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2010 16: 241 originally published online 12 August 2010Food Science and Technology InternationalI. Flores, V. Cabra, M.C. Quirasco, A. Farres and A. Galvez

Emulsifying Properties of Chemically Deamidated Corn (Zea Mays) Gluten Meal  

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Emulsifying Properties of Chemically Deamidated Corn

(Zea Mays) Gluten Meal

I. Flores, V. Cabra, M.C. Quirasco, A. Farres and A. Galvez*

Departamento de Alimentos y Biotecnologıa. Facultad de Quımica, Universidad Nacional Autonomade Mexico. Circuito de la Investigacion Cientıfica s/n. Mexico. D.F. 04510 Mexico

Corn gluten meal is a by-product of starch production that is readily available. Corn protein isolates havelimited applications due to their hydrophobic nature, low solubility and limited functionality as emulsifiers.In this study, a mild acidic treatment of corn gluten meal was performed in order to achieve deamidation ofasparagine and glutamine residues and modify the interfacial behavior of this byproduct. A 0.1N HCl

treatment for 6 h at 70 �C rendered a deamidation degree of 20.4%, which increased the emulsificationactivity index of corn gluten meal from 6.8 to 16.8m2/g protein, with a remarkable increase in emulsionstability from 0 to 90.6% oil retention. Proteins participating in the emulsion were separated by SDS-

PAGE and the main polypeptides were identified as alpha and beta-zeins. After deamidation, proteindissociation and unfolding due to the obtained negative charges resulted in enhanced functionality.

Key Words: corn gluten meal, acidic deamidation, emulsifying properties, Zea mays

INTRODUCTION

In the past 5 years, the demand for food-grade pro-

teins has begun to compete progressively with other

applications, such as the production of biofuels

(von Braun et al., 2008). This situation has increased

the cost of traditional vegetable and animal protein

sources. Consequently, there is a growing need for an

integral use of less expensive protein sources. The main

challenge is still to modify nontraditional proteins to

achieve the required functional properties and render

them appropriately for their use in food formulations.

Corn gluten meal (CGM), a co-product of the corn wet-

milling industry, is an example of a high protein content

source (60% w/w) with potential food uses (May, 1987).

Dry-milling processes have been developed for corn-

starch conversion into bioethanol or high fructose

syrups, which also generate protein co-products at

approximately 28% of the total weight. These bypro-

ducts are typically known as dry distilled grains

(DDGs) with or without solubles as well as corn con-

densed distiller’s solubles that are normally used as

animal feed and are mainly composed of corn gluten

(Xu et al., 2008). The increasing demand for starch in

bio-fuel production will account for a growing availabil-ity of these protein co-products, with limited use due tothe disadvantage of having a poor functionality in thefood area. Considering CGM content in corn as of5�6% (May, 1987), the current availability of corngluten protein from the American corn industry wouldbe approximately of 19 million tons in 2008 (USDA,2009). Corn products have other potential added valueapplications, such as functional ingredients in food, rawmaterial for the production of biodegradable plasticsand films and as a source of the nutraceuticals luteinand zeaxanthin (Lu et al., 2005; Miyoshi et al., 2005).

Among the proteins available in the market, milk andsoy are widely used as food ingredients because of theirfunctionality. As for vegetable proteins, most of themrequire structural modifications in order to expand theiruse in food formulations. Several chemical and enzy-matic modification methods have been described forthe improvement of their solubility, emulsification andfoaming properties (Riha et al., 1996; Yong et al., 2006).CGM has low solubility in aqueous systems at the pHand ionic strength of most food products; consequently,attempts to improve its functional properties haveincluded modifications related to pH adjustment, parti-cle size reduction and freeze-drying (Wu, 2001; Singhet al., 2005) as well as controlled enzymatic hydrolysis(Mannheim and Cheryan, 1992). However, none of thementioned experiments has yielded a potential industrialprocess. Zeins constitute 68% of the total protein con-tent in CGM, while 27% and 1.2% correspond to glu-telins and globulins, respectively (Wu, 2001). Zeins arerich in hydrophobic amino acids, especially aliphaticamino acids, such as leucine, isoleucine and alanine

*To whom correspondence should be sent(e-mail: galvez@unam.mx).Received 29 January 2009; revised 22 June 2009.

