两种鱼皮胶原蛋白的比较及其降解物中多肽的识别

本研究从鳕鱼皮和罗非鱼皮中提取酶促溶性胶原蛋白(PSC),采用SDS-聚丙烯酰胺凝胶电泳、傅立叶变换红外光谱、X-射线衍射、氨基酸组成分析、旋转流变仪以及高效液相色谱-串联质谱等技术,分析了鳕鱼皮PSC(G-PSC)和罗非鱼皮PSC(O-PSC)的分子结构、热稳定性以及降解产物中多肽的序列。结果表明:两种来源的胶原蛋白均符合典型Ⅰ型胶原蛋白的组成,具有相似的二级结构和三级结构,但氨基酸组成存在差异,其中O-PSC的脯氨酸羟基化率(49.1%)大于G-PSC的脯氨酸羟基化率(33.3%);O-PSC的热变性温度(24.7 ℃)要高于G-PSC(13.8 ℃),两种鱼皮胶原蛋白中脯氨酸羟基化率越高,则胶原蛋白的热稳定性越强;两种胶原蛋白经相同的酶解处理后,特征性肽片段被检测识别。结论:不同来源的胶原蛋白高级结构方面具有相似性,但经相同的酶解后会生成差异性肽片段,可以利用它们进行胶原蛋白来源的鉴别,这为胶原蛋白快速分析方法的建立提供依据。     

虹鳟鱼皮胶原蛋白提取及其性质研究

采用0.5 mol/L醋酸提取以及NaCl沉淀的方法从虹鳟鱼皮中提取胶原蛋白,分析其部分理化特性。虹鳟鱼皮胶原蛋白的最大吸收波长为234 nm,通过红外光谱证实了它的3股螺旋结构。SDS-PAGE电泳结果表明,该胶原蛋白分子由两条α链及其聚合物(β链和γ链)组成。虹鳟鱼皮胶原蛋白的变性温度区间为19.2~28.7℃,相变曲线峰顶温度为45.5℃。其氨基酸组成以甘氨酸、脯氨酸、丙氨酸为主,不含胱氨酸,脯氨酸的羟基化程度为38.55%。根据凝胶电泳图谱及氨基酸组成判断:虹鳟鱼皮胶原蛋白为Ⅰ型胶原蛋白。其变性温度低于温水性鱼类,而略高于典型冷水性鱼类。     

氨基酸组成对胶原与整合素α2β1结合能力的影响

以不同物种来源的胶原作为研究对象，测定各胶原的氨基酸组成，利用酶联免疫吸附测定实验和细胞黏附实验研究各胶原样本与整合素α2β1及HT1080细胞的结合能力，探讨氨基酸组成对胶原配体-细胞受体α2β1结合能力的影响。结果表明，不同来源的胶原样本在与整合素α2β1和HT1080细胞结合能力方面均存在显著差异，其中，哺乳动物胶原的结合能力明显大于鱼类胶原。氨基酸组成对胶原-整合素α2β1及HT1080细胞结合能力的影响具有相似性，其结合能力均与羟脯氨酸、不带电荷极性氨基酸、亚氨基酸含量和羟基化率呈正相关，而与谷氨酸、甘氨酸、带电荷极性氨基酸和酸性氨基酸含量呈负相关（P<0.01）。同时对胶原与整合素α2β1高亲和力结合位点的信息氨基酸含量进行多元逐步回归分析，建立了羟脯氨酸和精氨酸含量与胶原-整合素α2β1结合能力之间的数学模型，经验证，该数学模型具有较好的预测准确性。     

云南鲷鱼皮酸溶性胶原蛋白的组成及理化特性

采用低温酸法提取云南鲷鱼皮酸溶性胶原蛋白(ASC),对ASC的氨基酸组成、亚基组成、结构特征、热稳定性以及溶解性等理化性质进行了研究,为云南鲷鱼皮的综合利用提供了理论依据。研究表明,ASC的主要氨基酸为甘氨酸、脯氨酸和丙氨酸,酪氨酸、蛋氨酸和半胱氨酸含量较低,脯氨酸的羟化率为35.47%;SDS-PAGE电泳图谱表明ASC主要由α_1、α_2以及β链组成,符合I型胶原蛋白的特征;ASC的最大紫外吸收波长为230 nm;红外光谱和X-射线图谱表明ASC分子排列规则紧凑,具有完整的三螺旋结构;差示热量扫描显示ASC的热变性温度分别为82.1和222.3℃,表明ASC的热稳定性较好;扫描电镜显示ASC分子呈三维的多片状结构,分布均匀,适合用作食品、药品和化妆品的载体和生物医学的基料;在pH值小于4或NaCl浓度低于40 g/L时,ASC具有良好的溶解性。     

