at SciVerse ScienceDirect

Journal of Cereal Science 56 (2012) 747e753

Contents lists available

Journal of Cereal Science

journal homepage: www.elsevier .com/locate/ jcs

Oat malt as a baking ingredient e A comparative study of the impact of oat, barleyand wheat malts on bread and dough properties

Outi E. Mäkinen, Elke K. Arendt*

Department of Food Science, Food Technology and Nutrition, University College Cork, Western Road, Cork, Ireland

a r t i c l e i n f o

Article history:Received 29 November 2011Received in revised form7 August 2012Accepted 9 August 2012

Keywords:GerminationConfocal laser scanning microscopyRheologyGluten

* Corresponding author. Tel.: þ353 21 490 2064; faE-mail address: e.arendt@ucc.ie (E.K. Arendt).

0733-5210/$ e see front matter � 2012 Elsevier Ltd.http://dx.doi.org/10.1016/j.jcs.2012.08.009

a b s t r a c t

Oat malt is a nutritionally rich ingredient mainly used in a small number of speciality products. The aimof this study was to evaluate the suitability of oat malt in wheat baking. The effect of oat malt on breadand dough properties at levels ranging from 0.5% to 5% was studied and compared with barley and wheatmalts. The addition of all malts increased loaf specific volumes. Barley and wheat malts at levels above2.5% led to a sticky and coarse crumb, but the effect of oat malt on the crumb grain was negligible.Rheological characterisation could not explain the superior baking performance of oat malt, as itincreased extensibility and decreased resistance extensively indicating weakening of the extensionalproperties of the gluten network. The high lipolytic activity may have compensated for the loss of doughstrength by improving the surface properties of gas cells. The results show that oat malt can be used inwheat baking to improve the loaf volume and nutritional quality without the detrimental effects asso-ciated with the excess amylolytic activity of barley and wheat malts.

� 2012 Elsevier Ltd. All rights reserved.

1. Introduction

Malting, the controlled germination of cereals ending witha heat treatment is a process that transforms oat into a morepalatable and nutritionally richer form (Kaukovirta-Norja et al.,2004). During germination, enzymes are synthesised or activatedto mobilise the storage compounds of the grain, leading to highenzyme activities and modification of nutrient bioavailability. Also,various bioactive compounds are formed in the metabolicprocesses. Larsson et al. (1996) reported an increased iron and zincabsorption from breakfast meals containing oat malt as a result ofhigh phytase activity. Also, the amount of phenolic compounds,avenanthramides and phytosterols increase during germination(Oksman-Caldentey et al., 2001). The total antioxidant activity ofoat malt is comparable to that of butylated hydroxytoluene (BHT),a common food antioxidant (Pike et al., 2007). Kilning reduces themoisture content to end the metabolic processes and leads to theformation of Maillard products, providing a unique flavour andaroma profile (Kaukovirta-Norja et al., 2004; Oksman-Caldenteyet al., 2001). Another interesting feature of oat malt is its poten-tial in dietetic products. Uncontaminated oat is well tolerated bymost celiac patients and the incorporation of “pure oat” in gluten

x: þ353 21 427 0213.

All rights reserved.

free diets is being recommended by many national coeliac associ-ations (Butzner, 2011; Kemppainen et al., 2007).

In regards to food formulations and processing, the enzymeactivity of oat malt may have both beneficial and detrimentaleffects on the product. Exogenous enzymes are widely used in thebaking industry to improve dough and bread quality and shelf life(Poutanen, 1997). They may, however, also have a negative impacton baking quality if not correctly dosed, as is shown by the largebody of literature on the effects of pre-harvest sprouting of breadwheat.

Amylases are routinely added to wheat flours to optimise thefalling number, as well as antistaling agents to retard crumbhardening caused by rearrangements in the starch network andchanges in water distribution. Mechanisms of action include theoverall weakening of the starch networks by endo-acting amylasesand the prevention of amylopectin recrystallization side chaincleavage by exo-acting amylases (Goesaert et al., 2009). The releaseof fermentable sugars increases the yeast activity and producesprecursors for theMaillard reaction (Poutanen,1997). In addition tothe many functions of amylases, also proteases, lipases, hemi-cellulases and oxidative enzymes improve bread volume andcrumb grain by the modification of bulk and surface rheologicalproperties (Poutanen, 1997; Primo-Martín et al., 2006).

The aim of this study was to evaluate the suitability of oat maltas an ingredient in wheat baking. The influence of oat malt ondough and bread properties was compared with barley and wheatmalts.

O.E. Mäkinen, E.K. Arendt / Journal of Cereal Science 56 (2012) 747e753748

2. Materials and methods

2.1. Malting

Barley malt was purchased from Greencore Malt. Wheat wasmalted according to the MEBAK method 2.5.3.1. The maltingprocedure of oats was as follows: the grains were steeped to 45%out-of-steep moisture by varying wet (13 �C) and air (20 �C) stagesand germinated in a proofer (Koma BV, Roremond, Netherlands) for5 d at 15 �C. The green malt was kilned in three stages (35 �C, 50 �Cand 60 �C) using a malting machine (Joe White Malting Systems,Perth, Australia). The malts were milled with a laboratory disc mill(Bühler GmbH, Braunschweig, Germany) and sieved to passa 0.25 mm screen.

