International Journal of Food Microbiology 193 (2015) 34–42

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Effect of air classification and fermentation by Lactobacillus plantarum VTT E-133328 on faba bean (Vicia faba L.) flour nutritional properties Rossana Coda a,1,⁎, Leena Melama a, Carlo Giuseppe Rizzello b, José Antonio Curiel b, Juhani Sibakov a, Ulla Holopainen a, Marjo Pulkkinen c, Nesli Sozer a a b c

VTT Technical Research Centre of Finland, Tietotie 2, 02044 VTT, Finland Department of Soil, Plant and Food Sciences, University of Bari, 70126 Bari, Italy Department of Food and Environmental Science, 00014, University of Helsinki, Finland

a r t i c l e

i n f o

Article history: Received 10 September 2014 Received in revised form 6 October 2014 Accepted 7 October 2014 Available online 12 October 2014 Keywords: Faba bean Lactic acid bacteria Air classification Antinutritional factors

a b s t r a c t The effects of air classification and lactic acid bacteria fermentation on the reduction of anti-nutritional factors (vicine and convicine, trypsin inhibitor activity, condensed tannins and phytic acid) and in vitro protein and starch digestibility of faba bean flour were studied. Free amino acid (FAA) profile analysis was also carried out. Air classification allowed the separation of the flour into protein and starch rich fractions, showing different chemical compositions and microstructures. Lactobacillus plantarum growth and acidification in faba bean flour and its fractions were assessed. The anti-nutritional compounds were separated mostly to the fine protein-rich fraction. Fermentation caused the decrease of vicine and convicine contents by more than 91% and significantly reduced trypsin inhibitor activity and condensed tannins (by more than 40% in the protein-rich fraction). No significant (P N 0.05) variation was observed for total phenols and phytic acid content. Fermentation increased the amount of FAA, especially of the essential amino acids and γ-aminobutyric acid, enhanced the in vitro protein digestibility and significantly lowered the hydrolysis index. This work showed that the combination of air classification and fermentation improved nutritional functionality of faba bean flour which could be utilized in various food applications. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Faba bean (Vicia faba L.) also known as field bean, broad bean, horse bean or tick bean, is an annual legume growing in different climatic zones, from Europe to Africa and Asia, where it represents one of the more ancient and common crops for food and feed (Jezierny et al., 2010; Li et al., 2009). Faba bean seeds are rich in proteins (about 30% of lysine-rich), vitamins, minerals and dietary fibre but also bioactive compounds, such as antioxidants, phenols and γ-aminobutyric acid (GABA) (Jezierny et al., 2010; Köpke and Nemecek, 2010; Li et al., 2009). Besides high protein and fibre content and energy supply, Abbreviations: ANF, anti-nutritional factor; FAA, free amino acid; FBF, faba bean flour; FBFict, incubated control of faba bean flour; fFBF, fermented faba bean flour; fPRF, fermented protein rich fraction; fSRF, fermented starch rich fraction; HI,hydrolysisindex; ict, incubated control; IVPD, in vitro protein digestibility; MF, mass yield of the fine fraction; PCF, protein concentration of the fine fraction; PCW, protein content; PRF, protein rich fraction; PRFict, incubated control of protein rich fraction; PSE, protein separation efficiency; SRF, starch rich fraction; SRFict, incubated control of starch rich fraction; TIA, trypsin inhibitor activity; TTA, total titratable acidity. ⁎ Corresponding author at: Department of Food and Environmental Sciences, Agnes Sjöbergin katu 2 FIN- 00014, University of Helsinki Finland. Tel.: +358 504486634. E-mail address: rossana.coda@helsinki.fi (R. Coda). 1 Rossana Coda is currently affiliated with University of Helsinki, Department of Food and Environmental Sciences.

http://dx.doi.org/10.1016/j.ijfoodmicro.2014.10.012 0168-1605/© 2014 Elsevier B.V. All rights reserved.

positive effects of faba beans also include the decrease in plasma LDL-cholesterol levels (Frühbeck et al., 1997). Nevertheless, secondary metabolites that may exert an anti-nutritional effect are also present, including pyrimidine glycosides called vicine and convicine, condensed tannins, protease inhibitors, alkaloids, lectins, and others (Liener, 1990). It has been shown that divicine and isouramil, active aglycone derivatives of vicine and convicine, are toxic to human carriers of a genetic deficiency of the erythrocyte located glucose-6-phosphate dehydrogenase, causing the haemolytic anaemia disease known as favism (Crépon et al., 2010). Divicine and isouramil are released in the digestive track by β-glycosidase activity. The reduced form of these compounds causes rapid oxidation of glutathione (GSH) in erythrocytes, altering several functions of the red blood cells (Crépon et al., 2010). To reduce the content of anti-nutritional factors (ANFs) in legumes, different methods have been applied such as dehulling, soaking, air classification, extrusion, or heat treatment (Jezierny et al., 2010; van der Poel, 1990). In addition, genetic improvements were shown to reduce tannins and/or vicine and convicine contents in faba bean seeds (Crépon et al., 2010). Also biological methods such as germination, enzymatic treatments, and fermentation have been applied (Alonso et al., 2000; Granito et al., 2002; Luo et al., 2009). The effect of fermentation on the ANF content has been studied in different legumes. In beans, a partial or complete elimination of α-galactosides, tannins, phytic acid and trypsin inhibitor activity (TIA)

