WO2008093378A1

WO2008093378A1 - Process of selective enzymatic enrichment of a mixture containing omega-3

Info

Publication number: WO2008093378A1
Application number: PCT/IT2008/000051
Authority: WO
Inventors: Giovanni Cotticelli; Raul Salvetti
Original assignee: Adorkem Technology SpA
Current assignee: Adorkem Technology SpA
Priority date: 2007-01-31
Filing date: 2008-01-29
Publication date: 2008-08-07
Anticipated expiration: 2009-07-31
Also published as: ITMI20070157A1

Abstract

A selective enzymatic enrichment procedure is described for preparing omega-3 starting from natural oils containing DHA (docosahexaenoic acid) and EPA (eicosapentaenoic acid), also suitably derivatised in ester form. The technique provides for the use of specific enzymes belonging to the lipase class which are capable of selectively hydrolysing such natural mixtures and that, after suitable purification, are capable of giving rise to mixtures enriched with EPA and DHA both in triglyceride form and ester form.

Description

Process of selective enzymatic enrichment of a mixture containing omega-3

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.

PRIOR ART

With the term "omega-3 acids" or more simply "omega-3", it is intended the polyunsaturated fatty acids having a double bond in position ω-3; they normally have from 18 to 22 carbon atoms and from 3 to 6 unsaturations. The most common omega-3 are α-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).

The omega-3 and their esters, in particular the ethyl 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.

The mixture commonly employed for such purposes is defined as ethyl esters of omega-3 90 acids and is characterised by (European Pharmacopoeia 5.0):

• a minimum content of 80% EPA and DHA ethyl esters, with a minimum of 40% EPA ethyl esters and a minimum of 34% DHA ethyl esters;

• a minimum content of 90% omega-3 ethyl esters.

The preparation of omega-3 and related alkyl esters is described in different patent documents and is known in the literature. In 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. By this technique, 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.

Over the years, obtainment processes have been described of such mixtures by means of the aid of enzymes. Jn patent application WO9626287, a chemo- enzymatic approach is described which provides for a transesterification reaction with the aid of specific and non-specific lipases; such approach permits obtaining a triglyceride mixture with 60% polyunsaturated fatty acids (PUFA) starting from a mixture which contains only 30% thereof. For the obtainment of a good PUFA purity level, however, saponification and crystallisation steps are necessary before the enzymatic transesterification.

In 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.

Moreover, these processes have the disadvantage of using chemical reagents, with the formation of potential impurities that can be damaging for the organism.

DESCRIPTION OF THE INVENTION

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.

With the expression "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 C₁-C₄ alcohol esters, having from 1 to 3 hydroxy groups. In particular, the process according to the present invention is aimed for the preparation of mixtures in accordance with the European Pharmacopoeia, or rather mixtures having:

• a minimum content of 90% omega-3 ethyl esters.

It was in fact surprisingly found that by using specific enzymes belonging to the lipase class, until now used for the transesterifϊcation reactions, one is able to carry out a selective hydrolysis reaction on the ester residues of saturated fatty acids or unsaturated fatty acids having a short chain, or less than 18 carbon atoms, maintaining the esters of the larger chain fatty acids unaltered, which are then isolated from the reaction medium by simple extraction or by distillation. 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. Preferably, 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 Fluorescβns, 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.

Preferably, 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.

According to the method of the present invention, 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. Subsequently, the esters of the fatty acids of interest are isolated from the reaction medium by means of distillation or chemical separation or chromatography. In the case of starting mixtures based on omega-3 triglycerides, 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. Preferably, 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.

In order to separate the esters from the hydrolysed acids, the mixture is subjected to extraction or distillation. Alternatively, 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. In addition, by using preferably water as reaction solvent, the ecological and environmental impact is improved, thus avoiding the use of solvents.

The following examples are given as merely illustrative and non-limiting examples of the invention.

Example 1 :

In a 25 ml reactor, 1 g is loaded of a mixture of ethyl esters of fatty acids containing EPA and DHA in a 40/20 by weight ratio, dissolved in a 1 M pH 7 phosphate buffer. 0.5 g of lipase from Pseudomonas Fluorescens 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 NaOH.

