effect of bioprocessing and particle size on the nutritional properties of wheat bran fractions

$: Effect of bioprocessing and particle size on the nutritional properties of wheat bran fractions$
Innovative Food Science and Emerging Technologies xxx (2013) xxx–xxx

INNFOO-01100; No of Pages 9

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Innovative Food Science and Emerging Technologies

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Effect of bioprocessing and particle size on the nutritional properties of wheatbran fractions

Rossana Coda a,⁎, Carlo Giuseppe Rizzello b, José Antonio Curiel b, Kaisa Poutanen a,c, Kati Katina a

a VTT, Tietotie 2, 02044 VTT, Finlandb Department of Soil, Plant and Food Sciences, University of Bari, 70126 Bari, Italyc Department of Clinical Nutrition, University of Eastern Finland, Kuopio Campus, P.O. Box 1627, FIN-70211 Kuopio, Finland

Abbreviations: as, Ash; BHT, butylated hydroxytoluentary fiber; EAA, Essential Amino Acid; EAAI, Essential Amacid; FID, flame ionization detector; GC, gas chromatofiber; NI, Nutritional Index; PA, phytase activity; PCA, PPER, Protein Efficiency Ratio; Pr, protein; RP, reducing poST, starch; DFt, total dietary fiber; TFAA, total free amintrifluoroacetic acid; Wx, water extractable arabinoxylans.⁎ Corresponding author. Tel.: +358 408428330.

E-mail address: [email protected] (R. Coda).

1466-8564/$ – see front matter © 2013 Elsevier Ltd. All rihttp://dx.doi.org/10.1016/j.ifset.2013.11.012

Please cite this article as: Coda, R., et al., EffecFood Science and Emerging Technologies (201

a b s t r a c t
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Article history:Received 22 July 2013Accepted 27 November 2013Available online xxxx

Editor Proof Receive Date 27 December 2013

Keywords:Lactic acid bacteriaYeastFermentationBranBioprocessingMicronizationFiberAntioxidant

In this work, the influence of bioprocessing on the nutritional quality and health effects ofwheat bran of differentparticle sizes (750, 400, 160, 50 μm)was evaluated. Bioprocessing was carried out by a 24 h-fermentation usingLactobacillus brevis E95612 and Kazachstania exigua C81116 as starters, with or without the addition of an en-zymemixture with specific carbohydrase activities. Bioprocessing clearly affected themicrostructure and chem-ical and nutritional features of wheat bran depending on the particle size. Bioprocessing significantly improvedthe antioxidant and phytase activities (up to 3.7 fold, respectively), peptides and total free amino acids and con-tent (up to 40%) and the in vitro digestibility of proteins. The antioxidant power and nutritional indexes werehigher for the bioprocessed brans compared to the native, mainly in bran having smaller particle size. In everycase, the addition of the enzymes further improved the positive effect of the microbial fermentation.Industrial relevance:Wheat bran is a source of nutritionally important compounds such as dietaryfibers,minerals,vitamins and phenolic acids. Commonly, processing of bran has mostly been performed for technological pur-poses, to facilitate its use as a DF rich ingredient in foods improving its structural and organoleptic features.The bioprocessing technology here applied offers a tool to enhance also the nutritional value of wheat bran, es-pecially of finer particle size. As a result, bioprocessed wheat bran showed higher potential compared to the na-tive bran, and qualified as a suitable ingredient for food preparations.

© 2013 Elsevier Ltd. All rights reserved.

1. Introduction

Worldwide, food and health authorities recommend an increase ofthe consumption of whole grain cereals due to the evidence that morewhole grains in the diet lead to a reduction of the risk of several chronicdiseases (Frølich, Åman, & Tetens, 2013). Besides the nutrient contentand the presence of compounds such as dietary fibers, minerals, andphenolic acids, also the structure and texture of cereal foods have longbeen recognized as parameters involved in the health benefits ofwhole grain foods (Frølich et al., 2013). Therefore, different novel tech-nologies were developed for transformation processes in order to betterexploit the cereal nutritional potential. Among these, micro- and nano-technologies are showing great potential in nutraceuticals and function-al food manufacture for human health improvement (Chen, Weiss, &

e; BV, Biological Value; DF, die-ino Acid Index; FAA, free aminography; DFi, insoluble dietaryrincipal Component Analysis;wer; DFs, soluble dietary fiber;o acid; TP, total phenols; TFA,

ghts reserved.

t of bioprocessing and particl3), http://dx.doi.org/10.1016/

Shahidi, 2006). Recently, the application of micronization in food re-search has shown that the reduction of the particle size of variousfiber-rich plantmaterials alters the structure, surface area and function-al properties of the particles (Hemery et al., 2011). In vitro digestionstudies of bran-enriched breads have shown that the bioaccessibilityof phenolic acids and minerals was improved with the decreasing ofbran particle size and with the increasing concentration of micronizedaleurone material (Hemery, Mabille, Martelli, & Rouau, 2010). Fromthe industry point of view the possibility to exploit bran fractions witha different granulometry is a useful opportunity to obtain new function-al ingredients for the preparation of several foods (Esposito et al., 2005).This is important considering that, besides the nutritional value mainlyrelated to dietary fibers, phenols andminerals, bran has negative effectson sensory and technological properties which limit its use as a food in-gredient in general and in breadmaking in particular. One possibility toovercome this effect is to pre-treat bran with bioprocessing techniquessuch as fermentation, using specific yeast and lactic acid bacteria startercultures and/or enzymes, such as cell wall degrading enzymes (Delcour,Rouau, Courtin, Poutanen, & Ranieri, 2012).

The use of bioprocessing techniques has been shown to be also agood approach to improve the bioaccessibility of health-promotingcompounds in bran (Coda et al., 2014; Katina et al., 2007; MateoAnson, van den Berg, Havenaar, Bast, & Haenen, 2009). Enzymatic and

e size on the nutritional properties of wheat bran fractions, Innovativej.ifset.2013.11.012

http://dx.doi.org/10.1016/j.ifset.2013.11.012

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2 R. Coda et al. / Innovative Food Science and Emerging Technologies xxx (2013) xxx–xxx

fermentation technologies offer an array of tools to modify the grainmatrix. During fermentation, the grain constituents are modified bythe action of both endogenous and bacterial enzymes, thereby affectingtheir structure, bioactivity, and bioavailability (Hole et al., 2012). Yeast-started fermentation improved the bioactivity and baking properties ofwheat bran prepared from peeled kernels, resulting in solubilization ofarabinoxylans (Katina et al., 2012). Fermentation can exert an impacton the nutritional quality and health effects of whole grain foods. For in-stance, fermentation with lactic acid bacteria has a well-known role inenhancing the nutritional properties as well as the texture and palat-ability of whole grain and fiber rich products (Poutanen, Flander, &Katina, 2009). It increases the level of bioactive peptides, dietary fibersolubility and mineral bioavailability, and decreases glycemic index(Rizzello, Cassone, Di Cagno, & Gobbetti, 2008). Lactic acid bacteria fer-mentation is a useful tool to obtain specific results, but selection of prop-er strains is an important pre-requisite for successful achievements. Thecapacity of lactic acid bacteria to release bioactive compounds duringcereal fermentation is very well known but few studies are availableon bran fermentation. Bran seems to have a great potential to improvethe technological performances and/or integrate foods with healthycompounds, if a proper processing technology is applied. In this study,the impact of the combination of milling procedure and bioprocessingon wheat bran having different particle sizes was evaluated. Chemicaland nutritional properties of fermented bran were investigated.

