Process for isolating high purity protein preparation from plant material, and products thereof

Abstract

To provide processes for preparing and purifying protein from plant materials, to provide compositions containing the same, and to provide uses thereof.SOLUTION: A process includes: a) providing a plant material in a buffer solution including a reducing agent; b) dissolving the plant material; c) separating the dissolved plant material into a solid phase and a liquid phase containing soluble protein and chlorophyll; d) coagulating the chlorophyll in the liquid phase by heating it to a first set temperature within about 30 min and subsequently cooling it to a second set temperature within about 30 min, the cooling being initiated when the liquid phase reaches the first set temperature; e) contacting the liquid phase of d) 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; f) separating the flocculated mixture of e) into a solid phase and a liquid phase; and g) filtering the liquid phase of f) to yield a filtrate containing a purified protein.SELECTED DRAWING: None

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit of U.S. Provisional Patent Application No. 62 / 872,917, filed on July 11, 2019, the entire contents of which are incorporated herein by reference.

[0002] Technical Field Disclosed are processes for making, extracting, and purifying protein extracts from plant materials, as well as products made from such processes.

Background Art

[0003] The production of meat for human consumption has a significant negative impact on the environment. The manufacturing industry is one of the largest emitters of greenhouse gases and is a major cause of water pollution and loss of biodiversity. It is known that Steinfeld et al, Livestock’s Long Shadow: Environmental Issues and O ptions, (2006), Food and Agriculture Organ ization of the United Nations; Machovina et al., Science of the Total Environment, (2016), 536:419 - 431; Godfray et al., Scienc e, (2018), 361(6399):eaam5324. As the world consumption of meat increases due to changes in population, wealth, and lifestyle, there is a growing need for alternatives to environmentally unfriendly meat. Erb et al., Nat.Comms., (2016 ), 7:11382.

[0004] ​​ Replacing meat in the diet requires a plant-derived high-protein substitute that can be produced on a large scale and sustainably. Protein ribulose-1,5-bisphosphate carboxylase / oxygenase (「RuBisCo」) constitutes up to 50% of the total protein in plants. It is the enzyme involved in the first major step of carbon fixation, the process by which carbon dioxide in the atmosphere is converted into high-energy molecules such as glucose by plants and other photosynthetic organisms. Due to its abundance in plants, it serves as an alternative source of protein for food production. Purified RuBisCo is usually a tasteless, odorless white powder.

[0005] Duckweed (subfamily Lemnoideae, genus Lemna) is the world's smallest flowering plant. Despite its small size, it has the ability to grow rapidly and double its biomass in about 16 - 48 hours depending on conditions. Mestameyer et al. , Spirodela punctata Aquatic Botany, (1984 ), 19:157 - 70. Lemna has a high protein content (about 30 - 3 5% of its dry mass is protein) and is used as animal forage. All of these characteristics make Lemna an attractive candidate for the large-scale production of plant-derived protein for food.

[0006] The ability to form protein emulsions, gels, and stable foams is also important in the production of various foods and forms the basis of texture in foods. For example, a foam with a uniform distribution of small bubbles imparts density, mouthfeel, and lightness to food. Protein standards ​ The ability of the product to form a foamy substance is related to its purity, and at least 80% purity may be required to form a stable foamy substance. Similarly, gels made from proteins yield foods with various rheological properties and appearances. The gel-forming ability of a protein may be measured by the amount of protein required to form the gel. Therefore, protein preparations with high purity, foaming ability, foam stability, and gel-forming ability are highly desirable for food use.

[0007] Currently, there is a need for an economical process for purifying and extracting proteins from plants, for example, from Lemnoideae or Lemna. Disclosed herein is a process capable of producing a purified protein preparation that is adaptable for processing into most foods. SUMMARY OF THE INVENTION

[0008] The present disclosure provides a) providing plant material in a buffer solution containing a reducing agent; b) dissolving the plant material; c) separating the dissolved plant material into a solid phase and a liquid phase, wherein the liquid phase contains soluble protein and chlorophyll; d) coagulating chlorophyll in the liquid phase by heating to a first set temperature within about 30 minutes and then cooling to a second set temperature within about 30 minutes, wherein the cooling begins when the liquid phase reaches the first set temperature; e) contacting the liquid phase of d) with a flocculant and / or an adsorbent and mixing for a time sufficient for the chlorophyll in the liquid phase to flocculate and / or adsorb to the adsorbent, thereby forming a flocculated mixture; f) separating the flocculated mixture of e) into a solid phase and a liquid phase; g) Filtering the liquid phase of f) to obtain a filtrate containing the purified protein, a process for producing a protein preparation purified from plant material is provided. The present disclosure a) providing plant material in a buffer solution containing a reducing agent; b) dissolving the plant material; c) separating the dissolved plant material into a solid phase and a liquid phase, wherein the liquid phase contains soluble protein and chlorophyll ; d) coagulating the chlorophyll in the liquid phase by adding one or more salts; e) contacting the liquid phase of d) with a flocculant and / or adsorbent and mixing for a time sufficient for the chlorophyll in the liquid phase to flocculate and / or adsorb to the adsorbent, thereby forming a flocculated mixture; ; f) separating the flocculated mixture of e) into a solid phase and a liquid phase; g) Filtering the liquid phase of f) to obtain a filtrate containing the purified protein, a process for producing a protein preparation purified from plant material is also provided.

[0009] The present disclosure a) providing plant material in a buffer solution containing a reducing agent; b) dissolving the plant material; c) separating the dissolved plant material into a solid phase and a liquid phase, wherein the liquid phase contains soluble protein and chlorophyll ; d) coagulating the chlorophyll in the liquid phase by adding one or more salts; e) contacting the liquid phase of d) with a flocculant and / or adsorbent and mixing for a time sufficient for the chlorophyll in the liquid phase to flocculate and / or adsorb to the adsorbent, thereby forming a flocculated mixture; ; f) separating the flocculated mixture of e) into a solid phase and a liquid phase; g) Filtering the liquid phase of f) to obtain a filtrate containing the purified protein, a process for producing a protein standard purified from plant material also provides.

[0010] The present disclosure a) Providing plant material in a buffer solution containing a reducing agent; b) Dissolving the plant material; c) Separating the dissolved plant material into a solid phase and a liquid phase, wherein the liquid phase contains soluble protein and chlorophyll ; d) Coagulating the chlorophyll in the liquid phase using a coagulant derived from a polymer; e) Contacting the liquid phase of d) with a flocculant and / or an adsorbent and mixing for a time sufficient for the chlorophyll in the liquid phase to flocculate and / or adsorb to the adsorbent, thereby forming a flocculated mixture; ; f) Separating the flocculated mixture of e) into a solid phase and a liquid phase; g) Filtering the liquid phase of f) to obtain a filtrate containing the purified protein, a process for producing a protein standard purified from plant material also provides.

[0011] The present disclosure a) Providing plant material in a buffer solution containing a reducing agent; b) Dissolving the plant material; c) Separating the dissolved plant material into a solid phase and a liquid phase, wherein the liquid phase contains soluble protein and chlorophyll ; d) Coagulating the chlorophyll in the liquid phase by electrocoagulation; e) Contacting the liquid phase of d) with a flocculant and / or an adsorbent and mixing for a time sufficient for the chlorophyll in the liquid phase to flocculate and / or adsorb to the adsorbent, thereby forming a flocculated mixture; ; f) Separating the flocculated mixture of e) into a solid phase and a liquid phase; g) Filtering the liquid phase of f) to obtain a filtrate containing the purified protein, and a process for producing a protein preparation purified from plant material is also provided.

[0012] In some embodiments, the present disclosure relates to the following embodiments. 1. A process for producing a protein preparation purified from plant material, comprising: a) providing the plant material in a buffer solution containing a reducing agent; b) dissolving the plant material; c) separating the dissolved plant material into a solid phase and a liquid phase, wherein the liquid phase contains soluble protein and chlorophyll; d) heating the liquid phase to a first set temperature within about 30 minutes and then cooling to a second set temperature within about 30 minutes to coagulate the chlorophyll in the liquid phase, wherein the cooling starts when the liquid phase reaches the first set temperature; e) contacting the liquid phase of d) with a flocculant and / or an adsorbent and mixing for a time sufficient to flocculate the chlorophyll in the liquid phase and / or adsorb it to the adsorbent, thereby forming a flocculated mixture; f) separating the flocculated mixture of e) into a solid phase and a liquid phase; g) filtering the liquid phase of f) to obtain a filtrate containing the purified protein. The process as described above. process.

[0013] 2. A process for producing a protein preparation purified from plant material, comprising: a) providing the plant material in a buffer solution containing a reducing agent; b) dissolving the plant material; c) separating the dissolved plant material into a solid phase and a liquid phase, wherein the liquid phase contains soluble protein and chlorophyll; d) Coagulating the chlorophyll in the liquid phase by adding a salt of 1 or more; ; e) Contacting the liquid phase of d) with a flocculant and / or an adsorbent and mixing for a time sufficient to flocculate the chlorophyll in the liquid phase and / or adsorb it onto the adsorbent, thereby forming a flocculated mixture; ; ; f) Separating the flocculated mixture of e) into a solid phase and a liquid phase; g) Filtering the liquid phase of f) to obtain a filtrate containing the purified protein, said process.

[0014] 3. A process for preparing a protein preparation purified from plant material, a) Providing the plant material in a buffer solution containing a reducing agent; b) Dissolving the plant material; c) Separating the dissolved plant material into a solid phase and a liquid phase, wherein the liquid phase contains soluble protein and chlorophyll; ; d) Coagulating the chlorophyll in the liquid phase using a coagulant derived from a polymer; ; e) Contacting the liquid phase of d) with a flocculant and / or an adsorbent and mixing for a time sufficient to flocculate the chlorophyll in the liquid phase and / or adsorb it onto the adsorbent, thereby forming a flocculated mixture; ; ; f) Separating the flocculated mixture of e) into a solid phase and a liquid phase; g) Filtering the liquid phase of f) to obtain a filtrate containing the purified protein, said process.

[0015] 4. A process for preparing a protein preparation purified from plant material, a) Providing the plant material in a buffer solution containing a reducing agent; b) Dissolving the plant material;​​ c) separating the dissolved plant material into a solid phase and a liquid phase, wherein the liquid phase contains soluble proteins and chlorophyll; and d) coagulating the chlorophyll in the liquid phase by electrocoagulation; and e) contacting the liquid phase of d) with a flocculant and / or an adsorbent and mixing for a time sufficient to flocculate the chlorophyll in the liquid phase and / or adsorb it onto the adsorbent, thereby forming a flocculated mixture; and f) separating the flocculated mixture of e) into a solid phase and a liquid phase; and g) filtering the liquid phase of f) to obtain a filtrate containing purified protein, the process.

[0016] 5. The process according to any one of embodiments 1 to 4, wherein the plant material is washed before a).

[0017] 6. The reducing agent is 2-mercaptoethanol (BME), 2-mercaptoethylamine -HCl, sodium metabisulfite, cysteine hydrochloride, dithiothreitol (DTT) , glutathione, cysteine, tris(2-carboxyethyl)phosphine (TCEP) , ferrous ions, nascent hydrogen, sodium amalgam, oxalic acid, formic acid, magnesium, manganese, phosphoric acid, potassium or sodium, the process according to any one of embodiments 1 to 4.

[0018] 7. The process according to any one of embodiments 1 to 4, wherein the reducing agent is a sulfite.

[0019] 8. The process according to any one of embodiments 7, wherein the sulfite is sodium sulfite, magnesium sulfite or sodium metabisulfite.

[0020] ​​​9. The process of any one of embodiments 7, wherein the sulfite is sodium bisulfite.

[0021] 10. The process of any one of embodiments 1 to 4, wherein the solution of a) contains one or more buffers.

[0022] 11. The process of any one of embodiments 1 to 4, wherein the solution of a) contains one or more chelating agents.

[0023] 12. The process of any one of embodiments 1 to 4, wherein the solution of a) contains one or more protease inhibitors.

[0024] 13. The process of any one of embodiments 1 to 4, wherein the solution of a) contains one or more buffers, one or more chelating agents, and / or one or more protease inhibitors.

[0025] 14. The process of any one of embodiments 1 to 4, wherein the pH of the solution of a) is from about pH 5.0 to about pH 9.0.

[0026] 15. The process of embodiment 14, wherein the pH of the solution is from about pH 6.0 to about pH 7.6.

[0027] 16. The process of embodiment 15, wherein the pH of the solution is about pH 6.8.

[0028] 17. The process of any one of embodiments 1 to 4, wherein the ratio of the plant material to the solution of a) is about 6:1.

[0029] 18. The process of any one of embodiments 1 to 4, wherein the ratio of the plant material to the solution of a) is about 3:1.

[0030] 19. Any one of Embodiments 1 to 4, wherein the plant material and the solution of a) are in a ratio of about 2:1 One process.

[0031] 20. Any one of Embodiments 1 to 4, wherein the plant material and the solution of a) are in a ratio of about 1:1 One process.

[0032] 21. Any one process of Embodiments 1 to 4, wherein the dissolution of the plant material includes adding one or more divalent ions (plural available) to the lysate and / or filtrate and / or adding chitosan to the lysate and / or filtrate

[0033] 22. Any one process of Embodiments 1 to 4, wherein the dissolution of the plant material includes adding calcium ions to the lysate

[0034] 23. Any one process of Embodiments 1 to 4, wherein the dissolution of the plant material includes adding calcium chloride to the lysate

[0035] 24. Any one process of Embodiments 1 to 4, wherein the plant material is dissolved chemically, mechanically, and / or enzymatically

[0036] 25. Any one process of Embodiments 1 to 4, wherein the plant material is dissolved chemically

[0037] 26. Any one process of Embodiments 1 to 4, wherein the plant material is dissolved chemically using one or more surfactants

[0038] 27. Any one process of Embodiments 1 to 4, wherein the plant material is dissolved chemically using CHAPS

[0039] ​​​​​​28. The process according to any one of Embodiments 1 to 4, wherein the plant material is enzymatically lysed using one or more enzymes. Any one of the processes.

[0040] 29. The process according to any one of Embodiments 1 to 4, wherein the plant material is lysed using cellulase or pectinase. Any one of the processes.

[0041] 30. The process according to any one of Embodiments 1 to 4, wherein the plant material is mechanically lysed.

[0042] 31. The process according to any one of Embodiments 1 to 4, wherein the plant material is mechanically lysed using a blender. Any one of the processes.

[0043] 32. The process according to any one of Embodiments 1 to 4, wherein the plant material is mechanically lysed using a mill, a homogenizer, a microfluidizer, mechanical pressure, or a Stephan cutter. Any one of the processes. Any one of the processes.

[0044] 33. The process according to any one of Embodiments 1 to 4, wherein the plant material is mechanically lysed using a press, an ultrasonic disruptor, a grinder, using a pulsed electric field, using nitrogen injection, using ultrasonic energy, or by freezing. Any one of the processes. Any one of the processes.

[0045] 34. The process according to any one of Embodiments 1 to 4, wherein the plant material is mechanically lysed using at least one mill. Any one of the processes.

[0046] 35. The process according to any one of Embodiments 1 to 4, wherein the plant material is mechanically lysed using at least two different types of mills. Any one of the processes.

[0047] 36. The separating in (c) is carried out by a screw press, a decanter, or a centrifuge. The process of any one of Embodiments 1 to 4 to be performed.

[0048] The separating in 37.c) is performed using a disk stack centrifuge, a continuous centrifuge, or The process of any one of Embodiments 1 to 4 to be performed using a basket centrifuge .

[0049] The separating in 38.c) is performed using filtration, any one of Embodiments 1 to 4 One process.

[0050] The separating in 39.c) is performed using a press, any one of Embodiments 1 to 4 One process.

[0051] The separating in 40.c) is performed using filtration, any one of Embodiments 1 to 4 One process.

[0052] The separating in 41.c) is performed using gravitational sedimentation, any one of Embodiments 1 to 4 One process.

[0053] The separating in 42.c) is performed using sieving, any one of Embodiments 1 to 4 One process.

[0054] The process of Embodiment 1, wherein the first set temperature in 43.d) is about 80 °C or lower.

[0055] The process of Embodiment 1, wherein the first set temperature in 44.d) is about 65 °C or lower.

[0056] The process of Embodiment 1, wherein the first set temperature in 45.d) is about 55 °C or lower.

[0057] The process of Embodiment 1, wherein the first set temperature in 46.d) is about 50 °C or lower.

[0058] The process of Embodiment 1, wherein the second set temperature in 47.d) is about 25 °C or lower.

[0059] The process of Embodiment 1, wherein the second set temperature in 48.d) is about 15 °C or lower.

[0060] The process of Embodiment 1, wherein the second set temperature in 49.d) is about 10 °C or lower.

[0061] The process of Embodiment 1, wherein heating to the first set temperature in 50.d) takes about 15 minutes or less. process.

[0062] The process of Embodiment 1, wherein heating to the first set temperature in 51.d) takes about 5 minutes or less. process.

[0063] The process of Embodiment 1, wherein cooling to the second set temperature in 52.d) takes about 15 minutes or less. process.

[0064] The process of Embodiment 1, wherein cooling to the second set temperature in 53.d) takes about 5 minutes or less. process.

[0065] The process of Embodiment 2, wherein the one or more salts in 54.d) include 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.

[0066] The process of Embodiment 2, wherein the one or more salts in 55.d) include potassium phosphate and / or calcium chloride. embodiment.

[0067] The process of embodiment 2, wherein the one or more salts of 56.d) are added at a concentration of 5 mM to 2 M .

[0068] 57. The flocculant is alkylamine epichlorohydrin, polydimethyl diallyl ammonium chloride, polysaccharide, polyamine, starch, aluminum sulfate, alum, polyacrylamide , polyacrylamide or polyethyleneimine, any one of embodiments 1 to 4 processes.

