Field of Invention

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

Background of the Invention

There are a variety of oils and oil-soluble molecules that are recovered from animal tissues for use as human and animal dietary supplements, including polyunsaturated fatty acids (omega-3 and omega- 6) and lipids containing them as well as steroids and other lipid soluble hormones. Animal fats (e.g., lard, goose and duck fat) provide key flavour and texture components used to improve the mouth feel or enhance the flavour of both human and pet processed foods. These materials are typically derived from the inedible wastes generated from slaughterhouses and fish processing plants; however, in some instances animals are harvested specifically as to supply these essential oils (e.g., greenshell mussels and krill), producing significant agricultural waste as a byproduct (e.g., animal protein and shells).

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 chloroforrmmethanol 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 muscle 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).

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, required heat. Given that 60-80% of most raw animal tissues are composed of water, however, drying added considerable cost to the oil extraction process. Most of the desirable essential oils are polyunsaturated, however, making them thermally-unstable and oxygen sensitive. Therefore, it was the advent of commercialscale freeze drying technology that really opened up the extraction possibilities. Freeze-drying, however, brings in both refrigeration costs to initially freeze the tissue, the cost of maintaining a vacuum, and still has the 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, made ethanol a preferred solvent for dry tissue extraction.

Ethanol extraction of dried tissues provides good yields of both neutral and phospholipids, but left the problem of driving off the ethanol solvent. Evaporation of the ethanol required heat and cooling for condensing the vapors. The resulting oils typically blackened by the process and not the desired golden yellow colour that the market desired. This is still a solid/liquid extraction process, and, as such, the size of the tissue particle directly effects the lipid recovery, which means the best extraction efficiency was obtained from the finest powdered tissues.

Supercritical fluids, particularly CO2, was ultimately shown to be an alternative extraction solvent for dried or freeze-dried animal tissues [Catchpole et al. 2018)]. Recovery of the extracted oil could be conducted cold by releasing the pressure instantly, evaporating the supercritical fluid and leaving droplets of lipid. However, the extraction efficiency of supercritical CO2 (scCCh) was low (below 50%) [Catchpole et al. (2018)] because only the neutral lipids (triglycerides) were recovered. While SCCO2 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 present inventor(s) believe that 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 essentially 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 matrix that is difficult for solvents to penetrate and create a tortuous d iffusio n a I path to get the lipids back out of the solid particles once they are solubilized.

MacCrides and Broadbent (2006) used protease enzymes in an aqueous solution to digest the proteins comprising the solid tissue into peptides and amino acids. Doing so, removed most of the solid phase that the tissue presented and converted what was a solid/liquid extraction process into a liquid/liquid extraction process in which any suitable water-immiscible solvent could be used to extract the lipids. Alternatively, the aqueous peptide solution could be dried or freeze-dried and extracted with ethanol, diethyl ether, and supercritical carbon dioxide. In yet a further embodiment, they also add lipase enzymes to liberate the fatty acids from the lipids present in the tissue to further enhance their extraction. Some of fatty acids and lipids would naturally phase separate from the aqueous digest and could be recovered by centrifugation. 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 enzymatic digestion of tissues using specific endoproteases have described previously in the literature to identify and quantify the proteins comprising tissues by liquid chromatography coupled to tandem mass spectrometry [Hunt, et al. (1986)]. For example, surfactants, like sodium dodecylsulfate [Laemmli (1970)] and cholamidopropyl-dimethylhydroxypropanesulfonate [Hockstrasser et al. (1988)], are often used to dissolve the proteins in tissues so that individual proteins can be separated, quantified, and identified. Aqueous solutions containing high or nearly- saturating concentrations of urea and thiourea have also been described to solubilize proteins prior to analysis by electrophoretic means [O'Farrell, P.H. (1975)]. But these protein solublization methods have not been applied to lipid extraction. Surfactants exhibit similar solubilities to lipids and fatty acids and would contaminate the lipid extracts of tissues solubilized by these materials. Furthermore, the co-extraction of surfactants and lipids would deplete the surfactant concentration in the aqueous phase and cause the proteins to precipitate. Urea and thiourea must be used at very high concentrations (e.g., 8-9 M = 50 % weight to volume) in water to solubilize proteins and are solid materials which are difficult to recover from the residual proteins, creating a larger process waste stream.

