Method for extraction and purification of non-denatured proteins

Abstract

The present application relates to methods of extracting and purifying non-denatured proteins. Materials and methods for extracting and purifying proteins are provided. For example, the materials and methods provided herein can be used to extract and purify proteins that denature at low temperatures.

Description

[0001] This application is a divisional application of the invention patent application filed on October 1, 2015, with application number 201580061566.8 and invention title "Method for Extracting and Purifying Undenatured Proteins". Cross-citation of related applications

[0002] This application claims priority to U.S. Provisional Application No. 62 / 060,400, filed October 6, 2014, and U.S. Provisional Application No. 62 / 058,211, filed October 1, 2014, both of which are incorporated herein by reference in their entirety. Technical Field

[0003] This article relates to materials and methods for the extraction and purification of proteins, and more specifically to materials and methods for the extraction and purification of proteins denatured at low temperatures. Background Technology

[0004] Low-temperature denatured proteins in their undenatured state are crucial for the success of food simulation products (such as cheese or meat simulation products, like beef simulation products). Existing commercial protein extraction processes involve unit operations and conditions that degrade proteins and are not suitable for manufacturing products containing these proteins. Furthermore, most proteins possess associated colors and odors, which may negatively impact their utility in food simulation products. Summary of the Invention

[0005] This article is based, at least in part, on the development of processes for extracting and purifying proteins from plant materials, such that the proteins retain their undenatured state without their associated color and odor. For example, this article is based, at least in part, on the unexpected discovery that hydrophilic polymers, and specifically polyethylene glycol (PEG), can adsorb colored and odorous compounds from protein solutions.

[0006] On one hand, this document provides a method for purifying proteins from biomass. The method may include extracting biomass with an aqueous solution containing PEG and optionally a flocculant to produce an extract slurry containing bulk solids and extract; optionally adjusting the pH of the extract slurry to pH 2 to 10; collecting the extract and adding salt to form a two-phase mixture; separating the two-phase mixture to produce a PEG phase and a product phase; and collecting and filtering the product phase to produce a filtered product phase containing the protein. The biomass may include plant material (e.g., flowers or leaves). Plants may be algae, corn, wheat, rice, sorghum, rye, canola, millet, barley, soybean, sunflower, safflower, alfalfa, potato, brassica, cotton, tomato, or tobacco. The protein may be ribulose-1,5-bisphosphate carboxylase. The molecular weight of PEG may be about 8000. The flocculant may include alkylamine epichlorohydrin. The salt may include magnesium sulfate. The separation step may include gravity sedimentation or centrifugation (e.g., using a disc stack centrifuge). Filtration may include microfiltration. The method may further include concentrating and permeating the filtered product phase to produce a product concentrate. Distillation may include using an ultrafiltration membrane system. The method may further include sterilizing the product concentrate to obtain a sterilized product concentrate. Sterilization may include UV irradiation, pasteurization, or microfiltration. The method may further include drying the sterilized product concentrate. Drying may include using a spray dryer or a freeze dryer under mild conditions.

[0007] On the other hand, this article provides a method for removing impurities from a protein solution. The method may include adding a hydrophilic polymer and a salt to the protein solution to produce a polymer-enriched phase and a protein phase, and separating the polymer-enriched phase from the protein phase. The hydrophilic polymer may be PEG. The molecular weight of PEG may be about 8000. The method may further include adding a flocculant to the protein solution. The flocculant may contain alkylamine epichlorohydrin. The salt may include magnesium sulfate. The method may further include adjusting the pH of the protein solution to pH 2 to 10. The protein may be ribulose diphosphate carboxylase. Separation may include gravity sedimentation or centrifugation (e.g., using a disc stack centrifuge). The method may further include filtering the protein phase to produce a filtered product phase (e.g., by microfiltration). The method may further include concentrating and perfiltration of the filtered product phase to produce a product concentrate. Perfiltration may include using an ultrafiltration membrane system. The method may further include sterilizing the product concentrate to obtain a sterilized product concentrate (e.g., by UV irradiation, pasteurization, or microfiltration). The method may further include drying the sterilized product concentrate (e.g., using a spray dryer or freeze dryer under mild conditions).

[0008] This document also provides a composition comprising ribulose diphosphate carboxylase and a hydrophilic polymer, wherein the hydrophilic polymer is present at a concentration of less than about 0.1% (w / w). The hydrophilic polymer may be PEG. The hydrophilic polymer may be present at a concentration of less than about 0.01% (w / w).

[0009] On the other hand, this document provides a method for purifying proteins, wherein the method includes providing a protein suspension from which solids have been removed; optionally adding salt to the protein suspension; optionally adjusting the pH of the protein suspension to pH 2 to 10; dialyzing the protein suspension against a PEG solution, or ultrafiltration the protein suspension with PEG on the permeate side of a membrane; and subjecting the dialyzed or ultrafiltered protein solution to one or more concentration or filtration steps to produce a product phase containing proteins. The protein suspension may contain one or more proteins derived from plant material. Plant material may include flowers or leaves. Plants may be algae, corn, wheat, rice, sorghum, rye, canola, millet, barley, soybean, sunflower, safflower, alfalfa, potato, brassica, cotton, tomato, or tobacco. The protein may be ribulose-1,5-bisphosphate carboxylase. The molecular weight of PEG may be about 8000. The steps may include extracting decomposed biomass to remove solids and produce a protein suspension, optionally adding a flocculant to the biomass. The flocculant may include alkylamine epichlorohydrin. Extraction may include using gravity sedimentation or centrifugation to separate solids from the protein suspension. Salt may include magnesium sulfate. One or more filtration steps may include dialysis. The method may include concentrating and filtering the dialysis or ultrafiltration-treated protein solution to produce a product concentrate. The method may further include sterilizing the product concentrate to obtain a sterilized product concentrate, and / or drying the sterilized product concentrate (e.g., using a spray dryer or freeze dryer under mild conditions).

