Field of Invention

The present invention relates to methods for extracting a lipidic component and/or oil soluble component from plant or algal material. The present invention also relates to such a lipidic component and/or oil soluble component extracted from plant or algal material.

Background of the Invention

The lipids of plant and algal tissues are being used as sources for biodiesel fuels and dietary supplements. The lipid and fatty acid content in most leafy plants and algae are high because of the thylakoid membrane of the chloroplasts. They represent an excellent replacement for fossil fuels because they are produced using sunlight as the energy source and both sequester atmospheric carbon dioxide and produce oxygen in the process. The oils of plants are also typically lower in sulfur and nitrogen than fossil fuels, hence are considered clean burning.

Plant and algal tissues are also rich in polyunsaturated oils (e.g., omega-3 and omega-6 fatty acids) and oil-soluble secondary metabolites such as astaxanthins and zeoxanthins which are taken as antioxidant dietary supplements to promote joint health and reduce inflammation, and added to feed to impart a pink color to the flesh of farmed fish [Guerin et al. (2003)]. Anthocyanin and lycopene are oil-soluble pigmented phenolic plant compounds used to enhance the immune system, inhibit macular degeneration, and to promote heart health [Ghosh and Konishi (2007)]. All of these polyunsaturated compounds are considered essential nutrients for animals and humans since they are sourced primarily through the animal's diet, not synthesized directly by animal tissues. The low extraction yields of these materials from plant and algal tissues means that there is a lot of associated agricultural waste from the production of these dietary supplement ingredients.

The primary challenge has been recovering the oils from these tissues. The extraction of oils and other components from algal and plant cells is typically low because of the polysaccharide rich cell walls that surround the contents of the plant cells and membranes. These are typically insoluble and crosslinked polysaccharide materials and often absorb and inhibit the diffusion of oils and oil-soluble compounds through the cell walls. These cell walls also prevent organic extraction solvents from entering the cell to dissolve and extract organically-soluble materials (e.g., lipids and fatty acids).

The highest recovery efficiencies from algae being reported to be less than 35% [Ramola et al. (2019 and Derakhsahn et al. (2014)], ), with the highest recoveries being reported after shredding the cell walls in a French Press. Recoveries from industrially-su ita ble methods being less than 16%. Over 90% recovery was reported in one study that used hot (microwave) and ultrasonic processing, which significantly altered the resulting oil composition. The more valuable polyunsaturated oils, however, are irrevocably damaged by shear intensive and high temperature processing shown to be conducive to higher oil recoveries. Lower recoveries are generally obtained from terrestrial plant tissues which have more structural polysaccharide material surrounding the tissues because they must support their own weight as they are not suspended in water like algae. Most terrestrial plant oils are extracted by pressing seeds and nuts (e.g., rape seed, linseed, sunflower seeds, hemp seed, peanuts, and olives) where the oil concentrations are sufficient enough to be free flowing when the pulp containing it is pressed with sufficient force. However, such oils are uniquely used in the seeds and nuts of plants as an high density energy storage vehicle to support the germination of the seed germ.

Folch et al. (1951) developed a solid/liquid extraction method to recover lipids and fatty acids along with other oil-soluble materials from animal brains involving homogenizing the tissue in a single phase in a 2:1 chloroforr methanol solution, filtering the solids from the solution, then breaking the solution into two phases by the addition of additional water containing salts. Later work [Folch, et al. (1957)] showed that the salt type and concentration affected the lipid recovery and extended the technique to liver and animal mussel tissues. Reported lipid yields, however, never exceeded 38.5% and varied considerably by tissue type. Bligh and Dyer (1959) adapted and optimized this method for oil recovery from other animal tissues, particularly fish and marine animals. The Bligh and Dyer method was similarly based on solid-liquid extraction, in which the animal tissues to be extracted where first macerated into small pieces in a monophasic solution of chloroform, methanol, and water (initial extraction). Sufficient methanol was used to draw the normally immiscible chloroform and water into a single combined phase. After a period of time, additional chloroform and/or water was added to the monophasic extraction solvent to dilute the methanol forcing a phase separation. The oils were recovered in the chloroform phase. Their inability to extract additional oil with a second extraction caused them to speculate that they had achieved complete (or near complete recovery). However, when the residual extracted tissue was assayed for lipids in subsequent work it was shown that this method only recovers about 35% of the total oil content of the animal tissues, similar to the best yields reported by Folch et al. (1951). These techniques were initially adapted for the extraction of oils from plant and algal tissues.

