Improved methods for producing omega-3 containing compositions from algae and related extracts

Abstract

A method for producing a highly bioavailable oil composition from biomass, particularly autotrophic algal biomass, and providing characterized, purified, mass-balanced degradation and component products from the oil composition without significant mass loss during extraction, such as by liquid-liquid extraction, polar separation, and the use of mechanical cartridges.

Description

[Technical field]

[0001] This application claims priority to U.S. Patent Application No. 17 / 718,009, filed April 11, 2022, which is a continuation-in-part of U.S. Patent Application No. 16 / 953,978, filed November 20, 2020, both of which are incorporated herein by reference.

[0002] [STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT] Not applicable. [Background technology]

[0003] Long-chain omega-3 oils are essential for human health, and their deficiency can have serious adverse health effects. These range from impaired development in the brain and neural networks to our cardiovascular health. Today, omega-3 oils are in high demand, as only about 800,000 tonnes of omega-3 fatty acids for human consumption in aquaculture, fisheries, and other marine sources are available annually. This is far below the human nutritional requirement of 1.4 million tonnes currently required to provide the world population with 500 mg of omega-3 fatty acids daily, and will be exacerbated by population growth. Deficiencies in omega-3 fatty acids have been observed worldwide, particularly affecting people living in North America, Central Europe, the Middle East, India, Brazil, and the United Kingdom, with regional and socio-economic differences within these countries. Three major omega-3 fatty acids are found in nature. Alpha linoleic acid (ALA), which has 18 carbons and three double bonds, is found, for example, in flaxseed, soybean oil, and olives. Docosahexaenoic acid (DHA), which has 22 carbons and 6 double bonds, and eicosapentaenoic acid (EPA), which has 20 carbons and 5 double bonds, are only produced in aquatic species (algae) and can be extracted by krill or fish that eat the algae, or from the algae themselves.

[0004] "Working algae", i.e., algae that grow using photosynthesis using sunlight or artificial light, often contain polar lipids that fall into the categories of glycolipids and phospholipids. By their nature, these species have hydrophobic tails and polar (hydrophilic) heads, i.e., they are essentially foreign natural detergents. These substances have a very high bioavailability and therefore easily enter the human bloodstream to achieve the health benefits mentioned above. It has been proven that these diverse and valuable substances are not naturally produced in other sources, which may be of questionable sustainability or have proven difficult to feasibly access. Furthermore, their extraction from microalgae efficiently and in a pure, natural, bioavailable form has not been effectively achieved commercially with success, as unattractive appearance, odor, and texture have hindered existing commercial products. While algae extracts have a higher bioavailability, they traditionally contain components that cause the extracted oil to look very dark, almost black overall, and feel highly viscous, causing the extracted oil to resemble a tarry black solid in which the concentration of omega-3 fatty acids remains relatively low.

[0005] Moreover, to date, existing crude products have not been fully or adequately analyzed and characterized. Indeed, despite decades of research, fully characterized mass balances have not been achieved or published without reporting the inclusion of high levels (e.g., up to 10%) of either "unknown" or "unidentified" substances, or by reporting results only "by difference," placing essentially uncharacterized substances in a bucket simply labeled, for example, "carbohydrates." This is particularly undesirable in the case of dietary supplement and pharmaceutical products.

[0006] In summary, a process for producing a highly bioavailable, highly concentrated omega-3, EPA, and polar lipid-rich composition, particularly a glycolipid composition, presented as a low viscosity, low chlorophyll content, light to dark amber oil for use in dietary supplement and pharmaceutical products, whose contents are fully or nearly fully characterized, is currently unavailable but highly desirable.Furthermore, a method for obtaining such highly desirable compositions directly from an abundant and highly sustainable algae source would be ideal. Summary of the Invention

[0007] The present disclosure describes a solution to the above problem by utilizing a separation process, which may include liquid-liquid reactors and / or mechanical cartridges, to extract an oil extract from the microalgal biomass. The crude extract is then fractionated into clean, well-characterized fractions, such as polar lipids, polysaccharides, carotenoids, etc., with high efficiency and very high recovery. The innovative fractionation process of the present disclosure allows for a perfect mass balance between the oil extract and the whole algal biomass.

[0008] In one embodiment, the disclosed process includes a method of producing an oil composition with low chlorophyll content, comprising obtaining a suitable algal biomass, such as an algal paste or dry powder, extracting the algal biomass with a polar solvent, such as an alcohol, such as ethanol, to form an extract of algal lipids, extracting the resulting extract (e.g., with an organic solvent, such as the hydrocarbons hexane or heptane) to separate the non-polar lipid fraction, e.g., transferring the alcohol layer containing pigments and polar lipids to additional processing steps, and adding water to the extracted (e.g.) alcohol layer (e.g., with heptane) followed by sequential extractions to extract the pigment fraction and separate the polar lipid fraction (e.g., with heptane). Polar lipids can then be obtained from the polar lipid-containing fraction by evaporation, and pigments can also be obtained by evaporation of the pigment-containing fraction.

[0009] In an alternative embodiment, the disclosed process includes a method of producing a low chlorophyll oil composition comprising obtaining an algal biomass containing both polar and non-polar lipid fractions and having a chlorophyll concentrate. The method includes substantially separating the chlorophyll concentrate along with the non-polar lipid fraction, and further includes using the polar properties of the polar and non-polar lipid fractions to separate polar components from non-polar components in the algal biomass. Additional steps include bleaching substantially all of the chlorophyll concentrate from the non-polar containing fraction, and recombining the polar and non-polar lipid fractions to produce a low chlorophyll LC-PUFA oil composition.

[0010] Embodiments of the above process produce attractive compositions for use in both the nutraceutical and pharmaceutical fields, particularly in that opacity and viscosity are reduced while high bioavailability is maintained.