[0010] On the other hand, this article provides a method for purifying proteins, comprising providing a protein suspension from which solids have been removed; optionally adding salt to the protein suspension; optionally adjusting the pH of the protein suspension to pH 2 to 10; applying the protein suspension to a support containing PEG; and collecting the protein phase not retained on the support. The protein suspension may contain one or more proteins derived from plant material. Plant material may include flowers or leaves. Plants may be algae, corn, wheat, rice, sorghum, rye, mustard, millet, barley, soybean, sunflower, safflower, alfalfa, potato, brassica, cotton, tomato, or tobacco. The protein may be ribulose-1,5-bisphosphate carboxylase. The molecular weight of PEG may be about 8000. The steps may include extracting decomposed biomass to remove solids and produce a protein suspension, optionally adding a flocculant to the biomass. The flocculant may include alkylamine epichlorohydrin. Extraction may include separating the solids from the protein suspension using gravity sedimentation or centrifugation. The salt may include magnesium sulfate. The method may include incubating a protein suspension with a PEG-containing support for 24 to 48 hours. The method may further include concentrating the protein phase to produce a product concentrate, and / or sterilizing the product concentrate to obtain a sterilized product concentrate, and / or drying the sterilized product concentrate (e.g., using a spray dryer or freeze dryer under mild conditions).

[0011] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although the invention may be practiced with similar or equivalent methods and materials to those described herein, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated herein by reference in their entirety. In case of conflict, this specification (including definitions) shall prevail. Furthermore, materials, methods, and examples are illustrative only and are not intended to be restrictive.

[0012] Details of one or more embodiments of the invention are set forth in the accompanying drawings and the following description. Other features, objectives, and advantages of the invention will become apparent from the detailed description, the drawings, and the claims. Attached Figure Description

[0013] Figure 1 A flowchart illustrating the steps of a general embodiment of the extraction and purification process provided herein.

[0014] Figure 2A A flowchart illustrating the steps of one embodiment of the extraction and purification process provided herein. Figure 2B To depict Figure 2A The flowchart shows an alternative embodiment of the method described herein.

[0015] Figure 3 A flowchart illustrating the steps of another embodiment of the extraction and purification process provided herein.

[0016] Figure 4 A flowchart illustrating the steps of one embodiment of a pH-based purification process.

[0017] Figure 5 A flowchart illustrating the steps of one embodiment of a chromatographic purification process.

[0018] Figure 6 This is a flowchart illustrating the steps of one embodiment of an expanded bed chromatography purification process.

[0019] Figure 7 A flowchart illustrating the steps of one embodiment of a purification process utilizing a membrane.

[0020] Figure 8 A flowchart illustrating the steps of one embodiment of a purification process utilizing immobilized PEG.

[0021] Figure 9 A flowchart illustrating the steps of one embodiment of the PEG recycling method.

[0022] Figure 10 A flowchart illustrating the steps of another embodiment of the extraction and purification process provided herein indicates any useful steps that may be added. Detailed Implementation

[0023] Low-temperature denaturation of proteins in their undenatured state is crucial for the success of food simulation products, such as beef simulation products. However, existing commercial protein extraction processes can lead to the denaturation of these proteins. Additionally, most proteins that may be functional in food simulation products have associated colors and odors, which may reduce or inhibit their application. For example, ribulose-1,5-bisphosphate carboxylase (ribulose-1,5-bisphosphate carboxylase oxygenase) represents approximately 30 to 50% of the soluble proteins in plant chloroplasts; its green color and grassy odor may inhibit its use in food simulation products. Ribulose-1,5-bisphosphate carboxylase also denatures at low temperatures (50°C to 60°C). Elsewhere reported methods for extracting ribulose diphosphate carboxylase include, for example, heat-based precipitation at 50 to 60°C, extraction with high concentrations of reducing agents, temperature-based precipitation of non-ribulose diphosphate carboxylase proteins, carbon adsorption, crystallization-based purification, and / or extraction at pH 11 followed by protein precipitation at pH 4.5 and large-scale washing with organic solvents (see, for example, U.S. Patent Nos. 3,959,246, 4,006,078, 4,334,024, and 4,588,691; PCT Publication No. WO 2011 / 078671; Lamsal et al., Proceedings of the American Society of Agricultural Engineers). Trans. ASAE ) 46(3):715-720, 2003); and Yang et al., Journal of Agricultural and Food Chemistry ( J. Agric. Food Chem. ) 52:2223-2225, 2004).

[0024] The materials and methods presented herein can be used to extract and purify sufficient quantities of cryogenically denatured proteins (such as ribulose-1,2-bisphosphate carboxylase) with suitable characteristics for use in food-simulated products. These methods can provide high-purity products in as few as one step, and in some cases, without the use of chromatography. The methods also offer the possibility of scale-up using conventional extraction equipment from the chemical industry, with low capital expenditure and improved manufacturing recoveries.

[0025] The methods described herein may include the use of hydrophilic polymers, such as polyethylene glycol (PEG). For example, one method may include steps of dispersing and extracting chromopeptides with PEG, followed by a two-phase separation step containing water. In some embodiments, the volume concentration of the PEG phase may be from 2% (w / v) to 50% (w / v) PEG (e.g., 2-10% (w / v), 10-25% (w / v), or 25-50% (w / v)), potentially salting out proteins. The affinity of chromopeptides for PEG may cause them to separate from the remainder of the mixture. The molecular weight of PEG is in the range of about 300 to about 300,000 (e.g., 300, 400, 500, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10,000, 20,000, 30,000, 40,000, 50,000, 60,000, 70,000, 80,000, 90,000, 100,000, 200,000 or 300,000, about 300 to about 3000, about 3000 to about 10,000, about 10,000 to about 30,000, about 30,000 to about 100,000, or about 100,000 to about 300,000). In some cases, for example, the molecular weight of PEG can be around 8000.

