Properties Of Protein Gels Research Articles

Optimizing Protein-Glutaminase Expression in Bacillus subtilis.

Protein-glutaminase (PG) is a promising protein deaminase. It only hydrolyzes the side chain amido groups of protein-bound to generate ammonia and protein-L-glutamic acid and does not catalyze any other undesirable changes in protein structures. Deamidation of proteins via PG can influence the solubility, emulsification, foaming, and gelation properties of proteins, which are important properties for some food proteins. Therefore, there is great potential for the application of PG in the food industry. PG is derived from Chryseobacterium proteolyticum (C. proteolyticum); however, wild strains are difficult to industrialize because of their low levels of enzyme production. In this article, we studied different strategies for PG expression in B. subtilis. Results showed that PG produced from C. proteolyticum could be successfully secreted in B. subtilis WB800N, and actively secreted in B. subtilis 168(BS168) or DB403 containing a pro-peptide (pro-PG). The secreted PG from B. subtilis WB800N was inactive unless digested by exogenous proteases, such as trypsin, alkaline protease, and neutral protease. However, active PG was secreted by the self-processing of BS168 and DB403. The specific activity of purified PG reached 20.9 U/mg. PG showed maximum activity at pH 5.5, 55°C and more than 80% of PG activity was retained within a range of pH 3.5-6.5. When Cbz-Gln-Gly was used as the substrate, PG activity was 31.1 ± 0.9μMmin-1mg-1. Mg2+, Ca2+, and Zn2+ stabilized and even activated PG activity. These strategies concerning PG expression in B. subtilis and the enzymatic properties of PG provide efficient alternatives for PG research and contribute to the industrial-scale production of PG.

Ultrasonic Freezing Reduces Protein Oxidation and Myofibrillar Gel Quality Loss of Common Carp (Cyprinus carpio) during Long-Time Frozen Storage.

Ultrasonic freezing (UF) is an effective method to increase the freezing speed and improve the quality of frozen food. The effect of UF on myofibrillar protein oxidation and gel properties of common carp (Cyprinus carpio) during frozen storage were investigated with air freezing (AF) and immersion freezing (IF) as controls. The results showed that the carbonyl and dityrosine content of UF samples were lower and the free amine content was higher than those of AF and IF samples during frozen storage indicating that UF inhibited protein oxidation caused by frozen storage. The particle size of UF myofibrillar protein was the smallest among all the groups indicating that UF inhibited the protein aggregation. The UF sample had higher G’, G” value, gel strength and gel water holding capacity than AF and IF groups showing that UF reduced the loss of protein gel properties. The gel microstructure showed that UF protein gel was characterized by smaller and finer pores than other samples, which further proves that UF inhibited loss of gel properties during frozen storage. The UF sample had shorter T2 transition time than other samples demonstrating that UF decreased the mobility of water. In general, UF is an effective method to reduce protein oxidation and gel properties loss caused by frozen storage.

Protein extraction pH and cross-linking affect physicochemical and textural properties of protein gels made from channel catfish by-products.

Channel catfish farming is very important aquaculture industry in the southern states of the USA. However, huge amounts of by-products are generated from catfish fillet-processing. The by-products (mostly heads and bone frames) are excellent sources of proteins. Currently, catfish by-product has little value, and is regarded as a 'waste', which if not utilized properly could cause serious environmental pollution. Therefore, to find a way to utilize those by-products is critical for the economy of aquaculture. Protein isolates were extracted from the mixture of catfish by-products (heads and frames) under different alkaline conditions (pH 7.5-11) and made into protein gels. Secondary structures of extracted protein isolates were studied. Microbial transglutaminase (MTGase, 0-4 U g-1 proteins) was incorporated to improve gel structure. Gelling, physicochemical, textural and thermal properties of protein gels treated with/without MTGase were investigated. Protein pattern changes of MTGase-treated protein gels were studied and the microstructure of the protein gels was analyzed. Alpha-helicity of protein isolates made at pH 11 was 21.5% lower than that extracted at pH 8.5. Storage modulus (G') of protein gel decreased with increasing extraction pH (pH > 9) of the corresponding protein isolate. MTGase treatment exhibited significant effects on denaturation temperature and enthalpy of protein gels. Excessive MTGase (>2 U g-1 ) could weaken the gel structure. Protein isolates can be extracted from catfish by-products and made into protein gels, which are a value-added product. © 2021 Society of Chemical Industry.

