Process for producing polyunsaturated fatty acids in ester form

Abstract

The present invention relates to an integrated process for the simultaneous production of polyunsaturated fatty acids, in particular in the form of esters, and palmites from algae. The compounds of the present invention are useful in the pharmaceutical industry (products for preventing cardiovascular diseases) and in the cosmetic industry (emollients and moisturizers). Specifically, the present invention relates to a process for obtaining eicosapentaenoic acid (EPA) esters and docosahexaenoic acid (DHA) esters from an algal material, comprising the steps of: a) treating the algal material with an extraction solvent to obtain a liquid phase containing said solvent and an algal oil and a solid phase containing cellulose, sugars and proteins; b) separating the solid phase from the liquid phase of step a); c) separating algal oil from the extraction solvent in the liquid phase of step b); d) subjecting the algae oil of step c) to a low temperature treatment to obtain a precipitate of saturated fatty acid triglycerides and monounsaturated fatty acid triglycerides and a liquid phase containing or consisting of polyunsaturated fatty acid triglycerides; e1) separation, segregation and purification of DHA triglyceride and EPA triglyceride from the liquid phase of step d), or e2) transesterification of DHA triglyceride and EPA triglyceride with ethanol and segregation of the respective DHA ethyl ester and EPA ethyl ester; f) optionally, isolating and purifying palmitic acid triglycerides from the phase containing saturated fatty acid triglycerides and monounsaturated fatty acid triglycerides of step d); g) optionally, the palmitic triglyceride of step f) is transesterified with a linear or branched C1-C4 alcohol to obtain a C1-C4 alkyl ester of palmitic acid and glycerol; h) optionally hydrogenating the glycerol of step g) to obtain isopropanol.

Description

Technical Field

[0001] The present invention relates to an integrated process for the simultaneous production of polyunsaturated fatty acids (particularly in the form of esters) and palmitate from algae. The compounds of the present invention can be used in the pharmaceutical industry (products for the prevention of cardiovascular diseases) and in the cosmetic industry (emollients and moisturizers). Background Art

[0002] Omega-3 polyunsaturated fatty acids (PUFA), mainly composed of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), are mainly produced by fisheries and, to a lesser extent, by krill fishing. Faced with the almost stagnant situation of fisheries (which does not ensure an adequate increase in the production of fish oils, and thus the extraction of PUFAs themselves), the use of PUFAs in the prevention of cardiovascular diseases has greatly increased, which in turn has increased the demand for these pharmaceutical products and led to the need to find new sources of supply. Such a source can be identified in some algae, where the PUFA content in algal bio-oil can even reach 15-20%.

[0003] Furthermore, the cultivation of oleaginous microalgae allows the conversion of CO2 into organic derivatives (such as sugars or fats) through chlorophyll photosynthesis, thus reducing the overall content of this greenhouse gas. Oleaginous microalgae are plant microorganisms that accumulate energy reserves from the sun in the form of vegetable oils through chlorophyll photosynthesis, with a higher energy efficiency than all other oleaginous plants in nature, accumulating up to 50% by mass of oil.

[0004] In fact, these microorganisms and animal organisms together create oil reserves that are thousands of years old. These microalgae can grow in places where edible plants cannot grow: in water (marine, fresh water), in worse arid and rocky soils, and even in deserts. Dry soil can also be used as a passive support for tanks for aquatic cultivation of microalgae, whereby the cultivation of microalgae does not harm normal agriculture, as happens with ethanol from wheat, rice, potatoes, etc. The bio-oil obtained from algal material is usually used to produce biodiesel.

[0005] Furthermore, the market demands the replacement of petrochemically derived active ingredients of the cosmetics industry with similar products of bio-origin. For example, alternatives to long-chain alkanes used as moisturizers and emollients are sought in creams containing products with similar bio-derived characteristics. Esters of palmitic acid with certain alcohols, derived from glycerol, can be effective substitutes for long-chain alkanes obtained by olefin oligomerization.

[0006] It is known from WO 2020 / 053118 of the present applicant to obtain bio-oil from algae via deep eutectic solvents (DES).

[0007] It is also known to extract PUFAs from algae by supercritical CO2.

[0008] A. Mendes et al. (DHA Concentration and Purification from the Marine Heterotrophic Microalga Crypthecodinium cohnii CCMP316 by Winterization and Urea Complexation, Food Technol. Biotechnol. 45(1)38-44(2007)) describe a method for obtaining DHA, which comprises the following steps: saponification and methylation of wet biomass, winterization and complexation with urea.

[0009] However, there are no known industrial processes that allow the simultaneous production of DHA and EPA in purified form from algae.

[0010] It is not known to obtain palmitic acid esters from algal materials that can be used in the cosmetic industry.

[0011] In other words, there is currently no method that can simultaneously utilize the fraction consisting of the unsaturated and saturated parts of fatty acid triglycerides (the saturated part can enhance both parts of algae oil) and make CO2 biofixation by algae more economically sustainable.

