Nutritional and functional effects of the lactic acid bacteria fermentation on gelatinized legume flours

Journal Pre-proof Nutritional and functional effects of the lactic acid bacteria fermentation on gelatinized legume flours

Ilaria De Pasquale, Erica Pontonio, Marco Gobbetti, Carlo Giuseppe Rizzello PII:

S0168-1605(19)30357-5

DOI:

https://doi.org/10.1016/j.ijfoodmicro.2019.108426

Reference:

FOOD 108426

To appear in:

International Journal of Food Microbiology

Received date:

7 June 2019

Revised date:

11 September 2019

Accepted date:

3 November 2019

Please cite this article as: I. De Pasquale, E. Pontonio, M. Gobbetti, et al., Nutritional and functional effects of the lactic acid bacteria fermentation on gelatinized legume flours, International Journal of Food Microbiology (2018), https://doi.org/10.1016/ j.ijfoodmicro.2019.108426

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© 2018 Published by Elsevier.

Journal Pre-proof Nutritional and functional effects of the lactic acid bacteria fermentation on gelatinized legume flours

Ilaria De Pasquale1, Erica Pontonio1, Marco Gobbetti2, Carlo Giuseppe Rizzello1*

Department of Soil, Plant and Food Science, University of Bari Aldo Moro, 70126 Bari, Italy;

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Faculty of Science and Technology, Free University of Bozen, 39100 Bozen, Italy.

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*Corresponding author. Email: [email protected]

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Journal Pre-proof Abstract Aiming at meeting recent consumers requirements in terms of high nutritional value and functional foods, the cereal food industry has proposed the use of legumes as wheat substitutes due to the high contents of proteins with high biological value and dietary fibers. Nevertheless, legumes contain several anti-nutritional factors which may limit the bio-availability of several nutrients. In this study, an integrate biotechnological approach, combining a thermal treatment (“gelatinization”) and fermentation with selected lactic acid bacteria, was set-up in order to improve the functional and

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nutritional quality of red and yellow lentils, white and black beans, chickpeas and peas flours. Gelatinization carried out at pilot-plant level on legume grains before milling, affected the

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nutritional properties of the flours by the increase of protein digestibility, resistant starch formation,

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the decrease of trypsin inhibitors, although negatively affecting the antioxidant activity. Fermentation with Lactobacillus plantarum MRS1 and Lactobacillus brevis MRS4 further

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enhanced the nutritional properties of processed legume flours through the increase of free amino

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acids concentration and protein digestibility, the degradation of phytic acid, condensed tannins and raffinose, and the decrease of the trypsin inhibitory activity and starch hydrolysis index. Moreover,

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processed legumes.

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fermentation also contributed to the increase of the radical scavenging activity of both raw and

Keywords: legumes, lactic acid bacteria, sourdough fermentation, nutritional profile, wheatalternatives

Abbreviations rF, raw flour doughs; gF, gelatinized flours doughs; F-rF, fermented raw flour doughs; F-gF, fermented gelatinized flour doughs; AACC, American Association for Clinical Chemistry; LAB, lactic acid bacteria; MRS, de Man Rogosa and Sharp; YPDA, Yeast Peptone Dextrose Agar medium; VRBGA, Violet Red Bile Glucose Agar; WSE, Water/salt-soluble extract; ME, methanol extract; TFAA, Total Free Amino Acids; DPPH, 2,2-diphenyl-1-picrylhydrazyl; TTA, Total 2

Journal Pre-proof titratable acidity; MCPA (2-methyl-4-chlorophenoxyacetic acid); QF; quotient of fermentation; OPA, o-phtaldialdehyde; BHT, butylated hydroxytoluene; IVPD, in vitro protein digestibility; HI,

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hydrolysis index; DF, dietary fibers; SDF, soluble dietary fiber; IDS, insoluble dietary fiber.

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Journal Pre-proof 1. Introduction The Leguminosae are the most important group of the Dicotyledonae, being one of the largest families of flowering plants with 18,000 species and, the second worldwide food crops, after cereals. Although legumes belong to the traditional dietary habit in many countries, their consumption gained popularity only recently due to the consumers’ awareness of the nutritional and functional values. Indeed, legumes are excellent sources of proteins with high biological value and dietary fibers, and supply relevant levels of vitamins, minerals, oligosaccharides and phenolic

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compounds (Roy et al., 2010). In the framework of a balanced diet, the frequent consumption of

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legumes is effective to prevent or decrease risks of cardiovascular diseases (Widmer et al., 2015),

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overweight and obesity (Mollard et al., 2012).

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type 2 diabetes (Jenkins et al., 2012), some types of cancer (Feregrino-Perez et al., 2008), and

Apart from the direct consumption as dishes, legumes have the potential to become fundamental

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ingredients, as cereals substitutes, in baked goods and pasta (Monnet et al., 2019). The use as

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fortifiers could increase their consumption meeting as strongly recommended by many dietary guidelines (Rizzello et al., 2018a). Nevertheless, legumes contain several compounds which can

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prevent some nutrients absorption and compromising mineral bio-availability and protein digestibility (Díaz-Batalla et al., 2006). These are often referred to as anti-nutritional factors (ANF) and correspond to raffinose, phytic acid, condensed tannins, alkaloids, lectins, pyrimidine glycosides (e.g., vicine and convicine), saponins, and protease inhibitors (Coda et al., 2014a; Liener, 1990). Most of these ANF are heat-labile (e.g., protease inhibitors and lectins), so that thermal treatments, prior consumption, would remove potential negative effects (Muzquiz et al., 2012). On the other hand, phytic acid, raffinose, tannins and saponins are heat-stable, and various methods, such as dehulling, soaking, air classification, extrusion, or cooking, were used to decrease their negative impact on foods nutritional value (Coda et al., 2014a; Jezierny et al., 2010; Rizzello et al., 2014a; Van der Poel, 1990). 4

Journal Pre-proof Aiming at improving the nutritional and technological characteristics of legumes flours, the food industry has recently introduced the use of various physical treatments, before or after grains milling. Microwaving, extrusion, steaming, boiling, roasting, and infrared, all associated with heating, cause a total or partial starch gelatinization improving emulsifying, foaming, and water retention capacity and thickening thus improving technological suitability of legumes flours to be used as ingredients in baked goods, pasta and snacks (Gòmez and Martìnez, 2015; Min et al., 2010; Neelam et al., 2012). Moreover, decreases of the ANF contents, with extent depending on the

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method used, were also suggested (Avil´es-Gaxiola et al., 2018; Khattab et al., 2009; Margier et al.,

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2018). Commercially, these flours are known as “pre-gelatinized” or “gelatinized”.

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Fermentation, spontaneous or carried out by selected lactic acid bacteria (LAB), has also been

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successfully used as pre-treatment of legumes (e.g., lentils, beans, chickpeas and peas) leading to partial or complete degradation of ANF as well as the improvement of the protein digestibility and

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the bio availability of fibers and total phenols (Curiel et al., 2015; Gobbetti et al., 2018; Rizzello et

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al., 2014, 2018a). Nevertheless, the effects of combined processes to improve these features need to be explored.

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This study investigated the effects on nutritional and functional features of red and yellow lentils (Lens culinaris), white and black beans (Phaseolus vulgaris), chickpeas (Cicer arietinum), and peas (Pisum sativum) flours using a combination of a thermal treatment (“gelatinization”) and lactic acid fermentation . The effects of both single and combined processes are also highlighted.

