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WO2015024055A1 - Séparation d'acides gras oméga-3 - Google Patents

Séparation d'acides gras oméga-3 Download PDF

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Publication number
WO2015024055A1
WO2015024055A1 PCT/AU2014/000825 AU2014000825W WO2015024055A1 WO 2015024055 A1 WO2015024055 A1 WO 2015024055A1 AU 2014000825 W AU2014000825 W AU 2014000825W WO 2015024055 A1 WO2015024055 A1 WO 2015024055A1
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WIPO (PCT)
Prior art keywords
oil
dpa
fatty acids
omega
lipase
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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PCT/AU2014/000825
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English (en)
Inventor
Colin James Barrow
Taiwo Olusesan AKANBI
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Deakin University
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Deakin University
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Filing date
Publication date
Priority claimed from AU2013903147A external-priority patent/AU2013903147A0/en
Application filed by Deakin University filed Critical Deakin University
Publication of WO2015024055A1 publication Critical patent/WO2015024055A1/fr
Anticipated expiration legal-status Critical
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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/20—Carboxylic acids, e.g. valproic acid having a carboxyl group bound to a chain of seven or more carbon atoms, e.g. stearic, palmitic, arachidic acids
    • A61K31/202—Carboxylic acids, e.g. valproic acid having a carboxyl group bound to a chain of seven or more carbon atoms, e.g. stearic, palmitic, arachidic acids having three or more double bonds, e.g. linolenic
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23D—EDIBLE OILS OR FATS, e.g. MARGARINES, SHORTENINGS OR COOKING OILS
    • A23D9/00—Other edible oils or fats, e.g. shortenings or cooking oils
    • A23D9/007—Other edible oils or fats, e.g. shortenings or cooking oils characterised by ingredients other than fatty acid triglycerides
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K20/00—Accessory food factors for animal feeding-stuffs
    • A23K20/10—Organic substances
    • A23K20/158—Fatty acids; Fats; Products containing oils or fats
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11C—FATTY ACIDS FROM FATS, OILS OR WAXES; CANDLES; FATS, OILS OR FATTY ACIDS BY CHEMICAL MODIFICATION OF FATS, OILS, OR FATTY ACIDS OBTAINED THEREFROM
    • C11C1/00—Preparation of fatty acids from fats, fatty oils, or waxes; Refining the fatty acids
    • C11C1/02—Preparation of fatty acids from fats, fatty oils, or waxes; Refining the fatty acids from fats or fatty oils
    • C11C1/04—Preparation of fatty acids from fats, fatty oils, or waxes; Refining the fatty acids from fats or fatty oils by hydrolysis
    • C11C1/045—Preparation of fatty acids from fats, fatty oils, or waxes; Refining the fatty acids from fats or fatty oils by hydrolysis using enzymes or microorganisms, living or dead
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/18—Carboxylic ester hydrolases (3.1.1)
    • C12N9/20—Triglyceride splitting, e.g. by means of lipase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
    • C12P7/6409—Fatty acids
    • C12P7/6427—Polyunsaturated fatty acids [PUFA], i.e. having two or more double bonds in their backbone
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y301/00—Hydrolases acting on ester bonds (3.1)
    • C12Y301/01—Carboxylic ester hydrolases (3.1.1)
    • C12Y301/01003—Triacylglycerol lipase (3.1.1.3)
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
    • C12P7/6409—Fatty acids
    • C12P7/6427—Polyunsaturated fatty acids [PUFA], i.e. having two or more double bonds in their backbone
    • C12P7/6432—Eicosapentaenoic acids [EPA]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
    • C12P7/6409—Fatty acids
    • C12P7/6427—Polyunsaturated fatty acids [PUFA], i.e. having two or more double bonds in their backbone
    • C12P7/6434—Docosahexenoic acids [DHA]

Definitions

  • the present disclosure provides for the use of a pancreatic lipase for separating n-3 docosapentaenoic acid (DPA) from other omega-3 fatty acids present in an omega-3 containing oil and to uses of n-3 DPA enriched by the method.
  • DPA docosapentaenoic acid
  • Omega-3 fatty acids are particularly useful in pharmaceutical and/or nutritional supplement products. Omega-3 fatty acids may regulate plasma lipid levels, cardiovascular and immune functions, insulin action, neuronal development and visual function. Omega-3 fatty acids have also been shown to have beneficial effects on the risk factors associated with cardiovascular disease, including hypertension and hypertriglyceridemia, and on the coagulation factor VII phospholipid complex activity. Omega-3 fatty acids may also lower serum triglycerides, increase serum HDL cholesterol, lower systolic and diastolic blood pressure and/or pulse rate. Furthermore, omega-3 fatty acids are generally well tolerated when consumed by subjects.
  • omega-3 rich foods such as fatty fish is important as a source of these compounds.
  • the growing nutritional supplement, food and pharmaceutical markets have a high demand for more concentrated omega-3 oils than those present in fish and therefore methods for the production of high quality omega-3 concentrates are industrially important.
  • Lipases can have positional or fatty acid specificities and have been investigated for ability to cleave some fatty acids from the glycerol backbone. They carry out hydrolysis or synthetic reactions on the ester bonds of triglycerides in living beings. Lipases offer particular advantages in that operating conditions are milder. Moreover, it is not necessary to eliminate impurities of any sort (whether acid or alkaline), simplifying phase separation.
  • Pancreatic lipase also known as triacylglycerol acylhydrolase (EC 3.1 .1 .3), is an important enzyme for the digestion of dietary fats (Arroyo R et al (1996) Lipids 31 (1 1 ):1 133- 1 139). It cleaves dietary triacylglycerol (TAG) derivatives into monoacylglycerols (MAGs) and free fatty acids (FFAs). The amino acid sequence of pancreatic lipase has been reported to be conserved among animals such as human, canine, porcine and rat (Kirchgessner TG et al (1987) Journal of Biological Chemistry 262(18):8463-8466).
  • pancreatic lipases Since positional selective lipases have been reported to be applicable in the production of omega-3 fatty acids such as DHA, it is possible that the selectivity of pancreatic lipase may be useful in studies aimed at concentrating omega-3 fatty acids. Several studies have also shown that pancreatic lipases can be successfully immobilized for repeated reuse, an important criteria for cost effective application of these enzymes. However, investigation into the relative selectivity of pancreatic lipase to a broad range of polyunsaturated fatty acids from diverse sources is lacking.
  • the inventors examined the hydrolysis activity of a pancreatic lipase on omega-3 polyunsaturated fatty acids in seal, anchovy and canola oil which are oil sources rich in omega-3 fatty acids. Determining the relative selectivity towards different omega-3 fatty acids is complicated by the differing positional distribution of each on the acyl glycerol backbone for fish and seal oils.
  • omega-3 fatty acids a-linolenic acid (ALA) and docosapentaenoic acid (DPA) were found to be better substrates for the lipase compared to stearidonic acid (STA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA).
  • pancreatic lipase was postulated to be useful for facilitating the separation of omega-3 polyunsaturated fatty acids, and in particular, facilitating separation of omega-3 DPA (hereinafter n-3 DPA) from other omega-3 fatty acids such as EPA and DHA (hereinafter n-3 EPA and n-3 DHA respectively).
  • omega-3 DPA omega-3 DPA
  • EPA and DHA omega-3 fatty acids
  • n-3 DPA form other omega-3 fatty acids
  • the separated n-3 DPA is thus an enriched source of n-3 DPA which can be utilised in pharmaceutical and non-pharmaceutical applications.
  • the separated n-3 DPA can be utilised in dietary supplements in subjects, particularly for vegetarians or those having low red meat diets
  • n-3 DPA has been found to have particular advantages that distinguish this fatty acid over other omega-3 fatty acids such as n-3 EPA and n-3 DHA, such as, for example in endothelial cell migration ability which may be important in wound healing processes, as well as inhibition of platelet aggregation, and enhanced cardiovascular benefits.
