Food Research International 44 (2011) 2505–2514

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Comparative study of functional properties of commercial and membrane processedyellow pea protein isolates

Ali R. Taherian ⁎, Martin Mondor, Joey Labranche, Hélène Drolet, Denis Ippersiel, François LamarcheAgriculture and Agri-Food Canada, Food Research and Development Center, 3600 Casavant West, St-Hyacinthe, Quebec, J2S 8E3, Canada

⁎ Corresponding author. Tel.: +1 450 768 3329; fax:E-mail address: ali.taherian@agr.gc.ca (A.R. Taherian

0963-9969/$ – see front matter. Crown Copyright © 20doi:10.1016/j.foodres.2011.01.030

a b s t r a c t

a r t i c l e i n f o

Article history:Received 9 September 2010Accepted 10 January 2011

Keywords:Membrane technologyPea protein isolateFunctional propertiesRheology

Functional properties of commercial and membrane processed pea protein isolates (PPI) prepared fromyellow peas were investigated. Four protein isolates were prepared from yellow pea flour using water and KClextractions at 25 °C followed by ultrafiltration and diafiltration (UF and DF) at pHs of 7.5 and 7.5 or 6respectively. Following assessment of compositional attributes; solubility, foaming, flow and dynamicrheology, emulsification ability and heat-induced textural and rheological properties of prepared PPIs and acommercially available PPI were tested and compared. Membrane purification of proteins resulted in 28% to68% reduction in phytic acid and enhanced, comparatively, the tested functional properties. Solubility ofmembrane processed PPIs, at all tested pHs, was superior and the lowest foaming stability and apparentviscosity were associated with commercial PPI. Gelling temperatures of water and KCl extracted PPIs, DFtreated at pH 6, trimmed down to 75.7±0.63 °C and 81.6±0.55 °C in contrast to that of commercial PPI atabove 90 °C. Similarly, the formation of firm gels, after 1 h heating at 90 °C, was associated with membraneprocessed PPIs whereas commercial PPI did not develop any gel.

Crown Copyright © 2011 Published by Elsevier Ltd. All rights reserved.

1. Introduction

Proteins and polysaccharides from a wide range of plant and animalsources are broadly used in human food because of their nutritionalbenefits and functional properties.With this regards, substantial studieshave covered proteins from animal origin (e.g., whey protein, sodiumcaseinate…) while plant proteins have received lesser attention. As aresult, challenges to replace animal proteinswith plant proteins in novelfood products have increased. Moreover, there is an economical reason,such as less energy usage for the production of plant proteins, thatincreases the use of plant proteins worldwide (Dijkink & Langelaan,2002; Makri, Papalamprou, & Doxastakis, 2005; Singh, Kaur, ChandRana, & Kumar Sharma, 2010).

Plant proteins in the form of isolate have widely been employed toimprove the texture and the nutritional quality of the food products andgenerate gel structures that give the same texture with reduced lipid inthe final product (Shand, Ya, Pietrasik, & Wanasundara, 2007). Amongplant proteins, pea proteins (Pisum sativum) with the worldwide totalproduction of 10.3 million metric tonnes are rather new industrialproteins (Simsek, Tulbek, Yao, & Schatz, 2009). Pea protein shows awellbalanced profile of amino acids, especially a high content in lysine(Nunes, Raymundo, & Sousac, 2006). Sun and Arntfield (2010) andGharsallaoui, Yamauchi, Chambin, Cases, and Saurel (2010) stated thatpea protein plays important roles in many foodstuffs, because of its

+1 450 773 8461.).

11 Published by Elsevier Ltd. All rig

nutritional value, contribution to the texture of food and the surfaceproperties of constitutive protein units: the storage globulins7S (vicilin)and 11S (legumin). Ducel, Richard, Popineau, and Boury (2004) showedthat peaproteins are able todecrease the interfacial tensionbetween thewater and oil and help to stabilize emulsions by forming a rigidmembrane at the oil–water interface. Study by Agboola, Mofolasayo,Watts, and Aluko (2010) pointed out that the functional properties ofwhole pea flour, high fibre, fibre–starch and high protein ingredients,derived from yellow field peas could contribute desirable functionalcharacteristics to a wide range of food products. In addition, high levelsof water and oil absorption, good gelation capabilities, and gel clarityshould make these ingredients useful in a variety of applications.However, factors such as pH, ionic strength, or the presence of otheringredients will affect the functional properties of pea protein.Therefore, knowledge of functional properties of individual protein is,particularly, valuable for the development of new food products(Braudo et al., 2001; Bilgiçli, Elgün, & Türker, 2006; Yu, Ahmedna,Goktepe, 2007; Tsumura et al., 2005; Wang, Hatcher, Warkentin, &Toews, 2010; Hua et al., 2005).

Despite the fact that peas are an excellent source of proteins,carbohydrates and essential minerals, its utility to humans is limited bythe presence of antinutrients, such as phytic acid, protease inhibitors,and tannins (Habiba, 2002). Consequently, this study was conductedwith a particular concern on using pea protein isolates with low phyticacid content produced by membrane technologies for the developmentof novel food products. The aims of this investigation were to study andcompare the functional attributes such as solubility, flow and dynamicrheology, heat-induced textural properties and foaming of membrane

hts reserved.

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processed and commercial pea protein isolates (PPIs) as affected bytemperature, pH, and salt content.

2. Materials and methods

2.1. Materials

Certified#1 Eclipse Yellowpeaswerepurchased fromWagonWheelSeed Corporation (Churchbridge, SK) and commercial PPI (Propulse™Pea Protein) was obtained from Nutri-Pea Ltd (Portage La Prairie, MB).Based on the information provided by Nutri-Pea the PPI contains82±2% protein (Dumas, N×6.25), 11000 ppm phosphorus, b4.0% ashand b3% fat.

