Methods and systems for isolating analytes

Abstract

An optical device includes a first optical resonator and a second optical resonator optically coupled to the first optical resonator. A first heater is thermally coupled to the first optical resonator and a second heater is thermally coupled to the second optical resonator. The second heater is configured to be controlled separately from the first heater. A photodetector is coupled to the optical device to monitor light output from the coupled resonators. A controller applies a common-mode dither to the first heater and the second heater. Prior to applying the common-mode dither, the controller reads a first output from the photodetector. Subsequent to applying the common-mode dither, the controller reads a second output from the photodetector. The controller adjusts voltages applied to the first heater and the second heater based on a comparison between the first output and the second output to compensate for variations between the resonators.

Description

[0001] METHODS AND SYSTEMS FOR ISOLATING ANALYTES

[0002] RELATED APPLICATIONS

[0003] This application claims the benefit of priority of U.S. Patent Application No. 63 / 754,655 filed February 6, 2025, the contents of which are incorporated herein by reference in their entirety.

[0004] FIELD AND BACKGROUND OF THE INVENTION

[0005] The present invention, in some embodiments thereof, relates to methods and systems for isolating and purifying analytes, including proteins and small molecules, from a solution.

[0006] The isolation and purification of proteins are essential processes in biochemistry and biotechnology, serving critical roles in research, diagnostics, and the production of pharmaceuticals, nutraceuticals, and industrial enzymes. Traditional methods for protein purification commonly rely on chromatography techniques, where ion-exchange resins play a fundamental role. Specifically, ion-exchange resins are widely utilized to separate proteins based on their charge, facilitating the isolation of target proteins with high purity. However, conventional resins are often derived from synthetic polymers, such as polystyrene sulfonate, which present environmental and economic drawbacks due to their reliance on petrochemicals and high production costs.

[0007] In recent years, there has been growing interest in sustainable and cost-effective alternatives to synthetic resins. Plant materials, abundant in nature and renewable, have shown promise as viable substitutes. Certain plant components, such as lignocellulosic biomass and polysaccharide-based structures, naturally carry functional groups (e.g., carboxyl and hydroxyl groups) that can be chemically modified to exhibit ion-exchange properties. These modifications allow plant components such as purified lignin and cellulose to function as an effective ionic exchange medium.

[0008] Additional related background art:

[0009] Kumla et al. Additional related background art:

[0010] Burapha Science Journal Volume 27 (No.3) September - December 2022;

[0011] Wang et al. Environmental Research Volume 52, Issue 1, June 1990, Pages 7-22;

[0012] Korner et al. J Environ Qual . 2003 Sep-Oct;32(5): 1583-90;

[0013] Ozengin et al. J Environ Biol 2007 Apr;28(2):307-14;Al-Hashimi Journal of King Saud University - Engineering Sciences Volume 22, Issue 1, January 2010, Pages 11-18;

[0014] Suppadit et al. ScienceAsia 34 (2008): 163-168;

[0015] Zhou et al. Plants (Basel) 2023 Jan 29; 12(3): 589;

[0016] Farid Chemosphere Volume 304, October 2022, 135262;

[0017] Abdul Aziz et al. Appl. Sci. 2020, 70(16), 5397;

[0018] Li et al. Bioresource Technology Volume 318, December 2020, 123858;

[0019] CN114560564

[0020] AU2022425698

[0021] SUMMARY OF THE INVENTION

[0022] According to an aspect of the invention, there is provided a method of isolating a protein from a solution, the method comprising contacting the solution which comprises the protein with a resin which comprises inactivated tissue of a plant under conditions which allow adsorption of the protein to the inactivated tissue of the plant, thereby isolating the protein.

[0023] According to embodiments of the invention, the method further comprises eluting the protein from the resin following the isolating, thereby isolating the protein from the solution.

[0024] According to an aspect of the invention, there is provided a method of isolating a small molecule from a solution, the method comprising:

[0025] (a) contacting the solution which comprises the small molecule with a resin which comprises inactivated tissue of a plant under conditions which allow adsorption of the small molecule to the inactivated tissue of the plant; and

[0026] (b) eluting the small molecule from the resin following the isolating, thereby isolating the small molecule from the solution.

[0027] According to embodiments of the invention, the small molecule is not ammonia or lactate. According to embodiments of the invention, the contacting is effected in an aseptic environment.

[0028] According to embodiments of the invention, the solution comprises a cell culture medium. According to embodiments of the invention, the solution comprises a Tris buffer, a citrate buffer, glycine buffer, phosphate buffer or an acetate buffer.

[0029] According to embodiments of the invention, the aseptic environment is a sterile environment.

[0030] According to embodiments of the invention, the contacting is for a time period which is 1 minute to 24 hours.According to embodiments of the invention, the protein is characterized by having an isoelectric point (pl) above 4.5.

[0031] According to embodiments of the invention, the protein is characterized by having an isoelectric point (pl) above 7.

[0032] According to embodiments of the invention, the small molecule is positively charged. According to embodiments of the invention, the small molecule is hydrophobic.

[0033] According to embodiments of the invention, the resin comprises inactivated crosslinked tissue of the plant.

[0034] According to an aspect of the invention, there is provided a kit for isolating a protein or a small molecule from a solution comprising:

[0035] (i) a resin which comprises inactivated tissue of a plant;

[0036] (ii) elution buffer; and

[0037] (iii) adsorption buffer,

[0038] wherein a salt concentration of the elution buffer is higher than a salt concentration of the adsorption buffer.

[0039] According to an aspect of the invention, there is provided a kit for isolating a protein from a solution comprising a resin which comprises particles of inactivated tissue of a plant, wherein a volume of each of the particles is less than 30 mm3.

[0040] According to an aspect of the invention, there is provided an edible article of manufacture comprising a resin which comprises particles of inactivated plant tissue, wherein the resin comprises an edible protein adsorbed thereto.

[0041] According to embodiments of the invention, each particle has a volume of less than 30 mm3. According to embodiments of the invention, the resin is pre-packed in a column.

[0042] According to embodiments of the invention, the resin comprises inactivated, crosslinked tissue of the plant.

[0043] According to embodiments of the invention, the plant is a pectin-rich plant.

[0044] According to embodiments of the invention, the inactivated tissue is a pectin-rich inactivated tissue.

[0045] According to embodiments of the invention, the inactivated tissue comprises a mixture of at least two polysaccharides.

[0046] According to embodiments of the invention, the inactivated tissue is not root tissue.

[0047] According to embodiments of the invention, the inactivated tissue comprises proteinaceous material.According to embodiments of the invention, the stiffness of the inactivated tissue is at least as that of a carrot sample of 50 mm diameter and 7700 mm2volume the minimum forces required for bruising, bending, and shearing of the carrot fruit being 71 N, 48 N, and 41 N, respectively.

[0048] According to embodiments of the invention, the inactivated tissue is hydrous comprising a water content of at least 50 % v / w.

[0049] According to embodiments of the invention, the inactivated tissue is of a fruit or a vegetable. According to embodiments of the invention, the plant is selected from the group consisting of carrot, apple, sugarbeet, soy and corn.

[0050] According to embodiments of the invention, the plant is carrot.

[0051] According to embodiments of the invention, the plant is Duckweed.

[0052] According to embodiments of the invention, the plant is Moringa.

[0053] According to embodiments of the invention, the Duckweed is selected from the group consisting of a Wolffia species and Lemna species.

[0054] According to embodiments of the invention, the Wolffia species is selected from the group consisting of Wolffia angusta, Wolffia arrhiza, Wolffia australiana, Wolffia borealis, Wolffia brasiliensis, Wolffia columbiana, Wolffia cylindracea, Wolffia elongata, Wolffia globose, Wolffia microscopica and Wolffia neglecta.

[0055] According to embodiments of the invention, the Wolffia species is Wolffia arrhiza (e.g., var. spotless watermeal).

[0056] According to embodiments of the invention, the Lemna species is selected from the group consisting of Lemna minor L., Lemna disperma L., Lemna ecuadoriensis L., Lemna gibba L., Lemna gibba L. , Lemna obscura L. and Lemna trisulca L.

[0057] According to embodiments of the invention, the inactivated tissue is cellularized.

[0058] According to embodiments of the invention, the inactivated tissue is at least partially decellularized.

[0059] According to embodiments of the invention, the inactivated tissue is diced, shredded or powdered.

[0060] According to embodiments of the invention, the method is for isolating a protein and a particle volume of the inactivated tissue is less than 30 mm3.

[0061] According to embodiments of the invention, the method is for isolating a small molecule, and a particle volume of the inactivated tissue is between 30 mm3-100 mm3.

[0062] According to embodiments of the invention, the inactivated tissue is immobilized on a solid support.According to embodiments of the invention, the cell culture medium is selected from the group consisting of PBS, Hanks’ BSS, Earle’s salts DPBS, HBSS, EBSS, DMEM, MEM, GMEM , RPMI 1640 Leibovitz L-15, TC 100 Graces insect medium Schneider's Insect medium, Ham F10, Ham F12, DMEM / F12 and Serum-Free Insect Medium 1.

[0063] According to embodiments of the invention, the protein is a recombinant protein.

[0064] According to embodiments of the invention, the inactivated tissue is GRAS.

[0065] According to embodiments of the invention, the inactivated tissue is inactivated by a treatment is selected from the group consisting of an ethanol fixation, sonication, autoclave, freeze-drying, oven-drying, steam sterilization, radiation (e.g., UV radiation), detergent treatment and a combination thereof.

[0066] According to embodiments of the invention, the inactivated tissue is inactivated by chemical treatment with a polycarboxylic acid.

[0067] According to embodiments of the invention, the polycarboxylic acid is citric acid.

[0068] According to embodiments of the invention, the inactivated crosslinked tissue of the plant is generated by:

[0069] contacting viable tissue of the plant with a crosslinker under conditions which generate inactivated crosslinked plant material; and

[0070] contacting the inactivated crosslinked plant material with an alkali under conditions that enhance binding capacity of the resin.

[0071] According to embodiments of the invention, the crosslinking agent used to carry out the crosslinking is an ionic crosslinking agent, an enzymatic crosslinking agent or a covalent crosslinking agent.

[0072] According to embodiments of the invention, the crosslinking agent is a covalent crosslinking agent.

[0073] According to embodiments of the invention, the covalent crosslinker is citric acid.

[0074] According to embodiments of the invention, the ethanol for the ethanol fixation is about 70 % v / v.

[0075] According to embodiments of the invention, the detergent is sodium dodecyl sulfate (SDS). According to embodiments of the invention, the method further comprises performing at least one additional round of isolating following the eluting.

[0076] Unless otherwise defined, all technical and / or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the invention, exemplary methods and / or materials aredescribed below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting.

[0077] BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

[0078] Some embodiments of the invention are herein described, by way of example only, with reference to the accompanying drawings. With specific reference now to the drawings in detail, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of embodiments of the invention. In this regard, the description taken with the drawings makes apparent to those skilled in the art how embodiments of the invention may be practiced.

[0079] In the drawings:

[0080] FIGs. 1 A-C are photographs of results of protein (BSA, insulin and erythropoietin) analysis following ion exchange chromatography as assayed using SDS-PAGE gels. A - proteins treated at pH 4. B- proteins treated at pH 7. C - elution samples by NaCl (lanes 4-6) or NaOH (lanes 7-9). Calibration curves (100%, 70%, 35%) for each protein were loaded on the left side of each gel in lanes 1-3. Abbreviations: t60 (after 60-minute incubation time); tl20 (after 120-minute incubation time).

[0081] FIG. 2 is a photograph of results of protein (lysozyme and BSA) analysis following ion exchange chromatography using carrots as the cation exchanger as assayed using SDS-PAGE gel. Calibration curves (100%, 70%, 35%, 10 %) for each protein were loaded on the left side of each gel in lanes 1-4. Abbreviations: t60 (after 60-minute incubation time); H20 (after 120-minute incubation time).

[0082] FIGs. 3A-F are photographs of results of protein analysis of lysozyme following ion exchange chromatography using carrots as the cation exchanger as assayed using SDS-PAGE gel. Figure 3A- 1,500 mg / L lysozyme t=60 min; Figure 3B - 1,500 mg / L lysozyme t=120 min; Figure 3C - 3,000 mg / L lysozyme t=60 min; Figure 3D - 3,000 mg / L lysozyme t=120 min. Figure 3E -1,500 mg / L lysozyme t=60 min; Figure 3F - 1,500 mg / L lysozyme t=120 min. Calibration curves (100%, 70%, 35%) for each protein were loaded on the left side of each gel in lanes 1-3. Abbreviations for lanes 4-8 - "con" lane 4 is a control, B 1 and B2 are big Carrot - duplicate samples and SI and S2 are small samples.

[0083] FIG. 4 is a graph illustrating the kinetics of adsorption or elution of lysozyme using ethanol-treated carrot as a percent of the initial concentration.FIGs. 5A-B are photographs of SDS PAGE gels illustrating lysozyme adsorption kinetics (Figure 5 A) and elution kinetics (Figure 5B). Top band is carbonic anhydrase. Bottom band is lysozyme.

[0084] FIG. 6 is a photograph of an SDS PAGE gel illustrating adsorption of lysozyme on inactivated (heat or ethanol) carrot resin.

[0085] FIG. 7 is a graph illustrating binding capacity of lysozyme to inactivated (heat or SDS) carrot resin.