Food Sci Tech Int 2010;16(3):0241–10� SAGE Publications 2010Los Angeles, London, New Delhi and SingaporeISSN: 1082-0132DOI: 10.1177/1082013210366750

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(Wilson, 1987). This feature is responsible for the highlyaggregated state of CGM that results in poor solubility.Like other cereal proteins, such as wheat and oatproteins, corn proteins have high levels of the amide-containing amino acids, glutamine and asparagine(Riha et al., 1996). The amide bond of these sidechains is susceptible to hydrolysis, which leads to therelease of ammonia and transformation into acidicgroups. The deamidation treatment can be performedby enzymatic or chemical methods. Enzymes such astransglutaminase, protease, peptidoglutaminase andrecently protein-glutaminase (Hamada, 1992; Yonget al., 2006; Cabra et al., 2007), have been reported inthe literature for food protein deamidation. However,nonenzymatic deamidation has captured the interest offood researchers because of the convenience and feasi-bility of such modifications (Matsudomi et al., 1985;Casella and Whitaker, 1990; Cabra et al., 2007), whichtake place by addition of acids or alkalis. Regardingmaize gluten proteins, Cabra et al. (2007) reportedthat for a better deamidation of the gummy and highlyinsoluble Z19 a-zein, an alkaline treatment was required(0.5N NaOH in 70% ethanol at 70 �C during 12 h).Degree of hydrolysis (DH) values as low as 5% wereobtained under these conditions. A number of factorsmay influence deamidation reaction rate and mecha-nism, such as pH, temperature, water activity, aminoacid sequence, available ions and nonionic catalysis(Riha et al., 1996). The present investigation shows anapproach to improve the functional properties of CGMby chemical deamidation. The effect of the deamidationdegree (DD) obtained by a mildly acidic treatment usingHCl on the emulsifying activity and emulsion stability ofCGM was studied.

MATERIALS AND METHODS

Materials

Commercial (Zea mays) CGM was kindly donated byArancia Corn Products, S. A. de C. V. (Tlalnepantla,Mexico). It was ground in a disk mill (Weber BROS.and White, Metal Works. Inc., USA) to generate parti-cles smaller than 425 mm. The CGM was then keptat 5 �C in nontranslucent, tightly closed containers inorder to avoid oxidation. The other food grade materialused was corn oil from Productos de Maız S.A. de C.V.,Mexico City, Mexico.

Methods

Unless otherwise stated, reagents were purchasedfrom Sigma Chemical Co. (St. Louis, MO). In order toasses the overall quality of raw materials, proximateanalyses of CGM were performed according to AOAC

methods (AOAC International, 2006) and microbiolog-ical analytical procedures were carried out according tothe Mexican Official Standard (1996). The pH of CGMwas determined according to AOAC method 943.02(2006), in which 10 g of CGM were homogenized with50mL of distilled water at pH 7 for 2min at 8000 rpm inan Ultraturrax T 25 (Janke and Kunkel GmbH and Co.KG � IKA-Labortechnik, Staufen, Germany) and werebrought up to a final volume of 100mL. This suspensionwas magnetically stirred for 30min at room temperatureand the pH value was registered using a pH meter(Beckman Instruments, 34 pHmeter, Irvine, CA).Amino acid content was determined by HPLC after6N HCl hydrolysis (105 �C, 24 h), at Silliker, Inc.Laboratories (TX, US).

Chemical Deamidation of Corn Gluten

Deamidation of CGM was achieved according to pre-vious reports on wheat gluten (Popineau et al., 1988)and corn gluten (Flores, 1997). Treatments with 0.1Nor 0.25N HCl were applied to 5% (w/v) protein suspen-sions. The reaction was performed at 70 �C for 0, 1, 3and 6 h. Protein suspensions were homogenized with90mL of HCl (0.1 or 0.25N), which had been heatedat 60 �C, for 1min at 8000 rpm in an Ultraturrax T 25.The homogenized suspension was brought up to a totalvolume of 100mL with the corresponding concentrationof HCl, preheated at 60 �C. Suspensions were subse-quently stirred at 150 rpm at 70 �C in a shaker (NewBrunswick Scientific Model R76, Edison, NJ) for eachtime period previously stated. The reaction was stoppedby neutralization (pH 7.0) using 0.1N NaOH(Mallinckrodt Baker, Phillipsburg, NJ) on an ice bath.

