Physicochemical, Nutritional and Antioxidant

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Physicochemical, Nutritional and Antioxidant Properties of Tempeh Flour from Common Bean ( Phaseolus vulgaris L.) M. Reyes-Bastidas, E.Z. Reyes-Fernández, J. López-Cervantes, J. Milán-Carrillo, G.F. Loarca-Piña and C. Reyes-Moreno Food Science and Technology International 2010 16: 427 originally published online 11 November 2010 DOI: 10.1177/1082013210367559 The online version of this article can be found at: http://fst.sagepub.com/content/16/5/427

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Physicochemical, Nutritional and Antioxidant Properties of Tempeh Flour from Common Bean (Phaseolus vulgaris L.) M. Reyes-Bastidas,1 E.Z. Reyes-Fernańdez,1 J. Lo´pez-Cervantes,2 J. Milań-Carrillo,1,3 G.F. Loarca-Pinã4 and C. Reyes-Moreno1,3,* 1

Facultad de Ciencias Quı´mico Biolo´gicas, Universidad Autońoma de Sinaloa, Ciudad Universitaria, AP 354, CP 80000 Culiacań, Sinaloa, Mexico 2 Departamento de Biotecnologia y Ciencias Alimentarias, Instituto Tecnolo´gico de Sonora, CP 85000, Cd. Obregon, Sonora, Mexico 3 Universidad Autońoma de Sinaloa, Ciudad Universitaria, AP 354, CP 80000 Culiacań, Sinaloa, Mexico 4 Facultad de Quı´mica, Universidad Autońoma de Quere´taro, 76010 Quere´taro, Quere´taro, Mexico The effects of solid state fermentation (SSF) on physicochemical, nutritional and antioxidant properties of common bean flour were studied. SSF increased protein content (21.7%) and decreased lipids (38.4%), carbohydrates (3.5%) and phytic acid (58.3%). Fermented (tempeh) flour showed higher dispersability, lower water solubility index and pH than unfermented flour. Fermentation also increased an average of 0.21 g/100 g protein, six of the essential amino acids (EAAs), including total sulfur (Met þ Cys), the limiting EAAs in unfermented flour (score ¼ 0.91); Lys and Trp decreased 0.21 and 0.09 g/100 g protein, respectively. SSF improved the in vitro protein digestibility and the calculated protein efficiency ratio. Tempeh flour had 2.2-fold more phenolics than the bean flour and exhibited antiradical activity (43%) and antioxidant activity (38%) correlated with total phenolics content. Common bean tempeh flour may be considered for the fortification of widely consumed legume-based food products and also for the prevention of pathologies associated with oxidative stress. Key Words: common bean, solid state fermentation, nutritional properties, antioxidant activity

INTRODUCTION Common bean (Phaseolus vulgaris L.) plays an important role in the diet of Latin-American people, providing 2040% protein, essential fatty acids, complex carbohydrates, vitamins and minerals (Reyes-Moreno and Paredes-Lo´pez, 1993). However, its wider use is somehow limited by the presence of antinutritional factors (e.g., enzyme inhibitors, lectins, phytates, cyanoglycosides and phenolic compounds), which might produce adverse effects for human and animal nutrition (Martin-Cabrejas et al., 2004). Consumption of common bean has been associated to reduce the risk of some diseases such as coronary hearth and cancer (Bazzano et al., 2001; Aparicio-Fernańdez *To whom correspondence should be sent (e-mail: [email protected]). Received 27 July 2009; revised 17 November 2009. Food Sci Tech Int 2010;16(5):0427–8 ß SAGE Publications 2010 Los Angeles, London, New Delhi and Singapore ISSN: 1082-0132 DOI: 10.1177/1082013210367559