[0086] FIG. 8 is a photograph of an SDS gel showing absorption of lysozyme on inactivated (heat or SDS) carrot resin.

[0087] Fanes 1-3 -control protein (100 %, 50 % and 25 %);

[0088] Lanes 4-5 - samples following adsorption on SDS-treated carrots;

[0089] Lanes 6-7 - elution samples following adsorption on SDS-treated carrots;

[0090] Lanes 8-9 - samples following adsorption on heat-treated carrots;

[0091] Lanes 10-11- elution samples following adsorption on SDS-treated carrots;

[0092] Lanes 12-13 - samples following adsorption on non-treated carrots;

[0093] Lanes 14-15 - elution samples following adsorption on SDS-treated carrots.

[0094] FIG. 9 is a graph illustrating binding capacity of lysozyme to inactivated (SDS or ethanol) Wolffia resin.

[0095] FIG. 10 is a photograph of an SDS gel showing absorption of lysozyme on inactivated (SDS or ethanol) Wolffia resin.

[0096] Lanes 1-3 - control protein (100 %, 70 % and 35 %);

[0097] Lanes 4-5 - samples following adsorption on SDS-treated Wolffia;

[0098] Lanes 6-7 - elution samples following adsorption on SDS-treated Wolffia;

[0099] Lanes 8-9 - samples following adsorption on ethanol-treated Wolffia according to the “old” method;

[0100] Lanes 10-11- elution samples following adsorption on ethanol-treated Wolffia according to the “old” method;

[0101] Lanes 12-13 - samples following adsorption on ethanol-treated Wolffia according to the “new” method;

[0102] Lanes 14-15- elution samples following adsorption on ethanol-treated Wolffia according to the “new” method.

[0103] FIG. 11 is a graph illustrating binding capacity absorption of lysozyme to treated moringa. FIG. 12 is a graph illustrating the binding capacity of lysozyme to carrot resin over 40 reuse cycles, as measured by the BCA assay.FIG. 13 is a graph illustrating the binding capacity of lysozyme to carrot resin over 40 reuse cycles as analyzed by SDS-PAGE.

[0104] FIG. 14 is a graph comparing the binding capacity of lysozyme to carrot resin as compared with beads that are fixing pectin powder resin.

[0105] FIG. 15 are images of control plant cells under TEM microscopy (69-70 K magnification). FIG. 16 are images of ethanol-inactivated plant cells under TEM microscopy (69-70 K magnification).

[0106] FIG. 17 are graphs illustrating the kinetic change in solution absorbance of dyes using viable vs inactivated carrot cubes compared to control (No exposure to tissue).

[0107] FIG. 18 are graphs illustrating the ability of the inactivated plant resin to deplete ammonium from a culture medium.

[0108] FIG. 19 is a graph illustrating binding capacity for lysozyme on crosslinked plant tissue resins.

[0109] FIG. 20 is a graph indicating static binding capacity (SBC) of the crosslinked resin after 100 cycles.

[0110] FIG. 21 is a graph illustrating the effect of temperature and percentage of crosslinker on the binding capacity of the crosslinked resin.

[0111] DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION

[0112] The present invention, in some embodiments thereof, relates to methods and systems for isolating and purifying proteins and small molecules from a solution.

[0113] Before explaining at least one embodiment of the invention in detail, it is to be understood that the invention is not necessarily limited in its application to the details set forth in the following description or exemplified by the Examples. The invention is capable of other embodiments or of being carried out in various ways.

[0114] Conventional resins for ion-exchange chromatography are often derived from synthetic polymers, such as polystyrene sulfonate, which present environmental and economic drawbacks due to their reliance on petrochemicals and high production costs.

[0115] This invention addresses these challenges by introducing a novel method for preparing and utilizing plant material as a cationic exchange resin for isolating proteins. Through this method, plant material is modified to enhance its ion-exchange properties, enabling it to effectively capture and isolate proteins under various conditions. This process not only provides a sustainable and economical alternative to synthetic resins but also expands the potential applications of plant-based materials in the field of protein purification.Whilst reducing the present invention to practice, the present inventors have shown that carrot tissue inactivated by heat (Figures 6-8), SDS treatment (Figures 7-8), ethanol treatment (Figure 4 and 6) could serve as an effective resin for selective adsorption of positively charged proteins under conditions above pH 4 (e.g. neutral pH). Likewise, Wolffia plant tissue inactivated by SDS (Figure 9-10) or ethanol (Figures 9-10) could also serve as an effective resin for isolation of positively charged proteins. Ethanol-inactivated Moringa tissue similarly adsorbed positively charged proteins at pH above pH 4.

[0116] In addition, the present inventors found that inactivated carrot tissue could effectively adsorb small molecules. The adsorption capacity was affected by both the carrot particle size, and the size, charge and hydrophobicity of the small molecule (Figure 17).

[0117] The present inventors showed that ethanol-inactivated resins produced according to embodiments of the invention were flexible and robust with a static binding capacity of 30-40 mg of lysozyme protein per packed ml (i.e. 50-60 mg per gram of resin). Such resins were shown to be reusable more than 50 times.

[0118] The present inventors further showed that crosslinked resins produced according to embodiments of the invention were shown to have a static binding capacity of 100 mg of lysozyme protein per packed ml. The resin also exhibits a dynamic binding capacity exceeding 100 mgP / ml with a contact time as short as five minutes. It was shown to be effectively regenerated with 1-2 M NaCl or NaOH (e.g 0.2 M or 0.5 M) solutions. Such resins were shown to be reusable more than 100 times.

[0119] Thus, according to an aspect of the invention, there is provided a method of isolating a protein from a solution, the method comprising contacting the solution which comprises the protein with a resin which comprises inactivated tissue of a plant under conditions which allow adsorption of the protein to the inactivated tissue of the plant, thereby isolating the protein.

[0120] According to another aspect of the invention, there is provided a method of isolating a small molecule from a solution, the method comprising:

[0121] (a) contacting the solution which comprises the small molecule with a resin which comprises inactivated tissue of a plant under conditions which allow adsorption of the small molecule to the inactivated tissue of the plant; and

[0122] (b) eluting the small molecule from the resin following the isolating, thereby isolating the small molecule from the solution.

[0123] In the context of a protein, the term “isolating” refers the act of separating or distinguishing a protein from a mixture or solution, through any means by which the protein is preferentially adsorbed, captured, or retained by the resin, regardless of the extent of purity achieved or thepresence of other components in the final product. The molecular weight of the protein following the isolating is typically identical to the molecular weight following the isolating. Thus, the process does not act to degrade the protein.

[0124] In some embodiments, proteins may be immobilized on the inactivated plant-derived resin or support described herein in a manner that preserves their biological activity.

[0125] In some embodiments the proteins bind directly to the inactivated plant-derived resin. In certain embodiments, immobilization is achieved via non-covalent interactions, including electrostatic adsorption mediated by surface charges present on the support, thereby enabling attachment without substantially disrupting the native structure of the protein. In further embodiments, one or more attachment-promoting agents are associated with the resin or support to facilitate binding of negatively charged proteins or biomolecules, for example by providing positively charged functional groups, wherein non-limiting examples of such agents include polycationic polymers such as polylysine.

[0126] In other embodiments, the support is chemically modified to enable covalent attachment of the biological agent, for example through so-called “zero-length” crosslinking techniques that form direct bonds between functional groups of the agent and the support. Non-limiting examples of suitable crosslinking agents include carbodiimides such as EDC, glutaraldehyde, and genipin.

[0127] Typically, 1 ml of plant resin is capable of adsorbing at least 10, at least 20, at least 30, at least 40, at least 50 mg of protein (e.g., lysozyme). For example, 1 ml of plant resin is capable of adsorbing between 10-60 mg protein.

[0128] In another embodiment, 1 gram of plant resin is capable of adsorbing at least 10, at least 20, at least 30, at least 40, at least 50 mg of protein (e.g., lysozyme). For example, 1 gram of plant resin is capable of adsorbing between 10-60 mg protein.

[0129] In still another embodiment, 1 ml of resin is capable of adsorbing between 10-10,000, between 10-5000 pmol of lysozyme protein.

[0130] According to a specific embodiment, at least 25 %, 30 %, 40 %, 50 %, 60 %, 70 %, 80 %, 90 % or more of the protein present in the solution is assimilated in (e.g. adsorbed or absorbed onto) the resin.

[0131] According to a specific embodiment, the treatment with the plant tissue decreases the protein content of the solution.

[0132] As used herein “protein content” refers to concentration and / or composition of the proteins present in the medium.

[0133] The present invention contemplates isolating any protein using the methods described herein.In one embodiment, the protein is an animal protein i.e. has an amino acid sequence which is of an animal species (e.g. human).

[0134] In another embodiment, the protein is a plant protein.

[0135] The term “protein” also referred to herein as a polypeptide, refers to a protein of any size, typically comprising at least 4 amino acids linked by peptide bonds.

[0136] In one embodiment, the protein is positively charged at working solutions.

[0137] In another embodiment, the protein is negatively charged at working solutions.

[0138] In still another embodiment, the protein has a molecular weight between 1-200 KDa, e.g. between 3-160 KDa.

[0139] In one embodiment, the protein is an edible protein.

[0140] In still other embodiments, the protein has an isoelectric point (pl) greater than 4, great than 4.5 or even greater than 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5 or 12.

[0141] According to other embodiments, the protein is a recombinant protein.

[0142] According to other embodiments, the protein is an antibody (e.g. a single domain antibody). Particular examples of proteins that may be isolated using the methods described herein include lysozyme, collagen, Cytochrome C, Ribonuclease, Lactoferrin Papain, Protamine, Myelin Basic Protein, Elastase, Aprotinin, Cytochrome b562, Avidin, Melittin, sweetening proteins such as Brazzein, Thaumatin, Monelin, Curculin, Mabinlin, Miraculin and Pentadin.

[0143] In the context of a small molecule, the term “isolating” refers to the act of separating or distinguishing a small molecule from a mixture or solution, through any means by which the small molecule is preferentially adsorbed, captured, or retained by the resin, regardless of the extent of purity achieved or the presence of other components in the final product. The molecular weight of the small molecule following the isolating is typically identical to the molecular weight following the isolating. Thus, the process does not act to degrade the small molecule.

[0144] The term “small molecule” refers to a molecule having a molecular weight which is less than 700 ((g / mol). In another embodiment, the small molecule has a molecular weight between 100-700 g / mol. In another embodiment, the small molecule has a molecular weight which is less than 100 g / mol.

[0145] In one embodiment, the small molecule is positively charged, at working solution.

[0146] In another embodiment, the small molecule is negatively charged, at working solution. In still another embodiment, the small molecule is positively charged, at working solution and hydrophobic.

[0147] Examples of contemplated small molecules include for example naringenin and dyes such as methylene blue, methyl orange, resazurin sodium, safranine, coumarin and IPTG.According to a particular embodiment, the small molecule is a food dye, a sweetener, a food preservative or a flavor enhancer.

[0148] According to another embodiment, the small molecule is a metabolite.

[0149] According to a particular embodiment, the small molecule is not ammonia or lactate. Irrespective of the component being adsorbed onto the resin, the plant tissue does not typically refer to isolated cells (single cells or up to cell clumps of less than 200 cells or less than 20 or 50 cells).

[0150] As used herein the term “plant tissue” refers to at least one of meristematic tissue and / or permanent tissue.

[0151] According to a specific embodiment, the plant tissue does not refer to a whole plant.

[0152] According to a specific embodiment, the plant tissue does not refer to a whole organ. According to a specific embodiment, the plant tissue does not refer to a single polysaccharide derived from a plant (e.g. pectin or cellulose). Typically, the plant tissue comprises at least 2, 3 or more different polysaccharides (e.g. combinations of pectin, cellulose, hemicellulose and lignin).

[0153] According to another embodiment, the plant tissue is not root tissue.

[0154] According to another embodiment, the plant tissue is not isolated cell walls.

[0155] According to a specific embodiment, the plant tissue refers to a whole plant.

[0156] According to a specific embodiment, the plant tissue refers to a whole organ.

[0157] In one embodiment, the plant tissue is cellularized.

[0158] In another embodiment, the plant tissue is partially decellularized. The present inventors contemplate that at least 10 %, 20 %, 30 %, 40 % or even 50 % of the cells of the tissue still remain in the resin. The present inventors further contemplates that at least 10 %, 20 %, 30 %, or even 40 % of the cells of the tissue are removed during the decellularization process.

[0159] Methods of decellularizing plant tissue are known in the art and include detergent based methods (e.g. SDS, Triton-X-100, Tween-20, Tween-80), enzyme based methods (e.g. use of trypsin, collagenase, DNAse, RNAse), physical methods (e.g. agitation, heat / cold shock).

[0160] A combination of these methods may be used to achieve optimal decellularization while maintaining ECM integrity. The choice of method depends on factors like plant tissue type, application (e.g., biomedical scaffolds, biohybrid materials), and the desired mechanical and biochemical properties of the final scaffold.

[0161] In still another embodiment, the plant tissue is not fully decellularized.

[0162] It will be appreciated that partial decellularization of the plant tissue renders the tissue more porous. Exemplary pore size of the resin for adsorbing protein is between 10-100 nm. Exemplarypore size of the resin for adsorbing small molecule is 0.01-10 nm (i.e. less than 10 nm). Preferably, the pore size diameter is not greater than 200 nm, 400 nm, 600 nm, 800 nm or even 1000 nm.

