TGase Cross-linking Research Articles

Unveiling the slow digestion and peptide profiles of polymerised whey gel via heat and TGase crosslinking: An in vitro/vivo perspective.

Polymerised whey is widely used in dairy products and can affect digestibility when its high-molecular-weight aggregates and gel structure are modified. This study investigated the digestibility, peptide profiles and satiety of modified whey protein isolate (MWPI) pre-heated with transglutaminase. Results showed that 43.06% of MWPI was digested during the 4-h in vitro digestion, indicating a slow digestion rate. Compared with whey protein isolate (WPI), MWPI yielded 103 peptides with higher abundance following in vitro digestion, including 17 angiotensin-converting enzyme inhibitors and 1 dipeptidyl peptidase-4 inhibitor. Visual analytics indicated differential peptides located at distinct α-helix and β-sheet of β-lactoglobulin, α-lactalbumin and bovine serum albumin. MWPI gavage extended stomach retention time, decreased intestinal propulsion rate from 75.60% (WPI group) to 33.72% in 30min and enhanced satiety within 120min compared with WPI. Overall, whey polymerisation modulates protein-enzyme interactions, releasing different peptides and enhancing satiety.

Impact of Transglutaminase-Mediated Crosslinking on the Conformational Changes in a Dual-Protein System and IgE Reactivity of Soy Protein.

Transglutaminase (TGase)-catalyzed crosslinking has gained substantial traction as a novel strategy for reducing allergenic risk in food proteins, particularly within the realm of hypoallergenic food production. This study explored the impact of TGase crosslinking on conformational changes in a binary protein system composed of soy protein isolate (SPI) and sodium caseinate (SC) at varying mass ratios (10:0, 7:3, 5:5, 3:7 (w/w)). Specifically, the immunoglobulin E (IgE) binding capacity of soy proteins within this system was examined. Prolonged TGase crosslinking (ranging from 0 h to 15 h) resulted in a gradual reduction in IgE reactivity across all SPI-SC ratios, with the order of IgE-binding capability as follows: SPI > SPI5-SC5 > SPI7-SC3 > SPI3-SC7. These alterations in protein conformation following TGase crosslinking, as demonstrated by variable intrinsic fluorescence, altered surface hydrophobicity, increased ultraviolet absorption and reduced free sulfhydryl content, were identified as the underlying causes. Additionally, ionic bonds were found to play a significant role in maintaining the structure of the dual-protein system after crosslinking, with hydrophobic forces and hydrogen bonds serving as supplementary forces. Generally, the dual-protein system may exhibit enhanced efficacy in reducing the allergenicity of soy protein.

Designing gelatin microgels by moderate transglutaminase crosslinking: Improvement in interface properties

In this study, gelatin microgels with various crosslinking degrees were facilely fabricated through moderate transglutaminase (TGase) crosslinking. The influences of crosslinking degree on the surface and interface properties of gelatin microgels were investigated. Furthermore, the influence on the stability of gelatin microgels emulsions was also explored. The crosslinking degree of gelatin microgels was gradually increased from 1.85% to 12.25% and positively correlated with the addition amount of TGase. With the increase of crosslinking degree, the high molecular weight components in gelatin microgels increased, and the wettability (32°–81°) and surface hydrophobicity of gelatin microgels gradually increased. The interfacial protein content also increased with the increase of crosslinking degree and reached equilibrium as the crosslinking degree exceeded 6.85%. The results of interfacial dynamic adsorption and interfacial dilatational viscoelasticity demonstrated the formation of a dense and strong interfacial structure, especially at relatively higher crosslinking degree (>6.85%). The liquid chromatography-tandem mass spectrometry (LC-MS/MS) results confirmed that TGase crosslinking increased the crosslinking between α2 chain in gelatin molecular chains, thus enhancing interfacial interaction and regulating interface microstructure. TGase crosslinking effectively improved the physical stabilities of gelatin microgel emulsions, suggesting a dominant contribution of the adjusted interface properties of gelatin microgels by moderate TGase crosslinking.

