It is generally acknowledged that despite the superior nutritional value of soymilk, the presence of off-flavours catalysed by lipoxygenase (LOX) activities has to some extent negatively influenced the overall acceptability of soymilk in consumers. In this current study, the effect of roasting soybeans at different temperatures (110–130 °C) and times (20–120 min) on physical, chemical properties and LOX activity of soymilk was investigated in order to establish optimum soybean roasting conditions for developing nutritious and LOX free soymilk. Results have revealed that subjecting soybeans to increased roasting temperature and time negatively affected physical chemical properties of soymilk by decreasing the yield, solid content, colour and nutritive value. On the other hand, roasting soybeans at a relatively low temperature of 110 °C for 80 min significantly inactivated LOX but retained adequate protein content in soymilk. The findings of this study have demonstrated that careful selection of roasting conditions can be a useful strategy for inactivating LOX activity in development of a more acceptable soymilk product.

•At high ionic strength, pHc occurred at the pH lower than isoelectric point of BBI.•The binding of BBI to ι-carrageenan formed a two-step binding.•The presence of nonelectrostatic interactions at the first binding step was proved.•rcritical was independent of protein concentration, ionic strength and temperature.Complex behavior of Bowman–Birk protease inhibitor (BBI) with ι-carrageenan (LC) as a function of pH, protein to polysaccharides ratio and salt concentration was studied by turbidimetric titration, dynamic light scattering (DLS) and isothermal titration calorimetry (ITC). At fixed BBI/LC weight ratio of 5:1, turbidity and DLS results showed that pHc and pHφ1 shift to the lower pH values with the increase in ionic strength (I), whereas the former occurred at the pH lower than isoelectric point (pI = 4.2) of BBI at I ≥ 100 mM NaCl. ITC results showed that BBI binding to LC involves a two-step process with an increasing exothermic enthalpy at the first binding step. The further insight of BBI-LC complexation was studied as a function of BBI concentration, ionic strength and temperatures using ITC. The critical molar ratio (rcritical) between two binding steps was independent of protein concentration, ionic strength and temperature, although the heat flow obviously decreased with the increasing I (0–200 mM) and slightly increased with the elevated temperature (25–45 °C). The negative heat capacity (ΔCp) and the gain in nonionic contribution (ΔGno) indicated the involvement of nonelectrostatic interactions (e.g., hydrophobic effect) for the first binding step.Download high-res image (271KB)Download full-size image

•Non-network proteins contained mainly acid polypeptides and undissociated AB.•Higher G′ of gel at higher temperature was due to higher network proteins ratio.•Larger protein aggregates resulted in higher G′ values of soy gel.•More basic polypeptides were observed as network proteins in glycinin-rich gel.The effects of heating temperature, ionic strength and 11S ratio on the rheological properties of heat-induced soy protein gels, particularly in relation to network proteins content and aggregates size were investigated. Results revealed that non-network proteins consisted of undenatured glycinin (11S) AB subunits, high levels of A and A3 polypeptides but low levels of β-conglycinin (7S) α and α′ subunits. Results further showed that a positive correlation between storage modulus (G′) and network proteins ratio of gels that were formed at different temperatures. Additionally, the G′ values of gels initially increased with increasing NaCl concentration from 0 to 0.25 M, but decreased with further increasing ionic strength from 0.25 to 0.50 M. This trend was consistent with the variation of heat-induced protein aggregates size in soy protein solution at the same ionic strength. Higher 11S/7S ratio resulted in higher storage modulus values of soy protein gels due to the formation of larger and compacter aggregates mainly by B polypeptides via hydrophobic bonds.Download high-res image (296KB)Download full-size image