  • the present disclosure provides for the use of a pancreatic lipase for separating n-3 docosapentaenoic acid (DPA) from other omega-3 fatty acids present in an omega-3 containing oil.
  • DPA docosapentaenoic acid
  • the omega-3 containing oil is one which comprises n-3 DPA.
  • the present disclosure provides for the use of a pancreatic lipase for separating n-3 docosapentaenoic acid (DPA) from n-3 eicosapentaenoic acid (EPA) and n-3 docosahexaenoic acid (DHA).
  • DPA docosapentaenoic acid
  • EPA eicosapentaenoic acid
  • DHA docosahexaenoic acid
  • the present disclosure also provides for the use of a pancreatic lipase for separating n-2 DPA from n-3 EPA, n-3 DHA and n-3 stearidonic acid (STA).
  • the separated n-3 DPA is in free fatty acid form.
  • the present disclosure provides a method for separating n-3 DPA from other omega-3 fatty acids present in an omega-3 containing oil, the method comprising:
  • the n-3 DPA glycerides are triglycerides.
  • preferentially hydrolyses it is meant that the lipase hydrolyses an ester linkage of an omega-3 fatty acid that is sensitive to the activity of the lipase.
  • omega-3 fatty acid glycerides e.g. triglycerides
  • the lipase hydrolyses n-3 DPA ester linkages more rapidly compared with hydrolysis of ester linkages of n-3 EPA and/or n-3 DHA.
  • the omega-3 containing oil according to the present disclosure may be native oil or a synthetic oil.
  • the native oil may be derived from animal or non-animal oils.
  • the oil is derived from at least one oil chosen from marine oil, single cell oil, algal oil, plant- based oil, microbial oil, mammalian oil and combinations thereof.
  • the mammalian oil is seal oil.
  • the marine oil is a fish oil, sardine oil, mackerel oil, herring oil, cod oil, tuna oil, saury oil, anchovy oil, krill oil, or other lipid composition derived from fish.
  • the oil is not whale oil.
  • Plant-based oils include for example, flaxseed oil, canola oil, mustard seed oil, and soybean oil.
  • Single cell/microbial oils include for example, products by Martek, Nutrinov and Nagase & Co. Single cell oils are often defined as oils derived from microbial cells and which are destined for human consumption (see e.g. Wynn and Ratledge, "Microbial oils: production, processing and markets for specialty long chain omega-3 polyunsaturated fatty acids” pg 43-76 in Breivik (Ed.) Long-Chain Omega-3 Specialty Oils, the Oily Press, P.J. Barnes and Associates, Bridgewater UK, 2007).
  • the oil according to the present disclosure may be chosen from among one or more glyceride esters.
  • the term "glyceride” as used herein refers to mono-, di- or triesters; i.e. one or more monoglycerides, diglycerides, triglyceride or mixtures of these.
  • the oil is rich in omega-3 polyunsaturated fatty acids.
  • the oil comprises n-3 DPA glycerides and/or phospholipids.
  • the oil comprises n-3 DPA triglycerides.
  • the oil mixture is selected from seal oil, anchovy oil, AHIFLOWERTM oil, fish oil or algal oil.
  • the oil is one in which n-3 DPA is enriched at sn-2.
  • the lipase according to the present disclosure is one which preferentially hydrolyses n- 3 DPA glycerides.
  • the lipase preferentially hydrolyses n-3 DPA triglycerides or diglycerides.
  • the lipase is a pancreatic lipase.
  • it is a porcine pancreatic lipase. Any type of porcine pancreatic lipase may be employed in the method of present disclosure, for instance a lipase selected from a commercial industrial porcine pancreatic lipase (PPL), an enzyme preparation containing it, such as for example Sigma's Pancreatin, or a pancreatic lipase extracted directly from pig pancreas.
  • PPL commercial industrial porcine pancreatic lipase
  • the lipase comprises the sequence shown in Figure 1 , or a sequence at least 80%, 85%, 87%, 90%, 92%, 95%, 97%, 98%, or 99% identical thereto. It will be appreciated by persons skilled in the art that activities of different lipases can vary and thus need to be standardised according to methods known in the art and as described for example in Akanbi T et al (2013) Food Chemistry 138:615-620. The degree of hydrolysis should be obtained as quickly as possible in order to minimise acyl migration and inactivation of the lipase enzyme.
  • a concentration of lipase is selected which results in a degree of hydrolysis of n-3 DPA glycerides of between about 1 5 to 80%, about 25 to 75%, about 30 to 70%, about 45 to 65%, about 50 to 60%, about 50 to 55% or about 50 to 52%. In another example, the degree of hydrolysis is about 50 + 5%. It will be appreciated that the degree of hydrolysis needs to be balanced against the concentration of lipase used since excess lipase load has been reported to be a major cause of undesirable acyl migration of fatty acids during hydrolysis (Fernandez-Lafuente (201 0) Journal of Molecular Catalysis B: Enzymatic 62(3- 4):197-212).
  • the lipase concentration may be 100, 200, 400, 600, 800, 1000, 1200, 1500, 3000, 5000, 7000, 8000, 9000 or 10000 U/g of oil.
  • the hydrolysis is carried out at a pH which does not cause substantial denaturation of lipase secondary structure, in particular a-helical content.
  • the hydrolysis is carried out at neutral or alkaline conditions.
  • the hydrolysis is carried out at a pH between about 4.5 and 12, a pH between about 5 and 8, a pH between about 6 and 7.5, a pH between about 6.2 and 7.2, or a pH at or about 7.
  • the pH is between about 7 and about 8.
  • the pH at which the hydrolysis reaction is carried out may be obtained through the addition of a base.
  • Suitable bases include the following: NaOH, KOH, NH 4 OH, Na 2 C0 3 , NaHC0 3 , K 2 C0 3 , KHC0 3 or a combination of these.
  • the lipase according to the present disclosure may be free or immobilised.
  • the lipase may be immobilised by attachment to an organic or inorganic support.
  • the lipase is adsorbed on an organic or inorganic support.
  • the lipase is used in immobilized form by chemical attachment of the enzyme to an inorganic solid that acts as a support, allowing for its easy recovery at the end of the process and its subsequent reuse.
  • the lipase is present in free or attached form in a proportion of 1 /1000 with respect to the weight of the oil used, making it possible to convert 1 mole of oil in 24 hours per gram of lipase.
  • Covalent immobilisation of pig pancreatic lipase has been previously reported Bautista FM et al (1999) Journal of Chemical Technology and Biotechnology 72(3) :249-254.
  • the hydrolysis reaction is carried out for a period of time sufficient to hydrolyse at least a portion of the n-3 DPA glycerides.
  • the period of time is such that about 30% to 80% of the glycerides present in the oil mixture are hydrolysed.
  • the period of time is such that about 35 to 70%, about 40-65%, about 45-60%, about 47-55%, or about 50-55% of the n-3 DPA glycerides are hydrolysed.
  • Suitable temperatures at which the hydrolysis reaction can occur include, but are not limited to from about ambient (e.g. about 20 ° C) to about " ⁇ ⁇ , from about 35 °C to about 80°C, or from about 37 ° C to about 50 ° C, or from about 37 ° C to about 45 ° C.
  • the reaction time can be adjusted by varying the temperature. In examples where pancreatic lipase is used, it is preferred that the temperature is between about 37 ° C and about 40 ° C.
  • the lipase may be recovered by methods known in the art including centrifugation, or chromatography.
  • the method of the present disclosure may also incorporate the additional use of other lipases.
  • one or more additional lipases may be included which do not hydrolyse n-3 DPA glycerides.
  • Such additional lipase may be employed prior to the use of PPL to facilitate removal of saturated and monounsaturated fatty acids present in the oil mixture.
  • examples of such lipases include Rhizomucor miehei lipase (RmL), Thermomyces lanuginosus lipase (TL 100L), and Candida Antarctic lipase (Cal B).
  • the one or more additional lipase(s) may be employed concurrently with the PPL.