2.2. Enzyme activities

Amylotic activities were determined by Ceralpha and Betamyl-3methods (Megazyme, Wicklow, Ireland) using non-reducing endblocked p-nitrophenyl maltoheptaoside (endo-activity/a-amylases)and p-nitrophenyl maltopentaoside (exo-activity/b-amylases) assubstrates. The hydrolysed substrate is cleaved to p-nitrophenyland glucose by a-glucosidase, the phenolate colour is developedunder alkalic conditions and the A400 is measured. One unit ofactivity corresponds to the liberation of 1 mmol p-nitrophenol min-1 under assay conditions (pH 5.2, T ¼ 40 �C).

Protease extracts were prepared by extracting freshly milledmalts for 30 min at 5 �C in 0.05 M acetate buffer with 2 mM L-cysteine at pH 5.0. Solids were removed by centrifugation(10 000 g � 15 min at 4 �C) and the extracts were assayed imme-diately. The extracts were used for the protease assay, class-specificinhibitor studies and the wheat protein digestion assay.

Proteolytic activities were determined by mixing 50 ml sampleextract and 450 ml 0.2 M acetate buffer (pH 4.0) containing bovinehaemoglobin (reaction concentration 0.5%) and incubating for150 min at 40 �C. The reaction was stopped with 400 ml cold 10%trichloroacetic acid (TCA) and centrifuged (10 000 g� 10 min). Freea-amino nitrogen was measured by incubating 25 ml supernatantand 225 ml TNBS for 20min at 50 �C and stopping the reaction using750 ml HCl (0.2 M). A340 was measured and quantified against L-leucine standard curve. One unit of activity corresponds to theenzyme activity that liberates 1 mg leucine/h/g under the assayconditions.

The effect of class-specific inhibitors on the azocasein hydrolysingactivity was determined at dough pH (5.8) using E-64 (cysteineprotease inhibitor), pepstatin A (aspartic), PMSF (serine) and o-phenanthroline (metallo) at concentrations 10 mM, 20 mM, 10 mMand 1 mM, respectively (Mikola and Jones, 2000a). 5 ml inhibitorsolutions were added to 250 ml sample extract and incubated for10 min before the addition of 350 ml 1.4% (w/v) azocasein dissolvedin 0.2 M sodium acetate buffer. Themixtures were incubated for 1 hat 40 �C and the reaction stopped with 0.5 ml cold 10% TCA.Samples were centrifuged (10 000 g � 10 min), and supernatantmixed with 0.5 M NaOH (1:1) and A340 measured after 20 min. Theinhibition was expressed as (A440 sample/A440 control) � 100%.

Lipase activities were determined using the dough method(Matlashewski et al., 1982). Samples were defatted using petroleumether (40e60�) and air dried. 330 ml TriseHCl buffer (0.05 M, pH7.5) with 1% Triton-X-100 and 90 mg glyceryl trioleate wereemulsified by shaking at 37 �C for 15 min. Reaction was started byadding 0.5 g sample and mixing the dough for 2 min with a glassrod. After a 60 min incubation at 37 �C, the reactionwas stopped bystirring 0.1 ml 1MHCl in the dough. Reaction blanks were preparedby acidifying the substrate buffer emulsion before adding thesample. To extract the free fatty acids, 5 ml 2,2,4-trimethylpentane

and glass beads were added in the tubes and they were boiled for10 min with occasional vortex mixing. The solvent was transferredinto clean tubes, the volumewas adjusted to 5ml and assayed usingthe copper soap method (Kwon and Rhee, 1986) and quantifiedagainst an oleic acid standard curve.

2.3. Malt protease induced changes in wheat proteins

The effect of malt proteases on wheat proteins was studied byincubating 5 g wheat flour in 10 ml malt protease extract (pH 5.8)under gentle shaking at 40 �C. Aliquots were collected, frozen andfreeze dried and ground for analysis using a mortar and pestle.Samples (40 mg) were washed with 0.4 ml 0.5 M NaCl, centrifuged10 000 g � 5 min and the supernatant was discarded. The ethanolsoluble fraction was obtained by extracting twice in 0.4 ml 60%ethanol for 10 min at room temperature. The supernatants werepooled and the pellet was extracted with 0.8 ml 1% SDS/DTT at65 �C for 5 min to yield the SDS/DTT soluble fraction. The proteinprofiles were analysed by lab-on-a-chip capillary electrophoresisusing a Protein230 kit with a molecular weight range 14e230 kDa(Bioanalyzer, Agilent Technologies, Palo Alto, USA) under reducingconditions according to manufacturer’s instructions. The 2100Expert software aligns the sample proteins to the molecular weightladder using internal standards and determines the concentrationsrelative to the peak area of the upper standard. For result evalua-tion, the data was rescaled to match the area of the upper markerwhen necessary.

2.4. Baking and bread characterisation

Wheat bread was baked with malt additions at concentrations0.5%, 1%, 2.5% and 5%. The baking trials were performed using botha constant water level (63%) giving a consistency of 500 BU for thewheat flour used in the trials and water levels optimised to givea consistency of 500 BU determined using the Farinograph. Theoptimised water level ranges were 63.9e67%, 63.9e67.5%, 63.7e66.8% for barley, oat and wheat malt doughs, respectively.