It is well-known that disulfide-mediated interactions are important when soy protein is heated, in which soy proteins are dissociated and rearranged to some new forms. In this study, the disulfide bond (SS) linked polymer, which was composed of the acidic (A) polypeptides of glycinin, basic (B) polypeptides of glycinin, and a small amount of α′ and α of β-conglycinin, was formed as the major product, accompanied by the formation of SS-linked dimer of B and monomer of A as minor products. The role of sulfhydryl (SH) of different subunits/polypeptides for forming intermolecular SS was investigated. The SH of B in glycinin (Cys298 of G1, Cys289 of G2, Cys440 of G4) was transformed into SS in polymer identified by mass spectrometry analysis. The SH content of polymer was lower than that of glycinin and β-conglycinin subunits when heated. The SH content of B in polymer was lower than that in glycinin subunit, and both of them were decreased by heating. The SH content of A in polymer was increased and higher than that of B in polymer and A in glycinin subunit when heated. These results indicated that the SH of B in glycinin subunit was subjected to not only SH oxidation but also SH–SS exchange (with SS of A) for forming intermolecular SS of polymer. The SH oxidation and SH–SS exchange were proven by the change of SH content for the first time. The SH of B was suggested to be reactive for forming intermolecular SS of polymer.

Heat-induced soy protein gels were prepared by heating protein solutions at 12%, 15% ,or 18% for 0.5, 1.0, or 2.0 h. The release of non-network proteins from gel slices was conducted in 10 mM pH 7.0 sodium phosphate buffer. SDS-PAGE and diagonal electrophoresis demonstrated that the released proteins consisted of undenatured AB subunits and denatured proteins including monomers of A polypeptides, disulfide bond linked dimers, trimers, and polymers of A polypeptides, and an unidentified 15 kDa protein. SEC-HPLC analysis of non-network proteins revealed three major protein peaks, with molecular weights of approximately 253.9, 44.8, and 9.7 kDa. The experimental data showed that the time-dependent release of the three fractions from soy protein gels fit Fick’s second law. An increasing protein concentration or heating time resulted in a decrease in diffusion coefficients of non-network proteins. A power law expression was used to describe the relationship between non-network protein diffusion coefficient and molecular weight, for which the exponent (α) shifted to higher value with an increase in protein concentration or heating time, indicating that a more compact gel structure was formed.

This study aimed to develop an optimal continuous procedure of immobilized hydroperoxide lyase (HPL)-catalyzed synthesis of hexanal. A central composite design was used to study the combined effect of substrate concentration and the residence time of the reactant on hexanal concentration. The optimum conditions for hexanal synthesis included a 13-HPOD concentration of 43.54 mM and a residence time of 60.99 min. The maximum hexanal concentration was 3560 ± 130 mg/L when 16 U of immobilized HPLwas used. Furthermore, the stability of immobilized HPL was significantly improved in the packed-bed reactor, as evidenced by the slowed enzyme inactivation and prolonged operation time. The immobilized HPL remained activity until 40 mL substrate solution flowed past the packed-bed reactor. The catalyst productivity of hexanal in the packed-bed reactor was 5.35 ± 0.34 mg/U, much higher than that in the batch stirred reactor. This study was greatly meaningful for providing a green method to the large-scale production of hexanal.

The adsorption of heat-denatured soy proteins at the oil/water (O/W) interface during emulsification was studied. Protein samples were prepared by heating protein solutions at concentrations of 1–5% (w/v) and were then diluted to 0.3% (w/v). The results showed that soy proteins that had been heated at higher concentrations generated smaller droplet size of emulsion. Increase in homogenizer rotating speed resulted in higher protein adsorption percentages and lower surface loads at the O/W interface. Surface loads for both unheated and heated soy proteins were linearly correlated with the unadsorbed proteins’ equilibrium concentration at various rotating speeds. With the rise in NaCl addition level, protein adsorption percentage and surface loads of emulsions increased, whereas lower droplet sizes were obtained at the ionic strength of 0.1 M. The aggregates and non-aggregates displayed different adsorption behaviors when rotating speed or NaCl concentration was varied.

