Extraction and refining of vegetable cuticular waxes from aqueous dispersion with the use of capture agent

Abstract

Extraction and refining of cuticular waxes from plants using an aqueous dispersion with a capturing agent. This is a method for extracting and refining wax from plant material.In summary, the method comprises the steps of (a) providing plant material comprising cuticular wax, (b) dissociating the cuticular wax from the plant material, thereby obtaining a sample comprising plant-derived cuticular wax and plant material with wax removed in an aqueous suspension, (c) solubilizing the plant-derived cuticular wax by increasing the sample temperature, (d) separating the suspension into a solid fraction and a liquid fraction comprising molten plant-derived cuticular wax, (e) mixing the liquid fraction with a capturing agent, wherein the capturing agent has a boiling point above 85 °C at ambient pressure, (f) separating the mixture into an aqueous fraction and a capturing fraction comprising capturing agent and plant-derived cuticular wax, (g) recovering the plant-derived cuticular wax from the capturing fraction.

Description

1 / 40 “EXTRACTION AND REFINING OF VEGETABLE CUTICULAR WAXES FROM AQUEOUS DISPERSION USING A CAPTURE AGENT” FIELD OF THE INVENTION

[0001] The present invention relates to a method for extracting and refining cuticular wax from plant material. BACKGROUND OF THE INVENTION

[0002] Vegetable waxes are typically supplied from two different processes, the first being a byproduct of vegetable oil production, to which group waxes such as soy wax, rapeseed wax, cottonseed stearin, rice bran wax and palm wax belong, and the second being a more or less artisanal production process of natural waxes such as candelilla wax, carnauba wax and ouricurium wax. Wax products such as jojoba wax or castor wax are also commercially available. Other commercially relevant wax sources are montana wax, beeswax, lanolin, synthetic wax and paraffin waxes, the latter being by far the largest in volume, originating as a byproduct of petrochemical refining.

[0003] Waxes derived from vegetable oil are frequently used in candle production because they are characterized by a medium / low melting point and are therefore less suitable for more demanding applications requiring heat resistance and shine / luster—such as car wax, boat wax, and cosmetics. These characteristics are met by paraffin waxes and synthetic waxes, complemented by premium natural waxes such as carnauba or candelilla wax.

[0004] Mineral / fossil waxes account for about 75% of global wax production, with synthetic waxes accounting for another 20%, totaling 95%. The remaining waxes represent less than 5% of global production, and this scarcity is a major barrier to increasing the use of natural waxes. Petition 870210113006, dated 06 / 12 / 2021, page 5 / 53 2 / 40

[0005] With the increased interest in fossil-free ingredients and materials, there is also a significant demand for wax produced from renewable sources. Natural waxes are, as mentioned above, scarce; in fact, the availability of such waxes is far from sufficient to replace paraffin, and attempts to increase the cultivation of plants that provide wax from Candelilla (Euphorbia antisyphilitica), Carnauba (Copernicia prunifera), and Ouricuri (Syagrus coronata) have not been successful so far; and the overexploitation of sources such as the Candelilla shrub is leading to a greater scarcity of natural waxes in demand.

[0006] When supply is limited, security of supply for high-volume applications, such as cosmetics, paints, and polishes, becomes a problem. To overcome this problem, a natural wax must be abundant and exhibit acceptable quality, defined for example by its melting point, hardness, and / or color.

[0007] The extraction of wax from lignocellulosic plant material, such as bark, has been previously described (US2781336), in which the wax is extracted using benzene - a volatile, explosive, toxic and flammable hydrocarbon.

[0008] It has been previously demonstrated that wax can be extracted from various plants, including cereals, grasses, etc. (WO 2015 / 185685). As an example, wheat straw has a wax content of 1 to 3%. The annual global production of wheat exceeds 700 million tons, which brings an estimated 3 to 400 million tons of straw. The potential global supply of wheat straw wax could therefore be 3 to 9 million tons, which are magnitudes greater than the current supply of natural waxes. Expanding this to other common agricultural crops, there is great potential to utilize a residual crop product and still meet the supply demand of industries wishing to introduce larger quantities of natural waxes into their product lines. Although the agricultural base is there to provide abundant quantities of wax, the very low wax content in Petition 870210113006, dated 06 / 12 / 2021, p. 6 / 53 3 / 40 plant biomass and subsequent dilution in the extraction process makes it very difficult to recover the wax in reasonable yields using conventional techniques, without resorting to solvent extraction methodologies.

[0009] The present invention relates to a method for refining wax from common agricultural plant material, such as cereal straw.

[0010] The methods previously described for removing wax from plant material by combining mechanical, thermal, and enzymatic methods have in common that an aqueous liquid is added along with enzymes during the wax removal process. The released wax is then diluted, dissolved, suspended, or otherwise present in a larger volume, therefore at lower concentrations. If, for example, a straw paste of 20% dry matter (DM) is used, the 1% wax in the straw becomes a 0.2% wax in the aqueous paste.

[0011] Currently, the main refining tool for natural waxes such as beeswax, carnauba wax, and candelilla wax is the filtration of molten wax (usually kept below 100 °C) using an industrial filter press. For example, with carnauba wax, filtration, centrifugation, and bleaching are generally performed: the raw wax is boiled in water, followed by filtration and separation of the wax from the water. The wax isolated from this is then melted and filtered again. As another example, with candelilla wax, the wax is melted and then filtered through a suitable matrix, such as "Fullers Earth" or activated charcoal; and / or optionally it can be further bleached using hydrogen peroxide. Yet another example, with beeswax, simple melting and filtration is performed.

[0012] Depending on the application of the extracted wax, the color of the wax can be of great relevance – for example, in cosmetics. To compare the color of waxes, the Gardner color method can be used. The Gardner color scale ranges from 1 (white) to 18 (dark brown). As Petition 870210113006, dated 06 / 12 / 2021, p. 7 / 53 4 / 40 as seen in Table 1, cereal straw waxes traditionally appear darker compared to refined commercial waxes. TABLE 1. GARDNER COLOR FOR COMMERCIAL AND CEREAL WAXES1 Wax Type Color Gardner Commercial Waxes Lanolin Beeswax Candelilla Wax Carnauba Wax 9 3 9 9 Cereal straw waxes Wheat straw wax 18 Barley straw wax > 18 Oat straw wax________________________________> 18_______________ 1Sin EHK 2012. PhD Thesis: The extraction and fractionation of biomass waxes, New York University.

[0013] The present invention provides an improved method for refining natural waxes, a method that overcomes the difficulties of isolating a product at low concentration and provides a highly pure wax product compared with current refining tools used for natural waxes. SUMMARY OF THE INVENTION

[0014] A first aspect of the invention relates to a method for extracting and refining cuticular wax from plant material, said method comprising the steps of

[0015] a. provide plant material comprising cuticular wax,

[0016] b. dissociate cuticular wax from said plant material provided in step (a), thereby obtaining a sample comprising plant-derived cuticular wax and plant material with wax removed in an aqueous suspension, Petition 870210113006, dated 06 / 12 / 2021, page 8 / 53 5 / 40

[0017] c. solubilize said vegetable-derived cuticular wax by increasing the temperature of the sample obtained in step (b) to a temperature higher than the melting point of said vegetable-derived cuticular wax,

[0018] d. separate the suspension obtained in step (c) into a solid fraction and a liquid fraction, wherein said liquid fraction comprises melted vegetable-derived cuticular wax,

[0019] e. mix said liquid fraction from step (d) with a capturing agent, wherein said capturing agent is a water-immiscible liquid in which said vegetable-derived cuticular wax is soluble,

[0020] f. separate the mixture obtained in step (e) into an aqueous fraction and a capture fraction, wherein said capture fraction comprises said capture agent and said vegetable-derived cuticular wax,

[0021] g. recover said vegetable-derived cuticular wax from said capture fraction of step (f) wherein the capture agent has a boiling point above 85 °C at ambient pressure.

[0022] A second aspect of the invention relates to a vegetable wax composition obtained by the method described above, wherein said vegetable wax composition comprises less than 3% of a capturing agent, less than 1% of a C1-C4 alcohol, such as ethanol.

[0023] A third aspect of the invention relates to a vegetable wax composition obtained by the method described above for use in cosmetics. BRIEF DESCRIPTION OF THE FIGURES

[0024] Figure 1: a flowchart outlining different stages of the invention process, as described in example 1

[0025] Figure 2: a GC chromatogram of wheat straw extracted with chloroform; peaks before 7.5 minutes are fatty acids (mainly C16 and C18), peaks at 9.5 to 12.5 minutes are mainly alkanes, aldehydes and fatty alcohols, peaks from 14 to 17.5 minutes are mainly Petition 870210113006, dated 06 / 12 / 2021, page 9 / 53 6 / 40 sterols and beta-diketone, while the peaks after 18 minutes are waxy esters.

[0026] Figure 3: a GC chromatogram of the wax product prepared in example 1.2; the peaks before 7.5 minutes mainly represent the residual capture agent.

[0027] Figure 4: a GC chromatogram of the wax product prepared in example 1.3; the peaks before 7.5 minutes mainly represent the residual capture agent.

