CN120981214A - Emulsifiers based on lignin fractions and their applications - Google Patents

Abstract

The application relates to an emulsifier comprising lignin fraction and its use in the preparation of emulsions. The application also relates to a cosmetic comprising such an emulsifier and to a method for the preparation thereof.

Description

Emulsifiers based on lignin fractions and their applications

Technical Field

This invention relates to emulsifiers and their use in cosmetics or food.

Background Technology

Stable colloidal dispersions or emulsions are found in a variety of products and commodities, especially cosmetics and pharmaceuticals, as well as household products, food and other industrial sectors.

Oil-in-water emulsions are thermodynamically unstable systems composed of oil droplets dispersed in an aqueous medium. They tend to decompose due to complex physicochemical mechanisms, including flocculation, coagulation, Ostwald ripening, emulsification, and sedimentation (1). In order to produce stable emulsions with long shelf life and resistance to environmental stress, it is necessary to add stabilizers such as emulsifiers, thickeners, or gelling agents (2).

Currently, the most commonly used emulsifiers in skincare products are petroleum-based surfactants. These include cationic surfactants (such as polyquaternium-10), anionic surfactants (such as sodium dodecyl sulfate (SDS) and linear alkylbenzene sulfonates (LAS)), and nonionic surfactants (such as fatty alcohol ethers and acrylic polymers grafted with hydrophobic alkyl acrylates).

The use of such surfactants poses environmental problems: when used in skincare products, they can be discharged into aquatic ecosystems and/or deposited in agricultural soils (3), causing environmental pollution. In fact, synthetic surfactants are difficult to remove through wastewater treatment (4). Furthermore, surfactants can reduce the effectiveness of microorganisms used to remove pollutants, thus contributing to environmental pollution, and their toxicity to organisms ranging from mammals to bacteria is well-known (5,6).

On the other hand, there is a growing demand for organic and/or natural cosmetics. Currently, only a limited number of surfactants are permitted in cosmetics labeled as organic or natural, such as COSMOS.

Therefore, there is a need for environmentally friendly and sustainable alternatives to traditional petroleum-based surfactants.

Therefore, various emulsifiers derived from biological sources have emerged as alternatives to synthetic emulsifiers. These natural emulsifiers can be derived from a variety of natural sources, such as plants, animals, or microorganisms, and are mainly divided into two categories: proteins and polysaccharides (7).

Proteins adsorb onto the oil-water interface via electrostatic interactions, thus acting as emulsion stabilizers. Most polysaccharides primarily act as emulsion stabilizers by forming dense networks in the continuous phase, thereby forming gels and slowing droplet movement by creating macromolecular barriers between dispersed droplets, preventing them from emulsifying (8,9). However, some polysaccharide derivatives can also adsorb onto the oil-water interface, such as gum arabic, guar gum, and chemically modified starch or cellulose derivatives. They possess surface/interfacial activity by adsorbing onto the surface of oil droplets and prevent flocculation and aggregation through steric or electrostatic repulsion. For gum arabic and guar gum, this is due to the presence of protein subunits in their structure (10). For cellulose derivatives, interfacial activity is due to the different hydrophobic and hydrophilic groups on the cellulose backbone. It provides a strong mechanical barrier against aggregation, which contributes to high stability (11).

Target emulsifiers also include synthetic compounds prepared from natural starting compounds, such as sugars and plant lipids, for example, lipids derived from palm, coconut, soybean, rapeseed, or sunflower. Examples include alkyl polyglycerol esters and sucrose esters of fatty acids. Unfortunately, due to competition from the food industry, the supply of these compounds in the cosmetics sector is limited.

Plant-based emulsifiers are attracting increasing attention due to their biocompatibility, biodegradability, low toxicity, and low production costs, especially when they are derived from by-products or symbiotic products.

However, sustainable and environmentally friendly surfactants still account for only a small portion of the surfactant market. New alternatives to synthetic surfactants are still needed, especially for use in cosmetics.

Summary of the Invention

In a first aspect, the present invention relates to an emulsifier in the form of lignin fractions, characterized in that it comprises:

- 6.0% to 8.5% by weight, preferably 7.0% to 8.2%, more preferably 7.2% to 8.0% lignin monomers.

- 14.0% to 20.0% by weight, preferably 15.0% to 20.0%, more preferably 15.5% to 18.0% of lignin dimer, and

- Lignin oligomers comprising 70% to 80% by weight, preferably 71.8% to 78.0%, more preferably 74.0% to 77.3%.

The weight percentage is relative to the total weight of lignin monomers, dimers, and oligomers in the emulsifier. Preferably, the percentage is determined by size exclusion chromatography.

Lignin fractions may further have at least one of the following characteristics:

- Weight-average molar mass (Mw) of 1200 g/mol to 1600 g/mol, such as 1200 g/mol to 1500 g/mol, and/or

- Number-average molar mass (Mn) of 500 g/mol to 800 g/mol, such as 600 g/mol to 700 g/mol, and/or

- The polydispersity index is 1.85 to 2.40, preferably 1.95 to 2.20.

In some embodiments, the emulsifier may comprise, or be composed of, the aforementioned lignin fraction dissolved in _C2 - _C6 alkyldiol (preferably propylene glycol).

In another embodiment, the lignin fraction in the emulsifier is obtained from industrial alkali lignin. Preferably, the lignin fraction is obtained from industrial alkali lignin through one-step extraction using a solvent selected from _C2 - _C6 alcohols and C3 _{- C8} ketones.

In a particular embodiment, the lignin fraction of the emulsifier is at least partially esterified at its aliphatic primary hydroxyl groups by _C2 - _C18 alkyl ester groups such as hexanoate groups.

In another embodiment, the present invention relates to the use of the lignin fraction as an emulsifier to stabilize oil-in-water emulsions as defined above, preferably in cosmetic or pharmaceutical compositions or food compositions. Typically, the lignin fraction constitutes 0.1% to 5% of the total weight of the oil-in-water emulsion, preferably 0.5% to 4.0%, such as 2.0% to 3.0% or about 2.5%.

In certain embodiments, lignin fractions are also used as pigments.

Another object of the present invention is a cosmetic, pharmaceutical, or food composition, preferably a cosmetic composition comprising the emulsifier defined above. For example, the cosmetic composition may be selected from creams such as daily cream or body cream, body lotion, makeup remover, conditioner, concealer stick or concealer cream, liquid soap, shampoo, and nourishing face mask.

In another embodiment, the present invention relates to a cosmetic product comprising the emulsifier defined above, wherein the cosmetic product is preferably selected from foundation, blush, eyeshadow, tinted body cream or face cream such as BB cream, mascara, eyeliner and concealer.

The present invention also relates to a method for preparing the emulsifier defined above from industrial lignin, the method comprising step (i) extracting the industrial lignin using a solvent selected from _C2 - _C6 alcohols, C3 _{- C8} ketones and combinations thereof, wherein the solvent is preferably selected from methyl ethyl ketone (MEK), acetone, isopropanol and ethanol, and more preferably selected from ethanol or MEK.

In some implementations, step (i) includes:

- Under conditions that allow for the extraction of target lignin molecules, industrial lignin is contacted with a solvent.

- Separate liquid and solid fractions and recover the liquid phase, and

- Remove solvent from liquid fraction to recover emulsifier in lignin fraction form.

The starting industrial lignin is preferably selected from organic solvent lignin or alkali lignin, preferably alkali lignin.

In some embodiments, the method does not subject industrial alkali lignin to any acid and/or alkali treatment prior to extraction, and/or the method includes a single step of extracting industrial alkali lignin using a polar solvent.

The method may also include:

- Step (ii), wherein the lignin fraction obtained in step (i) is subjected to enzymatic transesterification with fatty acid methyl ester or ethyl ester, preferably ethyl hexanoate, in the presence of Candida Antarctica lipase-B (CAL-B), and/or

- The step of dissolving the optionally esterified lignin fraction in a solvent selected from alkyldiols, ketal levulinates such as methyl levulate or ethyl levulate, glycerol ketal levulinates, lower alcohols (such as _C2 - _C6 alcohols), preferably ethanol and isopropanol, and lower alcohol/ _H2O mixtures, preferably _C2 - _C6 alkyldiols such as propylene glycol.

This invention also relates to a method for preparing an oil-in-water emulsion, the method comprising:

(a) An emulsifier provided in the form of a lignin fraction as defined above, at least partially soluble in a polar solvent, preferably a _C2 - _C6 alkyldiol.

(b) The lignin solution is added to the aqueous phase with stirring, and the pH of the aqueous phase is readjusted to a value of 5.5-7.0, preferably about 6.0, and

(c) Under high shear, the oil phase is added to the solution obtained in step (b) to obtain an oil-in-water emulsion.

This invention also relates to a method for preparing cosmetics (such as color cosmetic products), comprising:

(a) Provide the emulsifier or oil-in-water emulsion described herein, and

(b) Combining the emulsifier or the oil-in-water emulsion with one or more cosmetically acceptable excipients.

Attached Figure Description

Figure 1A shows the most common lignin monomer alcohols (also known as lignin alcohol precursors), which correspond to the three different aromatic structural units of lignin: coniferol (also known as the G unit), sinigrin (also known as the S unit), and p-coumarol (also known as the H unit).

Figure 1B shows examples of lignin monomers produced during the pulping process, which can be found in alkali lignin. _R1 and _R2 are independently H, OH, or _OCH3 .

Figure 1C shows an example of lignin dimers produced during pulping, which can be found in alkali lignin. _R1 and _R2 are independently H, OH, or _OCH3 . (Adapted from C. Crestini, et al. Biomacromolecules , vol.12, no. 11, pp. 3928–3935, Nov. 2011, doi: 10.1021/bm200948r)

Figure 1D shows an example of lignin oligomers that can be produced during the pulping process. (Adapted from Prothmann, J., et al. Anal Bioanal Chem 410, 7803–7814 (2018). https://doi.org/10.1007/s00216-018-1400-4.)

Figure 2A shows the size exclusion chromatography of fractionated lignin LFI-1 and starting industrial lignin. Dashed line: corresponding to the chromatogram of starting industrial lignin; solid line: corresponding to the chromatogram of lignin fractionated using MEK (LFI-1 - of this invention); the peak at 20.2 min represents toluene as a reference peak in THF, mixed E column, 2 x 300 mm x 7.5 mm, THF 1 mL/min, detection UV 280 nm. MEK fractionation removes high molecular weight polymers while increasing the proportion of lignin dimers and monomers in the recovered fraction.

Figure 2B shows size exclusion chromatograms of lignin LFI-1 fractionated with MEK and lignin LFI-2 fractionated with ethanol. Solid line: corresponding to the chromatogram of alkali lignin fractionated with ethanol (LFI-2 - this invention); dashed line: corresponding to the chromatogram of alkali lignin fractionated with MEK (LFI-1). The peak at 20.2 min represents toluene as a reference peak in THF, mixed E column, 2 x 300 mm x 7.5 mm, THF 1 mL/min, detection UV 280 nm. Fractionation with EtOH or MEK yields similar lignin fractions.