et al., 2006). These physiological effects of common bean may be due to the presence of abundant phytochemicals including polyphenolics, which possess both anticarcinogenic and antioxidant properties (Aparicio-Fernańdez et al., 2006). Solid state fermentation (SSF) represents a technological alternative for processing a great variety of legumes and/or cereals to improve their nutritional and nutraceutical properties and to obtain edible products with palatable sensory characteristics. Tempeh is a nutritious oriental fermented food produced by SSF of soybeans, although several other substrates have been used to produce it, including common bean, chickpea, maize and mixtures of legumes/cereals (Hachmeister and Fung, 1993; Sharma and Khetarpaul, 1997; CuevasRodrı´ guez et al., 2004; Angulo-Bejarano et al., 2008). In general, SSF can be performed with Rhizopus sp. fungi; an important function of the fungus during fermentation is the synthesis of enzymes, which hydrolyze some of the substrate constituents and contribute to the development of a desirable texture, flavor and aroma of the product. Enzymatic hydrolysis may also decrease or eliminate antinutritional factors; consequently, the nutritional quality of the fermented food may be


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improved (Hachmeister and Fung, 1993). The potential of using SSF to improve the nutritional value of cereals and legumes has been evaluated (Cuevas-Rodrı´ guez et al., 2006; Angulo-Bejarano et al., 2008). Fermentation of soybean causes increased antioxidant activity (AA), which is associated with increased glucosidase and glucuronidase activities that release potent antioxidant substances by transformation of flavonoids (McCue et al., 2003). The 6,7,40 -trihydroxyisoflavone (factor 2) was isolated from soybean tempeh and exhibited marked AA (Gyo¨rgy et al., 1964). More recent studies have documented that not only Rhizopus generates polyhydroxylated isoflavones during fermentation but also several bacterial strains (Klus and Barz, 1995). In addition to the role of antioxidants in food protection against oxidative spoilage, antioxidants in soybean tempeh are of medical interest because of their protective role against oxidative stress, which is related to the pathogenesis of various chronic degenerative diseases. The objective of this investigation was to evaluate the effect of SSF on physicochemical, nutritional and antioxidant properties of common bean flour.

MATERIALS AND METHODS Common bean (P. vulgaris L., cv Azufrado Higuera, light yellow testa) was grown and harvested in 2007 at the Culiacan Valley Experimental Station of the National Research Institute for Forestry, Agriculture and Livestock (INIFAP), Sinaloa, Me´xico. This cultivar was selected because it is highly produced and consumed in the northwest region of Mexico. Grains were cleaned and stored at 4 C in tightly sealed containers until used. The Rhizopus oligosporus NRRL 2710 strain was obtained from the American Type Culture Collection, Manassas, USA. Methods Manufacture of Fermented (Tempeh) Common Bean Flour Tempeh flour was prepared using the procedure described by Reyes-Moreno et al. (2004) with minor modifications. Common bean seeds were soaked with an acetic acid solution (pH 3.0) for 16 h at 25 C. Seeds were then drained and their seed coats removed manually. The cotyledons were then cooked in acetic acid solution (pH 3.0) at 90 C for 30 min, cooled at 25 C for 4 h, inoculated with a suspension of R. oligosporus NRRL 2710 (1 106 spores/mL), and packed in perforated polyethylene bags (15 15 cm2). SSF was carried at 34.9 C for 51 h. The resulting common bean tempeh was dried at 50 C for 24 h, cooled at room temperature (25 C) and milled (UD, Cyclone