2. Materials and methods

2.1. Raw materials

Commercial wheat bran (Fazer Mills, Lahti, Finland) was ground byTurboRotor technology (Mahltechnik Görgens GmbH, Dormagen,Germany) to three different levels of fineness. The median particlesizes of the four brans obtained, and analyzed by sieving, were: 750(unground), 400, 160 and 50 μm, as provided by the supplier. All thefour brans were used in bioprocessing. Total DF contents of the branswere 48.0 ± 1.3% (750 μm), 48.9 ± 1.9% (400 μm), 47.9 ± 1.9%(160 μm) and 48.4 ± 1.7% (50 μm), respectively, as determined ac-cording to the method AOAC 9852.

2.2. Bran bioprocessing

Lactobacillus brevis E95612 and Kazachstania exigua C81116 belong-ing to VTT Culture Collection (VTT, Technical Research Centre ofFinland) were used as starters for fermentation. L. brevis E95612was cultivated for 24 h at 30 °C on MRS (Oxoid LTD, Basingstoke,Hampshire, United Kingdom) in anaerobic conditions, while the yeastwas cultivated for 24 h at 25 °C in YM (3 g/L malt extract, 3 g/L pep-tone, 10 g/L dextrose). After the late exponential phase of growth wasreached, cells were recovered by centrifugation (10,000 ×g for10 min), successively washed twice in 0.05 M phosphate buffer,pH 7.0, and re-suspended in tap water (ca. 15% of the initial volume ofthe culture). Wheat bran and water (ratio 20/80) were mixed using aBamix blender (Bamix, Switzerland). Lactic acid bacteria and yeastwere both inoculated at cell density of ca. 106 cfu/g. Enzyme prepara-tions Depol 740L (Biocatalyst Ltd., Great Britain) and Grindamyl 1000(Danisco, Denmark) were added at the beginning of fermentation. Inparticular, Depol 740L (liquid preparation) was dissolved in water,while Grindamyl 1000 (powder) was added to bran. The enzymesused contained a variety of hydrolytic enzymes, mainly xylanase,endoglucanase and β-glucanase in Depol 740L (Mateo Anson et al.,2009), and alpha-amylase in Grindamyl. Enzyme dosages were:161 nkat xylanase/g of bran for Depol 740L (xylanase activity accordingto Bailey, Biely, and Poutanen (1992)), and 75 nkatα-amylase/g of branfor Grindamyl 1000 (n α-amylase activity according to MegazymeCeralpha method). Bioprocessing of bran was carried out inoculatingthe two starterswithout andwith the addition of enzymes, as described

Please cite this article as: Coda, R., et al., Effect of bioprocessing and particlFood Science and Emerging Technologies (2013), http://dx.doi.org/10.1016/

above. Fermentations were carried out using Termarks incubators,KBP6151, Norway at 20 °C for 24 h. After fermentation, bran wasfreeze-dried and characterized.

2.3. Microbiological analyses and pH determination

Bran samples (10 g) were homogenized with 90 mL of sterile salinein a Stomacher 400 lab blender (Seward Medical, London). Serial dilu-tions were made and enumeration of lactic acid bacteria and yeastswas carried out by plating on MRS and YM agar after incubation for48 h at 30 °C or 25 °C respectively. The pH value was measured by aTitroLine autotitrator (Alpha 471217, Schott, Mainz, Germany)suspending an aliquot of 10 g of fermented bran in 100 mL of distilledwater. All samples were analyzed in duplicate.

2.4. Microscopic analysis

Aliquots of the different bran fractions were collected beforeand after bioprocessing, embedded in 2% agar and fixed in 1%glutharaldehyde dissolved in 0.1 M phosphate buffer (pH 7.0),dehydrated in a graded ethanol series, and embedded in hydroxyethylmethylacrylate, as recommended by the embedding kit manufacturer(Leica Historesin, Heidelberg, Germany). Polymerized sampleswere sectioned (2 mm sections) in a rotary microtome HM 355(MicromLaborgeräte GmbH, Walldorf, Germany) using a steel knife.Protein was stained with aqueous 0.1% (w/v) Acid Fuchsin (Gurr, BDHLtd., Poole, U.K.) in 1.0% acetic acid for 1 min, and β-glucan was stainedwith aqueous 0.01% (w/v) Calcofluor White (fluorescent brightener 28,Aldrich, Germany) for 1 min. The sections were also stained with LightGreen (BDH Chemicals Ltd, Poole, Dorset, UK)/Lugol's iodine solution.When imaged in bright field, Light Green stains protein green/yellow,whereas Lugol's iodine solution stains the amylose component of starchblue and amylopectin brown. Most starch appears dark blue becauseamylose masks the amylopectin. The samples were examined withan Olympus BX-50 microscope (Tokyo, Japan). In exciting light(epifluorescence, 400 and 410 nm; fluorescence, N455 nm) intact cellwalls were stained with Calcofluor that appear in blue and proteinswere stained with Acid Fuchsin that appear in red. Starch is unstainedand appears black. Micrographs were obtained using a SensiCam PCOCCD camera (Kelheim, Germany) and the Cell^P imaging software(Olympus).

2.5. Dietary fiber and arabinoxylan analysis

Total dietary fiber (DF) content was analyzed. Soluble and insolubleDF of the bran and bread samples was determined by the enzymaticgravimetric AOAC Method 2009 with an assay kit (Total DF K-TDFR,Megazyme, Ireland), following manufacturer's instructions.

The water-extractable arabinoxylan fraction was obtained byextracting 1 g of the cereal sample with 7 mL of cold water (4 °C)(Santala, Nordlund, & Poutanen, 2013). Successively, the water-soluble fraction was hydrolyzed with 1.2 mL of 7.5 N H2SO4 in a boilingwater bath for 2 h (Santala et al., 2013). Tomeasure the total amount ofarabinoxylans, 50 mg of cereal sample was pre-hydrolyzed with1.56 mL of 72% (w/w) H2SO4 at room temperature (25 °C) for 30 min(Blakeney, Harris, Henry, & Stone, 1983). Samples were then dilutedwith 15.6 mL of Milli-Q water (Millipore, Billerica, MA) and hydrolyzedin a boiling water bath for 2 h. After cooling, the solutions wereneutralized by adding appropriate volume of 4 MNaOH. The sugars ob-tained from the hydrolysis steps and the monosaccharide standards(50 mg/mL; glucose, arabinose, xylose, galactose, and mannose) wereanalyzed as their alditol acetates, as described by Blakeney et al.(1983). Standard curves were obtained analyzing dilutions obtainedby thesemonosaccharide solutions. Myo-inositol was used as the inter-nal standard (0.5 mg/mL sample). The acetylated monosaccharideswere analyzed with gas chromatography (GC) using an Agilent 6890



3R. Coda et al. / Innovative Food Science and Emerging Technologies xxx (2013) xxx–xxx

GC (Palo Alto, CA) equipped with a flame ionization detector (FID). Thecolumn was DB-225 (30 m × 0.32 mm, film thickness 0.15 μm,Agilent). Helium was used as the carrier gas at 1.2 mL/min. Split injec-tion (1:3) was performed at 250 °C, and the FID was operated at250 °C. The analyteswere separated at 220 °C for 15 min. Themonosac-charides were identified according to their retention times and quanti-fied with a standard curve. Free hexose sugars were corrected by afactor of 0.9 to anhydro sugars, and pentose sugars by factor of 0.88.All analyses were made in duplicate.

