酪蛋白的转谷氨酰胺酶氨基葡萄糖修饰与功能性变化

在37℃、pH值为7.5和氨基葡萄糖存在下,利用转谷氨酰胺酶(EC 2.3.2.13)对酪蛋白进行交联修饰制备修饰酪蛋白;用十二烷基磺酸钠-聚丙烯酰胺凝胶电泳和高效液相色谱分析证实酪蛋白同时发生交联与糖基结合,且反应4 h时每摩酪蛋白可结合1.2摩葡萄糖.与酪蛋白相比,交联酪蛋白的溶解性质和起泡性质受损,而修饰酪蛋白产品的溶解性质得到改善,起泡性质尤其是泡沫稳定性质显著提高.在蛋白质质量浓度为1 g/L时,修饰酪蛋白的起泡能力和泡沫稳定性分别比酪蛋白提高8.6%和21%;质量浓度为100 g/L的修饰酪蛋白分散液表现出非牛顿流体特性,表观黏度显著高于交联酪蛋白或酪蛋白.     

酪蛋白与明胶的酶促交联与产物的流变学性质

利用转谷氨酰胺酶对酪蛋白和明胶进行酶促混合交联。在酪蛋白与明胶比例为4︰1(质量比)时,以交联产物中羟脯氨酸质量分数为指标,采用单因素试验研究酶添加量、反应时间和温度对交联反应的影响。优化后的适宜交联条件为:底物质量浓度固定为50 g/L,酶添加量为每克蛋白质20 U,反应时间为4 h,温度为45℃。SDS-聚丙烯酰胺凝胶电泳分析表明产物中含有蛋白质聚合物。与原料酪蛋白、转谷氨酰胺酶促交联的酪蛋白相比,所得到的产物的分散液表观黏度和黏弹性均有显著的改变,表明转谷氨酰胺酶催化的酪蛋白和明胶混合可以用于改善其流变学性质。     

玉米醇溶蛋白酶法糖基化修饰及产物性质研究

以转谷氨酰胺酶为催化剂,壳寡糖为酰基受体,通过糖基化反应修饰玉米醇溶蛋白。以修饰产物中氨基葡萄糖的导入量为指标,采用单因素试验优化糖基化反应条件,并对修饰产物的部分功能性质进行研究。结果表明:壳寡糖糖基化修饰玉米醇溶蛋白的最优条件为:反应初始pH 7.7,温度37℃,底物质量分数3%,酰基供体与酰基受体的物质的量比1∶3,加酶量60 U/g蛋白,反应时间8 h。在此条件下,壳寡糖的接入量最大,为101.47 mg氨基葡萄糖/g蛋白。与玉米醇溶蛋白和交联玉米醇溶蛋白相比,糖基化修饰玉米醇溶蛋白的溶解性和体外消化性均明显改善。     

TGase催化玉米谷蛋白与壳寡糖糖基化修饰的研究

在壳寡糖存在的条件下,利用转谷氨酰胺酶(Transglutaminase,TGase)对玉米谷蛋白进行糖基化交联修饰,以玉米谷蛋白糖基化修饰产物中的单糖导入量为指标,采用单因素试验优化糖基化反应条件,并比较了玉米谷蛋白、玉米谷蛋白交联产物和玉米谷蛋白糖基化修饰产物的溶解性。结果表明,最适糖基化反应条件为p H 8.0、酶添加量15 U/g玉米谷蛋白、玉米谷蛋白质量浓度3%、反应温度47℃、时间2 h、玉米谷蛋白中酰基供体与壳寡糖中酰基受体物质的量比1︰3。与玉米谷蛋白相比,玉米谷蛋白糖基化修饰产物的溶解性显著改善。     

玉米肽的酶法糖基化修饰及产物溶解性的研究

以玉米醇溶蛋白为原料,先利用Alcalase碱性蛋白酶酶解制备玉米肽,再以转谷氨酰胺酶为催化剂,氨基葡萄糖为酰基受体,通过糖基化反应修饰玉米肽,以接糖量为指标,采用单因素实验和正交实验优化糖基化反应条件,并对修饰产物的溶解性进行研究。结果表明,氨基葡萄糖糖基化修饰玉米肽的最优条件为:反应初始pH 7.7,反应温度44℃,玉米肽质量分数3.5%,玉米肽与氨基葡萄糖质量比1∶3,酶添加量55 U/g(以玉米肽质量计),反应时间7 h。在最优条件下,接糖量为149.60 mg/g。与玉米肽相比,玉米糖肽的溶解性显著增加,尤其是pH 6.0时,玉米糖肽的溶解性增加16.31%。     

