Food Chemistry 131 (2012) 1076–1085

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Food Chemistry

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Transglutaminase treatment of brown rice flour: A chromatographic,electrophoretic and spectroscopic study of protein modifications

Stefano Renzetti a,b,⇑, Juergen Behr c, Rudi F. Vogel c, Alberto Barbiroli d, Stefania Iametti d,Francesco Bonomi d, Elke K. Arendt a

a Department of Food Science, Food Technology and Nutrition, National University of Ireland, Cork, Irelandb Biotransfer Unit, University College Cork, Irelandc TU-München, Lehrstuhl für Technische Mikrobiologie, D-85350 Freising, Germanyd Dipartimento di Scienze Molecolari Agroalimentari, Università degli Studi di Milano, Italy

a r t i c l e i n f o a b s t r a c t

Article history:Received 30 December 2010Received in revised form 20 June 2011Accepted 9 August 2011Available online 16 August 2011

Keywords:Brown riceProteinTransglutaminaseSE-HPLCTwo dimensional gel electrophoresisFront face fluorescenceDynamic light scattering

0308-8146/$ - see front matter � 2011 Elsevier Ltd. Adoi:10.1016/j.foodchem.2011.08.029

⇑ Corresponding author at: TNO, Expertise GroUtrechtseweg 48, 3704 HE, Zeist, The Netherlands. Te30 6944466.

E-mail address: stefano.renzetti@tno.nl (S. Renzett

Recently, it was shown that transglutaminase (TGase) treatment of brown rice (BR) flour results in tex-tural improvements of gluten-free bread. In this study, changes in the protein profiles of BR flour and pro-tein fractions induced by TGase treatment were investigated to better understand the activity andspecificity of the enzyme. Size-exclusion HPLC (SE-HPLC) profiles of flour extracts, under reducing condi-tions, revealed the presence of macromolecular protein complexes, as well as low molecular weight pro-teins. After TGase treatments (10 U/g of proteins) a general reduction in peak intensities indicated thepolymerisation of BR proteins into larger, insoluble complexes. Microchip capillary electrophoresis andtwo-dimensional (2D) gel electrophoresis revealed that the a and b glutelin subunits were primary sub-strates for the polymerisation reaction, whereas albumins and globulins were only slightly affected. SE-HPLC of the protein fractions revealed glutelins’ polymerisation into high molecular weight structuresafter TGase treatment. Dynamic light scattering measurements showed that new supramolecular aggre-gates of glutelins co-existed with the macromolecular complexes already present in the untreated frac-tion. Front-face fluorescence approaches indicated that TGase treatment caused a decrease in proteinsurface hydrophobicity of BR flour, but not of the glutelin suspensions. It is concluded that the large pro-tein complexes resulting from glutelin polymerisation and the stronger hydrophobic interactions amongproteins result in the improved textural properties of TGase-treated BR bread.

� 2011 Elsevier Ltd. All rights reserved.

1. Introduction

Coeliac disease is an immune-mediated enteropathy triggeredby the ingestion of wheat, rye, and barley in genetically susceptiblepersons and is currently estimated to affect 1% of the worldwidepopulation (Catassi & Fasano, 2008). At present, the only effectivetreatment is a permanent withdrawal of gluten from the diet.The worldwide diffusion of the disease and the lack of good qualitygluten-free products have increased the interest of food research-ers in the development of gluten-free products, including bread(Clerici, Airoldi, & El-Dash, 2009; Hüttner, Dal Bello, & Arendt,2010; Korus, Witczak, Ziobro, & Juszczak, 2009; Marco & Rosell,2008; Schober, Bean, Boyle, & Park, 2008; Van Riemsdijk, Van DerGoot, Hamer, & Boom, 2011).

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The use of gluten-free flours in breadmaking is technologicallydifficult due to the fact that, after hydration and mixing, proteinsin gluten-free flours do not develop into a visco-elastic networkas wheat proteins do. The use of protein cross-linking enzyme,i.e. transglutaminase (TGase), has been investigated to improvethe textural quality of gluten-free breads by promoting formationof a stable, covalently linked protein network (Gujral & Rosell,2004; Moore, Heinbockel, Dockery, Ulmer, & Arendt, 2006).

TGase is a protein-glutamine c-glutamyl-transferase(EC 2.3.2.13), which catalyses an acyl-transfer reaction betweenthe c-carboxyamide group of peptide-bound glutamine residuesand the e-amino groups of peptide-bound lysine residues, resultingin the formation of covalent bonds between two peptide chains(Folk & Finlayson, 1977). We have recently demonstrated thatTGase treatment of brown rice (BR) flour enhances the consistencyand elasticity of BR batters and results in significant improvementof the textural quality of BR breads (Renzetti, Behr, Vogel, & Arendt,2008). These effects were related to the formation of large proteinaggregates which were visualised in both batters and bread

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crumbs by confocal laser scanning microscopy. However, no inves-tigations were conducted on the nature of the proteins involved inthe action of TGase on BR, on the changes in protein solubility pro-files, or on the nature and extent of non-covalent interactions thatare expected to accompany the formation of an interprotein net-work stabilized by TGase-formed covalent bonds.

The aim of the present work was to investigate the changes inprotein profile induced by TGase treatment of BR flour and gatherinformation on the contribution of BR protein fractions to the for-mation of protein complexes. Size exclusion-HPLC (SE-HPLC),microchip capillary electrophoresis (MCE) (Dolnik and Liu, 2005;Wang, Taylor, Jemere, & Harrison, 2010) and two dimensional(2D) gel electrophoresis (Westermeier & Schickle, 2009) were usedto address events occurring in a system resembling a BR batter, aswell as to investigate the reactivity of the isolated protein frac-tions. Front-face fluorescence was used to address changes in pro-tein conformation and in inter-protein interactions associated withthe polymerisation reaction. In the studies on isolated proteins, dy-namic light scattering (DLS) was added to the array of methodolo-gies already used in the first part of this study to assess changes inTGase-treated BR flour.

