JP2021521882A - A novel process for providing eicosapentaenoic acid oil compositions - Google Patents

Abstract

A method for producing an oil composition having 20-70% eicosapentaenoic acid, measured as a weight percent, is provided by using a simple technique and minimizing the refining process.
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Description

The present invention provides an eicosapentaenoic acid (EPA) oil composition comprising approximately 20-70% eicosapentaenoic acid (EPA) in natural or synthetic form. It relates to the field of industrial processes of algal preparations derived from Oculata (nannochloropsis). Such oil compositions are useful for many purposes, including food supplements or cosmetics.

Eicosapentaenoic acid (EPA) is a fatty acid that contains all 20 carbon atoms and 5 double bonds in a cis configuration. The double bond is located at positions 5, 8, 11, 14 and 17, and the official chemical name is all cis-5, 8, 11, 14, 17-eicosapentaenoic acid. A common abbreviation is EPA.

The human body synthesizes it from the essential fatty acid α-linolenic acid. As a result, supplementation of EPA levels is often required. This can be achieved by receiving EPAs directly from food sources such as oily fish, or by dietary supplements containing oil compositions containing EPAs.

EPAs are available from natural sources in the form of phospholipids, triglycerides, diglycerides and monoglycerides, amides, esters, or many other different types, salts and / or other compounds. In each case, the EPA moiety can usually be separated from the complex molecule into a free acid form and then optionally reattached to another complex molecule. This makes it possible to provide a synthetic form of EPA.

Dietary supplements containing oil compositions containing EPA may be provided from natural sources such as fish, krill or algae.

Fish and krill are used to obtain such oil compositions. However, microalgae have been recognized as a promising alternative source for lipid production. Some species of microalgae can be induced to produce specific lipids and fatty acids by relatively simple manipulation of the physical and chemical properties of their culture medium. Microalgaes can produce and accumulate significant amounts of lipids (about 20-50% of dry weight). The accumulation of lipids in microalgae results from the consumption of sugars at a rate faster than the rate of cell formation, facilitating the conversion of excess sugars to lipids.

In addition, algae are sustainable of biomass, oil, and other components as a result of combining many advantages, including their fast growth rate, carbon dioxide consumption, non-competitiveness with agriculture, and underwater growth. Considered to be the source.

Among the many algae species used to obtain fatty acids, especially EPAs, N. Oculata is considered an advantageous species as it produces more EPA, especially docosahexaenoic acid (DHA). In particular, N. More than two-thirds of the fatty acids produced by Oculata consist of EPA, palmitic acid and palmitoleic acid.

There are many methods available to provide an oil composition comprising EPA in one of the forms described above. These methods are a combination of two main parts: oil extraction from algae (usually oil extraction with the help of solvents) and methods such as degumming, caustic neutralization, bleaching, deodorization, dewaxing, etc. Including purification of. In contrast to fish oil, algal oil contains a significant amount of chlorophyll, the removal of which is an important step associated with the provision of EPA oil compositions suitable for use in food supplements, cosmetics and the like. Other techniques such as silica chromatography or the formation of urea adducts can be further utilized to provide the final product with enhanced EPA content.

WO 20141055576 discloses methods for providing highly bioavailable EPA formulations. In those formulations, EPA is available as a free fatty acid or glycolipid, or a phospholipid conjugate. As disclosed herein, an algae paste to be used for extraction with an organic solvent is provided. After standard procedures to remove non-lipid organics, triglycerides, including free fatty acids, diglycerides and triglycerides, are extracted and separated from polar lipids using _{supercritical CO 2.} The extract is then hydrolyzed to give free fatty acids, which are then fractionated to produce concentrates of EPA free fatty acids. This concentrate is combined with a previously isolated polar lipid in subsequent steps to provide a patent-pending formulation.

Alternatively, the patent application discloses a method similar to that described above, in which the algal paste is extracted with ethanol, treated with an alcan solvent, and then the neutral and polar lipids are separated by elution from a silica gel adsorbent. As described above, the triglyceride is hydrolyzed and further eluted from the silica gel adsorbent.