Effect of transglutaminase crosslinking on the protein structure and potential allergenicity of dual-protein system, with a focus on β-conglycinin

Transglutaminase (TGase)-mediated crosslinking serves as a promising strategy to mitigate soy antigenicity. β-Conglycinin is recognized as a key allergen among soy proteins. Nonetheless, our understanding of the underlying relationship between immunoreactivity and protein structure remains inadequate. This study aimed to examine the impact of TGase-mediated crosslinking on the immunoreactivity of β-conglycinin in a dual-protein system consisting of soy protein isolate (SPI) and whey protein isolate (WPI) (with SPI-WPI ratios of 10:0, 7:3, 5:5, 3:7 (w/w)). Conformational changes were also examined. Results revealed that β-lactoglobulin and α-lactalbumin in WPI were less susceptible to TGase crosslinking compared to the 7S and 11S subunits in SPI. The conformation of proteins in the dual-protein system after TGase crosslinking were altered, as demonstrated by increased ultraviolet absorption intensity, fluctuant intrinsic fluorescence intensity and surface hydrophobicity, and decreased free sulfhydryl content. As the crosslinking time of TGase increased from 1 h to 18 h, the allergenicity of β-conglycinin gradually decreased in all four SPI-WPI ratios. Notably, the SPI3-WPI7 ratio exhibited the lowest allergenicity of 77.21 ± 1.89% after 18 h of TGase catalyzation. Overall, the dual-protein system proved to be more effective in reducing the allergenicity of β-conglycinin.

Designation and characterization of cold-set egg white protein/dextran sulfate hydrogel for curcumin entrapment.

This study aimed to design a cold-set hydrogel of egg white protein (EWP) with good mechanical properties for encapsulating curcumin. Dextran sulfate (DS) and transglutaminase (TGase) were used to control the aggregation and gelation behavior of EWP at preheating step and gelation step, respectively. The optimum soluble protein aggregate size was obtained in the EWP/DS mixture at a mass ratio of 10 under 85°C preheated (HED10). The presence of TGase further enhanced the cross-linking degree between protein aggregates during the gelation step. The highest gel hardness was found in HED10 hydrogel with TGase, which is almost 10 times the pure EWP gel. Besides, the HED hydrogels effectively slowed down the release rate of curcumin in gastrointestinal digestion. This work provides a theoretical basis for the development of cold-set EWP hydrogel with good mechanical strength by sulfated polysaccharide addition and TGase cross-linking as encapsulation delivery systems.

Fabrication of emulsion gels based on sonicated grass pea (Lathyrus sativus L.) protein as a delivery system for β-carotene: Kinetic modeling and release behavior

In this study, the release behavior and encapsulation efficiency of β-carotene in emulsion gels fabricated by sonicated grass pea protein isolate (GPPI) under simulated gastrointestinal conditions were investigated. Therefore, the emulsion gels stabilized by sonicated GPPIs (8% w/v) and corn oil (20% volume fraction and containing 0.5% w/w of β-Carotene) were induced by transglutaminase. The emulsion gels prepared by sonicated GPPIs exhibited sustained release of β-carotene. The Ritger-Peppas model best described the release behavior of β-carotene from the emulsion gels, indicating that the predominant release mechanism was non-Fickian diffusion. Sonicated GPPI-based emulsion gels exhibited higher β-carotene encapsulation efficiency (97.75%) and loading capacity, attributed to increased protein surface hydrophobicity, TGase cross-linking between ϵ-(γ-glutamyl) lysine residues, and the formation of smaller protein particles through ultrasonic treatment. Incorporation of β-carotene within emulsion gels was confirmed by FTIR analysis. Emulsion gels prepared using sonicated GPPIs at 75% amplitude demonstrated approximately fivefold improved bioaccessibility and enhanced retention of β-carotene during storage (20 days) compared to emulsion gels prepared using native GPPI. These findings suggest that structural modification of protein and gel structure can significantly affect the bioaccessibility and stability of β-carotene in the product.

Improving gel properties of soy protein isolate through alkaline pH-shifting, mild heat treatment, and TGase cross-linking

Improving the functional properties of soybean protein isolate (SPI) is essential for expanding its applications in food-related fields, given its poor solubility and gelation. This study investigated the effect of alkaline pH-shifting and mild heating on the structure of SPI, as well as the gel properties after TGase cross-linking. Solubility of SPI increased with increasing alkalinity from pH 7.0 to 12.0 at 25 °C, accompanied by decreased particle size and increased surface hydrophobicity. At pH 12.0 and 50 °C, SPI showed the highest solubility, smallest particle size, and best surface hydrophobicity. Intrinsic fluorescence spectra revealed that the protein structure unfolded after combined treatment, exposing more hydrophobic groups. TGase cross-linking modified SPI exhibited significantly increased gel hardness and water holding power. Therefore, alkaline pH transfer combined with mild heating treatment has great potential for generating functional modified SPI and provides a simple and easy method for preparing SPI with diverse gel and structural properties.

