Tunable solvents and methods of use

Abstract

A two-phase natural deep eutectic solvent (NADES) generally includes a hydrophilic phase that has at least one hydrophilic component and a hydrophobic phase that has at least one hydrophobic component. In one or more embodiments, the two-phase NADES includes hydrophilic components and hydrophobic components present in a mass ratio of up to 100:1.

Description

[0001] TUNABLE SOLVENTS AND METHODS OF USE

[0002] CROSS-REFERENCE TO RELATED APPLICATION

[0003] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 650,156, filed May 21 , 2024, which is incorporated herein by reference in its entirety.

[0004] SUMMARY

[0005] This disclosure describes, in one aspect, a two-phase natural deep eutectic solvent (NADES). Generally, the two-phase NADES includes a hydrophilic phase that has at least one hydrophilic component and a hydrophobic phase that has at least one hydrophobic component. In one or more embodiments, the two-phase NADES includes hydrophilic components and hydrophobic components present in a mass ratio of up to 100: 1.

[0006] In one or more embodiments, the hydrophilic phase includes choline chloride, glycerol, or both.

[0007] In one or more embodiments, the hydrophobic phase includes thymol, menthol, or both.

[0008] In another aspect, this disclosure describes a method of extracting protein, oil, or both, from a plant-based matrix. Generally, the method includes adding a sample of full fat flour from the plant-based matrix to a two-phase natural deep eutectic solvent (NADES), extracting the sample, centrifuging the sample, collecting supernatant and subjecting the supernatant to phase separation conditions to produce a protein rich phase and a lipid rich phase and collecting the protein rich and lipid rich phase.

[0009] In one or more embodiments, the centrifugation step and the phase separation step may be replaced with a single step involving the use of a three-phase decanter.

[0010] In one or more embodiments, the extracting step includes agitating the sample at 100 rpm to 400 rpm at a temperature ranging from 20 °C to 35 °C.

[0011] In one or more embodiments, centrifuging the sample includes centrifuging the sample at 8000 rpm to 12,000 rpm for 15 to 60 minutes at room temperature.

[0012] In one or more embodiments, the phase separation conditions include incubating the supernatant for one to 16 hours at a temperature ranging from 4 °C to 25 °C. In one or more embodiments, collecting the protein rich phase includes dialysis or membrane fdtration.

[0013] In one or more embodiments, the two-phase NADES has a pH from 6.0 to 9.5.

[0014] In one or more embodiments, the plant-based matrix is not subjected to a defatting step.

[0015] In one or more embodiments, the method lacks a desolventization step.

[0016] The above summary is not intended to describe each disclosed embodiment or every implementation of the present invention. The description that follows more particularly exemplifies illustrative embodiments. In several places throughout the application, guidance is provided through lists of examples, which examples can be used in various combinations. In each instance, the recited list serves only as a representative group and should not be interpreted as an exclusive list.

[0017] BRIEF DESCRIPTION OF THE FIGURES

[0018] FIG. 1. Hemp protein extraction yield and protein composition (protein purity, lipid content and ash) for Trials 1 to 3, with different ratios of hydrophilic:hydrophobic NADES components (100:0, 100:5, and 100: 15, (w / w)) and control sample from hexane defatted sample. (A) Extraction yield. (B) Protein Composition. Extraction conditions were as follows: 1 :20 solids to liquid ratio (5 g of full-fat hemp flour in 100 g of hydrophilic NADES + hydrophobic NADES (Trial 1: 0 g; Trial 2: 5 g; Trial 3: 15 g), pH 7.0, 25 °C, and one-hour extraction time.

[0019] FIG. 2. Chickpea protein extraction yield and protein composition (protein purity and lipid content) for Trials 1 to 3, with different ratios of hydrophilic: hydrophobic NADES (100:0, 100:5, and 100:10 (w / w)) and control sample from a defatted material. (A) Extraction yield. (B) Protein Composition. Extraction conditions were as follows: 1 :20 solids to liquid ratio (5 g of full-fat chickpea flour in 100 g of hydrophilic NADES + hydrophobic NADES (Trial 1 : 0 g;

[0020] Trial 2: 5 g; Trial 3: 15 g), pH 7.5, 25 °C and one-hour extraction time.

[0021] FIG. 3. Protein solubility of the control and two-phase NADES-assisted hemp protein isolate under heated (H) and non-heated (NH) and in acidic and neutral conditions. Control samples were extracted from a defatted hemp flour and alkaline extraction at pH 8.0 followed by isoelectric precipitation.

[0022] FIG. 4. Off notes detected in hemp protein isolates produced using two-phase NADES assisted extraction, reference (control) sample produced using defatted hemp flour and extraction at under the same pH used for the NADES-assisted extraction followed by isoelectric precipitation. (A) Aldehydes; (B) Alcohols; (C) Ketones; (D) Terpenes.

[0023] FIG. 5. Characterization of hemp protein extraction. (A) yield (B) protein purity of enzyme-assisted, control, and reference samples at pH 7, pH 8, pH 9, pH 10, and pH 11 for hemp defatted flour. Control (*, middle): sample heat treated without enzyme along with alkaline extraction and isoelectric precipitation. Reference (A, right): Alkaline extracted protein.

[0024] FIG. 6. Conventional lipid extraction strategies. (A) Solvent-based method. (B) Mechanical pressing method.

[0025] FIG. 7. Natural Deep Eutectic Solvents.

