Food Chemistry 187 (2015) 280–289

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Wheat milling by-products and their impact on bread making Sami Hemdane, Sofie Leys, Pieter J. Jacobs, Emmie Dornez, Jan A. Delcour, Christophe M. Courtin ⇑ Laboratory of Food Chemistry and Biochemistry and Leuven Food Science and Nutrition Research Centre (LFoRCe), KU Leuven, Kasteelpark Arenberg 20 – box 2463, 3001 Leuven, Belgium

a r t i c l e

i n f o

Article history: Received 19 October 2014 Received in revised form 10 February 2015 Accepted 13 April 2015 Available online 22 April 2015 Keywords: Wheat Coarse bran Coarse weatings Fine weatings Low grade flour Bread making

a b s t r a c t This study investigates the relationship between the properties of dietary fiber (DF) rich wheat milling by-products and their impact on bread making. From coarse bran over coarse and fine weatings to low grade flour, the content of starch and lipids increased, while that of DF and ash decreased. Enzyme activity levels differed strongly and were not related to other by-product properties. Average particle size of the by-products was positively correlated with DF and ash contents and their hydration properties. When meals from flour and by-products were composed on the same overall starch level to compensate for differences in endosperm contamination in the by-products, bread specific volume was more strongly depressed with fine weatings and low grade flour than with coarse bran and weatings. This suggests that the properties of the former were intrinsically more detrimental to bread making than those of the latter. Ó 2015 Elsevier Ltd. All rights reserved.

1. Introduction Wheat (Triticum aestivum L.) bran, recovered in a number of byproducts of the wheat milling industry, is a good source of dietary fiber (DF), antioxidants, B vitamins and minerals (Delcour & Hoseney, 2010; Shewry, 2009). Epidemiologic evidence exists that the daily consumption of bran-enriched cereal products may reduce the risk of welfare diseases like obesity (de Munter, Hu, Spiegelman, Franz, & van Dam, 2007), cardiovascular disease (Jacobs & Gallaher, 2004) and colorectal cancer (Jacobs, Marquart, Slavin, & Kushi, 1998). Furthermore, the European Food Safety Authority recently recognizes two health claims concerning wheat bran, allowing to state that wheat bran increases fecal bulk and accelerates the intestinal transit (EFSA Panel on Dietetic Products Nutrition and Allergies (NDA), 2010). Therefore, incorporation of wheat bran in cereal-based products is an interesting and low-cost strategy to improve their nutritional properties and physiological effects. However, incorporation of bran-containing milling byproducts in cereal-based foods generally results in inferior endproduct quality. In bread, such incorporation results in a decreased bread loaf volume, an increased crumb firmness and a decreased sensory acceptance (Moder, Finney, Bruinsma, Ponte, & Bolte, 1984; Pomeranz, Shogren, Finney, & Bechtel, 1977). Nowadays, the detrimental effect of wheat bran in bread making is counteracted by use of strong wheat flour and addition of ⇑ Corresponding author. Tel.: +32 16 321917; fax: +32 16 321997. E-mail address: [email protected] (C.M. Courtin). http://dx.doi.org/10.1016/j.foodchem.2015.04.048 0308-8146/Ó 2015 Elsevier Ltd. All rights reserved.

bread improvers, rather than working on bran itself. Although several studies investigated the detrimental effect of wheat bran in bread making, a clear view on the contribution of different aspects of bran on this effect is lacking. Nevertheless, several hypotheses on the underlying causes of quality loss exist. Firstly, it is generally accepted that the replacement of flour by wheat bran causes a dilution of wheat gluten proteins. Pomeranz et al. (1977), for instance, reported that the addition of up to 5% wheat bran decreased bread volume to an extent expected from dilution of gluten proteins. However, when 7% bran or more was added, the volume decrease could no longer be explained by the dilution of gluten proteins only. Also the competition for water between meal constituents might explain the detrimental effect of wheat bran on bread properties. More recently, Bock, Connelly, and Damodaran (2013) observed a redistribution of water among dough components when wheat bran is added, resulting into partial dehydration and structural changes of the gluten proteins. A third possible explanation of the impact of bran on bread properties is its steric hindrance during dough development, disturbing the formation of an optimal gluten network (Gan, Ellis, Vaughan, & Galliard, 1989). Wheat bran might furthermore not only hinder the formation of a gluten network, but may also destabilize gas cells, resulting into a decrease in gas retention (Gan, Galliard, Ellis, Angold, & Vaughan, 1992; Pomeranz et al., 1977). Finally, Wootton and Shams-Ud-Din (1986) noticed that bran extract is more detrimental to bread volume than the washed residue, suggesting the involvement of deleterious bran-related enzymes or reactive chemical components like glutathione or ferulic acid, which might

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react with sulfhydryl groups of gluten proteins (Noort, van Haaster, Hemery, Schols, & Hamer, 2010). When studying the impact of bran addition on bread making, it should be recognized that the use of the term ‘‘bran’’ can lead to confusion. From a botanical point of view, bran is defined as the outer layers of the wheat kernel, consisting of the pericarp, seed coat and nucellar epidermis. Bran defined by the miller contains besides the botanical bran also aleurone and starchy endosperm remnants which were not removed during the milling process (Delcour & Hoseney, 2010). It should also be noted that botanical bran is distributed over a range of different by-products formed during milling, namely coarse bran, coarse weatings, fine weatings and low grade flour. Coarse bran (which is usually defined as regular bran) and coarse weatings (or fine bran) are separated in the early breaks of the milling process, while fine weatings (also referred to as middlings or shorts) are smaller particles of endosperm and bran, which were not separated after grinding (Delcour & Hoseney, 2010). This latter by-product has also been reported to contain more aleurone and germ than (coarse) bran (Lai, Hoseney, & Davis, 1989b). Low grade flour (or red dog) is the flour like material remaining after the last grinding steps and contains a high level of non-endosperm material (Delcour & Hoseney, 2010). Although all of the above mentioned milling by-products can be reconstituted with their flour complement to form whole meal, little information can be found on their functionality relative to each other in bread making. This is unfortunate from a scientific point of view as comparison of their properties with their impact on bread making might yield insight in the detrimental impact of bran in general and the different by-products in particular on bread making. Also from a practical point of view this is unfortunate, as each of the fractions is a possible candidate for addition to bread formula to enhance dietary fiber levels in bread. Therefore, in this study, we try to gain more insight in the phenomena underlying the detrimental effect of wheat bran on bread making by studying the properties and functionality of different wheat milling by-products (coarse bran, coarse weatings, fine weatings and low grade flour) in bread making. To this end, differences in bran chemical composition, enzyme activity levels and physical properties were investigated and related to dough and bread properties.