2. Materials and methods 2.1 Raw and gelatinized legume flours Yellow and red lentils, white and black beans, chickpeas, and peas grains were cleaned, sieved, dehulled by abrasion through a dehulling machine and subjected to cooking (wet gelatinization) at the pilot plant of Molino Favero (Padova, Italy). Gelatinization was followed by drying and milling steps. Raw flours, obtained from the same legume batches, were used as controls. 5

Journal Pre-proof Protein (total nitrogen ×5.7), lipids, ash and moisture were determined according to the AACC approved methods 46-11A, 30–10.01, 08–01 and 44-15A, respectively (AACC, 2010). Carbohydrates were determined by difference. Proteins, lipids, carbohydrates and ash were expressed as % of dry matter (d.m.). For microbiological analysis, 10 g of raw and gelatinized flours were homogenized with 90 ml of sterile peptone water (1% [w/v] of peptone and 0.9% [w/v] of NaCl) solution. LAB were enumerated using De Man, Rogosa and Sharpe (MRS) (Oxoid, Basingstoke, Hampshire, UK) agar

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medium, supplemented with cycloheximide (0.1 g/l). Plates were incubated, under anaerobiosis

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(AnaeroGen and AnaeroJar, Oxoid), at 30 °C for 48 h. Cell densities of yeasts and molds were

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estimated on Yeast Peptone Dextrose Agar medium (YPDA) (Sigma-Merck, Darmstadt, Germany)

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supplemented with chloramphenicol (0.1 g/l), through pour and spread plate enumeration, respectively, and incubated at 30 °C for 72 h. The identification of yeast or mold was done by visual

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analysis of colony morphology. Enterobacteriaceae were determined on Violet Red Bile Glucose

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Agar (VRBGA, Oxoid) at 37 °C for 24 h. 2.2 Microorganisms and fermentation

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Aiming at increasing the microbial diversity, starters were selected among 9 LAB strains previously isolated from matrices (raw or spontaneously fermented quinoa, hemp, chickpea and wheat germ) with different chemical composition and sharing either most of the functional compounds and ANF with legumes. Lactobacillus plantarum T0A10 (Rizzello et al., 2016), L. plantarum 18S9, Pediococcus acidilactici 10MM0, Leuconostoc mesenteroides 12MM1 (Nionelli et al., 2018), L. plantarum LB1, Lactobacillus rossiae LB5 (Rizzello et al., 2010), L. plantarum MRS1, MR10 and Lactobacillus brevis MRS4 (Rizzello et al., 2014a), belonging to the Culture Collection of the Department of Soil, Plant and Food Sciences (University of Bari, Italy) were found to be the most best performing strains when used for the fermentation of their own food matrices (Nionelli et al., 2018; Rizzello et al., 2010, 2014a, 2016) and thus were used in this study (Supplementary Table S1). 6

Journal Pre-proof LAB strains were propagated into MRS broth at 30 °C for 24 h. Cells were harvested by centrifugation (10,000 ×g, 10 min, 4°C), washed twice in 50 mM sterile potassium phosphate buffer (pH 7.0), re-suspended in tap water at the cell density of ca. 9.0 log10 cfu/ml and used as starters for raw and gelatinized legume flour doughs (rF and gF, respectively) fermentation (initial cell density in the dough, ca. 7.0 log10 cfu/g), aiming at investigating the main pro-technological characteristics. The ratio water:flour of the doughs was chosen according to the water absorption data obtained by Brabender Farinograph analyses, and expressed as dough yield (DY, dough weight

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×100 / flour weight) (Supplementary Table S2). Fermentation was carried out in triplicate at 30 °C

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for 24 h.

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Kinetics of growth and acidification were determined as reported by Nionelli et al. (2018). The pH

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was determined on-line by a pHmeter (Model 507, Crison, Milan, Italy) with a food penetration probe.

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Based on the kinetics parameters, two LAB strains (L. plantarum MRS1 and L. brevis MRS4) were

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selected and used as mixed starter (ratio 1:1) for raw and gelatinized fermentations. Cell suspensions and doughs were prepared as described above. Fermentation was carried out in

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triplicate at 30 °C for 24 h. Fermented raw flour (F-rF) and gelatinized flour (F-gF) doughs were characterized and compared to unfermented rF and gF (doughs before fermentation, at t0). 2.3 Organic acids, fermentation quotient, free amino acids Water/salt-soluble extracts (WSE) used to analyze total free amino (TFAA) and organic acids concentrations from legume doughs were prepared according to the method originally described by Osborne (1907) and modified by Weiss, Vogelmeier, and Gorg (1993). TFAA were analyzed by a Biochrom 30 series Amino Acid Analyzer (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 in the WSE were determined 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, 7

Journal Pre-proof Richmond, CA), and an UV detector operating at 210 nm. Elution was carried out at 60 °C, with a flow rate of 0.6 ml/min, using H2SO4 10 mM as mobile phase. The fermentation quotient (FQ) was determined as the molar ratio between lactic and acetic acids. 2.4 Dietary fiber and resistant starch Insoluble (IDF) and soluble (SDF) dietary fibers were determined according to the procedure previously described by Goñi et al. (2009) for solid samples. Resistant starch of flours and sourdough was determined according to the AACC approved methods 32-40.01 3 (AACC, 2010).

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2.5 Total phenols and antioxidant activity

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Total phenols were determined on the methanolic extract (ME) of doughs both prior and after the

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fermentation. Five grams of each sample were mixed with 50 ml of 80% methanol to get ME. The

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mixture was purged with nitrogen stream for 30 min, under stirring condition, and centrifuged at 4,600 ×g for 20 min. MEs were transferred into test tubes, purged with nitrogen stream and stored

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at ca. 4°C before analysis. The concentration was determined as described by Slinkard and

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Singleton (1977) and expressed as gallic acid equivalent. The radical DPPH% was used for determining the free radical scavenging activity (Rizzello et al., 2010). The synthetic antioxidant

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butylated hydroxytoluene (BHT) was included in the analysis as the reference (75 ppm). 2.6 Antinutritional factors

Raffinose, phytic acid (and phytase activity), condensed tannins, trypsin inhibitors and saponins were determined on legume doughs both prior and after the fermentation. Raffinose and phytic acid concentration was measured using Raffinose/D-Galactose Assay Kit K-RAFGA and Megazyme kit K-PHYT 05/07 (Megazyme International Ireland Limited, Bray, Ireland) respectively, following the manufacturer’s instructions. Phytase activity was determined on the WSE of legume doughs, by monitoring the rate of hydrolysis of p-nitrophenyl phosphate (p-NPP) (Sigma, 104–0). The assay mixture contained 200 μl of 1.5 mM p-NPP (final concentration) in 0.2 M Na-acetate, pH 5.2, and 400 μl of WSE. The mixture was incubated at 45 °C and the reaction was stopped by adding 600 μl of 0.1 M NaOH. The p-nitrophenol released was determined by measuring the absorbance at 405 8

Journal Pre-proof nm (Rizzello et al., 2010). One unit (U) of activity was defined as the amount of enzyme required to liberate 1 μmol/min of p-nitrophenol under the assay conditions. Condensed tannins were determined using the acid butanol assay, as described by Hagerman (2002). Trypsin inhibitors were determined as described by Alonso et al. (2000), using α-N-benzoyl-DL-arginine-pnitroanilidehydrochloride (BApNA) as the substrate for trypsin. Trypsin inhibitor activity (TIA), expressed as trypsin inhibitor units/mg sample, was calculated from the absorbance read at 410 nm against a reagent blank. One trypsin unit was determined as the increase by 0.01 absorbance unit at

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410 nm of the reaction mixture. Total saponins in flour and sourdough were determined as reported

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by Lai et al. (2013) with minor modifications. Briefly, the freeze-dried samples (0.5 g) were mixed

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with 10 ml of petroleum ether by shaking for 4 h. The residues (20 mg) were then extracted with

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5 ml of 80% (v/v) aqueous methanol with shaking for 4 h. The extracts were kept at 4 °C in the dark until they were subjected to analysis. Total saponin content (TSC) was determined using the

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2.7 In vitro protein digestibility

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spectrophotometric method (Lai et al., 2013). All data are expressed on dry weight basis.