Figure 2C shows size exclusion chromatography of lignin CLF (comparative) fractionated with MEK and ethyl acetate and starting industrial lignin. CLF refers to the lignin fraction obtained by a two-step sequential extraction with ethyl acetate (AcOEt) followed by methyl ethyl ketone (MEK). Solid lines: corresponding to the chromatogram of alkali lignin; dashed lines: corresponding to the chromatogram of CLF. CLF (comparative) consists of lignin oligomer fractions, containing almost no lignin monomers or dimers. The peak at 20.2 min represents toluene as a reference peak in THF, mixed E column, 2 x 300 mm x 7.5 mm, THF 1 mL/min, detection UV 280 nm.

Figure 3 illustrates the optimized method for preparing emulsions according to the present invention.

Figure 4 shows the sensory stability and optical microscopy analysis of emulsions prepared using industrial lignin, lignin fraction CLF (comparative), and the lignin fractions of the present invention (LFI-1 and gLFI-1) according to Example 1 before and after storage at 25°C or 40°C. Figure 4 also shows the results after one day using the comparative lignin fraction PF570 prepared as described in Example 8 (*: not studied).

Figure 5 shows the evaluation results of different emulsions prepared with LFI-1 using the optimized or alternative methods according to the present invention before and after storage at 25°C or 40°C. Sensory stability, sedimentation, and microstructure were evaluated.

Figure 6 shows the evaluation results of the two emulsions of the present invention prepared with LFI-1 using the optimized or alternative methods according to the present invention before and after storage at 25°C or 40°C. Sensory stability, sedimentation, and microstructure were evaluated.

Figure 7 shows the sensory characteristics of two oil-in-water emulsions prepared using the lignin fraction of the present invention and the lignin fraction of a commercial product (reference product) obtained in Example 7.

Figure 8 shows size exclusion chromatography of the lignin fraction of the present invention (LFI-1), the starting industrial lignin fraction, and the comparative lignin fractions (PF568, PF569, and PF570) prepared according to US2015/0141628 (see Example 8 below). The peak at 20.2 min represents toluene as a reference peak in THF, mixed E column, 2 x 300 mm x 7.5 mm, THF 1 mL/min, detection UV 280 nm.

Figure 8 shows the size exclusion chromatography of the lignin fraction of the present invention (LFI-1), the starting industrial lignin, and the comparative lignin fraction (PF575D) obtained by extraction with EtOH after alkali-acid treatment (see Example 8). The peak at 20.2 min represents toluene as a reference peak in THF, mixed E column, 2 x 300 mm x 7.5 mm, THF 1 mL/min, detection UV 280 nm.

Detailed Implementation

Lignin is the second most abundant biopolymer in nature after cellulose, accounting for 30% of the organic matter in the biosphere. As a symbiotic product or byproduct of the papermaking and biorefining industries, a large amount of lignin is produced annually. Therefore, the efficient utilization of lignin is receiving increasing attention due to concerns about reducing resource waste.

Lignin is a complex polyphenol polymer composed of three phenylpropane monomers (also known as lignin monomer alcohols, namely coniferyl alcohol, sinigrin, and p-coumaryl alcohol), which are cross-linked in different ways via ether or carbon-carbon bonds. The relative amounts of the precursor “monomers” (lignin monomer alcohols) vary depending on the plant source. As described in Crestini et al. (Biomacromolecules, 2011, 12, 3928-3935), the lignin structure is the result of a complex biosynthetic pathway. This biosynthetic pathway is achieved through the oxidative radicalization of the lignin monomer alcohols, followed by radical coupling of two monomer radicals to form a dehydrogenated dimer. Coupling at the β-position of the lignin monomer alcohols leads to the formation of dimers of arylglycerol-β-aryl ether (β-O-4’), pinoresinol (β-β′), phenylcoumaranthone (β-5′), spirodienone (SD), or diphenylethane (β-1′). In a subsequent step, the dimer is newly dehydrogenated to a phenoxy radical, which can then be coupled to another monomer radical in an end-coupling mode. The two lignin oligomers are coupled at the 4- and/or 5-positions, resulting in the formation of a diaryl ether (4-O-5′) and a diphenyl (5-5′) lignin subunit. In turn, the 5-5′ subunit can undergo α-β-O-4-4′ coupling to a dibenzodioxanone (DBDO) unit. The DBDO and 4-O-5′ coupling modes constitute the branching points of lignin. See Scheme 1 in Crestini (13), which shows the coupling modes of lignin monomer alcohols and oligomeric lignin chains; Figure 1 in Crestini shows an example of inter-unit bonds in lignin.

Due to its phenolic function, lignin exhibits interesting properties such as antioxidant activity, UV resistance, and property stabilization. Existing technologies propose the use of lignin as an emulsion stabilizer, for example, Rojas et al., 2007 (Materials, Chemicals and Energy from Forest Biomass, Argyropoulos, ACSsymposium Series, Chapter 12, 182-198), or coupled with nanoparticles, such as international application WO2017/093185, or coupled with PEG molecules, such as Perkins et al., 2017 (Colloids and Surfaces A: Physicochemical and Engineering Aspects, 530, 200-208).

However, despite the interesting properties of lignin, its value-added applications remain limited to low-value applications, such as energy production through combustion.

This is due to the challenging structure of lignin. Lignin exhibits a heterogeneous structure because it is polymerized from three different monomers (so-called lignin alcohol precursors), which can be cross-linked in different ways (e.g., via radical-radical coupling). Furthermore, lignin obtained from industrial production (so-called industrial lignin) has a wide molecular weight distribution. In fact, in pulping or organic solvent processing, the natural lignin present in lignin-cellulose biomass is altered through the breaking of chemical bonds and the formation of new ones, ultimately producing a variety of molecules, including long-chain lignin, medium-molecular-weight oligomers, and low-molecular-weight molecules (such as lignin dimers and monomers).

Molecular weight significantly influences the physical and chemical properties of lignin, especially in applications seeking surface interactions. Furthermore, due to its high polydispersity, polarity, and network structure, industrial lignin exhibits very limited solubility in common solvents, hindering its growth. In particular, lignin precipitates in aqueous media, limiting its application in cosmetics. To address this issue, industrial lignin fractionation methods have been developed to obtain lignin fractions with lower polydispersity and more uniform molecular weight. This method requires multiple steps, including precipitation and solvent extraction.

In this context, the inventors attempted to conceive of an emulsifier from lignin. Surprisingly, they found that lignin with low polydispersity did not exhibit optimal emulsifying properties. Instead, the inventors demonstrated that a specific lignin fraction comprising well-defined proportions of lignin monomers, dimers, and oligomers with a polydispersity of approximately 1.9 to 2.2 exhibited superior emulsifying properties compared to starting alkali lignin or highly fractionated lignin obtained through multi-step fractionation methods. Without adhering to any particular theory, the inventors believed that a combination of well-defined amounts of lignin monomers and dimers with lignin oligomers is crucial for obtaining highly efficient emulsifiers. Indeed, the inventors showed that the fraction of the present invention, combining oligomeric and short-chain lignin (LFI-1 and LFI-2), is more effective than starting industrial alkali lignin and oligomeric lignin fractions (CLF) in stabilizing the oil/water interface over time. In fact, compared to other lignin forms tested in the prepared oil-in-water emulsions (Examples 3 and 5), less sedimentation and no oil droplet aggregation were observed using the lignin fraction of the present invention. The lignin fraction of the present invention can also provide an oil-in-water emulsion with satisfactory sensory properties: as demonstrated in Example 7, the emulsion is easy to apply and has a soft, powdery and non-greasy effect on the skin.

The inventors further demonstrate that the stability and emulsifying effect of the lignin fractions of the present invention can even be improved by enzymatic transesterification with ethyl hexanoate.

Furthermore, the lignin fractions of the present invention are obtained through a very simple extraction method based on a single rapid extraction step of industrial lignin with a polar solvent (preferably a cosmetically acceptable solvent). The polar solvent is preferably selected from short-chain ketones and alcohols. The polar solvent can be a green solvent, such as an agricultural solvent. Therefore, the lignin fractions of the present invention can be obtained through a cost-effective and environmentally friendly method. It is worth noting that prior art grading methods (such as the dual extraction step or liquid-liquid extraction described in US2015/0141268) result in differences in the distribution of monomers, dimers, and oligomers in the lignin fractions and impair emulsification properties. Furthermore, acid and/or alkali treatment of industrial lignin prior to extraction with a polar solvent (as described in CN11226159) also leads to a reduction in monomer and dimer content, thereby affecting the composition of the final lignin fraction (see Example 8).

Finally, the inventors demonstrated that the pH value of the aqueous phase affects the resulting emulsion. Therefore, the inventors conceived a method for optimizing the formation of oil-in-water emulsions, based on pre-dissolving the lignin fraction in alkyldiol, then diluting it in water, readjusting the pH to approximately 6.0, and then adding the oil phase under stirring.

Therefore, the present invention relates to a novel lignin fraction comprising lignin oligomers and lignin monomers/dimers and its use as an emulsifier.

The present invention also relates to a method for preparing such an emulsifier, and a method for preparing an oil-in-water emulsion based on the use of said emulsifier.

In another aspect, the present invention also relates to a cosmetic, more preferably a colored cosmetic comprising the lignin fraction of the present invention, such as a skin-care foundation. In such cosmetics, the lignin fraction of the present invention may have two functions, namely, acting as an emulsifier and a pigment in the product.

In another aspect, the present invention also relates to a method for preparing cosmetics comprising the lignin fraction of the present invention as an emulsifier and/or pigment.

- Methods for preparing emulsifiers from industrial lignin

This invention relates to a method for preparing the emulsifier described herein from industrial lignin. The emulsifier described herein can be obtained by any suitable method to reproduce the aforementioned monomer, dimer, and oligomer distribution. For example, the emulsifier can be obtained by solid-liquid extraction of industrial lignin using a suitable solvent (such as MEK). Other methods are also contemplated, such as dissolving the industrial lignin in a suitable solvent (aqueous or hydroalcoholic) followed by ultrafiltration and/or diafiltration.

In one particular aspect, the emulsifiers described herein are obtained by a solid-liquid extraction step of industrial lignin using a suitable solvent (preferably selected from _C2 - _C6 alcohols, _C3 - _C8 ketones and mixtures thereof, such as methyl ethyl ketone (MEK and ethanol)).

In one particular aspect, the present invention relates to a method for preparing an emulsifier from industrial lignin, the method comprising step (i) directly extracting the industrial lignin using a polar solvent.

In some embodiments, the polar solvent is selected from short-chain ketones and alcohols. The polar solvent can also be a green solvent, such as an agricultural solvent.

In a preferred embodiment, the solvent is selected from _C2 - _C6 alcohols, _C3 - _C8 ketones and mixtures thereof, such as _C2 - _C4 alcohols, _C3 - _C6 ketones and mixtures thereof.

As used herein, _C2 - _C6 alcohols include _C2 , _C3 , _C4 , _C5 , and _C6 alcohols. _C2 - _C6 alcohols include ethanol, propanol, butanol, pentanol, hexanol, and their isomers, such as isopropanol, tert-butanol, isobutanol, and isoamyl alcohol.

