Hayat et al., The Journal of Animal & Plant Sciences, 24(3): 2014, Page:J.

860-868
Anim. Plant Sci. 24(3):2014
ISSN: 1018-7081

EXPLORING THE POTENTIAL OF RED KIDNEY BEANS (PHASEOLUS VULGARIS L.)

TO DEVELOP PROTEIN BASED PRODUCT FOR FOOD APPLICATIONS
I. Hayat, A. Ahmad*, A. Ahmed*, S. Khalil** and M. Gulfraz***

Department of Food Technology, The University of Poonch Rawalakot, Azad Kashmir, Pakistan
*
Department of Food Technology, PMAS-Arid Agriculture University Rawalpindi, Pakistan
**
Grain Quality Testing Laboratory, National Agriculture Research Center Islamabad, Pakistan
***
Institute of Biochemistry & Biotechnology, PMAS-Arid Agriculture University Rawalpindi, Pakistan
Corresponding author E-mail: [email protected]

ABSTRACT
Protein isolate was prepared from red kidney beans and its functional properties were evaluated at different pH levels to
access its suitability for food applications. Carbohydrates, crude protein, crude fiber, crude fat and ash contents of red
kidney bean seeds were found to be 53.02±1.14%, 25.78±0.77%, 6.82±0.31%, 1.92±0.15% and 4.34±0.20%,
respectively. Magnesium, calcium, sodium, potassium and iron were observed as macro elements in red kidney bean
seeds. Protein solubility, emulsification, gelation as well as foaming properties of the bean protein isolate were
significantly (P≤0.05) affected by different pH levels. The solubility, emulsifying activity and stability as well as foam
capacity of the protein isolate were dependant with minimal values observed at pH 4 while maximum at pH 10.
Contrarily, the stability of foam was highest at pH 4 while a decreasing trend in foam stability was observed with
increase of pH. Gelation properties improved at acidic pH with maximum gelation capacity observed at pH 4 while these
properties decreased at alkaline conditions. Conclusively, red kidney beans can be utilized to prepare protein isolate
whose functional properties can be modified by changing the pH of the environment for better utilization in the food
formulation systems.
Key words: Kidney bean, protein isolate, protein solubility, emulsification, gelation, foaming.

INTRODUCTION lysine which is undersupplied by the cereals. Therefore,

the combined consumption of beans and cereals can
Grain legumes are an important source of ensure a balanced protein diet due to the nutritional
nutrients such as proteins, carbohydrates as well as complementation of essential amino acids. (Tang et al.,
certain minerals and vitamins (Boye et al., 2010). 2009). Due to this nutritional complementation, red
Legumes are renowned as poor man’s meat due to higher kidney beans can be utilized as an economical source of
protein content and have the potential to combat the dietary proteins, especially for the people of low income
problems of protein energy malnutrition, especially in group in the developing countries (Yin et al., 2010).
developing countries (Qayyum et al., 2012). Red kidney However, red kidney beans are also reported to contain
bean is also an important legume crop which is different antinutritional factors such as trypsin inhibitor,
extensively produced in Asian, South American and lectins and heamagglutinins which impede proteolytic
African regions (Wani et al., 2010). The botanical and enzymes resulting in reduced digestibility and absorption
linguistic evidences clearly manifested that red kidney of proteins (Kumar et al., 2013). Therefore, it is
beans have been originated in America and belong to the imperative to eliminate these anti-nutritional factors to
family phaseoleae (Bressani, 1993). Beans are mostly improve the digestibility and bioavailability for efficient
consumed as dry seeds but their utilization as green utilization of proteins. This can be achieved by extracting
shelled seeds and green pods is also possible. These are the proteins to produce isolates or concentrates which
also utilized as staple foods in Maxico, South American will not only contain high protein contents but also have
and African countries with per capita consumption upto better protein digestibility due to the elimination of anti-
40 kg per year (Tang et al., 2009). Although red kidney nutritional factors. The protein isolate can be utilized as a
beans contain high amounts of dietary fiber, starch, promising food ingredient in the industry to improve the
vitamins, minerals as well as an extensive array of functional properties of the products as well as to
phytochemicals but the most important component of enhance the nutritional status of the diets.
nutritional significance is their high protein content The most widely used method for the
which is 2-3 times that of cereal grains (Mundi and preparation of protein isolates on commercial scale
Aluko, 2012). Red kidney bean proteins are the superior involve alkaline extraction of proteins, following by
source of certain indispensable amino acids particularly which the extracted proteins are precipitated at their
isoelectric point (Kaur and Sing, 2007). The plant

