刺参胶原蛋白的结构特征与聚集行为的耦合性研究

采用X-射线衍射、傅里叶红外光谱、扫描电镜、流变学分析以及芘荧光探针等技术分析酶促溶性刺参胶原蛋白的结构特征、流变学特性及聚集特性,探讨不同浓度、pH和离子强度对其聚集特性的影响。结果表明:酶促溶刺参胶原蛋白呈完整的三螺旋结构,其聚集结构为疏松多孔状;在不同温度下,刺参胶原蛋白溶液的弹性模量G′和黏性模量G″呈剪切频率依赖性,而在浓度不同时则没有此现象,在低浓度时,大分子均不发生缠结和聚集;通过聚集实验发现随着质量浓度的增加,胶原蛋白会出现明显的聚集行为,当pH达7.6时易发生聚集,且Na Cl的加入使其聚集性发生滞后;随着聚集时间的增加,表征其聚集程度的芘荧光强度(Ⅰ_1、Ⅰ_3及Ⅰ_1/Ⅰ_3值)降低,说明聚集程度随着时间的延长而增加;其临界聚集质量浓度CAMC为0.813 g/L。试验表明,刺参胶原蛋白的聚集行为与其在溶液中的形态及氢键有关。     

刺参体壁酶促溶性胶原蛋白的热变性

从刺参（Stichopus japonicus）体壁中提取酶促溶性胶原蛋白（pepsin-solubilized collagen，PSC），并对其热处理过程中的降解规律及结构变化进行研究，发现PSC变性温度为35.3 ℃，当加热温度为40～70 ℃时，PSC三螺旋结构解缠绕，α-螺旋结构破坏严重，但在冷却后依靠分子间氢键和二硫键的相互作用仍可形成强凝胶体系；当加热温度超过70 ℃时，PSC的α-肽链开始逐渐降解成小分子多肽，冷却后分子间交联作用较差，不易形成凝胶体系。     

草鱼皮胶原蛋白的理化性质及其自组装的影响因素分析

以草鱼皮为原料,提取酶溶性胶原蛋白和酸溶性胶原蛋白,对这2种胶原蛋白的理化性质及自组装影响因素进行了研究。研究结果表明,草鱼皮酶溶性胶原蛋白和酸溶性胶原蛋白均由3条不同的肽链β、α1和α2组成,是典型的Ⅰ型胶原蛋白。这2种胶原蛋白在波长(200~225)nm均具有较强的紫外吸收。NaCl浓度、pH和胶原蛋白浓度对这2种胶原蛋白的自组装行为均有明显影响,在NaCl浓度75 mmol/mL、pH7.2和胶原蛋白浓度1.348 mg/mL条件下这2种胶原蛋白自组装程度均达到最高。此外,研究发现添加家蚕丝素蛋白酶解产物也能提高草鱼皮酶溶性胶原蛋白和酸溶性胶原蛋白的自组装程度。     

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。     

兔皮胶原蛋白的微观结构及不同因素对其聚集特性的影响

以兔皮胶原蛋白为原料,通过紫外光谱(UV)、红外光谱(FTIR)和扫描电镜(SEM)对兔皮胶原蛋白的微观结构进行了初步研究,在该基础上进一步研究了温度、胶原蛋白浓度、pH值和离子强度对其聚集特性的影响。紫外光谱和红外光谱的峰型及对应波长均符合Ⅰ型胶原蛋白的特征,电镜扫描观察到兔皮胶原蛋白为不规则的致密片状膜,部分表面褶皱;聚集特性研究表明,胶原蛋白浓度与测定温度升高,胶原蛋白的聚集速度和聚集程度均增加;在酸性环境中,胶原蛋白聚集时间较长;pH由7增至8的过程中,胶原蛋白的聚集程度先增加后降低,在pH 7.2时,聚集程度和速度均最高;随着离子浓度的增加,聚集程度和聚集速度先增加后降低,在NaCl浓度为120mmol/L时聚集速度最快,且自组装程度最高。     

大豆球蛋白自组装聚集及凝胶特性

研究了在低pH值、低离子强度下,分别加热诱导不同浓度11S(大豆球蛋白)和7S(大豆伴球蛋白)自组装纤维化聚集体的形成。通过平均粒径和Thioflavin T(硫磺素T)荧光光谱,对自组装纤维化聚集体的性质进行表征,并对其热致凝胶的流变学,硬度和微结构特性进行考察。结果表明:在低pH值、低离子强度的诱导条件下,蛋白浓度对自组装聚集的形成起着关键作用,随着诱导浓度的增大,蛋白的纤维化聚集越明显,7S比11S更容易形成纤维化聚集。在酸性环境下,大豆球蛋白的纤维化聚集程度越高,越有利于热致凝胶网络结构的形成。在相同的预处理条件下,11S自组装凝胶硬度强于7S。扫描电镜结果显示7S自组装凝胶的网络结构较11S致密,但有序性较11S低。     

