EP1409418A1 - Floculants - Google Patents

Floculants

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Publication number
EP1409418A1
EP1409418A1 EP02742639A EP02742639A EP1409418A1 EP 1409418 A1 EP1409418 A1 EP 1409418A1 EP 02742639 A EP02742639 A EP 02742639A EP 02742639 A EP02742639 A EP 02742639A EP 1409418 A1 EP1409418 A1 EP 1409418A1
Authority
EP
European Patent Office
Prior art keywords
proteins
protein
seed
oil
seeds
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP02742639A
Other languages
German (de)
English (en)
Inventor
Ian William Marison
Stephan Christos DÖRRIES
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Optima Environnement SA
Original Assignee
Optima Environnement SA
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Optima Environnement SA filed Critical Optima Environnement SA
Publication of EP1409418A1 publication Critical patent/EP1409418A1/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F1/00—Treatment of water, waste water, or sewage
    • C02F1/52—Treatment of water, waste water, or sewage by flocculation or precipitation of suspended impurities
    • C02F1/5272—Treatment of water, waste water, or sewage by flocculation or precipitation of suspended impurities using specific organic precipitants
    • C—CHEMISTRY; METALLURGY
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F1/00—Treatment of water, waste water, or sewage
    • C02F1/52—Treatment of water, waste water, or sewage by flocculation or precipitation of suspended impurities
    • C02F1/5263—Treatment of water, waste water, or sewage by flocculation or precipitation of suspended impurities using natural chemical compounds