Solvent extraction remained the industry standard although different water-immiscible extraction solvents were substituted over time, such as hexane for chloroform and ethanol for methanol, because of the toxicity of chloroform. The intrinsic problem was dealing with the water presented by working with raw tissues. Oils and water don't mix, so a cosolvent that would allow an oil dissolving phase to intermix with the water in the tissue was necessary to aid the release and solubilization of tissue-embedded lipids into the liquid phase for extraction.

This limitation was finally overcome when the tissues were dried or freeze-dried prior to extraction, which allowed more solvents to be used for the extraction. Drying, however, requires heat. Leafy terrestrial plant tissues are composed of more than 50% water, typically wilting if the relative water content drops below 40-60% [Mullan and Piet raga I la (2012)]. The dilute nature of the harvested microalgal cultures (with typically more than 99.6% water on a weight basis) makes dewatering a very costly step [Mata et al. (2012)] - dewatering and drying adds considerable cost to the oil extraction process. Further, most of the desirable essential oils (those used for dietary supplements) are polyunsaturated making them thermally-unstable and oxygen sensitive.

Therefore, commercial-scale freeze drying technology is more typically used before the extraction of dietary supplement oils, raising the processing costs further than the 20-30% of total processing costs reported [Grimma et al. (2003)] for biodiesel production. Freeze-drying, however, adds refrigeration costs to initially freeze the tissue, costs to maintaining a vacuum, and heating costs to drive the sublimation process. Sublimation is also a slow process lengthening batch cycle times, which means additional capital investment to maintain production rates. Once the tissues are dry, however, many different organic solvents can be used to extract the lipids since water no longer has to be excluded and the solvent is now able to penetrate the dried tissue particles to directly contain the lipids without the entrained water presenting a phase barrier. Because it is low boiling, making it easy to evaporate off from the extracted lipids, and any residue left in the oil is food safe, ethanol is a preferred solvent for dry tissue extraction.

Refined fossil fuel hydrocarbons are typically used to extract the oils from dried algae and plants for biodiesel production, since the end goal of biodiesel production is the combustion value of the oil and the natural oils can merely be left in the hydrocarbons for this purpose. Ethanol is now more typically used for dietary supplement oil extraction since it is more easily removed from the final dietary supplement product by evaporation and it is intrinsically food safe. This is still a solid/liquid extraction process, and, as such, the size of the dried tissue particle directly effects the lipid recovery, which means the best extraction efficiency is obtained from the finest powdered solids.

Supercritical fluids, particularly CO ₂ , are now often used as alternative extraction solvents for dried or freeze-dried plant tissues [Catchpole et al. 2018)]. Unlike ethanol which must be thermally- evaporated, sometimes causing thermal damage to the nutrient value or market appeal of polyunsaturated oils, oil extraction by supercritical carbon dioxide can be recovered cold and instantaneously by releasing the pressure, evaporating the supercritical fluid and leaving droplets of oil to be collected. However, the extraction efficiency of supercritical CO ₂ (scCO ₂ ) was low (below 50%) [Catchpole et al. (2018)] because only the neutral lipids (triglycerides) were recovered. The extraction efficiency of supercritical carbon dioxide alone was significantly lower than that obtained for ethanol extraction of cannabinoids from Cannabis sativa [Moreno et al. (2020)] While scCO ₂ eliminated many of the problems of working with solvents (e.g., toxicity, flammability, evaporative effects on product quality), this was at the expense of considerable capital costs for the required high-pressure processing equipment and operating costs for the re-compressing the cooling the scCO ₂ for recycle.

The major challenge in both wet tissue and dried tissue extraction is the ability of a lipid-compatible solvent to penetrate the solid matrix. Lipids are naturally hydrophobic and are effectively insoluble in water. The large amounts of water inside of animal tissues prevent the lipid-compatible, water- immiscible solvents from penetrating the wet tissue particle, inhibiting lipid extraction. When the tissues are dried or freeze-dried, the lipids become encased in a hard proteinaceous or polysaccharide matrix that is difficult for solvents to penetrate and create a tortuous diffusional path to get the lipids back out of the solid particles once they are dispersed.

