Processes for separating products from plant biomass

Abstract

The present disclosure provides processes for relates to processes for separating one or more water-soluble products from non-woody plants of the genus Hesperaloe. The processes described herein sequentially separates one or more water-soluble products from non-woody plants of the genus Hesperaloe. The process may include sequential separation and enrichment of different saponins, polyphenols, and / or organic acids from a juice derived from non-woody plants of the genus Hesperaloe. The sequential separation of water-soluble products from non-woody plants of the genus Hesperaloe provides multiple high-value products.

Description

KT Ref.109296-1449036 PROCESSES FOR SEPARATING PRODUCTS FROM PLANT BIOMASS CROSS- REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of and priority to U.S. Provisional Patent Application No.63 / 519,150, filed on August 11, 2023, and entitled “PROCESSES FOR SEPARATING PRODUCTS FROM PLANT BIOMASS”, the contents of which is hereby incorporated by reference in its entirety for all intents and purposes. FIELD

[0002] The present disclosure relates to processes for separating products from plant biomass. More particularly, the present disclosure relates to processes for separating and enriching solids, particularly water-soluble solids, such as saponins, polyphenols, and organic acids, derived from non-woody plants of the genus Hesperaloe. BACKGROUND

[0003] Plants produce a vast and diverse assortment of organic compounds, the great majority of which do not appear to participate directly in their growth and development. These substances, traditionally referred to as secondary metabolites or plant natural products, often are distributed among limited taxonomic groups within the plant kingdom. The functions of secondary metabolites remain largely unknown, although a number of compounds have been associated with attributes useful to the plants, e.g., protection against herbivores and protection against microbial infection, as attractants for pollinators and seed- dispersing animals, and as compounds that influence competition among plant species (allelochemicals). There is a growing interest in plant natural products, since these products often have a wide range of applications in different kinds of industries, including pharmaceutical industries, cosmetic industries, food industries, detergent industries, and the like.

[0004] Secondary metabolites or natural products found in plants are of general interest within a wide range of different industries. However, low yields of secondary metabolites or natural products are obtained from processing plant biomass despite being both capital-KT Ref.109296-1449036 intensive and energy-intensive. Additionally, products separated from plants include impurities, which limits their use in industries, such as cosmetics and pharmaceuticals. There is therefore a growing need for processes that can efficiently separate and purify compounds derived from plant sources with high yields. SUMMARY

[0005] The present disclosure relates to processes for separating and enriching solids, particularly water-soluble solids, such as saponins, polyphenols, and organic acids, derived from non-woody plants of the genus Hesperaloe.

[0006] In some embodiments, the present disclosure relates to processes for separating products from a non-woody plant of the genus Hesperaloe, the process comprising: providing a crude extract obtained from a non-woody plant of the genus Hesperaloe, wherein the crude extract comprises water-soluble products including saponins, polyphenols, and organic acids; removing saponins from the crude extract to produce a first residuum stream comprising polyphenols and organic acids, wherein the first residuum stream has a lower concentration of saponins than the crude extract; removing polyphenols from the first residuum stream to produce a second residuum stream comprising organic acids, wherein the second residuum stream has a lower concentration of polyphenols than the first residuum stream; and removing organic acids from the second residuum stream to produce a third residuum stream, wherein the third residuum stream has a lower concentration of organic acids than the second residuum stream.

[0007] In some embodiments, the present disclosure relates to processes for separating products from a non-woody plant of the genus Hesperaloe, the process comprising: providing a juice obtained from a non-woody plant of the genus Hesperaloe, wherein the juice comprises water-soluble products including saponins, polyphenols, and organic acids; filtering the juice to remove solids and produce a filtered juice; foaming the filtered juice to produce a foam product, wherein the foam product comprises saponins; extracting polyphenols from the filtered juice; and extracting organic acids from the filtered juice. DESCRIPTION OF THE DRAWINGS

[0008] FIG.1 provides a schematic diagram of a process flow for separating products from plant biomass according to some embodiments of the present invention.KT Ref.109296-1449036

[0009] FIG.2 provides a process flow diagram for separating and purifying compounds from plant biomass according to some embodiments of the present invention.

[0010] FIGS.3A-D show images of foam fractionation of a juice extracted from plant biomass according to some embodiments of the present invention.

[0011] FIG.4A show images of juice extracted from plant biomass at different dilution levels and FIG.4B shows the foaming of the diluted juice according to some embodiments of the present invention.

[0012] FIG.5 show images of desorption solutions including water-soluble solids extracted from a resin column using ethanol and hot water according to some embodiments of the present invention.

[0013] FIG.6 shows a schematic for clarifying and deashing a juice extracted from plant biomass according to some embodiments of the present invention.

[0014] FIG.7 shows an ion exchange column for deashing a clarified juice extracted from plant biomass according to some embodiments of the present invention. DETAILED DESCRIPTION

[0015] The present disclosure relates to processes for separating products from plant biomass. In particular, the present disclosure relates to processes for separating products from non-woody plants and more particularly the non-woody plants of the genus Hesperaloe. In some embodiments, the present disclosure is directed towards separating water-soluble solids derived from non-woody plants of the genus Hesperaloe including, for example, Hesperaloe funifera, Hesperaloe nocturna, Hesperaloe parviflora, and Hesperaloe chiangii. Water soluble solids that may be recovered may include, for example, saponins, polyphenols, organic acids, and minerals.

[0016] The processes described herein sequentially separates one or more water-soluble products from non-woody plants of the genus Hesperaloe. For example, the process may include sequential separation and enrichment of different water soluble solids from non- woody plants of the genus Hesperaloe. In some embodiments, the water soluble solids can be saponins, polyphenols, and / or organic acids. The sequential separation of water-soluble products from non-woody plants of the genus Hesperaloe provides multiple high-value products.KT Ref.109296-1449036

[0017] In some embodiments, Hesperaloe biomass may be processed to separate and purify solids, particularly water-soluble solids, such as saponins, polyphenols, and organic acids. In some embodiments, the process includes providing a crude extract derived from a non- woody plants of the genus Hesperaloe. For example, the crude extract can be obtained by milling biomass derived from a non-woody plants of the genus Hesperaloe. In some embodiments, the process may include washing the milled biomass with an aqueous solvent to yield a juice and separating water insoluble solids from the juice.