[0163] Preferably, the pore size diameter is not greater than 200 nm, 400 nm, 600 nm, 800 nm or even 1000 nm.

[0164] The term '"plant" as used herein encompasses tissues from plants or plant parts, including seeds, fruit, root, leaves, shorts, bark, tubers and fronds. The plant tissue can be differentiated. According to other embodiments, the plant tissue is undifferentiated e.g., callus or meristems. Plants that are particularly useful in the methods of the invention include all plants which belong to the superfamily Viridiplantee, in particular monocotyledonous and dicotyledonous plants including a fodder or forage legume, ornamental plant, food crop, tree, or shrub selected from the list comprising Acacia spp., Acer spp., Actinidia spp., Aesculus spp., Agathis australis, Albizia amara, Alsophila tricolor, Andropogon spp., Arachis spp, Areca catechu, Astelia fragrans, Astragalus cicer, Baikiaea plurijuga, Betula spp., Brassica spp., Bruguiera gymnorrhiza, Burkea africana, Butea frondosa, Cadaba farinosa, Calliandra spp, Camellia sinensis, Cannabaceae, Cannabis indica, Cannabis, Cannabis sativa, Hemp, industrial Hemp, Capsicum spp., Cassia spp., Centroema pubescens, Chacoomeles spp., Cinnamomum cassia, Coffea arabica, Colophospermum mopane, Coronillia varia, Cotoneaster serotina, Crataegus spp., Cucumis spp., Cupressus spp., Cyathea dealbata, Cydonia oblonga, Cryptomeria japonica, Cymbopogon spp., Cynthea dealbata, Cydonia oblonga, Dalbergia monetaria, Davallia divaricata, Desmodium spp., Dicksonia squarosa, Dibeteropogon amplectens, Dioclea spp, Dolichos spp., Dorycnium rectum, Echinochloa pyramidalis, Ehraffia spp., Eleusine coracana, Eragrestis spp., Erythrina spp., Eucalypfus spp., Euclea schimperi, Eulalia vi / losa, Pagopyrum spp., Feijoa sellowlana, Fragaria spp., Flemingia spp, Freycinetia banksli, Geranium thunbergii, GinAgo biloba, Glycine javanica, Gliricidia spp, Gossypium hirsutum, Grevillea spp., Guibourtia coleosperma, Hedy s arum spp., Hemaffhia altissima, Heteropogon contoffus, Hordeum vulgare, Hyparrhenia rufa, Hypericum erectum, Hypeffhelia dissolute, Indigo incamata, Iris spp., Leptarrhena pyrolifolia, Lespediza spp., Lettuca spp., Leucaena leucocephala, Loudetia simplex, Lotonus bainesli, Lotus spp., Macrotyloma axillare, Malus spp., Manihot esculenta, Medicago saliva, Metasequoia glyptostroboides, Musa sapientum, Nicotianum spp., Onobrychis spp., Ornithopus spp., Oryza spp., Peltophorum africanum, Pennisetum spp., Persea gratissima, Petunia spp., Phaseolus spp., Phoenix canariensis, Phormium cookianum, Photinia spp., Picea glauca, Pinus spp., Pisum sativam, Podocarpus totara, Pogonarthria fleckii, Pogonaffhria squarrosa, Populus spp., Prosopis cineraria, Pseudotsuga menziesii, Pterolobium stellatum, Pyrus communis, Quercus spp., Rhaphiolepsis umbellata, Rhopalostylis sapida, Rhus natalensis, Ribes grossularia, Ribes spp., Robinia pseudoacacia, Rosaspp., Rubus spp., Salix spp., Schyzachyrium sanguineum, Sciadopitys vefficillata, Sequoia sempervirens, Sequoiadendron giganteum, Sorghum bicolor, Spinacia spp., Sporobolus fimbriatus, Stiburus alopecuroides, Stylosanthos humilis, Tadehagi spp, Taxodium distichum, Themeda triandra, Trifolium spp., Triticum spp., Tsuga heterophylla, Vaccinium spp., Vicia spp., Vitis vinifera, Watsonia pyramidata, Zantedeschia aethiopica, Zea mays, amaranth, artichoke, asparagus, broccoli, Brussels sprouts, cabbage, canola, carrot, cauliflower, celery, zucchini, cucumber, collard greens, flax, kale, lentil, oilseed rape, okra, onion, potato, rice, soybean, straw, sugar beet, sugar cane, sunflower, tomato, squash, tea, trees. According to a specific embodiment, the plant tissue is of a fruit or a vegetable.

[0165] According to a specific embodiment, the tissue is of a plant selected from the group consisting of artichoke, asparagus, broccoli, Brussels sprouts, cabbage, canola, carrot, cauliflower, celery, zucchini, cucumber, collard greens, flax, kale, lentil, oilseed rape, okra, onion, potato, rice, soybean, straw, sugar beet, sugar cane, sunflower, tomato and squash.

[0166] According to another embodiment, the plant tissue is selected from the group consisting of carrot, apple, sugarbeet, soy and corn.

[0167] In one embodiment, the plant material comprises or consists of plant waste material. Plant waste materials suitable for use herein include any non-edible or discarded portions of plant matter, including outer coverings, internal fibrous tissues, and processing residues.For example, suitable plant waste materials include citrus rinds, fruit skins, vegetable peels, sugarcane bagasse, corn husks, and pith from stalks or stems.

[0168] According to a specific embodiment, the tissue is of a pectin-rich plant.

[0169] A pectin-rich plant typically has a pectin content of at least 5 % of the plants dry weight in the tissues where it is most abundant.

[0170] Thus, for example, a carrot can be considered a pectin-rich plant since the amount of pectin in carrot cell walls is typically greater than 5 % of the cell wall plants dry weight. Another example of a pectin rich plant is Moringa which contains pods comprising pods and leaves with very high pectin content.

[0171] According to a specific embodiment, the fruit is a fleshy fruit (e.g., berry, drupe, pome, hesperidium or cucurbiteae).

[0172] According to a specific embodiment, the vegetable is selected from the group consisting of a leafy vegetable, root vegetable, tuber vegetable, flower vegetable, stem vegetable, bulb vegetable, pod or seed vegetable and sea vegetable.According to a specific embodiment, the plant tissue (which is used for isolating small molecule) is hydrous following inactivation and comprises a water content of at least 75 %, 80 %, 85 %, 90 % v / w.

[0173] According to a specific embodiment, the plant tissue (prior to inactivation) (which is used for isolating small molecules) is hydrous and comprises a water content of at least 75 % v / w.

[0174] According to a specific embodiment, the plant tissue (following inactivation) which is used to isolate proteins comprises a water content of at least 20 % v / w.

[0175] It will be appreciated that following manipulation of the plant material (e.g. inactivation, crosslinking etc.), the water content of the plant material may be lowered, as further described herein below.

[0176] Methods of determining water content of a plant tissue are well known in the art. For example, measuring the amount of water present in the tissue relative to its dry weight. The most common method used for this purpose is the gravimetric method, which involves drying the tissue and measuring the weight loss.

[0177] According to a specific embodiment, the plant tissue is of a root vegetable e.g., carrot. According to a specific embodiment, the plant is a duckweed.

[0178] According to a specific embodiment, the Duckweed is selected from the group consisting of a Wolffia species and Lemna species.

[0179] According to a specific embodiment, the Wolffia species is selected from the group consisting of Wolffia angusta, Wolffia arrhiza, Wolffia australiana, Wolffia borealis, Wolffia brasiliensis, Wolffia columbiana, Wolffia cylindracea, Wolffia elongata, Wolffia globose, Wolffia microscopica and Wolffia neglecta.

[0180] According to a specific embodiment, the Wolffia species is Wolffia arrhiza (e.g., var. spotless watermeal).

[0181] According to a specific embodiment, the Lemna species is selected from the group consisting of Lemna minor L., Lemna disperma L., Lemna ecuadoriensis L., Lemna gibba L., Lemna obscura L. and Lemna trisulca L.

[0182] According to a specific embodiment, the plant is Moringa (i.e. of the species Moringa Oleifera).

[0183] According to a specific embodiment, the stiffness of the plant tissue is at least as that of a carrot sample of 50 mm diameter and 7700 mm2volume the minimum forces required for bruising, bending, and shearing of the carrot fruit being 71 N, 48 N, and 41 N, respectively.

[0184] According to a specific embodiment the plant tissue is generally recognized as safe (GRAS).Once the plant tissue is selected, it can be used as viable material, or subjected to treatment that renders it inactive.

[0185] According to a specific embodiment, the plant tissue is diced, shredded or powdered. For adsorption of proteins, contemplated sizes of plant particles (e.g. carrot particles) are between 0.0002-80 mm3in volume (e.g. diameter of 40-6000 pm). In one embodiment, the particle size is less than 30 mm3in volume.

[0186] For adsorption of small molecules, contemplated size of plant particles (e.g. carrot particles) are between 3-100 mm3in volume (e.g. have a diameter greater than 1000 pm, such as between 1000-6000 pm and a surface area between 0.1 to 3 m2 / gr). Exemplary shapes of plant particles for adsorption of small molecules include box shaped, spherical shaped, cylindrical shaped. 3-100 mm3in volume.

[0187] According to a specific embodiment, the plant tissue is granulated.

[0188] According to a specific embodiment, the plant tissue is diced.

[0189] According to another embodiment, the plant tissue is minced.

[0190] According to a specific embodiment, the plant tissue is juiced and the remaining pulp is used.

[0191] According to a specific embodiment, the plant tissue is viable. Of note, viable and alive are interchangeably used.

[0192] According to another embodiment, the plant tissue is subjected to a treatment which renders it inactive.

[0193] According to a specific embodiment, the treatment is selected from the group consisting of an ethanol fixation, polycarboxylic acid (e.g. citric acid - 0.1-2% w / v) treatment, sonication, autoclave, freeze-drying, oven-drying, steam sterilization, radiation (e.g., UV radiation) and detergent treatment or any combination of the above.

[0194] Inactivation can be effected for a suitable length of time - e.g. 0.5 - 24 hours.

[0195] According to a specific embodiment, inactivation is in ethanol such as at a ratio of 1-4 g (tissue) in 10 ml ethanol for 30 min.

[0196] According to a specific embodiment, ethanol for the ethanol fixation is about 50-100 % v / v, e.g., 70 % (v / v).

[0197] According to some embodiments, the detergent can be ionic, non-ionic or zwitterionic. According to a specific embodiment, the detergent is Sodium Dodecyl Sulfate (SDS): A synthetic organic compound used as a detergent and surfactant. According to a specific embodiment SDS is used at a concentration of 0.1-5 %, e.g., about 1 %. For instance, soaking the plant tissue biomass in x 3 volume of 1 % Sodium dodecyl sulfate (SDS), e.g., for 2 hr. and thenwash with deionized water. According to some embodiments the following conditions may be used: Triton X-100 a non-ionic detergent with moderate detergent power, used at a concentration of 0.1-3 %. Sodium Deoxycholate, an ionic detergent, can be used at a concentration of 0.5 % to 2 % (w / v). CHAPS (3-[(3-Cholamidopropyl) dimethylammonio]-l -propanesulfonate), a zwitterionic detergent can be used at a concentration of 0.1-0.5% (w / v). Tween-20 (polysorbate) non-ionic detergent can be used at a concentration of 0.05% to 0.5% (v / v).

[0198] According to a specific embodiment, the plant tissue is inactivated.

[0199] As used herein “inactivated” or “inactive” refers to a plant tissue which is non-dividing yet still comprises enzymatic activity. As used herein “viable” or “live” refers to a plant tissue which has not undergone inactivation and therefore its cells are still dividing. Methods of determining plant tissue viability include but are not limited to (Triphenyl) Tetrazolium Chloride (TTC) staining, Evans blue staining, fluorescein diacetate staining, neutral red staining.

[0200] An exemplary method of preparing a resin according to embodiments of the invention comprises processing the plant material to obtain particles of the desired size and treating with ethanol (as described herein above).

[0201] The present inventors further contemplate treating the tissue with a crosslinking agent prior to isolating (e.g. prior to, concomitant with or following the inactivation step) which is optionally followed by an alkali step, wherein the combined treatment promotes formation of a crosslinked polymer and subsequent partial hydrolysis thereof, so as to generate and expose carboxyl groups making them more available to adsorb the target molecule. In one embodiment, the plant material is dried prior to the crosslinking step (e.g. subjected to a heat treatment of 37 °C-90 °C for 3-24 hr). The crosslinking step serves to rehydrate the material.

[0202] In another embodiment, the crosslinker is added to the plant material whilst it is still wet. The solution is then heated so as to promote crosslinking (e.g. between 90-200 °C).

[0203] Examples of crosslinking agents contemplated by the present inventors include ionic crosslinking agents, enzymatic crosslinking agents and covalent crosslinking agents.

[0204] Ionic crosslinkers include calcium ions, magnesium ions and zinc ions.

[0205] Exemplary covalent crosslinkers include glutaraldehyde, genipin and polycarboxylic acids including, but not limited to citric acid, tartaric acid, succinic acid, malic acid and fumaric acid.