[0069] 58. The flocculant is chitosan, any one of embodiments 1 to 4 processes.

[0070]

[0071] 60. The flocculant is activated chitosan at 1 to 20% w / w in solution, embodiment 1 to 4 any one of the processes.

[0072] 61. The adsorbent of e) is a resin, any one of embodiments 1 to 4 processes.

[0073] 62. The adsorbent of e) is activated carbon, activated charcoal or activated fossil charcoal, embodiments 1 to 4 any one of the processes.

[0074] 63. The adsorbent of e) has a surface area of more than 250 m / g, a weight average diameter of 1 to 1000 μm, an iodine value of 400 to 1,400 mg / g, a molasses number in the range of 100 to 550 , and / or activated carbon having at least 10 g / 100 g of methylene blue adsorption, any one of embodiments 1 to 4 processes.

[0075] The process according to any one of Embodiments 1 to 4, wherein the separation in 64.f) is carried out at 25°C or lower. Process.

[0076] The process according to any one of Embodiments 1 to 4, wherein the separation in 65.f) is carried out at 15°C or lower. Process.

[0077] The process according to any one of Embodiments 1 to 4, wherein the separation in 66.f) is carried out at 10°C or lower. Process.

[0078] The process according to any one of Embodiments 1 to 4, wherein the separation in 67.f) is carried out using filtration. Process.

[0079] The process according to any one of Embodiments 1 to 4, wherein the separation in 68.f) is carried out using a press, using gravitational sedimentation, or by sieving. Process.

[0080] The process according to any one of Embodiments 1 to 4, wherein the separation in 69.f) is carried out using a centrifuge or a decanter, or by microfiltration. Process.

[0081] The process according to any one of Embodiments 1 to 4, wherein all steps of the process excluding 70.e) are carried out at 25°C or lower. Process.

[0082] The process according to any one of Embodiments 1 to 4, wherein all steps of the process excluding 71.e) are carried out at 15°C or lower. Process.

[0083] The process according to any one of Embodiments 1 to 4, wherein all steps of the process excluding 72.e) are carried out at 10°C or lower. Process.

[0084] The process according to any one of Embodiments 1 to 4, wherein the filtering in 73.g) is carried out by a membrane filter. Process.

[0085] The process according to any one of Embodiments 1 to 4, wherein the filtering in 74.g) is performed by a 0.7 μm membrane filter.

[0086] The process according to any one of Embodiments 1 to 4, wherein the filtering in 75.g) is performed by a 0.2 μm membrane filter.

[0087] The process according to any one of Embodiments 1 to 4, wherein the filtering in 76.g) is performed by diatomaceous earth and / or activated carbon.

[0088] The process according to any one of Embodiments 1 to 4, wherein the filtering in 77.g) is performed by activated carbon up to about 10% at most.

[0089] The process according to any one of Embodiments 1 to 4, wherein the filtering in 78.g) is performed by activated carbon up to about 2% at most.

[0090] The process according to any one of Embodiments 1 to 4, wherein the filtering in 79.g) is performed by a 0.2 μm membrane filter and about 2% of activated carbon.

[0091] Furthermore, the process according to any one of Embodiments 73 to 79, comprising filtering the filtrate of g) through a 0.2 μm membrane filter after g).

[0092] The process according to any one of Embodiments 1 to 4, wherein one or more liquid phases and / or one or more filtrates contain one or more defoaming agents and / or one or more foam suppressants.

[0093] One or more liquid phases and / or one or more filtrates to remove small solids and / or microorganisms. ​​​​​​​​The process of any one of Embodiments 1 to 4 in which the filtrate is filtered.

[0094] Any one of Embodiments 1 to 4 in which the liquid phase of 83.1 or more and / or one or more filtrates are sterilized One process.

[0095] 84. Further, any one of the processes of Embodiments 1 to 4 including concentrating the filtrate Process.

[0096] 85. The process of Embodiment 84 in which concentrating the filtrate is carried out by ultrafiltration .

[0097] 86. The ultrafiltration is through polyethersulfone, polypropylene, polyvinylidene fluoride, polyacrylonitrile, cellulose acetate or polysulfone, Embodiment 8 5 process.

[0098] 87. The process of Embodiment 85 in which the ultrafiltration is carried out using an ultrafiltration filter with a cut-off of 100 kDa or less Process.

[0099] 88. The process of Embodiment 85 in which the ultrafiltration is carried out using an ultrafiltration filter with a cut-off of 50 kDa or less Process.

[0100] 89. The process of Embodiment 85 in which the ultrafiltration is carried out using an ultrafiltration filter with a cut-off of 10 kDa or less Process.

[0101] 90. The process of any one of Embodiments 1 to 4 in which the yield of the purified protein is at least about 10% of the soluble protein in the liquid phase in step c) One process.

[0102] 91. The yield of the purified protein is the soluble protein in the liquid phase in step c) A process according to any one of Embodiments 1 to 4, which is at least about 20% in quality.

[0103] 92. A process according to any one of Embodiments 1 to 4, wherein the yield of the purified protein is at least about 25% of the soluble protein in the liquid phase in step c). A process according to any one of Embodiments 1 to 4, which is at least about 25% in quality.

[0104] 93. A process according to any one of Embodiments 1 to 4, wherein the yield of the purified protein is at least about 40%. A process according to any one of Embodiments 1 to 4, which is at least about 40% in quality.

[0105] 94. A process according to any one of Embodiments 1 to 4, wherein the yield of the purified protein is at least about 60%. A process according to any one of Embodiments 1 to 4, which is at least about 60% in quality.

[0106] 95. A process according to any one of Embodiments 1 to 4, wherein the yield of the purified protein is at least about 80%. A process according to any one of Embodiments 1 to 4, which is at least about 80% in quality.

[0107] 96. A process according to any one of Embodiments 1 to 95, wherein the weight ratio of chlorophyll to protein in the purified protein standard is less than about 1:1000, about 1:1500, about 1:2000 or about 1:2500. A process according to any one of Embodiments 1 to 95, which is less than about 1:1000, about 1:1500, about 1:2000 or about 1:2500 in weight ratio of chlorophyll to protein in the purified protein standard. A process according to any one of Embodiments 1 to 95, which is less than about 1:1000, about 1:1500, about 1:2000 or about 1:2500 in weight ratio of chlorophyll to protein in the purified protein standard.

[0108] 97. A process according to any one of Embodiments 1 to 96, wherein one or more agents (plural possible) in the purified protein standard that impart or are related to one or more functional properties are reduced or removed compared to the original plant material. A process according to any one of Embodiments 1 to 96, wherein one or more agents (plural possible) in the purified protein standard that impart or are related to one or more functional properties are reduced or removed compared to the original plant material. A process according to any one of Embodiments 1 to 96, which is reduced or removed compared to the original plant material in one or more agents (plural possible) in the purified protein standard that impart or are related to one or more functional properties.

[0109] 98. A process according to any one of Embodiments 1 to 96, wherein one or more agents (plural possible) in the purified protein standard that impart or are related to one or more functional properties are 5%, 10%, 15% compared to the original plant material. 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65% , 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95% , reduced by 96%, 97%, 98%, or 99%, any one of Embodiments 1 to 96 of the process.

[0110] 99. The purified protein standard is essentially odorless, any one of Embodiments 1 to 96 of the process.

[0111] 100. The purified protein standard is odorless, the process of any one of Embodiments 1 to 96 of the process.

[0112] 101. The purified protein standard has an essentially unbiased taste, the process of any one of Embodiments 1 to 100 of the process.

[0113] 102. The purified protein standard has an unbiased taste, the process of any one of Embodiments 1 to 100 of the process.

[0114] 103. The protein is RuBisCo, the process of any one of Embodiments 1 to 102 of the process.

[0115] 104. The plant material is derived from Lemna, the process of any one of Embodiments 1 to 103 of the process.

[0116] 105. The plant material is derived from Lemnoideae, the process of any one of Embodiments 1 to 103 of the process.

[0117] 106. A product produced by the process of any one of Embodiments 1 to 105.

[0118] 107. A food containing a purified protein standard derived from plant material, wherein the protein standard is The food containing impurities of 80% or less.

[0119] 108. The food according to embodiment 107, wherein the protein standard contains RuBisCo.

[0120] 109. The food according to embodiment 107 or 108, wherein the plant material is derived from Lemna.

[0121] 110. The food according to embodiment 107 or 108, wherein the plant material is derived from Lemnoideae thereof.

[0122] In some aspects of the present disclosure, the plant material is washed before the process is started. Several In some embodiments, the reducing agent in a) is 2-mercaptoethanol (BME), 2 -mercaptoethylamine-HCL, sodium metabisulfite, cysteine hydrochloride, dithi othreitol (DTT), glutathione, cysteine, tris(2-carboxyethyl ) phosphine (TCEP), ferrous ions, nascent hydrogen, sodium amalgam, acetic acid, formic acid, magnesium, manganese, phosphoric acid, potassium and sodium. Several In some embodiments, the reducing agent is sulfite. In some embodiments, the sulfite is sulfurous acid sodium, magnesium sulfite, or sodium metabisulfite. In some embodiments the sulfite is sodium bisulfite.

[0123] In some embodiments, the pH of the solution in a) is from about pH 5.0 to about pH 9.0 is. In some embodiments, the pH of the solution is from about pH 6.0 to about pH 7.6. Several In some embodiments, the pH of the solution is about pH 6.8.

[0124] In some embodiments, the ratio of plant material to the solution of a) is about 6:1. In some embodiments, the ratio of plant material to the solution of a) is about 3:1. In some embodiments, the ratio of plant material to the solution of a) is about 2:1. In some embodiments, the ratio of plant material to the solution of a) is about 1:1.

[0125] In some aspects of the present disclosure, the plant material is mechanically dissolved. In some embodiments, the plant material is mechanically dissolved using a blender. In some embodiments, the plant material is mechanically dissolved using a mill, a homogenizer, a microfluidizer, mechanical pressure, or a Stephan cutter.

[0126] In some aspects of the present disclosure, the separation of c) is performed using a screw press, a decanter, or a centrifuge.

[0127] In some aspects of the present disclosure, the first set temperature of d) is about 80 °C or lower. In some aspects of the present disclosure, the first set temperature of d) is about 65 °C or lower. In some embodiments, the first set temperature of d) is about 55 °C or lower. In some embodiments, the first set temperature of d) is about 50 °C or lower. In some embodiments, the second set temperature of d) is about 25 °C or lower. In some embodiments, the second set temperature of d) is about 15 °C or lower. In some embodiments, the second set temperature of d) is about 10 °C or lower. In some embodiments, heating to the first set temperature of d) takes about 15 minutes or more. In some embodiments, heating to the first set temperature of d) takes about 5 minutes or more. In some embodiments, cooling to the second set temperature of d) In some embodiments, cooling to the first set temperature in d) takes no more than about 5 minutes. .

[0128] In some aspects of the present disclosure, one or more salts in d) include potassium phosphate and / or calcium chloride. In some aspects of the present disclosure, one or more salts in d) are added at a concentration of 5 mM to 2 M. .

[0129] In some aspects of the present disclosure, the flocculant is chitosan. In some aspects of the present disclosure, the flocculant is activated chitosan. In some embodiments, the flocculant is 1 to 20% w / w activated chitosan in solution. In some embodiments, the adsorbent in e) is activated carbon, activated charcoal or activated fossil charcoal. In some embodiments, the adsorbent is a hydrophobic adsorbent. .

[0130] In some aspects of the present disclosure, the separation in f) is carried out at 25°C or lower. In some embodiments, the separation in f) is carried out at 15°C or lower. In some embodiments, the separation in f) is carried out at 10°C or lower. In some embodiments, the separation in f) is carried out using a centrifuge or a decanter, or by microfiltration.

[0131] In some aspects of the present disclosure, all steps of the process except e) are carried out at 25°C or lower. In some embodiments, all steps of the process except e) are carried out at 15°C or lower. In some embodiments, all steps of the process except e) are carried out at 10°C or lower.

[0132] In some aspects of the present disclosure, the filtering in g) is carried out by a membrane filter. . In some embodiments, filtering in g) is performed by a 0.7 μm membrane filter. . In some embodiments, filtering in g) is performed by a 0.2 μm membrane filter. . In some embodiments, filtering in g) is performed by diatomaceous earth and / or activated carbon. In some embodiments, filtering in g) is performed by up to about 10 % activated carbon. In some embodiments, filtering in g) is performed by up to about 2 % activated carbon. In some embodiments, filtering in g) is performed by a 0.2 μm membrane filter and 2% activated carbon. . In some embodiments, the process further includes filtering the filtrate of g) through a 0.2 μm membrane filter after g). . In some embodiments, the process further includes concentrating the filtrate. . In some embodiments, concentrating the filtrate is performed by diafiltration. . In some embodiments, concentrating the filtrate is performed by ultrafiltration. In some embodiments, ultrafiltration is performed using an ultrafiltration filter with a cut-off of 100 kDa or less. . In some embodiments, ultrafiltration is performed using an ultrafiltration filter with a cut-off of 50 kDa or less. . In some embodiments, ultrafiltration is performed using an ultrafiltration filter with a cut-off of 10 kDa or less. .

[0133] . In some aspects of the present disclosure, 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% of the soluble protein in the liquid phase of step c). . In some In an embodiment, the yield of the purified protein is at least about 25% of the soluble protein in the liquid phase of step c). In some embodiments, the yield of the purified protein is at least about 40%. In some embodiments, the yield of the purified protein is at least about 60%. In some embodiments, the yield of the purified protein is at least about 80%.

[0134] In some aspects of the present disclosure, the protein is RuBisCo. In some embodiments the plant material is derived from Lemna. In some embodiments, the plant material is Lem onoideae.

[0135] Also disclosed herein is a product made by the disclosed process.

[0136] Also disclosed herein is a food comprising a purified protein preparation derived from plant material, wherein the protein preparation contains 80% or less impurities. BRIEF DESCRIPTION OF THE DRAWINGS

[0137]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7A

Figure 7B

Figure 8

Figure 9

Figure 10

Figure 11

Figure 12

Figure 13

Figure 14

Figure 15

Figure 16

Figure 17

Figure 18

Mode for Carrying Out the Invention

[0138] The disclosed processes and compositions may be more readily understood by reference to the following detailed description in conjunction with the accompanying figures which form a part of this disclosure.

[0139] Throughout this specification, the description refers to processes and the compositions made by the processes. When the present disclosure discloses or claims features or embodiments related to compositions, Such features or embodiments are equally applicable to the process of making the composition. If the disclosure discloses or claims features or embodiments related to a process for making a composition, When a range of values ​​is expressed, such features or embodiments are equally applicable to the composition. When a range is stated, it includes embodiments using a specific value within that range. Reference to a stated value includes each and every value within that range. When expressed as approximations, by use of the present application, it is understood that the particular values ​​form alternative embodiments. It will be understood that the use of "or" means "and" unless the specific context of its use dictates. All references cited herein are for any purpose. In the event of a conflict between the references and the specification, the specification shall prevail. The present disclosure, which is disclosed herein in the context of separate embodiments for clarity, will control. Certain features of the disclosed processes and compositions may be provided together in a single embodiment. It should be understood that the present invention may be practiced in a variety of ways. Conversely, the present invention may be described in the context of a single embodiment for the sake of brevity. As shown, the various features of the disclosed processes and compositions can be used separately or in any subset. It should be understood that a combination of the above may be provided.

[0140] As used herein, the singular forms "a," "an," and "the" are used where the context clearly indicates. The terms "about" and "approximately" include the plural form unless otherwise indicated. When used in context, the embodiments will be apparent to those skilled in the art from the teachings contained herein. is quoted as being acceptable for purpose up to plus or minus approximately 10% Refers to a value or range that approximates or is close to a value or range. In some embodiments ,"about" means plus or minus 10% of a numerical value.

[0141] Disclosed herein is a process for making protein preparations from plant materials. As used herein, the term "plant" refers to organisms belonging to the plant kingdom. Examples of plants suitable for use in the disclosed process include trees, herbs, shrubs, grasses, vines, ferns, mosses, and green algae. The term "plant material" refers to any biomass derived from a plant. Plant material may be derived from any part of a plant, such as a stem, root, fruit, leaf or seed. In some embodiments, the plant material is derived from leaves. In some embodiments, the plant material is derived from stems. The plant material may be obtained from one or more species of plants. For example, in some embodiments, the plant material is derived from duckweed, algae, sugar beet leaves, spinach, kale beet, chard, sugar beet, Swiss chard, fodder beet, soybean or tobacco. In some embodiments, the plant material is derived from duckweed. In some embodiments, the plant material is derived from Lemna. In some embodiments, the plant material is derived from L emnoideae.

[0142] As used herein, the term "protein" refers to a compound composed of amino acid residues covalently bonded by peptide bonds. Proteins usually contain at least two amino acids or amino acid variants and there is no limit to the maximum number of amino acids that can make up a protein sequence. The term "protein preparation" refers to an isolate of a protein. At that time, the protein is substantially separated from the non-protein components of the mixture. Protein The "purity" of the protein standard refers to the amount of protein relative to the total amount of the standard. In some embodiments the purity of the protein standard is expressed as a percentage of the total dry mass. In some embodiments the protein standard contains at least about 40%, 45%, 50%, 55%, 60%, 65%, 7 0%, 75%, 80%, 85%, 90%, 95%, 97%, or 99% protein In some embodiments, the purity of the protein standard is at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, or 99% protein. The protein standard may contain one or more types of proteins and may contain the same protein in different sizes. For example, in some embodiments the protein standard may contain edestin, gluten, legumin or vicilin In some embodiments, the protein standard contains RuBisCo. The processes disclosed herein separate proteins from other compounds found in plant material For example, the process may remove chlorophyll, volatile compounds, acids, bases, sugars, salts and / or lipids. In some embodiments, the processes disclosed herein remove chlorophyll from plant material to produce a dechlorophyllated protein standard. For example, in some embodiments the weight ratio of chlorophyll to protein in the protein standard is less than about 1:1000, 1:1500, 1:2000 or 1:2500.

