Bioresource Technology 101 (2010) 21672172

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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
iban, Nada J. Petrovic Mirjana G. Antov *, Marina B. c
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
The ability of coagulation active proteins from common bean (Phaseolus vulgaris) seed for the removal of water turbidity was studied. Partial purication of protein coagulant was performed by precipitation with ammonium sulphate, dialysis and anion exchange chromatography. Adsorption parameters for ion-exchange process were established using dialysate extract. Results revealed that the highest values 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 puried coagulant at initial turbidity 35 NTU expressed the highest value of coagulation activity, 72.3%, which was almost 22 times higher than those obtained by crude extract considering applied dosages. At the same time, the increase in organic matter that remained in water after coagulation with puried protein coagulant was more than 16 times lower than those with crude extract, relatively to its content in blank. 2009 Elsevier Ltd. All rights reserved.

Article history: Received 30 July 2009 Received in revised form 2 November 2009 Accepted 5 November 2009 Available online 30 November 2009 Keywords: Natural coagulant Common bean Ion-exchange Coagulation activity

1. Introduction Coagulation/occulation 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, synthetic organic polymers or coagulants from natural sources. Aluminium sulfate (alum) is a common coagulant globally used in water treatment. In spite of its undoubtfull effusiveness in turbidity removal, alum increases concerns towards ecotoxicological impact when introduced into the environment as post-treatment sludge having large volumes. Regarding the application of synthetic 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 development 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 relatively 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 voluminous sludge that amounts only 2030% 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). 0960-8524/$ - see front matter 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2009.11.020

In recent numerous studies variety of plant materials as a source of natural coagulants has been reported (Raghuwanshi et al., 2002; Diaz et al., 1999; Miller et al., 2008) but the most studied is Moringa oleifera whose efciency has been reported for turbidity 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 synthetic ones, major disadvantage of their application as crude extracts 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 purication of the coagulant (Ghebremichael et al., 2006). During the course of plants screening program in our labora iban et al., 2005, 2009), crude extract from common bean tory (c (Phaseolus vulgaris) seed showed the ability to act as a natural coagulant. Seed from common bean as potential source of coagulant 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 benecial for both economic and environmental reasons. The objective of the study was partial purication of the coagulation active components extracted from common bean seed. Optimal conditions for ion-exchange chromatographic purication of coagulant protein regarding the process of adsorption were established. In addition, application of the partially puried common bean coagulant was evaluated as well as its suitability in comparison to crude extract regarding organic load.

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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 ne 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 ltered through a rugged lter paper (Macherey-Nagel, MN 651/120) to obtain ltrates crude extracts of active 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 redissolved 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 1214 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 exchange capacity P 1.00 eq/L (Cl form). In order to nd the optimum 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, TrisHCl 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 varying 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 extract in 10 mL phosphate buffer (50 mmol/L, pH 7.5). Protein solution was added to 10 mL IEX matrix and mixed in magnetic stirrer at 100 rpm. Samples were collected in certain time intervals, centrifuged 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 unadsorbed protein concentration was measured and amount of adsorbed protein was calculated from a mass balance.

Maximum adsorption capacity of the matrix and the dissociation constant of the adsorption were determined form the Langmuir adsorption model (Faust and Aly, 1987):

Ce 1 Ce ; 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 adsorbent (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. Purication of active component Dialysate extract was loaded onto column (10 mm 150 mm glass column) packed with 10 mL of AmberliteTM IRA-900 Cl previously 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 ow 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 allowed 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 solution, in accordance with previous investigations (Okuda et al., iban et al., 2005). 2001a; c 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. Samples 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 sedimentation. After 1 h of sedimentation, an aliquot of 100 mL of claried sample was collected from the top of the beaker and residual 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. Coagulation activity was calculated as:

Coagulation activity %

RT B RT S 100: 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 partially puried protein were determined as chemical oxygen demand (COD) according to Standard Methods (APHA, 1998). All experiment were run in duplicate (the accuracy is considered to be 5%) and the mean value is presented herein.

