Bioresource Technology 101 (2010) 2167–2172

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Bioresource Technology
journal homepage: www.elsevier.com/locate/biortech

Proteins from common bean (Phaseolus vulgaris) seed as a natural coagulant

for potential application in water turbidity removal
Mirjana G. Antov *, Marina B. Šćiban, Nada J. Petrović
Faculty of Technology, University of Novi Sad, Blvd. Cara Lazara 1, 21000 Novi Sad, Serbia

a r t i c l e i n f o a b s t r a c t

Article history: The ability of coagulation active proteins from common bean (Phaseolus vulgaris) seed for the removal of
Received 30 July 2009 water turbidity was studied. Partial purification of protein coagulant was performed by precipitation
Received in revised form 2 November 2009 with ammonium sulphate, dialysis and anion exchange chromatography. Adsorption parameters for
Accepted 5 November 2009
ion-exchange process were established using dialysate extract. Results revealed that the highest values
Available online 30 November 2009
of the adsorbed protein were achieved in 50 mmol/L phosphate buffer at pH 7.5 and the maximum
adsorption capacity was calculated to be 0.51 mg protein/mL matrix. Partially purified coagulant at initial
Keywords:
turbidity 35 NTU expressed the highest value of coagulation activity, 72.3%, which was almost 22 times
Natural coagulant
Common bean
higher than those obtained by crude extract considering applied dosages. At the same time, the increase
Ion-exchange in organic matter that remained in water after coagulation with purified protein coagulant was more
Coagulation activity than 16 times lower than those with crude extract, relatively to its content in blank.
Ó 2009 Elsevier Ltd. All rights reserved.

1. Introduction In recent numerous studies variety of plant materials as a

Coagulation/flocculation as a step in water treatment processes
is applying for removal of turbidity in raw water that comes from
suspended particles and colloidal material. Materials that are used
in this stage of water treatment can be inorganic coagulants, syn-
thetic organic polymers or coagulants from natural sources. Alu-
minium sulfate (alum) is a common coagulant globally used in
water treatment. In spite of its undoubtfull effusiveness in turbid-
ity removal, alum increases concerns towards ecotoxicological im-
pact when introduced into the environment as post-treatment
sludge having large volumes. Regarding the application of syn-
thetic polymers, the presence of residual monomers is undesirable
because of their neurotoxicity and strong carcinogenic properties
(Mallevialle et al., 1984).
A part of possible solution of these problems might be develop-
ment of new coagulants, preferably from natural and renewable
sources, which have to be safe for human health as well as
biodegradable. Because their production relies on local materials,
renewable resources and food grade plant material, and is rela-
tively inexpensive, it can contribute to achieving sustainable water
treatment technologies. By using natural coagulants considerable
savings in chemicals and sludge handling cost may be achieved
along with production of readily biodegradable and less volumi-
nous sludge that amounts only 20–30% that of alum treated
counterpart (Narasiah et al., 2002).
* Corresponding author. Tel.: +381 21 485 3647; fax: +381 21 450 413.
E-mail address: mantov@uns.ac.rs (M.G. Antov).
source of natural coagulants has been reported (Raghuwanshi
et al., 2002; Diaz et al., 1999; Miller et al., 2008) but the most stud-
ied is Moringa oleifera whose efficiency has been reported for tur-
bidity removal (Ndabigengensere and Narasiah, 1998; Okuda
et al., 2001a; Ghebremichael et al., 2006) as well as antimicrobial
properties (Ghebremichael et al., 2005). Apart of all previously
mentioned preferences of using natural coagulants instead of syn-
thetic ones, major disadvantage of their application as crude ex-
tracts in water treatment is an increase of organic matter in
water. This complicates further processing and adversely affects
water quality but could be overcome by purification of the coagu-
lant (Ghebremichael et al., 2006).
During the course of plants’ screening program in our labora-
tory (Šćiban et al., 2005, 2009), crude extract from common bean
(Phaseolus vulgaris) seed showed the ability to act as a natural
coagulant. Seed from common bean as potential source of coagu-
lant for water treatment would be promising considering its food
grade nature. Moreover, it offers a few advantages over M. oleifera
seed – because of no oil present in it there is no need for extraction
by organic solvents thus avoiding delipidation step which is bene-
ficial for both economic and environmental reasons.
The objective of the study was partial purification of the
coagulation active components extracted from common bean seed.
Optimal conditions for ion-exchange chromatographic purification
of coagulant protein regarding the process of adsorption were
established. In addition, application of the partially purified
common bean coagulant was evaluated as well as its suitability
in comparison to crude extract regarding organic load.