MacCrides and Broadbent (2006) used protease enzymes in an aqueous solution to digest the proteins comprising the solid tissue into peptides and amino acids. However, this method does nothing to remove the polysaccharide cell walls of algae and plants. Xylanase and cellulase enzymes have been applied to reduce the polysaccharides of woody tissues into their constitutive sugars [Srivastava et al. (2018)], which can be subsequently fermented to produce biofuels (e.g., methane by anaerobic bacteria, and lipid membranes in aerobic bacteria). However, this approach is very inefficient for digesting woody tissues since the saccharase enzymes typically work from the outside in as they can't efficiently penetrate the cellulose fibers of the cell wall.

A known advantage of liquid/liquid extractions is that the higher the intensity of mixing (higher shear) reduces the diffusional path and increases the efficiency and rate of mass transport between the aqueous and solvent-rich phases. This does not happen in solid/liquid extraction where the diameter of the solid particle determines the rate and efficiency of mass transport.

The dissolution of cellulose contained in woody biomass is a necessity both for paper making and viscous cellulose ("Visose") production. Several methods for the solubilization of cellulosic materials have been cited in the literature. Paper was traditionally made from raw wood fiber (cellulose) that was first solubilized in hot sulfuric acid so that the lignin that glued the individual cellulosic fibers together could be extracted into the aqueous phase [Bajpa (2015)]. The purified cellulosic fibres (pulp) are separated from the lignin by filtration and can be further bleached to produce raw cellulosic fiber. The heat and concentrated sulfuric acid, however, are incompatible with many thermal- and acid-labile lipids. In the production of reconstituted cellulose (viscose), the cellulosic fibres are dissolved by reaction with carbon disulfide to produce a viscous cellulosic polymer solution in hot caustic [Bradley and Newcomb (2018)]. While this process does result in a fully soluble cellulose xyanthanate fiber the heat and sodium hydroxide also destroy many of the valuable cellular components to be extracted.

Environmental and occupational health issues associated with these hot caustic digestion processes have led to the development of alternative approaches to the dissolution of cellulosic materials [Chegolya et al. (1989)]. Those suitable for low temperature use are: phosphoric acid, derivatization with nitrogen tetroxide (N ₂ O ₄ ) in various water miscible organic solvents (dimethylformamide, dimethylsulfoxide, dimethylacetamide, aniline, and methyl-, ethyl-, and butyl-acetate). However, phosphoric acid still causes acidic damage to lipids and N ₂ O ₄ is a strong oxidizing agent spontaneously generating hydroxyl radicals in the presence of water, which is again damaging to the polyunsaturated materials [Gert et al. (1993)]. The effect of N ₂ O ₄ on cellulose is the production of soluble nitrocellulosic materials (gun cotton), which is explosive, when dried. N-methylmorpholine- N-oxide (NMMO) is a milder oxidizing agent than N ₂ O ₄ that operates through a reversible physical solubilization process [Lang et al. (1986)]. However, this is still prone to undesirable side reactions that require the addition of stabilizers to be prevented [Noe and Chanzy (2011) and Rosenau et al. (2002)]. Ionic liquids have also been reported to solubilize cellulose [Sirvio and Heiskanen (2020), Mohd et al. (2017), and Abe et al. (2015)]. Tetraalkylammonium and tetraalkylphosphonium salts appear to be able to solubilize cellulose rapidly in aqueous solution at moderate temperatures.

It is an object of the invention to provide an efficient method of extracting a lipidic component and/or oil soluble component from plant or algal material.

Alternatively, it is an object of the invention to at least provide the public with a useful choice.

Summary of the Invention

In a first aspect the invention provides a method for extraction of a lipidic component and/or oil soluble component from plant or algal material, the method including the steps of: i) providing plant or algal material including a lipidic component and/or oil soluble component; ii) mixing the plant or algal material with a tetraalkylphosphonium or tetraalkylammonium salt in the presence of water to form a mixture; ill) extracting the lipidic component and/or oil soluble component from the mixture using an organic solvent; wherein the organic solvent is within a Hansen Euclidean radius of 25 MPa ⁰⁵ from the Hansen solubility parameters of: dispersive = 11; polar = 13.7; and hydrogen-bonding = 6.3.

It has now been found that the use of a tetraalkylphosphonium or tetraalkylammonium salt in the presence of water, followed by extraction using an organic solvent, leads to an efficient extraction of a lipidic component and/or oil soluble component from plant or algal material.

