Effects of protein solubilisation and precipitation pH values on the functional properties of defatted wheat germ protein isolates

Original article

Effects of protein solubilisation and precipitation pH values on

the functional properties of defatted wheat germ protein isolates

Fengru Liu, Zhengxing Chen,* Li Wang & Ren Wang

National Engineering Laboratory for Cereal Fermentation Technology, School of Food Science and Technology, Jiangnan University, 1800

Lihu Avenue, Wuxi, 214122, Jiangsu Province, China

(Received 11 October 2012; Accepted in revised form 22 January 2013)

Summary Wheat germ protein isolates were prepared from defatted wheat germ flour using alkaline solubilisation

and acid precipitation. A central composite design with two independent variables (solubilisation pH and

precipitation pH) and bivariate correlations was selected for the correlation analysis of the protein separa-

tion conditions and the functional properties. The results showed that the protein yield (Y) and functional

properties of isolates, such as water absorption (WA) and fat absorption (FA), were sensitive to both sol-

ubilisation pH and precipitation pH, whereas the emulsification was sensitive to only solubilisation pH.

Emulsifying activity (EA) and FA of isolates showed a high positive correlation with yield. Gel electro-

phoresis analysis of protein fractions gave evidence to the compositional changes between proteins iso-

lated under different conditions, highly alkaline conditions result in the degradation of protein chains and

formation of toxic compounds. Surface hydrophobicity suggested that proteins tend to be more denatured

when solubilised at highly alkaline conditions. These conformational and compositional changes due to

different protein separation conditions have contributed to the changes in functional properties of protein

isolates.

Keywords Acid precipitation pH, alkaline solubilisation pH, correlation analysis, defatted wheat germ protein, functional properties.

Introduction

Wheat germ is the embryo of the wheat plant andbelongs to raw material of biological value. In wheatflour milling industry, it contains a high-fat portionand has to be removed to extend the flour shelf life.The main by-product of oil extraction process is adefatted wheat germ (DWG), which is a highly nutri-tive protein material with a relatively high protein con-tent (27.8–30 g/100 g) (Arshad et al., 2007), and is oneof the most attractive sources of vegetable proteins.Wheat germ protein was found to be even superior toother first-class proteins, with respect to the sameamino acids (Waggle et al., 1967). Among vegetableproteins, wheat germs have probably the best essentialamino acids combination, which is well comparablewith that of egg protein (Miladi et al., 1972). Thenutritional qualities of the protein in wheat germsapproach the value of many animal proteins (Grewe &LeClerc, 1943). However, the wheat germ proteinsource has poor utility for human applications.

Wheat germ protein isolates are generally preparedby alkaline extraction and isoelectric precipitation—a traditional procedure to plant protein isolatesextraction. Protein extraction from meal/flour isaffected by various factors such as particle size, previ-ous thermal history, solvent-to-meal ratio, extractiontime and temperature, pH and ionic strength of thesolution (Kinsella, 1979). The pH of the solvent is oneof the most sensitive and highly influential parametersamong these factors (Aluko & Yada, 1997; Bora &Ribeiro, 2004; Lawal, 2004). In protein isolation pro-cedures, both solubilisation (extraction) and precipita-tion (purification) steps contribute to the proteinyield. Therefore, protein recovery should be maxi-mised at each step to maximise the overall proteinyield. Generally, high solubilisation pH values (around9.5) and low precipitation pH values (around 4.0) areexpected to increase the extraction and purification ofwheat germ proteins (Hettiarachchy et al., 1996; Geet al., 2000; Zhu et al., 2006). The extraction and pre-cipitation pH values may also have an effect on thebehaviour of the protein isolates in food and non-foodapplications, but reports on such impacts are rare inliterature.

*Correspondent: Fax: +86-510-85197856; e-mail: zxchen_2008@

126.com

International Journal of Food Science and Technology 2013 48, 1490–1497

doi:10.1111/ijfs.12117

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1490

Several reports have stated that pH plays a signifi-cant role in determining the functionality (waterabsorption, emulsification and gelation) of differentprotein sources such as pea (Sun & Arntfield, 2011),cowpea (Aluko & Yada, 1997) and soy (Jiang et al.,2009). In this study, the effects of protein solubilisationand precipitation pH values on the yield and func-tional properties of wheat germ protein isolate weredetermined.