2.6. Phytase and antioxidant activities

Phytase activity was measured in doughs and expressed as theamount of inorganic orthophosphate released from the phytic acid byphytase (Shimuzu, 1992). One unit (U) of phytase activity was definedas the amount of enzyme required to liberate 1 μmol of phosphate/minunder the conditions of the assay.

For the determination of antioxidant activities, 5 g of each bran wasmixed with 50 mL of 80% methanol. The mixture was purged with ni-trogen stream for 30 min in stirring condition and centrifuged at4600 ×g for 20 min. Extracts were transferred into test tubes, purgedwith nitrogen stream and stored at ca. 4 °C before the determinationof total phenols, free radical scavenging activity and Fe3+ reducingpower. Analysis of total phenols was carried out in agreement withthe method of Slinkard and Singleton (1997) on methanolic extracts.The concentration of total phenols was expressed as gallic acid equiva-lent. The free radical scavenging capacity was determined using the sta-ble 2,2-diphenyl-1-picrylhydrazyl radical (DPPH•) as reported by Yu,Perret, Harris, Wilson, and Scott Haley (2003). The reaction was moni-tored by reading the absorbance at 517 nm every 2 min for 30 min. Ablank reagent was used to verify the stability of DPPH• over the testtime. The absorbance valuemeasured after 10 minwas used for the cal-culation of the μmol DPPH• scavenged by the extracts. The absorbancevalue in the presence of the extract was also determined over 30 minand compared with butylated hydroxytoluene (BHT) at the concentra-tion of 75 ppm as the antioxidant reference.

The Fe3+ reducing power of the methanolic extracts was deter-mined by the method of Oyaizu (1986) with slight modifications. Theextracts were mixed with 0.75 mL of phosphate buffer (0.2 M, pH 6.6)and 0.75 mL of potassium hexacyanoferrate [K3Fe(CN)6] (w/v 1%),followed by incubating at 50 °C in a water bath for 20 min. The reactionwas stopped by adding 0.75 mL of trichloroacetic acid (TCA) solution(10%) and then centrifuged at 3000 r/min for 10 min. 1.5 mL of the su-pernatant wasmixed with 1.5 mL of distilled water and 0.1 mL of ferricchloride (FeCl3) solution (0.1%, w/v) for 10 min. The absorbance at700 nm was measured as the reducing power. Higher absorbance ofthe reaction mixture indicated greater reducing power.

2.7. Peptide profiles and free amino acid analysis

The water/salt-soluble extract of samples, prepared according toWeiss, Vogelmeier, and Gorg (1993), was used to analyze peptidesand free amino acids. Peptide profiles were obtained by reversed-phase fast protein liquid chromatography (RP-FPLC), using a ResourceRPC column and ÄKTA FPLC equipment, with a UV detector operatingat 214 nm (GE Healthcare Bio-Sciences AB, Uppsala, Sweden). The pep-tide concentration in water/salt soluble extracts was determined bythe o-phtaldialdehyde (OPA) method (Church, Swaisgood, Porter, &Catignani, 1983). Aliquots of each extract were added to 0.05% (vol/vol)trifluoroacetic acid (TFA), centrifuged at 10,000 ×g for 10 min, and thesupernatant was filtered through a Millex-HA 0.22 μm pore size filter(Millipore Co.) and loaded onto the column. Gradient elution was per-formed at a flow rate of 1 mL/min using a mobile phase composed ofwater and acetonitrile (CH3CN), containing 0.05% TFA. The CH3CN con-tent was increased linearly from 5 to 46% between 16 and 62 min.


Free amino acids were analyzed by a Biochrom 30 series Amino AcidAnalyzer (Biochrom Ltd., Cambridge Science Park, England) with a Na-cation-exchange column (20 by 0.46 cm internal diameter) as de-scribed by Rizzello, Nionelli, Coda, De Angelis, and Gobbetti (2010).

2.8. Nutritional characterization

The in vitro digestibility of samples was determined by the methodof Akeson and Stahmann (1964). A known amount of sample was incu-bated with 1.5 mg of pepsin, in 15 mL of 0.1 M HCl, at 37 °C for 3 h.After neutralizationwith 2 MNaOH and addition of 4 mg of pancreatin,in 7.5 mL of phosphate buffer (pH 8.0), 1 mL of toluene was added toprevent microbial growth, and the solution was incubated for 24 h at37 °C. After 24 h, the enzyme was inactivated by addition of 10 mL oftrichloroacetic acid (20%, wt/vol), and the undigested protein was pre-cipitated. The volume was made up to 100 mL with distilled waterand centrifuged at 3200 ×g for 20 min. The concentration of protein ofthe supernatant was determined by the Bradford method (Bradford,1976). The precipitate was subjected to protein extraction, accordingtoWeiss et al. (1993), and the concentration of proteinwas determined.The in vitro protein digestibility was expressed as the percentage of thetotal protein, which was solubilized after enzyme hydrolysis.

The supernatant, which contained the digested protein, was freeze-dried and used for further analyses. The modified method of AOAC982.30a (AOAC, 1990) was used to determine the total amino acid pro-file. The digested protein fraction, which is derived from 1 g of sample,was added of 5.7 M HCl (1 mL/10 mg of proteins), under nitrogenstream, and incubated at 110 °C for 24 h. Hydrolysis was carried outunder anaerobic conditions to prevent the oxidative degradation ofamino acids. After freeze-drying, the hydrolysate was re-suspended(20 mg/mL) in sodium citrate buffer, pH 2.2, and filtered through aMillex-HA 0.22 μmpore size filter (Millipore Co.). Amino acidswere an-alyzed by a Biochrom30 series AminoAcid Analyzer as described above.Since the above procedure of hydrolysis does not allow the determina-tion of tryptophan, it was estimated by the method of Pintér-Szakácsand Molnán-Perl (1990). One gram of sample was suspended in10 mL of 75 mM NaOH, and shaken for 30 min at room temperature.The sample was centrifuged (12,800 ×g for 10 min), and 0.5 mL of thesupernatant was mixed with 5 mL of ninhydrin reagent (1 g of ninhy-drin in 100 mL of HCl 37%: formic acid 96%, at the ratio 2:3) and incubat-ed for 2 h at 37 °C. The reaction mixture was cooled at roomtemperature and made up to 10 mL with the addition of diethyl ether.The absorbance at 380 nm was measured. A standard tryptophancurve was prepared using a tryptophan (Sigma Chemicals Co.) solutionin the range 0–100 μg/mL.

Essential Amino Acid Index (EAAI) estimates the quality of the testprotein, using its EAA content as the criterion. EAAI was calculated ac-cording to the procedure of Oser (1959). It considers the ratio betweenEAA of the test protein and EAA of the reference protein, according tothe following equation:

EAAI ¼ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi

EAA1 � 100ð Þ EAA2 � 100ð Þ …ð Þ EAAn � 100ð Þ sample½ �EAA1 � 100ð Þ EAA2 � 100ð Þ …ð Þ EAAn � 100ð Þ references½ �

n

s:

The Biological Value (BV) indicates the utilizable fraction of the testprotein. BV was calculated using the equation of Oser (1959):BV = ([1.09 ∗ EAAI] − 11.70). The Protein Efficiency Ratio (PER)estimates the protein nutritional quality based on the amino acid profileafter hydrolysis. PER was determined using the model developedby Ihekoronye (1981): PER = −0.468 + (0.454 ∗ [Leucine]) −(0.105 ∗ [Tyrosine]). The Nutritional Index (NI) normalizes the qualita-tive and quantitative variations of the test protein compared to its nutri-tional status. NI was calculated using the equation of Crisan and Sands(1978), which considers all the factors with an equal importance:NI = (EAAI ∗ Protein(%)/100).