2. Materials and methods 2.1. Materials Ten wheat milling by-products were kindly provided by different industrial milling companies. In addition, four milling by-products were produced on laboratory scale with a Bühler MLU-202 laboratory mill (Uzwil, Switzerland) by milling wheat cultivars Akteur and Apache (harvest 2012) obtained from AVEVE (Landen, Belgium), resulting into coarse bran 5 and fine weatings 3, and coarse bran 6 and fine weatings 4, respectively. Akteur is a cultivar with excellent baking properties and a flour protein content of 14.6 ± 0.1%, while Apache gives flour of inferior quality (flour protein content of 10.3 ± 0.2%). Milling yields were 76.3% and 75.8%, respectively. The reason to produce the latter by-products was twofold. First, it allowed to assess the difference in the properties and functionality of milling by-products obtained from wheat cultivars with superior and inferior baking properties. Secondly, the production of these samples on laboratory scale resulted in better insight in how the by-products obtained by laboratory milling differ from those obtained by industrial milling. The flour used in this study was Crousti (Dossche Mills, Deinze, Belgium) with following composition (dry matter basis): 2.3 ± 0.1% DF, 83.2 ± 1.1%

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polymeric glucose, 13.6 ± 0.2% proteins, 0.63 ± 0.01% ash and 1.5 ± 0.1% lipids. Vital wheat gluten was from Tereos Syral (Aalst, Belgium) and fresh baker’s yeast from AB Mauri (Merelbeke, Belgium). Also commercial sugar and salt were used. All chemicals, solvents and reagents were of analytical grade and purchased from Sigma–Aldrich (Bornem, Belgium), unless specified otherwise. 2.2. Chemical composition of the milling by-products DF content was analyzed according to the AOAC Official Method 991-43 (AOAC International., 1995). Total starch (measured as polymeric glucose), total arabinoxylan (TOT-AX) and water-extractable arabinoxylan (WE-AX) contents were analyzed in triplicate using a gas chromatography based method as described by Courtin, Van den Broeck, and Delcour (2000). Saccharides were hydrolyzed to their constituting monosaccharides with 2.0 M trifluoroacetic acid, reduced to alditols with NaBH4 and derivatized to alditol acetates using acetic acid anhydride. Polymeric glucose was calculated as the level of glucose multiplied by 0.90, arabinoxylan content was calculated as the sum of the arabinose and xylose levels multiplied by 0.88 (Courtin et al., 2000). Protein content was measured in duplicate by the Dumas combustion method, an adaptation of the AOAC Official Method 990-03 (AOAC International, 1995) to an automated Dumas protein analysis system (EAS VarioMax N/CN, Elt, Gouda, The Netherlands). The detected nitrogen was converted to protein content using 6.25 as conversion factor. Total lipid content was determined gravimetrically after extraction with water-saturated butanol as described by Gerits, Pareyt, and Delcour (2013). Moisture, ash and damaged starch contents were determined in triplicate following AACCmethods 44-19.01, 08-01.01 and 76-31.01, respectively (AACC, 2000). 2.3. Enzyme activity levels of the milling by-products

a-Amylase and endoxylanase activity levels in bran extracts were determined with the Amylazyme and Xylazyme AX methods, respectively (Megazyme, Bray, Ireland). Enzymes were extracted by suspending 1.0 g of sample in 10.0 mL maleate buffer (100 mM, pH 6.0, 5 mM CaCl2) for the Amylazyme method and in 10.0 mL sodium acetate buffer (25 mM, pH 5.0) for the Xylazyme AX method and shaking (Laboshake, VWR International, Leuven, Belgium) during 30 min at room temperature. An azurine crosslinked amylose or AX tablet was added to 1.0 mL pre-equilibrated sample at 40 °C. After an appropriate incubation time (generally 15 min for amylase activity levels and 6 h for xylanase activity levels), the reaction was stopped by adding 10.0 mL of Tris(hydroxymethyl)aminomethane (Tris) base solution (2.0 w/v% for the Amylazyme method, 1.0 w/v% for the Xylazyme AX method). After filtration of the suspensions, the filtrates were cooled to room temperature and the extinction values at 590 nm (E590) [Ultraspec III UV/vis spectrophotometer (GE Healthcare, Uppsala, Sweden)] were measured against a control, prepared by incubating the extracts without the tablet. Correction was made for non-enzymic color release by the substrate tablets. Activities were expressed in a-amylase and endoxylanase units (AU and XU, respectively) per gram dry matter (dm). One unit is defined as the increase of E590 per hour of incubation, under the conditions of the assay. Endopeptidase activity levels were determined using azocasein as substrate (Feller & Erismann, 1978). Enzymes were extracted by suspending 1.0 g sample in 10.0 mL maleate buffer (100 mM, pH 6.0, 5 mM CaCl2) and shaking (Laboshake, VWR International, Leuven, Belgium) during 30 min at room temperature. Azocasein solution (350 lL, 1.4 w/v%) was added to 250 lL enzyme extract and incubated for 4 h at 40 °C. After incubation, 500 lL of cooled

