Isolation and Characterization of Pepsin-Solubilized Collagen from the Skin of Black Carp (<i>Mylopharyngdon piceus</i>)

To make more effective use of underutilized resources, pepsin-solubilized collagen (PSC) was successfully extracted from the skin of black carp (Mylopharyngdon piceus) with a yield of 45.7% based on dry weight. The PSC comprising two identical α1-chains and one α2-chain with no disulfide bond was characterized as type I, and it contained 195 imino acid residues vs. 1000 amino acid residues. The collagen showed an absorption edge around 218 nm, which was lower than the maximum absorption wavelength of other PSC. The denaturation temperature of PSC was 25.6°C, which was lower than that of porcine collagen by approximately 11°C. The isoelectric point (pI) was estimated to be 8.23, and the collagen was soluble at an acidic pH as well as below 40 g/L NaCl. It is thought that the high yield and stability of PSC from the skin of black carp warrant its application as a new source of collagen for industrial purposes.

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.

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.

罗非鱼鳞胶原蛋白的提取及其酶解产物的抗氧化性

以罗非鱼鳞为原料,提取酸溶胶原蛋白(acid-soluble collagen,ASC)和酶促酸溶胶原蛋白(pepsin-soluble collagen,PSC),并对PSC的理化性质及其酶解产物抗氧化性进行探讨。实验结果表明:0.1 mol/L HCl提取ASC和PSC的得率分别为3.52%和14.88%;PSC氨基酸组成中,每1000个氨基酸残基甘氨酸为351个,亚氨基酸——脯氨酸和羟脯氨酸为180;PSC强吸收峰在205 nm处,磷酸盐缓冲液检测PSC的性质,与I型胶原蛋白相符。PSC经胃蛋白酶水解3 h,过氧化抑制率达到82.8%,抗氧化效果略优于等量的Vc。

鲤鱼鱼皮和鱼骨酶溶性胶原蛋白性质异同的分析

本研究对以鲤鱼鱼皮和鱼骨为原料得到的两种酶溶性胶原蛋白(PSC)的性质进行比较分析。结果表明,两种胶原蛋白中脯氨酸和羟脯氨酸含量均低于牛皮酸溶性胶原蛋白;黏度法测定鱼皮PSC的变性温度为28.1℃,鱼骨PSC的变性温度为30.0℃;DSC分析的结果也说明鱼骨PSC的热稳定性高于鱼皮PSC;电泳结果表明,鱼皮PSC和鱼骨PSC的肽链组成和多肽链的分子量相近,二者具有很大的相似性。

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。

鮟鱇鱼皮胶原蛋白酶提取最优工艺研究与结构表征

为了研究提取鮟鱇鱼皮胶原蛋白的最优工艺,以鮟鱇鱼皮为研究对象,通过选取液料比、加酶量和酶解时间3个变量进行单因素试验,再根据Box-Behnken中心组合试验设计原理,在单因素基础上采用三因素三水平响应面分析法(RSM),制备胃蛋白酶酶促溶性胶原蛋白(PSC)并对其进行理化性质分析。结果表明:鮟鱇鱼皮胶原蛋白最优提取工艺条件是4℃、液料比18:1、加酶量3%、提取时间48h,此条件下鮟鱇鱼皮胶原蛋白提取率为2.11%;氨基酸分析得鱼皮中Arg和Tyr含量分别为16%和18%;差示扫描量热仪(DSC)测定PSC的热收缩温度为48.9℃;SDS-PAGE电泳显示胶原蛋白基本保留了三股螺旋结构;电子显微镜(SEM)扫描显示PSC呈多孔网状结构。

鲍鱼腹足胶原蛋白的提取及性质研究

测定了鲍鱼腹足的基本组成成分,对皱纹盘鲍酶促溶性胶原蛋白(PSC)的提取及相关性质进行了研究。结果表明,用酶法提取的鲍鱼腹足PSC得率为8.72%(以干基计)。圆二色谱分析表明,鲍鱼腹足PSC溶液在221和199nm分别有一正、负峰,是具有典型三螺旋结构的胶原蛋白特征圆二色谱峰型。鲍鱼腹足PSC等电点为pH 5.17;质量浓度0.3 mg/mL、pH 3.0～3.5的PSC溶液起泡性达110%,泡沫稳定性较差,乳化性好,为100%;但乳化稳定性一般,放置5h后,仅保持在50%左右。鲍鱼腹足PSC具有较高的Fe2+螯合活性,其IC50值为0.348mg/mL,当质量浓度为1.2mg/mL时Fe2+螯合能力可达91.62%。

