MTGase聚合大豆蛋白及其改性机理(I)MTGase催化大豆蛋白研究

采用SDS-PAGE结合凝胶扫描技术，研究了微生物转谷氨酰胺酶(MTGase)对大豆酸沉蛋白(SAPP)的聚合作用，以及不同酶量、加热或蛋白酶预处理对该聚合反应的影响。结果显示：(1)MTGase较易催化SAPP的大豆球蛋白(glycinin)聚合，而不易使7S球蛋白聚合，而且只能使大豆球蛋白中的酸性亚基聚合，几乎不能使其碱性亚基聚合；(2)随着酶量的增加，MTGase对大豆球蛋白的聚合效果逐渐递增，10～20U／g酶量范围内的聚合效果差不多；(3)加热预处理(100℃，O～45s)可显著地提高MTGase对SAPP的聚合效果；(4)适度的蛋白酶降解处理有利于MTGase对SAPP的聚合，而深度的蛋白酶降解处理则不利。     

转谷氨酰胺酶聚合改性大豆分离蛋白的功能特性研究

研究了转谷氨酰胺酶(MTGase)聚合改性大豆分离蛋白的持水性、吸油性、溶解性、乳化性、发泡性及凝胶强度等功能特性。结果显示,与大豆分离蛋白相比,MTGase改性的大豆分离蛋白(MSPI)具有更高的凝胶性和乳化稳定性,但其溶解性、持水性、吸油性、起泡性与泡沫稳定性和乳化性明显减弱。     

超声波改性与MTGase诱导的大豆分离蛋白凝胶强度的研究

以大豆分离蛋白为原料,采用超声波物理改性处理手段与利用MTGase进行诱导制备凝胶、采用单因素与正交试验设计的方法优化凝胶过程,并对比改性与未改性大豆分离蛋白凝胶强度的差别。试验结果表明:经MTGase诱导未通过经超声处理的SPI样品,诱导时间3 h、诱导温度50℃、p H7.0、MTGase添加量30 U/g,所得大豆分离蛋白的凝胶强为154.32 g;经时间20 min、功率200 W的超声波处理后SPI,诱导时间3 h、诱导温度50℃、p H7.0、MTGase添加量30 U/g、所得凝胶的凝胶强度为165.84 g;经超声改性处理的SPI其凝胶强度明显高于未改性的SPI凝胶强度。     

转谷氨酰胺酶对大豆分离蛋白凝胶性的影响

以微生物来源的转谷氨酰胺酶(MTGase)对大豆分离蛋白(SPI)进行改性,主要考察了凝胶性的变化.结果显示,MTGase对SPI的凝胶性有明显的改善作用,且加酶量、pH、反应温度、底物蛋白浓度及反应时间均对凝胶性影响显著.改性SPI在加酶量为5 U/g、pH 8.0、反应温度为37℃、蛋白浓度为12%时凝胶性改善明显,随着MTGase作用时间的延长,SPI凝胶性也呈增加趋势.MTGase的作用使SPI凝胶的蛋白质分子间形成了空间的网络交错结构.     

微生物谷氨酰胺转移酶对大豆分离蛋白凝胶性能的影响

研究了底物浓度、pH、酶浓度、温度、时间、离子浓度和二巯基苏糖醇(DTT)的添加对微生物谷氨酰胺转移酶(MTGase)诱导的大豆分离蛋白(SPI)凝胶强度的影响。结果表明,在SPI溶液中加入MTGase,可以使体系在低温下形成凝胶;SPI低于8%不能形成凝胶;pH7.0,酶量为30U/g蛋白,50℃水浴加热1h,NaCl为0.6N时,均可获得最高的凝胶强度;添加DTT,对体系无影响。     