The in vitro protein digestibility (IVPD) was determined on legume doughs both prior and after the

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fermentation by the method proposed by Akeson and Stahmann (1964) with some modifications (Rizzello et al., 2016). Samples were subjected to a sequential enzyme treatment mimicking the in vivo digestion in the gastro-intestinal tract and IVPD was expressed as the percentage of the total protein which was solubilized after enzyme hydrolysis. The concentration of protein of digested and non-digested fractions were determined by the Bradford method (Bradford, 1976). 2.8 Starch hydrolysis The analysis of starch hydrolysis was carried out on doughs. The procedure mimicked the in vivo digestion of starch (De Angelis et al., 2009; Liljeberg et al., 1996). Aliquots of sourdough, containing 1 g of starch, were subjected to enzymatic process and the released glucose content was measured with D-Fructose/D-Glucose Assay Kit (Megazyme). The degree of starch digestion was expressed as the percentage of potentially available starch hydrolyzed after 180 min. Wheat flour 9

Journal Pre-proof bread (WB) leavened with baker's yeast was used as the control to estimate the hydrolysis index (HI =100). 2.9. Statistical analysis All data of biochemical analyses were obtained at least in duplicates and each replicate was analyzed twice. Data were subject to one-way ANOVA, using the IBM SPSS Statistics 26 (IBM Corporation, New York City, NY, United States) software. Results

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3.1 Proximate composition and microbiological analysis of raw and gelatinized flours

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The proximate composition of raw flours is reported in Supplementary Table S3. Moisture was

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lower than ca. 11% for all the rF. Protein content of the flours obtained from red and yellow lentils

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and white beans was in the range 23.4±0.6 - 25.8±0.4% of dry matter (d.m.), while significantly (P<0.05) lower values were found in black beans, chickpeas and peas flours (ca. 19 % of d.m.). Fat

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concentration was lower than 3.4% in all the flours, except for chickpeas flour, that contained a

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slightly but significantly (P<0.05) higher amount (4.0±0.1). Ash content was lower than ca. 3.0% of d.m., except for black beans and chickpeas flours, having significantly (P<0.05) higher content

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(Supplementary Table S3).

The results of the microbiological analysis of raw and gelatinized flours are reported in Table 1. Overall, all microbial groups investigated were found in all the samples at a density lower than ca. 4 log10 cfu/g. As regard to rF, white bean contained the highest cell density of LAB and yeasts, while, molds and Enterobacteriaceae were more abundant in red lentils and black beans, respectively (Table 1). The gelatinization process mainly affected yeasts and Enterobacteriaceae. Indeed, a significant (P<0.05) decrease of the cell density of both microbial groups was found, regardless of the type of flour investigated (Table 1). The lowest decreases of yeasts and Enterobacteriaceae, corresponding to ca. 2 log10 cfu/g, were found in white and black beans. LAB and molds remained almost constant during the gelatinization in all the legume flours (Table 1). 3.2 Starter selection 10

Journal Pre-proof Raw and gelatinized flours were mixed to water, aiming at obtaining homogeneous and workable soft doughs. Due to the different water absorption of the flours, DY ranged from 160 to 176 for rF, while a higher amount of water was used for gF (DY values ranged from 192 to 208) (Supplementary Table S2). All the doughs were singly inoculated with the 9 LAB strains, and growth and acidification monitored during 24 h of incubation. During the incubation, a growth of ca. 2 log10 cycles was found in all the matrices (final LAB cell density ranged from 9.1±0.5 to 9.6±0.4 log10 cfu/g of dough) (data not shown). Aggregated data of the kinetics parameters

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obtained for the strains in rF and gF were evaluated (Figure 1A-C).

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L. plantarum MRS1 showed the highest acidification capacity (dpH median value of 2.42±0.3, with

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the highest values observed in red lentil-rF and pea-gF). The other strains generated a dpH ranging

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from 1.53±0.07 to 2.08±0.01 (Figure 1A). The latency phase of acidification ranged in a wide range for L. plantarum LB1 and P. acidilactici 10MM0 (Figure 1B), while λ median values of all the

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other strains were lower than 0.53±0.02 h (Figure 1B). L. brevis MRS4 was characterized by the

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highest Vmax (median value of 0.21±0.01 dpH/h), showing the same behavior on all the matrices considered (Figure 1C). Based on these considerations, L. plantarum MRS1 and L. brevis MRS4

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were selected and used as mixed starter for further experiments. 3.3 Legume flours fermentation

Doughs inoculated with the selected starters were incubated for 24 h at 30 °C. A growth of ca. 2 log10 cycles of the LAB was observed in all the samples (final cell density ranged from 9.54 ± 0.05 to 9.82 ± 0.09 log10 cfu/g without significant differences among the fermented samples) (data not shown). As the consequence of the lactic fermentation, pH decreased to values ranging from 4.35±0.21 to 4.70±0.11 for rF, while values slightly higher were observed for gF (4.31±0.24 to 4.50±0.25) (Figure 2A). No significant (P>0.05) differences were found among the different legume flours within the F-rF or F-gF groups, respectively. Lactic acid, which was not found in doughs prior fermentation, reached concentration up to ca. 100 mmol/kg in fermented doughs. Median values for fermented rF and gF were 83.4±4.2 and 86.4±3.9 11

Journal Pre-proof mmol/kg, respectively (Fig. 2B). The highest concentrations were found in chickpeas doughs. Although only slight differences were found in final lactic acid concentration of F-rF and F-gF, the synthesis of acetic acid significantly (P<0.05) differed, for any legume considered, between rF and gF (Fig. 2C). In F-rF, acetic acid concentration ranged from 9.0±0.4 to 23.7±0.3 while it ranged from 16.4±1.2 to 39.7±3.0 mmol/kg in F-gF. The widest differences were observed for doughs obtained with red and yellow lentils and black beans gF, characterized by acetic acid concentrations 2- to 4-fold higher than those found in corresponding F-rF. FQ ranged from 4.23 ± 0.21 to 8.04 ±

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0.40 for F-rF while values ranging from 1.93 ± 0.09 to 4.80 ± 0.24 were found for r-gF.

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3.4 Functional and nutritional features

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Data of the nutritional characterization of rF and F-gF were elaborated and compared through

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Principal Component Analysis (PCA) (Figure 3). The two PCs explained ca. 72% of the total variance of the data. Factor 1 clearly differentiated rF from the corresponding F-gF; indeed, the

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former and the latter are scattered on the left and right part of the plane, respectively. As expected,

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rF were separated from F-gF due to the highest concentrations of ANF. On the contrary, F-gF differentiated from the corresponding rF due to the highest concentration of TFAA, values of IVPD

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as well as the lowest amount of ANF and lowest values of starch HI. Factor 2 highlighted the differences between black beans and the other legumes. Indeed, lentils, peas, white beans, and chickpeas were characterized by the highest presence of raffinose and phytase activity while black bean differentiated due to the highest TIA and saponins, and condensed tannins, respectively. 3.4.1 Total phenols and antioxidant activity Total phenols concentration was determined on rF and gF subjected to methanolic extraction. Among rF, red lentil and chickpea had the lowest and highest concentrations, respectively (Table 2). Overall, gF showed a total phenols concentration slightly lower (up to 21%) than the corresponding rF, except for red lentils that showed similar (P>0.05) values prior and after the gelatinization. The antioxidant activity followed the same trend (Table 2). Indeed, the values of radical scavenging activity, determined on DPPH, were up to 30% lower in gF as compared to rF. 12

Journal Pre-proof The antioxidant activity was higher than 38% in all gF except for chickpeas, characterized by a lower value. The highest activity was found in black beans, both rF, and gF (Table 2). Increases of total phenols during fermentation were found in both rF and gF. In detail, the concentrations were from ca. 2- to 3-fold higher in fermented compared to the unfermented doughs. The highest increases were found in red and yellow lentils F-rF, however chickpeas F-rF contained the highest phenols amount (2.21±0.03 mmol/kg). Although the antioxidant activity of both F-rF and F-gF was in any case significantly (P<0.05) higher than the corresponding unfermented doughs,

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the highest increases were observed for F-rF (Table 2). Overall, the highest scavenging activity was

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observed for black beans, both F-rF and F-gF (88.3±1.7% and 76.2±1.1%, respectively).