2.2. Preparation of flour

Yellow peas werewashed, dehulled and ground to produce flour. A5 kg yellow pea was first soaked in 40 kg water at room temperaturefor 90 min. After soaking for 90 min in water, sodium hypochlorite(NaOCl) was added to the mixture until a concentration of 0.7 g/L wasreached and the soaking was carried out for an additional 30 min. Thesolution was then drained, and the peas were rinsed with water(40 kg) to remove any hypochlorite residue. The peas weresubsequently dried in a four-tray dryer (model 52979; NationalDrying Machinery Co., Philadelphia, PA) at room temperature forabout 21 h. Upon removal from the oven, the peas were ground usinga Quadro Comil model 196 (Quadro Engineering Inc., Ontario, Canada)equipped with screen 002 for a first pass and with screen 009 for asecond pass. The broken peas and their hulls were then passedthrough a multi aspirator (Kice, Wichita, Kansas, USA, model 6F6),where the hulls were removed from the top while the dehulled peasfell into the reservoir through the force of gravity. The dehulled peaswere ground again using the Quadro Comill equippedwith screen 106to produce flour. The resultant flour was sealed in aluminium pouchesand stored at 4 °C prior to use.

2.3. Preparation of isolates in the pilot plant

Preparedflourwas dispersed in anappropriate extraction solution ata ratio of 1:15 (w/w), i.e. 7 kg flour in 105 kg extraction solution (wateror 0.06 M KCl). The dispersion was agitated for 30 min to allowhydration of the flour followed by adjustment of the pH at 7.5 using 2 MNaOH. Extractionwas performed at room temperature for 45 minunderhigh speed agitation and continuous adjustment of the pH if required.Once the extraction was completed, the solid material was removedusing a basket centrifuge (model STM-1000; The Western StatesMachine Co., Hamilton, OH) at rotational speed of 3000 rpm through2 passes and cut-offs of 60 μm and 1 μm for the first and second passesrespectively. The resultant solution was then purified/concentrated byultrafiltration/diafiltration using a home-made module equipped withtwo 50 kDa hollow fibres membranes with a surface of 2.3 m2 for eachmembrane (Romicon, model CTG 3 , HF25-60-PM50; KOCHMembraneSystems, Inc.,MA). Ultrafiltration (UF) stepwasfirst carried out throughthe application of volume concentration ratio (VCR) of 5 and pH of 7.5.Further purification was obtained by the application of discontinuousdiafiltration (DF) step consistingof theadjustmentof solution volume toits initial volumewith tapwater and re-VCR 5 at either pH of 7.5 or 6. Atthe end, the isolates were freeze-dried, placed in aluminium pouches,sealed, and stored at 4 °C prior to further examination.

2.4. Determination of protein content

Total nitrogen content was estimated using a Kjeldahl digestionsystem (Tecator, Höganäs, Sweden) and a conversion factor of 6.25(Owusu-Apenten, 2002) was employed to estimate the total protein

content. A 0.3 g of dried or 5 ml of liquid samples was used andmeasurements were carried out in duplicates.

2.5. Determination of total solids, ash and total phosphorus

Total solids, ash content and total phosphorus weremeasured basedon the methods provided by Mondor, Ali, Ippersiel, and Lamarche(2010). By comparing the mass of the samples before and after dryingusing a vacuumoven at92 °C for approximately 16 h the amount of totalsolids was determined. Dried samples were then burned in a mufflefurnace at 550 °C for about16 h for themeasurementof ash content. Theresultant ash was dissolved in a 1.86 M HCl solution to measure thephosphorus content. Total phosphorus was then estimated usingspectrophotometry at 400 nm. Themethodwas based on the formationof a molybdivanadophosphoric complex, which absorbs in the visiblespectrum. A monobasic potassium phosphate solution was used as astandard. All above measurements were conducted in duplicates.

2.6. Estimation of phytic acid—Wade reagent method

Phytic acid estimationwas based on themethod by Gao et al. (2007)with slight modification. A 0.4 g of each isolate was dispersed in 10 g of2.4% HCl in a 50 ml Falcon tubes and vigorously agitated for 16 h. Thesample was centrifuged at 1000 g for 20 min at 10 °C and thesupernatant was transferred into a 50 ml Falcon tubes holding 1 gNaCl following by agitation for 20 min. After storing at 4 °C for 1 h, thetube was centrifuged at 1000 g for 10 min at 10 °C and 1 g of thesupernatant was diluted 25 times with bi-distilled water. Three gramsof the diluted solution was then combined with 1 g of the modifiedWade reagent containing 0.03% FeCl3·6H2O and 0.3% sulphosalicylicacid, in a Falcon tubes. The resultant was agitated in vortex and oncemore centrifuged at 1000 g for 20 min at 10 °C. Ultimately, the absor-bance of the supernatants was measured at 500 nm and compared tocalibration curve which was plotted using sodium phytate standards.The results were then expressed as milligrams of phosphorus fromphytic acid (PA-P) per gram of proteins.

2.7. Assessment of proteins

An approximate amount of 1 mg PPI was weighed and added to aknown volume of buffer (Bio-Rad no. 161-0791) in order to obtain aconcentration of 1 mg protein/ml buffer. A 50 μl of 2-mercaptoethanol(Bio-Rad no. 161-0792) was added to the mixture. The resultingsolution was then mixed and placed into the boiling water for 5 min. Inorder to remove any precipitated proteins, samples were centrifugedprior to analysis. Later, a Bio-Rad Criterion Cell was used with Bio-Rad4–12%Bis–Tris Gel as themedium, anddilutionwasperformedusingXTMOPS buffer (Bio-Rad No. 161-0788) at a constant voltage of 200 V inaccordance with the manufacturer's recommendations. A volume of20 μl of the samplewas used, and themolecularweightswere estimatedwith a low-range standard. The proteins were visualized by stainingwith Coomassie Blue R-250 (Bio-Rad No. 161-0436).

