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Journal of Cereal Science 56 (2012) 181e188

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Journal of Cereal Science

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Influence of phenolic acid content on sensory perception of bread and crackersmade from red or white wheat

Carolyn A. Challacombe a, El-Sayed M. Abdel-Aal b, Koushik Seetharaman a, Lisa M. Duizer a,*aUniversity of Guelph, Department of Food Science, 50 Stone Rd E, Guelph, ON N1G 2W1, CanadabGuelph Food Research Centre, Agriculture and Agri-Food Canada, 93 Stone Rd W, Guelph, ON N1G 5C9, Canada

a r t i c l e i n f o

Article history:Received 9 August 2011Received in revised form15 March 2012Accepted 26 March 2012

Keywords:Phenolic contentsRed and white wheatBreadCrackers

* Corresponding author. Tel.: þ1 519 824 4120x534E-mail address: lduizer@uoguelph.ca (L.M. Duizer)

0733-5210/$ e see front matter � 2012 Elsevier Ltd.doi:10.1016/j.jcs.2012.03.006

a b s t r a c t

Despite the health benefits of wholegrain, consumer acceptance of wholegrain products remains an issuedue to the presence of characteristic flavours that some consumers consider to be unacceptable. It washypothesized that phenolic acids could be contributing to the perceived unacceptable flavours describedin wholegrain products. The purpose of this study was to examine the relationship between totalphenolic acid content (TPAC) and phenolic acids as quantified by HPLC, to the sensory properties ofwholegrain products using partial least squares (PLS) mapping. Red and white wheat flours wereinvestigated in an intermediate (bread) and low (cracker) moisture product system. Red and white wheatdemonstrated different phenolic acid profiles despite having similar TPAC. Within the bread crumb, thefree and bound phenolic acids provided the best predictive scores; whereas only bound phenolic acidsprovided high predictive scores in crackers. This suggests that the contribution of phenolic acids toflavour characteristics of wholegrain products varies depending upon the product moisture.

� 2012 Elsevier Ltd. All rights reserved.

1. Introduction

There has been increasing interest in the use of wholegrains infood products due to numerous health benefits associated withwholegrain consumption. These health benefits are attributed to,amongst others, the phenolic acids inherent within wheat.Phenolic acids are naturally occurring secondary plant metabolitesthat are required for normal plant functioning. Phenolic acids actas antioxidants, scavenge free radicals and inhibit lipid oxidation(Shahidi and Wanasundra, 1992). While there are a number ofphenolic acids present within wheat, ferulic acid appears to bepredominant and is stated to be a major contributor to this anti-oxidant activity (Anson et al., 2008; Kim et al., 2006; Mpofu et al.,2006; Zhou et al., 2004).

Within wheat, the majority of phenolic acids are located in thebran portion of the kernel (Abdel-Aal et al., 2001; Adom et al., 2005;Rybka et al., 1993), and thus, these acids have been investigated asa contributing factor to seed coat colour. Results regarding thisissue, however, are contradictory. While some researchers haveobserved that red bran contains higher levels of phenolic acids thanwhite bran (Kim et al., 2006), others have found no correlations

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between seed coat colour and total phenolic content (TPC) (Betaet al., 2004). Additionally, published values for the types ofphenolic acids present in red and white wheat bran vary. Kequanet al. (2005) reported that red wheat bran contains greaterconcentrations of ferulic and syringic acids when compared towhite wheat bran. Others, however, have reported that red wheatcontains a higher concentration of sinapinic but lower concentra-tions of ferulic acid (Liu et al., 2010). Phenolic acid levels areinfluenced by genetics, environmental factors and other stressors(Bravo, 1998; Mpofu et al., 2006). These factors, particularlygenetics, could be responsible for the varying phenolic acid profileswhich are observed between red and white wheat.

From a sensory attribute perspective, it is reported that phenoliccompounds elicit unacceptable flavours within plant foods andtheir products (Drewnowski and Gomez-Carneros, 2000). Whole-grain products have been described as havingmore intense flavoursthan their refined counterparts and, within rye based products, thishas been associated with the increased concentration of phenolicacids within the bran portion of the grain (Heiniö et al., 2003,2008). Furthermore, a number of specific sensory properties havebeen associated with phenolics within grain products and includebitter taste (Busch et al., 2006; Heiniö et al., 2008; Huang and Zayas,1991; Kobue-Lekalake et al., 2007; Robichaud and Noble, 1990),astringency (Huang and Zayas, 1991; Kobue-Lekalake et al., 2007;Robichaud and Noble, 1990), sour taste (Huang and Zayas, 1991),cereal flavour and germ-like flavour (Heiniö et al., 2008). The

C.A. Challacombe et al. / Journal of Cereal Science 56 (2012) 181e188182

association between phenolic acids and sensory properties of manygrain-based products has been studied, including corn (Huang andZayas, 1991), rye (Heiniö et al., 2008), oats (Molteberg et al., 1996)and sorghum (Kobue-Lekalake et al., 2007). There is, however,limited research into this association as it relates to wheat basedproducts. The objective of this paper was to quantify specificphenolic acids commonly present withinwholegrain red and whitewheat products and to determine if a relationship existed betweenthe sensory properties of intermediate moisture (bread) productsand lowmoisture (cracker) products madewith flours from red andwhite wheat and phenolic acid content of the wheat as well as theproducts.