As used herein, _C3 - _C8 ketones include _C2 , _C3 , _C4 , _C5 , _C6 , _C7 , and _C8 ketones. _C3 - _C8 ketones include acetone, methyl ethyl ketone (also known as butanone), pentanone, hexanone, heptanone, octanone, and their isomers.

In a preferred embodiment, the solvent is selected from methyl ethyl ketone (MEK), acetone, isopropanol, and ethanol, and mixtures thereof, preferably ethanol and/or MEK. MEK is the preferred solvent.

Ethanol is typically used as anhydrous ethanol or ethanol with a water content of up to 5% (preferably up to 2%) per volume (i.e., 98° ethanol).

As used herein, “emulsifying agent or emulsifier” refers to a compound or substance used as an emulsion stabilizer to prevent or limit aggregation phenomena such as “coagulation” or “flocculation” (droplet coalescence leading to an increase in size over time) and/or gravitational separation (such as sedimentation), especially in oil-in-water emulsions.

As used herein, "technical lignin" refers to the lignin derivatives obtained during the delignination process of lignin-cellulose biomass. For a given lignin-cellulose biomass, the structure of technical lignin varies depending on the processes and chemical reactions used in the biomass/starting material treatment. Different types of processes can be used to recover lignin. A well-known chemical pulping process is the kraft pulping process, in which lignin is separated from cellulose and hemicellulose using sodium hydroxide and sodium sulfide at 170°C. Another process is the bisulfite process, in which lignin is produced as lignin sulfonate. The lignin produced by both of these processes contains sulfur. There are also sulfur-free pulping processes, including soda pulping and organic solvent processes, which produce alkali lignin (also known as soda lignin) and organic solvent lignin, respectively. Alkali lignin is generally obtained through the soda pulping process (also known as the alkali pulping process). This well-known process treats the wood pulp with an alkaline solution (such as sodium hydroxide or _{Na₂CO₃ )} as a cooking chemical. Alkali lignin is typically recovered from black liquor produced during alkaline pulping processes via acid precipitation. Organic solvent lignin is obtained through an organic solvent process, a pulping technique in which lignin-cellulose feedstock is suspended in an aqueous organic solvent, usually in the presence of acid at temperatures above 140°C.

In all these cases, native lignin is decomposed by the cleavage of numerous aryl ether bonds, the dissolution of lignin fragments, and the formation of condensation structures with stable C-C bonds.

In certain embodiments, the industrial lignin is organic solvent lignin or alkali lignin. In more specific embodiments, the industrial lignin is alkali lignin.

The starting lignin cellulose biomass used to prepare industrial lignin can be of any type.

In some embodiments, industrial lignin is prepared from plant biomass selected from softwoods (such as pine), hardwoods, herbaceous plants (such as plants belonging to the Graminae family), and their derivatives. Derivatives include by-products or symbiotic products of industrial or agricultural food processing, such as bagasse, straw, or rice straw.

In some embodiments, industrial lignin is prepared from lignin-cellulose biomass derived from corn, rice, wheat, rye, oats, sorghum, sarkanda, and combinations thereof.

For example, industrial lignin can be prepared from wheat straw, sweetgrass ( Saccharum munja ) and combinations thereof.

For example, industrial lignin can be prepared from beech wood using an organic solvent process. Alternatively, industrial lignin can also be prepared from wheat straw and sweetgrass ( Saccharum munja ) via alkali pulping.

In a particular embodiment, the industrial lignin is an alkali lignin with a pH value below 5, preferably 2.5 to 4.5, such as 3.0 to 4.0.

In certain embodiments, the average weight-average molar mass (Mw) of the industrial lignin is from 1800 g/mol to 4500 g/mol, such as from 1800 g/mol to 4000 g/mol or from 2500 g/mol to 4000 g/mol, and/or the number-average molar mass (Mn) is from 600 g/mol to 1100 g/mol, such as from 650 g/mol to 1000 g/mol or from 750 g/mol to 1000 g/mol.

In certain embodiments, the particle size of the industrial lignin is less than 300 µm, preferably less than 250 μm. In some other aspects, the particle size distribution of the industrial lignin is characterized by a d90 of less than 250 μm, preferably about 200 μm, and a d50 of 50 μm to 80 μm, preferably about 65 μm.

As used herein, “about X” means “X of X ± 10%”, preferably “X of X ± 5%”.

In a preferred embodiment, the industrial lignin used as a starting material in the method of the present invention is alkali lignin (i.e., obtained through an alkali pulping process), characterized by having one or more (preferably all) of the following characteristics:

- Alkali lignin has a pH value of 3.0 to 4.0, and/or

- The Mw of alkali lignin is 1800 g/mol to 4000 g/mol, and/or

- The Mn of alkali lignin is 600 g/mol to 1000 g/mol, and/or

- The particle size distribution of alkali lignin is characterized by d90 less than 250 μm, preferably 180 μm to 220 μm, and d50 of 50 μm to 80 μm, preferably 60 μm to 75 μm.

In some embodiments, the polydispersity index (PI) of the industrial lignin is about 2.80 to 3.10, such as about 2.95. In some embodiments, the maximum molar mass (Mmax) of the industrial lignin is at least 14,000 g/mol, such as 14,000 g/mol to 18,000 g/mol.

The Mw, Mn, PI, and Mmax of the starting industrial lignin can be determined by any suitable method known to those skilled in the art, such as size exclusion chromatography (SEC) as further described below.

In the method of this invention, industrial lignin is directly extracted using a target solvent, meaning that no fractionation or extraction steps are performed on the industrial lignin prior to step (i). Specifically, no solvents other than the solvent used in step (i), including ethyl acetate, are used to extract the industrial lignin. Furthermore, no additional acid or base treatment, such as using acidic or alkaline aqueous solutions, is performed on the industrial lignin prior to step (i). More generally, the method of this invention does not include any treatment of the industrial lignin with aqueous solutions, whether the solution is acidic or alkaline. In particular, the method of this invention does not include the step of suspending or dissolving the industrial lignin or lignin fractions in aqueous solutions (including acidic or alkaline aqueous solutions).

In certain embodiments, the method of the present invention does not include any extraction or fractionation of industrial lignin with ethyl acetate, or more generally with any ester solvent, before or after step (i). In a preferred embodiment, step (i) is the only step of solvent extraction or solvent fractionation performed in the method of the present invention.

In some implementations, no pretreatment steps are performed on the industrial lignin prior to step (i).

Extraction step (i) typically involves resuspending the industrial lignin in the target solvent in a suitable container. No aqueous solutions, including water, are added in this step. The mixture is stirred by rail agitation or by mechanical or magnetic stirring for at least 5 minutes, such as at least 10 minutes, 30 minutes, or 60 minutes, such as 60 minutes to 2 hours or 2 hours to 4 hours. The extraction duration may depend on the amount of industrial lignin to be extracted.

Typically, at least 1 liter of solvent can be used per kilogram of industrial lignin, for example, 5 to 60 liters of solvent per kilogram of industrial lignin, especially 5 to 50 liters of solvent per kilogram of lignin, for example, 5 to 10 liters, 10 to 20 liters, 20 to 30 liters or 30 to 40 liters of solvent per kilogram of lignin.

Extraction is typically carried out at temperatures between 15°C and 40°C, such as room temperature. Preferably, the mixture of lignin and solvent is not heated.

After extraction, the liquid phase containing soluble lignin fractions is separated from the solid phase, which is mainly composed of insoluble long-chain lignin polymers, and recovered. This separation step can be performed using any method known to those skilled in the art, such as decantation, filtration (e.g., on a glass filter), or centrifugation. Preferably, the liquid phase is recovered by filtration.

The solvent in the liquid phase is then removed, for example, by vacuum evaporation, to obtain the graded lignin of the present invention (also known as lignin fraction). Optionally, the lignin fraction can be recovered from the liquid phase, for example, by first concentrating the liquid phase and then precipitating it using a hydrophobic solvent such as hexane. Optionally, the concentrated lignin fraction can be freeze-dried.

Lignin fractions are usually obtained in solid form such as powder.

As fully explained above and below, the resulting lignin fraction is characterized by a specific composition of lignin monomers, dimers, and oligomers in well-defined proportions. Without being bound by any theory, the inventors believe that this specific composition is the reason for the improved emulsifying properties of the lignin fraction.

In some embodiments, the resulting lignin fractions can be used as emulsifiers (e.g., directly) to stabilize oil-in-water emulsions.

The inventors have demonstrated that esterification with short-chain fatty acids (such as _C2 - _C18 fatty acids) can improve the emulsifying properties of the lignin fraction obtained in step (i). In fact, lignin monomers, dimers, and oligomers all contain aliphatic and phenolic hydroxyl groups, which can all be esterified.

In some embodiments, the method of the present invention further includes step (ii), which involves esterifying the lignin fraction obtained in step (i).

Typically, step (ii) causes the free aliphatic primary hydroxyl groups in the lignin molecule to form alkyl esters, preferably _C2 - _C18 alkyl esters. The _C2 - _C18 alkyl ester chain can be straight or branched, preferably straight.

Step (ii) can be carried out by any esterification technique known to those skilled in the art.

In a preferred embodiment, step (ii) is carried out by enzymatic transesterification. In other words, the transesterification is catalyzed by a suitable enzyme. Preferably, the enzyme is a lipase belonging to enzyme classification (EC) 3.1.1.3, and the reaction is carried out in the presence of an ester, preferably a fatty acid methyl ester or fatty acid ethyl ester (usually a methyl/ethyl ester of a _C2 - _C18 carboxylic acid, preferably a _C4 - _C10 carboxylic acid, which may be linear or branched, preferably linear).

In fact, this enzymatic transesterification can selectively esterify aliphatic primary hydroxyl groups without producing side reactions on phenolic hydroxyl groups. Since phenolic groups contribute to the antioxidant properties of lignin, it is highly advantageous to keep these groups in a free state.

There are various lipases available on the market for industrial applications. The enzyme can be immobilized on a solid support (such as polymer beads, like acrylic beads) to allow for recycling.

Target enzymes include, but are not limited to: Candida antartica lipase B (CAL-B), Rhizopus oryzae lipase, Candida rugosa lipase, Pseudomonas fluorescens lipase, porcine pancreatic lipase, and their variants. Lipases can be isolated from naturally occurring materials or produced recombinantly.

Preferably, the lipase is Candida antarcticis lipase B (CAL-B). For example, CAL-B immobilized on acrylic resin can be used, such as CAL-B (≥5,000 U/g, recombinant, expressed in Aspergillus niger ) sold by Sigma Aldrich.

For example, the ester can be methyl hexanoate or ethyl hexanoate.

Preferably, the esterification of the lignin fraction is carried out via enzymatic transesterification, using CAL-B as the lipase and ethyl hexanoate as the ester.