[0143] In some embodiments, the processes disclosed herein result in a purified protein standard One or more agents that impart or are related to one or more sensory properties is reduced or removed. Non-limiting examples of such sensory properties include odor (e.g., off-odor or undesirable odor) and taste (e.g., off-taste or undesirable taste). In some embodiments, the processes disclosed herein reduce one or more agents (plural) by 5%, 10%, 15%, 20%, 25%, 30% compared to the original plant material, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80% 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%. In some embodiments, the processes disclosed herein completely reduce one or more agents (plural). In some embodiments, the processes disclosed herein reduce or remove one or more agents (plural) that impart or are related to odor to produce a protein preparation that is odorless or essentially odorless . In some embodiments, the processes disclosed herein reduce or remove one or more agents (plural) that impart or are related to taste to produce a protein preparation with a balanced taste or essentially balanced taste . In some embodiments, the processes disclosed herein reduce or remove one or more agents (plural) that impart or are related to odor and / or taste to produce a protein preparation that is odorless and has a balanced taste, essentially odorless and has a balanced taste, odorless and essentially has a balanced taste, or essentially odorless and essentially has a balanced taste . In some embodiments, the agent is a volatile compound . . . . . . . . It is. In some embodiments, the agent is a non-volatile compound. In some embodiments the agent is a polyphenol, polyphenol oxidase, lipoxygenase, phenol nol, lipid, alcohol, aldehyde, sulfide, peroxide, terpene, albumin (e.g. lectin or protease inhibitor), a substrate for oxidase activity (e.g., fatty acid, e.g., C14:0 (methyl myristate), C15:0 (methyl pentadecanoate), C16:0 methyl palmitate, C16:1 methyl palmitoleate, C17:0 hepta decanoate methyl, C18:0 methyl stearate, C18:1 methyl oleate, C18 :2 methyl linoleate, C18:3α methyl linoleate, C20:0 methyl eicosanoate , and C22:0 methyl behenate), and / or an enzyme that reacts with the lipid substrate. In some embodiments, the purified protein preparation has reduced oxidase activity compared to the protein source. In some embodiments, the purified protein preparation has a 5%, 10%, 15%, 20% or 25% reduction in oxidase activity compared to the protein source. In some embodiments, the protein source is RuBisCo and the purified protein preparation has a 5%, 10%, 15%, 20% or 25% reduction in oxidase activity compared to RuBisCo. In some embodiments, the oxidase activity is lipoxygenase activity. In some embodiments, the purified protein preparation has lower oxidation of lipids or residual lipids compared to the protein source due to reduced lipoxygenase activity. In one aspect of the present disclosure, the process (Figure 1) for making a purified protein preparation from plant material is

[0144] In one aspect of the present disclosure, the process (Figure 1) for making a purified protein preparation from plant material is a) Provide plant material in a solution containing a reducing agent; b) Dissolve the plant material; c) Separate the dissolved plant material into a solid phase and a liquid phase, where the liquid phase contains soluble proteins and chlorophyll; d) Heat to a first set temperature within about 30 minutes and then cool to a second set temperature within about 30 minutes to coagulate the chlorophyll in the liquid phase, where the cooling starts when the liquid phase reaches the first set temperature; e) Contact the liquid phase of d) with a flocculant and / or adsorbent and mix for a time sufficient for the chlorophyll in the liquid phase to flocculate and / or adsorb to the adsorbent, thereby forming a flocculated mixture; f) Separate the flocculated mixture of e) into a solid phase and a liquid phase; g) Filter the liquid phase of f) to obtain a filtrate containing the purified protein.

[0145] In some embodiments, the plant material is harvested and cleaned before the process is started. For example, in some embodiments, the plant material is chemically washed before the process is started. In some embodiments, the plant material is washed with water before the process is started. The plant material may be washed multiple times before the process is started.

[0146] In some embodiments, the plant material is mixed in a solution containing a reducing agent. Examples of reducing agents suitable for use in the disclosed process include 2-mercaptoethanol (BME ), 2-mercaptoethylamine-HCL, sodium metabisulfite, cysteine hydrochloride , dithiothreitol (DTT), glutathione, cysteine, tris(2-carboxy ethyl)phosphine (TCEP), ferrous ions, nascent hydrogen, sodium amalgam, Examples include, but are not limited to, oxalic acid, formic acid, magnesium, manganese, phosphoric acid, potassium, or sodium. In some embodiments, the plant material is mixed in a solution containing more than one reducing agent. In some embodiments, 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, the reducing agent is thought to act to regulate and / or inhibit the activity of polyphenol oxidase. A solution containing a reducing agent may be formulated to improve the stability of its components. For example, the pH, ionic strength, or temperature of the solution may be adjusted. In some embodiments, the solution may contain a buffer. Examples of buffers for use in the disclosed processes include alkali metals (e.g., Na or K ), NaCl, ammonium ions (NH ), nitrates, acetates (e.g., sodium acetate), chlorates, perchlorates (NO ), metal binary compounds with halogens (e.g., Cl

[0147] ), bromides (e.g., Br ), or iodides (e.g., I ), sulfates (SO4 ), ammonium sulfate, alkaline earth metal hydroxides (e.g., + OH + ), Ca 4 ), or Sr ), sulfides (S 3- ), carbonates (CO 2 H 3 O 2- ), chlorides (CI O 3- ), chlorites (CIO 4- ), binary compounds of halogens with metals (e.g., Cl - ), bromides (e.g., Br - ), or iodides (e.g., I - ), sulfates (SO4 2- ), ammonium sulfate, alkaline earth metal hydroxides (e.g., OH - ), Ca 2+ ), or Sr 2+ ), sulfides (S2- ), hydroxide (OH - ), carbonates (e.g. , sodium carbonate), oxalates, chromates (CO 3 2- , C 2 O 4 2- , CrO 4 2- ), Phosphorus Acid salt (PO 4 3- ) (e.g., sodium phosphate, monopotassium phosphate (KH 2 PO 4 ), Phosphorus Dipotassium phosphate (K 2 HPO 4 ), monosodium phosphate (NaH 2 PO 4 ), disodium phosphate M(Na 2 HPO 4 ), ammonium phosphate (NH 4 ) 3 PO 4 , calcium phosphate (Ca 3 ( PO 4 ) 2 ), magnesium phosphate, monomagnesium phosphate, dimagnesium phosphate, and Trimagnesium phosphate), Tris-HCl, HEPES, ACES, ADA, BES, Isopropyl alcohol Midazole-HCl, MES, MOPS, MOPSO, PIPES, TES, Bis-T Trisine, tricine, bicine, 3-{[tris(hydroxymethyl)methyl]amino}propane sulfonic acid, N,N-bis(2-hydroxyethyl)glycine, tris(hydroxymethyl) N-tris(hydroxymethyl)methylamine, N-tris(hydroxymethyl)methylglycine, 3-[N-tri (hydroxymethyl)methylamino]-2-hydroxypropanesulfonic acid, 4-2- Hydroxyethyl-1-piperazineethanesulfonic acid, 2-{[tris(hydroxymethyl {[2-(methyl)amino]ethanesulfonic acid, 3-(N-morpholino)propanesulfonic acid} , piperazine-N,N'-bis(2-ethanesulfonic acid), dimethylarsinic acid, sodium citrate, physiological saline sodium citrate, 2-(N-morpholino)ethanesulfonic acid, chloramine T, acetamido glycine, tricine, glycine amide, and ammonium carbonate are included, but not limited thereto.

[0148] In some embodiments, the solution for use in the disclosed process is a chelating agent. Examples of chelating agents for use in the disclosed process include, but are not limited to, chloride, cyanide, organic acids (including citric acid, glycolic acid, lactic acid, malic acid, malonic acid, oxalic acid, and succinic acid), deferoxamine, deferiprone, deferasirox, penicillamine, honey, sodium pyrophosphate, sodium hexametaphosphate, sodium tripolyphosphate, BAL, EDTA, dexrazoxane, Prussian blue, ALA, BAPTA, DTPA, DMPS, DMSA, EGTA, ribose, deoxy ribose, glucose, fructose, glucosamine, sucrose, lactose, maltose, cellulose, starch, pectin, gum, alginic acid, chitin, chitosan, lactic acid, pyruvic acid, citric acid, acetic acid, lipids, monoglycerides, diglycerides, triglycerides, phosphatidylcholine, phosphatidylethanolamine, ceramide, sphingomyelin, xanthophyll, vitamin A, cortisone, cortisol, cholanic acid, deoxycholic acid, taurocholic acid, glycine, alanine, valine, leucine, isoleucine, phenyl L-alanine, tryptophan, serine, threonine, tyrosine, aspartic acid, glutamic acid, lysine, arginine, asparagine, glutamine, histidine, cysteine, methionine, threonine, proline, histidine, adrenaline, insulin, ATP, NAD, FMN, FAD, coenzyme A, DNA, RNA, carbonate, bicarbonate, cyanide, glycolic acid, oxalic acid, lactic acid, citric acid, orthophosphate, pyrophosphate, metaphosphate, polyphosphate salt, phytic acid, MDP, HMDP, HEDP, hemoglobin, chlorophyll, plant alkaloid, anthocyanin, tannin, sulfate, sulfuric acid, chondroitin sulfate, vitamin B 12, ascorbic acid, and water are included, but are not limited thereto.

[0149] In some embodiments, the solution for use in the disclosed process contains protease inhibitors. Examples of protease inhibitors for use in the disclosed process include, but are not limited to, PMSF, sodium fluoride, β-glycerophosphate, sodium pyrophosphate, leupeptin, and E-64.

[0150] In some embodiments, the pH of the solution is from about 5.0 to 9.0. In some embodiments the pH of the solution is from 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 from about 6.5 to 7.0.

[0151] The solution containing the plant material and the reducing agent may be mixed in a ratio that enhances the proximity of the plant material to the reducing agent. For example, the solution containing the plant material and the reducing agent may be prepared in a ratio of about 6:1, 3:1, 2:1 or 1:1.

[0152] As used herein, the term "lysing" refers to breaking cells apart from plant material and exposing the contents of the cells. For example, lysing may include breaking the cell wall, disrupting the cell membrane, and / or exposing the cytoplasm. Methods for lysing plant material are known in the art and may include mechanical lysis, chemical lysis, and / or enzymatic lysis. In some embodiments, the plant material is mechanically lysed. Examples of mechanical lysis suitable for use in the disclosed processes include, but are not limited to, mechanical agitation, pressure, grinding, pressing, and shearing. In some embodiments, the plant material is mechanically lysed using a blender. In some embodiments, the plant material is mechanically lysed using a mill, such as a knife mill, high shear mill, colloid mill, ball mill, Boston shear mill, hammer mill, grinder, Rietz mill, wet mill, or high shear mill. In some embodiments, the plant material is mechanically lysed using at least two different types of mills (e.g., via serial milling). Without wishing to be bound by theory, in some embodiments, mechanically lysing the plant material using at least two different types of mills is thought to result in more effective lysis of the plant material. In some embodiments, the plant material is mechanically lysed using a sonicator, using nitrogen jetting, using ultrasonic energy, or by freezing. In some embodiments, the plant material is mechanically lysed using a press (e.g., a screw press or a French press). In some embodiments, the plant material is mechanically lysed using a sonicator, using nitrogen jetting, using ultrasonic energy, or by freezing. In some embodiments, the plant material is mechanically lysed using a press (e.g., a screw press or a French press). In some embodiments, the plant material is mechanically lysed using a sonicator, using nitrogen jetting, using ultrasonic energy, or by freezing. In some embodiments, the plant material is mechanically lysed using a press (e.g., a screw press or a French press). In some embodiments, the plant material is mechanically lysed using a sonicator, using nitrogen jetting, using ultrasonic energy, or by freezing. In some embodiments, the plant material is mechanically lysed using a press (e.g., a screw press or a French press). In some embodiments, the plant material is mechanically lysed using a sonicator, using nitrogen jetting, using ultrasonic energy, or by freezing. In some embodiments, the plant material is mechanically lysed using a press (e.g., a screw press or a French press). In some embodiments, the plant material is mechanically lysed using a sonicator, using nitrogen jetting, using ultrasonic energy, or by freezing. In some embodiments, the plant material is mechanically lysed using a press (e.g., a screw press or a French press). In some embodiments, the plant material is mechanically lysed using a sonicator, using nitrogen jetting, using ultrasonic energy, or by freezing. In some embodiments, the plant material is mechanically lysed using a press (e.g., a screw press or a French press). In some embodiments, the plant material is mechanically lysed using a sonicator, using nitrogen jetting, using ultrasonic energy, or by freezing. In some embodiments, the plant material is mechanically lysed using a press (e.g., a screw press or a French press). In some embodiments, the plant material is mechanically lysed using a sonicator, using nitrogen jetting, using ultrasonic energy, or by freezing. In some embodiments, the plant material is mechanically lysed using a press (e.g., a screw press or a French press). In some embodiments, the plant material is mechanically lysed using a sonicator, using nitrogen jetting, using ultrasonic energy, or by freezing. In some embodiments, the plant material is mechanically lysed using a press (e.g., a screw press or a French press). In some embodiments, the plant material is mechanically lysed using a sonicator, using nitrogen jetting, using ultrasonic energy, or by freezing. In some embodiments, the plant material is mechanically lysed using a press (e.g., a screw press or a French press). In some embodiments, the plant material is mechanically lysed using a sonicator, using nitrogen jetting, using ultrasonic energy, or by freezing. In some embodiments, the plant material is mechanically lysed using a press (e.g., a screw press or a French press). In some embodiments, the plant material is mechanically lysed using a sonicator, using nitrogen jetting, using ultrasonic energy, or by freezing. In some embodiments, the plant material is mechanically lysed using a press (e.g., a screw press or a French press). In some embodiments, the plant material is mechanically lysed using a sonicator, using nitrogen jetting, using ultrasonic energy, or by freezing. In some embodiments, the plant material is mechanically lysed using a press (e.g., a screw press or a French press). The plant material is mechanically lysed using a homogenizer (e.g., a high-pressure homogenizer or a microfluidizer). In some embodiments, the plant material is mechanically lysed using a grinder. In some embodiments, the plant material is mechanically lysed using pulsed electric fields (PEF). In some embodiments, the plant material is mechanically lysed using mechanical pressure. In some embodiments, the plant material is mechanically lysed using one or more of the techniques for mechanical lysis disclosed herein.

[0153] In some embodiments, the plant material is chemically lysed. In some embodiments, the plant material is chemically lysed using one or more surfactants. In some embodiments the one or more surfactants are ionic surfactants. In some embodiments, the one or more surfactants are cationic surfactants. In some embodiments, the one or more surfactants are anionic surfactants. In some embodiments, the one or more surfactants include sod ium dodecyl sulfate (SDS). In some embodiments, the one or more surfactants are nonionic surfactants such as, for example, Triton X-100, NP-40, digitonin, and / or s aponin. In some embodiments, the one or more surfactants are zwitterionic surfactants such as, for example, Triton, NP, Brij, Tween, octyl-β-glucoside, octylthioglucoside, SDS, CHAPS, and / or CHAPSO. In some embodiments, the one or more surfactants are hypotonic surfactants. In some embodiments, the one or more surfactants are hypertonic surfactants. In some embodiments, the one or more surfactants are isotonic surfactants. In some embodiments ​In some embodiments, the one or more surfactants are isotonic surfactants. In some embodiments the plant material is chemically dissolved using one or more of the techniques for chemical dissolution disclosed herein .

[0154] In some embodiments, the plant material is enzymatically lysed using one or more enzymes . In some embodiments, the one or more enzymes include cellulase. In some embodiments the one or more enzymes include pectinase

[0155] In some embodiments, the plant material is chemically and mechanically lysed. In some embodiments the plant material is chemically and enzymatically lysed. In some embodiments the plant material is mechanically and enzymatically lysed. In some embodiments, the plant material is chemically, mechanically and enzymatically lysed

[0156] In some embodiments, lysis of the plant material includes adding one or more divalent ion(s) to the lysate . In some embodiments, lysis of the plant material includes adding chitosan to the lysate . In some embodiments, lysis of the plant material includes adding one or more divalent ion(s) to the lysate and adding chitosan to the lysate. In some embodiments lysis of the plant material includes adding calcium ions to the lysate In some embodiments, lysis of the plant material includes adding calcium ions to the lysate and adding chitosan to the lysate. In some embodiments, lysis of the plant material includes adding calcium chloride to the lysate. In some embodiments, lysis of the plant material The solution includes adding calcium chloride to the lysate and adding chitosan to the lysate. .