2. Materials and methods

2.1. Materials

BR flour (Doves Farm Foods Ltd., Berkshire, UK) was treatedwith a microbial transglutaminase (100 units/g, Ajinomoto Co.,Hamburg, Germany). The BR flour had moisture, protein, and ashcontent of 12.8%, 7.8% and 1.4% (m/m), respectively.

2.2. Methods

2.2.1. Flour and protein fraction analysesCrude protein (N � 6.25) was determined by combustion (AACC

International, 2000), using a Leco FP-528 nitrogen analyser (St. Jo-seph, MI, USA). Moisture and ash contents were determined, fol-lowing Approved Methods 44–15A and 08–01, respectively(AACC International, 2000).

Total amino acid profiles of protein fractions were determinedaccording to Moore and Stein (1963). Amino acids were quantifiedusing a Jeol JLC-500/V amino acid analyser (Jeol UK Ltd., GardenCity, UK) fitted with a Jeol Na + high performance cation-exchangecolumn.

2.2.2. Protein fraction extractionsBR flour (10 g) was defatted with hexane by Soxhlet extraction

for 4 h and air-dried in 24 h at room temperature. Protein fractionswere then extracted according to Landry and Moureauc (1980). BRglutelins were extracted using a borate buffer (0.0125 M Na2B4O7 �12H2O, 0.02 M NaOH, pH 10), containing 0.6% 2-mercaptoethanol(v/v) and 0.5% SDS (m/v). After extraction, proteins were exten-sively dialysed and then freeze-dried.

2.2.3. Flour and protein fraction treatmentsBR batters were obtained by mixing 1 g of flour with 1.25 ml of

water containing enough TGase to give a final concentration of10 U per gramme of protein. Samples were then vortexed for 30 sand incubated at 30 �C for 30 and 180 min in a water bath. Sampleswithout enzyme addition were used as a control. After incubation,samples were immediately cooled in icy water and freeze-dried.

For each protein fraction, dispersions (5 mg/ml) prepared inphosphate buffer (0.05 M NaH2PO4�H2O, 0.15 M NaCl, pH 7.6) weretreated with 10 U of TGase per gramme of fraction, and incubatedfor 60 min at 50 �C in a water bath. Aqueous N-ethylmaleimide

(NEM) (0.1 ml, 0.1% v/v) was added to stop the enzymatic reaction.A control, with no addition of the enzyme and addition of NEM,was used for each fraction.

In the case of the glutelin fraction, the polymerisation reactionwas also monitored by taking an aliquot of the dispersion at 0, 5,10, 15, 30 and 60 min after the addition of TGase. The enzymaticreaction was stopped by adding NEM (0.1 ml, 0.1% v/v). The NEMsolution was added prior to the enzyme in the control run at time 0.

2.2.4. MCE of untreated and TGase-treated flour and protein fractionsProteins were extracted from untreated and TGase-treated BR

batter samples by dissolving 100 mg of freeze-dried material in1 ml of phosphate buffer (0.05 M NaH2PO4�H2O, 0.15 M NaCl, pH7.6) containing 6 M urea and 20 mM DTT. Samples were shakenfor 90 min and then centrifuged at 16,000g for 15 min. After centri-fugation, the supernatant was collected and loaded with Agilentsample buffer on a 230 Protein LabChip (14–230 kDa molecularrange) into the Agilent 2100 Bioanalyzer (Agilent Technologies,Palo Alto, CA), following the manufacturer’s instructions. For eachsample, the relative concentration of the polypeptides separated,according to size, was calculated against the internal standardpresent in the Agilent sample buffer.

For protein fraction analyses, 100 ll of untreated and TGase-treated protein solutions were freeze-dried and re-dissolved inphosphate buffer (0.05 M NaH2PO4�H2O, 0.15 M NaCl, pH 7.6). con-taining 6 M urea and 20 mM DTT, and analysed on the Agilent 2100Bioanalyzer.

2.2.5. SE-HPLC of untreated and TGase-treated BR batters and proteinfractions

Proteins were extracted from untreated and TGase-treated BRbatter samples, using the procedure previously described. Aftercentrifugation, the supernatant was collected and filtered (Dura-pore, 0.45 lm, Millipore, Italy). Then, 0.2 ml of the filtered solutionwas loaded onto a Superose 6 column (Pharmacia, Italy), connectedto a Waters 490E dual wavelength detector (Waters Co., Milford,MA, USA). Separation was performed at room temperature at0.4 ml/min. The mobile phase was phosphate buffer (0.05 M NaH2-

PO4�H2O, 0.15 M NaCl, pH 7.6) containing 4 M urea and 1 mM DTT.The eluate was monitored, at both 220 and 280 nm. The columnwas calibrated using lysozyme (14 kDa) and bovine serum albumin(66 kDa).

Untreated and TGase-treated BR protein fractions (10 ll) wereapplied to a Superdex 200 column (Amersham Biosciences, Upp-sala, Sweden) to achieve fractionation in the 10–600 kDa range,as previously described (Renzetti, Dal Bello, & Arendt, 2008). Sam-ples were eluted with phosphate buffer (0.05 M NaH2PO4�H2O,0.15 M NaCl, pH 7.6) at 0.6 ml/min. The UV detector was set to210 nm. The column was calibrated using aprotinin (6.5 kDa), cyto-chrome c (12.4 kDa), carbonic anhydrase (29 kDa) and bovine ser-um albumin (66 kDa).

