WO2021007484A1 - Process for isolating a high purity protein preparation from plant material and products thereof - Google Patents

Process for isolating a high purity protein preparation from plant material and products thereof Download PDF

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Publication number
WO2021007484A1
WO2021007484A1 PCT/US2020/041525 US2020041525W WO2021007484A1 WO 2021007484 A1 WO2021007484 A1 WO 2021007484A1 US 2020041525 W US2020041525 W US 2020041525W WO 2021007484 A1 WO2021007484 A1 WO 2021007484A1
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WO
WIPO (PCT)
Prior art keywords
plant material
liquid phase
purified protein
lysed
chlorophyll
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
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PCT/US2020/041525
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English (en)
French (fr)
Inventor
Tony MARTENS
Cesar Flores
Eduardo REYNOSO MORENO
Roland VAN DER MOLEN
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Plantible Foods Inc
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Plantible Foods Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority to US17/625,468 priority Critical patent/US20220259259A1/en
Priority to EP20837203.7A priority patent/EP3997102A4/en
Priority to CN202411910916.0A priority patent/CN120081893A/zh
Priority to CN202080061713.2A priority patent/CN114364688A/zh
Priority to AU2020310192A priority patent/AU2020310192A1/en
Priority to MX2022000315A priority patent/MX2022000315A/es
Priority to MYPI2021007839A priority patent/MY208163A/en
Priority to JP2022500839A priority patent/JP7646624B2/ja
Application filed by Plantible Foods Inc filed Critical Plantible Foods Inc
Priority to CA3146551A priority patent/CA3146551A1/en
Publication of WO2021007484A1 publication Critical patent/WO2021007484A1/en
Anticipated expiration legal-status Critical
Priority to US17/722,893 priority patent/US20220232877A1/en
Priority to US18/121,226 priority patent/US20230217981A1/en
Priority to JP2025034337A priority patent/JP2025084972A/ja
Ceased legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/17—Amino acids, peptides or proteins
    • A23L33/185—Vegetable proteins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/145—Extraction; Separation; Purification by extraction or solubilisation
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
    • A23J1/00—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites
    • A23J1/006—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites from vegetable materials
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
    • A23J3/00—Working-up of proteins for foodstuffs
    • A23J3/30—Working-up of proteins for foodstuffs by hydrolysis
    • A23J3/32—Working-up of proteins for foodstuffs by hydrolysis using chemical agents
    • A23J3/34—Working-up of proteins for foodstuffs by hydrolysis using chemical agents using enzymes
    • A23J3/346—Working-up of proteins for foodstuffs by hydrolysis using chemical agents using enzymes of vegetable proteins
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/105—Plant extracts, their artificial duplicates or their derivatives
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/30—Extraction; Separation; Purification by precipitation
    • C07K1/32—Extraction; Separation; Purification by precipitation as complexes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/34—Extraction; Separation; Purification by filtration, ultrafiltration or reverse osmosis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/88—Lyases (4.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y401/00—Carbon-carbon lyases (4.1)
    • C12Y401/01—Carboxy-lyases (4.1.1)
    • C12Y401/01039—Ribulose-bisphosphate carboxylase (4.1.1.39)
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs

Definitions

  • RuBisCo protein ribulose-l,5-bisphosphate carboxylase/oxygenase
  • the protein ribulose-l,5-bisphosphate carboxylase/oxygenase (“RuBisCo”) comprises up to 50% of the total protein in plants. It is the enzyme involved in the first major step of carbon fixation, a process by which atmospheric carbon dioxide is converted by plants and other photosynthetic organisms to energy -rich molecules such as glucose. Due to its abundance in plants, it serves as an alternative source of protein for food production.
  • Purified RuBisCo is typically a tasteless, odorless, white powder.
  • the duckweed (subfamily Lemnoideae, genus Lemna) is among the smallest flowering plants in the world. Despite its diminutive size, it has the ability to grow quickly, doubling its biomass in about 16 to 48 hours depending on the conditions. Mestameyer et al., Spirodela punctata Aquatic Botany, (1984) 19: 157-70. Lemna has a high protein content (about 30-35% of its dry mass being protein) and thus has been used as in animal feedstock. All these properties make Lemna an attractive candidate for large scale production of plant-based protein for food.
  • the ability of proteins to form emulsions, gels and stable foams are also important in the production of a variety of foods, forming the basis for texture in the food products. For example, foams with a uniform distribution of small air bubbles impart body, smoothness and lightness to the food.
  • the ability of a protein preparation to form a foam is related to its purity, and a purity of at least about 80% may be needed to form a stable foam.
  • gels made from proteins give rise to foods of different rheological properties and appearances.
  • the gelling capacity of a protein may be measured by the amount of protein needed to form a gel.
  • protein preparations with high purity, foaming capacity, foam stability and gelling capacity are desired for use in food products.
  • the present disclosure provides a process for making a purified protein preparation from a plant material, comprising:
  • the present disclosure also provides a process for making a purified protein preparation from a plant material, comprising:
  • the present disclosure also provides a process for making a purified protein preparation from a plant material, comprising:
  • the present disclosure also provides a process for making a purified protein preparation from a plant material, comprising:
  • the present disclosure also provides a process for making a purified protein preparation from a plant material, comprising:
  • the present disclosure relates to the following embodiments:
  • a process for making a purified protein preparation from a plant material comprising:
  • a process for making a purified protein preparation from a plant material comprising:
  • a process for making a purified protein preparation from a plant material comprising:
  • a process for making a purified protein preparation from a plant material comprising:
  • reducing agent is 2-mercaptoethanol (BME), 2-mercaptoethylamine-HCL, sodium metabisulfite, cysteine hydrochloride, dithiothreitol (DTT), glutathione, cysteine, tris(2-carboxyethyl)phosphine (TCEP), ferrous ion, nascent hydrogen, sodium amalgam, oxalic acid, formic acid, magnesium, manganese, phosphorous acid, potassium, or sodium.
  • BME 2-mercaptoethanol
  • 2-mercaptoethylamine-HCL sodium metabisulfite
  • cysteine hydrochloride dithiothreitol (DTT)
  • glutathione glutathione
  • cysteine cysteine
  • TCEP tris(2-carboxyethyl)phosphine
  • ferrous ion ferrous ion
  • nascent hydrogen sodium amalgam
  • oxalic acid formic acid
  • magnesium manganese
  • the one or more salts of d) comprise one or more calcium salts, one or more magnesium salts, one or more beryllium salts, one or more zinc salts, one or more cadmium salts, one or more copper salts, one or more iron salts, one or more cobalt salts, one or more tin salts, one or more strontium salts, one or more barium salts, and/or one or more radium salts.
  • the flocculant is an alkylamine epichlorohydrin, polydimethyldiallylammonium chloride, a polysaccharide, a polyamine, starch, aluminum sulphate, alum, polyacrylamide, polyacromide, or polyethyleneimine.
  • a food comprising a purified protein preparation from a plant material, wherein the protein preparation contains no more than 80% impurities.
  • the plant material is washed before the process is started.
  • the reducing agent in a) is 2-mercaptoethanol (BME), 2-mercaptoethylamine-HCL, sodium metabisulfite, cysteine hydrochloride, dithiothreitol (DTT), glutathione, cysteine, tris(2- carboxyethyl)phosphine (TCEP), ferrous ion, nascent hydrogen, sodium amalgam, oxalic acid, formic acid, magnesium, manganese, phosphorous acid, potassium, and sodium.
  • the reducing agent is a sulfite.
  • the sulfite is sodium sulfite, magnesium sulfite, or sodium metabisulfite.
  • the sulfite is sodium bisulfite.
  • the pH of the solution in a) is about pH 5.0 to about pH 9.0. In some embodiments, the pH of the solution is about pH 6.0 to about pH 7.6. In some embodiments, the pH of the solution is about 6.8.
  • the plant material and solution of a) is at a ratio of about 6: 1. In some embodiments, the plant material and solution of a) is at a ratio of about 3: 1. In some embodiments, the plant material and solution of a) is at a ratio of about 2: 1. In some embodiments, the plant material and solution of a) is at a ratio of about 1 : 1.
  • the plant material is lysed mechanically.
  • the plant material is lysed mechanically using a blender.
  • the plant material is lysed mechanically using mills, homogenizers, microfluidizers, mechanical pressure or Stephan cutter.
  • the separating of c) is performed with a screw press, a decanter or a centrifuge.
  • the first set temperature of d) is no more than about 80°C. In some aspects of the disclosure, the first set temperature of d) is no more than about 65°C. In some embodiments, the first set temperature of d) is no more than about 55°C. In some embodiments, the first set temperature of d) is no more than about 50°C. In some embodiments, the second set temperature of d) is no more than about 25°C. In some embodiments, the second set temperature of d) is no more than about 15°C. In some embodiments, the second set temperature of d) is no more than about 10°C. In some embodiments, heating to a first set temperature of d) takes no more than about 15 min.