[0157] Separation of the dissolved plant material into a solid phase and a liquid phase may be carried out by any solid-liquid separation technique known in the art. Such separation techniques suitable for use in the disclosed process include, for example, sieving, filtration, centrifugation, and decantation. In some embodiments, separating the dissolved plant material into a solid phase and a liquid phase is carried out by a screw press, a decanter, or a centrifuge. In some embodiments, separating the dissolved plant material into a solid phase and a liquid phase is carried out using a disk stack centrifuge, a decanter centrifuge, a continuous centrifuge, or a basket centrifuge. In some embodiments, separating the dissolved plant material into a solid phase and a liquid phase includes using a dead-end filtration system, using ultrafiltration, using a tangential flow filtration system, or using a flat sheet filter, including but not limited to filtration. In some embodiments, separating the dissolved plant material into a solid phase and a liquid phase includes using a screw press machine, a French press machine, a belt press machine, a filter press machine, a fan press machine, a finishing press machine, or a rotary press machine, including but not limited to the use of a press machine. In some embodiments, separating the dissolved plant material into a solid phase and a liquid phase includes using gravity sedimentation. In some embodiments, separating the dissolved plant material into a solid phase and a liquid phase includes sieving, including but not limited to a circular vibration separator or a linear / tilted motion shaker. In some embodiments, the liquid phase includes soluble proteins and chlorophyll. In some embodiments, the solid phase includes but is not limited to these. contains insoluble proteins, lignin, fibers, etc.

[0158] Separation of the dissolved plant material into a solid phase and a liquid phase may give rise to substances useful for various applications including, but not limited to, agricultural applications. For example, the liquid phase obtained from the separation of the dissolved plant material may contain soluble proteins, chlorophyll, phenolic compounds, cell membranes (e.g., lipids), carbohydrates (including but not limited to pectin), nucleic acids, and / or light-harvesting complexes / photosystems. For example, the solid phase obtained from the separation of the dissolved plant material may contain plant fibers, cellulose, hemicellulose, pectin, intact plant cells, organelles, insoluble proteins, chlorophyll, and / or fats. In some embodiments, the solid phase may be used, for example, as animal feed or biofuel. In some embodiments, the solid phase may contain levulinic acid, a precursor in the production of biofuels. In some embodiments, the chlorophyll obtained from the solid phase obtained from the separation of the dissolved plant material may be used, for example, as a pigment and / or in human and / or animal nutrition in cosmetic applications. The process of making a purified protein preparation may also include a step of using heat treatment to coagulate undesired components (e.g., components other than RuBisCo) and leaving the desired protein component (e.g., RuBisCo) in the liquid phase. Without being bound by theory, heating causes unfolding of the higher-order structure of the amino acid chain and results in aggregation of some proteins. Depending on the amino acid sequence and higher-order structure state, the proteins may have various

[0159] The process of making a purified protein preparation may also include a step of using heat treatment to coagulate undesired components (e.g., components other than RuBisCo) and leaving the desired protein component (e.g., RuBisCo) in the liquid phase. Without being bound by theory, heating causes unfolding of the higher-order structure of the amino acid chain and results in aggregation of some proteins. Depending on the amino acid sequence and higher-order structure state, the proteins may have various components other than RuBisCo) and leaving the desired protein component (e.g., R uBisCo) in the liquid phase. Without being bound by theory, heating causes unfolding of the higher-order structure of the amino acid chain and results in aggregation of some proteins. Depending on the amino acid sequence and higher-order structure state, the proteins may have various unfolding of the higher-order structure of the amino acid chain and results in aggregation of some proteins. Depending on the amino acid sequence and higher-order structure state, the proteins may have various aggregation of some proteins. Depending on the amino acid sequence and higher-order structure state, the proteins may have various ​​​​has an unfolding temperature, and when exceeding it, it begins to unfold and aggregate Begin to. By carefully controlled heating and cooling conditions, proteins with an unfolding temperature lower than the unfolding temperature of the desired protein product can be coagulated even if they coagulate. In some embodiments, heating is also carried out under mild conditions to prevent the target protein from aggregating is carried out. In some embodiments, the liquid phase is heated to a first set temperature . In some embodiments, the first set temperature is about 40°C, 45°C, 50°C, 55°C , 60°C, 65°C, 70°C, 75°C or 80°C or lower. In some embodiments , heating is carried out rapidly. In some embodiments, the heating to the first set temperature takes about 30 minutes or more. In some embodiments, the heating to the first set temperature takes about 15 minutes or more . In some embodiments, the heating to the first set temperature takes about 5 minutes or more . In some embodiments, after the liquid phase is heated to the first set temperature, it is cooled to a second set temperature . In some embodiments, it is about 30°C, 25°C, 20°C, 15°C, 10°C or 5 °C or higher. In some embodiments, cooling starts as soon as the first set temperature is reached . In some embodiments, cooling is carried out rapidly. In some embodiments, it takes about 30 minutes or more to cool to the second set temperature . In some embodiments, it takes about 15 minutes or more to cool to the second set temperature . In some embodiments, it takes about 5 minutes or more to cool to the second set temperature . The process of making a purified protein sample coagulates undesired components (

[0160] for example, components other than RuBisCo) by adding one or more salts, and the desired protein component (e.g In the case of RuBisCo, components other than RuBisCo) are coagulated to make the desired protein component in the liquid phase (e.g For example, the process may also include a step of leaving RuBisCo. In some embodiments, 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 a combination thereof. In some embodiments, the salt is calcium chloride, calcium nitrate, or iron carbonate. In some embodiments, the added salt is potassium phosphate. In some embodiments, the added salt is calcium chloride. In some embodiments, the added salts are potassium phosphate and calcium chloride. In some embodiments, one or more salts are added at a concentration of 5 mM to 2 M. In some embodiments, the process of making a purified protein preparation may also include a step of coagulating undesired components (e.g., components other than RuBisCo) by adding one or more coagulants to leave the desired protein component (e.g., RuBisCo) in the liquid phase. At this time, the coagulant is a quaternary ammonium species including, but not limited to, protonated tertiary, secondary, or primary ammonium species. In some embodiments, the coagulant is selected from epi-amine, polytannin, polyethyleneimine, polylysine, and cationic polyacrylamide. In some embodiments, the coagulant is a coagulant derived from a polymer. In some embodiments, 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 beads. In some embodiments, the polymer is a bead.

[0161] In some embodiments, the process of making a purified protein preparation may also include a step of coagulating undesired components (e.g., components other than RuBisCo) by adding one or more coagulants to leave the desired protein component (e.g., RuBisCo) in the liquid phase. At this time, the coagulant is a quaternary ammonium species including, but not limited to, protonated tertiary, secondary, or primary ammonium species. In some embodiments, the coagulant is selected from epi-amine, polytannin, polyethyleneimine, polylysine, and cationic polyacrylamide. In some embodiments, the coagulant is a coagulant derived from a polymer. In some embodiments, 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 beads. In some embodiments, the polymer is a bead. In some embodiments, the process of making a purified protein preparation may also include a step of coagulating undesired components (e.g., components other than RuBisCo) by adding one or more coagulants to leave the desired protein component (e.g., RuBisCo) in the liquid phase. At this time, the coagulant is a quaternary ammonium species including, but not limited to, protonated tertiary, secondary, or primary ammonium species. In some embodiments, the coagulant is selected from epi-amine, polytannin, polyethyleneimine, polylysine, and cationic polyacrylamide. In some embodiments, the coagulant is a coagulant derived from a polymer. In some embodiments, 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 beads. In some embodiments, the polymer is a bead. In some embodiments, the polymer is less than 1% up to 100% In some embodiments, the polymer has a theoretical molar charge density of up to 500 Daltons. In some embodiments, the polymer has a molecular weight of from about 10,000,000 to about 20,000,000 daltons. has a molecular weight of more than 20,000,000 daltons.

[0162] In some embodiments, the process for producing a purified protein preparation is by electrocoagulation. The method may also include a step of solidifying undesired components (e.g., components that are not RuBisCo). In some embodiments of the electrocoagulation method, water is passed through an electrocoagulation cell, whereupon metal The ion(s) enter the water, and at the cathode surface the water is hydrated to hydrogen gas and hydroxyl groups. The electrons are then free to flow, destabilizing the surface charges on the suspended solids and emulsified oils, causing the floating Large floccules form which trap free solids, heavy metals, emulsified oils, and other impurities. is removed from the water in downstream solids separation and / or filtration operation(s).

[0163] The process for producing a purified protein preparation also involves contacting the liquid phase with a flocculating agent and / or an adsorbent. and for a period of time sufficient to aggregate the chlorophyll in the liquid phase or to adsorb it onto the adsorbent. The method may also include the step of mixing the mixture for a period of time, thereby forming a coagulated mixture. When used in combination, the term "flocculant" refers to a substance that destabilizes colloids and causes them to come out of suspension. The term "adsorption" refers to the attachment of molecules to a solid surface or "adsorbent." The process of flocculation is well known in the art, and exemplary flocculating agents include alkyl Amine epichlorohydrin, polydimethyldiallylammonium chloride, polysaccharides (e.g., tosan), polyamine, starch, aluminum sulfate, alum, polyacrylamide , polyacrylamide or polyethyleneimine may be mentioned, but are not limited thereto . In some embodiments, the flocculant is activated chitosan. In some embodiments , the flocculant is 1-20% w / v activated chitosan in solution. To activate chitosan , the method of dissolving chitosan in solution is well known in the art, and any method of preparing activated chitosan in solution may be used. An exemplary method may include dissolving 1 % chitosan in 20% acetic acid and 79% water. Adsorbents are known in the art, and exemplary adsorbents include activated carbon, graphite, silica gel, zeolite, clay, polyethylene, etc . Exemplary adsorbents may also include resins. In some embodiments , the resin for use in the disclosed process is an ion exchange resin including, but not limited to, strong cation exchangers, weak cation exchangers, strong anion exchangers, weak anion exchangers, mixed bed resins, chelating resins, and polymer catalysts. In some embodiments , the resin for use in the disclosed process is a size exclusion chromatography (SEC) resin including, but not limited to, Sephacryl, Sepha dex, Sepharose, and Superdex (GE Healthcare B io-Sciences Corp, Westborough, Massachusetts ts). In some embodiments, the resin for use in the disclosed process is a matrix analog, antibody-antigen, polysaccharide (e.g., lectin), complementary base sequence (e.g., nucleic acid), receptor (e.g., hormone), avidin-biotin, calmodulin, poly-A, glut . In some embodiments, the resin for use in the disclosed process is a resin that is a matrix analog, antibody-antigen, polysaccharide (e.g., lectin), complementary base sequence (e.g., nucleic acid), receptor (e.g., hormone), avidin-biotin, calmodulin, poly-A, glut amine, etc It has an affinity for thion, protein A and protein G, and / or metal ions. In some embodiments, the resin for use in the disclosed process is, for example, a resin containing phenyl, butyl, octyl, hexyl, ether and / or PPG, and has hydrophobic interactions.

[0164] In some embodiments, the adsorbent is activated carbon, activated charcoal or activated fossil charcoal. In some embodiments, the activated carbon has a surface area exceeding 250 m / g, a weight average diameter of 1 - 1000 μm, an iodine value of 400 - 1,400 mg / g, a Molasses number in the range of 100 - 550, and / or has a methylene blue adsorption of at least 10 g / 100 g. In some embodiments, the liquid phase is contacted with the polymer. In some embodiments, the polymer is nonionic. In some embodiments, the polymer is anionic. In some embodiments, the polymer is cationic. In some embodiments, 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 beads. In some embodiments, the polymer is uncharged. In some embodiments, the polymer has a charge density ranging from less than 1 to a maximum of 100%. In some embodiments, the polymer has a molecular weight of 500 Daltons to 20,000,000 Daltons. In some embodiments, the polymer has a molecular weight exceeding 20,000,000 Daltons. Without being bound by theory, chlorophyll in the liquid phase aggregates and settles. to form particles of a relatively large size and then adsorb them on the surface of activated carbon, activated charcoal or activated fossil charcoal may be adsorbed. The aggregated mixture includes a solid phase containing a flocculant, an adsorbent, an insoluble protein and chlorophyll and a liquid phase containing a soluble protein in solution.

[0165] Next, the aggregated mixture may be separated into a solid phase and a liquid phase. The separation of the aggregated mixture may be carried out by a solid-liquid separation method known in the relevant technical field. Examples of such separation methods suitable for use in the disclosed process include sieving, filtration, centrifugation, and decantation are included. In some embodiments, the separation of the aggregated mixture into a solid phase and a liquid phase is carried out by a screw press , a decanter, or microfiltration. In some embodiments, the separation of the aggregated mixture into a solid phase and a liquid phase includes centrifugation, such as the use of a decanter centrifuge, a disk stack centrifuge, or a continuous centrifuge. In some embodiments, the separation of the aggregated mixture into a solid phase and a liquid phase includes filtration, such as the use of a dead-end filtration system, the use of ultrafiltration , a tangential flow filtration system, or a flat sheet filter. In some embodiments, the separation of the aggregated mixture into a solid phase and a liquid phase includes the use of a press, such as a screw press , a French press, a belt press, a filter press, a fan press, a finishing press machine or a rotary press. In some embodiments, the separation of the aggregated mixture into a solid phase and a liquid phase includes gravitational sedimentation. In some embodiments, the separation of the aggregated mixture into a solid phase and a liquid phase includes sieving. are included. In some embodiments, the separation of the aggregated mixture into a solid phase and a liquid phase includes the use of a hydrophobic adsorbent are included. In some embodiments, the separation of the aggregated mixture into a solid phase and a liquid phase includes the use of a hydrophobic adsorbent are included. In some embodiments, the separation of the aggregated mixture into a solid phase and a liquid phase includes the use of a hydrophobic adsorbent machine or a rotary press. In some embodiments, the separation of the aggregated mixture into a solid phase and a liquid phase includes gravitational sedimentation. In some embodiments, the separation of the aggregated mixture into a solid phase and a liquid phase includes sieving. are included. In some embodiments, the separation of the aggregated mixture into a solid phase and a liquid phase includes gravitational sedimentation. In some embodiments, the separation of the aggregated mixture into a solid phase and a liquid phase includes sieving. are included. In some embodiments, the separation of the aggregated mixture into a solid phase and a liquid phase includes gravitational sedimentation. In some embodiments, the separation of the aggregated mixture into a solid phase and a liquid phase includes sieving.

[0166] In some embodiments, the separation of the aggregated mixture into a solid phase and a liquid phase involves taking a hydrophobic adsorbent including removal. In some embodiments, removing the hydrophobic adsorbent may involve, for example, using a disc stack centrifuge, a continuous centrifuge, or a basket centrifuge, such as centrifugation. In some embodiments, removing the hydrophobic adsorbent may involve using a dead-end filtration system, using ultrafiltration, using a tangential flow filtration system, or using a flat sheet filter, including but not limited to filtration. In some embodiments, removing the hydrophobic adsorbent may involve using a screw press, a French press, a belt press, a filter press, a fan press, a finishing press, or a rotary press, including but not limited to using a press. In some embodiments, removing the hydrophobic adsorbent may involve using gravity sedimentation. In some embodiments, removing the hydrophobic adsorbent may involve screening. In some embodiments, removing the hydrophobic adsorbent may involve column filtration. Separation of the solid and liquid phases of the aggregated mixture may result in substances useful in various applications, including but not limited to agricultural applications. For example, if the adsorbent is activated carbon, activated charcoal, or activated fossil charcoal, the liquid phase may contain soluble proteins, and the solid phase may contain activated carbon, activated charcoal, or activated fossil charcoal and phenols, pigments, and / or cell membranes. Phenols derived from the solid phase may be used in human and / or animal nutrition. For example, in some embodiments, phenols may include, for example, carotenoids that may be used in dietary supplements. In some embodiments, phenols may be used in sunscreen. In some embodiments, the activated Activated carbon, activated charcoal, or activated fossil charcoal derived from a solid phase can be applied to plants to, for example, improve moisture retention. In some embodiments, activated carbon, activated charcoal or activated fossil charcoal derived from a solid phase has applicability in biofuel technology. For example, in some embodiments activated carbon, activated charcoal, or activated fossil charcoal can be used in the production of biochar.

[0167] In some embodiments, the liquid phase and / or filtrate for use in the disclosed process may contain an antifoaming agent and / or a foam inhibitor. In some embodiments, the antifoaming agent and / or the foam inhibitor is an oil-based antifoaming agent. In some embodiments of the oil-based antifoaming agent, the oil is any oil that is insoluble in the foaming medium except for mineral oil, vegetable oil, white oil, or silicone oil. In some embodiments, the oil-based antifoaming agent contains wax and / or hydrophobic silica. In some embodiments, the wax is ethylene bis-stearamide (EBS), paraffin wax, ether wax, and fatty alcohol wax. selected from. In some embodiments, the antifoaming agent and / or the foam inhibitor is a powder antifoaming agent. In some embodiments, the powder antifoaming agent is an oil-based antifoaming agent on a specific carrier such as silica. In some embodiments, the powder antifoaming agent is added to powder products such as, for example, cement, plaster, and surfactants. In some embodiments, the antifoaming agent and / or the foam inhibitor is an aqueous antifoaming agent. In some embodiments, the aqueous antifoaming agent contains one or more oils and / or waxes in an aqueous base, such as, for example, mineral oil, vegetable oil, long-chain fatty alcohols, and fatty acid esters or fatty acid esters. In some embodiments, the antifoaming agent and / or the foam inhibitor is. The defoaming agent is a silicon-based defoaming agent. In some embodiments, the silicon-based defoaming agent is a polymer of a silicon main chain In some embodiments, the silicon-based defoaming agent is delivered as an oil-based emulsion or an aqueous -based emulsion. In some embodiments, the silicon compound comprises, or consists of, hydrophobic silica dispersed in silicone oil. In some embodiments, the defoaming agent and / or foam inhibitor is a silicon-based defoaming agent containing an emulsifier. In some embodiments the defoaming agent and / or foam inhibitor is a silicon-based defoaming agent containing silicone glycol and / or a modified silicone fluid. In some embodiments, the defoaming agent and / or foam inhibitor is an EO / PO-based defoaming agent. In some embodiments the defoaming agent and / or foam inhibitor is an EO / PO-based defoaming agent containing a copolymer of polyethylene glycol and / or polypropylene glycol In some embodiments, the defoaming agent and / or foam inhibitor is an EO / PO-based defoaming agent delivered as an oil, an aqueous solution, or an aqueous emulsion. The separation of the agglomerated mixture into a solid phase and a liquid phase may result in substances useful for various applications, including but not limited to agricultural applications. For example, the liquid phase may contain soluble proteins, excess aggregating agents (e.g., chitosan), other linked, branched, or linear polysaccharides (including but not limited to ionic, non-ionic

[0168] and / or neutral polysaccharides), vitamin B-12, calcium chloride or other divalent ions (e.g., magnesium chloride), RuBisCo a light-harvesting complex / photosystem, soluble proteins, cell membranes, phenolic compounds, carotenoids lutein, and / or xanthophyll. For example, the solid phase may contain chlorophyll ​​​​It may contain leu, calcium phosphate, cell membrane, light-harvesting complex / photosystem, and / or chitosan. It may also be used. In some embodiments, the solid phase may be used, for example, as animal feed or as biofuel. In some embodiments, this solid phase may contain levulinic acid, which is a potential biofuel precursor. In some embodiments, chlorophyll obtained from the solid phase obtained from the separation of dissolved plant material may be used, for example, as a pigment in cosmetic applications and / or in human and / or animal nutrition. It may also be used.