[0206] Enzymatic crosslinkers include but are not limited to laccases such as fungal laccases (from Trametes versicolor, Pycnoporus cinnabarinus, Pleurotus ostreatus) or bacterial laccases (from Bacillus subtilis, Streptomyces cyaneus), peroxidases and polyphenol oxidases (PPOs). The enzymatic crosslinker may be a transglutaminase (e.g. microbial transglutaminase (mTG, from Streptomyces mobaraensis).In one embodiment, the crosslinking agent is capable of crosslinking pectin. Examples of contemplated pectin crosslinking agents include divalent ions such as Ca2+(about 1000 mg / L solution in pH range of 5-7, for 2-24 hours) citric acid or UV exposure.

[0207] An exemplary method of preparing a crosslinked resin according to embodiments of the invention is set forth below.

[0208] Liquid plant material (e.g. pectin rich plant, such as carrot) is obtained (e.g. pulp material after juicing) and contacted with a crosslinker (e.g. citric acid). The liquid is removed (e.g., by filtration) and the remaining solid material may optionally be dried. The dried material may used to prepare a powder having a particle size between 50-250 micron. The particulate plant material is subjected to a heat treatment at (e.g. 90-200 °C) which promotes crosslinking of the plant material (e.g. creates a crosslinked polymer network via ester bonds between citric acid and hydroxyl groups on the pectin) thereby generating inactivated, crosslinked material. It will be appreciated that the temperature and duration of heating may be optimized depending on plant type and the properties of the resin. The heated plant material may then be rehydrated and treated with a functional group regeneration solution which partially hydrolyzing ester linkages, (e.g. 0.1- IN NaOH, or other suitable alkali for 10 minutes to 2 hours) thereby generating new charged groups such as COO- and OH. This increases the availability of active binding sites on the resin surface. Following washing, the wet resin may optionally be passed over a sieve to achieve a smaller particle size e.g. having a particle size between 50-250 micron. The obtained resin may be stored in ethanol for future use.

[0209] Typically, the inactivated plant material holds less water following the crosslinking step as compared to the inactivated plant material prior to the crosslinking step. This serves to increase the binding capacity of the plant material per volume. In one embodiment, the inactivated plant material holds up to 20 %, 30 %, 40 %, 50 %, 60 %, 70 %, 80 % or even 90 % less water following inactivation (optionally including the crosslinking) as it holds prior to inactivation.

[0210] According to a specific embodiment, the plant tissue used in the method or kit described herein is immobilized on a solid support (e.g. beads, films, membranes. Glass, ceramics, etc.). The solid supported may be fabricated from materials known in the art including but not limited to agarose, sepharose, dextran, cellulose or chitosan. The beads may be generated by embedding the plant material into the matrix (e,g. agarose matrix) havin a size range between 200-500 microns.

[0211] According to another embodiment, the plant tissue is not immobilized to a solid support and in fact remains in a "free" state. Instead of being fixed or attached to a solid support or matrix, the tissue remains dispersed and freely moving within the medium.The plant tissue serves as a resin for adsorbing protein / small molecules from a solution and can be packed as an article of manufacture for that purpose optionally attached to a solid support as described herein and / or packed as a column.

[0212] As mentioned, the contacting is performed under conditions which allow adsorption or absorption of the protein to the inactivated plant material.

[0213] These conditions refer to any one of or all of: temperature, pH, contact time, shaking. For example, temperature which can be 10-42 °C, e.g., 15-42 °C, 20-42 °C, 10-38 °C, 10-37 °C, 10-36 °C, 10-25 °C, 37-42 °C, 25-38 °C, 25-37 °C, 25-36 °C, 10-20 °C; contact time range from 1 min-24 hr, 1-5 min, 5 min to 24 hr, 1 min to 12 hrs, 1 min to 6 hrs, 1 min to 2 hrs, 1 min to 1 hr, 1 min to 30 min, 1 min to 20 min, 1 - 10 min, 2-10 min, 2-20 min, 2-5 min, 5 - 20 min, 5-15 min, 5-10 min.

[0214] The pH of the solution is typically between 3-10, 6-8, 6.5-7.5, 6.8-7.2. It will be appreciated that the exact pH of the solution is selected according to the pl of the protein being isolated. Thus, for example, the pH is typically lower than the pl of the protein.

[0215] Typically, the solution which is used when isolating the protein is a buffered solution -including for example buffers such as phosphate (PBS), HEPES, Tris, citrate, glycine or acetate.

[0216] In one embodiment, the inactivated tissue and the conditions of use allow for selective and specific adsorption of a particular protein (e.g. based on its pl) but do not substantially (+ / - 10 % w / v) remove lactate and / or ammonia from the medium.

[0217] In another embodiment, the inactivated tissue and the conditions of use allow for selective and specific adsorption of a particular protein (e.g. based on its pl) but also allow for substantial removal of lactate and / or ammonia from the medium.

[0218] In still other embodiments, the inactivated tissue and the conditions of use allow for selective and specific adsorption of a particular protein or proteins (e.g. based on its / their pl) but do not substantially remove small molecules from the medium.

[0219] In another embodiment, the inactivated tissue and the conditions of use allow for selective and specific adsorption of a particular group of proteins (e.g. based on their pl) but also allow for substantial removal of small molecules from the medium.

[0220] In another embodiment, the inactivated tissue and the conditions of use allow for selective and specific adsorption of a particular small molecule (e.g. based on its size, charge and / or hydrophobicity), but do not allow for substantial removal of protein from the medium.

[0221] In one embodiment, the protein is comprised in a culture medium.

[0222] As used herein, the term “medium” or “cell culture medium” refers to a medium as known in the art, and compositions comprising ingredients of biological origin. Such media and culturesmay contain nutrients, supplements and feeds, amino acids, peptides, proteins and growth factors (such as albumin, e.g., BSA, catalase, transferrin, insulin, erythropoietin, fibroblast growth factor (FGF), TGF beta, IGF, and others), vitamins, polyamines, sugars, carbohydrates, lipids, nucleic acids, hormones, fatty acids, trace materials, certain salts (such as potassium salts, calcium salts, magnesium salts), as well as waste materials such as ammonia, lactate, toxins and sodium salts. The medium is typically an aqueous based solution that promotes the desired cellular activity, such as viability, growth, proliferation, differentiation of the cells cultured in the medium.

[0223] In cell culturing, various types of media are employed to provide the necessary nutrients and environment for cell growth and maintenance. These media can be broadly categorized into natural and synthetic media, each of which is contemplated herewith as a separate embodiment. Natural media include complex substances like serum, plasma, and tissue extracts, which contain a mixture of growth factors, hormones, and other nutrients. Serum, often derived from fetal bovine sources, is particularly rich in growth factors but can vary in composition. Synthetic or defined media, on the other hand, are precisely formulated to include specific concentrations of salts, amino acids, vitamins, glucose, and other essential nutrients. These media may also be supplemented with growth factors, hormones, and antibiotics to promote cell proliferation and prevent contamination. A common synthetic medium is Dulbecco's Modified Eagle Medium (DMEM), often used in conjunction with fetal bovine serum (FBS) and antibiotics. Other specialized media, such as RPMI 1640, Minimal Essential Medium (MEM), and Roswell Park Memorial Institute (RPMI) medium, are tailored to specific cell types and applications, ensuring optimal conditions for diverse cellular processes, each of which is contemplated herewith as a separate embodiment.

[0224] According to a specific embodiment, the isolation method described herein may be operated under aseptic conditions. Sterile implies the absence of all life, desired or undesired. Aseptic implies life in the absence of foreign pathogens. A bioreactor / fermenters is sterile before a batch starts, and is operated aseptically after inoculation. Piping and connected equipment outside the aseptic bioreactor remain sterile. Sanitary implies that pathogens may be present, but have been reduced to levels where they cannot cause disease. Within these definitions, there exists an important distinction between aseptic industrial fermentation and aseptic animal cell culture: While industrial fermentation of a commodity bioproduct can generally be carried out in the presence of small numbers of foreign organisms — as long as these are not pathogenic to the production cells or users of the end product — any stray microbial life is functionally pathogenic to animal cells in culture. At neutral pH, elevated temperature, and (according to cultured-meat messaging) in the absence of antibiotics, a cell-culture bioreactor will readily harbor and be overcome by thecontaminating microbe, which proliferates much faster than animal cells. Typically, animal cell cultures can thus be further classified as axenic, i.e., free of any foreign organisms.

[0225] According to a specific embodiment, the aseptic environment is a sterile environment. The protein may be expressed in cells and secreted into the medium. For example, the protein may be expressed in bacterial cells or fungal cells (e.g. yeast cells) and secreted into the medium.

[0226] Once sufficient time has passed for absorption of the protein to the inactivated plant tissue, the absorption media is isolated (i.e. removed) from the plant tissue.

[0227] The protein may be then eluted from the inactivated plant tissue.

[0228] Prior to elution, the plant material may undergo washing to remove contaminations.

[0229] As used herein “washing” refers to reduction of osmolality of the tissue, e.g., by washing the plant tissue with a pre-determined volume e.g., x 5, x 10, x 20 x 5-20, x 5-10, e.g., about xlO volumes of deionized water for 1-20 min.

[0230] Filtration may be used to separate the plant tissue from the wash water by any separation methods such as but not limited to, vacuum filter, microfiltration / ultrafiltration, filter press or gravity sedimentation.

[0231] It will be appreciated that if the inactivated plant system is packed as a column, the wash medium is allowed to flow through the column for a sufficient length of time so as to remove contaminants.

[0232] Elution from the inactivated plant material is typically carried out using an appropriate solution (e.g. buffer). Elution may be carried out either by applying continuous gradients (linear or exponential) or in discrete steps.

[0233] In one embodiment, the elution buffer has a higher ionic strength (i.e. salt concentration) than the adsorption buffer. Exemplary salts that can be included in the elution buffer include, but are not limited to sodium chloride, sodium citrate, sodium phosphate, potassium chloride, ammonium sulfate, or calcium chloride at concentrations of between 0.1-2 M.

[0234] Additionally or alternatively, the elution buffer has a lower pH than a pH of the adsorption buffer. Without being bound to theory, the H+ions act to displace the target protein by altering the target protein’s net charge and thus its binding affinity to the inactivated plant material. Still alternatively, the elution buffer has a higher pH than a pH of the adsorption buffer.

[0235] In one embodiment, the inactivated tissue and the conditions of elution allow for selective and specific elution of a particular protein or group of proteins.

[0236] In still another embodiment, the inactivated tissue and the conditions of elution allow for elution of small molecules but do not substantially elute proteins.Sometimes more than one cycle of treatment (i.e., at least 2 times) with the plant tissue may be needed to isolate as much protein / small molecule as possible (or to deplete the medium from a sufficient amount of the protein / small molecule). In such a case fresh plant tissue (e.g., inactivated as described herein) may be used though this is not obligatory.

[0237] According to a specific embodiment, the at least 2 times of treatment are separated by using the medium resultant of one time of the at least 2 times in cell culturing.

[0238] According to a specific embodiment, the at least one time of the at least 2 times is effected with fresh plant tissue.

[0239] The use of “fresh” means a plant tissue not having been used in the methods described herein. Such a tissue is naive to the treated medium (wasn’t exposed to media before).

[0240] According to a specific embodiment, at least one time of the at least 2 times is effected with plant tissue having been used according to the method.

[0241] Thus, improved isolation of the protein / small molecule may be achieved by subjecting the medium more than once to treatment (more than one cycle) and / or by adding a new batch of plant tissue.

[0242] In one embodiment, the plant tissue of the resin is recycled after use. For example, the plant tissue may be regenerated using 1-2 M NaCl or 0.1-1 M NaOH solutions.

[0243] The methods may be carried out using kits which are dedicated towards isolating a protein. In one embodiment, the kit comprises:

[0244] (i) a resin which comprises inactivated tissue of a plant;

[0245] (ii) elution buffer; and

[0246] (iv) adsorption buffer.

[0247] In another embodiment, the kit is for isolating a protein from a solution and comprises: a resin which comprises particles of inactivated tissue of a plant, wherein a volume of said particles (e.g. the volume of each of the particles) is less than 30 mm3, less than 30 mm3, less than 10 mm3, less than 0.1 mm3, less than 0.05 mm3. For example, each of the particles may be between 0.0002-30 mm3in volume and have an average diameter of 40-1000 pm.

[0248] In one embodiment the particles have an average diameter of about 250-400 pm following rehydration.

[0249] In another embodiment the particles have an average diameter of about 50-200 pm or 100-200 pm following rehydration.

[0250] The resins described herein may be prepacked in a column.

[0251] The column can be a microcolumn, between 0.1-0.5 mL (used for small-scale screenings and high-throughput purification), a MiniSpin Column (between .5-2 mL), a standard laboratorycolumn (1 mL (commonly used in FPLC / HPLC systems, 5 mL (larger sample capacity) or 10 mL (for preparative work)). Alternatively, the column can be a mic-scale column (e.g. between 10-50 mL, for medium-scale protein or biomolecule purification or between 100-500 mL. The column can also be a large-scale column used in industrial procedures and bioprocessing, including for example 1-5 L columns and 10 L columns or greater.

[0252] Each of the components of the kit are described herein above.

[0253] In one embodiment, the salt concentration of the elution buffer is higher than a salt concentration of the adsorption buffer. In another embodiment, the pH of the elution buffer is lower than a pH of the adsorption buffer.

[0254] In another aspect there is provided an edible article of manufacture comprising a resin which comprises particles of inactivated plant tissue, wherein the resin comprises an edible protein adsorbed thereto.

[0255] The resins of this aspect of the present invention are generated from edible material and used to adsorb edible proteins.

