WO2008093378A1 - Processus d'enrichissement enzymatique sélectif d'un mélange contenant des oméga trois - Google Patents

Processus d'enrichissement enzymatique sélectif d'un mélange contenant des oméga trois Download PDF

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Publication number
WO2008093378A1
WO2008093378A1 PCT/IT2008/000051 IT2008000051W WO2008093378A1 WO 2008093378 A1 WO2008093378 A1 WO 2008093378A1 IT 2008000051 W IT2008000051 W IT 2008000051W WO 2008093378 A1 WO2008093378 A1 WO 2008093378A1
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WO
WIPO (PCT)
Prior art keywords
process according
mixture
range
lipase
esters
Prior art date
Application number
PCT/IT2008/000051
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English (en)
Inventor
Giovanni Cotticelli
Raul Salvetti
Original Assignee
Adorkem Technology Spa
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Adorkem Technology Spa filed Critical Adorkem Technology Spa
Publication of WO2008093378A1 publication Critical patent/WO2008093378A1/fr

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    • 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
    • C12P7/6436—Fatty acid esters
    • C12P7/6445—Glycerides
    • C12P7/6458—Glycerides by transesterification, e.g. interesterification, ester interchange, alcoholysis or acidolysis
    • 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
    • C12P7/6436—Fatty acid esters
    • C12P7/6445—Glycerides
    • C12P7/6472—Glycerides containing polyunsaturated fatty acid [PUFA] residues, 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
    • 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
    • C12P7/6436—Fatty acid esters