  • heating to a first set temperature of d) takes no more than about 5 min. In some embodiments, cooling to a second set temperature of d) takes no more than about 15 min. In some embodiments, cooling to a second set temperature of d) takes no more than about 5 min.
  • the one or more salts of d) comprise potassium phosphate and/or calcium chloride. In some aspects of the disclosure, the one or more salts of d) is added at a concentration of 5 mM to 2 M.
  • the flocculant is chitosan. In some aspects of the disclosure, the flocculant is activated chitosan. In some embodiments, the flocculant is 1-20% w/v activated chitosan in solution. In some embodiments, the adsorbent of e) is activated carbon, activated charcoal, or activated coal. In some embodiments, the adsorbent is a hydrophobic adsorbent.
  • separation of f) is performed at no more than 25°C. In some embodiments, the separation of f) is performed at no more than 15°C. In some embodiments, the separation of f) is performed at no more than 10°C. In some embodiments, the separation of f) is performed using a centrifuge, or a decanter, or by microfiltration.
  • all steps of the process except for e) are performed at no more than 25°C. In some embodiments, all steps of the process except for e) are performed at no more than 15°C. In some embodiments, all steps of the process except for e) are performed at no more than 10°C.
  • the filtering of g) is performed with a membrane filter. In some embodiments, the filtering of g) is performed with a 0.7 um membrane filter. In some embodiments, the filtering of g) is performed with a 0.2 um membrane filter. In some embodiments, wherein the filtering of g) is performed with diatomaceous earth and/or an activated carbon. In some embodiments, the filtering of g) is performed with up to about 10% activated carbon. In some embodiments, the filtering of g) is performed with up to about 2% activated carbon. In some embodiments, the filtering of g) is performed with a 0.2 pm membrane filter and a 2% activated carbon.
  • the process further comprises, after g), filtering the filtrate of g) through a 0.2 pm membrane filter. In some embodiments, the process further comprises concentrating the filtrate. In some embodiments, concentrating the filtrate is performed by diafiltration. In some embodiments, concentrating the filtrate is performed by
  • the yield of the purified protein is at least about 10% of the soluble protein in the liquid phase of step c). In some embodiments, the yield of the purified protein is at least about 20% the soluble protein in the liquid phase of step c). In some embodiments, the yield of the purified protein is at least about 25% the soluble protein in the liquid phase of step c). In some embodiments, the purity of the purified protein is at least about 40%. In some embodiments, the purity of the purified protein is at least about 60%. In some embodiments, the purity of the purified protein is at least about 80%.
  • the protein is RuBisCo.
  • the plant material is from Lemna. In some embodiments, the plant material is from Lemnoideae.
  • foods comprising a purified protein preparation from a plant material, wherein the protein preparation contains no more than 80% impurities.
  • FIG. 1 Flow chart of one embodiment of the process.
  • FIG. 2 Flow chart of a second embodiment of the process.
  • FIG. 3 Flow chart of a third embodiment of the process.
  • FIG. 4 Flow chart of a fourth embodiment of the process.
  • FIG. 5 Depiction of Fractions 1, 2, 3, and 4 of Example 5 after micro filtration.
  • FIG. 6 Depiction of Fractions 4, 3, 2, and 1 of Example 5 after microfiltration.
  • FIG. 7A Depiction of Fractions of Example 5.
  • FIG. 7B Depiction of Fractions of Example 5.
  • FIG. 8 Depiction of samples of Fractions of Example 6 after calcium chloride and phosphate addition and benchtop centrifugation.
  • FIG. 9 Depiction of samples of Fractions 1-6 of Example 6 after removal of activated carbon and chitosan.
  • FIG. 10 Depiction of results of SDS-PAGE Coomassie staining analysis for samples of Fractions of Example 6.
  • FIG. 11 Depiction of SDS-PAGE gel for various samples of Example 7.
  • FIG. 12 Depiction of Fractions of Example 7 after removal of activated carbon and chitosan.
  • FIG. 13 Depiction of SDS-PAGE gel for various samples of Example 8.
  • FIG. 14 Depiction of SDS-PAGE gel for various samples of Example 9.
  • FIG. 15 Depiction of Fractions 1-5 of Example 10 after microfiltration.
  • FIG. 16 Depiction of a chromatogram of final protein product and protein standard.
  • FIG. 17 Depiction of an SDS-PAGE gel of final plant protein product.
  • FIG. 18 Depiction of an absorbance spectrum of materials of Example 16.
  • plants refers to an organism belonging to the kingdom Plantae. Examples of plants suitable for use in the disclosed processes include trees, herbs, bushes, grasses, vines, ferns, mosses, and green algae.
  • plant material refers to any biomass derived from a plant. Plant material may be derived from any part of a plant, e.g., stem, root, fruit, leaves, or seeds. In some embodiments, plant material is derived from leaves. In some embodiments, plant material is derived from stems. The plant material may also be obtained from one or more species of plants.
  • plant material may be derived from duckweed, algae, sugar beet leaves, spinach, kale, beet, chard, sugar beet, sea beet, Mangel beet, soy, or tobacco.
  • plant material is derived from duckweed.
  • plant material is derived from Lemna.
  • plant material is derived from Lemnoideae.
  • the term“protein” refers to a compound comprised of amino acid residues covalently linked by peptide bonds.
  • a protein typically contains at least two amino acids or amino acid variants, and no limitation is placed on the maximum number of amino acids that can comprise a protein sequence.
  • the term“protein preparation” refers to an isolate of proteins, wherein the proteins has been substantially separated from non-protein components of a mixture.
  • The“purity” of a protein preparation refers to the amount of protein relative to the total amount of preparation. In some embodiments, the purity of the protein preparation is expressed as a percentage of the total dry mass. In some
  • a protein preparation comprises at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%,
  • the purity of the protein preparation is at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, or 99% protein.
  • a protein preparation may comprise one or more types of protein and may comprise different sizes of the same protein. For instance, in some embodiments, the protein preparation may comprise edestin, gluten, legumin or vicilin. In some embodiments, the protein preparation comprises RuBisCo.
  • the processes disclosed herein separates proteins from other compounds found in plant material.
  • the process may remove chlorophyll, volatilized chemical compounds, acids, bases, sugars, salts, and/or lipids.
  • the processes disclosed herein remove chlorophyll from plant material, producing protein preparations that are dechlorophyllized.
  • the weight ratio of chlorophyll to protein in the protein preparation is less than about 1:1000, 1: 1500, 1:2000, or 1:2500.
  • the processes disclosed herein reduce or remove one or more agent(s) that imparts or is associated with one or more organoleptic properties in the purified protein preparation.
  • organoleptic properties include odor (e.g., off-odor or undesirable odor) and taste (e.g., off-taste or undesirable taste).
  • the processes disclosed herein reduce the one or more agent(s) by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% relative to the source plant material.
  • the processes disclosed herein completely reduce the one or more agent(s). In some embodiments, the processes disclosed herein reduce or remove one or more agent(s) that imparts or is associated with odor and produce protein preparations that are odorless or essentially odorless. In some embodiments, the processes disclosed herein reduce or remove one or more agent(s) that imparts or is associated with taste and produce neutral-tasting protein preparations or essentially neutral-tasting protein preparations.
  • the processes disclosed herein reduce or remove one or more agent(s) that imparts or is associated with odor and/or taste and produce protein preparations that are odorless and neutral-tasting, essentially odorless and neutral-tasting, odorless and essentially neutral-tasting, or essentially odorless and essentially neutral-tasting.
  • the agent is a volatile compound. In some embodiments, the agent is a non-volatile compound.
  • the agent is a polyphenol, polyphenol oxidase, lipoxygenase, a phenol, a lipid, an alcohol, an aldehyde, a sulfide, a peroxide, a terpene, an albumin (e.g., a lectin or a protease inhibitor), a substrate for an oxidative enzymatic activity (e.g., a fatty acid, such as (04:0 (methyl myristate), 05:0 (methyl pentadecanoate), 06:0 methyl palmitate, 06: 1 methyl palmitoleate, 07:0 methyl heptadecanoate, 08:0 methyl stearate, 08: 1 methyl oleate, 08:2 methyl linoleate, 08:3 methyl alpha linoleate, C20:0 methyl eicosanoate, and C22:0 methyl behenate, and/or an enzyme that react
  • the purified protein preparation has a reduced oxidative enzymatic activity relative to the source of the protein. In some embodiments, the purified protein preparation has a 5%, 10%, 15%, 20%, or 25% reduction in oxidative enzymatic activity relative to the source of the protein. In some embodiments, the source of the protein is RuBisCo, and the purified protein preparation has a 5%, 10%, 15%, 20%, or 25% reduction in oxidative enzymatic activity relative to RuBisCo. In some embodiments, the oxidative enzymatic activity is lipoxygenase activity. In some embodiments, the purified protein preparation has lower oxidation of lipids or residual lipids relative to the source of the protein due to reduced lipoxygenase activity.