0960-8524/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.biortech.2009.11.020
2168 M.G. Antov et al. / Bioresource Technology 101 (2010) 2167–2172
2. Methods
2.1. Extraction of active component from common bean seed
The locally obtained common bean (P. vulgaris) dry seed was
ground to a fine powder by using a laboratory mill and sieved
through 0.4 mm sieve. The fraction with particle size less than
0.4 mm was used in experiments. Fifty grams of seed powder
was suspended in 1 L of 0.5 mol NaCl/L water. The suspensions
were stirred using a magnetic stirrer for 10 min to accomplish
extraction and then filtered through a rugged filter paper (Mache-
rey-Nagel, MN 651/120) to obtain filtrates – crude extracts of ac-
tive component.
2.2. Precipitation of active component
The coagulation active component from common bean was
further processed by precipitation and dialysis. Crude extracts
were saturated to 80% by addition of (NH4)2SO4 and centrifuged
at 4000g (5804-R, Eppendorf) for 10 min. Precipitate was redis-
solved in 10 mmol/L appropriate buffer (i.e. some of the buffers
listed in the following text) and dialysed overnight at 4 °C
against Millipore water in dialysis bag with molecular cut-off
12–14 kDa.
2.3. Adsorption studies
Adsorption studies were conducted using dialysate extracts
obtained above in series of buffers in batch ion-exchange (IEX)
experiments with AmberliteTM IRA 900 Cl (Rohm and Haas) as
matrix. AmberliteTM IRA 900 Cl is a macroreticular polystyrene
type 1 strong base anion exchange resin containing quarternary
ammonium groups whose shipping weight is 700 g/L and total ex-
change capacity P 1.00 eq/L (Cl form). In order to find the opti-
mum pH of the buffer for the adsorption, dialysate extract was
diluted in universal Britton and Robinson (I) buffer having pH from
7 to 9 with an increment of increase of pH 0.5. The choice of the
buffering substance was made by measuring the amount of bound
protein in phosphate, Tris–HCl or ammonium acetate buffer at pH
7.5. The effect of ionic strength of the buffer on the adsorption of
active compounds to anion exchange resin was evaluated by vary-
ing concentration of phosphate buffer (10, 25, 50, and 100 mmol/L)
at pH 7.5.
In order to estimate optimum volume of IEX matrix, adsorption
experiments were carried out by adding 0.33 mg dialysate extract
in phosphate buffer (50 mmol/L, pH 7.5) to different volumes of the
matrix ranging from 0.33 mL to 1.0 mL.
2.4. Kinetic studies and adsorption isotherm
Kinetics of adsorption was studied using 3.3 mg dialysate ex-
tract in 10 mL phosphate buffer (50 mmol/L, pH 7.5). Protein solu-
tion was added to 10 mL IEX matrix and mixed in magnetic stirrer
at 100 rpm. Samples were collected in certain time intervals, cen-
trifuged immediately and supernatants were analysed for protein
content. Blanks were carried out without matrix to check out if
any measurable loss of protein came out from reasons other than
its adsorption to matrix.
Adsorption capacity of the matrix was estimated using 1.78–
6.44 mg dialysate extract in 10 mL phosphate buffer (50 mmol/L,
pH 7.5). Protein solution were added to 10 mL of IEX matrix and
mixed at 100 rpm for 90 min at room temperature. After that un-
adsorbed protein concentration was measured and amount of ad-
sorbed protein was calculated from a mass balance.
Maximum adsorption capacity of the matrix and the dissocia-
tion constant of the adsorption were determined form the Lang-
muir adsorption model (Faust and Aly, 1987):
Ce 1 Ce
¼ þ ; ð1Þ
qe b  X m X m
where Ce is the concentration of protein in solution in equilibrium
(mg/mL); qe is the amount of protein adsorbed per volume of adsor-
bent (mg/mL); b is a constant that is related to the enthalpy of
adsorption (mL/mg) and Xm is the maximum adsorption capacity
(mg/mL).
2.5. Purification of active component
Dialysate extract was loaded onto column (10 mm  150 mm
glass column) packed with 10 mL of AmberliteTM IRA-900 Cl previ-
ously equilibrated with 50 mol/L phosphate buffer, pH 7.5. Active
components were eluted from resin by linear gradient of ionic
strength of NaCl solution from 0 to 1 mol/L at a flow rate 1 mL/
min. Protein content and coagulation activity of fractions (2 mL)
were determined.
2.6. Preparation of turbid water
Turbid water for coagulation tests was prepared by adding 1 g
kaolin to 1 L tap water. The suspension was stirred for 1 h to
achieve uniform dispersion of kaolin particles, and then it was al-
lowed to remain for 24 h for completing hydration of the particles.
This suspension was used as the stock suspension. Turbid water
with 50 mg/L kaolin (about 35 nephelometric turbidity units –
NTU) was prepared by diluting 50 mL of stock suspension to
1000 mL tap water just before the coagulation test. The initial pH
of the synthetic water was adjusted to 9.0 with 1 mol/L NaOH solu-
tion, in accordance with previous investigations (Okuda et al.,
2001a; Šćiban et al., 2005).
2.7. Coagulation test
Coagulation activity of the fraction eluted from column as well
as crude extract was evaluated in jar tester VELP, model FC6S. Sam-
ples were added to the beakers at different dosages (0.5, 1.0 or
2.0 mL/L turbid water) and the content was stirred at 200 rpm
for 2 min. The mixing speed was then reduced to 80 rpm and
was kept for 30 min. Then, the suspensions were left to allow sed-
imentation. After 1 h of sedimentation, an aliquot of 100 mL of
clarified sample was collected from the top of the beaker and resid-
ual turbidity was measured. The residual turbidity of sample was
RTS. The same coagulation test was performed with no coagulant
as the blank. The residual turbidity in the blank was RTB. Coagula-
tion activity was calculated as:
RT B  RT S
Coagulation activity ð%Þ ¼  100: ð2Þ
RT B
2.8. Analytical methods
Protein concentration was measured according to Bradford
(1976) with bovine serum albumin as standard. Turbidity was
measured using a turbidimeter (TURB 550 IR) and it was expressed
in nephelometric turbidity units (NTU). The amount of organic
matter released from common bean seed crude extract and par-
tially purified protein were determined as chemical oxygen de-
mand (COD) according to Standard Methods (APHA, 1998).
All experiment were run in duplicate (the accuracy is consid-
ered to be ±5%) and the mean value is presented herein.
 M.G. Antov et al. / Bioresource Technology 101 (2010) 2167–2172 2169

