Impact of ultrafiltration/diafiltration sequence on the production of soy protein isolate by membrane technologies

In previous studies, the advantages of combining electrodialysis using a bipolar-cationic membranes configuration to acidify a soy protein extract to pH 6 with ultrafiltration/diafiltration (UF/DF) using a 100 kDa membrane to produce a soy protein isolate with low phytic acid content and improved solubility between pH 2 and 4 was demonstrated, when compared to the production of soy protein isolates by isoelectric precipitation and by UF/DF of a soy protein extract at pH 9. However, limited work was done to establish the impact of the UF/DF sequence for the purification of the pH 6 extract. Therefore, the purpose of this work was to study the impact of four different UF/DF sequences with a total permeate volume of 1.5–1.6 times the initial volume, on membrane fouling and permeate flux, as well as on the isolate protein, ash and phytic acid contents and solubility profile. Of the investigated UF/DF sequences, the VCR 5, VD 4 sequence was shown to be the one with the most severe fouling and consequently the most severe permeate flux decline. At the same time, it was also the VCR 5, VD 4 sequence which was the most efficient in terms of ash and phytic acid removal, followed by the VCR 5, re-VCR5 sequence, the VCR 2, VD 2 sequence and the VCR 2, (re-VCR 2)X 2 sequence, respectively. It was also observed that isolate with low phytic acid content resulted in narrower protein solubility profiles around the isoelectric point and higher protein solubility for the pH range of 2 to 4.Industrial relevancePlant proteins have made up a higher proportion of the human diet in recent years. Soybeans are the most important source of plant protein ingredients accounting for some 68% of global plant protein consumption in the world. Soy protein isolate is traditionally prepared by isoelectric precipitation process. This process has high productivity, however, it results in products with poor functional properties due to protein denaturation and to the presence of phytic acid (1–3% w/w) which alters the solubility of the isolates especially for the pH below the proteins' isoelectric point. In this work, we combined electrodialysis using a bipolar-cationic membranes configuration to acidify a soy protein extract to pH 6 with ultrafiltration/diafiltration (UF/DF) using a 100 kDa membrane to produce soy protein isolates with low phytic acid content. The impact of four different UF/DF sequences on membrane fouling, permeate flux, isolate composition and solubility profile was studied. Of the investigated UF/DF sequences, the VCR 5, VD 4 sequence was shown to be the one with the most severe fouling but at the same time the most efficient in terms of ash and phytic acid removal. It was also observed that the isolate produced by the VCR 5, VD 4 sequence shows narrower protein solubility profiles around the isoelectric point and higher protein solubility for the pH range of 2 to 4 than isolates produced by alternative UF/DF sequences. This isolate could be considered as a valuable ingredient for the formulation of fruit juice beverages or power juices, considering that the pH of these liquid food products is around 3.5.     

Production of soy protein isolates with low phytic acid content by membrane technologies: Impact of the extraction and ultrafiltration/diafiltration conditions

The content of the antinutrient, phytic acid, of soy protein was analyzed during their extraction and purification by a series of ultrafiltration and diafiltration steps. The phosphorus content of the extracts was used as an indication of their phytic acid content and their ash content as an indication of their mineral content. The extraction of soy proteins was conducted by using a 2³ factorial experimental design, pH (7.5 or 9), solvent (0.06 M KCl or water), and temperature (25 °C or 50 °C). The most promising extraction conditions were 0.06 M KCl/pH 9.0/25 °C for the lowest phosphorus to protein ratio (12.2 ± 0.1 mg P/g protein) and H₂O/pH 9.0/50 °C for the combination of low phosphorus to protein ratio and the lowest ash content (13.9 ± 1.2 mg P/g protein, 9.6 ± 0.8% w/w ash content). After extraction, soy proteins were purified by sequential ultrafiltration (UF) with a volume concentration ratio (VCR) of 5 and diafiltration (DF) with volume diafiltration ratio (VD) of 4. Extracts were purified with no pH adjustment or with pH adjustment to 6.5 between the UF and the DF steps. The extraction conditions 0.06 M KCl/pH 9.0/25 °C and the purification conditions UF pH 9.0/DF pH 6.5 showed the lowest phosphorus to protein ratio (4.4 ± 0.3 mg P/g protein) and reduced membrane fouling when compared to extraction conditions with water.     