Methods have been developed for extracting plantprotein based on acid-, alkaline- and enzyme-assistedextraction, and physical methods such as reversemicelles, microwave-assisted membrane. However,these advanced technologies are not yet mature. Thealkali treatment is a common process for high proteinrecovery (>80%) by dissolving the insoluble plantproteins in dilute alkaline solution and subsequentlyprecipitating protein under acidic conditions (the iso-electric point of protein). Recently, it has beenreported that this method effectively improves in vitroand in vivo digestibility of rice protein, which is asso-ciated with the modification of protein body structureand amino acid composition (Kumagai et al., 2006;Kubota et al., 2010; Xia et al., 2012). Otherwise, theprotein isolates use as food ingredients depends largelyon their functional properties, which are related totheir hydration, degree of unfolding, and their compo-sition at various alkaline pH values (Tomotake et al.,2002; Tang et al., 2006). Several molecular parame-ters, such as mass, conformation, flexibility, net chargeand hydrophobicity, as well as interaction with otherfood components, have already been shown to playan important part in water absorption (Abugochet al., 2008), emulsifying (Karaca et al., 2011) andfoaming properties (Aluko et al., 2009). The existingliteratures, however, focus on the improvement of pro-tein yield and content (Jiamyangyuen et al., 2005; Sariet al., 2013). It is not desirable that the impact ofextraction conditions on the functional properties ofprotein has been generally ignored. To obtain thegreatest economic benefit, therefore, the effects andcorrelations of protein solubilisation and precipitationpH values on the yield and functional properties ofwheat germ protein isolate were simultaneously con-sidered by a central composite design with two inde-pendent variables and bivariate correlations in thisstudy.

Materials and methods

Sources of materials and chemicals

Defatted wheat germ with protein content (N 9 5.45)of 27 g/100 g (wet weight basis) was purchased fromthe Anyang Mantianxue Food Co., Ltd. (Henan,china). The material was milled by a small-scale

hammer mill (DFY-600, Zhejiang province, China),and the resulting flour was sieved through a 200-meshscreen. All other reagents used in the experiments wereof analytical grade and obtained from Guoyao Chemi-cal Reagent Co., Ltd. (Shanghai, China).

Protein extraction and isolation

For protein extraction, 100 g of defatted meal/flourwas dispersed in 1000 mL of distilled water (1:10, w/v)and stirred for 30 min at ambient temperature (about25 °C). The pH of the suspension was adjusted torequired solubilisation pH using 1M sodium hydroxide(NaOH) and stirred for 1 h. The protein-rich superna-tant was centrifuged at 2420 g for 20 min at 4 °C toremove the fibre and other suspended solids and wasthen precipitated by 1M hydrochloric acid (HCl) to therequired precipitation pH. The precipitated proteinswere recovered by centrifugation at 2420 g for 20 minat 4 °C and freeze-dried. Lyophilised protein isolateswere used for functional property testing. The percent-age yield was based on the mass (g) of overall lyophi-lised protein extraction produced from 100 g of DWGflour.The processes followed for the overall protein

recovery by an experimental design matrix with arandomised treatment order (Myers et al., 2009) areillustrated in Table S1. A central composite design(CCD) (Li et al., 2011) with two independent variables(solubilisation pH and precipitation pH) was selectedfor model development. The design consisted of 4 fac-torial runs, 4 axial runs and 5 replicates at the designcentre point. The centre point (solubilisation pH 9.5and precipitation pH 4.0) was the point of maximumprotein yield found in previous work. The highest andlowest levels for solubilisation and precipitation pHvalues were set as 1 pH unit above and below the cen-tre point, respectively.

