酶法糖基化修饰对玉米醇溶蛋白部分结构及流变学性质的影响

为了提高玉米醇溶蛋白的生物利用率,用谷氨酰胺转氨酶(TGase)和低聚氨基葡萄糖对玉米醇溶蛋白进行酶法糖基化修饰,研究了TGase催化的低聚氨基葡萄糖的共价结合反应对玉米醇溶蛋白部分结构性质及流变学性质的影响。试验结果表明,与玉米醇溶蛋白相比,低聚氨基葡萄糖的共价结合使玉米醇溶蛋白的游离巯基含量降低32.37μmoL/g,热变性温度和变性焓分别增加11℃和72.3J/g,即糖基化反应使玉米醇溶蛋白的热稳定性增加。在剪切速率为1～100s~(-1)条件下,糖基化玉米醇溶蛋白分散液的表观黏度最高,并表现出剪切稀化的特性。在剪切频率0.1～10 Hz时,交联玉米醇溶蛋白和糖基化玉米醇溶蛋白分散液的弹性模量(G′)均大于黏性模量(G″),表现为类固体特征。     

大豆球蛋白酸、碱性链分离及其酶交联特性的研究

本文以大豆为原料,分离大豆球蛋白中酸、碱性链,并探讨酸、碱性链在微生物转谷氨酰胺酶(MTG)介导下参与交联反应的差异性。利用等电点沉淀、凝胶过滤层析以及亲和层析分离得到纯度大于94%的大豆球蛋白,再通过热变性、β-巯基乙醇(β-ME)还原和等电点沉淀分离大豆球蛋白酸和碱性链,并对其进行了电泳鉴定和氨基酸分析;进一步以MTG催化大豆球蛋白及其酸、碱性链交联,利用SDS-PAGE和溶解性试验探讨大豆球蛋白酸、碱性链交联特性和交联产物的溶解性。结果表明大豆球蛋白酸、碱性链都是MTG的有效底物,均可参与交联反应并形成高聚物,但碱性链反应率较酸性链低,两者参与交联反应差异受其谷氨酰胺和赖氨酸残基含量的影响较小,而受其疏水性氨基酸含量影响较大。     

肌原纤维蛋白转谷氨酰胺酶交联程度对鱼糜凝胶及其风味释放影响的研究进展

转谷氨酰胺酶可以催化肌原纤维蛋白发生交联反应。随着交联程度的增加,鱼糜凝胶从弹黏体变为弹脆体,风味也随之改变。风味物质的扩散、释放与凝胶网络的交联程度密切相关,因此深入了解交联程度与食品品质的关系及风味物质在凝胶网络中的扩散行为显得尤为重要。本文总结转谷氨酰胺酶催化肌原纤维蛋白的交联机理,归纳了国内外对肌原纤维蛋白交联程郭彩霞度的测定方法与影响因素,探讨交联程度对鱼糜蛋白的凝胶特性与风味释放的影响,并对今后的研究方向提出思考与展望。     

Stability of casein micelles cross-linked by transglutaminase

In this study, caseins micelles were internally cross-linked using the enzyme transglutaminase (TGase). The integrity of the micelles was examined on solubilization of micellar calcium phosphate (MCP) or on disruption of hydrophobic interactions and breakage of hydrogen bonds. The level of monomeric caseins, determined electrophoretically, decreased with increasing time of incubation with TGase at 30°C; after incubation for 24 h, no monomeric β- or κ-caseins were detected, whereas only a small level of monomeric α_S1-casein remained, suggesting near complete intramicellar cross-linking. The ability of casein micelles to maintain structural integrity on disruption of hydrophobic interactions (using urea, sodium dodecyl sulfate, or heating in the presence of ethanol), solubilization of MCP (using the calcium-chelating agent trisodium citrate) or high-pressure treatment was estimated by measurement of the L*-value of milk; i.e., the amount of back-scattered light. The amount of light scattered by casein micelles in noncross-linked milk was reduced by >95% on complete disruption of hydrophobic interactions or complete solubilization of MCP; treatment of milk with TGase increased the stability of casein micelles against disruption by all methods studied and stability increased progressively with incubation time. After 24 h of cross-linking, reductions in the extent of light scattering were still apparent in the presence of high levels of dissociating agents, possibly through citrate-induced removal of MCP nanoclusters from the micelles, or urea- or sodium dodecyl sulfate-induced increases in solvent refractive index, which reduce the extent of light-scattering.     