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3.4.2 Proteolysis and IVPD

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Aiming at investigating the degree of proteolysis occurring during gelatinization and fermentation, the concentration of TFAA was determined in all the samples. Overall, the thermal treatment of the

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gelatinization led to a significant increase of the TFAA concentration in all the legumes considered

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(Table 2). Except for red and yellow lentils, for which only a slight and not significant (P<0.05) increase was found as the consequence of gelatinization, significantly (P<0.05) higher

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concentrations were found in the other gF as compared to the corresponding rF (Table 2). The highest increase was found in peas gF (ca. 36%), however chickpeas gF contained the highest amount (2207±61 mg/kg). Fermentation caused a further significant (P<0.05) increase of the TFAA concentration, in both rF and gF. Compared to unfermented rF and gF, increases ranging from 13 to 42% and up to 60% were found in F-rF and F-gF, respectively (Table 3). Except for peas, all the FgF were characterized by TFAA concentrations higher than ca. 2 g/kg. IVPD of the rF ranged from 72±3 (red lentils) to ca. 80% (chickpeas and white beans). Overall, the proteolysis extent reflected on the IVPD. Indeed, gelatinization caused moderate increases (up to 14%) of the IVPD values in gF compared to corresponding rF (Table 2). Among the gF, the highest IVPD was found in white beans, followed by chickpeas and black beans.

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Journal Pre-proof After fermentation, a further increase of IVPD was found in all the samples. According to the higher IVPD of the gF compared to rF observed prior fermentation, the highest values at the end of the incubation were observed for F-gF. Indeed, digestibility ranged from 82±3 to 91±2 for F-rF, while F-gF were characterized by IVPD from 89±2 to 95±2% (Table 2). 3.4.3 Resistant starch, starch hydrolysis and dietary fiber The thermal process affected the resistant starch concentration regardless of the legume considered. Overall, the resistant starch fraction was significantly (P<0.05) higher in gF compared to the

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corresponding rF. The highest concentrations of resistant starch were found in black beans

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(9.13±0.08% and 11.62±0.03%, respectively in rF and gF). Except for black and white bean flours,

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resistant starch was lower than ca. 4% in all the other unfermented doughs (Table 2). After

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fermentation, significantly (P<0.05) higher values of resistant starch were found in F-rF compared to rF. Increases ranged from 30 to 85% were found. Although red lentils rF was subjected to the

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highest increase of the resistant starch during fermentation, the highest value was found in black

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beans F-rF. Overall, F-gF were characterized by the highest resistant starch concentration (5.41±0.07-15.92± 0.53%) (Table 2).

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Both gelatinization and fermentation caused a decrease of the HI, regardless of the legume used. Indeed, values up to ca. 9% lower were found in gF and F-rF as compared to the corresponding rF. The combination of the treatments led to a further decrease of the HI values in F-gF. Overall, chickpeas and white and black beans F-gF were characterized by the lowest (ca. 40) and highest (ca. 58) values, respectively. The thermal treatment did not cause significantly (P>0.05) changes in SDF concentration (Figure 4A), while increases (up to ca. 15%) were found in IDF concentration of gF compared to rF (Figure 4B). With the only exception of red lentil and chickpea, the gelatinization produced a slight increase of the TDF concentration in all the doughs (Figure 4C). Overall, values ranged from 21.1±0.3% to 29.4±0.31% and from 21.5±0.21% to 30.8±0.29%, in rF and gF, respectively. The highest contents of TDF were found in gF chickpea and black bean (>30%). 14

Journal Pre-proof Overall, the fermentation caused an increase of SDF, IDF and TDF (Figure 3A-C) in all the samples, although the highest increases were found in gelatinized samples. SDF in F-rF and F-gF ranged from 5.4±0.3 to 10.5±0.5% and from 5.5±0.3 to 11.0±0.5% respectively and were significantly (P<0.05) higher than the corresponding rF and gF (Figure 3A). In detail, the highest increases (63-72%) were found for yellow lentils and white beans doughs, while the lowest increases (23-24%) were observed when chickpeas and peas flours were used. With different magnitudes, according to the legume considered, IDF were also influenced by the

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fermentation (Figure 3B). Compared to the unfermented rF and gF, the highest increase of IDF (ca.

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18%) was found in white beans F-gF, which also reached the highest concentration (29.1±0.3%)

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(Figure 3B). TDF in F-rF varied from 24±0.3 to 32.3±0.3%, with black and white beans flours

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having the highest content (32.1±0.3 and 32.3±0.3 %, respectively). Higher values, in the range of 25.2±0.3 - 34.8±0.2%, were found in F-gF with black and white beans flours having the highest

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3.4.4 Raffinose

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contents (34.8±0.2 and 34.5±0.3%, respectively) (Figure 3C).

Raffinose in rF was found at concentrations ranging from 1.62±0.07 to 2.90±0.15 g/kg, with beans

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(white and black) and red lentils having the lowest and highest values, respectively (Table 3). Overall, both gelatinization and fermentation led to a decrease in the concentration of raffinose as compared to rF. In detail, values from 18 to 45% lower were found in gF as compared to rF. The most relevant increase was found in black beans gF which also contained the lowest content (Table 3). Lentils flours, both red and yellow, contained the highest amount of raffinose both prior and after the gelatinization process. When rF were fermented, relevant decreases in the raffinose were found. Indeed, values from 62 to 81% lower were found in F-rF as compared to rF. White beans were subjected to the highest decrease and contained the lowest concentration of raffinose (Table 3). The concentration of raffinose in F-gF was from 41 to 73% lower than the corresponding gF, with beans doughs (white and black) subjected to the highest decrease and containing the lowest concentration. When the gelatinization and fermentation were combined (F-gF) the concentration of 15

Journal Pre-proof raffinose was significantly lower than the corresponding F-rF only in peas and black beans doughs (16 - 40%). Moreover, a significantly (P<0.05) higher value (ca. 28%) was found in chickpeas F-gF as compared to the corresponding F-rF. No significant (P>0.05) differences were found between lentils (red and yellow) and white beans F-rF and F-gF (Table 3). 3.4.5 Condensed tannins The concentration of condensed tannins in rF varied from 0.72±0.03 to 3.01±0.07 mg/g, with white and black beans doughs containing the lowest and highest amounts. The condensed tannins were

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not influenced by the gelatinization process. Indeed, no significant (P>0.05) differences were found

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between rF and gF (Table 3). After fermentation, significant (P<0.05) decreases of the

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concentrations were found in both F-rF and F-gF, and the magnitude of the variation depended on

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the legume used. Indeed, decreases from 23 to 62% were found in F-rF and F-gF as compared to the corresponding rF and gF, respectively (Table 3). Black beans, both F-rF and F-gF contained the

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3.4.6 Trypsin inhibitor activity

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highest concentrations of condensed tannins.

A trypsin inhibitor activity varying from 0.41 ± 0.01 to 0.82 ± 0.04 U was found in legumes rF.