[0026] FIG. 8. Natural Deep Eutectic Solvents (NADES). Model hydrogen bond acceptors and model hydrogen bond donors that may be combined to form NADES.

[0027] FIG. 9. Protein yield and purity of hemp protein from a defatted flour and alkaline extraction at pH 7, pH 8, pH 9, pH 10, and pH 11.

[0028] DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

[0029] This disclosure describes compositions and methods that involve two-phase natural deep eutectic solvents (NADES) for concurrent extraction of oil and protein from oil-bearing materials. The use of a two-phase NADES for simultanous extraction of oil and protein has not been previously reported. The extraction of oil and protein simultaneously using NADES allows the production of sustainable and highly functional food ingredients with improved functional properties and allows one to do so without the use of flammable solvents.

[0030] The development and popularity of plant-based protein diets have spurred research efforts to extract dietary proteins from many plant sources as alternatives to animal protein. The rapid growth of the plant protein market has spurred additional interest in developing and improving processing technologies to promote sustainable production of functional and nutritious proteins. However, widespread adoption of plant-based proteins is hindered by their limitations in functional behavior and flavor within food systems. Therefore, the food industry is actively seeking effective strategies to enhance the functionality and flavor of pea and chickpea proteins for successful incorporation in various applications.

[0031] Plant protein production requires upstream processing, such as dehulling, milling, and defatting in the case of oil-bearing materials. Protein extraction from oil bearing materials requires a lipid removal step with organic solvents, such as hexane (FIG. 6). Even when mechanical pressing is used for oil extraction, there is a need to use solvent to further defat the meal generated prior to protein production. Solvent defatting is efficient and cost-effective, however, concerns regarding its health and environmental impact, and consumer perception have led to the exploration of alternative processing strategies for producing plant proteins. On the other hand, a common commercial process to produce plant protein isolates (> 80% protein) uses harsh alkaline conditions to extract the protein. This approach results in protein denaturation and aggregation, contributing to detrimental effects on the functional behavior in food systems and can generate off flavors. Therefore, there is a strong need to develop novel, sustainable, and green alternative extraction processes that reduce (and even eliminate) the use of flammable solvents and / or preserve or enhance the functional and flavor properties of the protein.

[0032] While described herein in the context of exemplary embodiments in which oil and protein are co-extracted from hemp and chickpeas as model matrix materials, the compositions and methods described herein can involve extraction of oil and protein from any suitable matrix material source (FIG. 7). Exemplary alternative matrix material sources include, but are not limited to oats, soybean, camelina, pennycress, canola, peanut, almond, and other oil-bearing materials. This strategy could also be applied to matrix materials including, but not limited to, pea, lentil, and bean to extract protein while removing residual lipids that can oxidize during processing and generate off flavors, therefore improving the flavor of the extracted protein.

[0033] Natural deep eutectic solvents (NADES) are bio-based deep eutectic solvents that include two or more compounds that are generally plant-based primary metabolites (FIG. 8). NADES can be derived from binary or ternary eutectic mixtures that include at least one hydrogen bond donor (HBD) and at least one hydrogen bod acceptor (HBA). Exemplary hydrogen bond donors include, but are not limited to, an organic acid, a sugar, an amino acid, or combinations of two or more members of any of the foregoing. Exemplary hydrogen bond acceptors include, but are not limited to, a non-toxic quaternary ammonium salt, an amino acid, or any combination of two or more members of any of the foregoing. Thus, NADES are made of nontoxic components that are recognized as safe for food applications. This diversity allows for numerous combinations of distinct polarity in NADES production, offering great potential for their application in various extraction processes Natural deep eutectic solvents (NADES) have recently drawn attention as innovative and versatile green solvents due to their tunability, eco-friendliness, and biodegradability. Since NADES can be prepared from a wide range of organic and inorganic compounds, NADES possess several advantageous properties including, but not limited to, low vapor pressure, thermal stability, adjustable viscosity, and excellent miscibility. NADES-assisted extraction is effective in extracting proteins and bioactive compounds from different sources. Importantly, current examples using NADES to extract protein are limited to the use of hydrophilic-NADES and defatted raw materials. Thus, NADES have never been applied to full-fat materials such as oilseeds. Further, NADES have not been used to simultaneously extract oil and protein to produce ingredients for food applications.

[0034] This disclosure describes a NADES-assisted extraction method that can be applied as a solvent in a two-phase system, allowing co-extraction of protein and oil from oil-bearing materials. The methods described herein therefore allow one to eliminate a defatting step that typically requires using one or more flammable solvents. Aqueous and enzyme-assisted extractions have been explored for the simultaneous extraction of oil and protein from several matrices, including almond and chickpea. However, such processes have been challenged by their low extraction yields, requiring harsh alkaline conditions to extract the protein, and by the formation of an emulsion phase, which needs to be broken down to allow oil recovery.