[0012] Therefore, there is a need to provide a process for producing DHA and EPA from algal material which is industrially applicable and economically advantageous and which preferably also allows obtaining palmitate which can be used in the cosmetics industry. Summary of the invention

[0013] Therefore, the present invention relates to:

[0014] 1) A method for obtaining eicosapentaenoic acid (EPA) esters and docosahexaenoic acid (DHA) esters from algae material, comprising the following steps:

[0015] a) treating the algae material with an extraction solvent to obtain a liquid phase containing the solvent and algae oil and a solid phase containing cellulose, sugars and proteins;

[0016] b) separating the solid phase from the liquid phase of step a);

[0017] c) separating the algae oil from the extraction solvent in the liquid phase of step b);

[0018] d) subjecting the algae oil of step c) to low temperature treatment to obtain a precipitate (precipitate) of saturated fatty acid triglycerides and monounsaturated fatty acid triglycerides and a liquid phase containing or consisting of polyunsaturated fatty acid triglycerides;

[0019] e1) separating, isolating and purifying DHA triglycerides and EPA triglycerides from the liquid phase of step d), or

[0020] e2) transesterifying DHA triglyceride and EPA triglyceride with ethanol and isolating the respective DHA ethyl ester and EPA ethyl ester;

[0021] f) optionally, isolating and purifying palmitic triglyceride from the phase containing saturated fatty acid triglycerides and monounsaturated fatty acid triglycerides of step d);

[0022] g) optionally, transesterifying the palmitic acid triglyceride of step f) with a linear or branched C1-C4 alcohol to obtain a C1-C4 alkyl ester of palmitic acid and glycerol;

[0023] h) Optionally, hydrogenating the glycerol of step g) to obtain isopropanol.

[0024] The present invention also relates to:

[0025] 2) The method as in point 1), wherein the algae material is selected from the group consisting of algae material from microalgae belonging to the genera Chlorella, Tetraselmis, Nannochloropsis and Scenedesmus, preferably microalgae from the genera Nannochloropsis and Scenedesmus;

[0026] 3) The method of point 1) or 2), wherein the extraction solvent is selected from a chloroform / methanol mixture (preferably in a ratio of 2:1), and DES (deep eutectic solvent), DES being the preferred extraction solvent;

[0027] 4) The method as in point 3), wherein the extraction solvent is a low melting point eutectic (DES type III) formed by an ammonium salt and a hydrogen bond donor, wherein the ammonium salt is selected from cholinium chloride, cholinium acetate, cholinium nitrate, betainium chloride, ethyl ammoniochloride, tetramethyl ammonium chloride, and the hydrogen bond donor is selected from carboxylic acid (e.g., acetic acid, formic acid, tartaric acid, oxalic acid, levulinic acid, citric acid or lactic acid), amine (e.g., urea, thiourea, 1,3-dimethylurea, 1,1-dimethylurea) or polyol (e.g., ethylene glycol, propylene glycol or glycerol) hydrogen bond, wherein the molar ratio of the ammonium salt to the hydrogen bond donor is preferably 1:1 to 1:3;

[0028] 5) The method as in point 4), wherein the DES consists of a mixture of choline chloride and lactic acid, or choline chloride and citric acid in a ratio of 1:3;

[0029] 6) The method according to any one of points 3) to 5), wherein step a) is carried out by treating the algae material with DES in an amount of 1:1 to 5:1 (preferably 1:1 to 2:1) relative to the weight of the algae material, stirring the dispersion at a temperature of 80°C to 130°C (preferably 90°C to 110°C) for 30 minutes to 5 hours (preferably 3 hours to 5 hours);

[0030] 7) The method according to any one of points 1) to 6), wherein, in step b), separation of the solid phase is carried out by filtration or centrifugation;

[0031] 8) The method according to any one of points 1) to 7), wherein, in step c), the separation of the algae oil from the extraction solvent is carried out by distillation or by treatment with a strong polar washing solvent that is immiscible with the algae oil but miscible with the extraction solvent;

[0032] 9) The method as in point 8), wherein the extraction solvent is DES, the washing solvent is water, and wherein the washing solvent is added to the algae oil solution in DES in a weight ratio of preferably 100 wt % to 300 wt %;

[0033] 10) The method as in point 9), wherein step c) comprises a further step c1) of recycling the extraction solvent in step a);

[0034] 11) The method according to any one of points 1) to 10), wherein step d) consists of "winterization", "wherein saturated fatty acid triglycerides and monounsaturated fatty acid triglycerides (solid phase) are crystallized at low temperature from a liquid phase containing polyunsaturated fatty acid triglycerides, wherein the algae oil obtained in step c) is treated at a temperature of 0°C to -30°C (preferably -2°C to -25°C), preferably in the presence of alcohol and / or urea, more preferably alcohol;

[0035] 12) The method as in point 11), wherein the alcohol is selected from linear or branched C1-C4 alcohols, and is preferably selected from ethanol, n-butanol and isopropanol, and wherein the weight ratio of the alcohol relative to the weight of the algae oil is preferably 13:1 to 25:1;

[0036] 13) The method according to any one of points 1) to 12), wherein step e1) is carried out by preparative HPLC;

[0037] 14) The method as in point 13), wherein step e1) is carried out by a reverse phase preparative HPLC system, using an acetonitrile-chloroform mixture with a gradient or a methanol-water mixture with a gradient as the mobile phase, and / or using a stationary phase consisting of a PMDVB (poly(m-divinylbenzene)) porous resin modified with 3,5-dinitrobenzoyl chloride (3,5-dinitrobenzoyl chloride) or with 2-chloroethanol, or a stationary phase consisting of silver(I)-mercaptopropyl (AgTCM);

[0038] 15) A process according to any of points 1) to 12), wherein alternative step e2) comprises converting the PUFA triglycerides (in particular DHA triglycerides and EPA triglycerides) present in the liquid phase into the corresponding ethyl esters by transesterification, wherein, when step d) is carried out in the presence of ethanol, the conversion is carried out by adding an acid catalyst to the liquid phase and heating to a temperature preferably of 70° C. to 90° C. until the reaction is complete, the acid catalyst being preferably selected from sulfuric acid, hydrochloric acid, an acidic resin or a zeolite;

[0039] 16) The method according to any one of points 1) to 15), wherein step f) of isolating palmitic acid triglyceride is carried out by preparative HPLC;