2.8. Assessment of protein solubility

Protein solubility was assessed at pH range of 2.0, 2.5, 3.0, 3.5, 4.0,4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 8.0 and 9.0 using 1.0% (w/w) protein. A 0.30 gof PPI was dispersed in 24 g of double-distilled water and allowed tostir for 1 h at room temperature to facilitate solubilization. Thedispersion pH was then adjusted at 2.0 using known volume of 0.1 NHCl and brought to 30 g with double-distilled water. For all other pH,dispersions were adjusted at desired pH using 0.1 N HCl or KOH. Inorder to maintain constant salt content 0.1 N KCl was also added intothe dispersion. The amount of added 0.1 N KCl corresponds to thedifference between the amount of added HCL for the adjustment at pH2.0 minus the amount of HCl (or KOH) added to adjust the desired pH.

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Later, the solution was allowed to stir for an additional 45 min. Thedispersion was then centrifuged at 14000 g for 30 min at roomtemperature to precipitate out insoluble deposit. An aliquot of thesupernatant, approximately 5 g, was taken and placed in falcon tubeand stored at −20 °C prior to analyses for nitrogen content. Nitrogencontent of the protein supernatant was measured using a Kjeldahldigestion system, according to AOAC Official Method. Protein relativesolubility (%) was determined by multiplying the fraction ofsupernatant protein content over total protein content by 100. Allmeasurements were performed in duplicates.

2.9. Foaming assessment

Foaming experiments were conducted based on the methoddescribed by Liu, Elmer, Low, and Nickerson (2010) with slightmodification. A 15 ml (Vli, initial volume) of each protein sample atselected concentration (solubilized for a minimum of 1 h) was pouredinto a 50 ml graduated cylinder in order to prepare the foam. The airwasthen dispersed in pea protein solutions using a Polytron Homogenizer(PT 10-35,withGenerator PTA1012 mm, Kinematica inc., Bohemia,NY,USA) at high speed for 5 min. Foam stability was evaluated andexpressed as (%FS) using Eq. (1):

%FS =Vli−Vltð ÞVli−Vloð Þ × 100% ð1Þ

where Vlo is the initial liquid volume immediately after foaming (t=0)andVlt is the liquid volumeafter time t. Foamstabilitywasmeasuredas afunction of time. All measurements were performed in duplicates.

Effects of protein concentration on foaming were evaluated bywhipping2, 6, and10% (w/v) PPI asdescribed above. Effect of salt (NaCl)on foaming was evaluated using 2% (w/v) protein and 0.25, 0.5 and 1%added NaCl salt concentrations. The pH of all PPI solutionswas adjustedat 7 using 0.1 MHCl or 0.1 M KOH. The pH effect was investigated on 2%(w/v) PPI and pH range of 2, 3.5, 5, 7, and 9 using either 0.2 M HCl or0.2 M KOH for the adjustment prior to whipping. In order to maintainthe salt at constant concentration, certain amount of 0.2 NKCl calculatedbased on the difference between the amount of added HCl to obtain pH2.0 and the amount of added HCl or KOH to obtain the desired pH wasadded into the solution.

2.10. Gel preparation

Gel preparation was based on the method by Makri et al. (2005)with slight modification. Pea protein isolate (PPI) at 20 wt.% was firstdispersed in phosphate buffer adjusted at pH 6.5 in order to have asteady pH environment. The solution was allowed to stir for 1 h atroom temperature to facilitate solubilization. Hydrated PPI was thencentrifuged at 14000 g for 30 min and room temperature to separateinsoluble deposit. Prior to heat induction each solution was degassedin a vacuumdesiccator under reduced pressure. The degassed solutionwas poured into a metallic cylindrical cell (16×25 mm), with sealedends, and immerged into boiling water for 60 min. Immediately afterheat induction, the protein gel was cooled inside ice water for 15 minand then stored at 4 °C for 24 h. Using a wire cutter, the gel was thencut in both ends to obtain a 16 mm×16 mm cylindrical sample. Inaddition, the pH influence on gelling capacity of 20 wt.% PPI wasassessed by adjusting the pHs at the range of 3.5, 7 and 9 using 2 MHCl or 2 M KOH and identical gelling procedure.

2.11. Texture profile analysis

Texture profile analysis (TPA) was conducted on 20 wt.% heat-induced 16 mm×16 mmcylindrical gel samples at pH 7 using a TA-TX2(Texture Technologies Corp., Scarsdale, NY, USA). Two cycle compres-sion was performed with the aid of a cylindrical probe (25 mm

diameter). Samples were dual compressed at 40% of height, 5 s waitingtime and 1 mm/s crosshead speed. The experiments were carried out24 h after preparation, in order to allow full maturation of the gels. Allsamples were tested in duplicates comprising a total of 10 measure-ments for each sample.

2.12. Rheological measurements

Measurements of flow and dynamic rheological parameters such asshear viscosity (η/γ

.) and (G'), (G˝) and delta degree (δ=G˝ /G') were

carried out using anAR1000Rheometer (TA Instrument, NewCastle, DE,USA). The instrument was equipped with a 60 mm cone of 1.59° andsolvent trap. Flow and dynamic rheological properties of 10 wt.%proteins solutions (pH 7) were measured at 0.1–100/s and 1–25 rad/srespectively. An oscillation stress of 0.5 Pa (found from linear region ofstress sweep test) and frequency of 1 Hz were used to measure thetemperature effect at increasing rate of 10 °C/min.

2.13. Preparation of emulsion

Buffer solutions at pHof 3.4 (juice beverage) and6.8 (milk beverage)were first prepared based on the method by Colowich and Kaplan(1995) using citric acid (0.1 M) and dibasic sodium phosphate (0.2 M)solutions mixed in appropriate ratios. Oil-in-water emulsion was thenpreparedby dispersion of 10 wt.% canola oil into the1 wt.% hydrated PPIin either buffer solution. The procedure consisted of the preparation of acoarse emulsion by blending canola oil and hydrated protein for 3 minfollowed by high pressure homogenization (Emulisiflex-C5, Avestin,ON, Canada) at 5000 psi for 3 passes.