[0011] In a related embodiment, a method is provided for fractionating and purifying an algal biomass into a clean, accurately characterized Liquid Extracted Biomass, i.e., residual biomass after extraction ("LEA"), polar lipids, pure neutral lipids, chlorophyll, polysaccharides, and carotenoids components with high overall recovery and reporting a complete algal biomass balance. The method includes obtaining an algal biomass such as a paste or powder, extracting the algal biomass with a polar solvent, such as an alcohol, such as ethanol, to form an alcoholic extract (for example) of algal lipids, extracting the resulting alcoholic extract (for example) with an organic solvent, such as the hydrocarbons hexane or heptane, to separate the non-polar lipid fraction, e.g., transferring the alcoholic layer containing pigments and polar lipids to additional processing steps, and adding water to the extracted alcoholic layer (for example, with heptane) followed by sequential extractions to extract the pigment fraction and separate the polar lipid fraction (for example, with heptane).

[0012] In a further aspect, a method is provided for extracting biomass oils, including but not limited to lipids, chlorophyll, sugars, carotenoids and cannabidiol (CBD), from a dried, preferably powdered biomass. The method includes obtaining a mechanical cartridge or extraction column with an internal bore and an inlet end and an outlet end configured to allow for solvent flow. The mechanical cartridge can be heated / cooled and pressurized, and can accommodate a heated / cooled and pressurized solvent to a range sufficient to separate the target biomass oil from the dried biomass. For example, the operating conditions can allow an operator to melt the lipids, thus extracting them from the dried biomass in which they were contained. The mechanical cartridge itself is loaded with the dried biomass, and the mechanical cartridge can be heated / cooled to a predetermined temperature (typically -30°C to 150°C). A temperature-controlled solvent is pumped through the mechanical cartridge loaded with the dried biomass at a temperature, pressure, retention time and flow rate sufficient to remove the target biomass oil. In the case of lipid-containing biomass, the hot solvent extracts lipids in liquid form from a portion of the biomass held in the mechanical cartridge under the same conditions by melting the lipids in the lipid-containing portion of the dry biomass.

[0013] The present disclosure further relates to bioavailable compositions, LC-PUFA compositions, omega-3 compositions, EPA compositions, polar lipid compositions, and glycolipid / phospholipid rich compositions, particularly suitable for use in dietary supplement and pharmaceutical compositions, which can be considered as health or pharmaceutical compositions and have other valuable end products. The starting material of the composition can be derived from the algae described herein. In one embodiment, a composition is disclosed that includes a polar lipid fraction of a total lipid concentration of at least 20% by weight of total lipid, wherein the polar lipid fraction includes at least 40% by weight of glycolipid, and the composition includes 4% or less by weight of the composition as chlorophyll concentrate.

[0014] Compositions produced as described above, in which at least 20% by weight of the total lipids is the polar lipid fraction and at least 40% by weight of said polar lipids is the glycolipid fraction, but the chlorophyll fraction is less than 4% by weight in the composition, may further comprise formulations containing additive non-polar lipids and / or nutraceutical oils such as DHA or EPA or DHA esters or salts (or mixtures thereof), or other beneficial additives as described in more detail below, which also serve to provide a particular beneficial combination of oils that are more bioavailable, more nutritious, lighter in color, and less viscous.

[0015] These and other features of the present disclosure are explained in greater detail in the detailed description that follows. [Brief description of the drawings]

[0016] [Figure 1] 1 discloses a schematic diagram of the extraction process steps for an exemplary method of producing oil according to embodiments of the present disclosure. [Diagram 2] 2 is a photographic depiction of an embodiment of a composition of the present disclosure produced by the exemplary method described in FIG. 1 having a desired target LC-PUFA concentrate, polar lipid concentrate, omega-3 concentrate, EPA concentrate, glycolipid-rich concentrate, and chlorophyll-depleted concentrate with the desired oil composition bioavailability, color, and viscosity, the embodiment of which is disclosed herein. [Diagram 3] FIG. 1 is a photographic representation of a powder obtained by an embodiment of the process of the present disclosure consisting essentially of non-lipid components, including (for example) polysaccharides, contained in a crude ethanolic algal extract produced by an embodiment of the process described herein. [Figure 4] FIG. 1 is a graph showing the spectral characterization of embodiments of an ethanol extract of Nannochloropsis and products after pigment removal as discussed in Example 1, where Y-axis=AU (Absorption Unit), X-axis=nm (nanometers, wavelength). [Diagram 5]FIG. 1 is a graph showing UV-Visible spectral characterization for an embodiment of an algae extract as discussed in Example 2, Y-axis=AU (Absorption Unit), X-axis=nm (nanometers, wavelength). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0017] The following detailed description is illustrative of the claimed disclosure and is not intended to be limiting. The description clearly enables one skilled in the art to make and use the claimed disclosure, and describes several embodiments, adaptations, variations, alternatives, and applications of the claimed disclosure. It is further understood that the claimed disclosure is not limited to its application only to the details and compositions specifically described in the following description or illustrated by the drawings. The claimed disclosure is capable of other embodiments and can be practiced or carried out in various ways. It is also understood that the words and terms used herein are for the purpose of description and should not be regarded as limiting.

[0018] As used herein, the term "lipid" refers to any of a variety of organic compounds that are insoluble in water. Lipids include fats, waxes, oils, hormones, and certain components of membranes.

[0019] As used herein, the term "polar lipid" refers to an amphipathic lipid having a hydrophilic head and a hydrophobic tail. Examples of polar lipids include phospholipids and glycolipids.

[0020] The term "non-polar lipid" as used herein refers to a fatty molecule in which the charge distribution is approximately uniform and the molecule does not have positive and negative ends. Examples of non-polar lipids include triglycerides of various fatty acids in oils, such as EPA and palmitoleic acid (including mixtures thereof, such as triglycerides (TAG) containing variations or combinations of such fatty acids).