[0035] The two-phase NADES system described herein overcomes those issues. In a bench-scale study using hemp full-fat flour, the use of two-phase NADES was effective in achieving high protein extraction yields (75-83%) and high protein purities (88-94%) (FIG. 1A), values significantly higher than the ones obtained by alkaline extraction using a defatted material (protein yield of 8% at pH 8 with protein purity of 76%) (FIG 9). Moreover, the extracted hemp protein isolates had low residual lipid (0.9-2.9%), and low ash content (~1%) (FIG. IB). When applying the same approach to chickpea full-fat flour, lower extraction yields from 38-46% were achieved (FIG. 2A), with protein purity ranging from 74-84%, and residual lipid content ranging from 1.5-6.4% (FIG. 2B). The differences in extractability and purity between hemp and chickpea proteins suggest that the NADES system can be tuned for each source matrix. The use of NADES can produce protein with improved functionality — e.g., increased solubility for NADES-extracted hemp protein compared to control protein extracted from defatted material (FIG. 3). The use of NADES can not only produce protein with improved functionality but also produce protein with a better flavor profile. FIG. 4 shows that NADES -extracted protein contains fewer compounds responsible for off notes: aldehydes, alcohols, ketones, and terpenes.

[0036] The two-phase NADES extraction process promotes the co-extraction of oil and protein and does not require the use of flammable solvents, enhancing the sustainable aspects of the process. Another potential advantage of the two-phase NADES co-extraction of oil and protein is the potential reduction in energy usage since upstream defatting processing steps, which include pressing and solvent extraction, are not required. Since the two-phase NADES co-extraction process does not require organic solvents, its use can eliminate the desolventization step that is typically required to remove an organic solvent from the final product. The desolventization step involves a thermal treatment, which can be detrimental to protein quality in the residual defatted meal. Developing processes that reduce (or even eliminate) the need for harsh conditions will contribute to high-quality ingredients.

[0037] Solvent Composition

[0038] The NADES system can include any combination of suitable NADES components. Generally, the NADES system includes at least one hydrogen bond acceptor and at least one hydrogen bond donor. Suitable hydrogen bond acceptors include, but are not limited to, an ammonium salt (e.g., trimethylammonium chloride, tetrabutylammonium chloride) choline chloride, betaine (also referred to as trimethylglycine) or a salt thereof (e.g., betaine hydrochloride), certain amino acids (e.g., glycine, alanine, proline, etc ), a fatty acid, nicotinic acid, a terpene, or any combination of two or more hydrogen bond acceptors. Suitable hydrogen bond donors include, but are not limited to, a sugar (e.g., sucrose, maltose, galactose, lactose, glucose, fructose, mannitol, mannose, sorbitol, etc.), an alcohol, (e.g., glycerol, ethylene glycol, propanediol, etc.), urea or a derivative thereof (e.g., dimethylurea), a carboxylic acid (e.g., oxalic acid, ascorbic acid, malic acid, benzoic acid, aconitric acid, lactic acid, citric acid, maleic acid, malonic acid, etc.), certain amino acids (e.g., alanine, proline, serine, etc.), a terpene (e.g., thymol, menthol, etc.) or any combination of two or more hydrogen bond donors.

[0039] In one or more embodiments, the NADES system can include two different solvents: one based on a more polar combination (e.g., choline chloride and glycerol) and a second based on a non-polar combination (thymol and menthol). Such an exemplary NADES system forms two phases since the differences in polarity of the two solvents favors phase separation. The NADES composition may be characterized in terms of the ratio of hydrophilic :hydrophobic ratio. In one or more embodiments, the hydrophobic:hydrophilic ratio mass ratio may be up to 100:0 such as, for example, up to 100: 1, up to 100:2, up to 100:3, up to 100:4, up to 100:5, up to 100:6, up to 100:7, up to 100:8, up to 100:9, up to 100: 10, up to 100: 15, up to 100:20, or up to 100:25. Thus, for example, in one or more embodiments, the hydrophilic:hydrophobic ratio may be 100:5. In an exemplary embodiment, the NADES composition includes choline chloride and glycerol as hydrophilic components and menthol and thymol as hydrophobic components having a cumulative hydrophilic:hydrophobic ratio of 100:5. This NADES composition possesses high extractability, with high protein purity and low residual lipid content. Hemp protein isolated using this exemplary NADES two-phase NADES composition possessed significantly higher solubility under acidic conditions that the control sample, highlighting its potential for food applications.

[0040] The NADES system can be tuned for use with particular matrix materials. In one or more embodiments, the NADES system may be tuned by altering the molar ratio of hydrophilic solvent, the hydrophobic solvent, or both solvents. For example, a choline chloride:glycerol mixture at a molar ratio of 1 : 1 (in water; 40% solvent mixture by weight) was used to extract protein from hemp. The same hydrophilic component was initially used to extract protein from chickpeas. Then, the molar ratio of the hydrophobic component was modified to enhance the extractability of chickpea protein. The ratio of glycerol was increased to molar ratios of 1 :2 or 1 :3 (choline chloride:glycerol) in the hydrophilic solvent, then tested at 100: 10 hydrophilic :hydrophobic mass ratio. Extraction of chickpea protein was significantly increased, thereby establishing that the NADES system can be tuned for use with a particular matrix material. The NADES system can, alternatively or additionally, be tuned to increase protein purity, improve protein functionality, and / or reduce off notes in flavor.

[0041] In one or more embodiments, the two-phase NADES has a pH of from 6.0 to 9.5. Thus, for example, the two-phase NADES can have a pH of 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, or 9.0.