Cationic Gelatin Cross-Linked with Transglutaminase and Its Electrospinning in Aqueous Solution.

Gelatin (GE) is a renewable biopolymer with abundant active groups that are beneficial for manufacturing functional biomaterials via GE modification. An antibacterial fibrous GE film was prepared by electrospinning the modified GE in an aqueous solution. The original GE was modified by reacting it with N,N-dimethyl epoxypropyl octadecyl ammonium chloride (QAS), and then it was cross-linked with transglutaminase (TGase). FTIR analysis illustrated that QAS was grafted onto GE through the epoxy ring-opening reaction, and the modification did not influence the main GE skeleton structure. The investigation of the solution properties showed that the grafted cationic QAS group was the main factor that decreased the surface tension of the solution, increased the electrical conductivity of the solution, and endowed GE with antibacterial activity. TGase cross-linking clearly influenced the rheological properties such that the flow pattern of the spinning solution varied from Newton-type to shear thinning, and the aqueous solution of GE-QAS-TGs transformed from liquid-like to solid-like and even induced gelatinization with increasing TGase content. A satisfactory fibrous morphology of 200-500 nm diameter was obtained using a homemade instrument under the optimized electrospinning conditions of a temperature of 35 °C, a distance between electrodes of 12 cm, and a voltage of 15 kV. The study of film properties showed that the antibacterial activity of the fibrous GE film depended only on the grafted quaternary ammonium, whereas the thermostability, wettability, and permeability were greatly influenced by both the TGase cross-linking and film-forming methods. Cytotoxicity was tested using the CCK-8 and live/dead kit staining methods in vitro, which showed that the modified GE had good biocompatibility.

Investigation of Consequences of High-Voltage Pulsed Electric Field and TGase Cross-Linking on the Physicochemical and Rheological Properties of Pleurotus eryngii Protein.

This study aimed to evaluate the effects of high-voltage pulsed electric fields (HPEF) and transglutaminase (TGase) cross-clinking on the physicochemical and rheological properties of Pleurotus eryngii protein (PEP). The results showed that HPEF increased α-helixes and β-turns but decreased β-folds. A HPEF at 1500 V/cm maximized the free sulfhydryl content and solubility of PEP. TGase formed high-molecular-weight polymers in PEP. TGase at 0.25% maximized the free sulfhydryl groups, particle size, and solubility; shifted the maximum absorption wavelength from 343 nm to 339 nm and 341 nm; increased α-helixes and β-turns and decreased β-folds; and showed better rheological properties. Compared with TGase cross-linking, HPEF-1500 V/cm and 1% TGase significantly reduced the free sulfhydryl groups, particle size, and solubility, produced more uniform network structures, and improved the rheological properties. These results suggest that HPEF can increase the cross-linking of TGase and improve rheological properties of TGase-cross-linked PEP by affecting the physicochemical properties.

Synergistic effect of microfluidization and transglutaminase cross-linking on the structural and oil–water interface functional properties of whey protein concentrate for improving the thermal stability of nanoemulsions

Generally, whey protein concentrate (WPC) undergoes high-temperature denaturation and aggregation, which reduces its emulsifying properties and is not conducive to as an emulsifier to maintain the thermal stability of emulsions. In this study, dynamic high-pressure microfluidization technology (DHPM) combined with TGase (TG) cross-linking was applied to prepare DHPM-TG-WPC, and the thermal stabilization mechanism of nanoemulsions prepared with DHPM-TG-WPC was explored. Results showed DHPM treatment could promote the formation of TG-crosslinked WPC polymers. Compared to WPC, the free sulfhydryl and free amino group content of DHPM-TG-WPC was significantly decreased (P < 0.05), the surface hydrophobicity and interfacial tension of DHPM-TG-WPC were increased by 45.23 % and 62.34 %, respectively. And its emulsifying stability index and interface protein adsorption was significantly enhanced (P < 0.05). Furthermore, compared to WPC, DHPM-WPC and TG-WPC, DHPM-TG-WPC-stabilized nanoemulsions showed the best 15 days of storage stability after thermal sterilization. This study provides a theoretical basis for the application of modified-WPC emulsion.

