Protein Quality and in Vivo Digestibility of Different Varieties of Bean ...

Protein Quality and in Vivo Digestibility
of Different Varieties of Bean (Phaseolus
vulgaris L.)
AUTORES
AUTHORS
Geralda Aldina Dias Rodrigues CRUZ
PhD student, Department of Biochemistry and Molecular
Biology - E-mail: gal@tdnet.com.br
Maria Goreti de Almeida OLIVEIRA
Professor, Department of Biochemistry and Molecular
Biology - E-mail: malmeida@mail.ufv.br
Universidade Federal de Viçosa.
CEP: 36571-000, Viçosa, MG, Brazil
Christiano Vieira PIRES
MsC student, Department of Biochemistry and Molecular
Biology - E-mail: ms33771@correio.ufv.br
Maria Regina de Araújo GOMES
Temporary professor, Department of Biochemistry and
Molecular Biology - E-mail: td41753@correio.ufv.br
SUMMARY
Qualidade da Proteína e Digestibilidade
in vitro de Diferentes Variedades de
Feijão (Phaseolus vulgaris L.)
The determination of the protein digestibility of a food is an important factor in
estimating its quality. One of the main limitations of beans is the low nutritional value of
their proteins due to low digestibility and a reduced content and bioavailability of sulphur
amino acids. This work evaluated the protein quality and digestibility of different bean cultivars
using a biological assay. The digestibility, Protein Efficiency Ratio (PER), Net Protein Ratio
(NPR) and Net Protein Utilization (NPU) were determined for the “A 774”, “Carioca”, “Diamante
Negro”, “Ouro Branco”, “RAO 33” and “Vermelho Coimbra” cultivars. The results obtained
showed that the values for digestibility, PER, NPR and NPU were higher than those quoted
in the literature, and that the “Ouro Branco” cultivar showed the highest nutritional
performance, compared to the other varieties analysed. The most coloured varieties, such
as “Vermelho Coimbra” and “Diamante Negro” showed lower values, indicating that the
pigments and/or the compounds related to them were responsible for reducing the protein
quality of the beans.
Neuza Maria Brunoro COSTA
Professor, Department of Nutrition and Health
E-mail: nmbc@mail.ufv.br
Maria Helena Namsser BRUMANO
Post-doctor da BIOAGRO
Maurílio Alves MOREIRA
Professor, Department of Biochemistry and Molecular
Biology - E-mail:moreira@mail.ufv.br
RESUMO
A determinação da digestibilidade protéica de um alimento é um importante fator
para estimar a sua qualidade. Um dos maiores problemas do feijão é representado pelo
baixo valor nutricional de suas proteínas, decorrente, por um lado, da sua baixa digestibilidade
e, de outro, dos reduzidos teor e biodisponibilidade de aminoácidos sulfurados. Este trabalho
visou avaliar, através de ensaio biológico, a qualidade protéica e digestibilidade de diferentes
cultivares, melhorados geneticamente. Foram feitas análises de digestibilidade, PER
(Coeficiente de Eficácia Protéica), NPR (Razão Protéica Líquida) e NPU (Utilização Líquida da
Proteína) para os cultivares “A 774”, “Carioca”, “Diamante Negro”, “Ouro Branco”, “RAO
33” e “Vermelho Coimbra”. Os resultados obtidos mostraram que os valores encontrados
para digestibilidade, PER, NPR e NPU foram mais altos que os valores encontrados na literatura
e que o cultivar “Ouro Branco” apresentou a melhor qualidade nutricional dentre as cultivares
analisadas, e as variedades de maior coloração como “Vermelho Coimbra” e “Diamante
Negro” tiveram a sua qualidade protéica reduzida.
PALAVRAS-CHAVE
KEY WORDS
Protein quality; Digestibility; Beans.
Qualidade protéica; Digestibilidade; Feijão.
Braz. J. Food Technol., v.6, n.2, p. 157-162, jul./dez., 2003 157 Recebido / Received: 24/04/2002. Aprovado / Approved: 22/01/2003.

CRUZ, G.A.D.R. et al.
Protein Quality and in Vivo Digestibility
of Different Varieties of Bean (Phaseolus
vulgaris L.)
1. INTRODUCTION
The legume seeds are widely consumed around the
world and the acceptability depends on the climatic conditions
in the area. In Latin America, beans (Phaseolus vulgaris) are the
most consumed legume, supplying considerable amounts of
nutrients to the diet of undernourished people (COELHO, 1991).