The baking procedure was as follows: the yeast (1.5%; Puratos,Belgium) was activated by dissolving it in 30 �C tap water. The yeastsuspension was mixed with wheat flour (protein (Nx6.25) 12.1%;Hagberg falling number 250; moisture 13.2%, Odlums, Ireland),malt (0, 0.5, 1, 2.5 or 5%), butter (2%; Glanbia plc, Kilkenny, Ireland)and salt (1.2%; Glacia British Salt Ltd., UK) for a total of 8 min usinga Kenwood Chef (Kenwood Manufacturing Co. Ltd., UK). The doughwas rested for 15 min at 30 �C and 85% RH in a proofer (Koma BV,Roremond, Netherlands), scaled into 400 g portions, moulded ina small scale moulder (Machinefabriek Holtkamp BV, Almelo,Holland), placed in tins and proofed for 75 min. The loaves werebaked for 45 min at 190 �C in a deck oven (MIWE, Arnstein,Germany) previously steamed with 700 ml and let cool for 2 h oncooling racks before analysis.

The volume of the loaves was measured using VolScan Profiler(Stable Micro Systems, Surrey, UK) and specific volume calculatedby dividing the volume by the mass of the loaf. Texture profileanalysis was carried out to monitor crumb hardening, usinga TAXT2i texture analyser (Stable Micro Systems, Surrey, UK)equipped with a 25-kg load cell and a 20-mm aluminium cylin-drical probe. The middle of a 25 mm bread slice was compressedtwice to 50% of its original height with a force of 0.98 N and a speedof 5 mm/s. The maximum force during the first compression wasdefined as hardness. Crumb structure was evaluated by imageanalysis using C-cell Imaging System and software (Calibre ControlInternational Ltd., UK). The parameters used were cells/cm2

(number of cells/slice area), wall thickness and net cell elongation(degree of overall elongation).

60%

80%

100%

hibi

tion

O.E. Mäkinen, E.K. Arendt / Journal of Cereal Science 56 (2012) 747e753 749

2.5. Dough rheology

Farinograph and Extensograph characteristics were determinedaccording to AACC methods (Brabender GmbH & Co, Duisburg,Germany). Mixing stability, extensibility and resistance to exten-sion were used in evaluation of the results.

For rheological measurements, the dough samples withoutyeast were mixed for 70 s with a Glutomatic (Falling Number AB,Huddinge, Sweden). The sample was placed in a sealed containerfor 10 min. After the rest, the sample was mounted on a controlledstress rheometer (MCR301, Anton Paar GmbH, Austria) witha cross-hatched parallel plate geometry (50 mm; gap 2 mm) andthe edges of the dough were trimmed and covered with a vacuumgrease/heptane mixture. The sample was covered with a chamberlined with a wet strip of cotton wool and rested for 5 min. A timesweep (f ¼ 1 Hz; g ¼ 0.01%; T ¼ 30 �C) was performed for 85 min,followed by a creep recovery test consisting of 5 min creep phase(s ¼ 250 Pa) and a 20 min recovery phase. By the end of the timesweep, the normal force of all samples was >1 N.

2.6. Confocal laser scanning microscopy (CLSM)

Dough samples were prepared with 0.05% Rhodamine B asdough liquid using the Glutomatic as described under 2.5 andincubated at 30 �C for 90 min. For microscopic observation a smallpiece of dough was placed on a cover slip and flattened by slidinganother cover slip on top. FV300 confocal laser-scanning system(Olympus, Germany) mounted on an Olympus IX80 invertedmicroscope with a 40� dry objectives was used. Fluorescenceimages of 25 optical sections were acquired by scanning the samplealong the optical axis with a 543 nm excitation wavelength anda 535e565 nm emission filter and a micrograph was taken of theprojection of the layers.

2.7. Statistical analysis

Means were compared using one way analysis of variance(ANOVA) at a significance level of p < 0.05 using Sigmaplot 11.0(Systat Software Inc., USA).

3. Results

3.1. Enzyme activities and effect of proteases on wheat proteins

3.1.1. Enzyme activitiesEnzyme activities of the malt flours are reported in Table 1.

Amylolytic activities of oat malt were lower than in barley andwheat malts, but the a-amylase activity was still moderate (49 U/gcompared to 134 and 119 U/g of barley and wheat malts). Reason-ably high a-amylase activity but low diastatic power (related to b-amylase activity) has been previously reported for oat malts(Peterson, 1998). Proteolytic activities of malts ranged from 8.9 for

Table 1Enzyme activities of ground and sieved malts, U/g � SD.

Oat Barley Wheat

a-amylasea 48 � 5.1 b 133.7 � 4.8 a 119.1 � 5.2 cb-amylasea 2.7 � 0.4 b 12.0 � 1.7 a 23.5 � 1.8 cProteaseb 11.9 � 0.3 b 9.4 � 0.3 a 8.9 � 0.5 aLipasec 29.5 � 3.46 b 3.0 � 0.64 a 6.9 � 1.61 a

a One unit of activity corresponds to the liberation of 1 mmol p-nitrophenol/min.b One unit of activity corresponds to the liberation of 1 mg L-leucine/h.c One unit of activity corresponds to the liberation of 1 mmol oleic acid/h under

assay conditions.

wheat malt to 11.9 U/g for oat malt. Oat malt had a high lipaseactivity compared to barley and wheat malts. The fate of lipolyticactivity in oats during germination has been studied previously butwith contradictory results (Kaukovirta-Norja et al., 2004). In thisstudy, the lipase activity of oat malt was higher than in ungermi-nated oat (30 vs. 16 U/g). However, the material used in this studywas flour which makes comparison with previous studies dealingwith the whole kernel difficult. Also, the mechanical properties ofungerminated and germinated grains differ and it is possible thatthe amount of grain layers high in enzyme activities is higher in themalt flour.

