Transglutaminase Cross-linking Research Articles

Improved properties of gelatin films involving transglutaminase cross-linking and ethanol dehydration: The self-assembly role of chitosan and montmorillonite

In this study, the chitosan and montmorillonite (MMT) were synergistically introduced to improve the properties of the gelatin films through self-assembly. The preparation of gelatin films (G), gelatin + MMT composite films (G + MMT), gelatin-chitosan bilayer films (G-CS), and gelatin + MMT-chitosan composite bilayer films (G + MMT-CS) involved transglutaminase (TG) cross-linking followed by ethanol precipitation and self-assembly process. The water contact angle of the composite bilayer films (G-CS and G + MMT-CS) was higher than 110° due to the formation of surface hydrophobic layer formed by self-assembly via hydrogen bonding and electrostatic interaction between gelatin and chitosan, but the MMT alone cannot improve the hydrophobicity of gelatin films. The water solubility (WS) of G + MMT, G-CS and G + MMT-CS films was significantly lower than that of G films. The mechanical and barrier properties were significantly improved due to the introduction of MMT and the surface deposition of chitosan, attributing to the increase of triple helix content and formation of bilayer structure, respectively. Moreover, MMT caused the decrease of sterilization thermal shrinkage from 49.54% to 34.00%. Interestingly, MMT facilitated the surface deposition of chitosan during self-assembly process. The mold growth of bread wrapped with G + MMT-CS was effectively inhibited during storage, implying potential application as antimicrobial food packaging materials. • Self-assembly of gelatin and chitosan improved hydrophobicity of gelatin (G) films. • Chitosan and montmorillonite improved mechanical and barrier properties of G films. • Sterilization thermal shrinkage of G films was reduced by montmorillonite. • Introducing montmorillonite facilitated the self-assembly deposition of chitosan.

Functional and physiochemical properties of the yoghurt modified by heat lactosylation and microbial transglutaminase cross-linking of milk proteins.

This study aimed to recognize the effect of Maillard reaction (MR) on the functional properties of milk proteins and the physiochemical, textural, and sensory properties of yoghurt. Heating at 100°C for 2h increased the carbohydrate ratio in caseins, whey proteins, and total milk proteins from 2.83%, 1.93%, and 1.8% to 4.15%, 3.58%, and 5.32%, respectively. Solubility of the lactosylated caseins, whey proteins, and total milk proteins is increased at low pH values compared to that of the control caseins, whey proteins, and total milk proteins. Lactosylation at 70 and 100°C increased the emulsion activity index (EAI) of caseins at all pH values, especially at pH below 6, and this increment was higher for casein samples treated at 100°C. Foam volume of whey proteins and total milk proteins also increased for samples lactosylated at 100°C compared to control samples. The combination of heating and microbial transglutaminase (MTGase) had a synergistic and enhancing effect on the pH values of yoghurt samples, especially in yoghurt samples produced by whole milk protein compared to control samples. Viscosity and hardness of yoghurt samples were enhanced by heat lactosylation, MTGase treatment, and also storage for 21 days at 7 ± 1°C.

Unaided efficient transglutaminase cross-linking of whey proteins strongly impacts the formation and structure of protein alginate particles

There is a dogma within whey protein modification, which dictates the necessity of pretreatment to enzymatic cross-linking of β-lactoglobulin (β-Lg). Here microbial transglutaminase (MTG) cross-linked whey proteins and β-Lg effectively in 50 mM NaHCO3, pH 8.5, without pretreatment. Cross-linked β-Lg spanned 18 to >240 kDa, where 6 of 9 glutamines reacted with 8 of 15 lysines. The initial isopeptide bond formation caused loss of β-Lg native structure with t1/2 = 3 h, while the polymerization occurred with t1/2 = 10 h. Further, cross-linking effects on protein carbohydrate interaction have been overlooked, leaving a gap in understanding of these complex food matrices. Complexation with alginate showed that β-Lg cross-linking decreased onset of particle formation, hydrodynamic diameter, stoichiometry (β-Lg/alginate) and dissociation constant. The complexation was favored at higher temperatures (40 °C), suggesting that hydrophobic interactions were important. Thus, β-Lg was cross-linked without pretreatment and the resulting polymers gave rise to altered complexation with alginate.