3. Results and discussion 3.1. Adsorption parameters

Our previous investigations showed that the highest values of
protein concentration and coagulation activity were achieved
when protein coagulant from common bean seed was extracted
by 0.5 mol/L NaCl in water (Antov et al., 2007). In the current study
the coagulation active components were precipitated by ammo-
nium sulphate and dialysed, and dialysate extract was used in
adsorption studies in order to maximise results of purification pro-
cess of common bean coagulant (CBC).
(a) 100
80
Adsorbed protein (%)
According to the literature, isoelectric points of proteins from P.
vulgaris are near to pH 4.5 (Belitz et al., 2004; Morales-de Leon
et al., 2007). Cloud point test (data not shown) conducted in our
laboratory indicated that isoelectric point (pI) of our dialysate ex-
tract of common bean seed was between pH 4 and 5.5. So, the ef-
fect of pH of buffer on the adsorption of dialysate extract on anion
resin was studied in batch experiments within pH range 7–9 by
monitoring the amount of protein bound to the matrix (Fig. 1a).
Results revealed that in the investigated pH range percentage of
adsorbed protein was varied in narrow range – from 75.6% to
84.6%. Maximum of protein adsorption was achieved, as expected,
at the highest investigated pH 9 considering the fact that net sur-
face charge of proteins increases with the increase in distance from
pH to pI. However, because the portions of adsorbed protein at pH
7.5 (84%) and pH 9 (84.6%) were just slightly different and also con-
sidering the following elution step, further experiments were con-

ducted at pH 7.5.
60 In the next experiments, several buffering substances were
tested at pH 7.5 in order to find the most appropriate one for the
adsorption of dialysate extract to IEX matrix. The highest percent-
40
age of protein, 80.7%, was bound to matrix in the presence of phos-
phate ions (Fig. 1b). So, the effect of ionic strength of buffer on the
20 adsorption was monitored by measuring the amount of protein
bound to matrix in phosphate buffer within concentration range
10–100 mmol/L at pH 7.5. With an increase of buffer ionic strength
0
amount of adsorbed protein increased and the highest value was
7 7.5 8 8.5 9
observed with 50 mmol/L phosphate buffer (Fig. 1c). However, at
pH 100 mmol/L amount of adsorbed protein was decreased which
was indicated by increased concentration of protein in un-bound
(b) 100 fraction. This result can be explained by the increased competition
toward adsorption sites at IEX matrix between protein-ions and
buffer-ions when they are present in higher concentrations
80
(Scopes, 1994).
Adsorbed protein (%)