Degree of Hydrolysis

Five mL of the diluted samples were added to 5mL of5% (w/v) trichloroacetic acid (TCA) (Merck Chemicals,Darmstadt, Germany). The amount of solubilized pro-tein in the supernatant, after high molecular weight pro-tein precipitation with TCA, was quantified with themodified Lowry method (Peterson, 1977). Total hydro-lysis was performed by an exhaustive 6N HCl treatmentat 100 �C (24 h). The DH was calculated as a ratiobetween the protein solubilized under each deamidationcondition and the protein quantified for the 100%hydrolyzed gluten.

Deamidation Degree

The amide groups that remained on the protein afterthe mild acidic treatments were quantified according toArntfield and Murray (1981). The ammonia releasedafter deamidation was quantified with an ammoniumelectrode (Orion Research, model 95-10, Beverly, MA).The maximum deamidation value (100%) was

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determined by exhaustively treating CGM with 2N HClat 110 �C for 3 h. Deamidated samples were dialyzed inorder to avoid interference with the ammonium elec-trode measurements. The degree of deamidation isexpressed as the ratio of ammonia released from theacid-modified CGM suspensions treated at differenttimes and HCl concentration, to that of the completelydeamidated CGM protein suspension (Matsudomi et al.,1985). Non-treated gluten is here referred to as nativeCGM.

Functional Properties Determination: Solubility

Suspensions of 1 g protein/100mL were prepared byadding treated and native CGM to water adjusted with1N HCl (Merck Chemicals, Darmstadt, Germany) or1N NaOH (Mallinckrodt Baker, Phillipsburg, NJ) tovarious pH values in the range of 2�12, followingPopineau’s method (Popineau et al., 1988). Protein sus-pensions were incubated at 30 �C, 175 rpm for 120min,with pH adjustments to the desired pH value performedevery 30min by potentiometric means. Afterwards, sus-pensions were centrifuged at 4 �C for 30min at10 000� g in a Beckman J2-MC centrifuge (BeckmanCoulter, Inc, Fullerton, CA) and soluble proteins werequantified in the supernatant as described by Peterson(1977), using bovine serum albumin as a standard.

Emulsifying Properties Evaluation

The emulsifying activity index (EAI) was determinedby the method of Pearce and Kinsella (1978). Corn oil(10mL) was dispersed into 30mL protein suspensions(1% (w/v)), pH 7.0, with an Ultraturrax T 25 homoge-nizer at 20 000 rpm for 2min. The emulsions obtainedwere immediately diluted 250-fold with a solution ofsodium dodecyl sulfate (0.1% w/v, pH 7) and 0.1Msodium chloride. The turbidity T (T¼ 2.303A/L, A,absorptivity of the emulsion; l cm, path length of thecuvette) of the dilutions was immediately measured at500 nm in a spectrophotometer (Perkin Elmer Corp.,Norwalk, CT). EAI was defined as:

EAI ¼ 2T=�C ð1Þ

where � was the volume fraction of the oil phase (here�¼ 0.25) and C was the protein concentration in theaqueous phase. The EAI, expressed in m2/g, was relatedto the interfacial area stabilized per unit weight ofprotein.

Stability Towards Coalescence

Freshly prepared emulsions, in 12mL volumes, wereimmediately centrifuged for 10min at 3000� g (CentraCL2, International Equipment Co., Needham Heights,MA). The volume (Vs) of the separated oil phase was

measured. The ratio of 100 Vs/Vi (where Vi¼ 3mL, totaloil volume in the emulsion) was taken as a measure ofcoalescence (Dagorn-Scaviner et al., 1987).

Electrophoretic Pattern of Proteins Participatingin Emulsions

Proteins were extracted from emulsions made withnative as well as with deamidated gluten, using a mod-ification of the method reported by Saito et al. (1993).The cream portion of the emulsion was washed threetimes with 0.1M phosphate buffer, pH 7.0, gentlymixed and recovered by centrifugation at 4 �C for45min at 10 000� g. Separated water and oil were dis-carded. The emulsion was destabilized by the addition ofan equal volume of 6% (w/v) SDS and 10% (v/v) glyc-erol and boiled for 5min. This mixture was then centri-fuged (15 000� g) at 4 �C for 30min. The aqueousportion was oil extracted with an equal volume ofether. After centrifugation, the remaining aqueousphase was lyophilized and further analyzed bySDS-PAGE. The participating proteins in the emulsionwere loaded with 500 mg of protein per lane and run indenaturing 10% acrylamide gels (LaemmLi, 1970) at250V and 60mA for 6 h in a Hoefer SE600 Series SElectrophoresis Unit, with a Bio-Rad molecular weightmarker.