Transglutaminase-Induced Free-Fat Yogurt Gels Supplemented with Tarragon Essential Oil-Loaded Nanoemulsions: Development, Optimization, Characterization, Bioactivity, and Storability.

There is a high demand for designing healthy-functional dairy gels with a newly structured protein network in the food industry. Non-fat yogurt gels enriched with stable tarragon essential oil-nanoemulsions (TEO-NEs) using crosslinking of microbial transglutaminase (MTGase) were developed. The gas chromatography-mass spectrometry analysis showed that methyl chavicol (85.66%) was the major component in TEO extracted by the hydrodistillation process. The storage-dependent droplet size and physicochemical stability data of samples at room temperature for 30 days revealed that the TEO-NE containing 0.5% tween-80 and 1:2 TEO/sunflower oil had the lowest peroxide value and droplet growth ratio. The response surface methodology-based formulation optimization of free-fat yogurt gels using MTGase (0.15–0.85 U/g) and the best TEO-NE (0.5–3.02%) using the fitted second-order polynomial models proved that the combination of 0.87% TEO-NE and 0.70 U/g MTGase led to the desired pH (4.569) and acidity (88.3% lactic acid), minimum syneresis (27.03 mL/100 g), and maximum viscosity (6.93 Pa s) and firmness (0.207 N) responses. Scanning electron microscopy images visualized that the MTGase-induced crosslinks improved the gel structure to increase the firmness and viscosity with a reduction in the syneresis rate. The optimal yogurt gel as a nutritious diet not only provided the highest organoleptic scores but also maintained its storage-related quality with the lowest mold/yeast growth and free-radical oxidation changes.

Effects of phosphorylation pretreatment and subsequent transglutaminase cross-linking on physicochemical, structural, and gel properties of wheat gluten

The presence of a large number of hydrophobic groups and non-polar amino acids in the wheat gluten (WG) is responsible for its poor water solubility, greatly limiting its industrial applications. Our results showed that the solubility and zeta potential of WG were significantly (P < 0.05) improved with the increasing concentration of sodium tripolyphosphate (STP), while the average particle size of WG was decreased. After WG was incubated with TGase, phosphorylation pretreatment significantly increased apparent viscosity of WG dispersant solution, suggesting that phosphorylation treatment promoted the generation of cross-linked polymers. In addition, phosphorylation pretreatment enhanced hydrophobic interactions and disulfide bond formation between TGase-induced WG gels, thus leading to a more homogeneous and dense three-dimensional network structure of gel, which was confirmed by SEM micrographs. To summarize, STP can be used as an effective additive for the modification of WG with an improved degree of TGase-mediated cross-linking for better rheological and gel properties.

Effect of transglutaminase crosslinking on the structural, physicochemical, functional, and emulsion stabilization properties of three types of gelatins

Molecular modification has attracted many attentions in the field of gelatin science. In this work, a transglutaminase (TG) modification method was developed and applied to modify bovine bone gelatin, porcine skin gelatin, and cold-water fish skin gelatin, and then the effect of TG crosslinking on the structural, physicochemical, functional, and emulsion stabilization properties of three types of gelatins were explored and compared. The results demonstrated that TG modification increased the nanoparticle formation behaviors and change the physicochemical and functional properties of three types of gelatins. Further, TG-modified aquatic gelatin showed comparable foaming properties (147% ± 6% for foaming capability and 55% ± 4% for foam stability) and higher emulsifying properties (18 ± 1 m2/g for emulsion activity index and 142 ± 11 min for emulsion stability index) to unmodified mammalian gelatins. In addition, TG-modified gelatins decreased the droplet sizes and increased creaming stability of fish oil-loaded emulsions compared with unmodified gelatins. All these results suggested TG modification was an efficient molecular modification method for gelatins. Moreover, TG-modified aquatic gelatin could be potential mammalian gelatin replacer. This work could provide useful information to understand the relationship among animal sources, tissue sources, extraction methods, amino acid compositions, molecular modification, molecular structure, physicochemical properties, functional properties, and emulsification stability of gelatins.