2. Materials and methods

2.1. Materials

Commercial hard and soft wheat flours were provided by KraftMississauga Mill (Mississauga, ON). Both the hard and soft flourswere sourced from either red wheat or white wheat and were thenmilled to different particle sizes (coarse and fine). This resulted infour treatments (red coarse, red fine, white coarse and white fine)for each flour type.

The majority (84%) of the fine wholegrain flour passed througha 149 mm sieve. The coarse flour had a particle size distributionwithless than 20% passing through the 149 mm sieve, less than 5%passing through the 840 mm sieve and a relatively even particle sizedistribution across the remaining five sieves (162e710 mm). Themoisture contents of the flours ranged from 10.6% to 13.0% for thehard flours and 10.3e12.1% for the soft flours. The protein content(N� 5.7) of the refined flours was 11.9% and 6.5% for hard and softflours, respectively. The protein content of the four wholegrainflours ranged from 11.8% to 12.4% for the hard flours and 8.2e9.7%for the soft wheat flours.

The four hard flour treatments were used tomake bread and thefour soft flour treatments were used to make crackers. Bread andcrackers were also made from refined flour of each type (hard orsoft) which was included as controls in the study.

2.2. Bread preparation

Breadwasprepared fromthehardfloursusing the standardAACCmethod 10-10.03 (AACC, 1999). Optimal water absorption wasdetermined by recording the amount of water required to reach the500 BU consistency line on a Farinograph�-E (C.W. Brabender, SouthHackensack,NJ). The averagemoisture of the crumbwas42% (�1.44)while the average moisture of the crust was 25% (�0.95). The crustwas separated by cutting 0.5 cm into the bread.

2.3. Cracker preparation

Chemically leavened crackers were produced at Reading BakerySystems (Reading, PA) with the soft flours. The crackers containedflour (100% flour basis; fb), 7% fb shortening, 4% fb sugar, 0.06% fbmonocalcium phosphate, 0.06% fb sodium bicarbonate, 0.8% fb saltand 40e44% fb water, depending upon the flour type. The doughwas sheeted to 0.2 cm thick and was baked using a 2-zone PRISMoven and dryer (Reading Bakery Systems, Reading, PA). Once atroom temperature, the products were sealed into foil bags. Finalproduct moistures were approximately 3% (�0.43).

2.4. Analysis of phenolic content

Both the flour used to prepare the products and the productsthemselves were analyzed for phenolic content. The bread and

cracker were prepared for phenolic content analysis by freezedrying the samples (Virtis Unitop 600 SL, Gardiner, NY). For thebread samples, the crust was separated from the crumb of thebread before freeze drying and each component was analyzedseparately.

Total phenolic acid content (TPAC) of free and insoluble boundphenolic acids was determined based on a modifiedFolineCiocalteu method (Beta et al., 2004). For extraction of thephenolic acids from the flour and from the baked products, 0.3 g(flour) or 0.5 g (baked products) were extracted using 5 mL 80:20MeOH:H2O. Samples were shaken at 350 rpm for 30 min usinga shaker (New Brunswick Scientific G24 Environmental incubatorshaker, Edison, NJ). The samples were centrifuged (Thermo Scien-tific, Sorvall RTI, Waltham, MA) at 10,000 rpm (w15,000 g) for10 min and the supernatant was decanted. This was repeateda second time and the supernatants were combined. A portion ofthe combined supernatants were evaporated under nitrogen andreconstituted to 1 mL with deionized water to constitute the freephenolic acids. The remaining residue was washed with 10 mL ofhexane before 5 mL of 2 M NaOH was added. The samples wereflushed with nitrogen before being shaken for 1 h at 350 rpm.Samples were left for approximately 20 h in darkness before beingacidified to a pH of 2 using HCl (2 M). The phenolic acids wereextracted three times using 1:1 ethyl ether and ethyl acetate. Theorganic layers were separated each time and pooled. The organicsolvents were evaporated to dryness under nitrogen and recon-stituted to 2 mL using deionized water to comprise the boundfraction of the phenolic acids.

For TPAC determination, 0.25 mL of the free or bound phenolicacid extracts was combined with 0.25 mL of freshly diluted (1:1)FolineCiocalteu reagent. The mixture was allowed to sit for 5 minbefore 0.5 mL sodium carbonate and 4 mL of water was added.Samples were vortexed and placed in darkness for 30 min. Sampleswere centrifuged (Thermo Scientific Sorvall RTI, Waltham, MA)before readings were taken at 725 nm using a Varian Inc. Cary 1 BioUV Visible spectrophotometer (Palo Alto, CA). Means and standarddeviations of duplicates are reported as ferulic acid equivalents ona flour dry weight basis.