[0040] 17) A method as described in any one of points 1) to 16), wherein the step g) of transesterifying the palmitic acid triglyceride of step f) with a linear or branched C1-C4 alcohol to obtain a C1-C4 alkyl ester of palmitic acid and glycerol is carried out in the presence of an acid catalyst, using a C1-C4 alcohol as a solvent, and heating to a temperature of 50° C. to 100° C. (e.g., at the boiling point of the alcohol), wherein the acid catalyst is preferably selected from sulfuric acid, hydrochloric acid, an acidic resin or a zeolite;

[0041] 18) The method of point 17), wherein palmitic acid triglyceride is transesterified with isopropanol in the presence of zeolite as a catalyst at about 80° C. or under reflux to obtain isopropyl palmitate and glycerol;

[0042] 19) A process as described in any one of points 1) to 18), wherein the step h) of reducing the glycerol obtained in step g) to obtain isopropanol is carried out in two steps: h1) reducing with hydrogen and a copper-ruthenium-based catalyst supported on clay to obtain 1,2-propanediol, h2) reducing the 1,2-propanediol of step h1) with hydrogen and a copper-cerium-based catalyst to obtain a mixture of propan-1-ol and propan-2-ol, followed by separation of the propan-2-ol by distillation;

[0043] 20) A process as in point 19), wherein the isopropanol obtained in step h) is recycled in the transesterification step g) to obtain isopropyl palmitate.

[0044] These and any further objects will be described in the following description as outlined in the accompanying claims.

[0045] Other characteristics and advantages of the invention will become apparent from the following description of a preferred embodiment given by way of non-limiting example. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 A block diagram illustrating the method of the present invention in a preferred embodiment is shown. DETAILED DESCRIPTION

[0047] The present invention relates to a method for obtaining eicosapentaenoic acid (EPA) esters and docosahexaenoic acid (DHA) esters from algae material, comprising the following steps:

[0048] a) treating the algae material with an extraction solvent to obtain a liquid phase containing the solvent and algae oil and a solid phase containing cellulose, sugars and proteins;

[0049] b) separating the solid phase from the liquid phase of step a);

[0050] c) separating the algae oil from the extraction solvent in the liquid phase of step b);

[0051] d) subjecting the algae oil of step c) to low temperature treatment to obtain a precipitate of saturated fatty acid triglycerides and monounsaturated fatty acid triglycerides and a liquid phase containing or consisting of polyunsaturated fatty acid triglycerides;

[0052] e1) separating, isolating and purifying DHA triglycerides and EPA triglycerides from the liquid phase of step d), or

[0053] e2) transesterifying DHA triglyceride and EPA triglyceride with ethanol and separating the respective DHA ethyl ester and EPA ethyl ester;

[0054] f) optionally, isolating and purifying palmitic triglyceride from the phase containing saturated fatty acid triglycerides and monounsaturated fatty acid triglycerides of step d);

[0055] g) optionally, transesterifying the palmitic acid triglyceride of step f) with a linear or branched C1-C4 alcohol to obtain a C1-C4 alkyl ester of palmitic acid and glycerol;

[0056] h) Optionally, hydrogenating the glycerol of step g) to obtain isopropanol.

[0057] The algae material used for the purposes of the present invention may be of various origins. Preferably, algae material from microalgae belonging to the genera Chlorella, Platymonas, Nannochloropsis and Scenedesmus will be used. Microalgae of the genera Nannochloropsis and Scenedesmus are preferred.

[0058] In the method of the present invention, wet algae biomass can be used, which is obtained by, for example, separation from an algae suspension in water or an aqueous solution (e.g., seawater) by, for example, microfiltration or centrifugation, or dry algae biomass, which is obtained from the aforementioned method by a further drying step. Wet algae biomass can be treated with DES according to the method of the present invention, and relative to the gross weight of the wet biomass, the wet algae biomass preferably contains up to 90% by weight, more preferably more than 5% by weight to up to 70% by weight of water. On the contrary, dry algae biomass can contain up to 5% by weight, preferably up to 3% by weight of residual moisture.

[0059] The algae material in step a) is finely ground and contacted with an extraction solvent. The extraction solvent can be any solvent capable of extracting bio-oil from the algae material. For example, a chloroform / methanol mixture in a ratio of, for example, 2:1 can be used.

[0060] Preferably, the extraction solvent is a solvent miscible in water.

[0061] In a preferred embodiment, the extraction solvent is a DES (deep eutectic solvent). The term DES generally refers to a mixture of two or more components, at least one donor and one hydrogen bond acceptor, which are combined to form a eutectic mixture, for example, by hydrogen bonding or even van der Waals interactions, and whose melting temperature is much lower than the melting temperatures of its components. They are widely used, economical, environmentally friendly and biodegradable. WO2020 / 053118 discloses the use of DES for extracting bio-oil from algae.

[0062] Low melting point eutectics (deep eutectic solvent (DES) type III) are conveniently used, which are formed by an ammonium salt (e.g., choline chloride, choline acetate, choline nitrate, betaine chloride, ethylammonium chloride, tetramethylammonium chloride) and a hydrogen bond donor selected from a carboxylic acid (e.g., acetic acid, formic acid, tartaric acid, oxalic acid, levulinic acid, citric acid or lactic acid) or an amine (e.g., urea, thiourea, 1,3-dimethylurea, 1,1-dimethylurea) or a polyol (e.g., ethylene glycol, propylene glycol or glycerol). The molar ratio of the ammonium salt to the hydrogen bond donor is preferably 1:1 to 1:3.

