Effect of aging at different temperatures on LAOS properties and secondary protein structure of hard wheat flour dough

The effects of aging from t = 0–108 h at two different temperatures (4 and 25 °C) on the non-linear viscoelastic rheological properties and secondary protein structure of hard wheat flour dough (HWD) were investigated using large amplitude oscillatory shear tests (LAOS) coupled with Fourier transform infrared spectroscopy (FTIR) and SDS-PAGE. Storage (G') and loss (G'') moduli rapidly decreased during aging at 25 °C. Subjecting HWD to progressively longer aging times at 25 °C caused dramatic changes in the non-linear viscoelastic properties demonstrated by strain softening (negative values of e₃/e₁) and shear thinning (negative values of v₃/v₁) behavior. Elastic Lissajous curves of the unaged control dough showed clockwise turn and wider elliptical trajectories as dough aging proceeds especially at higher temperatures. Other non-linear LAOS parameters (G'_M-G''_L, η'_M-η'_L, S and T) supported that aging process at higher temperature caused a progressive change in dough structure from strain stiffening to strain softening behavior while dough samples aged at 4 °C showed fairly close behavior with the control dough sample. FTIR spectra indicated that the relative content of β-sheet and β-turn structures decreased while the content of α-helix structure increased for all dough samples as a result of dough aging. SDS-PAGE results supported the breakdown of high molecular weight (HMW) and low molecular weight (LMW) glutenin subfractions. Aging at the higher temperature of 25 °C decreased the HMW/LMW ratio from 0.77 to 0.59, while the ratio was 0.73 for the dough aged at 4 °C which is fairly close to the control sample. Our results show that the degradation rate of gluten/starch network was triggered by aging at higher temperature, longer aging time, and natural fermentation which resulted in increasing acidity and increase in endogenous proteolytic and amylolytic activity, and also increasing gluten solubility and break down of intermolecular disulfide bonds at acid pH.     

湖南永州尖吻蝮蛇蛇毒的蛋白组分及毒性分析

采用Nano–LC–ESI–MS/MS蛋白鉴定技术对湖南永州尖吻蝮蛇蛇毒蛋白组分进行质谱分析,应用常规体外试管法对尖吻蝮蛇蛇毒体外溶血值进行检测,采用改良寇式法对尖吻蝮蛇蛇毒LD_(50)进行测定。结果显示:尖吻蝮蛇蛇毒含有50种蛋白,蛋白相对分子质量集中在1.0×10~4～2.0×10~4(46.9%)、4.0×10~4～5.0×10~4(22.4%)、2.0×10~4～3.0×10~4(10.6%)和7.0×10~4～8.0×10~4(7.6%),其中代表性的高丰度蛋白有蛇毒金属蛋白酶A(11.7%)、蛇毒金属蛋白酶H1(9.8%)、蕲蛇类凝血酶–2(7.3%)、抗凝血酶A–A亚基(6.8%),低丰度蛋白(0.1%)有Ecto–5'–核苷酸酶、碱性磷脂酶A2 DAV–N6、生长分化因子11;蛇毒引起红细胞溶血的最低质量浓度为60.00μg/mL,尖吻蝮蛇蛇毒对KM小鼠的LD_(50)为7.1796mg/kg,攻毒小鼠出现呼吸急促、躁动不安、注射部位出现奇特瘙痒和大面积溃烂,至死亡。     