High performance liquid chromatography (HPLC) was utilized toquantify specific free and bound phenolic acids. The free and boundextracts used in the HPLC analysis were prepared using the sametechniques as those previously described. An Agilent Series 1100HPLC equipped with a diode array detector (DAD) and a SupelcosilLC-18 column (SigmaeAldrich, Oakville, ON) was utilized for theseparation and quantification of the phenolic acids. Detection wasperformed at five wavelengths (260, 275, 300, 320, 330 nm) inwhich the phenolic acid was quantified at its maximum absorptionwavelength. A gradient elution using 6% formic acid and 6% formicacid in acetonitrile was used at a flow rate of 1 mL/min. Standardsprocured from SigmaeAldrich (Oakville, ON) were run during eachrun to verify retention times and the UV/Vis spectra.

2.5. Sensory analysis

The sensory attributes of the baked products were evaluatedusing a trained descriptive analysis panel. Participants for thedescriptive panel were recruited from Guelph, Ontario andthe surrounding community. Ethics approval was obtained fromthe University of Guelph Research Ethics Board (REB#09MY20).Responses were collected using computerized data collectionsoftware Compusense Five � 2008 (Compusense, Guelph, ON). Twoseparate panels, utilizing the same panelists, were conducted forthe bread and crackers. Panelists were selected based on their tasteacuity, availability and interest in the project. Thirteen panelistsparticipated on the bread panel and were trained for 20 sessions.

C.A. Challacombe et al. / Journal of Cereal Science 56 (2012) 181e188 183

For the cracker panel, the panel consisted of 10 participants whowere trained for 13 sessions. A shorter time frame was needed forthe cracker training, as the panelists involved in this panel haddeveloped expertise in sensory techniques during training andtesting for the bread samples. At the beginning of the training,panelists generated and defined terminology related to the taste,flavour and texture for each product. For the bread, 11 attributesrelated to tastes and flavours of the breads while 14 attributes weregenerated for tastes and flavours as well as textures of the crackers.Additional training involved familiarization with the attributes aswell as practicing with the scales.

Between tastings, a slice of Gala apple and a glass of water wereprovided to cleanse the palate. Samples were designated randomthree digit codes and presented in a randomized order with a 3 minbreak between each sample. As there were visible differencesbetween the products, all testing was conducted under red light.Samples were evaluated on a 15 cm line scale.

2.5.1. BreadThe breads to be tested by sensory evaluation were baked 24 h

in advance of each testing day. On the day of testing, the productswere sliced into 2.3 cmwidths using aWaring Pro FS150 food slicer(Torrington, CT). The crust was removed from the crumb and eachcomponent was evaluated by the panel separately. The crumb wassliced into 2.3 cm� 2.3 cm� 1.3 cm pieces while the crust wassliced into 1.3 cm� 2.3 cm� 0.5 cm pieces. Three samples of thecrumb and crust were placed in individual sealed plastic bags foreach panelist for not more than 1 h prior to testing. A total of fourrepetitions of testing were completed.

Table 1Differences in bound phenolic acids for red and white wheat based wholegrain flours ancracker from soft flour).

Hard flour Bread crumb Bread

Red White Red White Red

Total phenolic content (mg FAE/g flour basis)Means 1218.5aa,b 1370.0a 1048.7b 1393.2a 1160St dev 174.22 55.21 184.60 188.51 90

Hydroxybenzoic acids (mg/g flour basis)p-Hydroxybenzoic acidMeans 1.7b 2.1a 1.2b 2.2a 2St dev 0.22 0.33 0.17 0.39 0

VanillicMeans 4.5a 4.7a 2.4b 3.6a 2St dev 1.09 1.08 0.40 0.30 0

SyringicMeans 2.4a 2.4a 1.9b 2.7a 2St dev 0.17 0.75 0.25 0.82 0

Hydroxycinnamic acids (mg/g flour basis)CaffeicMeans 1.3a 0.8a 2.2a 3.0a 8St dev 0.84 0.77 0.85 0.60 0

p-CoumaricMeans 9.4b 11.8a 4.5b 14.0a 9St dev 1.32 1.49 0.81 1.39 0

FerulicMeans 582.7a 560.5a 308.0b 635.7a 544St dev 116.40 103.96 48.13 80.99 37

SinapinicMeans 4.2a 2.7b 3.9b 7.4a 10St dev 2.56 0.78 2.56 1.39 5

FAE¼ ferulic acid equivalents.a Means for red and white wheat within a product type (hard flour, crumb, crust, soft flb n¼ 4 (2 particle sizes * 2 reps).

2.5.2. CrackerTwo portions of each sample 4.6 cm� 4.6 cm� 0.3 cm were

provided in sealed plastic bags during the testing period. Theproducts were placed in the plastic bags for not more than 1 h priorto testing. Samples prepared from each of the five flours wereevaluated in one session and a total of three repetitions of testingwere completed.

