Effects of transglutaminase treatment on the thermal properties of soy protein isolates

The effects of covalent cross-linking of microbial transglutaminase (MTGase) on the thermal properties of soy protein isolates (SPI), including the thermal denaturation and glass transition were investigated by conventional and modulated differential scanning calorimetry (DSC). The MTGase treatment significantly increased the thermal denaturation temperatures (including the on-set temperature of denaturation, T_m and the peak temperature of denaturation, T_d) of glycinin and β-conglycinin components of SPI (P ⩽ 0.05), and the thermal pretreatment of SPI further increased the extent of this improvement. The MTGase treatment also improved the ability of SPI to resist the urea-induced denaturation. Modulated DSC analysis showed that there were two glass transition temperatures (T_g) in the reversible heat flow signals of native SPI (about 5% moisture content), approximately corresponding to 45 and 180 °C, respectively. These T_g values of SPI were significantly decreased by the MTGase treatment (at 37 °C for more than 2 h) (P ⩽ 0.05). The improvement in the hydration ability of protein and the formation of high molecular biopolymers may account for the changes of thermal properties of soy proteins caused by the MTGase cross-linking.     

Effect of processing parameters on the properties of transglutaminase-treated soy protein isolate films

The effects of processing parameters, including the applied amount of microbial transglutaminase (MTGase), the pH of film-forming solution, air-drying temperature, as well as the additional pre-incubation, on the properties of MTGase-treated soy protein isolate (SPI) films were investigated. The treatment with low concentration of MTGase (4–10 units per gram of SPI, U g^− 1) significantly increased the tensile strength (TS) values of SPI films, while high concentration of MTGase (over 20 U g^− 1) resulted in significant decrease in the TS values (P ≤ 0.05). The elongation at break (EB) values of corresponding films gradually decreased, and the contact angle values persistently increased with the enzyme concentration. At alkaline pH range, the TS and EB values of MTGase-treated SPI films were significantly higher than that at pH 7.0 (P ≤ 0.05). Meanwhile, the contact angle values significantly decreased with increasing pH from 7.0 to 10.0 (P ≤ 0.05). The TS, EB and contact angle values of MTGase-treated films gradually but insignificantly decreased with increasing the air-drying temperature from 18 to 50 °C (P > 0.05). The properties of MTGase-treated films were also affected by the additional pre-incubation of film-forming solutions with MTGase before casting. Furthermore, the aggregation of SPI or its components induced by MTGase has been proved to mainly account for the influence of processing parameters on the properties of SPI films (MTGase-treated). Thus, low concentration of enzyme, alkaline pH range and low air-drying temperature, at which conditions the MTGase-induced aggregation of SPI in film-forming solutions could be in some extent inhibited or delayed, might facilitate the improvement of the properties of SPI films by MTGase, especially the mechanical and surface hydrophobic properties.Industrial relevanceThe development of biodegradable protein film has attracted a lot of attention worldwide. The enzymatic cross-linking induced by transglutaminase has been confirmed to improve mechanical and surface hydrophobic properties of cast films from most of food proteins, including soy proteins. Results of this study show that, the improvement of properties of cast films of soy proteins by transglutaminase treatment is largely dependent upon many processing parameters, e.g., enzyme concentration, the pH of film-forming solution and temperature.     

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.     

Comparative study of gelation and cross-link formation during enzymatic texturisation of leguminous proteins