2.9. Statistical analysis

Data (three replicates) from microbiological, rheological and chem-ical analyses were subjected to one-way ANOVA; pair-comparison oftreatment means was obtained by Tukey's procedure at P b 0.05,using the statistical software Statistica forWindows (Statistica 8.0,Win-dows). The following variables were analyzed with Principal Compo-nent Analysis (PCA) using covariance matrix, with the softwareStatistica 8.0, Windows (Dijksterhuis, 1997): total DF (DFt), insolubleDF (DFi), soluble DF (DFs), water extractable arabinoxylans (Wx),starch (ST), protein (Pr), ash (as), total free amino acids (TFAAs),phytase activity (PA), total phenols (TP), reducing power (RP), EssentialAmino Acid Index (EAAI), Biological Value (BV), Protein Efficiency Ratio(PER), Nutritional Index (NI).

3. Results

3.1. Microbiological, microscopic and chemical characterization ofbioprocessed bran

After 24 h of fermentation, cell density of lactic acid bacteria variedfrom 8.5 ± 0.3 to 8.9 ± 0.2 log cfu/g. The highest variation observed(ca. 2 log cycles) occurred for 160 and 50 μmbrans. Yeast final cell den-sity varied from 8.0 ± 0.1 to 8.5 ± 0.1 log cfu/g, showing the highestincrease in the 160 and 50 μm brans (ca. 1.8 log cycle). When enzymeswere added, the final cell density of lactic acid bacteria further increasedof 0.5–0.7 log cycle, mainly in the case of 50 μm brans, while no signif-icant variationwas observed in yeast growth. The values of pH for nativebranwere in the range 6.2 ± 0.1–6.7 ± 0.1. After 24 h, fermented branshowed pH values in the range 5.6 ± 0.2–5.8 ± 0.3. When enzymeswere added, the pH further decreased to 5.0 ± 0.2–5.3 ± 0.2. Microbi-ological results are in accordance with those previously reported byCoda et al. (2014).

The microstructure of the bran material before and afterbioprocessing was analyzed using epifluorescence microscopy (Fig. 1).The four native brans differed in both size and composition of their par-ticles. As expected, the reduction of particle size corresponded to differ-ent distribution and size of pericarp, starchy endosperm, and aleuronelayer. In general, they appeared separated and clearly recognizablein their native structure in the coarse fraction, disaggregated and

Fig. 1.Microstructure of native bran having particle size of 750 (A1) and 50 μm (B1) after biopaleurone cell walls and endosperm is stained in blue, the pericarp layer is stained in green, andaleurone layer) is stained in orange. Starch is unstained and appears black. (For interpretation ofarticle.)


homogeneously mixed in the finest bran. Bioprocessing greatly influ-enced the microstructure of the bran samples, irrespective of their par-ticle size. After fermentation, a partial disruption of the structures of thedifferent layers occurred.Moreover, when enzymeswere added, amoreextensive breakdown of the cell wall structures was observed.

In native bran, the amount of total DF varied from 47.9 ± 1.9 to48.9% ± 1.9 showing slight differences between the fractions havingdifferent particle sizes (Table 1). In general, bioprocessing did not affectthe content of total DF. However, a significant (P N 0.05) decrease wasobserved after bioprocessing for 24 h with the addition of enzymesin the case of bran having particle size of 50 μm. Solubility ofarabinoxylans was enhanced by fractionation, showing the highest con-tent in the finest bran (50 μm). During bioprocessing, solubility ofarabinoxylans increased irrespective of the particle size, mainly whenenzymes were added, reaching a double content in comparison tobran fermented with the starters alone (Table 1). The enhancementwas the highest in the coarsest bran, showing a concentration morethan 11-fold in comparison to its native control. Starch content of thenative finest brans (160 and 50 μm)was higher than the coarsest. How-ever, after bioprocessing, a reduction of starch contentwas observed forall the samples, in particular the highest starch content reduction wasobserved for the bran having particle size of 160 μm (Table 1). Overall,the protein content showed a slight but significant increase afterbioprocessing, irrespective of the particle size while no significant(P N 0.05) variation was observed for ashes (Table 1).

Peptide concentration of the samples was determined on thewater/salt soluble extracts. This type of extract contains low-molecular-masspeptides naturally present in samples or deriving from the degradationof the different wheat proteins during bioprocessing. Native bran frac-tions were characterized by peptide concentration ranging from33.48 ± 5.20 to 50.80 ± 4.82 mg/kg for 750 and 50 μm, respectively.Peptideswere at the highest concentration in the 50 μmfractionwhere-as their concentration decreased with the increase of the particle size.Bioprocessing caused a marked increase in peptide concentration; inparticular, fermentation with the selected starters corresponded to in-creases of 7–22 mg/kg compared to native brans. When enzymeswere added, the increase in peptide concentration was in the range31–36 mg/kg compared to the native brans. The highest increase wasfound for the 160 μm fraction, which was characterized by a peptideconcentration of 62.77 ± 5.62 mg/kg when fermented for 24 h by the

rocessing without (A2 and B2) and with the addition of enzymes (A3 and B3). β-glucan inproteins are stained in red and reddish brown. The pigment strand (between pericarp andthe references to color in this figure legend, the reader is referred to theweb version of this


image of Fig.�1


Table 1Chemical composition of native and bioprocessed bran fractions (24 h, without or with the addition of enzymes). Data are expressed as % on dry weight, with the exception of peptides(expressed as mg/kg).

Bran fractions(μm)

Fermentationconditions

Dietary fiber (%) Water extractablearabinoxylans (%)

Starch (%) Protein (%) Peptides (mg/kg) Ash (%)

Insoluble Soluble Total

750 0 44.9 ± 1.5a 3.1 ± 0.1c 48.0 ± 1.3a 0.2 ± 0.1e 13.9 ± 0.2c 18.7 ± 0.5b 33.48 ± 5.20e 6.2 ± 0.224 h 46.0 ± 2.0a 2.9 ± 0.1c 48.9 ± 0.4a 1.2 ± 0.2c 13.6 ± 0.1c 19.5 ± 0.3a 45.93 ± 4.43d 6.3 ± 0.124 h E* 42.7 ± 2.5b 3.6 ± 0.2b 46.4 ± 2.0a 2.3 ± 0.1b 13.1 ± 0.5a 20.4 ± 0.3a 67.06 ± 6.02b 6.3 ± 0.2

400 0 45.4 ± 1.8a 3.5 ± 0.2b 48.9 ± 1.9a 0.3 ± 0.1e 14.9 ± 0.2b 18.4 ± 0.2b 36.51 ± 3.62e 6.2 ± 0.124 h 44.9 ± 1.7a 3.0 ± 0.3c 47.9 ± 2.0a 1.3 ± 0.1c 13.0 ± 0.5c 19.4 ± 0.2a 43.44 ± 6.11d 6.3 ± 0.124 h E 44.7 ± 1.6a 3.5 ± 0.2b 48.2 ± 1.5a 2.1 ± 0.2b 12.6 ± 0.2d 19.9 ± 0.1a 64.85 ± 3.60b 6.6 ± 0.2