It is an object of the invention to provide an efficient method of extracting a lipidic component and/or oil soluble component from animal 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 animal material, the method including the steps of: i) providing animal material including a lipidic component and/or oil soluble component; ii) mixing the animal material with an organic acid in the presence of water to form a mixture; iii) 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 = 11.2; polar = 10; and hydrogen-bonding = 7.8.

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 tissue.

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 animal material using an organic solvent that is within a Hansen Euclidean radius of 25 MPa ⁰⁵ from the Hansen solubility parameters.

Without wishing to be bound by theory it is believed that the step of mixing the animal material with an organic acid in the presence of water to form a mixture helps to facilitate the extraction of the lipidic component and/or oil soluble component from the mixture using the organic solvent. In particular, it is believed that the organic acid aids with denaturing the protein, rather than digesting the protein in the animal material.

The present invention now enables a high yield (otherwise known as a recovery) of the lipidic component and/or oil soluble component from wet animal material. That high yield would certainly not have been expected based on previous approaches in the field.

For example, the present invention may preferably use aqueous acetic acid solution to dissolve the animal material (such as animal tissue). An immiscible solvent may then be extract the lipidic component and/or oil soluble component. For instance, from 40-60% (v/v) acetic acid in water, such as an approximately 50% by volume acetic acid in water solution, exhibits Hansen solubility parameters [Barton (1991) and Abbott et al. (2008)] nearly identical to that of 8-9 M urea and will generally dissolve the individual proteins in animal tissues. Such aqueous acetic acid solutions are especially preferred. Some proteins in animal tissues are covalently crosslinked (e.g., connective tissues and membranes) and will remain attached to one another a solubilized state. However, and without wishing to be bound by theory, the 40-60% (v/v), such as 50% (v/v) acetic acid solution is believed to cause these crosslinked connective tissues and membranes to swell exponentially making them highly porous, such that the protein matrix does not present a significant barrier to solvent penetration and molecular diffusion.

In some examples a premixed water/organic acid (such as acetic acid) solution can be mixed with previously dried or freeze-dried animal material to rehydrate and dissolve that material prior to extraction of the mixture. The organic acid may be provided with water and/or the animal material may be provided with water. In any case, mixing the animal material with an organic acid will occur in the presence of water and all such modes are contemplated herein.

The organic acid may be selected so as to help facilitate the extraction of the lipidic component and/or oil soluble component from the mixture using the organic solvent. Examples of organic acids that may be suitable include benzenesulfonic acid, croconic acid, methanesulfonic acid, ascorbic acid, p-toluenesulfonic acid, thioacetic acid, trifluoromethanesulfonic acid, although preferably the organic acid will be a carboxylic acid. In some examples the organic acid will have a pKa of between 3.5 and 5. Examples of carboxylic acids (and some representative pKa values) include acetic acid 4.74), propionic acid (4.87), oxalic acid, formic acid (3.75), lactic acid, glycolic acid, malic acid, maleic acid, malonic acid, succinic acid, trifluoroacetic acid, difluoroacetic acid, chloroacetic acid, dichloroacetic acid, trichloroacetic acid, bromoacetic acid, tribromoacetic acid, chlorodifluoroacetic acid, fumaric acid, tartaric acid, gallic acid, nitrilotriacetic acid, phthalic acid, sorbic acid, 2- hydroxybenzoic acid, palmitic acid, benzoic acid, citric acid. Preferably the organic acid is acetic acid. The organic acid may also be provided with one or more (co)solvents during the step of mixing the animal material with an organic acid in the presence of water to form a mixture. Examples of such (co)solvents include methanol and acetonitrile.