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trichloroacetic acid (7 °C) was added and precipitated proteins were removed by centrifugation (10,000g, 10 min). Finally, 500 lL of NaOH (500 mM) was added to 500 lL supernatants and the extinction at 440 nm (E440) was measured. Endopeptidase activity levels were expressed as endopeptidase units (PU) per gram dm, with one PU defined as the increase of E440 per hour of incubation. 2.4. Particle size distribution of the milling by-products Particle size distribution of samples was determined in triplicate by sieving 20.0 g of sample on a Vibratory Sieve Shaker AS 200 control (Retsch, Aartselaar, Belgium) for 30 min, with intervals every 30 s and a shaking amplitude of 0.75 mm. Sizes of the sieve pores were 2000 lm, 1000 lm, 710 lm, 500 lm, 250 lm, 125 lm and 50 lm. The weight retained on the different sieves was expressed as w/w%. Mean particle size (MPS) was also calculated, using the formula of Ensor, Olson, and Colenbrander (1970):

Pn 1

MPS ¼ 10

pffiffiffiffiffiffiffiffiffi d d Þ Pn i iþ1

ðW i log

1

Wi

ð1Þ

With Wi defined as the mass of sample retained on sieve i (g), di as the pore diameter of sieve i (lm) and di+1 as the pore diameter of the upper sieve following sieve i (lm). 2.5. Hydration properties of the milling by-products The procedure for water retention capacity (WRC) determination was optimized based on the method described by Mongeau and Brassard (1982). Wheat bran (1.0 g) was weighed in a 50 mL tarred centrifuge tube and 10.0 mL deionized water was added. After vortex stirring, the sample was left for 60 min at room temperature, followed by 10 min of centrifugation at 4,000g. The supernatant was carefully removed and a draining step of 15 min was performed in order to remove residual non-retained water. The centrifuge tube was weighed and the WRC was expressed as g water retained per g dm bran and as g water per g by-product DF (WRCDF). Swelling capacity (SC) was determined by the bed volume technique as described by Kuniak and Marchessault (1972) with slight adaptations. Approximately 500 mg of sample was weighed in a 10.0 mL graduated glass cylinder and steeped for 60 min at room temperature in 5.0 mL deionized water. SC was expressed as volume occupied by the swollen sample (mL) per g dm bran. 2.6. Composing meals For the investigation of the impact on wheat milling by-products on dough and bread making properties, meals were enriched in different bran types by following two different approaches. In a first approach, meals were composed on a 15% bran basis by mixing 80% Crousti flour, 15% milling by-product and 5% vital wheat gluten. In a second approach, meals were composed on an overall 70% starch basis as starch was considered to be a good measure for the endosperm contamination of the bran. Therefore, depending on the starch content of the milling by-products, different levels of sample were mixed with Crousti flour. The reason for the latter approach was that the different milling by-products analyzed in this study significantly differ in starch content, and thus consequently in botanical bran content. In order to investigate the functionality of the botanical bran component in the milling by-products, we decided to also compose meals on a same overall starch level of 70%. As a result of this approach, the endosperm content was equal for all meals. Also in the latter approach, 5% vital

wheat gluten was mixed. In both approaches, a control flour composed of 95% Crousti flour and 5% gluten was used as reference. 2.7. Dough properties For the determination of dough properties, doughs of the composite meals were made in triplicate using a Brabender E330 Farinograph (Duisburg, Germany) with a 10 g stainless steel mixing bowl, according to AACC method 54-21.02 (AACC, 2000). Farinograph water absorption (FWA) and dough development time (DDT) were determined. FWA was expressed on a 14% meal moisture basis and corrected to 500 Farinograph Units. 2.8. Bread making Breads enriched in the different bran types were prepared in triplicate according to the straight dough procedure of Shogren and Finney (1984). The composite meal (10.00 g, moisture content 14.0%), 0.53 g compressed fresh yeast, 0.60 g sucrose, 0.15 g salt and 6.8 mL water were mixed with a 10 g pin mixer (National Manufacturing, Lincoln, NE, USA) for 4 min 15 s. For the breads with low grade flour and composed on a 15% bran basis, a water volume of 6.6 mL was used. For the white control bread (95% Crousti flour, 5% gluten), 6.2 mL of water and a mixing time of 4 min were used. Preliminary bread making trials led to the use of the amount of water and mixing times described here. Fermentation and final proofing were performed in a fermentation cabinet (National Manufacturing) at 30 °C and 90% relative humidity for 90 and 36 min, respectively. Doughs were punched after 52 and 77 min fermentation and punched and molded after 90 min. Baking was performed for 13 min at 232 °C in a rotary oven (National Manufacturing). Bread volume was measured by rapeseed displacement, according to AACC method 10-05.01 (AACC, 2000). Breads were weighed and their specific volumes calculated. 2.9. Statistical analysis Statistical analysis of the results was performed using the Statistical Analysis Software 9.3 (SAS Institute Inc., Cary, NC, USA). One-way analysis of variation (ANOVA) was performed to analyze significant differences between mean values of several variables. After a positive omnibus test, post hoc analyses were conducted to detect differences among experimental settings. A Tukey multiple comparison procedure was used with a 5% family significance level. Pearson’s correlation coefficients (P < 0.05) for linear correlations between mean values were also calculated with SAS 9.3. Principal Component Analysis (PCA), a multivariate data analysis method, was conducted with the Unscrambler software 9.1.2 (CAMO Technologies, Woodbridge, NJ, USA). In PCA, the high number of original variables is reduced to a smaller number of new variables called principal components (PCs), which are linear combinations of the original variables. These PCs are independent variables and describe in decreasing order the variability of the data. All variables were centered and scaled to unit variance prior to the multivariate analyses. 3. Results 3.1. Chemical composition of the milling by-products Table 1 shows the chemical composition of the milling by-products under study. On average, coarse bran contained 51.1% DF, of which 25.5% TOT-AX, 22.0% starch, 18.9% proteins, 6.4% ash and 2.8% lipids. These values are comparable to data reported in