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3. Results and discussion 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 ammonium sulphate and dialysed, and dialysate extract was used in adsorption studies in order to maximise results of purication process of common bean coagulant (CBC).

3.1. Adsorption parameters 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 extract of common bean seed was between pH 4 and 5.5. So, the effect of pH of buffer on the adsorption of dialysate extract on anion resin was studied in batch experiments within pH range 79 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 surface 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 considering the following elution step, further experiments were conducted at pH 7.5. In the next experiments, several buffering substances were tested at pH 7.5 in order to nd the most appropriate one for the adsorption of dialysate extract to IEX matrix. The highest percentage of protein, 80.7%, was bound to matrix in the presence of phosphate ions (Fig. 1b). So, the effect of ionic strength of buffer on the adsorption was monitored by measuring the amount of protein bound to matrix in phosphate buffer within concentration range 10100 mmol/L at pH 7.5. With an increase of buffer ionic strength amount of adsorbed protein increased and the highest value was observed with 50 mmol/L phosphate buffer (Fig. 1c). However, at 100 mmol/L amount of adsorbed protein was decreased which was indicated by increased concentration of protein in un-bound 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 (Scopes, 1994). Estimate of optimum volume of IEX matrix that is required for adsorption and purication of protein extracted from common bean was based on the experiments conducted with constant amount of dialysate extract but varying volumes of matrix (data not shown). Results revealed that increase in matrix volume from 0.33 mL to 1 mL led to an increase in percentage of adsorbed protein from 50% to nearly 85% added protein, respectively, and that the highest investigated volume of matrix was sufcient for

(a)
Adsorbed protein (%)

100

80

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0 7 7.5 8 8.5 9

pH

(b)
Adsorbed protein (%)

100

80

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Phosphate buffer

Tris-HCl buffer

Ammonium acetate buffer Adsorbed protein (mg/mL resin)

0.35 0.30 0.25 0.20 0.15 0.10 0.05

(c)
Adsorbed protein (%)

100 80 60

40 20 0 10 25 50 100

0.00 0 20 40 60 80 100 120

Cbu ffer (mmol/L)

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 anion exchange matrix AmberliteTM IRA 900 Cl.

Time (min)
Fig. 2. Adsorption kinetics of dialysate extract from common bean seed on anion exchange matrix AmberliteTM IRA 900 Cl at 50 mmol/L phosphate buffer, pH 7.5.

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0.7 0.6 0.5

C e/q e

0.4 0.3 0.2 0.1 0 0 0.05 0.1 0.15 0.2 0.25 0.3

R = 0.9675

Ce (mg/mL)
Fig. 3. Linearized form of Langmuir model for adsorption of dialysate extract from common bean seed on anion exchange matrix AmberliteTM IRA 900 Cl.

Equilibrium parameters were determined from the Langmuir isotherm model considering the smooth continuous time course of protein adsorption (Fig. 2) that suggests the formation of protein monolayer on surface. From a linear relationship Ce/qe vs. Ce (Fig. 3) the maximum adsorption capacity was calculated to be 0.51 mg protein/mL matrix. It should be noticed that this result was based on dialysate extract and not on the puried protein. Obtained value for Xm is signicantly lower than those for coagulant protein from M. oleifera (Ghebremichael et al., 2006) even considering that different IEX matrixes were used. This might be also explained by the differences in molecular weight of coagulation active proteins from P. vulgaris and M. oleifera. Namely, it is estimated that coagulant protein from M. oleifera has molecular weight 6.5 kDa (Ghebremichael et al., 2005) i.e. 13 kDa for dimer (Ndabingengensere et al., 1995) while Mw of subunit of major storage protein (which is trimer) in common bean seed is $50 kDa (Belitz et al., 2004; Montoya et al., 2008). It is known fact that small macromolecules have higher adsorption per unit of weight (volume) of adsorbent (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). 3.3. Purication of coagulation active components The chromatogram of dialysate extract on anion exchange matrix is shown in Fig. 4. Dialysed extract (containing 3.3 mg protein) was loaded to column and then bound proteins were eluted by linear gradient NaCl. Coagulation activity of each fraction was determined 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 measured 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 (12 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 heterogeneity 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 puried common bean coagulant (CBC) was further analysed in coagulation study.

