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0733-5210/$ - se

doi:10.1016/j.jc

Abbreviations

CLSM, confoca

basis; HPMC, h

teff; TGase, tra�Correspond

University Coll

Tel.: +353 21 4

E-mail addr

Journal of Cereal Science 48 (2008) 33–45

www.elsevier.com/locate/jcs

Microstructure, fundamental rheology and baking characteristics ofbatters and breads from different gluten-free flours treated with a

microbial transglutaminase

Stefano Renzettia,b, Fabio Dal Belloa,b, Elke K. Arendta,�

aDepartment of Food Science, Food Technology and Nutrition, National University of Ireland, Cork, IrelandbBiotransfer Unit, University College Cork, Cork, Ireland

Received 28 March 2007; received in revised form 9 July 2007; accepted 16 July 2007

Abstract

Gluten is a fundamental component for the overall quality and structure of breads. The replacement of the gluten network in the

development of gluten-free cereal products is a challenging task for the cereal technologist. The functionality of proteins from gluten-free

flours could be modified in order to improve their baking characteristics by promoting protein networks. Transglutaminase (TGase) has

been successfully used in food systems to promote protein cross-linking. In this study, TGase was investigated for network forming

potential on flours from six different gluten-free cereals (brown rice, buckwheat, corn, oat, sorghum and teff) used in breadmaking.

TGase was added at 0, 1 or 10U/g of proteins present in the recipe. The effect of TGase on batters and breads was evaluated by

fundamental rheological tests, Texture Profile Analysis and standard baking tests. Three-dimensional elaborations of Confocal Laser

Scanning Microscopy (CLSM) images were performed on both batters and breads to evaluate the influence of TGase on microstructure.

Fundamental rheological tests showed a significant increase in the pseudoplastic behaviour of buckwheat and brown rice batters when

10U of TGase were used. The resulting buckwheat and brown rice breads showed improved baking characteristics as well as overall

macroscopic appearance. Three-dimensional CLSM image elaborations confirmed the formation of protein complexes by TGase action.

On the other side, TGase showed negative effects on corn flour as its application was detrimental for the elastic properties of the batters.

Nevertheless, the resulting breads showed significant improvements in terms of increased specific volume and decreased crumb hardness

and chewiness. Under the conditions of this study, no effects of TGase could be observed on breads from oat, sorghum or teff. Overall,

the results of this study show that TGase can be successfully applied to gluten-free flours to improve their breadmaking potentials by

promoting network formation. However, the protein source is a key element determining the impact of the enzyme.

r 2007 Elsevier Ltd. All rights reserved.

Keywords: Transglutaminase; Gluten-free; Microstructure; Rheology; Bread

1. Introduction

Celiac disease (CD) is an immune-mediated enteropathytriggered by the ingestion of gluten in genetically suscep-tible persons (Catassi et al., 2007). Despite the advances

e front matter r 2007 Elsevier Ltd. All rights reserved.

s.2007.07.011

: BR, brown rice; BW, buckwheat; CD, celiac disease;

l laser scanning microscopy; CR, corn; Fwb, flour weight

ydroxypropylmethylcellulose; OT, oat; SG, sorghum; TF,

nsglutaminase; TPA, texture profile analysis

ing author. Department of Food and Nutritional Sciences,

ege Cork, Western Road, Cork, Ireland.

902064; fax: +353 214270213.

ess: e.arendt@ucc.ie (E.K. Arendt).

which have been made in the understanding of CDpathogenesis and the potential development of noveltherapies, at present the only safe and effective treatmentfor CD sufferers is the avoidance of gluten-containingfoods such as wheat, rye, barley (Ciclitira et al., 2005) aswell as durum wheat, spelt wheat, kamut, einkorn andtriticale (Kasarda, 2001). Since wheat is one of the maincomponents of a daily diet worldwide, as well as the basicingredient of most baked goods, a gluten-free diet iscomplex and can easily overwhelm patients (Kupper,2005). One of the major issues for CD sufferers tocompletely adhere to a gluten-free diet is finding goodquality gluten-free foods (Case, 2005). Despite the fact that

ARTICLE IN PRESSS. Renzetti et al. / Journal of Cereal Science 48 (2008) 33–4534

several gluten-free products are nowadays available on themarket, baked products from gluten-free ingredients aregenerally of poor quality due to the lack of the glutennetwork (Arendt et al., 2002). In wheat the gliadins(prolamins) are responsible for dough’s cohesiveness, whilethe glutenins (glutelins) are apparently responsible forthe dough’s resistance to extension (Hoseney, 1994). Thecombination of these two proteins, which results inthe gluten complex, confers the dough unique viscoelasticproperties and the ability to retain gasses, resulting in goodquality breads. Such properties are not found in proteinsfrom gluten-free flours. To date, the main approach for thedevelopment of leavened products from gluten-free flours,particularly breads, has been the addition of polymericsubstances such as xanthan gum and hydroxypropyl-methylcellulose (HPMC) in order to mimic the propertiesof gluten (Ahlborn et al., 2005; Gallagher et al., 2003;Moore et al., 2004; Schober et al., 2004).

The functionality of proteins from gluten-free flourscould be modified, e.g. by enzyme action, in order topromote protein networks and improve their bakingcharacteristics. Among the enzymes used in the foodindustry, Transglutaminase (TGase) has been successfullyapplied in several food systems (Kuraishi et al., 2001) forits unique ability of modifying protein functionality andpromote protein cross-linking (Babiker, 2000; Babin andDickinson, 2001; Basman et al., 2002a; Il Jun Kang et al.,1994; Motoki et al., 1984; Nai-Chi Siu et al., 2002a, b).