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Table 1 Dietary fiber, total arabinoxylan (TOT-AX), total and damaged starch, protein, ash and lipid contents of wheat milling by-products (coarse bran, coarse weatings, fine weatings and low grade flour).a

a Values in grey represent averages of the by-products from a different origin but of a same type. The individual samples are compared to each other for significant differences and are grouped using small letters. The averages are compared to each other as well and are grouped using capital letters. Values within the same column not sharing a same letter are significantly different (P < 0.05).

previous studies (Seyer & Gélinas, 2009; Zhang & Moore, 1997). The average composition of coarse weatings was 43.2% DF, of which 20.5% TOT-AX, 28.8% starch, 18.8% proteins, 4.6% ash and 3.7% lipids. Fine weatings consisted of 31.9% DF, of which 16.1% TOT-AX, 40.5% starch, 19.5% proteins, 3.8% ash and 5.0% lipids and low grade flour contained 14.5% DF, of which 9.1% TOT-AX, 54.4% starch, 18.8% proteins, 2.8% ash and 5.7% lipids. WE-AXlevels were relatively low in all samples, ranging from 0.53% to 1.37% (results not shown), indicating that arabinoxylan in bran is mainly water-insoluble. The damaged starch content increased

from 2.5% for coarse bran to 12.8% for low grade flour (P < 0.05, Table 1). This fivefold increase in damaged starch content in the finer bran types can only be partly explained by the increase in total starch content, as the ratio of damaged starch over total starch only increased twofold, from 11.4% for coarse bran up to 23.5% for low grade flour (P < 0.05). Overall, when comparing the chemical composition of milling by-products of the same type, differences in composition were rather small. For protein contents, there was even little or no variability over the different milling by-product types (Table 1).

Table 2

a-Amylase, endoxylanase and endopeptidase activity levels of wheat milling by-products (coarse bran, coarse weatings, fine weatings and low grade flour).a

Milling by-product Coarse bran 1 Coarse bran 2 Coarse bran 3 Coarse bran 4 Coarse bran 5 Coarse bran 6 Average Coarse weatings 1 Coarse weatings 2 Coarse weatings 3 Average Fine weatings 1 Fine weatings 2 Fine weatings 3 Fine weatings 4 Average Low grade flour Average a

α-Amylase activity AU/g dm 6.3 ± < 0.1b 70.5 ± 0.3 a 14.4 ± 0.2 i 4.5 ± 0.1 k 6.2 ± 0.1 j 24.8 26.1 34.3 21.8 28.2 28.1 17.7 28.1 9.1

± 0.1 ± 24.8 ± 0.2 ± 0.1

e

± ± ± ± ±

d

A c f

0.1 6.2 A 0.1 h 0.1 d 0.3 i

20.0 ± 0.3 18.7 ± 7.8

g A d

28.0 ± 0.2 28.0 ± 2 / A

Endoxylanase activity XU/g dm 2.4 ± < 0.1 h 4.6 ± < 0.1 d 4.7 ± < 0.1 c 1.2 ± < 0.1 k 1.4 ± < 0.1 j 5.6 3.3 0.9 3.2

± < 0.1 ± 1.9 ± < 0.1 ± < 0.1

5.4 3.2 0.7 2.0 1.0

± ± ± ± ±

< 0.1 2.3 < 0.1 < 0.1 < 0.1

2.6 ± < 0.1 1.6 ± 0.9 2.5 ± < 0.1 2.5 ± 1/9

a A m e b A n i l f A g A

Endopeptidase activity PU/g dm 3.8 ± 0.1 j 6.3 ± 0.1 d 4.7 ± 0.1 h 3.2 ± 0.2 k 5.1 ± 0.1 g 6.4 4.9 5.4 6.2

± ± ± ±

0.1 d 1.3 A 0.1 f < 0.1d

7.5 6.4 4.1 9.6 5.9

± ± ± ± ±

0.1 b 1.0 A < 0.1i 0.1 a 0.1 e

6.6 ± 0.1 6.5 ± 2.3

c

6.9 ± 0.1 6.9 ± 1/3

c

A

A

Values in grey represent averages of the by-products from a different origin but of a same type. The individual samples are compared to each other for significant differences and are grouped using small letters. The averages are compared to each other as well and are grouped using capital letters. Values within the same column not sharing a same letter are significantly different (P < 0.05).

284

A

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high variability in a-amylase activity levels was noticed, varying from 4.5 AU/g up to 70.5 AU/g, but no relation was found between the type of bran and the a-amylase activity level. Indeed, the average a-amylase activity levels of the milling by-products were 26.1, 28.1, 18.7 and 28.0 AU/g for coarse bran, coarse weatings, fine weatings and low grade flour, respectively. Endoxylanase activity levels ranged from 0.7 XU/g to 5.6 XU/g and endopeptidase activity levels varied between 3.2 PU/g and 9.6 PU/g.

80% 60%

wt% 40% 20% 0% CB 1

B

CB 2

CB 3

CB 4

CB 5

CB 6

3.3. Particle size distribution of the milling by-products

80% 60%

wt% 40% 20% 0% CW 1

C

CW 2

CW 3

FW 2

FW 3

80% 60%

wt% 40% 20% 0% FW 1

FW 4

LGF

2000 µm

Fig. 1. Particle size distribution of coarse bran (CB, A), coarse weatings (CW, B), fine weatings (FW, C) and low grade flour (LGF, C) as determined by the sieving method. The Y-axis shows the weight percentage of the particles between the corresponding sieve pore diameter. Error bars are shown for each sieve.

