Comparative study of flocculation and adsorption behavior of peregrina and seeds proteins

Shirin Nouhi1, Habauka M. Kwaambwa2, Philipp Gutfreund3 and Adrian R. Rennie1* 1. Centre for Neutron Scattering, Uppsala University, Box 516, 751 20, Uppsala, Sweden. 2. University of Science and Technology, Faculty of Health and Applied Sciences, Private Bag 13388, 13 Storch Street, Windhoek, Namibia. 3. Institut Laue - Langevin, 71 avenue des Martyrs, F-38000 Grenoble, France

* Corresponding author, E-mail address: [email protected]

Abstract

Trees of Moringa oleifera are the most widely exploited species of Moringa and proteins extracted from its seeds have been identified as the most efficient natural coagulant for water purification. Largely for climatic reasons, other Moringa species are more accessible in some regions and this paper presents a comparative study of the adsorption to different materials of the proteins extracted from seeds of and Moringa oleifera as a means to explore their use as flocculating agents in regions where each are more readily accessible. Results showed that Moringa peregrina seed proteins had higher adsorption to alumina compared to silica, in contrast to the opposite behavior for Moringa oleifera. Both species provide proteins that can act as effective coagulants for the various impurities in water with different surface potential. Despite considerable similarity in the amino acid composition, Moringa peregrina seed proteins have significantly different adsorption to those from Moringa oleifera and this presents the opportunity to improve processes by choosing the optimal species or combination of species depending on the type of impurity and the possible development of separation processes.

Keywords: Alumina, Coagulation, Colloidal particles, Flocculation, Moringa oleifera, Moringa peregrina, Silica, Water purification,