[0018] The process includes processing the crude extract or juice to separate or concentrate water-soluble solids. For example, the process may include separating saponins, polyphenols, organic acids, or combinations thereof, from the crude extract. In some embodiments, saponins, polyphenols, and organic acids are sequentially extracted from the crude extract. In some embodiments, the process may include foam fractionation to separate and enrich saponins, a first resin separation step to extract polyphenols, and a second resin separation step to extract organic acids. Each of these process steps provide a high-purity water soluble product from the plant biomass. In some embodiments, the process includes a separation cascade to separate and purify water soluble products from the plant biomass. Each stage in the cascade can separate a specific water-soluble product in the plant biomass. For example, a first cascade can separate saponins from the crude extract, a second cascade can separate polyphenols from the remainder of the first cascade, and a third cascade can separate organic acids from the remainder of the second cascade. In some embodiments, the remainder from the third cascade can be further processed to separate minerals and mixed solid portions.

[0019] In some embodiments, a foam fractionation step may be used to extract saponins from the crude extract and / or the juice derived from the plant biomass. In the foam fractionation step, the crude extract is agitated with a gas (e.g., air or an inert gas) to produce a first saponin-rich foam and a first residual stream. The first residual stream can be the remaining crude extract and / or the juice after the first saponin-rich foam is separated. The first saponin-rich foam can be processed in a second foam fractionation step to produce a second saponin-rich foam and a second residual stream. The second residual stream can be the remaining first saponin-rich foam after the second saponin-rich foam is separated. The second saponin-rich foam includes a higher concentration of saponins than the first saponin- rich foam. The first residual stream and / or the second residual stream can be processed in one or more extraction columns (e.g., resin extraction column) to separate polyphenols fromKT Ref.109296-1449036 one or both of the residual streams. In some embodiments, each of the residual streams can be combined and then processed in the one or more extraction columns to separate a polyphenol product stream. The first residual stream and / or the second residual stream processed in the one or more extraction columns is depleted of polyphenols to produce a third residual stream. The third residual stream from the extraction column can be processed in a second extraction column to separate organic acids. Process of Separating Water-Soluble Products

[0020] FIG.1 provides a schematic diagram of a process flow for separating products from plant biomass according to some embodiments of the present invention. The process 100 may include one or more separation and purification steps to recover water-soluble products from a crude extract derived from plant biomass. The crude extract can be washed to produce a juice 110 that includes the water-soluble products. For example, the crude extract derived from a non-woody plant of the genus Hesperaloe can be washed with an aqueous solvent (e.g., water) to yield a juice 110. In some embodiments, the juice 110 can be diluted or dehydrated to achieve a desired solids concentration prior to separation.

[0021] The process 100 may include one or more separation steps to separate and enrich water-soluble products from the juice 100. In some embodiments, the process 100 provides a cascade of separation processes to recover a plurality of water-soluble products from the juice 100. For example, the process may be a sequential separation process to separate water- soluble products at different stages. The sequential separation process may separate a first water-soluble product from the juice 100 in a first stage, a second water-soluble product from the juice 100 in a second stage, a third water-soluble product from the juice 100 in a third stage, and so on. In this configuration, the residual stream from each stage can be the initial input stream for the next stage. For example, after the first water-soluble product from the juice 100 is separated in a first stage, the remaining juice that is depleted of the first water-soluble product can be processed in the second stage to separate the second water- soluble product.

[0022] FIG.1 is a process flow diagram 100 for a process of separating water-soluble products from juice 110. As described above, the juice 110 is derived from a non-woody plant of the genus Hesperaloe. The juice comprises at least one, preferably two or more, and still more preferably three or more, water soluble solids selected from saponins, polyphenols, and organic acids. In some embodiments, the water-soluble products includes saponins, polyphenols, and polyphenols. The process 100 includes a first stage 120, a second stageKT Ref.109296-1449036 130, and a third stage 140. The first stage 120 is configured to separate saponins from the juice 110. The second stage 130 is configured to separate polyphenols from the remainder of the juice processed in the first stage 120. The third stage 140 is configured to separate organic acids from the remainder of the juice processed in the second stage 130.

[0023] The juice 110 optionally can be filtered and prepared 115 prior to being supplied to the first stage 120. For example, the juice 110 can be filtered and diluted prior to being supplied to the first stage 120. The first stage 120 may include a first foaming step that is carried out in a first foam fractionator 122 and a second foaming step that is carried out in a second foam fractionator 124. In the first foaming step, the juice 110 can be supplied to the first foam fractionator 122 to produce a foam enriched with saponins and a remainder depleted of saponins. As saponins are natural surfactants that include hydrophobic groups, the saponins preferentially separate from the juice 100 in rising columns of foam. In the foam fractionator, a gas is mixed with the juice to produce the foam fraction including the saponins. In some embodiments, the gas is mixed into the juice in the foam fractionator using a stirrer or the gas can be sparged through the juice. The gas can be air, such as ambient air, or an inert gas. During foam fractionation, the saponins are preferentially removed from the juice, and the juice becomes progressively depleted in saponins. As a result, the juice is enriched in other components (e.g., polyphenols or organic acids). In some embodiments, the juice 110 can be filtered prior to the first stage 120. The juice can be filtered to remove suspended solids in the juice 110. The filtration of the juice 110 prior to foam fractionation produces a high-quality foam, whereas unfiltered juice does not achieve good foaming and the foam collapses without sufficient aeration.