Potential mechanism of different gelation properties of white and red muscle fibre from crocodile (Crocodylus siamensis) meat: Study of myofibrillar protein

The present study was performed to investigate the effects of myofibrillar proteins (MPs) extracted from crocodile (tail, legs, torso) on gel formation mechanism and gel characteristics. The results showed that protein compositions and ratios were different in the three body parts. The tail MPs had a higher surface hydrophobicity than the legs and torso MPs, while the leg MPs had more reactive sulfhydryl groups than the tail and torso MPs (P < 0.05). According to the Raman spectroscopy results, before heating, the leg MPs' β-sheet content was significantly higher than that of tail MPs (P < 0.05). Heating significantly reduced the contents of α-helices in the legs and torso, and there were significant differences in β-sheet content between the legs and tail (P < 0.05). Moreover, with increasing myosin content, a denser microstructure of the tail gel was observed by scanning electron microscopy, leading to its gel strength and water-holding capacity being significantly higher than those of other parts. The tail MPs revealed a higher storage modulus (G′) and spin–spin relaxation time (T21) than those in the other two parts. In conclusion, the tail of crocodile (white muscle) has stronger protein gelation properties than other parts (red muscle), which is conducive to producing gel products.

Improving sweet potato protein gel properties through ε-(γ-glutamy)-lysine isopeptide cross-link catalyzed by transglutaminase

Production of sweet potato protein (SPP) gels with enhanced viscoelasticity and mechanical properties through transglutaminase (Tgase) catalyzed SPP ε-(γ-glutamy) - lysine cross-link was investigated. Tgase induced cross-link of SPP increased the protein molecular weight, and slightly reduced its gelling point. Gel viscoelasticity was significantly enhanced with the maximum storage modulus (Gʹmax) increasing from 2,990 Pa to 30,700 Pa as Tgase concentration increased from 0% to 5.0%. The exponent (zʹ) of the power law (Gʹ = kʹωzʹ), decreased from 0.024 (control SPP gel) to zʹ in the range 0.018–0.022 for the SPP ε-(γ-glutamy) - lysine cross-link (SPP – GL) gels. SPP gel network structure showed increased covalence interaction with increase in the level of Tgase induced cross-linking. SPP gel had no fracture stress but suffer deformation under little stress, while SPP – GL cross-linked gels formed with 1.6, 3.2 and 5.0% Tgase could withstand higher stress and had fracture stress and strain in the range 97.3–235.5 kNm−2 and 0.065–0.448 respectively. Tgase induced SPP ε-(γ-glutamy) - lysine cross-link enhanced SPP gel hardness, resilience and adhesiveness.

Influence of amino acids on thermal stability and heat-set gelation of bovine serum albumin

This study investigated the parallels in the influence of amino acid additives on thermal denaturation temperature (Td) and heat-set gelation of bovine serum albumin (BSA). Complete denaturation of BSA occurred only when the gelation temperature (TG) was 14 °C above Td. Under these conditions, the relative effects of various amino acid additives on elevation of Td and gel strength followed a particular order. Further, while zwitterionic amino acids increased both the strength of junction zones and participation of protein in the gel network, sucrose increased the gel strength primarily by strengthening the existing junction zone. The net increase in Td of BSA was linearly correlated with the net increase in gel strength (ΔGS), indicating that the underlying molecular mechanism in both cases might be same. The results suggest that the rheological properties of protein gels can be enhanced by using amino acids, instead of polyols and sugars, as additives.

Combined effects of ionic strength and enzymatic pre-treatment in thermal gelation of peanut proteins extracts

Peanut proteins are mostly composed of arachins and conarachins, globular proteins that can form gels under thermal denaturation or enzymatic treatment. We explored here how ionic strength (0.5 M or 0.8 M) and gelation process (a thermal treatment preceded or not by an enzymatic pre-treatment) could affect peanut protein gel properties. Gel formation and final properties were characterized by rheology, and gel structure was observed by confocal microscopy. We found that the ionic strength imposed during protein extraction determines the arachins/conarachins ratio, and that conarachins-rich samples give stronger gels, which is attributed to their higher content in free thiol groups and lysine residues. The gel storage modulus exhibited a power-law dependence with the protein concentration, which exponent depended on the gelation process. Rheological results, together with confocal microscopy imaging, showed that an enzymatic pre-treatment resulted in denser structures than when a simple thermal treatment was applied.