2.6. Statistical analysis

To evaluate the effect of wheat colour and particle size on TPACand individual phenolic acids, a 2-way ANOVA with interaction(wheat colour, particle size and wheat colour * particle size) wasconducted (SAS version 9.2; SAS Institute Inc., Cary, NC). Wheresignificant differences were observed amongst the main effects,a post hoc Tukey’s Honestly Significant Difference (HSD) test wasconducted. Separate analyses were conducted on phenolic datacollected for the flours, the crackers, the bread crumb and the breadcrust. Given that the TPAC of the refined flour was low and fewphenolic acids were detected in the refined flour and productsmade from the flour, the refined flour data were not included in thestatistical analysis of the phenolic data.

To determine if significant differences in sensory propertiesexisted amongst products manufactured from the wholegrain (redand white wheat at different particle sizes) and refined flours, thesensory data were analyzed by a 3-way ANOVA (sample, replica-tions, judges). Where significant differences amongst samples wereobserved, a post hoc Tukey’s HSD was conducted.

d baked products made from the flours (bread crumb and crust from hard flour and

crust Soft flour Cracker

White Red White Red White

.3b 1442.4a 1489.3a 1349.8b 1283.2b 1403.5a

.41 227.83 98.23 204.90 172.83 111.04

.0b 2.8a 1.9a 1.6a 2.3a 1.8b

.08 0.39 0.10 0.45 0.42 0.15

.8b 3.8a 3.9a 3.7a 4.6a 3.5b

.17 0.38 0.64 0.73 1.20 0.47

.5a 2.6a 2.6a 2.1b 3.2a 2.5b

.41 0.33 0.66 0.92 0.64 0.60

.0a 4.2b 0.8a 0.7a 3.5a 1.6b

.53 0.60 0.40 0.17 0.67 0.20

.3b 15.8a 7.8a 9.0a 17.8a 9.4b

.72 1.00 0.43 1.49 1.53 0.94

.2b 667.3a 426.4a 411.0a 716.6a 429.1b

.10 56.50 23.20 71.50 98.82 25.11

.8a 12.9a 0.9a 0.7a 3.5a 5.4a

.56 6.21 0.30 0.15 2.01 2.43

our or cracker) in a row with the same letter are not significantly different p� 0.05.

C.A. Challacombe et al. / Journal of Cereal Science 56 (2012) 181e188184

To relate the phenolic acid content, including bound and freephenolic acids as well as TPAC, to the descriptive sensory data,a Partial Least Squares (PLS) mapping was conducted. The meansof all sensory attributes and phenolic measures of the four treat-ments as well as the refined control were used in this analysis. Thebound and free phenolic acids were included in the analysis indi-vidually as well as together to find the best relation with sensorydata. Statistical analysis was completed using XLSTAT 2010(New York, NY).

3. Results and discussion

3.1. Total phenolic acid content (TPAC)

Analysis of the total phenolic content of the wholegrain floursshowed no significant interactions between wheat colour andparticle size. Additionally, particle size did not have an effect onTPAC (data not shown). With regard to wheat colour, only thebound phenolic content was significantly different between thesoft red and soft white flours with no difference in bound TPACobserved between the hard red and hard white flours (Table 1).

The soft red wheat flour contained 1489.3 mg ferulic acidequivalents (FAE)/g of TPAC while soft white wheat flour had1349.8 mg FAE/g TPAC. These results follow those of Kim et al.(2006) who reported that wheat colour was correlated with TPCand that red wheat contained a greater concentration of TPC thanwhite wheat. Others, however, have found no correlation betweenwheat colour and total TPC (Beta et al., 2004). Free TPAC was notsignificantly different between the red and white wheat for eitherthe hard or the soft flours (Table 2).

Table 2Free phenolic acids for red and white wheat based wholegrain flours and baked products

Hard flour Bread crumb Bread

Red White Red White Red

Total phenolic content (mg FAE/g flour basis)Means 524.8aa,b 529.5a 701.2a 656.5b 1525St dev 24.05 44.38 22.05 19.79 61.08

Hydroxybenzoic acids (mg/g flour basis)p-Hydroxybenzoic acidMeans 0.3a 0.4a 0.9b 1.5a ndSt dev 0.09 0.08 0.06 0.33

VanillicMeans 1.1b 1.4a 3.9a 4.1a 5.0aSt dev 0.14 0.04 0.24 1.42 0.38

SyringicMeans 0.3b 0.5a 1.0b 1.6a ndSt dev 0.07 0.09 0.13 0.09

Hydroxycinnamic acids (mg/g flour basis)CaffeicMeans 0.2a 0.3a 0.6a 0.6a ndSt dev 0.09 0.07 0.06 0.05

p-CoumaricMeans 0.2b 0.5a 0.7a 0.2b ndSt dev 0.12 0.03 0.04 0.02

FerulicMeans 4.3a 4.8a 23.4b 42.2a 21.1bSt dev 1.47 1.81 3.73 9.28 3.82

SinapinicMeans nd nd 1.2a 1.5a 1.0aSt dev 0.17 1.30 0.69

FAE¼ ferulic acid equivalents, nd¼ not detectable.a Means for red and white wheat within a product type (hard flour, crumb, crust, soft flb n¼ 4 (2 particle size * 2 reps).