壳寡糖糖基化修饰对酪蛋白分子特性及流变性质的影响

研究利用转谷氨酰胺酶催化酪蛋白与壳寡糖发生糖基化交联反应,并控制反应时间(1、2 h和4 h)制备3种糖基化交联酪蛋白。经SDS-聚丙烯酰胺凝胶电泳、傅里叶红外光谱和游离氨基含量分析,表明酪蛋白同时发生了分子交联和糖基导入反应;经流变学分析,表明产物的表观黏度显著提高(剪切速率为1 s~(-1)时,反应4 h的修饰产物的表观黏度增加了约10倍),且表现出剪切稀释特性;同时,频率扫描实验表明,产物分散液由类固体性质转变为类流体性质。     

糖基化交联反应对酪蛋白胶凝和乳化性质的影响

利用转谷氨酰胺酶催化酪蛋白与壳寡糖发生糖基化交联反应,控制反应时间(1、2 h和4 h)制备3种糖基化交联蛋白质(修饰酪蛋白),分析糖基化交联反应对酪蛋白胶凝和乳化性质的影响。结果表明:修饰酪蛋白的凝胶时间显著缩短(约50%);凝胶的持水能力为99%(800 r/min条件下离心10 min),较高;凝胶的微观结构发生了显著的变化,而且随着反应时间的延长,凝胶的空间网络结构更加规则;糖基化交联反应对酪蛋白的乳化活性及乳化稳定性影响较大。     

转谷氨酰胺酶催化壳寡糖糖基化修饰玉米谷蛋白

以转谷氨酰胺酶(Transglutaminase,TGase)催化壳寡糖(1 ku)糖基化修饰玉米谷蛋白的最优条件为初始反应条件,利用TGase催化壳寡糖(3 ku)对玉米谷蛋白进行糖基化修饰。用高效液相色谱法(High Performance Liquid Chromatography,HPLC)确认糖基化交联反应的发生,以玉米谷蛋白中的单糖导入量为指标,采用单因素试验优化糖基化反应条件。结果表明:在转谷氨酰胺酶存在的条件下,壳寡糖能够与玉米谷蛋白发生糖基化交联反应;玉米谷蛋白与壳寡糖(3 ku)糖基化的最适反应条件为:玉米谷蛋白质量浓度为2%(w/v)、酶添加量为10 U/g玉米谷蛋白、玉米谷蛋白中酰基供体与壳寡糖中酰基受体物质的量比为1:3、反应时间为2 h。在此条件下,玉米谷蛋白糖基化修饰样品的单糖导入量为(488.99±19.11)mg/g玉米谷蛋白。     

酶法糖基化修饰对酪蛋白体外消化能力的影响

利用转谷氨酰胺酶（transglutaminase,TGase）催化制备壳寡糖（1 kDa）糖基化酪蛋白;十二烷基硫酸钠-聚丙烯酰氨凝胶电泳和反相-高效液相色谱证实糖基化修饰反应的发生;体外消化实验（胃蛋白酶＋胰蛋白酶）分析糖基化修饰反应对酪蛋白消化性的影响。结果表明：TGase成功催化壳寡糖连接到酪蛋白分子上,所制备的糖基化酪蛋白中氨基葡萄糖的导入量为6.86 g/kg;糖基化酪蛋白经体外模拟消化后,其水解度和三氯乙酸可溶性氮均低于酪蛋白对照物,糖基化酪蛋白消化物中出现了更多大分子质量的肽段,研究表明TGase途径糖基化修饰降低了酪蛋白的体外消化能力。     

糖基化交联酪蛋白乳液凝胶特性

采用转谷氨酰胺酶催化酪蛋白与壳寡糖发生交联反应制备糖基化交联酪蛋白。考察了由该蛋白制备的乳液凝胶性质,表征了小变形流变性质(凝胶点及弹性模量,G')、凝胶强度、凝胶的持水性及容重变化。流变分析结果表明:相对于酪蛋白制备的乳液凝胶,糖基化交联酪蛋白制备的乳液凝胶时间缩短,而且G'值增加。凝胶的质构发生了显著变化,凝胶强度显著增加。但是凝胶的持水性和容重没有发生显著变化。糖基化交联修饰对酪蛋白乳液凝胶的形成时间和质构影响较大。     