  • hydrolysed products prior to the addition of PPL may be removed according to known methods.
  • the method of the present disclosure further comprises combining the oil with a lipase that does not preferentially hydrolyse n-3 DPA glycerides contained in the oil.
  • the present disclosure also provides a method for enriching n-3 DPA from other omega-3 glycerides present in an omega-3 containing oil or an oil comprising omega-3 and omega-6 glycerides, the method comprising:
  • the hydrolysis reaction may be carried out either in an agitated- tank reactor or in a flow reactor.
  • the method of the present disclosure is not used in biodiesel production.
  • the method may comprise esterification of free fatty acids with an alcohol(s), in particular n- and iso-alcohols.
  • the esterification may be carried out simultaneously with the hydrolysis reaction.
  • n-3 DPA is primarily converted to its free fatty acid form whilst n-3 EPA and n-3 DHA remain on the glyceride backbone.
  • n-3 EPA and n-3 DHA remain on the glyceride backbone.
  • hydrolysis reaction a phase separation occurs where the n-3 EPA and n-3 DHA which are resistant to hydrolysis by the lipase, are retained in the organic fraction whilst hydrolysed n-3 DPA as free fatty acids form is in the aqueous fraction.
  • These phases can be separated for example by solvent extraction according to methods known in the art based on pH adjustment.
  • the degree of hydrolysis can be determined by methods known to those skilled in the art.
  • the degree of hydrolysis can be determined using capillary chromatography, for example iatroscan.
  • free fatty acids in the aqueous fraction may be extracted with a solvent such as hexane after adjusting the pH to acidic conditions.
  • a solvent such as hexane
  • Short chain fatty acids can be removed by methods known to persons skilled in the art. For example, short chain fatty acids can be enzymatically removed as free fatty acids prior to DPA hydrolysis. Alternatively, the short chain free fatty acids and n-3 DPA as free fatty acid can be removed after the hydrolysis reaction and then separated using industrial methods such as for example, fractional distillation or urea complexation.
  • the method further comprises extracting and concentrating the n-3 EPA and n-3 DHA containing fraction.
  • Such methods are known in the art.
  • distillation methods such as fractional distillation or thin film distillation, urea complexation or chromatographic methods can be used.
  • n-3 EPA can be further separated from n-3 DHA using known methods for example chromatographic methods, distillation methods, enzymatic methods, chemical methods, low-temperature crystallisation and supercritical fluid extraction, or by methods described in for example US 6846942, US 4792418, WO 2004043894.
  • n-3 EPA and n-3 DHA may be separated by use of the Thermomyces lanuginosus lipase as described in Akanbi T et al (2013) Food Chemistry 138:615-620.
  • n-3 DPA free fatty acids may be further separated from other omega-3 free fatty acids by methods known in the art.
  • urea complexation may be used to separate n-3 DPA free fatty acids from the other free fatty acids in the free fatty acid fraction as described in for example Hidajat K et al ( 995; J Chromatogr A 702:21 5; Gamez-Meza N et al (2003) Food Research International 36:721 -727).
  • chromatographic separation may be used e.g. gas chromatography.
  • the method further comprises separating n-3 DPA free fatty acids from other omega-3 free fatty acids contained in the aqueous free fatty acid fraction.
  • the present disclosure provides an oil composition enriched in n-3 DPA free fatty acids obtained according to a method of the present disclosure.
  • n-3 DPA free fatty acids may be further modified as desired for intended use as a pharmaceutical, food supplement, or cosmetic.
  • the n-3 DPA may be retained in free fatty acid form.
  • the n-3 DPA free fatty acids may be converted to a methyl ester.
  • n-3 DPA free fatty acids may be derivatised for example by chemical modification, conjugates, salts thereof or mixtures of any of the foregoing. Such derivatives include alkyl ester, ethyl ester, methyl ester, propyl ester, or butyl ester.
  • the n-3 DPA free fatty acids may be converted to acylglycerol forms.
  • n-3 DPA free fatty acids can be prepared in the form of ethyl-DPA, lithium-DPA, mono-, di-, or triglyceride DPA or any other ester or salt of DPA.
  • n-3 DPA may also be in the form of a 2-substituted derivative or other derivative which slows down its rate of oxidation but does not otherwise change its biological action to any substantial degree.
  • the oil composition enriched in n-3 DPA free fatty acids comprises at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.8% by weight.
  • the present disclosure also provides a pharmaceutical composition
  • a pharmaceutical composition comprising an oil composition enriched in n-3 DPA free fatty acids and/or derivatised n-3 DPA free fatty acids as described herein, together with a pharmaceutically acceptable carrier or excipient.
  • the enriched n-3 DPA oil compositions may be utilised in a number of pharmaceutical and non-pharmaceutical applications as described herein.
  • the enriched n-3 DPA compositions may be used in a weight loss supplement for treating or preventing obesity in a subject.
  • the term "obesity" as used herein is understood to mean a subject whose body mass index (BMI) is above the recommended range (i.e. above 25).
  • the present disclosure also provides a food additive comprising an enriched n-3 DPA oil composition or a pharmaceutical composition as described herein.
  • the food additive is added to a solid food.
  • the food additive is added to liquid food.
  • the food additive is added to animal feed.
  • the enriched n-3 DPA oil composition is used as a food additive in a food selected from a functional food, nutrient- supplementing food, formula suitable for feeding infants or premature infants, baby foods, foods for expectant or nursing mothers, and geriatric foods.
  • the enriched n-3 DPA composition is combined with carnitine and/or fibrates.
  • the present disclosure also provides an enriched n-3 DPA oil composition or pharmaceutical composition as described herein for use in treating or preventing hypertriglyceridemia or a disorder associated with hypertriglyceridemia in a subject.
  • the present disclosure also provides a method of treating or preventing a disorder selected from hypertriglyceridemia, a cardiovascular-related disease or disorder, obesity, or platelet aggregation disorder in a subject, comprising administering to a subject in need thereof an oil composition, a pharmaceutical composition, or a food additive according to the present disclosure.
  • the enriched n-3 DPA oil composition a pharmaceutical composition is administered to a subject from one to about four times per day.
  • the present disclosure also provides for the use of an oil composition enriched in n-3 DPA free fatty acids and/or derivatised n-3 DPA free fatty acids in the manufacture of a medicament for treating or preventing a disorder selected from hypertriglyceridemia, a cardiovascular-related disease or disorder, obesity, or platelet aggregation disorder in a subject.
  • the present disclosure also provides an enriched n-3 DPA oil composition or pharmaceutical composition as described herein for use in promoting wound healing in a subject in need thereof.
  • the present disclosure also provides an enriched n-3 DPA oil composition as described herein, in the manufacture of a medicament for promoting wound healing in a subject in need thereof.
  • the present disclosure also provides use of an enriched n-3 DPA composition or pharmaceutical composition as described herein in a cosmetic formulation.
  • the cosmetic formulation is a topical formulation.
  • the topical formulation is a moisturising cream or lotion, bar soap, lipstick, shampoo or therapeutic skin preparation for dryness, eczema and psoriasis.
  • purified n-3 DPA or pharmaceutical composition according to the present disclosure is administered to a subject in a therapeutically effective amount.
  • omega-6 fatty acids it may be necessary to remove omega-6 fatty acids if present in the oil mixture. If required, omega-6 and other saturated and monounsaturated fatty acids can be removed using reverse phase high performance liquid chromatography (HPLC). This can be performed following the hydrolysis reaction.
  • HPLC reverse phase high performance liquid chromatography
  • composition of matter, group of steps or group of compositions of matter shall be taken to encompass one and a plurality (i.e. one or more) of those steps, compositions of matter, groups of steps or group of compositions of matter.
  • SEQ ID No:1 refers to the sequence of porcine pancreatic lipase.
  • n-3 DPA or "docosapentaenoic acid” as used herein is intended to refer to the omega-3 ( ⁇ 3 or n-3) fatty acid and depending on the context can include the free fatty acid form or the natural forms, being the triglyceride form, or the phospholipid form.