adsorption of 0.28 mg protein/mL matrix. Higher efciency of protein adsorption at higher matrix volume can be attributed to the greater number of active sites at matrix which increases the probability 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 (Ghebremichael 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 simultaneously blanks without matrix were carried out. Experiments conrmed 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 rst 10 min and after that incremental increase started to decline (Fig. 2). The time needed to reach the equilibrium was estimated to 6090 min when maximal values of adsorbed protein were determined.

0.15

100

1.0

Protein concentration (mg/mL)

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0.8

60

0.6

40 0.05 20

0.4

0.2

0.00
2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34

0.0

V(mL)
Fig. 4. Elution diagram of dialysate extract from common bean seed from anion exchange matrix AmberliteTM IRA 900 Cl.

C NaCl (mol/L)

0.10

Coagulation activity (%)

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3.4. Coagulation study The primary goal of coagulation/occulation is turbidity removal and this was investigated at conditions regarding initial turbidity 35 NTU according to Okuda et al. (2001b). Results of coagulation test of crude extract and the fraction containing puried 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 puried CBC and amounted 1 mL/L turbid water. Calculated on the base of protein concentration, the optimal coagulation dosage of puried CBC was 0.081 mg/L turbid water which expressed the highest coagulation activity 72.3%. Starting from the same initial turbidity, crude extract at its optimal dosage 0.73 mg/L turbid water exhibited the highest efciency in turbidity removal which resulted in coagulation activity 30%. Hence, calculated on the base of protein which was added in turbid water, the highest obtained coagulation activity of partially puried 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-

sides, the use of natural coagulants may increase the organic load in water which may result in increased microbial activity (Ndabigengensere 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, purication of the active components 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 extract and the partially puried 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 puried 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 puried CBC in comparison to crude extract was in accordance with lower protein concentration in tested fraction (9 times) but additionally it might be as well a consequence of the diminished load of other organic compounds from crude sample. Chemical oxygen demand value when partially puried CBC was applied was in the range of those ones obtained when puried 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 puried 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).

(a)
Coagulation activity (%)

100

4. Conclusion Coagulation activity of the protein from common bean seed obtained after optimization of purication procedure and the increase in organic load were about 22 times higher and more than 16 times lower, respectively, than that of crude extract. Purication of natural coagulant from common bean seed has no delipidation step, that is usual for coagulants from oily seed, which make its application in water turbidity removal potentially more economical and environmental friendly. Acknowledgement

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1.5

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Dosage (mL/L)

The nancial support from Ministry of Science and Technological Development, Republic of Serbia (Project No. 20064) is greatly acknowledged. References
iban, M., Adamovic , S., Klanja, M., 2007. Investigation of isolation Antov, M., c conditions and ion-exchange purication of protein coagulation components from common bean seed. APTEFF 38, 310. APHA (American Public Health Association), 1998. Standard Methods for the Examination of Water and Wastewater, 20th ed. APHA, AWWA, WEF, Washington DC, USA. Belitz, H.-D., Grosch, W., Schieberle, P., 2004. Food Chemistry, third ed. Springer, Berlin. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248254. Commission Directive 98/83/EC of 16 February 1998 on the quality of water intended for human consumption, Ofcial J. L, 123, 2640. Diaz, A., Rincon, N., Escorihuela, A., Fernandez, N., Chacin, E., Forster, C.F., 1999. A preliminary evaluation of turbidity removal by natural coagulants indigenous to Venezuela. Process Biochem. 35, 391395. Faust, S.D., Aly, O.M., 1987. Adsorption Processes for Water Treatment. Butterworths, Boston. Ghebremichael, K.A., Gunaratna, K.R., Dalhammar, G., 2006. Single-step ion exchange purication of the coagulant protein from Moringa oleifera seed. Appl. Microbiol. Biotechnol. 70, 526532.