Preparation of Natural Food-Grade Core-Shell Starch/Zein Microparticles by Antisolvent Exchange and Transglutaminase Crosslinking for Reduced Digestion of Starch.

The purpose of this study was to slow down the digestibility of starch granules by encapsulating it in zein shells. Drop of the preformed swollen corn starch (CS) granule suspension into thermal-treated zein ethanolic solution enables antisolvent precipitation of thermal-treated zein on the surface of the preformed swollen CS granules, leading to the formation of core-shell starch/zein microparticles. Confocal laser scanning microscopy images showed that the preformed swollen CS granules were coated by thermal-treated zein shells with a thickness of 0.48–0.95 μm. The volume average particle diameter of core-shell starch/zein microparticles was 14.70 μm and reached 18.59–30.98 μm after crosslinking by transglutaminase. The results of X-ray diffraction and Fourier transform infrared spectroscopy demonstrated that an interaction occurred between the preformed swollen CS granules and the thermal-treated zein. The results for thermodynamic characteristics, pasting properties, and swelling power indicated that the compact network structure of core-shell starch/zein microparticles crosslinked by transglutaminase could improve starch granule thermal stability and resistance to shearing forces. Compared to native CS, the peak gelatinization temperatures of core-shell starch/zein microparticles increased significantly (p < 0.05), with a maximum value of 76.64°C. The breakdown values and the swelling power at 95°C of core-shell starch/zein microparticles significantly (p < 0.05) decreased by 52.83–85.66% and 0.11–0.28%, respectively. The in vitro digestibility test showed that the contents of slowly digestible starch and resistant starch in the core-shell starch/zein microparticles increased to ∼42.66 and ∼34.75%, respectively, compared to those of native CS (9.56 and 2.48%, respectively). Our research supports the application of food-grade core-shell starch/zein microparticles to formulate low-digestibility food products.

Combination of microwave heating and transglutaminase cross-linking enhances the stability of limonene emulsion carried by whey protein isolate

In this study, we examined the effect of transglutaminase (TG) cross-linking time (0 h, 2 h, 4 h, 8 h, and 12 h) on the stability of limonene emulsion loaded by microwave-heated WPI (MWPI). Size exclusion chromatography results showed that molecular weight of TG-treated MWPI and WPI was enhanced with the increase in TG cross-linking time (0–12 h). Microwave and TG cross-linking decreased particle size (7.55%) and increased ζ-potential (5.33%) and protein adsorption (4.58%) of WPI emulsion at TG cross-linking time of 12 h. CLSM results showed smaller droplet size of MWPI-TG emulsion and its uniform size. The obtained limonene emulsion loaded by MWPI-TG at 12 h showed the lowest creaming index and the strongest oxidative stability under aerobic conditions after 10thdays of storage. Thus, the combination of microwave heating and TG cross-linking effectively enhanced the stability of limonene emulsion stabilized by WPI and facilitating the application of WPI in the fields of food, medicine, cosmetics, and other areas.

Improving functional properties of pea protein through “green” modifications using enzymes and polysaccharides

Pea proteins have gained significant interest in recent years. The objective of this study was to enhance pea protein functional properties through enzymatic and/or conjugation modifications and understand the physicochemical properties of the modified proteins. Molecular changes of the proteins were characterized, and protein functionality, in vitro digestibility, and sensory properties were analyzed. The proteins crosslinked with transglutaminase showed significantly improved water holding capacity (5.2–5.6 g/g protein) compared with the control pea protein isolate (2.8 g/g). The pea proteins conjugated with guar gum showed exceptional emulsifying capacity (EC) and stability (ES) of up to 100% compared with the control protein (EC of 58% and ES of 48%). Some sequentially modified pea proteins, such as transglutaminase crosslinking followed by guar gum conjugation had multiple functional enhancement (water holding, oil holding, emulsifying, and gelation). The functionally enhanced pea proteins had comparable sensory scores as the control protein.

