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。     

海蜇伞部酶促溶性胶原蛋白的热变性动力学

为阐明海蜇伞部酶促溶性胶原蛋白(pepsin-solubilized collagen,PSC)的热变性反应机理,以保持完整三螺旋结构的PSC为研究对象,通过微量热仪测定不同升温速率条件下PSC的变性温度,以及采用34、35、36、37、38、39℃加热不同时间后的PSC残存率,并进行热变性动力学分析。结果表明,海蜇伞部PSC对热变化敏感,随着加热温度升高,单位时间内提高的热量增加,使海蜇伞部PSC变性速率加快,完成变性时间缩短;随着升温速率的减慢,吸热峰逐渐向低温区移动,即变性温度随升温速率的减慢而降低,但升温速率的变化对反应热并无显著影响。反应级数为0.9的回归方程能够较好地描述PSC热变性过程,在恒温34、35、36、37、38℃及39℃的条件下,PSC变性的D90值(90%蛋白变性所需时间)分别为53.76、26.11、15.75、4.89、4.26、2.55 min,Z90值(D值降低90%的温度变化)为3.69℃,表观活化能为481.90 k J/mol。研究结果可为海蜇胶原蛋白的进一步开发利用提供理论参考。     

鲟鱼皮胶原蛋白的理化特性研究

本文以俄罗斯鲟鱼皮为原料,采用酸法和酶法提取胶原蛋白,研究了酸溶性胶原蛋白(ASC)和酶促溶性胶原蛋白(PSC)的蛋白类型、结构、热变性温度和溶解度等理化性质,并与牛跟腱I型胶原蛋白(BATC)进行比较。SDS-PAGE电泳图显示ASC和PSC均包含了两条α链(α1链和α2链),为I型胶原蛋白;傅立叶红外光谱图表明提取的2种胶原蛋白均保存了完整的三螺旋结构,但ASC的有序度相对较高;ASC和PSC的热变性温度分别为32.48℃和32.68℃,低于BATC;在酸性pH条件下(pH 1~4),ASC和PSC溶解度较高,当Na Cl浓度大于2%时,PSC的溶解度较高;扫描电镜显示2种胶原蛋白均为网状结构,ASC的孔径较均匀,且孔径较小。上述结果表明提取方法不同,导致2种胶原蛋白的理化性质具有一定的差异,但都具有较好的热稳定性、溶解性,能形成较好的网状结构,有潜力的作为胶原蛋白的替代来源。     

大鲵骨酸溶性和胃蛋白酶溶性胶原蛋白的提取与表征

大鲵为一种珍贵的两栖动物,营养丰富。为了充分且有效利用其加工副产物,分离并表征了来自大鲵骨的酸溶性胶原蛋白 (ASC) 和胃蛋白酶溶解性胶原蛋白 (PSC)。大鲵骨ASC和PSC的产率分别为10.71%和13.11%。通过傅里叶红外光谱揭示胶原蛋白稳定的三螺旋结构。SDS-PAGE电泳图显示两个样品均为I型胶原蛋白,同时具有高水平的亚氨基酸(179~187/1 000个残基)。样品在pH 6时溶解度最高。热变性温度(45.78~46.51 ℃)显著高于海洋和淡水鱼物种。在SEM下,胶原蛋白显示出致密的片状和不规则的孔洞状。这些结果表明大鲵骨胶原蛋白特性与哺乳动物胶原蛋白相似,具有成为其替代品的潜力,为其后续的研究和开发利用提供了基础数据。     

鮟鱇鱼皮中胶原蛋白的提取及性质研究

采用0.5 mol/L醋酸提取,结合NaCI沉淀法从鮟鱇鱼皮中提取酸溶性胶原蛋白(ASC).并对鮟鱇鱼皮胶原蛋白与Sigma公司的牛跟腱I型胶原蛋白(BATC)进行比较,探讨二者在热变性温度、溶解度、黏度及氨基酸组成等方面的差异.SDS-PAGE结果显示ASC有2条α链(α1和α2链)、β链和γ链,表明该胶原为典型的I型胶原蛋白.DSC分析显示两种胶原的热变性温度分别为22.09℃和38.13℃.氨基酸组成分析结果是亚氨基酸含量ASC低于BATC,分别为14.69%和21.59%;甘氨酸含量相近,分别占所有氨基酸总数的40.05%和38.97%.溶解度结果显示ASC和BATC分别在pH 3和pH 4时达到最大值,在pH 7时两者都呈现最小溶解度;NaCl含量对两者溶解度的影响相同,当其含量低于2%时对溶解度的影响不明显,而随着NaCl舍量的增加,溶解度迅速下降.     