TGase is a protein-glutamine g-glutamyl-transferase (EC2.3.2.13), which catalyses an acyl-transfer reaction betweenthe g-carboxyamide group of peptide-bound glutamineresidues and a variety of primary amines (Motoki andSeguro, 1998). When the e-amino group of a peptide boundlysine residue acts as substrate, the two peptide chains arecovalently linked through an e-(g-glutamyl)lysine bond(Folk and Finlayson, 1977). Thus, the enzyme is capable ofintroducing covalent cross-links between proteins (Nonakaet al., 1989). In the absence of primary amines in thereaction system, water becomes the acyl-acceptor and theg-carboxy-amide groups of glutamine residues are deami-dated, becoming glutamic acid residues (Ando et al., 1989).With regards to cereal proteins, TGase application hasshown positive effects on wheat-based baked goods:increased volume and improved structure of breads(Wijngaards et al., 1997), improved strength of breadcrumb (Gerrard et al., 1998), height increase in puff pastryand greater croissants volume (Gerrard et al., 2001),improvement of dough stability (Gottmann and Sproessler,1992), and improvement of baking quality of weak wheatflours (Basman et al., 2002b).

Recently, the application of TGase in gluten-free systemsmodified the viscoelastic properties of the batters, improv-ing the quality of the resulting gluten-free breads bypromoting a protein network (Gujral and Rosell, 2004;Moore et al., 2006). Nevertheless, in these studies xanthangum and HPMC were added in order to compensate for theabsence of the gluten network. Moore et al. (2006)

investigated the application of TGase on a complex andoptimised gluten-free recipe. Therefore, little informationcould be extrapolated on the impact of TGase on eachindividual protein source present in the recipe. Gujral andRosell (2004) evaluated the partial replacement of HPMCwith TGase in a white rice formulation. However, noinvestigations were conducted on the impact of the enzymeon the microstructure of the batters and breads.The aim of the present study was to investigate the

effectiveness of TGase application in improving the bakingperformances of gluten-free flours without addition of anyhydrocolloids, and thus get a better insight on the extent ofcereal proteins modifications and network forming promo-tion for breadmaking. Several cereal flours were investi-gated in order to assess the impact of TGase on differentprotein sources. Additionally, Confocal Laser ScanningMicroscopy (CLSM) was extensively applied to unravel themodifications caused by TGase on the microstructure ofbatters and breads.

2. Materials and methods

2.1. Materials

Buckwheat flour (Doves Farm Foods Ltd., Berkshire,UK), brown rice flour (Doves Farm Foods Ltd., Berkshire,UK), corn flour (Smiths Flour Mills, England), oat flour(Flavahans, Co. Waterford, Ireland), sorghum flour(USDA-ARS, GMPRC, Manhattan, KS, USA) and teffflour (Soil & Crop Improvement, Assen, NL) were used inconjunction with instant dried yeast (Mauripan, BurnsPhilip Food Ltd., UK). Salt (Salt Union, West Point, UK),sugar (Suicra, Ireland) and tap water were also incorpo-rated into the batters.

2.2. Methods

2.2.1. Flour analysis

Crude protein (N� 6.25) was determined by combustion(Approved method 46-30, AACC International, 2000)using a Leco FP-528 nitrogen determinator (St. Joseph,MI). Flour moisture was determined using the air-ovenmethod (Approved method 44-15A, AACC International,2000).For total amino acid profile, flour samples were analysed

according to the method of Moore and Stein (1963).Amino acids were quantified using a Jeol JLC-500/V aminoacid analyser (Jeol (UK) Ltd., Welwyn Welwyn GardenCity, Herts, UK) fitted with a Jeol Na+ high performancecation exchange column.For the amino acid profile of soluble protein fractions,

flour samples were resuspended in water to a concentrationof 1mg/ml, aliquoted in 100 ml amounts, in duplicate, into8mm clear conical crimp vials and freeze dried overnight ina Virtis Advantage Freeze-Drier (Virtis, New York, USA).Samples were then hydrolysed to their amino acidconstituents via vapour phase hydrolysis in a CEM MARS

ARTICLE IN PRESSS. Renzetti et al. / Journal of Cereal Science 48 (2008) 33–45 35

5 microwave (CEM, North Carolina, USA) equipped witha protein hydrolysis accessory kit. The hydrolysis condi-tions used were as follows: microwave output 300W, rampto 150 1C in 20min, hold for 5min and cool to 30 1C beforeremoving samples. Following hydrolysis the samples wereresuspended in 125nm/ml norleucine, the internal standard,and the amino acids quantified using a Jeol JLC-500/Vamino acid analyser (Jeol (UK) Ltd., Welwyn Garden City,Herts, UK) fitted with a Jeol Na+ high performancecation exchange column.

2.2.2. Transglutaminase application

TGase (100units/g, Ajinomoto Co., Hamburg, Germany)was added to the ingredients prior to mixing. The enzymewas dissolved in half of the amount of water required inthe recipe. The addition of the enzyme was calculated on thebasis of the amount of crude protein present in each recipe.The levels tested in the present study were 1 and 10U ofTGase/g of protein. A formulation without any additionof enzyme was used as control.