Phytate is an important antinutritional factor in food products. In this study, a phytase-assisted processing method was used to produce low-phytate soybean protein isolate (SPI) samples, and their physicochemical and functional properties were examined. Hydrolysis condition at low temperature (room temperature) and pH 5.0 was better than that recommended by manufacturer (pH 5.0, 55 °C) at keeping the properties of SPI, so the former condition was selected to prepare SPI samples with phytate contents of 19.86–0.11 mg/g by prolonging hydrolysis time (0 (traditional method), 5, 10, 20, 40, and 60 min). Ash content (R2 = 0.940), solubility (R2 = 0.983), ζ-potential value (R2 = 0.793), denaturation temperatures (β-conglycinin, R2 = 0.941; glycinin, R2 = 0.977), emulsifying activity index (R2 = 0.983), foaming capacity (R2 = 0.955), and trypsin inhibitor activity (R2 = 0.821) of SPI were positively correlated with phytate content, whereas protein content (R2 = 0.876), protein recovery (R2 = 0.781), emulsifying stability index (R2 = 0.953), foaming stability (R2 = 0.919), gel hardness (R2 = 0.893), and in vitro digestibility (R2 = 0.969) were negatively correlated with phytate content. Simulated gastrointestinal digestion and subsequent dialysis showed that percentages of dialyzable Zn and Ca were increased with decreasing phytate content, whereas the amounts of dialyzable Zn and Ca revealed different behaviors: the former was increased and the latter was decreased. Circular dichroism spectra showed that secondary structure of SPI was changed by phytase. Compared with traditional processing method, the phytase-assisted processing method could produce SPI with lower phytate and higher protein contents, which had better in vitro digestibility and could be used to prepare gels with higher hardness by partially losing some other functional properties.

After oil bodies (OBs) were extracted from ungerminated soybean by pH 6.8 extraction, it was found that 24 and 18 kDa oleosins were hydrolyzed in the extracted OBs, which contained many OB extrinsic proteins (i.e., lipoxygenase, β-conglycinin, γ-conglycinin, β-amylase, glycinin, Gly m Bd 30K (Bd 30K), and P34 probable thiol protease (P34)) as well as OB intrinsic proteins. In this study, some properties (specificity, optimal pH and temperature) of the proteases of 24 and 18 kDa oleosins and the oleosin hydrolysis in soybean germination were examined, and the high relationship between Bd 30K/P34 and the proteases was also discussed. The results showed (1) the proteases were OB extrinsic proteins, which had high specificity to hydrolyze 24 and 18 kDa oleosins, and cleaved the specific peptide bonds to form limited hydrolyzed products; (2) 24 and 18 kDa oleosins were not hydrolyzed in the absence of Bd 30K and P34 (or some Tricine-SDS-PAGE undetectable proteins); (3) the protease of 24 kDa oleosin had strong resistance to alkaline pH while that of 18 kDa oleosin had weak resistance to alkaline pH, and Bd 30K and P34, resolved into two spots on two-dimensional electrophoresis gel, also showed the same trend; (4) 16 kDa oleosin as well as 24 and 18 kDa oleosins were hydrolyzed in soybean germination, and Bd 30K and P34 were always contained in the extracted OBs from germinated soybean even when all oleosins were hydrolyzed; (5) the optimal temperature and pH of the proteases were respectively determined as in the ranges of 35–50 °C and pH 6.0–6.5, while 60 °C or pH 11.0 could denature them.

Proteins in soybean whey were separated by Tricine–SDS-PAGE and identified by MALDI-TOF/TOF-MS. In addition to β-amylase, soybean agglutinin (SBA), and Kunitz trypsin inhibitor (KTI), a 12 kDa band was found to have an amino acid sequence similar to that of Bowman–Birk protease inhibitor (BBI) and showed both trypsin and chymotrypsin inhibitor activities. The complex behavior of soybean whey proteins (SWP) with chitosan (Ch) as a function of pH and protein to polysaccharide ratio (RSWP/Ch) was studied by turbidimetric titration and SDS-PAGE. During pH titration, the ratio of zeta potentials (absolute values) for proteins to chitosan (|ZSWP|/ZCh) at the initial point of phase separation (pHφ1) was equal to the reciprocal of their mass ratio (SWP/Ch), revealing that the electric neutrality conditions were fulfilled. The maximum protein recovery (32%) was obtained at RSWP/Ch = 4:1 and pH 6.3, whereas at RSWP/Ch = 20:1 and pH 5.5, chitosan consumption was the lowest (0.196 g Ch/g recovered proteins). In the protein–chitosan complex, KTI and the 12 kDa protein were higher in content than SBA and β-amylase. However, if soybean whey was precentrifuged to remove aggregated proteins and interacted with chitosan at the conditions of SWP/Ch = 100:1, pH 4.8, and low ionic strength, KTI was found to be selectively complexed. After removal of chitosan at pH 10, a high-purity KTI (90% by SEC-HPLC) could be obtained.