不同方法提取红鳍笛鲷鱼鳞胶原蛋白的结构和理化特性研究

为探明不同提取方法对红鳍笛鲷(Lutjanus erythropterus)鱼鳞胶原蛋白理化特性的影响,以红鳍笛鲷为原料,分别提取酸溶性胶原蛋白(acid soluble collagen, ASC)、酶溶性胶原蛋白(pepsin soluble collagen, PSC)及热水溶性胶原蛋白(hot water soluble collagen, HSC),对比分析3种胶原蛋白提取率、得率及纯度的差异,并测定其氨基酸组成、分子质量、二级结构、Zeta电位、溶解性以及微观结构等理化特性。结果表明,热水法提取率最高,为45.25%,其次为胃蛋白酶法;HSC、ASC、PSC的亚氨酸含量分别为18.28%、17.54%和18.30%。HSC、ASC和PSC均为I型胶原蛋白,包含两条α链(α1和α2),ASC比PSC的β链含量高,HSC、PSC的γ带消失;ASC、PSC具有相似的红外光谱,且保留了完整的三螺旋结构,HSC的三螺旋结构被破坏;HSC、ASC、PSC的净电荷零点为5～6;ASC、PSC在pH 5～11中溶解度较低;扫描电镜显示,ASC呈无序纤维状结构,PSC呈致密多孔网状结构,HS...     

草鱼皮酸溶性和酶溶性胶原蛋白的提取及性质

本文研究了从草鱼皮中提取酸溶性胶原蛋白(ASC)和酶溶性胶原蛋白(PSC)及其部分性质。草鱼皮中ASC和PSC的提取得率以干基计分别为8.0%和18.6%;对草鱼皮ASC和PSC的紫外光谱分析,最大吸收峰都接近223nm;SDS-PAGE电泳图谱显示草鱼皮胶原蛋白是由两条不同的α链组成,分子量都在100kDa以上,与猪皮I型胶原蛋白相似;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。结果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。     

鲽鱼骨胶原蛋白的结构及流变学特性

采用酸法和酶法从鲽鱼骨中提取制备得到酸溶性胶原蛋白(ASC)和酶溶性胶原蛋白(PSC),对ASC与PSC进行紫外吸收(UV)、红外吸收(FTIR)、亚基组成(SDS-PAGE)、热稳定性(DSC)、微观结构(SEM)以及流变学特性(DHR)分析。UV显示:ASC与PSC分别在227 nm与226 nm处有最大紫外吸收峰。FTIR图谱表明:ASC与PSC保持了三螺旋结构。SDS-PAGE电泳图表明:ASC与PSC是Ⅰ型胶原蛋白。DSC测定ASC与PSC的热变性温度分别为52.24℃与49.65℃。SEM显示:ASC与PSC呈现交织状纤维网状结构。DHR表明:1%的ASC溶液或PSC溶液在振荡频率0.1~10 Hz范围主要表现为弹性且具有较高的凝胶稳定性。     

草鱼鱼鳞酶溶性胶原蛋白粘度特性及变性温度研究

采用旋转流变仪系统考察了浓度、pH、剪切速率、NaCl、CaCl2、丙三醇、乙醇和保温时间对草鱼鱼鳞酶溶性胶原蛋白(PSC)粘度的影响。结果显示,PSC溶液粘度随浓度增大呈指数增加;pH 3时粘度最大,pH 4和pH6~9时粘度下降,pH 10时粘度又陡然回升;粘度随剪切速率的增大呈对数下降;NaCl和CaCl2的添加都会使PSC溶液粘度下降;丙三醇和乙醇添加量在10%以内,其添加浓度与粘度成正相关;保温时间长短对PSC溶液的粘度影响不大。通过粘度变化考察了不同处理下的变性温度,结果表明,随温度升高,PSC乙酸溶液粘度降低,在28.0℃左右粘度急剧下降,胶原蛋白变性,这与乌氏粘度计测定结果(胶原蛋白乙酸溶液的变性温度为32.0℃)存在较大差异。添加4%丙三醇或4%乙醇对PSC溶液的变性温度基本没有影响,而添加1.5%NaCl或2%CaCl2时,都使变性温度下降到22.0℃左右;PSC水溶液粘度明显大于其柠檬酸和乙酸溶液,且变性温度(32.0℃)高于其柠檬酸(26.0℃)和乙酸(28.0℃)溶液。     

