Physicochemical, Nutritional and Antioxidant Properties of

Tempeh Flour from Common Bean (Phaseolus vulgaris L.)

M. Reyes-Bastidas,1 E.Z. Reyes-Fernández,1 J. López-Cervantes,2

J. Milán-Carrillo,1,3 G.F. Loarca-Piña4 and C. Reyes-Moreno1,3,*
1
Facultad de Ciencias Quı´mico Biológicas, Universidad Autónoma de Sinaloa, Ciudad Universitaria,
AP 354, CP 80000 Culiacán, Sinaloa, Mexico
2
Departamento de Biotecnologia y Ciencias Alimentarias, Instituto Tecnológico de Sonora, CP 85000,
Cd. Obregon, Sonora, Mexico
3
Universidad Autónoma de Sinaloa, Ciudad Universitaria, AP 354, CP 80000 Culiacán, Sinaloa,
Mexico
4
Facultad de Quı´mica, Universidad Autónoma 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, respec-
tively. 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 preven-
tion of pathologies associated with oxidative stress.

Key Words: common bean, solid state fermentation, nutritional properties, antioxidant activity

INTRODUCTION et al., 2006). These physiological effects of common

Common bean (Phaseolus vulgaris L.) plays an impor-
tant role in the diet of Latin-American people, providing
20—40% protein, essential fatty acids, complex carbohy-
drates, vitamins and minerals (Reyes-Moreno and
Paredes-López, 1993). However, its wider use is
somehow limited by the presence of antinutritional
factors (e.g., enzyme inhibitors, lectins, phytates, cyano-
glycosides 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-Fernández
*To whom correspondence should be sent
(e-mail: creyes@uas.uasnet.mx).
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
bean may be due to the presence of abundant
phytochemicals including polyphenolics, which possess
both anticarcinogenic and antioxidant properties
(Aparicio-Fernández et al., 2006).
Solid state fermentation (SSF) represents a technolog-
ical alternative for processing a great variety of legumes
and/or cereals to improve their nutritional and nutra-
ceutical 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; Cuevas-
Rodrı́guez 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 hydro-
lyze 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
427
428 M. REYES-BASTIDAS ET AL.
improved (Hachmeister and Fung, 1993). The potential
of using SSF to improve the nutritional value of cereals
and legumes has been evaluated (Cuevas-Rodrı́guez
et al., 2006; Angulo-Bejarano et al., 2008).
Fermentation of soybean causes increased antioxidant
activity (AA), which is associated with increased gluco-
sidase 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 exhib-
ited marked AA (György et al., 1964). More recent
studies have documented that not only Rhizopus gener-
ates 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 degener-
ative diseases.
The objective of this investigation was to evaluate the
effect of SSF on physicochemical, nutritional and anti-
oxidant 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, México. 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. oligos-
porus 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
200—400 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 centrifuga-
tion 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 chloride-
saturated 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.
Dispersability
It was determined according to Mora-Escobedo et al.
(1994). One gram of flour sample was added into a grad-
uated 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 multi-
plied by 10 to give the percentage dispersability.
Amino Acid Analysis
Total amino acid composition was determined using
the method described by López-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 con-
nected to an HPLC system (GBC, Dandenong,
429
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 López-Cervantes et al. (2006).
Fluorescence detection was at 270 and 316 nm for exci-
tation 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 qual-
ity based on the amino acid composition. It was calcu-
lated 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
(2—5 years; FAO/WHO, 1991).
CS ¼ ðContent of the most limiting EAA=REAARÞ  100
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) trichloro-
acetic 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 nitro-
gennon-protein nitrogen)  6.25.
430 M. REYES-BASTIDAS ET AL.
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 chymo-
trypsin 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 follow-
ing 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 vanillin—hydrochloric 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 concentra-
tions 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-Martı́nez 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 absor-
bance 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-carotene-
linoleate model system (Fukumoto and Mazza, 2000)
adapted for microplates (Cardador-Martı́nez 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.
 Properties of Tempeh Flour 431