[0063] In a particularly preferred embodiment, the DES consists of a mixture of choline chloride and lactic acid, or choline chloride and citric acid in a ratio of 1:3.

[0064] DES is obtained by mixing a quaternary ammonium salt and a hydrogen bond donor together in a molar ratio of 1:1 to 1:3, and heating the mixture thus obtained at a temperature of 80-100° C. (e.g., for about 30 minutes) to obtain a transparent colorless liquid, which remains transparent and colorless once the DES reaches a temperature close to room temperature.

[0065] In this preferred embodiment, step a) is carried out by treating the algae material with DES in an amount of 1:1 to 5:1 (preferably 1:1 to 2:1) relative to the weight of the algae material, stirring the dispersion at a temperature of 80°C to 130°C (preferably 90°C to 110°C) for 30 minutes to 5 hours (preferably 3 hours to 5 hours).

[0066] In step b), separation of the solid phase can be carried out by filtration or centrifugation. The cellulose contained in the solid phase can be recovered and used, for example, as a precursor for industrial cellulose derivatives (e.g. nitrocellulose or cellulose acetate), or for obtaining cellulose-based end products (e.g. filters or paper materials).

[0067] In step c), separation of the algae oil from the extraction solvent can be performed by known methods, such as distillation or treatment with a strong polar washing solvent that is immiscible with the algae oil but miscible with the extraction solvent.

[0068] In a preferred embodiment, when the extraction solvent is DES, the washing solvent is water. The washing solvent is added to the bio-oil solution in the DES in a weight ratio of preferably 100 wt % to 300 wt %, and the extraction is preferably carried out by stirring at room temperature. The upper organic phase consisting of the bio-oil is then separated.

[0069] In certain embodiments, when the extraction solvent is DES, step c) comprises a further step c1) of recycling the extraction solvent in step a). Such step c1) comprises separating the extraction solvent from the washing solvent, for example by evaporation or distillation.

[0070] Step d), also called "winterization", consists of low-temperature fractional crystallization for separating triglycerides of saturated and monounsaturated fatty acids (solid phase) from triglycerides of polyunsaturated fatty acids (liquid phase). The algae oil obtained in step c) is treated at a temperature of 0°C to -30°C (preferably -2°C to -25°C), preferably in the presence of alcohol and / or urea, more preferably alcohol. The alcohol is preferably chosen from linear or branched C1-C4 alcohols, more preferably from ethanol, n-butanol and isopropanol. The weight ratio of alcohol relative to the weight of the algae oil is preferably from 13:1 to 25:1.

[0071] Step e1) can be preferably carried out by preparative HPLC. For example, a reverse phase preparative HPLC system can be used, using a gradient acetonitrile-chloroform mixture or a gradient methanol-water mixture as a mobile phase. In certain embodiments, a stationary phase (WK Zhang et al. April 2001) composed of a porous resin PMDVB (poly-m-divinylbenzene) modified with 3,5-dinitrobenzoyl chloride or modified with 2-chloroethanol can be used, or a stationary phase composed of silver (I)-mercaptopropyl, AgTCM (JT Billon et al., J. of Chromatography A 1312, August 2013). Preparative HPLC systems suitable for the present invention are described, for example, in Oh et al., Appl. Biol. Chem., (2020), 63: 56, or MP Mansour, J. of Chromatography A, 1097 (2005) 54-58.

[0072] Alternative step e2) instead includes converting the triglycerides of PUFA (particularly DHA triglycerides and EPA triglycerides) present in the liquid phase into the corresponding ethyl esters by transesterification. When step d) is carried out in the presence of ethanol, this conversion can be advantageously carried out by adding an acid catalyst (e.g., sulfuric acid, hydrochloric acid, acidic resin or zeolite) to the liquid phase and heating at a temperature preferably of 70°C to 90°C until the reaction is complete. The reaction and gas chromatography analysis can be carried out as described in, for example, Oh et al., Appl.Biol.Chem., (2020), 63:56.

[0073] DHA ethyl ester and EPA ethyl ester can then be separated and purified by preparative HPLC as described in step e1) above.

[0074] In certain embodiments, if desired, PUFA in the form of free fatty acids can be obtained by saponifying the obtained PUFA esters with alkali metal hydroxides or alkaline earth metal hydroxides, for example, by treating with aqueous NaOH or KOH. By neutralizing the sodium or potassium soap thus obtained, the corresponding fatty acids can be obtained in free form.

[0075] Step f) of isolating the palmitic triglycerides is preferably performed by preparative HPLC, for example using one of the systems described in steps e1) and e2).

[0076] Step f) is performed when the method of the invention provides for the production of the above-mentioned palmitate from algae with simultaneous production of polyunsaturated fatty acids, in particular in the form of esters (simultaneous production).

[0077] The separation conditions and the selection of the stationary phase, the mobile phase and the gradient thereof in steps e1), e2) and f) can be selected by a person skilled in the art according to conventional "trial and error" methods.

[0078] Step g) of transesterifying the palmitic acid triglyceride of step f) with a linear or branched C1-C4 alcohol to obtain a C1-C4 alkyl ester of palmitic acid and glycerol is carried out in the presence of an acid catalyst, using a C1-C4 alcohol as a solvent, and heating to a temperature of 50° C. to 100° C. (e.g., at the boiling point of the alcohol).

[0079] The acid catalyst may be sulfuric acid, hydrochloric acid, an acidic resin or a zeolite.

[0080] In a preferred embodiment, palmitic acid triglyceride is transesterified with isopropanol to obtain isopropyl palmitate and glycerol. This reaction is preferably carried out in the presence of zeolite as a catalyst at about 80° C. (boiling point of isopropanol) or under reflux.