Enzymatic transesterification is carried out under standard conditions. For example, particularly when the lipase is CAL-B, the lignin fraction obtained in step (i) is dissolved in a suitable solvent, such as MEK (e.g., 10 to 50 g of lignin fraction per liter of solvent). After dissolution, the enzyme (i.e., CAL-B) and the ester (e.g., ethyl hexanoate) are added, and the mixture is reacted, preferably under stirring, until sufficient conversion is obtained. The mixture can be heated or even placed under reflux conditions (e.g., in a DeanStark apparatus). Transesterification can continue for several hours. In the case of CAL-B, the reaction can continue for about 48 hours to obtain a suitable grafting yield. The mass ratio between the alkyl reagent and lignin can be from 0.5 to 2.0, such as about 1. The amount of enzyme added is from 50 U to 3000 U of enzyme per gram of lignin, such as 250 U to 2500 U of enzyme per gram of lignin, such as about 1000 U of enzyme per gram of lignin. Advantageously, the enzyme can be immobilized on a support so that it can be easily recovered at the end of the reaction.

After the reaction is complete (and after cooling), the esterified lignin fraction can be recovered using any standard method. For example, the mixture can be filtered as needed and concentrated under reduced vacuum. The esterified lignin fraction can be recovered by precipitation (e.g., in n-hexane), followed by washing, filtration, and vacuum drying.

Typically, enzymatic transesterification can esterify aliphatic hydroxyl groups present in lignin fractions to 5% to 25% mol (e.g., esterification of aliphatic hydroxyl groups present in lignin fractions is 20% mol/mol).

The resulting esterified lignin fractions may be sold and/or used as emulsifiers, or after additional steps such as dispersion or dissolution in a suitable carrier and packaging.

In this regard, the inventors have shown that pre-dissolving the lignin fraction obtained in step (i) or its esterified form obtained in step (ii) in a suitable solvent can promote the acquisition of an oil-in-water emulsion.

Therefore, in some embodiments, the method of the present invention further includes the step of dissolving the lignin fraction (optionally esterified) in a solvent.

Solvents are typically chosen to facilitate the dissolution of lignin fractions while also being cosmetically acceptable.

Target solvents include: alkyl glycols, alkyl levulinates (such as methyl levulinate or ethyl levulinate), ketal levulinates (e.g., glyceryl ketal levulinate), methyl levulinate propylene glycol ketal (methyl-LPK), ethyl levulinate propylene glycol ketal (ethyl-LPK), n-butyl levulinate propylene glycol ketal (n-butyl-LPK), lower alcohols (e.g., _C2 - _C6 alcohols, preferably ethanol and isopropanol), and lower alcohol/ _H2O mixtures (e.g., EtOH/ _H2OH from 99/1 v/v to 50/50 v/v).

As used herein, "alkanediol" refers to an alkyl group having two hydroxyl groups, which can be branched or straight-chain alkyl. The general formula for alkanediol is C _{_n} H( _2n+2 )O _₂ , where n is an integer. Preferably, n is an integer from 2 to 8, and more preferably an integer from 2 to 6.

Specifically, the target alkyldiols include _C2 - _C6 alkyldiols, such as ethylene glycol, propylene glycol, butanediol, pentanediol, and their isomers, such as methylpropanediol. Preferred alkyldiols are ethylene glycol, such as ethane-1,2-diol (also known as ethylene glycol), and propylene glycol, such as propane-1,3-diol. A preferred alkyldiol is propane-1,3-diol.

In this case, the lignin fraction optionally dissolved in the alkyldiol corresponds to the emulsifier.

Typically, the weight ratio of lignin fraction to alkyldiol can be 0.01 to 0.8, such as 0.1 to 0.5, or about 0.25.

The dissolution process in the solvent can be carried out using standard procedures. For example, the lignin fraction is added to the solvent with stirring. If necessary, the mixture can be lightly heated (<50°C) to promote dissolution.

The method of the present invention may include additional steps, such as packaging the emulsifier in a suitable vial under a protected environment, decolorizing the emulsifier, and sterilizing the emulsifier (e.g., by ultraviolet or gamma rays, water vapor, pulsed light, or combinations thereof).

The lignin fraction obtained in step (i) or optionally step (ii) may be colored, which may be detrimental to its use in light-colored compositions. Therefore, in some embodiments, the method of the present invention may include a decolorization step in order to obtain a lighter-colored lignin fraction.

This step can be performed by any method known to those skilled in the art, such as ozone, hydrogen peroxide and/or ultraviolet treatment, activated carbon filtration, bentonite treatment, and combinations thereof. This step can be performed after step (i) and before the optional dissolution and packaging steps.

In some specific embodiments, particularly when the lignin fraction is used in colored cosmetics, a decolorization step is not required. In fact, the lignin fraction of the present invention is a natural brown or beige color and can be used both as an emulsion product and as a pigment in cosmetics, such as color cosmetics (e.g., foundation, blush, eyeshadow, colored creams such as BB cream, mascara, eyeliner, concealer).

In some embodiments, the present invention relates to a method for preparing an emulsifier from industrial lignin selected from organic solvent lignin or alkali lignin (preferably alkali lignin), the method comprising: step (i) directly extracting the industrial lignin using a polar solvent selected from ethanol, MEK, isopropanol and acetone (preferably ethanol or MEK), which includes dissolving the industrial lignin in the solvent under conditions capable of extracting target lignin molecules, separating the liquid phase and the solid phase, recovering the liquid phase, and removing the solvent from the liquid phase, thereby obtaining an emulsifier in lignin fraction form.

In certain embodiments, the method of the present invention further includes one or all of the following steps:

- A step of enzymatic transesterification of the lignin fraction obtained in step (i) by a lipase (such as CAL-B) belonging to enzyme classification 3.1.1.3, in the presence of _C2 - _C18 methyl or ethyl esters (preferably _C4 - _C8 carboxylic acids, such as ethyl hexanoate), and/or

- The step of dissolving the lignin fraction (optionally esterified) in _C2 - _C6 alkyldiol (preferably propylene glycol).

Furthermore, the method of the present invention may further include a step of decolorizing the lignin fraction and/or a step of packaging the lignin fraction.

Needless to say, the present invention also relates to emulsifiers that are obtained or available through the methods of the present invention.

- Lignin fractionation according to the present invention

The method for preparing emulsifiers of the present invention can obtain lignin fractions exhibiting specific polydispersity, namely, a specific quantitative distribution of lignin monomers, lignin dimers and lignin oligomers.

In fact, the method of the present invention can remove the long polymer chains of lignin while retaining oligomers and short-chain lignin derivatives.

Without being bound by any theory, the inventors believe that the unique composition of the lignin fraction of the present invention helps to improve its emulsifying properties compared with industrial lignin and highly graded lignin.

Therefore, the present invention also relates to an emulsifier in the form of lignin fractions, wherein the lignin fractions are characterized in that they comprise:

- Lignin monomers comprising 6.0% to 8.5% by weight, preferably 7.0% to 8.2%, more preferably 7.2% to 8.0% by weight.

- 14.0% to 20.0% by weight, preferably 15.0% to 20.0%, more preferably 15.5% to 18.0% of lignin dimer, and

- Lignin oligomers comprising 70% to 80% by weight, preferably 71.8% to 78.0%, more preferably 74.0% to 77.3%.

The weight percentage is relative to the total weight of lignin monomers, dimers, and oligomers in the emulsifier.

In a particular embodiment, the lignin fraction is substantially composed of lignin monomers, dimers and oligomers in the amounts defined above, which means that the total weight percentage of lignin monomers, dimers and oligomers accounts for at least 95%, preferably at least 96%, 97%, 98% or 99% of the total weight of the lignin fraction.

In some embodiments, the lignin fraction consists of the amounts of lignin monomers, dimers and oligomers as defined above, wherein the sum of the weight percentages of lignin monomers, dimers and oligomers is 100%.

In a preferred embodiment, the lignin fraction comprises or is substantially composed of the following:

- 7.4% to 7.9% lignin monomer by weight,

- 15.6% to 17.8% lignin dimer by weight, and

- 74.3% to 77.0% lignin oligomers by weight,

The weight percentage is relative to the total weight of lignin monomers, dimers, and oligomers in the emulsifier.

For example, the lignin fraction consists of approximately 7.6% lignin monomers, approximately 17.3% lignin dimers, and approximately 75.1% lignin oligomers.

As another example, the lignin fraction consists of approximately 7.7% lignin monomers, approximately 16.1% lignin dimers, and approximately 76.2% lignin oligomers.

As used herein, "lignin monomer" refers to a monophenolic derivative produced during the pulping process of lignin-cellulose biomass, which can be found in industrial lignins such as alkali lignin. The lignin monomers present in the lignin fraction of this invention have a molecular weight of up to 350 g/mol, preferably from 100 g/mol to 350 g/mol. Figure 1B shows an example of the target lignin monomers. GC-MS analysis of the starting alkali lignin showed that the main lignin monomers included acetylsuccinone, syringaldehyde, vanillin, ferulic acid, coumaric acid, acetylsuccinone, syringic acid, benzoic acid, vanillic acid, and benzaldehyde.

As used herein, a "lignin dimer" refers to a molecule composed of at least two structural units selected from lignin monomers and/or lignin monomer alcohols, which may be identical or different. Lignin dimers are also derived from protolignin during the pulping process of lignin-cellulose biomass by breaking down lignin and/or through covalent coupling of two structural units. A general characteristic of lignin dimers is the presence of five different types of bonds between phenolic units, including direct 5,5'-ring-ring bonds, and β-O-4, β,1-diketone, α,1-monoketone, α,5-monoketone, and α,2-methyl side-chain ring couplings. Figure 1C shows an example of a lignin dimer. The molecular weight of the lignin dimers present in the lignin fractions of the present invention is generally up to 700 g/mol, preferably from 350 g/mol to 700 g/mol.

As used in this article, “lignin monomer alcohol” (also known as lignin precursor) refers to naturally occurring molecules involved in lignin biosynthesis. The three main precursors of protolignin are coniferol (also known as the G unit), sinigrin (also known as the S unit), and p-coumarol (also known as the H unit) (Figure 1A).

As used herein, lignin oligomers refer to oligomers composed of at least three structural units selected from lignin monomers and/or lignin monomer alcohols, which may be the same or different. Lignin oligomers are also produced from native lignin during the pulping process of lignin-cellulose biomass.

For examples of oligomers that may be generated during industrial pulping, see Prothmann, J., et al. Anal Bioanal Chem 410, 7803–7814 (2018). https://doi.org/10.1007/s00216-018-1400-4.

The molecular weight of the lignin oligomers present in the lignin fraction of the present invention is generally at most 10,000 g/mol, preferably from 700 g/mol to 9,900 g/mol. The average molecular weight of the lignin oligomers is preferably from 900 g/mol to 2,300 g/mol, such as about 1,200 g/mol.

Typically, lignin oligomers have 3 to 20 monomers and/or lignin monomer alcohols, preferably 3 to 10 monomers and/or lignin monomer alcohols, such as 3 to 6 lignin monomers and/or lignin monomer alcohols, for example 3, 4, 5 or 6 monomers and/or lignin monomer alcohols.