2.2.3. Breadmaking

The same basic formulation was used for the breadmak-ing experiments with the following flours: buckwheat(BW), brown rice (BR), oat (OT), sorghum (SG), teff(TF) and corn (CR). The formulation used consisted of 100parts flour (relative mass), 125 parts of water, 2 parts ofsalt, 2 parts of sugar and 3 parts of dried yeast. Theamount of flour was interpreted as flour weight basis (fwb).Dried yeast was dissolved in a solution of water and sugarat 22–26 1C and pre-fermented in a proofer (Koma BV,Roermond, The Netherlands) at 30 1C and 85% rh for10min. All dry ingredients were placed in the bowl of aKenwood Major mixer (Kenwood, Hampshire, UK). Thepre-fermented yeast first and then the enzyme solution wereadded to the remaining ingredients prior to mixing. Mixingwas performed for 2min with a paddle tool (K beater) atslow/medium speed (level 2 out of 6). After 30 s of mixing,the mixer was stopped and the mixer bowl was scraped.Mixing was then continued for the remaining time. Thebatters were scaled to 400 g into baking tins (930mlvolume; 7.3 cm height; 9.5� 15.2 cm top; 7.5� 13.2 cmbottom) and proofed at 30 1C and 85% rh for 30min.Baking was performed at 190 1C top and bottom heat for35min in a deck oven (MIWE, Arnstein, Germany). Theoven was pre-injected with steam (0.3 l of water) and afterloading the oven was steamed again with 0.7 l of water.After baking, the loaves were depanned and cooled for90min on cooling racks at room temperature.

2.2.4. Batter pH

The pH of each batter was measured using a suspensionof batter (10 g), acetone (5ml) and distilled water (95ml)according to a standard method (ArbeitsgemeinschaftGetreideforschung e.V., 1994).

2.2.5. Batter fundamental rheology

Rheological measurements were performed on a con-trolled stress rheometer (CS-50, Bohlin Instruments Ltd.,Cirencester, UK), fitted with a coaxial geometry consistingof a 25mm diameter bob fitted in a 27mm cup. Samples(based on 300 g of flour) were prepared as previouslydescribed but without yeast. The batters were incubated for30min at 30 1C as described for breadmaking. Sampleswere placed into the cup and batter excess was carefullytrimmed. The whole system was covered. Water-saturatedcotton strips were placed on the inner side of the cover tocreate an atmosphere with high relative humidity toprevent drying out. The batter was allowed to rest for5min in order to allow relaxation of residual stresses.Frequency sweep from 0.1 to 10Hz was performed with atarget strain of 10�3 (0.1%). Preliminary tests indicatedthat the strain was well within the linear visco-elasticregion. Ten measuring points were recorded. Temperaturewas kept constant at 30 1C. Every result is the average offour measurements. Single frequency oscillation tests wereperformed at 10Hz with a target strain of 10�3 (0.1%).Temperature was kept constant at 30 1C. Sample moisturewas evaluated using the air-oven method (Approvedmethod 44-15A, AACC International, 2000) at thebeginning and end of test in order to evaluate drying.Results of single frequency test are the average of threemeasurements.

2.2.6. Flow behaviour

Shear measurements were performed to evaluate the flowbehaviour of batters. Apparent viscosity was measured as afunction of shear rate over the range 0.06–5.0 s�1.Apparent viscosity is reported as the mean of fourreplicates for each batter.

2.2.7. Bread evaluation

Standard baking tests were conducted on three loaves(n ¼ 3) from each bread type: control breads (BW0, BR0,OT0, SG0, TF0, CR0), 1 unit of TGase breads (BW1, BR1,OT1, SG1, TF1, CR1) and 10 units of TGase breads(BW10, BR10, OT10, SG10, TF10, CR10). Loaves wereweighed and loaf volume was measured by rapeseeddisplacement. Loaf specific volume (ml/g) and bake loss(%) were then calculated. Texture Profile Analysis (TPA)(Bourne, 1978) of the crumb was performed on two slicestaken from the centre of each loaf. The slices were obtainedby cutting the bread transversely using a slice regulator andbread knife to obtain uniform slices of 25mm thickness.TPA was performed with a universal testing machineTA-XT2I (Stable Microsystems, Surrey, UK) equippedwith a 25-kg load cell and a 35-mm aluminium cylindricalprobe. Pre-test speed, test speed and post-test speed were2mm/s, trigger force was 20 g, distance was 10mm (40%compression) and wait time between first and secondcompression cycle was 5 s. Bread moisture was determinedaccording to the Approved method 44-15A (AACCInternational, 2000). All measurements obtained with the

ARTICLE IN PRESSS. Renzetti et al. / Journal of Cereal Science 48 (2008) 33–4536

three loaves from one batch were averaged into one value(one replicate). TPA was repeated with three loaves at days2 and 5 (50 and 122 h post-baking, respectively).

2.2.8. Confocal laser scanning microscopy

CLSM is a valuable tool for a deeper understanding ofthe microstructure of cereal products. The advantage ofthis technique is its ability to produce optical sectionsthrough a three-dimensional specimen and, with applica-tion of staining procedures, to select and differentiateparticular structures in the food system. For these reasonsCLSM was applied in order to visualise the formation ofprotein networks in the TGase supplemented batters andbreads.

Batters for microscopy were prepared as described forbaking tests without yeast in a Glutomatic 2200 (PertenInstruments AB, Huddinge, Sweden), which allowedmixing of small quantities of batter. Mixing speed was120 rpm. Addition of washing solution was avoided and amodified mixing chamber without a bottom perforationwas used. Mixing was performed for 2min. Rhodamine B(Sigma-Aldrich, St. Louis, MO, USA) was added to therespective recipes at a rate of 0.09% (fwb). The dye wassolubilised in water before mixing to ensure homogeneousdistribution. The control batters were incubated for 30minat 30 1C prior to analysis, while the TGase supplementedbatters were incubated for 30 and 180min at 30 1C.