Statistical Data Treatment

The data from all runs represented an average valueof at least three replicates and in most cases, the coeffi-cient of variation (c.v.) was lower than 10%. An analysisof variance was performed (�¼ 0.05) with the StatGraphics Plus 5.1 Program.

RESULTS AND DISCUSSION

Proximate Composition and Microbial Analyses

CGM showed the following composition: protein58.32±0.25, fat 1.80±0.09, ash 1.42±0.01, crude fiber2.23±0.25, moisture 10.44±0.09 and carbohydrates(determined by difference) 25.77 g/100 g of sample.CGM protein content may vary between 60% and73%. In this particular case, lower protein content wasfound; carbohydrate content contributed to one-fourthof the sample weight and all other parameters werewithin the intervals reported by other research groups(May, 1987; Mannheim and Cheryan, 1992; Wu, 2001).The pH of CGM was 3.81±0.02, resulting from wetmilling conditioning, which uses bisulfite in order toenhance the release of starch and insoluble proteinfrom the original granule matrix (Yang et al., 2005).CGM microbial content was within the interval requiredfor cereal products, flours and meals, according to the

Emulsifying Properties of Chemically Deamidated Corn 243

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Mexican Official Standard (NOM-147-SSA1-1996), atlevels of less than 100 000, 100 and 1000UFC/g formesophilic bacteria, coliforms and fungi, respectively.These values confirm that the proteinic raw material isappropriate for human consumption, resulting fromgood manufacturing practices.

Acidic Treatment Deamidation and Protein Hydrolysis

In plant storage proteins, as it is expected in CGM,almost all b- and g- carboxyl residues of Asp and Gluacids are amidated (Casella and Whitaker, 1990; Wonget al., 2009). During amino acid analyses, Asn and Glnare converted into Asp and Glu, rendering impossiblethe individual quantification of the original contents ofthe amidated forms. Amino acid contents of 20.82 and5.81 g/100 g protein for Glu and Asp, respectively, werefound (Table 1). Both the high amidated amino acidcontent and low solubility limit the usage of CGM infood formulations.

Considering the previous reports of wheat and corngluten deamidation and the need to reevaluate the usesof the by-products from the new corn industrializationsuch as DDGs, the amidation value determined in this

work reinforces the feasibility of an acidic deamidationtreatment. The expected result is the improvement offunctional properties by increasing the negative chargecontent and hydration (Wong et al. 2009). After anexhaustive deamidation, which implies the total conver-sion of amide groups from Asn and Gln residues intocarboxyl groups, the ammonium released led to the cal-culation of an experimental amidation value of24.98±0.04. Total elimination of amide groups was con-sidered as a 100% of DD.

Based on previously established conditions (Flores,1997), acidic treatments were performed on CGM pro-tein. Statistically significant high experimental DDvalues were obtained with longer incubation times andhigher HCl concentrations. The highest DD valuereached was 53.4% at 6 h, with 0.25N HCl (Table 2).

Compared to other deamidation treatments reportedon different protein sources, DD values were of the sameorder of magnitude: Mimuni et al. (1994) reported asimilar deamidation behavior versus time of acidictreatment for wheat gluten, in a protein suspension of2.5% w/w, using 0.1N HCl at 70 �C. Okara, a soy milkby-product, showed 41% DD after a 48 h treatmentusing 0.1N HCl at 65 �C (Chan and Ma, 1999). Otheracids have also been used on wheat gluten, such as8.75M acetic acid, as reported by Berti et al. (2007),which was utilized to obtain 13.2% ammonia releasefrom 5% (w/v) wheat gluten at 90 �C after a 3 h treat-ment. There are some reports on the chemical deamida-tion of corn proteins (Casella and Whitaker, 1990;Cabra et al., 2007). Casella and Whitaker (1990) treatednative zein with 0.05N HCl at 95 �C for 30min, reach-ing a DD of 3.4%. DD was improved to 9.5% whenSDS was used as a catalyst. In contrast, the resultspresented herein were performed at a lower temperature(70 �C), with HCl concentrations increased to 0.1 and0.25N, in order to obtain higher DD values. Thismethod has the advantages of a less stringent thermaltreatment, as higher temperatures might cause hydroly-sis of peptide bonds as well as the avoidance of alkalinedamage to Trp and the generation of undesirable com-pounds resulting from high pH treatments.