[0026] In some embodiments, biomass (e.g., flowers, leaves, or other plant material) may be ground using a grinder and / or a high-shear mixer, followed by water extraction with a flocculant containing PEG (e.g., PEG with a molecular weight of 8000) and optionally one or more flocculants containing alkylamine epichlorohydrin, polydimethyldiallylammonium chloride, or polyamines (e.g., MAGNAFLOC®, SUPERFLOC®, or TRAMFLOC®) to form an extract slurry. One or more flocculants can help reduce the amount of fine solids remaining in the centrifuged liquid and slightly reduce the color of the centrifuged liquid. The presence of PEG can increase protein solubility and can increase yield; PEG has also been observed to reduce the amount of fine solids remaining in the extract.

[0027] The slurry can then be decanted (e.g., using a decanting centrifuge) to separate the bulk solids from the extract. The extract can be collected in a stirred tank reactor, where salt can be added to form a two-phase mixture. The salt can cause the PEG to form a discontinuous phase and separate from the solution. Suitable salts include, for example, salts of metals (e.g., sodium, potassium, calcium, magnesium, zinc, iron, cobalt, or aluminum) and counterions (e.g., chloride, bromide, sulfate, nitrate, cyanide, citrate, carbonate, acetate, or phosphate). In some embodiments, the salt is NaCl or MgSO4. Mixtures of salts can also be used. The salt concentration can be from about 100 mM to about 2 M (e.g., 100 to 200 mM, 200 to 500 mM, 500 to 750 mM, 750 mM to 1 M, or 1 to 2 M).

[0028] Colored compounds and reaction products (e.g., enzymatic, oxidative, or browning reaction products) composed of small molecules such as chlorophyll, carotenoids, and flavonoids can be retained in the PEG phase by volume rejection, while proteins remain in the saline solution. Odor compounds (e.g., grassy or green odor compounds) can also be retained in the PEG phase by volume rejection. The two-phase mixture can then be separated into the PEG phase and the product phase (e.g., by gravity sedimentation or using a centrifuge, such as a disc stack centrifuge). The recovered PEG layer can be returned to the extraction tank for subsequent extraction until it is saturated with the colored component. At this point, the PEG phase can be heat-treated to separate pure PEG from the colored components in solution. It should be noted that in some embodiments, PEG (and optionally a flocculant) can be added to the phase separation step after the solids separation step.

[0029] The product phase separated from the two-phase mixture can be microfiltered (e.g., via a tangential flow filtration system or a one-pass empty-end microfiltration system) to remove any residual fine solids and microorganisms. The filtered product phase can be concentrated using an ultrafiltration membrane system or a combination of chromatography and ultrafiltration and then permeated with water to produce a product concentrate. This step allows for the separation of residual PEG and salts from the filtered product phase. The product concentrate can also undergo microbial reduction steps, which may include, for example, pasteurization (e.g., high-temperature short-time pasteurization or autoclaving), UV irradiation, or gamma irradiation. In some cases, non-thermal methods may be applicable. The product concentrate (sterilized or non-sterilized) can be dried under mild conditions using a spray dryer or freeze dryer, ensuring that the protein does not denature, producing a decolorized and deodorized pure protein. It should be noted that the product may contain low levels of PEG (e.g., less than about 1% (w / w), 0.1% (w / w), 0.01% (w / w), about 0.001% (w / w), about 0.0001% (w / w), or about 0.00001% (w / w)).

[0030] In some embodiments, the hydrophilic polymer (e.g., PEG) does not necessarily need to be in physical contact with the protein suspension, but can be separated from the protein suspension through a permeable membrane with a sufficiently small pore size to prevent polymer or target protein transfer. For example, PEG and the protein suspension can be separated through a membrane with a pore size of about 3 kDa to about 500 kDa (e.g., 3 to 5 kDa, 5 to 10 kDa, 10 to 30 kDa, 30 to 50 kDa, 50 to 100 kDa, or 100 to 500 kDa). The concentration of the PEG phase can be 2-50% (w / v) PEG (e.g., 2-10%, 10-25%, or 25-50%). The protein suspension may contain one or more salts to promote the transfer of colored and odorous compounds. In some embodiments, the pH of the protein solution can also be adjusted (e.g., adjusted to a pH between 2 and 10) to promote the transfer of colored and odorous compounds. As described above, protein suspensions may include salts, such as (but not limited to) salts of metals (e.g., sodium, potassium, calcium, magnesium, zinc, iron, cobalt, or aluminum) and counterions (e.g., chloride, bromide, sulfate, nitrate, cyanide, citrate, carbonate, acetate, or phosphate) (e.g., NaCl or MgSO4), or mixtures of salts. Similarly, salt concentrations can range from about 100 mM to about 2 M. The affinity of chromophores for PEG may cause them to separate from the rest of the mixture. As described above, the molecular weight of PEG can range from about 300 Da to about 300,000 Da (e.g., about 8,000 Da). Using PEG to indirectly remove chromophores and off-flavor compounds is particularly suitable for proteins that may precipitate at low PEG concentrations (thus making subsequent phase separation problematic). For example, PEG can be used across membranes to remove off-flavors from (but not limited to) soybean 7S and pea albumin proteins.

[0031] Using a membrane to maintain the separation of the protein suspension from the PEG phase eliminates the need for a phase separation step. The protein and PEG suspensions can be contacted with the permeation membrane using various methods, such as dialysis, backflow filtration, ultrafiltration, microfiltration, or nanofiltration. The membrane can be made of any of a variety of materials, including polymers such as polyethersulfone, polypropylene, polyvinylidene fluoride, polyacrylonitrile, cellulose acetate, and polysulfone. In some embodiments, the membrane can be incorporated into a physical matrix, such as ceramic or steel.

