COAGULATION AND GELATION OF SOY PROTEIN ISOLATES INDUCED BY MICROBIAL TRANSGLUTAMINASE

The reaction process and corresponding mechanism of coagulation and gelation of native soy protein isolates (SPIs) induced by microbial transglutaminase (MTGase) were investigated. The protein constituents of SPIs, including a majority of subunits of β‐conglycinin and acidic subunits of glycinin, could be polymerized by MTGase to form high weight molecular (WM) biopolymers. Both the coagulation and gelation reactions of native SPI solutions induced by MTGase were dependent upon the initial protein substrate concentration ([C] ₀ ). In the coagulating reactions, the turbidity of SPI solutions continually increased with increasing [C] ₀ in the range from 0.25 to 3.0%. As for the gelation reactions, with the concentration increasing from 3 to 8% (w/v), the onset time of gelation of native SPIs induced by 0.8 units/mL of MTGase at 37C shortened by ∼5‐fold, and the storage modulus (G′) of finally formed gels (after 4 h) increased from ∼1 to 1300 Pa. Both the coagulation and gelation reactions of SPI solutions were promoted remarkably by increasing the enzyme concentration. Sodium Dodecyl Sulfate‐Polyacrylamide Gel Electrophoresis analysis showed that the protein constituents of MTGase‐induced aggregates of SPI (2% w/v) were mainly composed of basic subunits of glycinin and some of newly cross‐linked high MW biopolymers. The solubility analysis of protein constituents indicated that the covalent cross‐linkage, hydrophobic and H bindings and disulfide bonds were mainly involved in the coagulation of SPI induced by MTGase.     

Preparation and characterisation of glyceollin‐enriched soya bean protein using solid‐state fermentation

Glyceollins, stress‐induced phytoalexins from parent soya bean isoflavones, were elicited with Aspergillus oryzae. This solid‐state fermentation facilitated the conversion of isoflavone glycosides into aglycones and glyceollins, which could mainly enrich in soya bean protein isolate (SPI) during protein preparation due to protein–polyphenol interactions. Glyceollin‐enriched SPI exhibited less flavour volatiles, higher solubility, lower whiteness and higher antioxidant activity than unfermented SPI. Fermented SPI was more easily to be digested during in vitro pepsin–trypsin digestion. This may be attributed to partial degradation of protein, especially α′ and α subunits of β‐conglycinin and acidic subunit of glycinin. The antioxidant activity of digestive products derived from fermented SPI was obviously enhanced with increasing digestion time due to simultaneous release of antioxidant peptides and glyceollins involved in the interior of protein molecule. These results suggest an effective technique for producing a nutrient‐enhanced SPI as novel functional ingredients applied in food industry.     

Structure,trypsin inhibitor activity and functional properties of germinated soybean protein isolate

The objective of this research was to investigate the effects of germination on functional and conformational properties as well as the in vitro trypsin digestibility of soy protein isolate (SPI). The influences of germination on the molecular properties of SPI were also evaluated. The germination degraded lipoxygenase, α, α' subunits of β‐conglycinin (7S) and acidic chains of glycinin (11S) of soybean. Concomitantly, the loss of tertiary structures of SPI occurred due to the germination. The trypsin inhibitor activity of germinated SPI presented a decreasing trend, followed by an increase in the growth of hypocotyls. The in vitro trypsin digestibility of germinated SPI followed a consistent trend with the trypsin inhibitor activity. The protein solubility (PS) and emulsifying properties of SPI were improved by the germination, in a hypocotyl length‐dependent manner. The data suggest that some selected functional properties and the in vitro digestibility of soy proteins can be improved by means of the germination.     