3.1.2. Effect of class-specific inhibitors on proteolytic activityMore detailed information about the proteolytic activities of the

malts was attained by using class-specific inhibitors at the doughpH (5.8). The distribution of activities between different classesvaried between the malts, but the predominant activity wascysteine protease inhibited by E-64 (Fig.1). E-64 reduced the azo-casein hydrolysing activity by 50.8%, 36.6% and 29.5% of barley, oatand wheat malts, respectively. It was clearly responsible for themajority of activity in barley malt, with serine (PMSF) and aspartic(pep A) protease activity explaining 10% of the total activity each.The pattern was similar in wheat malt with a little more emphasison serine proteases compared to barley. In oat malt, serine andmetallo (o-phen) proteases contributed another 30% of the activity,but aspartic proteases were responsible for only 6.3% of activity.AlsoMikola and Jones (2000a) reported similar figures for green oatmalt, but in their study, aspartic proteases were not present at pH6.2.

3.1.3. Malt enzyme induced proteolysis of wheat proteinsThe degradation of wheat proteins was studied with protease

extracts and long incubation times, as no changes in the proteinpatterns in doughs treated as in the baking trials were observed.The electropherograms of the SDS/DTT soluble fraction is presentedin Fig.2. The main changes visible in that fraction are that bothLMW-GS (44 and 58 kDa) and HMW-GS (140e220 kDa) showedextensive degradation compared to the control (Balázs et al., 2011).The ethanol soluble fractions were nearly identical, except the twopeaks with MW 207 and 220 kDa that were higher in malt proteasetreated samples than in the control. In the SDS/DDT soluble

0%

20%

40%

Barley Oat Wheat

In

Fig. 1. Inhibition of azocasein hydrolysing activity of malts by class-specific inhibitorsE-64 (light grey), pepstatin A (black), PMSF (white) and o-phen (grey) and non-inhibited (striped).

O.E. Mäkinen, E.K. Arendt / Journal of Cereal Science 56 (2012) 747e753750

fraction, the respective peaks were lower than the control, espe-cially in the oat malt digested samples. This indicates a change insolubility rather than lowering of the MW, possibly also in unex-tractable protein fractions.

The malt proteases showed different affinities towards wheatproteins. The peak at 44 kDawas higher in the barley malt proteasetreated samples thanwheat and oat, while oat had the least impacton the 207 and 220 kDa peaks. According to these data, wheat maltproteases have the overall strongest degrading effect on wheatproteins, which was expected as it is their natural substrate.

3.2. Baking results

3.2.1. Optimised vs. constant waterLoaves were baked with both constant water level (63%) used

for all malt addition levels and water levels optimised to 500 BU foreach recipe. The effects of malts on crumb grain and hardness weremore pronounced with optimised recipes compared to constantwater levels, as the increase of drymatter compensated the effect ofthe malt enzymes on dough and bread when a constant water levelwas used (data not shown).

When using optimised water levels, the maximum maltconcentration was 1%, as higher doses of wheat and barley maltsproduced loaves with extremely sticky crumbs and even hollowloaves. The constant water level approach was chosen for furthertrials, as it enabled the addition of malts at higher levels without anenzyme overdose.

3.2.2. Bread propertiesThe addition of barley and wheat malt darkened the bread crust

but the effect of oat malt was negligible (not shown). The loafspecific volume increased with increasing malt levels (Table 2).Barley and oat malts had the largest effect with 2.5% additionincreasing the specific volume from3.04ml/g to 3.36 and 3.44ml/g,respectively. The effect of wheat malt was significant only at 5%malt with 3.21 ml/g. Loaves baked with 2.5e5% wheat malt and 5%barleymalt had sticky crumbs but this was not observed in oat maltbreads due to much lower amylotic activities. The crumb stickinesswas most pronounced in the wheat malt breads and is associatedwith dextrins produced as a result of a-amylase activity (Every andRoss, 1996).

Image analysis of the bread crumb showed that the addition ofbarley and wheat malts led to a more open crumb grain, whereasoat malt had little effect (Table 2). The number of cells per cm2

decreased significantly at levels >1% of barley or wheat malt,indicating cell coalescence leading to larger and fewer cells. Higherlevels of malts increased the wall thickness and decreased the cellelongation value meaning rounder cells. The effects of barley andwheat malts on the crumb grain were nearly identical. The desired

Fig. 2. Electropherogramms of SDS/DTT (a) and ethanol (b) soluble fractions of wheat flourbarley (grey) and wheat (grey dotted).

crumb grain characteristics depend on the product type. In theproduction of white pan bread, a fine crumb with small to inter-mediate cells and thin cell walls is preferred and thus the additionof barley andwheatmalts can be considered as deteriorating for thecrumb grain (Hayman et al., 1998).