Table 3 Mean particle size (MPS), water retention capacity (WRC) and swelling capacity (SC) of wheat milling by-products (coarse bran, coarse weatings, fine weatings and low grade flour).a

Fig. 1 shows the particle size distribution of the milling byproducts, with their corresponding MPS shown in Table 3. Coarse bran samples (Fig. 1A) had a MPS varying between 936 lm and 1344 lm (Table 3). The distribution patterns of coarse and fine weatings shifted to smaller sizes (Fig. 1B and C, respectively) compared to the coarse bran samples (P < 0.05), with MPS-values varying between 348 lm and 523 lm for coarse weatings and between 168 lm and 304 lm for fine weatings (Table 3). For low grade flour, the highest proportion of particles had a particle size between 125 lm and 250 lm (Fig. 1C), resulting in a MPS of 173 lm. Some variations in particle size distribution were also observed between the industrial and the laboratory samples within one type of milling by-product. The laboratory samples had a wider size distribution than the industrial samples, resulting in a lower MPS for coarse bran compared to industrial coarse bran and a higher MPS for fine weatings compared to industrial fine weatings.

3.4. Hydration properties of the milling by-products The variability in WRC and SC of the different milling by-products was investigated (Table 3). Coarse bran generally had a high WRC and SC (average values of 5.3 g H2O/g and 10.6 mL/g, respectively), followed by coarse weatings (3.7 g H2O/g and 7.1 mL/g), fine weatings (2.8 g H2O/g and 6.7 mL/g) and low grade flour (1.8 g H2O/g and 6.0 mL/g). Assuming that DF was the main chemical component influencing the WRC, WRCDF-values were calculated: coarse bran had an average WRCDF of 10.2 ± 1.4 g H2O/g DF, coarse weatings had a WRCDF of 8.7 ± 1.2 g H2O/g DF, fine weatings 8.9 ± 1.2 g H2O/g DF and low grade flour 12.2 ± 0.7 g H2O/g DF. Here, no trend could be observed anymore among the different milling by-products

3.5. Relationships between chemical composition, enzyme activity levels and physical properties of the milling by-products

a Values in grey represent averages of the by-products from a different origin but of a same type. The individual samples are compared to each other for significant differences and are grouped using small letters. The averages are compared to each other as well and are grouped using capital letters. Values within the same column not sharing a same letter are significantly different (P < 0.05).

3.2. Enzyme activity levels of the milling by-products

a-Amylase, endoxylanase and endopeptidase activity levels of the milling by-products were also determined (Table 2). A very

To establish the relationship between chemical constituents, enzyme activity levels and physical properties of wheat milling by-products, PCA was performed (Fig. 2). The first principal component (PC 1) explained 62% of the variability in the dataset, while PC 2 explained 16% of the variability. On the left side of the correlation loading plot (Fig. 2A), total starch, damaged starch and lipids cluster together, while on the right side of the plot, DF and ash are pooled. When observing the corresponding score plot, the different milling by-products could clearly be distinguished (Fig. 2B), with low grade flour on the left side of the score plot and the coarse bran samples on the other side of the plot. Next, MPS and the hydration properties (WRC and SC) are located close to the DF and ash contents in the correlation loading plot (Fig. 2A), indicating that they are positively correlated with these parameters. The second PC seemed to be a measure for the level of a-amylase and endoxylanase activity in the bran, though not of endopeptidase activity.

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on a same overall starch level of 70%. Results of the addition of the milling by-products under study on dough and bread properties considering these two approaches are here presented. 3.6.1. Dough and bread made with meals containing 15% milling byproduct Results on FWA and DDT of the doughs prepared from meals in which 15% of the flour was replaced by milling by-products are summarized in Table 4A. FWA and DDT increased from 62.2% and 2.6 min for white flour up to a maximum average value of 68.7% and 8.6 min for coarse bran. Compared to this latter value, a slight decrease in FWA was noticed when flour was replaced by finer bran types (P < 0.05). FWA values went from 68.7% for coarse bran down to 66.8% for low grade flour. Finally, a shorter DDT was observed when finer bran types were added, going from 8 to 9 min for coarse bran down to 5 min for low grade flour. Table 4A equally shows the results of the bread specific volumes obtained with the standard flour (5.9 cm3/g) and meal in which 15% of flour was replaced by different milling by-products. Besides the expected decrease in specific volume when wheat flour was replaced by bran containing milling fractions, some differences between the breads with different milling by-products could also be noticed. Indeed, although no significant differences were observed between the breads with coarse bran, coarse weatings and fine weatings (3.6, 3.9 and 3.7 cm3/g, respectively), replacement of flour by low grade flour resulted in bread with a significantly higher specific volume compared to breads containing other milling by-products (4.4 cm3/g).

Fig. 2. PCA correlation loading plot (A) and score plot (B) of chemical composition, enzyme activity levels and physical properties of different wheat milling byproducts. PC 1 (horizontal axis) explained 62% of the variability. PC 2 (vertical axis) explained 14% of the variability. Abbreviations used: dietary fiber (DF), total arabinoxylan (TOT-AX), water-extractable arabinoxylan (WE-AX), damaged starch (DStarch), a-amylase activity level (Amy), endoxylanase activity level (Xyl), endopeptidase activity level (Pep), mean particle size (MPS), water retention capacity (WRC) and swelling capacity (SC), coarse bran (CB), coarse weatings (CW), fine weatings (FW) and low grade flour (LGF).

