Physicochemical properties of native and recombinant mungbean (Vigna radiata L. Wilczek) 8S globulins and the effects of the N-linked glycans

We have previously cloned and characterized the cDNAs of three isoforms of the 8S globulin of mungbean, expressed the major 8Salpha isoform in Escherichia coli, and purified and successfully crystallized it (Bernardo, A. E. N.; Garcia, R. N.; Adachi, M.; Angeles, J. G. C.; Kaga, A; Ishimoto, M.; Utsumi, S.; Tecson-Mendoza, E. M. J. Agric. Food Chem. 2004, 52, 2552-2560). Herein, we report the physicochemical and emulsifying properties of the native 8S and recombinant 8Salpha globulin or vicilin. The circular dichroism spectra analysis of the native 8S and recombinant 8Salpha globulins revealed that the recombinant 8Salpha formed a secondary structure close to that of the native 8S. Further, gel filtration analysis showed that 8Salpha was able to assemble into trimers. The native 8S and recombinant 8Salpha globulins were soluble at pH 3.4 and at pH 7.4-9.0 at low ionic strength, mu = 0.08. Interestingly, the native 8S was more soluble at pH 7.0 and pH 7.4 than the recombinant 8Salpha at mu = 0.08. Both forms were very soluble at pH 3.4-9.0 at high ionic strength, mu = 0.50. The native form exhibited a higher T(m) (69.2, 79.5, and 83.8 degrees C) than the recombinant form (65.6, 71.6, 77.5 degrees C) at mu = 0.1, 0.2, and 0.5, respectively. The recombinant form was found to have greater surface hydrophobicity than the native form. There was little difference in the emulsifying ability between the native 8S and 8Salpha at pH 3.4 and pH 7.6. The results indicate that the presence of N-linked glycans is not essential in the assembly and stable conformation of the mungbean vicilin. However, the N-linked glycans might have contributed to the higher solubility at low ionic strength, greater thermal stability, and decreased surface hydrophobicity of the native vicilin as compared to the recombinant 8Salpha. On the other hand, the N-linked glycans showed little effect on the emulsifying ability of the protein.     

Surface functional properties of native, acid-treated, and reduced soy glycinin. 2. Emulsifying properties

Emulsifying properties of native and chemically modified soy glycinins were studied. The influence of ionic strength, protein sample composition and concentration, and assay conditions on the flocculation-creaming process and coalescence resistance was analyzed. Differences in these emulsifying properties were exhibited by native glycinins, which have a variable content of 4S, 11S, and 15S forms. Structure and functionality of native glycinin were modified by means of combined treatments: mild acidic treatments without heating or with heating at variable time and with or without disulfide bonds reduction. Modified glycinins presented different degrees of deamidation, surface hydrophobicity, and molecular mass. A slight enhancement of emulsifying stability at moderated deamidation degrees was observed. In different protein samples, a positive relationship between the flocculation-creaming rate constant and equilibrium oil volume fraction of emulsions with surface hydrophobicity was detected. A remarkable difference was observed between reduced and nonreduced samples, mainly with respect to behavior at low or high ionic strength.     

Comparison of protein chemical and physicochemical properties of rapeseed cruciferin with those of soybean glycinin

Rapeseeds contain cruciferin (11S globulin), napin (2S albumin), and oleosin (oil body protein) as major seed proteins. The effects of oil expression and drying conditions on the extraction of these proteins from rapeseed meal were examined. The conditions strongly affected the extraction of oleosin and only weakly affected the extraction of cruciferin and napin. The protein chemical and physicochemical properties of cruciferin, the major protein present, were compared with those of glycinin (soybean 11S globulin) under various conditions. In general, cruciferin exhibited higher surface hydrophobicity, lower thermal stability, and lower and higher solubility at mu= 0.5 and mu = 0.08, respectively, than did glycinin. At the pHs (6.0, 7.6, and 9.0) and ionic strengths (mu= 0.08 and 0.5) examined, the emulsifying ability of cruciferin was worse than that of glycinin, except at mu= 0.08 and pH 7.6. The emulsifying abilities of cruciferin and glycinin did not correlate with thermal stability and surface hydrophobicity. Higher protein concentration, higher heating temperature, higher pH, and lower ionic strength were observed to produce harder gels from cruciferin. Gel hardness partly correlated with the structural stability of cruciferin.     