[0032] In some embodiments involving the use of membranes, biomass (e.g., flowers, leaves, or other plant material) may be ground using a grinder and / or a high-shear mixer, followed by extraction with water containing one or more flocculants (e.g., MAGNAFLOC®, SUPERFLOC®, or TRAMFLOC®) to form an extract slurry. The flocculant helps reduce the amount of fine solids remaining in the centrifuged liquid and can also reduce the color of the centrifuged liquid. The extract slurry can then be decanted using a decanter centrifuge or passed through a spiral pressure unit to separate bulk solids from the centrifuged liquid. The centrifuged liquid can be microfiltered via a tangential flow filtration system or a one-time pass through an empty-end microfiltration system to remove any residual fine solids and microorganisms. The microfiltration permeate can be collected in a tank and salt can be added to achieve specific conductivity. This solution can then be permeated with an 8% PEG solution using a UF membrane. Once the product is decolorized, PEG permeation can be stopped, and the resulting material can be concentrated and permeated with water to obtain a low-salt concentrated decolorized protein (e.g., ribulose-1,5-bisphosphate carboxylase). In some embodiments, the sample may be centrifuged to remove solids. The product concentrate may also undergo microbial reduction steps, which may include, for example, pasteurization (e.g., high-temperature short-time pasteurization or autoclaving), UV irradiation, or gamma irradiation. In some cases, non-thermal methods may be applicable. The product concentrate can then be dried under mild conditions using a spray dryer, freeze dryer, or similar methods to ensure that the protein does not denature, thus producing pure protein that is decolorized and deodorized.

[0033] In some embodiments, the hydrophilic polymer may be immobilized before contact with the protein suspension. Similarly, biomass (e.g., flowers, leaves, or other plant material) may be milled using a grinder and / or a high-shear mixer, and optionally one or more flocculants containing alkylamine epichlorohydrin, polydimethyldiallylammonium chloride, or polyamines (e.g., MAGNAFLOC®, SUPERFLOC®, or TRAMFLOC®) may be added to form an extraction slurry, and the suspension may optionally be clarified, for example by decantation, sedimentation, or centrifugation, to remove solids. The protein suspension may then be exposed to the immobilized hydrophilic polymer (e.g., PEG). In some embodiments, the immobilized PEG may be in resin form. The resin may be constructed from a solid core having a PEG molecular coating. The solid core may contain any of a variety of materials, such as SEPHAROSE®, agarose, polycarbonate, hydroxyapatite, glass, metal, charcoal, silica, alumina, ceramics, polypropylene, polystyrene, or divinylbenzene. As described above, the molecular weight of PEG can range from about 300 Da to about 300,000 Da (e.g., about 8,000 Da). In some embodiments, PEG can be immobilized by crosslinking to form a resin, such as NovaPEG (EMD Millipore; Billerica, MA). In other embodiments, PEG can be immobilized on a membrane. For the schemes described above, the protein suspension may contain one or more salts to promote chromosome transfer (e.g., salts present in the range of 100 mM to 2 M, including salts of metals (e.g., sodium, potassium, calcium, magnesium, zinc, iron, cobalt, or aluminum) and counterions (e.g., chloride, bromide, sulfate, nitrate, cyanide, citrate, carbonate, acetate, or phosphate).

[0034] In some embodiments of the method utilizing a fixed hydrophilic polymer (e.g., PEG), the protein suspension can be exposed to the polymer on the resin by adding the resin to the protein suspension. After a sufficient period of time (e.g., 1 minute to 48 hours, e.g., 1 to 10 minutes, 10 to 30 minutes, 30 to 60 minutes, 60 minutes to 2 hours, 2 to 4 hours, 4 to 6 hours, 6 to 12 hours, 12 to 24 hours, or 24 to 48 hours) at a temperature sufficient to allow association between the chromopeptides and PEG, the protein can be separated from the fixed PEG by a variety of methods, including gravity filtration, vacuum or centrifugal filtration, sedimentation and decantation, or centrifugation and separation of the centrifuged liquid. The fixed PEG may optionally be washed to remove any entrained proteins.

[0035] In some embodiments, immobilized PEG can be used as a chromatographic resin. The protein suspension can pass through the resin bed under conditions where chromosomes bind to the resin but the target protein flows through the bed and is collected. Similarly, the protein phase may contain one or more salts to promote chromosome binding.

[0036] Decolorized and deodorized protein suspensions can be further processed to produce decolorized and deodorized protein powders. For example, decolorized proteins can be concentrated and permeated. In some embodiments, the decolorized protein suspension can be centrifuged to remove solids. The concentrate can also undergo a microbial reduction step. As mentioned above, such procedures can include pasteurization (e.g., high-temperature short-time pasteurization or autoclaving), UV irradiation or gamma irradiation, or non-thermal methods. The product concentrate can then be dried under mild conditions (e.g., using a spray dryer, freeze dryer, etc.) to ensure that the protein does not denature, producing decolorized and deodorized pure protein.

[0037] Now let's look at the diagram. Figure 1 This is a flowchart depicting the general steps in one embodiment of the method provided herein. Generally, extraction step 110 (e.g., using decomposed / grinded / homogenized biomass and buffer) may be followed by solid separation step 120 (e.g., by decantation and addition of flocculant, and optionally PEG), color removal step 130 (e.g., by addition of PEG and salt, and optionally flocculant, followed in some embodiments by chromatography, pH precipitation and resolubilization), sterilization step 140 (e.g., by microfiltration, pasteurization or UV irradiation), and concentration step 150 (e.g., by ultrafiltration or membrane evaporation, followed by drying).