大豆蛋白多孔水凝胶机械性能的研究

本研究以大豆分离蛋白为原料,通过微生物转谷氨酰胺酶交联结合高速均质制备了大豆蛋白多孔凝胶,采用X射线断层扫描技术对所成凝胶的微结构进行观察,并考察了不同制备条件对制得凝胶机械性能的影响。结果表明,通过对凝胶的制备条件-酶添加量、蛋白热变性温度及均质速度进行调控,可制得具有不同孔隙度及机械性能的多孔凝胶。当对分离蛋白溶液在95℃热处理30min,添加6.67 U MTGase/g SPI,在8000 r/min均质30 s,可制得空隙大小分布均匀,具有较好应变、较强应力以及杨氏模量的大豆蛋白多孔凝胶。     

温度及酶量对MTGase催化聚合WPC质构特性的影响

以乳清浓缩蛋白(WPC)为底物,探讨了不同的温度及酶量微生物转谷氨酰胺酶(MTGase)催化聚合WPC对其质构特性的影响.结果表明:MTGase催化聚合WPC,在有还原剂DTT存在的条件下,酶/蛋白质比为10U/g左右时,25～45 ℃的反应温度范围内,可以获得质构特性较好的凝胶.酶/蛋白质比及温度过低或过高,都不利于蛋白凝胶的形成.     

亲水多糖对谷氨酰胺转氨酶交联大豆分离蛋白凝胶特性的影响

目的 基于谷氨酰胺转氨(transglutaminase, TG)酶交联法研究亲水多糖对TG酶交联大豆分离蛋白凝胶特性的影响。方法 以大豆分离蛋白为主要原料,TG酶交联法为基础进行响应面优化,得到最优凝胶弹性的工艺参数,在此参数条件下将大豆分离蛋白与亲水多糖混合,制备亲水多糖-大豆分离蛋白复合凝胶,并对凝胶的质构特性、持水性、热力学性质以及结构进行表征。结果 在酶交联pH 7.3、酶交联时间2.3 h、酶交联温度48℃条件下,制备的大豆分离蛋白凝胶弹性最佳。添加了亲水多糖后,凝胶的质构特性和持水性显著提高,热稳定性增强,蛋白质二级和三级结构发生变化,凝胶的微观结构变得更致密,孔径变小。结论 亲水多糖的添加能够改善大豆分离蛋白的凝胶特性,该研究为大豆分离蛋白凝胶深加工提供了理论基础。     

微生物转谷氨酰胺酶(MTGase)的蛋白质底物催化特性及其催化机理研究 (I)MTGase催化单底物蛋白质的聚合特性

采用SDS-PAGE结合凝胶成像技术比较了MTGase在还原状态下对酪蛋白酸钠(SC)、牛血清蛋白(BSA)、大豆球蛋白(glycinin)和β-伴豆球蛋白(β-conglycinin)、β-乳球蛋白(β-LG)和α-乳白蛋白(α-LA)等单底物蛋白的聚合效率。结果表明MTGase较易催化SC和BSA聚合,其次为大豆球蛋白,而β-伴豆球蛋白、β-LG和α-LA最不易。根据MTGase催化不同单底物蛋白质的聚合速率的差异,对MTGase催化单底物蛋白质的聚合特性进行了探讨,指出:①底物蛋白的分子结构对MTGase催化活性的重要性;②蛋白质表面疏水度对MTGase催化活性的重要性;③对蛋白质进行一定的预处理可增强MTGase对蛋白质(特别是球蛋白)的催化活性。     

菜豆属豆类蛋白酶促凝胶性能比较研究

研究比较芸豆(KPI)、红豆(RPI)和绿豆(MPI)3 种菜豆属类分离蛋白和大豆分离蛋白(SPI)在微生物转谷氨酰胺酶(MTGase)作用下的凝胶性能,并对其凝胶形成机理加以分析。SDS-PAGE 电泳和哈克流变分析结果表明：菜豆类分离蛋白是MTGase 的良好作用底物,初始成胶所需时间：KPI ＜ MPI ＜ SPI ＜ RPI。而酶反应后的凝胶G′值：RPI ＞ MPI ＞ KPI ＞ SPI,即KPI 形成凝胶最快,而RPI 最终成胶的强度最大;通过对不同溶剂下胶体溶解性的比较发现,除了酶共价交联外,静电相互作用,疏水作用及氢键都是形成凝胶的主要作用力,由于菜豆属类蛋白主要富含7S 球蛋白(不含二硫键),二硫键对其凝胶的稳定性作用不如SPI 明显。     