1 Introduction pH whereas cationic polymers are regarded as environmental pollutants that are toxic to fish (Liber, Challenges in water purification 2005). For these reasons, there has been increasing attention to find alternative flocculants particularly Remediation and purification of water remains a from natural products. A range of biopolymers have major challenge for environmental engineering in been identified that include polysaccharides and many areas: there is a continuing pressure to provide proteins (Bodlund, 2013; Muthuraman and Sasikala, safer water supplies. Costs must be carefully 2014; Teixeira et al., 2017; Verma et al., 2012; Choy controlled, and it is highly desirable to avoid the use et al., 2015) as potentially useful, however, feasibility of treatment chemicals that could be toxic if used of use of particular products is affected by lack of incorrectly or in excess. Furthermore, there is a need availability in some areas. to develop processes that do not require the supervision of trained technical personnel Background particularly in rural areas in developing countries. These reasons are some of the powerful drivers to The most widely exploited natural material for water investigate the exploitation of non-traditional treatment has been crushed seeds from Moringa technology in water purification. oleifera trees. This was first observed in use as a In treatment of water for human consumption or for traditional process in the valley of the Nile river but remediation of wastewater, a common first step is work by Broin et al. (2002), for instance, identified clarification by coagulation or flocculation of that the protein from the seeds is the effective particulate impurities that can then settle or float coagulant. Adsorption to various materials has been according to their density. This step is important both reported (Santos et al., 2012; Araujo et al., 2010; to remove potentially toxic material but also to enable Kwaambwa et al., 2010; Kwaambwa et al., 2015) and further processes such as disinfection where the it was shown that although the molecules are small, required amounts of oxidizing agents such as chlorine they bind effectively to many potential impurities as or ozone would be substantially increased if there well as associating so that flocculation occurs. The were too much organic residue. Even ultraviolet material effectively clarifies water with impurities irradiation requires adequate water clarity. Many that include mineral particles, bacteria and algae specifications for water treatment therefore impose (Hellsing et al., 2014; Lürling et al., 2010; Barrado- standards of sufficiently low turbidity not only Moreno et al., 2016). Engineering tests have been because discolored water is unattractive. made with various scales of process equipment from The process of flocculation of the impurities has been small scale jar-tests (Muyibi and Evison, 1995) up to described extensively (Chong, 2012) and involves trials (e.g. de Paula, 2014; Beltran-Heredia and controlled stirring and reduction the stability of Sanchez-Martin, 2009). particles. Often particulate impurities are negatively Although other seed proteins could be exploited to charged. In brief, colloidal stability is usually some extent in a similar manner, systematic reduced by adding polyvalent salts such as aluminum comparison of over a hundred natural coagulants sulfate or iron (III) chloride (Duan and Gregory, have shown that those of Moringa oleifera trees are 2003), or by adding synthetic polymers that are often the most effective coagulant (Bodlund et al., 2014; cationic (Gregory and Barany, 2011). The Bodlund, 2013; Conaghan, 2013; Kwaambwa, 2013; mechanism in the first case depends on screening the Kansa and Kumari, 2014; Muthuraman and Sasikala, charged repulsion that imparts colloidal stability to 2014). Seeds from Moringa trees are also a rich the particles. In the latter case, the polymers are source of oil and the widespread use of different parts chosen to adsorb readily and are usually sufficiently of the tree for nutrition and medicine in many large that they may link to several particles. Choice countries gives ready acceptance for use in the of flocculating agents and mixing conditions that treatment of drinking water. Moringa seed proteins produce large flocs that are compact or can be have been proposed for wider use as a biodegradable, dewatered readily is valuable for efficient processing. low-toxicity, low-cost and sustainable replacements There are several difficulties with conventional to the usual synthetic materials and they can be used treatment that arise from potential toxicity if high in a simple way without trained technical supervision concentrations of metal ions remain in the water. For (Stohs and Hartman, 2015). example, aluminum has been implicated in There are thirteen known species of Moringa trees Alzheimer’s disease (Flaten, 2001; Mirza et al., 2017; that are native to , Asia, Middle East and Exley, 2014). In the case of iron salts, it is often (Olson, 2002; Price, 1985; Shindano and necessary to precipitate excess them by increasing the Kasase, 2009) but some are grown in various regions 2 of the world, including regions with little rainfall. study provides an example of a heterogeneous Although Moringa oleifera is the most widely used, interface with both hydrophobic regions and some it is interesting to consider the possibility to exploit groups that carry negative charge. It provides a other varieties. There is much less known about the model for typical particles dispersed in water. alternative species and there are few comparative Neutron reflectometry was chosen as a technique that studies showing whether other species can be as allows determination of the structure and effective as Moringa oleifera in water treatment. The composition of interfacial layers as well as the species native to East Africa, , amount of material bound to the surface (Thomas and has been investigated (Hellsing et al., 2014). In Penfold, 1996). The reflected intensity measured as respect of the Moringa oleifera, there have been a function of wavelength and angle at low grazing several reports that the proteins are a complex angles can be analyzed to provide the molecular mixture. Moulin (2018) has reported extensive mass- composition profile. Neutron reflection is valuable spectrometry studies of the material that was obtained for studies of adsorption as it is straightforward to from Africa and the study clarified that certain apply, and the results are simple to interpret. components may bind differently to various However, it requires the use of large, flat substrates materials. that are transparent to neutrons so that the signal Moringa peregrina is another widely grown species reflected at low grazing angles of incidence can be of Moringa and it is native to various regions such as measured is sensitive to the adsorbed layer onto the Iran, the , , parts of Africa and surface (see Figure S1 and S2 in supporting as far north as the Dead Sea (Moustafa et al., 2012). information). Both silicon, which forms a native This geographical spread includes many areas where silica oxide layer on top, and alumina have the population suffer from limited access to clean insignificant absorption of neutrons and are readily water supplies. In the present study, we have chosen available as large and flat substrates. This provides seeds from two different varieties of Moringa trees another motivation to use silica and alumina for the from different regions, to explore their use as a adsorption behavior of proteins in this study. The flocculating agent, and to correlate that with flocculation caused by the seed proteins was tested molecular properties and the adsorption of the with a dispersion of polystyrene latex particles; and proteins to different