Both elution from supercritical carbon dioxide and silica gel adsorbents is a sophisticated and complex technique. On the other hand, oil compositions containing EPA are useful and are known to be useful for a variety of purposes when they contain EPA in 20-70% by weight.

Therefore, there is a need to produce oil compositions with 20-70% EPA, measured as a weight percent, by a cost-effective method that utilizes simple techniques to provide a final product with the desired EPA content. There is.

(Definition)
As used herein, the term "EPA" means the natural or synthetic form of EPA. These forms can be esters of EPA, salts of EPA, monoglycerides, diglycerides or triglycerides EPA, or free acid forms of EPA. The ester of EPA may be an ester with an alcohol, particularly a lower alkyl alcohol, more specifically an ethyl ester. The salt of EPA may be a salt with an amino acid, particularly lysine. The salt of EPA may be a salt of an alkali metal or an alkaline earth metal. Monoglyceride, diglyceride or triglyceride EPA, in some cases, means a glycerol molecule in which one, two, or all of its three available positions in one or more EPA molecules are esterified.

Preferred forms of "EPA" are esters of EPA with lower alkyl alcohols, salts with amino acids, free acids and monoglycerides of EPA, diglycerides or triglycerides EPA. More preferably, it is an ester of EPA and ethyl alcohol or a salt of lysine.

An "acid" is any compound that contains hydrogen and dissociates in water or solvent to produce positive hydrogen ions, as well as Lewis acids (eg, hydrochloric acid, sulfuric acid, phosphoric acid, acetic acid, trihaloacetic acid (eg, eg). TFA), sulfonic acids such as hydrogen bromide, maleic acid, toluene sulfonic acid and Drosophila sulfonic acid, propionic acids such as (R) -chloropropionic acid, N-[(R) -1- (L-naphthyl) ethyl] Means phthalamic acids such as phthalamic acid, tartrate acids such as L-tartrate and dibenzyl-L-tartrate, lactic acid, gypsum acid, aspartic acid, citroneric acid, BCl ₃ , BBr ₃ and other acids). do. Thus, the term includes weak acids such as ethaneic acid and hydrogen sulfide; strong organic acids such as methanesulfonic acid, trifluoroacetic acid and the like.

As used herein, "base" includes compounds such as hydroxides or alkoxides, hydrides, or amines and derivatives thereof, which accept protons in water or solvent. Thus, exemplary bases are alkali metal hydroxides and alkoxides (ie, MOR, where M is an alkali metal such as potassium, lithium, or sodium, and R is hydrogen or alkyl as defined above. Yes, more preferably R is a linear or branched C _1-5 alkyl, and thus, but not limited to, potassium hydroxide, potassium tert-butoxide, potassium tert-pentoxide, sodium hydroxide, sodium tert. -Including butoxide, lithium hydroxide, etc.); Other hydroxides such as _{magnesium hydroxide (Mg (OH) 2} ) or calcium hydroxide (Ca (OH) ₂ ), barium hydroxide (Ba (OH) ₂₎ Alkali metal hydrides (ie, MH, where M is as defined above, and thus includes, but is not limited to, sodium, potassium, and lithium hydride); potassium hexamethyldisilazide. And alkylated disilazides such as lithium hexamethyldisilazide; carbonates such as potassium carbonate (K ₂ CO ₃ ), sodium carbonate (Na ₂ CO ₃ ), potassium bicarbonate (KHCO ₃ ), sodium bicarbonate (NaHCO ₃ ). , Alkylammonium hydroxides such as tetrabutylammonium hydroxide (TBAH) and the like are included. Examples of the aqueous base include metal hydroxides, for example, hydroxides of Group 1 / Group 2 metals such as Li, Na, K, Mg, and Ca (for example, aqueous LiOH, NaOH, KOH, etc.), and alkylammonium water. Includes oxides and aqueous carbonates. The non-aqueous base, amines and their derivatives, for example, trialkylamine (e.g., _Et 3 N, diisopropylethylamine and the like), and aromatic amines _{(e.g., Ph-NH 2, PhN (} Me) H , etc.); an alkali Includes, but is not limited to, metal alkoxides; alkali metal hydrides; alkylated disilazides; and non-aqueous carbonates.