Isopeptide bonds that resulted from protein cross-linking, catalysed by a microbial transglutaminase (MTG), substantially contributed to the physicochemical modification of leguminous proteins. For the development of texturised vegetable protein (TVP)-based foodstuffs using MTG, valid methods for an efficient control of the gelation process are a prerequisite. Formation of ε-(γ-glutamyl)lysine cross-links in a simple food model system, containing proteins from soy [Glycine max (L.) Merr.] or pea (Pisum sativum L.), was monitored by quantification of the ε-(γ-glutamyl)lysine bond via HPLC-MS and by determining the depletion of free amino groups during cross-linking spectrophotometrically after derivatisation with o-phthaldialdehyde (OPA). Increasing gel strengths during incubation with MTG were measured via texture analysis. The OPA method proved too unspecific for controlling the enzymatic gelation process of leguminous proteins. Specifically for each substrate, the levels of isopeptide cross-links, detected via HPLC-MS analysis, correlated well with the gel strength of the texturised proteins (R² = 0.961–0.994). Rapidly measurable, gel strength was shown to be a reliable command variable for managing MTG-induced gelation. Its use also allowed indirect estimation of the degree of feasible cross-linking.Industrial relevanceLeguminous proteins represent a valuable alternative to animal proteins for the manufacture of texturised foodstuffs. However, due to the poor gelling properties of the native proteins, their potential is still unexploited. For the development of TVP using MTG, simple gel strength measurements were shown to be a valid tool for the control of the gelation process. For this purpose, unlike OPA determination of free amino groups and LC-MS analysis of isopeptide bonds, tedious sample preparation is not required.     

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

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

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.     

The effect of pH on the gelling behaviour of canola and soy protein isolates

The present study investigates the gelation mechanisms of a canola protein isolate (CPI) as a function of a pH (3.0–9.0), and compares it to that of a commercial soy protein isolate (SPI). A rheological investigation found that CPI was non-gelling at pH 3.0, and then formed a gel with increasing strength as pH was raised from pH 5.0 to 9.0. In contrast, the commercial SPI ingredient was found to be non-gelling at pH 9.0, but formed the strongest networks at pH 5.0 near its isoelectric point (pI = 4.6). Denaturation temperature as determined by differential scanning calorimetry were found to occur at ~ 78 °C for CPI at pH 5.0, then shifted to higher temperatures (~ 87 °C) at pH 7.0/9.0, whereas detection of SPI denaturation could not be obtained due to instrument sensitivity. Gelling temperatures were similar for both CPI and SPI (~ 82–86 °C) at all pHs, with the exception of SPI at pH 5.0 (~ 46 °C). Overall CPI networks were stronger than SPI, since the latter had weaker inter- and intramolecular junction zones. Confocal laser scanning microscopy images indicated that CPI gels became denser with lower lacunarity values as pH increased from 3.0 to 9.0. Moreover, the fractal dimension of CPI gels was found to increase from ~ 1.5-1.6 to ~ 1.8 as pH increased from 5.0/7.0 to 9.0, respectively suggesting diffusion-limited cluster-cluster aggregation. Images of SPI networks were not concurrent with fractal analysis under the conditions examined. Despite CPI having excellent gelling properties that are comparable to SPI, its need for alkaline pH conditions will limit its applicability in foods.     

Subunit,amino acid composition and in vitro digestibility of protein isolates from Chinese kabuli and desi chickpea (Cicer arietinum L.) cultivars

The subunit, amino acid composition and in vitro digestibility of the two protein isolates (GCPI and ZCPI) from one kabuli and one desi chickpea cultivars, grown extensively in Xinjiang Autonomous Region of China, were investigated and compared with those of soy protein isolate (SPI). SDS–PAGE showed that GCPI and ZCPI had almost the same band components under the reduced and unreduced conditions, with only minor difference in relative quantity for some bands, but different from that of SPI. The sulphur-containing amino acids were the first limiting amino acids for all three protein isolates of GCPI (2.11 g/100 g), ZCPI (2.20 g/100 g) and SPI (1.99 g/100 g). Amino acid score of the three protein isolates could reach the FAO/WHO requirement (1990) for the essential amino acids for preschool children. The order of in vitro digestibility was GCPI (87.47%) > ZCPI (80.82%) > SPI (71.04%). Our results indicated that, compared with soybean protein isolate, Chinese kabuli and desi chickpea protein isolates had higher digestibility value, and chickpea protein, especially for kabuli protein, could be utilized as a good source of protein for human nutrition.     