The untreated and TGase-treated glutelin samples were ana-lysed by resuspending 0.2 mg of the freeze-dried material fromeach treatment in 0.2 ml of phosphate buffer (0.05 M NaH2-

PO4�H2O, 0.15 M NaCl, pH 7.6) containing 6 M urea and 20 mMDTT, prior to filtration and SE-HPLC analysis carried out on a Supe-rose 6 column, as described above.

2.2.6. Light scattering measurementsDynamic light scattering measurements were performed by

using a DAWN HELEOS II multi angle light scattering (WyattTechnology Corporation, Santa Barbara, CA, USA) set up for 90-de-grees quasi-elastic light scattering measurement, and connectedon-line with the HPLC system. Signals were elaborated by Wyattsoftware (Astra V 5.1.9.1), that allowed us to calculate thedistribution of hydrodynamic radius by means of a regularization

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template. Buffer viscosity was calculated by the software SEDN-TERP v. 1.09 (University of New Hampshire).

2.2.7. Two dimensional gel electrophoresisUntreated and TGase-treated (10 U TGase, 60 min at 50 �C)

globulin and glutelin fractions were analysed by 2D gel electropho-resis, carried out as previously described (Renzetti, Behr et al.,2008). IEF was carried out using a Multiphor II DryStrip kit system(Amersham Biosciences Europe GmbH, Freiburg, Germany) with18 cm IPG 3–10 strips (Amersham Biosciences) at 20 �C. In total,0.1 ml of protein extract was applied by anodic cup loading.SDS–PAGE was performed on a vertical system (11% m/v acrylam-ide) at 15 �C. The proteins were visualised by colloidal Coomassiestaining (Roti-Blue, Carl Roth GMBH & Co., Karlsruhe, Germany).

2.2.8. Protein surface hydrophobicityFront-face fluorescence measurements were carried out at room

temperature in a LS-50 B Perkin-Elmer instrument. A dispersion of un-treated and TGase-treated BR flour (7 mg/ml, corresponding to 0.5 mgprotein/ml) was placed in a quartz cuvette and stirred throughout theexperiment. Emission spectra of 1,8-anilinonaphthalene sulphonate(ANS) were taken, from 400 to 600 nm, with excitation at 390 nm.Emission and excitation bandwidths were set at 5 nm. Titration withANS was performed by adding increasing appropriate volumes of con-centrated ANS (from stock 1, 10 and 20 mM solutions) to each sample.Results were analysed by standard binding algorithms, that allowed usto estimate the overall binding capacity of the protein samples for theprobe (given as fluorescence at saturating ANS, Fmax) and the apparentdissociation constant of the protein-ANS complex (Kd). The overallbinding capacity was then corrected for the total protein content ofeach sample. The same titration experiments were performed with dis-persions (0.5 mg/ml) of untreated and TGase-treated glutelins after a60 min incubation.

3. Results and discussion

3.1. Electrophoretic pattern of untreated and TGase-treated batters

In a previous study, we reported the formation of large proteinaggregates in BR batters after treatment with TGase (10 U/g of pro-tein) at 30 �C for 30 and 180 min. The aggregates were visualisedby confocal laser scanning microscopy (Renzetti, Dal Bello et al.,2008), but changes in the protein profile of the TGase-treated bat-ters were not investigated.

The protein profile of the untreated brown rice flour after 0, 30and 180 min of incubation were identical (data not shown), thusindicating that no proteolytic activities could be detected underthe conditions of this study. The electrophoretic pattern of the flour(Fig. 1A) was characterised by polypeptides at 14, 17, 22–25, 34–40,53, 60, 85, 95 and 103 kDa. Most of rice endosperm proteins belongto the glutelin fraction, which is characterised by a and b subunits atca. 32 and 25 kDa, respectively, linked by an intermolecular disul-phide bond (Van Den Borght, Vandeputte, Derycke, Brijs, Daenen,& Delcour, 2006) and which characterise the electrophoretic patternof the flour. This dimeric unit further aggregates through additionaldisulphide bonds and hydrophobic interactions to form large macro-molecular complexes (Sugimoto, Tanaka, & Kazai, 1986; Utsumi,1992; Van Den Borght et al., 2006). These protein complexes cannotbe entirely reduced due to the presence of inaccessible disulphdebonds or other binding forces (Van Den Borght et al., 2006), andprobably account for the dimeric unit (ca. 60 kDa) and the proteincomplexes larger than 80 kDa which can be observed in the elec-tropherograph. The LMW subunits, around 14 kDa, are most proba-bly due to albumins (Iwasaki, Shibuya, Suzuki, & Chikubu, 1982; Pan& Reeck, 1988; Ukada, Koga, Tsuji, Kimoto, & Takumi, 2000) and

prolamins (Bietz, 1982; Kumagai, Kawamura, Fuse, Watanabe, Saito,& Masumura, 2006; Ogawa, Kumamuru, Satoh, Iwata, Omura, &Kasai, 1987; Ukada et al., 2000), whereas globulins account for thesubunits at 17 and 22–25 kDa (Iwasaki et al., 1982; Kumagai et al.,2006; Morita and Yoshida, 1968; Pan & Reeck, 1988).

After 30 and 180 min of incubation, at 30 �C, of the TGase-trea-ted flour, a similar decrease in the relative concentration of all sub-units was found, which suggests polymerisation of these subunitsby TGase action already after short incubation periods (Fig. 1A). Nonew polypeptides were detected in the TGase-treated flour, whichmight suggest that the products of the polymerisation reaction arelarger than 230 kDa. However, polymerised proteins are difficult tosolubilise, and others have reported an increase in the amount ofunextractable proteins after TGase treatment of cereal flour(Marco, Pérez, León, & Rosell, 2008).