[0024] As shown in FIG.1, the first stage 120 may include a first fractionator 122 and a second fractionator 124. The juice 110 can processed in the first fractionator 122 in a first foaming step to produce a first foam product and a first residuum stream. The first foam product is enriched in saponins and may comprise minor amounts of other water-soluble components (e.g., polyphenols and organic acids). The first residuum stream comprises the remainder of the juice 110 depleted of the first foam product including the saponins and minor amounts of other water-soluble components. The first foam product has a higher concentration of saponins than the juice 110. The first foam product can be processed in the second fractionator 124 in a second foaming step to produce a second foam product and a second residuum stream. When the process includes a second foaming step, the process may also include a breaking step in which the first foam product is collected and collapsed beforeKT Ref.109296-1449036 it is aerated again in the second forming step. The second foam product has a higher concentration of saponins than the first foam product. The second residuum stream comprises the remainder of the first foam product depleted of the second foam product including the saponins. The first residuum stream and / or the second residuum stream can be supplied to the second stage 130. In some embodiments, the first residuum stream and the second residuum stream can be combined and then supplied to the second stage 130.

[0025] In some embodiments, the temperature of the foam fractionation unit and the flow rate of gas (e.g., air) can affect the foam formation. The temperature of the foam fractionation unit can range from 50° C to 100° C (e.g., from 55° C to 95° C, from 60° C to 90° C, from 65° C to 90° C, from 70° C to 90° C, or from 80° C to 100° C). For example, the temperature of the foam fractionation unit can be 50° C, 55° C, 60° C, 65° C, 70° C, 75° C, 80° C, 85° C, 90° C, 95° C, or 100 C. In some embodiments, the flow rate of gas into the foam fractionation unit to produce the foam can range 5 mm / s to 20 mm / s (e.g., from 6 mm / s to 18 mm / s, from 7 mm / s to 16 mm / s, from 8 mm / s to 15 mm / s, from 8 mm / s to 14 mm / s, or from 8 mm / s to 12 mm / s). In some embodiments, the flow rate of gas into the foam fractionation unit to produce the foam can be 5 mm / s, 6 mm / s, 7 mm / s, 8 mm / s, 9 mm / s, 10 mm / s, 11 mm / s, 12 mm / s, 13 mm / s, 14 mm / s, 15 mm / s, 16 mm / s, 17 mm / s, 18 mm / s, 19 mm / s, or 20 mm / s.

[0026] In some embodiments, the foam products resulting from the foregoing separation process may be subjected to further extraction or purification to obtain saponin in the form of a crude saponin extract or in a substantially purified form comprising saponins at a concentration greater than 50 wt. % (e.g., greater than 60 wt. %, greater than 70 wt. %, greater than 75 wt. %, greater than 80 wt. %, or greater than 90 wt. %). In some embodiments, the foam product stream(s) can be processed in an extraction process. For example, the foam product can be subjected to liquid-liquid extraction with a water- immiscible polar solvent. Suitable water-immiscible polar solvents include, for example, alcohols having from 4 to 6 carbon atoms, such as butyl, amyl, hexyl and cyclohexyl alcohols. Extraction of the foam product with a water-immiscible polar solvent generally removes impurities such as proteins, carbohydrates, and organic acids, which remain in the aqueous phase, the saponin being transferred to the solvent phase.

[0027] The solvent phase containing the saponin may be subjected to further treatment to separate the saponin from the polar solvent phase. This can be accomplished in various ways including, for example, by cooling, by dehydrating the solvent extract, or by adding anKT Ref.109296-1449036 organic solvent which is miscible with the polar solvent but in which the saponin is insoluble. Suitable precipitating solvents include, for example, diethyl ether, petroleum ether, acetone, and chloroform.

[0028] In some embodiments, the saponin is separated from the polar solvent by flash evaporation. Flash evaporation is a technique known in preparative chemistry for the rapid removal of a volatile component from a liquid mixture. The volatile liquid is removed from solution by rapid conversion to a vapor phase by creating a thin film of the solution over a large surface area under reduced pressure often accompanied by an increase of temperature of the solution above ambient but less than the boiling point of the solution at atmospheric pressure. The actual thickness of the film and the area over which it is applied is chosen to provide optimum evaporation and ease of use, but evaporation may be substantially instantaneous (hence the name “flash” evaporation). Flash evaporation avoids the prolonged use of high temperatures that may degrade the intended product and has the ability to remove almost all of the polar solvent component (which makes the remaining solution suitable for the preferred practice of spray drying employed in the next step). The polar solvent may be recovered from this step and re-used in a subsequent extraction process.

[0029] The saponin content of the polar solvent extract can be further increased by passage over an ultrafiltration membrane without significant alteration to or loss of the saponin composition. This concentrated saponin fraction where the saponin content is in the range of 85-90%, can then be further purified in a liquid state or reduced to a dry state. Individual saponins may be recovered by a combination of reversed-phase solid phase extraction and preparative reversed-phase HPLC. Alternatively, the polar solvent extract containing saponins can be fractionated directly by a combination of reversed-phase solid phase extraction and preparative reversed-phase HPLC. In some embodiments, the saponins in the foam product may be purified using pH adjustment and filtration.

[0030] In the second stage 130, the one or more residuum streams from the first stage 120 may be processed to separate polyphenols. In the second stage 130, the one or more residuum streams from the first stage 120 can be supplied to a resin column. The resin column may be packed with a resin that preferentially extracts polyphenols from the first stage residuum streams. In some embodiments, the resin column is an ion-exchange resin column. As the residuum stream flows through the column, the polyphenols binds to oppositely charged sites in the resin / stationary phase. The column eluent, which is the second stage residuum stream, is thus depleted of polyphenols. After the first stage residuumKT Ref.109296-1449036 stream is fully processed through the column, a resin wash can be supplied to the resin column to strip the column of the bound polyphenols and obtain a product stream enriched in the polyphenols.

[0031] Useful ion exchange resins have positively or negatively charged functional groups covalently linked to a solid matrix. Some of the factors affecting resin choice are anion or cation exchanger, flow rate, weak or strong ion exchanger, particle size of the resin, and binding capacity. The stability of the polyphenols of interest dictates the selection of an anion or a cation exchanger – either exchanger may be used if the stability is of no concern.

[0032] In some embodiments, the pH of the residuum streams can be adjusted prior to resin extraction of the polyphenols. In some embodiments pH of the extraction solvent can be between about pH 5.0 and 8.0, such as, for example, between about pH 6.0 and about pH 8.0, between about pH 6.5 and about pH 7.5. The extraction may be carried out at temperatures between about 25 and about 90°C, such as, for example, between about 30 and about 80°C, between about 35 and about 75°C, between about 40 and about 70°C, between about 45 and about 65°C or between about 50 and about 60°C.