大豆蛋白的体外模拟消化过程及热处理的影响

本研究探讨了天然大豆分离蛋白(SPI)的体外胃蛋白酶消化过程,以及热处理对该消化过程的影响。SDS-PAGE分析表明,天然SPI的大豆球蛋白最易为胃蛋白酶所消化,而β-伴大豆球蛋白则较难。β-伴大豆球蛋白的不同亚基对胃蛋白酶消化的敏感程度也有所不同,其中α-亚基最为敏感。TCA-NSI法分析显示,在一定蛋白浓度下,随酶/底物之比的增加,天然SPI受胃蛋白酶的作用释放氮的过程呈现出较为典型的酶浓度依赖性。另外,不同热处理对SPI的体外消化过程产生不同的影响。一定的干热处理(80℃,30～60min)几乎不影响SPI的体外胃蛋白酶消化过程,而同样条件下的湿热处理则显著提高胃蛋白酶及胰蛋白酶对SPI的消化效果。这结果意味着SPI的体外消化效果取决于其变性程度,热变性程度越高,其消化效果越好。     

Improvement of microbial transglutaminase‐induced gelation of β‐conglycinin by conjugation with dextran

This work focused on the effect of glycosylation on the gelation ability of β‐conglycinin induced by microbial transglutaminase (MTGase). Rheological results indicated that the gels of β‐conglycinin‐dextran conjugate products exhibited higher G′ value (172.2 ± 8.6 Pa) compared with those of dry‐heated β‐conglycinin (75.2 ± 5.1 Pa), β‐conglycinin (53.3 ± 4.0 Pa) and β‐conglycinin‐dextran mixture (38.6 ± 2.6 Pa) after 4 h incubation with MTGase. The gels prepared from β‐conglycinin‐dextran conjugate products had higher hardness, fracturability, springiness and cohesiveness values determined by textural profile analysis. The turbidity of β‐conglycinin‐dextran conjugate products solution incubated with MTGase increased faster than those of the other three protein samples. The conjugated dextran in β‐conglycinin‐dextran conjugate products could inhibit extensive protein–protein interactions which might result in the formation of more ordered and stronger gel network structures during MTGase cross‐linking process. A compact and homogeneous gel networks in β‐conglycinin‐dextran conjugate products gels were also observed by scanning electron microscopy.     

Changes in the structure,digestibility and immunoreactivities of glycinin induced by the cross‐linking of microbial transglutaminase following heat denaturation

This study aimed to evaluate the effects of microbial transglutaminase (MTG)‐mediated modification on the structure, digestibility and immunoreactivities of glycinin. Glycinin was separated from soya bean and cross‐linked with MTG, and the sulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE) showed that the molecular weight of cross‐linked glycinin was higher than that of native glycinin. Individual MTG cross‐linking could maintain stable secondary structures and spatial structure. Sequential heat denaturation and MTG cross‐linking could promote the unfolding of protein structures and reduce their hydrophobicity. The digestibility of glycinin was decreased, and its immunoreactivities were increased because of MTG‐induced structural alteration, including primary and spatial structures.     

Gelation of Food Protein Induced by Recombinant Microbial Transglutaminase

ABSTRACT: The recombinant microbial transglutaminase from Streptoverticillium mobaraense var. (rMTGase) was expressed in Escherichia coli. Specific enzyme activity of rMTGase was comparable to native MTGase. However, the gelation of a sodium caseinate solution induced by rMTGase was slower than that induced by native MTGase. In addition, the mechanical property of kamaboko prepared with rMTGase was weaker than that with native MTGase. In SDS‐PAGE analysis, α‐casein monomers decreased more slowly during the incubation with rMTGase than MTGase. These results confirmed the difference of cross‐linking activity between the 2 enzymes. Furthermore, thermal stability of rMTGase was lower compared to native MTGase. These results suggest that the difference of cross‐linking activity and thermal stability between the 2 enzymes cause differences in gelation activity of protein.     