The above is distilled under vacuum and a mixture is obtained with EPATDHA ratio equal to 2.5 and an EPA+DHA content equal to 74 % by weight.

Example 2:

In a 25 ml reactor, 1 g is loaded of ethyl esters of fatty acids EPA and DHA in

40/20 ratio, dissolved in a 1 M pH 7 phosphate buffer. 0.5 g of lipase from

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

NaOH.

The above is distilled under vacuum and a mixture is obtained with EPA/DHA ratio equal to 3.0 and an EPA+DHA content equal to 77 % by weight.

Example 3:

In a 25 ml reactor, 1 g is loaded of ethyl esters of fatty acids EPA and DHA in

40/20 ratio, dissolved in a 1 M pH 7 phosphate buffer. 0.5 g of lipase from

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

NaOH.

The above is distilled under vacuum and a mixture is obtained with EPA/DHA ratio equal to 1.8 and an EPA+DHA content equal to 78 % by weight.

Example 4:

In a 25 ml reactor, 1 g is loaded of ethyl esters of fatty acids EPA and DHA in

40/20 ratio, dissolved in a 1 M pH 7 phosphate buffer. 1 g of lipase from Candida antartica is loaded, immobilised on Eupergit C,^" 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 NaOH. The above is distilled under vacuum and a mixture is obtained with EPA/DHA ratio equal to 1.5 and an EPA+DHA content equal to 82 % by weight.

Claims

1. Process for increasing the omega-3 acid ester content in a mixture of fatty acids and/or their esters, characterised in that said mixture is subjected to hydrolysis in the presence of a lipase.

2. Process according to claim 1, characterised in that said esters are C₁-C₄ alcohol esters, having from 1 to 3 hydroxy groups.

3. Process according to claim 1, characterised in that said esters are ethyl esters and/or triglycerides.

4. Process according to claim 1, characterised in that said mixture is a natural oil or a mixture of natural oils.

5. Process according to claim 4, characterised in that said natural oil is chosen from among fish oils, algae oils, mushroom oils, microorganism oils and/or vegetable oils.

6. Process according to claim 1, characterised in that said mixture contains between 40% and 70% by weight of EPA and DHA esters.

7. Process according to claim 1, characterised in that the EPA/DHA ratio in said mixture is in the range of 3 - 0.5, preferably in the range of 1.5 - 0.9.

8. Process according to claim 1, characterised in that said lipase is a lipase from Pseudomonas Fluorescens, Pseudomonas cepacea and/or Candida antartica.

9. Process according to claim 1, characterised in that said hydrolysis is carried out in water or in a mixture of water and organic solvents miscible therewith.

10. Process according to claim 9, characterised in that the water or said mixture of water and organic solvents miscible therewith is in a quantity in the range of 1 - 50 volumes, preferably 1-5 volumes, per mixture volume.

1 1. Process according to claim 1, characterised in that the lipase is in a quantity in the range of 0.2 - 2.0 parts by weight with respect to the mixture, preferably in a quantity in the range of 0.2 - 1 parts by weight.

12. Process according to claim 1, characterised in that said hydrolysis is carried out at a temperature in the range of 0 - 100° C, preferably in the range of 20 - 35°C, and its duration is in the range of 2 - 96 hours, preferably in the range of 5 - 15 hours.

13. Process according to claim 1, characterised in that the lipase is in purified form or immobilised on a solid support or in crystallised enzyme form.

14. Process according to claim 13, characterised in that the lipase is immobilised on a solid support by means of absorption methods, ion interaction methods or covalent interaction methods.

15. Process according to claim 14, characterised in that the lipase is immobilised on polymer resins.

16. Process according to claim 15, characterised in that said polymer resins are functionalised with epoxy groups.

17. Process according to claims 1-16, characterised in that, after the hydrolysis, said mixture is subjected to hydrolysis, distillation, chromatography and/or purification on resins and/or silica gel.