PREPARATION OF BIOPOLYMER PARTICLES

Abstract

This disclosure provides a method for preparing biopolymer particles. The method comprises: membrane emulsification of a dispersed phase in a continuous phase, where the dispersed phase comprises the biopolymer in a solvent, and where passage of the dispersed phase through the membrane forms an emulsion of the biopolymer in the continuous phase; and phase inversion with an antisolvent to form biopolymer particles; wherein, prior to step (b), the emulsion is cooled to temperature T1. Biopolymer particles obtained from the method are also provided.

Description

PREPARATION OF BIOPOLYMER PARTICLES FIELD The present invention relates generally to a method for preparing biopolymer particles, particularly to a method that produces an improved yield of such biopolymer particles, where yield is defined herein. The present invention also provides biopolymer particles obtained by the inventive method. BACKGROUND Biopolymers represent a significant advance in reducing the environmental footprint of consumer products. Materials produced from polymers are widely used due to their adaptability, durability, and price; so much so that it is difficult to identify consumer products that do not contain any polymeric material. However, many synthetic polymers that have been developed are primarily derived from petroleum and coal as raw materials, meaning they are incompatible with the environment and dependent on an unsustainable resource. In particular, polymer particles pose serious ecological problems because they often remain in ecosystems after the consumer disposes of the product. Therefore, biopolymers and biopolymer particles contribute significantly to addressing these problems, as they are often biodegradable and derived from renewable and sustainable raw materials.However, the production of biopolymer particles can prove to be a challenge. One method that has been used to prepare biopolymer particles is membrane emulsification, accompanied by phase inversion. In the membrane emulsification process, a dispersed phase of a biopolymer is forced through the pores of a microporous membrane directly into a continuous phase to form an emulsion from which the particles can be extracted. However, several problems arise in this process, including coalescence or aggregation of particles in the continuous phase. Phase inversion involves exposing the emulsion to an antisolvent, for example, by immersing the emulsion in the antisolvent, and deformation can occur during exposure / immersion, as well as further aggregation and coalescence. In general, a method for preparing biopolymer particles that avoids the aforementioned problems remains in the technical field. Specifically, a method that prevents coalescence, aggregation, and / or deformation of biopolymer particles, and thus offers improved performance, is needed. BRIEF DESCRIPTION OF THE INVENTION An objective of the present invention is to provide a method for preparing biopolymer particles, wherein said method comprises: (a) membrane emulsification of a dispersed phase in a continuous phase, wherein the dispersed phase comprises the biopolymer in a solvent, and wherein passage of the dispersed phase through the membrane forms an emulsion of the biopolymer in the continuous phase; and (b) phase inversion with an antisolvent to form biopolymer particles. The method includes, prior to step (b), cooling the emulsion to a temperature, Ti, wherein Ti is greater than the pour point of the continuous phase (Tcont), but equal to or less than the transition temperature of the dispersed phase (Tdisp). The transition temperature of the dispersed phase is selected from the group consisting of the freezing point, the glass transition temperature, and the pour point. Ti is defined as: Tcont < Ti < Tdisp; where Tdisp > Tcont. In several embodiments of the present invention, the antisolvent is aqueous, i.e., it comprises water. In several embodiments of the present invention, the antisolvent comprises an organic solvent, for example, an alcohol, acetone, and the like. In several embodiments, the antisolvent comprises an organic solvent and water. In several embodiments of the present invention, the antisolvent is cooled. For example, the antisolvent can be cooled to a temperature T2 for phase inversion (b), where T2 is less than Tdisp. Preferably, T2 is substantially equal to Ti; preferably, T2 is equal to Ti. In several embodiments of the present invention, the membrane emulsification is selected from the group consisting of cross-flow membrane emulsification, rotational membrane emulsification, vibratory membrane emulsification, and combinations thereof. Preferably, the membrane emulsification involves cross-flow membrane emulsification. In several embodiments of the present invention, the emulsion is cooled at the membrane outlet, for example, as the continuous-phase dispersed emulsion forms. In several embodiments of the present invention, the continuous phase comprises a nonpolar solvent. In several embodiments of the present invention, the dispersed phase comprises a polar solvent. In several embodiments of the present invention, phase inversion is carried out under shear. In several embodiments, the phase inversion comprises a filtration process. In several embodiments, phase inversion is carried out under shear and involves a filtration process. In several embodiments of the present invention, the method is continuous. In several embodiments of the present invention, after phase inversion (b), the continuous phase / antisolvent mixture is removed from the particles. In several embodiments of the present invention, the biopolymer is a polysaccharide. Preferably, the biopolymer is cellulose. Another objective of the present invention is to provide biopolymer particles obtained by the method described herein. Therefore, the characteristics described herein in the context of the method are also applicable to the biopolymer particles obtained by this method. The biopolymer particles obtained by the method