Assessment of Cross-Linking in Combined Cross-Linked Soy Protein Isolate Gels by Microbial Transglutaminase and Maillard Reaction

ABSTRACT:  Soy protein isolate (SPI) gels were produced using single cross-linking agents (SCLA) of microbial transglutaminase (MTG) via incubation for 5 or 24 h (SCLA-MTG). When powdered SCLA-MTG gels were heated for 2 h with ribose (R2) (2 g/100 mL), dark brown gels were formed, and these were designated as combined cross-linking agent (CCLA) gels: MTG5(R2) and MTG24(R2). The results showed that the levels of Maillard-derived browning and cross-links of MTG5(R2) and MTG24(R2) gels were significantly ( P  < 0.05) lower than a control gel produced without MTG (SCLA-R2) even though the percentage of ribose remaining after heating of these gels was similar, indicating that a similar amount of ribose was consumed during heating. ɛ-(γ-glutamyl)lysine bonds formed during incubation of SPI with MTG may have reduced the free amino group of SPI to take part in the Maillard reaction; nevertheless, ribose took part in the Maillard reaction and initiated the Maillard cross-linkings within the CCLA gels.     

Citric acid cross-linking of heat-set whey protein hydrogel influences its textural attributes and caffeine uptake and release behaviour

Heat-induced whey protein gels were either pre-loaded with caffeine, then cross-linked with citric acid, or vice versa. Based on the results of Fourier transform infrared spectroscopy, the pre-loading then cross-linking procedure caused less cross-linkage formation than the cross-linking then caffeine loading (post-loaded) procedure. The latter also resulted in a firmer gel. The caffeine-to-protein ratio was approximately 8.5 μg mg⁻¹ in the non-cross-linked hydrogel; those of the caffeine-loaded then cross-linked and the cross-linked then caffeine-loaded hydrogels were 4.5 and 2.9 μg mg⁻¹, respectively. Cross-linking decreased swelling of the freeze-dried gels upon subsequent rehydration. It also influenced the extent of caffeine release from the protein hydrogel. Pre-loaded and post-loaded samples retained ∼78% and ∼88%, respectively, of the entrapped caffeine after immersion in water for 4 h; the non-cross-linked gel retained <55% of the initially loaded caffeine. Caffeine loading decreased gel water-holding capacity; subsequent citric acid cross-linking increased water-holding capacity.     

Gelling of microbial transglutaminase cross-linked soy protein in the presence of ribose and sucrose

Soy protein isolate (SPI) was incubated with microbial transglutaminase (MTGase) enzyme for 5 (SPI/MTG(5)) or 24 (SPI/MTG(24)) h at 40 °C and the cross-linked SPI obtained was freeze-dried, and heated with 2% (w/v) ribose (R) for 2 h at 95 °C to produce combined-treated gels. Longer incubation period resulted in more compact and less swollen SPI particle shape when reconstituted with sugar solution. Thus, this MTGase treatment affected samples in terms of flow behaviour and gelling capacity. Rheological study showed different gelling profiles with the cross-linking treatments and combined cross-linked SPI gave a higher G′ value compared to single treated samples. These are due to the formation of additional ε-(γ-glutamyl)lysine bonds and “Maillard cross-links” within the SPI protein network during the MTGase incubation and heating in the presence of ribose (i.e. reducing sugar). Network/non-network protein analysis found that network protein increased with cross-linking treatment, which also resulted in different SDS–PAGE profiles. As in non-network protein fraction, A₄ subunit was suggested to become part of the network protein as a result of combined cross-linking.     