[0028] Figure 5: a GC chromatogram of the wax product prepared in example 1.4. DETAILED DESCRIPTION OF THE INVENTION DEFINITIONS

[0029] “Capturing agent” (abbreviated CA) means a liquid with the following properties: (i) it is a liquid that is not miscible in water, (ii) the wax is soluble in the capturing agent, such as a liquid solvent for all components of natural waxes, (iii) it is a liquid that is miscible with C1-C4 alcohol.

[0030] “Plant material with wax removed” means plant material that has been treated in a way that removes / disassociates the cuticular wax from the plant material, such as more than 50, 55, 60, 65, 70, 75, 80, 85, 90% or even more than 95% of all plant wax has been removed, wherein the wax content is determined by the method provided in section II of this application.

[0031] “Plant or lignocellulosic material” or “plant or lignocellulosic biomass” means a broad and varied group of plant parts from many species. The terms plant material and lignocellulosic are used interchangeably. The plant material that can be used as starting material in the present invention comes from macroscopic multicellular plants comprising stems and leaves that are (at least one of them) covered by an outer layer or epidermis that is coated with a waterproof waxy protective layer, which is punctuated by Petition 870210113006, dated 06 / 12 / 2021, page 10 / 53 7 / 40 specialized pores, known as stomata, that regulate gas and water exchange.

[0032] “Cereal straw” means the stalk and leaves of the cereal plant that remain after the grains of the cereal have been harvested.

[0033] “Wax” or “wax components” means all the various forms of wax coating the surface of plant material. It is used collectively to describe the waxy components of the cuticles (cuticular wax) covering the aerial parts of plants, including wax on the surface of the plant (epicuticular wax) as well as wax just below the surface of the plant (intracuticular wax). Wax comprises very long linear chain (VLC) compounds, which include varying rates of fatty acids, primary and secondary alcohols, esters, aldehydes, free fatty acids, alkanes, and ketones. In addition, cyclic compounds such as pentacyclic triterpenoids, alkylresorcinols, sterols, and steryl esters occur in the wax of many species.The lipids that constitute the cell walls of macroscopic or microscopic (unicellular) plants are not considered "wax" as in the present context, but may be present in a small quantity in the final wax product if released during mechanical and / or enzymatic treatment. THE INVENTION

[0034] The present invention relates to the refining of cuticular wax from plant material. I. Wax Refining Method

[0035] Figure 1 provides an illustrative example of the present invention, which describes the different steps of the process to arrive at the desired products. All steps of the process can be carried out as illustrated, some steps can be omitted, some steps can be combined, and additional steps can be added. A detailed description is provided in the following sections. Petition 870210113006, dated 06 / 12 / 2021, page 11 / 53 8 / 40

[0036] In one aspect, the present invention relates to a method for refining cuticular wax from plant material, which produces an enhanced wax product with desired properties for further downstream processing. In a preferred embodiment, the present invention provides a wax refining method comprising the steps of:

[0037] (a) supplying plant material comprising cuticular wax,

[0038] (b) dissociate said cuticular wax from said plant material provided in step (a), in order to obtain a sample comprising plant-derived cuticular wax and plant material with wax removed in an aqueous suspension,

[0039] (c) solubilize said vegetable-derived cuticular wax by increasing the temperature of the sample obtained in step (b) to a temperature higher than the melting point of said vegetable-derived cuticular wax,

[0040] (d) separate the sample obtained in step (c) into a solid fraction and a liquid fraction, wherein said liquid fraction comprises melted vegetable-derived cuticular wax,

[0041] (e) mixing said liquid fraction from step (d) with a capturing agent, wherein said capturing agent is a liquid immiscible in water in which said vegetable-derived cuticular wax is soluble,

[0042] (f) separate the mixture obtained in step (e) into an aqueous fraction and a capture fraction, wherein said capture fraction comprises said capture agent and said vegetable-derived cuticular wax,

[0043] (g) recover said vegetable-derived cuticular wax from said capture fraction of step (f).

[0044] In another embodiment, the present invention provides a method as described above in steps (a)-(g), which further comprises the step of: Petition 870210113006, dated 06 / 12 / 2021, page 12 / 53 9 / 40

[0045] (h) bleaching of said vegetable-derived cuticular wax recovered in step (g).

[0046] In yet another embodiment, the present invention provides a method as described above in steps (a)-(g), optionally including step (h), which further comprises the step of:

[0047] (i) to formulate said vegetable-derived wax recovered in step (g) or said bleached wax obtained in step (h) into valuable products.

[0048] According to step (a) of the method of the present invention, a plant material comprising cuticular wax is provided. In one embodiment of the invention, the plant material originates from agricultural crops, such as cereals, sugar cane, palm trees, high-energy grasses. In a preferred embodiment, the wax-removed lignocellulosic material of the invention originates from cereals, selected from the group consisting of wheat, rye, barley, oats, sorghum, rice, triticale, etc., and combinations thereof. In another embodiment, the wax-removed lignocellulosic material of the invention originates from a high-energy grass, such as Miscanthus. The plant material can be provided in different forms, such as untreated natural plant material, or processed, such as in the form of, for example, pellets.

[0049] In one embodiment, cereal and grass straws, rapeseed straw, corn stalks, carnauba wax-producing plants (e.g., Copernicia prunifera, Copernicia cerifera), candelilla wax-producing plants (e.g., Euphorbia antisyphilica, the candelilla plant) or cactus are preferred plants for wax extraction and rendering by the present invention. Pineapple leaves and banana leaves are also preferred. In fact, most known waxy leaves are excellent sources of plant material for the method of the present invention. In a preferred embodiment, the lignocellulosic wax-removed material of the invention originates from cereal straw, selected from the group consisting of wheat, rye, barley, oats, Petition 870210113006, dated 06 / 12 / 2021, p. 13 / 53 10 / 40 sorghum, rice, triticale, etc. and combinations thereof; more preferably wheat straw. This cereal straw is the stems and leaves of the plant that remain after the harvest of the cereal grains.

[0050] According to step (b) of the method of the present invention, the wax is dissociated from the plant material provided in step (a), thus obtaining a sample comprising plant-derived wax and plant material with wax removed in an aqueous suspension. In one embodiment, the plant material was in step (b) treated in such a way that more than 50% of the wax was dissociated from the remaining plant material, such as treated in such a way that provides a sample comprising plant-derived wax and plant material with wax removed, in which more than 55, 60, 65, 70, 75, 80, 85, 90%, or even more than 95% of the wax in the original plant material was dissociated from the plant material, but is still present in the sample.

[0051] In step (b), the wax can be dissociated from the plant material by any method known in the art, such as by mechanical removal of the wax from the surfaces or even by hydrothermal and wet oxidation pretreatment.

[0052] In one embodiment, the wax is dissociated from the plant material by a mechanical method. In another embodiment, the wax is dissociated from the plant material by enzymatic treatment using enzymes suitable for degrading proteins associated with cuticular wax in the plant material. In a preferred embodiment, the wax is dissociated from the plant material by a method that uses a combination of mechanical and enzymatic treatment, wherein the enzymatic treatment is facilitated by enzymes suitable for degrading proteins associated with wax in the plant material. A similar method for removing wax from plant material is described in WO 2015 / 185685.

[0053] In one embodiment, the plant material is subjected to a dry mechanical treatment. Thus, in one embodiment of the present invention, the dry mechanical treatment comprises cutting, grinding Petition 870210113006, dated 06 / 12 / 2021, page 14 / 53 11 / 40 and / or crushing, as a mechanical treatment, is selected from the group consisting of grinding, hammer mill, disc mill, and combinations thereof. In some embodiments, the plant material may need to be dried prior to dry mechanical treatment. As part of the dry mechanical treatment, the plant material may be cut into suitable lengths for subsequent treatment in a mill suitable for deforming the plant material. Primary grinding may result in cuts between approximately 5 and 20 cm in length, between 5 and 15 cm, or between 5 and 10 cm in length. Grinding further chops the plant material into pieces less than 5 cm in length, less than 3 cm, less than 2 cm, or less than 1 cm. The processing equipment may be adjusted to optimize the sizes of the plant material according to the downstream use of the mechanically treated plant material.

[0054] Dry mechanical treatment can serve to deform the outer surface of plant material, preferably after drying, so that the wax coating is cracked and released, obtaining a plant material with partially removed wax and to open the surface of the plant material to help facilitate water penetration during subsequent wet processing.

[0055] The material obtained after dry mechanical treatment is optionally fractionated by size. In one embodiment of the present invention, the fractionation is done by a sieving treatment in order to obtain two fractions, the first fraction passing through the sieve mesh and the second fraction being retained by the sieve mesh. The mesh size of this sieve is in the range of 2 to 12 mm, such as in the range of 4 to 10 mm, for example, in the range of 6 to 8 mm. In a preferred embodiment, the mesh size is 8 mm. The sieving treatment may comprise one or more sieves having equal or different mesh sizes. The sieving treatment may be carried out in order to separate a fraction enriched in broken and liberated wax (the first fraction passing through the sieve) from a Petition 870210113006, dated 06 / 12 / 2021, page 15 / 53 12 / 40 fraction of partially removed plant wax material (the second fraction retained by the sieve).