described herein differ from those of the prior art due to their increased yield and uniformity of size and shape. This uniformity can be seen, for example, in Figure 5(B). These objectives and modalities are set forth in the accompanying independent and dependent claims. It should be noted that the features of the dependent claims may be combined with features of the independent claims in configurations other than those explicitly stated in the claims. Furthermore, the approaches described herein are not limited to specific modalities such as those set forth below, but include and contemplate any combination of features set forth herein. The foregoing, as well as other objectives, features, and advantages of the invention, will be discussed in greater detail below, along with the accompanying figures. However, it should be expressly stated that the figures are for illustrative purposes only and should not be interpreted as defining the limits of the invention. BRIEF DESCRIPTION OF THE FIGURES Figure 1 is a schematic representation of membrane emulsification. Figures 2(A) to 2(D) contain four photographs of particles showing an example of a desired particle size (< 50 pm) and shape (spherical) (Figure 2(A)), along with the deformation and aggregation problems that occur in the prior art (Figures 2(B) to 2(D)). Figures 3(A) and 3(B) contain a schematic representation of a prior art process (Figure 3(A)), as well as a representation of an emulsion cooling modality according to the present invention (Figure 3(B)). Figure 4 is a schematic representation of one modality of the method in accordance with the present invention. Figures 5(A) and 5(B) include two photographs; Figure 5(A) is a photograph of cellulose particles obtained from a comparative example without emulsion cooling in accordance with the present invention, and Figure 5(B) is a photograph of cellulose particles obtained by the example described below in accordance with the present invention. DETAILED DESCRIPTION OF THE INVENTION While this disclosure describes or suggests several examples of embodiments, the general inventive concepts encompass other examples of embodiments that use a variety of methods and materials similar or equivalent to those described or suggested herein. Those aspects and features of conventionally implemented embodiments may not be analyzed or described in detail for the sake of brevity. Therefore, it will be appreciated that aspects and features of the apparatus and methods described herein that are not described in detail can be implemented in accordance with any conventional technique for applying such aspects and features. The overall inventive concept focuses on increasing the yield of biopolymer particles through a membrane emulsification and phase inversion process, where the term "yield" refers to the mass of spherical particles or beads within a defined size distribution. Membrane emulsification is a known process in the art; it is a technique in which a dispersed phase is forced through the pores of a microporous membrane directly into a continuous phase, where emulsified droplets form and separate at the pore ends via a drop-by-drop mechanism. A schematic representation of a membrane emulsification process is shown in Figure 1, where the arrow indicates the flow direction. The dispersed phase typically includes a first liquid containing the biopolymer dissolved in a solvent, and the continuous phase includes a second liquid that is immiscible with the first. The interaction of the two liquids when the dispersed phase is forced or transported through the membrane is called the dispersion process, and their non-homogeneous mixture is called an emulsion—that is, droplets of the dispersed phase surrounded by the continuous phase. The advantages of membrane emulsification over conventional emulsification are well-established in the art; these include the ability to produce very fine emulsions with controlled droplet sizes and narrow droplet size distributions. Furthermore, successful emulsification can be achieved with significantly less emulsifier and energy consumption, and due to the reduced shear stress effect, membrane emulsification allows the use of shear-sensitive ingredients such as starch and proteins. However, to expand the industrial application of membrane emulsification, it has been recognized that the productivity of this method must be increased. In the context of biopolymer production, dispersed-phase droplets in continuous-phase emulsions have been successfully isolated using phase inversion. In the context of cellulose, this process is described in ACS Sustainable Chem. Ing. 2017, 5, 7, 5931-5939, which is incorporated herein by reference. Phase inversion is a chemical phenomenon that has been exploited in the fabrication of artificial membranes and is carried out by removing the solvent from a polymer-liquid solution. Several phase inversion methods exist, including immersing the polymer solution in a third liquid called an antisolvent. The use of antisolvent-based phase inversion has proven particularly effective in precipitating biopolymer droplets onto particles from a dispersed / continuous-phase emulsion. However, unfortunately, the dispersed phase droplets in the emulsion are at risk of participating in undesirable processes that reduce performance: the performance mentioned in this disclosure refers to the mass of spherical particles or beads within a defined size distribution. The droplets can interact irreversibly with each other (a process referred to as coalescence or aggregation in this disclosure) and / or become irreversibly deformed, for example, during phase inversion. Figures 2(A) to 2(D) show four biopolymer particle shapes. Figure 2(A) shows an example of a particle shape and size that may be desirable in certain applications: a single spherical bead with a diameter <50 pm; Figure 2(B) shows an undesirable shape deformation: a single teardrop-shaped particle; Figure 2(C) shows an undesirable coalescence of multiple spherical particles with a diameter >200 pm; and Figure 2(D) shows an undesirable asymmetric aggregation of multiple beads. Deformation and aggregation affect both the size and shape distribution of the biopolymer particles, and this has a negative effect on the performance of the biopolymer particles. In situ inspection of particles during formation presents a challenge, and therefore it is only possible to theorize where and how deformed shapes, coalescing structures, aggregated structures, etc., are generated. Without limiting themselves to any particular theory, the inventors believe that dispersed phase droplets can interact undesirably with each other when flowing in the apparatus typically used for membrane emulsification or in downstream process piping, fittings, and equipment. These droplets can clump together, for example, where there are changes in the fluid transport flow regime, such as at laminar-to-turbulent transition points, recirculation zones, changes in flow direction, etc.Another theory is that dispersed-phase droplets can be deformed, for example, by shear forces during the phase inversion process (e.g., as the emulsion flows through the antisolvent), and that these deformed shapes (e.g., teardrop-shaped) can be preserved by the antisolvent. Dispersed-phase droplets can also interact during the phase inversion process before or during contact with the antisolvent, and can aggregate to create a larger droplet or join together to create a larger structure that is then preserved by the antisolvent. Other mechanisms may also exist, including the consumption of smaller phase inversion particles by larger droplets during the phase inversion process and the subsequent preservation of these structures by the antisolvent. The present invention remarkably avoids these problems of deformation and aggregation and thus improves overall performance. This performance improvement is achieved by cooling the emulsion formed by membrane emulsification to a temperature Ti before phase inversion. In particular, Ti is greater than (i.e., higher than) the pour point of the continuous phase and equal to or less than (i.e., lower than) a transition temperature of the dispersed phase as defined herein, provided that the transition temperature of the dispersed phase is greater than / higher than the pour point of the continuous phase.Since deformation and aggregation are believed to occur when dispersed phase droplets are in a liquid state, it is conjectured that cooling the emulsion to or below the pour point of the dispersed phase temporarily changes it, at least partially, from the colloid category of an emulsion (i.e., liquid in liquid) to a colloidal solution (solid in liquid), and thus results in the dispersed phase being easier to use in subsequent processes. Furthermore, the fact that the dispersed phase has a transition temperature (the transition temperature selected from the group consisting of the freezing point, glass transition temperature, and pour point) higher than the pour point of the continuous phase means that the continuous phase surrounding the solidified dispersed phase can still function as a transport medium. A schematic representation of an emulsion undergoing cooling and temporary conversion to a colloidal solution within a cooling coil heat exchanger is shown in Figure 3(B). Figure 3(A) is a representation of the prior art; the continuous phase forms an emulsion with the dispersed phase droplets (microdroplets in this example; see definition below), and stagnation and turbulence in the flow produce unwanted coalescence and reduced yield. Therefore, Figure 3(B) is an example in which the emulsion is cooled within a coil heat exchanger to a temperature below the transition temperature of the dispersed phase, but above the pour point of the continuous phase, so that the continuous phase remains mobile and able to carry the transitioning droplets. The illustrative embodiment in Figure 3(B) avoids the coalescence, deformation, particle aggregation, and consequent reduction in yield that occur with the process of the prior art in Figure 3(A). Unless otherwise defined, all technical and scientific terms used in this disclosure have the same meanings they normally have to a person skilled in the art to which this invention pertains. The term biopolymer refers to a polymer produced by living organisms. In other words, a polymeric biomolecule. There are three main classes of biopolymers, classified according to the monomeric units used and the structure of the resulting biopolymer: polynucleotides (RNA and DNA), which are polymers composed of 13 or more nucleotide monomers; polypeptides, which are polymers of amino acids; and polysaccharides, which are typically polymeric carbohydrate structures. Other examples of biopolymers include rubber, suberin, melanin, chitin, and lignin, primarily chitin and lignin. In several embodiments of the present invention, the biopolymer is selected from the group consisting of polynucleotides, polypeptides, and polysaccharides. Preferably, the biopolymer is selected from the group consisting of polypeptides and polysaccharides. Preferably, the biopolymer is a polysaccharide, for example, starch, cellulose, chitin, chitosan, or glycogen. Preferably, the biopolymer is starch or cellulose. The term particle is used interchangeably with bead in this disclosure and refers to a solid formed after the phase inversion of a dispersed phase droplet. In several embodiments of the invention, the particles or beads are microparticles or microbeads. As is known to a person skilled in the art, microparticles or microbeads are particles / beads with a diameter of between 1 and 1000 micrometers (µm). Such particles can be readily identified, for example, by using optical microscope images and image analysis software with a suitable detection algorithm (e.g., ImageJ, using a contour detection algorithm), laser diffraction with commercially available instruments such as the Malvern Panalytical Mastersizer (e.g., Mastersizer 3000), or with a filter of appropriate size. The membrane