In the red and white flour-based products, both bound and freeTPAC differed based on wheat colour. Within the crumb and thecrust of the bread, bound phenolics were significantly higher inproducts made with white wheat flour in comparison to thosemade with red wheat flour (Table 1). The opposite trend wasobserved for free phenolics, with significantly higher free phenolicsin the crumb and crust of products from red wheat as compared towhite wheat (Table 2). Within the crackers, both bound and freeTPAC were significantly higher for those products made fromwhitewheat in comparison to those made from red wheat.

3.2. Phenolic acid profile: bound phenolic acids

Previous studies have reported that ferulic acid is the mainphenolic acid in wheat (Hatcher and Kruger, 1997). In this study aswell, ferulic acid was the major (free and bound) phenolic acidfound within both the hard and soft wheat varieties.

Within the bound fraction of phenolic acids, the hard whitewheat flour contained higher concentrations of p-hydroxybenzoic,and p-coumaric acid whereas the hard red wheat flour had a highersinapinic acid content (Table 1). The soft red wheat flour containedhigher concentrations of bound syringic acid than the soft whitewheat flour (Table 1).

In the products made with red and white wheat, the quantitiesof bound phenolic acids remained fairly constant for the majority ofphenolic acids when the baked products were compared to theirrespective flours (Table 1). Caffeic acid generally increased inbreads and crackers compared to the flours. It has been reportedthat within wheat bran, an increase in specified phenolic acids wasobserved with storage at 100 �C. The increase in phenolic acids was

from the flours (bread crumb and crust from hard flour and cracker from soft flour).

crust Soft flour Cracker

White Red White Red White

.6a 1247.1b 484.4a 482.5a 403.6b 458.9a9.80 14.40 25.35 16.70 40.79

nd nd nd 1.0a 1.1a0.11 0.23

4.7a 1.4b 2.7a 2.3a 3.1a0.94 0.26 1.21 0.35 0.81

nd 0.6b 2.2a 0.8a 1.0a0.04 1.77 0.22 0.24

nd nd nd 0.4a 0.3b0.08 0.01

nd 0.4b 0.7a 1.0b 1.2a0.08 0.23 0.26 0.05

29.6a 7.7b 9.1a 20.1a 16.0b4.39 0.52 1.68 2.66 1.42

1.9a nd nd nd nd0.80

our or cracker) in a row with the same letter are not significantly different p� 0.05.

Table 3Means and standard deviations (St dev) of sensory attributes for bread crumb.

Refineda Red coarse Red fine White coarse White fine

SweetMeans 7.4abb,c,d 6.8ab 7.6a 6.4b 7.1abSt dev 1.82 2.24 2.12 2.27 2.39

SaltMeans 6.7a 6.9a 6.5a 6.6a 6.9aSt dev 2.08 1.72 2.14 2.17 1.97

SourMeans 6.7a 7.5a 7.3a 7.5a 7.8aSt dev 2.60 1.94 2.29 1.99 2.01

BitterMeans 4.1b 5.4ab 5.6ab 6.5a 5.7aSt dev 2.55 2.85 2.61 2.82 2.94

AstringentMeans 4.8a 5.8a 5.5a 6.0a 5.7aSt dev 2.73 2.48 2.41 2.25 2.59

GrainlikeMeans 4.1c 6.4b 6.3b 8.2a 6.4bSt dev 2.36 2.39 1.99 2.21 2.37

WheatMeans 6.0b 7.2ab 7.3a 6.9ab 7.6aSt dev 2.62 2.11 2.06 2.45 2.06

ToastMeans 4.6a 5.6a 5.6a 5.9a 5.8aSt dev 2.69 3.00 2.80 2.96 2.92

YeastMeans 7.3a 6.4a 6.2a 6.2a 6.5aSt dev 2.24 2.14 2.14 1.93 2.21

MaltedMeans 4.4b 5.7a 5.7a 5.5ab 5.7aSt dev 2.05 5.14 2.09 1.83 2.13

MolassesMeans 3.3a 3.8a 4.0a 3.8a 3.9aSt dev 1.89 1.73 1.67 1.83 1.63

a Refers to flour treatments used to manufacture the bread where refined isrefined flour; red/white refer to wheat colour; fine/coarse are particle sizes.

b Means in a row with the same letter are not significantly different p� 0.05.c All attributes evaluated on a 15 cm line scale where a higher score indicates

a stronger intensity of the attribute.d n¼ 52 (13 panellists * 4 reps).

Table 4Means and standard deviations (St dev) of sensory attributes for cracker.