Water absorption, fat absorption activity of proteinisolates (WA and FA)

WA and FA of protein isolates were determined bythe modified methods reported by Ahmedna et al.(Ahmedna et al., 1999) and Tomotake et al. (Tomo-take et al., 2002), respectively. Three replicates wereused for each test.To determine water absorption (WA), the lyophi-

lised protein isolate samples (1 g) were suspended in10 mL of distilled water in 15 mL preweighted centri-fuge tubes. For each sample, distilled water was addedin small increments to a series of tubes under continu-ous stirring with a glass rod. After the mixture wasthoroughly wetted, samples were centrifuged at 1360 gfor 15 min and remove free water. WA was calculatedas grams of water per gram of sample:

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International Journal of Food Science and Technology © 2013 Institute of Food Science and Technology

International Journal of Food Science and Technology 2013

Effects of protein extraction conditions on functional properties F. Liu et al. 1491

WA ¼ w2 � w1

w0

W0 is the weight of the dry sample (g), W1 is theweight of the tube plus the dry sample (g), and W2 isthe weight of tube plus the sediment (g).

To determine FA, the protein isolate samples (1 g)were weighed into 15-mL centrifuge tubes preweightedand thoroughly mixed with 10 mL of soybean oil. Theprotein–oil mixture was centrifuged at 3000 rpm for15 min and remove free oil. FA was calculated asgrams of oil per gram of protein:

FA ¼ F2 � F1

F0

F0 is the weight of the dry sample (g), F1 is the weightof the tube plus the dry sample (g), and F2 is theweight of tube plus the sediment (g).

Emulsifying activity and emulsifying stability (EA and ES)

Emulsifying activity (EA) and emulsifying stability (ES)for samples were determined by a modified method ofPearce and Kinsella (Pearce & Kinsella, 1978). For theemulsion formation, a volume of 30 mL of the 0.2%(w/v) protein solution (in 0.01 M phosphate buffer, pH7.0) and 10 mL of soybean oil were homogenised in aT18 Ultra Turrax (Wilmington, NC, USA) for 1 min ata speed of 30 000 rpm. Immediately after the emulsionformation, 50 lL of the emulsion was taken from thebottom (to ensure the consistency of each samplingposition) of the homogenised emulsion and diluted witha 0.1% (w/v) sodium dodecyl sulphate (SDS) solution(1:100). Then, the diluted emulsion was shaken in avortex mixer for a moment, and the absorbance (A) at500 nm was read in the spectrophotometer andexpressed as EA of samples.

To determine the ES, the prepared emulsions werelet stand for 24 h, heated at 80 °C for 30 min, cooledat room temperature and the absorbance (A80) wasalso measured as described earlier. ES was calculatedfrom the relationships below:

ESð%Þ ¼ A80

A� 100

Foaming capacity and foaming stability (FC and FS)

Foaming capacity (FC) and foaming stability (FS)were determined by the method of Naczk et al. (Naczket al., 1985) with some modifications. Aqueous proteinsolutions (50 mL, 2%, w /v) in 0.05 M phosphate buf-fer (pH 7.0) were homogenised in a T18 Ultra Turrax(Wilmington, NC, USA) at a speed of 30 000 rpm for1 min at room temperature (about 25 °C). The mix-ture was immediately transferred to a 100 mL gradu-

ated cylinder and the total volume was recorded after0 and 60 min. FC and FS were calculated from:

FCð%Þ ¼ vt � v0v0

� 100

FSð%Þ ¼ FC

FC0� 100

V0 is the original volume of sample (50 mL), Vt is thetotal volume after different times (mL) and FC0 is theFC at 0 min.

Surface hydrophobicity (H0)

Surface hydrophobicity (H0) was determined using1-anilino-8-naphthalene sulphonate (ANS) as a fluores-cence probe, as reported by Kato and Nakai (Kato& Nakai, 1980). The protein samples (4 mg mL�1 pro-tein in 10 mM phosphate buffer, pH 7.0) were stirred for2 h at room temperature and centrifuged at 9680 g for30 min at 15 °C. The protein concentration in thesupernatant was determined by the Folin–phenolmethod (Lowry et al., 1951). Each supernatant was seri-ally diluted with the same buffer to obtain protein con-centrations ranging from 0.5 to 0.005 mg mL�1. Then, avolume of 4 mL of each diluted sample was added with50 lL of ANS (8.0 mM in 0.1 M pH 7.0 phosphate buf-fer solution). Fluorescence spectra for the samples wererecorded on a Hitachi F-7000 Fluorescence Spectrome-ter at wavelengths of 365 nm (excitation) and 484 nm(emission). The initial slope of the fluorescence intensityversus protein concentration plot was used as an indexof protein surface hydrophobicity (H0).