[0081] The isopropyl palmitate obtained in step g) can be purified by distillation to obtain cosmetic grade.

[0082] The step h) of reducing the glycerol obtained in step g) to obtain isopropanol is preferably carried out in two steps: h1) reduction with hydrogen and a copper-ruthenium-based catalyst supported on clay to obtain 1,2-propanediol (a method described in Green Chemistry 2009, 11, 1000-1006) and h2) reduction of the 1,2-propanediol of step h1) with hydrogen and a copper-cerium-based catalyst (a method described in Green Chemistry 2016, 18, 782-791) to obtain a mixture of propan-1-ol and propan-2-ol, followed by separation of the propan-2-ol by distillation;

[0083] If isopropyl palmitate is to be obtained, the isopropanol obtained in step h) can be used directly in the transesterification step g).

[0084] It is also possible to use in step g) only a part or an aliquot of the total amount of isopropanol obtained in step h) and to use the remainder to 100% of the isopropanol obtained as a gasoline additive known in the gasoline additive field.

[0085] In one embodiment of the process according to the invention, the triglycerides of saturated and monounsaturated fatty acids obtained in step d) in the form of a precipitate are fed to step f) and the subsequent optional steps g), h) only as a part relative to the total amount of the precipitate obtained in step d), for example in the case where the market demand for isopropyl palmitate is very low and high yields of PUFA are required, thus resulting in a reduced yield of isopropyl palmitate alone from the process according to the invention.

[0086] In this case, the remainder of the triglycerides of saturated and monounsaturated fatty acids to 100% (which are not subjected to step f)) can optionally be stored for subsequent use or can be subjected as feed to another process downstream of the present process, for example a process for obtaining biodiesel by hydrodeoxygenation and hydroisomerization of said feed.

[0087] Examples of methods for obtaining biodiesel from renewable feeds are eco-refinery methods as described in patent applications WO2008113492, EP2134817A1, WO2015 / 181744, WO2020053118A1, which are incorporated herein by reference in their entirety.

[0088] In the eco-refining process, the feed of a mixture of triglycerides containing saturated fatty acids and monounsaturated fatty acids (i.e. the remainder relative to the portion subjected to step f)) can undergo a first catalytic hydrodeoxygenation reaction, for example using mixed sulfides of cobalt and molybdenum supported on alumina, or Ni-Mo, Ni-W, Co-W as catalyst; in this step, the triglycerides are converted into alkanes and water, while the glycerol present in the triglycerides is hydrogenated into propane.

[0089] The catalytic hydrodeoxygenation reaction can be carried out by using hydrogen at a pressure of 25 bar to 75 bar (preferably 30 bar to 50 bar); the temperature of the catalytic hydrodeoxygenation reaction can be 240°C to 450°C (preferably 270°C to 430°C). Preferably, in the reaction, the LHSV is 0.5h -1 Up to 2h -1 , more preferably 0.5h -1 Up to 1h -1 .H 2 / The feed rate is preferably 400 Nl / l to 2000 Nl / l.

[0090] Subsequently, in the second step of the eco-refining method, the alkanes obtained from the above hydrodeoxygenation reaction are hydroisomerized using an acidic solid catalyst in the presence of hydrogen, thereby obtaining branched alkanes that can be used as a diesel fuel component with renewable origin.

[0091] An example of an acidic solid catalyst that can be used in the hydroisomerization step is a catalytic system comprising a metal component containing one or more Group VIII metals, optionally mixed with one or more Group VIB metals, and an acidic support (i.e., carrier) comprising completely amorphous microporous-mesoporous silica alumina, preferably having a SiO2 / Al2O3 molar ratio of 30 to 500, greater than 500 m 2 / g surface area, 0.3 mL / g to 1.3 mL / g pore volume, and an average pore size of less than 40 angstroms, such as Pt / MSA, as described in EP2134817A1, which is incorporated herein by reference in its entirety.

[0092] The hydroisomerization can be carried out at a temperature of 250°C to 450°C (preferably 280°C to 380°C); the operating pressure can be 25 bar to 70 bar (preferably 30 bar to 50 bar). Preferably, in the reaction, the LHSV is 0.5h -1 Up to 2h -1 The ratio of H2 / HC may preferably be 200 Nl / l to 1000 Nl / l.

[0093] The amount of the proportion of the precipitate obtained in step d) which is not subjected to step f) does not limit the scope of the present invention and it can vary in the range of 0.5% to 90% (preferably 10% to 50%) relative to the total amount of the precipitate obtained in step d), and it can also be up to 100% (i.e. no aliquot is subjected to step f)) and is therefore not outside the scope of the present invention.

[0094] Figure 1 A block diagram of the apparatus and method of the present invention according to a preferred embodiment is shown, wherein:

[0095] 1 indicates mechanical pretreatment of the bio-oil for extraction with DES (e.g. by screw);

[0096] 2 represents the phase consisting of bio-oil + DES and any water;

[0097] 3 represents the phase consisting of cellulose + sugars and proteins (residue of extraction with DES);

[0098] 4 represents the DES / bio-oil hydrolysis and separation reactor with water;

[0099] 5 represents the phase composed of bio-oil;

[0100] 6 represents the selective separation process between the saturated fatty acid phase and the polyunsaturated fatty acid phase (winterization);

[0101] 7 represents an evaporator of a DES+water mixture;

[0102] 8 represents a phase composed of saturated fatty acid triglycerides;

[0103] 9 represents purification of saturated fatty acids to obtain palmitic acid triglycerides (preparative HPLC);

[0104] 10 represents the phase composed of saturated fatty acid triglycerides;

[0105] 11 represents separation and purification of PUFA triglycerides from a mixture of unsaturated fatty acid triglycerides;

[0106] 12 represents a transesterification reactor between saturated fatty acid triglycerides (e.g. palmitic acid) and an alcohol (e.g. isopropanol) from the hydrogenation process of reactor 15;

[0107] 13 represents a separation and purification unit for the produced ester (e.g. isopropyl palmitate);

[0108] 14 represents the phase composed of glycerol from the transesterification reactor;

[0109] 15 represents a catalytic hydrogenation reactor for glycerol to alcohol (isopropanol);

[0110] 16 represents the phase consisting of alcohol (isopropanol) from the hydrogenation reactor;

[0111] 17 represents a unit for distilling and separating alcohol (isopropyl alcohol) from water and by-products.