[0033] In the third stage 140, the residuum stream from the second stage 130 may be processed to separate organic acids. In the third stage 140, the residuum streams from second stage 130 can be supplied to another resin column 142. The resin column 142 may be packed with a resin that preferentially binds organic acids from the second stage residuum stream. The column eluent, which is the third stage residuum stream, is thus depleted of organic acids. In some embodiments, the pH of the residuum stream can be adjusted prior to resin extraction of the organic acids. The third stage residuum stream can be further processed to separate any remaining minerals or solid portions. After the stream is fully processed through the column, a resin wash can be supplied to the column to strip the column of the bound organic acids and obtain a product stream enriched in the organic acids.

[0034] Alternatively, the third stage 140 may include other separation process to separate organic acids from the residuum streams. For example, the third stage 140 may be a liquid extraction step, a membrane separation step, or a distillation step. In some embodiments, the residuum stream from the second stage 130 may be processed according to any combination of the aforementioned methods to separate organic acids.

[0035] In some embodiments, the third stage 140 includes electro-membrane processes to recover organic acids. Electro-membrane processes, including electrodialysis (ED), electrometathesis (EMT), electro-ion substitution (EIS), electro-electrodialysis (EED),KT Ref.109296-1449036 electrodialysis with bipolar membrane (EDBM), and electrodeionization (EDI), are promising technologies for the recovery of organic acids. In the electro-membrane processes, organic acids are separated from water and other impurities based on the electro-migration of ions through ion-exchange membranes. These processes can recover various types of organic acids from the fermentation broth with high recovery yield and low energy consumption. In addition, the integration of fermentation and the electro-membrane process can improve the acid recovery with lower byproduct concentration and energy consumption.

[0036] In some embodiments, the first stage 120, the second stage 130, and the third stage 140 can be arranged in any configuration. For example, in some embodiments, the filtered juice can initially be processed in the second stage 130 to extract polyphenols. The residuum stream from the second stage 130 can then be supplied to the first stage 120 or the third stage 140. In some embodiments, a portion of the residuum stream from the second stage 130 is supplied to each of the first stage 120 and the third stage 140. In some embodiments, the filtered juice can initially be processed in the third stage 140 to extract organic acids. In this embodiment, the residuum stream from the third stage 140 can then be supplied to the first stage 120 or the second stage 130. In some embodiments, a portion of the residuum stream from the third stage 140 is supplied to each of the first stage 120 and the second stage 130.

[0037] FIG.2 provides an exemplary process flow diagram 200 for separating and purifying compounds from plant biomass according to some embodiments of the present invention. As discussed herein, a juice derived from a non-woody plant of the genus Hesperaloe can be processed to separate and enrich the water-soluble products. The process 200 is configured to separate a plurality of water-soluble products from the juice 202. The process 200 can be a continuous process or a semi-batch process. Prior to any separation processes, the juice 202 can be provided to a separation apparatus 205. The separation apparatus 205 can separate suspended solids 204 from the juice 202. The suspended solids 204 can be removed from the process 200 as a waste stream. The suspended solids, also referred to herein as the water insoluble fraction, may be removed from the crude extract by separation processes including, for example, clarification, filtration, centrifugation, or a combination thereof.

[0038] In some embodiments, the juice 202 can be heated and / or acidified prior to separation of the water insoluble fraction. For example, the juice 202 can be heated to a temperature of 100° C and the juice 202 can be acidified to a pH from 3 to 5 prior to filtration. Heating the juice and / or acidifying the juice prior to filtration can aid in filtration of solids from the juice. For example, heating and / or acidifying the juice prior to filtrationKT Ref.109296-1449036 202 denatures proteins in the juice for separation. Additionally, heating the juice prior to filtration and / or acidifying the juice can coagulate solids in the juice (e.g., minerals and divalent ions) for more effective separation when removing the water insoluble fraction from the juice (e.g., via centrifugation).

[0039] In some embodiments, the juice 202 can be purified prior to separation of the water insoluble fraction. The juice 202 can be purified prior or subsequent to heating and / or acidifying the juice. For example, the juice 202 can be subjected to centrifugation, deashing, or combinations thereof. The juice 202 can be centrifuged to produce a clarified juice followed by deashing to produce a deashed juice. Deashing the clarified juice may include by processing the clarified juice through one or more resins. For example, the resin can be a deash resin that removes salts and organic compounds from the juice. The clarified juice can be supplied to an ion exchange column including the one or more resins. In some embodiments, the clarified juice can be processed through a cation exchange resin to remove cations, for example, sodium, potassium, iron, magnesium, and calcium, as well as some crude proteins (e.g., amphoteric proteins). The clarified juice can then be processed through an anion exchange resin that removes acids and polyphenols from the clarified juice to obtain a filtered juice.

[0040] The filtered juice 206 can be provided to a foam fractionation unit 210. In some embodiments, the filtered juice 206 can be heated to a target temperature in an optional heating unit 208 prior to being supplied to the foam fractionation unit 210. The temperature of the filtered juice 206 can aid in foam formation in the foam fractionation unit 210. A gas 212 can be sparged through the filtered juice 206 in the foam fractionation unit 210 to produce a first foam product 215 that is enriched in saponins. For example, air can be sparged adjacent or near the bottom of the foam fractionation unit 210 to produce the first foam product 215 from the filtered juice 206. In some embodiments, a gas can delivered to other regions of the foam fractionation unit 210. For example, an inert gas can be supplied to the foam fractionation unit 210 anywhere below the feed of the filtered juice 206 to the foam fractionation unit 210. In this embodiment, the gas can be supplied parallel with the flow of the filtered juice 206. The filtered juice 206 that does not foam or is not removed from foam fractionation unit 210 as a foam product 215 can be removed from the foam fractionation unit 210 as a first residuum stream 214. The first residuum stream 214 is depleted in saponins in relation to the filtered juice 206.KT Ref.109296-1449036

[0041] The first foam product 215 can be withdrawn from the foam fractionation unit 210. In some embodiments, the foam can be continuously withdrawn from the top of the foam fractionation unit 210 and sent to a foam breaker 220. The first foam product 215 can be broken down by mechanical or chemical means in the foam breaker 220. For example, the foam breaker 220 can be a vessel including a high-speed rotor that breaks the bubbles in the foam to produce a liquid. The foam breaker 220 processes the foam product enriched in saponins to produce a liquid stream 222. The gas or air trapped in the foam product can be vented from the foam breaker 220 in stream 226.