Sample Mill, UD Corp., Boulder, CO, USA) to pass through a 80-US mesh (0.180 mm) screen. The tempeh flour was kept at 4 C in tightly sealed containers until used. Proximate Composition The following AOAC (1999) methods were used to determine proximate composition: drying at 105 C for 24 h for moisture (method 925.098); incineration at 550 C for ashes (method 923.03); defatting in a Soxhlet apparatus with petroleum ether during 4 h for lipids (method 920.39C); and micro-Kjeldahl for protein (N 6.25; method 960.52). Carbohydrate content was estimated by difference. All determinations were made in triplicate. Phytic Acid Phytic acid was determined by the method of Latta and Eskin (1980). Samples were ground (UD Cyclone Sample Mill, UD Corp., Boulder, CO, USA) to pass a 100-US mesh (0.150 mm) screen and extracted by placing 1 g of flour in a 20 125 mm2 screw-top tube to which 20 mL of 0.65 M HCl were added. The tubes were capped tightly and shaken with an orbital shaker (Cole-Parmer Model 51704-10, Cole Parmer International, USA) at 400 rpm and 25 C for 1 h. Following centrifugation (5000 g, 25 C, 30 min) with a refrigerated centrifuge (Sorvall RC-2, Ivan Sorvall Inc, Norwalk, CT, USA), 3 mL of supernatant were added into an ion-exchange column packed with 0.5 g of 200400 mesh AG1-X8 chloride anion exchange resin (BioRad, Hercules, CA, USA). Inorganic phosphorus and impurities were eluted with 10 mL of 0.1 M NaCl, while phytate was eluted with 10 mL of 0.7 M NaCl. To each tube containing 10 mL of eluant, 3.3 mL of Wade reagent (0.03% [w/v] FeCl3.6H20 and 0.3% [w/v] sulfosalicilic acid) were added; the tubes were covered with ParafilmTM and mixed. After centrifugation for 30 min (5000 g, 25 C), the absorbance of the supernatant was measured at 500 nm with an UV-Visible spectrophotometer (Spectronic 21D, Model 1146, Milton Roy, USA). pH The pH of the flour samples was recorded using a pH meter. Each flour sample (10 g) was suspended in 100 mL of boiling distilled water. After cooling, the slurry was shaken (1500 rpm, 25 C, 20 min) using an orbital shaker (Cole Parmer Model 21704-10, Cole Parmer International, Vernon Hills, IL, USA). Total Color Difference (E) The surface color of the samples was measured using a Minolta color difference meter Model CR-210


Properties of Tempeh Flour

(Minolta LTD, Osaka, Japan). The parameters L (0 ¼ black, 100 ¼ white), a (þvalue ¼ red, value ¼ green) and b (þ value ¼ yellow, value ¼ blue) were recorded. The L, a and b values of a white standard tile used as reference were 97.63, 0.78 and 2.85, respectively; E was calculated as E ¼ [(L)2 þ (a)2 þ (b)2]1/2, where L ¼ Lstd L sample, a ¼ astd asample and b ¼ bstd bsample. Water Activity (Aw) This parameter was determined in 5 g flour samples tempered at 25 C using a Hygrometer Aqua Lab Model CX-2 (Decagon Devices Inc., Pullman, WA, USA), which was calibrated with a potassium chloridesaturated solution (Aw ¼ 0.841 at 25 C). Readings were taken after leaving the sample for 1 h to attain headspace equilibrium. Water Absorption Index and Water Solubility Index Water absorption index (WAI) and water solubility index (WSI) were assessed as described by Anderson et al. (1969). Each flour sample (2.5 g) was mixed with 30 mL of distilled water in a tared 50 mL centrifuge tube. The slurry was stirred with a glass rod for 1 min at room temperature and centrifuged at 3000 g for 10 min. The supernatant was then poured carefully into a tared evaporating dish. The WAI was calculated from the weight of the remaining gel and expressed in g gel/g solids (db). The WSI (g solids/100 g original solids) was calculated from the weight of dry solids recovered by evaporating the supernatant overnight at 110 C.