[0038] Figure 2 is a flowchart depicting the steps of an exemplary embodiment of the extraction and purification method provided herein. The protein-enriched solid undergoes a decomposition step 210, followed by extraction 220 using an aqueous solution containing PEG and optionally one or more flocculants. The solid is separated in step 230. Salt is added to the extract (e.g., via recycling step 265), and a phase separation step 240 separates the PEG from the protein product phase. The PEG is recycled in step 245 for future extraction steps. The protein product phase undergoes a filtration step 250, followed by a concentration step 260, during which salt is removed and recycled via step 265 for future phase separation steps. The concentrated protein product undergoes a sterilization step 270 and a drying step 280 to produce a decolorized, deodorized, non-denatured protein product. Examples of the use of this method are provided in Example 1 below.

[0039] Figure 2B It is a display Figure 2AA flowchart of the steps in an alternative embodiment of the method depicted is provided. In this embodiment, PEG and optionally a flocculant are added to phase separation step 240, and the PEG is recycled to a further phase separation step.

[0040] Figure 3 This is a flowchart depicting the steps of another embodiment of the extraction and purification process provided herein, wherein PEG is added after the initial extraction step. The protein-enriched solid undergoes a decomposition step 310, followed by an extraction step 320 using an aqueous solution optionally containing one or more flocculants. The solid is separated in step 330, and the extract is filtered and concentrated in steps 340 and 350, respectively. PEG and salt are added to produce a multiphase mixture. The phases are separated in step 360. The PEG is recycled in step 365 for future separation steps. The protein product phase is concentrated in step 370, during which salt is removed and recycled in step 375 for future phase separation and / or concentration steps. The concentrated protein product undergoes a sterilization step 380 and a drying step 390 to produce decolorized and deodorized non-denatured protein.

[0041] Figure 4 This is a flowchart depicting the steps of one embodiment of a pH-based purification process. The protein-enriched solid undergoes a decomposition step 410, followed by an extraction step 420 using an added extraction buffer (e.g., an aqueous solution optionally containing a flocculant). The solid is separated in step 430, and the remaining solution is filtered in step 440 and concentrated in step 450. A dilute acid is mixed with the concentrate in step 460, and the protein is separated in step 470. A dilute alkali is mixed with the protein in step 475, followed by a sterilization step 480 and a drying step 490 for the protein product. Examples of the use of this method are provided in Example 3 below.

[0042] Figure 5 A flowchart illustrating the steps of one embodiment of the chromatographic purification process is provided. The protein-enriched solid undergoes a decomposition step 510, followed by an extraction step 520 using an extraction buffer (e.g., an aqueous solution optionally containing a flocculant). The solid is separated in step 530, and the remaining solution is filtered in step 540 and concentrated in step 550. In step 560, the concentrate is applied to a chromatographic column (e.g., a gel filtration column) and non-protein and unbound material is eluted. Proteins are eluted and then concentrated in step 570, followed by sterilization in step 580 and drying in step 590. Examples of the use of this method are provided in Example 4 below.

[0043] Figure 6This is a flowchart illustrating the steps of one embodiment of an expanded bed chromatography purification method. The protein-enriched solid undergoes a decomposition step 610, followed by an extraction step 620 using an added extraction buffer (e.g., an aqueous solution optionally containing a flocculant). The solid is separated in step 630. In step 640, the remaining solution is applied to a chromatographic column (e.g., an ion-exchange column, or a system based on hydrophobic interactions or pure adsorption, such as activated carbon), and non-protein and unbound material is eluted. The eluted protein is then concentrated in step 660, followed by a sterilization step 670 and a drying step 680.

[0044] Figure 7 This is a flowchart illustrating the steps of one embodiment of a method for separating a protein suspension from a PEG solution using a membrane. Protein-enriched solids undergo a decomposition step 710, followed by an extraction step 720 using water and, optionally, a flocculant. Solids are removed in step 730, and microfiltration step 740 is used to remove fine residual solids and / or microorganisms. In step 750, the filtrate is permeated across the PEG membrane; this may include steps such as dialysis, backflow filtration, ultrafiltration, microfiltration, or nanofiltration. The protein solution is then concentrated in step 760, salts are removed by perfiltration in step 770, and sterilization and drying steps are performed in steps 780 and 790.

[0045] Figure 8 This is a flowchart illustrating the steps of one embodiment of a method for removing colorants and odorous compounds using immobilized PEG. The protein enrichment solid undergoes a decomposition step 810, followed by an extraction step 820 using water and, optionally, a flocculant. In step 830, the solid is removed, and the protein suspension is added to immobilized PEG (e.g., in a chromatographic column or as a batch chromatography step) and incubated in step 840. In step 850, the protein solution is concentrated, followed by a sterilization step 860 and a drying step 870.

[0046] Figure 9 This is a flowchart depicting the steps of one embodiment of the PEG recycling method. After the addition of PEG and phase separation (e.g., depicted in Figures 2 and 3 and described above), the PEG in the PEG layer can be resuspended in step 910, followed by a carbon adsorption step 920. PEG may optionally be sterilized in step 930 (e.g., by microfiltration, pasteurization, or UV irradiation).

[0047] Figure 10A flowchart illustrating the steps of one embodiment of the extraction and purification method provided herein includes additional useful steps. The extracted solids and solvent from the separation step (e.g., in a PEG-based method as described herein) are subjected to an extraction step 1010, followed by a separation step 1020. In step 1030, the solvent is evaporated to obtain compounds such as carotenoids, chlorophyll, flavonoids, and prolysin. The fiber-rich waste solids are pelleted in step 1040 and used, for example, in products such as pet food, or subjected to an enzymatic digestion step 1050, followed by a fermentation step 1060 and a solids separation step 1070. The fermented waste solids are removed. The solvent is evaporated in step 1080 to obtain unfermented soluble compounds, as well as other materials that can be used, for example, as biofuels.

[0048] The present invention will be further described in the following examples, which do not limit the scope of the invention as described in the claims.