[0021] The term "biomass" as used herein means the total mass of biological life, including, for example, plants and microorganisms, and from a biological perspective can include cellulose, lignin, lipids, sugars, and proteins. Although aspects of the process of the present invention relate directly to the extraction, fractionation, and purification of algal biomass, "biomass" should be considered to include other types of life, such as of fungal origin or other origins, or "biomass" from a given area or volume, unless expressly limited to algal biomass as the sole or primary source.

[0022] The acronyms "EPA" and "DHA" used herein refer to eicosapentaenoic acid and docosahexaenoic acid, respectively, as well as to the salt and ethyl ester forms of each compound. In its naturally occurring acid form, EPA is an unsaturated chain with 20 carbons, resulting in a carboxylic acid functional group. However, those skilled in the art will appreciate that natural variants of this acid form include alkali salts, in which the deprotonated carboxylic acid is stabilized by a counter anion, and ethyl esters, in which two more carbons are covalently bonded singly to the SP3 hybridized oxygen to produce an ester. In the ethyl ester form, EPA has 22 carbons. Similarly, in the acid form, DHA is an unsaturated chain with 22 carbons, resulting in a carboxylic acid functional group, and is understood to have natural variants, including alkali salts and ethyl ester forms. In the ethyl ester form, DHA has 24 carbons, as a result. The alkali salt forms of these compounds may appear naturally as a result of the particular chemical environment in which they exist. Conversion to the ethyl ester variants is similarly easy, and these variants are also used as drugs, for example, to treat high blood triglyceride levels. Any use of the acronyms "EPA" and "DHA" in this application should not be construed as excluding alkali salt or ethyl ester variants of either compound, unless expressly excluded.

[0023] To produce the disclosed embodiments of the clarified compositions of algal biomass, an algal paste provided as a dark green or even black highly viscous oil can be obtained using standard steps known to those skilled in the art. See, for example, the production of algal paste and various useful algae that can be employed as described in U.S. Patent No. 8,591,912 B1 (hereinafter "Kadam and Goodall"), which is incorporated herein by reference and further discussed herein. Furthermore, such algal biomass useful for extraction can be dry and presented in the form of an algal powder or other suitable form.

[0024] The procedure for obtaining an algal biomass extract, the algae initiation and extraction procedure for producing an algal biomass, can include the following steps as part of the extraction.

[0025] Obtain or produce a biomass such as an algae paste or powder (e.g., produced from such an algae paste) from a suitable species suitable for producing such target biomass. For example, but not limited to, dried algae powder can be produced from the algae paste by, for example, drum dryers, powder dryers, refractance window dryers, freeze dryers, ovens, and the like, by procedures known to those skilled in such drying techniques. Generally, the microalgae can be harvested by conventional means (including, but not limited to, filtration, air flotation, centrifugation), and the algae paste is produced by concentrating the harvested microalgae to a desired weight percent solids. In certain embodiments, the microalgae used with the methods of the present invention belong to one of the phyla Chlorophyta, Cyanophyta (Cyanobacteria), and Heterokontophyta. In certain embodiments, the microalgae used with the methods of the present invention belong to one of the classes Bacillariophyceae, Eustigmatophyceae, and Chrysophyceae. In certain embodiments, the microalgae used with the methods of the present invention belong to one of the genera Nannochloropsis, Chiarella, Dunaliella, Scenedesmus, Selenastrum, Oscillatoria, Phormidium, Spirulina, Amphora, Trachydiscus, and Ochromonas. Non-limiting examples of microalgae species that can be used with the methods of the present invention include Achnanthes orientalis, Agmenellum spp.), Amphiprora hyaline, Amphora coffeiformis, Amphora coffeiformis var. linea, Amphora coffeiformis var. punctata, Amphora coffeiformis var. taylori, Amphora coffeiformis var. tenuis, Amphora delicatissima, Amphora delicatissima var. capitata, Amphora sp., Anabaena, Ankistrodesmus, Ankistrodesmus falcatus, Boekelovia hooglandii, Borodinella sp., Botryococcus braunii, Botryococcus sudeticus, Bracteococcus minor, Bracteococcus medionucleatus, Carteria, Chaetoceros gracilis, Chaetoceros muerellii muelleri, Chaetoceros muelleri var. subsalsum, Chaetoceros sp.), Chlamydomus perigranulata, Chlorella anitrata, Chlorella Antarctica, Chlorella aureoviridis, Chlorella Candida, Chlorella capsulate, Chlorella desiccate, Chlorella ellipsoidea, Chlorella emersonii, Chlorella fusca, Chlorella fusca var. vacuolata, Chlorella glucotropha glucotropha, Chlorella infusionum, Chlorella infusionum var. actophila, Chlorella infusionum var. auxenophila, Chlorella kessleri, Chlorella lobophora, Chlorella luteoviridis, Chlorella luteoviridis var. aureoviridis, Chlorella luteoviridis var. lutescenslutescens, Chlorella miniata, Chlorella minutissima, Chlorella mutabilis, Chlorella nocturna, Chlorella ovalis, Chlorella parva, Chlorella photophila, Chlorella pringsheimii, Chlorella protothecoides, Chlorella protothecoides var. acidicola, Chlorella regularis, Chlorella regularis var. minima regularis var. minima, Chlorella regularis var. umbricata, Chlorella reisiglii, Chlorella saccharophila, Chlorella saccharophila var. ellipsoidea, Chlorella salina, Chlorella simplex, Chlorella sorokiniana, Chlorella sp., Chlorella sphaerica, Chlorella stigmatophora, Chlorella vannierii vanniellii), Chlorella vulgaris, Chlorella vulgaris forma tertiatertia, Chlorella vulgaris var. autotrophica, Chlorella vulgaris var. viridis, Chlorella vulgaris var. vulgaris, Chlorella vulgaris var. vulgaris fo. tertia, Chlorella vulgaris var. vulgaris fo. viridis, Chlorella xanthella, Chlorella zofingiensis zofingiensis, Chlorella trebouxioides, Chlorella vulgaris, Chlorococcum infusionum, Chlorococcum sp., Chlorogonium, Chroomonas sp., Chrysosphaera sp., Cricosphaera sp., Crypthecodinium cohnii, Cryptomonas sp., Cyclotella cryptica, Cyclotella meneghiniana, Cyclotella sp.), Dunaliella sp.), Dunaliella bardawil, Dunaliella bioculata, Dunaliella granulate, Dunaliella maritime, Dunaliella minuta, Dunaliella parva, Dunaliella peircei, Dunaliella primolecta, Dunaliella salina, Dunaliella terricola, Dunaliella tertiolecta, Dunaliella viridis, Dunaliella tertiolecta, Eremosphaera viridis, Eremosphaera sp., Ellipsoidodon spp. Ellipsoidon sp., several Euglena spp., Franceia sp., Fragilaria crotonensis, Fragilaria sp., Gleocapsa sp., Gloeothamnion sp., Haematococcus pluvialis, Hymenomonas sp., Isochrysis aff. galbana, Isochrysis galbana galbana, Lepocinclis, Micractinium, Micractinium, Monoraphidium minutum, Monoraphidium sp., Nannochloris sp., Nannochloropsis salina, Nannochloropsis sp., Navicula acceptata, Navicula biskanterae, Navicula pseudotenelloides, Navicula pelliculosa, Navicula saprophila, Navicula sp.), Nephrochloris sp., Nephroselmis sp.), Nitschia communis, Nitschia alexandria, Nitschia closterium, Nitschia communis, Nitschia dissipata, Nitschia frustulum, Nitschia hantzschiana, Nitschia inconspicua, Nitschia intermedia, Nitschia microcephala, Nitschia pusilla, Nitschia pusilla elliptica, Nitschia pusilla monoensis monoensis, Nitzschia quadrangular, Nitzschia sp., Ochromonas sp., Oocystis parva, Oocystis pusilla, Oocystis sp., Oscillatoria limnetica, Oscillatoria sp., Oscillatoria subbrevis, Parachlorella kessleri, Pascheria acidophila, Pavlova sp., Phaeodactylum trichotomum tricomutum, Phagus, Phormidium, Platymonas sp.), Pleurochrysis carterae, Pleurochrysis dentate, Pleurochrysis sp., Prototheca wickerhamii, Prototheca stagnora, Prototheca portoricensis, Prototheca moriformis, Prototheca zopfii, Pseudochlorella aquatica, Pyramimonas sp., Pyrobotrys, Rhodococcus opacus, Sarcinoid chrysophyte, Scenedesmus armatus, Schizochytrium, Spirogyra, Spirulina platensis, Stichococcus sp., Synechococcus sp., Synechocystisf, Tagetes erecta, Tagetes patula, Tetraedron, Tetraselmis sp., Tetraselmis suecica, Thalassiosira weissfrogii weissflogii, Trachydiscus, and Viridiella fridericiana.