[0056] In one embodiment, the dry mechanically treated material or a selected fraction of the dry mechanically treated material is suspended in an aqueous solution comprising one or more protease and / or pectinase enzymes, and the temperature and pH are preferably adjusted to optimize the activity of the added enzyme(s).

[0057] Proteases are involved in the digestion of long protein chains into shorter fragments, which break down the peptide binders that link amino acid residues. In one embodiment, proteases may be selected from proteases that separate terminal amino acids from the protein chain (exopeptidases, such as aminopeptidases, carboxypeptidase A). In another embodiment, proteases may be selected from pectinases that attack the internal peptide binders of a protein (endopeptidases, such as trypsin, chymotrypsin, pepsin, papain, elastase); or from the group consisting of serine proteases, threonine proteases, cysteine ​​proteases, aspartate proteases, glutamic acid proteases, and metalloproteases.In yet another embodiment, proteases can be selected from commercially available proteases, such as those selected from the group consisting of Alcalase® (a protease from Bacillus licheniformis), Neutrase® (a protease from Bacillus amyloliquefaciens, both available from Novozymes, Denmark), and Promod® (a protease from Ananas comosus, available from BioCatalysts, UK). In yet another embodiment, a combination of two or more commercial protease enzymes or protease enzyme products can be used to degrade plant proteins.

[0058] Pectinases are involved in the breakdown of pectin, a polysaccharide found in the cell walls of plants, in which, for example, cellulose fibrils are often embedded. In Petition 870210113006, dated 06 / 12 / 2021, page 16 / 53 13 / 40 In one embodiment, pectinases can be selected from a group consisting of (I) pectin hydrolases that hydrolyze the pectic acid main structure into pectins (endopolygalacturonase, EC 3.2.1.15; exopolygalacturonase, EC 3.2.1.67), (II) pectin lyases that degrade pectic acid via elimination (endopolygalacturonase lyase, EC 4.2.2.2; exopolygalacturonase lyase, EC 4.2.2.9; endopolymethyl-d-galactosiduronate lyase, EC 4.2.2.10) and (III) pectin esterases, which cleave the methyl ester clump (pectin methyl esterase, EC 3.1.1.11). Pectinases are widely available on the market, and most are mixtures that incorporate all three types of enzymes mentioned.In another embodiment, pectinases may be selected from a group consisting of Pectinex® (a mixture of pectinases from Aspergillus Niger, available from Novozymes, Denmark) and Pectinase 947 L® (a mixture of pectinases available from BioCatalysts, UK; Pektozyme, a range of active pectin enzyme blends supplied by DuPont). In yet another embodiment, a combination of two or more pectinase enzymes or commercial pectinase enzyme products may be used to degrade vegetable pectins.

[0059] A combination of two or more protease(s) and / or pectinase(s) and / or commercial protease product(s) and / or commercial pectinase product(s) that can be applied to degrade plant proteins and / or pectins.

[0060] In one embodiment, one or more enzymes may be added to the mixture to obtain an enzyme concentration in the range of 0.01 to 2% w / w, such as in the range of 0.03 to 1.8% w / w, for example, in the range of 0.05 to 1.6% w / w, such as in the range of 0.07 to 1.4% w / w, for example, in the range of 0.09 to 1.2% w / w. The enzyme concentration depends on the enzyme activity; however, it may be preferred that the enzyme concentration in the mixture be from 1 to 2% w / w. In one embodiment of the present invention, it may be preferred that the enzyme activity be in the range of 1,000 to 12,000 U / g, such as in the range of 2,000 to 10,000 U / g, for example, in the range of 3,000 to Petition 870210113006, dated 06 / 12 / 2021, p. 17 / 53 14 / 40 9,000 U / g, as in the range of 4,000 to 8,000 U / g, for example, in the range of 5,000 to 7,000 U / g.

[0061] In order to benefit as much as possible from enzymatic treatment, the conditions for enzymatic activity, such as temperature, pH, salt concentration, etc., must be optimized in relation to the enzyme(s) used. The addition of acid or base to the paste / mixture may be necessary to achieve the ideal pH conditions.

[0062] The ideal temperature during enzyme treatment is selected to suit the enzyme(s) used. The temperature can be 25, 30, 35, 40, 45, 50 °C or even higher if thermostable enzymes are used. In one embodiment, the temperature during enzyme treatment is adjusted in the range of 30 to 70 °C, such as in the range of 35 to 65 °C, for example, in the range of 40 to 60 °C, for example, in the range of 45 to 55 °C, preferably in the range of 45 to 65 °C, more preferably in the range of 50 to 60 °C to optimize the activity of the enzymes used in carrying out the targeted hydrolysis of cell wall components.

[0063] In another embodiment, the pH during enzyme treatment is in the range of 3.5 to 7.0, such as in the range of 4.0 to 7.0, for example, in the range of 4.0 to 6.0, preferably in the range of 4.5 to 6.0 to optimize the activity of the enzymes used in carrying out the targeted hydrolysis of cell wall components. The pH can be adjusted by adding at least one acid and / or buffer selected from the group consisting of phosphoric acid, hydrochloric acid, sulfuric acid, phosphate buffers, acetate buffers and combinations thereof. In a preferred embodiment, the acid is phosphoric acid.

[0064] In order to obtain ideal exposure of the biomass components to the enzymes, agitation is preferably applied and can be selected from the group consisting of mechanical agitation and / or agitation by bubbling gas or compressed air and / or vessel rocking. Applicable mechanical agitators can be selected from the group Petition 870210113006, dated 06 / 12 / 2021, page 18 / 53 15 / 40 consisting of anchor agitators, blade agitators, K agitators, paddle agitators or any combination thereof.

[0065] In a preferred embodiment, hydrolysis under agitation is carried out for 0.5 to 5.0 hours, such as in the range of 0.5 to 4.0 hours, for example, in the range of 0.5 to 3.0 hours, for example, in the range of 1.0 to 2.5 hours, for example, in the range of 1.0 to 2.0 hours, for example, preferably in the range of 1.0 to 1.5 hours, preferably for 1.5 hours.

[0066] Mechanically and enzymatically treated dried material can be subjected to wet mechanical treatment. Wet mechanical treatment can be simultaneous with enzymatic treatment, periodically / intermittently during enzymatic treatment, or a subsequent mechanical treatment. In one embodiment of the invention, the wet mechanical treatment is selected from the group consisting of cone refiners, disc-type refiners, atmospheric refiners, pressurized refiners and combinations thereof; or wet grinding, such as toothed colloid milling. Such wet refining or grinding can be repeated as many times as desired: 1, 2, 3 or 4 repetitions will normally be sufficient. Alternatively, or additionally, very powerful agitation can be applied.

[0067] In a preferred embodiment of the present invention, the dissociation of the cuticular wax from the plant material in step (b) to obtain a sample comprising plant-derived cuticular wax and plant material with wax removed is carried out by a method comprising the step of:

[0068] (i) subjecting the plant material to a dry mechanical treatment,

[0069] (ii) optionally fractionate the material obtained in step (i) by size, Petition 870210113006, dated 06 / 12 / 2021, page 19 / 53 16 / 40

[0070] (iii) suspend the material obtained in step (i) or a selected fraction obtained in step (ii) in an aqueous liquid comprising one or more protease and / or pectinase enzymes,

[0071] (iv) optionally, subject the mixture obtained in step (iii) to wet mechanical treatment,

[0072] According to step (c) of the method of the present invention, the temperature of the aqueous sample obtained in step (b) is increased to solubilize the vegetable-derived wax. The temperature is increased to melt and liquefy the wax, so that the vegetable material with wax removed and other solids can be separated from a liquid part comprising water, water-soluble vegetable material and the melted waxes. The wax may be totally or partially liquefied, depending on the composition of the wax and the temperature.

[0073] Table 2 provides the melting temperature of a wide variety of waxes. In a preferred embodiment of the invention, the temperature is increased to a temperature higher than the melting point of the vegetable-derived wax in question based on its origin, as specified by Table 2. Petition 870210113006, dated 06 / 12 / 2021, page 20 / 53 17 / 40 TABLE 2 · MELTING POINT OF WAXES Type of wax Source Melting point (°C) Animal and insect wax Beeswax Bees 62 to 64 Lanolin Sheep wool 36 to 42 Spermaceti Sperm whale skull 42 to 50 Vegetable waxes Candelilla Euphorbia cerifera 68 to 72 Carnauba Copenicia cerifolia 62 to 86 Castor Hydrogenated oil of Rianus communis 62 Cottonseed Stearin Cottonseed 68 to 71 Jojoba Hydrogenated oil of Simmonds ia californica 60 to 70 Ouricury Syagrus coronata (palm) 81 to 84 Palm oil wax Vegetable oil byproduct 62 to 60 Rapeseed wax Vegetable oil byproduct 36 to 39 Rice bran wax Rice bran oil byproduct 77 to 86 Soybean Byproduct Vegetable oil 56 to 60 Wheat straw wax ^64 Barley straw wax -65 Oat straw wax ^64 Mineral / fossil waxes Montan Lignite / Coal 84 to 90 Vaseline Paraffin 63 Microcrystalline Slack wax Paraffin 49 Microcrystalline Vaseline Paraffin 58 Synthetics Polyethylene wax Ethylene various Fischer Tropsch Gas straight chain hydrocarbonsVarious synthesis methods: Synthetic ester waxes; Fatty acid + fatty alcohol synthesis (various).