Effects of moderate electric fields on cold-set gelation of whey proteins – From molecular interactions to functional properties

Whey protein gelation and final gel properties are dependent of the gel forming solution characteristics (e.g. protein concentration, pH, ionic strength), and physical variables involved in the method used for gel preparation. Ohmic heating (OH) is an emerging technology in food processing and its application in heat-induced gelation of whey proteins has demonstrated its capacity to influence the physicochemical properties of protein gels. In this work, we studied the OH process and its inherent moderate electric field (MEF) variables - i.e. electric field (EF) strength and frequency - in order to establish their influence in protein aggregation and gelation during WPI cold-set gels formation. The presence of the EF during OH, particularly at higher EF strengths conjugated with lower frequencies, contributed to the formation of smaller aggregates with lower content of reactive thiol groups and lower viscosity. The cold-set gels produced from the aggregates’ suspension presented distinctive properties, influenced by the EF variables. – i.e. higher EF strength and lower frequency. EF treatments give rise to more fine-stranded gels with lower disulphide crosslinking but higher number of hydrophobic interactions and hydrogen bonds. The EF effects during the treatments resulted in weaker, more elastic gels with higher water retention and swelling capacity. These results open novel perspectives for the use of OH as a tool for fine-tuning protein gel networks aiming at enhanced functionality for various applications (e.g. use as texturizer or encapsulating agents).

Effect of cold atmospheric plasma on the physicochemical and functional properties of myofibrillar protein from Alaska pollock (Theragra chalcogramma)

SummaryThis study aimed to investigate the effect of cold atmospheric plasma (CAP) on the physicochemical and functional properties of myofibrillar protein isolated from Alaska pollock (Theragra chalcogramma) muscle. Free sulfhydryl content, surface hydrophobicity, turbidity, differential scanning calorimetry, SDS‐PAGE, heat‐induced myofibrillar protein gel properties and water holding capacity were determined to evaluate the effect of CAP treatment on protein properties. CAP treatment from 10 to 60 kV for 10 min significantly (P &lt; 0.05) increased the water holding capacity, textural properties and colour properties of heat‐induced myofibrillar protein gel. As treatment voltage increased, free sulfhydryl content decreased significantly, while the turbidity and surface hydrophobicity of myofibrillar protein were increased. DSC data showed enhancement of thermal stability of the treated samples; however, the electrophoretic profile displayed a significant reduction in the band's intensity of protein at a higher voltage. These results suggest that cold atmospheric plasma (CAP) treatment could be used as an alternative approach to enhance the gelling properties of myofibrillar protein from Alaska pollock.

The beneficial effects of rutin on myofibrillar protein gel properties and related changes in protein conformation

Effects of different levels of rutin (0, 10, 50, 100 and 200 μmol/g protein) on the conformational changes and gel properties of myofibrillar protein (MP) were investigated. Rutin at 200 μmol/g caused the greatest carbonyl content. The incorporation of rutin caused the losses of thiol, free amine and α-helix contents, reduction in tryptophan intrinsic fluorescence intensity, and enhanced exposure of hydrophobic groups and protein cross-linking. When compared with control, the MP gels with 10, 50 and 100 μmol/g rutin had higher gel strength but slight lower water-holding capacity; the gels appeared to have compact microstructure with few visible pores. However, 200 μmol/g rutin was detrimental to gel properties. All the gels with rutin presented higher final storage modulus and converted to elasticity-dominant gel types. The results indicate that a slightly high concentration of rutin could improve MP gel properties which are related to the protein conformational changes induced by rutin.