Another approach for deamidation is an enzymatictreatment with peptidoglutaminases and transglutami-nases, which improved ammonia release after a certain

Table 2. Emulsifying properties of deamidated corn gluten meal treated with acid.

Deamination degree (%) Emulsifying actividy indexa (m2/g) Emulsified oila (%)

Deamidation time (h) 0.1 N HCl 0.25 N HCl 0.1 N HCl 0.25 N HCl 0.1 N HCl 0.25 N HCl

0 0 0 6.8±1.1 6.8±1.1 0 01 7.3±0.1 18.0±1.6 6.9±0.9 6.0±0.4 0 9.4±1.23 11.4±0.5 47.6±0.0 9.5±1.0 11.1±2.8 34.8±3.1 58.7±2.86 20.4±0.0 53.4±0.7 16.8±5.0 25.1±4.8 90.6±3.4 82.4±2.8

aThe protein samples were dispersed in 0.01 M sodium phosphate buffer, pH 7 at a concentration of 1% (w/v).

Table 1. Amino acid composition ofcorn gluten meala.

Amino acid g/100 g protein

Asp 5.81Glu 20.82Cys 0.29Ser 4.77Hys 1.79Gly 3.36Thr 3.43Arg 3.00Ala 8.36Tyr 9.44Met 2.06Val 3.29Phe 6.62Ile 2.65Leu 17.68Trp 1.62

aDetermined by HPLC; Lys and Pro: not determined.

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extent of hydrolysis (Hamada, 1992; Yong et al., 2006;

Cabra et al., 2007). The chemical deamidation of corn

gluten tested in this work rendered results similar to the

ones obtained with such enzymatic treatments.

However, the enzymatic process has not been scaled-

up (Hamada, 1991), due to the fact that these enzymes

are not commercially available at an industrial scale.A certain DH is expected after the acidic treatment.

Excessive peptide bond cleavage during hydrolysis could

render a more soluble product. However, it could result

in undesirable properties, such as bitter taste and

reduced functionality (Agboola and Dalgleish, 1996;

Riha et al., 1996). DH was determined in deamidated

and native CGM samples, as previously described.

Results are shown in Figure 1. An initial DH value of

2.7% was found in the raw material. An initial hydro-

lysis was expected in native CGM due to the acidic con-

ditions during the wet milling process, which involves

sulfurous and lactic acids. Sulfurous acid is produced

by the reaction of SO2 with water; while lactic acid

can be produced in vivo by fermentation of sugars by

Lactobacillus bacteria present in corn, or it can be added

to the process (Yang et al., 2005). In all samples treated

with mild acidic conditions, resulting DH values were

limited. HCl 0.1N caused a significant change, from

2.7% to 3% DH, after a 1-h treatment; longer reaction

times produced no significant changes. Although hydro-

lysis was proportional to the stringency of the treatment,

a low DH value (4.4%) was also obtained in the more

severe conditions tested (6 h and 0.25N HCl). The

hydrolytic effect was more important during the first

three hours of reaction and no significant changes

were observed after a 6-h treatment.

Mimuni et al. (1994) and Popineau et al. (1988) inves-tigated wheat gluten deamidation, with the conclusionthat protein solubilization was mainly due to deamida-tion and that resulting hydrolysis was limited in acidictreatments at 70 �C and 0.1N HCl. Other reports havedemonstrated that mild acidic treatments, such as thoseemployed, render a negligible degree of hydrolysis fordifferent vegetable proteins, such as soy and oat(Matsudomi et al., 1985; Ma and Khanzada, 1987).Exclusion chromatography has allowed demonstratingthat deamidation conditions can dissociate oligomericproteins, due to the introduction of negative charges inside chains that cause repulsion among peptides. Thedissociation of proteins is more important on solubiliza-tion than the effect caused by hydrolysis (Matsudomiet al., 1985; Ma and Khanzada, 1987). In addition, alimited DH (less than 10%) may even enhance func-tional properties of proteins, mainly emulsifying andfoaming capacities (Foegeding et al., 2002).