  • Docosapentaenoic acid is also known as clupanodonic acid and hence reference to one term includes reference to the other.
  • disorder associated with hypertriglyceridemia is intended to refer to a disorder cause by elevation in plasma or serum triglyceride levels above fasting levels and typically refers to high blood levels of triglycerides.
  • High triglyceride levels are typically in the range of about 200 to about 499 mg/dl.
  • Very high triglyceride levels are typically >500 mg/dl.
  • Baseline triglycerides are typically measured when the subject is in a fasting state, that is, the subject has fasted for a period of between 8 and 12 hours.
  • Non-limiting examples of such disorders include atherosclerosis, cardiovascular disease, acute pancreatitis, xanthomas, lipemia reinalis, and hepatosplenomegaly.
  • cardiovascular-related disease or disorder refers to any disease or disorder of the heart or blood vessels (i.e. arteries and veins) and any symptom thereof.
  • cardiovascular-related disease and disorders include hypertriglyceridemia, hypercholesterolemia, mixed dyslipidemia, coronary heart disease, vascular disease, stroke, athersclerosis, arrhythmia, hypertension, myocardial infarction and other cardiovascular events.
  • the "subject” according to the present disclosure shall be taken to mean any subject, including a human or non-human subject.
  • the non-human subject may include non-human primates, ungulate (bovines, porcines, ovines, caprines, equines, buffalo and bison), canine, feline, lagomorph (rabbits, hares and pikas), rodent (mouse, rat, guinea pig, hamster and gerbil), avian, and fish.
  • the subject is a human.
  • the subject consumes a traditional Western diet.
  • fatty acid refers to a molecule that is derived from a triglyceride or phospholipid and is comprised of a carboxylic acid with a long aliphatic tail (chain) which is either saturated or unsaturated. When not attached to other molecules, they are known as "free" fatty acids. Most naturally occurring fatty acids have a chain of an even number of carbon atoms, from 4 to 28. Short chain fatty acids (SCFA) are fatty acids with aliphatic tails of fewer than six carbons.
  • MCFA Medium chain fatty acids
  • MCFA are fatty acids with aliphatic tails of 6-12 carbons which can form medium chain triglycerides.
  • LCFA Long chain fatty acids
  • VLCFA Very long chain fatty acids
  • the fatty acid or the ester thereof can comprise at least 10, at least 12, at least 14, at least 16, at least 18, or at least 20 carbon atoms.
  • the fatty acid or the ester thereof can contain 10, 1 1 , 12, 13, 14, 15, 1 6, 17, 18, 1 9, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, or 45 carbon atoms, where any of the stated values can form an upper or lower endpoint when appropriate.
  • the fatty acid or the ester thereof can comprise a mixture of fatty acids or the esters thereof having a range of carbon atoms.
  • the fatty acid or the ester thereof can comprise from 10 to 40, from 12 to 38, from 14 to 36, from 16 to 34, from 18 to 32, or from 20 to 30 carbon atoms.
  • polyunsaturated fatty acids or PUFAs as used herein are intended to refer to fatty acids that contain more than one double bond in their backbone. Unsaturated refers to the fact that the molecules contain less than the maximum amount of hydrogen. Polyunsaturated fatty acids may be divided into omega 3 and omega 6 type fatty acids. Omega 3 fatty acids have a double bond that is three carbons away from the methyl carbon.
  • omega 3 polyunsaturated fatty acids examples include hexadecatrienoic acid (16:3 (n-3)), alpha-linolenic acid (18:3 (n-3)), stearidonic acid (18:4 (n-3)), eicosatrienoic acid (20:3 (n-3)), eicosatetraenoic acid (20:4 (n-3)), eicosapentaenoic acid (20:5 (n-3)), heneicosapentaenoic acid (21 :5 (n-3)), docasapentaenoic acid (22:5 (n-3)), docosahexaenoic acid (22:6 (n-3)), tetracosapentaenoic acid (24:5 (n-3)), tetracosahexaenoic acid (24:6 (n-3)).
  • omega-3 fatty acid(s) includes natural as well as synthetic omega-3 fatty acids, as well as pharmaceutically acceptable esters, free acids, triglycerides, derivatives or conjugates, precursors, salts or mixtures thereof (see for example US 2004/0254357 or US 624581 1 ).
  • omega-3 fatty acid oils includes, but are no limited to omega-3 polyunsaturated fatty acids such as a-linolenic acid (ALA 18:3n-3), octadecatetraenoic acid (i.e.
  • esters of omega-3 fatty acids with glycerol such as mono-, di- triglycerides and ester of the omega-3 fatty acids and a primary, secondary and/or tertiary alcohol such as for example fatty acid methyl esters and fatty acid ethyl esters.
  • glycolide also known as acylglycerol
  • acylglycerol refers to a diglyceride, a triglyceride or combinations thereof. They are esters formed from glycerol and fatty acids.
  • the glyceride in the oil can comprise a plurality of fatty acids saturated, unsaturated and even short chain carboxylic acids.
  • triglyceride refers to an ester derived from glycerol and three fatty acids.
  • the triglycerides of the present disclosure may be saturated or unsaturated.
  • the term "therapeutically effective amount” shall be taken to mean a sufficient quantity of n-3 DPA to reduce, inhibit or prevent one or more symptoms of a clinical disorder associated with elevated triglyceride levels to a level that is below that observed and accepted as clinically diagnostic or clinically characteristic of that disorder.
  • the term also means that the substance in question does not produce unacceptable toxicity to the subject or interaction with other components in the composition. The skilled artisan will be aware that such an amount will vary depending on, for example, the particular subject and/or the type or severity or level of disorder.
  • this term is not to be construed to limit the composition of the disclosure to a specific quantity, e.g., weight or amount of n-3 DPA and/or derivative(s), rather the present disclosure encompasses any amount of n-3 DPA and/or derivative(s) sufficient to achieve the stated result in a subject.
  • the term "treating" includes administering a therapeutically effective amount of n-3 DPA described herein sufficient to reduce or eliminate at least one symptom of a specified disorder.
  • the treatment involves administering a therapeutically effective amount of n-3 DPA to reduce plasma triglyceride levels.
  • the reduction is measured over a specific time period against a baseline level of fasting plasma triglycerides.
  • the reduction in plasma triglyceride levels is at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 60%, at least about 70%, at least about 80% or at least about 90% compared to baseline.
  • treatment also refers to prophylactic treatment.
  • preventing include administering a therapeutically effective amount of n-3 DPA described herein sufficient to stop or hinder the development of at least one symptom of a specified disorder.
  • administration of n-3 DPA or derivative thereof prevents elevation of plasma triglycerides.
  • Figure 1 shows the sequence of porcine pancreatic triacyglycerol lipase.
  • Figure 3 shows percentage hydrolysis and capillary chromatography (latroscan) profile of lipid classes from hydrolysed (a) anchovy and (b) seal oils using 10,000 units of porcine pancreatic lipase at 37 °C and pH 7.0.
  • Figure 4 shows fatty acid composition of canola oil before and after hydrolysis.
  • Figure 5 shows GC profile of (a) anchovy and (b) seal oil before and after hydrolysis.
  • Figure 6 shows chemical structures of ALA; C18:3n3 (A9,12,15-octadecatrienoic acid), STA; C18:4n3 ( ⁇ 6,9,12,1 5-octadecatetraenoic acid), EPA; C20:5n3 ( ⁇ 5.8.1 1 ,14,17- eicosapentaenoic acid), DPA; C22:5n3 (A7,10,13,16,19-docosapentaenoic acid) and DHA; C22:6n3 (A4,7,10,13,16,19-docosahexaenoic acid).
  • Figure 7 shows Progressive hydrolysis of (a) anchovy and (b) seal oils by porcine pancreatic lipase as measured using gas chromatography. EPA and DHA retention in the glycerol portions increased as DPA is progressively removed in the FFA portions.