In a specific embodiment, the oligomers present in the lignin fraction of the present invention have one or more of the following characteristics (preferably all of them):

- Weight-average molar mass (Mw) of 1400 g/mol to 1700 g/mol, such as 1400 g/mol to 1650 g/mol or 1500 g/mol to 1600 g/mol, and/or

- Number-average molar mass (Mn) of 900 g/mol to 1300 g/mol, such as 1000 g/mol to 1200 g/mol or 1050 g/mol to 1150 g/mol, and/or

- The multi-dispersion index is approximately 1.4.

The Mw, Mn, and PI of oligomers can be determined by HP-SEC analysis, as described below, for example.

Generally, the lignin fraction of the present invention comprises lignin molecules with a molecular weight of 100 g/mol to 10,000 g/mol.

In some embodiments, the lignin fraction of the present invention comprises lignin molecules with a weight-average molar mass (Mw) of 1200 g/mol to 1600 g/mol (e.g., 1200 g/mol to 1500 g/mol), and/or lignin molecules with a number-average molar mass (Mn) of 500 g/mol to 800 g/mol (e.g., 600 g/mol to 700 g/mol).

In a specific embodiment, the polydispersity index of the lignin fraction of the present invention is 1.85 to 2.40, preferably 1.95 to 2.20.

In some embodiments, the maximum molar mass (Mmax) of the lignin fraction of the present invention is less than 10,000 g/mol, for example 7,000 g/mol to 9,000 g/mol, or 7,500 g/mol to 8,500 g/mol, for example about 8,000 g/mol.

The molecular weight range, Mw, Mn, Mmax and polydispersity index (PI) of the lignin fractions of the present invention can be determined by HP-SEC, for example based on the relative areas of the elution peaks of each target compound.

For example, the quantitative composition of lignin fractions can be determined by high-performance size exclusion chromatography (HP-SEC) under the following conditions:

- Stationary phase: Polystyrene-divinylbenzene

- Eluent: Tetrahydrofuran

- Detection: UV absorption rate at 280 nm.

Typically, HP-SEC analysis is performed as follows: The lignin fraction to be analyzed is dissolved at a concentration of 1 mg/mL in a suitable solvent (e.g., THF containing 1% toluene (internal standard), optionally filtered if necessary. The lignin fraction is then injected into a typical polystyrene-divinylbenzene column (e.g., a Mixed E column from Polymer Laboratories, 3 µm 600 x 7.5 mm) and eluted at a constant flow rate with a suitable solvent (e.g., THF stabilized with BHT, at 1 mL/min). The eluted lignin species are detected using a UV absorbance detector (e.g., at 280 nm or 320 nm).

A chromatogram corresponding to ultraviolet absorption was obtained.

Porous beads in the stationary phase separate molecules based on their hydrodynamic volume, which is typically estimated from their molar mass. Therefore, molecules with a hydrodynamic volume smaller than the pore size migrate into the beads, extending their elution path and increasing their retention time in the column. Molecules larger than the pore size cannot diffuse into the beads; instead, they migrate with the mobile phase without entering the pores and reach the end of the column faster than smaller molecules.

In most cases, retention time is linearly related to the logarithm of the molecular weight of the eluted species, so the molar mass (in g/mol) of each eluted species can be obtained from the calibration curve. On the other hand, the percentage of each eluted species in the lignin fraction can be determined by integrating the area under the UV absorption peak obtained from SEC analysis.

In some embodiments, the lignin fraction is characterized by the chromatogram shown in Figure 2B, which was obtained by the SEC analysis described in Example 1. In the SEC analysis, the percentages of monomers, dimers, and oligomers were determined by peak area integration, with peak area integration times of 17 to 19.5 minutes for monomers, 16 to 17 minutes for dimers, and less than 16 minutes (11 to 16 minutes) for oligomers.

The inventors also demonstrate that the lignin fractions of this invention may have a specific content of hydroxyl groups. Lignin molecules comprise three types of hydroxyl groups: aliphatic hydroxyl groups, phenolic hydroxyl groups, and acidic alcohols (i.e., the OH group in the -COOH group). Each hydroxyl group can be identified and quantified by ³¹ pNMR after derivatization using 2-chloro-4,4,5,5-tetramethyl-1,3,2-dioxophosphazenecyclopentane.

Therefore, in a particular embodiment, the lignin fraction of the present invention (before any possible esterification) contains 1.25 to 1.65 mmol/g, for example 1.40 ± 0.05 mmol/g, aliphatic OH groups, as confirmed by ³¹ P NMR after derivatization with 2-chloro-4,4,5,5-tetramethyl-1,3,2-dioxophosphazenecyclopentane.

As described above, lignin fractions can be esterified, preferably via enzymatic transesterification. Therefore, in some embodiments, the emulsifier in the form of the lignin fraction of the present invention is esterified with _C2 - _C18 alkyl ester groups (such as hexanoate groups). In some embodiments, the lignin fraction is characterized by the esterification of 5% to 25% mol/mol (e.g., about 20% mol/mol) of its aliphatic hydroxyl groups, where the percentage refers to the number of moles of esterified aliphatic hydroxyl groups relative to the initial number of moles of aliphatic hydroxyl groups present in the lignin fraction.

The emulsifier of the present invention can be in a dry form, such as a powder. Alternatively, the emulsifier of the present invention can be in a liquid form, for example, the lignin fraction can be dispersed (preferably dissolved) in a suitable carrier or solvent, such as _C2 - _C6 alkyldiols, such as propylene glycol. As described above, other solvents are contemplated, such as alkyl levulinates, ketal levulinates (e.g., glyceryl ketal levulinate), methyl levulinate propylene glycol ketal (methyl-LPK), ethyl levulinate propylene glycol ketal (ethyl-LPK), n-butyl levulinate propylene glycol ketal (n-butyl-LPK), lower alcohols (e.g., _C2 - _C6 alcohols, preferably ethanol and isopropanol), and lower alcohol/ _H2O mixtures (e.g., 99/1 v/v to 50/50 v/v EtOH/ _H2OH ).

Typically, the lignin fraction/solvent weight ratio can be from 0.01 to 0.8, such as from 0.1 to 0.5, or about 0.25.

In some embodiments, the emulsifier may further comprise excipients, such as cosmetically acceptable excipients. For example, the emulsifier may further comprise stabilizers, such as antioxidants, preservatives, and/or suspending agents. Such excipients may comprise 0.01% to 5% of the total weight of the emulsifier.

-Method of using the emulsifier of this invention

As shown in the Examples section, the lignin fractions (unesterified and esterified forms) of the present invention can effectively stabilize oil-in-water emulsions with an oil content of up to 80% by weight or even higher. This stabilizing effect can last for several months during storage.

Therefore, this invention also relates to the use of the lignin fractions of this invention (both non-esterified and esterified forms), and more generally to the use of the emulsifiers of this invention in stabilizing emulsions (especially oil-in-water emulsions). More generally, this invention relates to the use of the lignin fractions provided herein as emulsifiers in the preparation of oil-in-water emulsions.

The lignin fraction of the present invention can be used in any field, such as pharmaceuticals, cosmetics, home care and detergents, and the food industry.

Therefore, the lignin fraction of the present invention, and more generally, the emulsifier or oil-in-water emulsion of the present invention, can be found in a variety of products, such as pharmaceuticals, cosmetics, household care products, detergents, and food. Cosmetics mainly include skin care products and hair care products.

Applications in the pharmaceutical and cosmetic fields are preferred.

In a preferred embodiment, the water-in-oil emulsion stabilized by the emulsifier of the present invention (such as the lignin fraction of the present invention) can be used in cosmetic compositions.

The target cosmetic composition includes, but is not limited to, creams such as face cream or body cream, body lotion, makeup remover, conditioner, makeup products (especially foundation, blush, eyeshadow, tinted creams such as BB cream, mascara, eyeliner, concealer), liquid soap, shampoo, nourishing mask, etc.

In fact, the inventors have demonstrated that the lignin fractions of the present invention provide highly satisfactory sensory properties in emulsions. Furthermore, emulsions prepared with the lignin fractions of the present invention are easy to apply and have a soft, powdery, and non-greasy effect on the skin.

In certain embodiments, the lignin fraction of the present invention, and more generally the emulsifier of the present invention, is also used to improve the sensory properties of the composition, preferably the sensory properties of a cosmetic composition.

Needless to say, the present invention also relates to a composition comprising the emulsifier of the present invention. More specifically, the present invention relates to a composition comprising an emulsion (particularly an oil-in-water emulsion stabilized by the emulsifier of the present invention).

As described above, the composition can be of any type. In addition to the lignin fractions provided herein, the composition may contain one or more other excipients (such as pharmaceutically or cosmetically acceptable excipients), optionally in combination with one or more active ingredients (such as one or more cosmetic active ingredients or one or more pharmaceutical active ingredients).

The emulsifier of the present invention is typically present in an oil-in-water emulsion in an amount of 0.1% to 5% by weight of the lignin fraction (present in the emulsifier) as a percentage of the total weight of the emulsion, preferably 0.5% to 4.0% (such as 2.0% to 3.0%, or about 2.5%).

To illustrate only, when the emulsifier is in the form of a lignin fraction dissolved in propylene glycol at a weight ratio of 0.25, the emulsifier may account for 12.5% by weight of the total weight of the emulsion (which is equivalent to 2.5% by weight of the lignin fraction).

Compared to the total weight of the emulsion, the composition (preferably a cosmetic composition) may contain 0% to 10% of the active ingredient and 70% to 99.9% of other excipients, expressed in percentages by weight.

This application also provides a method for preparing an oil-in-water emulsion based on the emulsifier of the present invention.

The method includes the following steps:

- Provides an aqueous phase comprising the emulsifier of the present invention.

- The aqueous phase and the oil phase are mixed to form an oil-in-water emulsion.

The inventors have demonstrated that pre-dissolving the lignin in an alkyl glycol before adding it to the oil phase, and then dissolving it in an aqueous phase with a pH of 5.5 to 7.5 (usually around 6.0), can promote emulsion formation and prevent lignin precipitation.

More specifically, the inventors have demonstrated that once pre-dissolved lignin (e.g., lignin fraction + propylene glycol) is added to the aqueous phase (e.g., at a neutral pH), the pH decreases and becomes acidic (e.g., around 3.0). Readjusting the pH of the aqueous phase to 5.5 to 7.5 facilitates the dissolution of the lignin fraction and prevents its precipitation upon addition to the oil phase. Once proper dissolution of the lignin fraction is achieved by adjusting the pH, the oil phase can be added. This method also yields emulsions with better time stability, i.e., no significant sedimentation or aggregation over time.

In one particular aspect, the present invention also relates to a method for preparing an oil-in-water emulsion, the method comprising:

(a) Providing the lignin fraction of the present invention that is at least partially dissolved in a solvent.

(b) The lignin solution is added to the aqueous phase with stirring, and the pH of the aqueous phase is readjusted to a value of 5.5-7.0, preferably about 6.0, and

(c) Add the oil phase to the solution obtained in step (b) to obtain an oil-in-water emulsion.

