酶法耦合膜技术制备鱼鳞胶原蛋白低聚肽的研究
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摘要
本文以罗非鱼鱼鳞为研究对象,采用复合酶水解结合膜技术制备和冷冻干燥技术具有较高附加值高生物活性的胶原蛋白低聚肽,旨在为充分利用鱼鳞资源,提供必要的科学依据。本文的主要研究内容有罗非鱼鱼鳞的基本成分、鱼鳞的前处理、复合酶的选择及酶解条件的优化、分离膜的选择及工作参数的确定和冷冻干燥工艺条件。本文的主要研究结果如下：
1、鱼鳞的脱钙和脱脂除杂工艺。酸碱的浓度、时间和料液比对前处理的效果有显著影响。实验结果表明：用3%的盐酸溶液,以1：15的料液比,浸泡4h,最终的脱钙率可达98%以上；用8%的氢氧化钠溶液,以1：15的料液比浸3h,处理后的鱼鳞脂类的含量降至0.16%～0.22%,鱼鳞中胶原蛋白量提高至93.6%-94.1%。
2、鱼鳞胶原蛋白低聚肽的提取工艺。确定了最佳的复合酶并优化了酶解条件。最优的酶解条件是：向经过预处理的鱼鳞中按动物蛋白水解酶比中性蛋白酶为1：1.2的比例加入2%的酶,选择温度在58～60℃和pH=6.8～7.0,以1：8的料液比,酶解4h,最终,胶原蛋白低聚肽的转化率可达94%～95%,胶原蛋白低聚肽的分子量分布在300～1000Dal的比例在86.5%～88.2%之间。
3、酶解液的膜除杂浓缩的工艺。对酶解液分别进行了100nm的微滤膜、截留分子量为2500Dal的超滤膜和截留分子量为300～500Dal的纳滤膜三级膜处理,使原蛋白低聚肽的纯度有提高到96%以上,分子量分布在300～1000Dal的比例由提高到95%以上,大大的改善了肽的品质。
4、浓缩液的冷冻干燥的工艺条件。通过正交试验表明：在冷冻干燥过程中,当加入物料的厚度为12mm,加热板的温度为65℃,真空度控制在50Pa左右时,冷冻干燥的效果最好,单位时间单位面积的出水量140L×h-1×m-2,平均冷冻时间为19.5～20h。所得产品为淡黄色粉末,分子量为300～1000Dal的肽的含量达95%以上,色泽和口感良好,并有很强的生物活性和溶解性。
In this study, study on preparing higher value-added and high biological activity collagen oligopeptide by complex enzyme hydrolysis technique and membrane technology from Tilapia scale were carried out, The whole researeh was aiming at providing a scientific basis for effective utilization of fish scale.In this work, basic components of Tilapia scale, pre-treatment of scale, the selection of complex enzyme and the optimization of enzyme hydrolysis Condition, the selection and work characteristics of membrane and technology of vacuum freeze-drying were investigated.
The main research results were as follows:
1. The decalcifying and degreasing process of fish scale were studied. The concentration of acid and alkali, time of processing, and the solid-liquid ratio significantly influenced the effect of pre-treatment. The results show that:decalcification rate reached above 98% when 3% hydrochloric acid was used with 1:15 solid-liquid ratio for 4h; when 8% Sodium Hydroxide was used with 1:15 solid-liquid ratio for 3h,the fat content decreased to 0.16%-0.22%, the collagen content can increase to 93.6%～94.1%.
2. The extraction technology of scale collagen oligopeptide was investigated. The complex enzyme and condition were determined. The results showed the optimal condition of enzyme hydrolysis was using 2% animal protease and neutral protease with a proportion of 1:1.2, ratio of solid-liquid 1:8, temperature between 58 60℃,pH=6.8～7.0,extraction time 4 hours. The conversion of collagen oligopeptide reached 94%-95%, the collagen oligopeptide of content is 86.6%～88.2%.
3. The impurity removal and concentration technology of enzyme solution was researched and discussed. The enzyme solution was done by there-order-membrane technology which were 100nmMF,MWCO2500DalUF and MWCO300-500NF.the purity of collagen oligopeptide raised to96%, the collagen oligopeptide with molecular weight between 300-1000Dal content increased to above 95%, quality of oligopeptide was greatly improved.
4. Vacuum freeze-drying technology of concentrated solution was studied. The analysis of the experimental results showed that the optimum technical conditions of the freeze-drying as follow:heating temperature of shell was 65℃, the material thickness was 12mm, the hothouse pressure was 50Pa, freeze-drying rate and time were 140 L×h-1×m-2 and 19.5-20h respectively.