Sodium dodecylsulphate polyacrylamide gelelectrophoresis (SDS-PAGE)

SDS-PAGE was performed on a discontinuous buffersystem according to the method of Laemmli (Laemmli,1970) using 12% separating gel and 5% stacking gel.The protein samples were solubilised in 0.125 M Tris–HCl buffer (pH 6.8), containing 1% (w/v) SDS, 2%(v/v) 2-mercaptoethanol (2-ME), 5% (v/v) glyceroland 0.025% (w/v) bromophenol blue, and heated for5 min in boiling water before electrophoresis. For eachsample, 10 lL was applied to each lane. After runningat a constant current of 20 mA for �3 h, the separat-ing gel was stained with 0.25% Coomassie brilliantblue (R-250) in the 50% trichloroacetic acid and thendestained in 7% acetic acid.

Statistical analyses

All determinations were carried out in triplicate, andresults were presented as mean � standard deviation.

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Effects of protein extraction conditions on functional properties F. Liu et al.1492

The analysis of variance (ANOVA) of data was car-ried out using SPSS 16.0 statistical software (SPSS Co,Chicago, IL, USA). Comparisons between means weredone using a duncan with a confidence interval of95%.

Results and discussion

Protein yield

The contour plot of the model for yield compared withprecipitation pH and solubilisation pH is shown inFig. S1. The results show that the yield is almostequally sensitive to solubilisation pH and precipitationpH. The highest yields (>36%) are predicted in a fairlylarge region near the centre point of the experimentalspace. Low pH solubilisation and low precipitation pHvalues (within the experimental space) result in thelowest protein yields; however, the low solubilisationefficiencies at higher pH values can be compensated tosome degree by carrying out the precipitation at ahigher pH. There is also more flexibility in terms ofyield with precipitation pH when solubilisation is doneat pH values near 9.5. Such information could beimportant depending on the impact of precipitation pHon other protein properties. Protein extractability generallyincreases with increase in solubilisation pH. At >10.0,non-protein components that interfere with proteinextraction are dissolved, resulting in improved proteinrecovery at isoelectric precipitation (Chen & Houston,1970). However, a higher solubilisation pH may dena-ture the proteins and form lysine-alanine complexes,resulting in a decreased nutritional value, and possiblyform toxic compounds (De Groot & Slump, 1969).

The significances of coefficients in the model areshown in Table S2, and the Pearson Correlation isshown in Table S3. The model shows the great signifi-cances of protein yield to solubilisation and precipita-tion pH, but there are no significances betweensolubilisation and precipitation pH; the Pearson Corre-lation reveals the significant positive linear correlationof protein yield to solubilisation pH, but there is nosignificances between precipitation pH and proteinyield.

Water absorption and Fat absorption of isolates

WA indicates the ability of protein isolates to physi-cally hold water against gravity (Kinsella, 1979). Theability of proteins to retain water is very important infood systems because it affects the flavour and textureof foods. The lower precipitation pH values andhigher solubilisation pH result in higher waterabsorption of protein isolates (Fig. S2). The linearterm of precipitation pH was significant in determin-ing the water absorption of wheat germ protein, and

it has a much greater impact than solubilisation pH(Table S2). Proteins are capable of binding largequantities of water because of their ability to formhydrogen bonds between water molecules and polargroups of polypeptide chains (Vani & Zayas, 1995).Low acidic pH affects the magnitude of the netcharge on protein molecules, and these net chargesprovide more binding sites for water, resulting inelectrostatic repulsion among molecules, hydration ofcharged residues and increased protein–solvent inter-actions (Hrynets et al., 2011). The rapid increase inWA on the acidic side compared to the alkalinemight be attributed to more ionisable groups withpKa values in acidic conditions than in alkaline con-ditions (Undeland et al., 2002). Figure S2 showed themaximum of WA (3.99 g g�1) at the extraction con-dition of 10.2/3.3, which is comparable to 3.55 g g�1