Impact of transglutaminase on the textural,physicochemical, and structural properties of chicken skeletal,smooth, and cardiac muscles

This study examined the effects of microbial transglutaminase (MTG; 3.1 mg/ml) on chicken skeletal, smooth, and cardiac muscles; the meat containing the different muscle types was shaped into sausages and treated at 40 °C and/or 78 °C for 30 min. Although the three muscle types were obtained from the same bird, the effects of MTG addition were not uniform. All the muscle types showed a significant increase in the breaking strength (P < 0.01), but skeletal muscle exhibited the maximum increase. All samples showed a decrease in the fluorescence intensity and a significant reduction in the concentration of proteins that were extracted in a high ionic strength solution (P < 0.05). Scanning electron microscopy images and histological studies revealed that different muscle types had different physical structures and frameworks after MTG treatment, which is a reflection of the differences in the reaction specificity of MTG with different muscle proteins. Histological studies revealed that the reactions of MTG with meat proteins are both exogenous and endogenous. Cooking loss data suggested that MTG did not have any negative effect on water retention during cooking. MTG appears to be a functional and contributive substance since the results suggest that MTG can function on all muscle types that are mechanically processed for different industrial applications. MTG aggregates muscle proteins in different ways that improve their organoleptic properties such as texture, appearance, and water retention.     

Influence of enzymatic cross-linking on milk fat globules and emulsifying properties of milk proteins

The enzyme transglutaminase (TGase) can modify dairy protein functionality through cross-linking of proteins. This study examined the effects of TGase treatment on milk fat globules and the emulsifying properties of milk proteins. The extent of TGase-induced cross-linking of caseins increased with incubation time, with no differences between whole and skim milk. Extensive clustering of fat globules in extensively cross-linked raw whole milk occurred on homogenisation at 400 or 800 bar. Considerably less clustering of fat globules was observed when recombined milk (90 g fat L^–1) was prepared from TGase-treated skim milk and homogenised at 400 or 800 bar. TGase treatment did not affect fat globule size in cream, but prevented coalescence of fat globules therein, possibly through cross-linking of milk fat globule membrane components. TGase-induced cross-linking of milk proteins affected their emulsifying properties and may increase the stability of natural milk fat globules against coalescence.     

Physicochemical and functional properties of soy protein isolate as a function of water activity and storage

The effect of storage temperature on the physicochemical characteristics, solubility and gelling properties of soy protein isolate (SPI) with different water activities (a_w) was investigated. SPI with a_w of 0.19 (SPI-0.19) was placed in environments with relative humidity (RH) of 33% and 74%. After reaching equilibrium, in 20 days, the samples were named SPI-0.33 and SPI-0.74. For SPI-0.74, modifications in the protein characteristics started during the equilibrium period, with a decrease in the solubility and alterations in both the electrophoretic profile of the soluble proteins and in the gelling characteristics. During the 180-day storage period, SPI-0.19 and SPI-0.33 showed similar behaviours: decrease in protein solubility and alteration in hardness, cohesiveness and microstructure of the gels. These alterations were more pronounced during storage at 45 °C than at 25 °C, and in SPI-0.33 more than in SPI-0.19. Results suggest that storage conditions – temperature and RH – affect the functional properties of the proteins and the use of the isolates as a functional ingredient.     

Modulation of mechanical and surface hydrophobic properties of food protein films by transglutaminase treatment

The effects of microbial transglutaminase (MTGase) treatment on the mechanical and hydrophobic properties of cast films from various food proteins were investigated. SDS–PAGE analyses confirmed that the MTGase treatment led to the formation of insoluble film network for most of food proteins. This enzymatic treatment significantly (P < 0.05) increased tensile strength (TS) values of cast films of all food proteins by 13–33% (except whey proteins). Meanwhile, the influence on the elongation at break (EB) values was mainly dependent upon the molecular nature of proteins. For most of food proteins, both increases in TS and EB values of films were obtained after the MTGase treatment. Furthermore, the surface hydrophobicity of most of protein films was also significantly improved. These results suggest that the enzymatic treatment by MTGase could be used as an effective technique to improve mechanical and surface hydrophobic properties of protein films.     