1、鱼鳞的脱钙和脱脂除杂工艺。酸碱的浓度、时间和料液比对前处理的效果有显著影响。实验结果表明：用3%的盐酸溶液,以1：15的料液比,浸泡4h,最终的脱钙率可达98%以上；用8%的氢氧化钠溶液,以1：15的料液比浸3h,处理后的鱼鳞脂类的含量降至0.16%～0.22%,鱼鳞中胶原蛋白量提高至93.6%-94.1%。
2、鱼鳞胶原蛋白低聚肽的提取工艺。确定了最佳的复合酶并优化了酶解条件。最优的酶解条件是：向经过预处理的鱼鳞中按动物蛋白水解酶比中性蛋白酶为1：1.2的比例加入2%的酶,选择温度在58～60℃和pH=6.8～7.0,以1：8的料液比,酶解4h,最终,胶原蛋白低聚肽的转化率可达94%～95%,胶原蛋白低聚肽的分子量分布在300～1000Dal的比例在86.5%～88.2%之间。
3、酶解液的膜除杂浓缩的工艺。对酶解液分别进行了100nm的微滤膜、截留分子量为2500Dal的超滤膜和截留分子量为300～500Dal的纳滤膜三级膜处理,使原蛋白低聚肽的纯度有提高到96%以上,分子量分布在300～1000Dal的比例由提高到95%以上,大大的改善了肽的品质。
4、浓缩液的冷冻干燥的工艺条件。通过正交试验表明：在冷冻干燥过程中,当加入物料的厚度为12mm,加热板的温度为65℃,真空度控制在50Pa左右时,冷冻干燥的效果最好,单位时间单位面积的出水量140L×h-1×m-2,平均冷冻时间为19.5～20h。所得产品为淡黄色粉末,分子量为300～1000Dal的肽的含量达95%以上,色泽和口感良好,并有很强的生物活性和溶解性。
In this study, study on preparing higher value-added and high biological activity collagen oligopeptide by complex enzyme hydrolysis technique and membrane technology from Tilapia scale were carried out, The whole researeh was aiming at providing a scientific basis for effective utilization of fish scale.In this work, basic components of Tilapia scale, pre-treatment of scale, the selection of complex enzyme and the optimization of enzyme hydrolysis Condition, the selection and work characteristics of membrane and technology of vacuum freeze-drying were investigated.
The main research results were as follows:
1. The decalcifying and degreasing process of fish scale were studied. The concentration of acid and alkali, time of processing, and the solid-liquid ratio significantly influenced the effect of pre-treatment. The results show that:decalcification rate reached above 98% when 3% hydrochloric acid was used with 1:15 solid-liquid ratio for 4h; when 8% Sodium Hydroxide was used with 1:15 solid-liquid ratio for 3h,the fat content decreased to 0.16%-0.22%, the collagen content can increase to 93.6%～94.1%.
2. The extraction technology of scale collagen oligopeptide was investigated. The complex enzyme and condition were determined. The results showed the optimal condition of enzyme hydrolysis was using 2% animal protease and neutral protease with a proportion of 1:1.2, ratio of solid-liquid 1:8, temperature between 58 60℃,pH=6.8～7.0,extraction time 4 hours. The conversion of collagen oligopeptide reached 94%-95%, the collagen oligopeptide of content is 86.6%～88.2%.
3. The impurity removal and concentration technology of enzyme solution was researched and discussed. The enzyme solution was done by there-order-membrane technology which were 100nmMF,MWCO2500DalUF and MWCO300-500NF.the purity of collagen oligopeptide raised to96%, the collagen oligopeptide with molecular weight between 300-1000Dal content increased to above 95%, quality of oligopeptide was greatly improved.
4. Vacuum freeze-drying technology of concentrated solution was studied. The analysis of the experimental results showed that the optimum technical conditions of the freeze-drying as follow:heating temperature of shell was 65℃, the material thickness was 12mm, the hothouse pressure was 50Pa, freeze-drying rate and time were 140 L×h-1×m-2 and 19.5-20h respectively.
引文
[1]http://www.cnfm.gov.cn/
[2]http://www.fish.cn/.
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[2]http://www.fish.cn/.
[3]李少华.我国水产品加工行业发展现状分析[J].企业技术开发,2009,28(2)：35-37
[4]马光霞.中国水产品市场形势分析与展望[J].农业展望,2009,9：15-19
[5]孙晓莲.低值水产品及加工废弃物的综合利用[J].食品研究与开发,2009,30(3)：158-160
[6]李兵兵.浅谈淡水水产品废弃物的加工利用[J].渔业致富指南,2009,6：12-14
[7]Kittiphattanabawon, Benjakul, Visessanguan,Nagai, Tanaka. Characterisation of acid-soluble collagen from skin and bone of bigeye snapper (Priacanthus tayenus) [J]. Food Chemistry,2005, 89(3):363-372
[8]王文彬.鱼类废弃物的加工与综合利用[J].渔业致富指南,2006,19,47-48
[9]Toshiyuki koma, Hisatoshi Kobayashi, Junzo Tanaka, Dominic Walsh, Stephen Mannc. Microstructure, mechanical, and biomimetic properties of fish cales from Pagrus major[J]. Journal of Structural Biology,2003,142:327-333
[10]段蕊,张俊杰,杜修桥.鱼鳞组成性质及其加利用的研究进展[J].食品与药品,2006,22(5)：128-132
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