[0112] Experimental Examples

[0113] Example 1 Extraction of bio-oil by mechanical grinding

[0114] 4 g of Nannochloropsis sp. CB-L2 were treated together with 6 g of DES consisting of choline chloride and lactic acid (weight ratio 1:3) in a mechanical grinder at a temperature of 100° C. for 210 minutes.

[0115] A fluid consisting of DES + bio-oil and a solid residue consisting of oil-poor algae material was obtained.

[0116] The DES+bio-oil fluid was placed in a glass reactor together with 50 g of distilled water, stirred for 10 minutes, and the oil phase supernatant was separated from the water phase.

[0117] The oil phase was separated and 840 g of algae oil were obtained (yield equal to 74% of theoretical yield).

[0118] Example 2 Extraction of bio-oil by mechanical grinding

[0119] 4 g of Nannochloropsis sp. CB-L2 were treated together with 6 g of DES consisting of choline chloride and citric acid (weight ratio 1:3) in a mechanical grinder at a temperature of 100° C. for 210 minutes.

[0120] A fluid consisting of DES + bio-oil and a solid residue consisting of oil-poor algae material was obtained.

[0121] The DES+oil fluid was placed in a glass reactor together with 50 g of distilled water, stirred for 10 minutes, and the oil phase supernatant was separated from the water phase.

[0122] The oil phase was separated and 744 g of algae oil were obtained (yield equal to 74% of theoretical yield).

[0123] Example 3 Extraction of bio-oil by mechanical grinding

[0124] 4 g of Scenedesmus SD-L1 were treated together with 6 g of DES consisting of choline chloride and lactic acid (weight ratio 1:3) in a mechanical grinder at a temperature of 100° C. for 210 minutes.

[0125] A fluid consisting of DES + bio-oil and a solid residue consisting of oil-poor algae material was obtained.

[0126] The DES+oil fluid was placed in a glass reactor together with 50 g of distilled water, stirred for 10 minutes, and the oil phase supernatant was separated from the water phase.

[0127] The oil phase was separated and 480 g of algae oil were obtained (yield equal to 100% of the theoretical yield).

[0128] Example 4 Extraction of bio-oil by mechanical grinding

[0129] 4 g of Scenedesmus SD-L1 were treated together with 6 g of DES consisting of choline chloride and citric acid (weight ratio 1:3) in a mechanical grinder at a temperature of 100° C. for 210 minutes.

[0130] A fluid consisting of DES + bio-oil and a solid residue consisting of oil-poor algae material was obtained.

[0131] The DES+oil fluid was placed in a glass reactor together with 50 g of distilled water, stirred for 10 minutes, and the oil phase supernatant was separated from the water phase.

[0132] The oil phase was separated and 345 g of algae oil were obtained (yield equal to 76% of theoretical yield).

[0133] Example 5 Isolation of PUFA-rich fraction

[0134] 440 mg of the algae bio-oil prepared as described in Example 1 was placed in a 50 cc glass flask, and then 6.8 g of anhydrous ethanol was added, followed by stirring to obtain a homogeneous solution.

[0135] The solution was placed in a refrigerator at -5°C for cooling and left to stand overnight (about 12 hours). In the morning of the next day, a solid precipitate was formed, which was cold filtered and consisted of a portion rich in saturated fatty acids and monounsaturated fatty acids, which was then dried and a weight of 100 mg (21.9% of the total amount) was obtained. The clarified solution was treated by evaporating ethanol under vacuum and a portion rich in PUFAs with a total weight of 334 mg (73.4%) was obtained. The relative composition was determined by H-NMR.

[0136] Example 6 Isolation of PUFA-rich fraction

[0137] 463 mg of the algae bio-oil prepared as described in Example 1 was placed in a 50 cc glass flask, and then 6.8 g of anhydrous ethanol was added, followed by stirring to obtain a homogeneous solution.

[0138] The solution was placed in a refrigerator at -20°C for cooling and left to stand overnight (about 12 hours). In the morning of the next day, a solid precipitate was formed, which was cold filtered and consisted of a portion rich in saturated fatty acids and monounsaturated fatty acids, which was then dried and a weight of 133 mg (29.3% of the total amount) was obtained. The clarified solution was treated by evaporating ethanol under vacuum and a portion rich in PUFAs with a total weight of 330 mg (72.8%) was obtained. The relative composition was determined by H-NMR.

[0139] Example 7 Isolation of PUFA-rich fraction

[0140] 499 mg of the algae bio-oil prepared as described in Example 1 was placed in a 50 cc glass flask, and then 7.5 g of anhydrous isopropanol was added, followed by stirring to obtain a homogeneous solution.