Transglutaminase Cross-Linking of Whey Proteins Affects Alginate Complexation

Transglutaminase Cross-Linking of Whey Proteins Affects Alginate Complexation

Effect of the Order of Transglutaminase Cross-Linking and Emulsification on the Stability and Digestion Characteristics of Limonene Emulsion Carried by Whey Protein Isolate

Effect of the Order of Transglutaminase Cross-Linking and Emulsification on the Stability and Digestion Characteristics of Limonene Emulsion Carried by Whey Protein Isolate

Effects of the Order of Transglutaminase Cross-Linking and Emulsification on the Stability and Digestion Characteristics of Limonene Emulsion Carried by Whey Protein Isolate

Effects of the Order of Transglutaminase Cross-Linking and Emulsification on the Stability and Digestion Characteristics of Limonene Emulsion Carried by Whey Protein Isolate

Influence of Transglutaminase Crosslinking on Casein Protein Fractionation during Low Temperature Microfiltration.

Low temperature microfiltration (MF) is applied in dairy processing to achieve higher protein and microbiological quality ingredients and to support ingredient innovation; however, low temperature reduces hydrophobic interactions between casein proteins and increases the solubility of colloidal calcium phosphate, promoting reversible dissociation of micellar β-casein into the serum phase, and thus into permeate, during MF. Crosslinking of casein proteins using transglutaminase was studied as an approach to reduce the permeation of casein monomers, which typically results in reduced yield of protein in the retentate fraction. Two treatments (a) 5 °C/24 h (TA) and (b) 40 °C/90 min (TB), were applied to the feed before filtration at 5 °C, with a 0.1 µm membrane. Flux was high for TA treatment possibly due to the stabilising effect of transglutaminase on casein micelles. It is likely that formation of isopeptide bonds within and on the surface of micelles results in the micelles being less readily available for protein-protein and protein–membrane interactions, resulting in less resistance to membrane pores and flow passage, thereby conferring higher permeate flux. The results also showed that permeation of casein monomers into the permeate was significantly reduced after both enzymatic treatments as compared to control feed due to the reduced molecular mobility of soluble casein, mainly β-casein, caused by transglutaminase crosslinking.

Effects of transglutaminase cross-linking process on printability of gelatin microgel-gelatin solution composite bioink

Three-dimensional (3D) bioprinting has emerged as a powerful engineering approach for various tissue engineering applications, particularly for the development of 3D cellular structures with unique mechanical and/or biological properties. For the jammed gelatin microgel-gelatin solution composite bioink, comprising a discrete phase of microgels (enzymatically gelled gelatin microgels) and a cross-linkable continuous gelatin precursor solution-based phase containing transglutaminase (TG), its rheological properties and printability change gradually due to the TG enzyme-induced cross-linking process. The objective of this study is to establish a direct mapping between the printability of the gelatin microgel-gelatin solution based cross-linkable composite bioink and the TG concentration and cross-linking time, respectively. Due to the inclusion of TG in the composite bioink, the bioink starts cross-linking once prepared and is usually prepared right before a printing process. Herein, the bioink printability is evaluated based on the three metrics: injectability, feature formability, and process-induced cell injury. In this study, the rheological properties such as the storage modulus and viscosity have been first systematically investigated and predicted at different TG concentrations and times during the cross-linking process using the first-order cross-linking kinetics model. The storage modulus and viscosity have been satisfactorily modeled as exponential functions of the TG concentration and time with an experimentally calibrated cross-linking kinetic rate constant. Furthermore, the injectability, feature formability, and process-induced cell injury have been successfully correlated to the TG concentration and cross-linking time via the storage modulus, viscosity, and/or process-induced shear stress. By combing the good injectability, good feature formability, and satisfactory cell viability zones, a good printability zone (1.65, 0.61, and 0.31 h for the composite bioinks with 1.00, 2.00, and 4.00% w/v TG, respectively) has been established during the printing of mouse fibroblast-based 2% gelatin B microgel-3% gelatin B solution composite bioink. This printability zone approach can be extended to the use of other cross-linkable bioinks for bioprinting applications.