Evaluation of microbial transglutaminase and ribose cross-linked soy protein isolate-based microcapsules containing fish oil

Soy protein isolate (SPI)-based microcapsules containing fish oil were prepared using a modified coacervation method followed by cross-linking treatments. The procedure yielded 95–98% microcapsules containing 0.5–0.6 g fish oil/g capsule with a volume mean diameter ranged from ~ 260 to ~ 280 μm. Four types of microcapsules produced were SPI with sucrose (MC-C/S), SPI with ribose (MC-C/R), SPI with sucrose and microbial transglutaminase (MC-MTG/S) and SPI with ribose and MTGase (MC-MTG/R). Protein cross-linking due to ε-(γ-glutamyl)lysine bonds and “Maillard cross-linking” were evidenced in the SDS-PAGE profiles of MC-C/R, MC-MTG/S and MC-MTG/R. Even though the microcapsules prepared with cross-linking treatments had significantly (P < 0.05) lower protein solubility as compared to that of the control, the results of fish oil release in pepsin solution at 37 °C indicated that the core release of all microcapsules prepared with ribose (MC-C/R and MC-MTG/R) was significantly (P < 0.05) lower than other microcapsules. During storage, microcapsules prepared with ribose had longer shelf life as compared to other microcapsules. This may be due to the release of antioxidative Maillard reaction products during heating and storage and a slower rate of gas permeability through the capsules.

Industrial relevance

The use of protein-based wall materials in the food industry for sensitive ingredients is limited because proteins are generally unstable with heating and damaged by organic solvents and the cross-linking agent is usually harmful. Therefore a novel method of combining two familiar cross-linking agents such as the microbial transglutaminase and ribose can convert SPI microcapsules into a stable form. The application of SPI in industry would be increased.     

Physicochemical properties and microstructure of soy protein hydrogels co-induced by Maillard type cross-linking and salts

The relationship between macroscopic (elasticity, swelling), microstructural and molecular properties was investigated in soy protein hydrogels formed by cross-linking to different degrees with a model Maillard type cross-linking agent (glutaraldehyde) in the absence or presence of 120 mM NaCl. Hydrogel macroscopic properties were not influenced by protein secondary structure. In contrast, rheological properties were strongly influenced by degree of cross-linking and microstructure. Cross-linking soy protein in the absence of salt led to the formation of a more porous network consisting of bigger flocs and/or bigger particles and to weaker gels. Gel swelling did not appear to depend on microstructural properties. The influence of cross-linking and salt on swelling would be connected to their respective impacts on elastic and ionic contributions to swelling.     

Influence of ultrasound and enzymatic cross-linking on freeze-thaw stability and release properties of whey protein isolate hydrogel

This study investigated the effect of ultrasound and enzymatic cross-linking on the freeze-thaw (FT) stability and release properties of whey protein isolate hydrogels. We evaluated the FT stability by the changes in the microstructure, riboflavin retention, syneresis, water holding capacity (WHC), and texture of gels subjected to 3 FT cycles. High-intensity ultrasound (HUS) and transglutaminase (TGase)-mediated cross-linking improved the FT stability of whey protein isolate hydrogels loaded with riboflavin (WPISAR), as demonstrated by a more uniform and denser porous structure, significantly higher riboflavin retention, WHC, and textural properties, and lower syneresis after 3 FT cycles than those of untreated hydrogels. Furthermore, HUS- and TGase-mediated cross-linking decreased protein erosion and swelling ratio of WPISAR in simulated gastrointestinal fluids (SGIF) and reduced the riboflavin release rate in SGIF both with and without the addition of digestive enzymes. After 3 FT cycles, faster riboflavin release occurred due to a more porous structure induced by ice crystal formation compared with their unfrozen counterparts as detected by confocal laser scanning microscopy. High-intensity ultrasound- and TGase-mediated cross-linking alleviated the FT-induced faster riboflavin release rate in SGIF. High-intensity ultrasound- and TGase-treated gel samples showed that both diffusion and network erosion were responsible for riboflavin release regardless of FT. These results suggest that HUS- and TGase-mediated cross-linking improved the FT stability of WPISAR with a high riboflavin retention, and might be a good candidate as a controlled-release vehicle for riboflavin delivery to overcome undesired FT processing.     