In step (a), solvents such as alkyl levulinates, ketal levulinates (e.g., glyceryl ketal levulinate), methyl levulinate propylene glycol ketal (methyl-LPK), ethyl levulinate propylene glycol ketal (ethyl-LPK), n-butyl levulinate propylene glycol ketal (n-butyl-LPK), lower alcohols (e.g., C2- _{C6 alcohols} , preferably ethanol and isopropanol), and lower alcohol/ _H2O mixtures (e.g., 99/1 v/v to 50/50 v/v EtOH/ _H2OH ). Preferably, the solvent is an alkyldiol, specifically a _C2 - _C6 alkyldiol, and more preferably propylene glycol. The weight ratio of the lignin fraction to the alkyldiol is 0.01 to 0.8, such as 0.1 to 0.5, e.g., about 0.25.

The pH of the aqueous phase in step (b) can be adjusted by using any organic or inorganic base and acid suitable for the intended use of the final emulsion, such as cosmetically or pharmaceutically acceptable bases and acids. Concentrated aqueous solutions of said base and acid, such as concentrated aqueous solutions of sodium hydroxide or citric acid, can be used.

Preferably, step (b) is carried out under mechanical or magnetic stirring, for example at a speed of about 100 rpm to 1000 rpm, such as about 500 rpm to 600 rpm.

In step (c), the oil phase is preferably added to obtain an emulsion under high-speed, high-shear stirring, for example using a rotor-stator device or a homogenizer. For example, a rotation speed of 10,000 rpm can be used. Alternatively, ultrasound can be used to promote emulsification.

The oil phase can account for up to 80% or more of the final emulsion weight.

For example, the oil-in-water emulsion obtained in step (c) may include:

- 20% to 99%, preferably 40% to 90% aqueous phase (including lignin fraction in solvent), and

- 1% to 80%, preferably 10% to 60% oil phase,

Percentages compared to the total weight of the emulsion are expressed by weight.

The aqueous phase is typically water, such as distilled water or ultrapure water. The aqueous phase may contain one or more additional excipients, such as suspending agents, antioxidants, preservatives, pigments, colorants, humectants, gelling agents, solubilizers, coemulsifiers, and combinations thereof.

The oil phase can be of any type, such as mineral oil, synthetic oil, vegetable oil, and animal oil. Preferably, an oil is chosen so that it is pharmaceutically or cosmetically acceptable.

In some embodiments, the oil phase is selected from vegetable oils, such as fatty esters, sweet almond oil, sunflower oil, calendula oil, olive oil, jojoba oil, coconut oil, argan oil, avocado oil, borage oil, and palm oil.

The oil phase may contain one or more components that can be incorporated into an oil-in-water emulsion and subsequently added to complex compositions such as cosmetics. For example, these components may be lipophilic active ingredients with cosmetic effects. Examples of active ingredients with cosmetic effects include, but are not limited to, caffeine, lipophilic plant extracts such as absolute oils or essential oils.

The oil phase may also contain one or more excipients, such as pigments, functional polymers, clays, fatty alcohols, fatty acids, coemulsifiers, and combinations thereof.

The method for preparing an oil-in-water emulsion according to the present invention may include one or more additional steps.

In some cases, due to the rising of oil droplets, an emulsification effect (two phases: a blocky phase and an emulsified layer) can be observed during emulsion formation. This phenomenon does not necessarily mean that the stability of the emulsion is affected. The formation of a creamy phase can be prevented by adding a suspending agent. Therefore, in some embodiments, the method of the present invention may include the step of adding an excipient, such as a suspending agent, to avoid the formation of an emulsified phase, preferably before step (b).

As used herein, a suspending agent is a substance added to promote the suspension and/or dispersion of particles and to reduce sedimentation. Suspension agents include (but are not limited to) carrageenan, xanthan gum, hard gum, kalube gum, guar gum, tara gum, konjac gum, and cellulose ethers and their derivatives, such as methylcellulose (MC), sodium carboxymethyl cellulose (CMC), hydroxypropyl methylcellulose (HPMC), and acrylate derivative polymers.

Typically, a suspending agent can be added to the aqueous phase in step (b).

In a further aspect, the present invention relates to a method for preparing cosmetics (such as color cosmetic products), comprising:

(a) Provide the emulsifier or oil-in-water emulsion described herein, and

(b) Combining the emulsifier or oil-in-water emulsion with one or more cosmetically acceptable excipients and/or one or more active ingredients with cosmetic effects.

In another aspect, the present invention also relates to a method for preparing cosmetics, comprising the following steps:

(i) Preparation of oil-in-water emulsions according to the methods provided herein, and

(ii) The oil-in-water emulsion is mixed with one or more excipients and/or one or more active ingredients with cosmetic effects.

In a further aspect, the present invention also relates to a cosmetic, such as a colored cosmetic comprising the lignin fraction or emulsifier provided herein.

In such cosmetics, lignin fractions can have two functions: as an emulsifier and as a pigment. Indeed, as mentioned above, the lignin fractions of this invention are naturally beige or brown, and therefore can be used as pigments.

The cosmetics of this invention include, but are not limited to, makeup products such as foundation, blush, eyeshadow, tinted body cream or face cream such as BB cream, mascara, eyeliner, concealer, etc.

Another object of the present invention is to use the lignin fraction provided by the present invention as an emulsifier and/or pigment in cosmetics.

The following examples are for illustrative purposes only and do not constitute a limitation.

Example Section

Chemicals and starting materials

Methyl ethyl ketone and ethyl acetate were purchased from Carlo Erba Reagents (France) and used as is. Grass (a mixture of wheat straw and sweetgrass), soda ash, and industrial lignin were purchased from Green Value (Switzerland). Ethyl hexanoate was purchased from Sigma-Aldrich (France), propylene glycol (Zemea) from DuPont (USA), and refined sweet almond oil from Jan Dekker (France). Other reagents and lipase from * Candida Antarctica * acrylic resin (…) were also used. 5,000 U/g, recombinant (expressed in Aspergillus niger ), named CAL-B, purchased from Sigma Aldrich.

Industrial alkali lignin has the following characteristics:

- pH value: 3 to 4

- Particle size <250 µm, d50: approx. 65 µm, d90: approx. 200 µm.

Example 1: Preparation and characterization of lignin fractions

The method of the present invention

Alkali lignin was magnetically stirred in MEK (40 L/Kg) for 2 hours at room temperature.

The mixture was filtered on a glass filter, and the filtrate was then evaporated under reduced pressure to remove MEK and recover the first lignin fraction of the invention (LFI-1) as a brown solid (58 % wt).

The same procedure was performed using ethanol as the extraction solvent. The second lignin fraction of the present invention (LFI-2) was obtained, which was a brown solid (68% wt).

Grading methods (comparison)

The fractionation method was based on that of Jin et al. (12). This method involves a two-step sequential extraction with ethyl acetate (AcOEt) followed by methyl ethyl ketone (MEK).

Alkali lignin (17 L/kg lignin) was magnetically stirred in ethyl acetate (AcOEt) at room temperature for 2 hours. The mixture was filtered through a glass filter, and the insoluble residue was recovered for further extraction. The insoluble residue was then magnetically stirred in MEK (17 L/kg) at room temperature for 2 hours. The mixture was filtered through a glass filter, and the filtrate was evaporated under reduced pressure to remove MEK, yielding a second comparative lignin fraction (CLF) as a brown solid (22% wt).

Characterization of lignin fractions

Size exclusion chromatography

The lignin fraction to be analyzed was dissolved in THF (containing 1% toluene) at a concentration of 1 mg/mL in vials and optionally filtered if necessary. The lignin fraction was then injected into a polystyrene-divinylbenzene column (Polymer Laboratories Mixed E column, 3 µm x 7.5 mm x 600 mm) and eluted with BHT-stabilized THF at a constant flow rate of 1 mL/min. The eluted lignin species were detected using a UV absorbance detector.

- ³¹ P NMR

A ³¹ P NMR sample (~25 mg) was prepared using 2-chloro-4,4,5,5-tetramethyl-1,3,2-dioxophosphazenecyclopentane as the phosphating agent (~50 μL) and a mixture of _CDCl₃ /pyridine (1/1.6, ~0.5 mL) as the solvent. A total of 128 scans were performed, with a delay of 6 seconds between two consecutive pulses. The following regions were integrated relative to triphenylphosphine (-5.0 ppm) as an internal standard (~20 mg): aliphatic hydroxyl (149.1–144.2 ppm), phenolic hydroxyl (143.8–137.0 ppm), and acidic region (136.6–133.6 ppm).

result

The chromatograms obtained by SEC chromatography are shown in Figures 2A-2C. Figure 2A shows the chromatogram of LFI-1 compared to that of the starting industrial lignin. Figure 2B shows the chromatograms of LFI-1 and LFI-2. Figure 3B shows the chromatograms of CLF and the starting industrial lignin.

Based on the area of each peak, the percentages of monomers, dimers, and oligomers are calculated by integration. The integration time for monomers is 17 to 19.5 minutes, for dimers it is 16 to 17 minutes, and for oligomers it is less than 16 minutes (11 to 16 minutes).

The SEC results showed that LFI-1 and LFI-2 consisted of approximately the same percentages of monomers, dimers, and oligomers. After fractionation with MEK or ethanol, both lignin fractions, LFI-1 and LFI-2, showed a decrease in the percentages of monomers and oligomers compared to the starting industrial lignin. In contrast, LFI-1 and LFI-2 showed an increase in the percentage of dimers compared to the starting industrial lignin. Therefore, single-step fractionation using EtOH or MEK resulted in an increase in dimers and a decrease in monomers and oligomers.

In contrast, CLF showed a significant reduction in the percentages of monomers and dimers compared to the starting industrial lignin. On the other hand, a significant increase in the percentage of oligomers was observed in CLF compared to the starting industrial lignin. The two-step fractionation resulted in a more monodisperse lignin fraction and an increased percentage of oligomers compared to the starting industrial lignin or the lignin fraction of the present invention.

Table 1: Mass percentage of monomers, dimers, and oligomers in lignin fractions characterized by SEC

Compared to other fractions, CLF exhibited the highest Mw and Mn values. LFI-1 and LFI-2 had very similar Mw and Mn values, both lower than the starting industrial lignin. These results indicate that CLF is primarily composed of high molecular weight lignin, while LFI-1 and LFI-2 are rich in low molecular weight lignin.

After single-step grading, the polydispersity index of the lignin fractions decreased (LFI-1 and LFI-2 compared to the starting industrial lignin), but remained higher than the polydispersity index of CLF.

It is worth noting that the maximum molecular _weight (Mmax) of the starting industrial lignin is 16,000 g/mol, while the maximum molecular _weight (Mmax) of LFI-1 is only 8,000 g/mol.

Table 2: Mw, Mn and polydispersity index

Table 2a: Mw, Mn and polydispersity index of oligomers present in each lignin fraction (elution time 11 to 16 minutes)

Compared to the aliphatic OH groups in the starting industrial lignin, a decrease in aliphatic OH groups was observed in both LFI-1 and CLF-1. Compared to LFI-1, CLF showed a slightly higher amount of aliphatic OH groups, which may be due to the higher amount of oligomers in CLF. No difference in phenolic OH groups or acidic OH groups was observed between LFI-1 and CLF compared to the starting industrial lignin.