大豆分离蛋白对微生物转谷氨胺酶诱导鲢鱼糜凝胶形成的影响

本实验通过对鲢鱼糜凝胶特性和溶解率的测定及SDS-PAGE、扫描电镜观察,研究大豆分离蛋白(SPI)对微生物转谷氨酰胺酶(MTGase)诱导鱼糜凝胶形成的影响及作用机理。结果表明：SPI 和MTGase 均能显著提高鱼糜凝胶特性,但SPI 的添加会阻碍MTGase 对肌球蛋白重链(MHC)的交联,降低鱼糜凝胶特性,增加溶解率。SPI改善鲢鱼糜凝胶特性的机理是自身的凝胶作用和抑制蛋白酶活性。     

Effects of ribose, microbial transglutaminase and soy protein isolate on physical properties and in-vitro starch digestibility of yellow noodles

Soy protein isolate (SPI), microbial transglutaminase (MTGase) and ribose (R) were used to modify physical properties and in-vitro starch hydrolysis of yellow noodle. Four types of noodles were produced; noodles with SPI (SPI/C noodles), noodles with SPI and ribose (SPI/R noodles), noodles with SPI and microbial transglutaminase (SPI/MTGase noodles) and noodles with SPI, ribose and MTGase (SPI/R/MTGase noodles). γ-glutamyl-lysine bonds by MTGase and ribose-induced Maillard reaction within SPI were induced by incubating the noodles for 5 h at 40 °C followed by steaming for 30 min. Cooked noodles were assessed for physical properties such as pH, color, tensile strength and elasticity, and in-vitro hydrolysis index (HI) and estimated glycemic index (GI). SPI/R/MTGase and SPI/MTGase noodles exhibited significantly (P < 0.05) higher tensile strength and elasticity than SPI/R and SPI/C noodles. HI and GI were in the order; SPI/R/MTGase < SPI/MTGase < SPI/R < SPI/C noodles. Incorporation of SPI that was treated with MTGase and ribose may be useful for controlling the texture and starch hydrolysis of yellow noodles. These attributes may be due to the formation of γ-glutamyl-lysine bonds during incubation of SPI, and ribose-induced Maillard reaction during steaming of the noodles.     

COAGULATION AND GELATION OF SOY PROTEIN ISOLATES INDUCED BY MICROBIAL TRANSGLUTAMINASE

The reaction process and corresponding mechanism of coagulation and gelation of native soy protein isolates (SPIs) induced by microbial transglutaminase (MTGase) were investigated. The protein constituents of SPIs, including a majority of subunits of β‐conglycinin and acidic subunits of glycinin, could be polymerized by MTGase to form high weight molecular (WM) biopolymers. Both the coagulation and gelation reactions of native SPI solutions induced by MTGase were dependent upon the initial protein substrate concentration ([C] ₀ ). In the coagulating reactions, the turbidity of SPI solutions continually increased with increasing [C] ₀ in the range from 0.25 to 3.0%. As for the gelation reactions, with the concentration increasing from 3 to 8% (w/v), the onset time of gelation of native SPIs induced by 0.8 units/mL of MTGase at 37C shortened by ∼5‐fold, and the storage modulus (G′) of finally formed gels (after 4 h) increased from ∼1 to 1300 Pa. Both the coagulation and gelation reactions of SPI solutions were promoted remarkably by increasing the enzyme concentration. Sodium Dodecyl Sulfate‐Polyacrylamide Gel Electrophoresis analysis showed that the protein constituents of MTGase‐induced aggregates of SPI (2% w/v) were mainly composed of basic subunits of glycinin and some of newly cross‐linked high MW biopolymers. The solubility analysis of protein constituents indicated that the covalent cross‐linkage, hydrophobic and H bindings and disulfide bonds were mainly involved in the coagulation of SPI induced by MTGase.     