Emulsifying properties of soy protein isolate fractions obtained by isoelectric precipitation

Soy protein isolate (SPI) fractions were produced by isoelectric precipitation based on results of isoelectric focusing carried out on the crude soy extract. The fractions were produced from crude protein extract (pH 9.0) sequentially and non‐sequentially at isoelectric points (pIs) of 5.6, 5.1 and 4.5. Emulsions stabilised by soy proteins with pIs between 5.6 and 5.1 had the highest (P < 0.01) emulsion stability index (ESI), while those stabilised with proteins having pIs between 5.1 and 4.5 resulted in the lowest ESI for sequentially precipitated fractions. Non‐sequential fractionation at pI 5.1 produced fractions with higher emulsifying activity index (EAI) than sequential fractionation. SDS‐PAGE profiles indicated that the fractions exhibiting high functionality in terms of ESI and EAI were also richer in 7S globulin protein subunits. © 2001 Society of Chemical Industry     

Functional properties and Angiotensin-I converting enzyme inhibitory activity of soy–whey proteins and fractions

Soy proteins when prepared to high purity can confer good functional properties and the whey by-product is a potential source for bioactivity. In this study, we determined the protein, moisture, fiber, solubility, foaming, emulsion properties, as well as Angiotensin-I converting enzyme (ACE-I) inhibitory activity of prepared soy–whey proteins and its fractions. The soy–whey proteins were fractionated into < 5, > 5, > 10, and > 50 kDa using ultrafiltration. The expanded AACC methods were used to determine protein, moisture, and fiber analyses of the whey and its fractions. Solubility method was conducted to determine the protein solubility profile of the soy–whey and its fractions at varying pHs. Turbidimetric method was used to evaluate emulsifying activity (EA) and emulsion stability (ES). There were significant differences observed in moisture, protein and salt contents between unfractionated, > 50 kDa and smaller sized fractions. No significant differences were observed with phytic acid and total dietary fiber contents among all samples. The unfractionated whey protein and > 50 kDa fraction showed better solubility than other fractions. Unfractionated whey protein had the highest foam capacity (42.7 mL) while the fraction > 5 kDa showed the greatest foaming stability (46 min). The highest emulsion activity (0.33 ± 0.1) and stability (825.1 ± 45.1) was obtained with the > 50 kDa fraction while the unfractionated whey protein had the highest ACE-I inhibition activity. The findings indicate that soy–whey protein fraction (> 50 kDa) had good solubility, emulsion activity and stability, while the unfractionated whey protein exhibited the strongest ACE-I inhibition percentage (19%).     

Preparation of canola protein materials using membrane technology and evaluation of meals functional properties

Suitable conditions for the extraction and precipitation of proteins from Iranian canola (Brassica napus, cv. Quantum, PF, and Hyola) meals were determined using a membrane-based process which consisted of extraction of hexane-defatted canola meals at pH 9.5–12.0 and precipitation, at pH values between 3.5 and 7.5, to recover a precipitated protein isolate (PPI). Acid soluble protein isolate (SPI) was then prepared by ultrafiltration (UF) followed by diafiltration (DF) and drying. The highest protein yield was obtained by alkaline extraction at pH 12.0 with all meals investigated. The maximum yield of precipitated protein was observed at pH values between 4.5 and 5.5, depending on variety and dehulling treatment. Almost 90% of the proteins were recovered in three products: PPI and SPI containing (81–98% protein, N*6.25), and the meal residue (35% protein). The glucosinolate content of all meals tested and their protein products was low, and in some cases they were below the detection limit of glucosinolates. Both isolates were low in phytic acid. Some functional properties (protein dispersibility index, water absorption, fat absorption, emulsifying activity, and foaming properties) were evaluated. Iranian canola meals were compared with soybean meal in terms of functional properties. All canola meals tested showed a high PDI and WA, and were superior to soybean meal in fat absorption, emulsifying activity and foaming properties.     

