棉籽7S及12S球蛋白的分离纯化及其物化性质的研究

以棉籽贮存蛋白为原料,采用蛋白纯化系统(配备凝胶层析柱)对棉籽7S和12S球蛋白进行分离纯化,对所得7S和12S球蛋白的蛋白质含量、氨基酸组成、变性温度及荧光强度等物化性质进行了测定分析。结果表明:棉籽7S及12S球蛋白的蛋白质含量分别为(91.59±0.53)%和(93.35±0.62)%,其SDS-PAGE谱图中除显示各自特征条带外无其他杂带,其氨基酸组成与绝大部分油料种子中球蛋白的基本一致,含有丰富的谷氨酸、天冬氨酸和精氨酸,而半胱氨酸和甲硫氨酸含量较低;差示扫描量热仪(DSC)分析棉籽7S及12S球蛋白的变性温度分别为93.3℃和107.7℃;棉籽7S及12S球蛋白的荧光发射光谱最大荧光强度均在325 nm左右。     

棉籽球蛋白凝胶特性研究

该研究以棉籽球蛋白为原料,考察了棉籽球蛋白溶解度随pH的变化,并于中性条件下加热诱导棉籽球蛋白聚集形成凝胶。采用物性仪对不同条件下制备的凝胶质构特性进行研究。通过正交试验考察蛋白质浓度、pH、加热温度、加热时间对凝胶形成的影响,得出形成凝胶较为适宜的条件。     

大豆11S球蛋白凝胶形成的影响因素分析

以低温脱脂大豆粕为原料,采用等电点冷沉法浸提11S球蛋白。对11S球蛋白凝胶形成过程中影响凝胶质构的因素进行了分析,研究表明,11S球蛋白凝胶形成时蛋白浓度、加热温度、加热时间、蛋白溶液pH对凝胶质构均有一定的影响。采用正交实验设计,通过质构仪进行物性测定比较其凝胶性,大豆11S球蛋白浓度14%、加热温度90℃、溶液pH为7、加热时间60min条件下形成的凝胶强度最好,其凝胶面积410.30g·s,凝胶力117.9g。     

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。     

醇变性大豆7S球蛋白结构特征研究

利用不同体积分数乙醇溶液对大豆7S球蛋白进行变性。分别对醇变性大豆7S球蛋白溶解度、表面疏水性、二级结构和聚集形态进行分析。大豆7S球蛋白经醇溶液变性后,溶解度在乙醇体积分数为55%~65%时基本达到最低值;表面疏水性在乙醇体积分数为25%和65%时分别达到最大值和最小值。傅立叶红外光谱表明,在乙醇体积分数较低时,β-折叠结构能稳定存在;在乙醇体积分数较高时,β-折叠和α-螺旋之间结构发生转变,呈现较高螺旋态。凝胶电泳表明,7S球蛋白亚基间以非共价相互作用力为主;经醇变性后,导致聚集体产生,且7S球蛋白分子间非共价相互作用随乙醇体积分数增大呈有逐渐增大趋势。     

高静压处理对醇变性大豆11S球蛋白影响研究

研究了高静压处理对醇变性大豆11S球蛋白溶解性及结构性质的影响.结果表明,大豆11S球蛋白以1:15的比例添加65%乙醇处理后溶解度降低为14.24%.利用300 MPa高静压处理醇变性大豆11S球蛋白.可使其溶解度恢复至95.5%.凝胶渗透色谱显示,醇变性大豆11S球蛋白在不同高静压下形成多种相对分子质量的可溶性聚集体.荧光光谱和圆二色谱对高静压处理的醇变性大豆11S球蛋白结构的测定证实其结构逐步被打开,形成柔性三级结构;醇诱导稳定的α-螺旋结构部分被破坏,高静压处理后蛋白的二级结构以β-折叠为主.     

胰蛋白酶限制性水解脱酚棉籽蛋白的研究

采用胰蛋白酶对脱酚棉籽蛋白进行限制性酶解,以改善棉籽蛋白在中性条件下的溶解性.以酶解棉籽蛋白水解度和溶解度为指标,对胰蛋白酶水解脱酚棉籽蛋白的条件进行了优化.结果表明,胰蛋白酶水解脱酚棉籽蛋白的优化条件为:酶底比2∶ 1(50 000 U/g),底物质量浓度100 g/L,pH 10,于20 ℃下酶解3 h.在此条件下,酶解棉籽蛋白的水解度为10.87%,溶解度为63.48%.与酶解前相比,溶解度提高了43.82%.溶解度分析结果表明,限制性酶解棉籽蛋白在pH 1～11范围内均具有较好的溶解度,仍然保持了棉籽蛋白酸溶性的特点.     