Table 3: Types of OH groups in lignin fractions analyzed by NMR ^31P

Example 2: Esterification

plan

Lignin was placed in a round-bottom flask and dissolved in MEK (25 g/L). Ethyl hexanoate (1:1 mass ratio to the loaded lignin resin) and CAL-B (20% of the mass of the loaded lignin resin) were added to the reaction flask. Enzymatic esterification was carried out under reflux using a DeanStark instrument with magnetic stirring for 48 hours. After cooling to room temperature, the mixture was filtered to recover the supporting lipase. The solution was concentrated under reduced pressure and then precipitated into hexane with magnetic stirring. The precipitated lignin was recovered by filtration through a glass filter. The washed precipitate of grafted lignin gLFI-1 was dried overnight under vacuum.

result

The grafting rate of gLFI-1 was 1.2% w/w, which is equivalent to 12% mol/mol of aliphatic hydroxyl esterification in the lignin fraction.

Example 3: Stability Comparison Study

The aim of these studies was to compare the stability of emulsions obtained using starting industrial lignin or different lignin fractions (i.e., gLFI-1, LFI-1 (in this invention), and CLF (comparative)).

-Emulsion preparation

The preparation process of the oil-in-water emulsion is shown in Figure 3, or more precisely, the preparation process is as follows:

Using a Turbotest® mixer (VMI, France) with its deflocculation turbine at 700 rpm, the lignin fraction was pre-dissolved in propylene glycol for 5 minutes. The resulting solution was then added to ultrapure water while agitated (550 rpm, 10 minutes). The pH of the aqueous phase was readjusted to a target pH of 6.0 while agitated (550 rpm, 5 minutes). The aqueous phase containing sweet almond oil (10%) was emulsified at 10,000 rpm over 10 minutes using a T25 digital Ultra-Turrax® (IKA, Germany) equipped with a rotor-stator turbine S25 N-25F.

Table 4: Composition of the test emulsion

-Stability assessment

An emulsion is considered stable when it does not exhibit aggregation (droplet coalescence leading to an increase in droplet volume over time) and shows little or no lignin precipitation (sedimentation). Emulsion separation (two phases: a bulk layer and an emulsion layer) may occur due to oil droplet migration, but this does not affect the emulsion's stability. As explained in the instructions, adding a suspending agent to the aqueous phase can prevent emulsion separation.

- To assess stability, the lignin emulsions were stored in 15 mL Eppendorf tubes at 25°C and 40°C, respectively. The stability of the emulsions was observed by sensory analysis and optical microscopy, and the results are as follows:

- The sensory stability of the emulsion was studied after storage at 25°C and 40°C for 1 day, 7 days, 14 days, 1 month and 3 months.

- Optical microscopy analysis was performed on the emulsion after it was stored at 25°C for 1 day, 7 days, 14 days, 1 month and 3 months (see below).

- Measure the emulsion layer in mL of 10 mL emulsion.

- Sedimentation is measured in mL units in 10 mL of emulsion.

Optical microscopy: A drop of dispersion or emulsion sample was placed on a microscope slide covered with a coverslip and observed at different magnifications in bright field using a Nikon optical microscope equipped with a DS-Fi3 digital camera (5.9 MP CMOS, 2880 x 2048, 15 images per second). The micrographs were stored at ambient temperature. The photomicrographs were analyzed using NIS-Elements Viewer 5.21.

-result

Figure 4 shows the sensory and optical microscopic results. Due to the lack of a suspending agent, emulsification occurred in all emulsions. After stabilization at 40°C for 3 months, the emulsion layers in the emulsions containing starting industrial lignin and those containing CLF became darker. After stabilization at 40°C for 3 months, no emulsion phase development was observed in the emulsions containing LFI-1 and gLFI. Severe sedimentation was observed in the emulsions prepared from starting industrial lignin, while sedimentation was less severe in the lignin fraction.

The sedimentation of fractions LFI-1 and gLFI-1 was significantly reduced, especially that of gLFI-1. In summary, the stability data over time at 25℃ and 40℃ indicate that, regardless of grafting, the emulsion prepared using LFI-1 fractions is the most stable, with the lowest sedimentation, the smallest droplet size, and the best size uniformity.

Example 4: Effect of the method on the stability of oil-in-water emulsions

Several oil-in-water emulsions were prepared using LFI-1 to evaluate the effect of the preparation method on stability. The different oil-in-water emulsions were prepared according to the general scheme shown in Figure 3.

- Test 1: Pre-dissolved in propylene glycol, target pH 6:

The lignin fraction (LFI-1) (2.5%) of this invention was pre-dissolved in propylene glycol (10%) at 700 rpm using a Turbotest® mixer (VMI, France) with its deflocculation turbine at 700 rpm for 5 minutes. The resulting solution was then added to ultrapure water with stirring (550 rpm, 10 minutes). The pH of the aqueous phase was readjusted to a target pH of 6.0 with stirring (550 rpm, 5 minutes). The aqueous phase containing sweet almond oil (10%) was emulsified at 10,000 rpm over 10 minutes using a T25 digital Ultra-Turrax® (IKA, Germany) equipped with a rotor-stator turbine S25 N-25F.

Table 5: Components of Test 1

Test 2: Pre-dissolved in propylene glycol, without pH readjustment:

The lignin fraction (LFI-1) (2.5%) of this invention was pre-dissolved in propylene glycol (10%) at 700 rpm using a Turbotest® mixer (VMI, France) with its deflocculation turbine at 700 rpm for 5 minutes. The resulting solution was then added to ultrapure water with stirring (550 rpm, 10 minutes) to obtain a pH of 3. The aqueous phase containing sweet almond oil (10%) was emulsified at 10,000 rpm over 10 minutes using a T25 digital Ultra-Turrax® (IKA, Germany) equipped with a rotor-stator turbine S25 N-25F.

Table 6: Components of Test 2

Test 3: No pre-dissolution in propylene glycol, target pH 6:

The lignin fraction (LFI-1) (2.5%) of the present invention was added to ultrapure water under stirring (550 rpm, 10 minutes). The pH of the aqueous phase was readjusted to a target pH of 6.0 under stirring (550 rpm, 5 minutes). The aqueous phase containing sweet almond oil (10%) was emulsified at 10,000 rpm over 10 minutes using a T25 digital Ultra-Turrax® (IKA, Germany) equipped with a rotor-stator turbine S25 N-25F.

Table 7: Components of Test 3

The stability of the three emulsions over time was evaluated as described in Example 3.

- result

Figure 5 shows the sensory and optical microscopic results. Pre-dissolving the lignin fraction and adjusting the pH of the aqueous phase before adding the oil phase resulted in a stable emulsion. When the lignin fraction (LFI-1) was added to the aqueous phase without any pre-dissolution, the resulting emulsion exhibited a heterogeneous microstructure, with fragile droplets and sedimentation. Unreplacing the pH and readjusting it to approximately 6.0 resulted in an unstable emulsion with large phase shifts, fragile droplets, and an inhomogeneous microstructure. Therefore, pre-dissolving the lignin in alkyldiol and readjusting the pH of the aqueous phase to 6 before adding the oil phase yielded the most stable lignin emulsion over time, with minimal sedimentation, the smallest droplet size, and the best size uniformity.

Example 5: Further Stability Studies

According to the optimization method described in Figure 3 and Example 3, the following emulsions were prepared using the non-grafted lignin fraction LFI-1 of the present invention:

Table 8: Composition of Emulsion 1

Table 9: Composition of Emulsion 2

The stability of the two emulsions over time was assessed as follows:

- The lignin emulsions were stored in 15 mL Eppendorf tubes at 25°C and 40°C. The stability of the emulsions was observed by sensory analysis and optical microscopy, and the results are as follows:

- The sensory stability of the emulsion was studied after storage at 25°C and 40°C for 1 day, 7 days, 14 days, 1 month and 3 months.

- Optical microscopy analysis was performed on the emulsion after it was stored at 25°C for 1 day, 7 days, 14 days, 1 month and 3 months.

- Measure the emulsion layer in mL of 10 mL emulsion.

- Sedimentation is measured in mL units in 10 mL of emulsion.

result

Figure 6 shows the sensory and optical microscopic results. The results indicate that both emulsions have a uniform microstructure. No sedimentation was observed after storage at 25°C for 3 months. In conclusion, the lignin fraction of this invention is an effective emulsifier that meets the requirements of the cosmetics industry.

Example 6: Cosmetic Formulation

Prepare the following cosmetics:

Table 10: Personal Care Formulas

The final pH value of the formulation was 6.5. The results showed that the formulation remained homogeneous and stable at 25°C or 40°C for at least three months.

Table 11: Foundation Compositions

The foundation has a final pH of 6.5 and a viscosity of 9000 Pa·s. It exhibits a uniform surface and suitable viscosity. Even without gelling or suspending agents in the formulation, the foundation remains stable over time. It further demonstrates good pigment uniformity and color payoff upon application. The foundation is non-greasy and feels soft on the skin.

Example 7: Sensory analysis of cosmetic and commercial products containing the lignin fraction of the present invention

Sensory analysis was performed on the two water-in-oil emulsions of the present invention, and they were compared with commercial products. The commercial product is a pigment-based sunscreen with an oil phase of less than 60% (water-in-oil emulsion). The emulsions of the present invention were prepared according to the methods provided in Example 3 and Figure 3, and their composition is as follows:

Table 12: Emulsion 1 (prepared with LFI-1)

Table 13: Emulsion 2 (prepared with gLFI-1)

The sensory analysis protocol is as follows:

- Group: 21 people

- Indoor temperature: 20℃

- Humidity: 34.9±3.9%

- The isolation enclosure conforms to AFNOR NF EN ISO 8589:2007 standard.

- Microbiological analysis and irritation test of lignin emulsion

- Statistical Analysis: Anova and Tukey's Tests

- Describe the choice of indicators:

Table 14: Selection of Description Indicators

result

Figure 7 provides a sensory profile. The sensory profile shows that the two emulsions of this invention are similar to commercial products in certain descriptive metrics, such as gloss, smoothness, lightness, and spreadability. The difference observed in coverage metrics is actually due to the absence of pigments in either of the lignin emulsions, while commercial products contain pigments.

In addition, the sensory properties of the two lignin emulsions were very similar, but the emulsion containing the grafted lignin fraction gLFI had slightly higher spreadability, indicating the added value of grafting.

It is noteworthy that such excellent sensory results are quite surprising, as the lignin emulsion tested in this invention consists of only four components, compared to commercial products containing more than 50 ingredients. Compared to commercial products, the lignin emulsion of this invention exhibits interesting sensory properties, demonstrating greater shape integrity, smoothness, and gloss on the skin.

Example 8: Comparative Measurement

To illustrate the effect of the preparation method of lignin fractions on their distribution in oligomers, dimers, and monomers, as well as their emulsifying properties, comparative experiments were conducted.

- Comparative determination of N°1

According to the method entitled "Purification with Limited Solubility Solvents" described in US2015/0141628 (see §[0278]-[0280]), several lignin fractions were prepared from the same alkali industrial lignin used to prepare LFI-1 and LFI-2. This method is based on liquid-liquid extraction. In this method, an aqueous solution of acidic lignin is mixed with a solvent of limited solubility (such as MEK) to obtain a two-phase system. After recovering the solvent phase, a purification step can be performed using a strong acid cation exchanger to remove cations.