Low temperature extrusion promotes transglutaminase cross-linking of whey protein isolate and enhances its emulsifying properties and water holding capacity

Impact of extrusion pretreatment (50, 70, 90, 110 and 130 °C) on physicochemical, emulsifying properties and water holding capacity (WHC) of transglutaminase (TGase) cross-linked whey protein isolate (WPI) were investigated in this study. The results of molecular size distribution and SDS-PAGE proved that there were more macromolecular polymers formed in TGase cross-linked WPI after extrusion pretreatment (E-WPI-TGase) and the consumption of free amino groups for E-WPI-TGase was the largest at the extrusion temperature of 50 °C and 70 °C. As the extrusion temperature increased from 90 °C to 130 °C, particle size of WPI after extrusion pretreatment (E-WPI) was not changed significantly, compared with that of E-WPI-TGase (p > 0.05). In addition, the emulsifying properties, surface hydrophobicity and WHC of E-WPI-TGase were higher than those of WPI cross-linked by TGase (WPI-TGase) (p < 0.05). Significantly, the emulsifying activity, surface hydrophobicity and WHC of E-WPI-TGase at the extrusion temperature of 50 °C was increased by 63.78%, 18.58% and 96.63% compared with that of unextruded WPI-TGase, respectively. Therefore, these results indicated that low temperature (50 °C and 70 °C) extrusion pretreatment could promote the cross-linking degree of TGase-catalyzed WPI, and improve its emulsifying properties, WHC and surface hydrophobicity.

Mass Spectrometric Identification of a Novel Factor XIIIa Cross-Linking Site in Fibrinogen.

Transglutaminases are a class of enzymes that catalyze the formation of a protein:protein cross-link between a lysine and a glutamine residue. These cross-links play important roles in diverse biological processes. Analysis of cross-linking sites in target proteins is required to elucidate their molecular action on target protein function and the molecular specificity of different transglutaminase isozymes. Mass-spectrometry using settings designed for linear peptide analysis and software designed for the analysis of disulfide bridges and chemical cross-links have previously been employed to identify transglutaminase cross-linking sites in proteins. As no control peptide with which to assess and improve the mass spectrometric analysis of TG cross-linked proteins was available, we developed a method for the enzymatic synthesis of a well-defined transglutaminase cross-linked peptide pair that mimics a predicted tryptic digestion product of collagen I. We then used this model peptide to determine optimal score thresholds for correct peptide identification from y- and b-ion series of fragments produced by collision-induced dissociation. We employed these settings in an analysis of fibrinogen cross-linked by the transglutaminase Factor XIIIa. This approach resulted in identification of a novel cross-linked peptide in the gamma subunit. We discuss the difference in behavior of ions derived from different cross-linked peptide sequences and the consequent demand for a more tailored mass spectrometry approach for cross-linked peptide identification compared to that routinely used for linear peptide analysis.

Effects of sequential enzymatic hydrolysis and transglutaminase crosslinking on functional, rheological, and structural properties of whey protein isolate

This work investigated the effect of transglutaminase (TGase) crosslinking condition on the functional, rheological, and structural properties of hydrolyzed whey protein isolate (WPI) at three hydrolysis degrees (DHs) (7.61%, 9.22%, 18.55%). Between three DHs crosslinking, it was found that low DH coexistent with higher fluorescence intensity, sulfhydryl content and emulsifying activity, and moderate DH had higher thermal stability and viscosity. In crosslinking process, increasing the temperature had significant effect on solubility, emulsification, and viscosity of TGase-induced hydrolysate with moderate DH (p < 0.05), while the sulfhydryl contents decreased to various degrees. And the best reaction temperatures of hydrolysate with moderate DH were identified for thermal stability (50 °C/30 °C), solubility (30 °C), emulsifying activity/emulsifying stability (EAI/ESI) (50 °C/40 °C) and viscosity (40 °C), respectively. With increasing TGase concentrations, hydrolysate with moderate DH had best thermal stability at 30 U/g, while the highest EAI and the lowest ESI was at 40 U/g. The process conditions adopted enzymatic hydrolysis coupled with TGase-induced crosslinking could significantly influence the protein conformation, consequently determining their specific functional properties. The understanding of hydrolysis and crosslinking effects on properties could assist in selecting the appropriate method to optimize the utilization of WPI in the food industry.

