Improvement of canola protein gelation properties through enzymatic modification with transglutaminase

Enzymatic modification with transglutaminase (TG) was used to enhance the gelation of canola protein isolate (CPI) and thus improve its potential as a food ingredient. The effects of CPI concentration, TG concentration, treatment temperature and treatment time on CPI gelation properties were evaluated. A texture analyzer, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and scanning electron microscopy were used to characterize the resulting networks. The protein concentration, amount of TG, and treatment temperature were found to significantly impact gel strength. It was found that gelation can be improved by increasing the amounts of protein and TG and by keeping the treatment temperature close to 40 °C. SDS-PAGE showed that cross-linking of subunits occurred through TG treatment thus helping to explain the increase in gel strength observed during texture analysis. Micrographs further corroborated the trends noted during rheological studies.     

超声-转谷氨酰胺酶改善红豆蛋白功能性质及结构

采用超声联合转谷氨酰胺酶（transglutaminase,TG）处理红豆分离蛋白（red bean protein isolate,RBPI）,对其功能性质和结构特征进行分析,以探究其结构修饰与功能性质的构效关系。结果表明：超声处理5 min能够使RBPI的乳化活性和发泡能力提高,但会降低泡沫稳定性,对乳液稳定性没有显著影响,同时增加表面疏水性和游离巯基含量;TG能够提高RBPI的乳化活性、乳化稳定性及泡沫稳定性,但会降低发泡能力、表面疏水性和游离巯基含量;超声-TG联合处理的RBPI具有更高的乳化活性和泡沫稳定性,更低的表面疏水性和游离巯基含量,且酰胺Ⅰ带处吸收峰强度增加,更多的无规卷曲结构转变为有序的β-折叠结构,这可能是导致RBPI功能性质改善的原因。超声处理5 min联合TG诱导的蛋白凝胶具有更加均匀、致密的微观结构,且凝胶硬度和黏附力增加,脱水收缩作用降低;峰值温度（Tp）和热焓变（ΔH）显著增加（P＜0.05）,改善了RBPI的热稳定性或三级结构稳定性。以上结果表明RBPI经过超声处理后更利于TG对蛋白质的交联作用,超声-TG联合处理促进了蛋白质功能性质的发挥。     

Effect of Trichoderma reesei tyrosinase on rheology and microstructure of acidified milk gels

The aim of this work was to study the potential of tyrosinase enzymes in structural engineering of acid-induced milk protein gels. Fat free raw milk, heated milk or a sodium caseinate solution were treated with tyrosinases from Trichoderma reesei (TrTyr) and Agaricus bisporus (AbTyr) and the reference enzyme transglutaminase (TG) prior to acid-induced gelation. TrTyr treatment increased the firmness of raw milk and sodium caseinate gels, but not that of heated milk gels, even though protein cross-linking was detected in heated milk. AbTyr did not cross-link proteins in any of the studied milk protein systems. TG was superior to TrTyr in gels prepared of heated milk. In acidified heated milk and sodium caseinate, TrTyr and TG treatment resulted in a decrease of the pore size. Scanning electron microscopy revealed more extensive particle interactions in the heated milk gels with TG than with TrTyr.     

Trypsin Treatment to Improve Freeze Texturization of Minced Bream

Mild trypsin proteolysis (enzyme concentration 0.05% at 15°C, pH 7.4; mixing 30 min at 20 rpm) of bream mince improved mince susceptibility to freeze texturization. Formation of porous-fibrous texture of heat-set, freeze-texturized minces was associated with strong fragmentation of myosin heavy chain (MHC), decrease in extractability of myofibrillar protein and increased insoluble protein and thermal drip. Some deamidation of protein was also observed. We concluded that limited trypsin proteolysis accelerated transglutaminase mediated cross-linking of protein by acyl transfer reactions. Protein polymerization through plastein reactions and intermolecular hydrophobic interaction probably also occurred.     