of commercial soy protein isolate (SPI) (Zhu et al.,2010), while the maximum of Y (36.64%) was at 9.5/4.0 (WA is 2.54 g g�1). Table S3 showed no correla-tion between yield and water absorption; thereforeproducer should be considered simultaneously proteinyield and high water absorption to determine theoptimum extraction conditions.The ability of wheat germ protein, like other proteins,

to bind fat is likely due to non-polar side chains thatbind hydrocarbon chains, thereby contributing toincreased oil absorption (Lin & Zayas, 1987). UnlikeWA, the FA showed a positive linear relationship withsolubilisation pH while showing a nonlinear relation-ship with precipitation pH (Table S2). FA increasedwith solubilisation pH (Fig. S3); however, the precipita-tion pH had a relatively smaller impact on FA of pro-tein isolates. A possible reason for increased FA is theincreased unfolding of proteins due to more aromaticand aliphatic amino acid residues that were exposed tothe surface at higher solubilisation pH values.Fat absorption is an important parameter in food

applications of protein isolates, where higher FAwould perform well in foods such as high-fat bakeryproducts and doughnuts, as well as in emulsion-typefoods. Extraction of proteins at higher pH values andprecipitation at either high or low pH values couldtherefore be beneficial for these applications. It is alsopossible to produce protein isolates with a range ofFA properties within the area of near-maximum yield.The wheat germ protein had the highest FA value of2.98 g g�1 at the extraction condition of 9.5 /3.0,which was similar with that of SPI (2.43 g g�1) (Zhuet al., 2010).

Emulsifying activity and emulsifying stability of isolates

Proteins are amphoteric substances, because they pos-sess both hydrophilic and hydrophobic characteristicsdue to the availability of polar and non-polar amino

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acids, respectively (Zhang et al., 2009). The ability ofproteins to interact with both oil and water determinesthe EA of proteins isolates. The influence extraction ofpH on the EA and ES of the proteins was very com-plex as shown in Fig. S4.

The results indicated that wheat germ protein wasbetter emulsifiers at pH 9.5 or 10.5 than that at acidicpH values. EA showed a positive relationship with thesolubilisation pH of the protein isolates, but theimpact of precipitation pH was not found to be signifi-cant (Table S3). Emulsification of proteins dependslargely on the ionic charge and hydrophobicity. Athigh pH values, unfolding and hydrolysis can exposemore hydrophobic groups in the protein chains (Zhanget al., 2009). It is tempting to suggest that the removalof the highly negatively charged oil droplets probablycontributed to the increased hydrophobicity of thesamples treated with alkaline pH (Jiang et al., 2009).However, the precipitation pH value results in minimalexcess charge and it is insensitivity to emulsification ofprotein (Manamperi et al., 2011).

The ability of food proteins to form and stabiliseemulsions is critical to their role as food ingredients inthe food industry to control emulsion stability. ES wasgreater for isolates at higher solubilisation pH andlower precipitation pH and steadily stayed in 70–80%(Fig. S4). The ability of a protein to form and make anemulsion stable depends on an adequate balancebetween its molecular size, charge, surface hydrophobic-ity and molecular flexibility. In general, high ES valuesof proteins could be attributed to their higher surfacecharge, higher surface hydrophobicity and higher solu-bility (Karaca et al., 2011). Differences in EA and ESvalues in the present study are thought to reflect differ-ences in protein composition and physicochemical prop-erties induced by the different extraction conditions.

Emulsifying activity and ES of wheat germ proteinisolate reached the highest value (0.207, 76.09%, respec-tively) at pH 9.5/3.0 and 10.5/4.0 in Fig. S4. It wasshown that EA and ES of DWGP were a little higherthan those of casein and close to those of Bovine SerumAlbumin (BSA) by Ge (Ge et al., 2000). Thus, wheatgerm protein isolate would be a useful functional pro-tein additive in emulsion-based food products.