Gel properties of potato protein and the isolated fractions of patatins and protease inhibitors – Impact of drying method, protein concentration, pH and ionic strength

The use of plant protein in food products is increasing. Potato protein gelation is scarcely documented, therefore potato protein gel properties were tested for two whole protein isolates (freeze dried or spray dried) and the purified sub-fractions, protease inhibitors and patatins. The effects of protein concentrations, 8 or 15% (w/w), pH-range of 3.0–7.5, and 2 different levels of ionic strength; low (15 mM NaCl) or high (200 mM NaCl) on heat induced gel properties was studied by oscillatory rheology and by uniaxial compression. Spray drying did not impair gel properties of the whole protein isolates, with the spray dried isolate actually showing higher gel strength at most conditions.For whole protein isolates, gel strength expressed as the storage modulus (G′) had a minimum at pH 4.7 at both high and low salt. The isolated patatin fraction showed a similar trend at low salt, but at high salt the gel strength was very low at pH 3, with progressively higher values at pH 5 and pH 7.5. The protease inhibitor fraction gelled best at low salt and pH values below pH 3.3, while higher pH resulted in very weak gels with a 10-fold decrease in G’. Gel appearance was affected by protein sample, pH, and ionic strength. Both whole protein isolates and the protease inhibitor fraction formed translucent gels at pH 3.0 and ionic strength 15 mM, while patatin formed translucent gels at both pH 3.0 and pH 7.5 at low salt concentration. All other conditions resulted in opaque gels.

Comparison of raw meat quality and protein-gel properties of turkey breast fillets processed by traditional or cold-batter mincing technology

This study was conducted to evaluate the effects of cold batter mincing on meat quality and protein functionality, using turkey fillets that were chill-boned (CB) or hot-boned (HB) with crust-freeze-air-chilling (HB-¼CFAC) at -12°C. For each of four replications, 48 toms (male) were raised and processed at Michigan State University Poultry Farm and Meat Laboratory, respectively. After evisceration, the turkeys were subjected to one of the four treatments: (1) traditional mincing of CB fillets after water immersion chilling (WIC); (2) cold batter mincing of WIC, CB, quarter-sectioned (¼), and CB-¼CFAC; (3) traditional mincing of HB-¼CFAC fillets; and (4) cold batter mincing of HB-¼CFAC fillets. Before mincing, the pH and R-values of turkey fillets in HB-¼CFAC were higher and lower, respectively, than those in CB fillets. During cold-batter mixing, the initial batter temperatures at -1.5 to -2.1°C reached 1.5°C and 14°C at 6 and 12min, respectively, and ended at 26 to 31°C at 24min. During traditional mincing, the initial batter temperatures at 3 to 4°C increased by ∼10°C every 6min, and ended at 32 to 35°C with higher batter temperatures seen for the 2% salt than the 1% salt batter. Dynamic rheological properties indicated that the cold-batter mincing showed elevated G' compared to the batters of traditional mincing, regardless of mixing time, indicating that the gel-setting temperature was reduced in the cold-batter mincing, potentially due to the different amounts of extracted protein and structural change. After cooking, improved cooking yield and protein functionality were observed in the batter of HB-¼CFAC fillets than the batter of CB fillets as well as in the batter of 2% salt than the batter 1% salt (P < 0.05). These results indicated that HB-¼CFAC fillets produced superior raw meat quality over the CB fillets, and cold batter mincing of HB-¼CFAC fillets significantly improved protein functionality compared with the traditional mincing of CB fillets (P < 0.05).

Effects of cold atmospheric plasma on squid proteases and gel properties of protein concentrate from squid (Argentinus ilex) mantle

The effect of cold atmospheric plasma (CAP) on protein concentrate from squid (Argentinus ilex) mantle has been evaluated in terms of gel properties, protease inhibition, texture profile, color attributes, and water holding capacity. Different exposure time (15, 60, 120, 180, 240 and 300 s) at 60 kV have been employed. Our results indicated that protease activity decreases with increasing treatment time. The highest reduction (p < 0.05) in protease activity (64%) was observed after 240 s of CAP treatment. Texture profile analysis, color properties and water holding capacity of the treated squid gel revealed a significant increase. Protein carbonyl and sulfhydryl group contents findings' showed a significant increase in carbonyl content (about three times of the original content), while the total sulfhydryl group decreased (up to about 40%) in the crude extract. Microstructure and SDS-PAGE analysis revealed a high degree of protein aggregation in the squid gel treated with CAP.