Definitions

  • the present invention relates to flocculating agents.
  • the most frequently employed inorganic flocculants include polyaluminium chloride, aluminium sulphate (Alum) and iron salts.
  • polyaluminium chloride aluminium sulphate (Alum)
  • iron salts such compounds have either been implicated in important human medical conditions, including Alzheimer's disease, or are suspected of having strong carcinogenic properties.
  • Synthetic organic polymers such as polyacrylamide have become increasingly used for water treatment, however in addition to cost there remains the problem of disposal of the flocculent residues after the treatment process.
  • flocculants may not be readily available in geographic environments, such as those in developing countries.
  • a major drawback to the application of the presently known Moringa seed protein extracts is that the flocculating activity is reported to be efficient for waters with high solids loading and not efficient for waters with low solids.
  • the exact nature of the flocculating activity is unclear with reports of non- protein, non- polysaccharide components of the seed extracts, with molecular weights of 3kDalton, being responsible.
  • oilseed crops such as soybean, rapeseed, sunflower and palm have been reported to contain a water insoluble oil fraction which is stored in discrete subcellular structures commonly referred to as oil bodies, oleosomes, lipid bodies or sphaerosomes.
  • oil bodies also comprise phospholipids and a number of proteins termed oil body proteins. It is believed that oil bodies can be considered to be an oil (triglyceride) matrix surrounded by a monolayer of phospholipids in which oil body proteins are embedded.
  • amphipathic proteins can act as flocculating agents.
  • amphipathic proteins such as oleosins, albumins and storage proteins
  • oleosins such as oleosins, albumins and storage proteins
  • storage proteins such as rapeseed, sunflower, peanut and jojoba.
  • proteins can be obtained according to specific processes. They can be extracted using aqueous solutions, in the presence and absence of salts, or via isolation of oil bodies. Crude and purified preparations of these extracts have been shown to have flocculation activity for the treatment of drinking and waste- waters and may be used alone or in conjunction with existing flocculating agents. Crude and purified preparations of these extracts have also been shown to have flocculation activity for the removal of cells, metals and organic compounds from fluids and may be used alone or in conjunction with existing flocculating agents in the preparation of feed, food, pharmaceutical and industrial products.
  • amphipathic is defined as proteins containing regions that are polar (or charged) and regions that are non- polar.
  • the polar, hydrophilic region interacts favorably with the solvent and tends to dissolve, but the non- polar, hydrophobic region tends to avoid contact with the water and cluster together or interact with other hydrophobic molecules or particles (reference: Lehninger: Principles of Biochemistry, 3rd edition (2001). Editors D.L. Nelson and M.M. Cox. Worth Publishers).
  • Examples include oil body proteins and oleosins in which the proteins are concentrated at the surface of oil droplets within the seeds, with the hydrophobic regions of the protein(s) associated with the hydrophobic oil and the hydrophilic, or charged, groups of the protein(s) are associated with the aqueous environment within the seeds or cells of the seeds.
  • the non- polar regions of the protein(s) cluster together to present the smallest hydrophobic area to the aqueous solvent, and the polar regions are arranged to maximize their interaction with the solvent.
  • Such stable structures in water are called micelles with the forces holding together the non-polar regions of the proteins termed hydrophobic interactions.
  • the flocculating activity of the proteins from oilseeds results from the unique amphipathic structure, in which particles within the aqueous phase (clays, silica, microorganisms etc) associate with either the non- polar or polar regions of the proteins to form complexes, which may involve micellar- like structures, with a density different from that of the water solvent, with the result that the complexes sediment or float and may be readily separated from the solvent.
  • the invention relates therefore to the use of amphipathic proteins as flocculating agents.
  • amphipathic proteins used are plant seed proteins such those from rapeseed, sunflower, soybean, peanuts, rice, tamarind, jojoba and others, as obtained according to specific extraction processes.
  • the method for the extraction of the proteins may involve de-husking of the seeds followed by grinding of the resulting de- husked seeds and extraction of the proteins using either hot or cold water, which may contain inorganic salts and reducing agents such as beta- mercaptoethanol or dithiothreitol.
  • extraction may involve mechanical homogenisation of whole or de- husked seeds, and the recovery of oil bodies via centrifugation or filtration.
  • the extraction procedure may contain water, inorganic salts, non- ionic detergents, organic solvents or combinations of these. The process, which is dependent on the raw plant material used, enables the production of crude protein extracts, which may or may not have associated oils or other lipid materials.
  • the crude extracts may be purified by standard chromatographic techniques, including size- exclusion, ion- exchange, hydrophobic interaction, reverse- phase or other, to isolate individual protein components with defined flocculating activity.
  • the aim is to produce natural protein materials, either as crude protein mixtures or as purified individual proteins, from a wide variety of commonly available agricultural crops for water and product purification, in order to replace the existing techniques involving the addition of aluminium sulphate (alum), other inorganic salts such as iron salts, or polymers composed of polyacrylamide, chitosan or others. It is believed that the protein extracts are innocuous to human and animal health, and may be readily biodegraded in the natural environment without a negative impact on environmental pollution loading, at the concentrations employed. Purification of the crude preparations can be used for the removal of contaminating plant materials which may impart a variety of undesirable properties, which may include allergenicity, undesirable odour, flavour or colour.
  • the proteins according to the invention are non- toxic, natural products, which have no adverse effects on the quality of the treated product, e.g. water.