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proteins should possess certain desirable characteristics percentages of the above mentioned parameters on
for their successful applications in different food moisture free basis from 100. The gross energy was
products. These characteristics are often designated as calculated by multiplying percentages of fat, protein and
functional properties which can influence the carbohydrates with their Atwater factors.
performance of protein in different food formulations For mineral analysis, the digestion of seed flour
(Moure et al., 2006). The solubility of the protein is an sample was carried out by using concentrated nitric acid
important functional attribute to access its suitability as and perchloric acid at 180-200oC while the digested
food ingredient because the solubility can also influence sample was subjected to atomic absorption
foaming, emulsifying and gelling properties of the spectrophotometer (GBC-932, Australia) by following
proteins. (Ragab et al., 2004). the standard procedure of AOAC (2000).
The functional attributes of the protein based
Preparation of Protein Isolate: By using hexane, red
ingredients are not only dependent upon type of
kidney bean seed flour was defatted under continuous
preparation, kind of protein and method of production but
shaking for 12 hours, following by which the removal of
are also affected by different environmental factors
hexane was carried out by the decantation process. The
(Sakamoto et al., 1994). The pH or ionic strength can
defatted flour was dried in air and used for the
influence the electrostatic and hydrophobic interactions
preparation of protein isolate. The proteins from defatted
within the matrix of protein molecules. Moreover, the
flour of red kidney beans were isolated by alkaline
hydrodynamics of proteins in the food formulations is
extraction and isoelectric precipitation method as
also affected by the prevalent pH (Aluko and Yada,
described by Kaur and Sing, (2007) with slight
1995). Therefore, by manipulating the pH, the functional
modifications. Different steps involved in the preparation
properties as well as structural conformation of the
of protein isolate are depicted Figure 1.
proteins can be modified for better utilization in the food
formulations. The objectives of this study were to explore Protein Solubility Profile: The solubility of the protein
the potential of underutilized red kidney beans to produce isolate was determined by mixing 1 g of the sample in 25
value-added protein product and to evaluate the ml of distilled water and adjusting the pH of dispersions
functional properties of the value-added product under in the range of 2-10 by either using 1N HCl or NaOH
different pH conditions to envisage its compatibility in solutions. After stirring the dispersions for 1 hour and
different food systems. centrifuging at 8000 g for 15 minutes, supernatants were
separated and their protein contents were determined
MATERIALS AND METHODS (Sai-Ut et al. 2009). Following equation was used to
calculate the solubility percentage of the isolate:
Raw material collection and preparation: The seeds of
indigenous cultivar of red kidney beans were obtained
from NARC, Islamabad. The seeds were screened and Emulsification Properties: The method of Neto et al.
rinsed with deionized water to remove the extraneous (2001) was followed to evaluate the emulsification
material. After drying the seeds in an oven at 50oC, these properties of protein isolate. The determination of
were ground and passed through a screen to get uniform emulsifying activity was carried out by blending 3.5 g of
size flour, which was stored in air tight containers until the isolate sample with 50 ml of water for 30 seconds and
used for analysis. The analytical grade chemicals were adjusting the pH (2-10) either by using 1N HCl or NaOH
used during the analysis. solutions. After the addition of 5ml of the canola oil, the
suspensions were again blended. The emulsions were
Chemical analysis of red kidney bean seeds: The
then centrifuged at 2000g for 5 minutes and the heights of
proximate analysis of red kidney bean seed flour for
the total contents and that of emulsified layer in the tubes
moisture, crude fat, crude fiber, ash protein and
were measured. Following equation was used to calculate
carbohydrate contents was carried out in triplicate by
the emulsion activity:
adopting the standard procedures of AOAC (2000). The
moisture content was determined by oven dry method by
drying the sample in hot air oven at 130oC till constant For the determination of emulsion stability,
weight was attained. For crude protein content, kjeldhal emulsions were heated at 80oC for 30 minutes and then
method was used to determine the nitrogen content which cooled under running tap water for 15 minutes prior to
was then multiplied with factor (6.25). The estimation of centrifugation. Emulsifying stability was calculated as
the fat content of the samples was carried out in soxhlet
apparatus by using petroleum ether. The estimation of ash
contents was carried out by using muffle furnace at a
temperature of 550 oC for 8 to 10 hours. Carbohydrate Gelation Properties: The method of Lawal et al. (2005)
content of the seed flour calculated by subtracting the was followed to determine the gelation properties of the