[0074] In one embodiment, the temperature of the suspension obtained in step (b) is increased to 60 to 90 °C, such as in the range of 65 to 90 °C, for example, in the range of 67 to 85 °C, such as in the range of 75 to 85 °C and preferably to 80 °C. In another embodiment, the temperature of the sample obtained in step (b) is increased to above 70 °C, preferably above 80, 90 or 95 °C.

[0075] The temperature can be increased by any standard means of raising the temperature of an aqueous solution. In a preferred embodiment, the temperature of the aqueous sample obtained in step (b) is adjusted by heat exchange, hot water injection or steam injection, or even a combination thereof. Petition 870210113006, dated 06 / 12 / 2021, p. 21 / 53 18 / 40

[0076] According to step (d) of the method of the present invention, the suspension obtained in step (c) is separated into a solid fraction and a liquid fraction comprising melted vegetable-derived wax.

[0077] In principle, any known method and device that can be applied to separate a solid fraction from an aqueous suspension can be applied. In one embodiment, the separation in step (d) is carried out by a method selected from the group consisting of decantation, centrifugation and filtration. In another embodiment, the removal of the solid plant material with wax removed from the aqueous composition is carried out using a mechanical device selected from the group comprising a centrifuge, a decanter, a filter, a press or an extruder.

[0078] In one embodiment, the separation is carried out using a centrifuge decanter, which produces a liquid upper phase comprising dissolved solids, including vegetable-derived wax in the form of a molten suspension and emulsion droplets, and a fibrous phase comprising the vegetable component with removed wax and residual insoluble material. In another embodiment, the separation can be carried out by any form of sieving / filtration, using any molecular size as desired, and the filtration device can be selected from a fine mesh filter, pressurized filter, belt filter, filter press, and combinations thereof, which similarly result in a fibrous product with removed wax and a liquid comprising the vegetable-derived wax.

[0079] In one embodiment, the temperature maintained during separation in step (d) is in the range of 65 to 95 °C, such as in the range of 65 to 90 °C, for example, in the range of 75 to 85 °C, such as in the range of 80 to 85 °C and preferably 80 °C. In another embodiment, the temperature maintained during separation in step (d) is above 70 °C, preferably above 80, 90 or 95 °C. Petition 870210113006, dated 06 / 12 / 2021, p. 22 / 53 19 / 40

[0080] The solid fraction comprising dewaxed fibrous plant material obtained after separation in step (d) has a dry matter content greater than 13%, preferably greater than 23%, even more preferably greater than 33%, with maximum preference greater than 40%. Additional water may be removed from this dewaxed fibrous material, for example, by using thermal or vacuum drying to increase the dry matter content. The dewaxed fibrous material may be used as biofuel. The dewaxed fibrous plant material may be pelletized or otherwise treated to facilitate handling of the material. Or it may be partially or totally suspended in an aqueous solution as a result of a previous treatment, such as described above.

[0081] The liquid fraction comprising vegetable-derived wax is further refined as described in the following steps.

[0082] According to step (e) of the method of the present invention, the liquid fraction of step (d) comprising melted cuticular wax is mixed with a capturing agent. The capturing agent is a water-immiscible liquid in which the wax is soluble, such as a liquid solvent for all components of natural waxes. The capturing agent also has the property of being a C1-C4 alcohol-miscible liquid. In one embodiment, the capturing agent is an organic component or a mixture of organic components, such as organic components selected from the group consisting of vegetable oil, modified vegetable oil, vegetable oil derivative(s), purified or not. In a preferred embodiment, the capturing agent is a methyl ester, preferably a fatty acid methyl ester, such as rapeseed methyl ester.In a more preferred embodiment, the capturing agent is a C10-C18 methyl ester preparation, such as C10, C12, C14, C16 or C18 methyl ester preparations or mixtures thereof.

[0083] The advantages of fatty acid methyl esters are that they are widely available, are not volatile at room temperature and slightly elevated, but can be distilled under Petition 870210113006, dated 06 / 12 / 2021, page 23 / 53 20 / 40 vacuum at temperatures lower than the decomposition temperature of the wax component. Therefore, in a preferred embodiment, the capturing agent is selected based on its physical / chemical properties, such that the capturing agent is a liquid in the temperature range of 0 to 200 °C, such as a liquid in the temperature range of 0 to 100 °C, with maximum preference in the temperature range of 10 to 85 °C. In other words, in a more preferred embodiment, the capturing agent has a boiling point above 85 °C at ambient pressure. In one embodiment, the capturing agent has a boiling point above 60, 70, 80, 85, 90, 100, 120, 140, 160, 180 or 200 °C at ambient pressure. In yet another embodiment, the capturing agent has a boiling point below 230 °C at any pressure < 10 mbar. At temperatures above 210 or 230 °C, the components of the wax may undergo thermal decomposition.Examples of boiling temperatures at different pressures of relevant capturing agents are given in Table 3. In a preferred embodiment, the capturing agent is a mixture of C16 and C14 methyl esters. TABLE 3. BOILING POINT OF THE AGENTS SELECTED CAPTURES___________________________ Capture agent________________Boiling point_______ Methyl oleate 210 °C at 10 mm Hg (C18 monounsaturated methyl ester) 218 ​​°C at 20 mm Hg 353 °C at ambient pressure Methyl stearate (saturated C18 methyl ester) Methyl palmitate (saturated C16 methyl ester) Methyl misitate (saturated C14 methyl ester) 215 °C at 10 mm Hg 443 °C at ambient pressure 164 °C at 4 mm Hg 185 °C at 10 mm Hg 417 °C at ambient pressure 160 °C at 10 mm Hg 323 °C at ambient pressure Petition 870210113006, dated 06 / 12 / 2021, page 24 / 53 21 / 40

[0084] Furthermore, the density of the trapping agent is preferably different from the density of water by more than 2%, such as by more than 10%, preferably by more than 20%.

[0085] The capture agent can be mixed with the liquid fraction from step (e) using a pump, an agitator, a static mixer injection nozzle, or any other standard method of mixing liquids. In one embodiment, the capture agent and the liquid fraction obtained in step (d) are mixed in a ratio of 1:20 to 1:60 (v / v), such as preferably in a ratio of 1:40.

[0086] In one embodiment, the temperature maintained during mixing in step (e) is in the range of 65 to 95 °C, such as in the range of 65 to 90 °C, for example, in the range of 75 to 85 °C, such as in the range of 80 to 85 °C and preferably 80 °C. In another embodiment, the temperature maintained during mixing in step (e) is above 70 °C, preferably above 80, 90 or 95 °C.

[0087] The molten cuticular wax from the liquid fraction of step (d) will, after mixing with the capturing agent in step (e), be in the capturing agent phase of the mixture. In one embodiment, this step in combination with the subsequent separation of the two phases is a means of increasing the wax concentration.

[0088] In a preferred embodiment, the capturing agent is a methyl ester and the capturing agent is mixed with the liquid fraction obtained in step (d) in a ratio of 1:40, at a temperature in the range of 80 to 90 °C.

[0089] According to step (f) of the method of the present invention, the mixture obtained in step (e) is separated into an aqueous fraction and a capture fraction comprising the capture agent and vegetable-derived cuticular wax. In principle, any known method and device that can be applied to separate two liquid solutions is not Petition 870210113006, dated 06 / 12 / 2021, page 25 / 53 The 22 / 40 miscible method can be applied, as well as known methods for separating two solutions that have different densities.

[0090] In one embodiment, separation in step (f) is performed by centrifugation. The capture fraction (upper phase), comprising capture agent, wax, and other soluble components, is thus separated from the aqueous fraction (water). This can be done in a single centrifugation step or in 2, 3, 4, or even more sequential centrifugations.

[0091] In one embodiment, the temperature maintained during separation in step (f) is in the range of 65 to 95 °C, such as in the range of 65 to 90 °C, for example, in the range of 75 to 85 °C, such as in the range of 80 to 85 °C and preferably 80 °C. In another embodiment, the temperature maintained during separation in step (f) is above 70 °C, preferably above 80, 90 or 95 °C.

[0092] According to step (g) of the method of the present invention, vegetable-derived cuticular wax is recovered from the capture fraction. The wax can be recovered by preferably removing all other components of the capture fraction, such as remaining water, solids, as well as the capture agent, from the wax.

[0093] In one embodiment, water can be removed from the capture fraction by further treatment, such as evaporation, distillation, membrane separation, molecular adsorption, or a combination thereof. For example, an evaporation chamber can be used, such as by mechanical agitation at a temperature of 70 to 80 °C, until a temperature increase is observed, indicating the absence of residual water.