Effect of cross-linking on the physicochemical and physiological properties of corn starch

Corn starch was chemically modified by cross-linking with STMP/STPP (99:1. w/w) and the physicochemical and physiological properties (in vitro and in vivo) of the cross-linked corn starch were investigated as a function of the degree of cross-linking. Cross-linking decreased the solubility, swelling factor, and paste clarity of corn starch. While the swelling factor was highly correlated with the degree of cross-linking (R² = 0.878), the X-ray diffraction patterns did not show any significant alteration in the crystallinity of corn starch. It was shown by SEM measurement that a black zone was observed on the surface of crossed-linked starch granules, which did not occur with native starch. When mice were fed the diets containing the corn starch with low (CLCS-5) and high (CLCS-12) degree of cross-linking (51.3 and 99.1%, respectively), significant effects on the final body weight, weight gain as well as perirenal weight of the mice (p < 0.05) were observed. Also, significant decreases in total lipid, triglyceride, and total cholesterol concentrations in serum were detected in CLCS-5 and CLCS-12 groups (p < 0.05). While total lipid level in the liver decreased with increasing degree of cross-linking, the triglyceride level was not affected by the supplementation with both of CLCS-5 and CLCS-12 corn starch samples.     

Heat-induced Maillard reaction of the tripeptide IPP and ribose: Structural characterization and implication on bioactivity

Maillard reaction products (MRPs) were prepared from aqueous model mixtures containing 60 g L^? 1 ribose and 30 g L^? 1 of the bioactive tripeptide IPP (Ile-Pro-Pro), heated at 98 °C. MRP and associated reactions with changes in IPP were observed within one hour of heat-treatment. The pH of MRPs decreased significantly during the heat treatment of IPP–ribose mixtures from 9.0 to 7.6 after one hour. The amino group content, IPP and ribose concentration decreased significantly during heat treatment. The fluorescence intensity of the IPP–ribose MRPs reached the maximum within 2 h. Modification of the UV/vis spectra for IPP–ribose MRPs was mainly due to a condensation reaction of IPP with ribose. Compounds with molecular weight between 300 and 650 Da were dominant while compounds smaller than 250 Da were also produced during the reactions, as characterized by size exclusion chromatography. Mass spectrometry revealed that IPP was conjugated to ribose at the N-terminal (m/z of 458.3) upon heat-treatment. The presence of ribose also promoted peptide degradation to dehydrated IP (m/z of 211.1). IPP–ribose MRPs lost the known angiotensin-I-converting enzyme (ACE) inhibitory activity of IPP; however, strong antioxidant properties were detected.     

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.     

Functional properties of milk proteins as affected by enzymatic oligomerisation

Milk proteins were enzymatically modified by transglutaminase, lactoperoxidase, laccase as well as glucose oxidase and analysed for changes in molar mass distribution, techno- and tropho-functional properties.The study revealed that high degrees of protein cross-linking were not only detected upon enzymatic modification of milk proteins by transglutaminase, but also upon incubation with oxidoreductases like lactoperoxidase, laccase as well as glucose oxidase. Due to different reaction mechanisms, oligomeric (20,000–200,000 g/mol) and polymeric (>200,000 g/mol) reaction products with different functional property profiles were synthesised. In contrast to transglutaminase-treated milk proteins, e.g., lactoperoxidase-treated milk proteins performed outstanding interfacial properties and glucose oxidase-treated milk proteins higher in vitro digestibility. Oxidoreductase-treated proteins were shown to exhibit increased antioxidative capacity. Laccase was demonstrated to generate oligomeric as well as polymeric protein/protein and protein/phenolic acid reaction products.The study characterises enzymatically cross-linked proteins and makes it possible to specifically select modified proteins for industrial applications according to the requirements towards food proteins, weighing changes in techno- and tropho-functional protein properties.     