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protein isolate. The isolate sample was suspended in accounted for 53.02±1.14% in the red kidney bean seeds.
distilled water at different concentrations (2-20%) and the Carbohydrates are the main source of energy which are
pH of suspensions was adjusted to the desired value (2- stored as glycogen and act as reservoir for glucose. The
10). Each sample suspension (10 ml) was taken into the carbohydrate value observed in this study is in
test tube and heated for 1 hr. in boiling water bath and conformity with the values of 51.5, 57.3 and 59.62% for
then cooled rapidly in cold water as well as further cooled tirga beans, pinto beans and white kidney beans,
at 4oC for 2 hrs. The test tubes were then inverted and respectively (Amir et al., 2006). The crude fiber content
analyzed for gelling ability. The concentration at which of red kidney beans was observed to be 6.82±0.31%.
the sample did not slip or fall from the inverted test tubes Although the crude fiber has little food value but it plays
was expressed as least gelation concentration. an essential role in the control of different diseases by
exerting certain physiological functions. The fiber
Foaming Properties: The determination of the foaming
content of red kidney beans is adequate in relation to diet.
properties of the protein isolate was carried out by
These findings are in accordance to Costa et al. (2006)
following the method of Lawal et al. (2005). For this
who observed crude fiber content of 6.26-8.98% in pea,
purpose, 3% sample dispersions were prepared in
chickpea and common beans. The crude fat content of red
distilled water and their pH was adjusted in the range of
kidney bean seeds was observed to be fairly low
2-10 either with HCl or NaOH solutions. After vigorous
(1.92±0.15%) which was within the range of 1-3% for
whipping of the dispersions in blender for 2 minutes,
most of the legumes (Costa et al., 2006). The ash content
foaming activity was calculated according to the
of bean seeds was 4.34±0.20% which falls within the
following equation:
range of 4-5% as reported for different varieties of beans
(Rui et al., 2011). The energy value of red kidney bean
seeds calculated by the use of Atwater factors was found
For the determination of foam stability, the
to be 332.48 Kcal/100g indicating these to be a fairly
volume of the foam was recorded after 30 minutes time
good source of energy. Previously, Khattab et al. (2009)
interval up to 90 minutes.
reported a caloric content range of 375.32-
Statistical Analysis: Results of three replications were 383.91Kcal/100g for Canadian and Egyptian cowpea and
articulated as mean±standard deviation. The data kidney beans.
regarding the effect of pH on functional properties was The results regarding mineral composition of red
subjected to analysis of variance (ANOVA) techniques kidney bean seeds are depicted in Table 2. These results
while the comparison of the means was performed by elucidated red kidney bean flour to be a good source of
using Duncun,s multiple range test as described by Steel minerals containing magnesium, calcium, sodium,
et al. (1997). potassium and iron in relatively higher amounts while
phosphorus, nickel, zinc, manganese and copper in
RESULTS AND DISCUSSION smaller quantities. Magnesium was found to be most
abundant mineral in the raw flour (762.7±6.61 mg/100g)
Chemical analysis of red kidney bean seeds: The which contradicts the findings of Olaofe and Sanni
results regarding the proximate composition of bean (1998) who reported potassium to be the abundant
seeds are depicted in Table 1. Moisture content of red element in agricultural products. The results regarding the
kidney beans was found to be 8.12±0.53%. Moisture mineral analysis substantiated the findings of Audu and
content is an important factor that dictates storage Aremu (2011), however, slight variations may be due to
stability as high moisture content in seeds may lead to varietal difference and geographic conditions.
deterioration and spoilage through bacterial and fungal Solubility profile of red kidney bean protein isolate:
attack. The red kidney bean seeds having low moisture The results regarding the solubility profile of the protein
content can be stored for a longer period of time. The isolate are illustrated in Figure 2. It is obvious from the
moisture content of red kidney bean seeds is comparable results that the protein solubility was experiential to be
to the values (8.9%) reported by Olaofe et al. (2010) for pH-dependant with minimum solubility (7.5%) was
common beans. The results regarding protein content observed at pH 4-5, beyond that subsequent change in pH
revealed red kidney beans to be an excellent protein on either side resulted in the progressive increase in
source containing 25.78±0.77% protein. Dietary proteins protein solubility. The protein solubility was higher in the
are essential for synthesis and repairing of body tissues, alkaline region with maximum solubilization (92%) was
hormones, enzymes as well as other substances required observed at pH 10. Similar variations in solubility with
for healthy functioning. Present results regarding the pH were previously reported for pigeon pea and lentil
protein content corroborated the findings of Rui et al. proteins (Mwasaru et al. 2000; Bora. 2002). Protein
(2011) who reported protein content of 22.36-28.50% in solubility may be influenced by the ratio of hydrophilic
different bean varieties. The carbohydrate content or hydrophobic amino acids. The low protein solubility at
calculated as nitrogen free extract by difference