  • a process (FIG. 1) for making a purified protein preparation from a plant material comprises the steps of:
  • the plant material is harvested and cleaned before the process is started.
  • the plant material is chemically washed before the process is started.
  • the plant material is washed with water before the process is started. The plant material may also undergo multiple rounds of washes before the process is started.
  • the plant material is mixed in a solution comprising a reducing agent.
  • reducing agents suitable for use in the disclosed processes include, but are not limited to, 2- mercaptoethanol (BME), 2-mercaptoethylamine-HCL, sodium metabisulfite, cysteine hydrochloride, dithiothreitol (DTT), glutathione, cysteine, tris(2-carboxyethyl)phosphine (TCEP), ferrous ion, nascent hydrogen, sodium amalgam, oxalic acid, formic acid, magnesium, manganese, phosphorous acid, potassium, and sodium.
  • the plant material is mixed in a solution comprising more than one reducing agent.
  • the reducing agent is a sulfite. In some embodiments, the reducing agent is at least one of sodium sulfite, magnesium sulfite, or sodium metabisulfite. In some embodiments, the reducing agent is sodium bisulfite. Without wishing to be bound by theory, it is believed that reducing agents act to regulate and/or inhibit the activity of polyphenol oxidase.
  • the solution comprising the reducing agent may be formulated to improve the stability of its components.
  • the pH, ionic strength or temperature of the solution may be adjusted.
  • the solution may comprise buffering agents.
  • buffering agents for use in the disclosed processes include, but are not limited to, alkali metals (e.g., Na + or K + ), NaCl, ammonium ions (NH 4 ), nitrates, acetates (e.g., sodium acetate), chlorates, perchlorates (NO 3- , C 2 H 3 O 2 , CIO 3 , CIO 4 ), binary compounds of halogens with metals (e.g., Cl , Br , or G ), sulfates (S04 2 ), ammonium sulfate, hydroxides of alkali earth metals (e.g., OH , Ca 2+ , or Sr 21 ), sulfides (S 2 ), hydroxides (OH-), carbonates (
  • solutions for use in the disclosed processes comprise chelating agents.
  • chelating agents for use in the disclosed processes include, but are not limited to, chloride, cyanide, organic acids (including but not limited to citric acid, glycolic acid, lactic acid, malic acid, malonic acid, oxalic acid, and succinic acid), deferoxamine, deferiprone, deferasirox, penicillamine, honey, sodium pyrophosphate, sodium hexametaphosphate, sporix, BAL, EDTA, dexrazoxane, Prussian Blue, ALA, BAPTA, DTP A, DMPS, DMSA, EGTA, ribose, deoxyribose, glucose, fructose, glucosamine, sucrose, lactose, maltose, cellulose, starch, pectins, gums, alginic acid, chitin, chitosans, lactic acid,
  • organic acids including but not limited to
  • solutions for use in the disclosed processes comprise protease inhibitors.
  • protease inhibitors for use in the disclosed processes include, but are not limited to, PMSF, sodium fluoride, beta-glycerophosphate, sodium pyrophosphate, leupeptin, and E-64.
  • the pH of the solution is about 5.0 to about 9.0. In some embodiments, the pH of the solution is about 6.5 to about 7.5. In some embodiments, the pH of the solution is about 7.5. In some embodiments, the pH of the solution is about 6.5 to about 7.0.
  • the plant material and the solution comprising the reducing agent may be mixed in proportions that increase the accessibility of the plant material to the reducing agent.
  • the plant material and the solution comprising the reducing agent may be fixed at a ratio of about 6: 1, 3: 1, 2: 1, or 1 : 1.
  • the term“lysing” refers to breaking up of the cells from the plant material and exposing the contents of the cell.
  • lysing may comprise breaking the cell wall, disrupting the plasma membrane, and/or exposing the cytoplasm.
  • Methods for lysing plant material are known in the art, and may comprise mechanical, chemical, and/or enzymatic lysis. In some embodiments, the plant material is lysed mechanically.
  • Examples of mechanical lysis suitable for use in the disclosed processes include, but are not limited to, mechanical agitation, pressure, grinding, squeezing, and shearing.
  • the plant material is lysed mechanically using a blender.
  • the plant material is lysed mechanically using a mill, e.g., by knife mill, high shear mill, colloid mill, ball mill, Boston shear mill, hammer mill, grinding mill, Rietz mill, wet mill or high shear mill.
  • the plant material is lysed mechanically using at least two different types of mills (e.g., via tandem milling).
  • the plant material is lysed mechanically using a sonicator, using nitrogen burst, using ultrasonic energy, or by freezing.
  • the plant material is lysed mechanically using a press (e.g., a screw press or a French press).
  • the plant material is lysed mechanically using a homogenizer (e.g., a high-pressure homogenizer or a micro fluidizer).
  • the plant material is lysed mechanically using a disintegrator.
  • the plant material is lysed mechanically using a pulse electric field (PEF). In some embodiments, the plant material is lysed mechanically using mechanical pressure. In some embodiments, the plant material is lysed mechanically using one or more of the techniques for mechanical lysis disclosed herein.
  • PEF pulse electric field
  • the plant material is lysed chemically. In some embodiments, the plant material is lysed chemically using one or more detergents. In some embodiments, the one or more detergents are ionic detergents. In some embodiments, the one or more detergents are cationic detergents. In some embodiments, the one or more detergents are anionic detergents. In some embodiments, the one or more detergents include sodium dodecyl sulfate (SDS). In some embodiments, the one or more detergents are non-ionic detergents, such as Triton X-100, NP-40, digitonin, and/or saponin.
  • SDS sodium dodecyl sulfate
  • the one or more detergents are zwitterionic detergents, such as Triton, NP, Brij, Tween, octyl-beta-glucoside, octylthioglucoside, SDS, CHAPS, and/or CHAPSO.
  • the one or more detergents are hypotonic detergents.
  • the one or more detergents are hypertonic detergents.
  • the one or more detergents are isotonic detergents.
  • the plant material is lysed chemically using one or more of the techniques for chemical lysis disclosed herein.
  • the plant material is lysed enzymatically using one or more enzymes.
  • the one or more enzymes include cellulase.
  • the one or more enzymes include pectinase.
  • the plant material is lysed chemically and mechanically. In some embodiments, the plant material is lysed chemically and enzymatically. In some embodiments, the plant material is lysed mechanically and enzymatically. In some embodiments, the plant material is lysed chemically, mechanically, and enzymatically.
  • the lysing of the plant material includes adding one or more divalent ion(s) to the lysate. In some embodiments, the lysing of the plant material comprises adding chitosan to the lysate. In some embodiments, the lysing of the plant material comprises adding one or more divalent ion(s) to the lysate and adding chitosan to the lysate. In some embodiments, the lysing of the plant material comprises adding calcium ions to the lysate. In some embodiments, the lysing of the plant material comprises adding calcium ions to the lysate and adding chitosan to the lysate.
  • the lysing of the plant material comprises adding calcium chloride to the lysate. In some embodiments, the lysing of the plant material comprises adding calcium chloride to the lysate and adding chitosan to the lysate.
  • Separation of the lysed plant material into a solid phase and a liquid phase may be performed by any solid-liquid separation techniques known in the art. Examples of such separation techniques suitable for use in the disclosed processes include sieving, filtration, centrifugation and decanting.
  • separating the lysed plant material into a solid phase and a liquid phase is performed with a screw press, a decanter or a centrifuge.
  • separating the lysed plant material into a solid phase and a liquid phase is performed using a disk stack centrifuge, a decanter centrifuge, a continuous centrifuge, or a basket centrifuge.
  • separating the lysed plant material into a solid phase and a liquid phase comprises filtration, including but not limited to using a dead-end filtration system, using ultrafiltration, using a tangential flow filtration system, or using a plate filter.
  • separating the lysed plant material into a solid phase and a liquid phase comprises use of a press, including but not limited to a screw press, a French press, a belt press, a filter press, a fan press, a finisher press, or a rotary press.
  • separating the lysed plant material into a solid phase and a liquid phase comprises using gravity settling.
  • separating the lysed plant material into a solid phase and a liquid phase comprises sieving, including but not limited to using a circular vibratory separator or a linear/inclined motion shaker.
  • the liquid phase comprises soluble proteins and chlorophyll.
  • the solid phase comprises insoluble proteins, lignin, fibers, etc.
  • the lysed plant material may comprise soluble proteins, chlorophyll, phenolic compounds, cellular membranes (e.g., lipids), carbohydrates (including but not limited to pectin), nucleic acids, and/or light harvesting complexes/photosystems.
  • the solid phase obtained from the separation of the lysed plant material may comprise plant fiber(s), cellulose, hemicellulose, pectin, intact plant cells, cellular organelles, insoluble proteins, chlorophyll, and/or fats.
  • this solid phase may be used as, for example, an animal feed or as a biofuel.
  • this solid phase may contain levulinic acid, which is a precursor in the manufacture of biofuels.