By way of further example, a highly concentrated organic acid, such as highly concentrated (glacial) acetic acid solution can be mixed in proportion to the amount of water contained within the animal material (such as animal tissue). The mixing step may include one or more other steps to enhance mixing, such as maceration and/or homogenisation.

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 = 11.2; polar = 10; and hydrogen-bonding = 7.8.

Examples of such organic solvents are given in Tables 1, 3 and 4 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 = 11.2; polar = 10; and hydrogen- bonding = 7.8) 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).

Optionally, some or all of the organic acid (such as acetic acid) may be separated and recovered from the residual biomass by evaporation or spray drying, and fractionally distilled for reuse.

Advantageously the method of the present invention may use a liquid edible oil (a food oil) as the organic solvent, and acetic acid as the organic acid, so that the entire process is food-safe.

The present invention also provides that the animal material (such as tissue proteins) is undigested and solubilized in their natural intact composition. The proteins or single proteins can then be recovered by chromatography, fractional precipitation, or immunoaffinity enrichment from the de- lipidated aqueous acetic acid solution. This allows the production of a second useful product from the original animal tissue, such as collagen or lysozyme from egg shell membranes.

The method of the present invention can be applied to animal material from a range of sources, including terrestrial, aquatic (fresh water and marine), and aerial animals. The animal material may include meat, fat, tissues, bone, eggs, skin, cartilage, shell, carcass, blood. The animal material may be animal cellular material. The animal material may be animal tissue. The animal material may include the soft tissues of marine molluscs such as Perna canaliculus, Mytilus galloprovinialis or Mytilus edulis. The present invention can be applied to the soft tissues of edible fish and meat processing facilities, such as the boney wastes from fish filleting operations and connective tissues and boney wastes from slaughterhouses. The present invention can be applied to the eggshell membranes, such as those produced by Gallus gallus.

Further aspects of the invention, which should be considered in all its novel aspects, will become apparent to those skilled in the art upon reading of the following description which provides at least one example of a practical application of the invention. Brief Description of the Drawings

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

Figure 2 shows the corrected 24 h organic solvent polyunsaturated lipid extraction yields (Yn _P id) 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 shows the estimated phase equilibrium constant (Ha _P id) 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, lipid extract may be prepared from animal tissues that have been substantially dissolved in an aqueous organic acid (such as acetic acid) solution followed by the recovery of the lipid and oil-soluble fraction by extraction with a water-immiscible solvent (which may include an oil).

In some examples, hydrated animal soft tissue may be fresh or frozen with bones or shells attached, and may be subjected to dissolution by mixing a sufficient amount of concentrated organic acid (such as acetic acid) to the natural water content of the soft tissues to reach a final organic acid (such as acetic acid) concentration of between 30-70% volume-to-volume and preferably 40-60% (v/v) organic acid (such as acetic acid). With the optimal acetic acid concentration being 50% volume-to-volume. The acetic acid is mixed with the animal meat and any associated bones or shells to homogenize the solution, dissolving the soft tissues and swelling the membranes in this process. The insoluble materials, including bones, shells, and hard tissues such as tendons and abductor muscle of mussels, may optionally be filtered from the remainder of the solubilized tissue solution.

In some examples, a sufficient amount of an organic acid (such as acetic acid) and water, as described above, may be added to previously dried, freeze-dried, or otherwise less than fully and/or normally hydrated animal material, in order to both rehydrate and solubilize the tissue. Where used, the acetic acid and water may be pre-mixed in the proper ratio before contacting the animal material (such as animal tissue). Alternatively, the water may be contacted first with the animal material (such as animal tissue), subsequently adding concentrated organic acid (such as acetic acid) to the proper ratio. The mixture is mixed to homogenize the solution and dissolve the animal material (such as animal tissue). Insoluble materials, including bones, shells, and hard tissues such as tendons and abductor muscle of mussels, may optionally be filtered from the remainder of the solubilized animal material solution.