Soy protein has been extracted and differently treated to yield products of diverse desirable characteristics. Generally, synthetic chemicals (NaOH and HCl) are used to extract the protein from n-hexane-defatted soy flour (DF). Some researchers have tried to extract it from full-fat flour (FF) using natural chemicals (NC) in response to the ever-increasing consumer preference for naturally processed foods. The effects of these chemicals on the structure of the resultant protein product are not known. This study examined oxidative and structural modification effects of NC and SC as they interact with proteins in the DF and FF systems. Significant differences (p < 0.01) were observed in all samples in lipoxygenase activity, degree of oxidation, free and total sufhydryl content and intrinsic fluorescence, which indicated increased structural changes in the FF-based samples as compared to those prepared from DF. Circular dichroism spectroscopy showed existence of α-helices and β-sheets protein secondary structures. However, DF samples showed increase in unordered structural conformation changes than the FF sample. Surface hydrophobicity and size exclusion chromatography results were more related to the extraction chemicals than the type of flour used, with samples prepared with SC showing an increase. That’s, the use of natural and synthetic protein extraction chemicals from FF and DF have different effects on the structure and degree of oxidation of the resultant proteins.

Chemical composition, molecular weight distribution, secondary structure and effect of sodium chloride concentration on functional properties of walnut protein isolates, concentrates and defatted walnut flour were study. Compared with walnut protein concentrates (75.6%) and defatted walnut flour (52.5%), walnut protein isolates contain a relatively high amount of protein (90.5%). The yield of walnut protein isolates and concentrates was 43.2% and 76.6%, respectively. In molecular weight distribution study, Walnut protein isolates showed one peak with molecular weight of 106.33 KDa (100%) and walnut protein concentrates showed four peaks with molecular weight of 16,725 KDa (0.8%),104.943 KDa(63.9%), 7.3 KDa (11.4%), 2.6 KDa (23.9%). The secondary structure of walnut protein isolates was similar to that of walnut protein concentrates, but was differ from that of defatted walnut flour. The addition of sodium chloride (0 ~ 1 M) could improve the functionality of walnut protein concentrates, isolates and defatted walnut flour. The maximum solubility, water absorption capacity, emulsifying properties and foaming properties of walnut protein isolates, concentrates and defatted walnut flour were at sodium chloride solutions of 1.0 M, 0.6 M, 0.4 M, 0.6 M, respectively. The solubility of walnut protein concentrates (32.5%) in distilled water with 0 M sodium chloride was lower than that of walnut protein isolates (35.2%). The maximum solubility of walnut protein isolates, concentrates and defatted walnut flour in solution were 36.8%, 33.7% and 9.6% at 1.0 M sodium chloride solutions, respectively. As compared with other vegetable proteins, walnut protein isolates and concentrates exhibited better emulsifying properties and foam stability.