谷氨酰胺转氨酶对山杏仁蛋白凝胶特性的影响

为提高山杏仁蛋白的凝胶特性,采用谷氨酰胺转氨酶(TG酶)作为交联剂,通过单因素实验研究了TG酶添加量、pH、交联温度、交联时间对蛋白凝胶硬度、弹性、内聚力的影响,并以凝胶强度为指标,利用响应面法对山杏蛋白形成条件进行优化。优化的TG酶交联山杏仁蛋白形成凝胶的条件如下:TG酶添加量17 U/g,交联温度43℃,pH为7.2,交联时间2.5 h时,此时凝胶硬度达到(135±7.14) g。结果表明利用TG酶交联山杏仁蛋白形成凝胶具有可行性,为提高山杏仁蛋白的功能性质和应用价值提供参考。     

Hydroxyl Radical and Ferryl-Generating Systems Promote Gel Network Formation of Myofibrillar Protein

ABSTRACT: The objective of the study was to examine how oxidatively induced protein cross-linking would influence the gelation properties of myofibrillar protein (MP) under meat processing conditions. MP suspensions in 0.6 M NaCl at pH 6 were treated with an iron-catalyzed oxidizing system (IOS: 10 μM FeCl₃, 0.1 mM ascorbic acid, 0.05 to 5 mM H₂O₂) or a H₂O₂-activated metmyoglobin oxidizing system (MOS: 0.01 to 0.1 mM metmyoglobin/H₂O₂) that produced hydroxyl radical and ferryl species, respectively. Both oxidizing systems promoted MP thermal gelation, which was evidenced by rapid protein–protein interaction and the enhancement in storage modulus (elasticity) of the gel network as revealed by dynamic rheological testing in the 20 to 74 °C temperature range. This gelation-enhancing effect was attributed to the shift of myosin aggregation in the early stage of heating from predominantly head–head association (nonoxidized control samples) to prevalently tail–tail cross-linking through disulfide bonds. However, both hardness and water-holding capacity of chilled gels tended to decline when MP was exposed to ≥1 mM H₂O₂ in IOS and to all concentrations of metmyoglobin in MOS. Microscopic examination confirmed a more porous structure in oxidized gels when compared with nonoxidized protein gels. The results demonstrated that mild oxidation altered the mode of myosin aggregation in favor of an elastic gel network formation, but it did not improve or had a negative effect on water-binding properties of MP gels. Practical Application: Mild oxidation promotes protein network formation and enhances gelation of myofibrillar protein under normal salt and pH conditions used in meat processing. This oxidative effect, which involves disulfide linkages, is somewhat similar to that in bakery product processing where oxidants are used to improve dough performance through gluten protein interaction.     

Effects of limited proteolysis and high-pressure homogenisation on structural and functional characteristics of glycinin

Glycinin was treated by limited proteolysis and/or high-pressure homogenisation in this work. The combined treatments significantly improved the solubility of glycinin, especially in acidic aqueous solution. Moreover, the modified glycinin had better emulsification capability and lower surface hydrophobicity. Electrophoresis analysis indicated more aggregates were formed after above treatments. But the laser scanning test showed the particle size of glycinin decreased. Compared with limited proteolysis, the combined treatments further increased the storage modulus and postponed the onset of glycinin gelation. The results of scanning electron microscopy showed that a fibrous curly structure was present in native glycinin. It was not found in modified glycinins. High-pressure homogenisation slightly shortened the onset of glycinin gelation, while limited proteolysis apparently postponed this behaviour. All the treatments formed weaker gels than native glycinin.     

酶法提高蛋清蛋白热稳定性的研究

蛋清蛋白由于蛋清蛋白质自身结构的关系使其具有良好的凝胶性,并且蛋清蛋白粉在加热过程中有利于凝胶性的提高。但超速溶蛋白粉制备过程中要求蛋白粉对热稳定,因此本文通过单因素试验和正交试验选出最优工艺破坏蛋清蛋白质的凝胶性,提高它的热稳定性。通过单因素试验选出最佳水解酶为碱性蛋白酶,水解时底物的最佳浓度为6%。并通过正交试验确定最佳工艺条件为:水解温度50~60℃、水解时间4h、pH值8和加酶量5U/mg。     

Xanthan Enhances Water Binding and Gel Formation of Transglutaminase-Treated Porcine Myofibrillar Proteins