[0026] Preferred algae are, for example, those that are rich sources of LC-PUFA oils. Exemplary of such algae strains are algae varieties of Nannochloropsis, Chlorella, or Trachydiscus minutus.

[0027] Algal biomass in the form of algal paste or other suitable form can be processed as follows: extracted (e.g., with a polar solution (such as an alcohol including ethanol)) to form an alcohol extract of algal lipids with low water content (e.g., to form an Ethanol Extract of Nannochloropsis Lipids (hereafter referred to as "EEN" for short)), and the resulting EEN is extracted (e.g., with an organic solvent such as the hydrocarbons hexane or heptane) to separate the non-polar lipid (e.g., triglycerides, waxes, carotenes) fraction, thereby forming a "Non-polar Lipid Fraction (F#1 in FIG. 1)" in the heptane layer. The alcohol layer contains the pigments and polar lipids and can be transferred to additional processing steps. See FIG. 1. Other solvents such as carbon dioxide or a mixture of carbon dioxide and ethanol are also contemplated.

[0028] Subsequent processing steps may include adding water to the extracted polar solvent layer (e.g., with heptane) and then sequentially extracting (e.g., with heptane) to extract the pigment fraction (see FIG. 1, heptane layer, F#3) and to separate the polar lipid fraction (see FIG. 1, water-alcohol layer, F#2). The polar lipids can be obtained by evaporation from the F#2 fraction, and the pigments can be obtained by evaporation from the F#3 fraction. The F#1 layer may contain a certain amount of chlorophyll and carotenoids, which can be removed by methods known to those skilled in the art, for example, methods known for the production of edible oils. Examples of these methods include adsorption-filtration using silica gel, bleaching clays such as B80, T41, and activated carbon. The selective removal of chlorophyll from the F#1 fraction results in a more or less liquid to semi-solid oil that is almost clear to dark amber in color, including red, in this embodiment.

[0029] In an alternative embodiment, a method for producing a low chlorophyll oil composition by extraction of an algal biomass, such as an algal paste or powder, so obtained, the oil composition so obtained contains both polar and non-polar lipid fractions and has a chlorophyll concentrate. The polar properties of the polar and non-polar lipid fractions of the biomass are used to separate the polar components from the non-polar components in the algal biomass, including substantially separating the chlorophyll concentrate along with the non-polar lipid fraction. As shown in Example 2, additional steps include bleaching substantially all of the chlorophyll concentrate from the non-polar containing fraction and recombining the polar and non-polar lipid fractions to produce a low chlorophyll oil composition.