3.2. SE-HPLC of untreated and TGase-treated batters

SE-HPLC separation of proteins extracted from the untreatedand TGase-treated BR batters under reducing conditions, was per-formed, in order to detect macromolecular protein complexes. Se-ven peaks (P1 to P7) were found in extracts from the sampleincubated in the absence of the enzyme (Fig. 1B). They were sepa-rated into two distinct regions of high and low molecular weightproteins (HMW and LMW, respectively) corresponding to P1(>66 kDa) and P2–5 (66–14 kDa) and P6–7 (<14 kDa).

P1 accounts for the macromolecular complexes formed by the aand b glutelin subunits which cannot be entirely reduced and wereeluted with the column void volume. Electophoretic analysis of thecollected peaks (data not shown) confirmed that LMW proteins inthe 66–14 kDa range include glutelin dimeric units, along with aand b glutelin subunits (P2 and P3), together with albumins (14–15 kDa), globulins (17–22 kDa), and prolamins (14 kDa) (P3 andP4). The protein profile in the absence of the enzyme did notchange after 180 min of incubation at 30 �C (data not shown).

Increasing incubation times in the presence of TGase resulted in atime-progressive decrease in the area of the large aggregates in P1(Fig. 1B). The enzymatic treatment apparently promoted the poly-merisation of the large macromolecules in P1, yielding aggregateswhich could not be solubilised. The participation of the LMW pro-teins in the polymerisation reaction may explain the decreased areaof peaks P2–7, as observed by MCE. However, it is also possible thatthe polymerisation reaction involving glutelins in P1 results in amore pronounced entrapment of LMW proteins in the aggregatednetwork of proteins in rice flour (Van Den Borght et al., 2006).

3.3. Protein fractionation

In order to gain insights of the contributions of individual frac-tions to the TGase-induced polymerisation reaction, protein frac-tions were separated. The flour contained 7.8% (m/m) protein,which is well within the range reported for commercial brown rice(Heinemann, Fagundes, Pinto, Penteado, & Lanfer-Marquez, 2005).About 87% (m/m) of the total nitrogen was recovered in the proteinfractions from the flour. Glutelins constituted the major proteinfraction, accounting for 86.6% (m/m) of the total protein extract,followed by globulins (10.8% m/m) and albumins (2.6% m/m). Onlysmall traces of prolamins were found. These figures are in therange of those previously reported for rice and brown rice flour(Cagampang, Cruz, Espiritu, Santiago, & Juliano, 1966; Juliano,1985; Mitra & Das, 1975; Ukada et al., 2000).

3.4. Amino acid profile of protein fractions

The cross-linking reaction catalysed by TGase is dependent onthe availability and accessibility of glutamine and lysine residues.

Fig. 1. (A) Electrophoretic pattern of protein extract from brown rice flour after TGase treatment: ( ) brown rice flour incubated for 30 min at 30 �C with no addition ofTGase ( ) brown rice flour treated with 10 U TGase for 30 min at 30 �C ( ) brown rice flour treated with 10 U TGase for 180 min at 30 �C. (B) SE-HPLC profile of proteinsextracted under denaturing and reducing conditions from control and TGase-treated BR batters: ( ) 30 min at 30 �C, no TGase; ( ) 30 min at 30 �C with TGase; ( )180 min at 30 �C with TGase (for interpretation of the references to color in this figure legend, the reader is referred to the web version of this article).

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The low lysine content is the limiting factor for TGase treatment ofcereal flours (Renzetti, Dal Bello et al., 2008). Therefore, the aminoacid composition of individual protein fractions was assessed as apossible indication of their reactivity. Albumins contained thehighest amount of lysine (45 mg/g), confirming previous reports(Iwasaki et al., 1982). Lysine content was high also in glutelins(36 mg/g), as previously reported (Tecson, Esmana, Lontok, & Juli-ano, 1971; Ukada et al., 2000; Wen & Luthe, 1985), and was con-firmed to be lowest in globulins (21 mg/g) (Iwasaki et al., 1982).

By considering only the availability of glutamine and lysine, itcan be suggested that the albumin and glutelin fractions shouldact as the best substrates for TGase activity.

3.5. Electrophoretic pattern of TGase-treated and untreated proteinfractions

The electrophoretic pattern of untreated and TGase-treated pro-tein fractions from BR flour under reducing conditions, is shown inFig. 2. At the level of addition used, TGase was not detected in thesystem (data not shown). The albumin fraction (Fig. 2A) was

mainly characterised by polypeptides around 15 kDa, as previouslyreported (Iwasaki et al., 1982; Pan & Reeck, 1988). Some polypep-tides, at 25 and 36 kDa, were also present. A similar protein profilewas observed after TGase treatment, thus indicating that the albu-min fraction is not a good substrate for the enzyme (Fig. 2A), de-spite its high lysine content.

The globulin fraction (Fig. 2B) was characterised by subunitsranging from 13 to 100 kDa, with major subunits at 14–19 and25 kDa. This is consistent with previous reports indicating highheterogeneity of the globulin fraction (Iwasaki et al., 1982) witha and b- globulins (ca. 25 and 15 kDa, respectively) being the mostprominent components (Cagampang, Perdon, & Juliano, 1976;Morita & Yoshida, 1968). The TGase-treated fraction (Fig. 2B)showed a similar profile, with the exception of a slight decreasein the relative concentration of the a-globulin subunit, that proba-bly slightly polymerised under the enzymatic action.