Drug release from hydrogel containing albumin as crosslinker

Albumin is the major plasma protein and acts as a physiological carrier for various compounds including drugs. To take advantage of the drug-binding ability of albumin for a drug delivery system, we have prepared hydrogels consisting of acrylamide (AAm) and bovine serum albumin (BSA) by introducing three to four vinyl groups into one BSA molecule and subsequently copolymerizing it with AAm. The resultant hydrogel was solubilized by trypsin treatment, since BSA served as a crosslinker in the hydrogel. The BSA-crosslinked hydrogel (BSA-AAm hydrogel) was loaded with salicylic acid or sodium benzoate and their release was investigated. The BSA-AAm hydrogel released much more salicylic acid than sodium benzoate. In addition, the amount of released salicylic acid increased with the BSA content of the hydrogel, despite a decrease in the swelling ratio of the hydrogel. On the other hand, the amount of released sodium benzoate increased with the swelling ratio. When a hydrogel crosslinked with N,N'-methylenebis (acrylamide) was used as a control, both drugs showed release tendencies similar to that of sodium benzoate from the BSA-AAm hydrogel. Furthermore, the salicylic acid release was sustained longer on the BSA-AAm hydrogel than the sodium benzoate release. Taken together, it is thought that albumin in the BSA-AAm hydrogel preferentially adsorbs salicylic acid and contributes to the high drug loading and the sustained release of salicylic acid.     

Formation of Elastic Whey Protein Gels at Low pH by Acid Equilibration

Abstract: Whey protein gels have a weak/brittle texture when formed at pH ≤ 4.5, yet this pH is required to produce a high-protein, shelf-stable product. We investigated if gels could be made under conditions that produced strong/elastic textural properties then adjusted to pH ≤ 4.5 and maintain textural properties. Gels were initially formed at 15% w/w protein (pH 7.5). Equilibration in acid solutions caused gel swelling and lowered pH because of the diffusion of water and H⁺ into the gels. The type and concentration of acid, and presence of other ions, in the equilibrating solutions influenced pH, swelling ratio, and fracture properties of the gels. Swelling of gels decreased fracture stress (because of decreased protein network density) but caused little change to fracture strain, thus maintaining a desirable strong/elastic fracture pattern. We have shown that whey protein isolate gels can be made at pH ≤ 4.5 with a strong/elastic fracture pattern and the magnitude of this pattern can be altered by varying the acid type, acid concentration, pH of equilibrating solution, and equilibrating time. Practical Application: Low-pH shelf-stable whey protein gels having the strong/elastic texture can be made by forming gels at high pH and equilibrating in acid solutions. Acid equilibration causes the gel to swell and lower the gel pH. Moreover, gel properties can be altered by varying the acid type, acid concentration, pH of equilibrating solution, and equilibrating time.     

Effect of soy protein substitution for sodium caseinate on the transglutaminate-induced cold and thermal gelation of myofibrillar protein

The influence of soy protein isolate (SPI) substitution for sodium caseinate (SC) on the properties of cold-set (4 °C) and heat-induced gels of pork myofibrillar protein (MP) incubated with microbial transglutaminase (TG) was investigated. The strength of cold-set MP–SC gels (formed in 0.45 M, NaCl, 50 mM phosphate buffer, pH 6.25) increased with time of TG incubation, but those gels with more than 66% SPI substituted for SC had a >26% reduced strength (P < 0.05). Upon cooking, both incubated and non-incubated protein sols were quickly transformed into highly elastic gels, showing up to 6000 Pa in storage modulus (G′) at the final temperature (72 °C). However, no differences (P < 0.05) in G′ were observed between heated samples with SPI and SC. Myosin heavy chain, casein and soy proteins gradually disappeared with TG incubation, contributing to MP gel network formation. Both cold-set and heat-induced gels had a compact protein matrix, attributable to protein cross-linking by TG.     