160 0 43.8 ± 1.8b 4.1 ± 0.2a 47.9 ± 1.9a 0.4 ± 0.1d,e 15.4 ± 0.2a 18.5 ± 0.3b 41.09 ± 1.61d 6.3 ± 0.324 h 45.1 ± 2.0a 3.0 ± 0.3c 48.2 ± 2.1a 1.5 ± 0.2c 12.8 ± 0.3d 20.4 ± 0.3a 62.77 ± 5.62b 7.0 ± 0.524 h E 42.6 ± 1.4b 3.7 ± 0.2b 46.3 ± 1.8a 2.5 ± 0.2b 12.5 ± 0.2d 20.2 ± 0.5a 77.28 ± 4.43a 6.9 ± 0.2

50 0 43.9 ± 1.8b 4.6 ± 0.2a 48.4 ± 1.7a 0.6 ± 0.1d 15.7 ± 0.2a 18.5 ± 0.3b 50.80 ± 4.82c 6.4 ± 0.224 h 42.2 ± 1.5b 3.5 ± 0.4b 45.7 ± 2.0a 1.8 ± 0.2c 13.9 ± 0.2c 20.0 ± 0.2a 62.88 ± 3.2b 6.7 ± 0.224 h E 39.9 ± 2.3c 4.7 ± 0.1a 44.6 ± 1.9b 3.0 ± 0.2a 13.5 ± 0.3c 20.1 ± 0.3a 81.38 ± 1.24a 6.8 ± 0.1

E* indicates bioprocessing with the addition of enzymes.a–eValues in the same column with different superscript letters differ significantly (P b 0.05).


starters, and of 77.28 ± 4.43 mg/kg when enzymes were added. Thedata were confirmed by the RP-FPLC analyses of the water/salt solubleextracts, which showed amarked increase of the peptide peak area dur-ing bioprocessing (Fig. 2). Compared to native brans, the main differ-ences found in the peptide profiles of bioprocessed brans concernedthe hydrophilic zone of the chromatogram and the elution intervalfrom 20 to 45% of the acetonitrile gradient (Fig. 2).

The four bran fractionswere characterized by concentration of TFAA,varying from 2309 ± 180 to 2993 ± 160 mg/kg and showing highervalue in the brans having smaller particle size (Table 2). Overall, theFAAs found at the highest concentration were Glu, Trp, Ser and Asp.After 24 h of bioprocessing, the increase of FAA content is of ca. 4–8%in the finest brans but arrives up to ca. 20% in the case of bran havingparticle size of 400 μm. When enzymes were added, the total amountof FAA ranged from 3705 ± 80 to 4448 ± 110 mg/kg, showing an in-crease of ca. 20–40% especially in the bran having bigger particle size(750 and 400 μm). Bioprocessing with the addition of enzymes

Fig. 2. Peptide profiles of the water/salt-soluble extracts of native and bioprocessed branfractions having particle size of 160 μm (24 h, without or with the addition of enzymes)as determined by RP-FPLC (214 nm).


particularly enhanced the amount of Pro and GABA, which were foundin the highest concentration in the bran having particle size of 160 μm(355 ± 13 and 406 ± 63 mg/kg, respectively).

3.2. Phytase and antioxidant activities of bioprocessed bran

The phytase activity of water soluble extracts of native brandecreased with particle size and ranged from 0.58 ± 0.1 to 1.62 ±0.01U (Table 3). Bioprocessing improved the phytase activity in allthe brans, in comparison to their control native counterpart. Afterbioprocessing, a significant improvement of the activity was observedespecially in the finer bran. Indeed, the addition of enzymescorresponded to a further increase of the activity compared to branfermented with the starters alone, especially for bran with particlesize of 160 and 50 μm (2.0 and 3.7 fold higher, respectively) (Table 3).

On the contrary, the reduction of particle size led to an increase ofthe antioxidant power of the fermented brans. The concentration oftotal phenols in methanolic extracts of native bran significantly(P b 0.05) increased with the decrease of particle size, rangingfrom 22.0 ± 0.5 to 27.0 ± 0.3 mM of gallic acid/kg (Table 3). Afterbioprocessing with enzyme addition, the concentration of total phenolsincreased of ca. 20% in all the extracts (Table 3).

The antioxidant properties of the fermented brans were determinedbased on the scavenging activity toward DPPH radical. During assay, thecolored stable DPPH radical is reduced to non-radical DPPH-H when inthe presence of an antioxidant or a hydrogen donor. Native branshowed a decrease of the value of remaining DPPH according to particlesize, and varying from 73.0 ± 0.3 to 58.0 ± 0.2% (data not shown).After 24 h of bioprocessing with the addition of enzymes, remainingDPPH value ranged from 9.3 ± 0.3 to 3.0 ± 0.2%, reaching values com-parable to BHT activity (5.0 ± 0.3%, data not shown).

The ferric-reducing power of native brans increased with the de-crease of particle size, varying from 1.18 ± 0.02 to 1.35 ± 0.02 for750 and 160 μm brans, respectively (Table 3). The ferric-reducingpower increased after 24 h of bioprocessing, but no significant increase(P N 0.05) was found when enzymes were added (Table 3).

3.3. In vitro protein digestibility and nutritional indexes

A multi-step protocol, which mimics the in vivo gastrointestinal di-gestion, was used to estimate the in vitro protein digestibility of bran.Native brans were characterized by in vitro protein digestibility in therange 28.51 ± 0.50 to 38.73 ± 0.38% (Fig. 3). The lowest value wasfound for the 50 μm fraction, while the highest value corresponded to400 μm fraction. No significant (P N 0.05) differences were found be-tween the 160 μm and the 750 μm fractions. In all the cases, the


image of Fig.�2


Table 2Concentration of free amino acids and their derivatives (mg/kg) of native and bioprocessed bran fractions (24 h, without or with the addition of enzymes).

Bran fractions (μm)

750 400 160 50

Amino acids 0 24 24E* 0 24 24E 0 24 24E 0 24 24ECysteic acid 11 ± 1d 26 ± 2b 34 ± 3a 12 ± 1d 22 ± 2c 28 ± 3b 15 ± 1d 28 ± 3b 29 ± 3b 35 ± 2a 13 ± 2d 29 ± 3b

Met sulphone 1 ± 0a 1 ± 0a 1 ± 0a 1 ± 0a 1 ± 0a 1 ± 0a 1 ± 0a 1 ± 0a 2 ± 0a 1 ± 0a 1 ± 0a 1 ± 0a

Asp 240 ± 16c 202 ± 12d 286 ± 23 b 280 ± 14b 169 ± 10e 250 ± 13b 343 ± 21a 210 ± 14c 287 ± 19b 153 ± 14e 268 ± 22b 227 ± 10c

Thr 43 ± 3c 45 ± 3c 59 ± 4a 54 ± 6b 37 ± 1d 49 ± 3c 58 ± 3a 46 ± 5c 54 ± 3b 31 ± 3e 56 ± 6b 39 ± 2d

Ser 252 ± 29b 180 ± 17d 252 ± 19b 292 ± 23b 154 ± 13d 215 ± 11c 317 ± 30a 191 ± 16d 244 ± 13c 143 ± 17e 350 ± 26a 190 ± 12d

Glu 431 ± 52c 404 ± 39c 623 ± 38a 508 ± 48b 338 ± 48d 566 ± 37b 559 ± 42b 415 ± 27c 653 ± 33a 347 ± 28d 521 ± 39b 577 ± 27b