ABSTRACT: In this study, the effect of xanthan on dynamic rheological properties, textural profile, and water binding of transglutaminase (TG)-treated myofibrillar protein (MP) gels was investigated. In experiment 1, MP suspensions (40 mg/mL protein, 0.6 M NaCl) at pH 6.45 with or without 0.05% xanthan were treated with 0%, 0.1%, 0.2%, 0.3%, 0.4%, and 0.5% TG; in experiment 2, MP suspensions (40 mg/mL protein, 0.6 M NaCl) at pHs 6.13, 6.30, 6.45, 6.69 with or without 0.05% xanthan were treated with 0.3% TG. Treated samples were analyzed with differential scanning calorimetry for thermal stability and oscillatory rheometry and Instron penetration tests for gelation properties. The TG treatments lowered the transition temperature (T_m) of MP by as much as 6 °C (P < 0.05) but increased apparent enthalpy of denaturation. However, there was no detectable thermal stability difference between MP samples with or without xanthan. The shear storage modulus (G′) of MP gels increased markedly upon treatments with ≥0.3% TG, and the presence of xanthan further enhanced the gel strength (P < 0.05). The addition of 0.05% xanthan decreased cooking loss of TG-treated MP gels by 17% to 23% when compared with gels without xanthan at all pH levels evaluated (6.13 to 6.67). Thus, the combination of TG and xanthan offered a feasible means to promote cross-linking and gelation of MP while reducing cooking losses.     

谷氨酰胺转氨酶对高水分豌豆蛋白挤出物结构及消化特性的影响

本实验以豌豆蛋白为原料,利用双螺杆挤出机制备挤出物,探究高水分挤出过程中添加不同比例的谷氨酰胺转氨酶（transglutaminase,TG）对挤出物结构及消化特性的影响。通过质构分析、组织化度分析和扫描电子显微镜观察等方法表征挤出物的宏观结构和微观结构。通过测定流变特性、蛋白质溶解度以及消化特性评价挤出物的结构性能及营养价值。结果表明,添加适量TG(0.25%～1.00%)促进了豌豆蛋白分子间交联,形成致密的网络结构,提高了挤出物的消化率,促进游离氨基酸释放。然而添加过量TG(2.00%)则对挤出物纤维的形成以及消化特性起到抑制作用。TG添加量为1.00%时,制备的豌豆蛋白高水分挤出物组织化程度与营养价值较好。综上,高水分挤压-TG法联用加工工艺能够有效改善植物蛋白肉的结构及消化特性。     

Effect of transglutaminase on the quality of wheat-based pasta products

Transglutaminase (TG) enzyme was tested in T. aestivum and T. durum wheat-based model systems to evaluate the quality improvement to pasta imparted by cross-linking. Quality was measured by the enhancement in sensory and cooking properties. In this process, tests were also performed to establish the biochemical structure-functions of the proteins involved at TG enzyme concentrations between 10 and 200 mg kg⁻¹. It was observed that the amounts of water/salt-, the alcohol- and the alkali-soluble protein fractions were reduced significantly. Change in the molecular weight distribution assessed by SDS PAGE was also noticed. The sensory properties were observed to improve from high water uptake and low cooking loss. The TG treatment changed the gluten structure in the modified pasta products. Immune-reactive gliadin fractions were detected by immunoblotting and independently measured by competitive indirect ELISA, using the anti-gliadin rabbit IgG.     

Standardized reaction times used to describe the mechanism of enzyme-induced gelation in whey protein systems

Whey protein isolate (WPI), either untreated or pretreated at 80 degrees C for 30 min, was incubated with a proteinase from Bacillus licheniformis until a gel was formed. Standardized reaction times, directly linked to the degree of hydrolysis, were obtained from plots of the relative amount of peptides released v. reaction time obtained under different conditions (enzyme concentration, temperature, pH, NaCl addition). This provided a connection between the gelation profile and the degree of hydrolysis. In the case of untreated WPI, gelation occurred at lower degrees of proteolysis when the enzyme concentration was decreased, demonstrating that a rate-limiting aggregation process occurred at the same time as the proteolysis in a manner similar to the renneting of milk. This was not the case for preheated WPI, when gelation was found to take place at a constant degree of proteolysis, independent of the enzyme concentration. In this case, the mechanism could be described by assuming the thermally induced aggregates present in this substrate had progressively more stabilizing peptide segments shaved off, resulting in increased attraction between individual aggregates that ultimately led to gelation. Results obtained at 40-60 degrees C supported this, as we found no effect of temperature on the degree of proteolysis at gelation for the untreated WPI, whereas the degree of proteolysis decreased with increasing temperature when heated WPI was hydrolysed. The effect of pH and NaCl addition on the process was to reduce repulsion between the aggregating species so that gelation was induced at a decreased degree of proteolysis.     