materials. the transmission of visible light through the sample was measured. The zeta potentials of the proteins Design of the experiment were measured and the amino acid compositions of The adsorption of proteins has been studied at three the different samples were determined. types of surfaces with different surface charge and properties, so that the efficiency of seed proteins as Experimental flocculating agents for various impurities could be Materials investigated. Although purified proteins were used to The seeds used for this study were provided by local identify differences between samples, the extraction suppliers in Iran and Africa. Seeds of Moringa procedure, e.g. using cold water, was chosen so that oleifera from Africa were collected from Botswana, the samples would resemble those that would be used Iranian Moringa oleifera came from Bushehr, in practical applications. It is known that the Bushehr province, and the Moringa peregrina from extracted material obtained with high concentrations Nikshahr, province of Sistan and Baluchestan. Figure of salt or with hot water can be different (Madrona et 1 shows a picture of the seeds before deshelling. al., 2010; Ali et al., 2010, Sánchez-Martín et al., Seeds of Moringa oleifera from Iran and Botswana 2010). Surfaces chosen for this study were are both winged and look very similar, whereas polystyrene colloidal particles and flat silica and Moringa peregrina seeds have an unwinged hard alumina surfaces. These provide model systems of shell. The Moringa oleifera from Botswana was the interfaces with different properties but characteristics same sample as that used in previous studies which can be relevant for water purification. Silica (Hellsing et al., 2015). and alumina are models for mineral particles that can The seeds were stored at room temperature prior to be found as impurities in water. At neutral pH, silica extraction of the proteins. The extraction procedure carries a negative charge when immersed in water was similar for all samples and started by deshelling whereas alumina is neutral or slightly positive and crushing the seeds using a kitchen blender. For (Kosmulski, 2009). Their comparison allows the Moringa peregrina because of the consistency of exploration of the effect of surface charge on the the oily product, it was necessary to use a pestle and adsorption of proteins. Polystyrene latex used in this mortar as well. 3 Charge stabilized polystyrene colloidal particles with 720 Å radius (polydispersity < 1%), PS3, were used as a model system for colloidal impurity in water. The particles were synthesized and characterized as described in previous articles (Hellsing et al., 2012; Rennie et al., 2013). The surface potential of the particles was determined to be about -30 mV at pH 7. Substrates for this experiment were 5 cm × 5 cm × 1 cm crystal of sapphire (alumina, Al2O3) and the amorphous silica (SiO2) layer formed on silicon crystal. The crystals were cleaned prior to the experiment by immersing in diluted Decon90 in a clean Petri dish for a few minutes and then rinsing with Milli-Q water. The cleaning was continued by spreading drops of concentrated sulfuric acid over the reflecting surface of the crystal and approximately the Figure 1. Moringa seeds used in this study include same amount of water for a few minutes before the Moringa peregrina (a), Moringa oleifera from Iran (b) and surface was then rinsed extensively with water. Moringa oleifera from Botswana (c). Cleaning with acid was repeated two or three times until the surface of the crystal was clearly hydrophilic Crushed seeds were then mixed with petroleum ether, with no measurable contact angle for water. 40-60 °C, (1:5 ratio) and stirred on a magnetic stirrer for about 2 hours to dissolve the oil from the seeds. Methods and procedures The paste was separated using vacuum filtration and The Zeta potential was measured for different the resulting powder was dried in air overnight. The proteins concentration in water using a Malvern powder was then dissolved in deionized water (each Zetasizer Nano ZS in disposable cells using the dip 50 g in about 200 mL water) using a magnetic stirrer accessory, ZEN1002. The analysis of amino acid for about an hour and the solution was filtered using composition was performed by Alphalyse, Denmark. 2-3 times until a relatively clear solution was The acid hydrolysis is performed at 110°C in 6M obtained. The proteins were then precipitated from HCl, 0.1% phenol and 0.1% thioglycolic acid under the solution as a sticky paste by adding ammonium reduced pressure in argon atmosphere. The amino sulfate to saturation. The paste was dissolved in acid separation was performed using ion exchange deionized water and filtered to remove large particles. chromatography, the derivatization with ninhydrin, The solution was dialyzed against high purity water and the detection of various components at 570 and using cellulose membrane tubing (Sigma-Aldrich 440 nm. As an internal control, the sample was D9527-100FT, molecular weight cut-off 14 kDa) to prepared with a known amount of sarcosine. remove ammonium sulfate. To further purify the Flocculation of particles caused by the proteins was proteins, dialyzed solution was added to a tested by measuring the ultraviolet (UV) transmission carboxymethyl cellulose packed column, to which spectra of dispersions of polystyrene latex particles in the proteins bind. The bound proteins were then water both in the absence and presence of Moringa released and eluted with a 1 M NaCl solution. Eluted seed proteins, using a Lambda 35 UV/VIS solution was dialyzed using dialysis tubing as above spectrophotometer. The sample holders for the UV in order to remove the remaining salt and the protein transmission measurements were 1 mm path length powder was finally obtained by freeze drying. fused quartz cuvettes as shown later in results. A further small difference in the extraction procedure Spectroscopy data is commonly shown as was the type of cellulose column used to purify the transmission of the beam through the sample at protein. Proteins from the seeds of Moringa oleifera different wavelengths. According to the Beer- from Botswana were extracted previously using CM- Lambert law, transmission (τ) is defined as: Cellulose microgranular 25-60 µm purchased from Sigma-Aldrich (C4021) which is now discontinued, τ = = (1) whereas the proteins from seeds from Iranian, used .. Ionsep CMC 52 preswollen carboxymethyl cellulose was purchased from Biophoretics. where ϵ is the absorptivity, l is the path length of beam and c is the concentration of the sample, I is the 4 intensity of the transmitted beam and Io is the Results and Discussion incoming beam. Flocculation of Particles The amount and the structure of the proteins adsorbed to different surfaces were determined as a function of The dispersion of 0.5 wt. % PS3 latex particles in the solution concentration using neutron deionized water with no additive is shown in Figure reflectometry. In this experiment, the intensity of the 3 (labelled as a) together with the other samples that reflected beam was measured at a given angle (where also contain 0.5 wt. % of particles dispersed in the angle of incoming and outgoing beam is the aqueous solutions that contain 0.2 wt. % of the same). The reflected intensities for different various different Moringa seed proteins as observed interfacial structural models were calculated and about 10-15 minutes after mixing. Sedimentation of fitted to the data using a least squares minimization particles due to flocculation by Moringa seed proteins to obtain the best fits. (Rennie, 2018). Figure 2 shows is evident for the samples b - d and looks similar for a schematic of specular neutron reflection experiment different Moringa seed proteins used in this study. where θi = θf (= θ).