[0094] Any separated aqueous fraction can be recycled and reused, such as, for example, heat exchange with the sample provided in step (b) to increase the temperature as specified in step (c).

[0095] In another embodiment, any solid particles can be removed from the capture fraction, such as by Petition 870210113006, dated 06 / 12 / 2021, page 26 / 53 23 / 40 filtration, preferably at a temperature where the wax is in liquid form. It may be an in-line filter or a separate filter, such as in the form of a stocking, flatbed, belt or band filter over which the suspension is pumped or poured. The filtration comprises a porous layer or perforated layer, fabric or a combination thereof, with or without a filter aid. The filter aid is selected from the group of diatomite, diatomaceous earth, coal, activated carbon, montmorillonite, bentonite, Fuller's earth, clay minerals, cellulose and perlite. Preferably, a filter band comprising a porous fabric of regenerated cellulose / viscose filter material or polypropylene filter material is used.

[0096] After separation of water and potentially solid particles from the capture fraction, the liquid capture fraction now mainly comprises plant-derived cuticular wax dispersed in the capture agent.

[0097] The capturing agent can be removed from the wax based on the properties of the capturing agent defined in step (e), such as the capturing agent being non-volatile at ambient and slightly elevated temperatures, but, for example, it can be distilled under vacuum at temperatures lower than the decomposition temperature of the waxy components. In one embodiment, the capturing agent is recovered by applying conditions in which the capturing agent is volatile, while the waxy components are not, such as vacuum distillation. For example, the capturing fraction comprising the capturing agent and wax is placed in a distillation vessel and the capturing agent is then removed by vacuum distillation, producing crude cuticular wax. In a preferred embodiment, vacuum distillation is carried out at a target distillation temperature of 160 °C, such as 170 °C, preferably up to 180 °C; and distillation will be complete as soon as the temperature starts to rise.

[0098] The capture agent is preferably recycled, as reused in step (e). Petition 870210113006, dated 06 / 12 / 2021, p. 27 / 53 24 / 40

[0099] In a further embodiment, the residual capture agent is removed from the crude wax using a suitable solvent. The solvent is a water-immiscible liquid. In one embodiment, the solvent is a C1-C4 alcohol. In a preferred embodiment, the solvent is selected from the group consisting of isomers of methanol, ethanol, propanol, isopropanol, butanol, isobutanol, tert-butanol, or a combination thereof. Depending on the temperature, the wax may dissolve in the solvent or crystallize and form a precipitate. In a preferred embodiment, ethanol is used as the solvent. Therefore, in a preferred embodiment, the crude wax is recovered from the distillation vessel as described above (discharged as a melt) and introduced into an excess of ethanol (at least 96% w / v, preferably 99%) during the final cleaning phase.

[0100] Two different approaches to removing the residual capture agent can be used:

[0101] (I) The molten crude wax is suspended in a solvent, selected from the list above, and allowed to recrystallize completely in the solvent as the temperature is reduced to between 2 and 25 °C, such as preferably lowered to between 10 and 20 °C. The precipitate (wax) is recovered, for example, by filtration or other means of separating an insoluble fraction from a liquid, and may optionally be washed with solvent to remove most of the residual trapping agent.

[0102] (II) The melted crude wax is dissolved in a hot / boiling solvent, selected from the list above, preferably ethanol. In one embodiment, the solvent temperature is above 60 °C, such as between 60 and 79 °C, preferably between 65 and 79 °C. The temperature is selected so that all components in the crude wax, including the residual capturing agent, dissolve / disperse in the hot solvent. The solution is then cooled to a temperature that leads to the precipitation of most of the waxy components (except some fatty acids), while the capturing agent remains in solution. In one embodiment, the temperature Petition 870210113006, dated 06 / 12 / 2021, p. 28 / 53 25 / 40 of the solution is reduced to less than 50 °C, such as less than 40, 35, 30 or 25, preferably less than 20 °C, such as reduced to between 2 and 25 °C, or preferably reduced to between 10 and 20 °C. The precipitate is recovered, for example, by filtration or other means of separating an insoluble fraction from a liquid. Finally, the recovered precipitate may optionally be washed with more cold solvent to remove any remaining traces of the trapping agent.

[0103] Alternative methods of removing the residual capture agent may be known and selected by those skilled in the art.

[0104] In a preferred embodiment, the residual capture agent is removed by approach (II), as described above, and the recovered precipitate is washed with cold solvent to extract the last traces of the capture agent, such as using 2, 4, 6, 8, 10, 12, 15, 20 or even more times the amount of solvent in excess of the amount of wax precipitated in the wash.

[0105] In both approaches I and II, the solvent can be recovered from the eluent and recycled.

[0106] In a further embodiment, the residual solvent is removed from the clean wax of approach I or II, such as by blowing hot air / stream into the wax, effectively evaporating the solvent; or removed by melting into the wax at a temperature that allows the solvent to evaporate, such as a temperature above 75 °C, preferably at a temperature in the range of 75 to 100 °C, more preferably in the range of 80 to 90 °C.

[0107] According to step (h) of the method of the present invention, the cuticular wax recovered in step (g) can be bleached. Bleaching is preferably achieved by exposure to a bleaching agent. In one embodiment, the bleaching agent is selected from the group consisting of oxidants, such as chlorine, hypochlorite, chloramine, chlorine gas, chlorine dioxide, sodium percarbonate, sodium perborate, molecular oxygen, ozone, peroxoacetic acid, Petition 870210113006, dated 06 / 12 / 2021, page 29 / 53 26 / 40 benzoyl peroxide and bromate. In a preferred embodiment, the wax is bleached with ozone.

[0108] Ozone treatment as a bleaching method is not limited to vegetable cuticular waxes treated according to steps (a) to (g) of the present invention, but can be applied to any wax product. Any wax composition can preferably be bleached using ozone as illustrated below.

[0109] With the use of ozone as a bleaching agent, the wax is preferably melted in a hot aqueous solution, such as at temperatures above the melting temperature of the wax selected from Table 1. In one embodiment, the wax is melted in an aqueous solution with a temperature in the range of 65 to 95 °C, such as in the range of 65 to 90 °C, for example, in the range of 75 to 85 °C, such as in the range of 80 to 85 °C and preferably at 85 °C. In another embodiment, the temperature is above 70 °C, preferably above 80, 85, 90 or 95 °C.

[0110] In one embodiment, the wax is dispersed in the aqueous solution using emulsion technology: the pH is increased, which affects the formation of soap from residual fatty acids in the wax, facilitating the formation of the emulsion. In one embodiment, the pH is increased above pH 9, such as increased to a pH in the range of 9 to 11, for example, in the range of 10 to 11. The pH adjustment of the solution can be carried out by adding a base selected from the group consisting of sodium hydroxide, potassium hydroxide, calcium hydroxide, ammonium hydroxide, sodium carbonate and combinations thereof.

[0111] In a preferred embodiment, the wax is dispersed in an aqueous solution at a temperature of 75 to 90 °C and pH 10 to 11. Mechanical stirring may be applied for optimal wax dispersion. Applicable mechanical stirrers may be selected from the group consisting of anchor stirrers, (multi)blade stirrers, K-stirrers, paddle stirrers, or any combination thereof. Petition 870210113006, dated 06 / 12 / 2021, page 30 / 53 27 / 40

[0112] Ozone (O3) is introduced into the dispersed wax, such as by bubbling it through the solution. In one embodiment, the ozone is bubbled through the dispersed wax for 1, 2, 3, 4, 5 hours, or even up to 6 hours.

[0113] The ozone dosing rate is approximately 20 g 400 g per hour output from the ozone generator.

[0114] In a preferred embodiment, ozone is bubbled through the dispersed wax for 1 to 4 hours at a dosing rate of 10 to 20 g per hour, maintaining the temperature at 80 to 90 °C and stirring throughout.

[0115] After ozone treatment, the pH is reduced to regenerate the fatty acids of their salts (soaps) and thus help to break down the remaining emulsion. In one embodiment, the pH is reduced to a value below pH 5, such as reduced to the pH range of 3 to 5, or even reduced to a pH value between pH 3.5 and 4. The pH adjustment of the solution can be carried out by adding an acid selected from the group consisting of phosphoric acid, hydrochloric acid, sulfuric acid, or acetic acid. At low pH, the bleached wax rises to the top as a separate layer. The mixture is preferably allowed to cool to room temperature and the wax can be recovered as a solid.

[0116] According to step (i) of the method of the present invention, the recovered (and optionally bleached) cuticular wax can be formulated into highly valuable products, such as in one embodiment formulated into cosmetics, medical additives and personal hygiene products; in another embodiment formulated into a food ingredient, food coating or even rodent bait; in yet another embodiment formulated into other surface coatings, for example, fertilizer coating; in yet another embodiment formulated into lubricants, molding, polishes, leather tanning, textile waterproofing, technical moisture barrier, clothing; in yet another embodiment formulated into an adhesive, Petition 870210113006, dated 06 / 12 / 2021, page 31 / 53 28 / 40 inks, paints, crayons, pencils; in yet another embodiment formulated into barbecue starter, matches, candles. In a preferred embodiment, the wax product is formulated into a cosmetic or other personal care product. The formulation may comprise process steps selected from the group consisting of granulation, flaking, beading, extrusion, grinding and melting. II. METHODS OF ANALYZING PRODUCTS OBTAINED BY THE PRESENT INVENTION II.i TOTAL WAX CONTENT

[0117] The total wax content of cereal straw can be determined gravimetrically as total lipids. Cereal straw containing dry wax is ground and then extracted with hot / boiling chloroform. This is carried out by either of two basic methods, where method 2 is preferred to method 1 if the apparent density of the plant material is high.