  • chlorophyll obtained from the solid phase obtained from the separation of the lysed plant material may be used in, for example, cosmetic applications, as a dye, and/or in human and/or animal nutrition.
  • the process for making the purified protein preparation may also comprise a step of coagulating components that are undesired (e.g., components that are not RuBisCO) using heat treatment, leaving the desired protein component (e.g., RuBisCO) in the liquid phase.
  • coagulating components that are undesired e.g., components that are not RuBisCO
  • desired protein component e.g., RuBisCO
  • heating causes conformational unfolding of amino acid chains, resulting in aggregation of some proteins.
  • proteins Depending on their amino acid sequence and conformational states, proteins have different unfolding temperatures, above which they will begin to unfold and aggregate. With carefully controlled heating and cooling conditions, proteins with unfolding temperatures lower than that of the desired protein product may be coagulated. In some embodiments, the heating is conducted under mild conditions to prevent the protein of interest from also aggregating. In some embodiments, the liquid phase is heated to a first set temperature. In some embodiments, the first set temperature is no more than about 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, 70°C, 75°C or 80°C. In some embodiments, the heating is performed rapidly. In some embodiments, the heating to the first set temperature takes no more than about 30 min.
  • heating to the first set temperature takes no more than 15 min. In some embodiments, heating to the first set temperature takes no more than 5 min. In some embodiments, the liquid phase is cooled to a second set temperature after heating to the first set temperature. In some embodiments, the second set temperature is no less than about 30°C, 25°C, 20°C, 15°C, 10°C, or 5°C. In some
  • the cooling is initiated immediately upon reaching the first set temperature. In some embodiments, the cooling is performed rapidly. In some embodiments, cooling to a second set temperature takes no more than about 30 min. In some embodiments, cooling to a second set temperature takes no more than about 15 min. In some embodiments, cooling to a second set temperature takes no more than about 5 min.
  • the process for making the purified protein preparation may also comprise a step of coagulating components that are undesired (e.g., components that are not RuBisCO) by addition of one or more salts, leaving the desired protein component (e.g., RuBisCO) in the liquid phase.
  • the salt is a calcium salt, a magnesium salt, a beryllium salt, a zinc salt, a cadmium salt, a copper salt, an iron salt, a cobalt salt, a tin salt, a strontium salt, a barium salt, a radium salt, or combinations thereof.
  • the salt is calcium chloride, calcium nitrate, or iron carbonate.
  • the salt added is potassium phosphate. In some embodiments, the salt added is calcium chloride. In some embodiments, the salts added are potassium phosphate and calcium chloride. In some embodiments, the one or more salt is added at a concentration of 5 mM to 2 M.
  • the process for making the purified protein preparation may also comprise a step of coagulating components that are undesired (e.g., components that are not RuBisCO) by addition of one or more coagulants, leaving the desired protein component (e.g., RuBisCO) in the liquid phase, wherein the coagulant is a quaternary ammonia species, including but not limited to a protonated tertiary, secondary, or primary ammonium species.
  • the coagulant is selected from epiamines, polytannines, polyethylene imines, polylysines, and cationic polyacrylamides.
  • the coagulant is a polymer-based coagulant.
  • the polymer is zwitterionic. In some embodiments, the polymer is in the form of a solution or an emulsion. In some embodiments, the polymer is granular. In some embodiments, the polymer is a bead. In some embodiments, the polymer is uncharged. In some embodiments, the polymer has a charge density from less than 1 and up to 100% theoretical mole. In some embodiments, the polymer has a molecular weight is from 500 Daltons to 20 million Daltons. In some embodiments, the polymer has a molecular weight that is greater than 20 million Daltons.
  • the process for making the purified protein preparation may also comprise a step of coagulating components that are undesired (e.g., components that are not RuBisCO) by electrocoagulation.
  • components that are undesired e.g., components that are not RuBisCO
  • electrocoagulation water passes through an
  • electrocoagulation cell wherein metal ion(s) are driven into the water, wherein, on a surface of a cathode, water is hydrolyzed into hydrogen gas and hydroxyl groups, wherein electrons flow freely to destabilize surface charges on suspended solids and emulsified oils, wherein large floes form that entrain suspended solids, heavy metals, emulsified oils, and other contaminants, and wherein the floes from removed from the water in downstream solids separation and/or filtration operation(s).
  • the process for making the protein preparation may also comprise a step of contacting the liquid phase with a flocculant and/or an adsorbent and mixing for a period of time sufficient to flocculate and/or adsorb chlorophyll in the liquid phase to the adsorbent, thereby forming a flocculated mixture.
  • a flocculant refers to substance that is added to destabilize colloids and cause them to come out of a suspension.
  • exemplary flocculants may include, but are not limited to, an alkylamine epichlorohydrin, polydimethyldiallylammonium chloride, a polysaccharide (e.g., chitosan), a polyamine, starch, aluminum sulphate, alum, polyacrylamide, polyacromide, or polyethyleneimine.
  • the flocculant is activated chitosan. In some embodiments, the flocculant is 1-20% w/v activated chitosan in solution.
  • Methods to activate chitosan and dissolve chitosan in solution are well known in the art, and any method to prepare the activated chitosan in solution may be used. Exemplary methods may involve dissolving 1% chitosan in 20% acetic acid and 79% water.
  • Adsorbents are known in the art, and exemplary adsorbents may include activated carbon, graphite, silica gel, zeolites, clay, polyethylene etc. Exemplary adsorbents may also include resins.
  • resins for use in the disclosed processes are ion-exchange resins, including but not limited to strong cation exchangers, weak cation exchangers, strong anion exchangers, weak anion exchangers, mixed bed resins, chelating resins, and polymeric catalysts.
  • resins for use in the disclosed processes are size exclusion chromatography (SEC) resins, including but not limited to Sephacryl, Sephadex, Sepharose, and Superdex (GE Healthcare Bio-Sciences Corp, Westborough, Massachusetts).
  • resins for use in the disclosed processes have an affinity for a substrate analogue, an antibody-antigen, a polysaccharide (e.g., lectin), a complementary base sequence (e.g., a nucleic acid), a receptor (e.g., a hormone), avidin-biotin, calmodulin, poly-A, glutathione, proteins A and G, and/or metal ions.
  • resins for use in the disclosed processes have a hydrophobic interaction, such as resins comprising phenyl, butyl, octyl, hexyl, ether, and/or PPG.
  • the adsorbent is activated carbon, activated charcoal, or activated coal.
  • the activated carbon has a surface area in excess of 250 m/g, a weight average diameter of 1-1000 mih, an iodine number of 400-1,400 mg/g, a molasses number in the range of 100-550, and/or a Methylene Blue adsorption of at least 10 g/100 g.
  • the liquid phase is contacted with a polymer.
  • the polymer is non-ionic.
  • the polymer is anionic.
  • the polymer is cationic.
  • the polymer is zwitterionic.
  • the polymer is in the form of a solution or an emulsion. In some embodiments, the polymer is granular. In some embodiments, the polymer is a bead. In some embodiments, the polymer is uncharged. In some embodiments, the polymer has a charge density from less than 1 and up to 100% theoretical mole. In some embodiments, the polymer has a molecular weight is from 500 Daltons to 20 million Daltons. In some embodiments, the polymer has a molecular weight that is greater than 20 million Daltons.
  • chlorophyll in the liquid phase may flocculate to form larger sized particles, which then adsorb to the surface of the activated carbon, charcoal or coal.
  • the flocculated mixture comprises a solid phase comprising the flocculant, adsorbent, insoluble proteins and chlorophyll, and a liquid phase that comprises soluble proteins in solution.
  • the flocculated mixture may then be separated into a solid phase and a liquid phase. Separation of the flocculated mixture may be performed by any solid-liquid separation techniques known in the art. Examples of such separation techniques suitable for use in the disclosed processes include sieving, filtration, centrifugation and decanting. In some embodiments, separation of the flocculated mixture into a solid phase and a liquid phase is performed with a screw press, a decanter or microfiltration. In some embodiments, separation of the flocculated mixture into a solid phase and a liquid phase comprises centrifugation, such as the use of a decanter centrifuge, a disk stack centrifuge, or a continuous centrifuge.
  • separation of the flocculated mixture into a solid phase and a liquid phase comprises filtration, such as the use of a dead-end filtration system, ultrafiltration, the use of a tangential flow filtration system, or a plate filter.
  • separation of the flocculated mixture into a solid phase and a liquid phase comprises use of a press, such as a screw press, a French press, a belt press, a filter press, a fan press, a finisher press, or a rotary press.
  • separation of the flocculated mixture into a solid phase and a liquid phase comprises gravity settling.
  • separation of the flocculated mixture into a solid phase and a liquid phase comprises sieving.
  • separating the flocculated mixture into a solid phase and a liquid phase comprises removing a hydrophobic adsorbent.
  • the removing of a hydrophobic adsorbent comprises centrifugation, such as the use of a disk stack centrifuge, a continuous centrifuge, or a basket centrifuge.