Estimate of optimum volume of IEX matrix that is required for

60 adsorption and purification of protein extracted from common
bean was based on the experiments conducted with constant
amount of dialysate extract but varying volumes of matrix (data
40 not shown). Results revealed that increase in matrix volume from
0.33 mL to 1 mL led to an increase in percentage of adsorbed pro-
20
tein from 50% to nearly 85% added protein, respectively, and that
the highest investigated volume of matrix was sufficient for

0
Phosphate Tris-HCl buffer Ammonium 0.35
buffer acetate buffer
Adsorbed protein (mg/mL resin)

0.30
(c) 100

0.25
80
Adsorbed protein (%)

0.20
60

0.15
40
0.10
20
0.05
0
10 25 50 100 0.00
Cbu ffer (mmol/L) 0 20 40 60 80 100 120
Time (min)
Fig. 1. The effect of (a) pH, (b) buffering substance and (c) concentration of
phosphate buffer on the adsorption of dialysate extract from common bean seed on Fig. 2. Adsorption kinetics of dialysate extract from common bean seed on anion
anion exchange matrix AmberliteTM IRA 900 Cl. exchange matrix AmberliteTM IRA 900 Cl at 50 mmol/L phosphate buffer, pH 7.5.
2170 M.G. Antov et al. / Bioresource Technology 101 (2010) 2167–2172

0.7 Equilibrium parameters were determined from the Langmuir

isotherm model considering the smooth continuous time course
0.6 of protein adsorption (Fig. 2) that suggests the formation of protein
monolayer on surface. From a linear relationship Ce/qe vs. Ce (Fig. 3)
0.5 the maximum adsorption capacity was calculated to be 0.51 mg
protein/mL matrix. It should be noticed that this result was based
0.4 on dialysate extract and not on the purified protein. Obtained value
C e/q e

for Xm is significantly lower than those for coagulant protein from

0.3 M. oleifera (Ghebremichael et al., 2006) even considering that dif-
2 ferent IEX matrixes were used. This might be also explained by
0.2 R = 0.9675
the differences in molecular weight of coagulation active proteins
from P. vulgaris and M. oleifera. Namely, it is estimated that coagu-
0.1
lant protein from M. oleifera has molecular weight 6.5 kDa (Ghe-
bremichael et al., 2005) i.e. 13 kDa for dimer (Ndabingengensere
0
et al., 1995) while Mw of subunit of major storage protein (which
0 0.05 0.1 0.15 0.2 0.25 0.3
is trimer) in common bean seed is 50 kDa (Belitz et al., 2004;
Ce (mg/mL) Montoya et al., 2008). It is known fact that small macromolecules
have higher adsorption per unit of weight (volume) of adsorbent
Fig. 3. Linearized form of Langmuir model for adsorption of dialysate extract from
common bean seed on anion exchange matrix AmberliteTM IRA 900 Cl.
(Kilduf et al., 1996). In addition, larger molecules can bind only
to the surface of the ion exchanger particles, so capacity for these
is very low (Scopes, 1994).

adsorption of 0.28 mg protein/mL matrix. Higher efficiency of pro-

tein adsorption at higher matrix volume can be attributed to the
greater number of active sites at matrix which increases the prob-
ability of the adsorption of protein molecules (under condition that
matrix was not overloaded like in these experiments, see Fig. 3) as
it was already found for M. oleifera protein coagulant (Ghebremich-
ael et al., 2006).
3.2. Kinetics of adsorption and equilibrium parameters
The kinetics of adsorption of proteins from dialysate extract on
matrix was studied during 2 h at room temperature and simulta-
neously blanks without matrix were carried out. Experiments con-
firmed that changes in protein concentration came out only as a
consequence of its adsorption to IEX matrix. The rate of protein
adsorption was high in the first 10 min and after that incremental
increase started to decline (Fig. 2). The time needed to reach the
equilibrium was estimated to 60–90 min when maximal values
of adsorbed protein were determined.
3.3. Purification of coagulation active components
The chromatogram of dialysate extract on anion exchange ma-
trix is shown in Fig. 4. Dialysed extract (containing 3.3 mg protein)
was loaded to column and then bound proteins were eluted by lin-
ear gradient NaCl. Coagulation activity of each fraction was deter-
mined at a dosage 1 mL/L turbid water. Results revealed existence
of several protein peaks that were not completely separated from
each other but coincided in some extent with the highest mea-
sured coagulation activities in fractions. Flocs formed during the
coagulation test of fractions eluted from 0.375 mol/L to
0.875 mol/L NaCl were visible to the naked eye (1–2 mm) during
the slow mixing part of the process. The presence of several protein
as well as coagulation active peaks might be explained by the het-
erogeneity of protein sample from common bean seed. The fraction
having the highest coagulation activity 72.3% was eluted by
0.875 mol/L NaCl. This fraction containing partially purified com-
mon bean coagulant (CBC) was further analysed in coagulation
study.