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Australia) equipped with a fluorescence detector LC 5100. The mobile phases used were as follows: A: 30 mM ammonium phosphate (pH 6.5) in 15 : 85 (v/v) methanol/water; B: 15 : 85 (v/v) methanol/water; and C: 90 : 10 (v/v) acetonitrile/water. The flow rate was 1.2 mL/min and the gradient program used was as reported in Table 1 by Lo´pez-Cervantes et al. (2006). Fluorescence detection was at 270 and 316 nm for excitation and emission, respectively. A calibration curve was constructed using a mix of standard amino acids. Tryptophan levels were determined using an alkaline hydrolysis. Twenty five milligrams of sample were mixed with 3 mL of 4.2 M NaOH and incubated in sealed tubes (N2 atmosphere) at 120 C for 4 h. After hydrolysis, the sample was adjusted to pH 9, washed with borate buffer (pH 9), vacuum filtered and then diluted to 50 mL with borate buffer. After centrifugation, the supernatant was filtered (0.45 mm) and then a 20 mL aliquot was analyzed as described above. Tryptophan was detected at 280 nm with an ultraviolet detector. Chemical Score The chemical score (CS) is a measure of protein quality based on the amino acid composition. It was calculated dividing the content of the limiting essential amino acid (EAA) in the sample by the content of the same amino acid in the standard amino acid reference mixture. The value was calculated using the FAO amino acid scoring pattern for pre-school children (25 years; FAO/WHO, 1991). CS ¼ ðContent of the most limiting EAA=REAARÞ 100

Dispersability It was determined according to Mora-Escobedo et al. (1994). One gram of flour sample was added into a graduated cylinder, suspended in distilled water and adjusted to 10 mL. The sample was vigorously stirred and allowed to settle for 30 min. The volume of settled particles was recorded and subtracted from 10. This value was multiplied by 10 to give the percentage dispersability. Amino Acid Analysis Total amino acid composition was determined using the method described by Lo´pez-Cervantes et al. (2006) with some modifications. Fifty milligrams of flour were mixed with 10 mL of 6 M HCl and incubated for 24 h at 100 C. The hydrolyzed sample was filtered and the extract diluted 200 times with milli-Q water. A 300 mL aliquot of the extract was dried and derivatized with 300 mL of 9-fluorenylmethyl-chloroformate (FMOC). A 20 mL aliquot was analyzed using an analytical scale (4.6 250 mm2) SGE Hypersil ODS C18 column (SGE, Dandenong, Australia) kept at 38 C and connected to an HPLC system (GBC, Dandenong,

where CS ¼ chemical score; EAA ¼ essential amino acid; REAAR ¼ recommended essential amino acid requirement. True Protein (TP) TP content was calculated from the difference between total nitrogen and non-protein nitrogen. For determination of non-protein nitrogen, about 0.5 g of sample was mixed with 20 mL of 10% (w/v) trichloroacetic acid (TCA) solution and shaken for 1 h (400 rpm, 25 C) using an orbital shaker (Cole Parmer Model 51704-10, Cole Parmer International, Melrose Park, IL, USA). The insoluble material was removed by centrifugation. The supernatant was made up to 50 mL with distilled water and an aliquot was taken for the determination of non-protein nitrogen. Nitrogen of the aliquot and total nitrogen were determinate by micro-Kjeldahl (method 960.52; AOAC, 1999). True protein (TP) content was calculated as (total nitrogennon-protein nitrogen) 6.25.