3.6. Functionality of wheat milling by-products in dough and bread making Usually, when wheat bran is added to white flour, a specific percentage of flour is replaced by bran. However, in the present study, significant differences in starch content, and thus in endosperm contamination, were observed between the analyzed milling by-products. When using a fixed replacement level, this implies that relatively less flour is replaced by botanical bran when the milling by-product contains more endosperm. This is for example the case for low grade flour compared to coarse bran. Consequently, with this approach, meals containing equivalent levels of coarse bran and low grade flour contain the same amounts of milling by-product, but differ in endosperm content. In order to investigate the functionality of the botanical bran component in the milling by-products, we also composed meals

3.6.2. Dough and bread made with meals having an overall starch content of 70% In a second part, meals were composed of flour and milling byproduct to obtain an overall starch level of 70% (Supplementary Table 1). With this approach, all of the composed meals had by approximation the same quantity of endosperm and non-endosperm material. The meals composed using this approach were analyzed for their dough properties (Table 4B). The FWA for by product containing meals varied between 69.5% and 73.8%. The highest values were observed for the dough containing fine weatings and low grade flour. Next, when the DDTs were investigated, some clear differences in development times could be observed (Table 4B). A significantly lower DDT was noticed for doughs containing wheat bran with a smaller average particle size. Indeed, the highest average DDTs were observed for doughs containing coarse bran (8.7 min), followed by doughs with coarse weatings (6.5 min), fine weatings (5.4 min) and finally doughs containing low grade flour (3.9 min). One exception was the coarse bran 5 sample, where a DDT comparable to the doughs with coarse weatings was observed (6.6 min). Table 4B also shows the differences in bread specific volume. A different trend was observed compared to breads composed on an equivalent bran sample basis (Table 4A). Indeed, when considering bread made with a recipe on a same starch basis (Table 4B), the specific volume of breads composed with coarse bran was comparable to the breads composed with coarse weatings (4.2 cm3/g and 4.0 cm3/g, respectively), but significantly higher compared to specific volumes of breads containing fine weatings and low grade flour (3.4 cm3/g and 2.8 cm3/g, respectively). 4. Discussion First, relations in the chemical composition, enzyme activity levels and physical properties between the milling by-products

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Table 4 Farinograph water absorption (FWA), dough development time (DDT) and bread specific volume for dough and bread made from meals containing 15% by-product (A) and composed on a 70% overall starch basis (B). The first column shows the milling by-product used in the meal (coarse bran, coarse weatings, fine weatings, low grade flour)a.

a Values in grey represent averages of the by-products from a different origin but of a same type. The individual samples are compared to each other for significant differences and are grouped using small letters. The averages are compared to each other as well and are grouped using capital letters. Values within the same column not sharing a same letter are significantly different (P < 0.05).

were investigated. When comparing the chemical composition of the different milling by-products, an increase in total starch, damaged starch and lipid content and a decrease in DF and ash content was observed when the by-product type was finer (P < 0.05). This is caused by a less efficient separation of starchy endosperm and bran in the finer fractions. Indeed, as mentioned earlier, it is common knowledge that low-grade flour and fine weatings consist of endosperm particles that still have bran attached after all the grinding steps (Delcour & Hoseney, 2010). The higher lipid content in the finer bran types are probably due to germ remnants or a higher amount of aleurone in these milling by-products. When relating these observations to the corresponding correlation loading plot (Fig. 2A), it can be noticed that constituents which are typically high in endosperm and/or germ are found to the left of the plot, while constituents that are typically high in the outer wheat kernel layers are found to the right of the plot. PC 1 hence represents the level of endosperm contamination in the bran. It is also clear that the protein content was not related to any other chemical component or bran property. Nevertheless, the protein composition would probably be different for the samples with a higher or a lower endosperm contamination, as previous research showed that the amino acid distribution differs between the different bran layers and the endosperm (Jensen & Martens, 1983). Among the different samples in one by-product category only relatively small variations in DF and starch contents were observed. These can easily be attributed to the efficiency of the milling process in different milling facilities. The largest difference in composition was observed between the fine weatings derived from industrial scale milling and laboratory scale milling. This results from the fact that the fine weatings obtained on laboratory scale also contains material that ends up in the low grade flour fraction in an industrial mill. Besides the strong correlation between the chemical components, it was clear that DF was also highly positively correlated to the by-product MPS, WRC and SC (Fig. 2A). The interaction between bran and water can be explained by molecular interactions like hydrogen bounds, ionic bounds or hydrophilic interactions and by the entrapment of water in the bran matrix (Chaplin, 2003). It is known that water strongly binds to DF components such as water-insoluble arabinoxylan (Chaplin, 2003; Courtin & Delcour, 2002), which is abundantly present in wheat bran (Table 1). The comparable calculated WRCDF-values for the

different by-products (shown in the Results section) support this. Indeed, from the WRCDF-values, we can deduce (i) that the amount of water bound per unit of DF is virtually the same for all milling by-product and hence (ii) that DF content is the main factor that determines water binding capacity. Indeed, no clear correlation could be found between the WRCDF and the type of milling by-product, while WRC was clearly depended on the type of by-product. The higher the overall level of DF in the by-product, the higher its water binding capacity. However, it should be taken into account that the by-products with higher DF-levels also had a higher MPS. Indeed, also the microporous structure and imperfect packing of bran particles are said to be important parameters for their capacity to retain water in the tests used (Jacobs, Hemdane, Dornez, Delcour, & Courtin, 2015; Mongeau & Brassard, 1982). Also damaged starch can absorb more water than for instance granular starch (Berton, Scher, Villieras, & Hardy, 2002). Nevertheless, damaged starch and WRC are placed on opposite sides of the PCA plots, indicating that damaged starch does not noticeably contribute to a high WRC in the presence of high botanical bran levels or DF levels as a proxy for such bran. A last set of bran properties analyzed are the enzyme activity levels. Although enzymes are highly concentrated in the outer layers of the wheat kernel (Poutanen, 1997), little is known about the contribution of bran related enzymes to its detrimental effects in bread making. Therefore, it seemed interesting to assess their variability in milling by-products and their relationship with either other by-product properties or with their functionality in bread making. None of the three analyzed enzyme activity levels (a-amylase, endoxylanase, endopeptidase) seemed to be related to the type of milling by-products. Previous studies reported for a-amylase that milling by-products were relatively high in a-amylase activity levels compared to the flour streams, but no clear relationship between the activity levels and the different milling by-product was noticed (Dornez, Gebruers, Wiame, Delcour, & Courtin, 2006). Furthermore, concerning endoxylanase, Dornez, Gebruers, et al. (2006) observed high endoxylanase activity levels in different milling by-products compared to the flour streams. But also in this study, it was difficult to notice clear relationships between the endoxylanase activity level and the type of bran. Finally, although a-amylase and endoxylanase activity levels cluster together in the PCA correlation loading plot (Fig. 2A), no strong correlations could be found between the different enzyme activity levels (P > 0.05).