The term "lipid" means a neutral or polar lipid. Polar lipids mainly include glycolipids and phospholipids. Neutral lipids mainly include fatty acids, triglycerides and diglycerides. Eicosapentaenoic acid (EPA) may be conjugated, part of any of their forms, or as a free fatty acid.

The term "weight percent" means the amount of EPA per 100 grams of substance. It is measured by gas chromatography and the results are provided as a% w / w assay (as is).

The term "purity" means the amount of each fatty acid ethyl ester observed by chromatography relative to the total amount of all compounds measured by gas chromatography and observed by chromatography. It is provided as the% purity of the compound being tested.

The present invention relates to N.I. Provided is a method for treating Oculata microalgae biomass in a minimal series of steps, thereby providing an oil composition containing from about 20% to about 70% EPA measured as a weight percent.

According to the first embodiment of the present invention, about 20% to about 70% N. is measured as a weight percent. A process for preparing an oil composition containing eicosapentaenoic acid (EPA) of the Oculata algae, which comprises the following steps is provided:
a) N. Provides microalgae biomass from Oculata algae;
b) Lipids are extracted from the microalgae biomass of step a by disrupting the cell wall and treating with a mixture of aqueous and organic phases or an organic phase to provide a crude oil extract containing EPA;
c) The crude oil extract of step b is subjected to transesterification conditions thereby transesterifying EPA and other fatty acids with their corresponding ethyl esters, thereby providing an oil phase;
d) One or more selected from treatment with bleached soil, dewaxing, and treatment with urea to provide an oil composition containing from about 20% to about 70% EPA measured as a weight percent. Purify the oil phase of step c by subjecting it to a purification method;
Alternatively, c') one or more selected from treatment with bleached soil, dewaxing, treatment with urea to provide an oil composition containing from about 20% to about 35% EPA measured as a weight percent. Purify the oil phase of step b by subjecting it to the purification method of.
d') The crude oil extract of step c'is subjected to transesterification conditions thereby transesterifying EPA and other fatty acids with their corresponding ethyl esters to provide an oil phase;
e) Optionally, to convert the EPA ethyl ester to another form of EPA, the oil composition is subjected to further treatment to provide a further oil composition.

N. provided in step a. Oculata microalgae biomass can be freeze-dried, lyophilized, or processed according to standard techniques known in the art. A useful form of microalgae biomass can be in the form of dry powder.

Drying the microalgae biomass is advantageous for facilitating further processing. Drying primarily means the removal of free surface water / water from untreated biomass, or the removal of surface water from a homogenized slurry of biomass (eg, by pulverization). In some cases, drying the biomass can facilitate a more efficient microalgae oil extraction process. Microalgae biomass can be dried by methods known to those of skill in the art. The drying method is not important to the performance of the present invention.

Extraction of lipids from microalgae biomass performed in step b is achieved by disrupting cell walls and treating cell debris with an organic phase or a mixture of aqueous and organic phases.

Many methods for cell destruction of algal cells are well known to those of skill in the art and are available in the art. Examples of such methods are mechanical press, sonication, microwave irradiation, osmotic shock, bead beating, high pressure homogenization, blending (solid shear fracture), laser, enzymatic digestion, solvent use, and the latter method. It is a combination of any of.

Cell wall disruption facilitates lipid extraction by treating the disrupted cells with an organic phase or a mixture of aqueous and organic phases. The aqueous phase may be an aqueous solution of an inorganic salt such as sodium chloride. Other aqueous phases suitable for this purpose are those containing a chelating agent such as EDTA or a mixture of a chelating agent and an inorganic salt such as NaCl. The organic phase may include one organic solvent or a mixture of organic solvents. Exemplary solvents used for lipid extraction from algae are dichloromethane, chloroform, methanol, isopropanol, ethoxyethane, acetone, 2-ethoxyethanol, ethanol.