Refineda Red coarse Red fine White coarse White fine

SweetMeans 8.0ab,c,d 6.8ab 7.1ab 6.1b 7.2abSt dev 1.83 1.62 1.84 1.81 1.52

SaltMeans 4.2a 3.9a 4.4a 4.1a 4.0aSt dev 2.22 2.10 2.50 2.10 1.84

SourMeans 2.4b 3.4a 2.7ab 3.6a 2.8abSt dev 1.47 2.22 1.67 2.68 2.00

BitterMeans 2.8c 4.2ab 3.6bc 4.9a 3.2bcSt dev 2.28 2.49 2.26 2.56 2.23

AstringentMeans 4.2a 5.0a 4.8a 4.8a 4.7aSt dev 3.10 2.26 2.40 2.09 2.93

GrainlikeMeans 5.8c 8.8a 7.4b 8.9a 7.0bSt dev 1.45 2.19 1.37 2.22 1.31

WheatMeans 6.7b 8.6a 8.1a 8.6a 7.7aSt dev 1.50 2.01 1.54 2.53 1.83

ToastMeans 5.0c 7.5a 7.0ab 7.5a 6.3bSt dev 1.81 1.53 1.40 1.70 1.67

BranlikeMeans 3.0c 8.2a 5.1b 8.2a 4.4bcSt dev 2.03 2.39 2.33 2.34 2.37

DairyMeans 9.1a 5.4c 6.9b 5.1c 7.2bSt dev 2.14 1.87 2.11 2.00 1.91

EarthyMeans 2.1d 6.7b 4.1c 8.7a 3.7cSt dev 1.53 2.34 2.23 2.32 2.24

HardMeans 7.1bc 5.9c 8.0b 6.0c 9.2aSt dev 1.88 1.14 2.34 1.60 2.41

CrispMeans 8.9a 7.0b 8.5a 6.6b 8.8aSt dev 1.60 1.32 1.72 1.48 2.41

CoarseMeans 3.1c 9.3a 4.6b 9.5a 4.3bcSt dev 1.78 1.92 2.06 1.97 1.91

a Refers to flour treatments used to manufacture the cracker where refined isrefined flour, red/white refer to wheat colour; fine/coarse are particle sizes.

b Means in a row with the same letter are not significantly different p� 0.05.c All attributes evaluated on a 15 cm line scale where a higher score indicates

a stronger intensity of the attribute.d n¼ 30 (10 panellists * 3 reps).

C.A. Challacombe et al. / Journal of Cereal Science 56 (2012) 181e188 185

attributed to the degradation of polyphenolics to extractable simplephenolics (Cheng et al., 2006). This may be a plausible explanationas to why an increase in some phenolic acids was observed post-baking. In the current study, vanillic acid significantly (F-val-ue¼ 17.50, p< 0.0001) decreased within the bread compared to theflour, but remained constant within the crackers (F-value¼ 2.86,p¼ 0.11).

It is known that red andwhitewheat differ in their phenolic acidprofiles, however results in the literature are contradictory (Kequanet al., 2005; Liu et al., 2010). The phenolic acid content and profile ofwheats are dictated by a number of factors, including genetics,environment and stressors (Bravo, 1998; Mpofu et al., 2006), andthat it is not unexpected that differences would appear betweencolour, as well as between hard and soft flour types. Furthermore,as commercial flours were utilized for this study, a diversity ofwheat varieties is included within the flours contributing to thevarying phenolic acids profiles. When the phenolic acids wereaveraged across colour, the differences between the fine and coarseparticle size flours were not significantly different. This suggeststhat wheat colour may have a greater impact on the phenolic acidcontent of wheat and their products when compared to particlesize.

3.3. Phenolic acid profile: free phenolic acids

Hard and soft white wheat flour contained higher concentra-tions of free vanillic, syringic, and p-coumaric acids whencompared to hard red wheat flour (Table 2). Additionally, theconcentration of ferulic acid in the soft white wheat flour washigher than in the soft red wheat flour (Table 2).

With few exceptions, the bread (crumb and crust) and crackerscontained higher levels of free phenolic acids in comparison to theoriginal flour. It has been reported that free phenolic acid contentincreases with baking and it is thought that thermal processingreleases some of the bound phenolic acids, resulting in an increasedconcentration in baked products. Ferulic acid content specifically

pHBA F

VA FCA F

Syr F

pCou F

FA F

Sin FTPAC F

pHBA B

VA BCA B

Syr B

pCou BFA B

Sin B

TPAC B Sweet

Salt

Sour

Grainlike

Yeasty

WheatyToasted

MaltMolasses

Bitter

Astringent

Refined crumb

Red fine crumb Red coarse crumb

White fine crumb

White coarse crumb

-1

-0.75

-0.5

-0.25

0

0.25

0.5

0.75

1

-1 -0.75 -0.5 -0.25 0 0.25 0.5 0.75 1

Axi

s 2 (9

%)

Axis 1 (68%)

Fig. 1. PLS map of bread crumb sensory attributes with free and bound crumb phenolic acids. - refers to phenolic acids where pHBA¼ p-hydrozybenzoic acid, VA¼vanillic acid,Syr¼ syringic acid, CA¼ caffeic acid, pCou¼ p-coumaric acid, FA¼ ferulic acid, Sin¼ sinapinic acid, TPAC¼ total phenolic acid content, F¼ Free, B¼ bound, X refers to sample wherered/white are wheat colour; fine/coarse are particle sizes, > refers to sensory attributes.