[0042] The two-phase NADES system generates fewer compounds responsible for off flavor in the protein isolates. Flavor is closely related with consumer acceptability. Thus, for certain applications, it may be desirable that the protein extraction process limits the likelihood and / or extent to which compounds that generate off flavors. The NADES extracted protein isolates had significantly lower aldehydes (e g., hexanal and nonanal), alcohols (e.g., 1 -hexanol and octen-3- ol) and ketones (e.g., 2-pentanone and 2-heptanone). Those compounds are known markers for off flavor in plant protein and are associated with beany, grassy, and green flavor. The use of the two-phase NADES system described herein efficiently removed the potential precursors for those off notes, polyunsaturated fatty acids. Those results indicate that the two-phase NADES process can reduce the presence of those compounds and generate a protein isolate with potentially improved flavor profile.

[0043] Protein Extraction Methods

[0044] In another aspect, this disclosure describes methods for extracting protein from a matrix material. In one or more embodiments, the method includes adding a sample of full fat flour from the plant-based matrix to a two-phase natural deep eutectic solvent (NADES), performing an extraction step, performing a centrifugation step, performing a phase separation step, and collecting the extracted protein.

[0045] The extracting step may be performed under a solids to liquid ratio of from 2% to 15%. Further, the extracting step may be performed at one or more temperatures ranging from 10 °C to 40 °C such as, for example, at room temperature (20 °C to 22 °C), at 10 °C, at 15 °C, at 25 °C, at 30 °C, at 35 °C, or at 40°C. The extraction step may be performed at temperatures that vary within a range having endpoints defined by (a) any temperature from 10 °C to 39 °C and (b) any temperature from 11 °C to 40 °C that is greater than the selected minimum endpoint of the range.

[0046] The extracting step generally includes agitating the sample at one or more speeds ranging from 100 rpm to 400 rpm such as, for example, at 300 rpm. The agitation may be performed for any suitable length of time such as, for example, at least 15 minutes, at least 20 minutes, at least 30 minutes, at least 40 minutes, at least 60 minutes, at least 90 minutes, or at least 120 minutes. In one or more embodiments, agitation for 40 minutes to 60 minutes is sufficient.

[0047] The centrifugation step may be performed by centrifuging the sample at one or more speeds ranging from 8000 rpm to 12,000 rpm such as, for example, at 9000 rpm. Similar centrifugation parameters can be translated to other centrifuge systems depending on the rotor size and other appropriate considerations. The centrifugation step may be performed at one or more temperatures ranging from 10 °C to 30 °C such as, for example, at room temperature (20 °C to 22 °C). The centrifugation step may be performed for any suitable length of time such as, for example, at least 10 minutes, at least 20 minutes, at least 30 minutes, at least 40 minutes, at least 60 minutes, at least 90 minutes, or at least 120 minutes. In one or more embodiments, the centrifugation step may be performed at 900 rpm for 20 minutes.

[0048] The phase separation step may be performed by allowing the two-phase NADES to separate into two phases: a protein rich hydrophilic phase and a lipid rich hydrophobic phase. In one or more embodiments, separation of phases may be achieved using a separatory funnel. Phase separation may be achieved by allowing the NADES to separate into phases over any appropriate duration of time such as, for example, from one hour to 48 hours. In one or more embodiments, the phase separation step involves allowing the NADES to separate for at least one hour, for at least two hours, for at least three hours, for at least four hours, for at least five hours, for at least six hours, for at least seven hours, for at least eight hours, for at least nine hours, for at least ten hours, for at least twelve hours, or for at least sixteen hours. The phase separation step may be performed at any suitable temperature such as, for example, one or more temperatures ranging from 4 °C to 25 °C. It is not necessary that the temperature be maintained constant throughout the duration of the phase separation step.

[0049] In one or more embodiments, the protein may be extracted in a one-step process in which the centrifugation step and the phase separation step are replace by the use of a three-phase decanter.

[0050] Finally, the method includes collecting the protein from the protein rich phase obtained from either from performing the phase separation step or the three-phase decanter. The protein may be collected using any suitable method. Suitable methods include, but are not limited to, dialysis, membrane filtration, or any combination of suitable protein collection methods. In one or more embodiments, the method can include collecting lipid from the lipid rich phase obtained from either from performing the phase separation step or the three-phase decanter. The lipid may be collected using any suitable method. Suitable methods include, but are not limited to, distillation (e.g., using Clevenger apparatus), pipetting, centrifugation, or solvent evaporation.

[0051] In the preceding description and following claims, the term “and / or” means one or all of the listed elements or a combination of any two or more of the listed elements; the terms “comprises,” “comprising,” and variations thereof are to be construed as open ended — i.e., additional elements or steps are optional and may or may not be present; unless otherwise specified, “a,” “an,” “the,” and “at least one” are used interchangeably and mean one or more than one.

[0052] The recitations of numerical ranges by endpoints include all numbers subsumed within that range, including the recited endpoints (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.). Further, unless otherwise expressly described, the value of a parameter may vary within the recited range — i.e., the value need not be kept constant so long as the variation remains within the recited range.

[0053] As used herein, the word “exemplary” means to serve as an illustrative example and should not be construed as preferred or advantageous over other embodiments.

[0054] As used herein, the terms “preferred” and “preferably” refer to embodiments of the invention that may afford certain benefits under certain circumstances. However, other embodiments may also be preferred under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful and is not intended to exclude other embodiments from the scope of the invention.

[0055] In the preceding description, particular embodiments may be described in isolation for clarity. Reference throughout this specification to “one embodiment,” “an embodiment,” “certain embodiments,” “one or more embodiments,” or “some embodiments,” etc., means that a particular feature, configuration, composition, or characteristic described in connection with the embodiment is included in at least one embodiment of the disclosure. Thus, the appearances of such phrases in various places throughout this specification are not necessarily referring to the same embodiment of the disclosure. Furthermore, the particular features, configurations, compositions, or characteristics may be combined in any suitable manner in one or more embodiments. Thus, features described in the context of one embodiment may be combined with features described in the context of a different embodiment except where the features are necessarily mutually exclusive.