Step b is typically carried out by disrupting the cell walls and bringing them into contact with an organic phase or a mixture of an aqueous phase and an organic phase, as described above.

Step b can optionally include the use of an antioxidant to protect the extracted oil from oxidation during the process. Suitable antioxidants for this purpose are tocopherols, BHT (butylhydroxytoluene), lecithin, oregano extract, rosemary extract, citric acid and / or ascorbic acid.

The extracted lipid fraction contains mainly lipids as defined above, along with trace components of biomass proteins, carbohydrates, minerals and fibers.

After extracting the lipids from the microalgae biomass, the mixture is subjected to standard procedures such as filtration and / or centrifugation and further treated with one or more aqueous phases to undissolve solids and other above. Remove trace components. Separation of the organic phase and removal of the organic solvent provides a crude oil extract.

The crude oil extract obtained in step b contains a lipid, which lipid contains a monoacylglycerol containing EPA, a fatty acid in the form of diacylglycerol or triacylglycerol, and a free acid, although other compounds may be present. .. The mixture is subjected to transesterification conditions in step c, thereby converting all fatty acids present as free fatty acids or in other forms as defined above to the corresponding ethyl esters. Under such conditions, the EPA is similarly converted to its corresponding ethyl ester. Well-known ester exchange conditions for those skilled in the art are catalytic amounts of strong bases (eg, sodium hydroxide or potassium hydroxide, or any other alkali metal hydroxide) at high temperatures or room temperature in the presence of large excess ethanol. It may involve treatment with, which leads to the conversion of the fatty acid triglyceride to the corresponding ethyl ester. Transesterification can also be achieved with an acidic catalyst. When the conversion is complete, the oil phase is obtained by quenching, extraction, filtration and solvent removal. In this step, the glycerol formed by the transesterification reaction is also removed.

Removal of any other unwanted oil impurities from the oil phase obtained in step c, namely chlorophyll and other pigments, polar lipids such as phospholipids, oxidation by-products, and other unwanted polar compounds. Achieved in step d. The type of oil with respect to polyunsaturated fatty acid content and impurity content affects the efficiency of the technique and the decolorization efficiency of the method used.

Therefore, the oil phase obtained in step c is subjected to one or more purification methods selected from treatment with bleached soil, dewaxing, and treatment with urea.

Bleaching is done with the appropriate bleach. Common bleaching agents are natural clay or soil, activated clay or soil and carbon. If the oil is easily bleachable, natural clay is usually used; if the oil is not easily bleachable, acid activated clay is used. Bentonite, sepiolite, attapargite, fuller soil, and montmorillonite are readily available bleaches used in oil refining methods.

Dewaxing or fractionation is a technique relating to the removal of solids formed under controlled crystallization when the oil is exposed to subzero temperatures. In particular, the degree of unsaturation of each fatty acid and the difference in melting point and solubility induced by a particular arrangement allows for the purification or enrichment of oils in polyunsaturated fatty acids.

Urea crystallization is a technique relating to the complex formed between urea and saturated or monounsaturated fatty acids in an alcohol solution. The complex crystallizes and is removed by filtration, but the free, uncomplexed unsaturated fatty acids remain soluble, thus enriching the solution. The content fraction depends on the degree of unsaturation of the fatty acid, not on the physical properties of the fatty acid, such as melting point and solubility.

Alternatively, a purification step in which one or more of the above purification methods are used can be performed immediately after step b and is therefore step c'. In this case, the transesterification step, i.e. d', is performed immediately after the purification step. After transesterification in step c or d'and purification in step d or c', an oil composition consisting of about 20% to about 70% ethyl ester form of EPA as measured by gas chromatographic analysis by weight percent Provided.