In this study, the sulfhydryl (SH) contents of unheated and heated (90 °C, 5 min) soy protein were detected under different conditions (pH, reagent addition order, SDS/GuHCl concentration, EDTA) using two aromatic disulfide reagents: 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) and 4,4′-dithiodipyridine (DPS). Two fluorescent alkylating reagents, monobromobimane (mBBr) and N-(1-pyrenyl)maleimide (NPM), were chosen due to their high sensitivity and were also used. Amino acid analysis was used to detect the SH (cysteine) contents of unheated (7.51 ± 0.45 μmol SH/g protein) and heated (1.47 ± 0.10 μmol SH/g protein) soy protein, and similar results were obtained using enzymatic hydrolysis-assisted DPS. The SH content detected by DTNB was affected by pH, denaturant species, and denaturant concentration, and the best results were obtained at pH 7.0 when 6 M GuHCl was added after DTNB. These results were lower than that of the amino acid analysis, however. The SH detected by DPS was not as affected as that of DTNB by pH, denaturant species, and denaturant concentration. Additionally, the results of the amino acid analysis were similar to that of DPS at pH 7.0 in 2% SDS and 4–6 M GuHCl when SDS and GuHCl were added after DPS. EDTA did not have a significant effect on SH detection when DTNB and DPS were added before SDS and GuHCl. Finally, although mBBr and NPM can detect SH in low protein concentrations (1/10 of that required for DTNB and DPS), mBBr and NPM overestimated the SH content of soy protein. Therefore, using DPS at pH 7.0 when it is added before SDS and GuHCl is the most reliable method for detecting the SH content of soy protein.

Mixtures of soy protein (SP) and gum arabic (GA) formed an electrostatic complex in a relatively narrow pH range at very low ionic strength. The conditions under which the complexes could be formed were determined using turbidimetric measurements first. In salt-free condition and 1:1 SP/GA mixture, critical pH values with the formation of soluble (pHc = 4.40), insoluble (pHφ1 = 3.55), and maximum (pHopt = 3.15) complexes were observed. As SP/GA ratios increased from 1:4 to 8:1, critical pH values shifted toward higher pH. Charge densities (ZN) for SP and GA were calculated from electrophoretic mobility using soft particle analysis theory. Results showed that a 1:1 charge ratio at pHφ1 was found at any SP/GA ratio, indicating that charge compensation was fulfilled for SP/GA insoluble complex formation. A SP–GA–water ternary phase diagram was built at pH 4.0. The influence of the total biopolymer concentration (0–10% w/w) and SP/GA ratio was represented in the phase diagram. At a total concentration of 0.10%, results were consistent with the turbidity measurement; that is, no phase separation occurred at an SP/GA ratio lower than 1:2 at pH 4.0. Salt effect (NaCl, 0–500 mmol/L) on SP/GA complexes was discussed. Results indicated that SP/GA complexing, which led to the formation of turbidity peaks at pH 3.2, was suppressed when NaCl concentrations were ≥50 mmol/L, whereas the remarkable increase in turbidity around pH 5.0 was caused by the aggregation of soy protein molecules on which gum arabic could be adsorbed.

Soybean oil bodies (OBs), naturally pre-emulsified soybean oil, have been examined by many researchers owing to their great potential utilizations in food, cosmetics, pharmaceutical, and other applications requiring stable oil-in-water emulsions. This study was the first time to confirm that lectin, Gly m Bd 28K (Bd 28K, one soybean allergenic protein), Kunitz trypsin inhibitor (KTI), and Bowman–Birk inhibitor (BBI) were not contained in the extracted soybean OBs even by neutral pH aqueous extraction. It was clarified that the well-known Gly m Bd 30K (Bd 30K), another soybean allergenic protein, was strongly bound to soybean OBs through a disulfide bond with 24 kDa oleosin. One steroleosin isoform (41 kDa) and two caleosin isoforms (27 kDa, 29 kDa), the integral bioactive proteins, were confirmed for the first time in soybean OBs, and a considerable amount of calcium, necessary for the biological activities of caleosin, was strongly bound to OBs. Unexpectedly, it was found that 24 kDa and 18 kDa oleosins could be hydrolyzed by an unknown soybean endoprotease in the extracted soybean OBs, which might give some hints for improving the enzyme-assisted aqueous extraction processing of soybean free oil.