[0056] For any method disclosed herein that includes discrete steps, the steps may be performed in any feasible order. And, as appropriate, any combination of two or more steps may be performed simultaneously.

[0057] In several places throughout the above description, guidance is provided through lists of examples, which examples can be used in various combinations. In each instance, the recited list serves only as a representative group and should not be interpreted as an exclusive list.

[0058] EXAMPLES

[0059] The present invention is illustrated by the following examples. It is to be understood that the particular examples, materials, amounts, and procedures are to be interpreted broadly in accordance with the scope and spirit of the invention as set forth herein.

[0060] NATURAL DEEP EUTECTIC SOLVENT (NADES)-ASSISTED EXTRACTION

[0061] Solvent preparation

[0062] Solvent systems were synthesized by mixing choline chloride and glycerol at 1 : 1, 1 :2, or 1 :3 (molar ratio) for the polar (hydrophilic) portion, and thymol and menthol (1 : 1, molar ratio) for the non-polar (hydrophobic) portion. The NADES mixtures (hydrophilic and hydrophobic) were heated to 80 °C and stirred at 500 rpm separately until a clear solution was obtained. The solutions were cooled to room temperature and water was added (40% (w / w)) to the hydrophilic solvent portion. The two portions of the solvent were mixed at the following mass ratio of hydrophilic:hydrophobic NADES (100 g:0 g, 100 g:5 g, 100 g: 15 g) and tested for oil and protein simultaneous extraction from hemp and chickpea.

[0063] Protein and oil co-extraction

[0064] Full fat hemp flour or chickpea flour were added to the three different solvents (100:0, 100:5, 100: 15) at 5% (w / v). The pH of the hemp slurry was adjusted to 7; the pH of the chickpea slurry was adjusted to 7.5. Extraction of oil and protein from each slurry was conducted at room temperature, agitated at 300 rpm, and one hour extraction time. Each slurry was then centrifuged for 20 minutes at 9000 rpm. Each supernatant was separated from the pellet (fiber rich phase) and then transferred to a separatory funnel and left for two hours at room temperature for oil and protein rich phase separation. The lower protein rich phase was separated from the upper lipid rich phase using the separatory funnel. The protein rich phase was then subjected to dialysis using a semi permeable membrane. After dialysis, each protein isolate was freeze dried and stored at -20 °C until further analysis. Assays were performed in triplicate. Proximate analysis and mass balances were conducted to determine total oil and protein extractability. Control protein isolate samples were prepared using a defatted flour, alkaline extraction, and isoelectric precipitation.

[0065] Alternatively, in triplicate, full-fat hemp flour (5 g) was dispersed in 100 g of hydrophilic solvent (5% total solids) with 0 g (100:0), 5 g (100:5), or 15 g (100: 15) of hydrophobic solvent. The pH was adjusted to 7 and the slurry was agitated for one hour at room temperature. The dispersion was centrifuged at 12,000* for 15 minutes. The fiber-rich pellet and the gums fraction right above the pellet (containing residual protein, lipids, and carbohydrates not extracted in the process) were collected individually and redispersed in double distilled water (DDW, 10% total solids), dialyzed against water, and lyophilized. The supernatant was poured into a separatory funnel to allow for the separation of the protein-rich and oil-rich phases overnight. The protein-rich solution was collected, dialyzed against water, and lyophilized. The oil layer was collected, and the separatory funnel was rinsed with a small amount of hexane to ensure proper oil recovery, which was then evaporated under nitrogen flux. All fractions were weighed for mass balance evaluation. To determine protein yield and purity, the protein content of each fraction was measured following the Dumas AO AC Method 990.03 using a nitrogen analyzer (LECO, St. Joseph, MI) and a protein conversion factor of 5.30 (FoodData Central Available online: https: / / fdc.nal.usda.gOv / fdc-app.html# / food-details / 170148 / nutrients (accessed on 22 September 2024)). Fat content was analyzed following the Mojonnier AO AC method 922.06 (Latimer, G.W. (Ed.), Official Methods of Analysis of AO AC INTERNATIONAL: 3- Volume Set; Twenty-Second Edition, Oxford University Press: Oxford, New York, 2023; ISBN 978-0-19-761013-8.). Moisture content was determined following the vacuum oven AACCI method 44-40.01 (AACC International Method 44-40.01. Moisture-Modified Vacuum-Oven Method. In AACC International Approved Methods; AACC International, 1999 ISBN 978-1- 891127-68-7 ). Ash content was measured following the dry ashing AOAC method 923.03 (Latimer, G.W. (Ed.), Official Methods of Analysis of AOAC INTERNATIONAL: 3-Volume Set; Twenty-Second Edition, Oxford University Press: Oxford, New York, 2023; ISBN 978-0- 19-761013-8 ). NADES-assisted extraction was repeated under the conditions that had the best yield and purity (at hydrophilic and hydrophobic solvent ratio of 100:5 g) to produce a sufficient amount of NADES-extracted hemp protein isolate (N-HPI) for structural, functional, and flavor characterization. ALKALINE -ASSISTED EXTRACTION COUPLED WITH ISOELECTRIC PRECIPITATION (AE-IEP) Preparation of defatted hemp meal (DHM)

[0066] Full fat hemp flour was defatted as previously described [Eckhardt et al., Current Research in Food Science 2024, 8, 100746, doi: 10.1016 / j.crfs.2024.100746], The defatted hemp meal (DHM) was milled to 50-mesh using a cyclone sample mill (UDY Corp., Fort Collins, CO). The milled DHM had a fat content of 1.5% on a wet basis (w.b.) as assessed by the Mojonnier method. The protein content was 62.5% w.b. as determined by the Dumas method.

