Isolation and Characterization of Acid-soluble Collagen and Pepsin-soluble Collagen from the Skin of Hybrid Sturgeon

By using the wastes fish skin of sturgeon processed as a raw material, a macromolecule biomaterial of collagen was extracted. Acid-soluble collagen(ASC) and pepsin-soluble collagen(PSC) were successfully isolated from the skin of hybrid sturgeon with two extraction methods. The yields of ASC and PSC based on the wet weight of skin were 5.73 ± 0.11% and 10.26 ± 0.39%, respectively. The denaturation and melting points of ASC(26.83 ℃ and 110.49 ℃) and PSC(26.54 ℃ and 102.99 ℃) were assessed by Circular dichroism(CD) and Differential scanning calorimetry(DSC). ASC and PSC appeared to be dense sheet-like film linked by random-coiled filaments under scanning electron microscopy(SEM). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) and Fourier transform infrared spectroscopy(FTIR) confirmed that both the ASC and PSC were Type I collagen and maintained a complete triple helix structure. These results indicated that both ASC and PSC possessed good biological activity and could be widely used in medical biomaterials and other fields.

Characterization of Turbot(Scophthalmus maximus) Skin and the Extracted Acid-Soluble Collagen

The biochemical composition of the turbot skin was investigated. The moisture level of the skin was found to be 51.4%. Based on dry matter content, there were relatively high protein(82.1%) and lipid(13.1%) concentrations in the turbot skin. Mineral element analysis revealed that the turbot skin had high Ca content(2069.0 mg kg^-1), and the concentrations of toxic heavy metals Hg and Pb were less than 0.005 mg kg^-1, which indicates that the turbot skin is a safe resource for collagen production. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) analysis showed that acid-soluble collagen(ASC) extracted from the turbot skin was type-I collagen. The imino acid content of the ASC was 241.6 per 1000 amino acids, which suggests a relatively high denaturation temperature. The Fourier transform infrared(FTIR) spectra of ASC reflected a highly stable structure, and the measured denaturation temperature of ASC was 29.5℃, which is higher than those from many temperate fishes. ASC was the most soluble at pH 4.0, and when the pH values were below or above 4.0, the solubility decreased rapidly. The ASC exhibited a relatively high solubility when NaCl concentration was lower than 2%. These results show that turbo skin can be employed as a source for producing collagen with high quality.

Preparation and Characterisation of Collagen from Freshwater Fish Scales

Acid-soluble collagen (ASC) and pepsin-solubilized collagen (PSC) were prepared from the waste freshwater carp fish scales. The results of SDS-PAGE showed that purified collagens were composed of at least two different chains which were in accordance with the type I collagen with α chain composition of (α1)2α2. Compared with the carp fish ordinary muscle type I collagen , porcine dermis type I collagen and other seawater fish collagens, freshwater carp fish scales collagen contained relative high half-cystine (Cys-s), but lower denaturation temperature(Td) than the porcine dermis type I collagen. These collagens had evident absorption at 230 nm by UV-Vis spectra. The spectrum X-ray diffraction showed that the collagen remained single-helix and tri-helix configuration with the minimum values of the repeat spacings (d) of about 4.48 ? and 11.87 ?. Therefore, to make more effective use of limited-resources, carp fish scales can be a potential resource for the extraction of type I collagen or gelatin.

鲤鱼鳞酸溶性胶原蛋白提取的研究

研究了以酸法提取鲤鱼鳞胶原蛋白过程中温度、时间、乙酸浓度的影响,并以SAS对影响因素进行了回归分析,得到了鲤鱼鳞胶原蛋白提取的最佳条件,温度18℃、提取时间38 h、乙酸浓度为1.3 m ol/L,在提取之前,采用Na2CO3处理对胶原蛋白提取有较大的提高。为淡水鱼综合加工利用提供理论依据。