Textural performance of Zea mays transglutaminase modified milk protein concentrate in stirred yoghurt

Zea mays transglutaminase (TGZ) crosslinking was investigated as ways to improve the textural performance of yoghurt with milk protein concentrate (MPC). TGZ modified MPC (TMPC) and non-modified MPC (NMPC) were each formulated into yoghurt with contents of 10, 20 and 30 g L−1 pH, texture, gel strength, apparent viscosity and water-holding capacity (WHC) were measured to compare the effect of TMPC and NMPC on the yoghurt. Yoghurt with TMPC were denser and more cohesive than those with NMPC. TGZ crosslinking improved yoghurt cohesiveness and increased WHC. Obvious improvements were observed for the yoghurt with TMPC when compared with those of NMPC. 20 g L−1 TMPC could produce yoghurt with the similar quality as the 30 g L−1 NMPC. In general, TGZ could indirectly improve the yoghurt textural performance by modified MPC, and this provide a novel method of TGZ applied in yoghurt.

Effects of microfluidization and transglutaminase cross-linking on the conformations and functional properties of arachin and conarachin in peanut

Functional properties of food protein can be modified by appropriate modification technology, which enable food proteins to have a abroad range of application in the food industry. In the present study, arachin and conarachin fractions of peanut protein were treated by microfluidization treatment (MT, 120 MPa) and/or transglutaminase (TGase) cross-linking (TC). We investigated the effects of these different treatments on the conformations and functional properties of arachin and conarachin. The results showed that both MT and TC could effectively unfold the structures of proteins along with increasing the exposed free SH contents and surface hydrophobicity (H0), and improve the emulsion stability. Interestingly, microfluidization was more effective in improving the functional properties (e.g., solubility or emulsifying properties) of arachin and conarachin than TGase cross-linking. Moreover, it was found that microfluidization pretreatment could facilitate TGase-induced cross-linking of arachin/conarachin. Compared with individual treatments, combined treatments could yield more unfolded protein structures of arachin/conarachin. In addition, the combined treatments could remarkably improve the emulsion stability for arachin and conarachin in comparison with individual MT, and also significantly enhanced the protein solubility and emulsifying activity compared with TC.

Effect of transglutaminase crosslinking on solubility property and mechanical strength of gelatin-zein composite films

The solubility (WS) of gelatin films in water limits its packaging application of high moisture content foods. Crosslinking by transglutaminase (TGase) and addition of zein were reported to reduce WS of gelatin films, but it is a challenge to incorporate TGase in a system of two immiscible materials of gelatin (water soluble) and zein (ethanol soluble) as well as in the environment of low pH when aqueous acetic acid is used to as a commonly used solvent. Zein in ethanol was added to gelatin in aqueous solution at a controlled rate. The final concentration of ethanol was 8.5% and this film forming solution provided the optimal pH for TGase while keeping both proteins soluble. Crosslinking was confirmed by the formation of higher molecular weight protein in SDS-PAGE. The optimum crosslinking degree (65%) was with 95% gelatin/5% zein. Tensile strength of the TGase treated films was improved from 5 to 36 times and proportional to the gelatin-zein crosslinking degree, while water solubility (~98% insoluble in water) depended on the type of crosslinking. The presence of zein improved the thermal properties (melting temperature increased up to 19 °C) but reduced the transparency as compare to pure gelatin film. TGase modified gelatin/zein composite films with improved water insolubility, water vaper permeability and mechanical properties are potential candidates for high moisture food packaging application.