摘要
以斑点叉尾鮰鱼皮为原料,提取了可溶性和不溶性胶原,并以胶原的性质为基础,优化了提取明胶的工艺条件。同时,以斑点叉尾鮰鱼皮明胶为原料,采用碱性蛋白酶和胰蛋白酶水解的方法生产出具有清除自由基能力的胶原蛋白多肽。利用小鼠皮肤的光老化模型,研究了胶原蛋白多肽清除光老化皮肤中自由基的能力。利用生物化学方法从胶原蛋白多肽中分离出一条具有高度抗氧化能力的多肽,经多级质谱鉴别为九肽。
采用乙酸溶解和胃蛋白酶分解的方法首次从斑点叉尾鮰鱼皮中分离得到可溶性和不溶性胶原,并对其部分定性。研究结果表明斑点叉尾鮰鱼皮胶原含有的甘氨酸约占23%,羟脯氨酸含量在7.3～7.59%。可溶性胶原的变性温度为34.8℃,比猪皮可溶性胶原变性温度低3℃。电泳结果表明斑点叉尾鮰鱼皮胶原含有两条分子量相同的α肽链,而不同于大部分真骨鱼含有的3条分子量不相同α肽链。可溶性胶原和不溶性胶原的得率分别为25.8%与38.4%。
分别利用饱和Ca(OH)2溶液和低浓度H2SO4溶液对鱼皮进行预处理,然后利用热水抽提的方法制取明胶。以凝胶强度为主要指标,运用响应面设计优化实验条件,得到优化工艺条件:预处理鱼皮经浓度为1.6g/L Ca(OH)2浸泡,浸泡时间为67h,提取pH为5.0～5.5,提取温度为43.2℃,提取时间为5.75h;浓度为0.5mL/L的H2SO4浸泡时间为17.3h,提取pH为3.5～4.0,提取温度为54.3℃,提取时间为5.89h。
分别对酸法明胶和碱法明胶的物理和化学性质进行了研究。傅立叶红外转换扫描结果表明,两种明胶均具有一定的与胶原相似的三级结构;在最佳工艺条件下制取的明胶具有很高的凝胶强度,酸法明胶的Bloom强度为271 Bloom,碱法明胶的Bloom强度为265 Bloom;酸法明胶和碱法明胶的等电点各为7.3和4.1。碱法明胶比酸法明胶都具有很高的粘度,分别为10.52±0.5 mPa.s和8.38±0.3 mPa.s。但是明胶的流变性表明碱法明胶的凝固点和熔点要低于酸法明胶。最佳工艺下的明胶的凝胶能力均好于试剂级猪皮明胶,这表明斑点叉尾鮰鱼皮明胶可以作为猪皮明胶的潜在替代品。
利用斑点叉尾鮰鱼皮明胶水解制取了护肤性的、具有自由基清除能力的胶原蛋白多肽。结果表明,鱼皮明胶的酶水解物的自由基清除能力高于用酸、碱水解的产物;碱性蛋白酶Alcalase水解碱法明胶可以在宽泛的时间范围内产生较高的自由基清除能力。经条件优化后,碱性蛋白酶Alcalase在底物浓度为45g/L、酶与底物比为2000U/g、水解液pH为7、水解温度为60℃的条件下,胶原蛋白多肽的·OH和O2·-清除率分别为88.33%和41.1%;经过胰蛋白酶的再次酶解,胶原蛋白多肽的·OH和O2·-清除率分别达到了95.24%和53.7%。胶原蛋白多肽具有很高的体外清除自由基的能力,平均分子量小于2,000Da,溶解性良好,具有一定的保湿性。
试验表明,胶原蛋白多肽具有减轻小鼠皮肤光老化的作用。确定了小鼠皮肤在UVB照射下的光老化模型。组织切片和组织内羟脯氨酸含量及各抗氧化体系活力的测定结果表明,模型组和空白组之间存在着显著差异。小鼠皮肤外观观察与组织切片表明,外涂高浓度胶原蛋白多肽组小鼠表皮要比模型组薄,说明其对光老化造成的表皮增生有一定治疗作用;外涂胶原蛋白多肽组小鼠皮肤中羟脯氨酸含量比模型组含量高,但是比空白组低。这说明胶原蛋白多肽对光老化皮肤中羟脯氨酸的减少又缓解作用,但是不能使损伤皮肤达到原有水平。涂抹胶原蛋白多肽组小鼠皮肤组织内SOD及GSH-Px活力均比模型组高,而MDA含量均低于模型组。可以推断胶原蛋白多肽可以清除自由基,通过抑制皮肤中的活性氧簇NOS和增强抗氧化酶的作用抑制皮肤损伤。
通过体外抗氧化测定系统,以·OH清除率为指标,依次以SP-Sephadex C-25离子柱、SP-Sephadex G-25体积排阻凝胶柱、中压反向液相纯化系统、高压液相分离纯化具有自由基清除能力的胶原蛋白多肽,得到序列为H- Gly - Pro - Leu - Ser - Gly - Pro - Gly -Hyp- Arg–OH(855.44Da)的9肽。
Collagen and gelatins were extracted from channel catfish (Ictalurus punctatu) skin, and properties of collagen and gelatins were studied. Furthermore, collagenous peptides were derived from channel catfish skin gelatin digested using Alcalse and trypsin. Those collagenous peptides showed protective effects on mice skin by removing free radicals for its strong antioxidant. A peptide with 9 amino acids was purified and characterized.