[0049] Example

[0050] Example 1 - Isolation of spinach ribulose diphosphate carboxylase

[0051] One kilogram of fresh spinach leaves was softened in a VITA-PREP® 3 blender (Vitamix Corp., Cleveland, OH) at a 1:1 (w / w) ratio with potassium phosphate buffer (pH 7.4) containing 8% (w / v) PEG (Carbowax Sentry PEG 8000; Dow Chemicals, Midland, MI) and 0.1% (w / v) cationic flocculant (863A; Tramfloc, Inc., Houston, TX). Extraction was performed for 3 minutes at the highest setting (3HP motor), always maintaining the temperature below 30°C. After grinding, the pH was adjusted to 7.4 using 10 M NaOH solution. The homogenate was centrifuged at 3500 g for 5 minutes using a benchtop centrifuge (Allegra X15R, SX4750 rotor; Beckman Coulter, Inc., Pasadena, CA). The centrifuge block was discarded, and the supernatant layer (approximately 1.6 L) was collected separately. Magnesium sulfate heptahydrate (K+SKALI GmbH, Kassel, Germany) was added to the supernatant layer to achieve a 1 M concentration. The solution was thoroughly mixed and centrifuged at 5451 g for 3 minutes using a benchtop centrifuge (Allegra X15R, SX4750 rotor; Beckman Coulter). Three layers formed in the centrifuge flask, and the remaining green solids were separated as a centrifuge block (approximately 0.1 L). The PEG layer (approximately 0.3 L) was separated, and the top layer was formed, with selective fractionation of the colored and odorous compounds. The remaining clarified protein product in the intermediate layer was then microfiltered using a hollow fiber type 0.2 µm modified polyethersulfone (mPES) membrane (KROSFLO® K02E20U-05N; Spectrum Laboratories, Inc., Rancho Dominguez, CA). The retention (approximately 0.25 L) was permeated with approximately 0.75 L of 1 M magnesium sulfate solution. The permeate from this filtration step (approximately 3 L) was concentrated to approximately 0.1 L using a 70 kDa mPES membrane (MiniKros N02E070-05N; Spectrum Laboratories, Inc.). This was further permeated with approximately 0.5 L of deionized water in 5 steps. The protein concentrate had a pH of approximately 7 and a conductivity of less than 5 mS / cm. The resulting protein concentrate was a clear, pale yellow color.The product was dried using a spray dryer or a freeze dryer. The material was analyzed using a standard 660 nm Pierce protein assay and SDS gel density determination. The dried solids were analyzed using an IR humidity analyzer. The flocculant and PEG concentrations in the final product were analyzed using titration. The protein concentration was approximately 91% (w / w), and the total solids were approximately 95% (w / w). PEG and flocculant concentrations were analyzed to be less than 0.2% (w / w). The product purity exceeded 90%, and the recovery rate throughout the process exceeded 90%. The obtained product was decolorized and retained its low-temperature denaturing properties.

[0052] Example 2 - Isolation of alfalfa ribulose diphosphate carboxylase

[0053] Two kiloliters of extraction buffer (pH 7.4, potassium phosphate) were prepared in a jacketed stirred tank containing 8% (w / v) PEG (Carbowax Sentry PEG8000; Dow Chemicals) and 0.1% (w / v) cationic flocculant (863A; Dumford). Five hundred kilograms of fresh alfalfa leaves were softened in a Corenco M12DA grinder (Corenco, Santa Rosa, CA), with the extraction buffer continuously recirculated during grinding to improve decomposition and the process temperature maintained below 40°C throughout. After grinding, the pH was adjusted to 7.4 using 10 M NaOH solution. The homogenate was centrifuged at 3500 g using a GEA Westphalian decanter GCE-345 (GEA Mechanical Equipment, New Jersey, NJ). Discard the centrifuge pellet. Add approximately 625 kg of magnesium sulfate (K+S KALI Ltd.) to the liquid centrifuge buffer (approximately 2200 L). Thoroughly mix the solution and centrifuge using a GEA Westphalia ESD-30 separator (GEA Mechanical Equipment) at a feed rate of approximately 5 gpm. The green solid is discharged as pellets, with a loss of approximately 10% (v / v) of the feed. Separate the PEG layer (approximately 20% v / v of the feed) and form the top layer, selectively fractionating colored and odorous compounds. The remaining clarified protein in the intermediate layer is then microfiltered using a 0.2 μm modified mPES membrane (KROSFLO® K02E20U-05N; Spectroscopy Laboratory Ltd.) in the form of hollow fibers. The retention (approximately 200 L) is permeated using approximately 400 L of 1 M magnesium sulfate solution. The permeate from this filtration step was concentrated to approximately 50 L using a 70 kDa mPES membrane (KROSFLO® KM-070E-300-01N; Spectroscopy Laboratory). It was then further permeated with approximately 250 L of deionized water in five steps. The protein concentrate had a pH of approximately 7 and a conductivity of less than 5 mS / cm. The resulting protein concentrate was a clear, pale yellow color. The product was dried using a spray dryer or freeze-dried using a freeze dryer. The material was analyzed using standard 660 nm Pierce protein assay and SDS gel density determination. The dried solids were analyzed using an IR humidity analyzer. The protein concentration was approximately 880 g / L, and the total solids were approximately 95% (w / w). The PEG and flocculant concentrations were analyzed to be less than 0.2% (w / w). The product purity exceeded 90%, and the recovery rate throughout the process exceeded 90%. The obtained product was decolorized and retained its low-temperature denaturing properties.