Definitions

  • the present invention has as object a process of selective enzymatic enrichment for the preparation of omega-3 and/or their esters; the process can be carried out starting from mixtures of natural oils where the omega-3 are already present in the form of esters.
  • omega-3 acids or more simply “omega-3”, it is intended the polyunsaturated fatty acids having a double bond in position ⁇ -3; they normally have from 18 to 22 carbon atoms and from 3 to 6 unsaturations.
  • the most common omega-3 are ⁇ -linolenic acid (C18:3), moroctic acid (C18:4), eicosatetraenoic acid (C20:4), eicosapentaenoic acid (C20:5; commonly called EPA), eneicosapentaenoic acid (C21:5), clupanodonic acid (C22:5) and docosahexaenoic acid (C22:6; commonly called DHA).
  • C18:3 ⁇ -linolenic acid
  • moroctic acid C18:4
  • eicosatetraenoic acid C20:4
  • eicosapentaenoic acid C20:5; commonly called EPA
  • omega-3 and their esters are normally employed in the food industry and in the pharmaceutical industry, in particular in the treatment and/or prevention of cardiovascular diseases and/or events such as heart attacks.
  • omega-3 and related alkyl esters are described in different patent documents and is known in the literature.
  • patent application WO870389 a purification process is described from marine oil by means of fractionation by crystallisation in the presence of urea and subsequent molecular distillation. Such procedure permits obtaining a mixture of fatty acids with total EPA and DHA content equal to about. 80-90% by weight.
  • the EPA and DHA content in the starting mixture constitutes an important factor, to be controlled, which affects the yield and quality of the process.
  • WO0073254 use is described of a lipase capable of giving selective transesterification and ethanolysis in the presence of alcohol alkoxide on natural fish oil.
  • Such process which uses subsequent molecular distillation steps, permits isolating mixtures rich with DHA or EPA (equal to about 80% by weight) in the form of esters.
  • EP 1300470 and WO2004043894 describe the use of lipases capable of giving rise to enrichment reactions of the PUFA fatty acid mixtures.
  • the yields obtainable with the processes known up to now are small and hard to reproduce, also due to the variability of the raw material that leads to the use of different purification and enrichment steps.
  • the object of the present invention is that of providing a process for preparing mixtures having a high omega-3 acid content, with a selective enrichment technique obtained through enzymatic hydrolysis which is free of the drawbacks of the prior art process.es.
  • mixtures having a high content of omega-3 acids it is intended mixtures of fatty acids and/or their esters having an omega-3 acid ester content, preferably ethyl esters, greater than 80% by weight; the esters are preferably C 1 -C 4 alcohol esters, having from 1 to 3 hydroxy groups.
  • the process according to the present invention is aimed for the preparation of mixtures in accordance with the European Pharmacopoeia, or rather mixtures having:
  • the starting mixture is preferably composed of natural triglyceride mixtures, such as natural oils; triglyceride mixtures can also be used in which the omega-3, in particular DHA and EPA, are already present as ethyl esters.
  • the natural oils can be chosen from among fish oils, algae oils, mushroom oils, microorganism oils and/or vegetable oils.
  • the reaction is carried out by using mixtures which contain between 40 and 70% by weight of EPA and DHA esters and in which the EPA/DHA ratio is in the ' range of 3 - 0.5, preferably in the range of 1.5 - 0.9.
  • the method provides for the use of enzymes belonging to the hydrolase class, specifically to the lipase class coming from different microbe sources; among the preferred lipases, those from Pseudomonas Fluoresc ⁇ ns, Pseudomonas cepacea and Candida antartica are particularly indicated.
  • the enzymes are preferably used in purified form, both free and immobilised on solid support, or in crystallised enzyme form, or they can be used in raw form, for example contained in cell pastes or in whole cells.
  • Immobilisations on solid support are particularly used, achieved by means of absorption methods, or ion interaction methods, or covalent interaction methods.
  • interactions are used of covalent type by means of the use of polymer resins functionalised with simply epoxy groups and/or suitably derivatised and/or stabilised.
  • the mixture containing the triglycerides, or the ethyl esters of the fatty acids, or the mixture of the two is placed in an aqueous environment; the lipase is added to the medium, and the reaction is carried out until the desired concentration of omega-3 is reached, in particular the desired concentration of EPA and DHA.
  • the esters of the fatty acids of interest are isolated from the reaction medium by means of distillation or chemical separation or chromatography.
  • the subsequent conversion into ethyl esters can be made by transesterification, according to methods well known in the art.
  • the reaction solvent is water, by itself or in a mixture with organic solvents miscible therewith, in the presence of a buffer or without a buffer, the pH preferably being in the range of about 4 to about 8 corrected by means of an alkaline salt and/or earth alkaline.
  • the solvent is normally used in a quantity in the range of 1 - 50 volumes, preferably between 1 - 5 volumes, per mixture volume to be treated.
  • the bioreaction is normally conducted at a temperature in the range of 0 - 100° C and its duration is normally in the range of 2 - 96 hours, as a function of the omega-3 concentration that one wishes to obtain.
  • the bioreaction is conducted at a temperature in the range of 20 - 35°C and has a duration in the range of 5 - 15 hours.
  • the lipase is normally used in a quantity in the range of 0.2 - 2.0 parts by weight with respect to the starting ethyl ester mixture, preferably in a quantity in the range of 0.2 - 1 parts by weight.
  • the mixture is subjected to extraction or distillation.
  • the product can be purified on resins or silica gel or through specific chromatographic techniques.
  • the mixture thus obtained can be subjected to new enrichment cycles, depending on the concentration of omega-3 in the starting mixture and the desired concentration.
  • This process is very advantages, since is permits selectively increasing the content of DHA and EPA and obtaining mixtures of fatty acids and/or their esters rich with omega-3, or with an omega-3 content equal to or greater than 80% by weight of the mixture and with a EPA/DHA ratio in the range of 0.9 - 1.5.
  • the technique provides for the use of a limited number of purification steps, which permits improved yields and process quality.
  • the ecological and environmental impact is improved, thus avoiding the use of solvents.
  • Pseudomonas cepacea is loaded and the mixture is left under stirring for 5 hours at a temperature of 25 ° C.
  • the pH is maintained constant by the addition of 0.1 N
  • Candida antartica is loaded and the mixture is left under stirring for 24 hours at a temperature of 25 ° C.
  • the pH is maintained constant by the addition of 0.1 N