[0067] Protein Extraction

[0068] Hemp protein isolates was produced from DHM following the AE-IEP as previously described (Eckhardt et al., Current Research in Food Science 2024, 8, 100746). The initial protein solubilization was performed at pH 7, pH 8, pH 9, pH 10, and pH 11 to select the extraction pH for the reference pH-extracted hemp protein isolate (R-HPI). In triplicate, DHM (5 g) was dispersed in 100 mL of DDW (5% w / v), and the pH was adjusted to 7, 8, 9, 10, or 11 with 2 N NaOH. The dispersion was stirred for one hour and centrifuged at 12,000* for 15 minutes. The supernatant was collected and neutralized. The pellet was redispersed in DDW (5% w / v) for a second round of solubilization at pH 7, 8, 9, 10, or 11, followed by stirring for another hour. After centrifugation, the supernatant was collected, neutralized, and combined with the first supernatant. The residual pellet was retained and lyophilized. The combined supernatant was adjusted to a precipitation pH of 5, followed by centrifugation. The supernatant was collected and lyophilized. The protein pellet was dispersed in DDW (1 :4 w / w) and neutralized before dialysis and lyophilization. All lyophilized fractions were weighed for mass balance determination. The protein yield and purity were determined by measuring the protein content of each fraction following the Dumas method. Fat, moisture and ash contents were determined as described above for NADES-assisted extraction.

[0069] Protein solubility

[0070] Protein solubility at 1% protein (w / v in DDW) was measured, in triplicate, as previously described (Boyle et al., 2018, J Am Oil Chem Soc 95:1049-1062). Protein dispersions were stirred for two hours and adjusted to either pH 3.4 or pH 7. The samples were either heated in a water bath at 80 °C for 30 minutes, or left without heat treatment, followed by centrifugation at 15,682xg. Protein solubility was calculated as the percentage of protein in the supernatant relative to the total protein in the initial sample as determined using the Dumas method. Results are shown in Table 1.

[0071] The protein isolates obtained using the two-phase NADES and the control samples were also analyzed regarding their solubility as previously described (Mitacek et al., 2024, Food Hydrocoll 154: 110152), and volatile profile by solid phase microextraction and gas chromatography-mass spectrometry (SPME-GC / MS). Results are shown in FIG. 4.

[0072] Table 1 cSPI: commercial soy protein isolate

[0073] N-HPI: NADES extracted hemp protein isolate

[0074] R-HPI: reference hemp protein isolate protein solution cPPI: commercial pea protein isolate protein solution

[0075] ",JMeans (n > 3) in each column with different lowercase letters are significantly different, according to the Tukey-Kramer multiple means comparison test (p < 0.05).

[0076] Alternatively, protein solubility of the hemp protein isolate samples produced at different solubilization pHs (pH 7, 8, 9, 10, 11) determined as previosuly described (Boyle et al., 2018, J Am Oil Chem Soc 95: 1049-1062; Bu et al., Food Chem 2022, 371, 131135). Protein solubility was assessed, in triplicate, at 5% (w / v) protein concentration, at pH 7 and pH 3.4, with and without heating at 80 °C for 30 minutes. Based on protein yield and purity and protein solubility results, the extraction pH of the R-HPI was determined to be pH 8. A sufficient amount of R-HPI was produced (pH 8 extraction) for structural, functional, and flavor characterization.

[0077] Color measurement

[0078] The color of R-HPI, N-HPI, cSPI, and cPPI samples was measured, in triplicate, following the CIE (International Commission on Illumination) 1976 L* a* b* color system using a Chroma Meter CR-221 (Minolta Camera Co., Osaka, Japan), as previously decribed (Eckhardt et al., Current Research in Food Science 2024, 8, 100746). L* represents lightness ranging from 0 (black) to 100 (white); a* denotes the red-green axis with positive values indicating redness and negative values indicating greenness; b* corresponds to the yellow-blue axis with positive values for yellowness and negative values for blueness.

[0079] PROTEIN STRUCTURAL PROPERTIES

[0080] Protein profiling by gel electrophoresis

[0081] The protein profile of R-HPI and N-HPI was monitored using sodium dodecyl polyacrylamide gel electrophoresis (SDS-PAGE) as previosuly described (Boyle et al., 2018, J Am Oil Che Soc 95: 1049-1062). The protein samples were dispersed in DDW (20 mg protein / mL) and solubilized for two hours. The solubilized samples were prepared under nonreducing (only Laemmli buffer) and reducing (Laemmli buffer with P-mercaptoethanol (PME) conditions. Protein samples (50 pg protein) and MW standard were loaded onto a 4-20% precast gradient gel. The gel was electrophoresed, stained, destained, and imaged (Molecular Imager Gel Doc XR system, Bio-Rad Laboratories, Inc., hercules, CA), as previously reported (Boyle et al., 2018, J Am Oil Chem Soc 95 : 1049- 1062).