TGase催化玉米醇溶蛋白糖基化改性

利用转谷氨酰胺酶（transglutaminase,TGase）催化玉米醇溶蛋白与氨基葡萄糖盐酸盐（glucosamine hydrochloride,GAH）发生交联反应。通过SDS-聚丙烯酰胺凝胶电泳确认玉米醇溶蛋白与GAH发生交联反应。以玉米醇溶蛋白糖基化修饰产物中GAH导入量为指标,优化糖基化反应条件,并对玉米醇溶蛋白糖基化修饰样品的溶解性进行了表征。结果表明,最适的糖基化反应条件为底物质量浓度5 g/100 mL、TGase添加量50 U/g（以玉米醇溶蛋白计）、玉米醇溶蛋白中酰基供体与GAH中的酰基受体物质的量比1∶6、初始pH 8.0、反应温度44 ℃、反应时间7 h;此反应条件下,玉米醇溶蛋白中GAH的最大导入量为（11.34±0.21） mg/g（以玉米醇溶蛋白计）。与玉米醇溶蛋白相比,玉米醇溶蛋白交联样品与糖基化修饰样品的溶解性均得到提高,玉米醇溶蛋白糖基化修饰样品的溶解性最高。     

基于图像灰度处理的谷氨酰胺转氨酶产生菌半定量-高通量筛选

以图像灰度处理软件Image J为测量手段,以微生物谷氨酰胺转氨酶（MTG）产生菌拟无枝酸菌（Amycolatopsis sp.）109.5为出 发菌株,经紫外诱变后,对突变株进行半定量测定和高通量筛选。 突变株在含有营养琼脂培养基的96孔板中30 ℃条件下培养3 d后进 行显色反应,利用Image J获得各孔灰度均值,根据灰度均值差异预测突变株的产酶水平。 结果表明,从334株突变株中筛选到一株 MTG活力为0.803 U/mL的正突变株80,较出发菌株MTG活力提高148.6%。 基于Image J图像处理软件与96孔板的固体培养法可以实 现MTG产生菌株的半定量测定和高通量筛选。     

燕麦麸皮球蛋白的糖基化结构修饰及功能性变化

在氨基葡萄糖存在的条件下,利用转谷氨酰胺酶(transglutaminase,TG)对燕麦麸皮球蛋白糖基化结构修饰,进而探讨糖基化蛋白功能性质与结构之间的关系。结果表明,糖基化交联球蛋白的溶解性、乳化稳定性、起泡性及泡沫稳定性相比于未修饰的球蛋白都有明显的改善,但表面疏水性明显下降;另外,酶促糖基化球蛋白的变性温度和焓变值都有所下降,其二级结构变化为:α-螺旋结构相对含量呈增加趋势,β-折叠和β-转角结构相对含量呈下降趋势,无规卷曲结构相对含量几乎没变。经糖基化处理的球蛋白酪氨酸分子主要呈现"暴露态",色氨酸相对拉曼强度更趋近于"包埋态"。酶促糖基化球蛋白二硫键振动模式为t-g-t。通过对球蛋白、修饰球蛋白的功能特性与空间构象的比较分析,明确TG催化葡萄糖结合在燕麦麸球蛋白上,进一步明晰修饰蛋白功能特性与空间构象之间的构效关系。结果可为延长杂粮产业链提供良好的理论依据,同时可以为今后制备燕麦蛋白特定产品进行分子设计和重组提供基础数据。     

聚丙烯微孔膜固定化转谷氨酰胺酶在大豆乳清废水处理中的应用

将固定化转谷氨酰胺酶酶膜应用到酶膜反应器中,对大豆乳清废水进行催化使其发生聚合并被截留,从而减轻大豆乳清废水对环境的污染,并确定其最佳影响条件,得出在最佳条件下进行处理的蛋白截留率为78.4%。对处理前后大豆乳清废水进行分析,其主要成分指标发生了很大变化,如蛋白质含量、生化需氧量(biochemical oxygen demand,BOD)值、化学需氧量(chemical oxygen demand,COD)值、灰分含量等指标较处理前发生显著下降。     

动态高压微射流和谷氨酰胺转氨酶交联技术对大豆分离蛋白凝胶性能的影响

采用动态高压微射流和谷氨酰胺转氨酶(TG酶)酶促交联技术对大豆分离蛋白进行改性研究。结果表明：随着动态高压微射流压力和处理次数的增加,交联效果呈现先增加后减小的趋势;随着底物质量分数和TG酶与底物比的增加交联效果增强,随温度和时间的增加交联效果先增强后减弱。     

Microencapsulation of Lactobacillus rhamnosus GG by Transglutaminase Cross‐Linked Soy Protein Isolate to Improve Survival in Simulated Gastrointestinal Conditions and Yoghurt

Microencapsulation is an effective way to improve the survival of probiotics in simulated gastrointestinal (GI) conditions and yoghurt. In this study, microencapsulation of Lactobacillus rhamnosus GG (LGG) was prepared by first cross‐linking of soy protein isolate (SPI) using transglutaminase (TGase), followed by embedding the bacteria in cross‐linked SPI, and then freeze‐drying. The survival of microencapsulated LGG was evaluated in simulated GI conditions and yoghurt. The results showed that a high microencapsulation yield of 67.4% was obtained. The diameter of the microencapsulated LGG was in the range of 52.83 to 275.16 μm. Water activity did not differ between free and microencapsulated LGG after freeze‐drying. The survival of microencapsulated LGG under simulated gastric juice (pH 2.5 and 3.6), intestinal juice (0.3% and 2% bile salt) and storage at 4 °C were significantly higher than that of free cells. The survival of LGG in TGase cross‐linked SPI microcapsules was also improved to 14.5 ± 0.5% during storage in yoghurt. The microencapsulation of probiotics by TGase‐treated SPI can be a suitable alternative to polysaccharide gelation technologies.