Modification of fermented whey protein concentrates: Impact of sequential ultrasound and TGase cross-linking

This study aimed to examine the impact of fermentation process on whey protein and improve the general properties of fermented whey protein concentrate (FWPC) recovered by a combined ultrafiltration-diafiltration (UF-DF) operation. Impacts of sequential ultrasound (US) pretreatment and transglutaminase (TGase) crosslinking on structural, functional, and physicochemical properties of FWPCs were investigated. Partially denatured and hydrolyzed fermented whey protein could replace heat denaturation prior to the TGase addition to a whey protein system. Sequential treatment increased the molecular weight of FWPCs as exhibited by both SEM and SDS-PAGE, which demonstrates that modification can lead to the polymers and oligomers production. The zeta potential value increased significantly after US treatment and enzyme catalysis, and all the modified FWPCs were strongly negatively charged. Compared with the secondary structure of untreated FWPCs, the percentage of α-helix and random coil in modified FWPCs significantly increased, while the percentage of β-sheet and β-turns reduced. Solubility, free sulfhydryl groups, and surface hydrophobicity of all FWPCs were significantly improved compared to non-fermented WPC (P < 0.05). Sequential treatment induced a substantial impact on the emulsifying activity and stability of modified samples in comparison with untreated FWPCs. Scanning electron microscope pictures confirmed the positive effects of sequential treatments on texture and void size reduction. Therefore, the application of recovering modified FWPCs is fully recommended as a commercially viable approach for enhanced protein production at the industrial scale.

Microfluidization treatment improve the functional and physicochemical properties of transglutaminase cross-linked groundnut arachin and conarachin

Improved gelation, thermal stability and water holding properties have been observed in transglutaminase (TGase) cross-linked (TC) proteins, however, TGase cross-linking would inevitably lead to a decrease in solubility and emulsifying properties. This study explored the effects of microfluidization treatment (MT) on the solubility, emulsifying and physicochemical properties of TC-arachin/conarachin. The results showed that the protein solubility, emulsion activity and stability of TC-arachin and TC-conarachin significantly increased after MT. Although the SDS-PAGE analysis revealed only slight changes in arachin (S2 - S5) and conarachin (S1) subunits, the molecular weight distribution and microstructure analysis showed a dissociation of large aggregates into small size clumps in TCMT-linkings after MT. It was found that the surface hydrophobicity ( H 0 ) values of TC-arachin/conarachin significantly increased from 2826.7 ± 49.3/4005.3 ± 41.5 to 3918.5 ± 79.5/4542.2 ± 68.4 ( p < 0.05) whereas the Z-average size declined from to 508.1 ± 4.5/175.7 ± 0.6 to 197.9 ± 1.1/155.9 ± 0.9 (nm, p < 0.05) after MT. Moreover, the increased free sulfhydryl (SH) content was observed in MT-treated TC-arachin/conarachin, which may be caused by the conformation changes of TC-arachin/conarachin. Circular dichroism spectroscopy showed a slightly decrease in α-helix and β-sheet, and a mild increase in β-turns and random coil of TCMT-arachin/conarachin. Together, our findings not only uncover the effects of microfluidization treatment on improving the poor solubility of crosslinking products with excellent functional properties, but also highlight the potential application prospects for arachin and conarachin as the emulsifying and water-retaining agents in processing and storage in the food industry. • Microfluidization improved the solubility of TGase cross-linked (TC) globulins. • Microfluidization enhanced the emulsifying activity of TC-arachin/conarachin. • Microfluidization had no effects on the subunits of TC-arachin/conarachin. • Microfluidization significantly increased the surface hydrophobicity of TC-globulins. • Microfluidization could effectively reduce the Z-average size of TC-globulins.