C.A. Challacombe et al. / Journal of Cereal Science 56 (2012) 181e188186

has been found to increase in baked pizza crust when comparedwith the original flour (Moore et al., 2009).

Within the bread crust, a number of phenolic acids were notdetectable. This may be due to interactions with Maillard reactionadducts or thermal degradation. Phenolic acids have beendemonstrated to react, via redox reactions, with intermediates ofthe Maillard reaction forming phenolic acid-Maillard adducts(Jiang and Peterson, 2010). As the bread crust has more favour-able conditions for the Maillard reaction to occur than incrackers, including moisture, pH and processing conditions(Capuano et al., 2008) the reaction of phenolic acids with Mail-lard intermediates may be a plausible reason as to why phenolicacids were not detected within the bread crusts, but detectablewithin crackers.

3.4. Sensory attributes

Mean sensory data for products (bread crumb and crackers)made from the wholegrain flours (red and white wheat at twoparticle sizes) as well as the refined control flour are presented inTables 3 and 4, respectively. Data from the bread crust is not shownas there were few significant differences in sensory properties.

The bread crumb was found to have significant differences insweet and bitter tastes as well as grainlike, wheat and maltedflavours (Table 3). Manufacturing the bread from refined flourproduced a crumb with low grainlike properties. Additionally, themalted note was significantly lower for the refined product whencompared to all other wholegrain products aside from the whitecoarse flour.

The crackers had many significant differences in sensory attri-butes amongst the refined and wholegrain samples, with all attri-butes except for salt taste and astringency differing amongst theproducts. The refined cracker displayed significantly lower wheat,toasted and earthy notes in comparison to thewholegrain products.A high dairy note was also perceived in the refined cracker(Table 4). The means of each sensory attribute for each productwere used in the creation of the PLS, as described below.

3.5. Relationship between phenolic acids and sensory data

In order to determine the relationship between phenolic acidconcentration and perception of the sensory properties of thebaked products, a PLS regression was conducted. This regressionwas carried out on two sets of data. First, the phenolic acid contentof the flours used to make the products was used in the analysisfollowed by the phenolic acid content of the baked productsthemselves. To find the best relationships between phenolic acidsand sensory properties in the analysis, bound and free phenolicacids were analyzed individually as well as together. The Q2cumcalculated during the PLS regression is a representation of theoverall general goodness of the fit of the model, whereas theR2Ycum represents the correlations between the independentvariables and the components of the PLS regression (Tenenhauset al., 2005). To determine the best model fit, both values must beconsidered.

The strength of the relationship between the independentvariables and the components of the PLS regression differed basedon product type (bread crumb, bread crust, or cracker). Withinbread crumb, good relationships were observedwhen both free andbound phenolic acids within the flour were included in the analysis(Q2cum¼ 0.545 and R2Ycum¼ 0.841). High Q2 and R2Ycum werealso observed when both free and bound phenolic acids from thecrumb of the bread were included in the regression analysis(Q2cum¼ 0.401 and R2Ycum¼ 0.767). The bread crust, however,displayed low Q2cum values, regardless of whether flour phenolics(Q2cum¼ 0.174) or crust phenolics (Q2cum¼�0.029) were used inthe regression. This indicates that the PLS model is not a good fit forthe crust data and that the model has little (if any) predictivepower. For this reason, the relations between phenolic acids andbread crust were not examined further. For crackers, the boundphenolic acids in both the flour and baked product produced thebest model fit. It has been reported that phenolic acids can interactwith Maillard reaction adducts, resulting in altered flavour andaromatic properties (Jiang and Peterson, 2010). Maillard reaction islimited within the crumb while the cracker and the bread crustundergo more Maillard browning during baking. This possibly

pHBA BVA B

CA B

Syr B

pCou B

FA B

Sin B

TPAC B

Sweet

Salt

Sour

WheatyToasted

GrainlikeBranlike

Dairy

EarthyBitter

Astringent

Hard

Crisp

Coarse

Refined

Red Fine

White Fine

Red Coarse

White Coarse

-1

-0.75

-0.5

-0.25

0

0.25

0.5

0.75

1

-1 -0.75 -0.5 -0.25 0 0.25 0.5 0.75 1

Axi

s 2 (1

3%)

Axis 1 (64%)

Fig. 2. PLS map of cracker sensory attributes with bound cracker phenolic acids. - refers to phenolic acids where pHBA¼ p-hydrozybenzoic acid, VA¼vanillic acid, Syr¼ syringicacid, CA¼ caffeic acid, pCou¼ p-coumaric acid, FA¼ ferulic acid, Sin¼ sinapinic acid, TPAC¼ total phenolic acid content, F¼ free, X refers to sample where red/white are wheatcolour; fine/coarse are particle sizes, > refers to sensory attributes.