The solution used is as follows:

In a beaker, industrial lignin (same as in Example 1 - 1 g) was resuspended in water (100 mL) to obtain an aqueous lignin solution with a pH of approximately 3.

The lignin aqueous solution was transferred to a separation funnel, and then a solvent with limited solubility, MEK (100 mL), was added. The system was stirred and decanted (once). The aqueous phase (orange/light brown) was discarded, and the solvent phase (dark) was recovered. The solvent phase was stirred for 1 hour and 30 minutes in the presence of a strong acid cation exchange resin (Amberlist IR120 28 g), and then filtered to remove the resin.

The solvent phase is divided into two parts:

- The first part was concentrated under reduced pressure and then freeze-dried to obtain sample PF568. Yield: 81% wt.

The second portion was added dropwise to hot water (85°C), stirred, and cooled overnight. The mixture was then filtered to recover the precipitated lignin. The precipitated lignin was washed with water, filtered, and then freeze-dried to obtain sample PF569: yield 52% wt.

Since the use of strong acid cation exchange resin treatment in US2015/0141628 is optional, an alternative approach was also implemented.

In a beaker, industrial lignin (same as in Example 1 - 2.5 g) was resuspended in water (250 mL) to obtain an aqueous lignin solution with a pH of approximately 3.

The lignin aqueous solution was transferred to a separation funnel, and then a solvent with limited solubility, MEK (100 mL), was added. The system was stirred and decanted (one round). The aqueous phase (orange/light brown) was discarded, and the solvent phase (dark) was recovered. The solvent phase was concentrated under reduced pressure and then freeze-dried to obtain the lignin fraction (sample PF570).

HP-SEC Analysis

The lignin fractions were compared as described in Example 1, analyzed by HP-SEC, and compared with industrial lignin and LFI-1.

Figure 8 shows the SEC chromatograms of the comparative fractions (PF569, PF570, and PF568) compared to industrial lignin and LFI-1. The comparative chromatograms are not superimposed on the LFI-1 chromatogram. All comparative fractions show an increase in the proportion of high molecular weight oligomers. Notably, sample PF569 shows an enrichment of oligomers, while monomer and dimer content decreases. The following table lists the composition of each lignin fraction:

Table 15: Percentages of monomers, dimers, and oligomers in lignin fractions characterized by SEC

Therefore, it can be seen that the preparation method of lignin fractions has a direct impact on their composition.

The composition of fraction PF570 appears to be similar to that of LFI-1. Therefore, this fraction was used for emulsion determination.

- Emulsion assay

Oil-in-water emulsions were prepared using the comparative lignin fraction PF570, as described in Example 3. The stability of the obtained emulsions was also evaluated as described in Example 3. After one day, the emulsions showed obvious signs of sedimentation, with a shiny oily surface. Sedimentation indicates that some of the extracted lignin was not located at the oil-water interface. The oily surface is the main state of instability in the emulsion. Compared with the "LFI-1" and "g-LFI-1" emulsions, the droplet size distribution observed under the microscope was larger (Figure 4).

Therefore, the method of preparing lignin fractions also has a significant impact on emulsification properties.

- Comparative determination of N°2

This determination was performed to evaluate the effectiveness of acid and alkali treatment prior to the extraction of industrial lignin using solvents such as ethanol. For example, CN11226159 describes such a pretreatment method.

Industrial lignin (same as in Example 1 - 2.5 g) was mixed with NaOH solution (1250 mL - 10% wt in water, 2.5 M) and magnetically stirred at room temperature for 20 hours. The mixture was then filtered through a glass filter. After recovery of the filtrate, it was acidified with 6 M HCl to a pH of 2-3. The precipitate residue was filtered and washed with deionized water until the pH of the effluent reached approximately 6. The powder was then lyophilized. 1 g of the powder was mixed with 50 mL of 96° ethanol. The mixture was vigorously shaken in a circular motion at room temperature for 30 minutes, then centrifuged (15 minutes, 4800 rpm, 5°C). The supernatant was collected, combined, and evaporated using a rotary evaporator to obtain sample PF575D (extraction yield 79.1% wt), which was analyzed by high performance size exclusion chromatography (HP-SEC) according to the method described in Example 1.

Figure 8 shows the HP SEC analysis results for PF575D. Compared with the fraction of this invention, the comparative fraction PF575D shows a significant reduction in both monomers and dimers.

Alkali treatment followed by acidification has a significant impact on the composition of the final lignin fraction.

Table 16: Percentages of monomers, dimers, and oligomers in lignin fractions characterized by SEC

References cited in the background section

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(2) McClements, DJ; Gumus, CE Natural Emulsifiers—Biosurfactants, Phospholipids, Biopolymers, and Colloidal Particles: Molecular and Physicochemical Basis of Functional Performance. Adv. Colloid Interface Sci. 2016, 234 , 3–26. https://doi.org/10.1016/j.cis.2016.03.002.

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(5) Ivanković, T.; Hrenović, J. Surfactants in the Environment. Arch. Ind. Hyg. Toxicol. 2010, 61 (1), 95–110. https://doi.org/10.2478/10004-1254-61-2010-1943.

(6) Holt, MS; Waters, J.; Comber, MHI; Armitage, R.; Morris, G.; Newbery, C. AIS/CESIO Environmental Surfactant Monitoring Programme. SDIASewage Treatment Pilot Study on Linear Alkylbenzene Sulphonate (LAS). Water Res. 1995, 29 (9), 2063–2070. https://doi.org/10.1016/0043-1354(95)00020-L.

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(8) Benna-Zayani, M.; Kbir-Ariguib, N.; Trabelsi-Ayadi, M.; Grossiord, J.-L. Stabilization of W/O/W Double Emulsion by Polysaccharides asWeak Gels. Colloids Surf. Physicochem. Eng. Asp. 2008, 316 (1–3), 46–54. https://doi.org/10.1016/j.colsurfa.2007.08.019.

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Claims (17)

1. An emulsifier in the form of a lignin fraction, characterized in that it comprises:

From 6.0% to 8.5%, preferably from 7.0% to 8.2%, more preferably from 7.2% to 8.0% by weight of monolignol,

-Lignin dimer in a weight percentage of 14.0% to 20.0%, preferably 15.0% to 20.0%, more preferably 15.5% to 18.0%, and

From 70% to 80%, preferably from 71.8% to 78.0%, more preferably from 74.0% to 77.3% by weight of lignin oligomer,

Wherein the weight percentages are relative to the total weight of monolignols, dimers, and oligomers in the emulsifier.

2. The emulsifier of claim 1 wherein the lignin fraction further has at least one of the following characteristics:

A weight average molar mass (Mw) of from 1200 g/mol to 1600 g/mol, such as from 1200 g/mol to 1500 g/mol, and/or

Number average molar mass (Mn) of from 500 g/mol to 800 g/mol, such as from 600 g/mol to 700 g/mol, and/or

The polydispersity index is from 1.85 to 2.40, preferably from 1.95 to 2.20.

3. An emulsifier according to claim 1 or 2, wherein the lignin fraction is dissolved in a C ₂-C₆ alkylene glycol, preferably propylene glycol.

4. An emulsifier according to any one of claims 1 to 3, wherein the lignin fraction is obtained from industrial alkali lignin by solid-liquid extraction using a solvent selected from the group consisting of C ₂-C₆ alcohols, C ₃-C₈ ketones and mixtures thereof, such as Methyl Ethyl Ketone (MEK) and ethanol.

5. The emulsifier according to any one of claims 1 to 4, wherein the emulsifier is at least partially esterified with C ₂-C₁₈ alkanoate groups such as caproate groups at its aliphatic primary hydroxyl groups.

6. Use of the lignin fraction according to any one of claims 1 to 5 as an emulsifier to stabilize an oil-in-water emulsion, preferably in a cosmetic or pharmaceutical composition or a food composition.

7. Use according to claim 6, wherein the lignin fraction comprises 0.1% to 5.0%, preferably 0.5% to 4.0%, such as 2.0% to 3.0% or about 2.5% of the total weight of the oil-in-water emulsion.

8. Use according to claim 6 or 7, wherein the lignin fraction is also used as pigment.

9. A cosmetic, pharmaceutical or food composition, preferably a cosmetic composition, comprising an emulsifier according to any one of claims 1 to 5.

10. Cosmetic composition according to claim 9, selected from creams such as day or body creams, body milks, make-up removers, hair conditioners, concealers or concealers, liquid soaps, shampoos and nourishing masks.

11. A make-up product comprising the emulsifier of any one of claims 1 to 5, preferably selected from foundations, blushes, eye shadows, coloured body creams or face creams such as BB creams, mascaras, eyeliners and concealers.

12. A method of preparing the emulsifier of any one of claims 1 to 5 from industrial lignin, the method comprising step (i) extracting the industrial lignin with a solvent selected from the group consisting of C ₂-C₆ alcohols, C _3-C₈ ketones, and combinations thereof, preferably selected from the group consisting of Methyl Ethyl Ketone (MEK), acetone, isopropanol, and ethanol, and more preferably selected from the group consisting of ethanol or MEK.

13. The method of claim 12, wherein step (i) comprises:

Contacting the technical lignin with a solvent under conditions capable of extracting the lignin molecule of interest,

-Separating the liquid and solid fractions and recovering the liquid phase, and

-Removing the solvent from the liquid fraction, thereby recovering the emulsifier in the form of lignin fraction.

14. The method of claim 12 or 13, wherein the industrial lignin is alkali lignin.

15. The process according to any one of claims 12 to 14, wherein the industrial alkali lignin is not subjected to an acid and/or alkali treatment prior to extraction and/or the process comprises a single step of extraction using a polar solvent.

16. The method of any one of claims 12 to 15, further comprising:

-step (ii) wherein the lignin fraction obtained in step (i) is subjected to enzymatic transesterification with fatty acid methyl or ethyl esters, preferably ethyl caproate, in the presence of candida antarctica lipase-B (CAL-B), and/or

-A step of dissolving the optionally esterified lignin fraction in a solvent selected from the group consisting of an alkylene glycol, a ketal levulinate such as methyl levulinate or ethyl levulinate, a glycerol ketal levulinate, a lower alcohol (e.g. a C ₂-C₆ alcohol), preferably ethanol and isopropanol, and a lower alcohol/H ₂ O mixture, preferably a C ₂-C₆ alkylene glycol such as propylene glycol.

17. A method of preparing an oil-in-water emulsion comprising:

(a) Providing an emulsifier in the form of a lignin fraction according to any one of claims 1 to 5 which is at least partially soluble in a polar solvent, preferably a C ₂-C₆ alkylene glycol,

(B) Adding the lignin solution to the aqueous phase with stirring and readjusting the pH of the aqueous phase to a value of 5.5-7.0, preferably about 6.0, and

(C) Adding the oil phase to the solution obtained in step (b) under high shear, thereby obtaining an oil-in-water emulsion.