emulsification step of the inventive method involves passing a dispersed phase through a membrane into a continuous phase to form an emulsion. The membrane is not limited; it can be any porous structure suitable for a membrane emulsification process. For example, the membrane can be a plate with holes that act as pores (e.g., holes the size of a pea), a perforated metal tube, or sintered porous glass. The term emulsion refers to a class of two-phase systems of matter in which both phases are liquid. Emulsions are a type of colloid and generally consist of two immiscible liquids. In several embodiments of the present invention, the emulsion may be a macroemulsion; that is, an emulsion in which the particles of the dispersed phase have diameters of approximately 1 to 1000 microns. The term colloidal solution refers to a general class of two-phase systems of matter where the continuous phase is liquid and the dispersed phase is solid. The term agglomerate refers to a structure composed of primary particles that can normally be redispersed. The term aggregate refers to a structure composed of primary particles that cannot be redispersed. The term pour point refers to the temperature below which a substance (e.g., a liquid) loses its flow characteristics. It is typically defined as the minimum temperature at which the liquid (e.g., oil) can flow from a beaker. The pour point can be measured using standard methods known in the art. For example, ASTM D7346 Standard Test Method for the Pour Point and Non-Flow Point of Petroleum Products and Liquid Fuels can be used. For commercially available materials, the pour point is often provided by the supplier or manufacturer. The term freezing point refers to the temperature at which a substance changes from a liquid to a solid state at standard atmospheric pressure (1 atmosphere). The freezing point can be measured using standard methods known in the art. For example, ASTM E794 Standard Test Method for Melting and Crystallization Temperatures by Thermal Analysis can be used. For commercially available materials, the freezing point is often provided by the supplier or manufacturer. The term glass transition point or glass transition temperature refers to the temperature at which a polymer structure transforms from a hard, glassy material into a soft, elastic material. This temperature can be measured by differential scanning calorimetry in accordance with ASTM E1356 Standard Test Method for Assignment of Glass Transition Temperature by Differential Scanning Calorimetry. For commercially available materials, the glass transition temperature is often provided by the supplier or manufacturer. For ease of reference, these and other additional features of the present invention are set forth below under the corresponding section headings. However, the guidelines in each section are not limited to the section in which they appear. Membrane emulsification As noted above, membrane emulsification is not limited and can correspond to any membrane emulsification process known in the prior art. For example, the membrane emulsification process could be cross-flow membrane emulsification, rotational membrane emulsification, vibratory membrane emulsification, or a combination thereof. The terms “cross-flow,” “rotational,” and “vibratory,” as used in the art, refer to the method used to generate shear on the membrane surface. A continuous phase could, for example, be displaced relative to a stationary membrane to create shear, or the membrane could be displaced relative to the stationary phases. Alternatively, the dispersed phase could be injected into a stationary continuous phase.Known process parameters, such as membrane type, average pore size and porosity, crossflow velocity, transmembrane pressure, and emulsifier, can also be used. In various embodiments of the present invention, membrane emulsification may involve a crossflow system, a stirred tubular cell membrane, a stirred flat cell membrane, a rotating flat membrane, a vibrating / rotating tubular membrane, and / or premixed membrane emulsification. International Patent Application No. WO 01 / 45830 describes an example of a rotational membrane emulsification. International Patent Application No. WO 2012 / 094595 describes an example of a cross-flow membrane emulsification. The article by Pedro S. Silva et al., “Azimuthally Oscillating Membrane Emulsification for Controlled Droplet Production,” AlChE Journal 2015 Vol. 00, No. 00, describes a vibrating membrane emulsification: specifically, a membrane emulsification system comprising a tubular metal membrane that is periodically oscillated azimuthalally in a smooth cross-flow continuous phase. Patent document WO 2019 / 092461 describes a cross-flow membrane emulsification. Each of these method descriptions is incorporated herein by reference. In several embodiments of the present invention, membrane emulsification is cross-flow membrane emulsification. Preferably, it is an emulsification process in which the continuous phase moves relative to a stationary membrane. As an expert in the field will observe, the dispersed phase and the continuous phase will depend on the biopolymer being produced. The dispersed phase will comprise a solvent that disperses or dissolves the biopolymer, and the continuous phase will comprise a solvent that is immiscible with the dispersed phase, so that an emulsion forms when the dispersed phase is forced through the porous membrane. The term solvent refers to any substance (for example, a liquid) that disperses or dissolves the biopolymer. The term “solvent” also includes mixtures of solvents. Identifying appropriate solvents for the dispersed and continuous phases falls squarely within the general knowledge of a person skilled in the art. As noted earlier, the only requirement is that the two phases (i.e., the dispersed and continuous phases) be immiscible. Therefore, it follows that the solvents for each phase must also be immiscible. Solvents can be, for example, aqueous solvents, ionic liquids (a salt in liquid form