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Table 5 Pearson’s correlation coefficients between the average Farinograph water absorption (FWA), dough development time (DDT), bread specific volume (Sp. Vol.) and dietary fiber (DF), total arabinoxylan (TOT-AX), water-extractable arabinoxylan (WE-AX), starch, damaged starch (D. Starch), proteins (Prot), ash, lipids, a-amylase activity (Amy), endoxylanase activity (Xyl), endopeptidase activity (Pep), bran mean particle size (MPS), bran water retention capacity (WRC) and bran swelling capacity (SC) in the doughs and breads containing 15% by-product (A) and composed on a 70% overall starch basis (B). Significant values (P < 0.05) are highlighted in bold. DF

TOTAX

WEAX

Starch

D. Starch

Prot

Ash

Lipids

Amy

Xyl

Pep

MPS

WRC

SC

FWA

DDT

Sp. Vol.

A

FWA DDT Volume

0.69 0.80 0.44

0.75 0.83 0.37

0.26 0.50 0.31

0.73 0.85 0.35

0.53 0.79 0.42

0.16 0.29 0.33

0.74 0.93 0.38

0.56 0.90 0.14

0.05 0.12 0.22

0.55 0.36 0.06

0.12 0.59 0.02

0.56 0.95 0.18

0.69 0.86 0.39

0.64 0.92 0.31

1.00 0.68 0.25

– 1.00 0.13

– – 1.00

B

FWA DDT Volume

0.19 0.68 0.60

0.09 0.70 0.71

0.52 0.58 0.66

– – –

0.35 0.78 0.80

0.53 0.84 0.68

0.19 0.65 0.50

0.56 0.86 0.79

0.23 0.75 0.59

0.28 0.25 0.31

0.56 0.81 0.80

0.58 0.89 0.72

0.40 0.88 0.67

0.44 0.88 0.67

1.00 0.46 0.59

– 1.00 0.71

– – 1.00

This suggests that higher or lower enzyme activities in the milling by-products could depend on the wheat varieties or the level of bran- or endosperm-related enzyme inhibitors or result from pre-harvest sprouting (Lunn, Kettlewell, Major, & Scott, 2001) or microbial contamination (Dornez, Joye, Gebruers, Delcour, & Courtin, 2006). For apparent endoxylanase activity levels, for instance, it is known that they strongly depend on the wheat variety and weather conditions, whereas the endoxylanase inhibitor levels are mainly genetically determined (Dornez, Joye, Gebruers, Lenartz, et al., 2006). In a second part of this study, the bread making functionality of the different milling by-products was investigated and related to their properties (Table 5 and Supplementary Fig. 2). To this end, meals were composed both on a constant by-product addition level and on a same starch level basis. Composing meals on a same starch level basis is not common. Nevertheless, this way, all breads contain the same level of non-endosperm compounds, which is not the case if a specific amount of flour is replaced by a milling byproduct. The impact of the milling by-products on dough and bread properties using both approaches is discussed here. In accordance with previous studies (Seyer & Gélinas, 2009; Shenoy & Prakash, 2002), FWA increased when flour was replaced by any milling by-product. This increase was expected as milling by-products are relatively high in DF, which can strongly bind water (Chaplin, 2003; Courtin & Delcour, 2002). However, it should also be noticed that doughs containing milling by-products are higher in damaged starch, which is reported to be highly positively correlated to the hydration capacities of regular flour (Berton et al., 2002). Nevertheless, no clear correlations could be found between FWA and either DF or damaged starch levels in the doughs as shown in Table 5. Also DDT increased when flour was replaced by milling byproducts. This increase was dependent on the type of by-product and highly correlated with bran particle size (Table 5, r = 0.95 for meals containing 15% milling by-product, r = 0.89 for meals having an overall starch content of 70%). Previous studies already observed a decreased DDT with decreasing bran particle size (Noort et al., 2010; Zhang & Moore, 1997), which Noort et al. (2010) attributed to a slower water uptake rate for coarser bran particles than for finer particles. This latter hypothesis would imply that prehydration of wheat bran decreases the DDT compared to doughs with untreated bran. This has been investigated by Nelles, Randall, and Taylor (1998), who indeed reported a decreased DDT when prehydrated bran was added. However, when observing the Farinograms in that study in more detail, we could not observe any clear DDT decrease, which makes it difficult to rely on their conclusions on the one hand and on the hypothesis of a slower water uptake for coarser particles on the other hand. It is thus more likely that the larger bran particles hinder the development of a proper gluten network more strongly than finer particles due to their more hindering physical structure. Therefore, more