胡子鲶鱼皮酸溶性胶原蛋白的理化性质研究

为提高水产加工副产品的高值化利用,本文以资源丰富且相对集中的一种淡水产品加工下脚料-胡子鲶鱼皮为原料,在4 ℃条件下,利用酸提法从鲶鱼皮中提取酸溶性胶原蛋白（ASC）,并对其进行相应的理化性质研究。通过测定羟脯氨酸含量得出了该鱼皮胶原蛋白的提取率（20.32%）,氨基酸组成分析得到含量最丰富的氨基酸是甘氨酸（32.59%）表明样品符合Ⅰ型胶原蛋白的氨基酸特点。SDS-PAGE电泳和紫外分析图谱表明样品为Ⅰ型胶原蛋白,且样品提取纯度较好。傅里叶变换红外光谱（FT-IR）表明该酸溶性胶原蛋白保留了天然的三螺旋结构。热变性温度（Td,26 ℃）和热收缩温度（Ts,53.01 ℃）的测定结果均显示,这种鲶鱼皮酸溶性胶原蛋白的热稳定性低于哺乳动物。X-射线衍射图谱显示了胶原蛋白分子的内部结构特点。     

胶原蛋白热稳定性研究进展

胶原蛋白是动物体内含量最丰富的蛋白质,独特的三螺旋结构和氨基酸组成使其具备独特的理化特性和生物学特性,被广泛应用于食品、皮革、制药和组织工程等领域。然而,由于胶原蛋白对热敏感,加热时易发生热变性,三螺旋结构被破坏,其特性随之改变,因此,了解胶原蛋白的热稳定性机理及调控措施对胶原蛋白的开发与应用极其重要。本文介绍了胶原蛋白的结构特征,归纳了近年来胶原蛋白热稳定性的研究进展和研究手段,重点综述影响胶原蛋白热稳定性的因素,探讨未来胶原蛋白热稳定性研究的重点和方向,以期为胶原蛋白热稳定性研究与应用提供参考和指导。     

云南鲷鱼骨胶原蛋白的制备及其理化性质

以云南鲷鱼骨为原料,采用低温酸法提取制备鱼骨中的酸溶性胶原蛋白（acid-soluble collagen,ASC）, 并对ASC的氨基酸组成、亚基组成、红外吸收、紫外吸收、热稳定性、X射线衍射、微观结构、多肽片段以及溶解 性进行了全面的分析。氨基酸组成表明ASC主要含甘氨酸、脯氨酸和丙氨酸,而酪氨酸、蛋氨酸和半胱氨酸含量 较低;十二烷基硫酸钠-聚丙烯酰胺凝胶电泳结果显示ASC为Ⅰ型胶原蛋白;ASC在230 nm波长处有最大紫外吸收 峰;傅里叶变换红外光谱和X射线衍射图谱表明ASC分子排列紧凑,保持了其原有的三螺旋结构;差示扫描量热分 析结果显示ASC的变性温度分别为86.5 ℃和226.2 ℃,有较好的热稳定性;扫描电子显微镜显示ASC分子分布均匀、 表面光滑呈三维立体结构;多肽片段分析结果显示鱼骨胶原的氨基酸构成主要为Gly-X-Y,符合胶原蛋白的一级结构 的特点;在pH值小于4的条件下,ASC溶解度较高,当NaCl质量分数大于4%时,ASC溶解度剧烈下降。     

鱼翅营养成分提取及定性分析

主要对鱼翅营养成分、氨基酸组成及胶原蛋白特性进行定性分析。结果表明,鱼翅中主要成分是由蛋白质组成,其中胶原蛋白含量占粗蛋白的92%以上;必需氨基酸占氨基酸总量的20.5%,不含蛋氨酸,色氨酸。非必需氨基酸含量丰富,甘氨酸含量最高,几乎占到氨基酸总含量的1/3,酪氨酸占氨基酸总量的6%。鱼翅的胶原蛋白是由三个相同的类α1链构成的拟弹性胶原蛋白,其热变性温度为67.98℃,分子量约为130kDa。     

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.     