2.14. Assessment of emulsion stability

Stability of PPIs stabilized emulsions was assessed by means ofoptical characterization based on the method by Taherian, Fustier, andRamaswamy (2007a, 2007b) with slight modification. A 6 ml of eachemulsionwas poured into a flat-bottom cylindrical glass tube (100 mmheight, 16 mm internal diameter) and subjected to an optical scanninginstrument (Quick Scan, Coulter Crop.,Miami, FL). The backscattering ofmonochromatic light (λ=850 nm) from the emulsions was measuredas a function of their height in order to quantify the separation rate byconducting a total of 10 scans (each scan was repeated 5 timesthroughout 10 min)within 7 days for each tube. This quantificationwasbasedon themigration rate of the oil droplets fromthebottomto the topof the sample which induces a progressive fall in concentration atthe sample bottom (clarification) and therefore decreases the backscat-tering. The resulting positive peaks were then transferred to MicrosoftExcel and separation ratewas calculatedas slopeof backscatteringmeanvalues over 7 day storage and emulsion stability was expressed as1/separation rate or (% creaming/h)−1.

3. Statistical analysis

The effect of pH, concentration, and salt on functionality ofcommercial and membrane processed PPIs were statistically testedusing the regression and ANOVA — two-factors with replication, andthe means were compared at significant level of 5% using a t-test.Statistical analysis was done using Microsoft Excel and experimentswere performed in duplicate.

4. Results and discussion

4.1. Constituents of pea protein isolates

Protein contents, ash, total phosphorus,moisture content and phyticacid in commercial pea protein isolate (CPPI) andmembrane processedpea protein isolates (MPPIs) are presented in Table 1. Overall, the

Fig. 1. SDS-PAGE patterns of tested PPIs: A) H2O/pH 7.5/25 °C/UF:DF pH 7.5:6, B) H2O/pH 7.5/25 °C/UF:DF pH 7.5:7.5, C) KCl/pH 7.5/25 °C/UF:DF pH 7.5:6, D) KCl/pH 7.5/25°C/UF:DF pH 7.5:7.5 and E) commercial ranging in size from 97 kDa at the top to14.4 kDa at the bottom as V = bands from vicilin proteins, La = legumin acidicsubunit and Lb = legumin basic subunit.

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protein content for CPPIwas lesswhereas the ash, total phosphorus, andphytic acid contents were greater compared to those of MPPIs. Thephytic acid content of MPPIs ranged from 2.45 mg phosphorus fromphytic acid (PA-P)/g protein to 5.57 mg phosphorus from phytic acid(PA-P)/g protein. The highest amount of phytic acid content wasassociated with CPPI (7.71 mg phosphorus from phytic acid (PA-P)/gprotein) and isolates produced by ultrafiltration and diafiltration at pH7.5. Isolates purifiedbydiafiltration at pH6.0 indicated the lowest phyticacid content.

The presence of phytic acid, which is regarded as an antinutritionalcompound of pulse crops (Bilgiçli et al., 2006; Roy, Boye, & Simpson,2010) and accounts for 60–90% of the total phosphorus in seeds (Reddy,Sathe, & Salunkhe, 1982; Torre, Rodriquez, & Saura-Calixto, 1991), wasdirectly related to the amount of total phosphorus and ash contents.Studies suggested that the presence of higher amount of phytic acid inproteinmight be due to the accumulation of storage proteins which arecomplexedwithphytic acid in the formof protein–phytate complex andincreases as thematurity of seeds and plant parts progressed (Chavana,McKenzieb, Amarowiczc, & Shahidi, 2003; Dai, Wang, Zhang, Xu, &Zhang, 2007; Igbedioh, Olugbemi, & Akpapunam, 1994).

Cheryan (1980) and Dai et al. (2007) reported that the directsalt-like linkage between phytic acid and the α-NH2 terminal groupsand ε-NH2 of lysine at intermediate pH could be the cause of complexformation which makes the proteins less soluble, and affect enzymaticdegradation andpeptic digestion.Our results demonstrate theefficiencyof the UF/DF processes in reducing the amount of phytic acid, when thePPI is processed at a pH of 7.5 with a VCR 5 (UF step) and a pH of 6re-VCR5 (DF step) using either water or 0.06 MKCl as extractionmedia.

It is worthy to note that, based on previously reported data the fatcontent ofmembrane processed PPIswas 2.01±0.28% and has not beenaffected by membrane processing (Boye et al., 2010; Mondor et al.,2009). Therefore, functional properties of PPI as an influence of phyticacid concentration is further considered in this paper.

4.2. SDS-PAGE analysis of protein isolates

The sodium dodecyl sulphate polyacrylamide gel electrophoresis(SDS-PAGE) technique is used to identify the protein subunits based ontheir molecular weights, under denaturation condition. With thisregards, SDS-PAGE patterns of PPIs: A) H2O/pH 7.5/25 °C/UF:DF pH7.5:6, B) H2O/pH 7.5/25 °C/UF:DF pH 7.5:7.5, C) KCl/pH 7.5/25 °C/UF:DFpH 7.5:6, D) KCl/pH 7.5/25 °C/UF:DF pH 7.5:7.5, E) commercial PPIand S) standard were examined and showed the range in size from97 kDa at the top to 14.4 kDa at the bottom (Fig. 1). Identification of thebands was based on the work by Shand et al. (2007) and, as illustrated,all the isolates have similar bands. Further summation and comparisonof the bands related to legumin or vicilin and the ratio of legumin/vicilinalso showed comparable results. Leguminwhich is a hexameric proteindissociates into two subunit peptides as acidic (legumin acidic) andbasic (legumin basic), was identified as acidic subunit “Lα” and basicsubunit “Lβ”. For isolates A the amounts of legumin (L), vicilin (V) andthe ratio of legumin to vicilin (L/V) were 29.25%, 49.98% and 0.59respectively. Isolate B indicated 31.88% L, 48.89% V and 0.65 L/V; isolateC showed 30.36% L,48.90% V and 0.62 L/V; and isolate D demonstrated

Table 1Composition of commercial and membrane treated pea proteins in different extraction con

Extraction conditions pH(UF/DF)

Moisture(%)

Proteins(N×6.25) % dry ba

H2O/pH 7.5/25 °C (A) 7.5/6.0 7.19±0.16 97.76±0.20H2O/pH 7.5/25 °C (B) 7.5/7.5 7.28±0.04 90.73±0.010.06 M KCl/pH 7.5/25 °C (C) 7.5/6.0 7.32±0.05 94.26±0.110.06 M KCl/pH 7.5/25 °C (D) 7.5/7.5 7.35±0.16 94.39±0.01Commercial pea protein (E) NA 7.18±0.03 88.69±0.23

a Measured by the WADE reagent method.b milligram phosphor from phytic acid/gram protein.