[0118] 1. A precisely weighed portion of ground biomass (oven-dried) is placed in a Soxhlet thimble and then subjected to 12-hour extraction in a Soxhlet extraction system using the standard Soxhlet methodology. After extraction, the thimble and the remaining solid material are dried at 103 °C, and the extracted wax is determined by the mass difference compared to the initial material. Or,

[0119] 2. A precisely weighed portion of approximately 30 g of dried, ground straw or other plant material is placed in a 2-liter round-bottom flask, to which 1 liter of chloroform is added. The flask is fitted with a reflux condenser, and the material is refluxed in chloroform for at least 3 hours. After this time, the remaining solids are quantitatively collected, then dried (103 °C) and weighed. The wax content is determined by the difference in mass compared to the input material. II.ii COMPOSITION OF THE WAX Petition 870210113006, dated 06 / 12 / 2021, p. 32 / 53 29 / 40

[0120] The composition of the wax is determined and monitored by gas chromatographic (“GC”) analysis. Wax samples are dissolved in chloroform (approximately 0.1 and 0.2 g of waxy solids per 25 g of chloroform) and analyzed using gas chromatography (GC) on an Agilent GC5890 system equipped with a Gerstel CIS4 input controlled by a C505 controller. Samples (25 microliters) are introduced, using an ALS 7683 autosampler, into a 15-meter-long J&W 123-5711E DB-5HT (containing 5% methyl silicon). The temperature ramp is ambient up to a maximum of 350 °C, with FID detection (375 °C). Figure 2 shows a GC trace of wheat straw wax extracted with chloroform (12 hours, Soxhlet method; 10 parts solvent to 1 part straw), which in this application is used as a “standard wax” for reference regarding purity.Peaks before 7.5 minutes are fatty acids (mainly C16 and C18), peaks from 9.5 to 12.5 minutes are mainly alkanes, aldehydes and fatty alcohols, peaks from 14 to 17.5 minutes are mainly sterols, beta-diketone, while peaks after 18 minutes are waxy esters. II.i ii PURITY OF THE WAX

[0121] The purity of the wax product is determined by the standard Soxhlet chloroform extraction method: 5 g of wax are placed in a pre-weighed extraction thimble and extracted with chloroform (250 ml reservoir) continuously for 12 hours (Soxhlet procedure), then the thimble is dried and weighed to determine the non-waxy residual components. The results are then evaluated in combination with the GC results, as described above (which allows estimation of the residual methyl ester content), to obtain a measure of purity. II.iv WAX COLOR

[0122] The color of wax can be described according to the Gardner scale index. The Gardner color is determined by comparing a test sample with a standard reference color, such as that determined by the Lico Spectral Colorimeter (by Hach), for example, Lico Petition 870210113006, dated 06 / 12 / 2021, page 33 / 53 30 / 40 690: The wax sample is melted and poured into an 11 mm disposable round cuvette to a depth of 2 cm. The outer glass of the cuvette must be clean, and it is important to ensure there are no air bubbles. The cuvette containing the wax is then inserted into the cuvette compartment, and the instrument performs a color measurement in the range of 0 to 18 with an accuracy of one decimal place. II. V MELTING POINT OF WAX

[0123] The melting point of wax can be measured by differential scanning calorimetry (DSC). III. PRODUCTS OBTAINED BY THE PRESENT INVENTION

[0124] The invention provides a refined wax product. The wax product comprises only a low amount of residual scavenging agent, such as less than 5%, 4%, 3%, 2%, preferably even less than 1%. The wax product further comprises only a small amount of residual ethanol, such as less than 3%, 2% or even less than 1%.

[0125] The texture of the dry wax product is hard and brittle to the touch, rather than soft and sticky.

[0126] The melting point (drop point) of the wax product is greater than 50 °C, such as greater than 52, 54, 56, 58 or 60 °C, depending on the origin of the wax. Preferably, the melting point is between 60 and 70 °C, as preferably 65 to 68 °C for cereal straw wax.

[0127] For cereal straw waxes, the wax product preferably comprises less than 1% of a capturing agent, less than 1% of alcohol and has a melting point between 65 and 68 °C.

[0128] The wax product may optionally be bleached, obtaining a bleached wax with Gardner color values ​​below 18, as well as a Gardner color between 8 and 18, preferably between 8 and 10, as well as below 8 for cereal straw waxes. IV. POTENTIAL USES OF PRODUCTS OBTAINED BY THIS INVENTION Petition 870210113006, dated 06 / 12 / 2021, p. 34 / 53 31 / 40

[0129] The present invention provides a highly valuable vegetable wax product. In one embodiment, the wax product can be used as a natural and “green” alternative to waxes from the petrochemical industry. In another preferred embodiment, the wax product can replace mineral oil-based waxes in numerous uses, including in cosmetics, medical additives, personal care products, food coatings, food ingredients, lubricants, polishes, molding, adhesives, surface coatings, fertilizer coatings, textile waterproofing, moisture barrier technology, leather tanning, inks, paints, clothing, crayons, pencils, barbecue starter, candles, matches, rodent bait. In a preferred embodiment, the waxes of the present invention – such as cereal straw waxes – are used in cosmetics. EXAMPLES EXAMPLE 1: WHEAT STRAW WAX REFINING 1.1 REMOVAL OF WAX FROM WHEAT STRAW

[0130] After the wheat grains were harvested from a wheat field in Vestsj^lland, Denmark, the remaining wheat straw was collected and transported to the treatment station where it was treated in a hammer mill and subsequently sieved through an 8 mm sieve. The fraction that passed through the sieve was then processed in a dust separator to remove the fine material (15 to 20% of the straw mass was removed as fine material).

[0131] The fines were suspended in water at 55 °C in a jacketed steel tank, with a load of 85 kg of straw (corresponding to approximately 77.5 kg of straw dry matter) per 1,400 liters of water. The pH of the resulting paste was adjusted to pH 5.4 using phosphoric acid, and the temperature was maintained around 55 °C. The paste was mechanically stirred using a Myers-type dispersion mixer to ensure good dispersion. 200 ml of protease-rich preparation (Promod 24 l (110 casein units / ml), BioCatalysts Ltd, UK) and 100 ml of preparation Petition 870210113006, dated 06 / 12 / 2021, pp. 35 / 53 32 / 40 of pectinase-rich enzyme (Pectinase 974 l (900 units / ml), BioCatalysts Ltd, UK) were added to break down the straw cuticle and help release wax. The paste was circulated through a Fryma-type wet mill (fitted with a toothed colloidal grinding head) with a large clearance in the mill head (> 2 mm), meaning the mill is acting as an effective pump mixer rather than a true mill, helping to ensure enzyme access to the straw cuticular surface. Wet grinding and agitation were applied during the enzymatic treatment, maintaining the pH and temperature profile as specified above. After about 1 hour, the paste temperature was raised to 80 °C to ensure all waxy components were in a molten state and to inactivate the enzymes; and the mixture was subsequently mechanically stirred for about 10 minutes.This process paste comprises molten wax along with water and water-soluble components and insoluble, solid material with wax removed. It was noted that a total of 2.9 kg of wax was released into the aqueous phase during the process (determined by the Soxhlet extraction method with standard chloroform). 1.2 REFINING OF WHEAT STRAW WAX USING AN EtOH CAPTURE AND EXTRACTION AGENT AND WASHING