Na~+、Ca~(2+)和pH值对鲸鲨皮胶原蛋白热变性温度的影响

通过差示扫描量热法(DSC)对不同浓度的Na+、Ca2+和pH值对鲸鲨皮I型胶原蛋白和鲸鲨皮Ⅱ型胶原蛋白热变性温度的影响进行系统研究。I型胶原蛋白的特征性紫外吸收波长位于233.05nm,Ⅱ胶原蛋白的特征性紫外吸收波长位于232.90nm和277.88nm。研究胶原蛋白的聚集动力学曲线,发现鲸鲨I型胶原蛋白和鲸鲨Ⅱ型胶原蛋白浊度随时间变化趋势均呈S型,其聚集过程均可分为初始阶段和生长阶段及趋于稳定阶段。通过高灵敏度差示量热扫描仪对鲸鲨I型胶原蛋白和鲸鲨Ⅱ型胶原蛋白的热稳定性进行表征。鲸鲨I型胶原蛋白的热变性温度为40℃,鲸鲨Ⅱ型胶原蛋白热变性温度为62℃。较低浓度的Na+、Ca2+使I型胶原蛋白和Ⅱ型胶原蛋白的热变性温度降低,这可能是由于金属离子的加入影响了胶原蛋白分子之间带电氨基酸残基的相互排斥作用,降低了胶原蛋白的热稳定性;较高浓度的Na+、Ca2+使I型胶原蛋白和Ⅱ型胶原蛋白的变性温度持续降低,这可能是由于金属离子与胶原蛋白分子竞争水分子,从而导致胶原蛋白的不稳定;继续增加Na+、Ca2+金属离子的浓度时,I型胶原蛋白和Ⅱ型胶原蛋白的变性温度由于发生盐析而增加。I型胶原蛋白在强酸强碱处理后,蛋白的吸热变...     

Thermal stability of fish myofibrils: a differential scanning calorimetric study

The variation in myofibrillar protein thermostability was compared for various fish species, using differential scanning calorimetry. The tropical fish, catfish ( Clarius gariepinus ), carp ( Cyprinus carpio ), Nile perch ( Lates niloticus ), red snapper ( Lutianus sebae ), red mullet ( Parpeneus barberinus ), sea bream ( Gymnocranius rivalatus ), and cold-water reared trout ( Salmo gairdneri ) and cod ( Gadus morhua ) were analysed. Onset temperature of myofibrillar protein denaturation occurred at up to 11°C higher for tropical species (43.5°C, catfish), than cod (32.6°C) at pH 7 and low ionic strength (I). As pH (6.0-8.0) and I (0.05-1.00) were increased, thermal denaturation temperatures of myosins from tropical, but not cold-water, species decreased. Enthalpies of myofibrillar denaturation decreased for all species with increasing pH and I. Only one thermal transition was detected for myosin at pH 6 and low I, increasing to three as pH and I were increased. Changes in thermal characteristics of myosin subunits over iced and frozen storage suggest more rapid deterioration in cold-water than in tropical fish. The differences in myofibrillar stability of fish from different habitat temperatures have implications for the processing and storage of tropical fish.     

Changes in myofibrillar structure of silver carp (Hypophthalmichthys molitrix) as affected by endogenous proteolysis under acidic condition

Effect of endogenous proteolysis on myofibrillar conformational changes under acidic condition was investigated by spectroscopic techniques. The result of intrinsic tryptophan fluorescence showed that tryptophan residues were exposed to polar environment by acid denaturation of protein and participation of endogenous proteolysis. Extrinsic fluorescence indicated that average surface hydrophobicity (S_hANS) decreased by reduction of pH but increased by endogenous proteolysis. Second‐derivative UV‐spectroscopy implied that the decrease of S_hANS under acidic condition may be due to burying of some aromatic amino acid residues into hydrophobic cores induced by acid‐induced aggregation, such as tyrosine, while S_hANS increased by exposure of some hydrophobic amino acids due to the effect of endogenous proteases. Raman spectra verified the changes in microenvironment of tryptophan and tyrosine residues, and showed that aliphatic amino acids were mainly exposed to polar solvent by both acid denaturation and endogenous proteolysis. In addition, new disulphide bonds were less likely to form by acid‐induced aggregation and proteolytic cleavage, while random coils increased and smaller water domains were found.     

草鱼皮胶原的体外自组装动力学研究

体外自组装是天然胶原的重要分子行为特征之一,并对胶原基产品性能产生显著影响。以草鱼皮酶溶性胶原蛋白为研究对象,重点开展胶原体外自组装动力学行为、影响因素、组装纤维的微观结构及其热稳定性能研究。浊度实验和自组装程度分析的结果表明,草鱼皮胶原蛋白具备体外自组装能力,其自组装进程受胶原质量浓度、pH值、离子强度、温度等因素的影响。在pH 7～8、胶原质量浓度3～5 mg/mL、体系温度25～30 ℃以及NaCl浓度0～200 mmol/L条件下胶原自组装进程较快、自组装程度较高;组装动力学分析的结果表明,在较高的离子强度（NaCl浓度300 mmol/L）和较低的组装温度（20 ℃）时,胶原组装进程表现为：成核、组装和平衡3 个阶段,而在较高组装温度（25～30 ℃）和较低离子强度时（NaCl浓度0～200 mmol/L）,胶原组装进程表现为：快速组装段、低速组装段和平衡段;胶原纤维形态学观察结果表明,草鱼皮胶原组装纤维具有典型的D周期特征但D周期长度值（64.6 nm）小于哺乳动物胶原纤维（约67 nm）;示差扫描量热法（differential scanning calorimetry,DSC）分析结果表明,经纤维重组后,草鱼皮胶原蛋白的热稳定性得到明显提升。     