The ratio of aqueous organic acid (such as acetic acid) solution to animal material (such as animal tissue, such as meat) should be sufficient to allow mixing to occur. In some examples, it is preferred to use animal material with a high solids content. In some examples the solids content can be increased or maximized using an extruder for mixing. Lower solids contents can be processed by tumble mixers and tank-based agitators. Without wishing to be bound by theory, it is believed that by lowering the volume of the aqueous phase animal material solution, it is possible to obtain a higher extraction efficiency.

Following the solubilization of the animal material (such as soft tissues), a volume of a water- immiscible organic phase can be added to extract the lipidic component and/or oil soluble component from animal material. Many different organic solvents (including 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 animal material.

The Euclidean distance formula is defined by Hansen for polymer solubility in organic solvents [Schneider (1991)] as follows: where, 6d _C = Hansen dispersive parameter of chloroform = 11.0 MPa ⁰⁵ .

6 _pc - Hansen polar parameter of chloroform - 13.7 MPa ⁰⁵ .

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

8 _ds = Hansen dispersive parameter for the solvent (including solvent blend).

6 _ps = Hansen polar parameter for the solvent (including solvent blend).

6 _hs = Hansen hydrogen-bonding parameter for the solvent (including 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/blends 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, 6, = refers to an individual Hansen solubility parameter (dispersive, polar, or hydrogen-bonding). i = the individual solvents comprising the miscible mixture.

The ratio of water-immiscible extraction solvent to aqueous acetic acid solution can be adjusted to that sufficient for the level of recovery desired. The lipidic component and/or oil soluble components will equilibrate between the organic-rich and aqueous organic acid (such as acetic acid) 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 ^eq ) at equilibrium. This equilibrium constant is a property of the organic phase. Hlipid. / ⁷

Therefore, the maximum concentration of lipid that can be reached in the organic phase (C _o ^e<? ) can be determined from the initial concentration of lipid (G) in the aqueous organic acid (such as acetic acid) homogenate as a function of Hn _P id and the volumes of aqueous (l/ ₀ ) and organic (l/ ₀ ) phases.

Therefore, the maximum lipid yield (Yu _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. Hn _P id 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, Jnpid = 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 (km) and the surface area of the included phase droplets (SAdrop) 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 extraction solvent, including: the ability to separate the lipid extracted from the extraction 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 acetic acid solution (approximately 10 °C) and the temperature at which the desired lipid or lipid- soluble products decompose. 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 organic acid (such as acetic acid) in the aqueous phase may also inhibit microbial growth, which similarly will not occur typically in the organic phase. Thus, the products, both lipid and residual protein, are protected from microbial contamination and digestion during processing.

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

Fresh frozen greenshell mussels (Perna canaliculus), with shells removed, were thawed and macerated in a blender. A measured quantity (25 mL) of the raw mussel macerate was removed and placed into a 50 mL polypropylene centrifuge tube. The natural water content of the frozen mussel meat was determined from the literature to be 82%. Therefore, an equal quantity (20.5 mL) of glacial acetic acid (Sigma) was then added to the centrifuge tube to create a final liquid concentration of 50% acetic acid by volume. The tube was capped and put on an end-over-end rotator overnight to mix the contents to homogeneity. Examination of the contents after overnight mixing showed that all of the mussel tissues had dissolved in the acetic acid to produce a viscous solution with the exception of pieces of the abductor muscles that attach the mussel meat to the shells.

Example 2

Freshly cracked chicken (Gallus gallus) eggshells were rinsed with water to remove any residual egg white. The inner membranes (about 2 g) were peeled from the calcified shells and placed into a 15 mL polypropylene centrifuge tube. A 5 mL quantity of a 50% (v/v) aqueous acetic acid solution was added to the tube. This was a sufficient quantity of liquid to fully immerse the membranes. The tube was capped and put on an end-over-end rotator overnight to mix the contents. The egg shell membranes were observed after the overnight incubation to have swollen considerably to become nearly transparent protein layers, which were found to be extremely fragile and easily broken up into smaller pieces.