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In vitro Protein Digestibility In vitro protein digestibility (IVPD) was determined following the method proposed by Hsu et al. (1977), which is based on the drop in pH that occurs when proteins are hydrolyzed by enzymes and protons are released from free carboxyl groups. A multi-enzyme system consisting of a mixture of porcine pancreatic trypsin type IX (1.6 mg/mL), bovine pancreatic chymotrypsin type II (3.1 mg/mL) and porcine intestinal peptidase grade III (1.3 mg/mL; Sigma Chemical Co., St. Louis, MO, USA) was freshly prepared, adjusted to pH 8.0 and kept on ice until used. Flours were mixed with distilled water to prepare 50 mL of an aqueous protein suspension (6.25 g protein/L), adjusting the pH to 8.0 while stirring in a water bath at 37 C. Five milliliter aliquots of the multi-enzyme solution were added to the protein suspension with stirring at 37 C. The rapid pH drop was recorded automatically over a 10 min period using a pH meter. IVPD was calculated from the equation IVPD ¼ 210.46 18.10X, where X ¼ pH after 10 min. Calculated Protein Efficiency Ratio Calculated protein efficiency ratio (C-PER) was calculated according to Satterlee et al. (1979) and summarized by the AOAC (1999), which is based on the IVPD and the EAA composition of the common bean flour samples (unfermented and fermented). Total Phenolics Assay Total phenolics contents of unfermented and fermented common bean flours were determined following the procedure of Deshpande and Cheryan (1985), adapted to use microplates. Flour samples (1 g) were extracted with methanol (10 mL) for 24 h at room temperature in the dark using a magnetic stirrer. After centrifugation (3000 g, 10 min), the supernatant was recovered and stored in the dark at 20 C until used. A 50 mL aliquot of sample extract was mixed with 200 mL of a vanillinhydrochloric acid solution (0.5% vanillin and 4% HCl) in a 96-well microtitration flat-bottom plate. The absorbance of the solution was monitored at 540 nm with a MRX plate reader (Multiskan, Thermo Electron Corporation, Lab-Tech Instrumentation, SA de CV) to measure total phenolics content. Catechin dissolved in methanol at concentrations ranging from 0 to 0.8 mg/mL was used as a standard. Phenolics content was expressed as milligram equivalent of (þ)-catechin/g of sample. 2,2-Diphenyl-1-picrylhydrazyl Method The 2,2-diphenyl-1-picrylhydrazyl (DPPH) method described by Fukumoto and Mazza (2000), adapted for microplates (Cardador-Martı´ nez et al., 2002), was

used to determine the AA of unfermented and fermented common bean flours. Reduction of DPPH by an antioxidant or by a radical species results in a loss of absorbance at 540 nm and the degree of discoloration of the solution indicates the scavenging efficiency of the added substance. A 150 mM solution of DPPH was prepared in 80% methanol in order to have a faster reaction rate for butylated hidroxytoluene (BHT) and lower evaporative losses. The procedure consisted of mixing 20 mL of the flour sample extracts (15 mg/mL) or BHT (25, 50, 100 and 1000 mM) with 200 mL of the DPPH solution in a 96-well microtitration flat-bottom plate. Samples and standards were prepared in triplicate. Absorbance readings (540 nm) were recorded from 0 to 90 min at 10 min intervals using a MRX plate reader (Multiskan, Thermo Electron Corporation, Lab-Tech Instrumentation, SA de CV). Plates were kept in the dark at room temperature between readings. A plot of A540 nm versus concentration was made for each time interval. The sample concentration with initial absorbance closest to that of the blank (DDPH þ solvent) was chosen for final calculation of antiradical activity (ARA) by the following equation: ARA ¼ 100 (1 Asample/Acontrol). -Carotene Bleaching Method The AA of unfermented and fermented common bean flour extracts was evaluated according to the b-carotenelinoleate model system (Fukumoto and Mazza, 2000) adapted for microplates (Cardador-Martı´ nez et al., 2002). Aliquots (20 mL) of each extract (15 mg/mL) or BHT (25, 50, 100 and 1000 mM) and 200 mL of the b-carotene solution were added to a well in a 96-well flat-bottom microtitration plate. The sample mixture was diluted by transferring 30 mL to another plate containing air-sparged distilled water (200 mL). Dilutions were done in triplicate since the b-carotene bleaching reaction was subject to noticeable variations. ADIBA (a,a0 azodiisobutyramidine dihydrochloride, 20 mL, 0.3 M), which generates peroxyl radicals from linoleic acid, was added to each well to initiate the reaction. Absorbance readings (540 nm) were recorded from 0 to 90 min at intervals of 10 min using a MRX plate reader (Multiskan, Thermo Electron Corporation, Lab-Tech Instrumentation, SA de CV). Plates were kept in the dark at room temperature between readings. AA was calculated as % inhibition relative to the control using the following relationship: AA ¼ Rcontrol Rsample 100=Rcontrol where Rcontrol and Rsample are the degradation rates of b-carotene in reactant mix without and with sample extract, respectively. The AA values for different times were averaged to give one AA value for each sample.