[0030] In addition to the low chlorophyll content aspect of the composition, the present invention also provides isolated oils derived at least in part from algae rich in LC-PUFAs, including at least one omega-3 fatty acid, such as, but not limited to, EPA or DHA, in the form of total and hydrolyzable phospholipids and total and hydrolyzable glycolipids extracted by the above process. The oils produced by such processes are unexpectedly high in polar lipids, such as phospholipids and glycolipids. Krill krill oil is known to have a higher bioavailability in mammals than fish oils, which contain almost exclusively neutral lipids (triglycerides), due to its phospholipids (polar lipids). See Jan Philipp Schuchardt et al., Lipids in Health and Disease, 2011, 10:145. Surprisingly, oils extracted from algae such as Nannochloropsis using the methods of the present invention have shown bioavailability to mammals that is even higher than krill oil.

[0031] In another embodiment of the lipid extraction aspect of the invention, the inventors have created a method for extracting biomass oils, including but not limited to lipids, chlorophyll, sugars, carotenoids and CBD, that utilizes a heated (or, in certain circumstances, cooled) and pressurized mechanical cartridge or, for example, in the case of lipids, an extraction column to extract lipids by melting them from a dry, preferably powdered biomass containing lipids (see above for various methods of producing powdered biomass). The powdered biomass containing lipids to which this aspect applies includes, but is not limited to, algal biomass, such as the preferred source (autotrophic, Nannochloropsis-derived, etc. algal biomass, described elsewhere in this application).

[0032] Extraction of biomass oils, e.g., lipids, using pressurized and temperature-controlled mechanical cartridges to melt the lipids contained in the dried biomass has several advantages. For example, its use significantly reduces the time required for lipid extraction to reach completion. The use of mechanical cartridges has been shown to reduce the processing time required by a factor of three compared to using standard reactors / mixing tanks. For another, the process allows for a significant reduction in the solvent required for extraction. It has been shown that the use of mechanical cartridges can reduce the amount of solvent required to a factor of four compared to using standard reactors / mixers.

[0033] The selected cartridge or extraction column is dimensioned to have an internal bore capable of accommodating the dried biomass and configured to allow and regulate solvent flow, and an inlet end and an outlet end. The internal diameter of the mechanical cartridge is constructed of a material, preferably stainless steel, that can be heated and pressurized and can accommodate the heated and pressurized solvent in a range sufficient to melt and separate lipids (or otherwise extract the target oil based on the differential reaction of temperature and pressure). The internal diameter of the mechanical cartridge can preferably be 25-1000 mm, and the linear length, not including the inlet and outlet connections, is 100-1000 mm. More preferably, the internal diameter can be 300-500 mm, and the linear length can be 150 mm-300 mm. Even more preferably, the internal diameter is 400 mm, and the linear length is 200 mm. The inlet and outlet ends of the mechanical cartridge that provide and regulate the solvent flow include grates, screens, gates, etc. that direct the solvent flow through the interior of the cartridge and the powdered biomass contained therein, allowing for control and regulation of the solvent flow rate and retention time. The inlet and outlet ends can also be used to restrict the remaining solid material that passes through and exits the mechanical cartridge.

[0034] The material chosen to fill the cartridge and its physical form will vary to some extent depending on the target biomass. When targeting algal biomass, the cartridge can be filled with the lipid-containing portion of dry biomass, preferably powdered dry biomass, and the cartridge is heated to a predetermined temperature to facilitate lipid melting. Non-limiting examples of methods for heating the cartridge itself include covering the cartridge, flowing a heated fluid around the outside, immersing the cartridge in a heated solution, as well as by electrical resistance or induction.

[0035] In this embodiment, the cartridge is loaded with the lipid-containing portion of the powdered biomass, and then a preheated solvent is pumped through the cartridge at a temperature, pressure, retention time and flow rate optimized to melt the lipids in the lipid-containing portion of the powdered biomass, thereby extracting lipids in liquid form from the portion retained in the cartridge (e.g., chlorophyll-containing) under the same conditions.

[0036] The solvent used in the cartridge method can be any applicable solvent. Non-limiting examples include alcohols such as ethanol, hexane, heptane, carbon dioxide, and solvent mixtures. Furthermore, this method is applicable to the full range of lipid profiles, i.e., from polar to non-polar, for associated crude extraction from dry biomass.

[0037] The operating principle for using the mechanical cartridge is the tolerance of extraction with a solvent at high temperature and pressure of the target (e.g., for algal biomass). The preferred temperature of the solvent may range from -30°C to 150°C. For lipid extraction, more preferably 70°C to 110°C, even more preferably 90°C. The preferred operating pressure of the solvent is 5 bar to 100 bar, more preferably 25 bar to 50 bar, even more preferably 30 bar. As mentioned above, depending on the material to be extracted, the solvent used, and the biomass from which the target material is extracted, furthermore, for preferred biomass material extraction where chilled solvent is more advantageous, chilled solutions may be employed as well.

[0038] Lipid extraction by such mechanical cartridges is easily combined with additional processing, for example by the procedures presented in Examples 1 and 2 below. [Example]

[0039] The following non-limiting examples illustrate the invention for purifying and determining specific components of an oil composition, and for producing a low chlorophyll, low polysaccharide oil rich in LC-PUFAs, as further described herein.