  • the oil according to the present disclosure can be any suitable oil composition that comprises omega-3 fatty acids and in particular comprises a docosapentaenoic acid (DPA).
  • the oil may be derived from animal oil(s) and/or non-animal oil(s).
  • the oil is a fatty acid oil mixture derived from at least one oil chosen from marine oil, single cell oil, algal oil, plant-based oil, microbial oil, and combinations thereof.
  • Marine oils include, for example, fish oil, krill oil, and lipid compositions derived from fish.
  • Plant- based oils include, for example, flaxseed oil, canola oil, mustard seed oil, and soybean oil.
  • Single cell/microbial oils include, for example, products by Martek, Nutrinova, and Nagase & Co.
  • Single cell oils are often defined as oils derived from microbial cells and which are destined for human consumption. See, e.g. , Wynn and Ratledge, "Microbial oils: production, processing and markets for specialty long-chain omega-3 polyunsatutrated fatty acids," pp. 43-76 in Breivik (Ed.), Long-Chain Omega-3 Specialty Oils, The Oily Press, P.J. Barnes & Associates, Bridgewater UK, 2007.
  • Additional oils include triglyceride vegetable oils, commonly known as long chain triglycerides such as castor oil, corn oil, cottonseed oil, olive oil, peanut oil, safflower oil, sesame oil, soybean oil, hydrogenated soybean oil, and hydrogenated vegetable oils; medium chain triglycerides such as those derived from coconut oil or palm seed oil, monoglycerides, diglycerides, and triglycerides.
  • long chain triglycerides such as castor oil, corn oil, cottonseed oil, olive oil, peanut oil, safflower oil, sesame oil, soybean oil, hydrogenated soybean oil, and hydrogenated vegetable oils
  • medium chain triglycerides such as those derived from coconut oil or palm seed oil, monoglycerides, diglycerides, and triglycerides.
  • oils such as esters of propylene glycol such as mixed diesters of caprylic/capric acids of propylene glycol, esters of saturated coconut and palm kernel oil-derived caprylic, linoleic, succinic, or capric fatty acids of propylene glycol.
  • the fatty acids of the oil may be esterified, such as alkyl esters, for example ethyl esters.
  • the fatty acids are in glyceride form, such as chosen from mono- , di-, and triglycerides. In other embodiments, the fatty acids are in free acid form.
  • Unsaturated fatty acids in the oil may be in cis and/or trans configuration.
  • omega-3 fatty acids in all-c/s configuration include, but are not limited to, (all-Z)-9,12,1 5- octadecatrienoic acid (ALA), (all-Z)-6,9,12,15- octadecatetraenoic acid (STA), (all-Z)-1 1 ,1 ,17- eicosatrienoic acid (ETE), (all-Z)- 8,1 1 ,14,17-eicosatetraenoic acid (ETA), (all-Z)-,7,10,13, 16,19-docosapentaenoic acid (DPA), (all-Z)-5,8,1 1 ,14, 17-eicosapentaenoic acid (EPA), (all- Z)-4,7,10,13,1 6,1 9- docosahexaenoic acid (DHA), and (all-Z)-6,
  • omega-6 fatty acids in all-cis configuration include, but are not limited to, (all-Z)-4,7,10,13,16-docosapentaenoic acid (osbond acid), (all- Z)-9, 12-octadecadienoic acid (linoleic acid), (all-Z)-5,8,1 1 ,14-eicosatetraenoic acid (AA), and (all-Z)-6,9,12- octadecatrienoic acid (GLA).
  • Examples of monounsaturated fatty acids in cis configuration include, but are not limited to, (Z)-9-hexadecenoic acid (palmitoleic acid), (Z)-9- octadecenoic acid (oleic acid), (Z)-1 1 -octadecenoic acid (vaccenic acid), (Z)-9- eicosenoic acid (gadoleic acid), (Z)-1 1 -eicosenoic acid (gondoic acid), (Z)-1 1 - eicoesenoic acid, (Z)-1 1 - docosenoic acid (cetoleic acid), Z-13-docosenoic acid (erucic acid), and (R-(Z))-12-hydroxy-9- octadecenoic acid (ricinoleic acid).
  • fatty acid oils include, but are not limited to, the fatty acids defined in pharmacopoeias such as the European Pharmacopoeia Omega-3 Acid Ethyl Esters 60, the European Pharmacopoeia Fish Oil Rich in Omega-3 Acids Monograph, the USP Fish Oil Monograph, the European Pharmacopoeia Omega-3 Acid Triglycerides, the European Pharmacopoeia Omega-3-Acid Ethyl Esters 90, and the USP Omega-3-Acid Ethyl Esters monograph.
  • the European Pharmacopoeia Omega-3 Acid Ethyl Esters 60 the European Pharmacopoeia Fish Oil Rich in Omega-3 Acids Monograph
  • the USP Fish Oil Monograph the European Pharmacopoeia Omega-3 Acid Triglycerides
  • the European Pharmacopoeia Omega-3-Acid Ethyl Esters 90 and the USP Omega-3-Acid Ethyl Esters monograph.
  • fatty acid oils comprising different fatty acids include, but are not limited to: IncromegaTM omega-3 marine oil concentrates such as IncromegaTM TG7010 SR, IncromegaTM E7010 SR, IncromegaTM TG6015, IncromegaTM EPA500TG SR, IncromegaTM E400200 SR, IncromegaTM E401 0, IncromegaTM DHA700TG SR, IncromegaTM DHA700E SR, IncromegaTM DHA500TG SR, IncromegaTM TG3322 SR, IncromegaTM E3322 SR, IncromegaTM TG3322, IncromegaTM E3322, IncromegaTM Trio TG/EE (Croda International PLC, Yorkshire, England); EPAX6000FA, EPAX5000TG, EPAX451 OTG, EPAX2050TG, EPAX5500EE, EPAX5500TG, EPAX5000EE, EPAX5000TG, EPAX6000EE, EPAX6000TG, EPAX6500EE
  • omega-3 fatty acids examples include marine, algae, microbial (single cell), and/or plant-based sources.
  • the fatty acid oil comprises n-3 EPA, n-3 DHA and n-3
  • the fatty acid oil may further comprise at least one other fatty acid, for example a polyunsaturated fatty acid (PUFA) other than n-3 EPA and n-3 DHA.
  • PUFA polyunsaturated fatty acid
  • examples of such PUFAs include, but are not limited to, other omega-3 fatty acids, such as C20-C22 omega-3 fatty acids other than n-3 EPA and n-3 DHA, and omega-6 fatty acids.
  • the hydrolysis step according to the present disclosure comprises the use of a lipase in liquid form to hydrolyse at least a portion of the n-3 DPA glyceride and provide a hydrolysed glyceride fraction and a free fatty acid fraction.
  • the lipase is a porcine pancreatic lipase obtained from a commercial supplier e.g. Sigma-Aldrich (Castle Hill, Australia).
  • the lipase has a sequence homology of at least 80%, at least 85%, at least 90%, at least 95%, at least 97% and at least 99% homology to porcine pancreatic lipase as described in Figure 1 .
  • the oil Prior to hydrolysis the oil can be optionally washed with water and/or a pH buffer such as pH 10 buffer (e.g. a potassium carbonate, potassium borate, potassium hydroxide buffer).
  • a pH buffer such as pH 10 buffer (e.g. a potassium carbonate, potassium borate, potassium hydroxide buffer).
  • An optional wash if performed can comprise one or more washes of the same of varying compositions and conditions.
  • an oil composition is washed with 60°C water.
  • an oil composition is twice washed with 60°C water.
  • the quantity of wash solution e.g., water, pH buffer, or other wash liquid
  • temperature, and duration of a wash can vary depending upon the starting oil composition and the desired outcome, such as, for example, purity, of a wash step.
  • a pH buffer of buffer solution can comprise any suitable buffer and/or buffer solution for use with the specific oil composition.