Step e is optional and further treatment to convert the ethyl ester of EPA contained in the oil composition into another form, eg, another ester, monoglyceride, diglyceride or triglyceride, salt, or free acid. It may be included. Therefore, additional oil compositions may be provided through this further treatment. This includes standard methods well known to those skilled in the art and compounds containing groups capable of forming an ester bond with a carboxylic acid group, such as alcohols, glycerol or amino acids, if another ester of glyceride is desired. May include acid or base treatment in the presence of. Alternatively, it may include treatment with a base to provide a salt of EPA or its free fatty acid form.

(experiment)
Assay and purity were measured by gas chromatography using methyl docosate as an internal standard.

Example 1: Extraction of Eicosapentaenoic Acid-Enriched Triglyceride In a two-necked 5 L round-bottom flask equipped with a mechanical stirrer, 400 g of Nannochloropsis oculata, 650 mg of α-tocopherol, 1.2 L of D.I. M. Charge with water (demineralized water), 0.4 L sodium chloride 20 wt% solution, 1.6 L dichloromethane and 0.8 L methanol. The mixture is flushed with argon and stirred at 20-25 ° C. for 2 hours. The solid is separated by filtration and the filtrate is collected. Next, 2 L of dichloromethane / methanol 2: 1 mixture is added and the mixture is washed with 3 L of sodium chloride 1 wt% solution. The two layers formed are carefully separated and the lower organic layer is dried over 1 g of anhydrous sodium sulphate by stirring for 30 minutes. The solvent is distilled off under reduced pressure and the oily residue is dried under high vacuum for 3-5 hours to give 110 g of eicosapentaenoic acid-enriched triglyceride as a viscous black-green oil (EPA as fatty acid methyl ester). Purity: 28.9%).

Example 2: Eicosapentaenoic acid-enriched ethyl ester crude mixture In a two-necked 1L round-bottom flask equipped with a magnetic stirring rod, a thermometer and a condenser, 100 g of eicosapentaenoic acid-enriched triglyceride and 0.3 L of anhydrous ethanol are placed in an inert atmosphere. Charge below. Next, 1.5 g of potassium hydroxide is added in one pot under an inert atmosphere of 20 to 25 ° C., and the reaction mass is continuously stirred at the same temperature for 2 to 3 hours. 30 mL D. M. Quench the reaction mass by adding water. Excess solvent is removed from the reaction mass under reduced pressure until the final volume is about 0.2 L. The residue was diluted with 1 L of cyclohexane and 0.4 L of D.I. M. Transfer to a 3 L separatory funnel with water and 0.1 L sodium chloride 20 wt% solution. The organic layer is washed, then separated and set aside. The aqueous layer is washed with 2 x 0.2 L cyclohexane. The extract and wash liquor are pooled and dried over anhydrous sodium sulfate. The solvent is distilled off with a rotary evaporator and the oily residue is dried under high vacuum for 3 to 5 hours to obtain 85 g of a crude mixture of eicosapentaenoic acid-enriched ethyl ester as a black-green oil (EPA purity: 30.3%). EPA GC assay: 20.8%).

Example 3: Purification of crude ethyl ester mixture by treatment with montmorillonite K10 bleached soil 85 g eicosapentaenoic acid enriched ethyl ester crude mixture, 0.85 L cyclohexane and in a 3-port 3 L round bottom flask equipped with a mechanical stirrer. Charge 425 g of montmorillonite K10. The mixture is flushed with argon and stirred at ambient temperature for 30 minutes to 1 hour under an inert atmosphere. The solid is then filtered by passing the mixture through a 425 g montmorillonite K10 filter pad under reduced pressure. The filter pad is washed with 0.85 L of cyclohexane and the filtrate and wash solution are pooled. The solvent is removed with a rotary evaporator and the oily residue is dried under high vacuum to give 56 g of an eicosapentaenoic acid-enriched ethyl ester mixture as a pale yellow oil (EPA purity: 28.9%; EPA GC assay: 25). .6%).