Crumb hardening was studied by following the increase of thecrumb hardness over 5 days. The use of barley and wheat malts at>2.5% led to a slightly softer crumb in fresh bread compared to thecontrol (Table 2). The anti-staling effect started to show after 2 d ofstorage at malt levels >0.5% for barley and wheat malts witha dependency on the level of malt (not shown). Interestingly, after5 d storage, all loaves with barley malt had a softer crumbcompared to the control but there were no significant differencesbetween the different levels of malt addition. Also in wheat maltbreads, the anti-staling effect was less influenced by the amount ofmalt, suggesting that even a low level of malt addition is enough todecrease the staling rate over a longer shelf life. Oat malt started toaffect the staling rate significantly only after 5 d storage.

3.2.3. Dough rheologyThe rheological properties of dough were studied using a creep

recovery test where the strain of the material is measured undera steady shear stress (creep phase) and after the stress is removed(recovery phase). These data were compared to the propertiesunder uniaxial extension measured using the Brabenderextensograph.

The creep recovery parameters evaluated were the maximumcreep strain (gc), maximum recovery strain (gr) and elastic recovery(R% ¼ gr/gc*100). gc increased as a result of malt addition, indi-cating higher deformability (Table 3). Wheat malt had the strongesteffect on gc resulting in an increase from 106% (control) to 315%with 5% malt. The effect of oat and barley malts were similar, bothyielding maximum values of ca. 200%. gr increased significantly indoughs containing 5% oat, �1% wheat and �2.5% barley malt.However, the consideration of gr alone as a measure of elasticitymay lead to erroneous conclusions because, with high creepdeformation, also the recovery values are high. Therefore theirratio, R%, gives a more accurate measure of elasticity, assuming thecreep strain has reached a steady state. According to Van Bockstaeleet al. (2011) there was no change in the amount of recoverablestrain over time and suggested 5 min as an adequate creep time forwheat doughs. All malts decreased R%, the minimum values being18, 22, 25 and 18% for wheat, oat and barley, respectively. Thus,wheat malt led to themost drastic loss of elasticity. Also the storagemodulus at the end of the time sweep (G’85 min) decreased in thesamples containing 2.5 and 5% wheat malt, but other changes werenot significant (not shown).

Resistance to extension (Rm) decreased only slightly in doughswith barley and wheat malt, but a large decrease from 810 to 530

digested for 24 h with protease extracts from malts: control (black), oat (black dotted),

Table 2Bread and crumb characteristicsa.

Volume (ml/g) Crumb hardness (N) Crumb structure

0 d 5 d Cells/cm2 Wall thickness (mm) b Elongation

Control 3.04 � 0.07 a 5.49 � 0.4 a 20.37 � 1.4 a 64.0 � 3.4 a 0.45 a 1.21 � 0.03 aOat 0.5% 3.11 � 0.09 a 5.3 � 0.5 a 18.0 � 1.7 ab 62.8 � 2.7 a 0.45 a 1.25 � 0.06 b

1% 3.09 � 0.05 a 4.8 � 0.6 a 18.2 � 2.4 b 61.8 � 2.4 ab 0.45 a 1.19 � 0.04 ac2.5% 3.44 � 0.09 b 4.6 � 0.6 a 16.6 � 2.0 c 61.2 � 4.1 ab 0.45 a 1.22 � 0.05 a

5% 3.44 � 0.07 b 4.8 � 0.3 a 15.9 � 1.2 c 58.7 � 2.1 b 0.46 a 1.16 � 0.05 cBarley 0.5% 3.13 � 0.11 a 5.1 � 0.6 a 15.0 � 1.2 b 65.5 � 3.1 a 0.45 a 1.16 � 0.05 a

1% 3.21 � 0.10 ab 5.0 � 0.5 a 15.6 � 0.9 b 59.1 � 2.5 b 0.47 a 1.17 � 0.05 b2.5% 3.36 � 0.15 b 4.0 � 0.7 b 14.8 � 1.1 b 54.7 � 2.9 c 0.48 b 1.14 � 0.04 b

5% 3.38 � 0.13 b 2.7 � 0.4 c 14.1 � 0.6 b 51.4 � 2.4 c 0.50 c 1.08 � 0.06 cWheat 0.5% 3.08 � 0.09 a 4.5 � 0.7 a 16.5 � 1.2 b 61.8 � 2.6 a 0.45 a 1.22 � 0.04 a

1% 3.15 � 0.08 a 4.4 � 0.7 a 15.7 � 1.3 b 59.5 � 2.7 b 0.46 a 1.17 � 0.05 a2.5% 3.16 � 0.10 a 3.2 � 0.4 b 13.7 � 1.1 c 54.4 � 2.0 c 0.48 b 1.15 � 0.03 b

5% 3.21 � 0.12 b 2.5 � 0.5 b 13.5 � 0.8 c 50.9 � 2.1 c 0.50 c 1.06 � 0.05 c

a The results are significant with a significance level of 95% (p < 0.05). Values are mean values of nine replicates (�standard deviations). Mean values followed by the sameletter are not significantly different from each other.

b The standard deviation was <0.012 in all samples.

O.E. Mäkinen, E.K. Arendt / Journal of Cereal Science 56 (2012) 747e753 751

BU occurred with only 0.5% oat malt and decreased further withhigher levels of added malt. Also, only oat malt had an increasingeffect on the extensibility (E). Rm or E did not correlate with creeprecovery parameters and in fact shows a very different trend amongthe samples than the creep recovery test. Also Van Bockstaele et al.(2008) reported no correlation between creep recovery parametersand Alveograph tenacity (biaxial equivalent for Rm), although therewas a significant correlation between those and extensibility.