[0042] The liquid stream 222 from the foam breaker 220 can be supplied to a second foam fractionation unit 230. In some embodiments, the liquid stream 222 can be heated prior to being supplied to the second foam fractionation unit 230. The liquid stream 222 supplied to the second foam fractionation unit 230 can be sparged with a gas to produce a second foam product 232. The second foam product 232 has a higher concentration of saponins than the first foam product 215. The second foam product 232 can be withdrawn from the foam fractionation unit 230. In some embodiments, the foam can be continuously withdrawn from a portion of the top of the second foam fractionation unit 230 and sent to a second foam breaker 238. The second foam breaker 238 processes the second foam product 232 enriched in saponins to produce a product stream 239 and gas or air from the second foam product 232 can be vented in stream 237. The product stream 239 can be further processed in an evaporator or dryer 280 to obtain a final saponin product 282.

[0043] The remaining liquid fraction that does not foam or is not removed from the second foam fractionation unit 230 as a second foam product 232 can be removed from the second foam fractionation unit 220 as a second residuum stream 234. For example, the second residuum stream 234 can be removed from the bottom of the second foam fractionation unit 220. The second residuum stream 234 is depleted in saponins in relation to the liquid stream 222 supplied to the second foam fractionation unit 230.

[0044] The first residuum stream 214 and the second residuum stream 234 can be further processed to separate polyphenols. The first residuum stream 214 can be removed from the bottom of the first foam fractionation unit 210 and supplied to a resin column 240. The second residuum stream 234 can be removed from the bottom of the second foam fractionation unit 230 and supplied to the resin column 240. In some embodiments, the first residuum stream 214 and the second residuum stream 234 can be combined and then supplied to the resin column 240. The first residuum stream 214 and the second residuumKT Ref.109296-1449036 stream 234 are depleted in saponins which is beneficial for separation of polyphenols due to the chemical similarities of saponins and polyphenols.

[0045] The resin column 240 may include a resin composition that preferentially binds polyphenols. In some embodiments, the resin column 240 can be an ion-exchange column. The ion-exchange resin is configured to capture the polyphenols while the remainder of the first residuum stream 214 and the second residuum stream 234 passes through the resin column 240, and elutes as residuum stream 242. In a subsequent step, the ion-exchange resin of column 240 can be washed with a solvent 244 to strip the captured polyphenols from the resin. In some embodiments, the solvent 244 for washing the resin may be water or an alcohol (e.g., ethanol). Ethanol was found to preferentially desorb polyphenols from the resin. In some embodiments, the resin can be any resin that has affinity to polyphenols. In some embodiments, resin can be PUROSORB PAD900, PUROLITE A502PS, PUROLITE MN202, PUROLITE A860S, AMBERLITE IRC-120(H), or SUPELITE DAX-8.

[0046] As shown in FIG.2, the combined first residuum stream 214 and the second residuum stream 234 are supplied to the resin column 240. The combined first residuum stream 214 and the second residuum stream 234 flow through the ion-exchange resin in the column and the polyphenols bind to the resin. The processed foam residuum streams are depleted of polyphenols as they flow through the column to produce a third residuum stream 242. A solvent 244 is then added to the resin column 240 to extract the polyphenols from the resin to produce a second product stream 246. The second product stream 246 can be removed from the resin column. The second product stream 246 has a higher concentration of polyphenols than the first residuum stream 214 and / or the second residuum stream 234 fed to the resin column 240. The second product stream 246 has a higher concentration of polyphenols than the filtered juice 206.

[0047] In some embodiments, the second product stream 246 can be further processed to further enrich polyphenols in the second product stream 246. For example, the second product stream 246 including the polyphenols and solvent can be supplied to an evaporator to concentrate the polyphenols. Additionally, the concentrated polyphenol stream can be further processed in a spray dryer to further concentrate and purify the polyphenols.

[0048] The third residuum stream 242 from the resin column 240 can be fed to a second resin column 250 to extract organic acids. In some embodiments, the organic acids include humic acid, fulvic acid, or combinations thereof. In some embodiments, the pH of the third residuum stream 242 is adjusted prior to feeding the third residuum stream 242 to the secondKT Ref.109296-1449036 resin column 250. For example, the third residuum stream 242 can be treated with an acid or a base to achieve a desired pH for ion-exchange in the second resin column 250. The third residuum stream 242 flows through the ion-exchange resin and the organic acids bind to the resin. In some embodiments, resin column can be loaded with PUROSORB PAD900, PUROLITE A502PS, PUROLITE MN202, PUROLITE A860S, AMBERLITE IRC-120(H), or SUPELITE DAX-8. The processed third residuum stream 242 is depleted of polyphenols as it flows through the second resin column 250 to produce a fourth residuum stream 252. A solvent 254 is then added to second resin column 250 to extract the organic acids from the resin to produce a third product stream 256.

[0049] The third product stream 256 can be removed from the resin column and further processed to purify the organic acids. For example, the third product stream 256 can be filtered to remove additional impurities. The third product stream 256 includes a higher concentration of organic acids than the first residuum stream 214, the second residuum stream 234, or the third residuum stream 242. The fourth residuum stream 252 can be further filtered in a filtration unit 260 to separate minerals or solid portions.