豌豆蛋白的功能特性研究

本论文对豌豆球蛋白(7S、11S)和豌豆分离蛋白(PPI)的物化和功能特性进行了分析和比较。结果表明,豌豆球蛋白具有良好的功能特性,其溶解度(PS)、乳化能力、乳化稳定性均显著高于PPI。荧光光谱和表面疏水性(H0)分析表明,PPI是部分变性的蛋白,其制备过程中的酸碱处理导致蛋白分子伸展、H0增加。DSC表明,11S热稳定性比7S要高,豌豆分离蛋白和豌豆7S出现不同程度的蛋白变性。     

籽瓜种子蛋白对肌原纤维蛋白功能的影响

本研究以籽瓜种子为原料,利用碱溶酸沉方法提取籽瓜种子蛋白,在40、60、80℃条件下获得不同籽瓜种子蛋白,分别进行分子质量、溶解度测定,并与肌原纤维蛋白形成凝胶,对凝胶作用力、弹性、回弹性、粘结性、水相分度、白度进行试验。结果表明,40~80℃范围内随着提取温度的升高,籽瓜种子蛋白溶解度先升高后降低差异显著(P0.05),并获得4个吸收峰,峰1为699~103ku的大分子蛋白聚集体,峰2、峰3分别为340、212 ku的球蛋白,及10~54 ku部分清蛋白、球蛋白的亚基,随着碱溶温度继续升高,峰2、峰3逐渐消失。籽瓜种子蛋白与肌原纤维蛋白形成凝胶的三维网状结构稳定性通过疏水键、氢键、以及二硫键维持,其中疏水键、氢键为主要作用力。碱溶温度为60℃时,WPS与MP形成凝胶弹性较好、保水性较好,差异显著(P0.05)。随着温度的增加,凝胶白度逐渐加,WPS结构发生改变、变性,差异显著(P0.05)。由此可知,温度对籽瓜种子蛋白结构、功能影响较大,不同碱溶温度提取的籽瓜种子蛋白对肌原纤维蛋白功能影响差异显著,碱溶温度为60℃时,获得籽瓜种子蛋白与肌原纤维蛋白形成的凝胶有利于改善对肉制品品质,可作为非肉蛋白稳定剂添加到猪肉制品中。     

转谷氨酰胺酶交联改性小米蛋白质的溶解特性

以小米分离蛋白为研究对象,通过转谷氨酰胺酶交联酶改性后,对其不同条件下的溶解性进行分析研究,通过SDS-PAGE电泳对小米酶改性蛋白组分进行亚基分析。结果表明,在不同处理情况下,小米酶改性蛋白在pH 5处溶解度最小,故等电点应在pH 5附近;随盐离子浓度升高,蛋白溶解度呈现先下降后上升再下降现象;温度升高,蛋白溶解度呈现先上升后趋于稳定现象;SDS-PAGE试验结果显示,小米酶改性蛋白亚基大部分分子质量大于66.2k Da,含二硫键的亚基占大部分,在不同pH下,等电点附近沉淀的亚基数量最多,进一步印证等电点附近蛋白的溶解度的变化规律;在不同离子强度下的溶解度差异,只是整体亚基的溶解度差异,并无亚基溶解度的特异差别,在不同温度下,最接近分子质量14.4 kDa的亚基不易聚集形成不溶性聚集体。     

Aggregation profile of 11S, 7S and 2S coagulated with GDL

The aggregation process of the proteins coagulated by glucono-δ-lactone (GDL) was monitored by using atomic force microscopy (AFM). Solutions of 11S, 7S and 2S proteins, after heating at 100 °C for 10 min, were mixed with GDL and formed aggregates with different aggregation profiles. When the three protein solutions (11S, 7S and 2S) were mixed with GDL and deposited onto the mica for 1, 2 and 4 min, 11S proteins formed the largest clusters of aggregates, 2S proteins formed smaller clusters of aggregates than 11S but bigger clusters of aggregates than 7S and 7S proteins formed the smallest cluster of aggregates. It was also found, by turbidity measurement, that when GDL was added to the three protein fractions, the level of turbidity was in the order of 11S > 2S > 7S. Both these results showed that, when GDL was added to the three protein fractions, the speed of aggregation was in the order of 11S > 2S > 7S.     