柞蚕胚胎发育期蛋白质的2D-PAGE图谱分析

采用SDS-PAGE和2D-PAGE技术分析柞蚕胚胎期不同发育阶段的蛋白质组成和含量变化,为从蛋白质水平探讨柞蚕胚胎发育过程中的基因顺序表达模式积累基础信息。SDS-PAGE电泳结果显示:在柞蚕胚胎的发育过程中共分离到相对明显的32条蛋白带,包括分子质量约70、60、30 kD的高丰度蛋白条带,随着胚胎的发育,这些蛋白质含量逐渐减少,而40、15 kD左右的蛋白质含量逐渐增加,到孵化成蚁蚕(324 h)时,分子质量约70、60 kD的蛋白条带基本消失。进一步分析柞蚕胚胎发育不同时期蛋白质的2D-PAGE图谱:蛋白点数量随胚胎发育逐渐增多,胚胎发育12 h检测到370个蛋白点,胚胎发育300 h的蛋白点达到422个;胚胎发育132 h及276 h的蛋白点与胚胎发育早期36 h蛋白点的匹配率分别为52.4%和28.4%。此外,孵化蚁蚕总蛋白2D-PAGE图谱与胚胎期相比存在较大差异。研究结果提示,柞蚕胚胎发育前期,从胚胎形成期到附肢发生期(12～60 h),蛋白质种类相对较少,且变化不大;胚胎发育的气管形成期(228～276 h)开始,蛋白质种类变化剧烈,偏酸性蛋白质增加,蛋白点的匹配率下降。     

Pericarp total protein profiles as molecular markers of tomato fruit quality traits in two segregating populations

The tomato fruit quality results of biochemical and physiological changes that occur during the ripening process. Although, the pericarp total protein profiles are less polymorphic than DNA-based markers the polymorphism in those could be directly associated with fruit quality traits. The aim of this work was to identify associations between polymorphic polypeptides from fruit pericarp at two ripening stages and fruit quality traits evaluated in two segregating populations of tomato. A cross between a normal ripening cultivar of Solanum lycopersicum (C, Caimanta) and a genotype carrying the nor (non ripening) gene (N) as well as a cross between Caimanta and a cherry type tomato of S. lycopersicum var. cerasiforme (Ce) showed genetic variance for several fruit quality traits such as fruit weight, shape, solids soluble content, acidity, color and fruit shelf life. The quantitative variations observed at phenotypic level had correspondence with the polymorphism detected in the protein profiles. Indeed, the polymophic polypeptides associated with quality fruit traits and fruit shelf life would be useful to assist tomato breeding programs as protein molecular markers.     

Variation among maize (Zea mays L.) accessions of Bendel State,Nigeria

Summary Variability in maize zein protein band mobilities in sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) was characterized to classify 27 maize accessions (OTUs) collected from Bendel State, Nigeria. The classification of the OTUs was achieved using two numerical procedures: average linkage cluster analysis and principal component analysis (PCA). Five clusters were delineated by the cluster analysis whereas the PCA complemented the cluster analysis by separating the OTUs with yellow kernels into one group and OTUs with early maturity into another. OTUs from the same geographical contiguity commonly grouped together. However some regional overlappings of the OTUs occurred. Results of the PCA revealed that zein bands that stained less intensely more strongly separated the OTUs into various clusters than did those that stained more intensely.     

猪笼草消化液中蛋白酶的活性初探

为了进。步分析猪笼草瓶状体内消化液中蛋白质的性质，本研究在不同的温度和pH条件下测定消化液中蛋白酶的活性，并且比较了3种不同沉淀方法对消化液蛋白进行的浓缩，通过SDS-聚丙烯酰胺电泳对消化液蛋白作了初步分离。结果表明，猪笼草消化液中蛋白酶的最适酶活温度为50℃，且稳定性最高；当pH为5时，该蛋白酶活性出现峰值，采用氯仿-正丁醇法（5：1）沉淀浓缩猪笼草消化液蛋白样品效果最佳。聚丙烯酰胺电泳结果表明，消化液至少包括三种蛋白组分，分子量分别为24.3kD、35．1kD和61．4kD，且35．1kD条带具有抗胰蛋白酶消化活性。本研究为综合开发利用猪笼草野生资源提供理论依据。     

鳖源嗜水气单胞菌M-96株表面抗原分析

用ＳＤＳ－ＰＡＧＥ、免疫印迹技术分析嗜水气单胞菌Ｍ－９６株表面抗原，表明该菌体表面抗原是由分子量为３０ＫＤ、３３ＫＤ、３６Ｋ和３９ＫＤ的蛋白质和脂多糖组成，其中部分对胃蛋白酶敏感。     