[0256] As used herein, the term “edible protein” refers to any protein or polypeptide that is suitable for human consumption and capable of being incorporated into an edible article without causing adverse health effects. Edible proteins may be derived from plant, animal, microbial, fungal, or recombinant sources and may include, without limitation, enzymes, structural proteins, binding proteins, storage proteins, immunoglobulins, peptides, and nutraceutical proteins. In some embodiments, the edible protein is selected from soy proteins, pea proteins, wheat gluten, rice proteins, dairy proteins, egg proteins, collagen, gelatin, whey proteins, caseins, algal proteins, yeast-derived proteins, and bacterially expressed recombinant proteins. The edible protein may be provided in native, denatured, modified, hydrolyzed, or crosslinked form, and may optionally be chemically or enzymatically treated to improve stability, solubility, bioavailability, or functional performance. In certain embodiments, the edible protein is adsorbed, immobilized, encapsulated, or otherwise associated with the resin as described herein, thereby enabling controlled release, enhanced retention, or improved interaction with target molecules in the edible article.

[0257] As used herein the term “about” refers to ± 10 %.

[0258] The terms "comprises", "comprising", "includes", "including", “having” and their conjugates mean "including but not limited to".

[0259] The term “consisting of’ means “including and limited to”.

[0260] The term "consisting essentially of" means that the composition, method or structure may include additional ingredients, steps and / or parts, but only if the additional ingredients, steps and / orparts do not materially alter the basic and novel characteristics of the claimed composition, method or structure.

[0261] As used herein, the singular form "a", "an" and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a compound" or "at least one compound" may include a plurality of compounds, including mixtures thereof.

[0262] Throughout this application, various embodiments of this invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

[0263] Whenever a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integral) within the indicated range. The phrases “ranging / ranges between” a first indicate number and a second indicate number and “ranging / ranges from” a first indicate number “to” a second indicate number are used herein interchangeably and are meant to include the first and second indicated numbers and all the fractional and integral numerals therebetween.

[0264] As used herein the term "method" refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts.

[0265] It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination or as suitable in any other described embodiment of the invention. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment is inoperative without those elements.

[0266] Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples.EXAMPLES

[0267] Reference is now made to the following examples, which together with the above descriptions illustrate some embodiments of the invention in a non-limiting fashion.

[0268] MATERIALS AND METHODS

[0269] Proteins:

[0270] Bovine serum albumin (BSA) (Sigma A8022)

[0271] Carbonic anhydrase from bovine erythrocytes (Ery) (Sigma C7025)

[0272] Insulin (Ins) (Sigma 10516)

[0273] Lysozyme (Lyz) (Sigma L7651)

[0274] Solution:

[0275] High-glucose DMEM with 15 mM HEPES (Sartorius).

[0276] 50 mM HEPES buffer (pH 7.2) (Biolab)

[0277] Tris HCL pH=8.8. (Biolab)

[0278] Plants:

[0279] Carrot (Daucus carota sativus).

[0280] Duckweed - Wolffia Arrhizal purchased from GreenOnyx Ltd. The duckweed, Wolffia arrhiza was cultivated as described in Appenroth, Klaus- J., et al. 2018 doi: 10.3389 / fchem.2018.00483. Medium was changed every 2 to 3 days to insure minimal microbial growth and healthy W. arrhiza biomass.

[0281] Moringa, specifically Moringa Oleifera

[0282] The following methods were used to determine protein concentration:

[0283] 1. Bradford assay, following established protocols by Sigma Aldrich (Sigma B6916-500ML) 2.Bicinchoninic Acid (BCA) protein assay, commonly used to measure total protein content in samples, (produced by Cyanogen QPRO™-(BCA) assay).

[0284] 3. Gel electrophoresis.

[0285] 4.SDS-PAGE using 4-20% gradient gels (Genscript).

[0286] Osmolarity analysis was performed using manufacturer's instructions with the Osmometer basic Type 7i device (LOSER). pH measured by Eutech pH150, conductivity by Eutech CON 150.

[0287] EXAMPLE 1

[0288] The following example was carried out in order to investigate the impact of pH and contact time of ethanol-treated carrots on four different proteins - insulin, lysozyme, carbonic anhydrase and bovine serum albumin (BSA), details of which are provided in Table 1, below.Table 1

[0289]

[0290] DMEM with 15 mM HEPES was titrated to two different pH (pH 4 and 7) after which the protein was added to a final concentration of 500 mg / ml

[0291] Two pH levels were tested:

[0292] 1. pH 7, which approximates the pH of a growing cell, and pH 4, which is lower than the isoelectric point of the tested proteins.

[0293] 2. pH 4 with 0.1 N HC1.

[0294] Carrot Particle size - 3 mm x 6 mm x 6 mm. The carrots that we used were treated with 50% ethanol for half an hour and then washed.

[0295] Experimental procedure

[0296] Medium ratio: 4 grams of carrots to 10 ml of medium were added in a 50 ml flask for each pH value, maintaining a 1:2.5 (w / v) volume ratio of carrots to medium.

[0297] Treatment duration: Samples were processed for 2 hours at ambient temperature. The specimens were placed in 50 ml Falcon tubes and agitated at 50 rpm. Measurements were taken at the start of the experiment, after 1 hour, and at the conclusion of the 2-hour period.

[0298] Control: A control sample containing only the medium was tested alongside the experimental setup for comparison.

[0299] Separation: After 2-hours, the carrots were separated from the medium by gravity.

[0300] Analytics: Protein quantification of the samples with SDS-PAGE gel electrophoresis. Washing: After separation, the carrots were washed with deionized water (DW) in a 1:1 volume ratio for 20 minutes to remove any loosely bound components.

[0301] Elution: The washed carrots were then subjected to an elution step to assess potential ion interactions between the treated carrots and the proteins under two different conditions:

[0302] a. High ionic strength elution - 0.5 M NaCl in a 1:2.5 (w / v) ratio of carrots to elution buffer for 60 minutes

[0303] b. High pH elution - The carrots were treated with a solution of 0.05 N NaOH in a 1:2.5 (w / v) ratio of carrots to elution buffer for 60 minutes.

[0304] The samples were analyzed after elution with SDS-PAGE gel electrophoresis.

[0305] RESULTS SDS-PAGE analysis was conducted on samples from the treatments and elution using a 16% gel with Tricine running buffer. Samples were diluted to 80 mg / L, with 10 pl loaded per well. Lysozyme was added as a normalization protein, and a protein ladder (250-5 kDa, Bio-rad) was included for size reference.

[0306] Results are presented in Figures 1A-C.Calibration curves (100%, 70%, 35%) for each protein were loaded on the left side of each gel. Figure 1 A shows the medium sample at pH 4, below the isoelectric point of the proteins, where they are positively charged. Protein bands for BSA, erythropoietin (Ery), and insulin weakened after 60 and 120 minutes of treatment, indicating reduced protein concentrations in the medium compared to the stable control.

[0307] The insulin band disappeared after 60 minutes, while BSA and erythropoietin concentrations decreased throughout the experiment, with an estimated final reduction of -65% from initial concentrations. No protein bands were observed in wash samples.

[0308] Elution samples at pH 4 (Figure 1C, lane 5) using the high ionic strength solution (0.5 M NaCl) showed small amounts of BSA and insulin, suggesting ionic interactions with the ethanol-treated carrots. However, the band intensity was insufficient for quantification. Elution at pH 4 using high pH (-12) 0.05 M NaOH (Figure 1C, lane 8) released substantial amounts of BSA, erythropoietin, and insulin, indicating that altering the proteins' net charge disrupts their ionic connections with tissue.

[0309] In contrast, at pH 7 (Figure IB), which is close to typical cell culture conditions and where the proteins are negatively charged, a different pattern emerged. The concentrations of BSA, erythropoietin (Ery), and insulin remained stable throughout the 120-minute experiment, with no observable changes in protein bands. Additionally, no protein was detected in the elution samples at this pH.

[0310] This stark difference in protein behavior between pH 4 and pH 7 conditions highlights the significant role of pH and protein charge in the interactions between these proteins and the plant tissue. The stability of protein concentrations at pH 7 suggests that the negative charge of proteins at this pH may prevent or significantly reduce their binding to the plant tissue, unlike the situation observed at pH 4.

[0311] EXAMPLE 2

[0312] Assessment of the binding capacity of inactivated carrot tissues on solution with lysozyme The following example was carried out in order to assess the binding capacity of lysozyme (pl = 10.7) at pH 7.2 to ethanol-inactivated carrots.

[0313] Carrot Particle size - 3 mm x 6 mm x 6 mm. The carrots were treated with 50% ethanol for half an hour and then washed.

[0314] Experiment Procedure: Lysozyme and bovine serum albumin (BSA) were dissolved in 50 mM HEPES buffer (pH 7.2) to a final concentration of 3 g / L each. Samples (4g) were incubated in 10 ml of this protein solution for 1 h at room temperature. The carrots were then separated fromthe treated medium and washed with deionized water. To elute any bound proteins, the carrots were incubated with 0.5 M NaCl for 1 hour at room temperature. The sample from the elution step and the incubation was checked for its protein content. SDS-PAGE analysis was conducted on samples from the treatments and elution using a 16% gel with Tricine running buffer. Samples were diluted to 80 mg / L, with 10 pl loaded per well, a protein ladder (250-5 kDa, Bio-rad) was included for size reference and carbonic anhydrase from bovine erythrocytes (Ery) (Sigma.C7025) was added as a normalizing protein.

[0315] RESULTS SDS-PAGE analysis demonstrated the selective binding of lysozyme to Carrot, while BSA concentration remained constant throughout the experiment (Figure 2, lanes 4-12). The elution fraction exhibited a single band corresponding to lysozyme (Figure 2, lanes 10-12). Quantitative analysis of band intensities revealed a 45% reduction in lysozyme after 30 minutes, indicating a binding capacity of 8.6 mgP / gr Carrot (mg of protein per gram of carrot). After 60 minutes, a 78% reduction in lysozyme band intensity was observed, corresponding to 11.1 mgP / gr Carrot., Quantitative analysis of band intensities using ImageJ software (version 1.54g, National Institutes of Health, USA). Elution with 0.5 mM NaCl at half the original volume resulted in a 95% recovery of lysozyme. These findings support the hypothesis that carrot selectively binds positively charged proteins (e.g., lysozyme) at pH 7.2, while negatively charged proteins (e.g., BSA) remain unbound. Furthermore, the results demonstrate that bound proteins can be effectively eluted from the matrix using high ionic strength solutions.

[0316] EXAMPLE 3

[0317] Assessment of the effect of lysozyme concentration on adsorption of carrots of varying particle sizes

[0318] Carrot was inactivated using 50% ethanol for 0.5 hour and washed with deionized water. The carrot was sliced into cubes categorized by size (B = Big, S = small) as presented in Table 2, herein below. The experimental medium consisted of a 50 mM HEPES buffer (pH=7.6-7.2). Carrot was exposed to various lysozyme concentrations (5,000, 3,000, or 1,500 mg / L) within this medium, a constant concentration of BSA was added as a normalized protein for quantification. Each size and concentration combination were replicated twice. Carrot was incubated with lysozyme for either 60 or 120 minutes.Experimental Procedure:

[0319] 1. Preparation: Duplicate experiments were conducted by adding four grams of ethanol- treated carrot to 10 ml of protein-containing medium in a 50 ml flask, maintaining a 1:2.5 gr to volume ratio of particles (i.e. interactive carrot plant tissue(gr) to medium (ml)).

[0320] 2. Incubation: The mixture was incubated at 50 rpm for 2 hours at room temperature~25C.

[0321] 3. Sampling: Samples were collected at 0, 1, and 2 hours.

[0322] 4. Control: A control sample containing only the protein-containing medium (without) was incubated alongside the experimental setup for comparison.

[0323] 5. Sample Preparation: The collected medium samples were prepared and diluted (-100 mg_p / L) for protein quantification using SDS-PAGE 16 % gel electrophoresis.

[0324] 6. Washing: After separation, the Carrot was washed with deionized water (DW) in a 1:1 volume ratio for 5 minutes to remove any loosely bound components.

[0325] 7. Analysis: The samples were analyzed after elution with SDS-PAGE gel electrophoresis.

[0326] Table 2

[0327]

[0328] Results:

[0329] As shown in Figures 3A-B (1500 mg / ml lysozyme), after 1 and 2 hours, no lysozyme was detected in the samples (lanes 4-8), indicating that the carrot' binding capacity exceeded the total lysozyme amount. The wash solution (lane 9) contained no lysozyme.

[0330] As shown in Figures 3C-D (3000 mg / ml lysozyme), after 1 hour, lysozyme was detected in the large Carrot particles (Bl and B2) but not in the small particles (SI and S2), suggesting faster binding kinetics for the higher surface area of the small particles. The wash solution (lane 9) was free of lysozyme. After 2 hours (Figure 3D), lysozyme levels were significantly reduced, with only faint bands remaining in the large Carrot samples.As shown in Figures 3E-F (5000 mg / ml lysozyme), a higher binding capacity and faster reaction is observed in the small carrot samples. However, at this higher concentration, the protein bands were more distinct, allowing for quantitative analysis of band intensities using ImageJ software (version 1.54g, National Institutes of Health, USA). Analysis results are presented in Table 3, the big Carrot reached 3,177 mgP / L reduction in lysozyme concentration in 2 hr while small Carrot particles reached 3,889 mgP / L at the same time.