R. Coda et al. / International Journal of Food Microbiology 193 (2015) 34–42

was obtained by natural fermentation (Granito et al., 2002). In the case of grass pea, reduction of phytic acid and TIA was obtained by fermentation with Lactobacillus plantarum strain (Starzyńska-Janiszewska and Stodolak, 2011). Bioprocessing with enzymes was also used, as in the case of fungal β-glucosidase from Aspergillus oryzae and Fusarium graminearum, to hydrolyse vicine and convicine from faba beans (McKay, 1992), and of α-galactosidases used to eliminate oligosaccharides in pinto bean flour (Song et al., 2009). In addition to a decrease of ANFs, fermentation enhanced the content of free amino acids and GABA and the in vitro protein digestibility of some legumes (Coda et al., 2010b; Granito et al., 2002). Fermentation has been traditionally applied to legumes in many countries, especially outside of Europe, where the mainly used legumes are soybean, chickpea and common bean (Humblot and Guyot, 2008). Even though fermented legume products are not as widely consumed in Western countries, some attempts have been made to use them also for our traditional foods, such as bread (Baik and Han, 2012; Coda et al., 2010b; Hallén et al., 2004). In recent years there has been a growing interest for including legumes in the diet and novel foods containing legume flour, protein or starch isolate were developed (Boye et al., 2010). The separation of the main starch and protein components of legume flours is commonly achieved by air classification, which represents a fast and economic process that separates light from heavy particles by using a stream of air (Vose et al., 1976). In this way, concentrates from legumes allow to obtain flours with high protein (20 to 30%) and starch (40 to 50%) contents, promoting their use as novel ingredients which may have functional food, feed, and bio-industrial applications (Gunawardena et al., 2010). Despite their nutritional profile and wide availability, faba beans have not been extensively used by the food industry. Few studies have considered the use of faba bean flour in gluten-free cake production, extruded snacks and spaghetti (Abou-Zaid et al., 2011; Giménez et al., 2013; Smith and Hardacre, 2011) for the positive repercussions on nutritional value and texture. The objective of this study is to set up a biotechnological protocol to enhance the functionality of faba bean flour by using the different fractions achievable by air classification and fermentation with lactic acid bacteria. Lactic acid bacteria fermentation is an established technology for improving the quality of different foods, and it has proven its potential also in the case of plant matrices and legumes (Coda et al., 2014). The effect of the strain L. plantarum VTT E-133328 as selected starter for faba bean flour fermentation was evaluated on the basis of technological performance and nutritional parameters. The decrease of the main anti-nutrients vicine, convicine, trypsin inhibitors, condensed tannins and phytic acid, and the increase of nutritional quality such as amino acid composition or in vitro protein and starch digestibility were investigated. 2. Materials and methods 2.1. Milling of faba bean flour and air classification Six faba bean (Vicia faba cv. Kontu) samples, cultivated in Finland (harvest year 2012), were purchased from local farmers and pooled. Dehulling was performed by rubbing the beans in a stone mill-type Supermasscolloider MKZA10-15J (Masuko Sangyo Co. Ltd., Kawaguchi, Japan) with a 0.5 mm gap. The hulls were then separated with an aspiration part of a spray drier (Mobile minor, GEA Niro A/S, Denmark). The particle size of the dehulled material was further reduced in a SM300 cutting mill (Retsch GmbH, Haan, Germany), using 1500 rpm rotor speed and 4 × 4 mm screen. Before air classification, faba bean flour (FBF) was ground twice in a 100UPZ fine-impact mill (Hosokawa Alpine AG, Augsburg, Germany) with stainless steel pin discs, using 17,800 rpm rotor speed. The fine ground material was separated into protein and starch rich fractions (PRF and SRF, respectively) by a MiniSplit air classifier (British Rema Manufacturing Company Ltd, Derbyshire, UK), using 15,000 rpm classifier wheel speed with an air flow rate of 220 m3/h and a feed speed of 30 rpm. The protein separation efficiency (PSE)

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was calculated for the fine fraction based on the Eq. (1) (Pelgrom et al., 2013), where PCF is the protein concentration of the fine fraction, MF is the mass yield of the fine fraction, and PCW is the protein content in the FBF. PSE ¼

PCF  MF PCF  MF PCF  MF PSE ¼ PSE ¼ PCW PCW PCW

ð1Þ

The volume based particle size distribution of the raw materials was analysed by a Beckman Coulter LS 230 (Beckman Coulter. Inc., CA, U.S.A.) using the liquid module and ethanol as a carrier. The particle size data was presented as cumulative undersize centiles (D50 and D90 values) with an average of triplicate analyses. 2.2. Chemical composition of flour and fractions Moisture was determined by oven drying the samples at 130 °C for 1 h according to the method 44-15.02 (AACC-International, 2013). Total protein concentration was analysed according to the method 4611A (AACC, 2003) with Kjeldahl autoanalyser (Foss Tecator Ab, Höganäs, Sweden). Total starch was quantified using Megazyme total starch assay kit according to the method 76-13.01 (AACC, 2003). Fat was determined using Soxhlet extractor (Büchi B-811, Labortechnik AG, Flawil, Switzerland). Ash content was determined with gravimetric method using Naber N11 ash oven (Nabertherm, Germany). Total dietary fibre was analysed with the enzymatic-gravimetric method 985.29 (AOAC, 1990). 2.3. Microorganisms and culture condition L. plantarum VTT E-133328 (also known as DPPMAB24W) deposited at VTT Culture Collection (Espoo, Finland) was previously selected based on its β-glucosidase activity toward p-nitrophenyl-β-Dglucopyranoside substrate (Di Cagno et al., 2010). The lactic acid bacteria strain was routinely propagated at 30 °C in MRS broth (Oxoid, Basingstoke, Hampshire, England). When used for fermentation, L. plantarum VTT E-133328 was cultivated until the late exponential phase of growth was reached (ca. 10 h) and, cells were recovered by centrifugation (10,000 ×g for 10 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 of the culture) used for the preparation of the dough. 2.4. Fermentation and chemical properties of doughs Each of the flour and flour fractions described above was mixed with water in a ratio of 50:50 (wt:vol), to prepare the doughs. FBF, PRF and SRF doughs were inoculated with the selected lactic acid bacteria (initial cell density of ca. 107 cfu/g of dough) and fermented (f) at 30 °C for 48 h (fFBF, fPRF, and fSRF). Other doughs of FBF, PRF and SRF were made as described above and incubated without bacterial inoculum under the same conditions (ict, incubated controls). All the doughs were obtained in triplicate and each of them was analysed twice. For each dough, 10 g-aliquots were suspended into a sterile physiologic solution (NaCl, 0.9% w/v) and homogenized with a Stomacher 400 lab blender (Seward Medical, London). Mesophilic lactic acid bacteria and yeasts were determined on MRS agar (Oxoid) containing 0.1% of cycloheximide (Sigma Chemical Co.) at 30 °C for 48–72 h under anaerobiosis, and on YM, added with 150 ppm chloramphenicol, at 30 °C for 72 h, respectively. Total mesophilic bacteria were determined on Plate Count Agar (PCA, Oxoid) at 30 °C for 48 h. Kinetics of growth and acidification were determined for 24 h and modelled in agreement with the Gompertz equation as modified by Zwietering et al. (1990): y = k + A exp{− exp [(μmax or Vmax e / A) (λ − t) + 1]}; where y is the growth expressed as log cfu/g/h or the acidification rate expressed as dpH/dt (units of pH/h) at the time t;