Characterization of amino cross-linked soy protein hydrogels

ABSTRACT:  This study focused on amino cross-linking as means of forming soy protein hydrogels with modifiable properties. The efficiency of glyceraldehyde, a potential food-grade cross-linking agent, was compared to glutaraldehyde, a well-known dialdehyde. The influence of the concentration of these agents on the degree of cross-linking as well as macroscopic and supramolecular properties was studied. The effect of increasing the cross-linker concentration was mainly an increase in degree of cross-linking and gel storage modulus ( G ') and a decrease in gel swelling ratio and increase in gel deswelling ratio. However, the cross-linking influence was less pronounced in the case of glyceraldehyde. Glutaraldehyde displayed greater ability to form hydrogels with modifiable properties. Finally, electron micrographs suggested that cross-linking agent type had no impact on gel microstructure.     

Physicochemical properties of gelatin gels from walleye pollock (Theragra chalcogramma) skin cross-linked by gallic acid and rutin

Gelatin gels were cross-linked by gallic acid and rutin. The gel strength, viscoelastic properties, thermal stability, swelling property, ultrastructure, X-ray diffraction patterns and FTIR spectra were determined to evaluate the physicochemical properties of the modified gels. The gel strength increased with increasing gallic acid concentration up to 20 mg/g dry gelatin, and then decreased at further elevated gallic acid concentration, while it continuously increased with increasing levels of rutin. Either cross-linking agent could enhance the elastic modulus (G′) and the viscous modulus (G″) of hydrogels, but the gelling and melting points didn’t show a notable improvement. Rutin boosted the thermal stability of xerogels, but decreased the equilibrium swelling ratio significantly, while as for gallic acid, there were no obvious effects on the thermal stability and equilibrium swelling ratio of xerogels. Scanning electron microscopy (SEM) was applied to observe the ultrastructure changes of the modified xerogels suggesting that gelatin xerogel at rutin concentration of 8 mg/g dry gelatin showed the highest cross-linking density. X-ray diffraction revealed that both gallic acid and rutin could enter the spacing of polypeptide chains of gelatin to reinforce the intermolecular interaction. And FTIR spectra verified that gallic acid and rutin molecules mainly interacted with skeletal C–N–C group and carboxyl group of gelatin molecules in the formation of gels. The results suggested that rutin was a better cross-linking agent for gelatin, and gels treated with rutin could be found with different physicochemical properties.     

三种离子物质对壳聚糖/果胶聚电解质复合物牛血清蛋白释放特性的影响

分别添加氯化钠、硫酸钠、氯化铁制备壳聚糖/果胶聚电解质复合物(PEC),研究其对牛血清蛋白(BSA)的负载能力及模拟释放行为,以探讨三种离子物质对壳聚糖/果胶PEC在蛋白缓释方面的影响。氯化铁PEC对BSA的包埋率最大,其次为硫酸钠、氯化钠,载药率为氯化钠PEC最大、其次为硫酸钠、氯化铁。在单一模拟环境中,各PEC在模拟胃液中的BSA累积释放率最小,在模拟结肠液中最大;氯化钠会使PEC的BSA释放率降低,而硫酸钠与氯化铁则可增大蛋白释放率;各PEC的蛋白释放行为属于Fick扩散。在连续模拟环境中,模拟胃液中BSA累积释放率较低,在模拟肠道环境中BSA大量释放,含有氯化钠的PEC累积释放率最低;在模拟结肠液中含有果胶酶时,各PEC的BSA累积释放率可达98%以上;各PEC的蛋白释放动力学属于非Fick扩散。含有三种离子物质的壳聚糖/果胶PEC均具有良好的p H敏感性和定点释放特性。     

环氧基交联剂对可食性大豆分离蛋白膜性能的影响

为了提高可食性大豆分离蛋白膜的性能,加入环氧基交联剂,考察了环氧基交联剂不同添加量对蛋白膜性能的影响。结果表明,环氧基交联剂的加入,可使大豆分离蛋白膜的机械性能增加显著,蛋白膜机械性能与交联剂的加入量显著关联。3种溶剂(甲苯、四氯化碳、四氢呋喃)溶胀实验表明,在实验条件下交联剂加入量在0.4~0.5 g时,蛋白膜交联程度较为理想。     