time is needed to develop an optimal dough if large bran particles are added compared to doughs containing smaller particles. Finally, the relationship between milling by-products properties and bread specific volume can be considered (Table 5 and Supplementary Fig. 2). Besides the expected decrease in specific volume when wheat flour was replaced by bran, differences between the breads with different milling by-products were also noticed. When flour was replaced by 15% of by-products, low grade flour resulted in bread with a higher specific volume compared to breads containing other milling by-products (P < 0.05). This observation could be explained by less dilution of gluten proteins in bread containing 15% of low grade flour compared to the other bran-enriched breads, due to the high endosperm contamination of low grade flour. This hypothesis is quite plausible, as bread volume is positively affected by gluten quantity (Delcour & Hoseney, 2010). However, based on this hypothesis alone, one could expect that the specific volume is higher when recipes containing byproducts with higher levels of endosperm remnants are considered. This is not entirely the case in present study. Breads containing fine weatings had a comparable specific volume to that of breads containing coarse bran or coarse weatings. The botanical bran of fine weatings seems hence inherently more detrimental to bread quality than the botanical bran of coarse bran and coarse weatings. This is confirmed by the results obtained with the breads composed with a same starch level. From these results, it can be concluded that some properties more associated with fine weatings are more deleterious to bread quality compared to those more associated with coarse bran and coarse weatings. They even show that some properties more associated with low grade flour have the most detrimental impact on bread quality. These properties are discussed in more detail later in this paper. One exception of the deleterious effect of fine weatings and low grade flour was the bread with the fine weatings 3 sample, which had a specific volume comparable to that of bread with coarse bran and coarse weatings. This is probably due to the excellent bread quality wheat cultivar from which the weatings originated and thus, to the high quality gluten proteins in its attached endosperm. Indeed, the proportion of gluten proteins originating from fine weatings 3 in the meal was estimated to be around 14% of the total gluten content in the meal, which was not negligible. The proportion of gluten proteins originating from coarse bran was, for instance, much lower (2.5% to 4% of the total gluten content in the meals composed on a 70% starch basis). The deleterious effect of bran in bread making might be either of a physical, chemical or biochemical nature. Regarding the differences in physical properties between fine weatings and low grade flour on the one hand and coarse bran and coarse weatings on the other hand, a first obvious dissimilarity was the MPS, which was smaller for the first two by-products than for the latter two. A considerable number of studies tried to investigate the importance of bran particle size in bread making. For instance, Noort et al. (2010)

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reported a larger decrease in bread quality when flour was replaced by finely ground bran compared to coarse bran. However, it has also been reported that particle size reduction of coarse bran slightly improved bread volume (Lai, Hoseney, & Davis, 1989a; Moder et al., 1984), which indicates that the impact of bran particle size on bread quality is still not very well understood. In this study, no correlation between MPS and bread specific volume was observed for breads with 15% by-product (Table 5). A slightly positive correlation was found for the breads made with meals formulated on a 70% starch (Table 5, r = 0.72). The latter correlation suggests that, if all breads contain the same level of nonendosperm compounds, smaller bran particles are more detrimental to bread volume than larger ones. This might be due to the higher contact surface of smaller bran particles, resulting into a more pronounced interaction between bran and flour components (Noort et al., 2010). Besides, it is also possible that the more detrimental effect of smaller particles is due to the fact that they have a higher abrasive effect on the gluten network during mixing, as more fine bran particles were added than coarse bran, to obtain breads with a same level of botanical bran. Nevertheless, the relative small correlation between MPS and the specific volume of breads made with meal containing 70% starch (Table 5, r = 0.72) suggests that the particle size of the by-products is not the only factor that affects bread volume. Next, a second difference in physical properties between the milling by-products are the hydration properties. However, no good correlations could be found between the hydration properties of the by-products and the bread specific volume (Table 5). Another explanation for the detrimental effect of fine weatings and low grade flour might be that these milling by-products contain higher levels of lipids or damaged starch. The specific volume of breads made with meal containing 70% starch was negatively correlated with lipids (Table 5, r = 0.79), which are known to negatively influence bread volume, especially non-polar lipids (Goesaert et al., 2005). Besides, a similar correlation was found between the specific volume of breads containing 70% starch and damaged starch levels (Table 5, r = 0.80). Although damaged starch is not a bran-related component, the abundant presence of this specific component is important to mention. Indeed, according to Barrera, Pérez, Ribotta, and León (2007), a highly negative correlation exist between the damaged starch level in flour and bread volume. Finally, a last possible reason for the decrease in bread quality upon bran addition are bran-related enzymes. Here, no good correlation could be found between the measured enzyme activity levels in the meals and the specific volume of either breads containing 15% by-product or 70% starch (Table 5), except between endopeptidase and the specific volume of breads containing 70% starch (Table 5, r = 0.80). Moreover, no clear relationship was observed between the type of by-product and a-amylase-, endoxylanase- and endopeptidase activity levels as shown in Table 2 and Fig. 2A. This does not imply that these bran-related enzymes are not functionally relevant in bread making, but implies that they do probably not contribute to a net volume increase or decrease. It can be concluded that depending on the type (coarse bran, coarse weatings, fine weatings or low grade flour), wheat milling by-products can strongly differ in chemical composition, enzyme activity levels and physical properties. They induce clear changes in dough and bread properties when flour is replaced by these different by-products. Bread specific volumes were more significantly decreased when fine weatings or low grade flour were added on an equal overall starch level, indicating that some properties more associated with these by-products were more detrimental to bread quality than properties more associated with coarse bran and coarse weatings. These properties can be related to particle size, lipid content or damaged starch content.

Acknowledgements This work was performed within the framework of a Flanders’ FOOD (Brussels, Belgium) project. P.J. Jacobs acknowledges the Institute for the Promotion of Innovation through Science and Technology in Flanders (IWT-Vlaanderen, Brussels, Belgium) for the financial support. E. Dornez acknowledges the ‘‘Fonds voor Wetenschappelijk Onderzoek – Vlaanderen’’ (FWO, Brussels, Belgium) for her postdoctoral fellowship. J.A. Delcour is W.K. Kellogg Chair in Cereal Science and Nutrition at KU Leuven. This study was also part of the KU Leuven Methusalem program ‘‘Food for the future’’ (20072021).

Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.foodchem.2015. 04.048.

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