[0040] [Example 1] 2.1 Process Protocol for this Example (see Figure 1) 1) Weigh out 100g of Nannochloropsis algae paste (22-27% solids in water). (Step 1, Figure 1) 2) The algae paste was placed in a 2L flask and 850ml of alcohol was added. (Step 1, Figure 1) 3) The algae was extracted at a temperature of 70° C. for 45 minutes with vigorous stirring (Step 1, FIG. 1). 4) The solid algae residue was filtered from the ethanol extract (vacuum filtration). (Step 2, FIG. 1) 5) The ethanol extract from the previous step was placed in a separatory funnel (2 L), 300 mL of heptane was added to the resulting extract, stirred vigorously for 2 minutes, the layers were separated, and the top layer was carefully screened and placed in a separate flask to obtain about 120 mL of green heptane layer. (Step 3, Figure 1) 6) An additional 100 mL of heptane was added to the ethanol layer, stirred vigorously for 2 minutes, the layers were separated (ethanol layer-bottom, and heptane layer-top), and the top layer was carefully selected and combined with the heptane layer from the previous step (yielding ~200-220 mL of combined green heptane layer, fraction F#1) (Step 4, Figure 1). 7) 1g of silica gel was added to the obtained total heptane layer, and after vigorously stirring for 5 minutes, the slurry was filtered through a layer of 1g of silica gel (instead of silica gel, activated carbon or T41 bleaching earth may be used). Due to the property differences of various silica gels, activated carbons, and bleaching earths, the actual amount of materials should be adjusted in the factory. (Step 8, Figure 1) 8) The bottom layer (ethanol layer) from step 6 of this protocol was removed, 350 mL of water and 200 mL of heptane were added, and the mixture was shaken vigorously for 2 min. The separated phases were allowed to settle for about 5 min, after which the top layer was carefully screened and placed into a separate flask (~400 mL of green heptane layer was obtained from this step). (Step 5, Figure 1) 9) Repeatedly extract the lower layer in the above step using 200 mL of heptane (3 x 200 mL). Carefully select the top layer and combine it with the heptane layer from the previous step (~1000-1020 mL of combined green heptane layer was obtained, fraction F#3). (Step 5, Figure 1) 10) The extracted bottom layer contained the purified polar lipids (F#2). This fraction can be purified, for example, by 3 g of Amaze-N bleach adsorbent from Helix Chromatography (15 E. Palantine Rd. #118, Prospect Heights, IL 60070; helixchrom.com) (or a similar adsorbent can be used, if necessary). 11) The resulting fraction was evaporated in vacuum with heating below 45°C.

[0041] The above exemplified extraction process is presented as a batch process. However, an automated process utilizing appropriate controls and / or liquid-liquid centrifuges to achieve the described protocol can be adopted by those skilled in the art as well. Automation of all presented examples / embodiments is possible using equipment known to those skilled in the art.

[0042] The above process steps and test results demonstrate a highly efficient and exemplary liquid-liquid extraction method for removing chlorophyll and carotenoid fractions from phototrophic (autotrophic) algae extracts such as Nannochloropsis or Chlorella lipids. By using the above embodiment of the disclosed method, not only was over 99% of chlorophyll a and b and pheophytin removed from the ethanol extract of Nannochloropsis (EEN), but also about 2 / 3 of the carotenoids (medium polar carotenoids) were removed. In so doing, at least 90% by weight, preferably more than 95% by weight, more preferably more than 97.5% by weight, even more preferably more than 98.0% by weight, and most preferably more than 99% by weight of the original mass balance was fully preserved and can be characterized for its major components without leaving the majority of the mass balance intact, whether characterized simply "by subtraction" or "by difference". See Table 1: Extraction Mass Balance (Composition) and Table 2: Major Components of Ethanol Extract of Nannochloropsis for Mass and Weight % Analysis of Composition. (By weight, this represents 99.86% of the total algal biomass.) See Figure 4 for spectral characterization of the Nannochloropsis ethanol extract after pigment removal.

[0043] [Table 1]

[0044] [Table 2]

[0045] While viscosity measurements may vary to some extent depending on factors such as temperature, compositional concentrations of various components in the formulation, as described herein, after combining the polar and neutral lipid fractions at 25°C, the viscosity reading measured for an embodiment of the Nannochloropsis extract produced as described was shown to be 165,000 mPa.'s.

[0046] In summary, the product shown in Example 1 can be decomposed into three main fractions of the ethanol extract of Nannochloropsis edulis (EEN): 1) fraction (F#1), non-polar lipids, mainly triglycerides; 2) fraction (F#2), polar lipids including glycolipids and phospholipids; and 3) fraction (F#3), a medium polar fraction including diglycerides and monoglycerides, free fatty acids (FFA's), carotenoids and chlorophyll. The purified F#1 and F#2 fractions can be used as valuable lipid sources rich in palmitoleic acid and eicosapentaenoic acid (EPA). F#3 is a concentrate of natural pigments including chlorophyll, astaxanthin, and zeaxanthin, etc., which are also valuable as food additives. Fractions F#1 to F#3 can be used as food additives, respectively, and are valuable raw materials with high biological potential.

[0047] [Example 2] A dark green paste sample of algal biomass was produced by the method generally described in U.S. Patent No. 8,591,912 B1 (see generally, column 6, line 62 to column 9, line 3) and discussed herein (see, e.g., paragraph 0021). The algal biomass paste was extracted with hot absolute ethanol. Specifically, 66 g algal paste, 3×250 mL ethanol, 75° C., 30′, each stirred at 500 rpm, and centrifuged at 4450 rpm for 10 min to obtain an algal extract sample.

[0048] Analysis of the oil extracts established that the predominant polar lipids in the algal samples were 1) glycolipids (monogalactosyldiglyceride (MGDG) and digalactosyldiglyceride (DGDG) and 2) phospholipids (phosphatidylcholine, phosphatidylethanolamine, and phosphatidylinositol) (see Table 3 herein).

[0049] [Table 3]

[0050] The bioavailable low chlorophyll content oils, polar lipid rich oils, LC-PUFA rich oils, and omega-3 rich oils embodiments of the present disclosure quantified herein were produced from the starting materials using the following additional steps.

[0051] 1) Polar lipids were separated from a mixture of nonpolar lipids, chlorophyll, and other components based on differences in polarity.

[0052] 2) Chlorophyll was bleached from the remaining non-polar lipid components using a well-developed protocol for bleaching vegetable oils in general, as described in Example 1 above, e.g., 1 g of silica gel was added to the total heptane layer obtained, and after vigorously stirring for 5 minutes, the slurry was filtered through a layer of 1 g of silica gel (activated carbon or T41 bleaching earth may be used instead of silica gel). Due to the property differences of various silica gels, activated carbons, and bleaching earths, the actual amounts of materials should usually be adjusted in the factory.