Combining Alcalase hydrolysis and transglutaminase-cross-linking improved bitterness and techno-functional properties of hypoallergenic soybean protein hydrolysates through structural modifications

Alcalase hydrolysis of soybean proteins was shown effective in degrading epitopes, but with a significant loss of tastes and functional properties. In this study, the combined use of controlled Alcalase hydrolysis and transglutaminase (TGase)-cross-linking was used to produce hypoallergenic soybean protein hydrolysates (SPHs) with improved flavor and techno-functional properties. Through establishing the kinetic model of heterogeneous enzymatic processes, SPHs with different degrees of hydrolysis (2.5–10.0%) were obtained and polymerized by TGase to yield products with various peptide components. Results showed that post-hydrolysis cross-linking rearranged the (poly)peptides in SPHs via glutaminyl modifications. Following TGase-treatments, due to the masking of bitter-active peptides and free amino acids, the bitterness of SPHs was significantly reduced with a slight improvement in the overall flavor. Furthermore, TGase–cross-linking rescued multiple techno-functionalities of SPHs, particularly their emulsifying and foaming properties. Using sera from soybean-allergic individuals, it was evidenced that TGase-addition did not generate neoallergens and the residual allergenicity of SPHs was not distinctly affected. The final products maintained 44–71% reduction in the IgE-binding levels in comparison to the unhydrolyzed protein. Combining limited Alcalase hydrolysis with TGase–cross-linking provides a feasible way to produce hypoallergenic soybean ingredients with desired flavor and functional attributes.

Properties of transglutaminase-induced myofibrillar/wheat gluten gels.

Gelation properties of myofibrillar protein (MP)/wheat gluten (WG) induced by glutamine transaminase (TGase) were studied. Results showed that the inclusion of transglutaminase increased the gel strength, water-holding capacity (WHC), and nonfreezable water (Wnf) of MP/WG mixture. Circular dichroism (CD) analysis showed that the β-sheet and random coil content of the MP/WG treated with TGase addition increased by 12.1% and 3.7%, while the α-helix and β-turn content decreased by 14.2% and 1.8%. Rheological measurements showed that TGase induced higher energy storage modulus value during the MP/WG gel heating-cooling cycle. the hydrogen bond and hydrophobic interaction content of the MP/WG gels increased by 80 and 120 ug/L, and the disulfide bond decreased by 200 ug/L, with TGase addition was increased from 0 to 120 U/g protein. Scanning electron microscope (SEM) showed that MP/WG gel with TGase had uniform and dense network structure. PRACTICAL APPLICATION: The properties of myofibrillar/wheat gluten gel induced by TGase crosslinking was studied. The gel structure and water holding capacity of MP/WG were improved by the cross-linking of TGase. The study of the gel properties of MP/WG induced by TGase crosslinking also can provide a theoretical basis for analyzing the effect of TGase on the application of gluten protein in complex meat emulsion system.

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.

Transglutaminase crosslinking promotes physical and oxidative stability of filled hydrogel particles based on biopolymer phase separation

In the present study, the effect of transglutaminase (TGase) concentration on the physical and oxidative stabilities of filled hydrogel particles created by biopolymer phase separation was investigated. The results showed that filled hydrogels had relatively smaller particle sizes, higher absolute zeta-potentials, higher interfacial layer thicknesses and lightness values with the increasing of TGase concentration (P < 0.05), as evidenced by the apparent viscosity and viscoelasticity behavior. However, the relatively higher TGase concentration promoted the protein aggregation, which weakens the protection of the surface protein layer, having the negatively impacted the physical stability of filled hydrogels. Microstructural images which obtained via cryo-scanning electron microscopy also verified the above results. In particular, it is noted that filled hydrogels displayed the lowest degrees of lipid and protein oxidation during 10 days of storage (P < 0.05) at TGase concentration of 10 U/g. Prevention against oxidation was attributed mainly to TGase crosslinking of protein molecules on the surface of droplets, which likely provided a denser interface around lipid droplets. Our results indicated that TGase was a favourable agent to crosslink protein on the surface of lipid and improve the physical and oxidative stability of filled hydrogel particles.