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With the only exception of pea rF (ca. 0.4 U), all samples were characterized by similar values of TIA (ca. 0.8 U) (Table 3). When rF were subjected to gelatinization, the activity of the trypsin inhibitors decreased from 20 to 49%. Pea presented the most relevant decrease and showed the lowest activity prior and after the gelatinization (rF and gF, respectively). A further decrease of the TIA was observed during fermentation. Indeed, F-rF were characterized by lower (23 - 44%) values as compared to the corresponding rF. Also, pea F-rF showed the lowest activity (0.23 ± 0.01 U). The combination of gelatinization and fermentation (F-gF) led to a further decrease of the TIA as compared to both gF and F-rF. Indeed, decreases up to 70% were found in F-gF. As expected, pea F-gF was characterized by the lowest value of TIA, corresponding to 0.07 ± 0.05 U (Table 3). Overall, regardless of the type of flour (rF, gF, F-rF and F-gF), the peas were characterized by a

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Journal Pre-proof significantly (P<0.05) lower TIA compared to the other legume’s doughs, all characterized by similar values (Table 3). 3.4.7 Phytic acid and phytase activity Phytic acid was found at concentrations ranging from 1.21±0.06 to 2.62±0.05 g/100g in rF. Chickpeas and peas rF contained the highest and lowest amounts, respectively. Phytic acid was affected by gelatinization, since gF were characterized by concentrations up to 60% lower than those of the corresponding rF. Although the highest decreases were found for red lentils and white

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beans flours, peas gF contained the lowest concentration (Table 3). When rF were fermented, phytic

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acid almost completely disappeared in all samples (F-rF) with decreases higher than 95%. A similar

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trend was found in F-gF; indeed, no significant (P>0.05) differences were found in the final

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concentration of phytic acid in F-rF and F-gF (Table 3).

The phytase activity significantly increased as results of both gelatinization and fermentation,

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reaching the highest values when the processes were combined (F-gF). In detail, phytase activity in

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gF, F-rF and F-gF was ca. 2- to 3-times higher than the corresponding rF. Red (rF and gF) and yellow (F-rF) lentils doughs were characterized by the highest phytase activity (Table 3).

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3.4.8 Total saponins

Total saponins in rF were in the range of 0.65±0.03 – 1.45±0.07 mg/g (Table 3). After gelatinization and fermentation, the concentration significantly decreased in all doughs. However, the latter process led to the highest decrease. In detail, decreases from ca. 20 to 50% and from 11 to 68% were found as a result of the gelatinization and fermentation, respectively (Table 3). Although black beans rF contained the highest content of total saponins, white bean gF and F-rF were characterized by the most abundant concentration after gelatinization and fermentation. When the gelatinization and fermentation were combined, a further decrease in the total saponins concentration was found in all legume doughs. White and black beans and peas F-gF contained the highest and lowest concentrations of total saponins, respectively (Table 3).

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Journal Pre-proof 4. Discussion As the perception of the importance of a healthy diet has become more evident, consumers and the food industry moved their interest towards foods with high nutritional value, which combine low glycemic index and fat content, high dietary fibers and natural antioxidants concentrations, presence of plant proteins with high biological value, low salt and synthetic additives contents. Aiming at meeting the demand of healthy and alternative products from an increasing niche of consumers, the cereal food industry has largely proposed the wheat replacement as a successful

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strategy (Bakke and Vickers, 2007; Coda et al., 2014b). Although the presence of ANF and absence

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of gluten limits their use as ingredients in cereal-like products (Díaz-Batalla et al., 2006), legumes

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are gaining great interest, due to the abundance of proteins, fibers, phenols and minerals (Campos-

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Vega et al., 2010; Roy et al., 2010). However, whether the levels of ANF will result in human health adverse effects will depend on frequency, amount and balancing of what else (containing the

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ANF) is consumed (Gemede and Ratta, 2014). Thermal treatments have been proposed to overcome

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the drawbacks of a gluten-free flour thanks to the induction of starch gelatinization which in turns improve the shear stress resistance, thermal decomposition, retrogradation and syneresis (Neelam et

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al., 2012); thus, making legume flours suitable as wheat replacers. On the other hand, the gelatinization guarantees palatability and nutritional improvements of the raw flours, thanks to the denaturation of the trypsin inhibitors due to the use of high temperature (Avilés-Gaxiola et al., 2018; Champ, 2002).

In this study, gelatinized flours of six different legumes, obtained at industrial pilot-plant level, were characterized and compared to the corresponding raw ones. Then, both raw and gelatinized flours were fermented with selected LAB to investigate the effect on the main nutritional and functional features. Gelatinization was performed at a temperature not exceeding 90 °C. Yeasts and Enterobacteriaceae significantly decreased, while thermo-resistant (thermoduric) LAB and molds were not affected by the treatment. Gelatinization induced an increase in water binding capacity of flours, as previously 18

Journal Pre-proof observed for wheat flour, thus hypothesizing a disruption of the internal structure of starch granules during thermal treatment (Wooton and Bamunuarachchi, 1978). Among the compounds of nutritional interest, total phenols were slightly but significantly affected by the thermal treatment. High temperature and pressure led to polymerization and ultimately decrease the bio-availability of phenolic compounds (Repo-Carrasco-Valencia et al., 2009; Wolosiak et al., 2011; 2018). Consequently, a proportional reduction of the antioxidant activity was found in gF compared to rF.

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According to the results, the thermal gradient occurring during the first step of the gelatinization

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process induced the activation of different endogenous enzymes. gF were characterized by higher

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concentration of TFAA, resulting from the proteolysis catalyzed by endogenous proteases and

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peptidases (Rizzello et al., 2018b). TFAA concentrations were, for all the gF, higher than 1400 mg/kg (more than double the wheat flour) (Minervini et al., 2010). Besides nutritional aspects, free

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amino acids are retained of great importance for the sensory properties (Pico et al., 2018). The

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moderate proteolysis operated by the endogenous enzymes probably affected the in vitro protein digestibility, however the degradation of the ANF factors, some of which responsible for binding

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proteins, must be considered as co-responsible for such increase. As previously reported (Sajilata et al., 2006), retrogradation occurring during steaming caused the formation of resistant starch, and consequently, the slight but significant increase of the insoluble fiber concentration.

The activation of the endogenous enzymatic activity can be also correlated to the degradation of raffinose and phytic acid. Although known as prebiotic compound, raffinose is also considered as ANF when present at high concentration, i.e., in legumes. Raffinose is not digestible by humans and monogastric animals, but it can be used as substrate by the intestinal microbiota, with a large production of gas (Udensi et al., 2007). Phytic acid interacts with proteins and minerals, reducing their solubility and bio-availability; it has also been linked to the inhibition of digestive enzymes such as trypsin and α-amylases (Chitra et al., 19

Journal Pre-proof 1996). Its degradation is catalyzed by phytases, that have been previously found and characterized in legumes (Frias et al., 2003; Steiner et al., 2007). Phytase activity in gF was up to 16% higher than that of the corresponding rF. Sourdough-like fermentation, carried out using selected LAB, has largely been reported as a suitable tool to enhance nutritional and functional properties of non-wheat flours (Coda et al., 2014a; Curiel et al., 2015; Gobbetti et al., 2014). In this study, the combination of gelatinization and fermentation has been studied. The pro-technological performances of strains of L. plantarum, P.

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acidilactici, Leuc. mesenteroides, L. brevis and L. rossiae previously selected as starters for

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fermentation of their own isolation matrices (Nionelli et al., 2018; Rizzello et al., 2010, 2014a,

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2016) were evaluated on legume flours. L. plantarum MRS1 and L. brevis MRS4 were selected and

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further used as mixed starter for rF and gF fermentation. Along with the commonly chemical changes typical of the sourdough fermentation (e.g., decrease of pH and increase of titratable

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acidity and organic acids concentration), functional and nutritional improvements were observed.