The breads for microscopy were prepared as describedfor the baking tests. Fresh crumb samples were cut fromthe centre of the loaves and immersed in a 2% agarsolution. After the agar solution had solidified, thin crumbslices were cut from the agar and placed on a welled slide.To stain proteins, a solution of 0.02% Fuchsin acid(Sigma-Aldrich, St. Louis, MO, USA) in 1% acetic acidwas added to the sample and kept for 15min. Afterstaining, excess of dye was removed by rinsing the samplewith deionised water for 30min. When the stainingprocedure was completed a glass coverslip was placed onthe sample.

An MRC-1024 confocal laser-scanning system (Biorad,Herts, UK) mounted on an upright microscope (Axioskop,Zeiss, Germany) with a 10� objective and a 40� waterimmersion objective was used. Fluorescence images (ex-citation ¼ 568 nm, emission ¼ 620 nm) of a number ofoptical sections were acquired by scanning the samplealong the optical axis. A micrograph was taken of the

Table 1

Protein content, moisture (%), total and soluble fractions amino acid profile

BW BR OT

Protein (N% � 6.25) 11.5 10.0 7.1

Moisture (%) 13.58 14.19 12.35

Glua 1.950a (0.887a) 1.511c (0.141c) 2.152a

Lysa 0.654a (0.250a) 0.368b (0.054c) 0.475c

aValues followed by the same letter in the same row are not significantly diff

the soluble fractions.

projection of the layers. To obtain 3D images, Volocity3.1.0 (Improvision Limited, Coventry, UK) was used.

2.2.9. Statistical analysis

Baking tests were performed on each flour and TGaselevel by using three replicates. For TPA analysis, eachbatch of nine bread loaves (one replicate of the same breadtype) was subdivided into 3� 3 loaves and these randomlyassigned to the three different storage times. Statisticalanalysis were performed with SPSS software (SPSS Inc.,Chicago, IL, USA) on all tests using a one-way ANOVAand Tukey’s post hoc test to detect significant differences.

3. Results

3.1. Flours characteristics

The protein content and amino acid profile for all theinvestigated flours are shown in Table 1. BW, SG and TFshowed higher protein content than BR and OT flours,whereas CR had the lowest protein content (5.6 g/100 g offlour). With regards to the amino acid profile, glutamineand lysine concentration were analysed as both aminoacids are the substrates of the cross-linking reactioncatalysed by TGase. All flours showed that glutaminewas the amino acid most present, even though significantdifferences were found among the flours. With regards tolysine, the highest content was present in BW (0.654 g/100 gof flour) compared to the other flours (Po0.05). OT alsoshowed a remarkable amount of lysine (0.475 g/100 g offlour), significantly higher than BR, SG, TF and CR(Po0.05). BR and TF had comparable levels while SG andCR showed the lowest amount of lysine with just 0.222 and0.171 g/100 g of flour, respectively. Considering the gluta-mine and lysine content for the soluble protein fractionsof all flours, BW showed the highest amounts of lysineand glutamine in solution (38% and 45% of total lysineand glutamine, respectively), followed by TF. BR, OT,SG and CR were comparable in both lysine and glutaminecontents of the soluble fractions (Table 1).

3.2. Batters pH

Since the pH could affect enzyme activity, which has anoptimum in the range 5–8, the pH of all batters wasmeasured (Table 2). All control batters showed comparable

of the investigated flours (g/100 g flour)

SG TF CR

11.7 12.3 5.6

12.16 10.71 14.53

(0.206c) 1.367c (0.075c) 3.085b (0.337b) 1.029d (0.048c)

(0.081c) 0.222d (0.026c) 0.332b (0.136b) 0.171d (0.012c)

erent (Po0.05); n ¼ 2. Values in brackets show the amino acid content of

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Table 2

pH and initial viscosity of the TGase-treated battersa

pH Z0 (Pa s)

0U/g 1U/g 10U/g 0U/g 1U/g 10U/g

BW 5.8270.17abA 6.1070.11a 5.7970.03b 117.2673.89a 121.8871.61a 212.4774.51b

BR 5.6070.27aA 5.6470.08a 5.5570.11a 5.3670.17a 5.2470.25a 10.2771.40b

OT 5.4670.15aAB 5.7070.11a 5.5770.08a 344722a 37775a 441716b

SG 5.5070.30aAB 5.5570.07a 5.3670.18a 54.7972.11a 69.4071.04b 72.4677.71b

TF 5.8070.06aA 5.7570.02a 5.6970.10a 17.0870.76a 18.1770.91a 22.6670.42b

CR 5.0070.20aB 5.1070.11a 4.6970.08b 18937180a 14027253a 12857241b

aMean values7standard errors of three replicates. Mean values followed by the same lower case letter in the same row are not significantly different

(Po0.05). For the pH column of control batters (0U/g) mean values followed by the same capital letter are not significantly different (Po0.05).

S. Renzetti et al. / Journal of Cereal Science 48 (2008) 33–45 37

pH values, with the exception of CR0 whose pH wassignificantly lower than BW0, BR0 and TF0 (Po0.05).

With addition of TGase, CR10 showed a significantdecrease in pH (Po0.05). No significant differences werefound for the other flours after TGase treatment.

3.3. Fundamental rheology

3.3.1. Frequency sweep

Batters were proofed for 30min at 30 1C and frequencysweeps were performed immediately after. Independentlyfrom the level of enzyme added, all batters showed that theelastic modulus (G0) was higher than the viscous modulus(G00), indicating that the batters had a solid, elastic-likebehaviour (data not shown). The results of the frequencysweep test for the investigated flours are shown in Fig. 1.Addition of 10U of TGase in BW batters increased thecomplex modulus (|G*|), indicating increased resistance todeformation compared to the control and 1U samples. Atthe same time the phase angle (d) of BW10 batter wasdecreased, indicating increased degree of elasticity. Similareffects were found with BR10, even though the effect wasless evident compared to BW10. OT10 did not show anydifferences in the |G*|, but the degree of elasticity wasincreased. In SG the addition of 1 and 10U of TGaseincreased |G*|, but the degree of elasticity was increasedonly for SG10. CR showed a completely opposite trend asthe addition of any level of enzyme decreased the resistanceto deformation (lower |G*|), while d was not affected. Noeffects were detected on TF batters at any enzyme leveltested.