处理方法对仿刺参体壁蛋白纤维超微结构的影响

目的:探讨不同处理方法对仿刺参体壁蛋白纤维超微结构的影响。方法:测定仿刺参的基本营养成分;利用差示热量扫描仪(DSC)、傅里叶变换红外光谱(FTIR)和扫描电镜对仿刺参的热反应变化过程、红外吸收光谱和超微结构进行分析。结果:仿刺参是一种高蛋白、低脂肪,营养价值极高的滋补品。DSC测试表明仿刺参的热收缩温度为51.4℃。红外光谱显示,仿刺参体壁具有胶原蛋白的特征吸收峰。电镜扫描显示,新鲜仿刺参纤维纤细,分布均匀,结构整齐有序。酸处理的纤维变粗、无序,结构变得致密;碱处理的纤维弯曲缠绕,纤维间空隙变大,整个形态发生明显变化;盐处理的纤维变化不明显,有少量纤维吸水膨胀溶解;酶处理的纤维随着作用时间的延长,呈现溶胀、断裂、局部聚集。     

刺参体壁及罗非鱼皮酶促溶性胶原蛋白特性的对比研究

本实验对刺参体壁酶促溶性胶原蛋白(pepsin soluble collagen from Stichopus japonicus,sjPSC)和罗非鱼皮酶促溶性胶原蛋白(pepsin soluble collagen from Oreochromis niloticus,onPSC)的理化特性进行对比研究。结果表明,两种胶原蛋白的紫外与傅里叶变换红外光谱分析结果都较为相似。由十二烷基硫酸钠-聚丙烯酰胺凝胶电泳图谱可知,两种胶原蛋白均以α-和β-链为主,在非还原和还原条件下,二者的电泳图谱非常相似,这表明蛋白中没有二硫键被还原。甘氨酸是sjPSC和onPSC中主要氨基酸,其中每1 000个氨基酸残基中分别含有(344.57±2.16)、(353.00±4.84)个甘氨酸残基。相比较而言,sjPSC中谷氨酸、天冬氨酸、丝氨酸、缬氨酸、异亮氨酸、酪氨酸含量较高,onPSC中L-羟脯氨酸、L-脯氨酸、亮氨酸、苯丙氨酸、组氨酸、赖氨酸含量较高。在V8酶和α-糜蛋白酶作用下,sjPSC和onPSC的肽谱分析表明二者的结构有一定区别,尤其是谷氨酸、天冬氨酸及羟脯氨酸残基。     

海参体壁酸溶性胶原提取及氨基酸组成分析

以海参体壁为原料,采用乙酸溶剂法提取酸溶性胶原。以酸溶性胶原得率为指标,通过均匀设计试验确定了乙酸溶剂法提取酸溶性胶原的优化条件:温度为4℃,乙酸浓度为0.5 mol/L,料液比为1∶1 000,提取时间为72 h,在此条件下酸溶性胶原的得率可达到胶原纤维原料的7.35%(质量分数)。氨基酸组成分析表明,海参体壁酸溶性胶原的甘氨酸(Gly)含量为33.6%,羟脯氨酸(Hyp)含量为7.04%。     

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。     

Physicochemical properties and radical scavenging capacities of pepsin-solubilized collagen from sea cucumber Stichopus japonicus

Pepsin-solubilized collagen (PSC) was successfully prepared from the body wall of sea cucumber Stichopus japonicus. Ultraviolet–visible and Fourier transform infrared spectroscopic analyses showed that the PSC was a high-purity collagen that maintained the intact triple-stranded helices. Physical and chemical characterization of the PSC showed a isoelectric point of 4.14, a superior moisture-absorption and retention capacities comparing to glycerol, a minimum solubility at pH 5.0 in 0.5 M acetic acid, and a sharp decrease in solubility in the presence of low concentration of NaCl. The viscosity of PSC was shown to decrease continuously as the temperature was increased. The radical scavenging activity of the PSC was characterized with two model systems: for hydroxyl radicals, the PSC was more active than vitamin C but less active than sulfourea; for 1,1-diphenyl-2-picrylhydrazyl radicals, the PSC was more active than vitamin E.     

不同盐渍条件下刺参体品质的变化及盐渍动力学研究

本文研究了在15%盐水、饱和盐水和干盐三种不同盐渍条件下,刺参品质(体壁组织构造、微观结构、流变学特性及质构特性)的变化情况,并探讨了刺参盐渍过程的动力学。采用饱和盐水和干盐盐渍的刺参,随着盐渍时间延长,刺参体壁胶原纤维发生收缩,结构排列趋于紧密,储能模量、损耗模量均逐渐增大,硬度、黏聚性、咀嚼度变大,弹性、回复性逐渐减小;而采用15%盐水盐渍的刺参,在盐渍初期,变化趋势与饱和盐水和干盐盐渍相似,盐渍后期刺参体壁胶原纤维膨胀,组织中空隙增大,储能模量、损耗模量下降,硬度、黏聚性、咀嚼度下降;动力学模型拟合结果表明,高盐度处理的刺参体壁,其盐渍过程符合单向正渗透原理。本研究表明了不同盐渍条件对刺参品质产生了明显的影响,可为刺参盐渍条件的确定提供科学依据。     

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.     