[0053] 3) The polar lipid fraction from step (1) above was combined with the bleached non-polar lipids from step (2) above.

[0054] Generally, a low viscous, nearly clear to light brown oil composition was obtained that was rich in bioavailable polar lipids and had a low chlorophyll content. See FIG. 5 for spectral analysis. The composition was a waxy solid at ambient temperatures of about 70° F. The composition melts when warmed and exhibits low viscosity when mixed with other oils such as triglycerides.

[0055] [Example 3] Use of extraction columns / mechanical cartridges. 1) 58.85 g of dried algae powder of Nannochloropsis (moisture content 1.2%, Karl Fischer) was placed into a stainless steel cartridge (inner diameter 25 mm, length 150 mm, volume 75 cm3).

[0056] 2) During extraction, the thermostatic extraction column packed with algae powder was maintained at 90°C.

[0057] 3) Dry ethanol (0.1% (w / w) in water, KF) at 90°C (preheated in a heat exchanger) was pumped through the extraction column at a flow rate of 4.0 ml / min (0.8 ml / min / cm2) for a total volume of 210 ml. The eluate (extract) was collected, observed as a dark green liquid. The total extract mass was 15.91 g and the dry residue weight (extracted algae) was 42.85 g. The mass % of the extract was 27.1%.

[0058] After separation of the lipid portion from the chlorophyll-containing fraction of the biomass, i.e. after the chlorophyll-containing fraction has been removed or significantly reduced from the lipid portion, as illustrated by the steps exemplified above, the resulting chlorophyll-depleted or chlorophyll-free extract can be further fractionated to obtain clean fractions of other components such as polar lipids, non-polar lipids, for example using liquid-liquid protocols as described in Examples 1 and 2.

[0059] Analysis of the oil composition of the bioavailable oil embodiments of the present disclosure produced using the above processes described herein demonstrated that an oil was obtained from the algal biomass having the following components and characteristics (column 1), and component ranges (column 2), as shown in Table 4.

[0060] [Table 4]

[0061] Minor variations in the weight percentages of the components and other properties of the oil disclosed in this application can be obtained by modifying the process used as known to those skilled in the art. However, preferably, the weight percentage of the polar lipid fraction in the total lipid concentrate of the oil produced is greater than 20% by weight, preferably greater than 30% by weight, more preferably greater than 40% by weight, even more preferably greater than 50% by weight, and even more preferably greater than about 70% by weight. Also, preferably, the weight percentage of chlorophyll concentrate in said oil product is less than 4% by weight of the total oil product, more preferably less than 3.0%, even more preferably less than 2.0%, even more preferably less than 1.0%, less than 0.75%, less than 0.50%, less than 0.2%, and even more preferably less than 0.1%. Also, preferably, the weight percentage of polysaccharide concentrate in said oil product is less than about 4% by weight of the total oil product, more preferably less than 3.0%, even more preferably less than 2.5%, less than 2.0%, even more preferably less than 1.0%, less than 0.5%, or lower. Furthermore, the weight percent of glycolipids as a weight percent of total polar lipids is greater than 20%, preferably greater than 30%, more preferably greater than about 40%, even more preferably greater than 50%, 60%, 70%, and even more preferably greater than 80%, and the weight percent of glycolipids of the total oil composition is greater than 10%, more preferably greater than 20%, and even more preferably greater than about 25%. Furthermore, the weight percent of phospholipids as a weight percent of polar lipids is greater than 20%, more preferably greater than 30%, and even more preferably greater than 35%, and the weight percent of phospholipids of the total oil composition is greater than 20%, more preferably greater than 30%, and even more preferably greater than 40%. Without being bound to any particular theory, applicants believe that the combination of these properties, including the very low chlorophyll concentration in the oil product, produces an attractive, light color, almost clear to amber, or even darker amber color, in a preferred embodiment, almost clear to amber. Additionally, the concentration of EPA in the total oil product content is preferably at least 20% by weight, more preferably at least 25% by weight, and even more preferably at least about 30% by weight or more.Similarly, the omega-3 content in the oil product is at least 20% by weight, more preferably at least 25% by weight, and even more preferably at least 30%, 40% by weight or more are produced. Taken as a whole, the weight percent LC-PUFA content of the oil product is at least 20% by weight, at least 25% by weight, 30% by weight, 40% by weight, and 50% by weight.

[0062] Enhanced bioavailability of the oil produced was also achieved. Additionally, unlike some prior art methods of extracting and fractionating microalgae, the disclosed methods do not involve harsh chemical reactions, such as the use of strong mineral acids, which often destroy polar lipids and can significantly degrade other valuable fractions of the algal biomass.

[0063] Additionally, embodiments of the disclosed process result in a powder of non-lipid components, such as polysaccharides, in the crude ethanolic algal extract. See FIG.

[0064] It should also be noted that the LC-PUFA-containing high polar content oil, omega-3-containing high polar content oil, and EPA-containing high polar content oil of the present disclosure have a significantly reduced weight percentage of chlorophyll, such as: 1) chlorophyll a, 2) protochlorophyll a, and 3) methyl chlorophyllide. Preferably, the weight percentage of chlorophyll in the composition is less than 4% of the total weight percentage of the composition, preferably less than 3%, more preferably less than 2%, even more preferably less than 1%, even more preferably less than 0.5%, 0.5%, and even more preferably less than about 0.1%.

[0065] In another embodiment of the composition of the present disclosure, the composition has an increased weight percentage of carotenoids, e.g., carotene (alpha and beta), and zeaxanthin (yellow pigments), and several other components including canthaxanthin and zeaxanthin (red pigments), with the total carotenoids comprising greater than 0.5% of the total weight of the extract, preferably 1% or more.