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Statistical analysis The results were analyzed using one-way analysis of variance (ANOVA), followed by Duncan’s multiple range test comparisons among means with a significance level of 5%.

RESULTS AND DISCUSSION Proximate Composition and Physicochemical Properties of Common Bean Flours Table 1 shows the proximate composition and some physicochemical properties of unfermented and fermented (tempeh) common bean flours. The SSF process increased (p < 0.05) the crude protein content (21.7%) and the TP content (9.13%), and decreased (p < 0.05) the lipid (38.4%), ash (42.7%), carbohydrate (3.5%) and phytic acid (58.3%) contents of raw beans. Previous studies of SSF with legumes have reported a significant increase in total protein content during the soaking, dehulling and cooking steps (Reyes-Moreno et al., 2004), as well as during the fermentation (Paredes-Lo´pez and Harry, 1989). Thus, the increase in protein content reflects the decrease of other constituents, which might have been lost by leaching during the initial steps of SSF or might have been consumed by the fungus for its growth. The lipids contents (Table 1) obtained in this study are similar to those reported by Paredes-Lo´pez and Harry (1989) for tempeh from fresh and hardened common

beans. A substantial reduction in crude lipids was also observed during the early stages of soybean fermentation (Ruiz-Terań and Owens, 1996), consistent with the fact that R. oligosporus produces lipases that release fatty acids, which are oxidized and used by the fungus as a source of energy. On the other hand, the decrease in phytic acid content after fermentation could be explained by the synthesis of phytase by Rhizopus, resulting in phytic acid hydrolysis (Sharma and Khetarpaul, 1997). Fermented flour had similar (p > 0.05) water activity (0.53 0.58), higher (p < 0.05) E (21.59 vs. 11.85), lower (p < 0.05) Hunter ‘L’ value (78.86 vs. 88.34) and pH (6.34 vs. 6.42) than unfermented flour (Table 1). Soaking and cooking significantly increased the Hunter ‘L’ value of the flour, meaning a whiter color, but fermentation resulted in a slightly darker color, probably due to the mycelia and the drying step. Despite tempeh flour had higher E than the unfermented sample, its color looked acceptable, although sensory evaluations were not conducted. Regarding the functional properties, tempeh flour showed higher (p < 0.05) WAI (3.37 vs. 2.34 g gel/g solids) and lower (p < 0.05) WSI (18.87 vs. 26.23 g solids/100 g original solids) than unfermented flour (Table 1); partial protein denaturing and starch gelatinization occurring during the cooking step may be responsible for these differences. The dispersability was higher (p < 0.05) in fermented than unfermented flour (95.65% vs. 43.47%). These results are in agreement with those of AnguloBejarano et al. (2008) who reported an increase in WAI and a decrease in WSI during the production of chickpea tempeh flour by SSF.

Table 1. Proximate composition and physicochemical properties of common bean flours. Common bean flour1 Property Proximate composition (%, db) Crude protein True protein Lipid Ash Carbohydrate Phytic acid (mg/g sample, db) Physicochemical Water activity Color Hunter ‘L’ value Total color difference pH Water absorption index, WAI (g gel/g solids, db) Water solubility index, WSI (g solids/ 100 g original solids, db) Dispersability (%) 1

Unfermented

Fermented

23.06±0.27 b 19.61±0.10 b 1.64±0.08 a 4.47±0.02 a 70.83±0.45 a 6.84±0.13 a

28.06±0.01 a 21.40±0.10 a 1.01±0.04 b 2.56±0.01 b 68.37±0.04 b 2.85±0.07 b

0.53±0.01 b

0.58±0.04 a

88.34±0.25 a 11.85±0.24 b 6.42±0.01 a 2.34±0.01 b

78.86±0.31 b 21.59±0.04 a 6.34±0.01 b 3.37±0.03 a

26.23±0.91 a

18.87±0.32 b

43.47±0.01 b

95.65±0.02a

Means followed by the same letter in the same row are not significantly different (Duncan, p 0.05). Downloaded from fst.sagepub.com at UPC (CSIC) on December 12, 2010