Comparison of physicochemical properties of 7S and 11S globulins from pea, fava bean, cowpea, and French bean with those of soybean--French bean 7S globulin exhibits excellent properties

Legume seeds contain 7S and/or 11S globulins as major storage proteins. The amino acid sequences of them from many legumes are similar to each other in the species but different from each other, meaning that some of these proteins from some crops exhibit excellent functional properties. To demonstrate this, we compared protein chemical and functional properties (thermal stability, surface hydrophobicity, solubility as a function of pH, and emulsifying properties) of these proteins from pea, fava bean, cowpea, and French bean with those of soybean as a control at the same conditions. The comparison clearly indicated that the 7S globulin of French bean exhibited excellent solubility (100%) at pH 4.2-7.0 even at a low ionic strength condition (mu = 0.08) and excellent emulsion stability (a little phase separation after 3 days) at pH 7.6 and mu = 0.08, although the emulsions from most of the other proteins separated in 1 h. These results indicate that our assumption is correct.     

Functional properties of purified vicilins from cowpea (Vigna unguiculata) and pea (Pisum sativum) and cowpea protein isolate

The major storage globulins (vicilins) of cowpea (Vigna unguiculata) and pea (Pisum sativum) seeds were purified by ammonium sulfate precipitation, and a semipurified cowpea protein isolate (CPI) was prepared by isoelectric precipitation. Some of the functional properties of these proteins, including solubility, foaming, and emulsifying capacities, were investigated and compared. The solubility of purified cowpea vicilin was reduced at pH 5.0, increasing markedly below and above this value. Pea vicilin exhibited poor solubility between pH 5.0 and pH 6.0, and CPI was little soluble in the pH range from 4.0 to 6.0. At neutral pH, the emulsifying activity indexes (EAI) of purified pea vicilin and CPI were 194 and 291 m(2)/g, respectively, which compare quite favorably to EAIs of 110 and 133 m(2)/g for casein and albumin, respectively. Remarkably, purified cowpea vicilin exhibited an EAI of 490 m(2)/g, indicating a very high emulsifying activity. Purified cowpea and pea vicilins exhibited lower foaming capacities and foam stablity indexes (FSI) than CPI. FSI values of 80 and 260 min were obtained for purified pea and cowpea vicilin, respectively, whereas a FSI value of 380 min was obtained for CPI. These results are discussed in terms of the possible utilization of purified vicilins or protein isolates from pea and cowpea in the food processing industry.     

高压微射流压力对大豆蛋白-大豆多糖体系功能性质的影响

为提升大豆分离蛋白（soy protein isolate,SPI）的功能性质,该文引入大豆可溶性多糖（soybean soluble polysaccharides,SSPS）,构建大豆分离蛋白-大豆可溶性多糖体系（SPI-SSPS）,研究动态高压微射流（dynamic high-pressure microfluidization,DHPM）处理对SPI-SSPS功能特性的影响。分别采用0,60,100,140和180 MPa的 DHPM压力处理SPI-SSPS,探究不同压力对SPI-SSPS起泡特性、乳化特性、溶解性、粒度分布和表面疏水性的影响。结果表明,DHPM处理能提高SPI的溶解性和起泡特性,且SSPS的存在能显著提高DHPM对SPI功能性质的改善效果（P<0.05）。100和60 MPa的DHPM处理能使SPI-SSPS呈现较高的起泡能力和起泡稳定性,分别为未处理样品的1.2和2.4倍。140 MPa的DHPM处理使SPI-SSPS溶解性较强,为未处理样品的1.8倍。然而,DHPM处理会显著降低SPI-SSPS的乳化特性、粒径和表面疏水性（P<0.05）。随着处理压力的增加,SPI-SSPS的粒度和表面疏水性逐渐降低,在180MPa的DHPM处理下SPI-SSPS具有较小的粒径和较低的荧光强度。综上所述,DHPM结合SSPS改性技术可用于改善SPI的功能性质（如溶解性、起泡性）,促进SPI在食品工业的应用。该文的研究结果可为SPI的功能性质改性提供参考。     