Foaming capacity and foaming stability of isolates

The results of effect of pH on FC and stability of wheatgerm protein are presented in Fig. S5. The maximum ofFC (150%) was at the extraction condition of 9.5 /3.0,which was similar to that of egg white standard thathave been reported by Hettiarachchy (Hettiarachchyet al., 1996). The results indicated 24% increase in theFC at pH 4.0 (corresponding to minimum FC) whereasthe maximum FC of 153% was recorded at pH 3.0. Sol-ubility, molecular flexibility and protein molecular

weight were important criterions for FC. The decreasein the molecular weight and the increase in the flexibilityprotein chains allowed proteins to partially unfold moreeffectively at the air/water interface, encapsulating airparticles and increasing FC. Therefore, the higher FCaway from isoelectric point was due to its high solubil-ity, small protein molecular and flexibility proteinchains (Omana et al., 2010).The FS of isolates being markedly higher in the

neighbourhood of the isoelectric pH of the proteinthan at any other pH maybe related to lack of repul-sive interactions between the interface and adsorbingmolecules. This may also have contributed to a suffi-cient intermolecular (protein–protein) interaction andformation of a viscous film at the interface, and thus ahigher viscoelasticity, resulting in the slower drainageand more stable foam (Adebowale & Lawal, 2003).

Correlation of yield and protein isolate properties

The functional properties of isolates generally did notshow high correlation between each other (Table S4).The functional properties (WA/FA/EA and FC) aregoverned by widely varying phenomena such as pro-tein conformation, hydrophobicity and ionic chargethat led to low correlation between each other. EAand FA of isolates, however, showed a high positivecorrelation with yield.Solubilisation pH was an influential parameter in all

properties (except FC), whereas precipitation pH wasless significant in most. This could be attributed to thefact that significant changes to the composition andquality of proteins (especially wheat germ proteins) takeplace in the high pH range. A higher extraction pH(close to pH 10.5) may significantly denature the pro-teins and possibly form toxic compounds, resulting in adecreased nutritional value (Hettiarachchy et al., 1996).However, when considering the precipitation pH

range (pH 3.0 to 5.0), yield was affected significantlywhile there were less significant changes to the proper-ties of the resulting protein isolates. With an expandedprecipitation pH range, however, properties of proteinisolates may exhibit more significant differences (atexpense of yield) with the changing precipitation pH.The fact that all functional properties (WA, FA, EA

and FC) of protein isolates were positively correlatedwith the solubilisation pH (P < 0.05) could be benefi-cially used in the protein-based food additives indus-try. By operating toward the higher end of theprecipitation pH range, the functional properties couldbe improved with smaller impacts on yield.

Gel electrophoresis of protein isolates

Electrophoresis (SDS-PAGE) of wheat germ proteinwas performed to obtain information on the molecular

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weight and distribution pattern of the protein compo-nents (Fig. S6). The SDS-PAGE analysis showed thatthe proteins recovered with different pH treatments didnot differ significantly. A wide band was observed ataround 55 kDa and another at 35 kDa. An apparentmolecular weights of several polypeptide protein subun-its were noticed at <21 kDa. The lack of lower migrat-ing proteins means that wheat germ does not containgluten-type proteins. All of the main bands of wheatgerm proteins are also visible but the protein fractionsabout 35–40 kDa and about 23 kDa are much moreintensive. This fact and the high rate of the globulinfractions mean that globulin is the main fraction ofwheat germ protein (T€om€osk€ozi et al., 1998).

The globulin bands intensities at higher solubilisa-tion pH values (9.5 to 10.5) were darker than thebands at lower solubilisation pH values (8.5 to 9.5).The relative intensities of the globulins bands (approxi-mately 35 kDa) revealed the increase in globulins withthe rising solubilisation pH. This indicates that ahigher fraction of more hydrophobic globulins com-pared to those extracted at lower pH values wasobtained. However, at the higher end of solubilisationpH, the higher molecular weight bands (>66 kDa)appear to be darker than those extracted at lower solu-bilisation pH values. This may be due to protein dena-turation and the formation of lysine-alaninecompounds in highly alkaline solutions (Hettiarachchyet al., 1996). Lysino-alanines as higher molecularweight subunits in the gel are formed by the combina-tion of smaller subunits of albumins and globulins viacross-linking of amino acid residues (Manamperiet al., 2011). This may result in a decrease in availablelysine and also affect functional properties due tocross-linking. Therefore, the formation of lysine-ala-nine can be reduced by appropriately decreasingextraction pH values for the proteins.