[0054] Example 3 - Isolation using pH-based purified spinach ribulose diphosphate carboxylase

[0055] One kilogram of fresh spinach leaves was softened in a Vitamix folding machine at a 1:1 ratio with potassium phosphate buffer (pH 7.4) containing 0.1 M NaCl. After grinding, the pH was adjusted to 7.4 using 10 M NaOH solution. The homogenate was centrifuged at 3500 g for 5 minutes. Solid pellets were discarded, and the liquid centrifuge buffer (approximately 1.6 L) was then microfiltered using a 0.2 μm mPES membrane. The retentate was permeated using approximately 1.5 L of extraction buffer. The permeate from this filtration step (approximately 3 L) was concentrated to approximately 0.1 L using a 10 kDamPES membrane. The protein concentrate from this step was approximately pH 7.4. An acid (e.g., HCl) was added to the concentrate to lower the pH to 5. The concentrate mixture was vigorously stirred for 30 minutes using a magnetic stir plate or homogenizer. This mixture was then centrifuged at 3500 g for 5 minutes to obtain off-white pellets and a brown centrifuge buffer. The supernatant was discarded, and the protein pellets were washed with deionized water. The pellets were resuspended in 0.05–0.1 L of deionized water. The solution was vigorously mixed to form a homogeneous slurry, and the pH was slowly raised to 11 using an alkali (e.g., NaOH). The resulting solution was a clear yellow color. The pH was then lowered to 9 to maintain the clarity of the mixture. The product was dried in this form using a spray dryer or by freeze-drying using a freeze dryer. The resulting product was slightly decolorized and retained its low-temperature denaturing properties.

[0056] Example 4 - Separation of spinach ribulose diphosphate carboxylase using chromatography

[0057] The concentrate prepared in Example 3 above was purified by size exclusion chromatography. A Superdex 200 column (26 / 600) was pre-equilibrated with 20 mM KPhos pH 7.4 and 100 mM NaCl. 0.4 g of protein (5 ml concentrate) was injected onto the column at 2.5 ml / min. The column was then eluted with 20 mM KPhos pH 7.4 and 100 mM NaCl buffer. High-resolution separation was observed between the aggregated protein, purified ribulose-1,2-bisphosphate carboxylase, colored components (pink, yellow), and salt in the interstitial volume.

[0058] Example 5 - Decolorization of spinach by ribulose-1,5-bisphosphate carboxylase via filtration

[0059] One hundred grams of fresh spinach leaves were softened in deionized water at a 1:4 (w / w) ratio in a VITA-PREP® 3 blender (Vitamix, Cleveland, Ohio). Extraction was performed for 3 minutes at the highest setting (3HP motor), maintaining the temperature below 30°C throughout. After grinding, the pH was adjusted to 7.4 using 10 M NaOH. Homogenization was performed using a benchtop centrifuge (Allegra X15R, SX4750 rotor; Beckman Coulter, Pasadena, CA) at 10,000 g for 30 minutes. The centrifuge pellets were discarded, and the supernatant was collected separately. Sodium chloride was added to the supernatant to achieve a concentration of 0.75 M. The solution was then subjected to dialysis using a 3 kDa UF membrane against an 8% PEG (Carbowax Sentry PEG 8000; Dow Chemical, Midland, Michigan) solution.

[0060] Color, along with some proteins and water, permeated through the membrane into the PEG phase. The product at the end of dialysis was 2-fold concentrated and colorless. This material was analyzed using standard 660 nm Pierce protein assay and SDS gel density determination.

[0061] Example 6 - Decolorization of spinach ribulose diphosphate carboxylase by batch chromatography

[0062] One hundred grams of fresh spinach leaves were softened in deionized water at a 1:4 (w / w) ratio in a VITA-PREP® 3 blender (Vitamix, Cleveland, Ohio). Extraction was performed for 3 minutes at the highest setting (3HP motor), with the temperature maintained below 30°C throughout. After grinding, the pH was adjusted to 7.4 using 10 M NaOH solution. Homogenization was performed using a benchtop centrifuge (Allegra X15R, SX4750 rotor; Beckman Coulter, Pasadena, California) at 10,000 g for 30 minutes. The centrifuge block was discarded, and the supernatant layer was collected separately. 20 mg of dried NovaPEG Wang resin (EMD Milipore, catalog number 855122) was added to each milliliter of suspension. The material was then tumbled to ensure agitation for 45 hours, followed by centrifugation at 10,000 × g for 5 minutes. The supernatant layer was separated, and UV-VIS analysis showed a significant decrease in absorbance at 320 nm. The protein content and composition of the clear layer were analyzed by standard 660 nm Pierce protein analysis and SDS gel density determination, showing no significant changes compared to the initial protein suspension.

[0063] Example 7 - Removing off-odors from soybean protein through PEG-targeted filtration

[0064] A 3% (w / v) soybean with soy globulin solution was adjusted to pH 8.5 using 2 N NaOH. The sample was then diluted to 0.75 M sodium chloride and dialyzed using a 3500 Da cutoff membrane against a 5% PEG 8000 solution (pH adjusted to 8.5) containing 0.75 M sodium chloride. After dialysis against the PEG salt, the protein sample was dialyzed against water to remove excess salt. The taste of the sample was evaluated after a 20-fold reduction in salt concentration. Tasters described the final sample as bitter, but with a complete removal of the cardboard and earthy odors. In contrast, tasters described the untreated soybean with soy globulin sample as bitter with a strong cardboard and earthy flavor.

[0065] Example 8 - Changes in Methods

[0066] The following are other variations of the method described above:

[0067] Method Change

[0068] ● An alternative is to add PEG after the decantation step, thereby minimizing the loss of PEG solids from the decantation. In this case, the decantation liquid is added to a pre-formed two-phase PEG-salt solution to fractionate the chromophores in the PEG phase.

[0069] ● Another option is to add solid PEG to the centrifuged liquid and add salt to form a phase separation when mixing is complete.

[0070] ● An alternative to the method described above is to utilize a two-phase extraction strategy instead of an extract of the UF concentrate. In this case, the entire process is carried out without the addition of PEG or magnesium sulfate until the UF concentrate is produced.

[0071] ● Another option is to centrifuge the PEG-treated solution before pasteurization or drying to remove any precipitate.