Example 3

Samples (2.5 mL) of the mussel tissue homogenate prepared in Example 1 were placed in 15 mL centrifuge tubes containing 2.5 mL of each of the solvents listed in Table 1. The samples were capped and placed on an end-over-end mixer for 24 hours. The aqueous and organic phases were separated by centrifugation with 0.75 mL of each phase removed to separate 1.5 mL polypropylene microfuge tubes. These 0.75 mL samples were centrifuged at 14,000 x g for 15 min to ensure that all particulates were pelleted before further analysis.

Example 4

An 8% methanolic HCI solution was prepared by mixing 362.5 ml of methanol (Sigma, technical grade) with 100 ml of 37% HCI in water (Sigma) to make 462.5 ml in a glass bottle. Methylation buffer was prepared by mixing 230 mL of this 8% methanolic HCI solution with 600 mL of methanol and 300 mL of toluene (Sigma). Example 5

The phase samples prepared in example 3 were saponified and methylated by the method described by Ichihara and Fukubayashi (2010). A 0.25 mL sample of each high-speed centrifuged sample from example 3 was transferred to a 1.5 mL polypropylene tube containing 1.13 mL of methylation buffer (Example 4). The sample was capped, mixed and incubated for 16 hours at 45 C in a heating block. The samples were again centrifuged to remove any particulates transferred to a glass gas chromatography vial and capped with a rubber gasket.

Example 6

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.

Table 1. GREENSHELL Mussel Solvent Extraction of polyunsaturated lipids

Table 1. Extraction yields and equilibrium constants determined for 24 h liquid/liquid extraction from solubilized green shell mussel meat in 50% aqueous acetic acid. The extraction variation by Hansen solubility parameters for each solvent and solvent blend are shown. Yield was calculated as the concentration of polyunsaturated fatty acids in the organic phase divided by the total in the organic and aqueous phases. The equilibrium constant for the extraction was determined as the ratio of the polyunsaturated fatty acid concentrations in the organic phase to that in the aqueous phase since the volumes of both phases were equal.

Example 7

The methylated fatty acid contents of the saponified and methylated fatty acid samples prepared in examples 5 and 6 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 were most desired, since they impart 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 8

Peak areas for the polyunsaturated fatty acid components (Table 1) were integrated for the gas chromatographic traces obtained from each sample. The integrated peak areas for each of the 7 polyunsaturated fatty acid methyl esters were summed for each sample (A _p ). The dilution factor and volumes of the aqueous and organic phases were identical (e.g., V _a = l/ ₀ ) so the ratio of these peak areas provided a way to estimate the polyunsaturated fatty acid recovery. The high acetic acid levels in the aqueous phase samples competed with the residual lipids for methylation by the method of example 5. It was possible to compensate for this interference by measuring the lipid content of a sample that was dissolved (example 1) but not extracted, which provided a biased estimate of C,, where Ci a b A _p The methylation efficiency factor (b) was assumed to be the same for both the unextracted and extracted samples. Therefore, from a material balance. where, A _pi is the sum of the peak areas for the sample that was not extracted,

A _pa is the sum of the peak areas for the aqueous phase sample after extraction.

A _po is the sum of the peak areas for the organic phase sample after extraction.

This can be solved for b since V _a = V _o by averaging the results over many extraction samples.

This compensation factor (b) was applied to all aqueous phase samples when calculating the lipid extraction yields (Yn _pi d) and equilibrium constants (Hn _pi d) for each solvent tested. The resulting corrected lipid extraction yields and phase equilibrium values for different solvents are presented in Table 1.