In some examples it has now been found that the closer the Euclidean distance (R) of the solubility parameters of the solvent is to those of chloroform, the better the extraction efficiency of lipids and oil-soluble components in the plant or algal material.

As used herein, "organic solvent" may refer to a solvent consisting of a single entity (such as chloroform), or may refer to a solvent consisting of a plurality of entities - namely a solvent system such as a mixture of trichloroethylene and dichloromethane, and even mixtures of components such as oils, such as food grade oil.

In a second aspect the invention provides lipidic component and/or oil soluble component extracted from plant or algal material using an organic solvent that is within a Hansen Euclidean radius of 25 MPa ⁰⁵ from the Hansen solubility parameters of: dispersive = 11; polar = 13.7; and hydrogenbonding = 6.3.

Without wishing to be bound by theory, it is believed that the advantages of the present invention are achieved using alkylphosphonium (typically tetraalkylphosphonium) and alkylammonium (typically tetraalkylammonium) salts (such as hydroxides and other strong bases) to solubilize the polysaccharide components of algal and (leafy) plant cell walls to increase the extraction efficiency (i.e., yield and speed) of the lipidic component and/or oil soluble component (e.g., polyphenols and pigments) by a (water-immiscible) organic solvent.

It has been realized by the present inventor(s) that lipidic component and/or oil soluble component extraction efficiencies increase with the concentration of the tetraalkylphosphonium and tetraalkylammonium ions above 15% by weight in the aqueous phase with maximum extraction efficiencies peaking near 20%, and declining as concentration of the tetraalkylphosphonium and tetraalkylammonium ions exceed 35% by weight in the aqueous solution. Preferably the tetraalkylphosphonium and tetraalkylammonium ions are present at a concentration of no more than about 50% by weight in the aqueous solution.

Examples of tetraalkylphosphonium and tetraalkylammonium ions that may be suitable for use in the present invention include tetramethylammonium, tetraethylammonium, tetrapropylammonium, benzyltrimethylammonium, tetrabutylammonium, tributylmethylphosphonium, tetrabutylphosphonium, and trioctylmethylphosphonium ions.

The preferred tetraalkylphosphonium and tetraalkylammonium ions are tetrabutylphosphonium and tetrabutylammonium ions.

Examples of counterions that may be used include hydroxide, fluoride, chloride, bromide, iodide, acetate, propionate, glycolate, oxalate, chloroacetate, formate, phosphate, hydrogen phosphate, dihydrogen phosphate, sulfate, nitrate, hydrogen sulfate, nitrite, thiosulfate, sulfite. In some examples the tetraalkylphosphonium and tetraalkylammonium salts include hydroxide or acetate counterions. In some examples, the acetate salts can be synthesized by mixing the tetraalkylphosphonium or tetraalkylammonium hydroxide salt with acetic acid (such as a 40-60% aqueous acetic acid solution). Tetraalkylphosphonium salts are believed to be more thermally stable than tetraalkylammonium salts, and are therefore preferred since they can be more readily recovered and recycled in the overall process.

In some examples, the present invention uses a tetraalkylphosphonium or tetraalkylammonium salt which: i) Is provided wherein the counterion is a hydroxide or other strong base; and/or ii) Is provided at a concentration of no more than about 50% by weight in the aqueous solution.

In some examples the present invention uses a tetraalkylphosphonium or tetraalkylammonium salt which: i) Is provided wherein the counterion is a hydroxide or other strong base; and ii) Is provided at a concentration of no more than about 50% by weight in the aqueous solution. In some examples the tetralkylphosphonium or tetraalkylammonium salts may be added directly to the harvested wet plant or algal material in proportion to the water content of the solid biomass (of the plant or algal material) to attain the desired aqueous phase concentration.

Where the plant tissues contain less than optimal residual water (e.g, previously dried or wilted), in some examples an aqueous solution containing tetraalkylphosphonium or tetraalkylammonium ions (such as more than 15% by weight or volume) can be added to the tissues in a sufficient quantity to promote mixing of the tissues and solubilization of the polysaccharides present.

In some examples, the step of mixing the plant or algal material with a tetraalkylphosphonium or tetraalkylammonium salt in the presence of water to form a mixture is conducted prior to a separate discrete step of adding the organic solvent to perform the extraction step. In other examples the organic solvent is added at the same time that the plant or algal material is mixed with a tetraalkylphosphonium or tetraalkylammonium salt in the presence of water to form a mixture.