Isoelectric solubilization/precipitation processing modified sarcoplasmic protein from pale, soft, exudative-like chicken meat

The sarcoplasmic protein from pale, soft and exudative -like chicken breast meat was modified using an isoelectric solubilization/precipitation process. After the modification, the sarcoplasmic protein obtained a larger particle distribution than the nontreated sample, indicating a severe aggregation. The isoelectric solubilization/precipitation-treated sarcoplasmic protein aggregates exhibited a flocculated and amorphous shape stabilized by the hydrophobic interactions. The hydrophobic interactions of alkali-treated sarcoplasmic proteins were relatively constant during heating, while they dramatically increased in nontreated sample. The alkali-treated sarcoplasmic protein could promote myofibrillar protein gelation properties, as proved by the higher breaking force and lower cooking loss. According to our results, it is hypothesized that the alkali-treated sarcoplasmic protein likely does not interfere in the gelation behavior of myofibrillar protein but does fill in the pores of the three-dimensional network, thereby strengthening its gelation elasticity. Overall, the alkali-treated sarcoplasmic protein exhibits the potential to improve the myofibrillar protein gelation properties.

Protein structure and sulfhydryl group changes affected by protein gel properties: process of thermal-induced gel formation of myofibrillar protein

ABSTRACT The study investigated the change in protein structure upon thermo-induced gelation of myofibrillar protein (MP, 2%) under different temperatures (30–80°C) at 0.6 M NaCl and pH 6.2. The results showed that heating from 45°C led to changes in physicochemical properties and intermolecular forces involved during MP gelation, which are related to each other. During the heating process, the free sulfhydryl content slowly increased and reached the highest value at 45°C (10.23 ± 0.13 μmol/g protein). When the temperature exceeded 50°C, Ellman’s titration and SDS-PAGE results indicated that MP gel began to form with the formation of disulfide bonds, which contributed to stabilize the gel structure. Gel strength of the heat-induced MP gel formed at 60°C was significantly higher than that at other temperatures (P < .05), reaching 1.12 ± 0.02 N. Strictly speaking, myofibrillar protein gelatinized after 60°C, and water-holding capacity of the MP gel decreased from 60-80°C, and reached a relatively low value at 80°C. Furthermore, Raman spectra showed that the α-helix content decreased with increasing temperature, β-sheets and random coil contents increased, and the content of the twist-twist-trans conformation increased steadily.

Gel properties of protein hydrolysates from trypsin-treated male gonad of scallop (Patinopecten yessoensis)

In order to provide a foundation for comprehensive utilization of scallop (Patinopecten yessoensis), the gel properties of hydrolysates from scallop male gonad (SMGHs) were studied. The hydrolysates were obtained from scallop male gonads (SMGs) by using trypsin. The changes in molecular mass of proteins were determined by SDS-PAGE and HPLC with a peptide separation column. The gel properties of SMGHs were investigated by using rheometer and texture instrument in comparison with that of ι-carrageenan and gelatin. The results showed that the proteins of SMGs were significantly degraded after hydrolysis with trypsin at a dosage of 3000 U/g protein. The SMGHs showed an appearance of gelation after hydrolysis for 30–180 min and cooling down. Under various sweep models, the values of storage modulus (G′), loss modulus (G″), viscosity (ƞ) of SMGHs were obvious higher than those of SMGs. The textural parameters containing firmness, cohesiveness and adhesive force of SMGHs at dosages of 50 and 75 mg/mL were similar to those of ι-carrageenan and gelatin at 25 mg/mL, respectively. For the mixtures, the gel property of SMGHs/ι-carrageenan SMGHs/gelatin increased significantly. The low-field NMR (LF-NMR) also reflected the T23 of SMGHs/ι-carrageenan, T21 of SMGHs/ι-carrageenan and SMGHs/gelatin mixture decreased significantly in comparison with SMGHs alone. These results suggest that SMGHs could be applied as a potential gelling and thickening agent in food formulations.

Effects of low fat addition on chicken myofibrillar protein gelation properties

This work aims to study the effects of low fat addition (0.05%–0.25%) on chicken myofibrillar protein (MP) gel properties and ultrastructure. The changes in sulfhydryl, surface hydrophobicity and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of MP solutions and MP gels were also determined. The results demonstrated that 0.20% fat addition increased MP gel hardness and water holding capacity, and the ratio between protein and fat was 15 to 1 at this time. The rheological curves revealed 0.20% fat treatment increased the G′ values during the heating and cooling stage. A dense and uniform network structure was also observed at 0.20% fat treatment. The changes in the main bands of SDS-PAGE indicated that the addition of fat reduced the rate of protein degradation during MP heating. The total sulfhydryl groups of MP gels showed a downward trend with the fat addition, and the surface hydrophobicity reached the maximum at 0.20% fat addition. The fat addition promoted the formation of disulfide bonds between the MP gel, and enhanced the hydrophobic interaction between MP gel molecules, thereby increased the gel properties of MP.