  • the present invention also includes methods of extraction of the seed proteins, since the present inventors have found that the flocculent activity of the crude extracted proteins is a function of the conditions employed. Crude seed protein extracts are defined as the protein mixtures obtained using the extraction procedure. The invention also covers the purification of these extracts to obtain purified individual proteins with defined flocculating activity.
  • the present invention provides for formulations of proteins, crude extracts or purified protein preparations, for applications in flocculation, e.g. of water to generate drinking water or in the preparation of industrial products.
  • Flocculation activity is defined as the ability of the protein extracts to combine with particles, such as glass beads, clay particles, diatomaceous earth particles, cells, including bacteria, yeast, fungi, algae, plant and animal, as well as metal ions, organic compounds and others present in fluids, with the ability to complex such particles or components and thereby facilitating removal of them.
  • the invention also provides for use of crude and purified seed protein extracts for the removal of such components from fluid sources other than used in water treatment, such as clarification of beer, wines and other fermentation broths, for removal of cells from fluids or for metal removal from fluids.
  • the present invention provides methods for extraction of the seed proteins and the application as flocculants in water treatment and in the treatment of other fluids such as blood, milk or any other similar liquid.
  • the methods for preparing the protein extracts from the plant seeds comprise:
  • reducing agents are added to the aqueous salt solution (2b) during protein extraction procedure for crude seed proteins.
  • the crude oil body and seed protein extracts (1 e) and 2(e) are stirred and heated for a defined time, subsequently cooled to room temperature, and insoluble components removed from the heated crude protein extracts.
  • the resulting heat- treated crude protein extracts are stable to storage at room temperature.
  • the crude and purified oil body and oil seed protein extracts can be added to a wide range of aqueous solutions containing suspensions of solid particles, such as glass particles, clay particles, and other particles commonly found in natural waters (rivers, lakes and other ground waters) in order to flocculate the particles and thereby facilitate removal by sedimentation, flotation or other.
  • the seed protein preparations are particularly suitable for the removal of all types of cells including bacteria, yeast and other fungi, microbial spores and animal cells as well as a wide range of metals and organic materials.
  • the protein extracts are suited for the removal of particles, metals, cells and organic compounds from a wide range of aqueous environments involved in the preparation of food and feed products, such as for the preparation of beverages such as beers and wines, pharmaceutical products and industrial products such as cell removal from cell culture broths.
  • Figure 1 shows a SDS-PAGE (polyacrylamide gel electrophoresis) of extracts of seed proteins, oil body proteins and synthetic peptides from rapeseed and Moringa oleifera.
  • Dry seeds of Moringa, rapeseed, sunflower and other seeds were dehusked manually and homogenized using a Polytron for 40 seconds at maximum power in 4 volumes of cold (4°C) homogenization buffer (0.15M Tricine buffer pH 7.5 containing 1 mM EDTA, 10mM KCI, 1mM MgCI 2 , 2mM dithiothreitol and 0.6M sucrose).
  • the homogenate was filtered through a nylon membrane (20 ⁇ m pore size) to remove large particles and seed debris.
  • Clarified homogenate was diluted with 1 volume flotation buffer (0.15 M Tricine pH 7.5 containing 0.4 M sucrose, 1 mM EDTA, 10mM KCI, 1mM MgCI 2 and 2 mM dithiothreitol) and centrifuged for 30 minutes at 10,000 g. Oil bodies were collected from the surface of the centrifuged suspension and added to 0.5 volumes of the homogenization buffer containing 2M NaCI to re- suspend. A further 0.5 volumes of homogenization buffer, containing 2 M NaCI and 0.25 M sucrose in place of 0.6 M sucrose, were added to the surface of the oil body suspension followed by centrifugation for 30 minutes at 10,000 g.
  • 1 volume flotation buffer (0.15 M Tricine pH 7.5 containing 0.4 M sucrose, 1 mM EDTA, 10mM KCI, 1mM MgCI 2 and 2 mM dithiothreitol
  • Oil bodies were collected from the surface of the centrifuged suspension and re- suspended in 0.5 volumes of homogenization buffer followed by re-centrifugation for 30 minutes at 10,000 g. The washing procedure was repeated and the oil bodies re- suspended in homogenization buffer to give a final concentration of 100 mg per liter (in general achieved by addition of 20 volumes of homogenization buffer to oil bodies and stored at 4°C.
  • the crude oil body protein extracts prepared in this way have been analyzed by SDS gel electrophoresis after the addition of SDS. Material prepared in this way can be employed directly as a crude oil protein preparation for flocculation of aqueous particle suspensions.
  • Crude oil body proteins prepared according to Example 1 could be purified by recovery of the oil bodies from the surface of the buffer after the final centrifugation step followed by the addition of an organic solvent such as acetone, hexane or other to remove the associated triacylglycerides. Solvent- treated oil body proteins were then recovered by centrifugation for 2 minutes at 13,500 g. Oil body proteins were recovered from the surface of the centrifuged samples, washed with organic solvent (acetone, hexane or other) and re- centrifuged under the same conditions. A second washing step was then carried out by resuspending the oil body proteins in diethyl ether and re- centrifuged for 2 minutes at 13,500 g.
  • organic solvent acetone, hexane or other
  • Oil body proteins were recovered form the last centrifugation step and resuspended in ultra- high purity (UHP) water containing 1.5 volumes of a 2:1 mixture of chloroform in methanol. The latter was centrifuged for 4 minutes at 10,000 g and the purified oil body proteins isolated from the water solvent interface. The isolated proteins were then washed twice with the water/chloroform/methanol solution, centrifuged for 4 minutes at 10,000 g. The purified oil body proteins were then recovered from the water- solvent interface and a dried protein preparation made by evaporation of the organic solvent under an atmosphere of nitrogen gas. The purified oil body proteins prepared in this way could be stored at 4 °C indefinitely.
  • UHP ultra- high purity
  • the purified oil body protein extracts prepared in this way have been analyzed by SDS gel electrophoresis after the addition of SDS.