Improving Functional Properties of Soy Protein Hydrolysate by Conjugation with Curdlan

ABSTRACT:  Soy protein hydrolysate was conjugated with curdlan through the naturally occurring Maillard reaction to extend its application in food processing. The gel-forming and emulsifying properties of soy protein hydrolysates were significantly improved ( P < 0.05) by conjugation with curdlan. The soy protein hydrolysate–curdlan conjugate (SPHCC)–soy protein isolate (SPI) mixed gel had a much thicker network than the soy protein hydrolysate and SPI mixed gel judged from scanning electron microscopic images. The improvement of gelling properties of soy protein hydrolysate by curdlan-conjugate was attributed to both the decrease in the repulsive forces among soy protein hydrolysates and the increase in the solubility. The covalent binding of soy protein hydrolysates and curdlan also showed a profound effect on the antioxidative activity of the soy protein hydrolysates. The higher antioxidative activity of SPHCC was related to the peptide reductants produced from the Maillard reaction and the higher emulsifying property of SPHCC. The conjugates of soy protein hydrolysate and curdlan can be used as a functional food additive having excellent gel-forming, emulsifying properties and antioxidative activity.     

Some functional properties of fractionated soy protein isolates obtained by microfiltration

Five soy proteins isolate (SPI) fractions were produced using two microfiltration membranes with different pore sizes. Fractionation was carried out on SPI produced by isoelectric precipitation of a crude protein extract. The five fractions were two retentates and two permeates from the two membranes, the fifth fraction was obtained as the retentate on the smaller-pore-sized membrane fed with the permeate from the larger-pore-sized membrane. Solubility, foaming and emulsifying properties of the collected fractionates were investigated. It was observed that in the pH range 3–8 the retentates featured superior solubility compared with permeates. There was no significant difference (p>0.01) in solubility between the retentates and SPI at pH6. Foaming characteristics of the fractions followed the same trend as solubility with regard to foam expansion. There was, however, no particular trend observed with regards to foam stability. Emulsions stabilised by the retentates exhibited higher values (p<0.01) of emulsion stability index (ESI) and emulsifying activity index (EAI) than those stabilised with permeates. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) profiles indicated that the fractions exhibiting high functionality in terms of solubility, foaming and emulsifying properties were also richer in 7S globulin soy protein subunits.Isoelectric focussing (IEF) profiles showed that retentates were richer in species with isoelectric points (pI) between 5.2 and 5.6 while permeates featured more prominently at pIs between 4.5 and 4.8.     

Influence of processing on composition and antinutritional factors of chickpea protein concentrates produced by isoelectric precipitation and ultrafiltration