Kunitz trypsin inhibitor (KTI) and Bowman–Birk inhibitor (BBI) have trypsin inhibitor activities (TIA), which could cause pancreatic disease if at a high level. It is not clear why some KTI and BBI lose TIA and some does not in the soymilk processing. This would be examined in this study. TIA assay showed residual TIA was decreased with elevated temperature and TIA was decreased quickly in the beginning and then slowly in boiling water bath. Interestingly, ultracentrifugation showed low residual TIA soymilk had more precipitate than high residual TIA soymilk and soymilk TIA loss had a high correlation coefficient (R2 > 0.9) with precipitate amount. In addition, the TIAs of floating, supernatant, and precipitate obtained by ultracentrifugation were assayed and >80% residual TIA was concentrated in the supernatant. Tricine-SDS-PAGE showed KTI in supernatant was mainly a noncovalent bound form which might exist as itself and/or incorporated into a small protein aggregate, while KTI in precipitate was incorporated into a protein aggregate by disulfide and/or noncovalent bonds. Chymotrypsin inhibitor activity (CIA) assay showed about 89% of the original CIA remained after 100 °C for 15 min. Ultracentrifugation showed that >90% residual CIA was concentrated in supernatant. Tricine-SDS-PAGE showed soymilk (100 °C, 15 min) BBI mainly existed in supernatant but not in precipitate. It was considered that BBI tended to exist as itself with its natural conformation. Thus, it was suggested residual TIA was mainly from the free BBI and TIA inactivation was mainly from KTI incorporation into protein aggregate. This study is meaningful for a new strategy for low TIA soymilk manufacture based on the consideration of promoting protein aggregate formation.

(2E)-Hexenal is widely used in flavors and perfumes; however, it is mainly derived from chemical synthesis. In this study, hydroperoxide lyase (HPL) naturally originated from Amaranthus tricolor was used to catalyze the reaction to generate (2E)-hexenal from 13-hydroperoxy-9Z, 11E, 15 Z-octadecatrienoic acid (13-HPOT), and salt-adding steam distillation was used for the separation of (2E)-hexenal. A maximum yield of (2E)-hexenal that reached 1,156.4 mg L−1 was obtained with a high substrate concentration (40 mM 13-HPOT) under the following conditions: 10 min, pH 7.5, 20 °C, HPL 16 U mL−1, butylated hydroxytoluene (BHT) 1 mM, and dithiothreitol (DTT) 15 mM. Then the separation of (2E)-hexenal was conducted by salt-adding steam distillation. It was found that AlCl3 had the greatest effect on the separation of (2E)-hexenal, followed by CaCl2 and NaCl. The distillate yield with the addition of AlCl3 was 93.2 %, while the distillate yield without the addition of salt was 81.2 %. The distillate concentration with AlCl3 was 6,464.2 mg L−1, while the distillate concentration without the addition of salt was 4,490.3 mg L−1. The addition of salt improved the efficiency of the steam distillation. This study was greatly meaningful for providing a green method to the large-scale production of (2E)-hexenal.

Hydroperoxide lyase (HPL) was extracted from amaranth tricolor leaves using Triton X-100, and purified to electrophoretic homogeneity by ammonium sulfate precipitation, ion-exchange chromatography, hydrophobic interaction chromatography and hydroxyapatite chromatography. The purified HPL preparation consisted of a single band and spot with a molecular mass of about 55 kDa as shown in SDS–PAGE and 2-D PAGE, respectively; the isoelectric point was found to be about 5.4. The maximum activity of the enzyme was observed at pH 6.0 and 25 °C, respectively. The HPL showed higher activity against 13-hydroperoxy-linolenic acid compared to 13-hydroperoxy-linoleic acid. Km value for 13-hydroperoxy-linolenic acid was 62.7 μM, and the corresponding Vmax was 178.5 μM min−1. The activity of HPL was significantly inhibited by nordihydroguaiaretic acid, HgCl2 and 2(E)-hexenal but not by EDTA and hexanal.