  • a pH 10 buffer comprises a potassium carbonate- potassium borate-potassium hydroxide buffer (0.05 M).
  • the aqueous fraction of the wash can optionally be separated and removed from the oil composition.
  • the pH of the oil Prior to the addition of the pancreatic lipase, it is important that the pH of the oil is brought to pH 7.2.
  • the oil can then be contacted with an aqueous solution of porcine pancreatic lipase.
  • Contacting of the oil composition and the lipase solution can be performed either separate from, combined with, or subsequent to an optional washing step.
  • an oil mixture is twice washed with water, washed with a pH 10 buffer solution, and then contacted with an aqueous solution of the lipase enzyme.
  • the aqueous solution of porcine pancreatic lipase is a phosphate buffered solution of pH about 7.2 (0.01 -0.1 M). The amount of liquid lipase used can vary.
  • An aqueous solution of porcine pancreatic lipase can be prepared by, for example, mixing a quantity of the lipase enzyme with water.
  • the amount of lipase enzyme mixed with water, and thus, the concentration of a resulting lipase solution, can vary.
  • the conditions for hydrolysis of the starting glyceride can vary, depending upon the desired extent of hydrolysis and the specific reaction components, provided that at least a portion of n-3 DPA on a glyceride are hydrolysed.
  • the phrase, "at a time and temperature sufficient to hydrolyze at least a portion of the glyceride” can be selected by one of skill in the art, depending on the desired amount of hydrolysis and the desired final product, while monitoring the reaction by known analytical techniques.
  • longer reaction times, higher temperatures (within the limits of the lipase), and/or more amounts of lipase can be used for more complete hydrolysis.
  • the resulting mixture can, in various examples, be sealed in an inert or substantially inert atmosphere, such as, for example, nitrogen or argon atmosphere.
  • the resulting mixture can also be optionally agitated for a period of time sufficient to allow a desired amount of hydrolysis.
  • an oil/lipase mixture is vigorously agitated for a period of from about 48 to about 72 hours at about 37°C.
  • Suitable temperatures at which the hydrolysis can occur include, but are not limited to, from about ambient to about 1 00°C, from about 35°C to about 80°C, or from about 40°C to about 50°C, preferably between ambient and 40 ° C.
  • the reaction time can be adjusted by varying the temperature. Thus, reaction times can vary from about 2 hours to about 72 hours or more, from about 24 hours to about 72 hours, from about 36 hours to about 72 hours, or from about 48 hours to 72 hours.
  • the hydrolyzed oil composition (comprising a glyceride fraction and free saturated fatty acid fraction) can optionally be washed with water one or more times.
  • the aqueous portion of the mixture can then be separated from the non-aqueous portion, and the non-aqueous portion dried. Drying conditions can vary and the disclosed methods are not intended to be limited to any particular drying conditions. In one example, the non-aqueous portion is dried under vacuum at about 80°C.
  • the hydrolysis step is conducted in water and in the absence of alcohol; thus the hydrolyzed product is a free polyunsaturated fatty acid and not a free polyunsaturated fatty ester.
  • these processes are normally carried out under nitrogen and/or with added antioxidants such as citric acid, ascorbic acid or BHT. Determining the degree of hydrolysis
  • capillary chromatography with flame ionisation detector may be used and the percent hydrolysis determined using appropriate software (e.g. SIC-480 II software) for multiple chromatogram processing by comparing the percentage peak areas of the unhydrolysed and hydrolysed triglycerides.
  • Capillary chromatography standards to identify each lipid class can be purchased from a commercial supplier. Examples of such methodology are described in, for example Luddy FE et al (1964) Journal of the American Oil Chemists' Society 41 (10):693-696; Gamez-Meza N et al (2003) Food Research International '36:721 -727).
  • Hydrolysed free fatty acid can be removed from acyl glycerol by methods known in the art.
  • wipe film evaporation and/or short path distillation can be used on the combined oil to selectively remove the more volatile free fatty acids via distillation.
  • water may be added to create an aqueous phase and the pH adjusted so that the free fatty acid exists primarily as unprotenated, charged, carboxylic acid dissolved in the aqueous phase.
  • the organic phase (containing glycerides) and aqueous phase (containing free fatty acids) may be separated according to methods known in the art e.g. by allowing the mixture to stand for a sufficient amount of time to obtain two substantially transparent phases, by centrifugation, by membrane technology, or by other suitable means.
  • the aqueous phase may be extracted with a displacement liquid, such as an organic solvent, resulting in formation of at least one extract.
  • suitable displacement liquids include, but are not limited to, alkanes, alkenes, cycloalkanes, cycloalkenes, dienes, aromates, and halogenated solvents.
  • the aqueous phase may be extracted more than once, i.e., at least two successive extractions.
  • the amount of displacement liquid for each extraction may range from about 0.1 to about 5 times by weight the amount of fatty acid that is dissolved in the aqueous phase.
  • Different displacement liquids and/or combinations of displacement liquids may be used according to the selectivity desired in concentrating n-3 DPA free fatty acids.
  • the aqueous phase and organic phase may be heated before they are separated. In such cases, the boiling point of the organic phase may be considered in determining the appropriate temperature.
  • the aqueous phase/organic phase mixture may be heated to a temperature ranging from about 30 °C to about 90°C.
  • the aqueous phase is heated after removing the organic phase, resulting in formation of at least one extract.
  • the aqueous phase may be heated to a temperature of at least 30 °C, such as a temperature ranging from about 30 °C to about 90 °C.
  • heating may cause the release of a fatty acid oil concentrated in omega- 6 fatty acids and/or specific omega-3 fatty acids, such as C20-C22 omega-3 fatty acids other than n-3 DPA, from the aqueous phase. Heating should be done carefully in the absence of oxygen and at sufficiently mild conditions to avoid oxidation, isomerization and/or degradation of the polyunsaturated fatty acids.
  • the process according to the present disclosure may concentrate or enrich n-3 DPA omega-3 free fatty acid while reducing the concentration of other omega-3 fatty acids in the oil.
  • the process may increase the ratio of n-3 DPA omega-3 to n-3 DHA and n-3 EPA.
  • the process concentrates or enriches n-3 DPA while reducing the concentration of C20-C22 omega-3 fatty acids other than n-3 DPA.
  • the total concentration of C 2o -C 2 2 omega-3 fatty acids other than n-3 DPA in the fatty acid fraction is less than 3% by weight, such as less than 2.5% by weight, such as less than 0.5% by weight.
  • the n-3 DPA free fatty acid may be concentrated or enriched using an aqueous silver salt according to known methods and as described for example in WO 2012/038833.
  • the n-3 DPA free fatty acid fraction may be purified by using at least one purification process.
  • the purification process may remove, for example, displacement liquid or lower-chain fatty acids or complexation compounds e.g. urea if urea complexation has been performed, cholesterol and/or vitamins.
  • Such purification processes include, but are not limited to, short- path distillation, molecular distillation, supercritical fluid extraction, enzymatic separation processes, iodolactonization fractionation, and preparative chromatography.
  • the process presently disclosed may be repeated to further concentrate the n-3 DPA omega-3 free fatty acid.
  • the fatty acid concentrate obtained from one or more concentration processes according to the present disclosure may comprise at least 80% of n-3 DPA omega-3 fatty acid, such as at least 90%, at least 95%, or even at least 98% of n-3 DPA omega-3 fatty acid.
  • the fatty acid concentration obtained according to the process of the present disclosure may also be treated by at least one conventional fractionation process such as short-path distillation, molecular distillation, iodolactonization fractionation, enzymatic fractionation processes, extraction, and/or chromatography.
  • the free fatty acid concentrate thus obtained may comprise at least 80% of n-3 DPA omega-3 free fatty acid, such as at least 90%, at least 95%, or even at least 98% of n-3 DPA omega-3 free fatty acid.