[0050] Generally, processing biomass according to the present invention removes at least about 50% of the water soluble solids from the biomass, such as from about 50 to about 98%, such as from about 50 to about 95%, such as from about 75 to about 90%. Illustrations

[0051] Illustration 1. A process for separating products from a non-woody plant of the genus Hesperaloe, the process comprising:providing a crude extract obtained from a non- woody plant of the genus Hesperaloe, wherein the crude extract comprises water-soluble products including saponins, polyphenols, and organic acids; removing saponins from the crude extract to produce a first residuum stream comprising polyphenols and organic acids, wherein the first residuum stream has a lower concentration of saponins than the crude extract; removing polyphenols from the first residuum stream to produce a second residuum stream comprising organic acids, wherein the second residuum stream has a lower concentration of polyphenols than the first residuum stream; and removing organic acids from the second residuum stream to produce a third residuum stream, wherein the third residuum stream has a lower concentration of organic acids than the second residuum stream.KT Ref.109296-1449036

[0052] Illustration 2. The process of Illustration 1, wherein providing the crude extract comprises milling a biomass derived from a non-woody plant of the genus Hesperaloe to yield a milled biomass and the crude extract.

[0053] Illustration 3. The process of Illustration 2, further comprising washing the milled biomass with an aqueous solvent to yield a juice comprising the water-soluble products.

[0054] Illustration 4. The process of any of Illustrations 1-3, wherein removing the saponins comprises foaming the crude extract in a first foaming step to produce a first foam product, wherein the first foam product has a higher concentration of saponins than the crude extract.

[0055] Illustration 5. The process of any of Illustrations 1-4, wherein the first foaming step comprises sparging a gas through the crude extract in a foam fractionation unit to produce the first foam product.

[0056] Illustration 6. The process of any of Illustrations 1-5, wherein the gas comprises air or an inert gas.

[0057] Illustration 7. The process of any of Illustrations 1-6, further comprising foaming the first foam product in a second foaming step to produce a second foam product, wherein the second foam product has a higher concentration of saponins than the first foam product.

[0058] Illustration 8. The process of any of Illustrations 1-7, wherein removing polyphenols from the first residuum stream comprises processing the first residuum stream in an ion- exchange column to extract polyphenols from the first residuum stream and produce the second residuum stream.

[0059] Illustration 9. The process of any of Illustrations 1-8, wherein processing the first residuum stream in the ion-exchange column comprises: flowing the first residuum stream through the ion-exchange column, wherein the polyphenols bind to a resin in the ion- exchange column; collecting the second residuum stream from the ion-exchange column, wherein the second residuum stream has a lower concentration of polyphenols than the first residuum stream; and washing the resin with a solvent to release the bound polyphenols from the resin and produce a polyphenol-rich product streamIllustration

[0060] Illustration 10. The process of any of Illustrations 1-9, wherein the solvent is an alcohol.

[0061] Illustration 11. The process of any of Illustrations 1-10, wherein removing organic acids from the second residuum stream comprises processing the second residuum stream in an ion-exchange column to extract the organic acids from the second residuum stream and produce the third residuum stream.KT Ref.109296-1449036

[0062] Illustration 12. The process of any of Illustrations 1-11, further comprising adjusting the pH of the second residuum stream prior to removing organic acids from the second residuum stream.

[0063] Illustration 13. The process of any of Illustrations 1-12, wherein processing the second residuum stream in the ion-exchange column comprises: flowing the second residuum stream through the ion-exchange column, wherein the organic acids bind to a resin in the ion-exchange column; collecting the third residuum stream from the ion-exchange column, wherein the second residuum stream has a lower concentration of organic acids than the second residuum stream; and washing the resin with a solvent to release the bound organic acids from the resin and produce an organic acid-rich product stream.

[0064] Illustration 14. The process of any of Illustrations 1-13, further comprising filtering the third residuum stream to remove inorganic salts, saccharides, lipids, or proteins.

[0065] Illustration 15. The process of any of Illustrations 1-14, wherein the non-woody plant is Hesperaloe funifera, Hesperaloe nocturna, Hesperaloe parviflora, Hesperaloe chiangii, or a mixture thereof.

[0066] Illustration 16. A process for separating products from a non-woody plant of the genus Hesperaloe, the process comprising: providing a juice obtained from a non-woody plant of the genus Hesperaloe, wherein the juice comprises water-soluble products including saponins, polyphenols, and organic acids; filtering the juice to remove solids and produce a filtered juice; foaming the filtered juice to produce a foam product, wherein the foam product comprises saponins; extracting polyphenols from the filtered juice; and extracting organic acids from the filtered juice.

[0067] Illustration 17. The process of Illustration 16, further comprising heating the juice to a temperature up to 100° C prior to filtering the juice.

[0068] Illustration 18. The process of Illustrations 16 or 17, further comprising acidifying the juice to a pH from 3 to 5 prior to filtering the juice.

[0069] Illustration 19. The process of any of Illustrations 1-18, wherein the polyphenols are extracted from the filtered juice prior to foaming.

[0070] Illustration 20. The process of any of Illustrations 1-19, wherein the organic acids are extracted from the filtered juice by liquid extraction, a membrane separation, electro- membrane membrane separation, or distillation. ExamplesKT Ref.109296-1449036 Examples of Foam Fractionation

[0071] Lab-scale experiments were conducted to test the foaming properties of a juice derived from a non-woody plant of the genus Hesperaloe. A juice derived from derived from a non-woody plant of the genus Hesperaloe was provided in a beaker. An aquarium air pump having a pump capacity up to 2.5 L / min and an aquarium aeration stone were used to generate bubbles in the juice. Two samples of juice were tested to determine foaming properties. The first sample was unfiltered juice, and the second sample was filtered to remove suspended solids from the juice. FIGS.3A and 3B show pictures of the foam produced for the filtered juice after 30 seconds of aeration and after 10 minutes of aeration, respectively. FIGS.3C and 3D show pictures of the foam produced for the unfiltered juice after 30 seconds of aeration and after 10 minutes of aeration, respectively. The filtered juice produced a denser foam that did not collapse when aeration stopped, whereas the unfiltered juice foamed poorly and the foam collapsed when aeration stopped. The experiments shown in FIGS.3A-3D demonstrates that suspended solids significantly inhibit foaming.