3.3. CLSM

The protein networks of the doughs appear white in themicrographs (Fig. 3). The micrographs of the control and doughsprepared with 5% barley and wheat malts looked very similar witha fibrous, continuous protein network (3a, c and b). The network inthe sample incubated with oat malt (3b) was finer and less ordered.Also the large protein fibrils visible in the other samples wereabsent.

4. Discussion

The incorporation of oat malt in cereal based foods improves thenutritional properties while providing flavour and aroma(Kaukovirta-Norja et al., 2004; Larsson et al., 1996). However, thehigh enzyme activities can cause problems during processing andin the final product quality. In this study, the effects of oat malt onwheat dough and bread properties were studied and compared

Table 3Rheological properties of the doughsa.

Creep recovery test

gc,max (%) gr,max (%) R%b

Control 106 � 9.4 a 42 � 2.3 a 39.61 �Oat 0.5% 135 � 6.1 a 43 � 0.6 a 31.88 �

1% 171 � 5.1 ab 47 � 0.6 ab 24.49 �2.5% 187 � 38.0 ab 46 � 3.7 ab 27.79 �

5% 234 � 51.0 b 50 � 2.5 b 21.71 �Barley 0.5% 124 � 21.9 a 43 � 3.4 a 35.28 �

1% 129 � 23.6 a 47 � 3.8 a 37.05 �2.5% 228 � 44.6 b 57 � 4.0 bc 25.71 �

5% 206 � 35.0 b 51 � 3.2 b 25.20 �Wheat 0.5% 151 � 30.7 ab 50 � 3.3 a 33.34 �

1% 232 � 19.9 bc 53 � 1.7 b 22.95 �2.5% 225 � 28.2 b 51 � 1.5 b 22.99 �

5% 315 � 52.9 c 56 � 4.6 b 17.98 �a The results are significant with a significance level of 95% (p < 0.05). Values are mean

letter are not significantly different from each other.b R% is the percentage of recovery from the maximum creep deformation gr,max/gc,max

with barley and wheat malts. The oat malt used was lipase high andamylolytic low, while barley and wheat malts dominated inamylolytic activities. Overall, proteolytic activity levels were similarin all malts and cysteine proteases were dominant. However, oatmalt had lower aspartic- and increased metalloprotease activitycompared to other malts. During germination the cysteine prote-ases become dominant, replacing the aspartic proteases of dormantcereals, thus playing a key role in the mobilisation of storageproteins (Capocchi et al., 2000; Koehler and Ho, 1990; Mikola andJones, 2000b).

Only oat malt increased the loaf specific volume without havinga detrimental effect on the crumb. In order to yield a loaf witha high volume and fine crumb, the membranes between the gascells have to be extensible enough to allow the expansion, yet havesufficient strength to prevent premature rupture of the cell wallsunder extension (Bloksma, 1990; Kokelaar et al., 1996; Vliet et al.,1992). A variety of rheological methods have been employed todetermine these qualities either directly or indirectly under verydifferent deformation conditions. Properties related to good bakingquality wheats are e.g. high elasticity, high resistance to extensionand extensibility and high maximum creep and recovery strains,(Bloksma, 1990; Kokelaar et al., 1996; Van Bockstaele et al., 2008).

In this study, malt addition increased the maximum creepstrain and decreased the recoverable strain in the orderwheat > oat > barley. Van Bockstaele et al. (2008) reporteda positive correlation between gc with a creep stress of 250 Pa but,in this studywheat with the highest impact on gc had a detrimental

Extensograph Farinograph

Rm (BU) E (cm) Stability (min)

1.42 a 810 � 70.7 a 13.63 � 0.53 a 5.37 � 0.68 a1.65 b 530 � 70.7 b 13.75 � 0.35 a 3.02 � 0 23 b0.91 bc 500 � 28.3 b 14.75 � 0.35 b 2.93 � 0.16 b4.59 bc 400 � 28.3 c 16.25 � 0.35 c 3 22 � 0.40 b3.52c 290 � 14.1 d 16.50 � 0.71 c 3.30 � 0.48 b3.89 a 810 � 14.1 a 13.50 � 0.71 a 4.25 � 0.35 b6.42 a 710 � 99.0 ab 14.50 � 0.71 a 3.63 � 0.18 bc6.86 b 630 � 127.3 b 13.50 � 1.50 a 2.75 � 0.35 c2.97 b 670 � 14.1 b 14.50 � 0.71 a 2.43 � 0.25 cd4.50 a 720 � 56.6 b 13.00 � 0.71 a 3.28 � 0.67 b1.22 b 570 � 14.1 c 13.75 � 0.35 a 2.88 � 0.18 bc2.08 b 600 � 28.3 c 13.00 � 1.41 a 1.93 � 0.25 cd1.84 b 590 � 42.4 c 13.75 � 0.35 a 1.73 � 0.04 d

values of three replicates (�standard deviations). Mean values followed by the same

*100.

Fig. 3. Confocal laser scanning micrographs of doughs with 5% malts after 90 min incubation: a. control, b. oat, c. barley, d. wheat. Magnification 40x, bar corresponds to 50 mm.

O.E. Mäkinen, E.K. Arendt / Journal of Cereal Science 56 (2012) 747e753752

effect on volume at high levels of addition. There is likely to bea range of gc values typical for good baking quality that wasexceeded due to excessive softening of the dough by wheat malt.The resistance to extension (Rm) showed a large decrease using oatmalt and little effect with other malts with only oat malt increasingthe extensibility (E).