Figure 3. 0.5 wt. % PS3 latex particles in water in the absence of Moringa seed proteins (a) and mixed with 0.2 wt. % of Moringa peregrina seed proteins (b), Figure 2. A schematic representation of neutron Moringa oleifera seed proteins from Iran (c), and reflectometry experiment. The reflection is Moringa oleifera seed proteins from Botswana (d). determined as the ratio of the intensities of the reflected and incident beam. In order to make a quantitative comparison of the Sample holders used for neutron reflectometry flocculation of particles, transmission spectra for experiment are described in detail by Rennie et al. visible light were recorded for the samples shown in (2015). In this design, the sample was contained by Figure 3. Figure 4 shows the light transmittance a 2 mm thick polytetrafluoroethylene (PTFE) gasket spectra for the samples in the absence and in the between two crystals. This allows measurements of presence of different seed proteins. The the same solution with two different surfaces by transmittance of dispersions with different Moringa rotation and translation to bring the different seed proteins measured by more than a factor of five interfaces into alignment. The crystals were clamped from the initial values for all three types of seed together with an aluminum frame that has channels proteins. This suggests that efficiency of the Moringa for circulating water that allows control of the sample peregrina seed protein as a flocculating agent is as temperature. For these measurements, samples were high as Moringa oleifera seed proteins. The results kept at 25°C. Data are reported as the ratio of the also showed that for the same species grown in reflected intensity to that of the incident beam as a different regions, water clarification is similar. function of Q that is given by Q = (4π/λ) sin θ. Amount and Structure of Bound Protein Layers The stock protein solutions were prepared at 0.1 or 0.2 wt. % concentration in water (highest measured The amount of protein bound to the two surfaces at concentration) by tumbling overnight. The pH (pD, various protein concentrations is shown in Figure 5. corresponding to the samples prepared in D2O for All proteins showed adsorption to both alumina and neutron reflection experiment) of 0.2 wt% solutions silica and the bound amount reaches a plateau at were found to be in the range 6 to 6.8. The stock concentrations of about 0.1 wt. %. solutions were diluted to the desired concentrations during the neutron experiment using a Knauer HPLC pump with a flow rate of 2 mL min-1. 5 extract proteins contain several proteins. It has been shown that the proteins extracted from seeds can vary according to the specific extraction procedure, explicit descriptions of various materials with different molecular mass have been described previously (Shebek et al., 2015; Freire et al., 2015; Nordmark et al., 2016). There are small differences, for example in the amount of material bound to silica (Kwaambwa et al., 2010; Nouhi et al., 2018) that have been reported for samples of Moringa oleifera extracted with the same procedure. However, the overall pattern of adsorption and coagulation behavior are normally the same. These results revealed that proteins from seeds of Moringa peregrina form a denser layer at the alumina Figure 4. Visible light transmission measured for the surface (70 % vol. proteins) compared to the silica dispersion of 0.5 wt. % of PS3 particles, 0.2 wt. % surface (50 % vol.), whereas the Moringa oleifera Moringa peregrina (MP), and a mixture of different seed proteins from Iran tree have a denser layer on the Moringa seed proteins species with particles in water. silica surface (70 % vol.) surface than the alumina MO stands for Moringa oleifera. surface (50 % vol.). For the seed proteins from both Moringa oleifera and Moringa peregrina collected Seed proteins from Moringa oleifera grown in from Iran, the width of the decaying density profile Botswana showed higher adsorption on both was twice as large on alumina (~ 20 Å) as compared surfaces. At the same concentrations, Moringa to silica (~ 10 Å). The different structure observed peregrina proteins showed more adsorption to for these materials might arise from different alumina than to silica. The Moringa oleifera seed compositions in the samples with some specific proteins from the trees grown in Iran showed higher protein molecules binding more to different surfaces. adsorption to silica, for the single concentration that could be measured in the allocated beam time, than to The structure of the layer close to the surface for the alumina but the overall amounts bound to both Moringa oleifera seed proteins from Iran and surfaces were lower than that seen for the sample Botswana were very similar, however, the seed from Botswana. This provides ideas as to which proteins from the later were observed to have a species could be more effective for aggregating slightly thicker dense layer on both surfaces and a certain types of impurity in water. region with a decaying density profile that was nearly The best fitting models to the neutron reflectivity data twice as wide on the silica surface. This suggests that (Figure S3, supporting information) showed that the these differences in source material might alter the adsorption of proteins to both silica and alumina capability to form multilayers and thus change the surfaces could be described with dense protein close amount required to give rise to saturated coverage of to the solid surface and a further region with an a particular surface. exponential decay of protein concentration to that in For the effect of rinsing with water for the three the bulk solution. The inset plot on the top right of samples of Moringa seed proteins, the reflectivity Figure 5 shows an example of the density profile of was studied by rinsing the surface that had been protein layers adsorbed to the solid surface as a exposed to the highest concentration of Moringa seed function of distance from the interface. It has been proteins with water. The cell was flushed with about shown previously that proteins extracted from the 30 mL of water (> 10 times volume of the cell). The seeds of Moringa oleifera contain several distinct reflectivity did not change from that seen in the proteins (Kwaambwa et al., 2010, Moulin, 2018), presence of the solution from the adsorbed layer at which is likely to be the origin of the observed high highest concentration after rinsing (see Figure S5 in adsorption in a form of multilayer to solid surfaces Supporting information). This indicates that the (Moulin et al., 2018). Multilayer adsorption was also protein layer is irreversibly bound to the interface or found in this study for the proteins extracted from the would require some other chemical change in the seeds of Moringa peregrina, suggesting that the solution to displace the adsorbed material.