Functional Properties

Protein Solubility

According to the results obtained from the solubilitycurves shown in Figure 2, native and deamidated CGMare both in a low solubility condition throughout theinvestigated pH range from 2 to 9, although a significantincreased solubility was found at pH values higher than9. This behavior could be explained by the electrostaticinteractions produced by the side chains with ionizingproperties as well as hydrogen bond formation with thesolvent. Hys is not charged; Arg and Lys are only par-tially protonated, while Asp and Glu are charged at

0.00

2.50

5.00

7.50

10.00

0 1 3 6

Treatment time (h)

Hyd

roly

sis

de

gre

e (

%)

Figure 1. Hydrolysis degree obtained with twodifferent HCl concentrations as a function of deamida-tion time: («) 0.1 N and (#) 0.25 N. Protein concentra-tion used: 5% (w/v). Acidic treatments performedat 70 �C.

0

10

20

30

40

50

60

2 3 4 5 6 7 8 9 10 11 12

pH

Sol

ubili

ty (

%)

Figure 2. Corn gluten meal (CGM) solubility as afunction of pH. CGM was deamidated for 6 h with:(�) 0.1 N HCl, (m) 0.25 N HCl, and (#) native. Proteinconcentration used 1% (w/v).

Emulsifying Properties of Chemically Deamidated Corn 245

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alkaline pH values. Such combinations of charged andnoncharged side chains provide native maize gluten witha maximum solubility value of 17.30%±0.75 (g of sol-uble protein per 100 g of sample) at pH 12. The curveprofile of native CGM, shown in Figure 2, is similar tothe one reported for zeins, with a low flat portion at pHbetween 4 and 7 and an increased solubility at pH valueshigher than 10 (Casella and Whitaker, 1990). Thisbehavior is expected, given the fact that CGM proteinfraction is composed by a pool of zeins and glutelins,with isoelectric points between 6.5�8.5 and 4.0�8.5,respectively (Wu, 2001). An isoelectric point value ofcommercial zein has been reported as 6.2 (Fu et al.,1999) and specifically for the 19 kD a-zein as 6.8(Cabra et al., 2006).

A marginal increase in solubility from the nativeCGM value of 5.3%±0.3 was obtained at pH 9, afterthe 0.1N and 0.25NHCl treatments, with values of9.9%±0.5 and 12.3%±0.5, respectively. At pHvalues� 10, solubility was 8-fold the value of nativegluten after deamidation with 0.25N HCl, while theless stringent treatment (0.1N HCl) promoted a slightincrease in solubility. This behavior could be explainedby the generation of negatively charged groups to someextent, because the degree of deamidation with thistreatment was only 20% (Table 2).

Emulsifying Properties: Emulsion Activity Index andEmulsion Stability

The role of proteins in emulsions involves molecularinteractions, which are constrained by their aggregationstate and their interface spanning capability. To evaluatetheir emulsifying properties, energy should be provided,through a high speed homogenization of the two immis-cible phases, with a resulting decrease in droplet size aswell as protein unfolding at the interface. In this process,protein chains quickly migrate to the oil droplet surfacewith consequent protection against coalescence. Theproteins that cannot be strongly adsorbed in an oil/water interface, whether because their side chains arestrongly hydrophilic or because they possess rigid struc-tures, will not be good emulsifiers. In order to assess theemulsifying capacity of a protein, EAI and emulsionstability (ES) have to be experimentally evaluated(Dalgleish, 2001).

EAI values, estimated in this study at pH 7 with a 0.25oil volumetric fraction, are shown in Table 2 as a func-tion of deamidation conditions. Native gluten has nota-bly limited emulsifying properties and a 0.1N HCltreatment for 6 h allowed a 2.6-fold increment in theEAI and an emulsion stability improvement from 0 to90.6% of oil retention capacity, as explained further inthe discussion. The EAI represents the interfacial surfaceproduced in an emulsion, which is expressed as theamount of square meters generated per gram of emulsi-fier (m2/g). A protein with good interfacial activity

causes a decrease in the surface tension between the oiland aqueous phase and allows for the production ofsmaller particle size emulsions, which is reflected in ahigher EAI value (Mangino, 1984; Tornberg et al.,1997), as was observed in the acidic treatment on CGMin this work. Although, the EAI exhibited the highestvalue under more stringent conditions (25.1m2/g),it was not in accordance with the highest emulsion sta-bility value, found for the 6-h treatment in the less acidicconditions. A possible explanation for this behavior isthat under stringent conditions, a certain increase inthe degree of protein hydrolysis was produced (from3.19 to 4.43DH, see Figure 1). Limited hydrolysiswould help the emulsifying capacity, because peptidesobtained would cover small interfacial surfaces wherehigh molecular weight proteins would not necessarilydo, resulting in fuller coverage of the oil droplet surface(Table 2). However, a more extensive hydrolysis wouldgenerate small peptides unable to cover large areas(Casella and Whitaker, 1990; Agboola and Dalgleish,1996; Dalgleish, 2001).