Acid-soluble and pepsin-soluble collagens (ASC and PSC) were extracted from the skin of channel catfish (Ictalurus Punctaus) and partially characterized. The collagen obtained in the experiment contained more than 23% glycine, 7.3% hydroxyproline. The denaturation temperature of acid-soluble collagen was 34°C, about 5°C lower than that of the porcine skin collagen. SDS-PAGE showed that the collagens were composed of two distinct alpha chains. This is similar to the porcine type I collagen. The contents of the skin ASC and PSC, on a dry weight basis, were 25.8% and 38.4%, respectively. These results suggest that channel catfish skin has protential as a supplement to the sources of vertebrate collagens.
The gelatin extraction from channel catfish (Ictalurus punctatus) skin using alkali and acid treatment methods was optimized. Response surface methodology was adopted in gelatin processing for extracting optimization. Concentration of alkali, treatment time, extracting temperature and extracting time were adopted as independent variables. The main dependent variable was gel strength, the secondary dependent variable were yield and viscosity. Optimal conditions of alkali pre-treatment process were the concentration of Ca(OH)2 =1.6g/L,treatment time=67h, extracting pH=5.0-5.5, extracting temperature=43.2°C and extracting time=5.75h. Optimal conditions of acid pre-treatment process were the concentration of H2SO4=0.5mL/L, treatment time=17.3h, extracting pH=3.5-4.0, extracting temperature= 54.3°C, extracting time= 5.89h.
Physical and chemical properties of gelatin extracted in the optimal conditions were determined. The Bloom strength of A-type and B-type gelatin were 271 Bloom and 265 Bloom, respectively. A-type and B-type gelatin have different isoionic points (pI) which were 7.3 and 4.1 respectively. Gelatin of type B exhibited higher viscosity (10.52±0.5 mPa.s) than gelatin of type A (8.38±0.3 mPa.s), but lower gelling and melting points than gelatin of type A. The gelatin from channel catfish skin also has a higher gelling ability and a lower thermo-stability than porcine skin gelatin. So we can assume that the skin of channel catfish can be a possible material for gelatin industry as the replacement of porcine and bovine skin.
Channel catfish skin gelatin was hydrolyzed to obtain antioxidant peptides and assessed for scavenging effects on·OH and O2·-. The results showed that scavenging effect on·OH and O2·- of hydrolysates digested by Alcalse was higher than those of hydrolysates produced by acid or alkali. The experiment deals with the effects of hydrolysis temperature, optimum pH, substrate concentration, enzyme amount and hydrolysis duration carried out with Alcalse on the hydrolysis proportion of the gelatin. The suitable hydrolysis conditions of this enzyme are hydrolysis temperature of 60°C, optimum pH 7, substrate concentration of 45g/L, enzyme amount of 2000U/g, hydrolysis duration of 45min. Under such conditions, the scavenging effects on·OH and O2·- were 88.33% and 41.1%, respectively. A great deal of activity peptide was prepared while hydrolysate was enzymatically hydrolyzed by trypsin for 45min, and the scavenging effects on·OH and O2·- roared to 95.24% and 53.7%, respectively. Hydrolysate has good solubility and ability of moisture retention, a lower molecular weight below 2,000 Da.
Protective effects on mice skin damaged by exposing to ultraviolet B were studied. The skin of mice was exposed to UVB to establish the oxidative damage model. The thickness of epiderm was observed after the experiment, and the activities of GSH-Px, SOD, contents of hydroxyproline and MDA were detected with biochemical methods. The epiderm in UVB exposure group became thick, however, that in protective group did not became thick obviously. Activities of GSH-Px, SOD and content of hydroxyproline in UVB exposure group were less than that in protective group. On the contrary, the MDA content in UVB exposure group was higher than that in protective group. It could be concluded that collagenous peptides from channel catfish skin showed some protective effects on photoaged skin, which might mediated by inhibiting the activity of NOS, by eliminating the free radicals and by increasing the activity of antioxidative enzymes to inhibit the oxidative damages of UVB.
Peptides derived from hydrolysate of channel catfish skin gelatin were assessed for their antioxidant properties in different in vitro assay systems. One representative peptide with comparatively higher antioxidant potency was separated by the consecutive chromatographic methods including ion-exchange chromatography (through SP-Sephadex C-25), gel filtration (through SP-Sephadex G-25) and reverse-phase high performance liquid chromatography. The isolated peptide was composed of Gly - Pro - Leu - Ser - Gly - Pro - Gly -Hyp- Arg (855.44Da).
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