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R. Coda et al. / International Journal of Food Microbiology 193 (2015) 34–42

k is the initial level of the dependent variable to be modelled (log cfu/g or pH units); A is the cell density or pH (units) variation (between inoculation and the stationary phase); μmax or Vmax is the maximum growth rate expressed as Δlog cfu/g/h or the maximum acidification rate expressed as ΔpH/h, respectively; λ is the length of the lag phase measured in hours. The experimental data were modelled by the nonlinear regression procedure of the Statistica 8.0 software (Statsoft, Tulsa, USA). The pH value was measured by a TitroLine autotitrator (Alpha 471217, Schott, Mainz, Germany) suspending an aliquot of 10 g of samples in 100 ml of distilled water. Total titratable acidity (TTA) was determined with the TitroLine Alpha autotitrator on 10 g of dough homogenized with 90 ml of distilled water and expressed as the amount (ml) of NaOH 0.1 M get pH of 8.5. For the chemical analyses, doughs were immediately freezed at −20 °C at the end of incubation or fermentation and freeze-dried.

Analysis of total phenolic compounds was carried out according to the method of Slinkard and Singleton (1977), using gallic acid as the standard. The concentration of total phenolic compounds was calculated as gallic acid equivalent (GAE). Condensed tannin concentration was measured using vanillin assay as described by Price et al. (1978). Flours or freeze dried samples were extracted with HCl:methanol (1:100) for 2.5 h at room temperature and centrifuged at 4000 rpm for 20 min. The calibration curve was obtained using catechin and the results were expressed as catechin equivalents. Vicine and convicine were extracted from flours or freeze dried samples by perchloric acid. Vicine and convicine concentrations were determined by HPLC based on the method of Marquardt and Frohlich (1981). Quantification by internal standard uridine was adapted from Quemener (1988).

2.5. Determination of organic acids and free amino acids

Trypsin inhibitor activity (TIA) was determined based on the method described by Kakade (1974) and modified by Alonso et al. (2000). For the analysis, flours or freeze dried samples were extracted over night at 4 °C with 0.15 M phosphate buffer (pH 8.1) and centrifuged for 15 min at 4000 rpm at 15 °C. Results were expressed as trypsin inhibitor units per 1 mg of sample. An increase of 0.01 absorbance units was determined as one trypsin activity unit. Phytic acid analysis was carried out by spectrophotometric method as described by Latta and Eskin (1980).

Water/salt-soluble extracts from faba bean flour, protein and starch rich fraction doughs were prepared following the method of Weiss et al. (1993). Organic acids were determined on water/salt-soluble extracts by High Performance Liquid Chromatography (HPLC), using an ÄKTA Purifier system (GE Healthcare, Buckinghmshire, UK) equipped with an Aminex HPX-87H column (ion exclusion, Biorad, Richmond, CA), and an UV detector operating at 210 nm. Elution was at 60 °C, with a flow rate of 0.6 ml/min, using H2SO4 10 mmol/l as mobile phase (Rizzello et al., 2010). Total and individual free amino acids were analysed by a Biochrom 30 series Amino Acid Analyser (Biochrom Ltd., Cambridge Science Park, England) with a Na-cation-exchange column (20 by 0.46 cm internal diameter) as described by Rizzello et al. (2010). Organic acids and amino acids used as standards were purchased from Sigma Chemical Co. (Milan, Italy). 2.6. Light microscopy The microstructure of faba bean flour, its protein- and starch-rich fractions and respective doughs fermented for 48 h was studied by light microscopy using an Olympus BX-50 microscope (Olympus Corp., Tokyo, Japan), a PCO SensiCam CCD colour camera (PCO AG, Kelheim, Germany) and the Cell^P imaging software (Olympus). Embedding and staining of samples was carried out as described by Curiel et al. (2014). Sections were treated with aqueous 0.1% (w/v) Toluidine Blue O (Sigma, St. Louis, MO, USA) that stains cell wall polymers different shades of blue. Protein and cell walls (cellulose) were stained with aqueous 0.1% (w/v) Acid Fuchsin (BDH Chemicals Ltd., Poole, Dorset UK) in 1.0% acetic acid, and with aqueous 0.01% (w/v) Calcofluor White (Fluorescent brightener 28, Aldrich, Germany) (Fulcher et al., 1994), respectively. In exciting light, intact cell walls stained with Calcofluor appear blue and proteins stained with Acid Fuchsin appear red. Starch is unstained and appears black. For visualisation of protein and starch, the sections were stained with aqueous 0.1% (w/v) Light Green (BDH Chemicals Ltd., Poole, England), followed by extra dye removing and pouring with Lugol's iodine solution (I2 0.33% (w/v) and KI 0.67% (w/v), diluted 1:10). Light Green stains protein green or yellow and iodine stains the amylose component of starch blue and amylopectin brown. 2.7. Total phenolic compound and condensed tannin analysis Extracts were prepared by weighing 5 g of flours or freeze dried samples and mixing with 50 ml of 80% methanol. The mixture was purged with nitrogen stream, mixed for 30 min and centrifuged at 6000 rpm for 20 min. Supernatants were transferred into test tubes, purged with nitrogen stream and stored at ca. 4 °C before analysis.