After the mixing step (providing the mixture, as referred to herein) extraction of the lipidic component and/or oil soluble component is facilitated by an organic solvent - such as a water- immiscible organic solvent. It has been found that optimal extraction is realised by using an organic solvent within a Hansen Euclidean radius of 25 MPa ⁰⁵ from the Hansen solubility parameters of: dispersive = 11; polar = 13.7; and hydrogen-bonding = 6.3.

Examples of such organic solvents are given in Table 1 below, however advantageously the skilled person will be able to prepare appropriate solvent and solvent mixtures from a range of known materials based on published or derived Hansen solubility parameters. Examples of solvents that may be used alone or in combination (so as to satisfy having a Hansen Euclidean radius of 25 MPa ⁰⁵ from the Hansen solubility parameters of: dispersive = 11; polar = 13.7; and hydrogen-bonding = 6.3) include: chlorinated solvents (such as chloroform, dichloromethane, carbon tetrachloride, 1,2- dichloroethane, chlorobenzene, trichloroethylene, perchloroethylene (tetrachloroethylene)); hydrocarbon solvents (such as cyclohexane, cyclohexene, benzene, toluene, butane, isobutane, pentane, isopentane, neopentane, hexane, 2-methylpentane, 3-methylpentane, 2,2,- dimethylbutane, 2,3-dimethylbutane, heptane, methylhexane, dimethylpentane, 3-ethylpentane, 2,2,3-trimethylbutane, octane, 2,2,4-trimethylpentane, nonane, decane, petroleum ether, xylene); ether and ester solvents (such as diethyl ether, ethyl acetate, methyl acetate, propyl acetate, methyl-tert-butyl ether, anisole); and liquid edible oils (such as peanut oil, vegetable oil, olive oil, or melted stearic acid). Advantageously, in some examples, plant or algal proteins that may be present may remain undigested in their natural intact composition. Such proteins may then be recovered (such as by chromatography, fractional precipitation, or immunoaffinity enrichment) from the de-lipidated tetraalkylphosphonium or tetraalkylammonium salt solution. This allows the production of a second useful product from the original plant or algal tissue, such as rubisco protein, which is a vegan alternative to egg whites.

The present invention can be applied to algae and microalgae species such as Chlorella sp. The present invention can be applied to seaweeds, such as Pyropia sp., The present invention can be applied to the leaves of terrestrial vining plants, such as Vitis sp. The present invention can be applied to leaves of grasses, such as Triticum sp. The present invention can be applied to leafy shrubs, such as Nicotiana sp. or Lactuca sp. The present invention can be applied to the leaves of root vegetables, such as Colocasia esculenta or Beta sp. The present invention can be applied to the leaves of trees, such as Maias domestica or Pranas dalcis. The present invention can be applied to the skins of fruits and berries, such as Sambucus sp. The present invention can be applied to the stamen and pedals of flowers, such as Echinacea purpurea.

Brief Description of the Drawings

Figure 1. Gas chromatogram of US National Institute of Standards SRM 3275 samples used to identify the polyunsaturated fatty acid methyl ester peaks.

Figure 2. The corrected 24 h organic solvent polyunsaturated lipid extraction yields (Yn _pi d) from aqueous acetic acid solubilized Perna canaliculus mussel meat as a function of the Euclidean distance of the extraction solvent system from the optimum Hansen solubility parameter coordinates.

Figure 3. The estimated phase equilibrium constant (Hn _pi d) for polyunsaturated lipids between a 50% aqueous acetic acid solution and various immiscible organic solvent systems. The equilibrium constant is estimated based on 24 h of equilibration between the phases. It is correlated with the Euclidean distance in Hansen solubility parameter space from the predicted highest equilibrium value. Detailed Description of the Invention

In accordance with the present invention, the lipid extract is prepared from algal or plant tissues that have been substantially dissolved in an aqueous tetraalkylphosphonium or tetraalkylammonium solution and the subsequent recovery of the lipid and oil-soluble fraction by extraction with a water- immiscible solvent or oil.

The hydrated plant tissues may be fresh or frozen with woody stems attached, is subjected to dissolution by mixing a sufficient amount of tetraalkylphosphonium or tetraalkylammonium ions added to the natural water content of the soft tissues to reach a final concentration of above 25% weight-to-volume and preferably above 15%. With the optimal tetraalkylphosphonium or tetraalkylammonium ion concentration being between 15-30% weight-to-volume. The acetic acid is mixed with the plant tissue and any associated woody stems, dissolving the softer primary photosynthetic tissues in this process. The insoluble materials, such as the woody stems, may optionally be filtered from the remainder of the solubilized tissue solution.