The glutelin fraction (Fig. 2C) was mainly characterised by the aand b- glutelin subunits at 22 and 34–40 kDa, respectively, and bya 14 kDa subunit which has been previously reported as a principalprolamin contaminant of glutelin extract from rice (Krishnan &

Fig. 2. Electrophoretic pattern of TGase-treated and untreated protein fractions from brown rice: (A) albumins (B) globulins (C) glutelins. ( ) untreated fraction; ( ) TGase-treated fraction. TGase treatment was performed with 10 U of enzyme per gramme of fraction incubating at 50 �C for 60 min (for interpretation of the references to color inthis figure legend, the reader is referred to the web version of this article).

1080 S. Renzetti et al. / Food Chemistry 131 (2012) 1076–1085

S. Renzetti et al. / Food Chemistry 131 (2012) 1076–1085 1081

Okita, 1986). However, glutelin subunits at ca. 15 kDa have beenpreviously reported (Padhye & Salunkhe, 1979). All glutelin sub-units showed a decrease in relative concentration (Fig. 2C), thusindicating that these proteins were good substrates for the cross-linking reaction, in agreement with their high lysine content.

The results of the electrophoretic investigation of BR fractionsseem to support the hypothesis of albumins and globulins beingentrapped in the polymerised glutelin complexes. This hypothesiswould explain the considerable TGase-induced decrease in the rel-ative concentration of the albumin and globulin subunits from theflour extracts (as observed by SE-HPLC and electrophoresis analysisof flour extracts and collected peaks), despite the low reactivity ob-served in the isolated fractions.

We have recently shown that TGase is not fraction specific to-wards buckwheat proteins, resulting in HMW aggregates with boththe albumin and globulin fractions (Renzetti, Behr et al., 2008). Onthe contrary, TGase specificity towards BR glutelins suggests thatdifferences in the accessibilities of the lysine and glutamine sub-strate residues determine the peculiar reactivity of BR proteins.

3.6. Two dimensional gel electrophoresis

In order to further investigate the specificity of TGase, 2D gelelectrophoresis (Fig. 3) was performed on untreated and

Fig. 3. 2D gel of globulins (A), TGase-treated globulins (B), glutelins (C) and TGase-treaterange. Isoelectric points are given at the top of each gel. Boxed spots on globulin 2D gelsubunit, 22 kDa and pI 5.47. Arrows on TGase treated globulin gel indicate new protein spthe glutelin gel (C) indicate a and b subunits (ca. 30 and 19 kDa, respectively) and Wx p

TGase-treated globulin and glutelin fractions, which together ac-counted for over 97% (m/m) of the proteins in the total extract.2D gel electrophoresis of BR globulin revealed size and IEF hetero-geneity of the fraction, although most proteins separated in the25–15 kDa range (Fig. 3A). In agreement with previous findings(Padhye & Salunkhe, 1979), most proteins separated in the 5.5–7.6 pI range, although basic subunits at pI P 9 were also detected.The globulin fraction was characterised by a subunit of 22 kDa andpI 5.85. Padhye and Salunkhe (1979) previously reported a broadand pronounced protein band focusing at pI 5.85, that probablycorresponds to the a-globulin identified by Houston and Moham-mad (1970). A less intense spot at 22 kDa and pI 5.47 could corre-spond to the minor a-globulin subunit reported by Pan and Reeck(1988).

The 2D gel of TGase-treated globulin was similar to that of theuntreated fraction, although three new protein spots were de-tected at 23, 22 and 18 kDa with pI values of 5.4, 6.68 and 5.62,respectively (Fig. 3B). The features of these ‘‘novel’’ LMW proteinsmay account for the slight reactivity of this fraction towards TGase,as detected by MCE.

The pattern observed for the untreated glutelin fraction wasdominated by proteins around 30 kDa, which separated in the pIrange 6–7.7, and by proteins around 19 kDa at pI 9.84 (Fig. 3C).This is similar to previous results reporting the glutelin fraction

d glutelins (D) from brown rice flour. Proteins were separated over the 15–250 kDa(A) indicate: (1) major a globulin subunit, 22 kDa and pI 5.85; (2) minor a globulinots of 23, 22 and 18 kDa with pI values of 5.4, 6.68 and 5.62, respectively. Arrows onroteins (56 kDa and pI 6.2–6.4).

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as composed of acidic (a) polypeptides with pI 6.5–7.5 (Wen &Luthe, 1985) and basic (b) polypeptides with pI > 9 (Juliano & Boul-ter, 1976; Wen & Luthe, 1985). The acidic subunits comprisedmany size and charge variants. These subunits show high homol-ogy with each other and can be classified into several cultivar-spe-cific types (Abe, Gusti, Ono, & Sasahara, 1996).

A group of proteins at 56 kDa and pI 6.2–6.4 were also presentin the glutelin fraction and may be related to the waxy (Wx) pro-tein (granule-bound starch synthase) identified by Sano (1984).The a and b subunits were highly reactive towards TGase, and their

Fig. 4. SE-HPLC profiles of protein fractions from brown rice incubated for 30 minat 30 �C in the absence ( ) or in the presence ( ) of TGase . Panel A, albumins;panel B, globulins; panel C, glutelins. Albumins and globulins were separated on aSuperdex 200 column, whereas a Superose 6 column was used for glutelins (forinterpretation of the references to color in this figure legend, the reader is referredto the web version of this article).

area decreased drastically upon TGase treatment (Fig. 3D). The Wx

protein, as well as minor protein spots in the 60–90 kDa range, alsoshowed reactivity towards TGase, that made them disappear. Thepresence of three new protein spots around 17–19 kDa and pI5.66–7.14 may be accounted for by the polymerisation of LMWproteins, as discussed above.