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Although lactic acid synthesis was comparable in the two types of legume doughs, acetic acid was found at higher concentrations in F-gF due to the use of fructose (released from raffinose first and

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sucrose later) as external acceptor of electrons and activating the acetate kinase route of the LAB (Frias et al., 2003; Gobbetti et al., 2005). LAB fermentation caused the increase of the total phenols concentration in both F-rF and F-gF. As previously reported (Coda et al., 2014a; Curiel et al., 2015; Gan et al., 2016; Gobbetti et al., 2014), the acidification improves their extraction, and feruloyl-esterase and β-glucosidase activities, already described for LAB (Nionelli et al., 2014), contributes to the release of phenols from bound and more complex forms. Lower concentration of total phenols was found in F-gF compared to F-rF according to the degradation occurring during the gelatinization step (Wolosiak et al., 2011). Thermal treatment induced a decrease of the total phenols due to (i) the polymerization and/or decomposition in aromatic ring structures (Wolosiak et al., 2010) and (ii) partial transfer into water

20

Journal Pre-proof (steam or brine) used for processing (Wolosiak et al., 2011). As expected, the radical scavenging activity in the ME, mainly dependent from the phenols concentration, followed the same trend. The combined effect of the flour endogenous proteases and LAB peptidases (Gänzle, 2014) led to a TFAA concentration ca. 16% (mean) higher in F-gF compared to F-rF due to the contribution of the endogenous proteases activated during the gelatinization. The markedly higher concentration of TFAA, meaning an intense proteolysis (Rizzello et al., 2014b) reflected on IVPD which was higher in F-gF than F-rF.

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As previously reported (Rizzello et al., 2017), also resistant starch markedly increased during

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fermentation, proportionally to the initial concentration of the flour (that was higher in gF).

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Resistant starch formation contributes to the increase of TDF concentration, nevertheless, the

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balance among soluble and insoluble fibers after fermentation is difficult since affected by the partial solubilization of the insoluble fraction (Rizzello et al., 2016). According to the increase of

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the resistant starch, the rate of starch hydrolysis decreased after gelatinization. Fermentation caused

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a further decrease of the HI. It was previously reported that the short-chain organic acids produced by LAB are responsible for the decrease of the glycemic response of fermented ingredients

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compared to the native (Ostman et al., 2002; Scazzina et al., 2008). Decreases of ANF in flours of different origin have largely been achieved with sourdough fermentation (Coda et al., 2015; Nionelli et al., 2018; Rizzello et al., 2010, 2016). The α-galactosides, such as raffinose, which are abundant in legumes, can be enzymatically hydrolyzed by LAB during fermentation (Coda et al., 2017), thus increasing product digestibility and reducing digestive discomfort (Waters et al., 2015). Fermentation with selected LAB significantly decreased the raffinose in both F-rF and F-gF, however a higher decrease was observed when gF were used as substrate. Condensed tannins and trypsin inhibitors are, among other ANF, responsible for the low bio-availability of legume proteins, and they followed same trends. According to the results previously reported for fermented legumes (Coda et al., 2015; Curiel et al., 2015), cereals (Roger et al., 2015) and other non-wheat flours (Nionelli et al., 2018), 21

Journal Pre-proof microbial activities and, especially, LAB fermentation consistently decreased the levels of condensed tannins, that were already affected by thermal treatment (Wolosiak et al., 2011). Tannins form insoluble tannin-protein complexes resistant to digestive enzymes (Champ, 2002) nevertheless, the ability of LAB, such as L. plantarum strains, to hydrolyze tannins through tannase activity has been reported (Pranoto et al., 2013). TIA decreased during fermentation, reaching the lowest values in F-gF, in which the thermal treatment already led to a relevant TIA decrease. The TIA reduction was already observed in fermented cereals, legumes (Starzyńska-Janiszewska and

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Stodolak, 2011), milling by-products (Rizzello et al., 2010), pseudocereals (Rizzello et al., 2016)

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and other non-conventional flours (Nionelli et al., 2018), depending on the specific capability of the

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LAB strains involved in fermentation (Waters et al., 2015).

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Fermentation caused a decrease of the phytic acid which was almost completely degraded especially in F-gF. It was previously reported that the acidification could enhance the endogenous

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phytase activity, although the activity of microbial phytases was previously reported for different

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LAB (Rizzello et al., 2018b; Zotta et al., 2007).

Together with the proteolytic activity of the LAB, the degradation of ANF contributed to the

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increase of the protein digestibility.

Saponins, biologically active glycosides, are present in legumes and known as ANF for their ability to hemolyze red blood cells and to form complexes with nutrients preventing their absorption in small intestine (Bolívar-Monsalve et al., 2017), although their anticarcinogenic and blood cholesterol-lowering effects have been also reported (Tarade et al., 2006). The saponins degradation was already observed at different extent during processing (soaking, cooking and drying) (Tarade et al., 2006). Compared to the gelatinization process, fermentation by LAB resulted more effective in saponins reduction. According to the results collected and above discussed, the combination of gelatinization and fermentation led to a relevant improvement of the nutritional features of legumes flours.

22

Journal Pre-proof 5. Conclusions This study combines the use of selected lactic acid bacteria and gelatinization to enhance the nutritional profile of legumes flours. The moderate thermal treatment activates, probably in the first step of the process, endogenous enzymes which flanked by the microbial activities led to a relevant degradation of the anti-nutritional factors and improvement of nutritional features. Although a slight decrease of the radical scavenging activity was found as results of the gelatinization, fermentation also contributed to the improvement of functional feature as compared to unfermented raw and

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gelatinized flours. Each fermented gelatinized flour has peculiar features, offering choices to fortify

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baked goods and pasta, which depend on specific nutritional aims. This study supplies a realistic

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option that combines wheat-alternatives and consumer expectations for healthy foods.

Acknowledgements

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Molino Favero (Padova, Italy) is gratefully thanked for having provided raw and gelatinized flours,

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and for making available the pilot plant for the gelatinization process.

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Author Contributions Statement

IDP, carried out the laboratory experiments and elaborated the results; EP contributed to the draft and critically revised the final manuscript; MG was the scientific advisor, also responsible for the research funding; CGR, designed the experimental work and coordinated the experimental activities. All authors read and approved the final manuscript.

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Rizzello, C. G., Lorusso, A., Montemurro, M., Gobbetti, M., 2016. Use of sourdough made with

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quinoa (Chenopodium quinoa) flour and autochthonous selected lactic acid bacteria for enhancing the nutritional, textural and sensory features of white bread. Food Microbiol. 56: 1-13.

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for enhancing nutritional and healthy properties of wheat based foods. Int J Food Microbiol.

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Rizzello, C. G., Coda, R., Wang, Y., Verni, M., Kajala, I., Katina, K., Laitila, A., 2018b. Characterization of indigenous Pediococcus pentosaceus, Leuconostoc kimchii, Weissella cibaria and Weissella confusa for faba bean bioprocessing. Int. J Food Microbiol. In press. Roger, T., Ngouné Léopold, T., Carl Moses Funtong, M., 2015. Nutritional properties and antinutritional factors of corn paste (Kutukutu) fermented by different strains of lactic acid bacteria. Int. J. Food Sci. Nutr. 502910: 1-13. Roy, F., Boye, J. I. Simpson, B. K., 2010. Bioactive proteins and peptides in pulse crops: pea, chickpea and lentil. Food Res. Int. 43: 432-442. Sajilata, M.G., Rekha Singhal, S., Pushpa Kulkarni, R., 2006. Resistant Starch—A Review. Compr Rev Food Scie Food Safety. 5: 1-17.

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Journal Pre-proof Scazzina, F., Del Rio, D., Pellegrini, N., Brighenti, F., 2008. Sourdough bread: Starch digestibility and postprandial glycemic response. J Cereal Sci. 49: 419-421. Slinkard, K., Singleton, V. L., 1977. Total phenol analysis: automation and comparison with manual methods. Am J Enol Vitic. 28: 49-55. Starzyńska-Janiszewska, A., Stodolak, B., 2011. Effect of inoculated lactic acid fermentation on antinutritional and antiradical properties of grass pea (Lathyrus sativus ‘Krab’) flour. Pol J Food Nutr Sci. 61: 245-249.

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activity, total phosphorus and phytate phosphorus in legume seeds, cereals and cereal by-

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products as influenced by harvest year and cultivar. Anim. Feed Sci. Technol. 133: 320-334.