Gly 85 ± 9e 215 ± 12c 319 ± 21a 104 ± 10e 185 ± 12d 297 ± 27b 113 ± 15e 220 ± 18c 327 ± 19a 187 ± 15d 108 ± 12e 291 ± 11b

Ala 145 ± 12e 222 ± 23c 328 ± 28a 171 ± 18d 178 ± 10d 278 ± 23b 186 ± 20d 217 ± 32c 320 ± 29c 166 ± 6d 179 ± 10d 221 ± 18c

Cys 21 ± 3d 43 ± 3b 53 ± 4a 23 ± 5d 35 ± 4c 49 ± 3b 27 ± 4d 39 ± 2c 51 ± 2a 30 ± 2c 24 ± 1d 35 ± 3c

Val 72 ± 9e 105 ± 10b 135 ± 9a 87 ± 8d 86 ± 10d 98 ± 8c 96 ± 8c 102 ± 7d 104 ± 5b 73 ± 4e 93 ± 6c 67 ± 4e

Met 24 ± 6e 38 ± 6c 50 ± 4a 31 ± 4d 30 ± 9d 41 ± 3b 33 ± 2d 36 ± 4c 42 ± 5b 25 ± 3e 31 ± 2d 28 ± 3e

Ile 35 ± 4d 50 ± 4b 66 ± 3a 43 ± 2d 39 ± 3d 52 ± 2b 46 ± 4c 48 ± 3c 54 ± 3b 32 ± 2c 45 ± 3c 35 ± 5d

Leu 62 ± 4e 110 ± 9b 143 ± 8a 77 ± 7d 83 ± 6c 105 ± 7b 82 ± 8c 99 ± 7b 112 ± 9b 67 ± 5e 80 ± 10c 69 ± 7d

Tyr 1 ± 0d 17 ± 2b 23 ± 4a 2 ± 0d 12 ± 1c 21 ± 2a 5 ± 2d 16 ± 2b 23 ± 2a 11 ± 2c 4 ± 1d 24 ± 3a

Phe 53 ± 4e 120 ± 4b 167 ± 12a 62 ± 4e 92 ± 13c 134 ± 8b 72 ± 2d 115 ± 10b 149 ± 8a 84 ± 5d 68 ± 2e 98 ± 6c

His 46 ± 8e 85 ± 10c 170 ± 9a 52 ± 9e 74 ± 4d 154 ± 16a 51 ± 3e 81 ± 6c 164 ± 12a 56 ± 3e 53 ± 4e 133 ± 8b

Trp 396 ± 54d 406 ± 66c 471 ± 59b 425 ± 53c 413 ± 55c 458 ± 37b 493 ± 69b 455 ± 53b 518 ± 37a 447 ± 42c 475 ± 34b 482 ± 51b

Orn 22 ± 9d 400 ± 35a 419 ± 65a 25 ± 6d 218 ± 23c 393 ± 41b 26 ± 3d 273 ± 32c 433 ± 33a 22 ± 2d 221 ± 12c 405 ± 31a

Lys 67 ± 5d 76 ± 6c 86 ± 5b 83 ± 9b 66 ± 7d 75 ± 9c 88 ± 3b 77 ± 11c 76 ± 8c 57 ± 4e 90 ± 12a 60 ± 7d

Arg 135 ± 6c 2 ± 0e 3 ± 0e 162 ± 8e 13 ± 2d 14 ± 1d 182 ± 22a 2 ± 0e 2 ± 0e 21 ± 1d 180 ± 15a 19 ± 3d

Pro 74 ± 13e 254 ± 32c 351 ± 42a 84 ± 7e 235 ± 32d 322 ± 28b 91 ± 12e 263 ± 28c 355 ± 13a 267 ± 12c 89 ± 6e 303 ± 20b

GABA 81 ± 33e 309 ± 27b 399 ± 48a 82 ± 21e 278 ± 53c 381 ± 35a 99 ± 24d 308 ± 12b 406 ± 63a 91 ± 12d 271 ± 32c 363 ± 33a

Total 2309 ± 20e 2678 ± 15d 3992 ± 24b 2669 ± 34d 3322 ± 31c 4449 ± 32a 2993 ± 27d 3253 ± 13c 4416 ± 25a 2850 ± 24d 2725 ± 23d 3705 ± 18b

E* indicates bioprocessing with the addition of enzymes.a–eValues in the same row with different superscript letters differ significantly (P b 0.05).


bioprocessing affected the digestibility of brans. In particular, the 24 hfermentation by the starters allowed the increase of the digestibilityvalues of 1.3–1.8%. Compared to samples bioprocessed only with thestarters, the use of the enzymes corresponded to increases higher than2%. Similarly to raw matrices, the lowest and the highest values forbioprocessed samples were found for 50 μm and 400 μm fractions,respectively.

The digestible protein fraction was further characterized. In particu-lar, the amino acid composition was determined and, to calculate nutri-tional indexes, the related chemical scoreswere calculated using the eggEssential Amino Acid (EAA) pattern as the FAO (Food and AgricultureOrganization) reference. EAA and BV indexes, which are commonlyused to estimate the quality of food proteins, were significantly(P b 0.05) higher for native 750 μm fraction in comparison to theothers. Native 160 μm fraction had the lowest values (Table 4). In allthe cases, the bioprocessing increased EAAI and BV of the samples. In

Table 3Nutritional properties related to phytase and antioxidant activities of native andbioprocessed bran fractions (24 h, without or with the addition of enzymes).

Bran fractions(μm)

Bioprocessingconditions

Phytaseactivity (U)⁎

Total phenols(mM gallic acid)

Reducingpower (Abs)⁎⁎

750 0 1.62 ± 0.01c 22 ± 2d 1.18 ± 0.02d

24 h 2.15 ± 0.07b 25 ± 2c 1.56 ± 0.01a

24 h E 2.60 ± 0.08a 29 ± 2b 1.55 ± 0.02a

400 0 1.46 ± 0.01c 24 ± 2c 1.26 ± 0.03c

24 h 2.09 ± 0.12b 28 ± 2c 1.43 ± 0.03a

24 h E 2.54 ± 0.04a 31 ± 1b 1.45 ± 0.02a

160 0 1.21 ± 0.17c 26 ± 3c 1.36 ± 0.02b

24 h 2.16 ± 0.03b 31 ± 2b 1.45 ± 0.03a

24 h E 2.34 ± 0.04a 34 ± 1a 1.45 ± 0.02a

50 0 0.58 ± 0.12d 27 ± 3c 1.24 ± 0.02c

24 h 1.9 ± 0.14b 31 ± 1b 1.36 ± 0.03b

24 h E 2.15 ± 0.07a 35 ± 2 a 1.38 ± 0.02b

E indicates bioprocessing with the addition of enzymes.a–dValues in the same column with different superscript letters differ significantly(P b 0.05).⁎ One unit (U) of phytase activity was defined as the amount of enzyme required to

release 1 μmol of phosphate/min under the assay conditions.⁎⁎ Absorbance at 700 nm. High absorbance value indicates high reducing power.


general, the enzyme addition allowed a further increase of the indexeswith respect to incubation with starters alone. Compared to raw branfractions, the activity of the starters allowed increases from 2.86 to7.08 of the EAAI and from 3.12 to 7.72 of the BV (Table 4). When en-zymes were added, the highest value of EAAI and BV was found for750 μm fraction (79.14 ± 0.48 and 74.56 ± 0.23, respectively), follow-ed by the 400 μm fraction (Table 4). The same trend was also found forthe PER. Raw bran fractions were characterized for values included inthe range 28.79 ± 0.21–40.74 ± 0.32 while after the incubation withstarters and enzymes together the range was from 32.30 ± 0.21 to41.65 ± 0.20. The PER of samples incubated with microorganismsalone was intermediate to raw brans and samples incubated also withenzymes (Table 4).