31.26% L, 50.93% V and 0.61 L/V. Examination of the bands related tocommercial PPI showed 28.41% L, 48.31% V and 0.59 L/V.

However, the two important protein fractions are legumin (11S) andvicilin/convicilin (7S). Legumin is a hexamer that breaks down into twopeptide subunits (alpha: acidic,molecularweight of 38–40 kDa; andbeta:basic, molecular weight of 19–22 kDa) in the presence of reducingconditions. Vicilin is a trimer composed of three heterogeneous unitswithmolecular weights of approximately 70 (convicilin), 50 and 33 kDa, aswell as a smaller quantity of peptideswithmolecularweights between 19and 12.5 kDa (Shand et al., 2007). Presence of Lypoxygenase, which has amolecular weight of about 90 kDa, was also reported by Shand et al.(2007). The above results confirm that the protein composition of theisolates was roughly the same and it can be assumed that the extractionprocedures used in the plant have the same effect on the proteincomposition and the state of the proteins in the different preparations.

4.3. Protein solubility–pH relationships

As is shown in Fig. 2 the pH–protein solubility profiles of testedPPIs were strongly pH-dependent and all had minimum solubilitybetween pH 4 and 6. Commercial PPI exhibited similar pattern butmuch lower solubility compared to themembrane processed PPIs. ThepH–protein solubility profiles of protein isolates purified by diafiltra-tion at pH 6 also showed more contracted range of pH for solubility.These differences in protein solubility–pH relationships have beenrelated to the dissimilarity of protein and surface hydrophobicity.Study by Yust et al. (2003) indicated that the basic subunits of the 11Sare very hydrophobic in nature and are situated inside the globulinmacromolecular assembly, while the less hydrophobic acidic domainsare situated at the surface. The balance of forces holding the subunitstogether is altered during the extraction and dehydration steps of theisolate preparation process, which in turn may affect the protein

ditions.

sisAsh%

Total phosphorus(mg P/g pr)

Phosphorus from phytic acida

(mg PA-P/g pr.)b

2.43±0.02 6.43±0.22 2.45±0.133.95±0.12 10.18±0.36 4.66±0.274.77±0.16 7.10±0.09 2.84±0.105.27±0.02 11.10±0.43 5.57±0.345.19±0.02 14.46±0.28 7.71±0.21

0

10

20

30

40

50

60

70

80

90

0 1 2 3 4 5 6 7 8 9 10

pH

So

lub

ility

(%

)

H2O/pH 7.5/25°C/UF:DF pH 7.5:6 H2O/pH 7.5/25°C/UF:DF pH 7.5:7.5

KCl/pH 7.5/25°C/UF:DF pH 7.5:6 KCl/pH 7.5/25°C/UF:DF pH 7.5:7.5

Commercial

Fig. 2. Effect of pH on solubility of pea protein isolates.

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surface hydrophobicity. The lower solubility of commercial PPI may,therefore, have arisen from an increase in exposed hydrophobicresidues, leading to increased hydrophobic interaction betweenproteins and/or peptides in the acidic pH region as stated by Tsumuraet al. (2005).

Pea proteins diafiltered at pH 6with lower phytic acid concentrationexhibitedhigher solubility compared to the other tested isolates. Studiesby Ali, Ippersiel, Lamarche, and Mondor (2010) and Cheryan (1980)have also emphasised the negative effect of phytic acid on solubility.Accordingly, our study showed that the effect of high concentration ofphytic acid on solubility of protein could be foremost.

4.4. Foaming properties—effect of pH, concentration and salt

Effect of pH on foaming stability of commercial and membraneprocessed PPIs after 15 min whipping is presented in Table 2. As isshown, foaming stabilities were pH-dependent for all tested PPIs.Commercial PPI indicated, significantly (Pb0.05), less foaming stabilitycompared to membrane processed PPIs. Moreover, the foam producedby whipping the commercial PPI vanished after 20 min. In general,superior foaming stabilities were associated with the acid and alkalineregions due to higher solubilities, increase in net charge of proteins andsurface hydrophobicity at these regions (Sorgentinia & Wagner, 2002).The inferior foam stabilities, for all tested PPIs, were associated withisoelectric (pI) region (between pH 4 and 6). The obtained results are inclose agreement with those reported for several legume proteins(Akintayo, Oshodi, & Esuoso, 1999).

Fig. 3 (a,b,c) shows the effect of salt concentration on foam stabilityof tested PPIs at time intervals of 0 to 60 min. Addition of salt up to 0.25%improved foam stability for all tested PPIs. However, further increase insalt concentration did not enhance foam formation of commercial andmembrane processed PPIs. Improvements of foam stability of testedproteins could be due to the higher protein solubility in salt solutionsand the ability ofNaCl to aid diffusion and spreadingof the protein at theinterface. However, these effects are concentration-dependent as highconcentrations of NaCl solutions above (0.25%, w/v) demonstrated noconsiderable enhancement in foam stability for all tested PPIs. This

Table 2Effect of pH on foaming stability of commercial and membrane processed pea protein isola

Foaming stability (%)

pH 2 pH 3.5

KCl–7.5–2–UF:DF 7.5:7.5 33.6±0.8 30.6±0.8KCl–7.5–25–UF:DF 7.5:6 48.0±1.2 45.3±1.1H2O–7.5–25–UF:DF 7.5:7.5 43.3±1.4 37.4±1.0H2O–7.5–25–UF:DF 7.5:6 51.0±1.3 46.0±0.7Commercial 13.8±0.3 7.6±0.1

phenomenon has also been related to the formation of denser foam atthe air/water interfaces (Akintayo et al., 1999). Moreover, study byMwasaru, Muhammad, Bakar, and Che Man (2000) showed that bothsalt and pH affect the yield and alter the solubility of proteins byaffecting protein–protein and protein–water interactions. Addition ofNaCl also presented several advantages derived from the ionic strengthand selective solubilization.