3.3.2. Single frequency oscillation

After mixing, batter samples were immediately placed onthe rheometer and a single frequency oscillation test wasperformed. BW10 showed a significant increase in G0 overtime compared to BW1 and BW0, until a steady level wasreached, while G00 was only slightly increased (Fig. 2).Similar results were found for BR10, even though the in G0

was less evident than that observed for BW10 (Fig. 2). Forall the samples moisture loss at the end of the test waslower than 3%.

3.4. Flow behaviour

Table 2 shows that TGase caused a significant increase ininitial viscosity for BW10, BR10, OT10 and TF10(Po0.05). The viscosity of SG batter was significantlyincreased with any level of enzyme. On the contrary, CR10showed a significant decrease in initial viscosity (Po0.05).

3.5. Baking tests

A basic recipe was applied to all flours used in this study.For each flour three levels of enzyme were examined (0, 1and 10UTGase/g of protein). Standard baking tests wereconducted on three breads (n ¼ 3) from each flour type andenzyme level. As shown in Table 3, 1U/g of TGasesignificantly decreased the bake loss for all flours, exceptOT and CR (Po0.05). Increasing the level of enzyme (i.e.10U) resulted in a further decrease in bake loss only for SGbread (Po0.05). The reduction in bake loss was remark-ably high for TF and BW breads (19.2% and 18.5%respectively). Despite the differences in bake loss, nosignificant differences in bread moisture were observed inany of the investigated breads (data not shown). Withregards to the specific volume, BW10 and BR10 showed asignificant decrease compared to their controls, while SG1and CR1 showed an increase in specific volume comparedto SG0 and CR0, respectively (Table 3). TGase treatmentdid not modify the specific volume of OT or TF breads.

3.6. Texture profile analysis

The results of TPA analysis are displayed in Table 3.BW10 showed a significant increase in crumb hardnesscompared to both BW1 and BW0 (Po0.05). Also BR10showed a significantly higher crumb hardness compared toBR1 (Po0.05). TPA results are not shown for BR0 as aproper crumb was missing in the centre of the bread slice(Fig. 3). Taking this aspect into consideration, a remarkableeffect of TGase could be seen on BR1 as a proper crumbappeared. TGase treatments did not influence the TPAprofile of OT, SG or TF breads, while a decrease in crumbhardness was found for CR1 compared to CR0. Overall,

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Frequency (Hz)

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Fig. 1. Effect of TGase on the complex modulus (|G*|) and phase angle (d) of the investigated flours batters: (A) buckwheat; (B) brown rice; (C) oat; (D)

sorghum; (E) teff; (F) corn. Phase angle (d): control batter ( ), 1U of TGase ( ) and 10U of TGase ( ). |G*|: control batter ( ), 1U of TGase

( ) and 10U of TGase ( ). Mean value of four replicates.

S. Renzetti et al. / Journal of Cereal Science 48 (2008) 33–4538

crumb chewiness showed a similar trend to hardness, i.e.crumb chewiness was significantly higher in BW10 comparedto both BW1 and BW0, as well as BR10 in comparison toBR1 (Po0.05), while CR1 showed a significant lowerchewiness than CR0 and CR10. The overall effect of TGaseapplication on the macrostructure of BW, BR and CRbreads is clearly showed by the appearance of the breadslices (Fig. 3). Crumb structure of BW, BR or CR breadsappears improved after TGase treatment.

The application of the enzyme did not show anysignificant effect on the overall characteristics of thecrumbs over 5 days of storage. Independently from the

presence or absence of enzyme treatment, staling of BWbreads was not observed, as no significant differences incrumb hardness were found over the 5 days of storage(data not shown). On the other hand, OT, SG, TF and CRbreads were significantly affected by storage time, as asignificant increase in crumb hardness, i.e. staling wasdetected over time (data not shown).

3.7. Confocal laser scanning microscopy

To gain a deeper insight on the effect of TGase on thefunctionality of the cereal proteins, CLSM was used to

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Time (hr) Time (hr)

0 1 7

G',

G''

(Pa

)

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6000

7000

G',

G''

(Pa

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2 3 4 5 6 0 1 72 3 4 5 6

Fig. 2. Effect of TGase on the elastic (G0) and viscous moduli (G00) of (A) buckwheat batters and (B) brown rice batters. G0: control batter ( ),1U of

TGase ( ) and 10U of TGase ( ). G00: control batter ( ), 1U of TGase ( ) and 10U of TGase ( ). Mean value of three replicates.