[0331] Table 3

[0332]

[0333] The binding capacity of Carrot presented in Table 3 was calculated according to the equation:

[0334]

[0335] The results demonstrated a correlation between increased surface area and enhanced binding kinetics and capacity of Carrot for the protein. This suggests an adsorption model where the protein binds to the carrot's surface. The ratio of surface areas between the two tested particle sizes is 1.227 (Table 2), which closely aligns with the binding capacity ratio of 1.223 between the two particle sizes.

[0336] EXAMPLE 4

[0337] Kinetics of adsorption and elution of lysozyme protein from the Carrot The aim of this example was to study the kinetics of adsorption and elution of the lysozyme from carrot.

[0338] Experimental Procedure:

[0339] • Carrot Preparation: Resin was prepared with fresh carrot, cut into cubes (1.2 mm x 1.2 mm x 0.8 mm) that were pre-treated with 50% ethanol for 30 minutes and then washed with deionized water.• triplicate experiments were conducted by adding two grams of ethanol-treated carrot to 10 ml of protein-containing medium in a 50 ml flask, maintaining a 1: 5 volume ratio of ethanol- treated carrot to medium.

[0340] • Incubation: The mixture was incubated at 50 rpm for 1 hour at room temperature.

[0341] • Sampling: Samples were collected at 10, 20, 30, 40, 50, 60 min (lOOpl).

[0342] • Sample Preparation: The collected medium samples were prepared and diluted (-100 mg p / L) for protein quantification using SDS-PAGE 16 % gel electrophoresis.

[0343] • Washing: After separation, the carrot was washed with deionized water (DW) in a 1:1 volume ratio for 5 minutes to remove any loosely bound components.

[0344] • Elution: The washed carrot was then subjected to an elution step to assess potential ion interactions. The Carrot was subjected to a solution of 0.5 M NaCl in a 1:5 (w / v) ratio of carrot to elution buffer for 60 minutes. Sample was collected every 10 minutes during the elution time (Ih).

[0345] The samples were analyzed after elution with SDS-PAGE 16% gel electrophoresis. Carbonic anhydrase from bovine erythrocytes (Ery) (Sigma.C7025) was added as a normalizing protein and tested with Bradford protein assay.

[0346] RESULTS

[0347] The results depicted in Figure 4 illustrate two key trends; the dark line represents the decreasing percentage of band intensity over time compared to the control (t=0 in Figure 5A, lane 2-8), indicating the adsorption process. Conversely, the light line shows the increasing band intensities during the elution step (using 0.5 M NaCl) relative to the control (t=0 in Figure 5B, lane 2,3-9). The data reveal that adsorption continues to increase even after 60 minutes, suggesting that longer durations could lead to higher adsorption percentages. In contrast, the elution process reaches a plateau after approximately 40 minutes. Notably, the adsorption and elution curves converge at the same point, indicating high recovery efficiency during elution. Figures 5A-B provides an example of one of the SDS-PAGE gels used in these experiments.

[0348] EXAMPLE 5

[0349] Assessment of the different inactivation methods on Carrot binding capacity to lysozyme protein

[0350] The purpose of this example was to examine the impact of different treatments on the binding capacity of carrot to lysozyme.

[0351] Materials and methods:

[0352] Samples of carrot were pre-treated (inactivated) using three different methods:1. Heat - the sample was heated in an autoclave or heating bath with heat temperature 85C° for 30 min.

[0353] 2. 50% Ethanol - sample was mixed with three volumes of ethanol for 30 min.

[0354] 3. Fresh (control) - washed with 3 volumes of deionized water (DW).

[0355] After the treatments all the samples were washed in deionized water with 10 volumes of carrot in the sample until the wash water showed no change in osmolarity.

[0356] Lysozyme was dissolved in 50 mm HEPES solution (pH between 7.2 and 7.6) at a concentration of 5,000 mg / L. Carrot was cut into sizes as detailed in Table 4. The dimensions and volumes of the pieces were measured with 30 replicates.

[0357] Table 4

[0358]

[0359] Experimental Procedure: Carrot samples, weighing 2g each, were combined with 5 ml of the lysozyme solution in 15 ml falcon tubes. These samples were incubated for 60 minutes, with agitation at 50 rpm on a horizontal shaker. The ratio of carrot to medium was 2:5. After incubation, the collected medium samples were diluted to approximately 100 mg p / L for protein quantification using SDS-PAGE with a 16% gel. Bovine serum albumin (BSA) (Sigma A8022) was added as a normalizing protein.

[0360] Quantification and Analysis: Calibration curves with varying protein concentrations (100%, 70%, 35%, 10%) were prepared and loaded in lanes 1-4 of the gel in Figure 6. The control sample, analyzed after 60 minutes (lane 5), showed no change in lysozyme band intensity. Ethanol-treated samples (lanes 6-8) exhibited a 75% reduction in lysozyme band intensity, corresponding to 9.3 mg P / g of Carrot. Fresh Carrot (lanes 9-11) showed a 19% reduction in lysozyme band intensity, indicating 2.5 mg P / g of Carrot. Heat-treated samples (lanes 12-14) demonstrated a 91% reduction in lysozyme band intensity, corresponding to 11.4 mg P / g of Carrot. Quantitative analysis of band intensities using ImageJ software (version 1.54g, National Institutes of Health, USA).

[0361] Results

[0362] The fresh samples exhibited minimal reduction in lysozyme band intensity. Ethanol treatment resulted in a 75% reduction, suggesting an increase in lysozyme extraction. Heat treatment led to the highest reduction at 91%, indicating the most effective lysozyme extraction.These findings suggest that while carrot has a basic ability to bind lysozyme, treatments with ethanol and heat significantly enhance this ability, likely by exposing additional binding sites.

[0363] Conclusion: The results indicate that both ethanol and heat treatments are effective in enhancing lysozyme extraction from tissues, with heat treatment proving to be the most efficient.

[0364] EXAMPLE 6

[0365] Comparison between two inactivation methods (Heat treatment and 1 % SDS) on carrot binding capacity to Lysozyme protein

[0366] Materials and Methods:

[0367] Pretreatment

[0368] ■ SDS: Carrots were treated with 1% SDS solution for 2 hours (1:15 sample to solution ratio).

[0369] ■ Heat: Carrots were immersed in an 85°C water bath for 30 minutes (1:3 sample to water ratio).

[0370] ■ Fresh (control): carrots were rinsed with sterile reverse osmosis (RO) water.

[0371] Experimental Procedure:

[0372] All the samples were washed with 10 volumes until the water reached osmolarity of OmOms / L. Lysozyme was dissolved in 50mM HEPES buffer at a lysozyme concentration of 5,000 mg / L. Carrot was cut into sizes as detailed in Table 4. The experiment executed two replicates of 1 g of Carrot in 5 ml of solution at a 1:5 ratio (w / v). The sample with the solution was stirred for 1 hour. The washing step: samples rinsed with deionized water at 1:5 ratio (w / v) for 5 min. The elution step was for 1 hour using 0.5 M NaCl with the same 1:5 ratio of carrot particles to elution solution.

[0373] Binding capacity was calculated utilizing the following equation:

[0374]

[0375] C = lysozyme concentration in the solution

[0376] AC = Ciyz initial- Ciyz after absorption = the change in lysozyme concentration in the solution during the contact time with the Carrot

[0377] V = the sample volume in liters

[0378] m = the mass in grams

[0379]

[0380] Results

[0381] Calibration curves with varying protein concentrations (100%, 50% and 25%) were prepared and loaded in lanes 1-3 of the gel (Figure 8). The control samples showed no change inlysozyme band intensity. SDS-treated samples (lanes 4,5) exhibited reduction in lysozyme band intensity, corresponding to 17.22 mg of protein per g of inactive carrot, SDS-treated eluted with 0.5 NaCl are in (lanes 6,7). Heat-treated samples (lanes 8 & 9) showed reduction in lysozyme band intensity, indicating 22 mg P / g of Carrot. Heat-treated elution (lanes 10 & 11) demonstrated a 91% in lysozyme band intensity compared to 100%, corresponding to 18.9+1.8 mg P / g of Carrot. RO wash carrot tissue showed 8.6 mg P / g (lanes 13 & 12) the elution (lanes 14 & 15). Quantitative analysis of band intensities using ImageJ software (version 1.54g, National Institutes of Health, USA).

[0382] EXAMPLE 7

[0383] Testing Wolffian plant tissue with different treatments for binding capacity of lysozyme

[0384] The purpose of this experiment was to compare the effect of heat and SDS treatment on the binding capacity of Wolffia to a solution that contains lysozyme and to quantify the protein using SDS-PAGE.

[0385] Materials and methods

[0386] Pretreatments

[0387] 1. SDS Treatment: Wolffia specimens were immersed in a 1% SDS solution at room temperature for 2 hours, using a 1:3 sample-to-solution ratio. This treatment affected the plant's hull while leaving the Wolffia's shape unchanged.

[0388] 2. Ethanol Treatment (New): fresh Wolffia plants were subjected to a 50% ethanol bath for 30 minutes, employing a 1:3 sample-to-water ratio. This method was designated as "New" in the experiment.

[0389] Reused Wolffia ethanol treatment (Old): This procedure involved first using Wolffia specimens as ion exchangers for 30 cycles. Subsequently, these reused Wolffia plants underwent the same 50% ethanol bath treatment for 30 minutes as described in the "New" method.

[0390] Experimental Procedure:

[0391] The experiment was performed in duplicate and tested at three times. All the samples were washed with 10 volumes until the rinse water reached 0 mOms / L.

[0392] Lysozyme was dissolved in a 50 mM HEPES buffer at a lysozyme concentration of 5,000 mg / L. The experiment executed two replicates of 1 g of Carrot in 5 ml of solution at a 1:5 ratio (w / v). The sample with the solution was stirred for 1 hour. Samples were rinsed with deionized water at 1 :5 ratio (w / v) for 5 minutes. The elution step was carried out during a 1 hour period using 0.5 M NaCl with the same 1:5 ratio of Wolffia to elution solution.Binding capacity was calculated as in Example 6.

[0393] Results

[0394] Calibration curves with varying protein concentrations (100%, 70% and 35%) were prepared and loaded in lanes 1-3 of the gel (Figure 10). The control sample, analyzed after 60 minutes, showed no change in lysozyme band intensity. SDS-treated samples (lanes 4,5) exhibited reduction in lysozyme band intensity, corresponding to 8.65 mg P / g of Wolffia, SDS-treated eluted with 0.5 NaCl are in (lanes 6,7). Reused Wolffia ethanol treatment (Old) samples (lanes 8 & 9) showed reduction in lysozyme band intensity, indicating 9.4 mg P / g of Wolffia. The “Old” elution sample (lanes 10 & 11) demonstrated corresponding to 11 mg P / g of Carrot. Ethanol Treatment (New), fresh Wolffia plants 7.66 mg P / g (lanes 13 & 12) the elution (lanes 14 & 15).

[0395] Conclusion

[0396] Wolffia Arrhiza treated with ethanol and SDS is an effective cation exchanger.

[0397] EXAMPLE 8

[0398] Evaluation of Lysozyme Binding Capacity in Ethanol-Treated Moringa Plant Tissues This example aims to assess and compare the lysozyme binding capacity of three distinct moringa plant parts (pod, bush stem, and tree branch) following treatment with 50% ethanol. The experiment results are analyzed by SDS-PAGE (4-20% Genscript) followed by image processing (image J) and the Bicinchoninic Acid (BCA) protein assay.

[0399] Sample Preparation:

[0400] o Fresh moringa tissues (pod, bush stem, and tree branch) were collected.

[0401] o Samples were immersed in 50% ethanol for 30 minutes, using a 1:3 sample-to-ethanol ratio, while preserving their structural integrity.

[0402] 2. Washing Protocol:

[0403] o All samples underwent thorough washing with 10 volumes of water until the conductivity of the wash water reached 0 mOsm / L.

[0404] 3. Lysozyme Binding Assay:

[0405] o A lysozyme solution (5,000 mg / L) was prepared using a 50 mM HEPES buffer. o For each plant part, 2 g of ethanol-treated tissue was combined with 5 mL of lysozyme solution (2:5 w / v ratio).

[0406] o The experiment was conducted in triplicate sets, with each set containing duplicate samples.

[0407] 4. Protein Quantification:o SDS-PAGE (4-20% gradient) and BCA assay were employed to quantify the bound lysozyme.

[0408] This experimental design allows for a comparative analysis of lysozyme binding capacity across different moringa plant tissues following ethanol treatment, providing insights into potential variations in protein-binding properties among plant parts.

[0409] Results

[0410] As shown in Figure 11, different parts of the Moringa plant have different binding capacity with the moringa pod and tree branch having -10 mg P / g, the bush stem has lower binding capacity. Without being bound by theory, it is proposed that the difference in the binding capacity is due to differences in cell wall composition.

[0411] EXAMPLE 9

[0412] Assessing lysozyme binding capacity of ethanol-treated carrots during multiple cycles Ethanol-treated carrots particles were used for multiple cycles of lysozyme absorption and elution. Lysozyme was absorpted from a 50 mM Hepes buffere solution. Lysozyme was eluted using a 0.5 M NaCl solution. Following each round of elution, the carrot particles were rinsed in IM NaCl solution.

[0413] Results:

[0414] Figure 12 presents the binding capacity of the absorption and the elution efficiency of 40 reuse cycles of the same ethanol-treated carrots. The binding capacity by the BCA assay is -15.5 ±0.26 mg P / g. Calculations were performed as in Example 6.