Hydrophobicity,Solubility, and Emulsifying Properties of Enzyme-Modified Rice Endosperm Protein

Rice endosperm protein was modified to enhance solubility and emulsifying properties by controlled enzymatic hydrolysis. The optimum degree of hydrolysis (DH) was determined for acid, neutral, and alkaline type proteases. Solubility and emulsifying properties of the hydrolysates were compared and correlated with DH and surface hydrophobicity. DH was positively associated with solubility of resulting protein hydrolysate regardless of the hydrolyzing enzyme, but enzyme specificity and DH interactively determined the emulsifying properties of the protein hydrolysate. The optimum DH was 6–10% for good emulsifying properties of rice protein, depending on enzyme specificity. High hydrophobic and sulfhydryl disulfide (SH-SS) interactions contributed to protein insolubility even at high DH. The exposure of buried hydrophobic regions of protein that accompanied high-temperature enzyme inactivation promoted aggregation and cross-linking of partially hydrolyzed proteins, thus decreasing the solubility and emulsifying properties of the resulting hydrolysate. Due to the highly insoluble nature of rice protein, surface hydrophobicity was not a reliable indicator for predicting protein solubility and emulsifying properties. Solubility and molecular flexibility are the essential factors in achieving good emulsifying properties of rice endosperm protein isolates.     

Effects of Different Lipase Inactivation Treatments on Physicochemical Properties of Naked Oat Globulins

Lipase inactivation is an essential treatment for oat processing, because of the negative effects of lipase on nutrient preservation and storage extension. The effects of different lipase inactivation treatments including hot air roasting, infrared roasting, normal‐pressure steaming, and high‐pressure steaming on the physicochemical properties of oat globulins were investigated. Results showed that normal‐pressure steaming had little effect on solubility of oat globulins; hot air roasting increased foaming capacity of oat globulins but did not change their foaming stability; and all the inactivation treatments increased the surface hydrophobicity and content of total sulfhydryls of oat globulin but decreased exposed sulfhydryl groups. In addition, oat globulin granules from the hot air roasting treatment were distributed more evenly in oat globulin powder compared with the control group. All treatments except normal‐pressure steaming changed the molecular weight of oat globulin subunits, which made the bands of 66,000 and 45,000 disappear from SDS‐PAGE. These results indicated that normal‐pressure steaming was ideal to maintain good solubility of oat globulins, and hot air roasting was ideal to maintain relatively good foaming properties. The treatments changed physicochemical properties of oat globulins by influencing protein aggregation and subunit composition that resulted in different content of sulfhydryl groups and surface hydrophobicity.     

Effect of low and high pH treatment on the functional properties of cod muscle proteins

The functional properties of cod myosin and washed cod mince (myofibrillar protein fraction) treated at high (11) and low (2.5) pH were investigated after pH readjustment to 7.5. The solubility of refolded myosin was essentially the same as the native myosin. The pH-treated myofibrillar proteins had increased solubility over the whole ionic strength range studied. Acid and alkali treatment gave myosin and myofibrillar proteins improved emulsification properties, which were correlated with an increase in surface hydrophobicity and surface/interfacial activity. Enhanced gel strength was observed with acid- and alkali-treated myosin compared to native myosin, while the same treatment did not significantly improve the gel strength of acid- and alkali-treated myofibrillar proteins. The acid- and alkali-treated protein samples unfolded and gelled at a lower temperature than did the native proteins, suggesting a less conformationally stable structure of the refolded proteins. Functional studies show that acid and alkali treatment, which leads to partial unfolding of myosin may improve functional properties of cod myosin and myofibrillar proteins, with the greatest improvement being from the alkali treatment. The results also show that improvements in functionality were directly linked to the extent of partial unfolding of myosin on acid and alkali unfolding and refolding.     