at temperatures between room temperature and 100 °C, for example, imidazolium-based ionic liquids such as 1-ethyl-3-methylimidazolium acetate, 1-butyl-3-methylimidazolium chloride, or similar), organic solvents, or non-aqueous inorganic solvents. In several embodiments of the present invention, the solvent for the dispersed phase comprises ionic liquids, non-aqueous inorganic solvents, aqueous solvents, or combinations thereof. In several embodiments of the present invention, the solvent for the dispersed phase comprises ionic liquids, non-aqueous inorganic solvents, or combinations thereof. In several embodiments of the present invention, the solvent for the dispersed phase comprises one or more ionic liquids. In other embodiments, the solvent for the dispersed phase comprises organic solvents. Non-limiting examples of solvents include: water, methanol, ethanol, ammonia, acetone, acetic acid, n-propanol, n-butanol, isopropyl alcohol, ethyl acetate, dimethyl sulfoxide, sulfuryl chloride, phosphoryl chloride, carbon disulfide, morpholine, N-methylmorpholine, NaOH with and without urea and thiourea association, bromine pentafluoride, hydrogen fluoride, sulfuryl chloride fluoride, acetonitrile, dimethylformamide, hydrocarbon oils and mixtures thereof, toluene, chloroform, carbon tetrachloride, benzene, hexane, pentane, cyclopentane, cyclohexane, 1,4-dioxane, dichloromethane, nitromethane, propylene carbonate, formic acid, tetrahydrofuran, diethyl ether, phosphoric acid, acetate of 1-ethyl-3-methylimidazolium, 1-butyl-3-methylimidazolium chloride, 1-methoxymethyl-3-methylimidazolium bromide, N-ethylpyridinium chloride, N-methylmorpholine oxide, 1-methylimidazole, Ν,Ν-dimethylformamide, N,N'-dimethylimidazolidana-2-one, N,Ndimethylacetamide, sulfolane,γ-valerolactone, γ-butyrolactone, N,N,N',N'-tetramethylurea, N-methylpyrrolidone, and methylene chloride. An expert will readily recognize which of these solvent examples are ionic liquids, organic solvents, and / or non-aqueous inorganic solvents. Preferably, the solvent used for at least one of the dispersed and continuous phases is environmentally friendly. Preferably, the solvent used for both the dispersed and continuous phases is environmentally friendly. The term "environmentally friendly" means that it is not harmful to the environment, so the solvent can be disposed of without the need for specialized equipment or processes; in other words, it is non-toxic. It is known in the prior art that polysaccharides have limited solubility in most common solvents. It is also known that those solvents that do dissolve polysaccharides are often toxic and / or highly selective. When the biopolymer is a polysaccharide such as cellulose, starch, chitin, glycogen, and / or chitosan, the solvent for the dispersed phase may therefore comprise an ionic liquid. The dissolution of cellulose with the ionic liquid 1-butyl-3-methylimidazolium chloride is discussed, for example, in Richard et al., J. Am. Chem. Soc. 2002, 124, 4974-4975. The solubility of cellulose in ionic liquids and ionic liquids with cosolvents is similarly discussed in Verma et al., Sustainable Chemistry and Pharmacy 13 (2019), 100162. Each of these disclosures is incorporated herein by reference. The concentration of biopolymer in the dispersed phase is not limited and can be any concentration suitable for membrane emulsification. The dispersed and / or continuous phase may also include optional components. These optional components include, but are not limited to, cosolvents, surfactants, porogens, active ingredients, air pockets, double emulsions, pigments, and dyes. The level of any of the optional components is not significant in the present invention. In several embodiments, the dispersed phase includes a cosolvent. The cosolvent is not limited and can be any solvent known in the art, including those described above for the continuous and / or dispersed phase. The cosolvent can also be a mixture of cosolvents. Examples of possible cosolvents include: water; 1-methylimidazole (1-MI); dimethyl sulfoxide (DMSO); N,N-dimethylformamide (DMF); N,N'-dimethylimidazolidine-2-one (DMI); N,N-dimethylacetamide (DMAc); sulfolane; γ-valerolactone (γ-val); γ-butyrolactone (γ-but); propylene carbonate (CP); N,N,N',N'-tetramethylurea (TMU); and N-methylpyrrolidinium (NMP). For example, 1-methylimidazole (1-MI); dimethyl sulfoxide (DMSO); N,N-dimethylformamide (DMF); N,N'-dimethylimidazolidin-2-one (DMI); N,Ndimethylacetamide (DMAc); sulfolane; γ-valerolactone (γ-val); γ-butyrolactone (γ-but); propylene carbonate (CP); Ν,Ν,Ν',Ν'-tetramethylurea (TMU); or N-methylpyrrolidinone (NMP). The surfactant can be any suitable surfactant known in the art, for example, any ionic or non-ionic surfactant. Ionic surfactants may include sulfates, sulfonates, phosphates, and carboxylates, for example, alkyl sulfates, ammonium lauryl sulfates, sodium lauryl sulfates, alkyl ether sulfates, sodium lauryl sulfate and sodium myristyl sulfate, sodium dioctyl sulfosuccinate, perfluorooctane sulfonate, perfluorobutane sulfonate, alkylbenzene sulfonates, alkyl aryl ether phosphates, alkyl ether phosphates, and alkyl carboxylates. Non-ionic surfactants may include polyethers, polyoxyalkylene derivatives of hexitol, partial esters of long-chain fatty acids such as sorbitan oleates, ethylene oxide derivatives of long-chain alcohols, ethoxylated vegetable oil, polydimethylsiloxanes, and ethylene oxide / propylene oxide copolymers. Cooling of the emulsion The advantages of the present invention derive primarily from the cooling of the emulsion formed by membrane emulsification prior to phase inversion. The emulsion is cooled to a temperature Ti, where Ti is greater than the pour point of the continuous phase (Tcont.), and equal to or less than a transition temperature selected from the group consisting of the freezing point, the glass transition temperature, and the pour point of the dispersed phase (Tdisp.): where Tdisp > Tcont. However, the absolute value of Ti is not critical to the present invention; rather, it is the