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Table 2. Essential amino acids (EEAs) contents and nutritional properties of common bean flours. Common bean flour Property

Unfermented

Fermented

Recommended1 (25 years)

2.45 b 3.09 b 7.21 a 6.52 a 2.28 a 8.55 b 3.52 b 1.23 a 3.53 b 38.38 b Met þ Cys 91 b 69.25 b 1.62 b

2.62 a 3.33 a 7.18 b 6.31 b 2.51 b 9.39 a 3.78 a 1.14 b 3.69 a 39.97 a 100 a 75.14 a 2.51 a

1.9 2.8 6.6 5.8 2.5 6.3 3.04 1.1 3.5 33.9

Essential amino acid (g/100 g protein) His Ile Leu Lys Met þ Cys Phe þ Tyr Thr Trp Val Total Limiting EAAs Essential amino acid score In vitro protein digestibility (%) Calculated protein efficiency ratio

1 Recommended essential amino acid requirements (FAO/WHO, 1991). Means followed by the same letter in the same row are not significantly different (Duncan, p 0.05).

Nutritional Properties of Common Bean Flours The EAA content of the common bean flour samples are shown in Table 2. Unfermented and tempeh flours contained 38.38 and 39.87 g EAA/100 g protein, respectively; these values are higher than those recommended by the FAO/WHO for children of age 25 years (33.9 g EAA/100 g of protein). When compared with the FAO/ WHO (1991) reference standards, proteins from unfermented common bean flour showed higher (p < 0.05) values of EAA for His, Ile, Leu, Lys, total aromatic (Phe þ Tyr), Trp, and Val; however, they had lower (p < 0.05) levels of total sulfur (Met þ Cys), the limiting EAA in legumes. In general, the EAA content of proteins from unfermented common bean flour was improved by the SSF process; the content of His, Ile, total sulfur (Met þ Cys), total aromatic (Phe þ Tyr), Thr, and Val increased (p < 0.05) in 0.17, 0.24, 0.23, 0.84, 0.26 and 0.16 g/100 g protein, respectively. However, Leu, Lys and Trp levels decreased (p < 0.05) 0.03, 0.21 and 0.09 g/100 g protein, respectively, although their final contents were still higher than those of the reference standards. It appears that the fungus does not depend upon a specific amino acid for growth and its effect on the amino acid composition depends on the substrate. Common bean proteins contain relatively high levels of Lys, but this amino acid decreased significantly during SSF. Paredes-Lo´pez and Harry (1988) reported that Lys and Met were released in higher amounts during fermentation; they suggested that the conversion of amino acids by the action of transaminases produced by the fungus could, in part, account for these changes. The EAA scores of proteins from unfermented and fermented (tempeh) common beans flours were evaluated taking into

account the suggested pattern of amino acid requirements for children 25 years old (FAO/WHO, 1991; Table 2). Total sulfur (Met þ Cys) was the limiting EAA in proteins from unfermented flour with an EAA score of 91; fermentation improved the EAA balance, resulting in a score of 100. The SSF process improved the IVPD of the bean flour from 69.25% to 75.14% (Table 2); this improvement in IVPD has been also observed using other substrates such as chickpea (Angulo-Bejarano et al., 2008). The increase in protein digestibility could be explained by the elimination of antinutritional factors (e.g. hydrolysis of phytic acid during fermentation) and denaturing of proteins during the cooking step, making them more susceptible to enzymatic hydrolysis. The C-PER of the beans was also increased by SSF in about 55% (Table 2), which reflects the increased digestibility and EAA content of the proteins. Total Phenolics Content The total phenolics contents of the common bean flour samples are shown in Table 3. Unfermented flour had 2.83 mg of catechin equivalents/g of sample, a value lower that those reported by Oomah et al. (2005) for different common bean cultivars with colored testa (3.316.6 mg/g). These differences can be attributed mainly to the cultivar (testa color) and the quantification method used. Interestingly, SSF increased the phenolics content of the beans to 6.09 mg/g (Table 3), a value that falls in the range observed for colored beans (Oomah et al., 2005). The effect of SSF on the phenolics content is consisted with the results reported by other researchers (Randhir et al., 2004; Lin et al., 2006); they suggested that fungal b-glucosidase catalyze the release