Functional Properties of Rice Bran Protein Isolate at Different pH Levels

The functional properties of proteins from Tarom and Shiroodi cultivars were determined and compared with technological aspects of food and nutraceutical applications. Shiroodi has higher protein content than Tarom, and the yields of protein obtained were 72.88 and 66.36%, respectively. Nitrogen solubilities of rice bran protein of Tarom were more than Shiroodi at all pH levels. In addition, higher solubility was found in acidic or alkaline conditions. Although the rice bran proteins had lower emulsifying properties than bovine serum albumin, they had similar foaming properties in comparison with egg white. Tarom isolates had a significantly higher solubility, emulsifying property, and foaming stability and greater surface properties than Shiroodi isolates. The results showed the surface hydrophobicities of rice bran protein were greater than casein and ovalbumin and lower than other proteins such as bovine serum albumin. Water and oil absorption capacities were 1.03 and 1.66 for Tarom and 87.3 and 75.3 for Shiroodi, respectively. The bulk densities of Tarom and Shiroodi were also 0.55 and 0.53 g/mL, which make them suitable for weaning food and other industrial applications. As a result, these rice bran proteins showed higher hydrophobicity than that of other rice bran protein varieties as well as more functionality. Thus, they have good potential in the food and pharmaceutical industries.     

Emulsifying and Foaming Properties of Okara Protein Hydrolysates

Okara is a low‐value coproduct of soy milk production. Its dry matter contains 25–30% protein that is of high nutritive quality, has an excellent efficiency ratio, and thus holds promise for applications in food systems. However, okara protein has low solubility. We here optimized its extraction and isolation from okara by using dilute sodium hydroxide and subsequent isoelectric precipitation. The obtained okara protein isolate (OPI) was hydrolyzed with different enzymes into a range of hydrolysates with different degrees of hydrolysis. Most hydrolysates had better emulsifying activity and produced more stable emulsions than OPI. In contrast, hydrolysis had no positive effect on foam‐forming and foam‐stabilizing activity of OPI proteins. Hydrolysis of OPI enhances the emulsifying capacity of the proteins. Furthermore, the emulsifying and foam‐forming capacities of most of the OPI hydrolysates were similar to or even better than those of the commercial (soy) protein hydrolysates used in this study.     

Structural Modifications of Gluten Proteins in Strong and Weak Wheat Dough During Mixing

The network‐forming attributes of gluten have been investigated for decades, but no study has comprehensively addressed the differences in gluten network evolution between strong and weak wheat types (hard and soft wheat). This study monitored changes in SDS protein extractability, SDS‐accessible thiols, protein surface hydrophobicity, molecular weight distribution, and secondary structural features of proteins during mixing to bring out the molecular determinants of protein network formation in hard and soft wheat dough. Soft wheat flour and dough exhibited greater protein extractability and more accessible thiols than hard wheat flour and dough. The addition of the thiol‐blocking agent N‐ethylmaleimide (NEM) resulted in similar results for protein extractability and accessible thiols in hard and soft wheat samples. Soft wheat dough had greater protein surface hydrophobicity than hard wheat and exhibited a larger decrease in surface hydrophobicity in the presence of NEM. Formation of high‐molecular‐weight (HMW) protein in soft wheat dough was primarily because of formation of disulfides among low‐molecular‐weight (LMW) proteins, as indicated by the absence of changes in protein distribution when NEM was present, whereas in hard wheat dough the LMW fraction formed disulfide interaction with the HMW fraction. Fourier transform infrared spectroscopy indicated formation of β‐sheets in dough from either wheat type at peak mixing torque. Formation of β‐sheets in soft wheat dough appears to be driven by hydrophobic interactions, whereas disulfide linkages stabilize secondary structure elements in hard wheat dough.     