Covalent chemical modification of myofibrillar proteins to improve their gelation properties: A systematic review

To improve the quality of meat products is a constant focus for both the meat industry and scientists. As major components in meat protein, the gelation properties of myofibrillar proteins (MPs) predominantly determine the sensory quality and product yield of the final product. Naturally or artificially occurring covalent modifications are known to largely affect MP functionality by changing the protein structure and forming aggregates, leading to both favorable and unfavorable outcomes. The review aims to summarize the mechanisms associated with several covalent modifications and the recent developments in enhancing MP gelation properties. Various extrinsic and intrinsic parameters controlling oxidation, phenolic–protein interactions, enzyme catalysis, glycation, and isoelectric solubilization/precipitation, and their effects on the characteristics of heat-induced MP gels are discussed. This article provides an improved understanding of the covalent modifications that occur mainly in the MP system and how they can be utilized to promote its gelation properties. Covalent modifications exhibited dose-dependent and dual-role manners for MP gelation properties. Mild oxidation, enzyme catalysis, and isoelectric solubilization/precipitation treatment would be beneficial to form more aligned and cross-linked three-dimensional networks for MP gels because of moderate protein aggregation. However, an excessive aggregate impedes the MP gelation behavior, leading to reduced gelation quality. Glycation effectively increased hydrophilicity of MPs and phenolic conjugation provides MPs with novel bioactivity. A proper utilization of such a process or even a rational combination of them allowed us to enhance the gelation properties of MP with assorted appreciated functionalities and further improve the quality of meat products.     

Processing and characteristics of canola protein-based biodegradable packaging: A review

Interest increased recently in manufacturing food packaging, such as films and coatings, from protein-based biopolymers. Among various protein sources, canola protein is a novel source for manufacturing polymer films. It can be concentrated or isolated by aqueous extraction technology followed by protein precipitation. Using this procedure, it was claimed that more than 99% of protein was extracted from the defatted canola meal, and protein recovery was 87.5%. Canola protein exhibits thermoplastic properties when plasticizers are present, including water, glycerol, polyethylene glycol, and sorbitol. Addition of these plasticizers allows the canola protein to undergo glass transition and facilitates deformation and processability. Normally, canola protein-based bioplastics showed low mechanical properties, which had tensile strength (TS) of 1.19 to 4.31 MPa. So, various factors were explored to improve it, including blending with synthetic polymers, modifying protein functionality through controlled denaturation, and adding cross-linking agents. Canola protein-based bioplastics were reported to have glass transition temperature, T_g, below ?50°C but it highly depends on the plasticizer content. Canola protein-based bioplastics have demonstrated comparable mechanical and moisture barrier properties compared with other plant protein-based bioplastics. They have great potential in food packaging applications, including their use as wraps, sacks, sachets, or pouches.     

肌原纤维蛋白氧化对谷氨酰胺转移酶介导的交联反应及凝胶性的影响

研究不同盐浓度下肌原纤维蛋白的氧化对后继的微生物谷氨酰胺转移酶(MTG)交联反应及凝胶能力的影响。分散在三种盐浓度(0.15、0.45和0.6mol/L NaCl)下的肌原纤维蛋白在4℃分别氧化2h和24h,然后在0.6mol/L NaCl条件下加入MTG于30℃交联2h。通过电泳和溶解度研究蛋白的氧化对MTG交联反应的影响,通过凝胶强度研究蛋白的氧化对MTG交联反应后凝胶性的影响。研究表明,三种盐浓度下的氧化都会促进MTG交联反应,特别是高盐条件下的氧化(提高量达47.8%)。所有样品经MTG交联后凝胶强度均显著增加(p<0.05),高盐条件下氧化的蛋白其凝胶强度明显高于低盐条件,然而在所有盐浓度下,氧化蛋白的凝胶强度增幅均小于未氧化的。结果表明过多的交联反应反而不利于蛋白成胶性。      