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Table 3. Phenolics contents and antioxidant activity of common bean flours. Antioxidant activity1

Common bean flour Unfermented Fermented BHT5 BHT BHT BHT

Phenolics content1 (mg catechin equivalent/g sample) 2.83±0.02 b 6.09±0.07 a

Concentration2 (mM)

Antiradical activity3 (%)

Antioxidant activity4 (%)

14.62 31.46 25 50 100 1000

20.09±2.33 c 42.99±1.34 b 3.20±0.23 f 6.02±0.71 e 12.60±1.13 d 79.22±0.26 a

19.85±0.95 e 37.83±1.29 c 14.59±2.46 f 24.21±2.09 d 49.38±1.48 b 68.08±0.70 a

1

Means followed by the same letter in the same column are not significantly different (Duncan, p 0.05). Concentration expressed as mM equivalent of (þ) catechin, obtained from the same concentration of unfermented and fermented common bean flour extracts (15 mg/mL). 3 DPPH method. 4 b-Carotene-linoleate method. 5 BHT ¼ butylated hidroxytoluene. 2

of aglycones from the bean substrate and consequently there is an increase in phenolics content. Antioxidant Activity The DPPH method was used to evaluate AA in unfermented and fermented common bean flour extracts. This assay provides stoichiometric information with respect to the number of electrons taken up by the tested compounds in the presence of the stable free radical. The ARA of unfermented bean flour (20.09%) was almost twice that obtained for the BHT standard at 100 mM. This result is in good agreement with those reported previously (Oomah et al., 2005; Xu et al., 2007). SSF increased (p < 0.05) the scavenging activity of the bean flour more than two times (ARA ¼ 42.99%, Table 3), which is consistent with the increase in the phenolics content. The b-carotene bleaching method was also used to evaluate AA in unfermented and fermented common bean flour extracts. This assay quantifies the ability of an antioxidant to function at a lipid/water interface. The AA value of the unfermented flour extract (19.85%) was between those obtained for the BHT standard at 25 and 50 mM, while the AA of the fermented flour extract was significantly higher (37.83%, Table 3); this value is close to those obtained for dark colored bean cultivars (Oomah et al., 2005). The above results demonstrate a positive effect of SSF on the AA of common bean. This is very relevant because it represents an alternative to provide added value to light colored common beans such as the azufrado used in this study, which is highly produced and consumed in the northwest region of Mexico.

a strong linear correlation with ARA and AA (r ¼ 0.995 and 0.998, respectively, p < 0.01), providing a good estimate of AA in common bean and therefore can be used as a good predictor of this property.

CONCLUSIONS This study showed that SSF could be used to improve the physicochemical, nutritional and antioxidant characteristics of common bean. Likewise, the correlation analyses showed that the increase in AA during the SSF of common bean was related to the increase in phenolics content. Thus, common bean tempeh flour may be considered for the fortification of widely consumed legume-based food products (bread, cookies and atoles), as well as in the prevention of pathologies in which free radicals play a key role. More research is needed to characterize the chemical composition and structures that contribute to the AA of common bean tempeh flour.

ACKNOWLEDGMENTS This research was supported by grants from Consejo Estatal de Ciencia y Tecnologı´ a (CECyT) - Sinaloa and Programa de Fomento y Apoyo a Proyectos de Investigacioń (PROFAPI) Universidad Autońoma de Sinaloa.

Correlation Between Phenolics Content and AA

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Phenolics content, expressed as milligrams equivalent of (þ)-catechin per gram of sample, showed

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