Physicochemical Properties of Rice Endosperm Proteins Extracted by Chemical and Enzymatic Methods

Rice proteins are nutritional, hypoallergenic, and healthy for human consumption. Efficient extraction with approved food‐grade enzymes and chemicals are essential for commercial production and application of rice protein as a functional ingredient. Rice endosperm proteins were isolated by alkali, salt, and enzymatic methods and evaluated for extractability and physicochemical properties. Alkali (RP_A) and salt (RP_S) methods extracted 86.9 and 87.3% of proteins with 65.9 and 58.9% yield, respectively. The enzymatic methods with Termamyl (RP_ET) and amylase S (RP_EA) extracted 85.8 and 81.0% proteins with 85.2 and 86.2% yield, respectively. Enthalpy values of RP_A (1.79 J/g), RP_S (1.22 J/g), RP_ET (nondetectable), and RP_EA (0.17 J/g), determined by differential scanning calorimetry, demonstrated that the varying level of denaturation of proteins depends on the method of extraction. Surface hydrophobicity data supported this observation. Alkali‐ and salt‐extracted proteins had higher solubility and emulsifying properties than those of enzyme‐extracted proteins. Comparatively, more favorable protein composition, lower surface hydrophobicity, higher solubility, and a lower degree of thermal denaturation of alkali‐ and salt‐extracted proteins contributed to higher emulsifying and foaming properties than those of enzyme‐extracted proteins; therefore, alkali‐ and salt‐extracted proteins can have enhanced functional use and a potential starting material for preparing tailored rice protein isolates.     

Preparation of Rice Endosperm Protein Isolate by Alkali Extraction

Rice endosperm extraction conditions were optimized by response surface methodology. The optimum alkali extraction conditions were pH 11.0 at 40°C for 3 hr with 8:1 solvent‐to‐solid ratio. The maximum protein yield was 43.1% at these conditions. As the extraction pH was increased from 9.0 to 12.0, protein extractability and content increased but the solubility and emulsifying properties of the extracted protein decreased. The extracted protein was recovered by either isoelectric precipitation (IEP) or ultrafiltration (UF). Ultrafiltering the supernatant with a 5‐kDa hollow fiber membrane concentrated the protein from 1.8 to 16% (dry basis) and the resulting solution was spray‐dried to produce a protein concentrate (RP_UF) with 71% protein. Although RP_IEP contained higher protein (86%) than RP_UF (71%), RP_UF showed higher solubility and emulsifying properties. The solubility of RP_UF was higher (37%) than RP_IEP (15%). RP_UF also demonstrated higher emulsion activity (0.414) and stability (22.4 min) compared with the emulsion activity (0.282) and stability (15.5 min) of RP_IEP. Higher solubility and the soluble nonprotein components of RP_UF contributed to higher emulsifying properties than RP_IEP. The UF provided milder extraction conditions with improved emulsifying properties than conventional IEP.     

离子强度及pH对不同类型土壤中胶体释放的影响

采用实验室土柱纵向淋溶法,研究了离子强度及pH变化对常熟乌栅土原状、扰动土柱以及东北黑土、江西红壤扰动土柱土壤胶体释放的影响。结果表明离子强度变化对我国不同类型土壤胶体释放影响不同：淋洗液电解质为NaCl时,对于常熟乌栅土以及东北黑土,离子强度减小促进胶体的释放,反之抑制胶体的释放;对于江西红壤,离子强度变化对土壤胶体释放则不产生影响。淋洗液电解质为CaCl2条件下,土壤胶体的释放量低于淋洗液为NaCl,且离子强度变化对三种土壤胶体的释放均不产生明显影响。相同条件下,常熟乌栅土及东北黑土胶体释放量远远大于江西红壤,pH变化则对上述类型土壤胶体释放的影响不明显。研究结果有助于进一步阐明水化学条件的变化对我国不同类型土壤胶体释放的影响规律。     

Enzymatic hydrolysis of brewers' spent grain proteins and technofunctional properties of the resulting hydrolysates