The responses of HMW polymer networks to large strain shearand extension are very different (Dobraszczyk and Morgenstern,2003). In the Extensograph measurement, the sample is extendeduntil it ruptures and Rm reflects the tensile strength and E thedeformation of the material. When tensile stress is applied on anelastic polymer network, extension is caused by breakage of non-covalent bonds and slippage of chains through entanglementnodes. If the rate of chain slippage exceeds the rate of deformation,thematerial yields high E and low Rmvalues. It has been shown thatmainly the unextractable polymeric proteins (high MW) in wheatcontribute to Rm and a shift of MW distribution to lower valuesdecreases the tensile strength of the dough (Southan andMacRitchie, 1999).

The electrophoretic study of wheat flour proteins digested withmalt protease extracts revealed that wheat proteases had thestrongest impact on overall protein hydrolysis despite the relativelylow amount of cysteine proteases. Capocchi et al. (2000) reportedthat wheat cysteine proteases are responsible for the hydrolysis ofgluten proteins contributing to elastic properties of the dough.However, samples digested with oat malt extract had the highestpeaks above 200 kDa in both ethanol and SDS/DTT soluble fractions,which may indicate changes in the solubility of the unextractableproteins determining the tensile properties of the dough (Southanand MacRitchie, 1999). Oat malt doughs also showed a verydifferent protein network structure compared to the other doughsas observed by CLSM. Even subtle protein changes can alter therheological properties but remain unobservable using molecularweight based separation methods (Dobraszczyk and Morgenstern,2003).

Despite the weakening effect of oat malt, it gave the highest loafvolumes and a superior crumb grain compared to wheat and barleymalts. This may be due to its high lipolytic activity capable of acting

on the gaseliquid interface, thus imparting a stabilising effect togas cells (Gan et al., 1995). Lipolytic enzymes alter the polarity oflipids and thus their surface properties and may have a similareffect in baking as surfactants (Primo-Martín et al., 2006). Theimpact of the high lipolytic activity in oat malt may compensate thepossible decrease in bubble stability caused by the weakening ofthe gluten. Also a high concentration of a lipid-binding protein(tryptophanin) on the gaseliquid interface of defatted oat foamshas been identified recently (Kaukonen et al., 2011). However, thepresence and functionality of tryptophanin in malted oat is not yetknown.

Despite the differences in rheological properties of wheat andbarley malt doughs, the undesired changes in crumb grain werenearly identical. Therefore, the changesmay be related to the actionof amylases during baking. Hydrolysis of the gelatinising starchduring early baking reduces the viscosity of the bulk phase andmaylead to coalescence of the gas cells. Indeed Hayman et al. (1998)reported that coalescence of cells occurs during early stages ofbaking when the loaf expands rapidly.

All malts retarded crumb hardening during storage, but toa lesser extent than commercially available thermostable microbialamylases. Amylolysis during proofing is limited to mostly damagedstarch (Goesaert et al., 2006). The mainwindow of activity is duringbaking between the starch gelatinisation (55e65 �C) and theenzyme inactivation temperatures (cereal a-amylases 75e80 �C).During this time, a-amylases rapidly cleave internal bonds ofamylopectin molecules, resulting in a rapid decrease in MW(Perten, 1964). This activity period is elongated when usingcommercial microbial amylases due to their thermostability andalso these are often enzymes with very specific activities, providingretardation of crumb hardening without the negative effects onbread quality (Poutanen, 1997).

5. Conclusion

Oat malt can be used in wheat bread at least up to a concen-tration of 5%. Oat malt weakened the extensional properties ofdough, but increased loaf specific volume and did not damage the

O.E. Mäkinen, E.K. Arendt / Journal of Cereal Science 56 (2012) 747e753 753

crumb grain, possibly because of an improvement of gas cellstability due to a high lipase activity. The addition of barley andwheat malts led to a sticky and coarse crumb as a result of highamylolytic activities.

Acknowledgements

The authors wish to thank Andrea Faltermaier (TUM) for malt-ing the wheat and Dr. Deborah Waters for proof reading themanuscript. This study was funded by the Food InstitutionalResearch Measure administered by the Department of Agriculture,Fisheries and Food (Ireland).

References

Balázs, G., Baracskai, I., Nádosi, M., Harasztos, A., Békés, F., Tömösközi, S., 2011. Lab-on-a-chip technology in cereal science: analytical properties and possibleapplication areas. Acta Alimentaria, 1e13.

Bloksma, A., 1990. Rheology of the breadmaking process. Cereal Foods World 35,228e236.

Butzner, J.D., 2011. Pure oats and the gluten-free diet. Journal of Parenteral andEnteral Nutrition 35, 447.

Capocchi, A., Cinollo, M., Galleschi, L., Saviozzi, F., Calucci, L., Pinzino, C.,Zandomeneghi, M., 2000. Degradation of gluten by proteases from dry andgerminating wheat (Triticum durum) seeds: an in vitro approach to storageprotein mobilization. Journal of Agricultural and Food Chemistry 48, 6271e6279.

Dobraszczyk, B.J., Morgenstern, M.P., 2003. Rheology and the breadmaking process.Journal of Cereal Science 38, 229e245.