[0072] Lab-scale experiments were conducted to determine the effects of dilution and temperature on the foaming properties of a juice derived from a non-woody plant of the genus Hesperaloe. Six portions of juice were diluted with water to produce samples having the concentrations provided in Table 1 below. Each of the samples was added to a beaker including an aquarium air pump having a pump capacity up to 2.5 L / min and an aquarium aeration stone to generate bubbles in the samples. The samples were aerated for 2 minutes. TABLE 1 Juice (mL) Diluent (mL) % Juice. s ows a p es - , , a , o e o g , p o o ae a o . . B shows the results of aeration for 2 minutes for each of Samples 1-6. For each sample shown in Table 1, the concentration of saponins in the juice (prior to foaming) and in the foam product under different operating conditions were measured as described further below. The concentration of saponins was measured in the diluted juice prior to foaming and the foam product by UV-Vis Spectroscopy. Table 2 provides the % difference between the saponinKT Ref.109296-1449036 concentration in the original juice compared to the foam product for Samples 1-6 when foaming was conducted at room temperature. Sample 4 (diluted to 10 % juice) had the best foaming properties for enriching saponins in the foam from the remainder of the juice. Sample 4 also achieved the highest levels of enrichment of saponins in the foam extract compared to Samples 1-3. TABLE 2 Enrichment of e

[0074] Table 3 provideeration and the wt. % of saponins in the foam product for Samples 1-6 when foaming was conducted at 60° C. The tests demonstrated that foaming at an elevated temperature (e.g., above room temperature) can substantially improve enrichment of saponins in the foam. Each of Samples 1-6 produced a foam enriched in saponins in comparison to the original juice. Sample 4 had the best foaming properties for separating saponins from the remainder of the juice. Sample 4 also achieved the highest levels of enrichment of saponins in the foam extract compared to Samples 1-3. Additionally, Sample 4 demonstrated the best foam properties. For example, Sample 4 produced a higher foam density (e.g., a stiffer foam) than the other samples which allowed for easier separation from the juice for foam breaking and further downstream processing. TABLE 3eExample of Extracting Polyphenols from Plant BiomassKT Ref.109296-1449036

[0075] Tests were conducted to determine the feasibility of extracting polyphenols from plant biomass using resin extraction with different elution solvents. Five columns were prepared with five different resins (Resins A-E). The Resins A-E were one of PUROSORB PAD900, PUROLITE A502PS, PUROLITE MN202, PUROLITE A860S, AMBERLITE IRC-120(H), or SUPELITE DAX-8. For each column, 50 mL of one of the resins were loaded into the column using deionized (DI) water. Five portions of 300 mL of 100% filtered juice derived from a non-woody plant of the genus Hesperaloe were each diluted using 300 mL of DI water to produce five 600 ml samples of 50% diluted juice. Each diluted sample was loaded into a column using a pump. Fractions of each sample were flowed through the column and were collected from the column. Then cold DI water was provided to the columns via the pump to flush out any residual juice. After removing all residual juice, elution solvents were provided to the columns to desorb components from the resin. In particular, analytical grade ethanol (greater than 99 % ethanol) and hot DI water (75° C) were used to desorb components from the resin in the columns to test the extraction efficiency of each elution solvent.

[0076] Table 4 provides the amounts of polyphenols and saponins desorbed from the resin column using ethanol as measured by UV-vis Spectrometry. The wt. % of saponins and polyphenols were based on the total weight of the eluent from the column. The hot DI water did not desorb detectable amounts of saponins and polyphenols trapped by the resin. Table 4 only provides the amounts of polyphenols and saponins desorbed from the resin column using ethanol. TABLE 4 Pol henols Sa onins

[0077] The hot DIwa e o e goo e ac o p ope es o solids when used as the desorption solvent. Ethanol desorbed far more solids from the resins than hot DI water. For example, ethanol desorbed a maximum of roughly 50 wt. % of the total solids trapped by resins A, C, and E from the diluted juice from the resin columns. Resins D and E showed the largest enrichment of saponins when using ethanol to desorb.KT Ref.109296-1449036

[0078] As discussed, different fractions were collected from the column when the hot DI water and ethanol were used to desorb solids from the resin. The different fractions are shown below in Table 5. FIG.5 shows the coloration for various fractions that eluted from the column. The color of the fractions demonstrates that ethanol desorbed more compounds than hot water. The darker color of the fractions taken from the column is indicative of the amounts of saponins and polyphenols desorbed from the resins. FIG.5 shows that desorption with ethanol separated higher amounts of saponins and polyphenols from the resin. The hot water desorption resulted in very little color change in each of fractions taken from the column demonstrating very little to no desorption of the water-soluble products. For example, the 0-20 mL fraction taken after water desorption was clear, whereas the 0-20 mL fraction taken after water desorption was darker. In each of fractions 1-4, the resins desorbed with ethanol had a darker color than resins desorbed with hot water. TABLE 5 Fraction Ethanol Hot DI waterExtracting Organic Acids from Plant Biomass

[0079] Tests were conducted to determine the feasibility of extracting organic acids from plant biomass using the Lamar method and resin extraction. Samples of solutions containing 600 mg / L were made using 80 mL of 10 % juice derived from a non-woody plant of the genus Hesperaloe and 420 mL 0.1M NaOH. The solution was stirred for 1 hour using a stir bar to ensure adequate mixing. The solution was transferred to a centrifuge tube and spun at 4000 x g for 10 mins. Insoluble precipitates were discarded and the supernatant returned to a 500 mL Erlenmeyer flask. Concentrated HCl was added to the Erlenmeyer flask until the solution reached a pH of 1.0 ±0.05, and solution was left to allow humic acid to precipitate

[0080] Fulvic acids were separated from other small organic acids in the acidified supernatant using a DAX-8 Column (Macroporusacrylic ester) by passing the acidified solution through the column at 7 mL / min. DI water was used to wash the column until theKT Ref.109296-1449036 column effluent was within 0.015 absorbance units of a DI water blank (~400 mL). Fulvic acid was desorbed by stirring column media in 0.1M NaOH overnight and subsequent filtering. A higher absorption was measured in the desorb solution compared to reference NaOH, indicating the presence of fulvic acid-like compounds.