Formation and properties of glycinin-rich and β-conglycinin-rich soy protein isolate gels induced by microbial transglutaminase

The gelation and gel properties of glycinin-rich and β-conglycinin-rich soy protein isolates (SPIs) induced by microbial transglutaminase (MTGase) were investigated. At the same enzyme and protein substrate concentrations, the on-set of gelation of native SPI and the viscoelasticity development of correspondingly formed gels depended upon the relative ratio of glycinin to β-conglycinin. The turbidity analysis showed that the glycinin components also contributed to the increase in the turbidity of SPI solutions incubated with MTGase (at 37 °C). Textural profile analysis indicated that the glycinin components of SPIs principally contributed to the hardness, fracturability, gumminess and chewiness values of corresponding gels, while the cohesiveness and springness were mainly associated with the β-conglycinin components. The strength of MTGase-induced gels of various kinds of SPIs could be significantly improved by the thermal treatment. The protein solubility analyses of MTGase-induced gels, indicated that besides the covalent cross-links, hydrophobic and H-bondings and disulfide bonds were involved in the formation and maintenance of the glycinin-rich SPI gels, while in β-conglycinin-rich SPI case, the hydrophobic and H-bondings were the principal forces responsible for the maintenance of the gel structure. The results suggested that various kinds of SPI gels with different properties could be induced by MTGase, through controlling the glycinin to β-conglycinin ratio.     

Physicochemical properties of soy protein isolate gels emulsified with various oils using a microbial transglutaminase

Soy protein isolate (SPI) gels emulsified with oils including soybean, olive, palm, and eicosapentaenoic acid (EPA) were prepared by a microbial transglutaminase (MTGase). The hardness of 10% SPI gel was greatly increased by adding higher amount of oils. The emulsion gels prepared with 10% SPI and 30% olive oil showed the highest hardness of 1,711 g. In the gelation with various oil content (5–30%), the higher concentration of oils indicated the drastic increase of elastic modulus G′ and viscous modulus G″ during initial gelation for 7 min. The G′ value of SPI emulsion gel showed the 150 (soybean oil 30%), 147 (olive oil 30%), 121 (palm oil 30%), and 61 Pa (EPA 25%), respectively. In the color value of SPI emulsion gel, addition of higher concentration of oils resulted in the increase of L value (brightness), indicating 99.14 (L value) at 30% palm oil. The micro-structure of SPI emulsion gel entrapped with various oils showed the homogeneous network with small porosity compared with that of SPI gel without oil. In particular, SPI emulsion gel with 10% palm oil showed the compact structure distributed evenly with small porosity. Conclusively, the functional and rheological properties of SPI emulsion gel produced by catalytic action of MTGase could be modulated by the type and content of oils fortified.     

干法糖基化改性提高大豆分离蛋白的乳化性

在干热条件下,大豆分离蛋白与葡聚糖两种大分子通过Maillard反应进行共价键合,以共价物的乳化活性为指标,确定影响糖基化蛋白乳化活性的因素依次为:反应温度＞反应时间＞pH＞底物配比,最佳工艺条件为:反应温度70℃,反应时间24 h,糖-蛋白(2:1),pH 8.0.以共价物的乳化稳定性为指标,确定了影响糖基化蛋白乳化稳定性的因素依次为:底物配比＞反应时间＞反应温度＞pH.最佳工艺条件为:糖-蛋白(3:1),反应时间24 h,反应温度70℃,pH8.0.通过聚丙烯酰胺凝胶电泳验证了大豆分离蛋白与葡聚糖发生了接枝反应.     