2.8. Determination of trypsin inhibitor activity and phytic acid content

2.9. In vitro protein and starch digestibility In vitro protein digestibility was determined using the method of Dahlin and Lorenz (1993) with modifications made by Coda et al. (2011). Flour or freeze dried sample suspensions containing 6.25 mg of total protein in 1 ml were rehydrated at 5 °C for 1 h. The pH was set to 8.0 with 0.1 N NaOH and 0.1 N HCl and the suspension were placed into 37 °C water bath. Freshly prepared trypsin solution (1.6 mg/ml trypsin, pH 8.0) was added to the suspensions under stirring and the pH was measured again after 10 min. The protein digestibility was calculated with the equation y = 210.4 − 18.1x, where x is the pH after 10 min (Hsu et al., 1977). In vitro starch digestibility was measured with the modified nonrestricted mincing method described by Germaine et al. (2008). Flour or freeze dried sample suspensions containing 1 g of starch in 0.05 M sodium potassium phosphate buffer (pH 6.9) were placed in water bath (37 °C) and the pH was adjusted to 6.9 with 1 M NaOH and 1 N HCl. Pancreatic amylase (110U) was added to the suspensions. Sample aliquots were removed before the addition and after 30, 60, 120 and 180 min. Removed aliquots were placed in boiling water bath for 5 min and then cooled in ice water. Samples were stored at − 20 °C till the reducing sugar analysis. Before the reducing sugar analysis with the method VTT-3783-3 (VTT, 2001) the samples were defrosted, centrifuged and diluted (sugar content of 0.1–0.5 g/l). DNS-reagent (2-hydroxy-3,5-dinitrobenzoic acid) was mixed with the samples and the reaction mixtures were placed to boiling water bath for 5 min. Absorbance was measured at 540 nm against the reagent blank. Calibration curve was determined with maltose. A commercial white wheat flour bread (Isopaahto, Vaasan Group, Espoo, Finland) was used as the control to estimate the hydrolysis index (HI = 100). Hydrolysis index (HI) was calculated as percentage of the value of ratio maltose (mg)/1 g soluble starch of the sample compared to control wheat bread. 2.10. Statistical analysis The results of the microbiological and chemical analyses and particle size measurements are presented as an average of two parallel measurements on three replicates.

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The data was subjected to one-way analysis of variance (ANOVA) and significant differences at P b 0.05 between the treatment means were identified by Tukey's test, using the IBM SPSS Statistics 20 (IBM Corporation, Somers, NY, U.S.A.) software.

Table 2 Main microbiological and chemical features of faba bean flour (FBF), protein rich fraction (PRF) and starch rich fraction (SRF). Results expressed as dry matter (dm) basis. FBF Microbiological analysis Total bacterial count (log cfu/g) Yeasts (log cfu/g)

3. Results 3.1. Physical and chemical characterisations of FBF, PRF and SRF Faba bean flour was separated into a fine protein-rich fraction and a coarse starch-rich fraction by air classification. Mass yield of the coarse fraction was higher compared to the fine fraction. Overall mass balance was 52.4% of coarse fraction, 46.7% of fine fraction and 0.9% of the flour was lost. The protein separation efficiency, which was the percentage of the total flour protein recovered in the protein fraction (Pelgrom et al., 2013), was 67%. Particle size distribution was not significantly different (P N 0.05) for FBF and SRF (90% of the particles have diameter of ca. 50 μm), while PRF was characterised by 90% of particles with 27.4 ± 0.9 μm diameter (Table 1). Chemical composition of FBF, PRF and SRF is reported in Table 2. The flour and its fractions differed mainly for protein, starch, dietary fibre, and ash content. FBF was characterised by 35.7 ± 0.4% of dry matter (dm) of protein and 42.1 ± 0.8% dm of starch. PRF had the highest amount of protein (51.5 ± 0.2% dm) but also significantly (P b 0.05) higher content of dietary fibre, ash and fat. SRF had the highest amount of total starch (65.8 ± 0.5% dm) and the lowest fat concentration. The abundance of proteins and fibre-rich cell wall structures in PRF and starch in SRF were also shown by light microscopy analysis (Fig. 1S). Fermentation slightly but significantly (P b 0.05) decreased the content of total starch, especially in fPRF (20.6 ± 0.1% dm) and fSRF (65.2 ± 0.0% dm), total fibre (6.6 ± 0.4, 9.8 ± 0.3 and 4.4 ± 0.0% dm for fFBF, fPRF and fSRF, respectively) and fat (0.9 ± 0.1; 1.0 ± 0.0 and 0.8 ± 0.0% dm in fFBF, fPRF and fSRF, respectively). No significant (P N 0.05) difference was observed for total protein amount (16.4 ± 0.01–52.3 ± 0.9% dm). The hydrolysis of cell wall structures in small fragments, the modification of the protein network in more porous structures, and the rupture of starch granules deriving from fermentation were observed by microscopy (Figs. 1S–3S). 3.2. Growth and acidification of L. plantarum during fermentation of faba matrices Microbiological data regarding total bacterial count and yeasts of FBF, PRF and SRF are reported in Table 2. Total bacterial count of the ict varied from 7.3 ± 0.2 to 7.6 ± 0.2 log cfu/g. When L. plantarum VTT E-133328 was inoculated, values of 9.4 ± 0.5–9.6 ± 0.2 log cfu/g were found at the end of the fermentation. Compared to the flour, yeasts increased of ca. 1 log cycle/g in FBFict. In details, after 48 h of fermentation, the cell densities of lactic acid bacteria were 9.3 ± 0.4, 9.4 ± 0.1, and 9.4 ± 0.1 log cfu/g, respectively for fFBF, fPRF, and fSRF. At the beginning of incubation, the pH was in the range of 6.6 ± 0.1–6.8 ± 0.1, while after fermentation the values were 4.1 ± 0.2, 4.6 ± 0.1, and 4.3 ± 0.1 for fFBF, fPRF, and fSRF, respectively. Table 1 Particle size distribution of faba bean flour (FBF), protein rich fraction (PRF) and starch rich fraction (SRF). D-values represent the diameter (μm) at which 10, 25, 50, 75, and 90% of the sample mass are comprised of smaller particles. Particle size distribution (D)

