高活性酪蛋白抗氧化肽的制备方法研究

采用木瓜蛋白酶对酪蛋白进行水解,得到抗氧化活性较好的酪蛋白水解物,并且水解物在木瓜蛋白酶作用下进行类蛋白反应制备出高活性酪蛋白抗氧化肽。第一步制备酪蛋白水解时酶添加量为500 U/g酪蛋白、温度45℃、底物浓度5%、反应时间2 h。第二步类蛋白反应的最优条件为：酶添加量为500 U/g水解物、温度30℃,底物浓度50%、作用时间5.5 h。毛细管电泳结果确认,类蛋白反应修饰后抗氧化肽的组成情况发生变化。抗氧化活性分析结果表明,类蛋白反应修饰后的酪蛋白抗氧化肽对两种自由基的清除能力显著提高。     

酪蛋白的转谷氨酰胺酶酶促糖基化交联条件及产物性质

在氨基葡萄糖存在的条件下,利用转谷氨酰胺酶(EC2.3.2.13)对酪蛋白进行糖基化交联修饰。以修饰酪蛋白产物中氨基葡萄糖的导入量为指标,采用单因素试验分别考察反应体系pH值、酶添加量、反应温度和时间对修饰反应的影响。优化后的适宜修饰条件为:酪蛋白底物质量浓度为30g/L,氨基葡萄糖添加量为3mol(每kg酪蛋白中)、pH值为7.5、酶添加量为10kU(每kg酪蛋白中)、反应温度为37℃、时间4h。与酪蛋白和转谷氨酰胺酶促交联的酪蛋白相比,修饰酪蛋白产物的乳化性质和胶凝性质得到显著改善,并且体外消化性能未受到影响,表明转谷氨酰胺酶催化的糖基化交联修饰可以用于改善酪蛋白的这些功能性质。     

糖基化酪蛋白乳液的流变性质及稳定性研究

考察转谷氨酰胺酶催化酪蛋白与壳寡糖发生的糖基化反应和交联反应对酪蛋白乳液流变性质及稳定性的影响,表征了乳液的平均粒径、力学谱图和表观黏度变化。结果表明:糖基的导入对酪蛋白乳液的平均粒径影响较小,但是电位值增加;糖基的导入导致乳液液滴的网络结构发生重排的速度减慢,稳定性提高,而交联反应破坏了乳液的稳定性;修饰反应导致酪蛋白乳液的表观黏度增加。     

κ-酪蛋白酶解物对双歧杆菌的促生长效果研究

研究了κ-酪蛋白的碱性蛋白酶、木瓜蛋白酶、凝乳酶酶解产物以及商业化酪蛋白糖巨肽(GMP)对双歧杆菌(BBMN68和Bb12)的体外促生长作用。结果表明,κ-酪蛋白的碱性蛋白酶、木瓜蛋白酶、凝乳酶酶解产物以及GMP对两株双歧杆菌均有促生长作用,其中作用最强的是碱性蛋白酶酶解产物,显著高于木瓜蛋白酶酶解产物、凝乳酶酶解产物和GMP的作用效果(P<0.05),并且GMP与κ-酪蛋白凝乳酶酶解产物的促生长作用无显著性差异(P>0.05)。     

酪蛋白水解物的酶法修饰与ACE抑制活性变化

利用枯草杆菌碱性蛋白酶水解酪蛋白制备酪蛋白水解物,其水解度为11.2%,IC50为47.1μg/mL。再应用相同的酶对酪蛋白水解物进行类蛋白反应修饰,考察底物浓度、温度和酶添加量对类蛋白反应的影响,并制备5个不同的修饰产物测定其ACE抑制活性和IC50值。结果表明,修饰产物的ACE抑制活性随修饰程度(游离氨基减少量)的增加而提高,并且都高于未经修饰的酪蛋白水解物。当游离氨基减少量为154.65μmol/g(蛋白)时,修饰产物的IC50值可降至0.6μg/mL。毛细管电泳分析结果显示类蛋白修饰后水解物的多肽组成情况发生明显变化。研究结果证明酪蛋白水解物的ACE抑制活性可以通过类蛋白反应的修饰作用而提高。     