  • the at least one fractionation process produces a free fatty acid concentrate comprising at least 90% cis-7, 10, 13, 1 6, 19-docosapentaenoic acid (DPA), such as at least 95% n-3 DPA, or for example, at least 98% n-3 DPA.
  • DPA deoxyribonucleic acid
  • the process presently disclosed may reduce the concentration of at least one environmental pollutant in the oil, such that the free fatty acid oil concentrate comprises a lower concentration of the at least one environmental pollutant than the oil mixture.
  • Environmental pollutants include, but are not limited to, polychlorinated biphenyl (PCB) compounds, polychlorinated dibenzodioxin (PCDD) compounds, polychlorinated dibenzofuran (PCDF) compounds, brominated flame retardants like polybrominated diphenyl ethers (PBDE), tetrabromobisphenol A (TBBP-A) and hexabromocyclododecane (HBCD), and pesticides like DDT (2,2 bis-(p-chlorophenyl)- 1 ,1 ,1 -trichloroethane) and metabolites of DDT.
  • the process presently disclosed may also reduce the concentration of total cholesterol (i.e., free and/or bound cholesterol) in the oil mixture, such that the fatty acid
  • n-3 polyunsaturated fatty acid concentrates Other methods of separation known in the art may be employed as required. For example high performance liquid chromatography and silver resin chromatography have been used for the production of n-3 polyunsaturated fatty acid concentrates. Solvent choice for separation of fatty acid esters depends on the desired purity of eluted fractions and their use as well as production requirements. Higher purity fractions of n-3 EPA and n-3 DHA can be obtained using tetrahydrofuran (THF) or ethanol and water.
  • THF tetrahydrofuran
  • Distillation has been used for partial separation of mixtures of fatty acid esters. This method takes advantage of the differences in boiling point and molecular weight of fatty acids under reduced pressure. The technique requires high temperatures of approximately 250 ° C. Short-path distillation or molecule distillation uses lower temperatures and short heating intervals. The most widely used distillation techniques is fractional distillation under reduced pressure (0.1 -1 mm HG). Even under these conditions moderately high temperatures are required sufficient to cause oxidation, polymerisation and isomerisation of double bonds of omega-3 polyunsaturated fatty acids.
  • Low temperature crystallisation may be used to separate fatty acids.
  • the solubility of fats in organic solvents decreases with increasing mean molecular weight and increases with increasing unsaturation.
  • Low temperature crystallisation process may be carried out in the absence of a solvent or in a selected solvent/solvent mixture.
  • the commonly used solvents are methanol and acetone which have been employed to separate stearic and oleic fractions. It has been reported that use of different organic solvents affects the concentration of polyunsaturated fatty acid. Therefore proper choice of solvent is necessary to achieve optimum concentration yield of omega-3 polyunsaturated fatty acid.
  • SPE Supercritical fluid extraction
  • any biologically acceptable dosage forms, and combinations thereof may be contemplated by the present disclosure.
  • dosage forms include, without limitation, chewable tablets, quick dissolve tablets, effervescent tablets, reconstitutable powders, elixirs, liquids, solutions, suspensions, emulsions, tablets, multi-layer tablets, bi-layer tablets, capsules, soft gelatin capsules, hard gelatin capsules, caplets, lozenges, chewable lozenges, beads, powders, granules, particles, microparticles, dispersible granules, cachets, douches, suppositories, creams, topicals, inhalants, aerosol inhalants, patches, particle inhalants, implants, depot implants, ingestibles, injectables, infusions, health bars, confections, cereals, cereal coatings, foods, nutritive foods, functional foods and combinations thereof.
  • the preparations of the above dosage forms are well known to persons of ordinary skill in the art.
  • compositions useful in accordance with the methods of the present disclosure are orally deliverable.
  • oral administration include any form of delivery of a therapeutic agent (e.g. n-3 DPA or a derivative thereof) or a composition thereof to a subject, wherein the agent or composition is placed in the mouth of the subject, whether or not the agent or composition is swallowed.
  • oral administration includes buccal and sublingual as well as oesophageal administration.
  • the purified n-3 DPA or derivative thereof is present in a capsule, for example a soft gelatin capsule.
  • compositions according to the present disclosure are not limited with regard to their mode of use.
  • Representative modes of use include foods, food additives, medicaments, weight supplements, additives for medicaments, and feedstuffs.
  • Examples of food compositions are functional foods, nutrient- supplementing foods, formula suitable for feeding infants, baby foods, foods for expectant or nursing mothers, and geriatric foods.
  • the composition may be added upon cooking such as soup, food to which oils and fat are used as heating medium such as doughnuts, oils and fat food such as butter, processed food to which oils and fat are added during processing such as cookies or food to which oils and fat are sprayed or applied upon completion of processing such as hard biscuits.
  • compositions of the present disclosure can be added to foods or drinks which do not normally contain oils or fat.
  • the definition of food also includes functional food.
  • Functional foods and medicaments may be provided in processed form such enteral agent for promoting nutrition, powder, granule, troche, internal solution, suspension, emulsion, syrup, capsule and such.
  • compositions according to the present disclosure can be formulated as one or more dosage units.
  • dose unit and “dosage unit” herein refer to a portion of a composition that contains an amount of a therapeutic agent suitable for single administration to provide a therapeutic effect.
  • dosage units may be administered one to a plurality (i.e. 1 to about 10, 1 to 8, 1 to 6, 1 to 4 or 1 to 2) of times per day, or as many times as needed to elicit a therapeutic response.
  • compositions of the present disclosure are administered to a subject over a period of about 1 to about 200 weeks, about 1 to about 100 weeks, about 1 to about 80 weeks, about 1 to about 50 weeks, about 1 to about 40 weeks, about 1 to about 20 weeks, about 1 to about 1 5 weeks, about 1 to about 12 weeks, about 1 to about 1 0 weeks, about 1 to about 5 weeks, about 1 to about 2 weeks, or about 1 week.
  • the compositions of the present disclosure comprise one or more antioxidants (e.g. tocopherol) or other impurities in an amount of not more than about 0.5%, or not more than 0.05%.
  • the compositions of the present disclosure comprise about 0.05% to about 0.4% tocopherol, or about 0.4% tocopherol, or about 0.2% by weight tocopherol.
  • compositions of the present disclosure include one or more additional excipients including, but not limited to gelatin, glycerol, polyol, sorbitol and water.
  • the n-3 DPA or derivative thereof is present in the composition in an amount of about 50 mg to about 5000 mg, about 75 mg to about 2500 mg, or about 100 mg to about 1000 mg, about 75 mg, about 100 mg, about 125 mg, about 150 mg, about 175 mg, about 200 mg, about 225 mg, about 250 mg, about 275 mg, about 300 mg, about 325 mg, about 350 mg, about 375 mg, about 400 mg, about 425 mg, about 450 mg, about 475 mg, about 500 mg, about 525 mg, about 550 mg, about 575 mg, about 600 mg, about 625 mg, about 650 mg, about 675 mg, about 700 mg, about 725 mg, about 750 mg, about 775 mg, about 800 mg, about 825 mg, about 850 mg, about 875 mg, about 900 mg, about 925 mg, about 950 mg, about 975 mg, about 1000 mg, about 1025 mg, about 1050 mg, about 1 075 mg, about 1200 mg, about 1250 mg, about 1300
  • compositions of the present disclosure comprise about 300 mg to about 1 g of the composition in a capsule.
  • the dosage form is a gel or liquid capsule and is packaged in blister packages of about 1 to about 20 capsules per sheet.
  • a composition of the present disclosure is administered to a subject once or twice per day. In another example, the composition is administered to a subject as 1 , 2, 3, or 4 capsules daily.
  • composition may be administered to a subject in need thereof immediately before a meal, during consumption of the meal or shortly following the meal.
  • composition of the present disclosure is formulated for topical application, for example in a cosmetic.
  • Topical products that may incorporate n-3 DPA according to the present disclosure include moisturizing creams and lotions, bar soaps, lipsticks, shampoos and therapeutic skin preparations for dryness, eczema and psoriasis.