Gelation properties of goose liver protein recovered by isoelectric solubilisation/precipitation process

SummaryIsoelectric solubilisation/precipitation (ISP) process was applied to goose liver (GL) for protein extraction. The gelation properties of proteins extracted by acid processes (ACP, pH 2.0, 2.5 and 3.0) and alkaline processes (ALP, pH 11.0, 11.5 and 12.0) were estimated, where the unextracted ground GL was set as the control. Nearly 58.39~79.00% of GL proteins were recovered by ISP treatments. High molecular weight (100~250 kDa) proteins were found to be partially hydrolysed by ACP, while few changes in proteins occurred during ALP. As evidenced by rheological and textural measurements, ALP proteins formed gels with high elasticity and superior texture, whereas ACP proteins had inferior gelation properties. Moreover, ALP proteins were able to form a highly interconnected and homogeneous three‐dimensional microstructure. Predominantly, gels produced by 11.0 had optimal texture and the lowest cooking loss (P &lt; 0.05). These results suggested that the ISP process (ALP) is a potential method to improve the economic value of GL.

γ-PGA and MTGase improve the formation of ε-(γ-glutamyl) lysine cross-links within hairtail (Trichiurus haumela) surimi protein

The present study investigated the mechanism of ε-(γ-glutamyl) lysine cross-links within hairtail (Trichiurus haumela) surimi protein via γ-polyglutamic acid (γ-PGA) and MTGase. The results indicated that the addition of MTGase and γ-PGA markedly improved the gelation properties of hairtail surimi protein, including its maximum breaking force and deformation, water holding capacity and gel strength. The maximum improvements were achieved by adding 0.5units MTGase/g meat paste in combination with 0.06% γ-PGA. SDS-PAGE showed that the band intensity of cross-linked proteins increased, whereas that of myosin heavy chain decreased after treatments. Further scanning electron microscopy (SEM) analysis showed the formation of a denser gel matrix, which was caused by much stronger and more inter- and intra-molecular cross-linking of proteins, via MTGase catalysing ε-(γ-glutamyl) lysine cross-links formed between lysine residues in the gel protein and glutamic residues in the hydrolytic γ-PGA. The results provide reliable guidance for the improvement of hairtail surimi protein gelation properties.

Precipitation and ultimate pH effect on chemical and gelation properties of protein prepared by isoelectric solubilization/precipitation process from pale, soft, exudative (PSE)-like chicken breast meat

Pale, soft, exudative (PSE)-like chicken breast is considered deteriorated raw material in the poultry meat industry that has inferior processing ability. The chemical and gelation properties of PSE-like chicken breast meat paste were studied. These pastes were prepared by the pH adjustment method and protein isolation using the isoelectric solubilization/precipitation (ISP) process from PSE-like chicken meat. The ISP-isolated samples were solubilized at pH 11.0 and recovered at pH 5.5 and 6.2. The ultimate pH of the ISP-isolated protein and meat paste was adjusted to 6.2 and 7.0. The ultimate pH in this article referred to the final pH of the extracted protein and meat paste. Higher reactive sulfhydryl content and surface hydrophobicity were found in the precipitation at pH 6.2 than at pH 5.5. However, various ultimate pH values showed no significant influence on the surface hydrophobicity. The hardness of gel, as measured by textural profile analysis, was improved using 6.2 as the precipitation pH compared with pH 5.5. The viscoelastic modulus (G′) of gel pastes prior to the thermal gelation was higher with ISP treatment. However, lower G′ was seen after thermal gelation compared with the control. Dynamic rheological measurement demonstrated a different gel-forming mechanism for protein precipitated at pH values of 5.5 and 6.2 compared with the meat paste. The cooking loss showed that the recovered protein failed to form a gel with good water-retention capacity unless the ultimate pH was adjusted to 7.0. Gels made from protein extracted by the ISP method had higher yellowness and lower redness values, probably due to protein denaturation. Precipitation at pH 6.2 formed a harder gel with lower water-retention ability than that at pH 5.5, and this result was possibly due to higher surface hydrophobicity and S-S bridge formation. Overall, network characteristics of ISP-treated protein gels were strongly dependent on precipitation pH and ultimate pH.