酶进入皮中状况的检验方法

在酶法制革过程中 ,通过组织切片的方法将进入皮内不同层域的蛋白酶移出 ,置入含酪蛋白的载体中 ,利用与酪蛋白进行降解反应产生乳白色斑的特征 ,了解不同蛋白酶品种、不同酶用量、不同酶作用时间及不同制革工艺中 ,酶从皮 2面进入皮内的状况以及酶在不同层域的分布状态     

酪蛋白水锯物的酶法修饰与ACE抑制活性变化

利用枯草杆菌碱性蛋白酶水解酪蛋白制备酪蛋白水解物,其水解度为11.2%,IC50为47.1μg/mL.再应用相同的酶对酪蛋白水解物进行类蛋白反应修饰,考察底物浓度、温度和酶添加量对类蛋白反应的影响,并制备5个不同的修饰产物测定其ACE抑制活性和IC50值.结果 表明,修饰产物的ACE抑制活性随修饰程度(游离氨基减少量)的增加而提高,并且都高于未经修饰的酪蛋白水解物.当游离氨基减少量为154.65 μmol/g(蛋白)时,修饰产物的IC50值可降至0.6 μg/mL.毛细管电泳分析结果显示类蛋白修饰后水解物的多肽组成情况发生明显变化.研究结果证明酪蛋白水解物的ACE抑制活性可以通过类蛋白反应的修饰作用而提高.     

重组黏玉米谷氨酰胺转氨酶的酶学性质及其对乳蛋白的交联作用

目的:从重组大肠杆菌中分离纯化黏玉米来源的谷氨酰胺转氨酶(TGZ),研究其酶学性质及对乳蛋白的交联作用。方法:采用荧光测定法研究重组TGZ的动力学参数、最适反应温度、温度稳定范围、最适反应p H值、p H稳定范围及金属离子对酶活性的影响,明确该重组酶的酶学性质。以微生物源TGase(MTG)为对照,通过SDS-PAGE分析TGZ对乳蛋白的交联作用。结果 :在p H 8.0,37℃的条件下,重组TGZ的反应动力学常数Km为1.55μmol/L,最大反应速率Vmax为155/min。该酶的最适反应温度为45℃,具有较稳定的耐热性。该酶的最适反应p H值为8.0,具有较强的耐碱性。K+、Ca2+、Na+和Ba2+对该酶的活性具有促进作用,Fe3+、Cu2+和Zn2+的抑制作用较强。对乳中酪蛋白和乳清蛋白的交联作用表明酪蛋白是TGZ的良好底物。结论:重组TGZ的酶学性质为其进一步在相关食品中的应用提供了理论依据。     

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。     

转谷氨酰胺酶对牛乳酪蛋白功能性质的影响

采用微生物转谷氨酰胺酶 (TG)对牛乳中的酪蛋白进行了酶法改性 ,并对改性后牛乳中酪蛋白功能性质如溶解性、起泡性、泡沫稳定性和持水性进行了研究 .结果表明 ,牛乳经TG作用后 ,其酪蛋白功能性质发生了较大变化 ,酪蛋白的起泡性、泡沫稳定性和持水性都有不同程度的增加 ,而溶解性略有下降 .实验中考察了TG作用的时间、温度、剂量等因素对酪蛋白功能性质的影响 ,结果显示 ,要获得综合性能优良的酪蛋白 ,TG最佳的作用条件为 :pH 7.0 ,5 0℃ ,6 0min ,酶的添加量0 .1～ 0 .2U/mL .     

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.     

Transglutaminase Inhibitor from Milk

ABSTRACT Cross-linking experiments of skimmed bovine milk with bacterial transglutaminase isolated from Streptoverticillium mobaraense showed only some degree of formation of high-molecular-weight casein polymers. Studies on the nature of this phenomenon revealed that bovine milk contains an inhibitor of transglutaminase activity. Removal of the casein and whey proteins from the milk resulted in a protein-poor fraction that still inhibited transglutaminase activity at cross-linking of β-casein and in several activity assays of transglutaminase. The inhibitor was partially purified by column chromatography and appeared to be a heat labile low molecular weight component. Inhibition of transglutaminase activity was observed with microbial transglutaminase, plasma transglutaminase and guinea pig liver transglutaminase. The inhibiting activity was found in bovine, goat, sheep, and human milk, but could not be detected in horse milk.     

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.     