Abstract

L'invention concerne un procédé d'enrichissement enzymatique sélectif destiné à préparer des oméga trois à partir d'huiles naturelles contenant du DHA (acide docosahexanoïque) et du DPA (acide eicosapentanoïque), qui peuvent aussi, si on le souhaite être dérivées sous une forme d'ester. Cette technique permet l'utilisation d'enzymes spécifiques appartenant à la classe des lipases, qui sont capables de sélectivement hydrolyser ces mélanges naturels et qui, après une purification adaptée, sont capables de donner des mélanges enrichis à l'OPA et au DHA sous forme de triglycéride et sous forme d'ester.
PCT/IT2008/000051 2007-01-31 2008-01-29 Processus d'enrichissement enzymatique sélectif d'un mélange contenant des oméga trois WO2008093378A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
IT000157A ITMI20070157A1 (it) 2007-01-31 2007-01-31 Processo di arricchimento enzimatico di una miscela contenente omega 3
ITMI2007A000157 2007-01-31

Publications (1)

Publication Number Publication Date
WO2008093378A1 true WO2008093378A1 (fr) 2008-08-07

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IT (1) ITMI20070157A1 (fr)
WO (1) WO2008093378A1 (fr)

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
ITMI20100961A1 (it) * 2010-05-27 2011-11-28 Erredue Spa Miscele ricche in esteri di acidi grassi omega-3, loro composizioni e loro processo di preparazione
WO2012087153A1 (fr) * 2010-12-23 2012-06-28 Marine Bioproducts As Enrichissement d'huiles d'animaux marins en acides gras oméga 3 polyinsaturés par une hydrolyse catalysée par une lipase
EP2578691A4 (fr) * 2010-05-28 2016-11-23 Nippon Suisan Kaisha Ltd Procédé de production d'huile ou de graisse contenant un acide gras fortement insaturé au moyen d'une lipase
CN106673998A (zh) * 2017-03-14 2017-05-17 山东禹王制药有限公司 一种从鱼油中提取epa＆dha的循环气提精馏系统
US20190071618A1 (en) * 2015-10-05 2019-03-07 Dsm Ip Assets, B.V. Oil compositions and methods of making

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS63105683A (ja) * 1986-10-22 1988-05-10 Japanese Res & Dev Assoc Bio Reactor Syst Food Ind 高度不飽和脂肪酸の分離方法
JPH02303492A (ja) * 1989-05-19 1990-12-17 Nippon Oil & Fats Co Ltd 長鎖高度不飽和脂肪酸グリセリドの濃縮法
JPH0319694A (ja) * 1989-06-16 1991-01-28 Nippon Oil & Fats Co Ltd ドコサヘキサエン酸グリセリドの濃縮法
WO1996026287A1 (fr) * 1995-02-24 1996-08-29 Goemar S.A. Procedes enzymatiques d'enrichissement en acides gras polyinsatures
WO1999015689A1 (fr) * 1997-09-24 1999-04-01 Enzymothec Ltd. Complexe surfactant-lipase immobilise sur une matrice insoluble
EP1300470A1 (fr) * 2000-07-13 2003-04-09 Nippon Suisan Kaisha, Ltd. Proc d de production de glyc rides l'aide de lipases
WO2003094625A1 (fr) * 2002-05-08 2003-11-20 Danmarks Tekniske Uni Technica Procede enzymatique simple en deux etapes destine a augmenter le contenu en acides gras polyinsatures d'huiles de poisson