relationship of Ti to the respective temperatures of the dispersed and continuous phases that is relevant. The cooling method is also not limited. The emulsion can be cooled by any means known in the art for removing heat (energy) from a system. The emulsion can be further cooled at any point before phase inversion. In various embodiments, this means that the emulsion is cooled simultaneously with or separately from the membrane emulsification process. The emulsion can be cooled, for example, as it forms (by means of a cooling medium located at the membrane outlet, for example). Alternatively, the emulsion can be cooled at a stage subsequent to membrane emulsification, for example, in a cooling apparatus separate from the membrane emulsification apparatus. Preferably, cooling should occur as soon as possible after emulsification takes place in order to reduce the possibility of the liquid phase droplets agglomerating and / or clumping. In various embodiments, the emulsion can be cooled by a cooling medium (e.g., water, ice, etc.) that at least partially surrounds the container where the emulsion is formed. In a preferred embodiment, the container (e.g., a tube) in which the emulsion is formed may have a cooling jacket containing a cooling medium. The cooling medium is not limited and includes any medium that has a lower temperature than the emulsion. In several embodiments, the emulsion can be cooled by a cooling apparatus connected to the membrane emulsion unit. The cooling apparatus can be a heat exchanger, such as an immersion heat exchanger. In an illustrative embodiment described below, a coil heat exchanger is immersed in a cooling medium (for example, a cold water bath), although the invention is not limited in this respect. Any type of heat exchanger could be used, such as a shell and tube heat exchanger, a plate and frame heat exchanger, or a jacketed tube. Additionally, an immersion heat exchanger could be used with another cooling medium, such as antifreeze, dry ice, or the like, to cool the emulsion to the specified temperature. Phase reversal Similarly, phase inversion is not limited, and any phase inversion process known in the art involving the use of an antisolvent may be employed. As noted above, phase inversion is carried out by removing the solvent from a polymer-liquid solution, which, in the present invention, is the continuous phase with the frozen dispersed phase contained within it. The antisolvent can be any suitable solvent or mixture of solvents known in the art. In several embodiments of the present invention, the antisolvent is aqueous. In several embodiments, the antisolvent is non-aqueous. The antisolvent may comprise, for example, an organic solvent such as an alcohol or acetone, or any other organic solvent known in the art. Suitable alcohols include ethanol and / or methane. In several embodiments, the antisolvent comprises an organic solvent, water, or a mixture thereof, for example, alcohol, acetone, water, or a mixture of the foregoing. In several embodiments of the present invention, the phase inversion is carried out at room temperature, i.e., between approximately 20 and 25 °C. In such embodiments, the antisolvent has a temperature between approximately 20 and 25 °C. Alternatively and preferably, the antisolvent is cooled to a temperature below room temperature, i.e., below approximately 20 °C. In several embodiments of the present invention, the antisolvent has a temperature T2 that is lower than the freezing point of the dispersed phase. The advantage of controlling the temperature of the antisolvent (T2) is to prevent premature thawing of the frozen droplets. In several embodiments of the present invention, T2 is equal to Ti, where Ti is defined previously. Without adhering to any particular theory, the inventors believe that by cooling the antisolvent to T2, the droplets remain in a frozen state (and are therefore spherical and non-agglomerated), while the surrounding continuous phase is removed by phase inversion. The antisolvent is then able to contact the droplet surface, causing the biopolymer to precipitate and the precipitate surface to harden. Furthermore, as a droplet of the frozen dispersed phase thaws, the antisolvent will convert the dissolved biopolymer droplet into a bead / particle, while it is leached via the solvent system into the antisolvent. In several embodiments of the present invention, phase inversion is carried out under shear; a person skilled in the art knows the appropriate shear conditions for phase inversion. Shear can be produced, for example, by using a stirred vessel (e.g., a mechanically stirred vessel) or a settling vessel (e.g., a gravity settling vessel). The term shear is used herein to refer to an external force acting on an object or surface parallel to the slope or plane on which it rests, where the stress tends to produce tension. Shearing is beneficial because it improves the rate at which the continuous phase is removed from the dispersed phase droplets and, therefore, the overall phase inversion rate. The phase inversion process is limited by the diffusion rate (Fickian diffusion), and shearing reduces the thickness of the continuous phase layer surrounding a dispersed phase droplet, thus reducing the distance an antisolvent molecule travels to reach the dispersed phase droplet surface and accelerating the phase inversion process. However, shearing is not typically used in current phase inversion processes due to its negative impact on particle shape and size. A moderate phase inversion step is currently employed, in which the emulsion is allowed to settle through the stationary antisolvent (at room temperature).Surprisingly, frozen-state dispersed phase droplets are more tolerant of other continuous phase separation methods, and this greater tolerance increases the efficiency of such separation. In several embodiments of the present invention, the phase inversion comprises a filtration process. The filtration process is not limited and may involve mechanical filtration or any other type