Every, D., Ross, M., 1996. The role of dextrins in the stickiness of bread crumb madefrom pre-harvest sprouted wheat or flour containing exogenous alpha-amylase.Journal of Cereal Science 23, 247e256.

Gan, Z., Ellis, P., Schofield, J., 1995. Gas cell stabilisation and gas retention in wheatbread dough. Journal of Cereal Science 21, 215e230.

Goesaert, H., Gebruers, K., Courtin, C., Brijs, K., Delcour, J., 2006. Enzymes inbreadmaking. In: Hui, Y.H. (Ed.), Bakery Products e Science and Technology.Blackwell Publishing, Ames, Iowa, USA, pp. 337e364.

Goesaert, H., Slade, L., Levine, H., Delcour, J.A., 2009. Amylases and bread firming e

an integrated view. Journal of Cereal Science 50, 345e352.Hayman, D.A., Hoseney, R., Faubion, J., 1998. Bread crumb grain development during

baking. Cereal Chemistry 75, 577e580.Kaukonen, O., Sontag-Strohm, T., Salovaara, H., Lampi, A.M., Sibakov, J., Loponen, J.,

2011. Foaming of differently processed oats: role of nonpolar lipids and tryp-tophanin proteins. Cereal Chemistry 88, 239e244.

Kaukovirta-Norja, A., Wilhelmson, A., Poutanen, K., 2004. Germination: a means toimprove the functionality of oat. Agricultural and Food Science 13, 100e112.

Kemppainen, T., Janatuinen, E., Holm, K., Kosma, V.-M., Heikkinen, M., Mäki, M.,Laurila, K., Uusitupa, M., Julkunen, R., 2007. No observed local immunologicalresponse at cell level after five years of oats in adult coeliac disease. Scandi-navian Journal of Gastroenterology 42, 54e59.

Koehler, S.M., Ho, T.H.D., 1990. A major gibberellic acid-induced barley aleuronecysteine proteinase which digests hordein: purification and characterization.Plant Physiology 94, 251e258.

Kokelaar, J.J., van Vliet, T., Prins, A., 1996. Strain hardening properties and extensi-bility of flour and gluten doughs in relation to breadmaking performance.Journal of Cereal Science 24, 199e214.

Kwon, D.Y., Rhee, J.S., 1986. A simple and rapid colorimetric method for determi-nation of free fatty acids for lipase assay. Journal of the American Oil Chemists’Society 63, 89e92.

Larsson, M., Rossander-Hulthén, L., Sandström, B., Sandberg, A., 1996. Improved zincand iron absorption from breakfast meals containing malted oats with reducedphytate content. British Journal of Nutrition 76, 677e688.

Matlashewski, G., Urquhart, A., Sahasrabudhe, M., Altosaar, I., 1982. Lipase activity inoat flour suspensions and soluble extracts. Cereal Chemistry 59, 418e422.

Mikola, M., Jones, B., 2000a. Electrophoretic and in solution analyses of endopro-teinases extracted from germinated oats. Journal of Cereal Science 31, 15e23.

Mikola, M., Jones, B.L., 2000b. Characterization of oat endoproteinases that hydro-lyze oat globulins. Cereal Chemistry 77, 572e577.

Oksman-Caldentey, K.-M., Kaukovirta-Norja, A., Heiniö, R.-L., Kleemola, T.,Lehtinen, P., Mikola, M., Sontag-Strohm, T., Pihlava, J.-M., Poutanen, K., 2001.Kauran prosessointi uusiksi elintarvikkeiksi (Biotechnical processing of oat fornovel food ingredients). In: Ryhänen, E.-L., Salo, R. (Eds.), Food Cluster ResearchProgramme, Final Report. MTT Publications, Jokioinen, Finland, pp. 11e22 (inFinnish).

Perten, H., 1964. Application of the falling number method for evaluating alpha-amylase activity. Cereal Chemistry 41, 127e140.

Peterson, D.M., 1998. Malting oats: effects on chemical composition of hull-less andhulled genotypes. Cereal Chemistry 75, 230e234.

Pike, P., Abdel-Aal, E., McElroy, A., 2007. Antioxidant activity of oat malt extracts inaccelerated corn oil oxidation. Journal of the American Oil Chemists’ Society 84,663e667.

Poutanen, K., 1997. Enzymes: an important tool in the improvement of the qualityof cereal foods. Trends in Food Science & Technology 8, 300e306.

Primo-Martín, C., Hamer, R.J., de Jongh, H.H.J., 2006. Surface layer properties ofdough liquor components: are they key parameters in gas retention in breaddough? Food Biophysics 1, 83e93.

Southan, M., MacRitchie, F., 1999. Molecular weight distribution of wheat proteins.Cereal Chemistry 76, 827e836.

Van Bockstaele, F., De Leyn, I., Eeckhout, M., Dewettinck, K., 2008. Rheologicalproperties of wheat flour dough and the relationship with bread volume. I.Creep-recovery measurements. Cereal Chemistry 85, 753e761.

Van Bockstaele, F., De Leyn, I., Eeckhout, M., Dewettinck, K., 2011. Non-linear creep-recovery measurements as a tool for evaluating the viscoelastic properties ofwheat flour dough. Journal of Food Engineering 107, 50e59.

Vliet, T., Janssen, A., Bloksma, A., Walstra, P., 1992. Strain hardening of dough asa requirement for gas retention. Journal of Texture Studies 23, 439e460.