Oxidative modification of soy protein by peroxyl radicals generated in a solution containing 2,2’-azobis (2-amidinopropane) dihydrochloride (AAPH) under aerobic condition was investigated. Incubation of soy protein with increasing concentration of AAPH resulted in gradual generation of protein carbonyl derivatives and loss of protein sulphydryl groups. Circular dichroism spectra indicated that exposure of soy protein to AAPH led to loss of α-helix structure. Effect of oxidation on tertiary structure was demonstrated by surface hydrophobicity and tryptophan fluorescence. Surface hydrophobicity steadily decreased, accompanied by loss and burial of some tryptophan residues, indicating that soy protein gradually aggregated. The results of the size exclusion chromatogram (SEC) implied that incubation caused an AAPH-dose-dependent increase of fragmentation and aggregation of oxidised soy protein. Sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) indicated that non-disulphide linkages were involved in aggregate formation, and β-conglycinin was more vulnerable to peroxyl radicals than glycinin.

To reveal the role of primary products of lipid peroxidation during soy protein oxidation process, oxidative modification of soy protein by 13-hydroperoxyoctadecadienoic acid (13-HPODE) generated by lipoxygenase-catalyzed oxidation of linoleic acid was investigated in this article. Incubation of soy protein with increasing concentration of 13-HPODE resulted in generation of protein carbonyl derivatives and loss of protein sulfhydryl groups. Circular dichroism spectra indicated that exposure of soy protein to 13-HPODE led to loss of α-helix structure. Effect of oxidation on tertiary structure was demonstrated by surface hydrophobicity and tryptophan fluorescence. Surface hydrophobicity gradually decreased, accompanied by loss and burial of some tryptophan residues. The results of surface hydrophobicity and tryptophan fluorescence implied that aggregation was induced by oxidation. Size exclusion chromatogram indicated that the extent of aggregation was increased in a 13-HPODE dose-dependent manner. Sodium dodecyl sulfate polyacrylamide gel electrophoresis indicated that non-disulfide linkages were involved in aggregate formation, and β-conglycinin was more vulnerable to 13-HPODE than glycinin.

Soybean lipoxygenase was inactivated to different degrees by dry heating of defatted soybean flour for 0, 5, 10, 15, 20 and 25 min and soy protein isolates were prepared thereof by isoelectric precipitation of the water extract of the defatted soybean flour. The fluorescence emission intensity at 420 nm of the chloroform–methanol extract of soy protein isolates, which was indicator of the existence of peroxidized lipid, varied in parallel with the lipoxygenase residual activity in defatted soybean flours. The dispersion of soy protein isolate showed an increasing turbidity with the increase of lipoxygenase residual activity in the starting defatted soybean flour, suggesting an elevated tendency to form insoluble aggregates during the preparation of soy protein isolate. Small deformation rheological test revealed that the gelling times were shorter for those soy protein isolates derived from low lipoxygenase activity defatted soybean flours than that of high lipoxygenase activity. Frequency sweep showed that G′ of soy protein isolate derived from low lipoxygenase defatted soybean flour was independent of oscillation frequency in contrast to that of derived from non dry-heated defatted soybean flour, the latter showed a marked frequency dependence. Large deformation test revealed that the gel hardness increased about 10 times after dry heating of defatted soybean flour for 20 min. As the increase of the lipoxygenase residual activity, the gel permeability increased markedly, suggesting that soy protein isolate from high lipoxygenase defatted soybean flour produced coarser textured gel, which corresponded well with the results of scanning electron microscopy.

The effects of molecular weight of dextran on the phase behavior and microstructure of preheated soy protein (heat-induced soy protein aggregate)/dextran mixtures have not been reported before. Hereby mixed systems of heat-induced aggregates with different size and dextran with different molecular weight have been investigated at room temperature (25 °C) and pH 7.0. Phase diagrams were established by centrifugation, chemical assays and visual observation. The mixture of dextran with larger molecular weight and larger aggregate phase separated at lower biopolymer concentration. The microstructures of the phase separated mixtures were described using confocal laser scanning microscopy (CLSM), which revealed the association of protein aggregates. The image analysis of the CLSM images showed that the histograms of the gray values were different significantly. Observations of small deformation rheology (G′, G′′) of the mixed system at concentrations corresponding to those of CLSM measurements provided additional information of the microstructures.