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(Sesquipedalis) seeds during thermal processing. Pak J Nutr. 6: 194-197. Van Der Poel, A. F. B., 1990. Effect of processing on antinutritional factors and protein nutritional

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value of dry beans (Phaseolus vulgaris L.). A review. Anim Feed Sci Techno.l 29, 179-208. Waters, D. M., Mauch, A., Coffey, A., Arendt, E. K., Zannini, E., 2015. Lactic acid bacteria as a cell factory for the delivery of functional biomolecules and ingredients in cereal based beverages: a review. Crit. Rev. Food Sci. Nutr. 55: 503-520. Weiss, W., Vogelmeier, C., Gorg, A., 1993. Electrophoretic characterization of wheat grain allergens from different cultivars involved in bakers’ asthma. Electrophoresis. 14: 805-816. Widmer, R. J., Flammer, A. J., Lerman, L. O., Lerman, A., 2015. The Mediterranean diet, its components, and cardiovascular disease. Am. J. Med. 128(3): 229-238. Wolosiak, R., Druzynska, B., Piecyk, M., Worobiej, E., Majewska, E. and Lewicki, P. P., 2011. Influence of industrial sterilisation, freezing and steam cooking on antioxidant properties of green peas and string beans. Int. J Food Sci Technol. 46: 93-100. 30

Journal Pre-proof Wolosiak, R., Druzynska B., Piecyk, M., Majewska, E., Worobiej, E., Lewicki, P.P., 2011. Influence of industrial sterilisation, freezing and steam cooking on antioxidant properties of green peas and string beans. Int J Food Sci Technol. 46: 93-100. Wolosiak, R., Druzynska B., Piecyk, M., Majewska, E., Worobiej, E., 2018. Effect of Sterilization Process and Storage on the Antioxidative Properties of Runner Bean. Molecules. 23: 1409. Wooton, M., Bamunuarachchi, A., 1978. Water Binding Capacity of Commercial Produced Native and Modified Starches. Starch. 9: 306-309.

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Cornetto di Matera sourdoughs. Int. J. Food Microbiol. 115: 165-172.

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Environ

Microbiol.

56:

1875-1881.

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curve.

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growth

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Zwietering, M. H., Jongeberger, I., Roumbouts, F. M., Van’t Riet, K., 1990. Modelling of bacterial

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Journal Pre-proof Legends to figures Figure 1. Aggregated data of the parameters of the kinetics of acidification (dpH, A; λ(h), B; Vmax (dpH/h), C) of the lactic acid bacteria strains used as starters to fermented legumes doughs at 30 °C for 24 h. The data are the means of three independent experiments (n = 3). The top and bottom of the box represent the 75th and 25th percentile of the data, respectively. The top and bottom of the error bars represent the 5th and 95th percentile of the data, respectively.

Figure 2. Box plot of the aggregated data of pH (A), lactic (B) and acetic acid (C) (mmol/kg) raw and gelatinized flour doughs prior (rF and gF) and after (F-rF and F-gF) the fermentation.

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Fermentations were carried out with Lactobacillus plantarum MRS1 and Lactobacillus brevis

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MRS4 at 30 °C for 24 h. Means of the data obtained in three independent experiments (n = 3) were used for the statistic elaboration. The top and bottom of the box represent the 75th and 25th

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percentile of the data, respectively. The top and bottom of the error bars represent the 5th and 95th

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percentile of the data, respectively.

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Figure 3. Principal Component Analysis (PCA) based on the concentrations of total phenols (Tp), radical scavenging activity (AA), total free amino acids (TFAA), in vitro protein digestibility

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(IVPD), resistant starch (RS), total and soluble dietary fibers (TDF/SDF), raffinose (Rf), condensed tannins (Ct), phytic acid (Pa), total saponins (S), hydrolysis index (HI) and trypsin inhibitors activities (TIA) of raw doughs prior (rF) and after (F-gF) the fermentation. Fermentations were

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carried out with Lactobacillus plantarum MRS1 and Lactobacillus brevis MRS4 at 30 °C for 24 h. (ᴏ) projection of the variables on the factor-plane; (◊) projection of the samples on the factor-plane; Lr: Red Lentil; Ly: Yellow Lentil; Cp: Chickpea; Bw: White Bean; Bb: Black Bean; Pe: Pea.

Figure 4. Soluble (panel A), insoluble (B) and total (C) dietary fibers (SDF, IDF, and TDF respectively) in raw and gelatinized flour doughs prior (rF and gF) and after (F-rF and F-gF) the fermentation. Fermentations were carried out with Lactobacillus plantarum MRS1 and Lactobacillus brevis MRS4 at 30 °C for 24 h. Data are expressed on dry weight basis. Error bars representing standard deviation are reported.

a-h

Values in the same panel with different superscript

letters differ significantly (P<0.05).

32

Journal Pre-proof Table 1. Microbiological characterization (log10 cfu/g) of raw and gelatinized legume flours.

LAB* Yeasts Molds Enterobacteriaceae

Red Lentil 2.8±0.1b,c 3.4±0.2b 3.4±0.1a 3.2±0.1b,c

Yellow Lentil 2.4±0.1c 1.3±0.2e 2.0±0.2d 2.9±0.2c

White Bean 3.6±0.1a 4.1±0.1a 2.5±0.2c 3.4±0.1b

Raw Black Bean 3.1±0.1b 3.8±0.2a,b 2.8±0.2b 4.0±0.1a

Chickpea

Pea

2.5±0.2c 2.0±0.1d 2.1±0.1d 2.3±0.1d

3.4±0.2a,b 4.0±0.1a 1.5±0.1f 4.2±0.2a

Red Lentil 3.0±0.2b 1.9±0.1d 3.4±0.1a 2.0±0.2d,e

Yellow Lentil 2.5±0.1c 1.0±0.2e 1.9±0.1e 1.9±0.1e

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Gelatinized White Black Beans Beans 3.8±0.1a 3.0±0.1b 1.9±0.1d 2.5±0.2c 2.5±0.1c 2.9±0.1b 1.5±0.1f 2.0±0.2d,e

Chickpea

Pea

2.5±0.2c 1.0±0.2e 2.2±0.1d 1.5±0.1f

3.5±0.2a 2.0±0.2d 1.8±0.2e 2.4±0.2d

LAB, lactic acid bacteria. a-f Values in the same row with different superscript letters differ significantly (P<0.05). The data are the means of three independent experiments ± standard deviations (n =3).

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Journal Pre-proof Table 2. Total phenols concentration, radical scavenging activity (DPPH), total free amino acids (TFAA), in vitro protein digestibility (IVPD), resistant starch and hydrolysis index (HI) of raw and gelatinized legume flour doughs prior (rF and gF, respectively) and after (F-rF and F-gF, respectively) the fermentation. Fermentations were carried out with Lactobacillus plantarum MRS1 and Lactobacillus brevis MRS4 at 30 °C for 24 h. rF

gF

Red Lentil

Yellow Lentil

White Bean

Black Bean

Chickpea

Pea

Red Lentil

Yellow Lentil

Total phenols (mmol/kg)

0.44±0.01f

0.51±0.01e

0.51±0.01e

0.68±0.02d

1.19±0.03a

0.85±0.02c

0.42±0.01f

0.41±0.01f

DPPH (%)

49.11±2.4 1de

55.72±1.21

45.69±2.2 8c

77.62±1.8 9a

42.66±2.09f

42.12±2.1 1f

47.13±1.27

51.88±1.57

c

e

d

1987±99b

1740±87c

1620±81d

1254±62e

1987±99b

1057±52f

2042±102b

1800±85c

72±3d

75±3c

79±4b

77±3c

80±4b

75±5c

79±4b

78±4b

2.22±0.15g

2.41±0.09g

8.81±0.07c

9.13±0.08b

2.7±0.09f

2.9±0.11f

58±2d

60±2c

67±2a

48±1f

65±2a,b

Red Lentil

Yellow Lentil

Total phenols (mmol/kg)