In another embodiment, a sufficient amount of aqueous tetraalkylphosphonium or tetraalkylammonium ions, as described above, may be added to previously dried, freeze-dried, wilted or otherwise inadequately-hydrated plant or algal tissue to both rehydrate and solubilize the tissue. The tetraalkylphosphonium or tetraalkylammonium ions may be pre-mixed in the proper ratio before contacting the tissue. Alternatively, the water may be contacted first with the tissue, subsequently adding concentrated tetraalkylphosphonium or tetraalkylammonium salts to the proper ratio. The mixture is mixed to homogenize the solution and dissolve the tissues. Insoluble materials, such as woody stems, may optionally be filtered from the remainder of the solubilized tissue solution.

The ratio of aqueous tetraalkylphosphonium or tetraalkylammonium solution to plant tissues should be sufficient to allow mixing to occur. Otherwise, the higher the tissue solids content the better. In one embodiment the solids content can be maximized using an extruder for mixing. Lower solids contents can be processed by tumble mixers and tank-based agitators. The lower the volume of the aqueous phase tissue solution the higher the extraction efficiency that can be obtained.

Following the solubilization of the plant or algal material, a volume of an immiscible organic phase can be added to extract the lipidic component and/or oil soluble component in the plant or algal material. Many different organic solvents and oils can be used for the extraction process. The choice of organic solvent can be optimized on the basis of Hansen solubility parameters. Hansen parameters are thermodynamic state properties of the organic solvent and measured values for many solvents are available in the literature [including Barton (1991) and Abbott et al. (2008) the entire contents of which are hereby incorporated herein by reference]. In addition to measured/known values for certain organic solvents, the Hansen parameters can be estimated using group contribution methods [Barton (1991)] for those other solvents whose solubility parameters have not been measured.

It has now been found that chloroform is an ideal solvent to optimise extraction. However, in some examples, it may be preferable to use a solvent other than chloroform. To that end, it is believed that the closer the Euclidean distance (R) of the solubility parameters of the solvent (including solvent blend) is to those of chloroform, the better the extraction efficiency of lipidic component and/or oil soluble components in the plant or algal material.

The Euclidean distance formula is defined by Hansen for polymer solubility in organic solvents [Schneider (1991)] as follows: R = where, 8dc = Hansen dispersive parameter of chloroform = 11.0 MPa ⁰⁵ .

6p _C = Hansen polar parameter of chloroform = 13.7 MPa ⁰⁵ .

She = Hansen hydrogen-bonding parameter of chloroform = 6.3 MPa ⁰⁵ .

6ds = Hansen dispersive parameter for the solvent or solvent blend.

6p _S = Hansen polar parameter for the solvent or solvent blend.

6hs = Hansen hydrogen-bonding parameter for the solvent or solvent blend.

Hansen solubility parameter values for miscible mixtures can be determined from the volumefraction average for each parameter [Schneider (1991)]. This principle enables the use of homogeneous solvent mixtures to mimic the behavior of other solvents by adjusting the volume fractions of the solvent mixture to match the solubility parameters of the target solvent. This technique can be used to swap extraction solvents based to lower cost, toxicity, increase volatility or otherwise enhance downstream separation from the extracted solute. where, 6j = refers to an individual Hansen solubility parameter (dispersive, polar, or hydrogen-bonding). i = the individual solvents comprising the miscible mixture. The ratio of organic solvent to the tetraalkylphosphonium or tetraalkylammonium ions, and plant or algal material, can be adjusted to that sufficient for the level of recovery desired. The lipidic component and/or oil soluble component will equilibrate between the organic-rich and aqueous ionic salt phases based on their phase equilibrium constant (Hn _P id), which relates the lipid concentration in the aqueous phase (C _a ^eq ) to that in the organic phase (C _o ^e<7 ) at equilibrium. This equilibrium constant is a property of the organic phase.

Hlipid = Ysi

Therefore, the maximum concentration of lipid that can be reached in the organic phase (C _o ^eq ) can be determined from the initial concentration of lipid (C,) in the tetraalkylphosphonium or tetraalkylammonium ions and tissue homogenate as a function of Hn _pi d and the volumes of aqueous (14) and organic (14) phases.