FBF (μm)

D10 D25 D50 D75 D90

5.22 9.55 17.58 30.15 52.83

PRF (μm) ± ± ± ± ±

0.55b 1.34b 1.76b 2.44a 8.43a

4.71 7.24 11.94 18.84 27.40

SRF (μm) ± ± ± ± ±

0.02c 0.03c 0.07c 0.24b 0.96b

14.02 18.17 23.81 32.58 51.05

± ± ± ± ±

0.06a 0.09a 0.17a 0.43a 1.51a

The data are the means of three independent experiments ± standard deviations (n = 3). a–e Values in the same row with different superscript letters differ significantly (P b 0.05).

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Chemical composition Moisture % Ash (% dm) Protein (% dm) Total starch (% dm) Total fibre (% dm) Fat (% dm)

5.0 ± 0.1a 2.3 ± 0.2a

9.45 3.98 35.66 42.21 7.17 1.53

± ± ± ± ± ±

0.07a 0.04b 0.38b 0.77b 0.32b 0.04b

PRF

SRF

5.2 ± 0.2a n.f

5.2 ± 0.3a n.f

7.66 ± 0.15c 5.38 ± 0.01a 51.49 ± 0.23a 23.38 ± 0.18c 10.16 ± 0.23a 2.00 ± 0.06a

8.53 ± 0.04b 2.22 ± 0.02c 16.73 ± 0.03b 65.82 ± 0.54a 4.64 ± 0.14b 0.85 ± 0.02c

The data are the means of three independent experiments ± standard deviations (n = 3). a–e Values in the same row with different superscript letters differ significantly (P b 0.05). n.f.: not found.

The parameters of the kinetics of growth and acidification are shown in Table 3. The highest growth of the starter (A) was observed in SRF (2.4 ± 0.1 log cfu/g), and PRF (2.3 ± 0.2 log cfu/g), while FBF fermentation was characterised by the lowest growth (A) and the highest latency phase (λ) and growth rate (μmax) (1.65 ± 0.3 log cfu/g, 6.22 ± 0.05 h, and 0.34 ± 0.4 Δlog cfu/g/h, respectively). Fermentation of PRF showed an intermediate behaviour between FBF and SRF, with the exception of μmax value, which was the lowest (Table 3). Kinetics of acidifications followed the same trend as kinetics of growth, showing higher ΔpH and faster acidification (Vmax) in SRF. In PRF, the starter showed the shortest λ but also the lowest Vmax value (5.12 ± 0.2 h and 0.12 ± 0.02 ΔpH/h). The pH of the ict was 6.3 ± 0.1, 6.5 ± 0.1, and 5.4 ± 0.1 for FBFict, PRFict and SRFict doughs, respectively. TTA values showed significant (P b 0.05) differences between the fermented doughs; the highest value was found for fPRF (33.8 ± 1.1 ml of NaOH 0.1 M/10 g of dough), followed by fFBF (25.5 ± 0.7 ml of NaOH 0.1 M/10 g of dough) and fSRF (19.1 ± 0.5 ml of NaOH 0.1 M/10 g of dough). Compared to not incubated controls, TTA was significantly (P b 0.05) higher also in the case of PRFict, varying from 14.6 ± 1.0 to 11.5 ± 0.4 ml of NaOH 0.1 M/10 g of dough. Lactic acid was the predominant organic acid in all the fermented samples, corresponding to 127.8 ± 0.4, 117.8 ± 0.7, and 102.9 ± 0.8 mmol/kg of dough, respectively for fPRF, fFBF, and fSRF. Acetic acid was produced in lower concentration and the highest amount was observed for fPRF (20.4 ± 0.2 mmol/kg of dough), followed by fFBF (11.6 ± 0.2 mmol/kg of dough) and fSRF (8.9 ± 0.1 mmol/kg of dough). Lactic and acetic acids were produced in concentration lower than 30 and 5 mmol/kg of dough, respectively, also in ict doughs (data not shown). 3.3. Free amino acids Free amino acid composition was analysed from faba bean matrices before and after fermentation. Overall, before incubation or fermentation, FBF and PRF had higher concentration of total FAA compared to SRF (7.10 ± 0.12 and 6.88 ± 0.07 vs. 5.94 ± 0.05 g/kg, respectively) and a similar amino acid profile (Fig. 1A). Total FAA concentration in ict ranged from 6.41 ± 0.03 g/kg (SRF) to 12.74 ± 0.02 g/kg (PRF). A moderate increase was observed for all the amino acids (data not shown), including GABA, which varied from 535 ± 20 to 626 ± 35 mg/kg. Fermentation by L. plantarum VTT E-133328 caused a marked increase in total FAA (from 2 up to 3.5 times, compared to the corresponding not incubated flours). The highest concentration of total FAA was observed in fPRF (24.27 ± 0.03 g/kg), while fFBF and fSRP were characterised by values of 17.66 ± 0.19 and 12.16 ± 0.24 g/kg, respectively. All the essential amino acids increased after fermentation from 3 up to 80 times. Cys varied from 267 ± 25 to 303 ± 10 mg/kg,