  • the removing of a hydrophobic adsorbent comprises filtration, including but not limited to using a dead-end filtration system, using ultrafiltration, using a tangential flow filtration system, or using a plate filter.
  • the removing of a hydrophobic adsorbent comprises use of a press, including but not limited to a screw press, a French press, a belt press, a filter press, a fan press, a finisher press, or a rotary press.
  • the removing of a hydrophobic adsorbent comprises using gravity settling.
  • the removing of a hydrophobic adsorbent comprises sieving.
  • the removing of a hydrophobic adsorbent comprises column filtration. Separation of the flocculated mixture into a solid phase and a liquid phase may yield materials that are useful in various applications, including but not limited to agricultural applications.
  • the liquid phase may comprise soluble proteins and the solid phase may comprise activated carbon, activated charcoal, or activated coal and phenolics, pigments, and/or cellular membranes.
  • Phenolics from the solid phase may be used in human and/or animal nutrition.
  • the phenolics include carotenoids that may be used in, for example, nutritional supplements.
  • the phenolics may be used in sunscreen.
  • the activated carbon, activated charcoal, or activated coal from the solid phase can be reused.
  • activated carbon, activated charcoal, or activated coal from the solid phase can be applied to plants to, for example, improve moisture retention.
  • activated carbon, activated charcoal, or activated coal from the solid phase has applications in biofuel technology.
  • activated carbon, activated charcoal, or activated coal can be used in the production of biochar.
  • the liquid phases and/or filtrates for use in the disclosed processes may comprise anti-foaming agents and/or defoaming agents.
  • the anti-foaming agents and/or defoaming agents are oil defoamers.
  • the oil is a mineral oil, a vegetable oil, a white oil, or any oil that is insoluble in a foaming medium except silicone oil.
  • an oil-based defoamer contains a wax and/or hydrophobic silica.
  • waxes are selected from ethylene bis-stearamide (EBS), paraffin waxes, ester waxes, and fatty alcohol waxes.
  • the anti-foaming agents and/or defoaming agents are powder defoamers.
  • powder defoamers are oil-based defoamers on a particulate carrier, such as silica.
  • powder defoamers are added to powder products such as cement, plaster, and detergents.
  • the anti-foaming agents and/or defoaming agents are water-based defoamers.
  • water-based defoamers comprise one or more oils and/or waxes in a water base, such as mineral oil, vegetable oils, long-chain fatty alcohol, and fatty acid soaps or esters.
  • the anti-foaming agents and/or defoaming agents are silicon-based defoamers.
  • silicon-based defoamers are polymers with silicon backbones.
  • silicon-based defoamers are delivered as an oil- or water-based emulsion.
  • the silicon compound comprises or consists of a hydrophobic silica dispersed in a silicone oil.
  • the anti-foaming agents and/or defoaming agents are silicon-based defoamers comprising emulsifiers.
  • the anti-foaming agents and/or defoaming agents are silicon-based defoamers comprise silicone glycols and/or other modified silicone fluids. In some embodiments, the anti-foaming agents and/or defoaming agents are EO/PO-based defoamers. In some embodiments, the anti-foaming agents and/or defoaming agents are EO/PO-based defoamers comprising polyethylene glycol and/or polypropylene glycol copolymers. In some embodiments, the anti-foaming agents and/or defoaming agents are EO/PO-based defoamers are delivered as oils, water solutions, or water-based emulsions.
  • the liquid phase may comprise soluble proteins, excess flocculant (e.g., chitosan), other linked, branched, or linear polysaccharides (including but not limited to ionic, non-ionic, and/or neutral polysaccharides), vitamin B-12, calcium chloride or other divalent ions (e.g., magnesium chloride), RuBisCo, light harvesting complexes/photosystems, soluble proteins, cellular membranes, phenolic compounds, carotenoids, lutein, and/or xanthophylls.
  • excess flocculant e.g., chitosan
  • other linked, branched, or linear polysaccharides including but not limited to ionic, non-ionic, and/or neutral polysaccharides
  • vitamin B-12 including but not limited to ionic, non-ionic, and/or neutral polysaccharides
  • calcium chloride or other divalent ions e.g., magnesium chloride
  • RuBisCo light harvesting complex
  • the solid phase may comprise chlorophyll, calcium phosphate, cellular membranes, light harvesting complexes/photosystems, and/or chitosan.
  • this solid phase may be used as, for example, an animal feed or as a biofuel.
  • this solid phase may contain levulinic acid, which is a potential biofuel precursor.
  • chlorophyll obtained from the solid phase obtained from the separation of the lysed plant material may be used in, for example, cosmetic applications, as a dye, and/or in human and/or animal nutrition.
  • the steps of the process may be advantageous to perform the steps of the process at low temperatures. Low temperatures may prevent denaturation of the soluble proteins.
  • separation of the flocculated mixture is performed at no more than about 35°C, 30°C, 25°C, 20°C, 15°C, 10°C, or 5°C.
  • all steps of the process for making a protein preparation except for the heating step is performed at no more than about 35°C, 30°C, 25°C, 20°C, 15°C, 10°C, or 5°C.
  • the liquid phase may be filtered to yield a filtrate containing the purified protein.
  • Methods of filtration are well known in the art, and may be performed by use of surface filters or depth filters, for example, by membrane filtration, column filtration, diafiltration, ultrafiltration, tangential flow filtration, etc.
  • filtration of the liquid phase of the flocculated mixture is performed with a membrane filter.
  • filtration of the liquid phase of the flocculated mixture is performed with a 5.0 pm, 4.0 pm, 3.0 pm, 2.0 pm, 1.0 pm, 0.7 pm, 0.5 pm, 0.22 pm membrane filter.
  • filtration of the liquid phase of the flocculated mixture is by surface or depth filtration with diatomaceous earth. In some embodiments, filtration of the liquid phase of the flocculated mixture is performed by surface or depth filtration with silt. In some embodiments, filtration of the liquid phase of the flocculated mixture is performed by surface or depth filtration with activated carbon. In some embodiments, filtration of the liquid phase of the flocculated mixture is performed with up to about 10%, 8%, 6%, 4%, 2%, or 1% activated carbon. In some embodiments, filtration of the liquid phase of the flocculated mixture comprises multiple steps or modes of filtration. For example, filtration may be performed with a membrane filter and an activated carbon bed.
  • filtering is performed with a 0.2 pm membrane filter and the proteinaceous liquid is exposed to about 2% of activated carbon.
  • the filtrate is further filtered through membrane filters, e.g., through a 5.0 pm, 4.0 pm, 3.0 pm, 2.0 pm, 1.0 pm, 0.7 pm, 0.5 pm, or 0.2 pm membrane filter.
  • small solids and/or microorganisms may be removed from liquid phases and/or filtrates.
  • small solids and/or microorganisms may be removed from liquid phases and/or filtrates by microfiltration, such as by using a one-pass dead end microfiltration system or a tangential flow filtration system.
  • liquid phases and/or filtrates may be sterilized.
  • liquid phases and/or filtrates are sterilized by microfiltration, such as by using a one-pass dead end microfiltration system or a tangential flow filtration system.
  • liquid phases and/or filtrates are sterilized by ultraviolet (UV) irradiation.
  • UV ultraviolet
  • liquid phases and/or filtrates are sterilized by gamma irradiation.
  • liquid phases and/or filtrates are sterilized by pasteurization, such as by high pressure pasteurization or high-temperature, short-time pasteurization.
  • the filtrate comprising the protein preparation may be further concentrated. Methods known in the art to concentrate solutes may be used. In some embodiments, concentrating the filtrate may be performed by ultrafiltration through a filter with as suitable cut-off filter. In some embodiments, concentrating the filtrate may be performed by ultrafiltration through polyethersulfone, polypropylene, polyvinylidene fluoride, polyacrylonitrile, cellulose acetate, or polysulfone. In some embodiments, concentrating the filtrate may be performed by evaporation concentrating the filtrate may be performed by reverse osmosis. Sizes of cut-off filter may be optimized depending on the protein of interest.
  • ultrafiltration is performed using a filter with a cut-off of no more than about 200 kDa, 150 kDa, 100 kDa, 75kDa, 50 kDa, 25 kDa, 10 kDa, or 5 kDa.
  • liquid phases and/or filtrates may be dialyzed.
  • dialysis may be performed using ultrafiltration.
  • dialysis may be performed using ultrafiltration through polyethersulfone, polypropylene, polyvinylidene fluoride, polyacrylonitrile, cellulose acetate, or polysulfone.
  • dialysis may be performed using reverse osmosis.
  • liquid phases and/or filtrates may be dried.
  • drying may be accomplished using a spray dryer, a freeze dryer, drum drying, film drying, bed drying, a flash dryer, or a rotary dryer.
  • the process disclosed herein enables the preparation of a high yield of purified protein.
  • the process disclosed herein may be used to prepare a high purity preparation of the purified protein.
  • the steps disclosed herein may produce a yield of at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, or 99% of the amount of soluble protein in the liquid phase after lysis of the plant material.