C.A. Challacombe et al. / Journal of Cereal Science 56 (2012) 181e188 187

explains why the free phenolic acids are correlated with thesensory properties in the crumb but not within the cracker or breadcrust.

Using the Q2 cum and R2Ycum data, PLS maps were generatedto examine the relations between phenolic acids and sensoryproperties. PLS maps generated from the flour phenolic acidsregressed with sensory attributes of bread crumb and crackers aresimilar to those generated when product phenolic acids areregressed with sensory attributes, therefore, only the PLS maps forthe latter regressions will be shown. Within these maps for breadcrumb and cracker, the majority of the phenolic acids was clus-tered close to the end of Axis 1 and in a space similar to thatoccupied by the sensory attributes associated with the wholegrainproducts (Figs. 1 and 2). On the opposite end of Axis 1 is the refinedcrumb that was best described by a yeasty note and was notassociated with any phenolic acid. Bound TPAC of the bread crumbwas linked with bitterness, grainlike, sour and wheaty character-istics (Fig. 1). Free TPAC was associated with a toasted note and,along with free sinapinic acid, fell into a similar space on the PLSmap as astringent, wheaty, grainlike and bitter notes. Free caffeicand vanillic acid were associated with malty and molasses notespresent within the crumb. It is noted that although the free andbound phenolic acids have the best model fit, the free phenolicacids are more aligned with the wholegrain sensory characteristicson the PLS map.

In the cracker PLS map, bound TPAC was associated with bran-like, grainlike and sour attributes (Fig. 2). Astringency was linkedwith bound p-hydroxybenzoic, vanillic, syringic and ferulic acids.Bound syringic acid was also associated with toasted and wheatyattributes. No bound phenolic acids were closely associated withbitterness in the cracker. Although ferulic acid is the main phenolicacid present in the wheat, it is not the only phenolic acid related tothe sensory attributes of wholegrain products. Other phenolic acidsas well as the total phenolic content also apparently contribute tothe flavour perception of wholegrain products.

Although it has been stated that free phenolic acids are the mostflavour active compounds (Heiniö et al., 2008), the present studysuggests that the bound fraction of phenolic acids may also becontributing to taste and flavour properties of wholegrain products.

It may be that during mastication, the bound phenolic acids arefreed by salivary enzymes, allowing them to interact with tastereceptors and other compounds within the mouth.

The inclusion of the TPAC as a variable in the PLS analysis wasbased on the fact that TPAC is nonspecific to the phenolic acidsquantified. If TPAC was found to be strongly related to the sensoryproperties of the products, other phenolic acid compounds whichwere not profiled in this study may be contributing to the sensoryproperties of the products. Results show that TPAC was linked withthewholegrain sensory properties in both bread crumb and cracker(Figs.1 and 2), suggesting that other phenolic acid compounds needto be profiled to have a more complete understanding of thecompounds involved in development of flavours and tastes ofwholegrain products. A recent study reported that bran flavour,dusty flavour, bitter taste and prickly sensation were correlatedwith fermentation, Maillard and lipid oxidation reactions in wholewheat bread (Jensen et al., 2011). The end products of theseprocesses may be contributing to the TPAC and may be a plausibleexplanation as to why TPAC was correlated with wholegrainsensory properties in the present study.

4. Conclusion

Overall, phenolic acid profiles varied between the red and whitewheat. TPAC, however, was similar. From the PLS analysis, althoughfree and bound phenolic acids were found to correlate with thesensory characteristics in the bread crumb, the bound phenolicacids were observed to have a better correlation. It is currentlyunknown as to the mechanism which bound phenolic acids arecontributing to sensory perception, and provides an area for futureresearch.

A strong correlation between TPAC and wholegrain sensoryattributes was also noted. As the TPAC test measures total phenolicacids, the strong association between TPAC and wholegrain char-acteristics suggests that there are phenolic acid compounds otherthan phenolic acids contributing to sensory properties of whole-grain products. Future research should also focus on identifyingother phenolic compounds present within wheat and wholegrainproducts that may be contributing to their sensory properties.

C.A. Challacombe et al. / Journal of Cereal Science 56 (2012) 181e188188

Acknowledgements

This research was supported by grants from the OntarioMinistry of Food, Agriculture and Rural Affairs (OMAFRA) andMathematics of Information Technology and Complex Systems(MITACS) in partnership with Kraft Mississauga Mill. We greatlyappreciate the support fromMr. Terry Groff, Ken Zvoncheck and thestaff at Reading Bakery Systems, Reading PA, who helped make thecrackers.

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