Incorporation of gelatin improves toughness of collagen films with a homo-hierarchical structure

In this study, gelatin (type A and type B) with/without transglutaminase (TGase) were added to collagen fiber films to form hierarchical structure and its effects on the film were investigated. The analysis of mechanical properties indicate that gelatin significantly increased the toughness of the collagen film, where the 10 wt% type A gelatin -contained films had highest tensile strength, elongation at break and work of fracture. However, TGase crosslinking compromised the benefits of type A gelatin greatly, while type B gelatin showed a slight improvement, due to the difference in crosslinking activity between them. In the meantime, the hydrogen bonds were formed between the collagen and gelatin according to the results of the Fourier transformation infrared. In general, it is expected that the hierarchical structure formed in the collagen/gelatin films can be used as an effective strategy to enhance the collagen matrix films’ mechanical properties.

Consequences of superfine grinding treatment on structure, physicochemical and rheological properties of transglutaminase-crosslinked whey protein isolate

Impacts of superfine grinding treatment (0, 2, 4, 6, 8 or 10 h) on structure, physicochemical and rheological properties of transglutaminase (TGase)-crosslinked whey protein isolate (WPI) were investigated. Size exclusion chromatography showed that high molecular weight polymers were formed in TGase-treated sWPI (WPI treated with superfine grinding), whereas its consumption of free amino groups reached the maximum at grinding 8 h and 10 h. With the milling time extended from 0 to 10 h, particle size of the TGase-crosslinked sWPI gradually increased. Emission spectrum shifted from 338.6 nm to 340.6 nm after sWPI treated with TGase. Meanwhile, TGase-crosslinked sWPI had higher apparent viscosity, emulsifying properties than TGase-crosslinked WPI, but the solubility of TGase-crosslinked sWPI was lower than sWPI. Superfine grinding treatment remarkably increased β-sheet and random compositions in TGase-crosslinked sWPI. These findings indicated that superfine grinding treatment could enhance the TGase cross-linking degree, and improve rheological properties in TGase-crosslinked WPI.

Improved freeze-thaw stability of o/w emulsions prepared with soybean protein isolate modified by papain and transglutaminase

Oil-in-water (o/w) emulsions stabilized by soybean protein isolate (SPI) and enzyme-modified soybean protein isolate (SPI + TGase, SPIH, SPIH + TGase) were subjected to three freeze-thaw cycles, and changes in the molecular weight distribution, secondary structure contents, particle size, oiling off (OF), creaming index (CI) and microstructures were investigated. The emulsions exhibited different freeze-thaw tolerance stabilities, which were dependent on the type of enzyme used to prepare the SPI as well as the cycle number of freeze-thaw treatments. Differential scanning calorimetry suggested that emulsion destabilization was mainly due to water crystallization. After three freeze-thaw cycles, the emulsion stabilized by SPIH + TGase showed a decrease of 78.89% in CI and 72.35% in OF compared with SPI. Correspondingly, the particle size decreased by 70.76% (in water) and 88.99% (in 1% SDS). The improvement in the freeze-thaw stability could be largely attributed to the increased molecular flexibility due to the increased α-helix and random coil contents after enzymatic hydrolysis and the formation of gel-like network structures after the addition of TGase cross-linking. The observation of the microstructure also indicated that SPIH + TGase was an effective food emulsifier. These findings could be of great importance for the formulation of food grade emulsions with excellent freeze-thaw stability.

Effect of ultrasound on the structure and functional properties of transglutaminase-crosslinked whey protein isolate exposed to prior heat treatment

Effects of ultrasound treatment (20 kHz, 41–45 W cm−2, 0, 20, 40 or 60 min) on the physicochemical, functional properties and elements of the secondary structure of transglutaminase (TGase)-crosslinked whey protein isolate (WPI) exposed to prior thermal treatment (75 °C, 15 min) were investigated. The largest molecular size of proteins in the TGase-crosslinked WPI was observed after the ultrasound and thermal pre-treatment (HU-WPI-TGase). HU-WPI-TGase had the maximum intrinsic fluorescence intensity, with highest loss of free amino groups. Ultrasound-treated WPI (U-WPI) showed more (13%) emulsifying activity and more (63%) foaming ability than untreated WPI, but HU-WPI-TGase had higher foam stability and lower emulsifying activity than U-WPI. FTIR analysis indicated that ultrasound, heat treatment and TGase cross-linking had effects on the β-sheet, β-turn and random coil of WPI. The outcomes from this study show a potential application in providing novel functional ingredients for the dairy industry.