1.26±0.04d

1.41±0.04c

1.04±0.01e

1.31±0.06c

DPPH (%)

51.62± 1.58e

60.54±1.39 c

59.48±1.9 3c

TFFA (mg/kg) IVPD (%)

2309±115c

2354±117c

83±3c

Resistant starch (%) HI (%)

TFFA (mg/kg) IVPD (%) Resistant starch (%) HI (%)

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2.71±0.09f

2.81±0.1f 55±2e

2.21±0.03a

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54±2e

88.34±1.6 6a

49.44±1.47

1951±97d,e

1778±88e

87±1a,b

84±2c

4.12±0.19e

4.32±0.07e

53±1c,d

55±2b,c

68±2a F-rF White Black Bean Bean

Red Lentil

Yellow Lentil

1.67±0.04b

0.76±0.02g

0.85±0.03f

51.49±1.1 2e

49.63±1.31

55.72±1.44

e

d

2243±122c,d

1435±71f

2567±128b

2738±136a

82±3c

91±2a,b

85±2c

90±1b

94±1a

12.1±0.61b

13.81±0.7 0b

4.23±0.06e

3.78±0.14f

5.63±0.13d

5.71±0.13d

62±2a

62±1a

44±1e

63±2a

50±2d

50±1d

Chickpea

l a

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e

Pea

White Bean 0.34±0. 01g 38.14± 1.91g 1996±9 9b 90±4a 9.52±0. 07b 63±1b White Bean 0.68±0. 01g 42.57± 1.61f 2109±1 05d 95±2a

Black Bean

Chickpea

Pea

0.51±0.01e

1.02±0.03 b

0.68±0.02d

72.89±1.09 b

31.32±1.6 1h

38.33±1.9 2g

1464±73e

2207±61a

1448±72e

86±2a 11.62±0.03

86±3a 3.39±0.08

79±3b

a

e

3.7±0.06d

45±1g

60±2c

63±2b F-gF Black Bean

Chickpea

Pea

0.85±0.01f

1.53±0.10 b

1.02±0.02e

76.22±1.11 b

39.24±1.6 2g

45.41±1.2 3f

2343±117c

2585±79b

1745±87e

89±2b

94±1a

90±2a,b

14.91± 0.75a

15.92±0.53

5.41±0.07

a

d

6.54±0.41c

58±1b

58±1b

41±2e

57±1b

a-h

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

34

Journal Pre-proof Table 3. Raffinose, condensed tannins, trypsin inhibitor activity (TIA), phytic acid and phytase activity, and total saponins of raw and gelatinized flour doughs prior (rF and gF, respectively) and after (F-rF and F-gF, respectively) the fermentation. Fermentations were carried out with Lactobacillus plantarum MRS1 and Lactobacillus brevis MRS4 at 30 °C for 24 h. rF

Raffinose (g/kg) Condense d tannins (mg/g)

2.90±0.15

Yellow Lentil 2.71±0.09

a

a

1.10±0.05

0.92±0.05

d

e

TIA* (U)

0.78±0.04

0.81±0.04

a

a

1.55±0.08

2.21±0.09

d

b

3.12±0.03

2.85±0.04

c

d

0.65±0.03

0.81±0.04

e

c

Red Lentil

Phytic acid (g/100 g) Phytase activity (U)** Total saponins (mg/g)

White Bean

gF Black Bean 1.62±0.08

Chickpea

Pea

Red Lentil

1.82±0.09

2.13±0.11

d

c

b

1.93±0.09e

0.72±0.03g

3.01±0.07

0.88±0.04

1.52±0.08

0.99±0.06d,

a

f

b

e

0.80±0.04a

0.82±0.04

0.78±0.04

0.41±0.01

a

a

e

2.52±0.10a

2.35±0.10

2.62±0.05

1.21±0.06

b

a

e

1.60±0.03i

2.08±0.04

2.08±0.02

h

h

1.02±0.05b

1.45±0.07 a

1.62±0.07d

F-rF

Raffinose (g/kg) Condense d tannins (mg/g)

0.71±0.04

Yellow Lentil 0.83±0.04

b

a

0.31±0.01f

0.75±0.07

0.67±0.05

b

b

TIA* (U)

0.58±0.04

0.62±0.04

a

a

0.08±0.04

0.09±0.02

a

a

4.12±0.07

5.70±0.05

d

a

Red Lentil

Phytic acid (g/100 g) Phytase activity ** (U)

White Bean

J

c

White Bean

Black Bean

Chickpea

Pea

1.22±0.07f

0.94±0.13h

1.5±0.08e,f

1.16±0.06g

0.67±0.05g

2.92±0.05a

0.90±0.05f

1.40±0.02c

f o

0.92±0.05e

0.62±0.11g

ro d

1.03±0.21f

1.43±0.21d

1.82±0.11c

0.72±0.16g

1.68±0.04i

3.73±0.09a

3.57±0.01b

2.64±0.02e

2.69±0.05e

2.58±0.03f

2.45±0.02g

0.98±0.03

0.72±0.02

b

d

0.33±0.02g

0.60±0.03e

0.81±0.04c

0.71±0.03d

0.70±0.04d

0.41±0.02f

White Bean

Black Bean

Chickpea

Pea

0.33±0.05f

0.31±0.04f

0.81±0.07a

0.68±0.02c

d

0.44±0.02f

2.24±0.07a

0.42±0.01f

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rn

u o

Black Bean 0.52±0.02

Yellow Lentil 2.02±0.11b,

0.50±0.03d

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0.57±0.03c

1.62±0.21c,

0.61±0.04b, c

0.66±0.03b

0.57±0.04c, d

0.21±0.01f

F-gF

Chickpea

Pea

Red Lentil

Yellow Lentil

0.71±0.03b

0.80±0.03a

0.63±0.03

0.81±0.04

e

d

a

0.45±0.02e,

2.31±0.01

0.49±0.02

0.58±0.02

f

a

e

d

0.60±0.04a

0.61±0.04

0.56±0.04

0.23±0.01

a

a

c

0.36±0.03b

0.40±0.03b

0.42±0.04b

0.41±0.03b

0.43±0.04b

0.07±0.05d

0.04±0.01b

0.02±0.01

0.03±0.01

0.05±0.03

0.04±0.01b,

0.04±0.01b,

0.04±0.01b,

0.04±0.02b,

0.04±0.04b,

0.06±0.03b,

c

c

b

c

c

c

c

c

c

2.92±0.05h

2.61±0.07i

3.21±0.08

4.92±0.03

g

c

5.51±0.09b

5.72±0.06a

3.83±0.03e

3.44±0.07f

5.51±0.08b

5.74±0.08a

0.66±0.03c

0.61±0.02c,

0.53±0.03d, e

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Journal Pre-proof Total saponins (mg/g)

0.21±0.01

0.34±0.02

e

d

0.91±0.05a

0.53±0.03

0.42±0.02

0.23±0.02

b

c

e

0.14±0.02f

0.16±0.03f

0.22±0.01e

0.25±0.03e

0.13±0.01f

0.11±0.04f

a-i

Values in the same row with different superscript letters differ significantly (P<0.05). The data are the means of three independent experiments ± standard deviations (n =3). * Trypsin inhibitor activity, expressed as trypsin inhibitor units/mg sample.** One unit (U) of activity was defined as the amount of enzyme required to liberate 1 μmol/min of p-nitrophenol under the assay conditions.

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Journal Pre-proof Highlights Raw and gelatinized flours were fermented with selected lactic acid bacteria Gelatinization decreased antinutritional factors Fermentation caused decrease of the starch hydrolysis index Gelatinization and fermentation increased in-vitro protein digestibility

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Combined treatment led to the most efficient degradation of antinutritional factors

37

Figure 1

Figure 2

Figure 3

Figure 4