An emulsifying protein, adsorbed in the interface,reduces the phase coalescence tendency that is producedto diminish the free interfacial energy caused by the sur-face increment during emulsification. The ES valueassesses the coalescence resistance and it is expressedas the percentage of the oil fraction that remains emul-sified after a centrifugal force is applied to the system.Figure 3 shows the stability of emulsions made withdeamidated gluten under different times of 0.1N HCltreatment, after centrifugation. The greater ES valuewas obtained after a 6-h treatment. A separated waterphase and a cream containing emulsified oil by the pro-tein fraction are shown; low stability emulsions areclearly visible in the cases where oil is separated (tubesA, B and C in Figure 3). In the photograph, the pellet attime zero shows the native CGM insoluble portion thatis not involved in the emulsion. With a progressive dea-midation (1, 3 and 6 h), a smaller pellet is observed and a

Oil phase

Creamedemulsion

Waterphase

Oil phaseCreamedemulsion

Waterphase

Insolublefraction

Figure 3. Emulsion stability after centrifugation.Emulsions were made with native (A) and deamidatedCGM: deamidation treatments were for 1 h (B), 3 h (C)and 6 h (D), at pH 7.0 and 1% (w/v) protein concentra-tions with 0.1 N HCl.

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large emulsified oil portion was obtained. The decreasein pellet volume may not only be due to a greater solu-bilization, which in turn was limited (3�9.5%), but alsodue to incorporation of the CGM into the emulsionas a result of a new dissociation and unfolded stateof resulting proteins. These results are in accordancewith other reports demonstrating that emulsifying prop-erties do not correlate directly to solubility (Figure 2)(Aoki et al., 1980). Previous studies (results not shown)demonstrated that the effect on ES of residual starchin CGM is negligible and that the increase on coales-cence resistance is mainly caused by deamidation(Cabra, 2002).The deamidation process of maize gluten produced

surface charges on the original protein through the gen-eration of deprotonated carboxyl groups that show neg-ative charges at neutral pH, given the pKaR values ofAsp and Glu. The repulsion between the new negativelycharged portions of the molecules, could be a factor forCGM proteins unfolding, exposing the hydrophilic aswell as hydrophobic groups, once buried in the aggre-gated native proteins (Bos and van Vliet, 2001). Besides,in an emulsion, the main driving forces are hydrophobicand electrostatic interactions in a process that gainsentropy, due to the conformational changes of the pro-tein during the adsorption (Bergenstahl and Claesson,1997). Therefore, the newly extended conformationallows the covering of a larger particle surface becausedeamidated gluten shows a better capacity for interfacespanning. Dalgleish (2001) reported an additional mech-anism for the stabilization of colloidal systems byproteins: the DLVO (Derjaguin�Landau�Verwey�Overbeek) theory, which encompasses the balance ofVan der Waals attractive forces and the electrostaticrepulsion of charges of identical signs, which are nega-tive in the case of deamidated gluten.In addition, the protein conformation in the interface

and protein surface activity could differ, depending onthe nature of the nonpolar phase that could be highlyheterogeneous in a food system (Rampon et al., 2004).

Electrophoresis Analysis of the Emulsifying Proteins

Considering the fact that CGM is composed of differ-ent protein fractions, mainly prolamines, glutelins andglobulins, these polypeptides might interact in differentways due to their structural characteristics. Figure 4shows SDS-PAGE analysis of proteins from nativeand deamidated CGM (lanes a and c), as well as theproteins extracted from emulsions made with bothCGM samples (lanes b and d), respectively. Given thefact that the amount of protein loaded on each gel lanewas standardized, the proteins that participate in theemulsion were determined, but not their concentration.The band pattern does not greatly vary from one treat-ment to another, indicating that the same protein frac-tions, from deamidated and non-deamidated gluten, do

participate in the emulsion. However, the difference intheir capacity to emulsify and more strikingly, to stabi-lize emulsions, was due to the newly generated chargesand consequently to the new states of association and/orfolding patterns.