Combination of NMR and MRI Techniques for Non-invasive Assessment of Sea Cucumber (<Emphasis Type="Italic">Stichopus japonicas</Emphasis>) Tenderization During Low-Temperature Heating Process

The purpose of this study was to develop and test the combination of nuclear magnetic resonance (NMR) and magnetic resonance imaging (MRI) method to assess the proton changes of sea cucumber body wall during low-temperature heating process. NMR relaxometry and MRI measurements indicated a significant proton change in the internal structure for sea cucumber body wall when the heating temperature increased from 45 to 55 °C. Differential scanning calorimetry (DSC) analysis revealed that the denaturation temperature of sea cucumber body wall was in the range of 45–55 °C with an endothermic peak at 51 °C, which is in accordance with the result observed in NMR and MRI. Rheological study showed similar trend to the DSC thermogram. The band change in amide I region of Fourier transform infrared (FTIR) spectra indicated the decrease in α-helix content and possible formation of other secondary structures. Scanning electron microscopy (SEM) further confirmed that the low-temperature heating did induce microstructure changes. The analysis of the Ringer?s soluble fraction (RSF), enzyme-labile fraction (ELF), and total unaltered fraction (TUF) for sea cucumber body wall during low-temperature heating provided more detailed information on the cause of structure change observed in NMR and MRI. The NMR parameters were highly correlated with the rheology storage modulus (G′), relative enzymatic assay parameters, RSF, ELF, and TUF. All these results demonstrated that it could be possible to use NMR and MRI to assess sea cucumber tenderization during low-temperature heating process.     

Characterization of a Main Extracellular Matrix Autoenzyme from the Dermis of Sea Cucumber Stichopus monotuberculatus: Collagenase

Sea cucumber is easy to undergo autolysis, causing serious economic loss in the aquaculture, fishery, and food processing. However, research on the autolysis of sea cucumber is limited. In the present study, the autoenzyme that could hydrolyze the structural protein of body wall was identified and characterized in sea cucumber Stichopus monotuberculatus. Although the main edible part of sea cucumber was the body wall, its microstructure was shown to be almost composed of collagen fibers belonging to extracellular matrix, indicating that the body wall of S. monotuberculatus belonged to dermis tissue. Starting from this perspective, crude collagen fibrils from dermis of S. monotuberculatus was isolated, and pepsin solubilized collagen was also obtained by hydrolyzing collagen telopeptides from crude collagen fibrils. Crude collagen fibrils and pepsin solubilized collagen were then acting as substrates, respectively, to identify dermis autoenzyme which had hydrolytic activity on dermis collagen. After that, a collagenase of 45 kDa was identified with the method of collagen zymograph, and the content of soluble protein was suppressed by collagenase inhibitor significantly in the autolysis of dermis. Finally, the effect of environmental conditions on collagenase activity was studied, results showed its best activity was at 40°C and pH 8, and divalent metal ion Mn²⁺ was essential for its activity. As a whole, our results showed that the dermis of sea cucumber was composed of collagen fibers, and collagenase was the main enzyme resulting in autolysis of sea cucumber.     

不同烹饪方式对海参体壁营养成分变化的对比分析

为了探究不同烹饪方式对海参体壁营养成分的影响,将鲜活海参分别使用常压烹饪、高压烹饪、重汤烹饪方式进行烹饪。对比不同烹饪方式下海参外观、体壁组织变化、海参失水率、失重率、收缩率、海参体壁营养成分及海参体壁质构变化。研究结果显示,高压烹饪组海参体壁水分、水溶性蛋白、胶原蛋白、多糖含量分别为84.27%、0.58%、1.73%、0.76%、2.73%,常压烹饪组海参体壁水分、水溶性蛋白、胶原蛋白、多糖含量分别87.58%、0.36%、1.92%、1.92%,重汤烹饪组海参体壁水分、水溶性蛋白、胶原蛋白、多糖含量分别为86.62%、0.46%、1.82%、0.81%。重汤烹饪组在海参体壁水分、水溶性蛋白、胶原蛋白、多糖含量上介于高压烹饪组、常压烹饪组之间,具有统计学差异(p<0.05)。结果表明重汤烹饪下的海参体壁组织较为完整,体壁营养成分保留较多,且对海参体壁的质构影响较小,符合海参烹饪的要求。     

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。     

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。     

Characterization and subunit composition of collagen from the body wall of sea cucumber Stichopus japonicus

Pepsin-solubilized collagen (PSC) without telopeptides was prepared from the body wall of the sea cucumber Stichopus japonicus and isolated by selective precipitation with NaCl. The PSC exhibited a maximum absorbance at 220 nm. The subunit of PSC was isolated by Sephacryl S-300 HR. The results of SDS-PAGE suggested that purified collagen from S. japonicus was a 1α trimer (about 135 kDa) while 1α chain resembling α₁ chain of type I collagen of vertebrate. The thermal stability temperature (T_s) was 57.0 °C as measured by DSC, about 5.0 °C lower than that of type I collagen of calf. Peptide mapping and amino acid analysis of PSC also revealed the difference between invertebrate and vertebrate. However, the presence of (α₁)₃ trimers was evident.