绿鳍马面鲀(Navodon septentrionalis)鱼头酸溶性与酶溶性胶原蛋白的分离纯化及理化性质研究

利用酸溶法和酶溶法制备马面鲀鱼头酸溶性胶原蛋白(ASC)和酶溶性胶原蛋白(PSC),并对所制备的ASC和PSC的氨基酸组成、SDS-PAGE、红外光谱、黏度和热变性温度,以及溶解度等性质进行分析。结果表明:马面鲀鱼头中ASC和PSC的提取率分别为0.94%±0.07%和3.91%±0.14%。Gly(265.0和251.8残基/1000残基)、Ala(111.0和107.6残基/1000残基)和Pro(87.0和83.1残基/1000残基)含量较高,而His和Tyr的含量较少,且未检测到Cys。ASC和PSC中亚氨基酸(Pro和Hyp)含量为169.3和160.6残基/1000残基。氨基酸组成分析、SDS-PAGE和红外光谱(FTIR)证实ASC和PSC均属于I型胶原蛋白,热变性温度分别为17.6°C和16.5°C。在酸性p H值范围内,ASC和PSC的溶解度较高;当Na Cl溶液浓度高于2%,ASC和PSC的溶解度会显著降低。综上,相对于哺乳动物类胶原蛋白,马面鲀鱼头ASC和PSC的亚氨基酸含量和热变性温度较低,结构稳定性差,易于降解,可作为胶原蛋白肽的制备原料进行开发。

响应面法优化鲫鱼鱼鳞酸溶性胶原蛋白提取工艺

在单因素实验基础上,采用响应面法对影响酸溶性胶原蛋白(acid-soluble collagen,ASC)提取效果的3个因素即乙酸浓度、液料比和提取时间进行了优化,建立并分析了各因素与ASC得率关系的数学模型。结果表明:ASC最佳提取工艺条件为:乙酸浓度0.41mol/L,液料比12mL/g,提取时间54.81h,在此工艺条件下,ASC最高得率(以干基计)理论值为20.97%,而实测值可达20.68%,与理论预测值相比,相对误差为1.38%。ASC的相对溶解度在pH4时最大,当NaCl浓度低于3%(mg/mL)时,ASC的相对溶解度基本不变,当NaCl浓度大于3%(mg/mL)时,相对溶解度迅速降低,但都在40%以上。

绿鳍马面鲀(Navodon septentrionalis)鱼皮胶原蛋白的分离纯化及理化性质研究

利用酸溶法和酶溶法分离纯化绿鳍马面鲀(Navodon septentrionalis)鱼皮酸溶性胶原蛋白(ASC)和酶溶性胶原蛋白(PSC),并对所得的ASC和PSC的氨基酸组成、亚基组成、红外光谱(FTIR)、黏度和热变性温度,以及溶解度等性质进行系统分析。研究结果表明:马面鲀鱼皮酸溶性胶原蛋白(ASC)和酶溶性胶原蛋白(PSC)的得率分别为0.87%±0.15%和9.52%±0.41%(按鱼皮干重计算)。ASC和PSC中含有的主要氨基酸为甘氨酸(Gly),含量分别为323.3残基/1000残基和321.7残基/1000残基;二者中所含的亚氨基酸含量分别为191.1/1000残基和183.4/1000残基。氨基酸组成分析、SDS-PAGE和FTIR证实马面鲀鱼皮酸溶性胶原蛋白(ASC)为I型胶原蛋白,且其SDS-PAGE图中的亚基组成(α1-肽链和α2-肽链)、氨基酸序列,以及胶原蛋白构型与PSC明显不同。ASC和PSC的热变性温度(Td)分别为21.5℃和18.9℃,显著低于哺乳动物来源的胶原蛋白。ASC和PSC的最大溶解度出现在p H 2—3时,当Na Cl的浓度低于2%时,二者的溶解度就开始急剧下降。此外,冻干的胶原蛋白显示出疏松多孔的超微结构。综上,相对于哺乳动物类胶原蛋白,马面鲀鱼皮ASC和PSC的亚氨基酸含量和热变性温度较低,结构稳定性差,易于降解,可作为胶原蛋白肽的制备原料进行开发利用。