[0169] To stabilize soluble proteins in the liquid phase, it may be advantageous to perform the steps of the process at low temperature. The low temperature may prevent the denaturation of soluble proteins. In some embodiments, the separation of the aggregated mixture is carried out at about 35 °C, 30 °C, 25 °C, 20 °C, 15 °C, 10 °C or 5 °C or lower. In some embodiments, all steps of the process of making a protein sample, excluding the heating step, are carried out at about 35 °C, 30 °C, 25 °C, 20 °C, 15 °C, 10 °C or 5 °C or lower.

[0170] After the separation of the aggregated mixture into the solid phase and the liquid phase, the liquid phase may be filtered to obtain a filtrate containing the purified protein. The method of filtration is well known in the art and may be carried out, for example, by the use of surface filters or depth filters, such as membrane filtration, column filtration, diafiltration, ultrafiltration, tangential flow filtration, etc. In some embodiments, the filtration of the liquid phase of the aggregated mixture is carried out by a thin film filter. In some embodiments, the filtration of the liquid phase of the aggregated mixture is carried out at 5.0 μm, 4.0 μm, 3.0 μm, 2.0 μm, 1.0 μm, 0.7 μ m, 0.5 μm, 0.4 μm, 0.3 μm, 0.2 μm, 0.1 μm, or lower. It is carried out by thin film filters of m, 0.5 μm, and 0.22 μm. In some embodiments the filtration of the liquid phase of the aggregated mixture is by surface filtration or depth filtration with diatomaceous earth. In some embodiments, the filtration of the liquid phase of the aggregated mixture is by surface filtration or depth filtration with sediment. In some embodiments, the filtration of the liquid phase of the aggregated mixture is by surface filtration or depth filtration with activated carbon. In some embodiments, the filtration of the liquid phase of the aggregated mixture is by surface filtration or depth filtration with up to about 10%, 8%, 6%, 4%, 2%, or 1% activated carbon. In some embodiments, the filtration of the liquid phase of the aggregated mixture includes multiple steps or modes of filtration. For example, the filtration may be carried out by a thin film filter and an activated carbon bed. In some embodiments, the filtration is carried out by a 0.2 μm thin film filter and the protein-like liquid is exposed to about 2% activated carbon. In some embodiments, the filtrate is further filtered through a thin film filter, for example, a thin film filter of 5.0 μm, 4.0 μm, 3.0 μm, 2.0 μm, 1.0 μm, 0.7 μm, 0.5 μm, or 0.2 μm. In some embodiments, small solids and / or microorganisms may be removed from the liquid phase and / or the filtrate. In some embodiments, small solids and / or microorganisms are removed from the liquid phase and / or the filtrate by microfiltration, for example, using a one-pass dead-end microfiltration system or a tangential flow filtration system. In some embodiments, the liquid phase and / or the filtrate may be sterilized. In some embodiments

[0171] In some embodiments, small solids and / or microorganisms may be removed from the liquid phase and / or the filtrate. In some embodiments, small solids and / or microorganisms are removed from the liquid phase and / or the filtrate by microfiltration, for example, using a one-pass dead-end microfiltration system or a tangential flow filtration system. In some embodiments, the liquid phase and / or the filtrate may be sterilized. In some embodiments

[0172] In some embodiments, the liquid phase and / or the filtrate may be sterilized. In some embodiments In a form, the liquid phase and / or the filtrate are sterilized by microfiltration, for example, by using a one-pass dead-end microfiltration system or a tangential flow filtration system. In some embodiments, the liquid phase and / or the filtrate are sterilized by ultraviolet (UV) irradiation. In some embodiments, the liquid phase and / or the filtrate are sterilized by gamma ray irradiation. In some embodiments, the liquid phase and / or the filtrate are sterilized by sterilization, for example, by autoclaving or high temperature short time sterilization.

[0173] The filtrate containing the protein sample may be further concentrated. Methods known in the art for concentrating solutes may be used. In some embodiments, concentrating the filtrate may be performed by ultrafiltration through a 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, and concentrating the filtrate may be performed by reverse osmosis. The size of the cut-off filter may be optimized according to the target protein. In some embodiments, ultrafiltration is performed using a cut-off filter of about 200 kDa, 150 kDa, 100 kDa, 75 kDa, 50 kDa, 25 kDa, 10 kDa, or 5 kDa or less.

[0174] In some embodiments, the liquid phase and / or the filtrate may be dialyzed. In some embodiments, dialysis may be performed using ultrafiltration. In some embodiments, dialysis Polyethersulfone, polypropylene, polyvinylidene fluoride, polyacrylonitrile This can be done using ultrafiltration through polysulfone, cellulose acetate or polysulfone. In some embodiments, dialysis may be performed using reverse osmosis.

[0175] The liquid phase and / or the filtrate may be dried. In some embodiments, the drying is by spray drying. dryer, freeze dryer, drum dryer, film dryer, bed dryer, flash dryer or rotary dryer This may be accomplished using a desiccator.

[0176] The processes disclosed herein allow for the preparation of high yields of purified protein. The process disclosed herein can be used to prepare high yield preparations of purified proteins. Advantageously, the process disclosed herein provides for the production of a liquid phase extract of plant material after lysis. At least about 5%, 10%, 15%, 20%, 25%, 30%, or more of the amount of soluble protein in the %, 35%, 40%, 50%, 60%, 70%, 80%, 90%, or 99% yield In some embodiments, the yield of the protein preparation is at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%.

[0177] The process disclosed herein extracts significant levels of protein from plant material. RuBisCo is an enzyme found in the chloroplasts of photosynthetic organisms. It is found in green leafy plant material and is used to catalyze the first major step in carbon fixation. Up to 50% of the total protein produced may consist of RuBisCo, which is In some embodiments, the protein preparation is Ru It is BisCo. In some embodiments, the plant material is duckweed, algae, beet root, spinach beet, common mallow, sugar beet, Swiss chard, Mangel beet, soybean or tobacco.

[0178] Another aspect of the present disclosure relates to products made by the processes disclosed herein. .

[0179] Yet another aspect of the present disclosure relates to foods comprising a purified protein preparation derived from plant material. Advantageously, the food comprising the protein preparation may contain 30%, 40%, 50%, 60%, 70%, 8 0%, 90%, or 95% or less impurities. In some embodiments, the food comprising the protein preparation comprises RuBisCo. In some embodiments, the protein is prepared from plant material derived from Lemna. In some embodiments, the protein is prepared from plant material derived from Lemonoideae.

[0180] The ability of proteins to form gels and stable foams is important in the manufacture of various foods. As used herein, a foam refers to a structure formed by trapping pockets of gas in a liquid or solid. The proteins in the foam contribute to the ability of the foam to form small bubbles and the stability to retain the structure. A foam with small bubbles uniformly distributed imparts texture, smoothness and lightness to the food. The ability of the protein preparation to form a foam is related to its purity, and a purity of at least about 80% may be required to form a stable foam. As used herein, a gel is a soft mass containing a large amount of aqueous phase. It is a solid. The protein gel is a protein with a continuous liquid phase throughout the matrix. It may include a three-dimensional network of protein fibers. Proteins with high gelling ability can form gels with less protein. The processes disclosed herein are advantageously highly pure, foaming ability, stability of the foaming substance, and gelling ability, which are suitable for use in foods. They may be used to prepare protein standards.

[0181] The present disclosure is further illustrated by the following limited examples.

Example

[0182] The soluble protein standards and lyophilized protein standards produced by the following processes detailed in Examples 1-4 and Comparative Example 1 were analyzed for their properties. Before lyophilization, the concentration of soluble protein in the solution was measured by Pierce 660 nm Protein Assay (Thermo-Scientific Inc.). The purity of the protein was measured by the Dumas method. Foaming substances were created from each of the soluble standards and the characteristics of the foaming were measured. The foaming ability (FC) was calculated as FC = (volume after foaming - volume before foaming) / volume before foaming × 100% The foaming stability at the time interval t after foaming was calculated as Foaming stability = volume of residual foaming substance at time t / volume of initial foaming substance × 100%

[0183] Example 1 1 kg of fresh *Cichorium intybus* was softened in a Vitamix Blender (Vitamix Corp , Cleveland, Ohio) at a ratio of 1:1 with a sodium carbonate buffer containing 0.3% w / v sodium bisulfite. The temperature was maintained below 30 °C and the medium speed setting was used. Extraction was carried out for 3 minutes at a fixed rate. Subsequently, a fine-mesh nylon filter bag (Natural Home Brands, Sun Valley, California) was used to filter the softened biomass, separating a highly fibrous solid filter cake from the liquid juice containing soluble proteins. The filtered homogenate was then centrifuged at a speed of 4000g / output (Allegra X15R, SX4750 rotor; Beckman Coul ter, Inc., Pasadena, California) for 10 minutes. The pellet was discarded and the supernatant was collected separately. The solution was heated to a temperature of 50°C in a water bath set at 55°C and then rapidly cooled to a temperature below 15°C after reaching the target temperature. Following the rapid cooling of the protein solution, 2% v / v activated chitosan and 4% w / v activated carbon (Ca bot Norit Americas Inc, Marshall, Texas) were added to the liquid juice. Subsequently, the solution was stirred for 5 minutes and then centrifuged at a speed of 5000g / output (Allegra X15R, SX4750 rotor; Beckman Coul ter, Inc., Pasadena, California) for 10 minutes. The green pellet in the centrifuge bottle was discarded, and the clear yellow supernatant was microfiltered precisely using a 0.7μm glass microfiber membrane (Whatma n 1825 - 047 Glass Microfiber Binder Free Filter, 0.7 Micron; Global Life Sciences So lutions USA LLC, Marlborough, Massachusetts s). Subsequently, the filtrate was filtered through a 0.2μm polyether membrane. s) and the clear yellow supernatant was microfiltered precisely. Subsequently, the filtrate was filtered through a 0.2μm polyether membrane. The solution was exposed to a Russulon membrane (polyethersulfone (PES) membrane filter, 0.2 micron; St erlitech Corporation Inc, Kent, Washington ) to remove the remaining unwanted particles including bacteria. Subsequently, a 70 kDa membrane ( MINIKROS® S02-E070-05-N; Spectrum Lab oratories, Inc., Rancho Dominguez, Californ ia) was used to concentrate the resulting pale yellow deodorized protein-like solution. Subsequently, the resulting concentrated solution was lyophilized (Harvest Right LLC, Salt La ke City, Utah), and the product was a white odorless soluble protein powder.

[0184] Example 2 Using a Vitamix Blender (Vitamix Corp, Cleveland, Ohio), 1 kg of fresh purslane was macerated at a 1:1 ratio with a potassium phosphate buffer solution containing 0.3% w / v ascorbic acid. Maceration was carried out at medium speed for 3 minutes to maintain a temperature below 30°C. Using a fine-mesh nylon filter bag (Natural Ho me Brands, Sun Valley, California), the dissolved biomass was filtered to separate the highly fibrous solid filter cake from the liquid juice containing soluble protein. The filtered homogenate was then centrifuged at a speed / output of 4000 g (Allegra X15R, SX4750 rotor; Beckman Coulter , Inc., Pasadena, California) for 10 minutes. The pellet was discarded and the supernatant was collected separately. Subsequently, the supernatant was mixed with 5% v / v activated chitosan (chitosan (Allegra X15R, SX4750 rotor; Beckman Coulter , Inc., Pasadena, California) for 10 minutes. The pellet was discarded and the supernatant was collected separately. Subsequently, the supernatant was mixed with 5% v / v activated chitosan (chitosan Hz (10 - 120 cps), of fungal origin (9012 - 76 - 4); Glentham Li fe Sciences Ltd., Corsham, Wiltshire, UK) and 10% w / v activated carbon (Cabot Norit Americas Inc, Mars hall, Texas) and mixed for 5 minutes. Subsequently, the mixed solution was centrifuged at a speed of 5000 g / output (Allegra X15R, SX4750 rotor; Beckman Coul er, Inc., Pasadena, California) for 10 minutes. The resulting pellet was discarded, and the deodorized and decolorized supernatant was microfiltered using a 0.2 μm polyethersulfone membrane (polyethersulfone (PES) membrane filter, 0.2 micron; Sterlitech Corporati on Inc, Kent, Washington). Subsequently, the resulting pale yellow deodorized protein-like solution was concentrated using a 70 kDa membrane (MINIKROS® S02 - E0 70 - 05 - N; Spectrum Laboratories, Inc., Ranch o Dominguez, California). Subsequently, the resulting concentrated solution was lyophilized (Harve st Right LLC, Salt Lake City, Utah), and the product was a white odorless soluble protein powder. Example 3

[0185] Using a Vitamix Blender (Vitamix Corp, Cleveland, Ohio), 1 kg of fresh Taraxacum officinale was softened at a ratio of 1:1 with distilled water containing 0.3% w / v sodium bisulfite and ascorbic acid. The softening was carried out at a temperature below 30°C ​​It was carried out at medium speed for 3 minutes to maintain. A fine-mesh nylon filter bag (Natura l Home Brands, Sun Valley, California) was used to filter the dissolved biomass and separate the highly fibrous solid filter cake from the liquid juice containing soluble proteins. Then, the filtered homogenate was centrifuged at 4000g speed / output for 10 minutes. The pellet was discarded and the supernatant was collected separately. Then, the supernatant was mixed with a solution containing 30 mM potassium phosphate and 20 mM calcium chloride for 5 minutes . Subsequently, the mixed solution was centrifuged at 5000g speed / output (Allegra X15R, SX4750 rotor; Beckman Coulter, Inc., Pasadena , California). The resulting pellet was discarded, and 5% w / v activated carbon (Cabot Norit Americas Inc, Marshall, Texas s) was added to the supernatant and the solution was stirred for 5 minutes. Subsequently, the activated carbon that adsorbed the remaining chlorophyll, polyphenols, and other particles that affect undesirable taste / sight / smell was removed by using a 0.2 μm polyethersulfone membrane filter (polyethersulfone (PES) membrane filter, 0.2 micron; Sterlitech Corporation , Kent, Washington) to microfilter the mixed solution containing activated carbon. Then, a 100 kDa membrane (Hollow Fiber Cartridge, 100,000 NMWC, 850 cm2; GE Healthcare e Bio-Sciences Corp, Westborough, Massachusetts) was used to concentrate the obtained light yellow deodorized protein-like solution. Subsequently ​​​​​​​​​ Then, the obtained concentrated solution was lyophilized, and the product was a white, odorless, and soluble protein powder.

[0186] Example 4 Using a Vitamix Blender (Vitamix Corp, Cleveland, Ohio), 1 kg of fresh *Portulaca oleracea* was soaked in distilled water containing 0.5% w / v sodium bisulfite in a 1:1 ratio. The soaking was carried out at medium speed for 3 minutes to maintain a temperature below 30 °C. The dissolved biomass was filtered using a fine-mesh nylon filter bag (Natural Home Br ands, Sun Valley, California) to separate fibrous solid filter cakes from the liquid juice containing soluble proteins. The filtered homogenate was then centrifuged at a speed / output of 4000 g (All egra X15R, SX4750 rotor; Beckman Coulter, Inc ., Pasadena, California) for 10 minutes. The pellet was discarded and the supernatant was collected separately. Then, the supernatant was mixed with a solution containing 30 mM potassium phosphate and 20 mM calcium chloride for 5 minutes. Subsequently, the mixed solution was centrifuged at a speed / output of 5000 g (Allegra X15R, SX4750 rotor; Beckman Coul ter, Inc., Pasadena, California) for 10 minutes . The obtained pellet was discarded. 2% w / v activated chitosan (chitosan (10 - 120 c ps), fungal origin (9012 - 76 - 4); Glentham Life Scienc es Ltd., Corsham, Wiltshire, UK) and 4% w / v activated carbon (Cabot Norit Americas Inc, Marshall, Texas (Cabot Norit Americas Inc, Marshall, Texas) ) was added to the supernatant and the solution was stirred for 5 minutes. Subsequently, the mixed solution was centrifuged at a speed of 5000 g (Allegra X15R, SX4750 rotor; Beckman Coulter r, Inc., Pasadena, California) for 10 minutes. The resulting pellet was discarded, and the deodorized and decolorized supernatant was microfiltered using a 0.7 μm polyethersulfone membrane (Whatman 182 5-047 Glass Microfiber Binder Free Filter, 0.7 micron; Global Life Sciences Solutions USA LLC, Marlborough, Massachusetts). Then, the supernatant was further microfiltered using a 0.2 μm polyethersulfone membrane ( Polyethersulfone (PES) membrane filter, 0.2 micron; Sterlitech Corporation Inc, Kent, Washington). Subsequently, the obtained pale yellow deodorized protein-like solution was concentrated using a 70 kDa membrane (MINIKROS® S0 2-E070-05-N; Spectrum Laboratories, Inc., Rancho Dominguez, California). Subsequently, the obtained concentrated solution was lyophilized (Harvest Right LLC, Salt Lake City, Utah), and the product was a white, odorless, and soluble protein powder. Results of Examples 1-4 The average purity of the protein standards prepared by the methods of Examples 1-4 was approximately 84.3%, and the concentration of soluble protein after ultrafiltration was 1.316 μg / mL. The achieved yield

[0187] Results of Examples 1-4 The average purity of the protein standards prepared by the methods of Examples 1-4 was about 84.3%, and the concentration of soluble protein after ultrafiltration was 1.316 μg / mL. The achieved yield ​​​​​​The foamability was 195%, and 92% stability was maintained after 1 hour. Gelation of the lyophilized substance The properties were verified, and it was sufficient to add 2% w / v of the lyophilized substance to form a gel.