[0081] Tests were conducted to determine the effects of dilution on extracting organic acids from plant biomass using the same experimental procedure described above. The Lamar method appears to separate fulvic acid like compounds based on the data in Table 7. Sample 8 with a 50 % dilution showed the highest concentration of solids. TABLE 7 Sample NaOH Dilution Absorbance Solids inur ca on o u ce er ve rom an omass

[0082] Lab-scale experiments were conducted to determine the effects of clarifying and deashing a juice derived from a non-woody plant of the genus Hesperaloe. The juice was clarified and deashed following the scheme shown in FIG.6. The juice was first centrifuged at 4696 G for 30 minutes to obtain a clarified juice. The clarified juice was then deashed in a resin exchange column shown in FIG.7 to obtain a deashed juice. The resin exchange column included a first region including a cation exchange resin and a second region including a weak base anion resin. The cation exchange resin comprised DupontTM AmberLiteTM FPC88 UPS Na ion exchange resin and the weak base anion resin comprised DupontTM AmberLiteTM FPA66.

[0083] During deashing, the cation exchange resin removes cations such as sodium, potassium, iron, magnesium, and calcium, as well as some crude proteins (amphoteric proteins). During this exchange process, hydrogen cations are released from the exchange sites. The weak base anion resin (free base form) removes some acids (e.g., organic acid) and colorant (e.g., polyphenol) from the juice. As shown in Table 8 below, the deashed juice is relatively free of ions.KT Ref.109296-1449036 TABLE 8 Raw Juice Clarified Juice Deashed Juice

[00] e c ar cat on process remove some suspen e so s n t e raw ju ce w ch is reflected in minor compositional changes, i.e., lowered crude proteins and significant iron removal. The deash resins removed all minerals to below a detectable limit. At the same time, the deash resins removed additional crude protein in comparison to the clarification step. The minerals captured by the ion exchange resins can be released during a regenerative stage to recover minerals.

Claims

KT Ref.109296-1449036 WHAT IS CLAIMED IS:

1. A process for separating products from a non-woody plant of the genus Hesperaloe, the process comprising: providing a crude extract obtained from a non-woody plant of the genus Hesperaloe, wherein the crude extract comprises water-soluble products including saponins, polyphenols, and organic acids; removing saponins from the crude extract to produce a first residuum stream comprising polyphenols and organic acids, wherein the first residuum stream has a lower concentration of saponins than the crude extract; removing polyphenols from the first residuum stream to produce a second residuum stream comprising organic acids, wherein the second residuum stream has a lower concentration of polyphenols than the first residuum stream; and removing organic acids from the second residuum stream to produce a third residuum stream, wherein the third residuum stream has a lower concentration of organic acids than the second residuum stream.

2. The process of claim 1, wherein providing the crude extract comprises milling a biomass derived from a non-woody plant of the genus Hesperaloe to yield a milled biomass and the crude extract.

3. The process of claim 2, further comprising washing the milled biomass with an aqueous solvent to yield a juice comprising the water-soluble products.

4. The process of claim 1, wherein removing the saponins comprises foaming the crude extract in a first foaming step to produce a first foam product, wherein the first foam product has a higher concentration of saponins than the crude extract.

5. The process of claim 4, wherein the first foaming step comprises sparging a gas through the crude extract in a foam fractionation unit to produce the first foam product.

6. The process of claim 5, wherein the gas comprises air or an inert gas.KT Ref.109296-1449036 7. The process of claim 4, further comprising foaming the first foam product in a second foaming step to produce a second foam product, wherein the second foam product has a higher concentration of saponins than the first foam product.

8. The process of claim 1, wherein removing polyphenols from the first residuum stream comprises processing the first residuum stream in an ion-exchange column to extract polyphenols from the first residuum stream and produce the second residuum stream.

9. The process of claim 8, wherein processing the first residuum stream in the ion-exchange column comprises: flowing the first residuum stream through the ion-exchange column, wherein the polyphenols bind to a resin in the ion-exchange column; collecting the second residuum stream from the ion-exchange column, wherein the second residuum stream has a lower concentration of polyphenols than the first residuum stream; and washing the resin with a solvent to release the bound polyphenols from the resin and produce a polyphenol-rich product stream.

10. The process of claim 9, wherein the solvent is an alcohol.

11. The process of claim 1, wherein removing organic acids from the second residuum stream comprises processing the second residuum stream in an ion-exchange column to extract the organic acids from the second residuum stream and produce the third residuum stream.

12. The process of claim 11, further comprising adjusting the pH of the second residuum stream prior to removing organic acids from the second residuum stream.

13. The process of claim 11, wherein processing the second residuum stream in the ion-exchange column comprises: flowing the second residuum stream through the ion-exchange column, wherein the organic acids bind to a resin in the ion-exchange column;KT Ref.109296-1449036 collecting the third residuum stream from the ion-exchange column, wherein the second residuum stream has a lower concentration of organic acids than the second residuum stream; and washing the resin with a solvent to release the bound organic acids from the resin and produce an organic acid-rich product stream.

14. The process of claim 1, further comprising filtering the third residuum stream to remove inorganic salts, saccharides, lipids, or proteins.

15. The process of claim 1, wherein the non-woody plant is Hesperaloe funifera, Hesperaloe nocturna, Hesperaloe parviflora, Hesperaloe chiangii, or a mixture thereof.

16. A process for separating products from a non-woody plant of the genus Hesperaloe, the process comprising: providing a juice obtained from a non-woody plant of the genus Hesperaloe, wherein the juice comprises water-soluble products including saponins, polyphenols, and organic acids; filtering the juice to remove solids and produce a filtered juice; foaming the filtered juice to produce a foam product, wherein the foam product comprises saponins; extracting polyphenols from the filtered juice; and extracting organic acids from the filtered juice.

17. The process of claim 16, further comprising heating the juice to a temperature up to 100° C prior to filtering the juice.

18. The process of claim 16, further comprising acidifying the juice to a pH from 3 to 5 prior to filtering the juice.

19. The process of claim 16, wherein the polyphenols are extracted from the filtered juice prior to foaming.KT Ref.109296-1449036 20. The process of claim 16, wherein the organic acids are extracted from the filtered juice by liquid extraction, a membrane separation, electro-membrane membrane separation, or distillation.

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