鲤鱼鱼皮和鱼骨酶溶性胶原蛋白的性质比较

以鲤鱼鱼皮和鱼骨为原料提取得到酶溶性胶原蛋白(PSC),对鱼皮PSC和鱼骨PSC的性质进行比较。电泳结果表明,鱼皮PSC和鱼骨PSC都属于Ⅰ型胶原;2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;粘度测定的结果显示,鱼皮PSC的变性温度为28.1℃,鱼骨PSC的变性温度为30.0℃;差热分析的结果说明,鱼骨PSC的热稳定性高于鱼皮PSC,这与氨基酸分析和粘度分析的结果相一致;傅立叶红外光谱分析结果显示,鱼皮PSC和鱼骨PSC结构具有很大的相似性。     

Isolation and characterisation of collagen extracted from the skin of striped catfish (Pangasianodon hypophthalmus)

Acid soluble collagen (ASC) and pepsin soluble collagen (PSC) from the skin of striped catfish (Pangasianodon hypophthalmus) were isolated and characterised. The yields of ASC and PSC were 5.1% and 7.7%, based on the wet weight of skin, respectively, with the accumulated yield of 12.8%. Both ASC and PSC comprising two different α-chains (α1 and α2) were characterised as type I and contained imino acid of 206 and 211 imino acid residues/1000 residues, respectively. Peptide maps of ASC and PSC hydrolysed by either lysyl endopeptidase or V8 protease were slightly different and totally differed from those of type I calf skin collagen, suggesting some differences in amino acid sequences and collagen structure. Fourier transform infrared (FTIR) spectra of both ASC and PSC were almost similar and pepsin hydrolysis had no marked effect on the triple-helical structure of collagen. Both ASC and PSC had the highest solubility at acidic pH. A loss in solubility was observed at a pH greater than 4 or when NaCl concentration was higher than 2% (w/v). T_max of ASC and PSC were 39.3 and 39.6 °C, respectively, and shifted to a lower temperature when rehydrated with 0.05 M acetic acid. Zeta potential studies indicated that ASC and PSC exhibited a net zero charge at pH 4.72 and 5.43, respectively. Thus, ASC and PSC were slightly different in terms of composition and structure, leading to somewhat different properties.     

酸法和酶法提取鳄鱼皮胶原蛋白及性质研究

对以鳄鱼皮为原料得到的酸溶性胶原蛋白(ASC)和酶溶性胶原蛋白(psc)的性质进行比较分析.紫外扫描结果表明所提取出的胶原蛋白在232nm波长处有显著吸收峰;SDS-PAGE结果表明ASC和PSC的肽链组成具有很大的相似性,均含有两种a肽链及其交联链(β链及γ链);溶解性分析表明鳄鱼皮胶原蛋白的等电点在pH7左右;ASC和PSC的保水性经过6h (25℃)仍然高于85％; ASC的吸油性(24mL/g)和PSC的吸油性(41 mL/g)差异较大.根据上述测定结果可知,鳄鱼皮胶原蛋白ASC和PSC组成类似,符合Ⅰ型胶原蛋白的特征,但二者具体的功能性质略有差异.     

中华鲟软骨Ⅱ型胶原蛋白的结构分析

采用胃蛋白酶辅助提取的方法提取了中华鲟鱼软骨中Ⅱ型胶原蛋白(Pepsin soluble collagens,PSCⅡ),并对其组成与结构进行了分析。结果显示,PSCⅡ在约110 kDa附近有单一的α1亚基,在200 kDa左右有一条二聚体β亚基。在229 nm出现最大紫外吸收峰,傅里叶变换红外光谱分析与圆二色谱结果均符合Ⅱ型胶原蛋白的特征。氨基酸分析表明PSCⅡ中甘氨酸的含量最高,约占总氨基酸残基数的1/3,富含丙氨酸和脯氨酸残基。热稳定分析表明,PSCⅡ热变性温度为31.5 ℃。扫描电镜显示PSCⅡ为不规则多孔状结构,可以作为良好的药物载体。     

Isolation and Characterization of Collagen from the Body Wall of Sea Cucumber Stichopus monotuberculatus