  • the purity of the protein preparation is at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%.
  • the process disclosed herein may be used to extract appreciable levels of protein from plant material.
  • RuBisCo is an enzyme found in the chloroplast of photosynthetic organisms, which is used to catalyze the first major step of carbon fixation. Up to about 50% of the total protein found in green plant material may consist of RuBisCo, making it the most abundant protein in leaves.
  • the protein preparation is RuBisCo.
  • the plant material is from duckweed, algae, beetroot, spinach beet, chard, sugar beet, sea beet, Mangel beet, soy, or tobacco.
  • Another aspect of the present disclosure relates to a product made by the processes disclosed herein.
  • a food comprising a purified protein preparation from a plant material.
  • the food comprising the protein preparation may contain no more than 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% impurities.
  • the food comprising the protein preparation comprises RuBisCo.
  • the protein is prepared from plant material from Lemna. In some embodiments, the protein is prepared from plant material from Lemnoideae.
  • foams refer to structures formed by trapping pockets of gas in a liquid or solid. Proteins in foams contribute to the foam’s ability to form small air cells and stability in holding the structure. Foams with a uniform distribution of small air bubbles impart body, smoothness and lightness to the food.
  • the ability of a protein preparation to form a foam is related to its purity, and a purity of at least about 80% may be needed to form a stable foam.
  • gels are soft solids comprising a high amount of an aqueous phase. Protein gels may comprise a three-dimensional network of protein fibers with a continuous liquid phase throughout the matrix. Proteins with higher gelling capacity require less protein to form a gel.
  • the processes disclosed herein may be used to prepare protein preparations with advantageously high purity, foaming capacity, foam stability, and gelling capacity that is suitable for use in food products.
  • FC (Volume after foaming - Volume before foaming)/Volume before foaming x 100%.
  • Foaming stability at a time interval t after foaming was calculated as:
  • Foaming Stability Residual Foam Volume at time t / Initial Foam Volume x 100%.
  • the filtered homogenate was then centrifuged for 10 minutes at a speed/force of 4000g (Allegra X15R, SX4750 rotor; Beckman Coulter, Inc., Pasadena, California). The pellet was discarded, and the supernatant was collected separately.
  • the solution was heated to a temperature of 50°C in a water bath that was set at a temperature of 55°C and was cooled rapidly to a temperature less than 15°C after reaching the target temperature.
  • 2% v/v of activated chitosan and 4% w/v of activated carbon is added to the liquid juice.
  • the solution was subsequently stirred for 5 minutes after which the solution was centrifuged for 10 minutes at a speed/force of 5000g (Allegra X15R, SX4750 rotor; Beckman Coulter, Inc., Pasadena, California).
  • the green pellet in the centrifuge bottle was discarded, and the clear yellow supernatant was micro filtered using a 0.7 pm Glass Micro fiber membrane (Whatman 1825-047 Glass Microfiber Binder Free Filter, 0.7 Micron; Global Life Sciences Solutions USA LLC, Marlborough, Massachusetts).
  • the filtrate was subsequently exposed to a 0.2 pm
  • Polyethersulfone membrane Polyethersulfone (PES) Membrane Filters, 0.2 Micron; Sterlitech
  • the obtained pale yellow and deodorized proteinaceous solution was then concentrated using a 70 kDa membrane (MINIKROS® S02-E070-05-N; Spectrum Laboratories, Inc., Collinso Dominguez, California).
  • the concentrated solution obtained was subsequently freeze dried (Harvest Right LLC, Salt Lake City, Utah) and the result was a white, odorless and soluble protein powder.
  • the filtered homogenate was then centrifuged for 10 minutes at a speed/force of 4000g (Allegra X15R, SX4750 rotor; Beckman Coulter, Inc., Pasadena, California). The pellet was discarded, and the supernatant was collected separately. The supernatant was then mixed with 5% v/v of activated chitosan (Chitosan (10 - 120 cps), fungal origin (9012-76-4); Glentham Life Sciences Ltd., Corsham, Wiltshire, UK) and 10% w/v of activated carbon (Cabot Norit Americas Inc, Marshall, Texas) for a period of 5 minutes.
  • the mixed solution was centrifuged at a speed/force of 5000g for 10 minutes (Allegra X15R, SX4750 rotor; Beckman Coulter, Inc., Pasadena, California).
  • the obtained pellet was discarded, and the deodorized and decolored supernatant was microfiltered using a 0.2 pm polyethersulfone membrane (Polyethersulfone (PES) Membrane Lilters, 0.2 Micron; Sterlitech Corporation Inc, Kent, Washington).
  • PES Polyethersulfone
  • the obtained pale yellow and deodorized proteinaceous solution was then concentrated using a 70 kDa membrane (MINIKROS® S02-E070-05-N; Spectrum
  • the concentrated solution obtained was subsequently freeze dried (Harvest Right LLC, Salt Lake City, Utah) and the result was a white, odorless and soluble protein powder.
  • the pellet was discarded, and the supernatant was collected separately.
  • the supernatant was then mixed with a solution containing 30 mM of potassium phosphate and 20 mM of calcium chloride for a period of 5 minutes. Subsequently the mixed solution was centrifuged at a speed/force of 5000g for 10 minutes (Allegra X15R, SX4750 rotor; Beckman Coulter, Inc., Pasadena, California). The obtained pellet was discarded. 5% w/v of activated carbon (Cabot Norit Americas Inc, Marshall, Texas) was added to the supernatant, and the solution was stirred for 5 minutes.
  • the mixed solution containing the activated carbon was micro filtered using a 0.2 pm polyethersulfone membrane filter (Polyethersulfone (PES) Membrane Filters, 0.2 Micron; Sterlitech Corporation Inc, Kent, Washington) in order to remove the activated carbon that had adsorbed the remaining chlorophyll, polyphenol and other unwanted taste/color/odor impacting particles.
  • PES Polyethersulfone
  • the obtained pale yellow and deodorized proteinaceous solution was then concentrated using a 100 kDa membrane (Hollow Fiber Cartridge, 100,000 NMWC, 850 cm2; GE Healthcare Bio-Sciences Corp, Westborough, Massachusetts). The concentrated solution obtained was subsequently freeze dried and the result was a white, odorless and soluble protein powder.
  • One kg of fresh Lemna minor was macerated using a Vitamix Blender (Vitamix Corp, Cleveland, Ohio) in a ratio of 1: 1 with distilled water containing 0.5% w/v of sodium bisulfite.
  • the maceration was performed for a period of 3 minutes at medium speed in order to maintain a temperature of less than 30 °C.
  • the lysed biomass was filtered by using a nylon straining bag (Natural Home Brands, Sun Valley, California) with a fine mesh to separate the fibrous high solids cake from the liquid juice containing the soluble protein.
  • the filtered homogenate was then centrifuged for 10 minutes at a speed/force of 4000g (Allegra X15R, SX4750 rotor; Beckman Coulter, Inc., Pasadena, California). The pellet was discarded, and the supernatant was collected separately. The supernatant was then mixed with a solution containing 30 mM of potassium phosphate and 20 mM of calcium chloride for a period of 5 minutes. Subsequently the mixed solution was centrifuged at a speed/force of 5000g for 10 minutes (Allegra X15R, SX4750 rotor; Beckman Coulter, Inc., Pasadena, California). The obtained pellet was discarded.
  • chitosan (Chitosan (10 - 120 cps), fungal origin (9012-76-4); Glentham Life Sciences Ltd., Corsham, Wiltshire, UK) and 4% of activated carbon (Cabot Norit Americas Inc, Marshall, Texas) were added to the supernatant, and the solution was stirred for 5 minutes. Subsequently the mixed solution was centrifuged at a speed/force of 5000g for 10 minutes (Allegra X15R, SX4750 rotor; Beckman Coulter, Inc., Pasadena, California).
  • the obtained pellet was discarded, and the deodorized and decolored supernatant was microfiltered using a 0.7 pm polyethersulfone membrane (Whatman 1825-047 Glass Microfiber Binder Free Filter, 0.7 Micron; Global Life Sciences Solutions USA LLC, Marlborough, Massachusetts).
  • the filtrate was then further microfiltered using a 0.2 pm polyethersulfone membrane (Polyethersulfone (PES) Membrane Filters, 0.2 Micron; Sterlitech Corporation Inc, Kent, Washington).
  • PES Polyethersulfone
  • the obtained pale yellow and deodorized proteinaceous solution was then concentrated using a 70kDa membrane (MINIKROS® S02-E070-05-N; Spectrum Laboratories, Inc., Collinso Dominguez, California).
  • the concentrated solution obtained was subsequently freeze dried (Harvest Right LLC, Salt Lake City, Utah) and the result was a white, odorless and soluble protein powder.
  • Lemna leaf proteins were extracted as described in WO2011/0778671 A1 (van de Velde et al.) with some modifications.