Table 3

Bake loss, specific volume and TPA profile of the TGase-treated breadsa

TGase level Bake loss (%) Specific volume (ml/g) Crumb hardness (N) Crumb chewiness (N)

BW 0U/g 12.0070.48a 2.1970.02a 7.2170.68a 4.4070.18a

1U/g 10.1670.08b 2.0970.05a 7.7470.81a 4.1270.43a

10U/g 9.7870.30b 1.8870.05b 12.2370.77b 7.1870.42b

BR 0U/g 12.4270.74a 1.8370.02a – –

1U/g 10.7370.25b 1.7970.06ab 9.0170.41a 4.3570.21a

10U/g 10.8970.20b 1.6870.08b 10.9971.04b 5.3870.65b

OT 0U/g 9.6470.22a 1.3770.02a 21.8371.50a 15.5570.94a

1U/g 9.6670.12a 1.3970.11a 24.3273.18a 16.8671.82a

10U/g 9.0970.74a 1.3370.08a 21.4471.18a 15.3870.87a

SG 0U/g 10.4370.19a 1.5270.03a 24.3970.94a 12.4571.61a

1U/g 9.9070.02b 1.7170.02b 23.1372.65a 10.7371.89a

10U/g 9.4870.19c 1.5770.06a 26.7373.85a 13.6872.49a

TF 0U/g 12.3170.97a 1.8270.08a 17.0172.77a 10.2471.60a

1U/g 9.9470.41b 1.8270.05a 17.8970.35a 10.6770.54a

10U/g 10.0570.42b 1.7970.04a 15.0472.05a 9.3671.10a

CR 0U/g 12.4170.91a 1.3570.09a 45.5175.10a 19.5270.78a

1U/g 11.3670.35a 1.6470.07b 36.6972.74a 11.3071.34b

10U/g 11.8970.44a 1.4670.12a 39.5176.23a 16.0973.95a

aMean values7standard errors of three replicates. Mean values followed by the same letter in the same column are not significantly different (Po0.05).

S. Renzetti et al. / Journal of Cereal Science 48 (2008) 33–45 39

investigate the microstructure of the batters and breadcrumbs. The microscopy of batters for BW0 and BR0 after30min of proofing revealed a homogeneous distribution ofproteins in the system (Fig. 4). After incubation of 10U ofTGase for 30min at 30 1C, BW and BR proteins appearedto be distributed in aggregates (Fig. 4). After 180min ofincubation larger protein agglomerates appeared in thesystem (Fig. 4). No differences could be detected for theother cereal batters (data not shown).

Analysis of bread crumb CLSM images revealed cleardifferences for BW and BR. The continuous protein phaseof BW0 appeared to be enhanced and reinforced by theactivity of TGase and the protein network of BW10 was

characterised by a finer mesh size (Fig. 5). In BR10 breadcrumb the continuity of the protein phase was enhancedcompared to BR0 (Fig. 5). On the contrary, no differencescould be detected in the crumb microstructure of theremaining cereals (data not shown).

4. Discussion

The replacement of the gluten network in the develop-ment of gluten-free cereal products is a challenging task forthe cereal technologist. This study shows that TGase can beused to improve the baking performances of gluten-freeflours. Addition of 10U/g of TGase to BW and BR batters

ARTICLE IN PRESS

Fig. 3. Bread slices from buckwheat (BW), brown rice (BR) and corn flour (CR) formulations treated with different TGase levels (0, 1 and 10U).

Fig. 4. 3D elaboration of CLSM images of buckwheat and brown rice batters (40� magnification): [A] BW control (0U of enzyme) after 30min at 30 1C;

[B] BW 10U of TGase batter after 30min at 30 1C; [C] BW 10U batter after 180min at 30 1C; [D] BR control (0U of enzyme) after 30min at 30 1C; [E] BR

10U of TGase batter after 30min at 30 1C; [F] BR 10U batter after 180min at 30 1C. Proteins appear red. With increasing incubation time the proteins in

the batter change from a homogeneous dispersion [A and D] to protein aggregates [B and C, E and F].

S. Renzetti et al. / Journal of Cereal Science 48 (2008) 33–4540

ARTICLE IN PRESS

Fig. 5. 3D elaboration of CLSM images of buckwheat and brown rice bread crumb (40� magnification): [A] BW control bread (0U of enzyme); [B] BW

10U of TGase bread; [C] BR control bread (0U of enzyme); [D] BR 10U of TGase bread. Proteins are stained red together with yeast cells which appear

round shaped. For buckwheat, the control bread shows a continuous protein network, characteristic only of buckwheat breads. The addition of the

enzyme results in a crumb with a strengthen, finer-meshed protein network. For brown rice, the control bread shows the absence of any kind of protein

network. The addition of the enzyme enhances the continuity of the protein phase.

S. Renzetti et al. / Journal of Cereal Science 48 (2008) 33–45 41

significantly increased the pseudoplastic properties andpositively influenced the baking performances of theseflours. Nevertheless, increased pseudoplastic behaviour didnot necessarily result in improved baking performances, aswas the case for OT and SG flours. Remarkably,improvements in the baking performances were detectedalso when the pseudoplastic behaviour of the batters wasdecreased, as was the case of CR flour.

BW10 and BR10 batters showed a higher resistance todeformation and a higher degree of elasticity compared tothe control or the batter treated with 1U of TGase (Fig. 1).At the same time the initial viscosity of BW10 and BR10was significantly increased (Table 2). Taken together, theseresults suggest the formation of a protein network and/orprotein agglomerates by covalent cross-linking. Proteinaggregation was monitored by single frequency oscillationtests and a significant increase in G0 over time was observedfor BW10 and BR10, while G00 was only slightly affected(Fig. 2). An increase in the average molecular weight ofproteins results in a more continuous protein phase, as

confirmed by the analysis of the microstructure of theBW10 and BR10 batters (Fig. 4), which reveals theformation of a protein network by aggregation of proteinswith increasing incubation time. Protein polymerisationimproved the baking performances of the flours. In fact,BW10 and BR10 breads showed a significant reduction inbake loss (Table 3), indicating an increased ability ofproteins to bind water. Improvement in the water holdingcapacity due to TGase activity has been previouslyreported in gels (Kuraishi et al., 2001) and in gluten-freesystems (Moore et al., 2006). The increased water holdingcapacity can be explained as a result of deamidation ofglutamine residues into glutamic acid, which decreases thehydrophobic environment (Gerrard et al., 1998). On theother hand, a strengthened protein network consequent toprotein cross-linking has improved ability to trap water(Lorenzen et al., 2002). Therefore, it can be suggested thatboth the deamidation and cross-linking reaction mightbe involved in the improvement of the water holdingcapacity of the BW and BR batters. Consequence of the