Effects of protein concentration on foam stability of PPIs, at constantpH of 7, are shown in Fig. 4 (a,b,c). As is illustrated, increase in proteinconcentrations enhances foam stability of membrane processed PPIs.Commercial PPI indicated a 32% raise in foaming when proteinconcentration increased from 2% up to 10% but foam stability decreasedfaster and vanished after 15 min. Among membrane processed PPIs,those diafiltered at pH 6 (H2O/pH 7.5/25 °C/UF:DF pH 7.5:6 “A” andKCl/pH 7.5/25 °C/UF:DF pH 7.5:6 “C”) indicated higher production offoamand superior foamstability compared to those diafiltered at pH7.5(H2O/pH7.5/25 °C/UF:DF pH 7.5:7.5 “B” andKCl/pH7.5/25 °C/UF:DF pH7.5:7.5 “D”). The greater production of foam has been associated withhigher concentration of flexible protein molecules that enables toreduce surface tension whereas highly ordered globular proteins whichare relatively difficult to surface-denature form lower amount of foam(Ganzevles, Stuart, vanVliet, & de Jongh, 2006;Makri et al., 2005).Makriet al. (2005) related the foamability to the rapid adsorbtion of protein atthe air/water interface during bubbling, aswell as, rapid conformationalchange and rearrangement of protein at the interface whereas theformation of a cohesive viscoelastic film via intermolecular interactionscontributes to the foamstability. The greater foamstabilityofmembraneproduced PPIs, therefore, may be due to the higher concentration offlexible proteins, faster film formation at the air/water interfaces andsuperior film viscoelasticity at the interfaces. Moreover, increased bulkphase viscosity is expected to contribute to foam stability due todifferences in total protein concentrations.

4.5. Flow and dynamic rheological properties of PPIs

The shear rate dependence of apparent viscosities for commercialand membrane processed PPIs is shown in Fig. 5a. The determinationcoefficients (R2) for all measurements were more than 0.98(not shown), indicating a high level of relation between measuringpoints. The apparent viscosities of tested proteins decreased in differentdegrees with increasing shear rate indicating shear-thinningbehaviours. The degree of decreases in apparent viscosity or flowbehaviour index (n) dependedupon, purity and solubility of theprotein.The PPIs diafiltered at pH 6, containing lower amount of phytic acid,indicated the lowestflowbehaviour indices of 0.69 and 0.73 for isolate Aand isolate C respectively. The apparent viscosities of these two PPIswere also higher over thewhole range of applied shear compared to theother tested proteins. The flow behaviour indices for isolates B, D andcommercial (E) were 0.81, 0.83 and 0.87 respectively. The shear-thinningbehaviour of a protein solution signifies its high viscosity at lowshear rates which decreases as the shear is increased. This property isimportant in emulsion stabilization because it means that the dropletsare prevented from creaming, but that the food emulsion still flowseasily when poured from a container (Taherian et al., 2007a).

tes during 15 min.

pH 5 pH 7 pH 9

20.2±0.3 27.2±0.6 31.8±0.222.8±0.3 28.8±0.8 43.5±0.321.3±0.3 26.9±0.5 41.2±0.825.5±0.4 29.6±1.1 46.5±0.70.0±0.0 12.1±0.2 14.0±0.1

2510 A.R. Taherian et al. / Food Research International 44 (2011) 2505–2514

The dynamic rheological parameters (G', G˝ and delta degree) of PPIssolutions were evaluated in the linear viscoelastic region. Therheological parameters were first measured as a function of stressamplitude at afixed frequency. After establishment of linear viscoelasticregion,measurementswere thenmade atfixed stress amplitude (0.5 Paand 1 Hz) as a function of temperature.

Fig. 5b compares the frequency dependence of delta degree (G˝/ G')for commercial andmembrane processed PPIs. The results indicate thatthe δ is not only frequency dependent, but also relies on the purificationpH of PPI; it decreases as both functions of the frequency and as ameaning of purity and solubility of PPI. The lowest values of δ at elevatedlevel of applied frequencywere associatedwith pea protein extracted inwater and diafiltered at pH 6 (27.2±1.0) followed by pea proteinextracted at KCl solution and diafiltered at pH6 (31.4±1.2). The valuesof δ at elevated level of applied frequency for PPIs extracted inwater andKCl solution followedbydiafiltration at pH7.5 and commercial PPIwere

(a) 0.25% NaCl

(b) 0.5% NaCl

(c) 1% NaCl

0

10

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30

40

50

60

70

80

0 10 20 30 40 50 60 70

Time (min)

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Time (min)

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Time (min)

Foa

m s

tabi

lity

(%)

0

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60

70

80

Foa

m s

tabi

lity

(%)

0

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Foa

m s

tabi

lity

(%)

Commercial

KCl/pH 7.5 /25°C/UF:DF pH 7.5:7.5

KCl/pH 7.5 /25°C/UF:DF pH 7.5:6

H2O/pH 7.5 /25°C/UF:DF pH 7.5:7.5

H2Ol/pH 7.5 /25°C/UF:DF pH 7.5:6

Commercial

KCl/pH 7.5 /25°C/UF:DF pH 7.5:7.5

KCl/pH 7.5 /25°C/UF:DF pH 7.5:6

H2O/pH 7.5 /25°C/UF:DF pH 7.5:7.5

H2Ol/pH 7.5 /25°C/UF:DF pH 7.5:6

Commercial

KCl/pH 7.5 /25°C/UF:DF pH 7.5:7.5

KCl/pH 7.5 /25°C/UF:DF pH 7.5:6

H2O/pH 7.5 /25°C/UF:DF pH 7.5:7.5

H2Ol/pH 7.5 /25°C/UF:DF pH 7.5:6

Fig. 3. Effect of salt on foamability and foam stability of commercial and membraneprocessed pea protein isolates at pH 6.5.