  • the supernatant obtained was subjected to two microfiltration steps. First, the supernatant was passed over a microfilter having a pore size of 0.7 pm (Whatman 1825-047 Glass Microfiber Binder Free Filter, 0.7 Micron; Global Life Sciences Solutions USA LLC, Marlborough, Massachusetts).
  • the filtrate was passed over a microfilter having a pore size 0.2 pm (Polyethersulfone (PES) Membrane Filters, 0.2 Micron; Sterlitech Corporation Inc, Kent, Washington).
  • PES Polyethersulfone
  • the purity of the protein was approximately 34.1% per unit of dry matter and the concentration of soluble protein prior to freeze-drying was 520 pg/mL.
  • the foaming properties of the freeze-dried material showed a total foaming strength of 92% with a stability of 62% after 1 hour. Gelation properties of the freeze-dried material were validated, and at least 7% w/v of freeze-dried material was needed to be added in order to form a gel. Table 1.
  • This example investigated the removal of chlorophyll using calcium chloride to coagulate chlorophyll-protein complexes.
  • Fraction 1 3.75g of phosphate buffer was added to get a concentration of lOmM in the fraction. The fraction was then run through the heat bath step set to 68 °C after which 15g activated carbon (Cabot Norit Americas Inc, Marshall, Texas) was added and mixed for 25 minutes. Following that 15g of 3% chitosan (Chitosan (10 - 120 cps), fungal origin (9012-76-4); Glentham Fife Sciences Ftd., Corsham, Wiltshire, UK) was added and mixed for an additional 5 minutes. The solution was then centrifuged and then filtered.
  • Fraction 2 3.75g of phosphate buffer was added to get a concentration of lOmM in the fraction. 15g of activated carbon (Cabot Norit Americas Inc, Marshall, Texas) was then added to the fraction and mixed for 15 minutes. Following that, 15g of 3% chitosan (Chitosan (10 - 120 cps), fungal origin (9012- 76-4); Glentham Fife Sciences Ftd., Corsham, Wiltshire, UK) was added and mixed for an additional 5 minutes. The solution was then centrifuged and then filtered.
  • Fraction 3 3.75g of phosphate buffer and 2.81g of calcium chloride solution was added to get a concentration of lOmM and 7.5mM respectively in the fraction. 15g of activated carbon (Cabot Norit Americas Inc, Marshall, Texas) was then added to the fraction and mixed for 15 minutes. Following that, 15g of 3% chitosan (Chitosan (10 - 120 cps), fungal origin (9012-76-4); Glentham Fife Sciences Ftd., Corsham, Wiltshire, UK) was added and mixed for an additional 5 minutes. The solution was then centrifuged and then filtered.
  • FIG. 5 depicts Fractions 1, 2, 3, and 4 after microfiltration.
  • FIG. 6 depicts Fractions, 4, 3, 2, and 1 after microfiltration.
  • coagulation with calcium chloride does not show a significant difference in the removal of chlorophyll relative to the control Fraction (Fraction 2).
  • the use of two different concentrations of calcium chloride in Fractions 3 and 4 did not result in a significant difference in chlorophyll removal.
  • FIG. 7 A depicts these Fractions.
  • the lysate obtained before solid/liquid separation was treated with phosphate (comprising potassium phosphate dibasic and potassium phosphate monobasic) and with EDTA.
  • FIG. 7B depicts these Fractions. As shown in FIG. 7A and FIG. 7B,
  • Fraction 2 from the second set of Fractions has a dramatically different color compared to Fraction 2 from the first set of fractions. Without wishing to be bound by theory, it is believed that these results suggest that adding EDTA to the filtrate leads to a greater removal of color compared to adding EDTA to the lysate.
  • This example investigated the use of calcium chloride to coagulate chlorophyll and/or chloroplast membranes as an alternative to using a heat bath.
  • Biomass was lysed in extraction buffer containing 0.1 M NaCl and 2% metabisulfite (without EDTA). Calcium chloride and phosphate (comprising potassium phosphate dibasic and potassium phosphate monobasic) were added to 375mL of post-basket centrifugation (Rousselet-Robatel Model RA20VxR Vertical Basket Centrifuge; Robatel Inc, Pittsfield, Massachusetts) filtrate in the amounts detailed in Table 4 below.
  • FIG. 8 depicts samples of the fractions after calcium chloride and phosphate addition and approximately five minutes of benchtop centrifugation. As depicted in FIG. 8, Fraction 4 demonstrated superior chlorophyll removal. As also depicted in FIG. 8, Fraction 5 contained a white pellet (content unknown) while the supernatant remained relatively chlorophyll-filled.
  • the results may suggest that phosphate is necessary for calcium chloride to remove chlorophyll effectively at a concentration of 75 mM.
  • the results may suggest that calcium chloride is less effective at a higher pH.
  • FIG. 9 depicts samples of Fractions 1-6 after removal of activated carbon and chitosan. As depicted in FIG. 9, chitosan and activated carbon function properly after pre treatment with calcium chloride and phosphate. As also depicted in FIG. 9, the color removal by activated carbon and chitosan were most effective on Fraction 6, which contained 75 mM calcium chloride and 100 mM phosphate.
  • FIG. 10 depicts results from the SDS-PAGE Coomassie staining analysis.
  • FIG. 10 depicts SDS-PAGE gel (Bio-Rad Laboratories, INC, Hercules, CA) results from the pellet and supernatant (“Sup”) after benchtop centrifugation for Fractions 5-7.
  • lane 9 shows that chlorophyll is still attached to a protein around 25 kDa
  • lane 6 shows that chlorophyll is detached.
  • the pH of the supernatant was then raised to 7.2 to check for remaining chitosan and sufficient colour removal.
  • Another 0.75g was added to the original solution to get a total of 10% of normal chitosan (1.5g in 375 mL). This was repeated until colour removal was sufficient or excess chitosan was observed.
  • the chitosan % at which this was observed was 10%. 25% of AC and 10% of chitosan were added to both fractions and spun down with the centrifuge.
  • FIG. 11 depicts an SDS-PAGE gel (Bio-Rad Laboratories, INC, Hercules, CA) on various samples. As depicted in FIG. 11, the detergent does not significantly increase the Rubisco in the filtrate, and there does not appear to be Rubisco in the activated carbon-chitosan pellet in either fraction.
  • FIG. 12 depicts the fractions after removal of activated carbon and chitosan. As depicted in FIG. 12, treatment with Chaps releases more polyphenols and/or polyphenol oxidase (PPO) during lysing.
  • PPO polyphenol oxidase
  • This example related to a calcium chloride baseline run, resuspension of a cake with 0.1% CHAPS, and a wash of second cake with 0.1% CHAPS.
  • Fraction 2 was then spun out with a basket centrifuge (Rousselet-Robatel Model RA20VxR Vertical Basket Centrifuge; Robatel Inc, Pittsfield, Massachusetts), and the fraction was collected as the filtrate.
  • the preparation of Fraction 2 comprised resuspending the basket centrifugation cake in 2L of resuspension buffer (70mM PO4 pH7.2, 0.1M NaCl, 0.1% CHAPS). The slurry was then Vitamix blended for 1 minute, and the filtrate (Fraction 2) and cake were separated by basket centrifugation. Very similar to Fraction 2, Fraction 3 was prepared by resuspending the basket centrifugation cake in 1L of resuspension buffer.
  • the slurry was mixed by hand, and allowed to sit for 10 minutes prior to basket centrifugation. 50g of activated carbon was added and mixed for 15 minutes followed by 20g of 3% chitosan (Chitosan (10 - 120 cps), fungal origin (9012-76-4); Glentham Life Sciences Ltd., Corsham, Wiltshire, UK) mixed for an additional 5 minutes per fraction. The solution was then spun down and microfiltered through a 0.2um filter (Polyethersulfone (PES) Membrane Filters, 0.2 Micron; Sterlitech Corporation Inc, Kent, Washington). Lastly, the solution was concentrated down with the 50 kDa ultrafiltration (MINIKROS® S02-E050-05-N; Spectrum Laboratories, Inc., Collinso
  • FIG. 13 depicts an SDS-PAGE gel for various samples, wherein“FI” refers to “Fraction 1,”“F2” refers to“Fraction 2,”“F3” refers to“Fraction 3,”“AC-C” refers to“activated carbon- chitosan,”“sup” refers to“supernatant,” and“Rubi” refers to“Rubisco.”
  • FI refers to “Fraction 1”
  • “F2” refers to“Fraction 2”
  • “F3” refers to“Fraction 3”
  • “AC-C” refers to“activated carbon- chitosan”
  • “sup” refers to“supernatant”
  • “Rubi” refers to“Rubisco.”
  • This example investigated treatment with calcium chloride, increased phosphate, and 0.25% CHAPS detergent.
  • Fraction 3 was lysed in the same phosphate buffer as Fraction 2 except a final concentration of 0.25% CHAPS detergent (Biovision Inc, Milpitas, California was added. Lysis occurred in the Vitamix blender using the same protocol as Fractions 1 and 2 (some foaming was observed).