INFLUENCE OF TRANSGLUTAMINASE-INDUCED CROSS-LINKING ON IN VITRO DIGESTIBILITY OF SOY PROTEIN ISOLATE

The influence of covalent cross‐linking by microbial transglutaminase (MTGase) on the sequential in vitro pepsin and trypsin digestion process and the digestibility of soy protein isolate (SPI), was investigated by sodium dodecylsulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) and nitrogen release analyses. Various subunits of β‐conglycinin and acidic subunits of glycinin were cross‐linked by MTGase to form high molecular weight (MW) biopolymers, while basic subunits of glycinin were unaffected. SDS‐PAGE analysis indicated that the cross‐linking mainly affected in vitro pepsin digestion pattern of various subunits of β‐conglycinin, while the trypsin digestion pattern of native SPI was nearly unaffected. Nitrogen release analysis showed that the in vitro pepsin or/and trypsin digestibility of native SPI (at the end of pepsin or trypsin ingestion) was significantly decreased (P ≤ 0.01) by the MTGase treatment (for more than 2 h). The cross‐linking by MTGase also significantly decreased the in vitro digestibility of preheated SPI. These results suggest that the cross‐linking by means of transglutaminase may negatively affect the nutritional properties of food proteins.     

MTGase聚合大豆蛋白及其改性机理 （II）MTGase聚合改性大豆蛋白研究

研究了微生物转谷氨酰胺酶(MTGase)聚合作用对大豆酸沉蛋白(SAPP)的溶解性能、乳化及起泡性能、凝胶性以及持水性能等功能特性的影响。结果显示：1)显著地提高了SAPP对pH的稳定性，MTGase催化SAPP(1％)聚合4h可获得较好的pH稳定性，然而降低了sAPP的溶解性能(除等电点附近有点增加之外)；2)降低了SAPP的乳化能力，然而其乳化稳定性稍有增加；3)对sAPP的起泡能力影响不大，然而可显著地改善泡沫稳定性。SAPP经MTGase聚合2h的样品泡沫稳定性最佳；4)显著地提高SAPP的凝胶性能；5)DSC分析结果表明。MTGase显著地提高SAPP的热变性温度，或者显著地改善SAPP的水化性能：     

糖基化大豆分离蛋白对肌原纤维蛋白功能特性的影响

选择70℃条件下反应4 h所得到的糖基化大豆分离蛋白样品(蛋白与葡萄糖质量比1∶1)与肌原纤维蛋白以不同比例(9∶1、8∶2、7∶3、6∶4、5∶5)进行复配,测定不同复合蛋白的乳化性能、浊度、表面疏水性、凝胶特性(质构特性、白度值、微观结构分析),探讨糖基化大豆分离蛋白对肌原纤维蛋白功能性质的影响及其机理。结果表明:复合蛋白的乳化性和表面疏水性均较肌原纤维蛋白显著提升(P0.05);复合蛋白的浊度随着加热温度的升高(30~80℃)而不断增加,随着糖基化大豆分离蛋白比例的加大,浊度不断变小;肌原纤维蛋白与糖基化大豆分离蛋白混合凝胶的硬度和弹性均显著优于其与天然大豆分离蛋白混合凝胶(P0.05),微观结构比其与天然大豆分离蛋白混合凝胶更加致密均匀;与纯肌原纤维蛋白凝胶相比,混合凝胶的白度值下降,但混合比例为9∶1时白度值下降不显著(P0.05)。     

Modification in physical properties of rice gel by microbial transglutaminase

BACKGROUND: Microbial transglutaminase (MTGase) may catalyse the cross‐linking between a peptide‐bound glutaminyl residue and an ε‐amino group of lysine residue in protein. MTGase has been used to modify many food proteins for improving the physical properties of products. However, its effect on the physical properties of rice products has not been investigated before. The present study aimed to investigate the effect of MTGase, as an additive in rice flour, on the rheological, textural and thermal properties of rice gel. RESULTS: Both the elastic and the viscous moduli of rice gel were increased as a result of the addition of MTGase to rice flour. The addition of MTGase at 0.01–0.3 U mg^?1 increased textural parameters (hardness and gumminess) and rheological properties. Differential scanning calorimetry also showed that MTGase treatment decreased the enthalpy change in gelation. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) confirmed that rice proteins were polymerised through the MTGase reaction. CONCLUSION: The present study proved that the cross‐linking of protein molecules in rice flour by the action of MTGase may improve the physical properties of rice gel. The addition of MTGase in rice flour in an adequate amount is essential for achieving appropriate physical properties of the product. Copyright © 2008 Society of Chemical Industry