38.1±0.7, 43.1±1.8, and 56.1±2.2 respectively. The delta degree formembraneprocessed PPIs at elevated level of applied frequency is lowerthan 45°, suggesting that solid like elastic behaviour dominates theseprotein solutions over liquid like viscous behaviour. Therefore, themembrane processed PPIs could provide greater emulsion stabilitywhen functioning as emulsifier.

4.6. Rheological and textural properties of heat-induced solution andgelation

Fig. 6 shows the effect of the temperature on the storage modulusG′ and the delta degree, δ, of commercial and membrane processedPPIs at pH 7 during a temperature ramp of 20 to 90 °C at 10 °C/min.

Depending upon the type of process applied for extraction andpurification of PPI, the point of sudden increase forG′ (decrease for deltadegree down to 45°) corresponding to transition of solution to gel (Tgel)was different. The PPIs extracted in water or KCl solution at pH 7.5 and25 °C followed by ultrafiltration at pH 7.5 and diafiltration at pH 6(A or C) indicated the lowest Tgel of 75.7±0.6 °C and 81.6±0.7compared to all other tested PPIs. These isolates contained lower

(a) 2% PPI

0

10

20

30

40

50

60F

oam

sta

bilit

y (%

)F

oam

sta

bilit

y (%

)F

oam

sta

bilit

y (%

)Commercial

KCl/pH 7.5 /25°C/UF:DF pH 7.5:7.5

KCl/pH 7.5 /25°C/UF:DF pH 7.5:6

H2O/pH 7.5 /25°C/UF:DF pH 7.5:7.5

H2Ol/pH 7.5 /25°C/UF:DF pH 7.5:6

(b) 6% PPI

0

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30

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70

80

90Commercial

KCl/pH 7.5 /25°C/UF:DF pH 7.5:7.5

KCl/pH 7.5 /25°C/UF:DF pH 7.5:6

H2O/pH 7.5 /25°C/UF:DF pH 7.5:7.5

H2Ol/pH 7.5 /25°C/UF:DF pH 7.5:6

(c) 10% PPI

0102030405060708090

100Commercial

KCl/pH 7.5 /25°C/UF:DF pH 7.5:7.5

KCl/pH 7.5 /25°C/UF:DF pH 7.5:6

H2O/pH 7.5 /25°C/UF:DF pH 7.5:7.5

H2Ol/pH 7.5 /25°C/UF:DF pH 7.5:6

0 10 20 30 40 50 60 70

Time (min)

0 10 20 30 40 50 60 70

70

Time (min)

0 10 20 30 40 50 60

Time (min)

Fig. 4. Effect of concentration on foam stability of commercial andmembrane processedPPIs at pH 6.5.

2511A.R. Taherian et al. / Food Research International 44 (2011) 2505–2514

amount of ash and phytic acid and showed better foam stability asmentionedearlier. The Tgel for PPIs extracted inwater andKCl solutionatpH 7.5 and 25 °C followed by ultrafiltration at pH 7.5 and diafiltration atpH 7.5 (B and D) were higher at 83.6±0.4 °C and 85.6±0.3 °Crespectively. Commercial PPI did not gel up to 90 °C suggesting thatthe corresponding Tgel must be above 90 °C. The high value of Tgel forcommercial PPI could be related to low protein concentration (lowsolubility) of this protein resulting in sole participation of subunits orpolypeptides in the gel network formation as reported by Kim,Renkema, and van Vlie (2001).

The mechanical characteristics of heat-induced gels comprisinghardness (the force necessary to attain a given deformation), spring-iness (the rate at which a deformed material goes back to its originalheight following removal of the applied force) and cohesiveness(the strength of internal bonds or the ratio of the first compressionarea over the second compression area, A1/A2) are compared in Fig. 7.

As expected, the membrane processed PPIs diafiltered at pH 6indicated relatively higher values of hardness (114.7±2.89 g for A and99.2±1.46 g for C), springiness (75.3±3.43% for A and 74.0±1.77%for C) and cohesiveness (0.66±0.02 for A and 0.65±0.01 for C)compared to the other tested PPIs. The values of hardness, springinessand cohesiveness for membrane processed PPIs diafiltered at pH 7.5were 85.6±1.1 g and 76.0±0.9 g, 66.2±1.5% and 61.2±0.8%,0.51±0.01 and 0.55±0.01 for B and D respectively.

(a)

1

10

100

10 100Shear rate (1/s)

Vis

cosi

ty (

mP

a.s)

H2Ol/pH 7.5/25°C/UF:DF pH 7.5:6

H2Ol/pH 7.5/25°C/UF:DF pH 7.5:7.5

KCl/pH 7.5/25°C/UF:DF pH 7.5:6

KCl/pH 7.5/25°C/UF:DF pH 7.5:7.5

Commercial

(b)

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Del

ta d

egre

e (G

"/G

')

H2Ol/pH 7.5/25°C/UF:DF pH 7.5:6

KCl/pH 7.5/25°C/UF:DF pH 7.5:6

H2Ol/pH 7.5/25°C/UF:DF pH 7.5:7.5

KCl/pH 7.5/25°C/UF:DF pH 7.5:7.5

Commercial0

10

20

30

40

50

60

70

80

90

100

Fig. 5. Comparison of shear viscosity and frequency dependence of delta degree (G˝/ G')of tested PPIs.

The differences in obtained mechanical parameters for PPIsdiafiltered at pH 6 or 7.5 could arise from the dissimilarities in size,shape, and configuration of protein molecules which developeddistinct protein networks even though the experimental conditionswere similar. This phenomenon has also been related to the higherreactivity of protein molecules towards the neighbouring moleculesto form a network when the well-defined secondary and tertiarystructures of the compact molecules become disrupted upon heating(Shand et al., 2007). Studies by Hamann and Lanier (1986) andBeveridge, Jones, and Tung (1984) showed that rheological andmechanical properties of proteins are influenced mainly by proteinquality and functionality, such as solubility, but not the processconditions of gel formation as well as increasing elasticity ordeformation of protein gels which probably result from increasingnumbers of cross-links that stiffen the structure of individualaggregates. Cross-link formation may strongly relate to the degreeof soluble aggregation of the protein molecules during the heatingprocess.