  • Fraction 2 The slurry was then Vitamix blended for 1 minute, and the filtrate (Fraction 2) and cake were separated by basket centrifugation. Very similar to Fraction 2, Fraction 3 was prepared by resuspending the basket centrifugation cake in 1L of resuspension buffer. The slurry was mixed by hand and allowed to sit for 10 minutes prior to basket centrifugation. 50g of activated carbon (Cabot Norit Americas Inc, Marshall, Texas) was added and mixed for 15 minutes followed by 20g of 3% chitosan (Chitosan (10 - 120 cps), fungal origin (9012-76-4); Glentham Life Sciences Ltd., Corsham, Wiltshire, UK) mixed for an additional 5 minutes per fraction.
  • the solution was then spun down and microfiltered through a 0.2 mih filter (Polyethersulfone (PES) Membrane Filters, 0.2 Micron; Sterlitech Corporation Inc, Kent, Washington). Lastly, the solution was concentrated down with the 50 kDa ultrafiltration (MINIKROS® S02-E050-05-N; Spectrum Laboratories, Inc., Collinso Dominguez, California), and diafiltered until salinity was below 0.1 ppt ( ⁇ 10L dH20). The last step was the 10 kDa ultrafiltration (MINIKROS® S02-E010-05-N; Spectrum Laboratories, Inc., Collinso Dominguez, California) after which it was freeze-dried (Harvest Right LLC, Salt Lake City, Utah). Parameters used during the process of control / Lraction 2 / Lraction 3 are provided in Table 6.
  • Lraction 2 was slightly darker than fraction 1 after basket centrifugation, fraction 3 was significantly darker green after basket centrifugation. Both fraction 2 and 3 were more turbid relative to fraction 1 after ultrafiltration.
  • the volume of Lraction 3 was 4.8L; however, it was treated as 3.4L due to the obviously increased amount of air that inflated the volume.
  • the volumes of the filtrates were all reduced to 2.5L before processing. Accordingly, 89% of Lraction 1, 100% of Lraction 2, and 96% of Lraction 3 were actually used.
  • This example investigated the effect of chitosan concentration on chlorophyll removal without using a heat bath.
  • a coffee filter was used followed by a Buchner funnel with a 0.45 mih cutoff filter sheet (Polyethersulfone (PES) Membrane Filters, 0.45 Micron; Sterlitech Corporation Inc, Kent, Washington) coated in DE (Dicalite Management Group Inc, Bala Cynwyd, Pennsylvania).
  • PES Polyethersulfone
  • Fraction 1 15g of activated carbon (Cabot Norit Americas Inc, Marshall, Texas) was then added to the fraction and mixed for 15 minutes. Following that, 15g of 1% chitosan (Chitosan (10 - 120 cps), fungal origin (9012-76-4); Glentham Life Sciences Ltd., Corsham, Wiltshire, UK) was added and mixed for an additional 5 minutes. The solution was then centrifuged (Allegra X15R, SX4750 rotor; Beckman Coulter, Inc., Pasadena, California) and then filtered (Polyethersulfone (PES) Membrane Filters, 0.2 Micron; Sterlitech Corporation Inc, Kent, Washington).
  • PES Polyethersulfone
  • Fraction 2 15g of activated carbon was then added to the fraction and mixed for 15 minutes. Following that, 15g of 2% chitosan was added and mixed for an additional 5 minutes. The solution was then centrifuged and then filtered.
  • Fraction 3 15g of activated carbon was then added to the fraction and mixed for 15 minutes. Following that, 15g of 3% chitosan was added and mixed for an additional 5 minutes. The solution was then centrifuged and then filtered.
  • Fraction 4 15g of activated carbon was then added to the fraction and mixed for 15 minutes. Following that, 15g of 4% chitosan was added and mixed for an additional 5 minutes. The solution was then centrifuged and then filtered.
  • Fraction 5 15g of activated carbon was then added to the fraction and mixed for 15 minutes. Following that, 15g of 5% chitosan was added and mixed for an additional 5 minutes. The solution was then centrifuged and then filtered. However, a mistake was made and the timer was not started after chitosan addition. Therefore, the results for Fraction 5 were not compared with the other Fractions.
  • FIG. 15 depicts Fractions 1-5 after microfiltration with the Buchner funnel. As depicted in FIG. 15, the color was removed seemingly equally well in Fractions 2, 3, and 4. Without wishing to be bound by theory, a comparison between Fraction 1 and Fraction 2 suggests that 1% chitosan did not remove chlorophyll as effectively as 2% chitosan. As depicted in FIG. 15, Fraction 5, which received approximately 2-4 minutes of additional exposure to activated carbon and chitosan relative to Fractions 1- 4, was clearer than Fractions 1-4.
  • This example investigated the use of activated bentonite clay (CC160 from EP Engineered Clays) to remove colored compounds from post-chitosan lysate in place of, or in conjunction with, activated carbon (Cabot Norit Americas Inc, Marshall, Texas).
  • activated bentonite clay CC160 from EP Engineered Clays
  • Fresh supernatant (post-chitosan spindown in the extraction process) was obtained, and its pH was increased to 7.0 with NaOH.
  • the optical density was measured by Pierce assay (PierceTM 660nm Protein Assay Reagent; Thermo Fisher Scientific, Waltham, Massachusetts), and the absorbance at 474 nm was measured to determine the starting point for [protein] and the amount of orange discoloration.
  • Pierce 660nm Protein Assay Reagent can be used to measure total protein concentration.
  • the Pierce 660nm Protein Assay is based on the binding of a dye-metal complex to protein that causes a shift in the dye’s absorption maximum, which is measured at 660 nm.
  • the dye-metal complex is reddish-brown and changes to green upon protein binding, and the color produced in the assay increases in proportion to increasing protein concentrations.
  • To a 100 mL sample was added 0.3% w/v activated carbon (Cabot Norit Americas Inc, Marshall, Texas). The mixture was stirred for 1 minute before being poured directly onto a Buchner funnel with a 0.45 m filter Polyethersulfone (PES) Membrane Filters, 0.45 Micron;
  • PES Polyethersulfone
  • Table 7 recites the concentrations of activated carbon or clay and the absorbances at 474 nm before and after treatment and filtration.
  • the mixture was stirred for 1 minute, flowed through a 0.45 m filter (Polyethersulfone (PES) Membrane Filters, 0.45 Micron; Sterlitech Corporation Inc, Kent, Washington) followed by a 0.2 m filter (Polyethersulfone (PES) Membrane Filters, 0.2 Micron; Sterlitech Corporation Inc, Kent, Washington) on a Buchner funnel, collecting the filtrate in a flask. A sample was collected for optical density measurements. To further 100 mL samples, resin was added in the amount (% w/v) indicated in Table 8, and, for each sample, the mixture was stirred for the time indicated in Table 8 prior to pouring the mixture through the Buchner setup. Optical density measurements were taken on each sample.
  • An aspect of the Example was to determine the binding time and concentration for which the orange OD is lowest while maintaining the highest possible Pierce assay reading, correlating to protein concentration. This can be seen graphically by the largest separation between the Pierce ratio line and the 474 ratio line. The experimental group with the largest separation was 20% for 10 minutes, with 83% of protein retained by Pierce assay and reduction of orange color to 33%.
  • This example investigated the effectiveness of a resin (Purolite MN200; Purolite Corporation, Kings of Prussia, Pennsylvania) at removing colored compounds, as compared to activated carbon.
  • a resin Purolite MN200; Purolite Corporation, Kings of Prussia, Pennsylvania
  • Final plant protein powder was diluted at a concentration of 10 mg/mL in deionized water.
  • the sample was analyzed using fast protein liquid chromatography (FPLC) using a gel filtration column (Superdex 200; GE Healthcare Bio-Sciences Corp, Westborough, Massachusetts).
  • a molecular weight standard (Bio-Rad Laboratories, INC, Hercules, CA) was subsequently run to approximate the molecular size of individual proteins and protein complexes in the sample of interest.
  • FIG. 16 depicts a chromatogram of final protein product and protein standard. Chromatogram protein peak analysis indicated that the protein of interest (Rubisco) was eluted from the column at a molecular weight near to but less than 670 kDa as measured by the molecular weight protein standard.
  • This example used SDS-PAGE electrophoresis to visualize Lemna plant protein purity and complexity via Coomassie staining.
  • a small sample of plant protein extract of final purified protein product was analyzed on a 4-15% SDS-PAGE gel (Bio-Rad Laboratories, INC, Hercules, CA).
  • FIG. 17 depicts the SDS-PAGE gel. Without wishing to be bound by theory, it is believed that these results suggest that the final protein product consists primarily of Rubisco enzyme and that the individual large and small subunits of Rubisco can be readily detected by SDS-PAGE Coomassie staining under denatured and reducing conditions.

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EP4767827A1 (en) 2024-12-24 2026-07-01 BK Giulini GmbH COMPOSITIONS FOR ACIDIFIED DAIRY PRODUCT ANALOGUES

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