[0188] Comparative Example 1 The protein of the cinquefoil leaf was extracted as described in WO2011 / 0778671 A1 (van de Velde et al.) with some modifications.

[0189] 1 kg of fresh cinquefoil was washed and macerated using a Vitamix Blender at a ratio of 2:1 with respect to 0.3% w / v of sodium bisulfite. Before heating to 60°C, the homogenate was filtered through cheesecloth. The filtrate was held at 60°C for 5 minutes and then cooled to 10°C. Following the heat treatment, the suspension was centrifuged at 5200 g for 5 minutes. Next, activated carbon was added to the supernatant in an amount of 5% w / w. Following the addition of activated carbon, the suspension was stirred for 5 minutes and then the supernatant was removed by decantation. The resulting supernatant was subjected to two microfiltration steps. First, the supernatant was passed through a microfilter (Whatman 1825-047 Glass Microfiber Binder Free Filter, 0.7 micron; Global Life S ciences Solutions USA LLC, Marlborough, Ma

[0190] ssachusetts) having a pore size of 0.7 μm. Subsequently, the filtrate was passed through a microfilter (polyethersulfone (PES) membrane filter, 0.2 micron; Sterl itech Corporation Inc, Kent, Washington) having a pore size of 0.2 μm. ​​​​​​​The liquid was passed through. Subsequently, the filtrate was lyophilized, and the product was a white, odorless powder.

[0191] Results of Comparative Example 1 The protein purity was approximately 34.1% per unit of the dried product, and the soluble protein concentration before lyophilization was 520 μg / mL. The foaming properties of the lyophilized material showed a total foaming strength of 92% and a stability of 62% after 1 hour. To verify the gelling properties of the lyophilized material, it was necessary to add at least 7% w / v of the lyophilized material to form a gel.

Table 1

[0192] Example 5 This example examined the removal of chlorophyll using calcium chloride to aggregate the chlorophyll - protein complex.

[0193] 2 kg of biomass was dissolved in an extraction buffer containing 2% metabisulfite and 0.1 M NaCl (Vitamix Corp, Cleveland, Ohio). Subsequently, four fractions all having a volume of 375 mL were made from the filtrate. All fractions were mixed at speed 4 and centrifuged at 5200 g for 5 minutes for all fractions. For filtration, a Buchner funnel with a 0.45 - μm cutoff filter sheet coated with DE (Dicalite Management Group Inc, Bala Cynwyd, Pennsylvania) (polyethersulfone (PES) membrane filter, 0.45 microns; Sterlitech Corporation Inc, Kent, Washington) was used.

[0194] ​​​​​​​​​​​Fraction 1: 3.75 g of phosphate buffer was added to obtain a concentration of 10 mM in the fraction. Next then, the fraction was passed through a hot bath process set at 68 °C, and then 15 g of activated carbon (Cabot Norit Americas Inc, Marshall, Texas) was added and mixed for 25 minutes. Subsequently, 15 g of 3% 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. Then the solution was centrifuged and then filtered.

[0195] Fraction 2: 3.75 g of phosphate buffer was added to obtain a concentration of 10 mM in the fraction. Next then 15 g of activated carbon (Cabot Norit Americas Inc, Marsh all, Texas) was added to the fraction and mixed for 15 minutes. Subsequently, 3% chitosan (chitosan (10 - 120 cps), fungal origin (9012 - 76 - 4); Glentha m Life Sciences Ltd., Corsham, Wiltshire, U K) 15 g was added and mixed for an additional 5 minutes. Then the solution was centrifuged and then filtered.

[0196] Fraction 3: 3.75 g of phosphate buffer and 2.81 g of calcium chloride solution were added to obtain concentrations of 10 mM and 7.5 mM respectively in the fraction. Next then 15 g of activated carbon (Ca bot Norit Americas Inc, Marshall, Texas) was added to the fraction and mixed for 15 minutes. Subsequently, 3% chitosan (chitosan (10 - 120 cps), fungal origin (9012 - 76 - 4); Glentham Life Scien 15 g of ces Ltd., Corsham, Wiltshire, UK) was added, and then The solution was mixed for 5 minutes and then centrifuged and filtered.

[0197] Fraction 4: 7.5 g of phosphate was added to obtain concentrations of 20 mM and 15 mM in the fraction, respectively. buffer and 5.63 g of calcium chloride solution were added. Then 15 g of activated carbon (Cabo t Norit Americas Inc, Marshall, Texas) was added to the fraction and mixed for 15 minutes. Subsequently, 15 g of 3% chitosan (chitosan (10 - 120 cp s), of fungal origin (9012 - 76 - 4); Glentham Life Science s Ltd., Corsham, Wiltshire, UK) was added and mixed for an additional 5 minutes. The solution was then centrifuged and filtered.

[0198]

Table 2

[0199] Figure 5 shows fractions 1, 2, 3, and 4 after microfiltration. Figure 6 shows fractions 4, 3, 2 and 1 after microfiltration. As shown in Figures 5 and 6, coagulation with calcium chloride shows no significant difference in chlorophyll removal compared to the control fraction (fraction 2). As also shown in Figures 5 and 6, the use of two different concentrations of calcium chloride in fractions 3 and 4 did not result in a significant difference in chlorophyll removal. removal.

[0200] Further experiments examined the point of adding EDTA. For the first setting of the fraction, the filtrate obtained after solid / liquid separation using a bench centrifuge was phosphate (dibasic ​​(treated with potassium phosphate and monobasic potassium phosphate), but not with EDTA by. Figure 7A shows these fractions. For the second setting of the fractions, the lysate obtained after solid / liquid separation was treated with phosphate (containing dibasic potassium phosphate and monobasic potassium phosphate) and also treated with EDTA. Figure 7B shows these fractions. As shown in Figures 7A and 7B, fraction 2 derived from the second setting of the fractions has a dramatically different color compared to fraction 2 derived from the first setting of the fractions. Without wishing to be bound by theory, these results are thought to suggest that adding EDTA to the filtrate results in a greater removal of color compared to adding EDTA to the lysate

[0201] Example 6 This example examined the use of calcium chloride to coagulate chlorophyll and chloroplast membranes as an alternative to using a heat bath

[0202] The biomass was lysed in an extraction buffer containing 0.1 M NaCl and 2% metabisulfite (without EDTA) . Calcium chloride and phosphate (containing dibasic potassium phosphate and monobasic potassium phosphate) were added to 375 mL of the filtrate after basket centrifugation (Rousselet-Robatel Model RA20VxR Vertical Basket Centrifug e; Robatel Inc, Pittsfield, Massachusetts ) in the amounts detailed in Table 4 below .

[0203]

Table 3

[0204] The filtrate was stirred at room temperature for 15 min. After calcium chloride treatment, 13 mL fractions were taken and Horizon Model 614B Centrifuge; cker Diagnostics LLC,Port Matilda,Pennsy The color of the supernatant and the weight of the pellet fraction were measured. A sample of the fractions after addition of calcium and phosphate and tabletop centrifugation for approximately 5 minutes is shown. As shown in Figure 8, fraction 4 showed excellent chlorophyll removal. 5 contained a white pellet (contents unknown), while the supernatant was relatively full of chlorophyll. Without wishing to be bound by theory, the results suggest that calcium chloride This suggests that phosphate is necessary to effectively remove chlorophyll at a concentration of 75 mM. As shown in FIG. 8, fraction 6 dissolved at pH 7.5 was significantly higher than fraction 4. Without wishing to be bound by theory, the results are The results may suggest that calcium chloride is less effective at higher pH levels.

[0205] The remainder of the lysate was then purified by standard procedures using activated charcoal (Cabot Norit Am Ericas Inc, Marshall, Texas) and chitosan (Chitosan (10 ~120cps), Fungal Origin (9012-76-4);Glentham Life S The samples were processed at Sciences Ltd., Corsham, Wiltshire, UK. The activated carbon-chitosan was centrifuged and the supernatant was further filtered through two coffee filters. The color of the supernatant was noted. Figure 9 shows samples of fractions 1-6 after removal of the activated charcoal and chitosan. As shown in Figure 9, chitosan and activated carbon showed excellent antibacterial properties after pretreatment with calcium chloride and phosphate. As shown in Figure 9, the color removal by activated carbon and chitosan was observed in fraction 6. was most effective in a 100 mM NaCl / 100 mM phosphate buffer solution, which contained 75 mM calcium chloride and 100 mM phosphate. Had.

[0206] SDS-PAGE Coomassie staining analysis was performed to identify RuBis in fractions 1, 5, 6, and 7. Co was visualized and its levels were determined. Figure 10 shows the results of SDS-PAGE Coomassie staining analysis. The results are shown in FIG. 10. The pellet and supernatant ("Su" in FIG. 10) after tabletop centrifugation for fractions 5 to 7 are SDS-PAGE gel (Bio-Rad Laboratories, Inc., Hercules, CA. As shown in FIG. 10, lane 9 shows chlorophyll still binds to a protein of around 25 kDa, whereas lane 6 Show that chlorophyll is detached. Not wanting to be bound by theory, These results suggest that the chlorophyll-binding protein is a subunit of approximately 25 kDa. It may also be suggested that the RuBisC has a size of 100 μm. o is predominant in the supernatants of fractions 5, 6 and 7, whereas the 25 kDa chlorophyll bond The combined protein is the majority in the pellet. Without wishing to be bound by theory These results suggest that calcium chloride with phosphate selectively binds chlorophyll-binding proteins. It may be suggested to selectively precipitate RuBisCo and leave the RuBisCo in solution.

[0207] Without wishing to be bound by theory, the results suggest that calcium chloride After separation, chlorophyll and cell membranes were efficiently removed from the green filtrate, and the RuBisCo was The portion remains in the supernatant, and precipitation induced by calcium chloride is thought to occur immediately, and it is considered that calcium chloride removes the 25 kDa chlorophyll-binding protein. It is considered that.

[0208] Example 7 This example examined the effect of a 0.5% surfactant on the recovery of proteins from filtering the lysate and the effect of the surfactant on downstream process steps. It examined the effect of a 0.5% surfactant on the recovery of proteins from filtering the lysate and the effect of the surfactant on downstream process steps.

[0209] 4 kg of biomass was dissolved in a buffer containing 0.1 M NaCl, 0.1 M phosphate (including dibasic potassium phosphate and monobasic potassium phosphate), and 2% metabisulfite (Vitamix Corp, Cleveland, Ohio). 2 L of the lysate was mixed with 200 mL of a 10% Chaps solution (Biovision Inc, Milpitas, Cali fornia) to obtain a concentration of 0.5% in the lysate. Then this was mixed for about 10 minutes and centrifuged in a basket centrifuge (Rousselet-Robatel M odel RA20VxR Vertical Basket Centrifuge; Robatel Inc, Pittsfield, Massachusetts). odel RA20VxR Vertical Basket Centrifuge; Robatel Inc, Pittsfield, Massachusetts). The remaining lysate was added with 200 mL of water to correct the dilution factor.

[0210] For both fractions, a fraction of 375 mL of the lysate was taken, 28 .1 mL of 1 M calcium chloride buffer was added to obtain a concentration of 75 mM. The mixture was centrifuged and the supernatants were compared. A control supernatant was taken, and 5% unbranched chitosan (chitosan (10 - 120 cps), fungal origin (90 12 - 76 - 4); Glentham Life Sciences Ltd., Cor (Sham, Wiltshire, UK) was added in an amount of (0.75 g to 375 mL). The solution was mixed for 5 minutes, after which a 13 mL aliquot was taken and centrifuged in a benchtop centrifuge (Horizon M odel 614B Centrifuge; Drucker Diagnostics LLC, Port Matilda, Pennsylvania). The pH of the supernatant was raised to 7.2 and the remaining chitosan and sufficient color removal were checked. Another 0.75 g was added to the original solution to obtain a total of 10% unbranched chitosan (1.5 g in 375 mL). This was repeated until sufficient color removal was achieved and excess chitosan was observed. The percentage of chitosan at which this was observed was 10%. 25% AC and 10% chitosan were added to both fractions and centrifuged in a centrifuge.

[0211] Figure 11 shows SDS-PAGE gels (Bio-Rad Laborator ies, INC, Hercules, CA) for various samples. As shown in Figure 11, the surfactant did not significantly increase RuBisCo in the filtrate and RuBisCo did not appear to be in the pellet of activated carbon-chitosan in either fraction.

[0212] Figure 12 shows the fractions after removal of activated carbon and chitosan. As shown in Figure 12, treatment with Chap s released more polyphenols and / or polyphenol oxidase (PPO) during lysis. Without wishing to be bound by theory, these results indicate that treatment with Chaps does not

[0213] significantly release more RuBisCo during lysis, but treatment with Chaps does The process may also suggest the release of even larger amounts of polyphenols and / or polyphenol oxidase (PPO) during dissolution. Also, without wishing to be bound by theory, these results may suggest that 25% activated carbon and 5% chitosan did not significantly remove color in any fraction. These results may also suggest the release of even larger amounts of polyphenols and / or polyphenol oxidase (PPO) during dissolution. Also, without wishing to be bound by theory, these results may suggest that 25% activated carbon and 5% chitosan did not significantly remove color in any fraction. These results may also suggest the release of even larger amounts of polyphenols and / or polyphenol oxidase (PPO) during dissolution. Also, without wishing to be bound by theory, these results may suggest that 25% activated carbon and 5% chitosan did not significantly remove color in any fraction. These results may also suggest the release of even larger amounts of polyphenols and / or polyphenol oxidase (PPO) during dissolution. Also, without wishing to be bound by theory, these results may suggest that 25% activated carbon and 5% chitosan did not significantly remove color in any fraction.

[0214] Example 8 This example relates to a baseline run of calcium chloride, resuspension of the filter cake with 0.1% CHAPS, and washing of a second filter cake with 0.1% CHAPS. This example relates to a baseline run of calcium chloride, resuspension of the filter cake with 0.1% CHAPS, and washing of a second filter cake with 0.1% CHAPS.

[0215] 2 kg of biomass was lysed in an extraction buffer containing 0.2 M NaCl, 0.1 M PO 4 pH 7.7, 2% metabisulfite, (recipe per kg: 20 mL of 5 M NaCl, 115 mL of 1 M NaOH, H O 115 2 mL, 50 mL of 1 M phosphate buffer pH 7.6, 200 g of ice) (Vitamix Corp, Cleveland, Ohio ). The 2 kg was ultimately divided into three fractions. For fraction 1, after lysis by Vitamix, 75 mM calcium chloride was added and manually mixed for 10 minutes while the pH was closely monitored. It was then centrifuged in a basket centrifuge (Rousselet-Robatel Model RA20VxR Vertical Basket Centrifuge; Robatel Inc, Pittsfield, Massachusetts ), and the fraction was recovered as the filtrate. The preparation of fraction 2 was carried out by resuspending the filter cake in 2 L of resuspension buffer (70 mM PO pH 7.2, 0.1 M NaCl, 0.1% CHAPS) in a basket centrifuge (Rousselet-Robatel Model RA20VxR Vertical Basket Centrifuge; Robatel Inc, Pittsfield, Massachusetts ), and the fraction was recovered as the filtrate. The preparation of fraction 2 was carried out by resuspending the filter cake in 2 L of resuspension buffer (70 mM PO 4 pH 7.2, 0.1 M NaCl, 0.1% CHAPS) in a basket It included resuspending the centrifugal filter cake. Subsequently, the slurry was mixed with a Vitamix, and the filtrate (fraction 2) and the filter cake were separated by basket centrifugation. Similar to fraction 2, fraction 3 was prepared by resuspending the basket centrifugation filter cake in 1 L of resuspension buffer. The slurry was mixed manually and allowed to stand for 10 minutes before basket centrifugation. 50 g of activated carbon was added and mixed for 15 minutes, followed by adding 20 g of 3% chitosan (chitosan (10 - 120 cps), fungal origin (9012 - 76 - 4); Glentham Life Sciences Ltd., Corsham, Wiltshire, UK), and mixing for an additional 5 minutes for each fraction. Then the solution was centrifuged and microfiltered precisely through a 0.2 - um filter (polyethersulfone (PES) membrane filter, 0.2 micron; Sterlitech Corporation Inc, Kent, Washington). Finally, ultrafiltration with a 50 - kDa cutoff (MINIKROS® S02 - E050 - 05 - N; Spectrum Laboratories, Inc., Rancho Dominguez, California) was used to concentrate the solution and diafiltered until the salt concentration was below 0.1 ppt (about 10 L of dH 2O). The final step was ultrafiltration with a 10 - kDa cutoff (MINIKROS® S02 - E010 - 05 - N; Spectrum Laboratories, Inc., Rancho Dominguez, California), and then lyophilized. The parameters used during the control / filter cake resuspension / second wash process are provided in Table 5. s), fungal origin (9012 - 76 - 4); Glentham Life Science s Ltd.,Corsham,Wiltshire,UK)20g was added and mixed for an additional 5 minutes for each fraction. Then the solution was centrifuged and microfiltered precisely through a 0.2 - um filter (polyethersulfone (PES) membrane filter, 0.2 micron; Sterlitech Corporation Inc, Kent, Washington). Finally, ultrafiltration with a 50 - kDa cutoff (MINIKROS® S02 - E050 - 05 - N; Spectrum Laboratories, Inc., Rancho Dominguez, California) was used to concentrate the solution and diafiltered until the salt concentration was below 0.1 ppt (about 10 L of dH 2O). The final step was ultrafiltration with a 10 - kDa cutoff (MINIKROS® S02 - E010 - 05 - N; Spectrum Laboratories, Inc., Rancho Dominguez, California), and then lyophilized. The parameters used during the control / filter cake resuspension / second wash process are provided in Table 5. ation Inc,Kent,Washington) was used to concentrate the solution and diafiltered until the salt concentration was below 0.1 ppt (about 10 L of dH 2O). The final step was ultrafiltration with a 10 - kDa cutoff (MINIKROS® S02 - E010 - 05 - N; Spectrum Laboratories, Inc., Rancho Dominguez, California), and then lyophilized. The parameters used during the control / filter cake resuspension / second wash process are provided in Table 5. Spectrum Laboratories,Inc.,Rancho Doming uez,California) was used to concentrate the solution and diafiltered until the salt concentration was below 0.1 ppt (about 10 L of dH 2O). The final step was ultrafiltration with a 10 - kDa cutoff (MINIKROS® S02 - E010 - 05 - N; Spectrum Laboratories, Inc., Rancho Dominguez, California), and then lyophilized. The parameters used during the control / filter cake resuspension / second wash process are provided in Table 5. 2 O) dialysis filtration was performed. The final step was ultrafiltration with a 10 - kDa cutoff (MINIK ROS® S02 - E010 - 05 - N; Spectrum Laborato ries,Inc.,Rancho Dominguez,California) and then lyophilized. The parameters used during the control / filter cake resuspension / second wash process are provided in Table 5. rameters used during the control / filter cake resuspension / second wash process are provided in Table 5. The parameters used during the control / filter cake resuspension / second wash process are provided in Table 5.