  • Quantitative 13 C NMR spectra of the unhydrolysed oils were recorded under continuous 1 H decoupling at 24 S C using a Brucker Avance 500 MHz. The spectra were collected on 0.5 g of the oil samples dissolved in 700 ⁇ _ CDCI 3 (99.8% pure). In order to quantify the residue of each fatty acid at different positions, peak area ratios were analysed by integration and presented in percentages (Akanbi et al (2013) Food Chemistry 138:615-620).
  • Enzymatic hydrolysis was carried out following the method of Luddy et al., (1963) Journal of the America Oil Chemists' Society 41 (10):693-696 with some modifications.
  • 100 ⁇ _ of 22% calcium chloride (CaCI 2 ) solution 200 ⁇ _ of 1 M tris(hydroxymethyl)aminomethane at pH 7.7 and 320 ⁇ _ of 0.1 % bile salts solution.
  • the mixture was gently flushed with nitrogen and warmed in a water bath at 37 S C for 5 min before the addition of 10,000 units of PPL.
  • Capillary chromatography with flame ionisation detector (latroscan MK5, latron Laboratories Inc., Tokyo, Japan) was used to determine the degree of hydrolysis. Portions of both the unhydrolysed and hydrolysed oil were analysed by latroscan as previously reported (Akanbi et al (2013) Food Chemistry 138:615-620). Percent hydrolysis was determined using SIC-480 II software for multiple chromatogram processing, by comparing the percentage peak areas of the unhydrolysed and hydrolysed triacylglycerol (TAG). Capillary chromatography standards purchased from Nu-Chek Prep were used to identify each lipid class. Analysis of fatty acid composition by gas chromatography
  • Fatty acids in both the unhydrolysed and hydrolysed portions of the oils were converted to methyl esters and the resulting fatty acid methyl esters (FAMEs) were analysed by an Agilent 6890 gas chromatograph with flame ionisation detector (FID), as previously described (Akanbi et al (2013) Food Chemistry 138:615-620).
  • FAMEs fatty acid methyl esters
  • DPA was highly enriched in the FFA fraction and so was selectively hydrolysed versus all other omega-3 fatty acids, including STA, EPA and DHA.
  • the selectivity of PPL toward DPA hydrolysis is not due to positional selectivity, since DPA is primarily at sn- 1 ,3 position in seal oil and enriched at the sn-2 position in anchovy oil.
  • DHA was resistant to PPL hydrolysis even though it is primarily at sn-1 ,3 in seal oil and enriched at sn-2 in anchovy oil.
  • EPA is enriched equally to DHA in anchovy oil, even though EPA is primarily at sn- 1 ,3 and DHA is primarily at sn-2, further indicating that the hydrolysis is fatty acid and not regiospecific.
  • ALA and DPA were more readily hydrolysed than DHA, EPA and STA, which is consistent with double bonds at C4-5 ( ⁇ 4 DHA), C5-6 ( ⁇ 5 EPA) and C6-7 ( ⁇ 6 STA) conferring greater resistance to hydrolysis than double bonds at C7-8 ( ⁇ 7 DPA) and C9- 10 ( ⁇ 9 ALA), irrespective of carbon chain length or position on the acyl glycerol backbone (Fig. 6).
  • Example 4 Use of PPL to separate DPA from EPA and DHA.
  • DPA is produced commercially via synthetic elongation from alpha linolenic acid (ALA) or stearidonic acid (STA), which is an expensive and difficult process (Kuklev DV et al (2006) Chemistry and Physics of Lipids 144(2):172).
  • ALA alpha linolenic acid
  • STA stearidonic acid
  • PPL perhaps after removal of abundant saturated and monounsaturated fatty acids using a lipase that doesn't hydrolyse DPA, it might be possible to enable the rapid isolation of DPA from seal oil or other DPA containing oil for commercial purposes.
  • porcine pancreatic lipase for a broad range of polyunsaturated fatty acids was investigated via hydrolysis of canola, anchovy and seal oils.
  • a combination of GC- FID and 13 C NMR was used to show that PPL discriminates against some polyunsaturated fatty acids regardless of their chain lengths, number of double bonds and position on the glycerol backbone.
  • PPL found ALA and DPA better substrates as it hydrolysed them more rapidly, while STA, EPA and DHA were highly discriminated against. Therefore, PPL enabled partial separation of DPA from EPA and DHA.

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Abstract

La présente invention concerne l'utilisation d'une lipase pancréatique pour séparer l'acide n-3 docosapentaénoïque (DPA) d'autres acides gras oméga-3 présents dans une huile contenant des acides gras oméga-3 et des utilisations de n-3 DPA enrichi selon le procédé de l'invention.
PCT/AU2014/000825 2013-08-20 2014-08-20 Séparation d'acides gras oméga-3 Ceased WO2015024055A1 (fr)

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US9474725B1 (en) 2014-06-11 2016-10-25 Poviva Tea, Llc Food and beverage compositions infused with lipophilic active agents and methods of use thereof
WO2017197453A1 (fr) * 2016-05-17 2017-11-23 Deakin University Compositions de glycérides d'acides gras polyinsaturés du type oméga-3 micro-encapsulés et leurs procédés de préparation
CN110029133A (zh) * 2019-03-12 2019-07-19 自然资源部第三海洋研究所 一种分离dha藻油中饱和脂肪酸和不饱和脂肪酸的方法
WO2020033712A1 (fr) * 2018-08-08 2020-02-13 Sobel Brands, LLC Compositions cosmétiques de base et compositions cosmétiques associées
US11311559B2 (en) 2020-04-20 2022-04-26 Poviva Corp. Compositions and methods for enhanced delivery of antiviral agents
JP2024004585A (ja) * 2022-06-29 2024-01-17 日清ファルマ株式会社 ω－３高度不飽和脂肪酸含有組成物の製造方法
NO20220910A1 (en) * 2022-08-25 2024-02-26 Epax Norway As Cetoleic acid composition
US12225913B2 (en) 2015-05-18 2025-02-18 5071, Inc. Cannabis compositions and methods of making the same

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Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9474725B1 (en) 2014-06-11 2016-10-25 Poviva Tea, Llc Food and beverage compositions infused with lipophilic active agents and methods of use thereof
US9839612B2 (en) 2014-06-11 2017-12-12 Poviva Tea, Llc Food and beverage compositions infused with lipophilic active agents and methods of use thereof
US9974739B2 (en) 2014-06-11 2018-05-22 Poviva Tea, Llc Food and beverage compositions infused with lipophilic active agents and methods of use thereof
US12225913B2 (en) 2015-05-18 2025-02-18 5071, Inc. Cannabis compositions and methods of making the same
WO2017197453A1 (fr) * 2016-05-17 2017-11-23 Deakin University Compositions de glycérides d'acides gras polyinsaturés du type oméga-3 micro-encapsulés et leurs procédés de préparation
WO2020033712A1 (fr) * 2018-08-08 2020-02-13 Sobel Brands, LLC Compositions cosmétiques de base et compositions cosmétiques associées
US11285092B2 (en) 2018-08-08 2022-03-29 Sobel Brands, LLC Cosmetic base compositions and associated cosmetic compositions
CN110029133A (zh) * 2019-03-12 2019-07-19 自然资源部第三海洋研究所 一种分离dha藻油中饱和脂肪酸和不饱和脂肪酸的方法
US11311559B2 (en) 2020-04-20 2022-04-26 Poviva Corp. Compositions and methods for enhanced delivery of antiviral agents
JP2024004585A (ja) * 2022-06-29 2024-01-17 日清ファルマ株式会社 ω－３高度不飽和脂肪酸含有組成物の製造方法
NO20220910A1 (en) * 2022-08-25 2024-02-26 Epax Norway As Cetoleic acid composition
NO348700B1 (en) * 2022-08-25 2025-05-05 Epax Norway As Cetoleic acid composition

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