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the isoelectric region is ascribed to the balance of observed which indicate optimum gelling ability of the
negative and positive charges resulting in the reduction of protein isolate at this pH, beyond that subsequent change
electrostatic repulsive interactions. High solubility of the of pH on either side resulted in an increase of LGC. At
red kidney bean protein isolate beyond isoelectric point pH 10 highest value (16%) of LGC was observed which
may be attributed to large net charges and electrostatic indicate poor gelling potential of the isolate at alkaline
repulsion due to the prevalence of cation III and anion II, conditions. The value of LGC (12%) in the present study,
respectively. The solubility is a valuable indices for the at pH 7 acting as control, was lower as compared to
suitability of the isolate for various food applications as mucuna bean and lupin proteins (Sathe et al., 1982;
low protein solubility over a wide range of pH indicate Adebowale and Lawal, 2003) signifying red kidney bean
severe protein denaturation and insolubilization which proteins to be better gelling food hydrocolloid than these
may also influence other functional properties. High legumes. Variations in gelation capacity of the protein
protein solubility of the isolate signify its potential for isolate at different pH levels may be ascribed to the
industrial application to develop high protein drinks and prevalence of surface charge on protein molecules. The
beverages. Moreover, high protein solubility of the balance of positive and negative charges at the isoelectric
isolate can be advantageously utilized to develop infant region minimizes repulsive interactions which promote
formulas, spray dried products and milk replacers. stronger interactions of protein molecules resulting in an
improvement of gelation capacity. Conversely, at pH
Emulsification properties of red kidney bean protein
levels beyond the isoelectric region, the interaction
isolate: The influence of pH on emulsification properties
among protein molecules weakens due to the prevalence
of protein isolate is shown in Figure 3 and 4, respectively.
of repulsive forces which prevents the formation of a
Minimum value of emulsion activity was observed at pH
well-organized protein network (Elofsson et al., 1997).
4 which increased with increasing the pH with maximum
The gelation properties of the protein isolate are
value (70.4%) was observed at pH 10. The emulsification
technologically advantageous for baked and pasta
activity was pH-dependant with alkaline pH improved
products, simulated ground meats as well as for dairy
this property more than acidic pH. The results regarding
products such as curds and cheese.
emulsion stability also followed similar pattern with
minimum value was observed at pH 4 while maximum Foaming properties of red kidney bean protein
emulsion stability was observed at pH 10. Different isolate: The results regarding the foaming properties of
factors such as protein conformation, interfacial tension, the isolate at different pH levels are depicted in Table 4.
droplet size as well as net protein charge can influence These results clearly indicated that foaming properties
the values of emulsion stability There is a direct were pH-dependant. Minimum value (33%) of foam
relationship between emulsification properties and capacity was found at pH 4 , which increased gradually
protein solubility. Low values of the emulsification by changing the pH on either side with maximum values
properties at the isoelectric region may be ascribed to low (84%) was found at pH 10. Acidic pH also favor the
protein solubility due to minimum electrostatic repulsion foaming capacity of the extracted protein isolate. The
among the particles which promotes coalescence and increased net charge on the protein molecules at the
flocculation (Hung and Zayas, 1991). At extremes of the acidic and alkaline regions weakens the hydrophobic
acidic and alkaline regions, unfolding of proteins exposes interactions, allowing quick spreading of proteins at the
majority of the obscured lipophilic functional groups with air-water interface as well as encapsulation of air
inadequate interfacial energy between aqueous and oil particles resulting in increased foam formation (Chau et
phases resulting in improvement of the emulsification al. 1997). Foam stability of all samples decreased with
properties (Chavan et al. 2001). The emulsification time at all pH levels. Contrary to foam capacity, highest
properties play an imperative role for the stability of high foam stability was found at the isoelectric region while
fat food products by reducing the rate of oxidative subsequent change in pH on either side decreased
rancidity. The emulsification properties of the protein stability with minimum values observed at pH 10.
isolate make it a promising food ingredient for utilization Highest foam stability in the acidic regions may be
in cake batters, bread, meat sausages, salad dressing, ascribed to minimum repulsive interactions leading to the
frozen desserts and dairy products. development of viscous film as well as stable molecular
layer at the interface, thus imparting stability to the foam.
Gelation Properties of red kidney bean protein
Similar variations in foaming properties were previously
isolate: The results regarding the gelling potential of the
reported for sesame and locust bean proteins (Inyang and
protein isolate are depicted in Table 3. The least gelation
Idhu 1996; Lawal et al. 2005). Foaming properties of the
concentration (LGC) was used as an indicator of gelling
protein isolate can be advantageously utilized in whipped
ability as lower values of LGC indicate better gelation
toppings, angel cakes, confectionary and chiffon mixes.
capacity. At pH 4 minimum value (8% w/v) of LGC was