BRESSANI et al. (1988) observed that besides increasing the
protein content of the meal, beans have contributed to
improving the protein quality of the diet by a factor of about 50
to 70%, when constituted predominantly of legumes and cereals,
because bean proteins are rich in lysine, complementing proteins
from cereals such as rice or corn, well known for being deficient
in this amino acid. This fact means beans are of exceptional
nutritional importance, especially when the restricted ingestion
of food of animal origin among the low income population is
considered, to whom the legume is a daily food.
Rice and beans are the staple foods of the Brazilian diet
and mixed in adequate proportion, they may improve the
biological value of the proteins consumed (VIEIRA, 1992).
The genus Phaseolus includes all species known as
beans. The species Phaseolus vulgaris L. is the most common
and includes many varieties such as “Carioca”, “Roxo” (Purple),
“Mulatinho” (Brown), “Preto” (Black) and others (KANAMORI
et al., 1982).
Although representing an important source of proteins,
starch, vitamins, minerals and fiber (COELHO, 1991; GEIL;
ANDERSON, 1994; BARAMPANA; SIMARD, 1994; MARTINEZ
et al., 1998) bean nutrients are not readily available for use by
the organism (EVANS; BAUER, 1978; SARWAR; PEACE, 1986;
RODRIGUES, 1995; CARBONARO et al., 1997).
Digestibility is the first factor affecting the efficiency of
diet proteins. When certain peptide links are not hydrolysed in
the digestive process, part of the protein is either excreted in
the faeces, or transformed into a metabolic product by
microorganisms present in the large intestine (SGARBIERI;
WHITAKER, 1982). One of the major limitations of beans is the
low nutritional value of their proteins, due to their poor
digestibility and reduced content of sulphur amino acids
(NIELSON, 1991; BRESSANI, 1993; GENOVESE; LAJOLO, 1996,
1998). Besides reduced sulphur amino acid contents, they also
show reduced bioavailability (SGARBIERI; WHITAKER, 1982).
The low biological usage of bean proteins is attributed
to many factors such as: compact structure of the native
proteins which may resist proteolysis; anti-nutritional
compounds which can modify the digestibility, altering the
liberation of amino acids and high excretion of endogenous
nitrogen (WU; KUNKEL, 1995).
In general, the digestibility of bean proteins can be
improved by thermal treatment (WU et al., 1974). However,
studies indicate that the digestibility is limited, even after
thermal treatment, due to the primary conformation of the
proteins and/or to the presence of some components of the
seed, such as thermo-stable protease inhibitors and
polyphenols which complex with digestive enzymes and/or
with bean proteins, reducing their susceptibility to hydrolysis
(RODRIGUES; LAJOLO, 1993).
An improvement of digestibility does not necessary
imply an increase in protein quality, such that the amino acids
can be better absorbed, but not take part in protein synthesis,
due to the deficiency of some essential amino acids. The
nutritional value of a food is indicated by its chemical
composition, although this does not characterize the food from
the nutritional point of view. Food nutrients are available to
the organism in their totality, only after their ingestion
(SGARBIERI, 1989). Hence, biological analyses such as PER, NPR
and NPU, which evaluate the protein quality based on the
organism’s response to the protein ingestion, are important
tools for better food characterization.
Several varieties of bean with great agricultural potential
have been obtained from genetic breeding studies. Among
these varieties are some that stand out due to the presence of
more favourable characteristics, and thus deserve more attention
for nutritional studies. Thus, the purpose of the present study
was to evaluate the protein quality of the cultivars, A774, Carioca,
Diamante Negro, Ouro Branco, RAO 33 and Vermelho Coimbra.
These cultivars were kindly supplied by the Centro Nacional de
Pesquisa de Arroz e Feijão da Empresa Brasileira de Pesquisa
Agropecuária (EMBRAPA – CNPAF).
2. MATERIALS AND METHODS
2.1 Genetic material
The bean samples (Phaseolus vulgaris L.) of the varieties
“A774” (brown group), “Carioca” (Carioca group), “Diamante
Negro” (black group), “Ouro Branco” (white group), “RAO”
33 (purple group) and “Vermelho Coimbra” (red group) were
obtained from the Empresa Brasileira de Pesquisa Agropecuária
(EMBRAPA/CNPAF), Goiânia, GO, Brazil.