[0082] Protein surface properties

[0083] Surface hydrophobicity and Zeta potential of R-HPI and N-HPI were measured, in triplicate, as previosuly described (Bu et al., Food Chem 2022, 371, 131135; Eckhardt et al., Current Research in Food Science 2024, 8, 100746).

[0084] Thermal denaturation by differential scanning calorimetry (DSC)

[0085] Denaturation temperature and enthalpy of the protein in R-HPI and N-HPI were determined, in triplicate, using a DSC instrument (DSC 1 STARe System, Mettler Toledo, Columbus, OH), as previosuly described (Eckhardt et al., Current Research in Food Science 2024, 8, 100746). Thermograms were collected and analyzed using Mettler Toledo's STARe Software version 11.00 to determine the peak denaturation temperature and enthalpy for each protein.

[0086] PROTEIN FUNCTIONAL PROPERTIES Protein solubility

[0087] The protein solubility of N-HPI, R-HPI, and commercial samples was measured as described above for AE-IEP.

[0088] Emulsification capacity

[0089] The emulsification capacity (EC) of R-HPI and N-HPI was determined, in triplicate, as previously described (Bu et al., Food Chem 2022, 371, 131135). In brief, protein solutions at 1% (w / v) concentration were homogenized using a digital mixer (IKA RW 20; IKA Works Inc., Wilmington, NC) equipped with a four-blade, 50 mm diameter shaft (IKA R 1342; IKA Works Inc., Wilmington, NC) rotating at 860-870 rpm. Corn oil colored with Sudan Red 7B was gradually titrated at a steady flow rate into the protein solution, while homogenizing. The addition of oil continued until phase inversion occurred. Emulsification capacity was calculated as the amount of oil (in grams) emulsified per gram of protein.

[0090] Gel strength and morphology

[0091] Protein solutions at concentrations of 15% or 20% (w / v in DDW) were prepared in triplicate, adjusted to pH 7, and stirred for two hours to ensure adequate dispersion. The protein solutions (1 mL aliquots) were heated at 95 °C (±2 °C) in a water bath for 15 minutes (cSPI) or 20 minutes (cPPI, R-HPI, N-HPI) and cooled to room temperature. Gel strength was measured using a texture analyzer (TA-XT PLUS; Stable Micro Systems LTD, Surrey, UK) with a 100 mm diameter probe, a 5 mm / s test speed, and a target distance of 0.5 mm from the plate. The force in Newtons (N) required to rupture each gel was recorded as gel strength. Scanning electron microscopy (SEM) was used to visualize the morphology of the lyophilized protein gels. The gels were sliced horizontally into 1 cm sections using a razor blade and were affixed onto aluminum stubs with double-sided carbon adhesive tabs. The samples were sputter-coated with 60 / 40 gold-palladium mixture. Scans were performed using a scanning electron microscope (S- 3500N; Hitachi, Ltd., Tokyo, Japan) at an accelerating voltage of 5 kV. Cross-sectional images were captured at 500* and 1000* magnification.

[0092] IMPACT OF RESIDUAL FAT ON OFF FLAVOR DEVELOPMENT

[0093] Fatty acid profile Fatty acid profile of hemp flour (HF), N-HPI, R-HPT, cSPI, and cPPI was determined as previosuly described (Dias et al., Prostaglandins, Leukotrienes and Essential Fatty Acids 2020, 152, 102040). Briefly, sample aliquots containing ~4 mg of fat were weighed, in triplicate, into glass tubes. Lipids were extracted using the Folch extraction method, and trans-esterified using methanolic HC1 to generate fatty acid methyl esters (FAME). The lipid extracts were spiked with 0.6 mg of the internal standard triheptadecanoin (Tri-C17:0, 15 mg / mL). The samples were analyzed using a gas chromatograph coupled with a flame ionization detector (6890N GC-FID, Agilent Technologies, Inc., Santa Clara, CA). One microliter of each sample was injected in split mode (1 :30). Separation was achieved using an FFAP column (30 m x 0.25 mm x 0.25 pm). The injector was set at 240 °C, and the detector at 300 °C. The oven temperature program was set at 50 °C for the first two minutes, increased to 180 °C at a rate of 10 °C / min, then ramped up to 240 °C at 5 °C / min, and was maintained at 240 °C for 13 minutes. Hydrogen was used as the carrier gas at a flow rate of 1 mL / min. Peak identification was carried out by comparing the retention times of the FAME 37 standards with those of the samples under the same conditions. Peak identification and integration were performed using CHEMSTATION software B.04.03 (Agilent Technologies, Inc., Santa Clara, CA). Relative quantification was performed using the internal standard.