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS63105683A (ja) * 1986-10-22 1988-05-10 Japanese Res & Dev Assoc Bio Reactor Syst Food Ind 高度不飽和脂肪酸の分離方法
JPH02303492A (ja) * 1989-05-19 1990-12-17 Nippon Oil & Fats Co Ltd 長鎖高度不飽和脂肪酸グリセリドの濃縮法
JPH0319694A (ja) * 1989-06-16 1991-01-28 Nippon Oil & Fats Co Ltd ドコサヘキサエン酸グリセリドの濃縮法
WO1996026287A1 (fr) * 1995-02-24 1996-08-29 Goemar S.A. Procedes enzymatiques d'enrichissement en acides gras polyinsatures
WO1999015689A1 (fr) * 1997-09-24 1999-04-01 Enzymothec Ltd. Complexe surfactant-lipase immobilise sur une matrice insoluble
EP1300470A1 (fr) * 2000-07-13 2003-04-09 Nippon Suisan Kaisha, Ltd. Proc d de production de glyc rides l'aide de lipases
WO2003094625A1 (fr) * 2002-05-08 2003-11-20 Danmarks Tekniske Uni Technica Procede enzymatique simple en deux etapes destine a augmenter le contenu en acides gras polyinsatures d'huiles de poisson

Non-Patent Citations (6)

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DATABASE WPI Week 198824, Derwent World Patents Index; AN 1988-166137, XP002485378 *
DATABASE WPI Week 199105, Derwent World Patents Index; AN 1991-033522, XP002485376 *
DATABASE WPI Week 199110, Derwent World Patents Index; AN 1991-070270, XP002485377 *
HALLDORSSON ARNAR ET AL: "LIPASE SELECTIVITY TOWARD FATTY ACIDS COMMONLY FOUND IN FISH OIL", EUROPEAN JOURNAL OF LIPID SCIENCE AND TECHNOLOGY, WILEY VCH VERLAG, WEINHEIM, DE, vol. 106, no. 2, 1 February 2004 (2004-02-01), pages 79 - 87, XP009073126, ISSN: 1438-7697 *
OKADA ET AL: "Production of n-3 polyunsaturated fatty acid concentrate from sardine oil by lipase-catalyzed hydrolysis", FOOD CHEMISTRY, ELSEVIER SCIENCE PUBLISHERS LTD, GB, vol. 103, no. 4, 27 March 2007 (2007-03-27), pages 1411 - 1419, XP022003148, ISSN: 0308-8146 *
YUKIHISA TANAKA ET AL: "CONCENTRATION OF DOCOSAHEXAENOIC ACID IN GLYCERIDE BY HYDROLYSIS OF FISH OIL WITH CANDIDA CYLINDRACEA LIPASE", JOURNAL OF THE AMERICAN OIL CHEMISTS' SOCIETY, SPRINGER, BERLIN, DE, vol. 69, no. 12, 1 December 1992 (1992-12-01), pages 1210 - 1214, XP000330808, ISSN: 0003-021X *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
ITMI20100961A1 (it) * 2010-05-27 2011-11-28 Erredue Spa Miscele ricche in esteri di acidi grassi omega-3, loro composizioni e loro processo di preparazione
EP2578691A4 (fr) * 2010-05-28 2016-11-23 Nippon Suisan Kaisha Ltd Procédé de production d'huile ou de graisse contenant un acide gras fortement insaturé au moyen d'une lipase
US10138502B2 (en) 2010-05-28 2018-11-27 Nippon Suisan Kaisha, Ltd. Method for producing oil containing polyunsaturated fatty acid using lipase
WO2012087153A1 (fr) * 2010-12-23 2012-06-28 Marine Bioproducts As Enrichissement d'huiles d'animaux marins en acides gras oméga 3 polyinsaturés par une hydrolyse catalysée par une lipase
US20190071618A1 (en) * 2015-10-05 2019-03-07 Dsm Ip Assets, B.V. Oil compositions and methods of making
CN106673998A (zh) * 2017-03-14 2017-05-17 山东禹王制药有限公司 一种从鱼油中提取epa＆dha的循环气提精馏系统

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