Therefore, the maximum lipid yield (Yn _P id) is determined from the aqueous to organic volume ratio and the equilibrium constant [Hn _P id} for the water-immiscible organic solvents chosen for the extraction. Hu _pid varying inversely the Euclidean distance of the organic solvent from chloroform.

The rate at which this maximum yield can be obtained depends on the rate of mass transfer between the two phases, which is determined by the law of mass transport. where, Jn _pi d = Total rate of lipid transfer from the aqueous to organic phase. k _m = mass transfer rate constant

SAdrop = the average surface area of a droplet in the discontinuous (included) phase.

In liquid/liquid extraction both the mass transfer rate constant (/< _m ) and the surface area of the included phase droplets (SAd _rop ) increase with the level of agitation of the mixer (shear rate), increasing the rate of mass transport over that possible in solid/liquid extraction since the surface area of the solid included phase is fixed by the average size of the solid particles, independent of the level of agitation of the mixer. Therefore, for a batch process, the concentration of lipid in the organic phase increases over time (t), asymptotically approaching the equilibrium concentration at long time.

Other considerations go into the selection of an organic solvent, including: the ability to separate the lipidic component and/or oil soluble component extracted from the organic solvent by evaporation, chromatography, winterization, affinity enrichment, precipitation, or zone recrystallization. In some examples, the extraction can be operated under pressure so that a low boiling point organic solvent (such as (liquid) butane or propane) can be used as the extraction solvent. When the pressure is released the butane and propane will vaporize and the lipid recovered. In another embodiment, the extraction can be performed with lauric acid as the extraction solvent at 45 °C. By then lowering the temperature below 44 °C, the lauric acid can be slowly solidified by winterization, leaving the lower melting lipids as a liquid. In another embodiment, any fatty acid that remains in the liquid state at the processing temperature can be separated by anion exchange chromatography from the extracted neutral lipids it contains.

The above process can be operated at any temperature above the freezing point of the aqueous and or organic phases, whichever is higher (approximately 0-10 °C) and the temperature at which the desired lipidic component and/or oil soluble component decomposes. For polyunsaturated fatty acids decomposition starts to occur at 45 °C. Another advantage of this process is that it operates at ambient temperatures and pressures, so requires no heat or refrigeration. The tetraalkylphosphonium and tetraalkylammonium salts are known anti-microbial agents [Melezhyk et al. (2015) and Kanazawa et al. (1994)]. In the absence of water contact, organic solvents and oils also do not support microbial growth. Thus, the products, both lipidic component and/or oil soluble component and residual protein, are protected from microbial contamination and digestion during processing.

Table 1. Hansen Solubility Parameters of Solvent mixtures for the extraction of polyunsaturated lipids. One or more embodiments of the invention will be described below by way of example only, and without intending to be limiting.

Examples

Example 1

A sample of 0.1 g of commercial dried, powdered, Chlorella vulgaris was added to 1.2 mL of each of the following aqueous solutions: 10%, 20%, 30%, and 40% tetrabutylphosphonium hydroxide (TBPOH) in separate experiments. These solutions were pressure-treated in a Barocycler (Pressure Biosciences, Cambridge, MA) from 1 to 2413 bar for 30 cycles of 45 sec at high pressure and 15 seconds at low pressure) to assist in the dissolution of the algal polysaccharides.

Example 2

After pressure treatment, the 1.5 mL of the resulting aqueous mixture was transferred to a 15 mL centrifuge tube and an equal volume of chloroform was added to the mixture to extract the lipids. The tubes were sealed and put on an end-over-end rotary mixer for 24 hr to accomplish the extraction. The 10% TBPOH was not observed to form a second lipid-rich chloroform phase. The 20%, 30%, and 40% TBPOH solutions all formed two phases.