[0094] Profile of volatile organic compounds

[0095] Volatile organic compounds were extracted, in triplicate, from HF, N-HPI, R-HPI, cSPI, and cPPI following a solid-phase microextraction (SPME), using A 2 cm divinylbenzene / carboxen / polydimethylsiloxane (DVB / CAR / PDMS) fiber as previously described (Oliveira, et al., Molecules 2024, 29, 4268). Analyses were conducted using a gas chromatograph (6890: Agilent Techniologies, Inc., Santa Clara, CA), equipped with an autosampler (PAL RSI 120; CTC Analytics AG, Zwingen, Switzerland) and a single quadrupole mass spectrometer (5973; Agilent Technologies, Inc., Santa Clara, CA) and a DB-5ms Ultra Inert column (30 m x 250 pm x 0.25 pm). Volatile organic compounds were identified using mass spectrometry (MS) with MASSHUNTER WORKSTATION UNKNOWN analysis software version 12.0.893.1 (Agilent Technologies, Inc., Santa Clara, CA) and the NIST 17 MS library. Compounds were identified if they had a signal-to-noise ratio greater than 3 and a match score above 80. To confirm the identities of the compounds, programmed temperature retention indexes (RIs) were calculated according to Van den Dool and Kratz methods by analyzing a solution of n-alkanes (C7-C20) under the same conditions. Relative quantification was performed as previously described (Xiao et al. Food Chemistry 2014, 151, 31-39) using four deuterated internal standards from different chemical classes (hexanal-d5, methylpyrazine-d6, hexyl alcohol-dl3, and heptanone-d5), each at a concentration of 0.5 mg / kg. The relative concentration was used to compare the profiles among the samples.

[0096] STATISTICAL ANALYSIS

[0097] SPSS Statistics software version 28 for Windows (International Business Machines Corp., Armonk, New York) or Statistica 14 software (StatSoft, Tulsa, OK) was used for one-way analysis of variance (ANOVA). Tukey-Kramer multiple means comparison test was used to determine the significant differences (p < 0.05) among the means of three or more samples. Independent-samples t-test was used to determine significant differences (p < 0.05) between the average of two samples. Principal component analysis (PCA) was performed to evaluate the differences among the variables and track trends among the samples. The data was auto-scaled and analyzed using MetaboAnalyst 5.0 https: / / www.metaboanalyst.ca. The PCA was performed using the concentration of each identified volatile compound.

[0098] The complete disclosure of all patents, patent applications, and publications, and electronically available material (including, for instance, nucleotide sequence submissions in, e.g., GenBank and RefSeq, and amino acid sequence submissions in, e.g., SwissProt, PIR, PRF, PDB, and translations from annotated coding regions in GenBank and RefSeq) cited herein are incorporated by reference in their entirety. In the event that any inconsistency exists between the disclosure of the present application and the disclosure(s) of any document incorporated herein by reference, the disclosure of the present application shall govern. The foregoing detailed description and examples have been given for clarity of understanding only. No unnecessary limitations are to be understood therefrom. The invention is not limited to the exact details shown and described, for variations obvious to one skilled in the art will be included within the invention defined by the claims.

[0099] Unless otherwise indicated, all numbers expressing quantities of components, molecular weights, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about.” Accordingly, unless otherwise indicated to the contrary, the numerical parameters set forth in the specification and claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

[0100] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. All numerical values, however, inherently contain a range necessarily resulting from the standard deviation found in their respective testing measurements.

[0101] All headings are for the convenience of the reader and should not be used to limit the meaning of the text that follows the heading, unless so specified.

Claims

What is claimed is:

1. A two-phase natural deep eutectic solvent (NADES) comprising: a hydrophilic phase comprising at least one hydrophilic component; and a hydrophobic phase comprising at least one hydrophobic component; wherein the hydrophilic components and hydrophobic components are present in a mass ratio of up to 100: 1.

2. The two-phase NADES of claim 1, wherein the hydrophilic phase comprises choline chloride, glycerol, or both.

3. The two-phase NADES of claim 2, wherein the hydrophilic phase comprises choline chloride and glycerol at a 1 :1 molar ratio.

4. The two-phase NADES of claim 2, wherein the hydrophilic phase comprises choline chloride and glycerol at a molar ratio other than a 1 : 1 molar ratio.

5. The two-phase NADES of any preceding claim, wherein the hydrophobic phase comprises thymol, menthol, or both.

6. The two-phase NADES of claim 5, wherein the hydrophobic phase comprises thymol and menthol at a 1 :1 molar ratio.

7. The two-phase NADES of claim 5, wherein the hydrophobic phase comprises thymol and menthol at a molar ratio other than a 1 : 1 molar ratio.

8. A method of extracting protein, lipid, or both protein and lipid from a plant-based matrix, the method comprising: adding a sample of full fat flour from the plant-based matrix to a two-phase natural deep eutectic solvent (NADES); extracting the sample; centrifuging the sample;collecting supernatant and subjecting the supernatant to phase separation conditions to produce a protein rich phase and a lipid rich phase; and collecting the protein rich phase and the lipid rich phase.

9. The method of claim 8, wherein the extraction is performed at room temperature.

10. The method of claim 8, wherein extracting the sample is performed under solids to liquid ratio from 2% to 15%, agitation at from 100 rpm to 400 rpm, and at a temperature ranging from 20 °C to 35 °C.

11. The method of any one of claims 8-10, wherein centrifuging the sample comprises centrifuging the sample at 8000 rpm tol2,000 rpm for 15 to 60 minutes at room temperature.

12. The method of any one of claims 8-11, wherein the phase separation conditions comprise incubating the supernatant for one to 16 hours at a temperature ranging from 4 °C to 25 °C.

13. The method of any one of claims 8-12, wherein collecting the protein rich phase comprises dialysis or membrane fdtration.

14. The method of any one of claims 8-13, wherein the two-phase NADES has a pH of from 6.0 to 9.5.

15. The method of any one of claims 8-14, wherein the plant-based matrix is not subjected to defatting.

16. The method of any one of claim 8-15, wherein the method lacks a desolventization step.

Images