of filtration (e.g., by using equipment known in the art, such as a hydrocyclone). A filtration process may also be included within the phase inversion carried out under shear, as described above. In various embodiments, a filtration medium (e.g., a filter) may be used to filter the emulsion through the antisolvent and thereby collect the biopolymer particles. In such embodiments, the emulsion may settle by gravity (shear) through the antisolvent and into the filter, while the continuous phase passes through the filter (the filtrate). The frozen droplets may be collected on the filter as a filtrate mass. If not collected as part of the phase inversion (e.g., by filtration or another method), the biopolymer particles can be separated from the continuous phase / antisolvent mixture, or the continuous phase / antisolvent mixture can be removed from the particles. The removal method is not limited. However, in several configurations, the removal method depends on whether the process is used in continuous or batch mode. When the method of the invention is used in batch mode, the phase inversion step can be carried out first in a closed vessel, and the resulting mixture is then transferred to a settling vessel and allowed to settle. Once settled, the layers can be sequentially removed from the bottom of the vessel. Typically, the order of the layers can be as follows: (1) a continuous phase, (2) an interface layer comprising moistened biopolymer particles, and (3) the remaining antisolvent. However, the invention is not limited in this respect, and a person skilled in the art will observe that the order of the layers will depend on their respective densities. In several embodiments of the present invention, the method is continuous and configured to operate in continuous mode. The phase inversion step can be carried out under the continuous inflow of emulsion and antisolvent, as well as the continuous outflow of the multiphase mixture into a decanter. Within the decanter, a steady-state separation of the mixture can occur, and there can be continuous and preferably simultaneous removal of each of the phases. For example, there can be continuous and preferably simultaneous removal of: (1) the continuous phase, (2) antisolvent, and (3) the moistened biopolymer particles. Of course, the order of these layers is variable, and the invention is not limited to any particular order. Alternatively, the multiphase (e.g., three-phase) mixture can be separated using processes known in the art, e.g., by a stack of disc separator (e.g., a centrifugal separator such as that produced by Andritz). To provide continuous cooling throughout a continuous phase inversion, the cooling medium (e.g., a medium surrounding the vessel containing the emulsion or used with a heat exchanger connected to the membrane emulsion unit) may need to be recycled or recirculated by means of a suitable device. A device such as a recirculating chiller (ThermoFlex, available through ThermoFisher Scientific) can, for example, be used to maintain the cooling medium at the desired temperature. Another advantage of the method according to the present invention is the flexibility in the sequence of steps. This flexibility is due to the fact that the dispersed phase droplets can be frozen within the emulsion. In several embodiments of the invention, the removal of the biopolymer particles, as described above, follows (or is involved in) phase inversion. After phase inversion, decantation can be carried out, followed by the removal of biopolymer particles from the mixture, and / or phase inversion can involve the mechanical filtration of the wetted particles from the antisolvent / continuous phase / particle mixture. Alternatively, the biopolymer particles can be removed from the continuous phase before phase inversion. In such methods, moistened frozen droplets can be removed from the colloidal solution (e.g., by filtration), and then phase inversion is carried out to precipitate the biopolymer and form beads / particles. Since the present invention has been described in general terms, further understanding can be obtained by reference to certain specific examples illustrated below, which are provided for illustrative purposes only and are not exhaustive or limiting unless otherwise specified. EXAMPLES A dispersed phase comprising 8 wt% cellulose in 70 wt% 1-ethyl-3-methylimidazolium acetate and 30 wt% dimethyl sulfoxide was prepared according to conventional methods known in the art. This dispersed phase had a transition temperature (e.g., freezing point) of approximately 11 °C. A continuous aqueous phase was also prepared according to conventional methods known in the art. The continuous phase had a pour point of -15 °C. iviA / a / zuzz / ui ¿z / o The dispersed and continuous phases were introduced into a membrane emulsion unit, as shown in Figure 4, and an emulsion was formed. The emulsion was then cooled to a temperature between 0 and 11 °C before being transferred to a phase inversion unit with an ethanol antisolvent to form cellulose particles. The emulsion was cooled using an immersion coil heat exchanger, as shown in the inset of Figure 4. An immersion coil heat exchanger was selected to maintain laminar flow and minimize flow disturbances as the emulsion cooled. The coil heat exchanger consisted of a length (L) of coiled tubing with a diameter D and pitch P, immersed in a cold water bath at 0°C, which proved sufficient to cool a 0.5 Umin emulsion below 11°C. The emulsion temperature was monitored with a thermometer at the outlet of the coil heat exchanger. A comparative example was also carried out in which cellulose particles were prepared without the cooling step. Figures 5(A) and 5(B) include two photographs (at 5x magnification) of the comparative example (a) and the example according to the invention (b). These 15 photographs show how the use of the cooling step before phase inversion significantly reduced the degree of decoalescence and aggregation of the cellulose particles. This results in improved performance.