Heat Gelling Properties of Hen's Egg Yolk Low Density Lipoprotein (LDL) in the Presence of Other Protein

Ovalbumin (OV), bovine serum albumin (BSA) and lysozyme (Lys) were added to egg yolk low density lipoprotein (LDL) and the heat-induced gel formation of LDL-protein mixture was studied. The gel formation of LDL-protein mixture was different between the acidic and alkaline pH region; fine stable gels were formed in the alkaline pH region, but weak paste-like gels were formed in the acidic pH region. The effect of added BSA or OV on the gel strength of LDL was much larger than that of Lys. Scanning electron micrographic observation showed that the microstructures of both LDL-BSA and LDL-OV gels were more uniform than that of LDL-Lys gel.     

Heat-Induced Gelation of Hen's Egg Yolk Low Density Lipoprotein (LDL) Dispersion

The heat-induced gelation of hen's egg yolk low density lipoprotein (LDL) dispersion (3.5%) was studied in comparison with ovalbumin (OV) and bovine serum albumin (BSA). Stable gels of both OV and BSA were formed only at the narrow pH regions, but LDL gels obtained at every pH between 4 and 9 were stable. The presence of large amounts of salts decreased the rigidity of both OV and BSA gels, especially on the acid side of the isoelectric point of the proteins, but did not affect that of LDL gels at every pH tested. The results indicated that the heat-induced LDL gels were more stable than those of OV and BSA under various environmental conditions.     

A COMPARISON OF THE VISCOELASTIC PROPERTIES OF CONVENTIONAL AND MAILLARD PROTEIN GELS

The properties of gels prepared by heating solutions of bovine serum albumin (BSA) for 30 min at 121C in the presence and absence of glucono-delta-lactone (GDL gels) and xylose (Maillard gels) were compared. During formation the pH of the Maillard and GDL gels decreased to 4.9 whereas the pH of the gels formed in the absence of GDL or xylose remained near neutral. Maillard gels show much less syneresis compared with the GDL gels and contained nondisulphide covalent crosslinks as evidenced by very low protein solubilities in mixtures of sodium dodecyl sulphate and β-mercaptoethanol. Both the GDL and Maillard gels could be formed at much lower protein concentrations than the neutral conventional gels. The stress relaxation of the gels in compression was measured and the response analyzed using Peleg's equation. The parameters in this equation were not strongly dependent on protein concentration or degree of deformation. The neutral pH gels were far more elastic than the low pH gels, but despite the difference in crosslinking mechanisms the viscoelastic behaviour of the Maillard and GDL gels was similar. However, the break strength and asymptotic residual modulus of the Maillard gels were higher. It is suggested that the stress relaxation occurs in weaker, noncovalently linked regions of the gel, whereas the nondisulphide covalent crosslinks in the Maillard gels reinforce strong regions already containing disulphide linkages.     

Effect of transglutaminase-induced cross-linking on gelation of myofibrillar/soy protein mixtures

Microbial transglutaminase (MTGase)-catalyzed interaction and gelation of mixed myofibrillar (MPI)/soy (SPI) protein isolates were investigated at varying ionic strengths and MPI:SPI ratios, with or without SPI being preheated (80?°C). MTGase treatments in deionized water converted myosin heavy chain and actin into lower molecular-weight polypeptides, which gradually diminished as the ionic strength increased up to 0.6 M NaCl. A reduced intensity in the electrophoretic bands of soy proteins (7S and 11S except the basic subunits) was observed in all treatments, suggesting cross-linking with MPI. The enzyme treatment slightly increased the thermal transition (denaturation) temperatures of MPI/SPI but greatly enhanced (P<0.05) the elasticity of the mixed protein gels when compared with untreated samples, independent of incubation time.     