紫蛇尾胶原蛋白提取工艺的研究

以紫蛇尾Ophiopholis mirabilis为原料检测其基本成分,并用盐酸对紫蛇尾进行脱钙处理,通过L9(34)正交试验对其胶原蛋白的提取工艺进行了优化。结果表明:紫蛇尾含水分51.80%,灰分42.91%,蛋白质1.68%,脂肪0.29%,总糖0.03%;酶促溶性胶原蛋白(PSC)提取工艺的优化条件为脱钙后的紫蛇尾干粉(g)与提取液(mL)的固液比为1∶15,胃蛋白酶加酶量3%,17℃下提取4 d,PSC得率为52.65%。对纯化后的酸促溶性胶原蛋白(ASC)和PSC进行紫外扫描分析,结果显示,ASC、PSC的最大吸收波长均在235 nm左右。对PSC的氨基酸组成分析表明,脯氨酸和羟脯氨酸的含量分别为10.1%和2.13%,是较典型的Ⅰ型胶原蛋白。

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。

Steatosis recovery after treatment with a balanced sunflower or olive oil-based diet: Involvement of perisinusoidal stellate cells

AIM： To analyze the relationship between perisinusoidal stellate cell （PSC） activation and the dietary fat quantity and composition in the treatment of hepatic steatosis. METHODS： Using an experimental rat model of steatosis based on the intake of a hyperlipidic diet （14% fat as olive oil or sunflower oil, HL-O and HL-S, respectively）, we analyzed the liver＇s capability of recovery after the treatment with a normal-lipidic diet （5% fat as olive oil or sunflower oil, NL-O and NL-S, respectively） by immunoo/tochemical and Western blot analysis of glial fibrillary acidic protein （GFAP） expression in PSCs, collagen quantification and serum aminotransferase determination. RESULTS： The fatty infiltration in the steatotic livers decreased after the treatment with both NL diets, indicating liver recovery. This decrease was accompanied with a lower collagen deposition and aminotransferase level as well as changes in the PSC population that increased the GFAP expression. The above-mentioned effects were more pronounced in animals fed on NL-O based diet. CONCLUSION： Treatment with a balanced diet enriched in olive oil contributes to the liver recovery from a steatotic process. The PSC phenotype is a marker of this hepatic-recovery model.

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.

动态压应力对生长板软骨前体干细胞PTHrP及细胞外基质mRNA表达的影响

目的 研究动态压应力对体外培养的大鼠生长板软骨前体干细胞甲状旁腺激素相关肽（PTHrP）以及软骨特性细胞外基质mRNA表达的影响.方法 免疫磁珠分选并体外培养大鼠软骨前体干细胞,用自行设计制作的可控细胞压力加载装置对细胞进行不同强度（30 mmHg、60 mmHg、90mmHg）和持续时间（1 h、6 h、24 h）的压应力刺激,未刺激组为对照组,采用逆转录-聚合酶链反应（RT-PCR）技术分别检测各组细胞的PTHrP以及软骨特性细胞外基质（Ⅱ型胶原、X型胶原以及aggreean）mRNA表达并进行半定量分析.结果 生长板软骨前体干细胞在30 mmHg、60 mmHg、90 mmHg动态压力培养环境下,各时间点PTHrP、Ⅱ型胶原以及aggrecan mRNA表达水平明显增强并高于对照组（P〈0.05）;Ⅱ型胶原以及aggrecan mRNA表达水平于6h达高峰,PTHrP mRNA的表达水平随时间增加而增强,24 h仍有较高水平的表达;且压力值越大,相同时间点mRNA相对表达量越高.X型胶原表达水平较低且与对照组比较差异无统计学意义（P〉0.05）.结论 适当的动态压应力能有效促进体外培养的大鼠软骨前体干细胞Ⅱ型胶原及aggreean mRNA表达,PTHrP在此力学信号转导过程中可能起重要的作用.

海蜇伞部不溶性胶原纤维及酶促溶性胶原蛋白的热稳定性对比研究

海蜇(Rhopilema esculentum)是我国传统渔业生产的重要大型经济水母,其伞部胶原蛋白含量丰富,但胶原蛋白对热敏感,对其热稳定性进行研究对海蜇资源的开发利用至关重要。本论文从海蜇伞部提取得到不溶性胶原纤维(ICF)及酶促溶性胶原蛋白(PSC),并对其热稳定性进行对比分析。结果表明,海蜇伞部ICF与PSC的氨基酸组成及分子质量类似于I型胶原蛋白,其高级结构差异亦造成热稳定性的不同。傅里叶变换红外光谱(FT-IR)研究发现,在加热至36℃时,PSC的酰胺I带红外吸收骤然降低,结构发生明显变化,而ICF温度升高至50℃时,酰胺I带红外吸收才开始略有变化,ICF热稳定性优于PSC。二维红外分析研究发现,PSC中310-螺旋结构与ICF中β-折叠结构(低频率)对温度变化最为敏感,除自动峰外,PSC与ICF均存在若干交叉峰,说明热处理过程中各个结构之间存在着分子内和分子间的相互作用。本研究为海蜇胶原蛋白的开发利用提供了一定的理论基础。