Cross-linking with microbial transglutaminase: Relationship between polymerisation degree and stiffness of acid casein gels

Acid casein in phosphate buffer was used as a simplified substrate for cross-linking experiments with microbial transglutaminase. The importance of the intensity of enzyme treatment on the rheology of acid gels was evaluated by preparing mixtures of casein that were cross-linked to a different extent and a native reference to obtain samples with a similar polymerisation degree. The stiffness of acid gels from caseins incubated longer then 3 h was always lower at identical polymerisation degree larger than approximately 30%. The quantification of isopeptides formed by the enzyme reveals a substantial increase of bonds at a stage of cross-linking where only 10% of monomers are available. This indicates that cross-linking must occur between and/or within already existing aggregates, which become larger and less flexible, and supports the hypothesis that, starting with a critical aggregate size the network formation is somewhat impaired by the size and reduced flexibility of the proteins.     

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.     

Characteristics of edible films made from dairy proteins and zein hydrolysate cross-linked with transglutaminase

The main objectives of this research were to develop whey protein or casein films incorporating zein hydrolysate and also cross‐linked by transglutaminase as to well as characterize the physical and mechanical properties of the film. Zein hydrolysate decreased the solubility of the whey protein film (P < 0.05), while treatment with transglutaminase did not change the solubility of the film significantly. Electrophoresis patterns demonstrated that casein molecules were cross‐linked by transglutaminase and the extent of this cross‐linkage was further increased when zein hydrolysate was added. In addition, the use of zein hydrolysate decreased the tensile strength of the whey protein film by 35–45%. The elongation of the casein film was increased by 41% because of the action of transglutaminase and zein hydrolysate (P < 0.05). The water vapour permeability of the films was not significantly different. As the addition of zein hydrolysate and treatment with transglutaminase improved the flexibility of the films, the level of plasticizer required to maintain film flexibility could be reduced without sacrificing their water vapour permeability.     

Spectrophotometric assay using o-phtaldialdehyde for the determination of transglutaminase activity on casein

In this work, the possibility of using a simple and quick method was tested for determining transglutaminase activity on casein using a spectrophotometric assay. The enzyme activity was estimated on the basis of the decrease of o-phthaldialdehyde reactive ε-amino groups of lysine following the formation of isopeptide bonds. The lysine residues involved in the formation of isopeptide bonds when the reaction reaches its plateau are equal to 0.126 μmol per mg of casein. This value results as equal to 0.205 μmol per mg of casein when N-carbobenzoxy-glutaminyl-glycine is added to the reaction medium as a small size acyl group donor. The electrophoretic analysis of the reaction products emphasised a different kinetic formation of casein polymers with the two substrate solutions used. This proposed method has resulted as accurate, with a mean coefficient of variation of 4.6%.     

辅料黏合剂的添加对调理猪排品质的影响

以猪颈背肉为主要原料,研究转谷氨酰胺酶(TG)、酪蛋白酸钠(SC)、复配胶(卡拉胶和魔芋胶1:1)对调理猪排品质的影响。通过单因素实验,研究转谷氨酰胺酶、酪蛋白酸钠、复配胶的添加对调理猪排剪切力、离心损失、蒸煮损失、水分含量、色差、出品率、感官评分的影响。在单因素实验的基础上,进行响应面试验,确定粘合剂的最优添加量。单因素结果表明,TG添加量为0.60%时,保水性及感官评分最高,酪蛋白酸钠添加量为0.10%,或复配胶(卡拉胶和魔芋胶1:1)添加量为0.20%时,既能保证适宜的嫩度与保水性,感官评分也最高。响应面试验结果表明,在转谷氨酰胺酶添加量为0.70%、酪蛋白酸钠添加量为0.07%、复配胶添加量为0.21%时,所加工的调理猪排的感官评分最高,总分60分时,得分为55.54。     

Behaviour of partially cross-linked casein micelles under high pressure

High pressure (HP) treatment of a casein micelle suspension at 250 and 300 MPa leads to an initial rapid increase of its light transmission, as measured in situ , indicating micellar disruption. Subsequently, a much slower, partial reversal of the HP-induced increase in light transmission is observed, indicating re-association of micellar fragments. Partial internal cross-linking of the casein micelles by the enzyme transglutaminase prior to pressure treatment slows down both the disruption and the reassociation process considerably. It is proposed that covalent cross-linking provides the micelle with extra stability against pressure-induced disruption and also prevents a molecular reorganization process required to induce reassociation of micellar fragments during prolonged pressure treatment.