3.7. Size exclusion chromatography of TGase-treated and untreatedprotein fractions

SE-HPLC was performed on each of the protein fractions iso-lated after TGase treatment, in order to detect newly formedcross-linked protein polymers, which could not be detected bythe electrophoretic methods. Both the albumin and globulin frac-tions were mainly constituted of LMW proteins, smaller than66 kDa (Fig. 4A and B).

Albumins separated in two peaks eluting at 33 and 47 min,respectively. Globulins also separated in two peaks eluting after21 and 31 min, respectively. After TGase treatment, only slight dif-ferences could be observed in the profiles of both albumin andglobulin fractions, confirming that the soluble fraction could notbe directly responsible for the formation of cross-linked macromo-lecular complexes in the flour extract. A new peak appeared in thealbumin fraction eluting after 38 min, corresponding to LMW pro-tein (mass < 6.5 kDa).

The glutelin fraction was characterised by four peaks (P1–4),eluting at 16, 26, 36 and 41 min (Fig. 4C), corresponding to HMW(>66 kDa) proteins (P1 and P2) and LMW (66–14 kDa range) pro-teins (P3 and P4). P1 eluted with the column void volume, indicat-ing the presence of macromolecular complexes larger than thecolumn cut-off. After TGase treatment, the peaks correspondingto LMW proteins (P3 and P4) had decreased areas, whereas P1and P2 had increased peak area (Fig. 4C). Most probably, the pro-tein subunits in P3 and P4 were cross-linked to yield new proteinaggregates, that eluted with the column void volume. This wasconfirmed by monitoring the time course of changes in the glutelinprofile during TGase treatment by SE-HPLC (Fig. 5). At incubationtimes up to 30 min, decreases in the areas of P3 and P4 wereaccompanied by an increase in the area of P1. The area of P1 thendecreased slightly, without affecting the areas of P3 and P4, sug-gesting that, at times longer than 30 min, some of the polymersin P1 may have formed aggregates too large to be eluted by the col-umn and/or solubilised. The area of P2 did not change after an ini-tial increase at 5 min of incubation, which might be indicative ofthe formation of reactive polymers from which larger aggregatesare formed.

Overall, these SE-HPLC studies showed evidence that novel mac-romolecular protein complexes are obtained by TGase treatment ofBR glutelins. By also taking into consideration the information gath-ered from MCE and 2D gel electrophoresis, it can be suggested thatglutelins are directly responsible for the formation of supramacro-molecular aggregates in TGase-treated BR flour. Instead, LMW pro-teins, i.e. albumins and globulins, do not appear to be directlyinvolved in the polymerisation reaction, but may end up entrappedin the glutelin-based macromolecular complexes.

3.8. Dynamic light scattering of untreated and TGase-treated glutelins

Dynamic light scattering was used to characterise the HMWprotein polymers resulting from the cross-linking reaction. Fig. 6shows the hydrodynamic radius of proteins present in P1, beforeand after 60 min of TGase treatment. The untreated glutelin frac-tion showed the co-existence of a major component with hydrody-namic radius between 10 and 100 nm, and traces of a much largercomponent with hydrodynamic radius between 1000 and10,000 nm. After 60 min, TGase treatment induced a substantial

S. Renzetti et al. / Food Chemistry 131 (2012) 1076–1085 1083

decrease in the area of the smaller component of 10–100 nmhydrodynamic radius, concomitantly with the appearance of alarge amount of a structure with a hydrodynamic radius around1000 nm. The persistence of these structures even in the dissociat-ing/reducing solvent used in SE-HPLC studies, confirms the solidityof the interactions within these networks.

By comparing the results of the time course changes in the glut-elin profile during TGase treatment and those of the dynamic lightscattering experiments, it is evident that the polymerisation mech-anism does not involve sequential addition of individual polypep-tides to a preformed chain/network. Rather, sizable aggregatesalready present in the system are stabilized by cross-linking inthe early reaction phases. These large polymers act as the enzymesubstrate in a later phase of the reaction, eventually leading to theformation of insoluble materials.

3.9. Protein surface hydrophobicity and solvation studies

ANS titration of BR flour, at 30 and 180 min of TGase treatment,showed a significant decrease in surface hydrophobicity (Fmax/

Fig. 5. Time course of TGase-dependent changes in the SE-HPLC profile of glutelins duri30 min; ( ) 60 min (for interpretation of the references to color in this figure legend,

Fig. 6. Hydrodynamic radius distribution of glutelin in fractio

protein), that went down to 50.6 and 48.1, respectively, from a va-lue of 56.5 in the untreated sample. The apparent dissociation con-stant of the protein/probe complex (Kd) also decreased withincreasing incubation time from 6.2 in the control to 5.7 and 4.7after 30 and 180 min of exposure to TGase treatment, indicatingan increased affinity of the residual sites on the protein for theprobe. These results indicate that the cross-linking reaction pro-moted closer hydrophobic interactions among proteins, and mayfurther support the hypothesis of LMW protein entrapment inthe polymerised glutelin complexes. The increased average sizeof the macromolecular structures and the increased compactnessof their hydrophobic regions involved in protein interactions maywell explain the increased elastic-like behaviour of BR batter uponTGase treatment and the consequent improvement in breadmak-ing performance (Renzetti, Dal Bello et al., 2008).

On the other hand, no changes in the number of hydrophobicsites available to the probe (not shown) were observed in TGase-treated glutelins (60 min incubation), although Kd decreased from5.2 to 4.8 in the treated sample. Apparently, the cross-linking ofglutelins did not promote the organisation of surface hydrophobic

ng TGase treatment: ( ) 0 min; ( ) 5 min; ( ) 10 min; ( ) 15 min; ( )the reader is referred to the web version of this article).

n P1 after 0 min (A) and 60 min (B) of TGase treatment.