[0216]

Table 4

[0217] After basket centrifugation, fractions 2 and 3 had a darker green color compared to fraction 1. However, after microfiltration, fraction 3 was almost colorless and fraction 2 was lighter in color than fraction 1. Figure 13 shows SDS-PAGE gels for various samples, where "F 1" refers to "fraction 1", "F2" refers to "fraction 2", and "F3" refers to "fraction 3". "AC-C" refers to "activated carbon·chitosan", "sup" refers to "supernatant", and "Rubi " refers to "RuBisCo". A yield of 94.25% was observed based on the soluble total protein content in the biomass of the soluble crude protein.

[0218] Example 9 This example examined treatment with calcium chloride, increased phosphate, and 0.25% CHAPS surfactant.

[0219] A total of 6 kg of biomass was processed (Vitamix Corp, Cleveland , Ohio). The 6 kg was divided into three batches of 2 kg each, and three experimental fractions were run. Fraction 1 was dissolved in an extraction buffer containing 0.2 M NaCl and 0.1 M phosphate (containing dibasic potassium phosphate and monobasic potassium phosphate), pH 7.7, and 2% metabisulfite (recipe per kg: 20 mL of 5 M NaCl, 120 mL of 1 M NaOH, H O 110 mL, 50 mL of 1 M phosphate 2 buffer pH 7.6, 200 g of ice). Dissolution was carried out for 3 minutes by Vitamixing at output 5. Fraction 2 was prepared in the same way as fraction 1.

[0220] Dissolution was performed by Vitamixing for 3 minutes at output 5. However, the extraction buffer contained more phosphate (recipe per kg: 5M NaCl 20 mL of 1M NaOH, 120 mL of H 2 Add 77 mL of 1M phosphate buffer solution Fraction 3 contained 0.25% CHA (83 mL of H7.6 and 200 g of ice). PS surfactant (Biovision Inc, Milpitas, California Fraction 2 was dissolved in the same phosphate buffer as fraction 2, except that a) was added. The same protocol was used in a Vitamix Blender (slight foaming). was observed).

[0221] Adjust the pH of the filtrate to pH 7.3 with 1M NaOH, never allowing it to drop below pH 6.8. After dissolution, 75 mM calcium chloride was added and mixed manually for 10 minutes. The pH was closely monitored at 20°C. It was then centrifuged in a basket centrifuge (Rousselet- Robatel Model RA20VxR Vertical Basket Ce ntrifuge;Robatel Inc, Pittsfield, Massachu The fraction 2 was prepared by centrifugation using a centrifuge and the filtrate was collected. 0 mM P04 pH 7.2, 0.1 M NaCl, 0.1% CHAPS) in a basket The centrifugation consisted of resuspending the cake in a centrifuge. The slurry was then mixed in a Vitamix for 1 minute. The mixture was mixed and the filtrate (fraction 2) and cake were separated by basket centrifugation. Fraction 3 was prepared by resuspending the basket centrifuge cake in 1 L of resuspension buffer. The slurry was mixed by hand and allowed to stand for 10 minutes before basket centrifugation. of activated charcoal was added and mixed for 15 min, followed by 3% chitosan (Chitosan (10-12 0 cps), fungal origin (9012 - 76 - 4); Glentham Life Scie nces Ltd., Corsham, Wiltshire, UK) 20 g was added and the fractions were further mixed for 5 minutes each. Then the solution was centrifuged and microfiltered through a 0.2 μm filter (polyethersulfone (PES) membrane filter, 0.2 micron; Sterlitech Cor poration Inc, Kent, Washington). Finally, the solution was concentrated by ultrafiltration with a 50 kDa cut - off (MINIKROS® S02 - E050 - 05 - N; Spectrum Laboratories, Inc., Rancho Dom inguez, California) until the salt concentration was below 0.1 ppt (about 10 L of dH 2O) diafiltered. The final step was ultrafiltration with a 10 kDa cut - off (MI NIKROS® S02 - E010 - 05 - N; Spectrum Labor atories, Inc., Rancho Dominguez, California 2 ), followed by lyophilization. The parameters used during the control / fraction 2 / fraction 3 processes are provided in Table 6. Fraction 2 was slightly darker than fraction 1 after basket centrifugation, and fraction 3 was significantly darker green after basket centrifugation. Both fraction 2 and 3 were more turbid than fraction 1 after basket centrifugation. The volume of fraction 3 was 4.8 L, but the volume of air added to increase the volume is presented in Table 5.

[0222]

Table 5

[0223] After basket centrifugation, fraction 2 was slightly darker than fraction 1, and fraction 3 was significantly darker green than fraction 1. Both fraction 2 and 3 were more turbid than fraction 1 after basket centrifugation. The volume of fraction 3 was 4.8 L, but the volume of air added to increase the volume is presented in Table 5. It was treated as 3.4 L due to an obvious increase in volume. The volume of the filtrate was reduced to 2.5 L before processing for all. Therefore, 89% of fraction 1, 100% of fraction 2, and 96% of fraction 3 were actually used.

[0224] Figure 14 shows SDS-PAGE gels for various samples, where "F1" refers to "fraction 1", "F2" refers to "fraction 2", "F3" refers to "fraction 3", "AC-C" refers to "activated carbon·chitosan", and "sup" refers to "supernatant". The yields based on the total soluble protein content in the biomass were as follows: control, 80.69%; fraction 2 : 89.43%; fraction 3: 95.20%

[0225] Example 10 This example examined the effect of chitosan concentration on chlorophyll removal without using a heat bath.

[0226] 2 kg of biomass was dissolved in an extraction buffer containing 2% metabisulfite, 10 mM EDTA, and 0.1 M NaCl (Vitamix Corp, Cleveland, Ohio). Five fractions were made from the filtrate, all having a volume of 375 mL. All fractions were mixed at speed 4 and centrifuged (Allegra X15R, SX4750 rotor; Beckman Coulter, Inc., Pasadena, California) for all fractions, with the process set at 5200 g and 5 minutes. For filtration, a coffee filter was used, followed by a 0.45 μm cut-off filter sheet coated with DE (Dicalite Management Group Inc, Bala Cynwyd, Pennsylvania) (polyethersulfone (PES)) ​ Membrane filter, 0.45 micron; Sterlitech Corporation I A Buchner funnel with nc, Kent, Washington was used.

[0227] Fraction 1: Then 15 g of activated carbon (Cabot Norit Americas Inc , Marshall, Texas) was added to the fraction and mixed for 15 minutes. Subsequently, 1% chitosan (chitosan (10 - 120 cps), fungal origin (9012 - 76 - 4); Gle ntham Life Sciences Ltd., Corsham, Wiltshi re, UK) 15 g was added and mixed for an additional 5 minutes. The solution was centrifuged (Allegra X15R, SX4750 rotor; Beckman Coulter, Inc., Pasadena, California), then filtered (Polyethersu lfone (PES) membrane filter, 0.2 micron; Sterlitech Corp oration Inc, Kent, Washington).

[0228] Fraction 2: Then 15 g of activated carbon was added to the fraction and mixed for 15 minutes. Subsequently, 2% chitosan 15 g was added and mixed for an additional 5 minutes. Then the solution was centrifuged and then filtered done.

[0229] Fraction 3: Then 15 g of activated carbon was added to the fraction and mixed for 15 minutes. Subsequently, 3% chitosan 15 g was added and mixed for an additional 5 minutes. Then the solution was centrifuged and then filtered done.

[0230] Fraction 4: Then 15 g of activated carbon was added to the fraction and mixed for 15 minutes. Subsequently, 4% 15 g of chitosan was added and mixed for an additional 5 minutes. The solution was then centrifuged and then filtered done.

[0231] Fraction 5: Then 15 g of activated carbon was added to the fraction and mixed for 15 minutes. Subsequently, 5% of 15 g of chitosan was added and mixed for an additional 5 minutes. The solution was then centrifuged and then filtered However, there was an error and the timer was not started after the addition of chitosan. Therefore the results for fraction 5 were not compared with those of the other fractions.

[0232] Summary of the exemplary variables · Range of chitosan: 1% - 5% · Without phosphate buffer · 15 minutes of 100% activated carbon mixing followed by 5 minutes of chitosan mixing

[0233] Figure 15 shows fractions 1 - 5 after microfiltration with a Buchner funnel. As shown in Figure 15, the colors appeared equal and were removed 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 the chloroform as effectively as 2% chitosan. As shown in Figure 15, fraction 5, which was exposed to activated carbon and chitosan for approximately 2 - 4 minutes more than fractions 1 - 4, was clearer than fractions 1 - 4. Without wishing to be bound by theory, these results may suggest that treatment with a 2% solution of chitosan

[0234] functions similarly to treatment with a 3% solution of chitosan or a 4% solution of chitosan. Without wishing to be bound by theory, these results may also suggest that, for example, treatment with a 5% solution of chitosan and a longer exposure time to activated carbon and chitosan Without wishing to be bound by theory, these results and longer exposure times to activated carbon and chitosan It may be suggested that treatment with may also improve chlorophyll removal.

[0235] Example 11 In this example, activated carbon (Cabot Norit Americas Inc., Mar. (shall, Texas) instead of or in conjunction with the use of chitosan from the post-dissolution Activated bentonite clay (EP Engineered Cl) for removing color compounds We investigated the use of CC160 derived from ays. Obtain fresh supernatant (after chitosan centrifugation in the extraction process) and adjust its pH to 7 with NaOH. The Pierce assay (Pierce™ 660 nm Protein Assay) was adjusted to 0. Assay Reagent; Thermo Fisher Scientific, Wal The optical density was measured by a 30-μm LCMS (Tham, Massachusetts) and the absorbance at 474 nm was The light intensity was measured to determine a starting point for the amount of [protein] and orange decolorization. As you will understand, the Pierce 660nm Protein Assay Reagent measures total protein concentration. Without wishing to be bound by theory, P The Ierce 660nm Protein Assay is a dye whose absorption maximum is measured at 660nm. It is believed to be based on the binding of a dye-metal complex to a protein that causes a change. The complexes are reddish brown and turn green upon protein binding, and the color produced in the assay is consistent with that of the TA. The concentration of activated carbon (C) in a 100 mL sample is increased in proportion to the protein concentration. abot Norit Americas Inc, Marshall, Texas) The mixture was stirred for 1 min and then filtered through a 0.45 μm filter (polyether ether sulphate). PES membrane filter, 0.45 micron; Sterlitech Corpor ation Inc, Kent, Washington), followed by a 0.2 μm filter (polyethersulfone (PES) membrane filter, 0.2 micron; Sterl itech Corporation Inc, Kent, Washington). It was poured directly into a Buchner funnel equipped with these and the filtrate was collected in a flask using vacuum. Pierce assay was used to measure the optical density and the absorbance at 474 nm was measured. In a separate 100 m L sample, 0.3% w / v of CC160 clay was added. The mixture was stirred for 1 minute and then poured directly into a Buchner funnel equipped with a 0.45 μm filter and then a 0.2 μm filter, and the filtrate was collected in a flask using vacuum. Pierce assay was used to measure the optical density and the absorbance at 474 nm was measured.

[0236] As cited in Table 7, the above procedure was repeated with samples using various concentrations of CC160 clay. Table 7 lists the concentration of activated carbon or clay before and after treatment and filtration and the absorbance at 474 nm.

Table 6

[0237] Without wishing to be bound by theory, these results may suggest that activated carbon is superior to bentonite clay in removing polyphenols.

[0238] Example 12 This example examined whether a resin could be used instead of activated carbon.

[0239] Fresh supernatant (after centrifugation following chitosan in the extraction process) was obtained and its pH was raised to 7.0 using NaOH. The starting optical density (「OD」) was measured by Pierce assay (Pierce™ 660nm Protein Assay Reagent; Thermo Fisher Scientific, Waltham, Massachusetts), and absorbance at 474nm was measured (Shimadzu PharmaSpec UV-1700; Shimadzu Scientific Instruments Incorporated, Columbia, Maryland). 0.3% w / v activated carbon (Cabot Norit Americas Inc, Marshall, Texas) was added to 100 mL of the sample. The mixture was stirred for 1 minute and passed through a 0.45μm filter (polyethersulfone (PES) membrane filter, 0.45 micron; Sterlitech Corporation Inc, Kent, Washington) followed by a 0.2μm filter (polyethersulfone (PES) membrane filter, 0.2 micron; Sterlitech Corporation Inc, Kent, Washington) using a Buchner funnel, and the filtrate was collected in a flask. Samples were collected for optical density measurement. To a further 100 mL of sample, the amounts (% w / v) of resin shown in Table 8 were added, the mixture was stirred for the times shown in Table 8 for each sample, and then the mixture was poured into a Buchner setup. The optical density was measured for each sample.

[0240]

[0241]

Table 7

[0241] The embodiments maintained the highest possible Pierce assay readings and determined the binding times and concentrations at which the orange OD was lowest while correlating with protein concentration. This was done by graphing the maximum separation between the Pierce ratio line and the 474 ratio line. The experimental group with the greatest separation was 20% at 10 minutes, and 83% of the protein was retained by the Pierce assay, with the orange color reduced to 33%.

[0242] Example 13 This example examined the effectiveness of a resin (Purolite MN200; Purolite Corporation, Kings of Prussia, Pennsylvania) in removing colored compounds compared to activated carbon.

[0243] Fresh supernatant (after centrifugation following chitosan in the extraction process) was obtained and its pH was raised to 7.0 with NaOH. The starting optical density ("OD") was measured by Pierce assay (Pierce™ 660nm Protein Assay Reagent; Thermo Fisher Scientific, Waltham, Massachusetts), and the absorbance at 474 nm was measured (Shimadzu PharmaSpec UV-1700; Shimadzu Scientific Instruments Incorporated, Columbia, Maryland). 0.3% activated carbon (Cabot Norit Americas Inc, Marshall, Texas) was added to 100 mL of the sample. The mixture was stirred for 1 minute and filtered through a 0.45 μm filter (polyethersulfone (PES) membrane filter, 0.45 micron; Sterlitech Corporation) ​​​​​​​​​​orporation Inc, Kent, Washington) and then 0.2 μm filter (polyethersulfone (PES) membrane filter, 0.2 micron; S terlitech Corporation Inc, Kent, Washingto n) and the filtrate was collected in a flask. Samples were collected for optical density measurement. To an additional 100 mL sample, the amounts of resin shown in Table 9 were added and the mixture for each sample was stirred for the time shown in Table 8 and then the mixture was poured into a Buchner setup. The optical density was measured for each sample (Pierce at 710 nm; Abs at 474 nm). (710 nm for Pierce; 474 nm for Abs).

[0244]

Table 8

[0245] Without wishing to be bound by theory, the large gap between the Pierce OD ratio and the orange OD ratio seen in the 1% activated carbon, 1-minute group may be an error as this was not observed in previous tests. Without wishing to be bound by theory, these results suggest that activated carbon is more efficient than the resins examined in removing color, but activated carbon may also remove more nitrogen compounds based on the Pierce assay. Without wishing to be bound by theory, these results suggest that activated carbon is more efficient than the resins examined in removing color, but activated carbon may also remove more nitrogen compounds based on the Pierce assay. These results suggest that activated carbon is more efficient than the resins examined in removing color, but activated carbon may also remove more nitrogen compounds based on the Pierce assay. These results suggest that activated carbon is more efficient than the resins examined in removing color, but activated carbon may also remove more nitrogen compounds based on the Pierce assay.