  • Dry seeds of Moringa, rapeseed, sunflower or other were dehusked manually and homogenized using a Polytron for 40 seconds at maximum power in 4 volumes of cold (4°C) homogenization buffer (0.15M Tricine buffer pH 7.5 containing 1 mM EDTA, 10mM KCI, 1 mM MgCI and 0.6M sucrose).
  • the homogenate was filtered through a nylon membrane (20 ⁇ m pore size) to remove triglycerides and oil bodies. The remaining solids material was collected and termed press- cake. Seed proteins were extracted by re- suspending the press- cake in 5 volumes of salt solution followed by stirring for 1 hour.
  • Extracted seed proteins were recovered by centrifugation for 5 minutes at 1 ,500 g followed by decantation through a fine cotton cloth. Decanted seed protein extracts were heated to 85°C with gently stirring and subsequently cooled to room temperature before centrifugation for 5 minutes at 1 ,500 g. The supernatant was collected and could be stored at room temperature.
  • the crude seed protein extracts prepared in this way have been analyzed by SDS gel electrophoresis after the addition of SDS.
  • Material prepared in this way can be employed directly as a crude seed protein preparation for flocculation of aqueous particle suspensions.
  • Example 3 The procedure is followed according to Example 3 except that a reducing agent, such as 1 % dithiothreitol (DTT) was added to the extraction salt solution.
  • a reducing agent such as 1 % dithiothreitol (DTT) was added to the extraction salt solution.
  • DTT dithiothreitol
  • Material prepared in this way can be employed directly as a purified oil protein preparation for flocculation of aqueous particle suspensions.
  • Figure 1 shows a SDS- PAGE (polyacrylamide gel electrophoresis) of extracts of seed proteins, oil body proteins and synthetic peptides from rapeseed and Moringa oleifera.
  • lanes 1 & 15 Low molecular weight protein standards (Sigma)
  • lane 2 Total oil body proteins extracted from M. oleifera under reducing conditions
  • lane 3 Total oil body proteins extracted from M. oleifera under non- reducing conditions.
  • lane 4 Total oil body proteins extracted from M. oleifera under reducing conditions in the presence of protease inhibitors.
  • lane 5 Synthetic peptide (sequence according to Gassenschmidt et al., 1995).
  • lane 6 Seed protein extract under non -reducing conditions from defatted M. oleifera seeds (press cake).
  • lane 7 Total oil body proteins extracted from rapeseed.
  • lane 8 BSA, reducing loading buffer 10 ⁇ g lane 9 Total oil body proteins extracted from rapeseed lane 10
  • lane 11 Total oil body proteins extracted from M. oleifera under non- reducing conditions.
  • lane 12 Total oil body proteins extracted from M. oleifera under reducing conditions in the presence of protease inhibitors
  • lane 13 synthetic peptide (sequence according to Gassenschmidt et al., 1995).
  • lane 14 Seed protein extract under reducing conditions from de- fatted M. oleifera seeds (press cake).
  • amphipathic protein extracts for the removal of glass particles from water
  • This method forms the basis of the flocculation assay test for the measurement of flocculation activity of amphipathic proteins (oil body proteins and others) extracted according to examples 1 and 2.
  • the method may also be applied using the same conditions and procedure except that the glass beads are replaced with diatomaceous earth particles.
  • spectrophotometer Into 3 ml spectrophotometer cuvettes were placed 2ml of 10mM sodium phosphate buffer pH 7.0 containing 7. 71 g/l sodium chloride which had been previously filtered through a 0.2 ⁇ m membrane filter. A small Teflon- coated magnetic stirring bar (diameter 3 mm, length 8 mm) is then placed in the cuvette. The cuvette is then placed in a spectrophotometer and the magnetic stirrer adjusted to give a stirring rate of 800 rpm. The spectrophotometer is set to a wavelength of 500 nm and the absorbance adjusted to give a value of zero.
  • a suspension of glass beads is prepared by addition of 100 mg of Spheriglass® 5000 (Potters- Ballotini) to 1 liter of 10mM sodium phosphate buffer pH 7.0 containing 7. 71 g/l sodium chloride which had been previously filtered through a 0.2 ⁇ m membrane filter.
  • the particle suspension is vortex agitated and 10 ⁇ l added immediately to the stirred cuvette.
  • the spectrophotometer absorbance values are recorded for 5 minutes before the addition of 1 to 50 ⁇ l of amphipathic protein extract and the spectrophotometer absorbance values recorded for a further 10 minutes.
  • the magnetic stirring is then stopped and the spectrophotometer absorbance values recorded for a further 15 minutes.
  • Flocculation activity is determined by calculation of the initial slope of the absorbance values as a function of time during the period after addition of the amphipathic protein extract. Flocculation activity is given as absorbance (or extinction) units per minute. The absorbance measured 25 minutes after addition of the amphipathic protein extract provides a value for the residual turbidity of the suspension.
  • the glass beads (Spheriglass® 5000, Potters- Ballotini) have an average diameter of 3.5 to 7.0 ⁇ m with 90% of the beads having a diameter within the range 0.5 to 19.3 ⁇ m.
  • Flocculation activity may also be determined using the same assay conditions and procedure except that the glass beads are replaced by acid- washed diatomaceous earth (98% Si0 2 , Sigma Chemical Co., St. Louis, MO, USA Product no.D-3977).
  • This method forms the basis of the flocculation assay test for the measurement of flocculation activity of seed proteins extracted according to examples 3 and 4.
  • the method may also be applied using the same conditions and procedure except that the glass beads are replaced with diatomaceous earth particles.
  • the procedure used is the same as that described by Example 5 except that the assay was initiated by the addition of 1 to 50 ml of seed protein extract instead of 1 to 50 ⁇ l of amphipathic protein extract.
  • Flocculation activity is determined by calculation of the initial slope of the absorbance values as a function of time during the period after addition of the amphipathic protein extract. Flocculation activity is given as absorbance (or extinction) units per minute. The absorbance measured 25 minutes after addition of the amphipathic protein extract provides a value for the residual turbidity of the suspension. Flocculation activity may also be determined using the same assay conditions and procedure except that the glass beads are replaced by acid- washed diatomaceous earth (98% Si0 2 , Sigma Chemical Co., St. Louis, MO, USA Product no.D-3977).