2.2 Preparation of bean flour
For the preparation of whole cooked flour, the grains
were cleaned and cooked in water in the proportion of 1:1.5
(w/v) in a pressure cooker for forty minutes. The grains were
then dried in an air circulating oven (24 h / 60 o C) and ground
in a mill.
2.3 Preparation of the diets
The composition of the experimental diets is shown in
Table 1. Diets 1 to 6 were prepared by using bean samples as
protein sources. Diet 7 (casein) was the standard and diet 8
was the protein free diet.
The nitrogen content of the mixture was determined
by the semi-micro Kjeldahl method using the factor 6.25 to
obtain the protein content (AOAC, 1984).
Braz. J. Food Technol., v.6, n.2, p.157-162, jul./dez., 2003 158

CRUZ, G.A.D.R. et al.
Protein Quality and in Vivo Digestibility
of Different Varieties of Bean (Phaseolus
vulgaris L.)
TABLE 1. Composition of the diets used in the experiments with rats (g/100g of the mixture).
Ingredients
D1
D2
D3
D4
D5
D6
D7
D8
1
Whole
and cooked Ouro Branco flour
50.82
- - - - - - -
1
Whole
and cooked Carioca flour
- 55.49
- - - - - -
1
Whole
and cooked RAO 33 flour
- - 52.14
- - - - -
Whole
and cooked A 774 flour1 - - - 46.00
- - - -
1
Whole
and cooked Verm. Coimbra flour
- - - - 43.49
- - -
1
Whole
and cooked Diamante Negro flour - - - - - 42.29
- -
Casein2 - - - - - - 12.30
-
2,
*
M . 5
ineral mix
3 3.
5 3.
5 3.
5 3.
5 3.
5 3.
5 3. 5
Vitamin
mix2 1.
0 1.
0 1.
0 1.
0 1.
0 1.
0 1.
0 1. 0
3
Soybean
oil
7.
0 7.
0 7.
0 7.
0 7.
0 7.
0 7.
0 7. 0
2
Choline
bitartrate
0.25
0.25
0.25
0.25
0.25
0.25
0.25
0.25
Cornstarch
(q.s.p)
3
8.93
4.26
7.61
13.75
16.25
17.46
42.45
54.75
L-cystine
2
0.
3 0.
3 0.
3 0.
3 0.
3 0.
3 0.
3 0. 3
2
Fibre
- - - - - - 5.
0 5. 0
Dextrinised
cornstarch2 13.
2 13.
2 13.
2 13.
2 13.
2 13.
2 13.
2 13. 2
Sucrose3 15.
0 15.
0 15.
0 15.
0 15.
0 15.
0 15.
0 15. 0
Proteins
(%)
9.96
9.68
9.66
9.33
9.99
9.54
9.25
-
1 Flour produced from the bean samples analysed 2 Obtained from RHOSTER- Industry and commercial Ltda
3 According to REEVES et al. (1993) 4 Obtained from a local supermarket in Viçosa
2.4 Biological assay
The evaluation of the protein quality of the experimental
diets was performed by a biological assay. 48 male recently
weaned Wistar rats, weighing 50 to 60g, from the Department
of Nutrition and Health, Universidade Federal de Viçosa, Brazil,
were used. The animals were divided into eight groups with
six animals each, so that the weight difference between the
groups did not exceed 5g. The rats were placed in individual
cages and received water and their experimental diets ad
libitum for 14 days (AOAC, 1975). The animals were maintained
at 22 ± 3 o C and the consumption of food was monitored
weekly.
The true digestibility (AOAC, 1984) was determined by
measuring the amount of nitrogen ingested in the diet, the
amount eliminated in the faeces, and the metabolic loss in the
faeces, which corresponds to the faecal nitrogen in the protein
free group.
For the digestibility determination, the diets were
labelled with indigocarmin in the proportion of 100mg/100g,
and given to the animals on the 7th and 13th days. The faeces
were collected from the 8th to the 13th days in individual
containers for each animal and maintained under refrigeration.
At the end of the experiment, the faeces were dried in
an air circulating oven at 105 o C for 24h, cooled, weighed and
ground in a food processor for the determination of nitrogen
concentration by the semi-micro Kjeldahl method (AOAC,
1984). The samples were analysed in triplicate (AOAC, 1984).