Functional properties of limited hydrolysed cross‐linked casein–gelatin composites

A casein–gelatin composite was prepared by cross‐linking of caseinate and bovine gelatin with a microbial transglutaminase, and the impact of limited proteolysis by trypsin on some functional properties of the composite was investigated in the present work. Two hydrolysed composites were prepared with degree of hydrolysis (DH) of 1 and 2% and analysed by SDS‐PAGE to reflect polypeptide profiles. Some functional properties of the two hydrolysed composites were evaluated, among which solubility, digestibility in vitro, surface hydrophobicity and emulsifying properties showed dependence on the DH. Limited proteolysis of the composite improved its solubility in pH 3–10, especially when the DH was 2%. Compared to the composite, the hydrolysed composites showed an increased emulsifying activity index (about 357–408%), emulsion stability index (about 23–28%) and digestibility in vitro (about 65–80% for pepsin, whereas 2–3% for pepsin–trypsin hydrolysis), together with a decreased surface hydrophobicity (about 55–62%) and oil absorption capacity (about 20–23%). The applied proteolysis also led to the aqueous dispersions of the two hydrolysed composites much lower apparent viscosity, storage and loss modulus.     

Changes in in vitro protein digestion of retort‐pouched pork belly during 120‐day storage

To investigate the effect of long‐term storage on in vitro protein digestion of the retort‐pouched pork belly. The products were stored at 25 °C for 0, 30, 60 and 120 days and digested with pepsin and trypsin at each time point. SDS‐PAGE and LC‐MS‐MS were applied to separate and identify proteins and their digested products. In vitro protein digestibility decreased from 47.7% after 60‐day storage to 25.4% after 120‐day storage for the samples digested by pepsin, and from 63.9% to 45.7% for the samples digested by pepsin and trypsin at corresponding time points. LC‐MS‐MS demonstrated changes in peptide composition and abundance with storage time. Long‐term storage could deteriorate nutritive values of meat products by reducing digestible protein content.     

Effect of pH and Pepsin Limited Hydrolysis on the Structure and Functional Properties of Soybean Protein Hydrolysates

Effects of limited enzymatic hydrolysis with pepsin on the functional properties and structure characteristics of soybean proteins were investigated. Hydrolysates with different incubation time (10 to 900 min) were prepared. Results showed that SPI hydrolyzed for 60 min exhibited the best emulsibility and the ability of resisting freezing/thawing. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis proved that pepsin can degrade glycinin but had little effect on the α’ subunit of β‐conglycinin. The structure unfolding reached the largest extent after incubation for 60 min and the soluble and flexible aggregates were formed. After 120 min, glycinin was degraded totally and β‐conglycinin formed insoluble aggregates. Moreover, 2 methods were applied for the deactivation of pepsin to obtain final hydrolysates at pH 2.0 and 7.0, respectively. The structure analysis revealed that the unfolding extent and structure characteristic were different in these 2 conditions. When adjusting the pH value from 2.0 to 7.0, the unfolding protein molecular would reaggregate again at pH 7.0 due to the charge neutralization, and the hydrodynamic diameter and λ_max absorbance decreased compared to pH 2.0. Moreover, some of the insoluble aggregates formed at pH 2.0 became soluble at pH 7.0, because of the salt‐in phenomenon.     

The use of anti‐soya globulin antisera in investigating soya digestion in vivo

Polyclonal antisera have been developed which recognise the soya globulins glycinin and β‐conglycinin. Their binding to proteolysed and processed globulins has been characterised with a view to using the antisera as probes to investigate the fate of soya globulins during digestion. Trypsinolysis reduced the immunoreactivity of β‐conglycinin but increased that of glycinin by threefold, as determined by enzyme‐linked immunosorbent assay (ELISA). However, both antisera recognised trypsinolysis products poorly by immunoblotting. These data are consistent with the fact that the anti‐glycinin antiserum was raised to digested glycinin and would therefore recognise both intact and proteolytic intermediates of glycinin. The latter would be disrupted into component polypeptides by sodium dodecyl sulphate–polyacrylamide gel electrophoresis and hence would only be present in the ELISA and not after blotting. The antibody preparations were used to investigate the fate of soya globulins during in vivo digestion of a processed soya ingredient in rats. Both glycinin and β‐conglycinin were digested rapidly, intact globulins disappearing from the gastrointestinal tract 3 h after dosing. Immunoreactive globulins were found in gut contents and associated with gut tissues but appeared to be in a semi‐intact form, probably comprising proteolytic intermediates. This study demonstrates the usefulness of antibody methods in following the digestion of individual components in a complex mixture, such as is found in food. The availability of antibody preparations which recognise heat‐stable epitopes will open the way for investigations into the effects of pre‐treatments, such as cooking, which are pertinent to human consumption of legumes. © 2000 Society of Chemical Industry     