The effect of chickpea processing (i.e. defatting, isoelectric precipitation vs ultrafiltration/diafiltration) on the composition, protein recovery and antinutritional factors of protein concentrates was studied for two varieties (Mylese and Xena). Defatting did not affect significantly the content of antinutritional factors in the flours. However, production of concentrates from defatted flours by isoelectric precipitation resulted in higher phosphorous and phenolic contents compared to the concentrates produced by the same process using the full fat flours as starting material, while trypsin inhibitor content was not affected. When processed by ultrafiltration/diafiltration, protein concentrates produced from defatted flour showed a slightly lower trypsin inhibitor content than the ones produced from full fat flours in most cases, while the inverse was true for the phosphorous content, and for the phenolic content; this effect was a function of chickpea variety. Overall, UF pH 9/DF pH 6 resulted in concentrates with the lowest phosphorous content, while isoelectric precipitation and UF pH 9/DF pH 9 resulted in concentrates with lower phenolic content compared to the ones produced by UF pH 9/DF pH 6; for both processes the trypsin inhibitor content of the concentrates remained high.Industrial relevanceChickpea production is one of the major agricultural sectors of significant importance to Canada. Although chickpeas are grown in Canada for export, very little is exported in the value-added or processed form. Development of new extraction technologies and value-added products such as the ones presented in this paper is of interest for Canada since it would have a significant impact on the growth of the industry domestically, thus, creating opportunities to strengthen rural development in Canada. Successful implementation of these technologies would make interested Canadian companies viable competitors in the global plant protein production industry and would put Canada in a good position to enjoy a large share of this market both locally and internationally.More specifically, we are the first research group to compare the use of isoelectric precipitation and ultrafiltration/diafiltration for the production of chickpea protein concentrates from full fat and defatted flours of Kabuli and Desi chickpea variety, and to quantify the effect of these processes on the composition and on the antinutritional factors (i.e. phytic acid, total phenolics and trypsin inhibitors) of the resulting concentrates. Overall, it was observed that isoelectric precipitation was effective to an extent in producing concentrates with low phosphorous and phenolic contents. UF pH 9/DF pH 9 was also effective to an extent in producing concentrates with low phenolic content, while UF pH 9/DF pH 6 was more efficient in producing concentrates with low phosphorous content. High-quality chickpea protein concentrates with improved nutritional properties and good functional properties could beneficially be combined with other protein sources, such as soy protein, or be used in the formulation of foods, such as meat analogues, dairy, and bakery products.     

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

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

Comparing the heat stability of soya protein and milk whey protein emulsions

The heat stability of emulsions stabilized by WPC or SPI or mixtures of the two are compared by following the change in oil droplet number during heating, and applying kinetic rate equations to calculate the rate constant (k) for destabilization. SPI emulsions were found to be unstable to heat at pH around the pI, whilst being stable at pH further from the pI. This is related to the pH dependent solubility of soy proteins. This determined that a pH close to the pI (pH 4.5) be used for further studies so as to give a heat labile emulsion. Both WPC and SPI emulsions showed a weak dependence of k on protein concentration at pH 4.5, and an increasing k as the temperature increased. Arrhenius plots for emulsions made with WPC were bilinear, whilst those for SPI followed a single straight line. The change in slope of the Arrhenius plots for the WPC emulsions occurred around 70 °C, lower than would be expected from the denaturation temperature of β-lactoglobulin, the protein that dominates the thermal behaviour of WPC. The activation energies for WPC and SPI emulsions calculated from the slopes of the Arrhenius plots are slightly lower for WPC and considerably lower for SPI than the equivalent values in the literature for these proteins in solution. This, and the apparent lower denaturation temperature of β-lactoglobulin in emulsions, we explain by hypothesizing that the WPC and SPI proteins are already partially denatured by surface adsorption when they are heated, and thus require less energy to denature, and unfold at lower temperatures than native non-adsorbed proteins.     

Co-processing Soy Proteins and Milk Proteins with Commercial Ultrafiltration Membranes

Ultrafiltration (UF) membranes were used to co-process soy protein extract and skim milk seeking to produce soy-milk food ingredients with protein efficiency ratios (PERs) of 2.5 or higher. Extracts from two commercial soy flours and ground soybeans were blended with commercial skim milk before and after ultrafiltration. Protein blends of 67% soy-33% milk, 50% soy-50% milk and 33% soy-67% milk were processed. Soy-milk products possessed high nitrogen solubility and light color. Lysine contents of soy-milk products ranged from 7.34–6.82%. PERs ranged from 2.36 for 33% soy-67% milk to 1.47 for UF soy isolate without milk. Soy and milk proteins exhibited some synergistic effects, but none of the soy-milk PERs reached the 2.5 level.     