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Table 1. Proximate analysis of red kidney bean seeds. Table 2. Minerals profile of red kidney bean seeds.

Parameters Composition (g/100g) Minerals Composition (mg/100g)

Moisture 8.12±0.53 Magnesium 762.7±6.61
Crude fat 1.92±0.15 Calcium 51.4±1.04
Crude protein 25.78±0.77 Sodium 39.2±0.25
Crude fiber 6.82±0.31 Potassium 19.6±0.62
Ash 4.34±0.20 Iron 14.2±0.40
Carbohydrate 53.02±1.14 Phosphorous 4.4±0.70
Energy value (Kcal/100g) 332.48 Nickel 3.76±0.08
Results of three determinations were reported as means ±SD Zink 3.62±0.04
Manganese 2.71±0.05
Copper 0.92±0.05
Results of three determinations were reported as means ±SD.

Table 3. Gelation properties of red kidney bean protein isolate at different pH levels.

Sample concentration pH 2 pH 4 pH 6 pH 7 pH 8 pH 10
(%)
2 (−)
( ( ( ( (
4 (−)
( ( ( ( (
6 (−)
( (
8 (±)
( ( ( ( (
10 (±)
( ( ( ( (
12 (+)
( ( ( ( (
14 (+)
( ( ( ( (
16 (+)
( ( ( ( (
18 (+)
( ( ( ( (
20 (+)
( ( ( ( (
LGC 12 8 10 12 14 16
Gelation levels: (+) gelation; (−) No gelation; (±) partial gelation

Table 4. Foaming properties of red kidney bean protein isolate at different pH levels.

pH Initial Volume after Foam Foam stability (%)

volume (ml) whipping capacity After time (minutes)
(ml) (%)
30 60 90
2 50 85.5±0.5 71±1c 82.14±1.03e 71.82±0.40d 61.96±0.54d
3 50 76.5±1 53±2e 84.28±0.63d 77.38±1.03c 64.76±0.65c
4 50 66.5±0.5 33±1g 90.89±0.27a 84.83±0.46a 72.70±0.82a
5 50 67.5±0.5 35±1g 88.56±0.32b 79.93±0.57b 68.55±0.90b
6 50 74±1 48±2f 86.81±0.72c 76.38±0.60c 59.7±0.89e
7 50 78±1 56±2d 82.12±0.63e 71.4±1.02d 54.74±0.90f
8 50 86.5±0.5 73±1c 76.70±0.32f 56.15±0.60e 36.97±0.86g
9 50 88.5±0.5 77±1b 72.71±0.35g 49.34±0.66f 29.85±0.91h
10 50 92±1 84±2a 66.65±0.79h 40.06±0.90g 19.03±0.73i
Results of three determinations were reported as means ±SD. Means sharing different alphabetic letters within the columns are
significantly (P≤0.05) different.

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Defatted RKB flour +H2O (1:10)

1N NaOH

Extraction for 1.5 hr, pH 10, 40oC

Recycled twice

Centrifugation, 8000g, 30 min. Residue

Supernatant

1N HCl