6

Figure 5. The bound amount (surface excess) for protein from Moringa peregrina, and Moringa oleifera seeds from Iran and Botswana on alumina (left) and silica (right) surfaces determined from the neutron reflection data. The adsorbed amount of Moringa oleifera seed proteins from Botswana on silica is taken from the work of Nouhi et al., (2018) and on alumina from Kwaambwa et al. (2015). The inset shows the density profile for Moringa peregrina on silica at 0.2 wt%.

Molecular and Surface Potentials of Moringa seed proteins used in this study are shown As mentioned above, the interfacial potentials for in Figure 6. The method used did not identify alumina and silica in pure water are known to be tryptophan and cysteine. Asparagine is included as different (Kosmulski, 2009). To understand the role aspartic acid, and glutamine as glutamic acid. of charge effects on binding, zeta potentials for all the Degradation of methionine, serine and threonine proteins were measured at different concentrations in could reduce the determined fractions by 10% of the reported values but significant differences were not H2O and they were all found to be +20±5 mV (see Figure S4 in supporting information). The positive reported for these components. zeta potential suggests that the differences observed in the adsorption behavior is unlikely to be simply due to the overall surface charge but other binding mechanisms and chemical influences could be important. Protein Amino Acid Composition Several studies have reported either the amino acid composition of Moringa oleifera seed proteins (Gassenschmidt et al., 1995; Oliveira, et al., 1999; Kwaambwa et al., 2010; Shebek, et al., 2015) or the sequence of a few specific molecules. There has also been one report of the composition for Moringa peregrina (Al Juhaimi et al., 2016). There are some differences in the reported compositions that may be due to region, harvesting conditions, climate, and/or the extraction and purification process. The amino Figure 6. The amino acid composition for samples of acid composition determined for the different species the different Moringa seed proteins used in this study.