响应面法优化狭鳕鱼鱼皮酸溶性胶原蛋白质的提取工艺

【目的】优化狭鳕鱼(Theragra chalcogramma)鱼皮中酸溶性胶原蛋白质的提取工艺,为实际生产提供工艺数据和理论依据。【方法】在乙酸浓度、提取时间和料液比的单因素实验基础上,以酸溶性胶原蛋白质提取率为响应值,采用Box-Behnken设计实验,建立提取率的二次多项式回归模型,考察3个因素对提取率的影响。【结果】对酸溶性胶原蛋白质提取率的影响为料液比>提取时间>乙酸浓度;交互项中"料液比-提取时间"项对提取率影响极其显著,"料液比-乙酸浓度"项对提取率影响显著,其他交互项对提取率影响不显著;通过实验数据结合回归模型进行数学分析,得到提取狭鳕鱼鱼皮酸溶性胶原蛋白质的最佳工艺参数:乙酸浓度0.5mol/L、提取时间40min、料液比1∶9。在优化参数条件下,酸溶性胶原蛋白质提取率的理论预测值为20.53%,实测值为20.10%;试验提取得到的胶原蛋白电泳图谱较为清晰,无杂条带出现。【结论】该回归模型合理可行,提取的产物基本保持了胶原蛋白质的原有结构。

Characterization of acid-and pepsin-soluble collagens from spines and skulls of skipjack tuna(Katsuwonus pelamis)

Acid-soluble collagen(ASC) and pepsin-soluble collagen(PSC) from the spine(ASC-SP and PSC-SP) and skull(ASC-SK and PSC-SK) of the skipjack tuna, Katsuwonus pelamis, were successfully isolated and characterized. The yields of ASC-SP, PSC-SP, ASC-SK and PSC-SK were(2.47 ± 0.39)%,(5.62 ± 0.82)%,(3.57 ± 0.40)%, and(6.71 ± 0.81)%, respectively, on the basis of dry weight. The four collagens contained Gly(330.2-339.1 residues/1 000 residues) as the major amino acid, and their imino acid contents were between 168.8 and 178.2 residues/1 000 residues. Amino acid composition, SDS-PAGE, and FTIR investigations confirmed that ASC-SP and ASC-SK were mainly composed of type I collagen, and had higher contents of high-molecular weight cross-links than those of PSC-SK and PSC-SP. The FTIR investigation also certified all the collagens had triple helical structure. The denaturation temperatures of ASC-SK, PSC-SK, ASC-SP, and PSC-SP were 17.8, 16.6, 17.6, and 16.5 °C, respectively. All isolated collagens were soluble at acidic pH(1-5) and lost their solubilities when the NaCl concentration was above 2%(W/V). The isolated collagens from the spines and skulls of skipjack tuna could serve as an alternative source of collagens for further application in food, cosmetic, biomedical, and pharmaceutical industries.

动物脂肪干细胞联合双重转化生长因子释放支架修复关节软骨损伤的实验研究

目的本实验拟应用壳聚糖/Ⅱ型胶原支架联合脂肪干细胞和两种转化生长因子治疗关节软骨损伤,观察及评价脂肪干细胞和转化生长因子对于软骨损伤修复过程的影响情况和意义。方法拟选用雄性新西兰大白兔12只,建立双侧后腿膝关节软骨缺损动物模型后随机分成3组,对软骨缺损处进行干预:A组,单纯放置壳聚糖/Ⅱ型胶原支架;B组,壳聚糖/Ⅱ型胶原支架搭载脂肪干细胞;C组,TGF-β2和BMP-7复合壳聚糖/Ⅱ型胶原支架搭载脂肪干细胞。手术4、8、12周后取材对比,行HE染色、苯胺蓝染色,并对各组关节软骨进行形态学及组织学观察和评分。结果手术8、12周后,形态学和组织学评分C组明显高于A、B组,HE染色、苯胺蓝染色可见关节软骨损伤修复水平C组明显优于A、B组。结论动物脂肪干细胞联合双重转化生长因子释放支架修复关节软骨具有积极的治疗意义。