Effect of transglutaminase on rheological properties and microstructure of chemically acidified sodium caseinate gels

The mechanical properties and microstructure of 2.7% and 4.5% sodium caseinate gels chemically acidified by glucono-δ-lactone (GDL) and cross-linked by microbial transglutaminase (TG) were studied. The acidification was performed at different temperatures. According to SDS–PAGE TG clearly caused polymerisation of caseinate irrespective of the treatment temperature (4–50 °C), The cross-linking of the proteins was more extensive at temperatures 22–50 °C. Low amplitude viscoelastic measurements showed that 4.5% caseinate gels acidified at 50 °C were formed much faster than gels acidified at 22 °C. TG only slightly increased the time of gelling. Control gels prepared without TG at temperatures of 4, 22, 37 and 50 °C were mechanically weak. Examination of the control gels with a confocal laser scanning microscope showed that gels formed at 37 and 50 °C were coarse and porous with large cavities between particle aggregates, whereas those formed at 22 °C were much more homogeneous. The TG-treated and acidified sodium caseinate dispersions formed firm gels, indicating cross-linking of casein proteins. Interestingly, the strongest gels were formed at 22 and 37 °C. TG treatment improved the homogeneity of the gel structure at temperatures of 37 and 50 °C. The hardness of TG-treated gels acidified at 4 °C increased during 1 week of storage.     

Effect of Maillard reaction conditions on browning and antiradical activity of sugar–tuna stomach hydrolysate model system

The antiradical activity of Maillard reaction products (MRPs) made from sugar–tuna stomach hydrolysate model system was tested. The antiradical activity of the MRPs derived from ribose was 11-fold higher than that of MRPs derived from glucose due to the acyclic form of the ribose. The activity reached the plateau at a 30 mg/mL ribose concentration. The ribose caramelization contributed to the antiradical activity and browning reactions at 95 °C and 115 °C. The increase in DPPH radical scavenging of MRPs is attributed not only to the temperature but also to the buffer type and buffer concentration. Phosphate buffer showed the most efficient compared to citrate or Tris–HCl buffers. A positive correlation (R² = 0.98) was observed between the antiradical activity, the browning and the phosphate concentration. The MRPs obtained under these mild experimental conditions exhibited no toxicity towards Vero cells and 3T3 cells, despite their high antiradical activity.     

Quercetagetin loaded in soy protein isolate–κ-carrageenan complex: Fabrication mechanism and protective effect

In the present study, the potential of soy protein isolate (SPI)–κ-carrageenan (κ-CG) complex as a protective carrier for quercetagetin was investigated at different pH values (pH 2.3 and 6.5). The particle size of the ternary aggregates was slightly increased at pH 2.3, yet dramatically decreased at pH 6.5 with increasing quercetagetin concentration. Moreover, the negative ζ-potential of the ternary aggregates was increased significantly (p < 0.05) at pH 6.5. The addition of quercetagetin to the SPI–κ-CG complex could highly quench the intrinsic fluorescence of SPI. Circular dichroism spectra further suggested that the bound quercetagetin could induce the rise of β-sheet and β-turn contents at the cost of α-helix and unordered coil fractions of SPI. In addition, quercetagetin could increase the viscoelasticity of the ternary aggregates at both pH. Furthermore, the SPI–κ-CG complex was found to be superior to single SPI or κ-CG in terms of improving light stability and radical scavenging ability of quercetagetin.     

Coating papers with soy protein isolates as inclusion matrix of carvacrol

Antimicrobial papers were prepared by coating paper with soy protein isolate (SPI) solution as inclusion matrix of carvacrol, an antimicrobial agent. Addition of carvacrol (30% w/w of SPI) to SPI solution (10% w/v) prepared at 25 °C induced soy protein aggregates and viscosity decrease. Heat treatment (50, 70, 90 °C) of SPI solutions and carvacrol addition improved homogeneity reduced particles size and increased viscosity of solutions. The aggregated structure of SPI in the presence of carvacrol at 25 °C may play the role of a trapping structure leading to low carvacrol losses during coating and drying process of paper (9.6% against 37% after heat treatment at 90 °C) and to lower release rates specially the first three days (0.04 g/m²/day and 0.31 g/m²/day when SPI coating solutions were prepared at 25 and 90 °C, respectively). Regardless of the heat treatments received by the SPI solutions, residual carvacrol quantities in the coated papers after 50 days ranged between 0.6 and 0.7 g/m².