Brewers' spent grain (BSG) is the insoluble residue of barley malt resulting from the manufacture of wort. Although it is the main byproduct of the brewing industry, it has received little attention as a marketable commodity and is mainly used as animal feed. Our work focuses on one of the main constituents of BSG, i.e., the proteins. The lack of solubility of BSG proteins is one of the limitations for their more extensive use in food processing. We therefore aimed to generate BSG protein hydrolysates with improved technofunctional properties. BSG protein concentrate (BPC) was prepared by alkaline extraction of BSG and subsequent acid precipitation. BPC was enzymatically hydrolyzed in a pH-stat setup by several commercially available proteases (Alcalase, Flavourzyme, and Pepsin) for different times and/or with different enzyme concentrations in order to obtain hydrolysates with different degrees of hydrolysis (DH). Physicochemical properties, such as molecular weight (MW) distribution and hydrophobicity, as well as technofunctional properties, such as solubility, color, and emulsifying and foaming properties, were determined. Enzymatic hydrolysis of BPC improved emulsion and/or foam-forming properties. However, for the hydrolysates prepared with Alcalase and Pepsin, an increasing DH generally decreased emulsifying and foam-forming capacities. Moreover, the type of enzyme impacted the resulting technofunctional properties. Hydrolysates prepared with Flavourzyme showed good technofunctional properties, independent of the DH. Physicochemical characterization of the hydrolysates indicated the importance of protein fragments with relatively high MW (exceeding 14.5 k) and high surface hydrophobicity for favorable technofunctional properties.     

Surface functional properties of native, acid-treated, and reduced soy glycinin. 1. Foaming properties

Foaming properties of native and chemically modified glycinin were evaluated. Effects of ionic strength and glycinin composition and concentration on foam formation and stabilization were studied. Glycinin was modified by means of combined treatments: cold or hot acidic treatments, with or without later disulfide bridges reduction. Modified proteins obtained from glycinin present different degrees of dissociation, deamidation, and as consequence, varied surface hydrophobicity and molecular size. Parameters of forming and stabilizing of foam were correlated with both deamidation and dissociation degrees of modified and native glycinin samples. A positive relationship was observed between surface behavior and foaming properties of different protein species. Results show that dissociation, deamidation, and reduction have produced structural changes on glycinin (increased surface hydrophobicity, increased net charge, decreased molecular size) which enhance the adsorption and anchorage of proteins at the air-water interface and, consequently, improve the foam forming and stabilizing capacities.     

Effects of ultrasound pretreatment on the enzymatic hydrolysis of soy protein isolates and on the emulsifying properties of hydrolysates

Soy protein isolate (SPI) was modified by ultrasound pretreatment (200 W, 400 W, 600 W) and controlled papain hydrolysis, and the emulsifying properties of SPIH (SPI hydrolysates) and USPIH (ultrasound pretreated SPIH) were investigated. Analysis of mean droplet sizes and creaming indices of emulsions formed by SPIH and USPIH showed that some USPIH had markedly improved emulsifying capability and emulsion stabilization against creaming during quiescent storage. Compared with control SPI and SPIH-0.58% degree of hydrolysis (DH), USPIH-400W-1.25% (USPIH pretreated under 400W sonication and hydrolyzed to 1.25% DH) was capable of forming a stable fine emulsion (d43=1.79 μm) at a lower concentration (3.0% w/v). A variety of physicochemical and interfacial properties of USPIH-400W products have been investigated in relation to DH and emulsifying properties. SDS-PAGE showed that ultrasound pretreatment could significantly improve the accessibility of some subunits (α-7S and A-11S) in soy proteins to papain hydrolysis, resulting in changes in DH, protein solubility (PS), surface hydrophobicity (H0), and secondary structure for USPIH-400W. Compared with control SPI and SPIH-0.58%, USPIH-400W-1.25% had a higher protein adsorption fraction (Fads) and a lower saturation surface load (Γsat), which is mainly due to its higher PS and random coil content, and may explain its markedly improved emulsifying capability. This study demonstrated that combined ultrasound pretreatment and controlled enzymatic hydrolysis could be an effective method for the functionality modification of globular proteins.