The Protein Efficiency Ratio (PER) was determined from
the ratio of weight gain of the animals and the consumption
of protein (AOAC, 1975).
The Net Protein Ratio (NPR) was determined by dividing
the weight gain of the test group plus the weight loss of the
protein free group by the consumption of protein of the test
group (BENDER; DOELL, 1957).
The Net Protein Utilization (NPU) was determined by
the nitrogen retention of the carcass of the animals. For the
calculation of NPU, the animals were sacrificed by inhaling CO 2
at the end of the experiment. The carcasses were dried in an
air circulating oven at 105 o C for 24h, cooled and weighed.
Fat was extracted in a Soxhlet apparatus for 4h using petroleum
ether as solvent. The nitrogen content of the carcasses was
determined by the semi-micro Kjeldahl method. The samples
were analysed in triplicate (AOAC, 1984).
The NPU was calculated according to the formula
(HEGSTED, 1977):
Nt Na 100
NPU
Ni
Nt = body nitrogen of test group
Na = body nitrogen of the free protein group
Ni = nitrogen intake of the test group
The results obtained were submitted to an analysis of
variance and the mean values compared by Tukey’s test at 5%
of probability.
Braz. J. Food Technol., v.6, n.2, p. 157-162, jul./dez., 2003 159

CRUZ, G.A.D.R. et al.
Protein Quality and in Vivo Digestibility
of Different Varieties of Bean (Phaseolus
vulgaris L.)
3.1 Digestibility
3. RESULTS AND DISCUSSION
The results for true digestibility are summarised in
Table 2.
It was observed that the true digestibility obtained from
the varieties analysed ranged from 85.66% for “RAO 33” to
93.97% for “Ouro Branco”.
Polyphenols, one of the pigments responsible for the
different colours of legumes, have been studied with the
purpose of evaluating their significance according to the
nutritional as well as the technological points of view (ELIAS,
1982). Their ability to form complexes and to precipitate
proteins makes them important from the nutritional point of
view. JAFFÉ (1950), quoted by SGARBIERI, WHITAKER (1982),
reported values of 76.8; 79.5 & 84.1% for in vivo digestibility
of proteins from black, pink and white beans (Phaseolus
vulgaris), respectively.
JAFFÉ; FLORES (1975) found in vivo and in vitro
digestibility values of 68.9 and 70.1%, respectively, for cooked
black beans.
Comparing the results obtained for the varieties
analysed with those of casein (Table 2), it was observed that
casein digestibility was significantly higher than that of the
varieties studied, except for the “Ouro Branco” cultivar, which
was not statistically different from casein. HUGHES et al. (1996)
found 80.9% digestibility for white beans, which was much
lower than the value obtained for the “Ouro Branco” variety.
TABLE 2. True digestibility (TD) of the casein diet and of the
whole cooked bean varieties in the experiment with rats.
Diet
G roup
DV (%)*
Casein
(D7)
- 9.00
Ouro
Branco (D1)
Diamante
Negro (D6)
Vermelho
Coimbra (D5)
A 774 (D4)
Carioca
(D2)
RAO 33 (D3)
9
a
White
3.97
9
a. b
Black
1.50
9
b. c
Red
0.44
9
b.c. d
Brown
8.67
8
b.c. d
Carioca
8.05
8
c. d
Purple
5.66
8
d
* Means within the same column followed by the same letter, are not
statistically different, according to Tukey test at 5% of probability.
BRESSANI (1989), in a review, showed that cooked
white beans were more digestible than the coloured cooked
beans, similar to the results found in the present study.
In general, the values for true digestibility observed here
were higher than those found in the literature for other varieties
and these results can be attributed to many factors such as :
low concentration of anti-nutritional compounds present in
the grain (NIELSON, 1991), different varieties analysed, sowing
conditions, storage time and heat treatments, which may have
produced, physicochemical changes in the proteins of the
legume seed, affecting their final nutritional properties
(ALONSO et al., 2000). These factors may have contributed to
increase the digestibility of these cultivars.
The improvement of digestibility is not necessarily
related to an increase in protein quality, particularly when the
protein is deicient in essential amino acids. For this reason,
biological assays such as PER, NPR and NPU were performed,
in order to evaluate the protein quality, based on the organism’s
response to the protein intake.