Surface hydrophobicity of protein

Hydrophobic interactions play a major role in definingthe conformation and structures of protein molecules.Therefore, surface hydrophobicity of proteins helps todetermine the rate of protein unfolding due to differentprocessing methods (Hrynets et al., 2011). Surfacehydrophobicity of different protein isolates are shownin Fig. S7. Surface hydrophobicity generally did notshow high response to varying extraction and precipi-tation pH values. However, a significant increase inprotein hydrophobicity was observed when the solu-bilisation pH values was carried out at upper to end,indicating that these proteins unfolded at this pH,which ultimately led to increased exposure of hydro-phobic groups on the surface (Omana et al., 2010).This agrees well with the higher FA and EA of isolatesseen at higher solubilisation pH. The WA and FC,

however, did not show positive correlations withincreasing surface hydrophobicity of the protein iso-lates at the higher solubilisation pH values.Meanwhile, treatments at pH 8.5 to 9.5 were found

to have light effect on wheat germ protein hydropho-bicity, indicating that the proteins retained more sec-ondary and tertiary structures at these pH values. Thesecondary and tertiary structures of proteins are usu-ally stabilised by multiple intra-molecular bonding.The partial unfolding of proteins due to the breakageof these intra-molecular linkages causes the exposureof hydrophobic groups (Lin & Park, 1998).

Conclusions

The functional properties of protein isolates showedhigh sensitivity to solubilisation pH and lesser, butstill significant, impact from precipitation pH. Theprotein yield showed high positive correlation withFA and EA but did not have significant correlationswith other properties. The yield was robust withrespect to both solubilisation and precipitation pHvalues showing a fairly large region for near-maxi-mum yield conditions. In addition, when wheat germprotein showed the highest Y (36.64%) in 9.5/4.0(solubilisation pH and precipitation pH), only FSshowed maximum (91.67%) and other functionalproperties were not necessarily the best, such as themaximum of WA (3.99 g g�1) was at the extractioncondition of 10.2/3.3, while FA, EA, FC were at 9.5/3.0 (2.98 g g�1, 0.207, 150%, respectively), and themaximum value of ES (76.09%) was at 10.5/4.0. Thisallows processers to adjust pH values according tothe protein isolate property requirements withoutaffecting the yields greatly. Upstream processing con-ditions, such as solubilisation pH and precipitationpH values, can be used as controllable parameters tomodify the properties of protein isolates according tospecific needs and applications.

Acknowledgments

This research was supported by the FundamentalResearch Funds for the Central Universities (JUD-CF10026), the National High Technology ResearchandDevelopment Program of China (863 Program) (No.2013AA102206 and 2013AA102204), special fund foragro-scientific research in the public interest(200903043-02), National Natural Science Foundationof China (31101383 & 31171785), and the Fundamen-tal Research Funds for the Central Universities(JUSRP51302A).

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Supporting Information

Additional Supporting Information may be found inthe online version of this article:

Figure S1. Contour plot showing the relationship ofisolate yield with precipitation pH and solubilizationpH.

Figure S2. Water absorption of wheat germ proteinat various extraction conditions (solubilization andprecipitation pH).

Figure S3. The fat absorption of wheat germ proteinat various extraction conditions (solubilization andprecipitation pH).

Figure S4. The emulsifying activity and emulsifyingstability of wheat germ protein at various extractionconditions (solubilization and precipitation pH).Figure S5. The foaming capacity and foaming stabil-

ity of wheat germ protein at various extraction condi-tions (solubilization and precipitation pH).Figure S6. Electrophoretic profile of wheat germ

protein fractions, MW molecular weight standard.Figure S7. The surface hydrophobicity of wheat

germ protein at various extraction conditions (solubili-zation and precipitation pH).Table S1. Coded levels and corresponding actual

values of the 2 factor central composite design.Table S2. Significances of coefficients between

extraction conditions (solubilisation and precipitationpH) and yield and properties of wheat germ protein.Table S3. Linear correlations between extraction

conditions (solubilisation and precipitation pH) andyield and properties of wheat germ Protein.Table S4. The correlation analysis between yield and

properties of wheat germ protein isolate.

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Effects of protein solubilisation and precipitation pH values on the functional properties of defatted wheat germ protein isolates