Example 3

The mixed phases in example 2 (with the exception of the 10% tetrabutylphosphonium hydroxide solution) were separated by centrifugation and samples of each phase recovered for fatty acid analysis. For the algal lipid extract, 0.2 mL of aqueous and organic layers were added separately into respective 15 mL centrifuge tubes by transfer pipette. Toluene (0.2 mL), 1.5 mL of methanol, and 0.3 mL of methanolic HCI (8% w/v) were added into each of the samples sequentially. Each tube was vortexed for 10 seconds to mix to homogeneity and incubated at 45 °C for 20 hours to both saponify the lipids and convert the resulting fatty acids into their corresponding methyl esters following the method of Ichihara and Fukubayashi (2010). The samples were again centrifuged to remove any particulates transferred to a glass gas chromatography vial and capped with a rubber gasket. Example 4

A three-sample set of Omega-3 and Omega-6 fatty Acids in Fish Oil acid methyl ester standard mixture (US National Institute of Standards, SRM 3275) was used to identify the components in the samples prepared in example 5. A 0.1 mL sample from each standard was diluted in 1 mL of a 30% methanol/70% toluene mixture. A 0.25 mL sample of this dilution was then transferred into 1.13 mL of methylation buffer (Example 4). The samples were capped, mixed and incubated for 16 hours at 45 °C in a heating block. The samples were centrifuged to remove any particulates transferred to a glass gas chromatography vial and capped with a rubber gasket.

Example 5

The methylated fatty acid contents of the saponified and methylated fatty acid samples prepared in examples 3 and 4 were analyzed on a Shimadzu GC-2014 gas chromatograph equipped with a 30 m Restek Stabiwax capillary column (0.25 mm ID, 0.25 micron film thickness) using a flame ionization detector (FID). The injection volume was 1 pL using a split injector at 220 °C with a split ratio of 1:186 using hydrogen at 50 cm/s as the carrier gas. The FID was operated at 250 °C. The column was operated with a temperature gradient consisting of 160 °C for 1 min. The temperature was increased to 185 °C at 5 °C/min then increased to 240 °C at 8 °C/min. Finally, the column was held at 240 °C for 10 min before returning to the starting temperature. Individual methylated fatty acid peaks in the samples from example 5 were identified by comparison to the elution times of the FAME standards (example 6). The identities of each of the polyunsaturated (omega-3 and omega-6) fatty acid methyl ester peaks in the standards were determined from the reported abundances and approximate elution times of each fatty acid methyl ester on the certificate of analysis (Figure 1 and Table 2). Only the polyunsaturated fatty acids (omega-3 and omega-6) were tracked as these are the molecules that imparted the known clinical utility.

Table 2. Retention times of and identities of omega-3 and omega-6 fatty acid methyl esters for the gas chromatography method of Example 7. Peak numbers correspond to those listed in Figure 1.

Example 6

Fatty acid methyl esters (FAMEs) were identified by their elution times by comparison to NIST 3275 Fish Oil standards run on the same column (Example 5). The abundances of each identified FAME were determined relative to baseline for each identified FAME. Since the volumes of the organic and aqueous phases were equal in the extraction, the yield of each FAME was calculated as the peak area of the FAME in the organic phase divided by the sum of the peak areas in both phases. The phase equilibrium constant was calculated as the ratio of the peak area in the organic phase to that in the aqueous phase sample. The Yields and Equilibrium constants for each experiment are shown in Tables 3-5. These data were determined from the averages of replicate experiments.

Table 3: Algal oil recovery by room temperature chloroform extraction from 20% TBPOH solubilized dried Chlorella vulgaris.

Table 4: Algal oil recovery by room temperature chloroform extraction from 30% TBPOH solubilized dried Chlorella vulgaris.

Table 5: Algal oil recovery by room temperature chloroform extraction from 40% TBPOH solubilized dried Chlorella vulgaris. Unless the context clearly requires otherwise, throughout the description and the claims, the words "comprise", "comprising", and the like, are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense, that is to say, in the sense of "including, but not limited to".

The entire disclosures of all applications, patents and publications cited above and below, if any, are herein incorporated by reference.

Reference to any prior art in this specification is not, and should not be taken as, an acknowledgement or any form of suggestion that that prior art forms part of the common general knowledge in the field of endeavour in any country in the world.

The invention may also be said broadly to consist in the parts, elements and features referred to or indicated in the specification of the application, individually or collectively, in any or all combinations of two or more of said parts, elements or features.

Where in the foregoing description reference has been made to integers or components having known equivalents thereof, those integers are herein incorporated as if individually set forth.

It should be noted that various changes and modifications to the presently preferred embodiments described herein will be apparent to those skilled in the art. Such changes and modifications may be made without departing from the spirit and scope of the invention and without diminishing its attendant advantages. It is therefore intended that such changes and modifications be included within the present invention.

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