Isoelectric precipitation, pH 4.5

Centrifugation 8000g, 20 min.

Protein precipitate

H2O, 1:3 w/v

Washing twice

1N NaOH

Neutralization, pH 7

Drying (vacuum oven), 45oC

Protein isolate
Figure 1: Different steps involved in the preparation of red kidney bean protein isolate

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Figure 2: Solubility profile of red kidney bean protein isolate at different pH levels

Figure 3: Emulsifying activity of red kidney bean protein isolate at different pH levels

Figure 4: Emulsion stability of red kidney bean protein isolate at different pH levels.

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Costa, G. E. A., K. S. Queiroz-Monici, S. M. P. M. Reis

Conclusions: Red kidney beans are an excellent source
and A. C. Oliveira (2006). Chemical
of proteins, fiber and essential minerals. Being a rich
composition, dietary fibre and resistant starch
source of proteins, red kidney beans can be utilized for
contents of raw and cooked pea, common bean,
the production of protein isolate for utilization as
chickpea and lentil legumes. Food Chem.
promising ingredient in the food industry. Variations in
94:327–330.
pH can exhibit modifications of functional properties.
Elofsson, C., P. Dejmek, M. Paulson and H. Burling
Red kidney bean protein exhibited higher solubility as
(1997). Characterization of a cold gelling whey
well as better emulsifying and foaming properties.
protein concentrate. Int. Dairy J. 7: 601–608.
Moreover, protein isolate was also found to be a better
Hung, S. C. and J. F. Zayas (1991). Emulsifying capacity
gelling hydrocolloid. Therefore, red kidney bean protein
and emulsion stability of milk proteins and corn
isolate has the potential to be utilized in different food
germ protein flour. J. Food Sci. 56: 1216–1223.
formulation systems.
Inyang, U. E. and A. O. Iduh (1996). Influence of pH and
Acknowledgements: The authors are grateful to Higher salt concentration on protein solubility,
Education Commission, Pakistan for financial support. emulsifying and foaming properties of sesame
protein concentrate. JAOCS 73(12): 1663-1667.
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