1084 S. Renzetti et al. / Food Chemistry 131 (2012) 1076–1085

patches into new regions capable of binding the probe, but proteinaggregation may have compacted existing hydrophobic sites onadjacent proteins, increasing their average affinity towards theprobe.

4. Conclusions

The results of this study suggest that the large protein com-plexes that are present in BR flour aggregate into highly polymer-ised structures under TGase treatment. Our results on isolatedprotein fractions indicate that TGase induces cross-linkage of glu-telins to form large aggregates. Instead, LMW proteins, namelyalbumins and globulins, do not extensively polymerise but mightbe indirectly involved in the polymerisation observed in the flourextract in which they become entrapped in the aggregated struc-tures. Evidence for the large protein complexes formed by glutelinsbeing the substrate of TGase action was corroborated by dynamiclight scattering studies, that do not offer evidence for chain-typeelongation of the polymers, but rather indicate that polymerisationinvolves large and apparently pre-formed complexes.

The a and b glutelin subunits appear to be the primary sub-strates for the polymerisation reaction, as indicated by SE-HPLC,MCE and 2D gel electrophoresis. The different reactivities of glute-lins and albumins cannot be explained on the basis of their lysinecontents, as albumins do not aggregate, despite having a muchhigher lysine content than glutelins. Therefore, the specificity ofTGase for BR glutelins may be related to higher accessibility (as op-posed to content) of their glutamine and lysine residues in compar-ison to the albumin and globulin fractions.

Surface hydrophobicity studies revealed that the polymerisa-tion reaction promoted hydrophobic interactions among proteinsin BR batters, increasing the number of surface hydrophobic sitesand their affinity towards the fluorescent hydrophobic probe usedin these studies. This may result from entrapment of albumins andglobulins in the polymerised glutelin matrix rather than fromstructural modification of the glutelins themselves, since TGasetreatment did not increase the number of surface hydrophobicsites on isolated glutelins.

From a more practical point of view, the supramolecular struc-tures resulting from TGase polymerisation of the glutelin com-plexes, and concomitantly from new and stronger hydrophobicinteractions among proteins, may well explain (in molecularterms) the improved textural properties of TGase-treated BR bread(Renzetti, Dal Bello et al., 2008).

Acknowledgements

This study was financially supported by the European Commis-sion (6th Framework Programme, Project HEALTHGRAIN (FP6-514008)). This publication reflects only author’s views and theCommunity is not liable for any use that may be made of the infor-mation contained in this publication. The authors acknowledgethat this research was also partly funded by the Food InstitutionalResearch Measure (National Development Plan 2007-2013). Theauthors would like to thank Dr. Patrizia Rasmussen (DISMA, Milan)for her support with front-face fluorescence experiments.

References

AACC International, 2000. Approved methods of the American Association of CerealChemists, 10th Ed. Methods 44-15A and 46-30. The Association: St. Pauli, MN.

Abe, T., Gusti, R. S., Ono, M., & Sasahara, T. (1996). Variations in glutelin and highmolecular weight endosperm proteins among subspecies of rice (Oryza sativa L.)detected by two-dimensional gel electrophoresis. Genes and Genetic Systems, 71,63–68.

Bietz, J. A. (1982). Cereal prolamin evolution and homology revealed by sequenceanalysis. Biochemical Genetetics, 20, 1039–1053.

Cagampang, G. B., Cruz, L. J., Espiritu, S. G., Santiago, R. G., & Juliano, B. O. (1966).Studies on the extraction and composition of rice proteins. Cereal Chemistry, 43,145–155.

Cagampang, G. B., Perdon, A. A., & Juliano, B. O. (1976). Changes in salt solubleproteins of rice during grain development. Phytochemistry, 15, 1425–1429.

Catassi, C., & Fasano, A. (2008). Celiac disease. In E. Arendt & F. Dal Bello (Eds.),Gluten-free cereal products and beverages. USA: Elsevier.

Clerici, M. T. P. S., Airoldi, C., & El-Dash, A. A. (2009). Production of acidic extrudedrice flour and its influence on the qualities of gluten-free bread. LWT – FoodScience and Technology, 42, 618–623.

Dolnik, V., & Liu, S. R. (2005). Applications of capillary electrophoresis on microchip.Journal of Separation Science, 28, 1994–2009.

Folk, J. E., & Finlayson, J. S. (1977). The e-(c-Glutamyl)lysine cross-link and thecatalytic role of transglutaminase. Advances in Protein Chemistry, 31, 1–133.

Gujral, H. S., & Rosell, C. M. (2004). Functionality of rice flour modified with amicrobial transglutaminase. Journal of Cereal Science, 39, 225–230.

Heinemann, R. J. B., Fagundes, P. L., Pinto, E. A., Penteado, M. V. C., & Lanfer-Marquez,U. M. (2005). Comparative study of nutrient composition of commercial brown,parboiled and milled rice from Brazil. Journal of Food Composition and Analysis,18, 287–296.

Houston, D. F., & Mohammad, A. (1970). Purification and partial characterization ofa major globulin from rice endosperm. Cereal Chemistry, 65, 5–12.

Hüttner, E. K., Dal Bello, F., & Arendt, E. K. (2010). Rheological properties and breadmaking performance of commercial wholegrain oat flours. Journal of CerealScience, 52, 65–71.

Iwasaki, T., Shibuya, N., Suzuki, T., & Chikubu, S. (1982). Gel filtration andelectrophoresis of soluble rice proteins extracted from long, medium andshort grain varieties. Cereal Chemistry, 59, 192–195.