3.2 PER, NPR and NPU
The results for PER and RPER (relative PER); NPR and
RNPR (relative NPR); NPU and RNPU (relative NPU) of the
experiment are summarised in Table 3.
The values for PER and RPER ranged from 1.2 to 2.36
and from 30.92% to 60.82%, respectively, and were statistically
different (P < 0.05) from the values obtained for casein.
Among the bean samples analysed, the “Ouro Branco”
variety showed the highest nutritional value based on its PER
and RPER values, which were significantly higher than the other
varieties. The “Diamante Negro” showed the lowest PER and
RPER values. These results are similar to those obtained by
BRESSANI; ELIAS (1984), where white beans showed better
nutritional performance than purple, black or brown beans.
In the present work, significant differences in RPER were
observed among beans and between the bean varieties and
casein diets.
TABLE 3. PER, RPER (relative PER), NPR, RNPR (relative NPR),
NPU, RNPU (relative NPU) of casein and whole cooked bean
diets.
DIET
G ROUP
P ER*
RPER
(%)
N PR
RNPR(%
)
N PU
RNPU%
Casein
(D7) - 3.88a 100a 4.56a 100a 66.73a 100a
Ouro Branco
(D1)
Carioca
(D2)
A 774 (D4)
RAO 33 (D3)
White
2.36b 60.82b 3.49b 76.53b 52.11a . b 78.09a.
b
b.
c
b.
c
b b a.
b a. b
Carioca
2.18
56.18
3.49
76.53
54.27
81.32
Brown
1.56b .c.d. e 40.20b .c.d. e 2.89b .c. d 63.37b .c. d 47.66b 71.42b
c.d.
e
c.d.
e
c.
d
c.
d
b b
Purple
1.44
37.11
2.76
60.52
46.38
69.50
Vermelho
Coimbra (D5)
Red
1.32d . e 34.00d . e 2.57d 56.35d 42.61b 63.85b
Diamante
Negro (D6)
Black
1.
2 e 30.92
d 2.53
d 55.48
b 49.61
b 74.34
Means within the same column followed by the same letter, are not
statistically different, according to Tukey’s test at 5% of probability.
PER and RPER were determined on the 14o day of the experiment.
The NPR and RNPR of the bean varieties were
significantly lower than for casein and ranged from 2.53 to
3.49 and 55.48% to 76.53%, respectively (Table 3), indicating
that statistically significant differences were detected only for
casein. This is explained by the fact that PER does not consider
the use of the protein for maintenance, only for growth
promotion, tending to underestimate the quality of inferior
proteins and, for this reason, enhances existing differences
Braz. J. Food Technol., v.6, n.2, p.157-162, jul./dez., 2003 160

CRUZ, G.A.D.R. et al.
Protein Quality and in Vivo Digestibility
of Different Varieties of Bean (Phaseolus
vulgaris L.)
among proteins of higher and smaller nutritional values,
contrary to the NPR value (SGARBIERI, 1989).
The “Ouro Branco”, “Carioca” and “A 774” varieties
showed the highest values for NPR. Although the “Diamante
Negro” variety showed the lowest value for NPR in the present
study, this value was higher than those observed by other
authors working with black beans (BRESSANI et al.,1981;
DURIGAN et al., 1987; CHIARADIA 1997).
The higher values for NPR and RNPR compared to those
found in the literature may be due to a decrease in antinutritional
factors during the heat treatment process or to
differences amongst varieties. Several studies confirm that heat
treatment can be efficient in reducing trypsin inhibitor activity
(KAKADE; EVANS, 1966; ANTUNES; SGARBIERI, 1980; MENDEZ
et al., 1993), although it may not be responsible for the reduced
protein digestibility in cooked beans.
The results for NPU and RNPU ranged from 49.61 to
52.11 and from 74.34% to 78.09%, respectively. The “Carioca”
and “Ouro Branco” varieties were statistically equal to casein.
The “Ouro Branco” variety showed the highest protein
quality, based on its digestibility, PER, NPR and NPU values.
No significant difference was observed between the NPU of
this variety and the casein diet. The most coloured varieties,
such as “Vermelho Coimbra” and “Diamante Negro” showed
lower values, indicating that the pigments and/or the
compounds related to them, were responsible for reducing
the protein quality of the beans.
ACKNOWLEGMENT
The authors thank FAPEMIG for the financial support.
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