To exploit a new collagen resource from the body wall of tropical sea cucumber, pepsin‐solubilized collagen of Stichopus monotuberculatus (PSC‐Sm) was isolated and characterized with UV‐vis spectra, sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE), amino acid composition, enzyme‐digested peptide maps, Fourier transform infrared spectroscopy (FTIR), maximum transition temperature (T_m), and solubilities. The maximum absorbance of PSC‐Sm was exhibited at 218 nm in UV‐vis spectra. The triple helical structure and activity of PSC‐Sm could be indicated by FTIR. SDS‐PAGE showed that the triple helix of PSC‐Sm was formed as (α₁)₃ by 3 α₁ chain homologous with molecular weight of 137 kDa. The T_m of PSC‐Sm and calf skin collagen (CSC) were 30.2 and 35.0 ºC, respectively, which consistent with the result of FTIR that CSC contained more stable triple‐helix than PSC‐Sm. Peptide maps were different between PSC‐Sm and CSC, indicating the differences in their amino acid compositions and sequences. The maximum and minimum solubilities of PSC‐Sm were observed at pH 2.0 and 4.0, respectively. A sharp decrease in solubility appeared when NaCl concentration was between 3% and 5%. These results showed that collagen from S. monotuberculatus had the type I collagen characteristics and good thermal stability, and therefore, it could be used as an alternative resource of collagen.     

鲟鱼和草鱼鱼鳔酶溶性胶原蛋白的理化性质比较

以鲟鱼鱼鳔和草鱼鱼鳔为原料,比较分析两种鱼鳔酶溶性胶原蛋白（PSC）的理化性质。通过胶原蛋白得率、氨基酸组成、十二烷基硫酸钠-聚丙烯酰胺凝胶电泳、紫外光谱、傅里叶红外光谱、圆二光谱、微量热仪分析两种鱼鳔PSC的组成及理化性质的差异。结果表明,鲟鱼鱼鳔基本营养成分中的粗脂肪和羟脯氨酸含量均显著高于草鱼鱼鳔（P<0.05）;鲟鱼鱼鳔PSC得率80.63%显著高于草鱼鱼鳔PSC得率49.85%（P<0.05）;两种鱼鳔PSC都属于I型胶原蛋白;鲟鱼鱼鳔PSC的α链相对分子质量和二聚体β链的含量低于草鱼鱼鳔PSC;氨基酸组成中鲟鱼鱼鳔PSC中的丝氨酸、蛋氨酸、异亮氨酸、亮氨酸、组氨酸和精氨酸的含量显著高于草鱼鱼鳔PSC（P<0.05）,草鱼鱼鳔PSC中的丙氨酸、苯丙氨酸、赖氨酸、脯氨酸和羟脯氨酸的含量显著高于鲟鱼鱼鳔PSC（P<0.05）;两种鱼鳔PSC具有相似的二级结构;鲟鱼鱼鳔PSC的变性温度29.26 ℃显著低于草鱼鱼鳔PSC变性温度36.01 ℃（P<0.05）。在两种鱼鳔PSC之间,草鱼鱼鳔PSC比鲟鱼鱼鳔PSC有更好的热稳定性,但是鲟鱼鱼鳔PSC比草鱼鱼鳔PSC有更高的得率。     

Characterization of acid- and pepsin-soluble collagens from flatfish skin

Acid-soluble collagen (ASC) and pepsin-soluble collagen (PSC) from flatfish skin were characterized. The yield of PSC was 85.5%, which was higher than that of ASC at 57.3%. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) patterns showed that both the ASC and PSC were consisted of α1 and α2 chains, and β-chain. According to the results of Fourier transform infrared (FTIR) spectroscopy and hydroxylation, no difference in the helical structure between ASC and PSC was found. Thermal denaturation temperature (TDT) of flatfish skin collagen was 26.6°C for ASC and 26.7°C for PSC, which were similar to those of temperate fishes, while were lower compared to tropical fishes, such as the conger eel and Nile perch. There was no solubility difference between ASC and PSC at various pH and NaCl concentrations. Therefore, PSC will be preferable compared to ASC for commercial uses because of its higher yield.     

Structure and characteristics of acid and pepsin-solubilized collagens from the skin of cobia (Rachycentron canadum)

Acid-solubilized collagen (ASC) and pepsin-solubilized collagen (PSC) were extracted from the skin of cobia (Rachycentron canadum). The yields of ASC and PSC were 35.5% and 12.3%, respectively. Based on the protein patterns and carboxymethyl-cellulose chromatography, ASC and PSC were composed of α1α2α3 heterotrimers and were characterised as type I collagen with no disulfide bond. Their amounts of imino acids were 203 and 191 residues per 1000 residues, respectively. LC-MS/MS analysis demonstrated the high sequences similarities of ASC and PSC. Fourier transform infrared spectroscopy spectra showed that the amide I, II and III peaks of PSC were obtained at a lower wave number compared with ASC. The thermal denaturation temperatures of ASC and PSC, as measured by viscometry, were 34.62 and 33.97°C, respectively. The transition temperatures (T(max)) were 38.17 and 36.03°C, respectively, as determined by differential scanning calorimetry (DSC). Both collagens were soluble at acidic pH and below 2% (w/v) NaCl concentration.