Juliano, B. O. (1985). Polysaccharides, proteins and lipids of rice. In B. O. Juliano(Ed.), Rice Chemistry and Technology (second ed., pp. 59–174). MN, USA: AACC.

Juliano, B. O., & Boulter, D. (1976). Extraction and composition of rice endospermglutelin. Phytochemistry, 15, 1601–1606.

Korus, J., Witczak, M., Ziobro, R., & Juszczak, L. (2009). The impact of resistant starchon characteristics of gluten-free dough and bread. Food Hydrocolloids, 23,988–995.

Krishnan, H. B., & Okita, T. W. (1986). Structural relationships among the riceglutelin polypeptides. Plant Physiology, 81, 748–753.

Kumagai, T., Kawamura, H., Fuse, T., Watanabe, T., Saito, Y., & Masumura, T. (2006).Production of rice protein by alkaline extraction improves its digestibility.Journal of Nutritional Science and Vitaminology, 52, 467–472.

Landry, J., & Moureauc, T. (1980). Distribution and amino acid composition ofprotein groups located in different histological parts of maize grain. Journal ofAgricultural and Food Chemistry, 28, 1186–1191.

Marco, C., Pérez, G., León, A. E., & Rosell, C. M. (2008). Effect of transglutaminase onprotein electrophoretic pattern of rice, soybean, and rice-soybean blends. CerealChemistry, 85, 59–64.

Marco, C., & Rosell, C. M. (2008). Functional and rheological properties of proteinenriched gluten free composite flours. Journal of Food Engineering, 88, 94–103.

Mitra, G. N., & Das, B. (1975). Nutritive value of some rice varieties grown in Orissa.I. Protein content and composition of protein. Journal of Research OrissaUniversity of Agriculture and Technology, 5, 51–57.

Moore, M. M., Heinbockel, M., Dockery, P., Ulmer, H. M., & Arendt, E. K. (2006).Network formation in gluten-free bread with application of transglutaminase.Cereal Chemistry, 83, 28–36.

Moore, S., & Stein, W. H. (1963). Chromatographic determination of amino acids bythe use of automatic recording equipment. Methods in Enzymology, 6, 819–831.

Morita, Y., & Yoshida, C. (1968). Studies on gamma globulin of rice embryo Part I.Isolation and purification of gamma globulin from rice embryo. Journal ofBiological Chemistry, 32, 664–670.

Ogawa, M., Kumamuru, T., Satoh, H., Iwata, N., Omura, T., & Kasai, Z. (1987).Purification of protein body-I of rice seed and its polypeptide composition. Plantand Cell Physiology, 28, 1517–1527.

Padhye, V. W., & Salunkhe, D. K. (1979). Extraction and characterization of riceproteins. Cereal Chemistry, 56, 389–393.

Pan, S. J., & Reeck, G. R. (1988). Isolation and characterization of rice a-globulin.Cereal Chemistry, 65, 316–319.

Renzetti, S., Behr, J., Vogel, R., & Arendt, E. K. (2008). Transglutaminasepolymerisation of buckwheat (Fagopyrum esculentum Moench) proteins.Journal of Cereal Science, 48, 747–754.

Renzetti, S., Dal Bello, F., & Arendt, E. K. (2008). Microstructure, fundamental rheologyand baking characteristics of batters and breads from different gluten-free flourstreated with a microbial transglutaminase. Journal of Cereal Science, 48, 33–45.

Sano, Y. (1984). Differential regulation of waxy gene expression in rice endosperm.Theoretical and Applied Genetics, 68, 467–473.

Schober, T. J., Bean, S. R., Boyle, D. L., & Park, S. H. (2008). Improved viscoelasticzein–starch doughs for leavened gluten-free breads: Their rheology andmicrostructure. Journal of Cereal Science, 48, 755–767.

Sugimoto, T., Tanaka, K., & Kazai, Z. (1986). Molecular species in the protein body II(PB-II) of developing rice endosperm. Agricultural and Biological Chemistry, 50,3031–3035.

Tecson, E. M. S., Esmana, B. V., Lontok, L. P., & Juliano, B. O. (1971). Studies on theextraction and composition of rice endosperm glutelin and prolamin. CerealChemistry, 48, 168–181.

Ukada, J., Koga, T., Tsuji, H., Kimoto, M., & Takumi, K. (2000). Efficient extraction andsome properties of storage proteins (prolamin and glutelin) in acient ricecultivars. Journal of Nutritional Science and Vitaminology, 46, 84–90.

S. Renzetti et al. / Food Chemistry 131 (2012) 1076–1085 1085

Utsumi, S. (1992). Plant food protein engineering. Advances in Food NutritionResearch, 36, 89–208.

Van Den Borght, A., Vandeputte, G. E., Derycke, V., Brijs, K., Daenen, G., & Delcour, J.A. (2006). Extractability and chromatographic separation of rice endospermproteins. Journal of Cereal Science, 44, 68–74.

Van Riemsdijk, L. E., Van Der Goot, A. J., Hamer, R. B., & Boom, R. M. (2011).Preparation of gluten-free bread using a meso-structured whey protein particlesystem. Journal of Cereal Science, 53, 355–361.

Wang, W., Taylor, J., Jemere, A. B., & Harrison, D. J. (2010). Microfluidic devices forelectrokinetic sample fractionation. Electrophoresis, 31, 2575–2583.

Wen, T. N., & Luthe, D. S. (1985). Biochemical characterization of rice glutelin. PlantPhysiology, 78, 172–177.

Westermeier, R., & Schickle, H. (2009). The current state of the art in high-resolutiontwo-dimensional electrophoresis. Archives of Physiology and Biochemistry, 115,279–285.