[0415] Figure 13 displays the binding capacity during absorption and elution efficiency over 40 reuse cycles of ethanol-treated bio resin. The results were analyzed using SDS-PAGE (4-20% Genscript) followed by image processing in ImageJ. Calculations were performed as in Example 6. The binding capacity was approximately 12.5 ± 1.26 mg P / g. The deviations observed in the SDS-PAGE results were higher than those in the BCA protein assay.

[0416] EXAMPLE 10

[0417] Comparison of shredded inactivated carrot with inactivated carrot powder fixed on agarose beads

[0418] Materials:

[0419] Shredded inactivated carrot:

[0420] Carrot was shredded using a food processor in deionized water (DW) at a ratio of 1:2 (carrot: DW based on volume) 300 w at 3200 RPM with 2 knives for 30 sec (E450 CH PILOT).The resultant particles had a size distribution between 0.5 mm - 3 mm and an undefined particle shape. The shredded carrot particles were inactivated with 1:3 ratio of carrot gr to 50% ethanol ml volume solution for 30 min.

[0421] Powder: Carrot tissue was cubed (6mm x 6mm x3mm) and inactivated with 1:3 ratio of carrot gr to 50% ethanol ml volume solution for 30 min. The inactivated carrot particles were dried in 70 °C incubator for 10 hr. The dried cubes were ground in an electric mill (300w pilot) and sieved to <149 micron (100 mesh). The dried powder was then used to create the beads.

[0422] Bead preparation: The powder was mixed with l%(w / v) CaCh with 3% (w / v) agarose (Biolab) and 5%(w / v) inactivated powder solution at 80 °C. The mixture was then dropped into mineral oil to form beads (668+204 micron diameter) name B831.

[0423] Preparation of commercial pectin agarose beads:

[0424] B23: The powder was mixed with a solution of l%(w / v) CaCh with 3%(w / v) agarose (Biolab) and 2%(w / v) commercial apple pectin and dropped into mineral oil to form beads (750+449 micron diameter).

[0425] B03: The powder was mixed with a solution of l%(w / v) Cal Mix with 3%(w / v) agarose (Biolab and then dropped into mineral oil to form beads (850+154 micron diameter).

[0426] Experimental procedure

[0427] All resins were dried on a Buchner funnel for 5 minutes and weighed separately. Lysozyme was dissolved in 50 mM HEPES buffer at a concentration of 5,000 mg / L. For each experiment, 0.5 mg of resin was added to a 15 ml tube together with 0.5 g of lysozyme 5 ml of 5mg / ml solution in each 15ml tube - 2 repeats of each type of resin - 0.5g with 5 mL of lysozyme 5 mgP / ml solution. The load of protein per gr of resin is 50 mgP per gr of beads or shredded inactivated carrots.

[0428] All the samples were washed with 2 volumes until the water reached osmolarity of OmOms / L. The experiment was carried out in two replicates of 0.5 g of Carrot in 5 ml of solution at a 1:10 ratio (w / v). The sample with the solution was incubated (whilst stirring) for 1 hour. The elution step was for 1 hour using 0.5 M NaCl with the same 1:5 ratio of carrot particles to elution solution.

[0429] Results:

[0430] The experimental results revealed differential binding capacities across various preparations (Figure 14). When analyzing lysozyme binding, the shredded inactivated material demonstrated the highest absorption capacity at 50 mgP / ml with an elution of 40 mgP / ml. The beads with inactivated powdered carrot showed a comparable absorption capacity of 49 mgP / ml but exhibited a lower elution of 35 mgP / ml. In contrast, both the plain agarose beads and thosecontaining commercial pectin displayed significantly lower absorption capacities, ranging between 13-16 mgP / ml, with minimal elution rates of approximately 3 mgP / ml.

[0431] Conclusion

[0432] The agarose and agarose-pectin beads exhibited lower binding capacities, and elution properties than the inactivated carrot resins.

[0433] Particle size reduction of the inactive plant tissue correlated with increased binding capacity (data not shown).

[0434] EXAMPLE 11

[0435] Carrot tissue characterization

[0436] Microscopic imaging: Carrot particles (dimension 3x3x3 mm3) underwent inactivation according to three different protocols:

[0437] Ethanol (50-70 %);

[0438] Heat (60, 70 or 85 °C for 30 minutes;

[0439] SDS (3 volumes of SDS 0.1 % - 1%) for 2-24 hours.

[0440] Following inactivation, the tissues were washed.

[0441] The measurements were obtained using Scanning Transmission Electron Microscopy (STEM) at magnifications of X70,000.

[0442] Results:

[0443] Figure 15 shows an image of a control sample (no inactivation) at X70,000 magnification. Figure 16 shows an image of an ethanol-treated sample (inactivated) at X70,000 magnification. The membrane in Figure 15 appears dense and the cell wall looks darker compared to the treated sample (Figure 16).

[0444] Pore size determination -.

[0445] Assessment of pore size and surface area was performed using a TriStar II Plus device according to [P. Klobes, K. Meyer, and R. G. Munro, “Porosity and Specific Surface Area Measurements for Solid Materials,” Mater. Sci., vol. 960-17, p. 79, 2006, [Online]]. Protocol of ISO 15901-2:2022(E) [Analysis of nanopores by gas adsorption,” 2022. doi: ISO 15901-2:2022] with nitrogen gas was conducted on a tissue sample. Samples were prepared and kept at -80°C overnight before undergoing lyophilization for 3 days until completely dehydrated. The sample treated with ethanol with 10-70% for 30 min at 25 °C. Samples were analyzed with BET (Brunauer-Emmett-Teller), surface area analyzer (NOVA 2000E produce by Anton Paar).

[0446] Results: The samples treated with 70% ethanol generally exhibited higher surface area than those treated with lower concentrations (50% or 10%; 1.17 m2 / g vs. 1.00 m2 / g and 0.47 m2 / g, respectively), suggesting that ethanol, particularly at higher concentrations, can effectivelyincrease the surface area and pore size of the plant tissue. Heat treatment increases surface area, indicating that the elevated temperature can induce structural changes.

[0447] Table 5

[0448]

[0449] EXAMPLE 12

[0450] Adsorption of small molecules

[0451] The adsorption of several dyes (summarized in Table 6) present in DMEM medium

[0452] (at a concentration of 20 mg / liter) to carrot tissue was analyzed. The study compared untreated (viable, live) carrot cubes and ethanol 50% inactivated carrot cubes against a control (no tissue exposure). Two grams of carrots were used per five milliliters of medium, and the kinetics of contact time were analyzed after 60 min by light spectrophotometer absorbance (KLAB model MRX A2000) each dye at its own wavelength.

[0453] Table 6

[0454] "

[0455]

[0456] Results

[0457] Experimental analysis of molecular interactions with tissues revealed distinct patterns based on molecular charge and structure, as illustrated in Figure 17.

[0458] For negatively charged molecules, Rose Bengal (1017 g / mol), a xanthene -based dye with halogen substitutions, showed minimal adsorption (<5%) in viable tissues during the 60-minuteexperimental period, with slightly higher absorption (-10%) in inactivated tissues. This suggests that while tissue diffusion is possible, it requires extended contact time.

[0459] Methyl orange (327 g / mol), another negative molecule containing an azo group linking benzene and naphthalene rings, demonstrated different absorption kinetics. Inactivated tissues showed approximately 25% absorption, matching expected diffusion values, while viable tissues exhibited less than 10% absorption over 60 minutes. This indicates that tissue inactivation alters the absorption kinetics for negative molecules.

[0460] In contrast, positively charged molecules showed markedly different behavior. Methylene blue (319 g / mol) exceeded expected diffusion values (26.8%) in both viable and inactivated tissues, suggesting additional interaction mechanisms, likely involving negatively charged pectin, lignin, and DNA / RNA residues. Notably, inactivated tissue depleted 55-70% of Methylene blue compared to 30-35% in viable tissue, indicating greater accessibility of binding sites. Similarly, Safranine (350.8 g / mol) showed approximately 60% reduction in both tissue types, with faster kinetics in inactivated tissue.

[0461] Disperse Orange 3 (242 g / mol), a hydrophobic dye, demonstrated 40% depletion in viable tissue and 80% in inactivated tissue, exceeding diffusion mechanisms. This suggests hydrophobic interactions with cellulose components, which become more accessible in inactivated tissue.

[0462] Conclusion:

[0463] 1. Molecular charge significantly influences tissue interaction, with positively charged molecules showing higher removal rates compared to negative ones, regardless of molecular weight or diffusion coefficient.

[0464] 2. Tissue inactivation generally enhances molecular interactions, particularly for positively charged and hydrophobic compounds.

[0465] 3. Multiple mechanisms, including charge-based interactions and hydrophobic binding, contribute to molecular uptake by tissues.

[0466] 4. Adsorption of negative molecules can be controlled by altering the contact time with the inactivated tissue.

[0467] EXAMPLE 13

[0468] Ammonium Removal

[0469] Ammonium (NHT) is a positively charged polyatomic ion found in numerous fertilizers and cleaning agents. Additionally, it is produced as a natural byproduct of protein metabolism within the body. In the medium context, it serves as one of the metabolites that tend to accumulate, altering pH levels and leading to delays in cell growth rate. The following experiment includedwater with 10 mM HEPES buffer and ammonium chloride concentrations range within 30-100 mg / L. The carrot to medium ratio was 2:5 m / v (i.e. 2 gr of carrots with 5 ml of medium). The contact time varied between 5-60 min, at RT. The experiment was carried out in triplicates. Expected reduction from diffusion is 26.8 %.

[0470] Results

[0471] As can be seen in Figure 18, the concentration of ammonium which remains in the medium is lower than what would be expected if the ammonium was removed from the medium by diffusion only.

[0472] In the next experiment, the effect of adding sodium chloride to the medium was analyzed. Sodium chloride dissociates into Na+(cation) and CT (anion). It was hypothesized that the addition of NaCl would increase the ionic strength of the solution, thereby altering the charge interaction of ions and potentially influencing the diffusion or charge binding of ammonium.

[0473] The experiment setup was as follows: 60-minute contact time, room temperature, carrot to medium ratio was (2:5 m / v).

[0474] The following solutions were tested:

[0475] • DW + 10 mM Hepes 7.4 pH + 34 ppm Ammonia

[0476] • DW + 10 mM Hepes 7.4 pH + 34 ppm Ammonia + 34 mg / L Sodium chloride

[0477] • DW + 10 mM Hepes 7.4 pH + 34 ppm Ammonia + 340 mg / L Sodium

[0478] • DW +10 mM Hepes 7.4 pH + 34 ppm Ammonia + 3,400 mg / L Sodium

[0479] Results

[0480] The results are summarized in Table 7, herein below.

[0481] Table 7

[0482]

[0483] Based on the results above (Table 7), it was concluded that in the presence of a competing cation, ammonium is solely absorbed through the diffusion mechanism.

[0484] The experiment aimed to monitor the reduction of ammonia concentrations through diffusion, anticipating a decrease of approximately 30%. However, in the absence of sodiumchloride (NaCl), the observed reduction was significantly higher at 57%. This suggests that another mechanism, beyond simple diffusion, affects the uptake of ammonium. It was hypothesized that this additional process involves binding sites for ammonium on the treated plant. As the concentration of NaCl increased, the ammonia reduction gradually decreased and stabilized between 35% - 37% for all tested NaCl concentrations. These results indicate that the added NaCl interacts with the ammonia binding sites on the plant, influencing the reduction of ammonium, bringing it closer to the expected diffusion-driven reduction of 30%. The interaction with NaCl indicates that the ammonium binding sites on the plant are based on electric charge.

[0485] EXAMPLE 14

[0486] Assessing lysozyme binding capacity of different crosslink plant tissue Methods

[0487] Preparation of crosslinked resins:

[0488] Carrot tops were removed and the carrots were juiced. The pulp was collected and mixed with 3 volumes of 2 % citric acid solution. The mixture was stirred using an overhead stirrer. Next, the liquid from the pulp mixture was filtered and spread thinly on trays. This was left to dry overnight at 90 °C. The dried mixture was then ground using a grinder so as to reduce to a fine powder. Next, the ground powder was sieved to obtain particles having a size between 100-230 mesh. The sieved powder was subjected to heat treatment at 150 °C for 10-120 minutes, depending on the quantity of sieved powder. The heat-treated powder was then rehydrated with ten volumes of reverse osmosis (RO) water and mixed for 10 minutes to ensure complete rehydration, followed by filtration using a Buchner funnel to separate liquid from solid material, washing of the retained resin with five volumes of RO water, and transfer of the semi-dry resin to an empty beaker. The collected semi-dry bioresin was treated with a 0.1 to 1 M sodium hydroxide solution for 20 minutes and subsequently washed on a Buchner funnel sequentially with five volumes of reverse osmosis water, a further five volumes of reverse osmosis water, and five volumes of 20 mM phosphate buffer at pH 6 to remove residual chemicals and colorants. The washed wet resin was then sieved to a particle size between 60 and 200 mesh to obtain a desired particle size distribution, and finally transferred to a suitable cylinder and filled with 20% ethanol.

[0489] Crosslinked resins prepared from apple pomace (without cores), soy pods, sugarbeet, beetroot peels, whole apple, apple peels and cores, corn and hemp powder were prepared as described herein above for carrots.