[0141] The solution was placed in a refrigerator at -5°C for cooling and left to stand overnight (about 12 hours). In the morning of the next day, a solid precipitate was formed, which was cold filtered and consisted of a portion rich in saturated fatty acids and monounsaturated fatty acids, which was then dried and a weight of 161 mg (32.2% of the total amount) was obtained. The clarified solution was treated by evaporating ethanol under vacuum and a portion rich in PUFAs with a total weight of 345 mg (69.1%) was obtained. The relative composition was determined by H-NMR.

[0142] Example 8 Isolation of PUFA-rich fraction

[0143] 374 mg of the algae bio-oil prepared as described in Example 1 was placed in a 50 cc glass flask, and then 7.5 g of anhydrous n-butanol was added, followed by stirring to obtain a homogeneous solution.

[0144] The solution was placed in a refrigerator at -5°C for cooling and left to stand overnight (about 12 hours). In the morning of the next day, a solid precipitate was formed, which was cold filtered and consisted of a portion rich in saturated fatty acids and monounsaturated fatty acids, which was then dried and a weight of 55 mg (15% of the total amount) was obtained. The clarified solution was treated by evaporating n-butanol under vacuum and a portion rich in PUFAs with a total weight of 319 mg (85%) was obtained. The relative composition was determined by H-NMR.

[0145] Example 9 Synthesis of Isopropyl Palmitate

[0146] In a 250 mL bubbling reflux flask, 25.6 g of palmitic acid (0.1 mol) (which was obtained by purifying the saturated portion of the precipitate as described in Example 5) was charged, and 30.1 g of 2-propanol (0.5 mol) was added thereto.

[0147] 2-Propanol is obtained by catalytically hydrogenating glycerol over copper-ruthenium supported on clay [as described in Green Chemistry 2009, 11, 1000-1006] to obtain 1,2-propanediol, followed by catalytically hydrogenating 1,2-propanediol with a copper-cerium catalyst to a mixture of 1-propanol and 2-propanol [as described in Green Chemistry 2016, 18, 782-791], and separating 2-propanol from the mixture of 1-propanol and 2-propanol by distillation.

[0148] 8 g of Zerolit 225 / H dried and cooled at 100°C was added to the mixture of palmitic acid and 2-propanol. + The exchange resin acts as an acid catalyst. The suspension is stirred and heated, refluxed for 4 hours, then cooled and filtered to separate the exchange resin.

[0149] The solution thus obtained was treated with a rotary evaporator to remove unreacted 2-propanol, and the residue thus obtained was then distilled by distilling isopropyl palmitate (T.eb 162°C).

[0150] 25.37 g of pure isopropyl palmitate were obtained (yield 85% of theoretical yield).

[0151] As can be clearly seen from the above, the method according to the present invention achieves the intended purpose. In fact, not only can high-purity PUFAs (especially DHA and EPA in the form of esters, which can be used to prepare drugs, supplements or foods for special medical purposes (FSMP) for cardioprotective purposes) be effectively isolated, but also palmitic acid esters (especially isopropyl palmitate, which is used as an emollient or moisturizer in the cosmetic industry) can be obtained at the same time.

[0152] The possibility of using glycerol obtained from the transesterification of palmitic triglycerides, reducing it to isopropanol also allows an optimization of energy and raw materials and a reduction of waste.

Claims

1. A method for obtaining eicosapentaenoic acid (EPA) esters and docosahexaenoic acid (DHA) esters from algae material, comprising the following steps: a) treating the algae material with an extraction solvent to obtain a liquid phase containing the solvent and algae oil and a solid phase containing cellulose, sugars and proteins; b) separating the solid phase from the liquid phase of step a); c) separating the algae oil from the extraction solvent in the liquid phase of step b); d) subjecting the algae oil of step c) to low temperature treatment to obtain a precipitate of saturated fatty acid triglycerides and monounsaturated fatty acid triglycerides and a liquid phase containing or consisting of polyunsaturated fatty acid triglycerides; e1) separating, isolating and purifying DHA triglycerides and EPA triglycerides from the liquid phase of step d), or e2) transesterifying DHA triglyceride and EPA triglyceride with ethanol and isolating the respective DHA ethyl ester and EPA ethyl ester; f) optionally, isolating and purifying palmitic triglyceride from the phase containing saturated fatty acid triglycerides and monounsaturated fatty acid triglycerides of step d); g) optionally, transesterifying the palmitic acid triglyceride of step f) with a linear or branched C1-C4 alcohol to obtain a C1-C4 alkyl ester of palmitic acid and glycerol; h) Optionally, hydrogenating the glycerol of step g) to obtain isopropanol.

2. The method according to claim 1, wherein the algae material is selected from the group consisting of algae material of microalgae belonging to the genera Chlorella, Platymonas, Nannochloropsis and Scenedesmus, preferably microalgae of the genera Nannochloropsis and Scenedesmus.

3. The method according to claim 1 or 2, wherein the extraction solvent is selected from a chloroform / methanol mixture and DES (deep eutectic solvent), the chloroform / methanol mixture preferably has a ratio of 2:1, and DES is the preferred extraction solvent.

4. The method according to claim 3, wherein the extraction solvent is a low melting point eutectic (DES type III) formed by an ammonium salt and a hydrogen bond donor, wherein the ammonium salt is selected from choline chloride, choline acetate, choline nitrate, betaine chloride, ethylammonium chloride, tetramethylammonium chloride, and the hydrogen bond donor is selected from carboxylic acids such as acetic acid, formic acid, tartaric acid, oxalic acid, levulinic acid, citric acid, or lactic acid or an amine such as urea, thiourea, 1,3-dimethylurea, 1,1-dimethylurea, or a polyol such as ethylene glycol, propylene glycol or glycerol hydrogen bond, and wherein the molar ratio of the ammonium salt to the hydrogen bond donor is preferably 1:1 to 1:

3.