Comparative study on protein cross-linking and gel enhancing effect of microbial transglutaminase on surimi from different fish

BACKGROUND: Microbial transglutaminase (MTGase) has been used to increase the gel strength of surimi. Nevertheless, its effectiveness varies with fish species. The aim of this study was to elucidate the effect of MTGase at different levels on protein cross‐linking and gel property of surimi from threadfin bream, Indian mackerel and sardine in the presence and absence of endogenous transglutaminase. RESULT: Breaking force of all surimi gels increased as MTGase levels (0–0.6 U g^?1) increased except for threadfin bream surimi gel, where the breaking force decreased at 0.6 U g^?1 (P < 0.05). In the presence of EDTA, the gel strengthening effect was lower, suggesting the combined effect of endogenous transglutaminase with MTGase. With the addition of MTGase, the gel with the highest increase in breaking force showed highest decrease in myosin heavy chain. When cross‐linking activity of MTGase on natural actomyosin (NAM) was determined, the highest decreasing rate in ε‐amino group content with the concomitant increased formation of cross‐linked proteins was found in NAM from threadfin bream. The reactivity of muscle proteins toward MTGase‐induced cross‐linking was in agreement with surimi gel strengthening. CONCLUSION: The composition and properties of muscle proteins of varying fish species more likely determined protein cross‐linking induced by MTGase, thereby affecting their gel properties. Copyright © 2011 Society of Chemical Industry     

A comparison study of the impact of boiling and high pressure steaming on the stability of soybean trypsin inhibitor

The aim of the study is to compare the effect of boiling and high pressure steaming (HPS) on the degradation, inhibitory activity reduction and gastric digestibility of soybean trypsin inhibitor (STI). Thermal stability analysis showed that HPS treatment was effective in eliminating the inhibitory activity of STI than boiling. SDS‐PAGE and Western blot analysis indicated that boiling has less impact on the gastric digestibility of STI than HPS. More importantly, boiling‐pretreated STI revealed high inhibitory activity against trypsin even after digestion by pepsin in simulated gastric fluid (SGF), while HPS treatment was more effective. SDS‐PAGE analysis further verified that after boiling, STI still revealed strong binding ability to trypsin, while STI could be completely degraded by trypsin after HPS treatment for 30 min.     

Modification in physical properties of rice gel by microbial transglutaminase

BACKGROUND: Microbial transglutaminase (MTGase) may catalyse the cross‐linking between a peptide‐bound glutaminyl residue and an ε‐amino group of lysine residue in protein. MTGase has been used to modify many food proteins for improving the physical properties of products. However, its effect on the physical properties of rice products has not been investigated before. The present study aimed to investigate the effect of MTGase, as an additive in rice flour, on the rheological, textural and thermal properties of rice gel. RESULTS: Both the elastic and the viscous moduli of rice gel were increased as a result of the addition of MTGase to rice flour. The addition of MTGase at 0.01–0.3 U mg^?1 increased textural parameters (hardness and gumminess) and rheological properties. Differential scanning calorimetry also showed that MTGase treatment decreased the enthalpy change in gelation. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) confirmed that rice proteins were polymerised through the MTGase reaction. CONCLUSION: The present study proved that the cross‐linking of protein molecules in rice flour by the action of MTGase may improve the physical properties of rice gel. The addition of MTGase in rice flour in an adequate amount is essential for achieving appropriate physical properties of the product. Copyright © 2008 Society of Chemical Industry     

Interaction of mulberry anthocyanins with soybean protein isolate: effect on the stability of anthocyanins and protein in vitro digestion characteristics