热处理对大豆11S球蛋白溶解性和二级结构的影响

采用差示扫描量热仪、Lowry法以及傅里叶变换红外光谱法分别对大豆11S球蛋白的热性质、溶解性和二级结构进行测定与分析,研究热处理对11S球蛋白溶解性和二级结构的影响。热处理能够使大豆11S球蛋白Td和ΔH降低,导致蛋白质发生部分或完全变性。80?℃热处理使大豆11S球蛋白的溶解性降低,这可能由于热处理改变了蛋白质的空间结构和表面电荷所致,此时蛋白质的α-螺旋结构逐渐转变为β-折叠和无规卷曲结构。而90?℃和100?℃热处理一定时间后,蛋白质溶解性又稍有升高,这可能是形成可溶性聚集体造成的。此时蛋白质的α-螺旋和β-折叠结构向β-转角和无规卷曲结构转变,表明β-转角和无规卷曲结构对热聚集体的形成具有重要作用。此外,蛋白质变性和氢键断裂是导致二级结构相互转变的重要因素。     

Gelation Mechanism of Soybean 11S Globulin: Formation of Soluble Aggregates as Transient Intermediates

Thermal association-dissociation behavior of soybean 11S globulin was investigated by sucrose density gradient centrifugation and polyacrylamide gel electrophoresis. Soluble aggregates with a molecular weight of 8 × 10⁶ were formed when 0.5% and 5% protein solutions were heated for 1 min at 100°C. At the lower protein concentration, subsequent heating caused disappearance of the soluble aggregate followed by complete dissociation into acidic and basic subunits. At the higher concentration, however, subsequent heating caused formation of highly polymerized aggregates, and gel was formed after 5 min of heating. The soluble aggregates appear to be transient intermediates in the course of gel formation of 11S globulin.     

大豆7S球蛋白凝胶光学性质的研究

本文深入地研究了大豆7S球蛋白凝胶光学和流变学性质与蛋白质浓度、加热温度和加热时间的关系。结果表明在形成蛋白质凝胶(蛋白质浓度≥7.5%)的前提下,低的蛋白质浓度有利于大豆7S球蛋白形成透明性凝胶,但凝胶强度较低;温度＞85℃有利于蛋白凝胶透明性和强度的提高;加热60min.较为适宜。扫描电子显微镜(SEM)观察显示大豆7S球蛋白透明凝胶具有有序微观结构;探讨了大豆7S球蛋白形成透明凝胶机理。可为进一步研究大豆蛋白凝胶的光学性质和研制透明的大豆蛋白产品提供理论依据。     

大豆球蛋白的纤维聚集体行为及其稳定性研究

为了明确蛋白质的纤维聚集行为,本研究以大豆球蛋白(soy globulin,11S)为原料,从亚基层面对酸性条件下热诱导的11S纤维聚集过程进行跟踪,监测蛋白及其亚基的水解过程、结构变化及其稳定性。结果表明,11S的纤维化是一个多步骤的过程,包括多肽链的水解、自组装成淀粉样纤维聚集结构及逐渐生长成宏观可见的具有扭曲螺旋结构的纤维聚集体。与11S纤维化过程的单指数增长相比,酸性亚基的纤维化过程存在迟滞期。酸性亚基在纤维化聚集的初期主要贡献于纤维聚集的成核过程,碱性亚基的加入改变其纤维聚集进程。蛋白质的纤维化过程会增加11S在等电点处的溶解度,降低中性和酸性pH下的溶解度。此外,碱性环境(pH值10.0)会导致11S纤维聚集体全部溶解、宏观纤维长度变小、结构发生改变。以上研究结果旨在为合理利用蛋白纤维化聚集体作为新的功能性食品配料提供理论依据。     