Cross-Linking and Glucosamine Conjugation of Casein by Transglutaminase and the Emulsifying Property and Digestibility In Vitro of the Modified Product

Microbial transglutaminase was applied as a biocatalyst and glucosamine as an acyl acceptor to modify casein by cross-linking and glucosamine conjugation. Electrophoretic analysis revealed that transglutaminase-induced cross-linking and glucosamine conjugation occurred simultaneously during reaction, and some polymers of glycoproteins with higher molecular weights were formed in the modified casein product prepared. HPLC analysis demonstrated that about 1.2 mol of glucosamine was conjugated to 1 mol of casein, under the preparation conditions as follows: casein concentration of 3% (w/v), molar ratio of acyl donor in casein to glucosamine acceptor of 1:3, transglutaminase addition level of 10 U/g casein, reaction temperature of 37°C, and reaction time of 4 h. The evaluation results also showed that the surface hydrophobicity of the modified casein product decreased. The emulsifying activity index and emulsifying stability index of the modified casein product were 100.9 m²/g and 84.3%, about 12 and 20% higher than that of original casein. The digestibility in vitro of the modified casein product was the same as that of original casein or had an increase of 9.8%. Meanwhile, cross-linking of casein by transglutaminase in the absence of glucosamine showed adverse impact on the emulsifying properties of the cross-linked casein prepared. The new modification method developed might have the potential as an effective approach to improve the functional properties of food proteins.     

酪蛋白在碎猪肉边角料重组中的应用研究

主要研究了以鲜猪肉分割过程中产生的碎精肉为原料,经腌制后添加转谷氨酰胺酶、酪蛋白进行重组的工艺.以转谷氨酰胺酶添加量、酪蛋白添加量和反应时间为影响因素,以坚实度和内聚性为指标进行重组工艺参数优化,结果表明,在原料肉中添加4g/kg TG和20g/kg酪蛋白,6℃条件下反应12h,重组肉的坚实度和内聚性分别为825g和0.334,研究结果为碎肉的重组和综合利用提供了理论依据.     

Transglutaminase-induced cross-linking and glucosamine conjugation in soybean protein isolates and its impacts on some functional properties of the products

In the presented work, we exploited microbial transglutaminase as a biocatalyst and glucosamine as an acyl acceptor to modify soybean protein isolates (SPI) by cross-linking and glucosamine conjugation and evaluated some functional properties of the modified product prepared. Electrophoretic studies revealed that transglutaminase-induced cross-linking and glucosamine conjugation occurred simultaneously during modification reaction, and some polymers of glycoproteins with higher molecular weights were formed in the modified product. HPLC analysis demonstrated that about 3.3 mol of glucosamine could be conjugated to 1 mol of SPI, under the preparation conditions as following: SPI concentration of 3% (w/v), acyl donor in SPI/glucosamine acceptor molar ratio of 1:3, transglutaminase addition level of 10 U g⁻¹ proteins, reaction temperature of 37 °C, and reaction time of 6 h. Compared to SPI and transglutaminase-induced cross-linked SPI, the modified product with glucosamine conjugation about 3.3 mol mol⁻¹ SPI clearly exhibited lower surface hydrophobicity, better interfacial properties (especially in emulsion and foaming stability), markedly increased apparent viscosity in the prepared dispersion, and higher enzymatic digestibility in vitro. Our results showed that this modification technique might have the potential as an effective approach to improve the functional properties of SPI.     

The preparation of an oligochitosan‐glycosylated and cross‐linked caseinate obtained by a microbial transglutaminase and its functional properties

A glycosylated and cross‐linked caseinate (GCC) with glucosamine amount of 4.74 g/kg protein was generated from caseinate and oligochitosan by a microbial transglutaminase. The applied temperature, pH and molar ratio of acyl donor/acceptor were 37 °C, 7.5 and 1:3, respectively; while caseinate concentration, transglutaminase addition and reaction time selected from single‐factor trials were 50 g/L, 10 kU/kg protein and 3 h, respectively. Electrophoretic analysis revealed the cross‐linking and glycosylation of caseinate. Compared with caseinate, GCC showed improved solubility in pH 4–11, higher digestibility in vitro and water binding capacity, about 3‐fold, but lower surface hydrophobicity and oil binding capacity (34%).     