Effect of soy protein substitution for sodium caseinate on the transglutaminate-induced cold and thermal gelation of myofibrillar protein

The influence of soy protein isolate (SPI) substitution for sodium caseinate (SC) on the properties of cold-set (4 °C) and heat-induced gels of pork myofibrillar protein (MP) incubated with microbial transglutaminase (TG) was investigated. The strength of cold-set MP–SC gels (formed in 0.45 M, NaCl, 50 mM phosphate buffer, pH 6.25) increased with time of TG incubation, but those gels with more than 66% SPI substituted for SC had a >26% reduced strength (P < 0.05). Upon cooking, both incubated and non-incubated protein sols were quickly transformed into highly elastic gels, showing up to 6000 Pa in storage modulus (G′) at the final temperature (72 °C). However, no differences (P < 0.05) in G′ were observed between heated samples with SPI and SC. Myosin heavy chain, casein and soy proteins gradually disappeared with TG incubation, contributing to MP gel network formation. Both cold-set and heat-induced gels had a compact protein matrix, attributable to protein cross-linking by TG.     

Spray-Dried Soy Protein Isolate Solubility, Gelling Characteristics, and Extractable Protein as Affected by Antioxidants

With the addition of antioxidants, spray-dried soy protein isolates (SPI) exhibited increased solubilities by 8% over the control in 0.1M NaCl. Increased solubilities corresponded to a 15% and 8% reduction in protein oxidation as determined by protein carbonyl contents. The greatest differences in solubility between a spray-dried SPI with antioxidant (66% protein solubility) and the control (54% protein solubility), as well as the minimum protein solubility, occurred in 0.2M NaCl. Following the initial solubilization step in SPI processing, amount of extracted proteins was increased by 4.5% over the control with addition of antioxidants. Gel fracturability, hardness and adhesiveness for heat set gels of 12% SPI processed with added antioxidants were increased by 26.3%, 23.5% and 24.6%, respectively.     

Physical and Chemical Characteristics of Extruded Rice Flour and Rice Flour fortified with Soybean Protein Isolate

Texturized products were produced from rice flour and rice flour fortified with Soy Protein Isolate (SPI) by extrusion cooking. Water absorption capacity (WA) of the extrudates was slightly depressed in the presence of SPI. Scanning electron microscopy revealed that the file structure of the textured products differed subtly from each other and that SPI formed fine strings in the starch matrix. Solubility tests indicated that thermoplastic extrusion processing increased noncovalent interaction and disulfide bonding resulting in decreased protein solubility. 7S subunit of the soy protein seemed more affected than the 11s component. Insolubilization was significantly less when rice flour and SPI were processed simultaneously indicating a sparing effect in their mutual presence.     

Modified Soy Proteins with Improved Foaming and Water Hydration Properties

Soy proteins were modified by alkali treatment at pH 10.0, followed by papain hydrolysis. Solubility, water hydration capacity (WHC), surface hydrophobicity, foaming and emulsifying properties of unmodified, alkali-treated, and papain-modified soy protein (PMSP) were compared. PMSP exhibited higher solubility (100% at pH > 7.0), WHC (3.13) and hydrophobicity (40.8) than unmodified soy protein which had solubility 68.5%, WHC 0.21, and hydrophobicity 8.1. The PMSP had foaming capacity (22.0 mL) similar to egg white (21.2 mL) at pH 7.0; and enhanced foam stability (36.4) compared to the unmodified control (32.9). In general, alkali-treated soy had lower functional properties. Emulsifying properties of PMSP and alkali treated soy were unchanged by the modification. PMSP could be used as an egg white substitute in foaming applications at neutral pH.     