This investigation characterized wettability and adhesive properties of the major soy protein components conglycinin (7S) and glycinin (11S) after urea modification. Modified 7S and 11S soy proteins were evaluated for gluing strength with pine, walnut, and cherry plywood and for wettability using a bubble shape analyzer. The results showed that different adhesives had varying degrees of wettability on the wood specimens. The 7S soy protein modified with urea had better wettability on cherry and walnut. The 11S soy protein modified with 1M urea had better wettability on pine. The 1M urea modification gave 11S soy protein the greatest bonding strength in all the wood specimens. The 3M urea modification gave 7S soy protein stronger adhesion on cherry and walnut than did 11S protein; but with pine, 11S soy protein had greater adhesion strength than 7S soy protein. Measurement of protein secondary structures indicated that the β-sheet played an important role in the adhesion strength of 3M urea-modified soy protein in cherry and walnut, while random coil was the major factor reducing adhesion strength of 7S soy protein modified with 1M urea.

•Chitosan hybrid hydrogel was a suit carrier matrix for HPL immobilization.•The introduction of hydrophobic spacer-arm was benefit to the immobilization of HPL.•Product yield of 2(E)-hexenal by immobilized HPL was higher than the free enzyme.•The amount of enzyme demanded in the catalytic reaction was lower for immobilized HPL.Five kinds of spacer arm attached chitosan hybrid hydrogels were tested for the possibility of being used as carriers for the immobilization of hydroperoxide lyase from amaranthus tricolor leaves. The 1,6-hexamethylenediamine attached chitosan-κ-carrageenan with biomimetic hydrophobic surface was proved to be the most suitable carrier. A maximum activity of 7.49 ± 0.19 U/g and a yield of 95% were obtained under optimized coupling condition. Meanwhile, the affinity between enzyme and substrates was not reduced after immobilization, as evidenced by the fact that the Km value of hydroperoxide lyase decreased from 108.6 to 79.97 μM for 13-hydroperoxy-linoleic-acid and almost unchanged for 13-hydroperoxy-linolenic-acid. Furthermore, the thermal, operational and storage stabilities of HPL were significantly improved after immobilization. Using the immobilized enzyme as the catalyst, the yield of 2(E)-hexenal and hexanal reached 1374.8 ± 51.8 mg/L and 1987.9 ± 67.9 mg/L, respectively, and the amount of immobilized enzyme needed in the reaction mixture was much lower than its free counterpart.Download full-size image

•At pHmax, KTI and BBI, rather than β-amylase and SBA complexed with chitosan.•KTI preferentially bound to carrageenan compared to BBI at pHmax.•Nonelectrostatic interactions will be involved in β-amylase/chitosan complexation.•A purified BBI with high TIA and CIA activity was obtained.Two successive and selective coacervations induced by chitosan (Ch) and carrageenan (CG) were applied to remove antinutritional protease inhibitors and purify Bowman–Birk protease inhibitor (BBI) from soybean whey. At the first coacervation induced by Ch (66.7, 200, and 510 kDa), only Kunitz trypsin inhibitor (KTI) and BBI complexed with Ch were extracted, while β-amylase and soybean agglutinin remained in supernatant. The binding constants for the interaction increased on the order Ch-66.7 < Ch-200 < Ch-510. At the second selective complexation, we observed a competitive binding behavior between KTI/BBI and CG. At a mixing weight ratio of 3:1 (pH 3.0 for ι-CG, and pH 3.11 for λ-CG), the preferential binding of KTI to CG led to the single enrichment of BBI in the supernatant. Our results indicated that the purified BBI was a good source for further study of its anti-carcinogenic properties, due to its high bioactivity (669.5 U/mg chymotrypsin-inhibitory activity and 2260 U/mg trypsin-inhibitory activity).