Effect of Ultra-high Temperature Treatment on the Enzymatic Cross-linking of Micellar Casein and Sodium Caseinate by Transglutaminase

ABSTRACT: It was found that ultra-high temperature (UHT) treatment of sodium caseinate and native whey protein-depleted micellar casein drastically increases the protein polymerization effect of an enzymatic treatment by microbial transglutaminase (TG). As a result the concentration of the isopeptide ε-(γ-glutamyl)lysine was increased significantly in UHT-treated micellar casein solutions after TG incubation compared with the unheated casein solution. Sodium caseinate was more susceptible to the cross-linking reaction as compared with the native casein micelles. The results demonstrate that the protein structure significantly affects the TG cross-linking reaction. The effect of an UHT treatment was considered to be related to a better TG accessibility due to a more open casein micelle structure and to the inactivation of a TG inhibitor substance. The results demonstrate that an unidentified component in the natural milk serum inhibits the TG reaction. The thermal inactivation of a TG inhibitor is the dominant effect explaining the improved cross-linking of UHT-treated casein micelles as well as sodium caseinate.     

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。     

酪蛋白湿法糖基化改性研究

研究了酪蛋白糖基化湿法改性工艺,分析了底物配比、浓度以及pH值对酪蛋白接枝度的影响,并确定了酪蛋白-葡聚糖接枝反应的优化工艺参数。研究结果表明:pH增大有助于该反应的进行,底物的配比与浓度对反应的影响是双向的。正交分析表明,最佳反应条件为:酪蛋白浓度2mg/mL,底物配比(酪蛋白与葡聚糖的质量比)0.1,pH7.8,接枝度(DG)可达到35.10%,且褐变指数仅为0.198。乳化性能测定结果表示随着接枝度的增大,乳化性逐渐增大,当接枝度为35%时,酪蛋白的乳化性提高了约1.5倍。SDS-PAGE结果证实酪蛋白经湿法改性生成了接枝共聚物。     

Effect of heat and transglutaminase on solubility of goat milk protein‐based films

The effect of heat, transglutaminase and combination of heat and transglutaminase treatments on the solubility of films prepared from goat milk casein, goat milk whey proteins and whole goat milk proteins was investigated. Goat milk casein films were less soluble when treated with transglutaminase and combination of heat with transglutaminase compare with heat‐treated caseins alone. Heat treatment was more effective at decreasing the solubility of whey protein films. SDS‐PAGE patterns demonstrated that goat milk caseins were better cross‐linked by transglutaminase, whereas whey proteins were better cross‐linked by heat. The extent of cross‐linking was further enhanced when a combination of heat and transglutaminase was used.     

Coupled Neutrase–Catalyzed Plastein Reaction Mediated the ACE-Inhibitory Activity In Vitro of Casein Hydrolysates Prepared by Alcalase

Casein hydrolysates with a degree of hydrolysis of 13.5% were prepared by hydrolyzing casein with an alkaline protease Alcalase, and showed ACE-inhibition in vitro with an IC₅₀ value of 45.2 μg/mL. The hydrolysates were modified by plastein reaction catalyzed by a neutral protease Neutrase to reveal the impact of the coupled Neutrase-catalyzed plastein reaction on the ACE-inhibition of the casein hydrolysates. The effects of addition level of Neutrase, substrate concentration, reaction temperature, and time on the plastein reaction of the casein hydrolysates were studied with the varying amount of free amino groups of the modified hydrolysates as index. The results illustrated that the amount of free amino groups of the modified hydrolysates increased in all occasions, and the addition level of Neutrase, substrate concentration, and reaction time had a clear impact on the plastein reaction. Six modified hydrolysates were prepared at a substrate concentration of 40% (by weight), Neutrase addition level of 3 kU/g peptides, reaction temperature of 35°C, and different reaction time. The assay results highlighted that the coupled Neutrase-catalyzed plastein reaction improved the ACE-inhibition of six modified hydrolysates with IC₅₀ values ranging from 15.6 to 20.0 μg/mL. Size exclusion chromatography analysis showed that some plasteins with a molecular weight of about 68 kDa existed in the modified hydrolysates. The results also demonstrated that it was the coupled Neutrase-catalyzed plastein reaction but not further hydrolysis of casein hydrolysates that enhanced the ACE-inhibition of the modified casein hydrolysates.