(b)

10

COD (mg/L)

0 Blank Crude extract Purified CBC

Fig. 5. (a) Coagulation activity of crude extract () and partially puried CBC (h) at different dosages and (b) COD values in water samples treated by crude extract and partially puried CBC in comparison to blank.

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M.G. Antov et al. / Bioresource Technology 101 (2010) 21672172 Ndabigengensere, A., Narasiah, K.S., 1998. Quality of water treated by coagulation using Moringa oleifera seeds. Water Res. 32, 781791. Ndabigengensere, A., Narasiah, K.S., Talbot, B.G., 1995. Active agents and mechanisms of coagulation of turbid waters using Moringa oleifera. Water Res. 29, 703710. Okuda, T., Baes, A.U., Nishijima, W., Okada, M., 2001a. Isolation and characterization of coagulant extracted from Moringa oleifera seed by salt solution. Water Res. 35, 405410. Okuda, T., Baes, A.U., Nishijima, W., Okada, M., 2001b. Coagulation mechanism of salt solution extracted active component in Moringa oleifera seeds. Water Res. 35, 830834. Raghuwanshi, P.K., Mandloi, M., Sharma, A.J., Malviya, H.S., Chaudhari, S., 2002. Improving ltrate quality using agrobased materials as coagulant aids. Water Qual. Res. J. Can. 37, 745756. Scopes, R., 1994. Protein Purication, third ed. Springer, New York. iban, M., Klanja, M., Antov, M., krbic , B., 2009. Removal of water turbidity by c natural coagulants obtained from chestnut and acorn. Bioresource Technol. 100, 66396643. iban, M., Klanja, M., Stojimirovic , J., 2005. Investigation of coagulation activity c of natural coagulants from seeds of different leguminose species. APTEFF 36, 8187.

Ghebremichael, K.A., Gunaratna, K.R., Henriksson, H., Brumer, H., Dalhammar, G., 2005. A simple purication and activity assay of the coagulant protein from Moringa oleifera seed. Water Res. 39, 23382344. Kilduf, J.E., Karanl, T., Chin, J.-P., Weber Jr., W.J., 1996. Adsorption of natural organic polyelectrolytes by activated carbon: a size exclusion chromatography study. Environ. Sci. Technol. 30, 13361343. Mallevialle, J., Brichet, A., Fiessinger, F., 1984. How safe are organic polymers in water treatment. J. Am. Water Works Ass. 76, 8793. Miller, S., Fugate, E., Craver, V.O., Smith, J., Zimmerman, J., 2008. Toward understanding the efcacy and mechanism of Opuntia spp. as a natural coagulant for potential application in water treatment. Environ. Sci. Technol. 42, 42744279. Montoya, C.A., Laterme, P., Beebe, S., Souffrant, W.B., Molle, D., Lalles, J.-P., 2008. Phaseolin type and heat treatment inuence the biochemistry of protein digestion in the rat intestine. Brit. J. Nutr. 99, 531539. Morales-de Leon, J., Vazquez-Mata, N., Torres, N., Gil-Zenteno, L., Bressani, R., 2007. Preparation and characterization of protein isolate from fresh and hardened beans (Phaseolus vulgaris L.). J. Food Sci. 72, 96102. Narasiah, K.S., Vogel, A., Kramadhati, N.N., 2002. Coagulation of turbid waters using Moringa oleifera seeds from two distinct sources. Water Sci. Technol. 2, 8388.