Claims

1. A method for preparing biopolymer particles; said method comprises: a. a membrane emulsification of a dispersed phase in a continuous phase, wherein the dispersed phase comprises the biopolymer in a solvent, and wherein the passage of the dispersed phase through the membrane forms an emulsion of the biopolymer in the continuous phase; and b. a phase inversion with an antisolvent to form particles of the biopolymer; wherein, prior to step (b), the emulsion is cooled to a temperature Ti, wherein Ti is greater than the pour point of the continuous phase (Tcont), and equal to or less than the transition temperature of the dispersed phase (Tdisp): Tcont < Ti Tdisp; wherein the transition temperature is selected from the group consisting of the freezing point, the glass transition temperature, and the pour point; and wherein Tdisp > Tcont.

2. The method according to claim 1, wherein the transition temperature of the dispersed phase is the freezing point.

3. The method according to claim 1, wherein the transition temperature of the dispersed phase is the pour point.

4. The method according to claim 1, wherein the transition temperature of the dispersed phase is the glass transition temperature.

5. The method according to any of claims 1 to 4, wherein the antisolvent is cooled to a temperature T2 for phase inversion (b), wherein T2 is less than Tdisp, preferably wherein T2 is equal to Ti.

6. The method in accordance with any of claims 1 to 5, wherein the cooling of the emulsion is at the outlet of the membrane.

7. The method in accordance with any of claims 1 to 6, wherein the phase reversal is carried out under shear.

8. The method according to any of claims 1 to 7, wherein the phase inversion comprises a filtration process.

9. The method according to any one of claims 1 to 8, wherein the membrane emulsification is selected from the group consisting of a cross-flow membrane emulsification, a rotational membrane emulsification, a vibratory membrane emulsification, and combinations thereof.

10. The method according to any of claims 1 to 9, wherein the antisolvent is aqueous and / or comprises an organic solvent.

11. The method in accordance with any of claims 1 to 10, wherein the method is continuous. 5 12. The method according to any one of claims 1 to 11, wherein, after phase inversion (b), the continuous phase / antisolvent mixture is removed from the particles.

13. The method according to any of claims 1 to 12, wherein the biopolymer is a polysaccharide, preferably wherein the biopolymer is cellulose.

14. Biopolymer particles prepared by the method in accordance with any one of claims 1 to 13.