Production of Whey Protein-Based Aggregates Under Ohmic Heating

Formation of whey protein isolate protein aggregates under the influence of moderate electric fields upon ohmic heating (OH) has been monitored through evaluation of molecular protein unfolding, loss of its solubility, and aggregation. To shed more light on the microstructure of the protein aggregates produced by OH, samples were assayed by transmission electron microscopy (TEM). Results show that during early steps of an OH thermal treatment, aggregation of whey proteins can be reduced with a concomitant reduction of the heating charge—by reducing the come-up time (CUT) needed to reach a target temperature—and increase of the electric field applied (from 6 to 12 V cm^?1). Exposure of reactive free thiol groups involved in molecular unfolding of β-lactoglobulin (β-lg) can be reduced from 10 to 20 %, when a CUT of 10 s is combined with an electric field of 12 V cm^?1. Kinetic and multivariate analysis evidenced that the presence of an electric field during heating contributes to a change in the amplitude of aggregation, as well as in the shape of the produced aggregates. TEM discloses the appearance of small fibrillar aggregates upon the influence of OH, which have recognized potential in the functionalization of food protein networks. This study demonstrated that OH technology can be used to tailor denaturation and aggregation behavior of whey proteins due to the presence of a constant electric field together with the ability to provide a very fast heating, thus overcoming heat transfer limitations that naturally occur during conventional thermal treatments.     

Ultrasonic Extraction of a Heat-Labile 7S Protein Fraction from Autoclaved, Defatted Soybean Flakes

Conventional stirring extraction of soybean proteins from autoclaved or heat-treated defatted flakes seldom reaches 30% of the total proteins. Using ultrasonic energy to disperse proteins in water from such treated flakes, one extraction can solubilize 80%. Gel electrophoresis of water extractable proteins in extracts prepared by the two methods of extraction reveals that extraction by stirring failed to extract a specific heat-labile protein fraction in 7S proteins that is extractable by sonication. Ultracentrifugation and gel filtration analyses show that sonication induced formation of aggregates, and the size of aggregates, rate of formation, and water solubility are different than those formed by heat treatment. The effects of heat and sonication treatments on isolated 7S proteins were also investigated.     

Modeling Gelation of Egg Albumen and Ovalbumin

The Flory gelation theory and a new protein gelation model were used to investigate the aggregation reactions of egg albumen and ovalbumin. Results showed that 178 to 267 ovalbumin monomers were required to establish the initial ovalbumin gel at the gel point temperature, 81°C. The gel point temperature for egg albumen was 72°C by the Instron and 60°C by protein solubility. The values of the activation energy rate constants for the formation of the different aggregates in the pre-gel stage suggested that the larger ovalbumin aggregates were more reactive than the smaller ones.     

热诱导菜豆属7S球蛋白纤维聚集研究

研究了菜豆属球蛋白(芸豆和绿豆)在低pH低离子强度条件下热诱导形成的自组装纤维聚集机理。通过对制备的球蛋白进行热性质及临界形成凝胶浓度分析,选定85℃和0.5%作为自组装纤维化的参数。通过硫代黄色素T(ThT)荧光法,动态光散射(DLS)和原子力显微镜(AFM)表征蛋白纤维化聚集程度及形态。结果表明:芸豆和绿豆球蛋白的Th T最大荧光强度在1h之内剧烈增大(分别是765和1093),表明芸豆和绿豆球蛋白自组装纤维化的"构筑单元"主要产生于加热的起始阶段。DLS结果显示芸豆自组装纤维聚集的能力比绿豆强。但是二者的自组装机理有所不同。AFM图清晰地显示了芸豆球蛋白在加热12 h时自组装形成规则的高度有序的"念珠串状"长线性纤维,绿豆则形成无规则的短棒状纤维和大量碎片状纤维。这为研究进一步研究超低固形物下基于自组装技术的纤维型植物蛋白凝胶的制备提供参考。     

Denaturation Kinetics and Aggregation Mechanism of the Sarcoplasmic and Myofibril Proteins from Grass Carp During Microwave Processing

Optimizing the physicochemical properties of microwave-cooked meat needs a better understanding of the mechanisms responsible for protein changes. This paper explored the causes of aggregation of the denatured fish protein in ready-to-eat food by determining the changes in sarcoplasmic and myofibril proteins, which includes the effects of different microwave power levels (300, 600, and 900 W) and heating times (0–60 s) on the aggregation kinetics and physiochemical properties. The determination of aggregation kinetics that accurately describes microwave cooking characteristics is crucial for the optimization of operating parameters, performance improvement of the cooked food system, and product quality. The aggregation rate and turbidity of proteins increased with increasing microwave power and time. A negative correlation was observed between the protein solubility and microwave power and time. Protein aggregation induced by microwave showed a reliable and reproducible characterization of particle size distributions. At longer microwave heating time or higher microwave power, these protein particles formed larger aggregates.