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Table 3 Parameters of the kinetics of growth and acidification of Lactobacillus plantarum VTT E-133328 during fermentation of faba bean flour (fFBF), protein rich fraction (fPRF) and starch rich fraction (fSRF) doughs (50% w/w) for 24 h at 30 °C. Doughs

A (log cfu/g)

λ (h)

μmax (Δlog cfu/g/h)

ΔpH (pH units)

λ (h)

Vmax (ΔpH/h)

fFBF fPRF fSRF

1.6 ± 0.2b 2.3 ± 0.2a 2.4 ± 0.1a

6.22 ± 0.05a 4.57 ± 0.10b 4.93 ± 0.17b

0.34 ± 0.4a 0.19 ± 0.3c 0.26 ± 0.5b

1.85 ± 0.04b 1.86 ± 0.07b 1.98 ± 0.04a

7.04 ± 0.03a 5.12 ± 0.20c 6.24 ± 0.16b

0.18 ± 0.07b 0.12 ± 0.02b 0.31 ± 0.14a

The data are the means of three independent experiments ± standard deviations (n = 3). a–e Values in the same column with different superscript letters differ significantly (P b 0.05).

while Met from 89 ± 35 to 316 ± 22 mg/kg. Arg, Leu, Lys, Glu, and Ala were the FAA found at the highest concentration in all the samples (Fig. 2B). 3.4. Anti-nutritional factors Anti-nutritional factors (vicine, convicine, TIA, total phenolic compounds, condensed tannins, and phytic acid) from FBF, PRF, and SRF were analysed before and after fermentation and in ict. The results are shown in Table 4. Compared to FBF, vicine and convicine concentrations were higher in PRF and lower in SRF. A decrease of vicine and convicine was observed in the ict; the lowest value was found in SRF (3.31 ± 0.03

and 1.50 ± 0.02 mg/g dm of vicine and convicine, respectively). After fermentation, the concentration of vicine and convicine decreased by more than 91% in almost all the samples compared to the ict, with the exception of fSRF, in which convicine concentration decreased by about 55% (Table 4). Compared to FBF, TIA was higher in PRF and lower in SFR. No significant (P N 0.05) variation in the activity was observed in the ict. After fermentation, TIA was significantly lower (P b 0.05) in all the samples. The lowest activity and the highest decrease were observed in fPRF (0.57 ± 0.02 and 86% TI unit/mg dm. respectively). The concentration of total phenolic compounds was higher in the PRF, followed by FBF and SRF. A slight but significant (P b 0.05) increase (ca. 8–15% of the total phenolic compounds) was observed in ict doughs. Compared to

Fig. 1. Free amino acids (FAAs) of faba bean flour (FBF), protein rich fraction (PRF) and starch rich fraction (SRF) (A) and fermented faba bean flour (fFBF), protein rich fraction (fPRF), and starch rich fraction (fSRF) doughs (mg/kg) (B).

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39

Fig. 2. Starch hydrolysis curves of a commercial white wheat bread used as control (WB), faba bean flour (FBF), protein rich fraction (PRF), starch rich fraction (SRF), fermented faba bean flour (fFBF), fermented protein rich fraction (fPRF), and fermented starch rich fraction (fSRF). The vertical axis reports the mg of maltose per 1 g of soluble starch (SS), and the horizontal axis displays time (min).

the ict doughs, fermentation caused a significant (P b 0.05) increase of the amount of phenolic compounds only in fFBF while no significant (P N 0.05) variation was observed in the other fractions (Table 4). Compared to the corresponding not incubated flours, condensed tannins showed a slight but significant (P b 0.05) decrease in FBFict and PRFict, while no variation was observed in SRFict (Table 4). Moreover, lactic acid fermentation caused a decrease of condensed tannins of ca. 50% in fFBF, ca. 42% in fPRF, and ca. 25% in fSRF, compared to the corresponding ict doughs. No significant variation (P N 0.05) in phytic acid content was observed in ict and fermented doughs in comparison with the corresponding not incubated flours (Table 4).