The slightly differing apparent molecular masses ofthe constituent proteins of CGM determined bySDS-PAGE, as reported by several authors, could beexplained by the intrinsic variability in protein compo-sition among the corn varieties analyzed, as well asextraction and electrophoretic conditions used by eachresearch group (Wilson, 1991). An important issue toconsider is that the starting raw material for this workis an industrial gluten meal, obtained from an unknownmix of corn varieties, which share a common family ofdivergent genes that are expressed as structurally similarproteins with slightly different molecular masses. Forthis reason, the unique and unequivocal identity ofeach of the bands observed in this study was difficultto ascertain. The SDS-PAGE analysis indicates that themain polypeptides found are b-zein of 18 kD and a-zeinsof 19, 21 and 24 kD. The 21 kD peptide has also beenidentified as g-zein. The bands of 27 and 31 kD, whichdemonstrated lower density, could be identified as g-zeinand glutelin, respectively. A lighter band located at10 kD could be d-zein (Wilson, 1991; Wang et al.,2003). Finally, some very faint bands could be observedaround 45 and 66 kD - these proteins could be oligomersof the 21 kD a-zein, as reported by Cabra et al. (2006).

Structurally, zeins are composed of nine adjacent,topologically anti-parallel helices that are clustered

97.4MWM (kDa) a b c d

66.2 66

50

44

31

28

24

22

1918

16

10

45

31

21.5

14.4

Figure 4. SDS-PAGE analysis of proteins from: (a)native corn gluten meal (CGM), (b) proteins extractedfrom the emulsion made with native CGM, (c) deami-dated CGM and (d) proteins extracted from the emul-sion made with the modified CGM.

Emulsifying Properties of Chemically Deamidated Corn 247

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within a distorted cylinder. The domains of the amphi-patic helices have been previously described as favoringgood emulsifying properties that contribute to the sur-face activity of the proteins (Krebs and Phillips, 1984).Being zeins the most abundant CGM proteins partici-pating in the emulsions analyzed, their hydropathyindex was calculated in an attempt to explain theiradsorption behavior at the interface. The computer pro-gram ProtScale tool, on the exPASY Server, systemati-cally evaluates the hydrophilic and hydrophobictendencies of a polypeptide chain according to the char-acteristics of the amino acid side chains. The programuses the Kyte and Doolittle hydropathy scale (Kyte andDoolittle, 1982) in which each amino acid has beenassigned a value reflecting its relative hydrophilicity(negative values) and hydrophobicity along the aminoacid sequence, reflected in a score (Y-axis) assigned toeach residue. Accordingly, protein hydropathicity valueswould reflect its capacity to span in the oil/waterinterface.

The analysis of b-zein (panel A) and Z22 a-zein (panelB) are displayed in Figure 5. As for Z19 a-zein (Cabraet al., 2006), three wide hydrophobic regions thatinclude most of the structure were separated by threesmall hydrophilic zones. b-zein showed two very widehydrophilic regions that were interspersed by twosmall hydrophobic ones and Z22 a-zein includes sixhydrophobic regions separated by four hydrophilicones. The alternating hydropathic behavior couldexplain the presence of the zeins at the interface, butthe difference in emulsion stability between native anddeamidated CGM, would be influenced mostly by pro-tein dissociation and unfolding, given that the Kyte andDoolittle scale considers Gln and Asn as hydrophilic asGlu and Asp.

CONCLUSIONS

Chemical treatments have proven to be still an optionfor functionality improvement with industrial potential,considering the increasing amounts of CGM and DDGsthat are obtained in the bio-fuel industry. A low costmild acidic deamidation was able to induce protein dis-sociation as well as unfolding, improving the interfacialfunctionality allowing the production of an added valuefood additive. The striking high emulsion stabilityachieved by the deamidated CGM was mainly basedon the repulsive negative forces generated. Among theprotein fractions involved in emulsification, zeins werethe most abundant at the oil-water interface. Theyencompass a good theoretical alternative pattern ofhydropathy, which may explain their presence in theemulsion, though surface hydrophobicity in native anddeamidated CGM should be evaluated experimentally.

ACKNOWLEDGMENT

Authors would like to acknowledge Pamela SuarezBrito for her technical support.

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