[0490] Experimental procedure:

[0491] Crosslinked plant tissue particles (50-250 pm), prepared as described above, underwent one cycle of lysozyme adsorption and elution, performed in triplicate. For each trial, 0.5 ml ofresin was exposed to 7 ml of lysozyme solution (7 mg / ml) prepared in 20 mM phosphate buffer at pH 6. The adsorption phase lasted 30 minutes, after which the bound lysozyme was eluted using 0.5 M NaCl solution for 30 minutes. After each elution cycle, the particles were washed with 1 M NaCl solution.

[0492] Results

[0493] Table 8 and Figure 19 present the result of test binding capacity for lysozyme, where the carrot resins (-120 mgP / (ml of peak resin)) that contain the high pectin content have the best results and the hemp with no pectin content preference poor binding capacity ((-20 mgP / (ml of peak resin)).

[0494] Table 8

[0495]

[0496] Fourier Transform Infrared (FTIR) measurements on dry powders of the crosslinked resins from the different plant tissues corroborate these results. The spectra of the crosslinked powders show prominent absorption bands around 1700 cm corresponding to C=O stretching vibrations of ester bonds, indicating successful crosslinking, with the carrot-derived sample exhibiting the highest intensity, which correlates with its superior binding capacity. Following activation with 1 N NaOH, the spectra of the treated display a marked decrease in the ester-associated band near 1700 cm1and a concomitant increase in absorption around 1600

[0497]

[0498] attributed to carboxylate or carboxylic acid C=O stretching, reflecting partial hydrolysis of ester groups. Again, the carrot-derived resin shows the highest intensity in this region, consistent with its enhanced binding performance.

[0499] EXAMPLE 15

[0500] Reusability and stability of crosslinked resins

[0501] Methods

[0502] Preparation of crosslinked resins: as described for Example 14.Experimental protocol: A 100-cycle study was conducted using pure lysozyme (5 mg / mL). Experimental conditions were designed to utilize approximately 70% of the resin's maximum potential capacity. Two conditions were evaluated using 0.5 mL of resin in triplicate batch replicates:

[0503] Standard Protocol: Regeneration with 0.2 N NaOH performed every 10 cycles.

[0504] Aggressive Cleaning Protocol: Regeneration with 0.2 N NaOH performed after every cycle.

[0505] Protein concentration was monitored at 280 nm using the extinction coefficient for lysozyme.

[0506] Results

[0507] Table 9 presents the results of the study.

[0508] Table 9

[0509]

[0510] As shown in Figure 20, the crosslinked bioresin demonstrated consistent performance over the course of 100 cycles. The average binding capacity was 72.7 ±1.26 mgP / mL for the standard protocol and 71.17 ±1.21 mgP / mL for the aggressive NaOH cleaning protocol. This indicates that the static binding capacity (SBC) remains stable even under rigorous cleaning conditions. Additionally, no significant changes in resin weight or volume were observed, and the recovery rate remained consistent at approximately 70%.

[0511] Conclusion

[0512] This example confirms that the column and resin are stable for the full 100-cycle duration. A temporary performance deviation observed between cycles 87 and 94 was attributed to a change in the protein feed source rather than resin degradation; performance metrics returned to baseline immediately thereafter.

[0513] The "NaOH Treatment" condition performed comparably to the "No Treatment" control at Cycle 100, proving that aggressive cleaning with 0.2 N NaOH does not damage the SBV resin.Consequently, it is recommended that the resin can be utilized beyond 100 cycles, as there are no signs of permanent degradation.

[0514] EXAMPLE 16

[0515] Effect of citric acid (CA) and temperature on the bioresin Methods

[0516] Preparation of resins: carrot resin was prepared as in Example 14, but with the following changes.

[0517] Treatment 1: untreated with citric acid - (tests the effect of temperature treatment only) Treatment 2: 2% w / v citric acid - 1:3 ratio (2% CA)

[0518] Treatment 3: 20% w / w citric acid in relation to dry carrot (20% CA).

[0519] Following CA treatment, the BioResin was dried, grinded and sieve and each treatment crosslinked at range of temperature: 90 °C, 110 °C, 120 °C, 130 °C, 140 °C, 150 °C.

[0520] Experimental protocol:

[0521] The capacity of the resin to capture lysozyme evaluated in two independent experiments, with each resin type analyzed in triplicate. For each trial, 0.5 ml of resin was exposed to 10 ml of lysozyme solution (6 mg / ml) prepared in 20 mM phosphate buffer at pH 6. The adsorption phase lasted 60 minutes, following which the bound lysozyme was eluted using IM NaCl solution for 30 minutes.

[0522] Results

[0523] Figure 21 presents the capture capacity test of lysozyme results. It can be observed that increasing the temperature to 110 °C leads to a significant, more than twofold increase in lysozyme binding capacity across all CA treatments. CA treatment (2% CA and 20% CA) further enhances this capacity by 10% to 20% at 110°C and above compared to the untreated resin (0% CA). However, crosslinking in temperatures above 130°C result in a decline in binding capacity for both 2% CA and 20% CA treatments.

[0524] Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims.

[0525] It is the intent of the Applicant(s) that all publications, patents and patent applications referred to in this specification are to be incorporated in their entirety by reference into the specification, as if each individual publication, patent or patent application was specifically and individually noted when referenced that it is to be incorporated herein by reference. In addition,citation or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the present invention. To the extent that section headings are used, they should not be construed as necessarily limiting. In addition, any priority document(s) of this application is / are hereby incorporated herein by reference in its / their entirety.

Claims

WHAT IS CLAIMED IS:

1. A method of isolating a protein from a solution, the method comprising contacting the solution which comprises the protein with a resin which comprises inactivated tissue of a plant under conditions which allow adsorption of said protein to said inactivated tissue of the plant, thereby isolating the protein.

2. The method of claim 1, further comprising eluting the protein from said resin following said isolating, thereby isolating the protein from the solution.

3. A method of isolating a small molecule from a solution, the method comprising: (a) contacting the solution which comprises the small molecule with a resin which comprises inactivated tissue of a plant under conditions which allow adsorption of said small molecule to said inactivated tissue of the plant; and(b) eluting the small molecule from said resin following said isolating, thereby isolating the small molecule from the solution.

4. The method of claim 3, wherein the small molecule is not ammonia or lactate.

5. The method of any one of claims 1-4, wherein said contacting is effected in an aseptic environment.

6. The method of any one of claims 1-5, wherein said solution comprises a cell culture medium.

7. The method of any one of claims 1-4, wherein said solution comprises a Tris buffer, a citrate buffer, glycine buffer, phosphate buffer or an acetate buffer.

8. The method of claim 5, wherein said aseptic environment is a sterile environment.

9. The method of any one of claims 1-8, wherein said contacting is for a time period which is 1 minute to 24 hours.

10. The method of claims 1 or 2, wherein said protein is characterized by having an isoelectric point (pl) above 4.5.

11. The method of claims 1 or 2, wherein said protein is characterized by having an isoelectric point (pl) above 7.

12. The method of claims 3 or 4, wherein said small molecule is positively charged.

13. The method of any one of claims 3, 4 or 11, wherein said small molecule is hydrophobic.

14. The method of any one of claims 1-13, wherein said resin comprises inactivated crosslinked tissue of the plant.

15. A kit for isolating a protein or a small molecule from a solution comprising:(v) a resin which comprises inactivated tissue of a plant;(vi) elution buffer; and(vii) adsorption buffer,wherein a salt concentration of said elution buffer is higher than a salt concentration of said adsorption buffer.

16. A kit for isolating a protein from a solution comprising a resin which comprises particles of inactivated tissue of a plant, wherein a volume of the particles is less than 30 mm3.

17. An edible article of manufacture comprising a resin which comprises particles of inactivated plant tissue, wherein the resin comprises an edible protein adsorbed thereto.

18. The edible article of manufacture of claim 17, wherein the particle has a volume of less than 30 mm3.

19. The kit of claims 15 or 16, wherein the resin is pre-packed in a column.

20. The kit or article of manufacture of any one of claims 15-19, wherein the resin comprises inactivated, crosslinked tissue of the plant.

21. The method, kit or article of manufacture of any one of claims 1-20, wherein the plant is a pectin-rich plant.

22. The method, kit or article of manufacture of any one of claims 1-20, wherein said inactivated tissue is a pectin-rich inactivated tissue.

23. The method, kit or article of manufacture of any one of claims 1-22, wherein said inactivated tissue comprises a mixture of at least two polysaccharides.

24. The method, kit or article of manufacture of any one of claims 1-23, wherein said inactivated tissue is not root tissue.

25. The method, kit or article of manufacture of any one of claims 1-24, wherein said inactivated tissue comprises proteinaceous material.

26. The method, kit or article of manufacture of any one of claims 1-24, wherein stiffness of said inactivated tissue is at least as that of a carrot sample of 50 mm diameter and 7700 mm2volume the minimum forces required for bruising, bending, and shearing of the carrot fruit being 71 N, 48 N, and 41 N, respectively.

27. The method of claims 3 or 4, wherein said inactivated tissue is hydrous comprising a water content of at least 50 % v / w.

28. The method, kit or article of manufacture of any one of claims 1-27 wherein said inactivated tissue is of a fruit or a vegetable.

29. The method, kit or article of manufacture of any one of claims 1-28, wherein said plant is selected from the group consisting of carrot, apple, sugarbeet, soy and com.

30. The method, kit or article of manufacture of any one of claims 1-28, wherein said plant is carrot.

31. The method, kit or article of manufacture of any one of claims 1-28, wherein said plant is Duckweed.

32. The method, kit or article of manufacture of any one of claims 1-28, wherein said plant is Moringa.

33. The method, kit or article of manufacture of claim 32, wherein said Duckweed is selected from the group consisting of a Wolffia species and Lemna species.

34. The method, kit or article of manufacture of claim 33, wherein said Wolffia species is selected from the group consisting of Wolffia angusta, Wolffia arrhiza, Wolffia australiana, Wolffia borealis, Wolffia brasiliensis, Wolffia columbiana, Wolffia cylindracea, Wolffia elongata, Wolffia globose, Wolffia microscopica and Wolffia neglecta.

35. The method, kit or article of manufacture of claim 33, wherein said Wolffia species is Wolffia arrhiza (e.g., var. spotless watermeal).

36. The method, kit or article of manufacture of claim 33, wherein said Lemna species is selected from the group consisting of Lemna minor L., Lemna disperma L., Lemna ecuadoriensis L., Lemna gibba L., Lemna gibba L., Lemna obscura L. and Lemna trisulca L.

37. The method or kit of any one of claims 1-30, wherein said inactivated tissue is cellularized.

38. The method, kit or article of manufacture of any one of claims 1-37, wherein said inactivated tissue is at least partially decellularized.

39. The method, kit or article of manufacture of any one of claims 1-38, wherein said inactivated tissue is diced, shredded or powdered.

40. The method of any one of claims 1-14, wherein the method is for isolating a protein and a particle volume of said inactivated tissue is less than 30 mm3.

41. The method of any one of claims 1-14, wherein the method is for isolating a small molecule, and a particle volume of said inactivated tissue is between 30 mm3-100 mm3.

42. The method, kit or article of manufacture of any one of claims 1-41, wherein said inactivated tissue is immobilized on a solid support.

43. The method of claim 6, wherein said cell culture medium is selected from the group consisting of PBS, Hanks’ BSS, Earle’s salts DPBS, HBSS, EBSS, DMEM, MEM, GMEM , RPMI 1640 Leibovitz L-15, TC 100 Graces insect medium Schneider's Insect medium, Ham F10, Ham F12, DMEM / F12 and Serum-Free Insect Medium 1.

44. The method, kit or article of manufacture of any one of claims 1-43, wherein said protein is a recombinant protein.

45. The method, kit or article of manufacture of any one of claims 1-44, wherein said inactivated tissue is GRAS.

46. The method, kit or article of manufacture of any one of claims 1-45, wherein said inactivated tissue is inactivated by a treatment is selected from the group consisting of an ethanol fixation, sonication, autoclave, freeze-drying, oven-drying, steam sterilization, radiation (e.g., UV radiation), detergent treatment and a combination thereof.

47. The method, kit or article of manufacture of any one of claims 1-46, wherein said inactivated tissue is inactivated by chemical treatment with a polycarboxylic acid.

48. The method, kit or article of manufacture of claim 47, wherein said polycarboxylic acid is citric acid.

49. The method, kit or article of manufacture of any one of claims 14 or 20, wherein said inactivated crosslinked tissue of the plant is generated by:contacting viable tissue of the plant with a crosslinker under conditions which generate inactivated crosslinked plant material; andcontacting said inactivated crosslinked plant material with an alkali under conditions that enhance binding capacity of the resin.

50. The method, kit or article of manufacture of any one of claims 14 or 20, wherein a crosslinking agent used to carry out said crosslinking is an ionic crosslinking agent, an enzymatic crosslinking agent or a covalent crosslinking agent.

51. The method, kit or article of manufacture of claims 49 or 50, wherein said crosslinking agent is a covalent crosslinking agent.

52. The method, kit or article of manufacture of claim 51, wherein said covalent crosslinker is citric acid.

53. The method, kit or article of manufacture of claim 46, wherein ethanol for said ethanol fixation is about 70 % v / v.

54. The method, kit or article of manufacture of claim 46, wherein said detergent is sodium dodecyl sulfate (SDS).

55. The method of claim 2, further comprising performing at least one additional round of isolating following said eluting.

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