Statistical analysis beans. A substantial reduction in crude lipids was also

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%), carbohy-
drate (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-López 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-López and Harry
(1989) for tempeh from fresh and hardened common
observed during the early stages of soybean fermenta-
tion (Ruiz-Terán 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 unfermen-
ted sample, its color looked acceptable, although sen-
sory 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 differ-
ences. The dispersability was higher (p < 0.05) in fer-
mented than unfermented flour (95.65% vs. 43.47%).
These results are in agreement with those of Angulo-
Bejarano 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 Unfermented Fermented

Proximate composition (%, db)

Crude protein 23.06±0.27 b 28.06±0.01 a
True protein 19.61±0.10 b 21.40±0.10 a
Lipid 1.64±0.08 a 1.01±0.04 b
Ash 4.47±0.02 a 2.56±0.01 b
Carbohydrate 70.83±0.45 a 68.37±0.04 b
Phytic acid (mg/g sample, db) 6.84±0.13 a 2.85±0.07 b
Physicochemical
Water activity 0.53±0.01 b 0.58±0.04 a
Color
Hunter ‘L’ value 88.34±0.25 a 78.86±0.31 b
Total color difference 11.85±0.24 b 21.59±0.04 a
pH 6.42±0.01 a 6.34±0.01 b
Water absorption index, WAI (g gel/g 2.34±0.01 b 3.37±0.03 a
solids, db)
Water solubility index, WSI (g solids/ 26.23±0.91 a 18.87±0.32 b
100 g original solids, db)
Dispersability (%) 43.47±0.01 b 95.65±0.02a
1
Means followed by the same letter in the same row are not significantly different (Duncan, p  0.05).
432 M. REYES-BASTIDAS ET AL.

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

Property Unfermented Fermented Recommended1 (2—5 years)

Essential amino acid (g/100 g protein)

His 2.45 b 2.62 a 1.9
Ile 3.09 b 3.33 a 2.8
Leu 7.21 a 7.18 b 6.6
Lys 6.52 a 6.31 b 5.8
Met þ Cys 2.28 a 2.51 b 2.5
Phe þ Tyr 8.55 b 9.39 a 6.3
Thr 3.52 b 3.78 a 3.04
Trp 1.23 a 1.14 b 1.1
Val 3.53 b 3.69 a 3.5
Total 38.38 b 39.97 a 33.9
Limiting EAAs Met þ Cys — —
Essential amino acid score 91 b 100 a —
In vitro protein digestibility (%) 69.25 b 75.14 a —
Calculated protein efficiency ratio 1.62 b 2.51 a —
1
Recommended essential amino acid requirements (FAO/WHO, 1991). Means followed by the same letter in the same row are not signif-
icantly 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, respec-
tively; these values are higher than those recommended
by the FAO/WHO for children of age 2—5 years (33.9 g
EAA/100 g of protein). When compared with the FAO/
WHO (1991) reference standards, proteins from unfer-
mented 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-López
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 require-
ments for children 2—5 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
(3.3—16.6 mg/g). These differences can be attributed
mainly to the cultivar (testa color) and the quantifica-
tion 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
 Properties of Tempeh Flour 433

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

Antioxidant activity1

Phenolics content1 (mg catechin Antiradical Antioxidant

Common bean flour equivalent/g sample) Concentration2 (mM) activity3 (%) activity4 (%)

Unfermented 2.83±0.02 b 14.62 20.09±2.33 c 19.85±0.95 e

Fermented 6.09±0.07 a 31.46 42.99±1.34 b 37.83±1.29 c
BHT5 25 3.20±0.23 f 14.59±2.46 f
BHT 50 6.02±0.71 e 24.21±2.09 d
BHT 100 12.60±1.13 d 49.38±1.48 b
BHT 1000 79.22±0.26 a 68.08±0.70 a
1
Means followed by the same letter in the same column are not significantly different (Duncan, p  0.05).
2
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.

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 unfer-
mented 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), provid-
ing 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 correla-
tion 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 Tecnologı́a (CECyT) - Sinaloa and
Programa de Fomento y Apoyo a Proyectos de
Investigación (PROFAPI) — Universidad Autónoma
de Sinaloa.

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