[0072] Unit operation changes

[0073] ● Use a homogenizer or high-shear grinder to enhance decomposition and cell disruption.

[0074] ● The centrifugation step for separating the three layers of disk stacks has been replaced with a step using a gravity settling tank.

[0075] ● Use a desalting column (e.g., G50 column) for salt separation.

[0076] Component changes

[0077] ● The method described was tested using spinach and alfalfa leaves, but can be extended to any ribulose diphosphate carboxylase source (e.g., algae, corn, wheat, rice, sorghum, rye, canola, millet, barley, soybean, sunflower, safflower, alfalfa, potato, brassica, cotton, tomato, or tobacco) or other proteins (e.g., other plant proteins).

[0078] ● The method can be used to remove color and / or odor from any protein suspension, including suspensions containing one or more proteins selected from the group consisting of (non-limiting) the following: leghemoglobin, non-symbiotic hemoglobin, hemoglobin, myoglobin, hemochloroglobin, invertebrate hemoglobin, neuroglobin, cytoglobin, protoglobin, truncated 2 / 2 globin, HbN, cyanobacterial globin, HbO, Glb3, and cytochromes, Hell's gateglobin I. I) Bacterial hemoglobin, ciliated myoglobin, flavin hemoglobin, ribosomal protein, actin, hexokinase, lactate dehydrogenase, fructose-1,5-bisphosphate aldolase, phosphofructokinase, triose phosphate isomerase, phosphoglycerate kinase, phosphoglycerate mutase, enolase, pyruvate kinase, protease, lipase, amylase, glycoprotein, lectin, mucin, glyceraldehyde-3-phosphate dehydrogenase, pyruvate decarboxylase, actin, translation elongation factor, histone, ribulose-1,5-bisphosphate carboxylase, ribulose-1,5-bisphosphate carboxylase oxygenase activator (ribulose-1,5-bisphosphate carboxylase activator), albumin, glycine, conglycine, globulin, pea globulin, conalbumin, gliadin, gluten White protein, glutenin, wheat glutenin, barley gliadin, gliadin, bean protein (protein), protein body, rye gliadin, extensin, wheat glutenin, collagen, corn protein, sorghum gliadin, oat protein, dehydrated protein, hydrophilic protein, late embryo-enriched protein, natural unfolded protein, any seed storage protein, tuber storage protein, potato glycoprotein, tuber-specific protein, protease inhibitor, animal protein, fish protein, egg protein, poultry protein, algal protein, fungal protein, recombinant protein, oil body protein, oil body calcium protein, oil body sterol protein or other oil body protein, nutrient storage protein A, nutrient storage protein B, mung bean seed storage 8S globulin, pea globulin, and pea albumin.

[0079] ● Use higher molecular weight PEG to further reduce the PEG concentration in the process.

[0080] ● Other hydrophilic polymers (such as polypropylene glycol, butanediol, hexanediol, glycerol, diglycerol, diethylene glycol, dipropylene glycol and mixtures thereof) are also potentially applicable.

[0081] ● Use a flocculant that provides 0.05–1 vol% cationic flocculant functionality in the extraction buffer (e.g., SUPERFLOC® 781G or MAGNAFLOC® LT 7989 from BASF, Florham Park, NJ). Other suitable flocculants include, for example, limestone, slaked lime, and salts of divalent or trivalent metals (e.g., ferric chloride, ferric sulfate, ferrous sulfate), aluminum sulfate, sodium aluminate, aluminum chloride, basic magnesium carbonate, calcium carbonate, calcium hydroxide, activated silicates, guar gum, starch, tannins, sodium alginate, polyaluminum sulfate, polyaluminum hydroxide, BIO-FLOCK®, and synthetic polyelectrolytes (e.g., ZETAG®, MAGNAFLOC®, and SUPERFLOC®).

[0082] ● Salts used for phase disruption are not limited to Epsom salt. Other suitable salts include potassium phosphate, sodium chloride, and ammonium acetate. Separation is better if ions are appropriately selected based on the Hoffmiester series; later salts in this series can saltify proteins.

[0083] ● The extraction method is improved by adding a reducing agent (e.g., metabisulfite (about 2% (w / v) solution or more)) to the initial extraction buffer and maintaining anaerobic conditions by the method described above.

[0084] Method efficiency

[0085] ● The separated PEG layer is recycled into another extraction buffer formulation, thereby maximizing the efficiency of the PEG used.

[0086] ● Permeate from UF membrane separation is concentrated using RO membrane to recover process water and salt concentrate.

[0087] Other embodiments

[0088] It should be understood that although the invention has been described in conjunction with its detailed description above, the description is intended to be illustrative and not to limit the scope of the invention, which is defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the appended claims.

Claims

1. A composition comprising ribulose diphosphate carboxylase and a hydrophilic polymer, wherein the hydrophilic polymer is present at a concentration of less than about 0.1% (w / w), and wherein the hydrophilic polymer comprises polyethylene glycol (PEG), butanediol, hexanediol, glycerol, diglycerol, diethylene glycol, dipropylene glycol, or a mixture thereof.

2. The composition of claim 1, wherein the hydrophilic polymer is present at a concentration of less than about 0.01% (w / w).

3. The composition according to claim 1, wherein the ribulose diphosphate carboxylase is derived from corn, wheat, rice, sorghum, rye, rapeseed, millet, barley, soybean, sunflower, safflower, tobacco, alfalfa, potato, brassica, cotton, tomato, or tobacco.

4. The composition according to claim 1, wherein the ribulose diphosphate carboxylase is derived from algae.

5. The composition according to claim 1, wherein the hydrophilic polymer is PEG with a molecular weight of about 8000.

6. The composition of claim 1, wherein the composition comprises a protein solution that has been concentrated, optionally sterilized, and optionally dried by dialysis or ultrafiltration.

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