5. The method according to claim 4, wherein the DES consists of a mixture of choline chloride and lactic acid, or choline chloride and citric acid in a ratio of 1:

3.

6. The method according to any one of claims 3 to 5, wherein step a) is carried out by treating the algae material with DES in an amount of 1:1 to 5:1, preferably 1:1 to 2:1, relative to the weight of the algae material, stirring the dispersion at a temperature of 80°C to 130°C, preferably 90°C to 110°C, for 30 minutes to 5 hours, preferably 3 hours to 5 hours.

7. The method according to any one of claims 1 to 6, wherein: In step b), separation of the solid phase is performed by filtration or centrifugation.

8. The method according to any one of claims 1 to 7, wherein: In step c), separation of the algae oil from the extraction solvent is performed by distillation or by treatment with a strong polar washing solvent that is immiscible with the algae oil but miscible with the extraction solvent.

9. The method according to claim 8, wherein the extraction solvent is DES, the washing solvent is water, and wherein the washing solvent is added to the algae oil solution in DES in a weight ratio of preferably 100 wt% to 300 wt%.

10. The process according to claim 9, wherein step c) comprises a further step c1) of recycling the extraction solvent in step a).

11. The method according to any one of claims 1 to 10, wherein step d) consists of "winterization" by low-temperature fractional crystallization of saturated fatty acid triglycerides and monounsaturated fatty acid triglycerides (solid phase) from a liquid phase containing polyunsaturated fatty acid triglycerides, wherein the algae oil obtained in step c) is treated at a temperature of 0°C to -30°C, preferably at a temperature of -2°C to -25°C, preferably in the presence of alcohol and / or urea, more preferably in the presence of alcohol.

12. The method according to claim 11, wherein the alcohol is selected from linear or branched C1-C4 alcohols, and is preferably selected from ethanol, n-butanol and isopropanol, and wherein the weight ratio of the alcohol relative to the weight of the algae oil is preferably 13:1 to 25:

1.

13. The process according to any one of claims 1 to 12, wherein step e1) is performed by preparative HPLC.

14. method according to claim 13, wherein step e1) is carried out by reverse phase preparative HPLC system, uses acetonitrile-chloroform mixture with gradient or methanol-water mixture with gradient as mobile phase, and / or uses the stationary phase consisting of PMDVB (poly-m-divinylbenzene) porous resin modified with 3,5-dinitrobenzoyl chloride or with 2-chloroethanol, or the stationary phase consisting of silver (I)-mercaptopropyl (AgTCM).

15. The process according to any one of claims 1 to 12, wherein alternative step e2) comprises converting the PUFA triglycerides present in the liquid phase, in particular DHA triglycerides and EPA triglycerides, into the corresponding ethyl esters by transesterification, wherein When step d) is carried out in the presence of ethanol, the conversion is carried out by adding an acid catalyst preferably selected from sulfuric acid, hydrochloric acid, acidic resins or zeolites to the liquid phase and heating to a temperature preferably from 70°C to 90°C until the reaction is complete.

16. The process according to any one of claims 1 to 15, wherein step f) of isolating palmitic triglycerides is preferably performed by preparative HPLC.

17. The method according to any one of claims 1 to 16, wherein the step g) of transesterifying the palmitic acid triglyceride of step f) with a linear or branched C1-C4 alcohol to obtain a C1-C4 alkyl ester of palmitic acid and glycerol is carried out in the presence of an acid catalyst, using a C1-C4 alcohol as a solvent, and heating to a temperature of 50°C to 100°C, for example at the boiling point of the alcohol, and the acid catalyst is preferably selected from sulfuric acid, hydrochloric acid, an acidic resin or a zeolite.

18. The process according to claim 17, wherein palmitic acid triglyceride is transesterified with isopropyl alcohol in the presence of zeolite as a catalyst at about 80°C or under reflux to obtain isopropyl palmitate and glycerol.

19. The process according to any one of claims 1 to 18, wherein the step h) of reducing the glycerol obtained in step g) to obtain isopropanol is carried out in two steps: h1) reduction with hydrogen and a copper-ruthenium based catalyst supported on clay to obtain 1,2-propanediol, and h2) reduction of the 1,2-propanediol of step h1) with hydrogen and a copper-cerium based catalyst to obtain a mixture of propan-1-ol and propan-2-ol, followed by separation of the propan-2-ol by distillation.

20. The process according to claim 19, wherein the isopropanol obtained in step h) is recycled in the transesterification step g) to obtain isopropyl palmitate.

21. The process according to any one of the preceding claims 16 to 20, wherein when step f) is carried out, only a portion of the total amount of saturated fatty acid triglycerides and monounsaturated fatty acid triglycerides obtained in the form of a precipitate in step d) is subjected to step f) and the subsequent optional steps g), h), The remainder to 100% of the saturated fatty acid triglycerides and monounsaturated fatty acid triglycerides are optionally used as feedstock, and a process for obtaining biodiesel is carried out by hydrodeoxygenation and hydroisomerization of the feedstock, preferably an eco-refining process.

22. An integrated process for the simultaneous production of eicosapentaenoic acid (EPA) esters and docosahexaenoic acid (DHA) esters, palmitate and biodiesel from algae material, comprising: - A method as claimed in any one of the preceding claims 1 to 21, wherein only a portion of the saturated fatty acid triglycerides and monounsaturated fatty acid triglycerides obtained in step d) is subjected to step f); followed by the subsequent - catalytic hydrodeoxygenation and hydroisomerization of the remainder up to 100% of the saturated fatty acid triglycerides and monounsaturated fatty acid triglycerides obtained in step d).

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