[0132] The paste from the aqueous enzymatic wax removal process described in Example 1.1 was centrifuged (using a decanter), producing a liquid “upper phase” (liquid fraction comprising wax) and a fiber phase with removed wax (insoluble fraction: bulk fiber residue from the wax removal step). This was carried out at approximately 80 °C and pH approximately 5 to 6. The liquid fraction comprised dissolved water-soluble plant material, some “solid fines,” and the dislodged wax in the form of a molten suspension and emulsion droplets. Maintaining the temperature at approximately 80 °C, the capture agent, rapeseed methyl ester, was rapidly stirred into the aqueous phase in a ratio of 1 part capture agent to 40 parts aqueous phase. After approximately 60 minutes, still at a temperature Petition 870210113006, dated 06 / 12 / 2021, page 36 / 53 At approximately 80°C, the mixture was centrifuged (8,000 rpm) using a GEA model SC 35-01-177 separator (disc stack, 3 phases, with self-cleaning bowl) in order to separate an "enriched upper phase" (capture fraction). This enriched upper phase (capture fraction) contained the capture agent, the waxy components of the straw, and a small amount of residual water and other partially soluble components (soluble or suspended in the capture agent and the overall mixture). To remove the water, the enriched upper phase was introduced into an evaporation chamber (sealed tank with a volume of 750 liters, equipped with exhaust and mechanical agitation), where it was agitated at a temperature of 70 to 80°C until the temperature was observed to rise, indicating the absence of residual water. The "dehydrated upper phase" was then filtered through a 200 to 300 μm mesh medium, removing the solid particles.This was done using a filter band (Model / type UF 1000, supplied by Union Oiltech ApS, Svendborg, Denmark), in which a mesh of porous polypropylene filter fabric on a roll was mounted, continuously wound as needed, resulting in a liquid filtrate comprising mainly waxy components extracted from the straw dissolved and dispersed in the capturing agent. The filtrate was collected and then placed in a distillation vessel (a 750-liter stainless steel tank equipped with mechanical stirring and vacuum installation), in which the capturing agent was removed by vacuum distillation at 180 °C, and the distillation was considered complete when the temperature began to rise. The crude wax was recovered from the distillation vessel (discharged as a hot melt) and introduced into an excess of 96% ethanol (1 part wax to 6 parts ethanol).The hot crude wax suspended in ethanol (now heated to about 30–50 °C by the wax) was stirred (manually, with a steel rod) to ensure proper mixing, then left for complete recrystallization of the wax in the ethanol as it cooled to room temperature (15 °C). The solution comprising recrystallized wax was then filtered using a second filter band configuration. Petition 870210113006, dated 06 / 12 / 2021, page 37 / 53 34 / 40 (Model / type UF 1000, supplied by Union Oiltech ApS, Svendborg, Denmark, equipped with a 200-300 μm mesh filter fabric), retaining the crystallized wax. The wax was then washed using 10 l of more cold ethanol (room temperature, approximately 15 °C) to remove most of the residual trapping agent (methyl esters). The washed wax was then melted and heated in an oven at 90 °C overnight to remove excess ethanol.

[0133] This resulted in an unbleached wax containing less than 3% residual methyl ester scavenging agent, as determined by GC monitoring of methyl ester peaks against the main peaks of the wax components (Figure 3). The residual ethanol content was measured to be less than 1%. The Soxhlet extraction method with standard chloroform, combined with the GC data, further showed that the wax product was at least 95% pure. Differential scanning calorimetry (DSC) measurement showed a maximum melting point for the wax at 65 °C. The wax was hard and brittle to the touch at room temperature. 1.3 REFINING OF WHEAT STRAW WAX USING A WASH-FREE CAPTURE AGENT WITH EtOH

[0134] The wax was prepared as in Examples 1.1 and 1.2, except that the recrystallized wax was not subsequently washed with cold ethanol, but simply filtered through the filter band to drain excess ethanol from the recrystallization; it was then melted and heated in an oven at 90 °C overnight to remove excess ethanol. This resulted in an unbleached wax containing 20–30% residual methyl ester scavenging agent, as determined by GC monitoring (Figure 4), and the peak melting point of the wax was found to be around 50 °C (as determined by DSC). The wax was soft and “sticky” to the touch at room temperature, rather than hard and brittle as in Example 1.2. 1.4 REFINING OF WHEAT STRAW WAX USING A CAPTURE AGENT AND HOT EtOH EXTRACTION Petition 870210113006, dated 06 / 12 / 2021, pp. 38 / 53 35 / 40

[0135] The wax was prepared as in Examples 1.1 and 1.2, except that the molten crude wax emanating from the distillation vessel was introduced into a 96% excess of ethanol (1 part wax discharge to 6 parts ethanol) in a steel vessel fitted with a mechanical stirrer and heating jacket. The temperature of the mixture was raised to 75 °C within the vessel, with continuous mechanical stirring, until the wax melted and redissolved and re-dissolved within the hot ethanol. The hot mixture was then transferred to an open cooling tank and the temperature was allowed to drop to room temperature (about 15 °C), and left for 1 hour at this temperature. The waxy material recrystallized and precipitated in the now cooled ethanol.The crystallized solid wax was recovered by filtration using a second filter band configuration (model / type UF 1000, supplied by Union Oiltech ApS, Svendborg, Denmark, equipped with a 200-300 micron mesh filter fabric) retaining the crystallized wax. The wax was finally washed using 5 liters of additional cold ethanol (room temperature, approximately 15 °C) per kg of precipitated wax to remove most of the methyl esters from the residual capture agent. The resulting moist ethanolic wax cake was manually pressed to remove a proportion of the ethanol through the filter, after which the wax cake was then melted and heated in an oven at 90 °C overnight to eliminate excess ethanol.

[0136] This resulted in an unbleached wax containing less than 1% residual methyl ester CA, as determined by GC monitoring of methyl ester peaks against peaks of the wax's main components (Figure 5). The residual ethanol content was also measured to be less than 1%. DSC measurement showed a maximum melting point for the wax at 68 °C. The wax was hard and brittle to the touch at room temperature.

[0137] Table 4 provides a comparison of the properties of the waxes obtained by the different methods described above. Petition 870210113006, dated 06 / 12 / 2021, pp. 39 / 53 36 / 40 TABLE 4. WAX PROPERTIES Residual Methyl Ester Method CA Residual EtOH Purity Melting Point Texture (at room temperature) Capture agent + EtOH extraction + cold EtOH wash Less than 3% Less than 1% 95 to 97 65 °C Hard and brittle Capture agent + EtOH extraction (without cold EtOH wash) 20 to 30% Less than 1% 70 to 80 50=0 Soft and sticky Capture agent + hot EtOH extraction + cold EtOH wash Less than 1% Less than 1% 98 to 99 68=0 Hard and brittle *Purity determined by the Soxhlet extraction method with standard chloroform EXAMPLE 2: TRADITIONAL WAX REFINING METHODS 2.1 Wax recovery by scalding

[0138] Wax removal from wheat straw was performed as described in Example 1.1, except that the wax content of the process slurry from the aqueous enzymatic wax removal process was increased by centrifuging the slurry (using a decanter), which produces a liquid “top phase” (liquid fraction comprising wax) and a fiber phase (insoluble fraction: bulk fiber residue from the wax removal step), wherein the liquid top phase was then reused as bulk process liquor for a second batch for wax removal. These three consecutive batch runs were performed. The dry matter content of the liquid decanter top phase from the three runs was determined by the standard method and found to be as follows: Run 1: 1.03%, Run 2: 1.76%, and Run 3: 3.30%. This confirmed that additional compounds (including wax) were indeed extracted with each additional run. Petition 870210113006, dated 06 / 12 / 2021, pages 40 / 53 37 / 40

[0139] 1.980 g of the upper phase liquid from the decanter of run 3 were carefully and quantitatively dried (oven at 80 °C). A total of 64.75 g of dry matter was obtained (confirming the 3.30% DM of run 3). 63.52 g of this dry matter were then extracted using the standard chloroform Soxhlet extraction method to determine the total extractable wax content. This yielded 5.73 g of wax material after evaporation of the CHCh. The chloroform extractable wax content of the upper phase liquid from the decanter of run 3 was thus determined to be 0.29%.

[0140] The wax product obtained by standard skimming was determined as follows: 121 liters of top-phase liquid from the decanter of run 3 into a processing vessel. The pH of the mixture was adjusted to 3.5 by adding phosphoric acid. Samples were then periodically scraped / skimmed from the surface as follows. In each case, a visible skin with a “greasy consistency” was observed and removed. The operation was performed 8 times over 2 days until no more waxy layer was observed on the surface. All collections were pooled, dried (oven at 80 °C, overnight) and weighed after drying. The dry weight of the pooled skimmed layer was 318 g. To determine the actual wax content of this layer, the standard chloroform Soxhlet extraction method using boiling chloroform was used (2 hours of reflux in 5x excess solvent).The CHCh and solubles were isolated by filtration and the solvent was then evaporated, the waxy residues were finally weighed and quantified: the wax extracted with total chloroform was 180 g.

[0141] As reported above, initial analysis of the top-phase liquid from the decanter of execution 3 showed that the chloroform-extractable wax content was 0.29%. Therefore, the total amount of wax in the 121 liters of top-phase liquid from the decanter is approximately 350 g. The skimming method, therefore, appears to yield only 51% of the available wax with a purity of only 57%. The skimmed wax product is not only crude. Petition 870210113006, dated 06 / 12 / 2021, pages 41 / 53 38 / 40 in composition and requires substantial additional extraction, the method is also quite time-consuming (1 to 2 days per batch) and is not considered a realistic commercial method for refining wheat straw wax. EXAMPLE 3: WAX WHITENING 3.1 Bleaching using ozone

[0142] Clean wax (1 kg dose), after evaporation of the ethanol (example 1.2), was added to a 10-liter jacketed vessel containing hot water (9 liters) at a temperature of 85 °C, with rapid stirring using a multi-blade mechanical stirrer. The wax melted and was dispersed using emulsion technology that increases the pH to approximately 10.5 by adding 3 M NaOH solution. Ozone (O3) was introduced (from an ozone generator) to the bottom of the vessel through a tube with multiple outlet holes to increase bubble formation and allowed to bubble through the liquid suspension for 4 hours, maintaining the temperature and stirring throughout. The ozone dosing rate was approximately 20 g per hour of output from the ozone generator. At the end of the treatment period, the pH was reduced to a value between 3.5 and 4 using phosphoric acid (which maintains the temperature and agitation) to help break down any remaining emulsion.The liquid suspension was quickly discharged from the container into a separate container, at which point the melted and bleached wax rose to the top as a separate layer. The mixture was allowed to cool to room temperature and the wax disc was removed as a solid. Residual water was dried by wiping with absorbent paper.