The NI of native 750 μm fraction was markedly higher compared tothe others. As the consequences of the bioprocessing especially whenenzymes were added, also NI values of all the samples significantly(P b 0.05) increased (Table 4).

Fig. 3. In vitro protein digestibility of native and bioprocessed bran fractions (24 h,withoutor with the addition of enzymes) as determined by the method of Akeson and Stahmann(1964). Data are the means of three independent analyses. a–hColumns with different su-perscript letters correspond to values that differ significantly (P b 0.05). Bars of standarddeviations are also represented.


image of Fig.�3


Table 4Nutritional indexes of native and bioprocessed bran fractions (24 h, without or with theaddition of enzymes).

Bran fractions (μm) Samples

0 24 24E

750 EAAI1 70.31 ± 0.62c 77.39 ± 0.41b 79.14 ± 0.48a

BV2 64.94 ± 0.22c 72.66 ± 0.30b 74.56 ± 0.23a

PER3 40.74 ± 0.32c 41.09 ± 0.29b 41.65 ± 0.20a

NI4 3.46 ± 0.10c 3.81 ± 0.07b 3.89 ± 0.02a

400 EAAI 61.21 ± 0.47b 66.90 ± 0.35b 65.31 ± 0.24a

BV 55.02 ± 0.21c 59.49 ± 0.19 b 60.86 ± 0.27a

PER 35.75 ± 0.30c 36.61 ± 0.12 b 37.86 ± 0.27a

NI 2.49 ± 0.04c 2.81 ± 0.09b 3.09 ± 0.10a

160 EAAI 54.99 ± 0.39c 57.85 ± 0.34b 62.18 ± 0.45a

BV 48.24 ± 0.35c 51.36 ± 0.26b 56.08 ± 0.32a

PER 28.79 ± 0.21c 30.97 ± 0.32b 33.95 ± 0.17a

NI 2.73 ± 0.23c 2.87 ± 0.13b 3.08 ± 0.10a

50 EAAI 57.35 ± 0.42c 61.52 ± 0.64b 64.69 ± 0.50a

BV 50.81 ± 0.30c 55.36 ± 0.50b 58.81 ± 0.23a

PER 29.74 ± 0.11c 31.59 ± 0.24b 32.30 ± 0.21a

NI 2.64 ± 0.12c 2.90 ± 0.11b 3.10 ± 0.08a

Data are themean of three independent fermentations twice analyzed; standarddeviationis reported.a–cValues in the same row with different superscript letters differ significantly (P b 0.05).

1 Essential Amino Acid Index.2 Biological Value.3 Protein Efficiency Ratio.4 Nutritional Index.


Data from the chemical composition and nutritional analysis of na-tive and bioprocessed brans were elaborated by Principal ComponentAnalysis (PCA) (Fig. 4). The first and second factors explained ca. 75–80% of the total variance. All the samples were well separated on theplane, and a sharp distribution between native (on the right) andbioprocessed brans (on the left) was obtained. Smaller particle size ap-peared on the upper part, while bigger fractions on the lowest. Betweennative brans, 160 and 50 μm fractions shared several characteristics incommon, as can be deduced by the relation on the plane. This tendencywas confirmed for the same bioprocessed samples, which showed com-mon features such as soluble DF content, total phenols, and protein con-tent. On the contrary, brans having bigger particle size were differentlydistributed, showing less similarity.

4. Discussion

In the present study, the nutritional features of brans of four differ-ent particle sizes obtained by micronization followed by fractionationthrough a dynamic separator and characterized, were investigated. In

Fig. 4. Principal component biplot of native and bioprocessed bran fractions (24 h, without orextractable arabinoxylans (Wx), starch (ST), protein (Pr), ash (as), total free amino acids (TFAIndex (EAAI), Biological Value (BV), Protein Efficiency Ratio (PER), Nutritional Index (NI).


particular, nutritional properties of brans were evaluated afterbioprocessing with selected starters and enzymes and compared to na-tive fractions. Two starters, belonging to L. brevis and K. exigua species,and two commercial enzyme preparations, were used since they ableto enhance the technological quality of bran enriched baked goods,allowing the obtainment of bread with appealing sensory properties(Coda et al., 2014). L. brevis and K. exigua were often isolated in sour-dough environment (Gobbetti, 1998).

The reduction of particle size of bran led to several modifications ofthe structure and surface area, changing the original characteristicsand developing new ones that the raw materials did not show before.Bioprocessingwas responsible of themost of the changes in the bran, ir-respective of particle size, especiallywhen enzymeswere added. As pre-viously shown (Coda et al., 2014; Nordlund, Katina, Aura, & Poutanen,2013), enzymes had an impact on microorganisms growth, leading toa series of changes such as lower pH, and enhanced microbial andbran endogenous enzymatic activities, that might be responsible ofthe more extensive modifications. It may be hypothesized that the in-crease of microbial growth and acidification was related to the higheravailability of fermentable carbohydrates, which were released frompolysaccharides through the different hydrolytic activities (Coda et al.,2014).

As showed bymicroscopy results, themilling process influenced theproportions of the different cell types in the bran, affecting also thechemical composition of the fractions collected through the dynamicseparation. Modifications induced by bioprocessing in cell wall struc-ture were mirrored by the changes in composition and solubility ofbran components as observed after microscopic and chemical analyses.The biggest effect of bioprocessing in the structure involved mainly thealeurone layer, especially after enzyme addition. As already observed inother studies, this might be due to the synergetic action of the multipleenzymatic activities of added and microbial and endogenous enzymesof bran, that is more active during fermentation (Katina et al., 2012;Nordlund et al., 2013). Total dietary fiber amount of native andbioprocessed brans did not show any significant variation after fraction-ation, while slight variations were found after bioprocessing, mainly inthefinest fraction. However, a different ratio between insoluble and sol-uble fibers is observed after fractionation and bioprocessing. Many fac-tors affected this ratio, especially, the soluble fiber concentration atthe end of the incubation. It was already observed that the decreaseof pH due to lactic acid bacteria fermentation, leads to the activationof endogenous degrading enzymes such as amylase, pentosanase, andβ-glucanase. Sugars released fromfibers become available for fermenta-tion by the starters (Katina et al., 2005). The addition of commercial en-zymes also affected, through different mechanisms, the balancebetween insoluble and soluble fibers. Arabinoxylan solubilization was

with the addition of enzymes). Total DF (DFt), insoluble DF (DFi), soluble DF (DFs), waterAs), phytase activity (PA), total phenols (TP), reducing power (RP), Essential Amino Acid


image of Fig.�4



slightly higher in the bran of smaller compared to the larger particlesize, as previously observed in other studies (Izydorczyk, Chornick,Paulley, Edwards, & Dexter, 2008), and it varied in relation to the acidi-fication and to the activity of xylan-degrading enzymes (Katina et al.,2007). Indeed, a marked increase of the extractability of arabinoxylans,which are known to exert important effects on digestion (Huges et al.,2007; Lopez et al., 1999), was observed after bioprocessing, in responseto combination of microbial activity and endogenous and added en-zymes, as previously observed by Katina et al. (2012).