In vitro glycation and antigenicity of soy proteins

Soy protein isolate (SPI) was glycated with fructose or fructooligosaccharides (FOS) through the Maillard reaction in powder and liquid systems. A reduction in primary free amino groups of SPI up to 85% and 96% was observed in the powder and liquid system, respectively. Following heating at 95 °C for 1 h under liquid conditions, the electrophoretic behavior of allergenic 7S (β-conglycinin) and 11S (glycinin) fractions of SPI was modified when glycated with FOS (molar ratio primary amino groups to FOS of 1:74) as shown by SDS-PAGE analysis. The antigenicity of this glycated protein was also largely reduced (up to ∼90%) compared with that of the unglycated form. Glycation reactions with fructose in a powder and liquid system also reduced the antigenicity of the glycated proteins.     

不同来源蛋白酶水解对大豆分离蛋白分散性及溶解性的影响

研究了动物(胰蛋白酶)、植物(木瓜蛋白酶与菠萝蛋白酶)、微生物(碱性蛋白酶和中性蛋白酶)三种来源蛋白酶的低限度水解对大豆分离蛋白(SPI)分散性和溶解性的影响。结果表明,三种来源蛋白酶轻度水解可显著提高SPI的分散性,但却使其溶解性有不同程度的降低。三种来源蛋白酶水解产物的分散度大小依次为:植物来源蛋白酶>微生物来源蛋白酶>动物来源蛋白酶,而其溶解性则相反:动物来源蛋白酶>微生物来源蛋白酶>植物来源蛋白酶。本文对采用酶解的方法制备高分散性与高溶解性SPI具有一定的参考价值。通过对木瓜蛋白酶水解沉淀物进行分析,可以推测酶解使SPI溶解度显著下降的原因可能是SPI被水解后通过疏水作用力和氢键相互聚集形成了不溶性的沉淀。     

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

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

Functional Properties of Protein Isolate from Cowpea (Vigna unguiculata L. Walp.)

ABSTRACT: Functional properties of protein isolates prepared from 3 cowpea varieties and 2 soybean varieties in each of 2 y were determined. Both cowpea protein isolate (CPI) and soy protein isolate (SPI) showed a u-shaped curve for solubility with the minimum solubility occurring at pH 4.5. The CPI had lower emulsifying activity than SPI but was similar in stability. Foaming capacity and foaming stability ranged from 58.6 to 60.2 mL and 63.7 to 64.4 min for CPI and from 31.9 to 33.0 mL and 43.4 to 45.0 min for SPI, respectively. Gels were formed at 70 °C for 40 min and 30 min for CPI (12%) and SPI (10%), respectively. The CPI needs modification to enhance functional properties for potential application in food products.     

Solubility,functional properties,ACE‐I inhibitory and DPPH scavenging activities of Alcalase hydrolysed soy protein hydrolysates

Soy proteins are less soluble at acidic pH value, which impedes their utilisation in acidic beverages. Soy protein isolate (SPI) was hydrolysed using varying Alcalase concentrations (0.0001–2.0 U g^?1 protein) at different pHs (3.0–4.0). Degree of hydrolysis (DH) of soy protein hydrolysates (SPH) at pH 3.0, 3.5 and 4.0 were 5.0–10.7%, 2.3–6.1% and 0–5.4%, respectively, while solubilities ranged from 70.7 to 74.9%, 18.8 to 51.2% and 7.1 to 40.4%, respectively. The highest solubility (74.9%) was observed at pH 3.0 with 1.5 U Alcalase per g protein (DH = 9.2%). Emulsifying activities of SPHs at pH 3.0 and 4.0 ranged from 0.49 to 0.63 AU and 0.19 to 0.24 AU, respectively, while the emulsifying stabilities were 12.2–14.7 min and 18.7–56.0 min, respectively. The foaming capacity at pH 3.0 and 4.0 was 44.9–46.3 mL and 31.2–41.3 mL, respectively, whereas the foaming stability was 25.5–35.2 min and 12.8–15.1 min, respectively. However, hydrolysates had an insignificant effect on ACE‐I inhibitory and DPPH scavenging activities in comparison with SPI.