[0143] The bleached wax was a light yellow color, as opposed to the dark brown color of the bleaching reactor feed wax. The light yellow hue was very similar to that normally seen for carnauba wax. Using the Gardner color index (Table 2), the bleached wheat straw wax was visually determined to have a Gardner value of around 8 to 10. Petition 870210113006, dated 06 / 12 / 2021, pages 42 / 53 39 / 40 3.2 Bleaching using hydrogen peroxide

[0144] The methodology for bleaching beeswax using hydrogen peroxide was adapted: The wax was emulsified as described for ozone in Example 2.1. 35 grams of 30% H2O2 per 100 grams of wax were added as a bleaching agent, maintaining the pH at 10.5 and the temperature at 80 °C, for 5 hours and 24 hours (two separate experiments). To recover the wax, the pH was rapidly reduced to 3.5 using phosphoric acid, maintaining mechanical agitation and the temperature at 80 °C, after which mechanical agitation was stopped and the wax phase rapidly separated to the top of the cup as a distinct layer. Upon cooling, this top layer was removed as a solid wax disc. Only very partial bleaching of the wax was observed, even after 24 hours of treatment. The wax mass remained a brown color, visually determined by having a Gardner value of around 18. 3.3 Bleaching with Chlorine

[0145] The wax was added to hot water (ratio of 1:10 based on mass), the mixture was heated to 85 °C with rapid mechanical stirring. The pH was reduced to 4.5 using acetic acid, with 10 g of sodium chlorite per 100 g of wax being added to the mixture and bleaching started for 1 hour. A transition of the wax from dark brown to light yellow was observed (Gardner value around 8 to 10). The method, therefore, works, but bleaching with chlorine is not desirable for most subsequent processing and commercial use of the wax. 3.4 Bleaching using ozone, in wax dissolved in chloroform

[0146] Crude wax was dissolved in hot chloroform (40 °C) at a 1:10 mass ratio. Ozone was bubbled into 1 liter of the mixture (rate of 10 g per hour from an ozone generator). The material changed Petition 870210113006, dated 06 / 12 / 2021, pages 43 / 53 40 / 40 visibly changes from dark brown to light yellow (Gardner value around 8 to 10) within 40 minutes of onset. However, ozone reacts with chloroform to release active chlorine species, and it is likely that bleaching was effected by these chlorine-derived oxidants along with ozone. Therefore, although effective bleaching was achieved, the “indirect” use of chlorine and chloroform is probably undesirable for most subsequent processing and commercial use of the wax. Petition 870210113006, dated 06 / 12 / 2021, pages 44 / 53

Claims

1 / 5 CLAIMS 1. Method for extracting and refining cuticular wax from plant material, wherein the method is characterized in that it comprises the steps of a. providing plant material comprising cuticular wax, b. dissociating cuticular wax from said plant material provided in step (a), thereby obtaining a sample comprising plant-derived cuticular wax and plant material with wax removed in an aqueous suspension, c. solubilizing said plant-derived cuticular wax by increasing the temperature of the sample obtained in step (b) to a temperature higher than the melting point of said plant-derived cuticular wax, d. separating the suspension obtained in step (c) into a solid fraction and a liquid fraction, wherein said liquid fraction comprises melted plant-derived cuticular wax, e. mixing said liquid fraction from step (d) with a capturing agent, f.to separate the mixture obtained in step (e) into an aqueous fraction and a capture fraction, wherein said capture fraction comprises said capture agent and said vegetable-derived cuticular wax, g. to recover said vegetable-derived cuticular wax from said capture fraction of step (f); wherein said capture agent has the particularity of (i) being a liquid immiscible in water in which said vegetable-derived cuticular wax is soluble, (ii) having a boiling point above 85 °C at ambient pressure, and (iii) being a liquid miscible with C1-C4 alcohol.

2. Method for extracting and refining cuticular wax from plant material, according to claim 1, characterized in that the capturing agent is selected from the group consisting of optionally purified vegetable oils, modified vegetable oil and vegetable oil derivative(s).

3. Method for extracting and refining cuticular wax from plant material, according to any one of claims 1 or 2, characterized in that the capturing agent is a methyl ester, such as methyl esters of fatty acids, such as C14-C18 methyl ester preparations, for example, rapeseed methyl ester.

4. Method for extracting and refining cuticular wax from plant material, according to any one of claims 1 to 3, characterized in that the plant-derived cuticular wax is recovered in step (g) g1. by removing the capturing agent from the cuticular wax by applying conditions in which the capturing agent is volatile, while the waxy components are not, such as vacuum distillation.

5. Method for extracting and refining cuticular wax from plant material, according to claim 4, characterized in that the residual capturing agent is removed from the cuticular wax by the steps of g2. suspending or dissolving the wax in alcohol, g3. cooling to a temperature that leads to precipitation of the wax, while the capturing agent remains in solution wherein said alcohol is a C1-C4 alcohol, such as selected from methanol, ethanol, propanol, isopropanol, butanol, isobutanol, tert-butanol or a combination thereof.

6. Method for extracting and refining cuticular wax from plant material, according to any one of claims 1 to 5, characterized in that the dissociation of the cuticular wax from the plant material in step (b) is done by a method comprising the steps of i. subjecting said plant material to a dry mechanical treatment, ii. optionally fractionating the material obtained in step (i) by size, Petition 870230035844, dated 28 / 04 / 2023, page 9 / 12 3 / 5 iii. suspending the material obtained in step (i) or a selected fraction obtained in step (ii) in an aqueous liquid comprising one or more protease and / or pectinase enzymes, iv. optionally subjecting the mixture obtained in step (iii) to a wet mechanical treatment.

7. Method for extracting and refining cuticular wax from plant material, according to any one of claims 1 to 6, characterized in that said plant material originates from agricultural crops, such as cereals, sugar cane, palm trees and high-energy grasses.

8. Method for extracting and refining cuticular wax from plant material, according to any one of claims 1 to 7, characterized in that said plant material is cereal straw.

9. Method for extracting and refining cuticular wax from plant material, according to any one of claims 1 to 8, characterized in that said cereal straw is selected from wheat, rye, barley, oats, sorghum, rice and triticale.

10. Method for extracting and refining cuticular wax from plant material, according to any one of claims 1 to 9, characterized in that it further comprises the step of h. bleaching said plant-derived cuticular wax recovered in step (g).

11. Method for extracting and refining cuticular wax from plant material, according to claim 10, characterized in that bleaching is obtained by exposing said wax to a bleaching agent selected from the group consisting of oxidants, chlorine, hypochlorite, chloramine, chlorine gas, chlorine dioxide, sodium percarbonate, sodium perborate, molecular oxygen, ozone, peroxoacetic acid, benzoyl peroxide and bromate; preferably ozone. Petition 870230035844, dated 04 / 28 / 2023, page 10 / 12 4 / 5 12. Method for extracting and refining cuticular wax from vegetable material, according to any one of claims 1 to 9, characterized in that it further comprises the step of i. formulating said cuticular wax derived from vegetable recovered in step (g) or said bleached cuticular wax derived from vegetable obtained in step (h) into valuable products selected from the group of cosmetics, medical additives, personal care products, food ingredient, food coating, rodent bait, surface coatings, fertilizer coating, lubricants, molding, polishes, leather tanning, textile waterproofing, technical moisture barrier, clothing items, adhesives, inks, paints, crayons, pencils, barbecue starter, matches, candles.

13. Vegetable wax composition characterized in that it is obtained by the method as defined in any one of claims 1 to 12, wherein said vegetable wax composition comprises less than 3% of a capturing agent and less than 1% of C1-C4 alcohol, such as ethanol.

14. Vegetable wax, according to claim 13, characterized in that it is a cereal straw wax and has a melting point (drop point) between 64 and 68 °C.

15. Cereal straw wax, according to claim 14, characterized in that the color is light yellow with a Gardner color scale value less than 12, such as less than 10, 9 or 8.

16. Vegetable wax, according to any one of claims 13 to 15, characterized in that it is for use in cosmetics.

17. Use of vegetable wax obtained by the method as defined in any one of claims 1 to 12, or of vegetable wax as defined in any one of claims 13 to 16, characterized by the fact that it is used in the preparation of selected products from the group consisting of: cosmetics, medical additives, personal hygiene products, food ingredients, food coatings, rodent bait, surface coatings, fertilizer coatings, lubricants, molding agents, polishes, leather tanning, textile waterproofing, technical moisture barrier, clothing items, adhesives, inks, paints, crayons, pencils, barbecue starter, matches and candles.

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