The interactions of anthocyanins and proteins might mutually influence each other on the physicochemical and functional properties. In this study, the interaction mechanism of soybean protein isolate (SPI) with mulberry anthocyanins (MA), and its effect on the thermal stability of anthocyanins and protein subunits’ digestibility were investigated through multiple spectroscopies and in vitro gastrointestinal models. Results showed that cyanidin-3-O-glucoside (C3G), the main anthocyanin monomer in MA, could bind to SPI through hydrophobic interactions, resulting in static fluorescence quenching of SPI. The secondary structure of SPI changed by binding to C3G, with an increase of β-sheet and a decrease of α-helix and random coil. The formation of the SPI-MA complexes improved the thermal stability of MA at 353 K, while, no significant protection occurred during heating at 368 K. Complexation with MA promoted the digestibility of SPI by pepsin, especially the α′ and α subunits of β-conglycinin and the basic subunit of glycinin, and slightly delayed the digestibility of SPI under intestinal fluid. These results provide an in-depth understanding of the influence of the protein-anthocyanin interactions on the stability of anthocyanins and protein digestibility.     

Characterization of proteins from kernel of different soybean varieties

BACKGROUND: Total soybean proteins, storage proteins, glycinin (11S) and β‐conglycinin (7S) fractions and their respective subunits in seven soybean varieties were analyzed. In this work we also present the correlation between concentration and activity of bioactive proteins, lipoxygenase and proteinase inhibitors. RESULTS: Glycinin and β‐conglycinin comprise about 750 g kg^?1 of the bean storage protein and as such account for both quantity and quality of the kernel protein. The 11S concentration of the varieties studied ranged from 503.4 to 602.9 g kg^?1 and those of 7S varied from 178.2 to 230.6 g kg^?1 of total extractable proteins. The ratio of 11S/7S proteins varied from 2.43 to 3.29 among the varieties. A very strong positive correlation was found between the concentration of Kunitz trypsin inhibitor and activity of total trypsin inhibitor (r = 0.96). However, lipoxygenase concentration did not show a strong correlation with lipoxygenase activity. CONCLUSION: It appears that among the seven ZP soybean genotypes there are genotypes with different amounts of subunits that should be bred in the future for a desired level of protein components. Copyright © 2010 Society of Chemical Industry     

Digestion of peanut allergens Ara h 1, Ara h 2, Ara h 3, and Ara h 6: A comparative in vitro study and partial characterization of digestion‐resistant peptides

Scope : There are differences in stability to pepsin between the major allergens in peanut; however, data are from different reports using different digestion models. This study provides a comprehensive comparison of the digestibility of the major peanut allergens. Methods and results : Peanut allergens Ara h 1, Ara h 2, Ara h 3 and Ara h 6 were incubated with pepsin to mimic the effect of gastric digestion. Samples were analyzed using SDS‐PAGE. To further investigate resistance to digestion, Ara h 2 was additionally subjected to digestion with trypsin and residual peptides were characterized. Ara h 1 and Ara h 3 were rapidly hydrolyzed by pepsin. On the contrary, Ara h 2 and Ara h 6 were resistant to pepsin digestion, even at very high concentrations of pepsin. In fact, limited proteolysis could only be demonstrated by SDS‐PAGE performed under reducing conditions, indicating an important role for the disulfide bridges in maintaining the quaternary structure of Ara h 2 and Ara h 6. Trypsin digestion of Ara h 2 similarly resulted in large residual peptides and these were identified. Conclusion : Ara h 2 and Ara h 6 are considerably more stable towards digestion than Ara h 1 and Ara h 3.     

