only 8%. In the conditions of 0.01 mol/L NaOH or 4℃for dealing with collagen, have less impact on the collagen extraction rate, so it can only choose lower concentrations of sodium hydroxide or low temperature skin.3. Key parameter temperature of extraction of collagen and pepsin have great impact on the extraction rate. At 4℃in the acetic acid+pepsin solution extraction rate more than double extraction rate in acetic acid, At 20℃and 30℃, the extraction rate of solution containing pepsin is far higher than that of acetic acid. The higher the temperature, the higher collagen extraction rate. However, SDS-PAGE showed that peptide chain of collagen extracted at 30℃degrade, indicating collagen denaturation. Collagen extracted at 20℃and below 20℃,triple helix structure keep intact with a complete biological activity.4. Acid-soluble and pepsin-soluble collagens （ASC and PSC） were extracted from the skin of grass carp and partially characterized. the UV spectra of ASC and PSC from grass carp showed that the distinct absorption was obtained near 223 nm; SDS-PAGE showed that acid-soluble collagen protein contain moreβ,γand polymer. Molecular weight of acid-soluble collagen higher than it of pepsin-soluble collagen, The denaturation temperature of ASC and PSC from grass carp was 33.8℃and 34.5℃, respectively, about 3℃lower than that of the porcine skin collagen（37℃）. These results suggest that grass carp skin has potential application in the functional food, healthcare, cosmetic, and pharmaceutical industries.5. Under the condition of pH 7.4, different salt concentrations fraction precipitate collagens from grass carp skin and pig skin.TypeⅢcollagen were completely precipitated at 1.8 mol/L salt concentration,at this time the majority of typeⅠcollagen dissolved in the solution, in the final were precipitated at 2.5 mol/L salt concentration. the samples purifying from varying salt concentration, analyzed by SDS-PAGE containing urea. It can make distinction between the electrophoresis mobility of a1（Ⅰ） and a1（Ⅲ） which used be the same. SDS-PAGE showed that grass carp skin samples without a1（Ⅲ）, however the pigskin contains a1（Ⅲ）. but purity of typeⅢcollagen protein is not high, only about 20%. TypeⅠcollagen reached electrophoresis purity. Conclusion: grass carp skin contains only typeⅠcollagen without proteinⅢcollagen, Pigskin main contain typeⅠcollagen protein also contain a small amount of typeⅢcollagen.更多还原