[0246] Example 14 The final plant protein powder was diluted with deionized water to a concentration of 10 mg / mL. Gel filtration column (Superdex 200; GE Healthcare Bio-Scien ces Corp, Westborough, Massachusetts) was used for high Samples were analyzed using fast protein liquid chromatography (FPLC). Subsequently, a molecular weight standard (Bio-Rad Laboratories, INC, Hercules, CA) was run to approximate the molecular sizes of the individual proteins and protein complexes in the sample of interest. Figure 16 shows the chromatograms of the final protein product and the protein standard. Analysis of the protein peaks in the chromatograms indicated that the protein of interest (RuBisCo) eluted from the column at a molecular weight close to, but below, 670 kDa as measured with the molecular weight protein standard.

[0247] Example 15 This example visualized the purity and complexity of Lemna plant proteins using SDS-PAGE electrophoresis and Coomassie staining. A small sample of the plant protein extract of the final purified protein product was analyzed on a 4-15% SDS-PAGE gel (Bio-Rad Laboratories, INC, Hercules, CA). Visualization of the proteins was performed by staining the proteins blue on the gel using Coomassie dye. Figure 17 shows the SDS-PAGE gel. Without wishing to be bound by theory, these results suggest that the final protein product consists mainly of the Rubisco enzyme and that the individual larger and smaller subunits of Rubisco can be readily detected by SDS-PAGE Coomassie staining under denaturing and reducing conditions.

[0248] Example 16 This example examined the removal of other light-absorbing molecules such as those characterized and quantified by chlorophyll, polyphenols, and spectroscopic analysis. Samples from each step of the purification process were ​​​​Characterized by a spectrophotometer (Shimadzu PharmaSpec UV-1700 ;Shimadzu Scientific Instruments Incorpo rated, Columbia, Maryland) through a process. The sample was scanned from 110 0 nm to 245 nm. Figure 18 shows the absorbance spectrum. The peak of fraction 1 ( "F1") corresponds to the signal detected from the filtrate after the first liquid / solid separation step. The peak of fraction 4 ("F4") corresponds to the sample taken after 0.2 μm precision filtration. Without wishing to be bound by theory, the absorbance peaks detected from the scanning spectrophotometer are thought to indicate the efficient removal of light-absorbing molecules throughout the process. (Item 1) A process for preparing a protein standard purified from plant material, comprising: a) providing the plant material in a buffer solution containing a reducing agent; b) dissolving the plant material; c) separating the dissolved plant material into a solid phase and a liquid phase, wherein the liquid phase contains soluble protein and chlorophyll; d) coagulating the chlorophyll in the liquid phase by heating to a first set temperature within about 30 minutes and then cooling to a second set temperature within about 30 minutes, (Item 2) A process for preparing a protein preparation purified from plant material, comprising: a) providing the plant material in a buffer solution containing a reducing agent; b) dissolving the plant material; c) separating the dissolved plant material into a solid phase and a liquid phase, wherein the liquid phase contains soluble protein and chlorophyll; d) coagulating the chlorophyll in the liquid phase by adding one or more salts; e) contacting the liquid phase of d) with a flocculant and / or an adsorbent, mixing for a time sufficient to flocculate the chlorophyll in the liquid phase and / or adsorb it to the adsorbent, thereby forming a flocculated mixture; f) separating the flocculated mixture of e) into a solid phase and a liquid phase; g) filtering the liquid phase of f) to obtain a filtrate containing the purified protein. (Item 3) A process for preparing a protein preparation purified from plant material, comprising: a) providing the plant material in a buffer solution containing a reducing agent; b) dissolving the plant material; c) separating the dissolved plant material into a solid phase and a liquid phase, wherein the liquid phase contains soluble protein and chlorophyll; d) coagulating the chlorophyll in the liquid phase using a coagulant derived from a polymer; e) contacting the liquid phase of d) with a flocculant and / or an adsorbent, mixing for a time sufficient to flocculate the chlorophyll in the liquid phase and / or adsorb it to the adsorbent, thereby forming a flocculated mixture; f) separating the flocculated mixture of e) into a solid phase and a liquid phase; g) filtering the liquid phase of f) to obtain a filtrate containing the purified protein. (Item 4) A process for preparing a protein preparation purified from plant material, comprising: a) Providing the plant material in a buffer solution containing a reducing agent; b) Dissolving the plant material; c) Separating the dissolved plant material into a solid phase and a liquid phase, wherein the liquid phase contains soluble proteins and chlorophyll; d) Coagulating the chlorophyll in the liquid phase by electrocoagulation; e) Contacting the liquid phase of d) with a flocculant and / or an adsorbent and mixing for a time sufficient to flocculate the chlorophyll in the liquid phase and / or adsorb it onto the adsorbent, thereby forming a flocculated mixture; f) Separating the flocculated mixture of e) into a solid phase and a liquid phase; g) Filtering the liquid phase of f) to obtain a filtrate containing purified protein, the process. (Item 5) The process according to any one of Items 1 to 4, wherein the plant material is washed before a). (Item 6) The reducing agent is 2-mercaptoethanol (BME), 2-mercaptoethylamine-HCL, sodium metabisulfite, cysteine hydrochloride, dithiothreitol (DTT), glutathione, cysteine, tris(2-carboxyethyl)phosphine (TCEP), ferrous ions, nascent hydrogen, sodium amalgam, oxalic acid, formic acid, magnesium, manganese, phosphoric acid, potassium or sodium, the process according to any one of Items 1 to 4. (Item 7) The process according to any one of Items 1 to 4, wherein the reducing agent is a sulfite. (Item 8) The process according to Item 7, wherein the sulfite is sodium sulfite, magnesium sulfite or sodium metabisulfite. (Item 9) The process according to Item 7, wherein the sulfite is sodium bisulfite. (Item 10) The process according to any one of Items 1 to 4, wherein the solution of a) contains one or more buffers. (Item 11) The process according to any one of items 1 to 4, wherein the solution of a) contains one or more chelating agents. (Item 12) The process according to any one of items 1 to 4, wherein the solution of a) contains one or more protease inhibitors. (Item 13) The process according to any one of items 1 to 4, wherein the solution of a) contains one or more buffers, one or more chelating agents, and / or one or more protease inhibitors. (Item 14) The process according to any one of items 1 to 4, wherein the pH of the solution of a) is from about pH 5.0 to about pH 9.0. (Item 15) The process according to item 14, wherein the pH of the solution is from about pH 6.0 to about pH 7.6. (Item 16) The process according to item 15, wherein the pH of the solution is about pH 6.8. (Item 17) The process according to any one of items 1 to 4, wherein the ratio of the plant material to the solution of a) is about 6:1. (Item 18) The process according to any one of items 1 to 4, wherein the ratio of the plant material to the solution of a) is about 3:1. (Item 19) The process according to any one of items 1 to 4, wherein the ratio of the plant material to the solution of a) is about 2:1. (Item 20) The process according to any one of items 1 to 4, wherein the ratio of the plant material to the solution of a) is about 1:1. (Item 21) The process according to any one of items 1 to 4, wherein the dissolution of the plant material includes adding one or more divalent ions to the lysate and / or filtrate and / or adding chitosan to the lysate and / or filtrate. (Item 22) The process according to any one of items 1 to 4, wherein the dissolution of the plant material includes adding calcium ions to the lysate. (Item 23) The process according to any one of items 1 to 4, wherein said dissolution of the plant material comprises adding calcium chloride to the lysate. (Item 24) The process according to any one of items 1 to 4, wherein the plant material is dissolved chemically, mechanically, and / or enzymatically. (Item 25) The process according to any one of items 1 to 4, wherein the plant material is dissolved chemically. (Item 26) The process according to any one of items 1 to 4, wherein the plant material is dissolved chemically using one or more surfactants. (Item 27) The process according to any one of items 1 to 4, wherein the plant material is dissolved chemically using CHAPS. (Item 28) The process according to any one of items 1 to 4, wherein the plant material is dissolved enzymatically using one or more enzymes. (Item 29) The process according to any one of items 1 to 4, wherein the plant material is dissolved using cellulase or pectinase. (Item 30) The process according to any one of items 1 to 4, wherein the plant material is dissolved mechanically. (Item 31) The process according to any one of items 1 to 4, wherein the plant material is dissolved mechanically using a blender. (Item 32) The process according to any one of items 1 to 4, wherein the plant material is dissolved mechanically using a mill, a homogenizer, a microfluidizer, mechanical pressure, or a Stephan cutter. (Item 33) The process according to any one of items 1 to 4, wherein the plant material is dissolved mechanically using a press, an ultrasonic disruptor, a grinder, using a pulsed electric field, using nitrogen jetting, using ultrasonic energy, or by freezing. (Item 34) The process according to any one of items 1 to 4, wherein the plant material is mechanically lysed using at least one mill. (Item 35) The process according to any one of items 1 to 4, wherein the plant material is mechanically lysed using at least two different types of mills. (Item 36) The process according to any one of items 1 to 4, wherein the separating in c) is carried out by a screw press, a decanter, or a centrifuge. (Item 37) The process according to any one of items 1 to 4, wherein the separating in c) is carried out using a disk stack centrifuge, a continuous centrifuge, or a basket centrifuge. (Item 38) The process according to any one of items 1 to 4, wherein the separating in c) is carried out using filtration. (Item 39) The process according to any one of items 1 to 4, wherein the separating in c) is carried out using a press. (Item 40) The process according to any one of items 1 to 4, wherein the separating in c) is carried out using filtration. (Item 41) The process according to any one of items 1 to 4, wherein the separating in c) is carried out using gravitational sedimentation. (Item 42) The process according to any one of items 1 to 4, wherein the separating in c) is carried out using sieving. (Item 43) The process according to item 1, wherein the first set temperature in d) is about 80 °C or less. (Item 44) The process according to item 1, wherein the first set temperature in d) is about 65 °C or less. (Item 45) The process according to item 1, wherein the first set temperature in d) is about 55 °C or less. (Item 46) The process according to item 1, wherein the first set temperature in d) is about 50 °C or less. (Item 47) The process according to item 1, wherein the second set temperature in d) is about 25 °C or lower. (Item 48) The process according to item 1, wherein the second set temperature in d) is about 15 °C or lower. (Item 49) The process according to item 1, wherein the second set temperature in d) is about 10 °C or lower. (Item 50) The process according to item 1, wherein heating to the first set temperature in d) takes about 15 minutes or more. (Item 51) The process according to item 1, wherein heating to the first set temperature in d) takes about 5 minutes or more. (Item 52) The process according to item 1, wherein cooling to the second set temperature in d) takes about 15 minutes or more. (Item 53) The process according to item 1, wherein cooling to the second set temperature in d) takes about 5 minutes or more. (Item 54) The process according to item 2, wherein the one or more salts in d) include 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. (Item 55) The process according to item 2, wherein the one or more salts in d) include potassium phosphate and / or calcium chloride. (Item 56) The process according to item 2, wherein the one or more salts in d) are added at a concentration of 5 mM to 2 M. (Item 57) The process according to any one of items 1 to 4, wherein the flocculant is alkylamine epichlorohydrin, polydimethyldiallylammonium chloride, polysaccharide, polyamine, starch, aluminum sulfate, alum, polyacrylamide, polyacylamide, or polyethyleneimine. (Item 58) The process according to any one of items 1 to 4, wherein the flocculant is chitosan. (Item 59) The process according to any one of items 1 to 4, wherein the flocculant is activated chitosan. (Item 60) The process according to any one of items 1 to 4, wherein the flocculant is 1 to 20% w / w activated chitosan in solution. (Item 61) The process according to any one of items 1 to 4, wherein the adsorbent in e) is a resin. (Item 62) The process according to any one of items 1 to 4, wherein the adsorbent in e) is activated carbon, activated charcoal or activated fossil charcoal. (Item 63) The process according to any one of items 1 to 4, wherein the adsorbent in e) is activated carbon having a surface area exceeding 250 m / g, a weight average diameter of 1 to 1000 μm, an iodine value of 400 to 1,400 mg / g, a Molasses number in the range of 100 to 550, and / or a methylene blue adsorption of at least 10 g / 100 g. (Item 64) The process according to any one of items 1 to 4, wherein the separation in f) is carried out at 25°C or lower. (Item 65) The process according to any one of items 1 to 4, wherein the separation in f) is carried out at 15°C or lower. (Item 66) The process according to any one of items 1 to 4, wherein the separation in f) is carried out at 10°C or lower. (Item 67) The process according to any one of items 1 to 4, wherein the separation in f) is carried out using filtration. (Item 68) The process according to any one of items 1 to 4, wherein the separation in f) is carried out using a press, using gravitational sedimentation, or by sieving. (Item 69) The process according to any one of items 1 to 4, wherein the separation in f) is carried out using a centrifuge or a decanter or by microfiltration. (Item 70) The process according to any one of items 1 to 4, wherein all steps of the process except e) are carried out at 25°C or lower. (Item 71) The process according to any one of items 1 to 4, wherein all steps of the process except e) are carried out at 15°C or lower. (Item 72) The process according to any one of items 1 to 4, wherein all steps of the process except e) are carried out at 10°C or lower. (Item 73) The process according to any one of items 1 to 4, wherein the filtering in g) is carried out by a membrane filter. (Item 74) The process according to any one of items 1 to 4, wherein the filtering in g) is carried out by a 0.7 μm membrane filter. (Item 75) The process according to any one of items 1 to 4, wherein the filtering in g) is carried out by a 0.2 μm membrane filter. (Item 76) The process according to any one of items 1 to 4, wherein the filtering in g) is carried out by diatomaceous earth and / or activated carbon. (Item 77) The process according to any one of items 1 to 4, wherein the filtering in g) is carried out by activated carbon up to about 10% at most. (Item 78) The process according to any one of items 1 to 4, wherein the filtering in g) is carried out by activated carbon up to about 2% at most. (Item 79) The process according to any one of items 1 to 4, wherein the filtering in g) is carried out by a 0.2 μm membrane filter and about 2% of activated carbon. (Item 80) Furthermore, the process according to any one of items 73 to 79, comprising filtering the filtrate of g) through a 0.2 μm membrane filter after g). (Item 81) The process according to any one of items 1 to 4, wherein one or more liquid phases and / or one or more filtrates contain one or more defoaming agents and / or one or more foam suppressants. (Item 82) The process according to any one of items 1 to 4, wherein one or more liquid phases and / or one or more filtrates are filtered to remove small solids and / or microorganisms. (Item 83) The process according to any one of items 1 to 4, wherein one or more liquid phases and / or one or more filtrates are sterilized. (Item 84) Furthermore, the process according to any one of items 1 to 4, comprising concentrating the filtrate. (Item 85) The process according to item 84, wherein concentrating the filtrate is carried out by ultrafiltration. (Item 86) The process according to item 85, wherein the ultrafiltration is through polyethersulfone, polypropylene, polyvinylidene fluoride, polyacrylonitrile, cellulose acetate or polysulfone. (Item 87) The process according to item 85, wherein the ultrafiltration is carried out using an ultrafiltration filter with a cut-off of 100 kDa or less. (Item 88) The process according to item 85, wherein the ultrafiltration is carried out using an ultrafiltration filter with a cut-off of 50 kDa or less. (Item 89) The process according to item 85, wherein the ultrafiltration is carried out using an ultrafiltration filter with a cut-off of 10 kDa or less. (Item 90) The process according to any one of items 1 to 4, wherein the yield of the purified protein is at least about 10% of the soluble protein in the liquid phase of step c). (Item 91) The process according to any one of items 1 to 4, wherein the yield of the purified protein is at least about 20% of the soluble protein in the liquid phase in step c). (Item 92) The process according to any one of items 1 to 4, wherein the yield of the purified protein is at least about 25% of the soluble protein in the liquid phase in step c). (Item 93) The process according to any one of items 1 to 4, wherein the yield of the purified protein is at least about 40%. (Item 94) The process according to any one of items 1 to 4, wherein the yield of the purified protein is at least about 60%. (Item 95) The process according to any one of items 1 to 4, wherein the yield of the purified protein is at least about 80%. (Item 96) The weight ratio of chlorophyll to protein in the purified protein standard is about 1:1 000, about 1:1500, about 1:2000 or less than about 1:2500, of any one of items 1 to 95 The process according to any one of the above items. (Item 97) One or more agents in the purified protein standard that confer or are related to one or more functional properties are reduced or removed compared to the original plant material, according to any one of items 1 to 96. (Item 98) One or more agents in the purified protein standard that confer or are related to one or more functional properties are reduced 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% compared to the original plant material, according to any one of items 1 to 96. (Item 99) The process according to any one of items 1 to 96, wherein the purified protein standard is essentially odorless. (Item 100) The process according to any one of items 1 to 96, wherein the purified protein standard is odorless. (Item 101) The process according to any one of items 1 to 100, wherein the purified protein standard has an essentially unbiased taste. (Item 102) The process according to any one of items 1 to 100, wherein the purified protein standard has an unbiased taste. (Item 103) The process according to any one of items 1 to 102, wherein the protein is RuBisCo. (Item 104) The process according to any one of items 1 to 103, wherein the plant material is derived from Lemna. (Item 105) The process according to any one of items 1 to 103, wherein the plant material is derived from Lemnoideae. (Item 106) A product produced by the process according to any one of items 1 to 105. (Item 107) A food containing a purified protein standard derived from a plant material, wherein the protein standard contains 80% or less impurities. (Item 108) The food according to item 107, wherein the protein standard contains RuBisCo. (Item 109) The food according to item 107 or 108, wherein the plant material is derived from Lemna. (Item 110) The food according to item 107 or 108, wherein the plant material is derived from Lemnoideae.

Claims

[Claim 1] The invention described in this specification.

Images