The hydrolysis index (HI) was significantly lowered (P b 0.05) by fermentation. Compared to control bread (WB, HI = 100), the values of HI for FBF, PRF and SRF were 11.3 ± 0.7; 14.9 ± 3.1 and 7.2 ± 0.6%, respectively. Fermentation significantly decreased HI values (P b 0.05) in fFBF and fPRF but did not affect fSRF (4.6 ± 0.7, 8.7 ± 1.6 and 8.5 ± 2.1%, respectively). The rate of starch hydrolysis (30–180 min) of samples is shown in Fig. 2. 4. Discussion In this study, faba bean flour was processed by air fractionation and fermentation with selected lactic acid bacteria starter. The aim was to reduce the anti-nutritional factors and improve the nutritional properties. To the best of our knowledge, the combined use of these technologies, which represent a simple and economic processing, were never reported before. Dry milling followed by air classification has previously been applied to faba bean allowing an easy fractionation of protein and starch in high yield (Gunawardena et al., 2010). Most of the faba bean flour (N 90%)

3.5. In vitro protein and starch digestibility The in vitro protein and starch digestibility were determined in FBF, PRF, and SRF and fermented doughs. The in vitro protein digestibility (IVPD) values are reported in Table 4. A slight but significant increase (P b 0.05) of IVPD was observed after fermentation in all the samples.

Table 4 Anti-nutritional factors and in-vitro protein digestibility (IVPD) of faba bean flour (FBF), protein rich fraction (PRF), and starch rich fraction (SRF) doughs, and of the corresponding incubated controls (FBFict, PRFict, SRFict) and fermented doughs (fFBF, fPRF, fSRF). Doughs

Vicine (mg/g dm)

FBF FBFict fFBF PRF PRFict fPRF SRF SRFict fSRF

11.46 7.76 0.67 15.85 12.41 0.77 5.17 3.31 0.47

± ± ± ± ± ± ± ± ±

0.22b 0.12c 0.01e 0.15a 0.13a 0.01e 0.07d 0.03d 0.01e

Convicine (mg/g dm) 6.24 3.62 0.56 8.54 6.91 0.77 2.71 1.50 1.22

± ± ± ± ± ± ± ± ±

0.14b 0.10c 0.01e 0.15a 0.14b 0.01e 0.05c 0.02d 0.02d

TI activity (TI unit/mg dm) 2.09 2.36 0.91 4.23 4.09 0.57 0.47 0.92 ND

± ± ± ± ± ± ± ±

0.09b 0.12b 0.04c 0.69a 0.19a 0.02c 0.09c 0.00c

Phytic acid (mg/g dm) 22.89 23.40 23.88 32.49 33.46 36.23 8.27 8.63 8.50

± ± ± ± ± ± ± ± ±

ND = not detected The data are the means of three independent experiments ± standard deviations (n = 3). a–e Values in the same column with different superscript letters differ significantly (P b 0.05).

1.30b 1.09b 0.35b 0.55a 0.17a 0.74a 0.92c 0.68c 0.67c

Total phenolic compounds (mg GAE/g dm) 3.86 4.76 5.21 5.37 5.84 6.30 2.32 2.85 2.86

± ± ± ± ± ± ± ± ±

0.22c 0.09b 0.73a 0.67a 0.35a 0.67a 0.07d 0.03d 0.19d

Condensed tannins (eq. cat/100 g) 27.10 23.08 13.71 35.02 31.10 14.92 17.04 16.80 12.80

± ± ± ± ± ± ± ± ±

1.30b 2.99c 0.79e 0.14a 2.34b 0.40e 0.34d 0.70d 0.07e

IVPD (%) 75.1 – 76.6 74.2 75.4 75.5 76.9

± 0.7b ± 0.4a ± 0.6c ± 0.6b ± 0.5b ± 0.3a

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was milled to a particle size b 100 μm, enabling a good separation of protein from starch granules (Gunawardena et al., 2010). Air classification separated the ANFs predominantly to the fine PRF while also increasing its dietary fibre content. In previous studies, lactic acid bacteria fermentation was successfully applied to legumes such as beans, soy, and chickpea, obtaining lower ANF content, increasing the protein digestibility, and producing γ-aminobutyric acid (Coda et al., 2010b; Doblado et al., 2003; Granito and Álvarez, 2006). It has also been reported that spontaneous fermentation of legumes improved the overall nutritional properties, including decreased amounts of some anti-nutritional compounds (Doblado et al., 2003; Granito et al., 2002). Even though spontaneous fermentation is an easy and effective process, it can also lead to failures due to the preponderance of undesirable organisms. In this work, L. plantarum VTT E-133328 was used as the starter since it was previously characterised for its high β-glucosidase activity (Di Cagno et al., 2010). The choice for this specific enzymatic activity was done with the main intention of decreasing the content of vicine and convicine. It should be however emphasized that, even if the β-glucosidase activity of the selected starter is very effective in reducing vicine and convicine contents, specific in vitro and in vivo analyses are still required in order to assess further transformation of these compounds during fermentation. It would be the next step to determine the potential toxicity of vicine, convicine and their derivatives. L. plantarum is ubiquitous species, found in several food ecosystems due to its versatile metabolism, and the capacity of large adaptation to different environmental conditions (Coda et al., 2014; Di Cagno et al., 2009). Compared to cereals, the growth of L. plantarum VTT E-133328 in faba bean matrix is slower and has significantly longer lag phase (Charalampopoulos et al., 2009; Coda et al., 2010a). The growth in PRF and SRF is overall more efficient than in FBF, probably due to an improved accessibility of the nutritive sources after air classification. The strain was however competitive in all the matrixes since its final cell density was 100-fold higher than that found in spontaneously fermented controls. A different behaviour of the three flours regarding acidification was shown by pH and TTA analyses. The shorter lag phase of the acidification kinetic in PRF, as well as the highest lactic and acetic acid production and TTA indicate a very strong buffering capacity, probably due to the higher content of protein. This highest production of organic acids could also be the consequence of a more efficient use of sugars deriving from the hydrolysis of fibres in PRF compared to SRF. Fermentation evidently changed the structure of faba bean matrices. The compact protein structure of FBF and PRF surrounded the starch granules, supporting the theory of increased interactions between protein and starch. The content of FAA and GABA significantly increased in all the samples after incubation and, especially, fermentation by L. plantarum VTT E-133328. In particular, the concentration of GABA also increased in ict, probably due to the flour endogenous glutamic acid decarboxylaseactivity (Coda et al., 2010b). GABA concentration in the fermented samples was 2-times higher compared to the ict, and from 40 to 70 times higher if compared to the corresponding flours. It has been shown that a daily intake of 10 mg of GABA for 12 weeks decreased blood pressure by 17.4 Hg in hypertensive patients (Inoue et al., 2003). The daily dose of GABA to get positive effects can be reached with an amount of 10 g of fermented faba matrices, which could be easily included in the diet as the ingredient of different foods. Legume seeds are rich in protein but contain low amount of the sulphur-containing amino acids, Met and Cys. To overcome this drawback the use of composite cereal/legume flours was also suggested, but the risk of deteriorating of processing and product quality occur in incorporation of legumes into cereal grain foods (Baik and Han, 2012; Dhingra and Jood, 2004; Hallén et al., 2004). In the fermented faba bean matrices the increase of Cys was ca. 3 times, while the content of the essential amino acid Met increased ca. 30–80 times, reaching values comparable with cereals (Di Cagno et al., 2002).