西藏白菜型油菜油体特征与营养品质及油体提取物的SDS-PAGE分析

【目的】探索白菜型油菜种子油体、油体蛋白表型与含油量和蛋白质、脂肪酸含量之间的关系,分析油体提取物蛋白亚基的多态性,为西藏白菜型油菜高油新品种的选育奠定基础。【方法】测定36份来自西藏不同地区的白菜型油菜农家种、野生近缘种种子的含油量和蛋白质、脂肪酸含量;利用Leica DM5000生物显微镜观察所提取油体的大小及油体蛋白的表型特征;利用SDS-PAGE对供试材料油体提取物进行凝胶电泳,分析供试材料的遗传多态性和进化关系。【结果】36份供试材料芥酸、油酸、亚油酸和烯酸含量分别为27.39~132.33,3.22~143.22,31.22~53.62和14.27~111.48mg/g,其他脂肪酸含量相对较低;36份材料的含油量为57.20~151.37mg/g,蛋白质含量为38.68~73.87mg/g。相关分析显示,含油量与蛋白质含量、棕榈酸、烯酸含量呈极显著负相关。根据含油量将供试材料划分为高油组(含油量≥138mg/g,HO)、中油组(110mg/g≤含油量138mg/g,MO)、低油组(含油量110mg/g,LO)3个类别,不同含油组材料油体和油体蛋白形状均以近圆形为主,HO油体平均粒径2.12μm,油体蛋白平均粒径5.24μm;MO油体平均粒径1.82μm,油体蛋白平均粒径4.58μm;LO油体平均粒径1.44μm,油体蛋白平均粒径3.81μm。相关分析表明,油体平均粒径与含油量呈极显著正相关,与蛋白质含量呈极显著负相关,而与脂肪酸含量相关性不大。油体粗提液SDS-PAGE电泳共分离出迁移率不同的蛋白谱带32条,出现频率为5.6%~100%;蛋白谱带的分子质量为25.5~128.7ku,且分布较集中区域在87.8~27.4ku;蛋白谱带N-J聚类分析表明,供试材料遗传相似系数为0.610~0.980,在遗传相似系数0.835处可将36份材料聚为9个类群。【结论】西藏白菜型油菜遗传变异丰富,在西藏高油白菜型油菜品种选育时可将油体粒径大且数量多、油体蛋白少且粒径大作为筛选亲本的条件。     

陕西省农家种大豆资源11S 和7S 球蛋白含量分析

利用SDS-PAGE分析了陕西省75份农家种大豆核心种质的11S和7S球蛋白亚基组成、相对百分含量和11S/7S比值,并对陕北、关中和陕南三个地区的资源材料进行了分析和比较。结果表明:大豆贮藏蛋白质由许多亚基组成,其中含量较高的有α′、α、β、A3、Acid和Basic;不同品种间大豆球蛋白组成几乎没有差异,但是各亚基的含量存在较大差异;75份供试材料的7S、11S球蛋白含量的平均值分别为36%和64%,变幅分别为28%~48%和52%~72%;11S/7S比值的变异幅度在1.09~2.53,平均值为1.78,高于2.0的种质有19个;陕北、关中、陕南三个地区农家种大豆群体11S/7S比值存在显著差异。     

奶牛早孕因子重组蛋白的原核表达、纯化及多克隆抗体的制备

本研究旨在表达、纯化奶牛早孕因子重组蛋白及制备多克隆抗体.本试验构建奶牛早孕因子重组蛋白原核表达载体pET32a-EPF,转化至大肠杆菌BL21(DE3)内进行诱导表达,SDS-PAGE切胶纯化的早孕因子重组蛋白免疫BALB/c小鼠制备多克隆抗体,并用Western blotting和ELISA对重组蛋白及抗体进行检测.结果显示,成功表达并纯化了奶牛早孕因子重组蛋白,重组蛋白分子质量约37 ku,表达量约占菌体总蛋白的48.6%,纯化后目的蛋白纯度可达93%,重组蛋白可与所制备的多克隆抗体结合,ELISA测定的抗体效价为1:12 800.结果表明,本研究成功表达并纯化了奶牛早孕因子重组蛋白,为研究奶牛早孕诊断奠定基础.