100 1.0
0.15

80 0.8
Protein concentration (mg/mL)

Coagulation activity (%)

0.10
C NaCl (mol/L)

60 0.6

40 0.4
0.05

20 0.2

0.00 0 0.0
2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34
V(mL)

Fig. 4. Elution diagram of dialysate extract from common bean seed from anion exchange matrix AmberliteTM IRA 900 Cl.
 M.G. Antov et al. / Bioresource Technology 101 (2010) 2167–2172
3.4. Coagulation study
The primary goal of coagulation/flocculation is turbidity re-
moval and this was investigated at conditions regarding initial tur-
bidity 35 NTU according to Okuda et al. (2001b). Results of
coagulation test of crude extract and the fraction containing puri-
fied CBC, that had protein concentrations 0.73 mg/mL and
0.081 mg/mL, respectively, in relation to coagulant dosage are
shown in Fig. 5a. The optimal dosage which was the minimum
one led to the lowest residual turbidity had the equal value for
crude extract and fraction with partially purified CBC and
amounted 1 mL/L turbid water. Calculated on the base of protein
concentration, the optimal coagulation dosage of purified CBC
was 0.081 mg/L turbid water which expressed the highest coagula-
tion activity 72.3%. Starting from the same initial turbidity, crude
extract at its optimal dosage 0.73 mg/L turbid water exhibited
the highest efficiency in turbidity removal which resulted in coag-
ulation activity 30%. Hence, calculated on the base of protein which
was added in turbid water, the highest obtained coagulation activ-
ity of partially purified CBC was almost 22 times higher than that
of crude extract.
3.5. Organic load in treated water
The crude extracts which are used as natural coagulants contain
biomolecules and inorganic substances which may be released in
water leading to increased COD (Ghebremichael et al., 2006). Be-
(a) 100
80
Coagulation activity (%)
60
40
20
0
0 0.5 1 1.5 2 2.5
Dosage (mL/L)
(b) 10
8
COD (mg/L)
6 Examination of Water and Wastewater, 20th ed. APHA, AWWA, WEF,
4 Berlin.
2 Commission Directive 98/83/EC of 16 February 1998 on the quality of water
0 preliminary evaluation of turbidity removal by natural coagulants indigenous
Blank Crude extract Purified CBC
Fig. 5. (a) Coagulation activity of crude extract () and partially purified CBC (h) at
different dosages and (b) COD values in water samples treated by crude extract and
partially purified CBC in comparison to blank.
2171
sides, the use of natural coagulants may increase the organic load
in water which may result in increased microbial activity (Ndabig-
engensere and Narasiah, 1998; Okuda et al., 2001a). In addition,
the organic matter might consume additional chlorine in the water
treatment plant and can acts as a precursor of byproducts during
the disinfection process. Thus, purification of the active compo-
nents is essential to minimize the value of unnecessary organic
material which might adversely affect quality of the water.
Organic matter in water before and after coagulation tests was
measured to establish the increase in organic load when crude ex-
tract and the partially purified CBC were added to turbid water at
the dose exhibiting the highest coagulation activity. Results
showed that the increase in organic matter that remained in water
after coagulation was 5.9 and 0.35 mg O2/L when crude extract and
purified CBC acted as coagulants, respectively, in comparison to its
content in blank (Fig. 5b). More than 16 times lower organic load
increase in water after turbidity removal by purified CBC in com-
parison to crude extract was in accordance with lower protein con-
centration in tested fraction (9 times) but additionally it might be
as well a consequence of the diminished load of other organic com-
pounds from crude sample. Chemical oxygen demand value when
partially purified CBC was applied was in the range of those ones
obtained when purified fractions of M. oleifera protein coagulant
were applied for turbidity removal (Ghebremichael et al., 2006).
In addition, it should be stressed that COD of water treated with
the partially purified CBC with applied coagulation dose was below
the maximum admissible concentration according to European
directive which is 5 mg/l (Commission Directive 98/83/EC, 1998).
4. Conclusion
Coagulation activity of the protein from common bean seed ob-
tained after optimization of purification procedure and the in-
crease in organic load were about 22 times higher and more than
16 times lower, respectively, than that of crude extract. Purifica-
tion of natural coagulant from common bean seed has no delipida-
tion step, that is usual for coagulants from oily seed, which make
its application in water turbidity removal potentially more eco-
nomical and environmental friendly.
Acknowledgement
The financial support from Ministry of Science and Technologi-
cal Development, Republic of Serbia (Project No. 20064) is greatly
acknowledged.
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