[0066] In another alternative embodiment of the present disclosure, the oil composition with high bioavailable polar lipids and low chlorophyll content including EPA, which is an embodiment disclosed herein, can be provided as a formulation to which other useful ingredients are added. These other useful ingredients can be added alone or in combination with one or more other ingredients, such as other essential oils, dietary supplements, and dietary supplements. Specific examples include, but are not limited to: 1) other omega-3-containing oils or ingredients, such as DHA and EPA (e.g., in the form of neutral lipids extracted as a product of the present invention or supplied exogenously), lysolipids from the present invention, or ethyl esters of DHA or EPA supplied exogenously; 2) antioxidants, such as carotenoids (including the carotenoid fractions of the present invention), including astaxanthin, lutein, zeaxanthin, lycopene, carotene (alpha and beta), cryptoxanthin, and mixtures thereof; 3) vitamins, such as vitamins C and D; 4) cannabinoids, such as cannabidiol (CBD); and 5) other combinations. It is understood that such formulations containing some of these species with less colored and / or less viscous compositional profiles can reduce the overall color profile and viscosity of formulations containing fatty acid compositions with high polar lipids, high glycolipids, and low chlorophyll concentrations produced by embodiments of the methods disclosed herein. Thus, this can be accomplished, for example, by including non-polar lipids re-added from the original biomass stock or an external source, or by producing formulations that exhibit such attributes.

[0067] An example of a dietary supplement formulation comprising a blend of the polar EPA fractions described above with DHA (omega-3) may advantageously be in a ratio of 10-90 or 90-10, where the preferred level of the polar EPA formulation component is mixed at 20-50%. Applications of such formulations include use as a delivery system for other neutral lipids and ingredients to be formulated with the primary dietary / health supplement for cardiovascular health, mood, antidepressant, etc. This may be DHA, other neutral forms of EPA, or mixtures thereof. Astaxanthin at levels of 0.04%-10%, preferably 0.1%-2%, more preferably 0.2%-1%, may also be advantageously formulated with the pure polar EPA lipids or blends thereof with neutral EPA and / or EPA. Another ingredient that may be advantageously added to such formulations is coenzyme Q10 at levels of about 1-50%, preferably about 2-20%, relative to the polar EPA, either pure or in said formulations.

[0068] In addition to other attributes, formulation blends with added neutral lipids may be useful for targeting various viscosity levels, such as, for example, less than 50,000 mPa·s, preferably less than 10,000 mPa·s, more preferably less than about 2,000 mPa·s, and most preferably less than about 300 mPa·s.

[0069] In view of the above, it will be seen that the several objects and advantages of the disclosure are achieved and other advantageous results attained.

[0070] Since various modifications may be made to the above configurations without departing from the scope of the present disclosure, all matter contained in the above description or shown in the accompanying drawings should be interpreted as illustrative and not in a limiting sense.

Claims

1. A method for producing an oil composition with a low chlorophyll content and rich in polar lipids, comprising: a. obtaining algal biomass; b. extracting the algal biomass with a polar solvent to form a polar extract of algal lipids with a low water content; c. substantially removing the non-polar lipid fraction in the organic solvent layer by further extracting the polar extract with an organic solvent to separate the non-polar lipid fraction; d. separating a polar layer containing pigments and polar lipids; e. adding water to the polar layer and sequentially adding the organic solvent for further extraction to substantially remove the pigment fraction and form a water-polar layer containing the polar lipids; f. separating the polar lipid fraction from the water-polar layer by evaporation to separate the produced oil composition with a low chlorophyll content and rich in polar lipids.

2. The method according to claim 1, wherein the polar lipid fraction contains about 1% or less chlorophyll by weight.

3. The method according to claim 1, wherein the polar lipid fraction contains LC-PUFA.

4. The method according to claim 3, wherein the LC-PUFA contains EPA.

5. The method according to claim 3, wherein the LC-PUFA contains DHA.

6. The method according to claim 3, wherein the polar lipid fraction contains about 0.1% or less chlorophyll by weight.

7. The method according to claim 1, wherein the algal biomass is obtained from autotrophic algal biomass.

8. The method according to claim 7, wherein the autotrophic algal biomass is obtained from algae such as Nannochloropsis, Thraustochytrium, or Chlorella.

9. The method according to claim 1, which does not include the step of adding strong mineral acid.

10. A method for characterizing or purifying an algal biomass composition, comprising: a. obtaining algal biomass; b. extracting the algal biomass with a polar solvent to form a polar solvent extract of algal lipids with a low water content; c. substantially removing the non-polar lipid fraction in the organic solvent layer by further extracting the polar solvent extract with an organic solvent to separate the non-polar lipid fraction; d. separating a polar layer containing pigments and polar lipids; e. adding water to the polar layer and sequentially adding the organic solvent for further extraction to substantially remove the pigment fraction and form a water-polar solvent layer containing the polar lipids; f. separating a polar lipid fraction from the water-polar solvent layer by evaporation; A method comprising the above. **Claim 11** The method according to claim 10, wherein the algal biomass is an autotrophic algal biomass. **Claim 12** The method according to claim 11, wherein the autotrophic algal biomass is obtained from algae of Nannochloropsis, Tetraselmis, or Chlorella. **Claim 13** The method according to claim 10, wherein the characterized or purified components of the algal biomass include non-polar lipids, mid-polar lipids, chlorophyll components, carotenoid components, polar lipids, and non-lipid components (selected from sugars and non-lipid polar components), the amount of the crude extract, and the remaining dried residue. **Claim 14** The method according to claim 10, wherein at least 95% by weight of the original biomass is retained after the characterization step or the purification step. **Claim 15** The method according to claim 10, wherein at least 99% by weight of the original biomass is retained after the characterization step or the purification step.

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