Beside xylanase activity, Depol 740L preparation also shows highβ-glucanase activity (Mateo Anson et al., 2009), able to cause thedepolimerization of β-glucans (Ninios et al., 2011). Probably due tothe β-glucan degradation, the increase of soluble fiber concentrationwas not strictly related to an increase of soluble arabinoxylan concen-tration. Starch content was lower in the finest fractions compared tothe others and a further reduction was obtained after bioprocessing.This might be due to an increased amylolytic activity as a consequenceof the pH drop occurring during lactic acid bacteria fermentation.

According to previous studies onwheat bran and germ, bioprocessingcauses an increase of free amino acid concentration as a consequence ofthehigh proteolytic activity of lactic acid bacteria and endogenous prote-ase activation at low pH (Rizzello, Coda, Mazzacane, Minervini, &Gobbetti, 2012; Rizzello et al., 2010).

The concentration of GABA, a functional nonprotein amino acid,also increased, as a consequence of the glutamate decarboxylase(GAD) activity commonly observed in wheat germ and bran(Rizzello et al., 2010, 2012), and also in sourdough lactic acid bacteriastrains. GABA synthesis in lactic acid bacteria is a response to acidicenvironment and increases with the lowering of pH (Rizzello et al.,2008, 2012).

Phytic acid, mainly located in the bran fraction of whole grain ce-reals, is an anti-nutritional factor because of its capability of stronglychelate minerals and trace elements (Muñoz, 1985). Degradation ofphytate is important because bran is a valuable source of minerals andhigh content of phytate can be responsible for a limited bioavailability.Reduction of particle size affected the original phytase activity, probablydue to the heat generated during the micronization and fractionationprocesses. Bioprocessing significantly improved it, especially in thebran having finest particle size and when enzymes were added, as aconsequence of the lower pH reached, very close to the optimal pH forendogenous phytase activity. This is in agreement with other studies,where treatment of bran with amylolytic and phytate-degrading en-zymeswas successful in overcoming its detrimental effect on theminer-al availability (Rizzello et al., 2012; Sanz Penella, Collar, & Haros, 2008).In addition, bioprocessing provides phytase by lactic acid bacteria(Rizzello et al., 2010, 2012).

Whole grains are a source of polyphenols, especially phenolic acidssuch as ferulic, vanillic, caffeic, syringic, sinapic and p-coumaric acids(Sosulski, Krygier, & Hogge, 1982) and cereal products have great anti-oxidant potentials in vitro (Coda, Rizzello, Pinto, & Gobbetti, 2012;Miller, Rigelhof, Marquart, Prakash, & Kanter, 2000). The total antioxi-dant capacity of native and bioprocessed bran extracts was evaluatedusing two different assays. Total phenolic compounds increased withthe reduction of particle size and especially after bioprocessingwith en-zymes. In native bran, DPPH scavenging activity and ferric reducingpower of smaller fractions were higher and further increased afterbioprocessingwith the addition of enzymes. The in vitro antioxidant ca-pacities of cereals are correlated with their phenolic acid contents, andthe content of phenolic compounds in cereal grains can bemarkedly af-fected by milling, extrusion, germination, and sourdough fermentation(Hole et al., 2012). Previous studies suggested that the antioxidant po-tential of cereals depends not only on their type but also on their particlesize (Fardet, Rock, & Rémséy, 2008). The enhanced properties of smallerparticle size bran could be related to an improved release of the com-pounds due to the breakage of the cells, as well as to a more accessiblesurface for microorganisms and enzyme activity which have a larger


contact area to access fermentable carbohydrates (Jenkins et al., 1999;Stewart & Slavin, 2009).

Proteins are one of the most critical components, which contributeto the nutritional value of foods. Besides the amount, the quality of pro-teins is another important attribute (Garcha, Khanna, & Soni, 1993).Usually, the quality of proteins is estimated through the determinationof their amino acid composition. The amino acid composition is not theonly predictor of the nutritive value, but this feature has to be combinedwith protein digestibility (Garcha et al., 1993chap. 24). The in vitro di-gestibility gives information on the stability of protein hydrolysates,and on how they withstand to digestive processes. Overall, it wasfound that the in vitro digestibility of the different bran fractions wasmarkedly lower than that of wheat flour (Rizzello et al., 2013). Never-theless, the digestibility of the bioprocessed brans was higher than thenative. According to previous findings (Clemente, 2000; Rizzello et al.,2013), this effect may be attributed to proteolysis by microorganism(mainly lactic acid bacteria) and endogenous or exogenous proteasesduring fermentation. The digestible protein fraction was used for thedetermination of the protein quality indexes. Indeed, it was shownthat the analysis of the total protein content should hide the effect ofthe proteolysis degree, which results otherwise in similar values forsamples that are instead characterized by different bioavailability andnutritional features of the protein (Floridi & Fidanza, 1975; Rizzelloet al., 2013). The EAA and BV indexes were the highest for thebioprocessed brans compared to the native matrices. In any case, theaddition of the enzymes further improved the positive effect of the mi-crobial fermentation. EAA indicates the ratio of Essential Amino Acids ofthe sample compared to the reference. BV estimates the nitrogenpoten-tially retained by human body after consumption. The PER, which re-flects the capacity of a protein to support the body weight gain, hadthe same trend. Within the indexes that are used to evaluate the nutri-tional value of foods, only the NI combines qualitative and quantitativefactors. Indeed, NI is considered a global predictor of the protein quality.Since the increased protein bioavailability, the value of NI of thebioprocessed brans was higher than the native counterparts, especiallywhen enzymes were added. Fractionation and bioprocessing clearlyaffected the chemical and nutritional features of wheat bran. Smallerparticle size brans were in fact differentiated from bigger, and thedifference was even marked after bioprocessing showing an improve-ment of the technological and health-promoting properties in compar-ison to native wheat bran. In particular, bran having particle size of160 μm showed very favorable characteristics from the chemical andnutritional point of view such as high arabinoxylans and low starch con-tent, high amount of FAA and GABA and good antioxidant propertiesand NI.

The effect of bran particle size on the regulation of colonic functions,constipation and related disorders is still debated. Some authors(Brodribb & Groves, 1978; Heller et al., 1980) found that coarse wasmore effective compared to finely ground bran, thanks to the higherwater holding capacity, whereas this latter showed higher mineral re-lease during colonic transit and increased fermentability by gutmicrobi-ota, with short chain fatty acid production. As the consequence, finewheat bran showed enhanced effectiveness in reducing risk factors ofseveral chronic diseases (Stewart & Slavin, 2009).

The need of new plant sources with high nutritional quality is in-creasing and bran potential as already been shown (Nordlund et al.,2013). The findings of this study support the use of small particle sizewheat bran to formulate high fiber foods that may confer additionalhealth advantages. However, these potential health benefits should beconfirmed in long-term trials.

Acknowledgments

The authors thank the Fundación Alfonso Martín Escudero (Madrid,Spain) for the postdoctoral fellowship for J.A. Curiel.




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http://refhub.elsevier.com/S1466-8564(13)00195-1/rf0010











































http://dx.doi.org/10.3402/fnr.v57i0.18503

http://dx.doi.org/10.3402/fnr.v57i0.18503















































http://www.icef11.org/content/papers/fpe/FPE1042.pdf























http://dx.doi.org/10.1016/j.fm.2013.06.017


























effect of bioprocessing and particle size on the nutritional properties of wheat bran fractions

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