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cross-linking reaction was the significant decrease inspecific volume for BW10 and BR10 breads (Table 3).Similar effects have been already reported in both gluten-free and wheat breads (Basman et al., 2002b; Moore et al.,2006; Salmenkallio-Marttila et al., 2004). Gujral and Rosell(2004) found an opposite trend with TGase, as an increasein specific volume was observed in a white rice breadformula. However, this trend was detected in a formulationcontaining 2% HPMC. Instead, without addition ofHPMC no significant effects were detected with up to12U of TGase, while a significant decrease in specificvolume was observed at higher enzyme concentrations.This might suggest that the interaction of TGase withhydrocolloids, and particularly with HPMC, could play amajor role on the overall effect of the enzyme. In terms ofcrumb characteristics, BW10 and BR10 showed a signifi-cant increase in hardness and chewiness (Table 3). Theincrease in hardness can be considered as an improvementin the structure of the crumb, as a proper structure waslacking in the control breads (Fig. 3). Comparison of themicrostructure of BW0 with BW10 as well as of BR0 withBR10 (Fig. 5) further confirms that the increase in crumbfirmness is the result of protein cross-linking. The additionof 10U of enzyme clearly strengthened and enhanced theprotein phase resulting in a finer-meshed protein network,as it was the case in BW bread. In BR bread 10U of TGaseenhanced the continuity of the protein phase. Increases incrumb firmness due to TGase activity were previouslyfound in wheat bread systems (Gerrard et al., 1998) and ingluten-free systems (Moore et al., 2006). The increase inchewiness can be explained as a consequence of theincrease in the number of covalent bonds, which increaseselasticity (Dickinson, 1997).

TGase addition affected the properties of OT batters.OT10 batter showed a higher degree of elasticity as well assignificantly increased initial viscosity in comparison toOT1 and OT0 batters, indicating that the cross-linkingreaction promoted by 10U of TGase affected the structureof the batter (Fig. 1). Nevertheless, none of the TGasesupplemented OT breads showed any significant differencein baking tests and TPA profile (Table 3). In bakingexperiments with a 1:1 oat-to-wheat flour proportion,Salmenkallio-Marttila et al. (2004) found that the additionof TGase decreased the specific volume of breads whileincreasing crumb hardness and chewiness. However, thepresence of wheat flour might account for such effectsrather than oat flour. It might be suggested that thepresence of b-glucan in oat flour, and thus the highviscosity of the batter (Table 2), could mask the effects ofTGase in the resulting bread. However, effects wereobserved for CR bread, even if CR batter showed aremarkably higher viscosity than OT batter (Table 2).Nai-Chi Siu et al. (2002b) reported that oat globulin, whichis the major protein fraction in oats (Peterson and Smith,1976), has lower reactivity to TGase compared to otherprotein substrates. Therefore, it can be suggested that thepolymerisation promoted by TGase is not as extensive as to

affect the resulting breads. This hypothesis is furthersupported by the results of CLSM investigations of bothOT batters and breads, as no protein aggregation neitherstrengthened protein network could be detected in theOT10 batter and bread, respectively (data not shown).The structural changes brought by TGase in terms of

increased pseudoplastic behaviour of SG1 and SG10batters were not reflected in significant changes in thebaking performances and TPA profile of the resultingbreads. However, a decreased bake loss with increasingTGase level and a higher specific volume for SG1 breadwere observed (Table 3). Sorghum protein has beenreported to be cross-linked by TGase, forming highmolecular weight aggregates (Babiker and Kato, 1998).Nevertheless, differences in the microstructure of SGbatters and breads supplemented with TGase could notbe detected by CLSM (data not shown), confirming that,under the conditions of this study, TGase activity could notsignificantly improve the baking performances of SG flour.TF flour revealed to be a poor substrate for TGase

application. Changes in the rheological properties of TFbatters were limited to an increase in the initial viscositywith 10U of TGase. The resulting bread showed asignificant decrease in bake loss, but no significant effectswere found on the specific volume or on the TPA profile ofthe crumbs.Remarkably, the effects of TGase on CR batters showed

an opposite trend to that observed for BW, BR, OT andSG batters. In particular, TGase activity resulted in batterswith reduced resistance to deformation and reducedviscosity (Table 2), while the degree of elasticity wasunaffected (Fig. 1). CR flour was found to have the lowestprotein content, and in particular the lowest lysine contentamong the investigated flours (74% and 54% lower thanBW and BR content, respectively). Taking these resultsinto consideration, it is tempting to speculate that areaction other than protein cross-linking, i.e. deamidation,is prevailing in the CR system. Deamidation would explainthe decrease in pH measured in the batters after TGasetreatment (Table 2), since glutamic acid is the end productof deamidation. It has been reported that deamidationlevels as low as 2–6% could enhance the functionalproperties of proteins (Matsumoodi et al., 1985), e.g.increases protein solubility and prevents protein coagula-tion (Babiker and Kato, 1998). Such effects can modify theprotein–protein and protein–starch interactions and possi-bly affect also the starch–starch interactions. Such inter-actions are measured during dynamic oscillations tests(Amemiya and Menjivar, 1992), and could account for thedifferences detected with TGase addition in CR batters.This hypothesis is further supported by the evidence thatno protein aggregation was detected by CLSM (data notshown). The results obtained with CR batters seem tocontradict the conclusion that increased elastic and viscousbehaviour improves the baking performances of the batters(Gujral and Rosell, 2004), as an increase in specific volumeand decreases in crumb hardness and chewiness were