Example 9

The 24 h extraction yields (Yn _P id) for each of the solvents and solvent blends tested were correlated with the Hansen solubility parameters reported in the literature for each of the extraction solvents and solvent blends tested (Table 1) to define the solubility coordinates that optimized the extraction efficiency. Chloroform was known by the inventors to be an effective lipid extraction solvent, and so its solubility coordinates were used as the initial guess of the coordinates that would produce the best extraction results and highest phase equilibrium constant. Figure 2 shows how the extraction yield ( Yupid) decreases with the Euclidean distance from the optimum solubility center point.

Example 10

Assuming that the extraction had approached equilibrium after 24 h of interphase contact, the phase equilibrium constant was calculated (Hn _pi d) for each of the solvents and solvent blends tested. These constants were correlated with the Hansen solubility parameters reported in the literature for each of the extraction solvents and solvent blends tested (Table 1) to define optimum solvent system to extract polyunsaturated lipids. Since chloroform has previously been recorded as the most effective lipid extraction solvent, its solubility coordinates were used as the initial guess of the coordinates that would produce the best extraction results and highest phase equilibrium constant. Figure 3 shows how the equilibrium constant (Hi _ipi d) decreases with the Euclidean distance from the optimum solubility center point determined in example 9.

Example 11

The solubility parameters of food-grade lipids have been reported in the literature and are generally within the range of the test extraction solvents (Table 3).

Table 3. The Hansen solubility parameters of various food-grade lipids [Pena-Gill et al. (2016)] with the calculated Euclidean distance (R) determined from the optimum polyunsaturated lipid yield point, but these lipid oils appear to be suboptimal for extraction. However, the Hansen solubility of food-grade fatty acids (e.g., the major constituents of olive oil and peanut oil) can be estimated from group contribution theory (Table 4) to be better suited for lipid extraction that the lipids based on their shorter Euclidean distances from the optimal yield Hansen solubility point (Figure 2).

Table 4. The Hansen solubility parameters estimated from group contribution methods [Barton (1991)] for food-grade fatty acids with the calculated Euclidean distance (/?) determined from the optimum polyunsaturated lipid yield point, but these lipid oils appear to be suboptimal for extraction.

A sample of solubilized Perna canaliculus meat prepared as described in Example 1. Was extracted with an equal volume of peanut oil as described in example 3 with the peanut oil rich phase recovered separately from the aqueous acetic acid phase. Because the fatty acids comprising the peanut oil extraction solvent would compete for the methylation reaction with the extracted lipids (Example 5) the peanut oil sample was diluted 1:100 in chloroform before methylation and analysis.

Several of the fatty acid methylation peaks of pure peanut oil were found to overlap with those of the mussel extract. Therefore, the portion of the polyunsaturated peak areas attributable to the mussel lipids was determined by the method of standard addition using 0, 10, and 20 % excess of peanut oil and back extrapolating the associated peak heights to the origin. When multiplied by the dilution factor (100) these could be directly compared to the corresponding fatty acid methyl esters remaining in the aqueous acetic acid phase. The associated lipid extraction yield (Yn _P id) was determined to be 82%.

Example 12

In another example, extraction conditions for the following solvent systems were adapted from those described elsewhere above as follows:

• Glacial acetic acid is added to 10 g Mussel Macerate in a 1:1 ratio with the determined water content of the sample volume to give a final concentration of acetic acid of 50%. • Organic solvent or blend shown in the table below were added to the Mussel and Acid aqueous system at a ratio of 1:2, aqueous:organic.

• Combined phases were shaken for 45 mins on an orbital shaker, before being left to phase separate at ambient temperatures.

• Following natural phase separation, the samples were centrifuged, and the solvent layer recovered into a pre-weighed rotary evaporator flask.

• After evaporation of Solvent, flasks containing the extracted oil were weighed.

• * Average Yields were calculated as ((amount of lipid extracted / total lipid present in the sample) x 100) based on the total lipid content obtained using accredited reference methods from an external Laboratory ( AOAC 948.15 OMA online (mod) ), from (n) replicates (such as 2 or 4).

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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