7 Noticeable differences in the amino acid composition Understanding the adsorption and association can are seen for glycine, glutamic acid/glutamine, help finding an effective aggregation agent native to alanine, arginine and proline (with a maximum of 5 some regions without having to access a specific % difference). Arginine is positively charged and species or produce what is needed for a specific glutamic acid is negatively charged at neutral pH. purpose and develop the purification technologies by Seed proteins from Moringa oleifera grown in Iran modifying the species of the or their growing showed slightly lower arginine and higher glutamic conditions. acid/glutamine compared to that grown in Africa (2- Moringa peregrina seed proteins have broadly 3 %). This may explain the higher adsorption of the similar surface charge and amino acid compositions proteins from trees grown in Botswana (Kwaambwa to Moringa oleifera seed proteins. The extracted et al., 2015) compared to that of the trees grown in material consists of several proteins and adsorb to Iran. Seed proteins of Moringa oleifera and Moringa mineral surfaces as a multilayer, in a similar way to peregrina from the trees grown in Iran, show a Moringa oleifera seed proteins. Despite the difference in the fraction of arginine residues. similarities, there are interesting differences such as Moringa oleifera seed proteins show a higher fraction in the adsorbed amount and the structure of layers at of arginine which can explain higher adsorption to solid surfaces. Seed proteins of Moringa peregrina silica compared to Moringa peregrina. There are offer unique adsorption properties as they bind more reports of different amounts of overall oil and protein to an alumina surface than a silica surface. This could as well as differences in composition for Moringa pave the way towards developing new methods for peregrina in a study evaluating its use as a food purifying certain impurities which cannot be removed source (Vaknin and Mishal, 2017). as effectively using Moringa oleifera seed proteins There is a possibility that these small changes in and separation procedures might be developed or use amino acid composition can be responsible for the of the optimum mixture of different proteins could differences seen in the adsorbed amount and the increase the efficiency of aggregation. Just like for structure of seed proteins adsorbed to different Moringa oleifera seed proteins, the adsorption of surfaces. However, there is also a possibility that Moringa peregrina seed proteins onto mineral there are other differences, for example, in the surfaces reaches a plateau at a concentration of sequence of the proteins that would not be identified approximately 0.1 wt. %. This knowledge of the by this simple analysis. It has been shown that amount needed in solution helps to optimize the different sequences of Moringa oleifera seed proteins amount used and avoid unnecessary residual material with differences in only a few amino acid residues being left in processed water. can show significantly different adsorption properties Seed proteins from same species grown in different (Moulin et al., 2018). To test whether these effects regions also showed a different adsorbed behavior, arise from differences between species, growth i.e. thicker layer with a longer decaying length was conditions or other factors and to understand these at observed for the protein of Moringa oleifera seeds the molecular level would require a much larger grown in Botswana. This suggests that by modifying variety of seed samples grown under controlled the trees and/or the growing conditions, proteins with conditions. the most effective properties can be obtained. However, to achieve this goal, further studies are Summary The present study provides a comparison between the required to understand and distinguish between the interactions of proteins extracted from seeds of various different effects, for example how the Moringa peregrina and Moringa oleifera tree, with properties of seeds can vary between different trees model mineral surfaces and colloidal particles. grown on the same field, same tree grown in different Moringa oleifera seed proteins have been suggested condition, harvesting season, extraction procedure, as the most effective natural coagulant/flocculant for etc. water purification purposes, however, they only grow Acknowledgments in specific regions. Moringa peregrina grows as The work was supported in part by the Swedish native species in different regions of the world but has Research Council (grants 348-2011-7241 and 621- been less exploited as a coagulant. The results of this 2012-4382). The authors would like to thank Dr study show that Moringa peregrina seed proteins can Amirhossein Mahvi for providing Moringa seeds provide an effective flocculent for colloidal particles from Iran and the Institut Laue Langevin, Grenoble, and that it acts in a similar way to Moringa oleifera France for the allocation of measurement time seed proteins in the clarification of water. (doi.ill.fr/10.5291/ILL-DATA.9-13-725). We also thank the Ångström Laboratory workshop for making 8 the sample holders, the polymer chemistry group for Duan, J., Gregory, J., (2003) Coagulation by hydrolysing the use of the UV spectrometer instrument and the metal salts. Advances in Colloid and Interface Science 100 – nanoscience group for Nanosizer instrument. 102, 475–502. Exley, C., (2014). What is the risk of aluminium as a References neurotoxin? Expert Rev. Neurother., 14, 589-591. Flaten, T. P., (2001). Aluminium as a risk factor in Ali E. N., Muyibi S. A., Salleh H. M., Alam M. Z., Salleh M. 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