Example 4: First enrichment of EPA by dewaxing the ethyl ester mixture A 100 mL round bottom flask equipped with a magnetic stir bar is loaded with 50 g of a crude mixture of eicosapentaenoic acid enriched ethyl ester and 2.5 mL of hexane. Enter. The solution is cooled and stirred at −20 ° C. for 5 hours. The formed crystals are then separated by cold filtration. The filtrate is concentrated under reduced pressure in a rotary evaporator to give 23.8 g of an eicosapentaenoic acid-enriched ethyl ester mixture as a yellow oil (48.9%; EPAGC assay: 46.6%).

Example 5: Second enrichment of EPA by urea treatment of ethyl ester mixture A 250 mL round bottom flask equipped with a magnetic stirring rod is charged with 69 mL of EtOH and 46 g of urea. The mixture is heated to 50-55 ° C. with stirring until a clear solution is formed. Then, 23 g of the eicosapentaenoic acid-enriched ethyl ester mixture is slowly added under an inert atmosphere at 50-55 ° C. with stirring. The mixture is then removed from heating and allowed to slowly reach ambient temperature (20-22 ° C.). Stirring in an inert atmosphere is maintained for 20 hours. The mixture is then extracted with 3 x 200 mL hexane. The upper hexane layer is pooled and washed with 200 mL DM water. The aqueous phase is backwashed with 2 x 50 mL of hexane, pooled with a hexane layer of extract and wash, and dried over 1 g of anhydrous sodium sulfate. The solvent is distilled off in a rotary evaporator under reduced pressure and the oily residue is dried under high vacuum for 3 hours to give 7.4 g of an ethyl ester mixture highly enriched with eicosapentaenoic acid as a yellow oil (EPA). Purity: 83.5%; EPA GC assay: 70.2%).

Claims (7)

Approximately 20% to approximately 70% N.I., measured as a weight percent. A process for preparing an oil composition comprising the Oculata algae eicosapentaenoic acid (EPA), the following steps:
a) N. Provides microalgae biomass from Oculata algae;
b) The cell wall is disrupted and treated with a mixture of aqueous and organic phases or an organic phase to extract lipids from the microalgae biomass of step a to provide a crude oil extract containing EPA;
c) The crude oil extract of step b is subjected to transesterification conditions thereby transesterifying EPA and other fatty acids with their corresponding ethyl esters, thereby providing an oil phase;
d) One or more selected from treatment with bleached soil, dewaxing, and treatment with urea to provide an oil composition containing from about 20% to about 70% EPA measured as a weight percent. Purify the oil phase of step c by subjecting it to a purification method;
Alternatively c') one selected from treatment with bleached soil, dewaxing, and treatment with urea to provide an oil composition containing from about 20% to about 70% EPA measured as a weight percent. By subjecting to the above purification method, the oil phase of step b is purified;
d') The crude oil extract of step c'is subjected to ester exchange conditions, whereby EPA and other fatty acids are ester exchanged with their corresponding ethyl esters to provide an oil phase; and e') optional. A process comprising the step of subjecting the oil composition to further treatment to provide the additional oil composition in order to convert the EPA ethyl ester to another form of EPA. Claimed that about 20% to about 70% eicosapentaenoic acid (EPA), measured as% by weight, is in the form selected from the group consisting of esters, salts, acids, monoglycerides, diglycerides or triglycerides, or mixtures thereof. Item 1. The process according to item 1. A group of about 20% to about 70% eicosapentaenoic acid (EPA), measured as a weight percent, consisting of esters with lower alkyl alcohols, salts with amino acids, free acids, monoglycerides, diglycerides or triglycerides, and combinations thereof. The process according to claim 2, which is in a form selected from. The process of claim 3, wherein the ester is an ethyl ester. The process of claim 3, wherein the salt is a salt with lysine. The process according to any one of claims 3 to 5, wherein the treatment in step b is performed by using a mixture of an aqueous phase and an organic phase. An oil composition that can be obtained by the process according to any one of claims 1 to 6.