The commercial PPI solution did not gel after heating in boilingwater for similar or longer heating time than the one applied for themembrane processed PPIs. Such phenomenon may be due to the factthat proteins of commercial PPI were partially or completelydenaturated during extraction which led to its reduced solubility. Inaddition the presence of phytic acid at high concentration incommercial PPI and those diafitered at pH 7.5 could allow theformation of protein–phytate complexation which may have changedtheir structures and caused decreases in solubility (Ali et al., 2010;Cheryan, 1980; Konietzny & Greiner, 2003).

4.7. Effect of pH on stability of PPIs stabilized emulsions

Although proteins are very efficient in preventing coalescence, anumber of collisions of the primary droplets are inevitable during alimited time of preparation. The total collision frequency betweenemulsion droplets is the contribution of the Brownian movement andthe shifting of the droplets under gravitational force (McClements,2005). The rate of separation is, therefore, related to the speedof proteinadsorption to the interface and the degree of the early stage coalescencecould control the separation rate (creaming) of the emulsion.The stabilizing effect of the PPIs could, somehow, be due to the kineticsof the early stages of their adsorption. Based on this assumption, theemulsifying capacity of tested PPIs could be ranked in the order of PPIsdiafiltered at pH 6NPPIs diafiltered at pH 7.5Ncommercial PPI (Fig. 8 aand b). Comparing the stability of emulsions prepared at pH3.4 (Fig. 8a)and those made at pH 6.8 buffer solutions (Fig. 8b) revealed that acidic

0

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90

20 30 40 50 60 70 80 90 100

TemperatureoC

Del

ta d

egre

e (G

"/G

')

H2O/pH 7.5/25°C/UF:DF pH 7.5:6 = 75.7 °C H2O/pH 7.5/25°C/UF:DF pH 7.5:7.5 =83.6 °CKCl/pH 7.5/25°C/UF:DF pH 7.5:6 =81.6 °C KCl/pH 7.5/25°C/UF:DF pH 7.5:7.5=85.6 °CCommercial > 90 °C

20 40 60 80 100TemperatureoC

G' (

Pa)

100

120

0

80

60

40

20

Fig. 6. Effect of temperature on dynamic rheological properties of commercial andmembrane processed PPIs.

Fig. 7. Comparison of textural properties of heat-induced pea protein isolate gels.

2512 A.R. Taherian et al. / Food Research International 44 (2011) 2505–2514

medium is unfavourable to emulsion stability. Overall, significantdifferences (pb0.001) were found for stability of emulsions preparedat pH 3.4 and those prepared at pH 6.8.

Study by Taherian, Britten, Sabik, and Fustier (2011) revealed thatthe ability of the protein to be adsorbed at the surface of oil droplets isrelated to its functional properties such as surface hydrophobicity,protein flexibility, solubility, degree of disulphide bonds, degree ofhydrogen interactions and other stabilizing forces. They reported thatthe stability of whey protein emulsions enhanced when pH wasincreased from 5 to 7 and noted that this is, most likely, due to anincrease in electrostatic repulsion of the charged proteins. Study byDagom-Scaviner, Gueguen, and Lefebvre (1987) also indicated thatthe differences in the efficiency of the proteins in retarding emulsionbreaking could reflect directly differences in the flocculation rates,which is related to the stronger repulsion forces between oil dropletsdue to the charge and the thickness of the adsorption layers.Moreover, the anionic phosphate groups of phytate could bindstrongly to the cationic groups of the protein to form insolublecomplexes in acidic pH (Cheryan, 1980; Konietzny & Greiner, 2003).Consequently, lower electrostatic repulsion in acidic pH and higher

concentration of phytic acid in commercial PPI and the PPIs diafilteredat pH 7.5 could be accounted as the factors causing reduction ofemulsion stability. Furthermore, our result indicated lower solubilityof pea protein at pH 3.4 compared to that of 6.8.

Jourdain, Leser, Schmitt, Michel, and Dickinson (2008) reportedthat higher viscosities of the continuous phases tend to inhibit the rateof creaming within emulsified systems. Therefore, the differences instability of PPIs stabilized emulsions could be due to the structure ofthe biopolymer interface and the continuous phase viscoelasticproperties. As a result, the greater stabilities of emulsions preparedwith PPIs extracted in water or KCl media followed by diafiltration atpH 6 could, partially, be due to the higher viscosity and elasticity ofthese proteins as shown earlier. The obtained results also demonstratethat there were close correlation between the solubility, phytic acidcontent, and foamability of the products and the emulsion stability.

5. Conclusions

This study shows that membrane processing is capable of loweringdown the phytic acid content and improves solubility of the pea

Fig. 8. Comparison of emulsion stability of commercial and membrane processed PPIs at pHs a) 3.4 and b) 6.8.

2513A.R. Taherian et al. / Food Research International 44 (2011) 2505–2514

protein. The emulsion stability, rheological andmechanical propertiesof solutions and heat-induced gels for membrane processed PPIs weresuperior compared to the commercial PPI. Diafiltration at pH 6granted further reduction of phytic acid and enhanced emulsionstability, vioscoelastic properties, strength and elasticity of heat-induced PPIs gels. Emulsions stabilized with commercial PPI illustrat-ed the lowest emulsion stability and the highest emulsion stabilitywas associated with PPI extracted in water and diafiltered at pH 6throughout the storage time. Overall, formation of insoluble com-plexes at acidic pH and reduction in protein solubility causedreduction of emulsifying capacity for emulsions prepared at pH 3.4.As a result of foaming, membrane processed PPIs were able to traphigher volumes of air bubbles which increased by increasing theconcentration or addition of NaCl up to 0.25%. Enhancement of foamstability, due to increase in concentration and addition of NaCl, wasalso observedwith all tested PPIs. At last, adjustment of extraction andpurification conditions could provide new opportunities to extend therange of functional properties of PPI in different food systems.

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