L. plantarum VTT E-133328 has the ability to synthesize aglycones and equol from isoflavones of soy bean which can be further transformed by dehydroxylation, reduction, C-ring cleavage, and demethylation during intestinal digestion (Setchell and Cassidy, 1999). Fermentation of legumes can modify the level of various phenolic compounds too. Overall, soluble phenols can be considered as antinutrients, although they also have a bioactive potential. In the case of grass pea, accumulation of phenols occurred as a result of L. plantarum fermentation (Starzyńska-Janiszewska and Stodolak, 2011). However, different activities were reported in different legume fermentations. For instance, in spontaneously fermented lentils, p-hydroxybenzoic and protocatechuic acids and (+)-catechin increase whereas hydroxycinnamic acids and procyanidin dimers decrease (Bartolomé et al., 1997). In this study, the content of total phenols did not significantly change after fermentation (with the exception of FBF), however the content of condensed tannins was reduced. Condensed tannins, composed of flavonoid units, are the most abundant form of tannins in faba bean and other legumes (Kosińska et al., 2011). The degradation of condensed tannins can follow different pathways involving several enzymes such as decarboxylases and oxygenases and it can also be carried out by microbial activity (Bhat et al., 1998; Deschamps, 1989). A similar mechanism might be hypothesized also in the case of faba beans. Since tannins are mostly concentrated in the hulls, previous dehulling was already useful to reduce their content. In this study, we hypothesize that a combined action of faba bean endogenous enzymes and lactic acid bacteria enzymes was responsible for the further condensed tannin reduction. Thus, it might be hypothesized that the degradation of condensed tannins into smaller molecules and the phenol transformation into different compounds are balancing the concentration of total phenolic compounds. The low digestibility of legume proteins has been related to the presence of ANFs, including protease inhibitors, widely distributed in legumes. The values of trypsin inhibitor activity reported here are in agreement with those previously found in faba bean, with the exception of PRF, which showed a content ca. 2 times higher (Alonso et al., 2000). Trypsin inhibitor activity was reduced by lactic acid fermentation in all samples, showing the highest drop in PRF. In agreement with previous studies on Bacillus spp. (Phengnuam and Suntornsuk, 2013) it can be hypothesized that the decrease of TIA is a consequence of the bacterial protease activity during fermentation. Fermentation was successful in decreasing phytic acid content in some legumes (Doblado et al., 2003; Luo et al., 2009). Faba bean was shown to exhibit endogenous phytase activity (Luo and Xie, 2013). In the conditions of this study, phytic acid was not affected by fermentation. It has been shown that the optimal conditions for phytate degradation between different plant species vary, and the optimal conditions for leguminous endogenous phytases are very different from the fermentation conditions here applied (Gustafsson and Sandberg, 1995; Scott, 1991). Phytic acid as well as condensed tannins and polyphenols form complexes with protein, decreasing their solubility and rendering them less susceptible to proteolysis (Cheryan and Rackis, 1980). The reduction of ANF in faba bean led to increase in protein digestibility (Alonso et al., 2000). The decrease of tannin content and TIA levels improved the IVDP as a consequence of natural fermentation of beans (Granito et al., 2002). This is in agreement with our results, particularly for SRF which contains the lowest amount of ANF. Accordingly, a decrease of starch digestibility after fermentation was observed only for FBF and PRF. The strict interactions between protein and starch might be the cause of the reduction of starch digestibility in the fermented FBF and PRF. This study showed that the combination of air classification and fermentation by a selected lactic acid bacteria strain was beneficial not only for diminishing/removing the ANF but also for improving FAA content, increasing the protein digestibility of faba bean matrices. Future research is needed to investigate on the presence of the aglycones of vicine and convicine, divicine and isouramil, in the fermented

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faba matrices. As already stated above, air classification separated the ANFs mostly in the PRF, thus it could be considered as a good technology option to obtain starch-rich fraction with a low amount of ANFs from faba beans. The major advantages of air classification are the relatively small changes in the chemical properties of the flour components, the retention of the functional properties of the fractions, and the lower energy applied compared to wet fractionation (Gunawardena et al., 2010; Pelgrom et al., 2013). Fermentation with the selected starter further enhanced the nutritional profile of faba bean flour and its fractions, which represent very promising ingredients for the production of several food products (e.g. baked products, protein beverages, high protein diet formulas, soups). Further studies on the potential of this matrix should be carried out to promote a more extensive use in the food industry. 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