Digestibility and allergenicity assessment of enzymatically crosslinked β‐casein

Crosslinking enzymes are frequently used in bioprocessing of dairy products. The aim of this study was to examine the effects of enzymatic crosslinking on IgE binding, allergenicity and digestion stability of β‐casein (CN). β‐CN was crosslinked by transglutaminase, tyrosinase, mushroom tyrosinase/caffeic acid and laccase/caffeic acid. The IgE binding to β‐CN was compared in vitro by CAP inhibition assay, ELISA inhibition as well as ex vivo by basophil activation assay. Crosslinked CNs were digested by simulated gastric fluid for 15 and 60 min and obtained digests analyzed for their ability to inhibit IgE binding by CAP inhibition assay and SDS‐PAGE. The ability of crosslinked CNs to activate basophils was significantly reduced in seven patients in the case of CN crosslinked by laccase and moderately reduced in the case of tyrosinase/caffeic acid crosslinked CN (in two cow's milk allergy patients tested with different allergen concentrations). The response to various crosslinked CNs differed individually among patients' sera tested by ELISA inhibition assay. The presence of caffeic acid hampered digestion by pepsin, and this effect was most pronounced for the tyrosinase/caffeic acid crosslinked CN. The laccase/caffeic acid and mushroom tyrosinase/caffeic acid had the highest potential in mitigating IgE binding and allergenicity of the β‐CN out of all investigated enzymes. The presence of a small phenolic compound also increased digestion stability of β‐CN.     

Characterization,amino acid composition and in vitro digestibility of hemp (Cannabis sativa L.) proteins

The protein constituents and thermal properties of hemp (Cannabis sativa L.) protein isolate (HPI) as well as 11S- and 7S-rich HPIs (HPI-11S and HPI-7S) were characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and different scanning calorimetry (DSC), and their amino acid composition and in vitro digestibility were also evaluated, as compared to soy protein isolate (SPI). SDS-PAGE analysis showed that the edestin (consisting of acidic and basic subunits, AS and BS) was the main protein component for HPI and HPI-11S, while HPI-7S was composed of the BS of edestin and a subunit of about 4.8 kDa. DSC analysis characterized thermal transition of the edestin component and the possible present form of different subunits. Except lysine and sulfur-containing amino acids, the essential amino acids of various HPIs met the suggested requirements of FAO/WHO for 2–5 year old infants. The proportion of essential amino acids to the total amino acids (E/T) for HPI (as well as HPI-11S) was significantly higher than that of SPI. In an in vitro digestion model, various protein constituents of various HPIs were much easily digested by pepsin plus trypsin, to release oligo-peptides with molecular weight less than 10.0 kDa (under reduced condition). Only after pepsin digestion, in vitro digestibility of HPIs was comparable to that of SPI, however after pepsin plus trypsin digestion, the digestibility (88–91%) was significantly higher than that (71%) of SPI (P < 0.05). These results suggest that the protein isolates from hempseed are much more nutritional in amino acid nutrition and easily digestible than SPI, and can be utilized as a good source of protein nutrition for human consumption.     

Formation and properties of glycinin-rich and β-conglycinin-rich soy protein isolate gels induced by microbial transglutaminase

The gelation and gel properties of glycinin-rich and β-conglycinin-rich soy protein isolates (SPIs) induced by microbial transglutaminase (MTGase) were investigated. At the same enzyme and protein substrate concentrations, the on-set of gelation of native SPI and the viscoelasticity development of correspondingly formed gels depended upon the relative ratio of glycinin to β-conglycinin. The turbidity analysis showed that the glycinin components also contributed to the increase in the turbidity of SPI solutions incubated with MTGase (at 37 °C). Textural profile analysis indicated that the glycinin components of SPIs principally contributed to the hardness, fracturability, gumminess and chewiness values of corresponding gels, while the cohesiveness and springness were mainly associated with the β-conglycinin components. The strength of MTGase-induced gels of various kinds of SPIs could be significantly improved by the thermal treatment. The protein solubility analyses of MTGase-induced gels, indicated that besides the covalent cross-links, hydrophobic and H-bondings and disulfide bonds were involved in the formation and maintenance of the glycinin-rich SPI gels, while in β-conglycinin-rich SPI case, the hydrophobic and H-bondings were the principal forces responsible for the maintenance of the gel structure. The results suggested that various kinds of SPI gels with different properties could be induced by MTGase, through controlling the glycinin to β-conglycinin ratio.