Nanoparticulation of enzymatically cross-linked whey proteins to encapsulate caffeine via microemulsification/heat gelation procedure

A co-surfactant free microemulsion was formulated with sunflower oil, span 80 and whey protein solution and used as nanoreactor to generate caffeine-enveloping capsules through heat gelation of protein. Transglutaminase-induced cross-linking of proteins prior to microemulsification decreased the mean diameter from 478 to 318 nm for core-free particles and from 232 to 118 nm for capsules. As well, the lower limit of capsules size decreased from 78 nm to 45 nm due to enzymatic cross-linking. Scanning electron microscopy showed that morphology of particles and capsules was not completely spherical which was attributed to the protrusion of protein molecules out of aqueous droplets during gelation. The enzymatic treatment yielded in particles with higher glass transition temperature due to the reinforced structure of particulate gel. Fourier transform infrared spectroscopy confirmed the structural changes in proteins by heat and establishment of covalent cross-linkages by the enzyme action manifested by a band at 1078 cm⁻¹.     

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。     

Transglutaminase effects on gelation capacity of thermally induced beef protein gels

Transglutaminase (TG) is an enzyme that catalyzes an acyl-transfer reaction between the γ-carboxyamide group of peptide or protein-bound glutaminyl residues, and primary amines. TG action on protein molecules, causes a cross-linking and polymerizing effect of these latter, through ε-(γ-glutamyl)lysine bonds. This TG-mediated protein cross-linking creates drastic physical changes in protein-rich foods.     

Invited review: Plasmin protease in milk: current knowledge and relevance to dairy industry

Plasmin is by far the predominant and most completely studied endogenous protease in bovine milk. Plasmin-induced proteolysis can have either beneficial or detrimental effects on the texture and flavor of dairy products, depending on the extent of hydrolysis and type of dairy product. In cheese, the breakdown of protein can help develop desirable flavors and texture during ripening, whereas in pasteurized milk and ultra-high-temperature milk, proteolysis causes undesirable gelation. Plasmin is part of a complex protease-protease inhibitor system in milk that consists of active and inactive forms of the enzyme, activators, and inhibitors. Considerable research has been done to isolate and characterize components of the plasmin system, determine how they interact, develop and compare quantitation methods, and determine how they are affected by cow characteristics, processing conditions, other milk components, storage conditions, and bacterial proteases. Considerable research has focused on enhancing or minimizing the activity of plasmin system components. The intent has been to control protease activity in casein and whey fractions, depending on the final food or ingredient application. Controlling the activity of plasmin has a great potential to improve dairy product quality and reduce their processing costs.     

Modification of rheological properties of whey protein isolates by limited proteolysis

Whey protein isolate (WPI) was subjected to limited tryptic hydrolysis and the effect of the limited hydrolysis on the rheological properties of WPI was examined and compared with those of untreated WPI. At 10% concentration (w/v in 50 mM TES buffer, pH 7.0, containing 50 mM NaCl), both WPI and the enzyme-treated WPI (EWPI) formed heat-induced viscoelastic gels. However, EWPI formed weaker gels (lower storage modulus) than WPI gels. Moreover, a lower gelation point (77 °C) was obtained for EWPI as compared with that of WPI which gelled at 80 °C only after holding 1.4 min. Thermal analysis and aggregation studies indicated that limited proteolysis resulted in changes in the denaturation and aggregation properties. As a consequenece, EWPI formed particulated gels, while WPI formed fine-stranded gels. In keeping with the formation of a particulate gel, Texture Profile Analysis (TPA) of the heat-induced gels (at 80 °C for 30 min) revealed that EWPI gels possessed significantly higher (p < 0.05) cohesiveness, hardness, gumminess, and chewiness but did not fracture at 75% deformation. The results suggest that the domain peptides, especially β-lactoglobulin domains released by the limited proteolysis, were responsible for the altered gelation properties.