Biochemical properties of skin collagens isolated from black carp (Mylopharyngodon piceus)

Acid-soluble collagen (ASC) and pepsin-soluble collagen (PSC) were extracted with the yield of 15.5 and 26.5% on the basis of dry weight from the skin of black carp (Mylopharyngodon piceus). ASC and PSC were similar in amino compositions and subunit compositions, but they were slightly different in the secondary structure. Both of ASC and PSC, either rehydrated in distilled water or in acetic acid, had a lower denaturation temperature than pig skin collagen (PPSC), but their denaturation temperature were higher than that of collagens from other freshwater fish. Intrinsic viscosity of different collagens decreased in the order of PPSC>PSC>ASC. Both of ASC and PSC showed only partial degradation in collagenase solution, but they revealed more sensitive to collagenase compared with PPSC. Black carp skin collagens did not induce a significant cytotoxic effect according to the results of in vitro cytotoxicity test.     

草鱼鱼鳞胶原蛋白体外自组装行为的研究

以草鱼鱼鳞为原料,分别提取鱼鳞中的酸溶性胶原蛋白(ASC)和胃蛋白酶溶性胶原蛋白(PSC),在对胶原蛋白分子结构表征的基础上,开展鱼鳞胶原蛋白体外自组装行为的研究,并与哺乳动物来源的猪皮胶原(PC)相比较。结果表明：制备所得的3种胶原蛋白均为典型的Ⅰ型胶原,3种胶原蛋白的分子质量和二级结构较为相似,但氨基酸组成存在一定差异。采用浊度实验的方法观察胶原蛋白的体外自组装行为,结果表明：在合适的温度和pH值条件下,3种胶原蛋白均可产生体外自组装行为,其自组装进程可划分为迟滞段、快速上升段和平稳段3个阶段,其出现时间受溶液pH值的影响;在相同条件下,PC的体外自组装程度最高,其次为鱼鳞PSC和ASC;环境温度、胶原蛋白质量浓度以及蛋白的热变性处理均会不同程度的影响胶原蛋白的体外自组装行为。     

Myosin thermal stability in fish muscle

The thermal stability of fish muscle proteins varies between species with considerable implications for storage and processing properties. Myosins were isolated from cod and snapper, cold- and tropical-water fish respectively, and their thermal melting characteristics were compared using differential scanning calorimetry over a range of pH and ionic strength conditions. At pH 6 and ionic strength 0.06 M, cod myosin exhibited a thermal melting transition 10 K lower than snapper myosin, reflecting a comparable differential in transitions attributed to myosin in intact muscle from the two species at 315 and 325 K, respectively. However, with increasing pH and ionic strength, conditions favourable to disaggregation and dissolution of myosin in vitro, the snapper myosin transition decreased to that exhibited by cod myosin, which remained essentially unaffected by the conditions. It is suggested that differences in the aggregation characteristics of fish myosins may explain in part the different thermal melting properties in vitro.     

Characterization of Fish Muscle Type I Collagen

Muscle Type I collagen of five teleosts was characterized with respect to its thermal stability and subunit composition. The muscle collagen exhibited a higher denaturation temperature, Td, in solution and a higher degree of proline hydroxylation, compared with skin Type I collagen of the respective species. Moreover, the Td values seemed to increase with the increasing upper limit of environmental temperature of fish habitats. The subunit composition also varied with fish species; an α1α2α3 heterotrimer existed in eel and common mackerel and an (α1)₂α2 heterotrimer in saury, while (α1)₂α2 and α1α2α3 heterotrimers appeared to be major and minor components, respectively, in chum salmon and carp.     

Thermal Stability and Gel-Forming Ability of Shark Muscle as Related to Ionic Strength

Thermal stability and gel-forming ability of silvertip, hammer-head and thresher shark as affected by ionic strength were investigated. Low temperature endothermic peaks (LTEP, between 30 and 44°C) were observed in differential scanning calorimetric (DSC) thermograms of the shark muscles and surimi samples. Increases in ionic strength reduced the thermal stability of surimi as shown by depression of transition temperature, denaturation enthalpies and LTEP. Although gel strength of heat-treated surimi increased with increasing NaCl concentration, the three species had different sensitivities to ionic strength. The highest NaCl-induced gel-forming ability of surimi was achieved at 0.5M salt concentration for silvertip and thresher; at 1.0M for hammer-head shark.