ARTICLE IN PRESSS. Renzetti et al. / Journal of Cereal Science 48 (2008) 33–45 43

observed for CR1 bread despite the detrimental effect ofTGase on the pseudoplastic properties of the batter. TGasesupplementation clearly improved the macroscopic appear-ance of CR breads (Fig. 3). These results can be explainedtaking into consideration the expansion of wheat doughs.In wheat doughs expansion is achieved in two stages.During the first stage, the expanding gas cells are supportedby the starch–gluten matrix which develops in a thinmembrane. In the second stage, it is the liquid film thatmaintains the integrity of the gas cells as discontinuities inthe starch–gluten matrix increase during expansion (Ganet al., 1995). In a batter system dominated by the starchphase and where a starch–gluten matrix and/or hydro-colloids are missing, the expansion of the dough reliesentirely on the stability of the liquid film. Therefore,assuming that the deamidation is the reaction prevailing inthe CR batters, it is tempting to speculate that byincreasing the number of polar groups of CR proteins,the deamidation might facilitate protein unfolding at thewater surface, thus stabilising the liquid lamellae andimproving the batter gas-holding capacity. Moreover, theability of proteins to stabilise emulsions and foams isdependent on their solubility (Tolstoguzov, 1991), which isincreased by deamidation. At the same time, it can besuggested that the decrease in viscosity brought by TGaseaddition might facilitate the expansion of the batters. Theuse of an enzyme which selectively deamidates glutamineresidues, e.g. peptidodeaminases, will help explaining theTGase activity observed on CR flour.

TGase can promote the formation of protein networks,but the availability of glutamine and lysine residues in theprotein matrix is essential, as these amino acids are thesubstrates for the cross-linking reaction. Investigation ofthe glutamine and lysine contents of the gluten-free floursused suggests that lysine can be the limiting factor for thecross-linking reaction (Table 1). Among the flours inves-tigated, the higher lysine content of BW and OT floursmight indicate these flours as the best substrates for TGaseapplication (Table 1). Positive effects of TGase on thebaking performances of the flours were clearly detected forBW breads, but no effects were detected on OT breads.Therefore, factors other than substrate availability affectedthe cross-linking reaction, i.e. accessibility of the substrateand protein solubility (Coussons et al., 1992; Il Jun Kanget al., 1994; Xiao-Qing Han and Srinivasan Damodaran,1996). In order to be reactive the residues must be in aflexible region or in regions with reverse turn or at theprotein surface, which in terms means that the substratemust be accessible to the enzyme (Coussons et al., 1992; IlJun Kang et al., 1994). Therefore, the different effects ofTGase on flours containing similar glutamine and lysinecontents, e.g. SG and CR or BR, OT and TF, can beexplained by the differences in substrates accessibility.Another factor, protein solubility, has also been suggestedto play a key role for TGase activity (Nai-Chi Siu et al.,2002b). However, this was not the case in our study. Forexample, the glutamine and lysine profiles for the soluble

fraction of BR flour were only 9.3% and 14.7%,respectively, of the total flour content (Table 1). The waterinsoluble fraction of BR has been reported to account forabout 73% of total proteins (Meesook and Jeong, 2002),therefore it can be assumed that the lysine residuesavailable for the cross-linking reaction were mainlydistributed in the water insoluble fraction. The significanteffects shown by TGase on BR10 batter and bread are thusmainly due to the reaction of the enzyme with the waterinsoluble proteins of the flour. This is further supported bythe evidence that despite being water insoluble, gluten itselfhas been reported to be significantly cross-linked by TGasewith the complete disappearance of the bands correspond-ing to the high molecular weight glutelins (Larre et al.,1998). Therefore, it can be concluded that, under thecondition of this study, it is the availability combined withthe specificity of the substrates rather than the proteinsolubility, which are the main factors affecting the cross-linking reaction.

5. Conclusion

The results of the present study show that thefunctionality of gluten-free flours in terms of breadmakingperformances can be successfully improved by the action ofTGase. BW and BR flours were the optimal substrates forTGase application among the investigated flours. Theimprovements in the pseudoplastic behaviour of BW andBR batters were reflected in significant improvements inthe textural and structural characteristics of the resultingbreads. The use of CLSM confirmed the improvementsbrought by TGase on the microstructure of batters andbreads. However, the protein source is a key elementdetermining the impact of the enzyme. In fact, OT, SG andTF flours were only slightly affected by the addition of theenzyme. Furthermore, the results of CR flour show thatTGase can also negatively affect the pseudoplastic beha-viour of batters, but that such effect can result in significantimprovements in the overall quality of the correspondingbreads. Further studies are required to fully understand themodifications brought by TGase on cereal proteins, andparticularly the role of the deamidation reaction.

Acknowledgements

This study was financially supported by the EuropeanCommission in the Communities Sixth Framework Pro-gramme, Project HEALTHGRAIN (FP6-514008). Thispublication reflects only authors’ views and the Commu-nity is not liable for any use that may be made of theinformation contained in this publication. The authorsacknowledge that this research was partly funded also byEnterprise Ireland (National Development Plan2000–2006). The authors would like to thank Mrs.Bereniece Riedewald and the Anatomy Department ofUCC for the use of CLSM and Volocity software and forthe technical assistance. The support of Mrs. Paula

ARTICLE IN PRESSS. Renzetti et al. / Journal of Cereal Science 48 (2008) 33–4544

O’Connor for amino acids analysis is gratefully acknowl-edged.

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