Abstract

L'invention se rapporte à l'utilisation de protéines bipolaires comme floculants.
EP02742639A 2001-07-19 2002-07-19 Floculants Withdrawn EP1409418A1 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
CH0100452 2001-07-19
WOPCT/CH01/00452 2001-07-19
PCT/CH2002/000402 WO2003008343A1 (fr) 2001-07-19 2002-07-19 Floculants

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EP1409418A1 true EP1409418A1 (fr) 2004-04-21

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EP02742639A Withdrawn EP1409418A1 (fr) 2001-07-19 2002-07-19 Floculants

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WO (1) WO2003008343A1 (fr)

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004092418A2 (fr) 2003-04-14 2004-10-28 Nugen Technologies, Inc. Amplification globale effectuee avec une amorce composite amorcee de maniere aleatoire
CN102674517A (zh) * 2012-05-23 2012-09-19 大理学院 水体蓝藻快速絮凝的方法
CN112340871B (zh) * 2021-01-11 2021-04-20 烟台雅米宠物食品有限公司 一种宠物食品加工用废水过滤净化装置

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Publication number Priority date Publication date Assignee Title
EP0872490B1 (fr) * 1997-04-16 2006-05-17 Sony Corporation Floculant à poids moléculaire élevé, son procédé de fabrication et procédé de traitement des eaux en utilisant ce floculant
GB0007829D0 (en) * 2000-03-31 2000-05-17 Optima Environment S A Process for preparing coagulants for water treatment

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Title
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