Papain-induced Gelation of Soy Glycinin (11S)

ABSTRACT:  The gelation of soy peptides produced by the action of papain enzymes on soy glycinin (11S) dispersions (4.7% w/v) was investigated. Cation-exchange chromatography was used to fractionate crude papain. The nonbinding fraction showed no gel-forming activity on the 11S dispersion. Two binding fractions showed gel-forming activity, and the gel strength of both 11S gels was similar. The activity of the crude papain on 11S dispersions produced a slightly stronger gel than one formed with either of the 2 binding fractions. With the crude papain, the rate of gel formation appeared to be strongly influenced by the enzyme concentration, but the maximum gel strength was independent of enzyme concentration. When the temperature was increased, the papain treatment of 11S soy protein produced weaker gels when the measurement was made at the temperature of formation. This dependence of maximum gel strength on temperature was found to be a function of only the measurement temperature and not the gel formation temperature. The degree of protein hydrolysis at maximum gel strength was similar (∼6%) for the gels formed at different temperatures. When the temperature was increased, the elastic modulus G', the viscous modulus G", and the degree of viscoelasticity (G"/G') decreased. This suggested that the gels were formed the by hydrophobic interactions among the peptides. This observation was supported by particle size measurements on samples of gels which were mixed with reagents known for their ability to disrupt hydrophilic/electrostatic, hydrophobic, or disulfide interactions.     

Flavonoid microparticles by spray-drying: Influence of enhancers of the dissolution rate on properties and stability

Naringenin (Nn) and Quercetin (Q) have numerous health benefits particularly due to their antioxidant properties. However, their low solubility, bioavailability and stability limit their use as components for functional foods, nutraceuticals and pharmaceutical agents. In this research, Nn- and Q-microparticles were produced by a spray-drying process using a combination of cellulose acetate phthalate (CAP) as coating gastroresistant polymer and swelling or surfactant agents as enhancers of dissolution rate. Raw materials and microparticles produced were all characterized by particle size analysis, differential scanning calorimetry, X-ray diffraction, and imaged by electron and fluorescence microscopy. During 12 months, storage stability was evaluated by analyzing drug content, HPLC and DSC profiles, as well as antioxidant activity (DPPH test). In vitro dissolution tests, using a pH-change method, were carried out to investigate the influence of formulative parameters on flavonoid release from the microparticles. Presence of a combination of CAP and surfactants or swelling agents in the formulations produced microparticles with good resistance at low pH of the gastric fluid and complete flavonoid release in the intestinal environment. The spray-drying technique and the process conditions selected have given satisfying encapsulation efficiency and product yield. The microencapsulation have improved the technological characteristics of the powders such as morphology and size, have given long-lasting storage stability and have preserved the antioxidant properties.     

Functional properties of caseinate glycoconjugates prepared by controlled heating in the ‘dry’ state

Glucose, ribose, fructose, lactose, fructo‐oligosaccharide (inulin) and a mixture of inulin and fructose were conjugated with caseinate via the Maillard reaction using controlled heating and low water activity conditions in order to improve the functional properties of caseinate for food purposes. Conjugation with ribose and glucose increased the viscosity of caseinate 28‐fold over that of the unmodified caseinate control but also produced the most browning. Glycoconjugates of caseinate–fructose reacted at a substrate ratio of 1:0.2 w/w and 60 °C at 67% relative humidity for 48 h increased the viscosity of caseinate 24‐fold with less browning. At 80% relative humidity the reaction occurred so rapidly that, within 24 h gels containing darkly coloured particulate matter were produced. Incorporation of inulin at 1:1:0.2 w/w/w caseinate/inulin/fructose prevented formation of gels and produced glycoconjugates having 15‐fold the viscosity of unmodified caseinate with minimal browning. Copyright © 2005 Society of Chemical Industry