WO2003094625A1

WO2003094625A1 - A facile two-step enzyme process for increasing the content of polyunsaturated fatty acids in fish oil

Info

Publication number: WO2003094625A1
Application number: PCT/DK2003/000296
Authority: WO
Inventors: Xuebing Xu
Original assignee: Danmarks Tekniske Universitet
Current assignee: Danmarks Tekniske Universitet
Priority date: 2002-05-08
Filing date: 2003-05-06
Publication date: 2003-11-20
Anticipated expiration: 2004-11-08
Also published as: AU2003222739A1

Abstract

A facile two-step enzyme process for the manufacture of fish oil product comprising triglycerides enriched in polyunsaturated fatty acids wherein the total content of polyunsaturated fatty acids is more than 55 wt%.

Description

A facile two-step enzyme process for increasing the content of polyun- saturated fatty acids in fish oil

FIELD OF THE INVENTION

The invention relates to nutritional foods, more particular fish oil and even more particular fish oil, where the triglycerides are enriched in the content of polyunsaturated fatty acids (PUFA), a process for modifying the fish oil and products containing the modified fish oil.

BACKGROUND OF THE INVENTION

All the citations in the following are from Babcock, et al. EPA: anti-inflammatory n-3 fat with potential clinical applications, Nutrition 16: 1116-1118, 2000; and Linko and Hayakawa, DHA: A valuable nutraceutical?, Trends in Food Science and Technology, 7: 59-63, 1996.

The omega-3 fatty acids, including eicosapentaenoic acid (EPA), docosapen- taenoic acid (DPA), and docosahexaenoic acid (DHA) also termed polyun- saturated fatty acids (PUFA), have been shown to have beneficial effects on the cardiovascular system and to have anti-inflammatory properties. Behavioural pharmacological studies of the central nervous system have been performed to determine the biological role of DHA. Several reports confirm the superior learning ability of rats fed DHA in a maze learning test and results suggest that fish oil, especially DHA, plays a very important role in the brain. DHA is essential for the growth and functional development of the brain in infants. DHA is also required for maintenance of normal brain function in adults. The inclusion of plentiful DHA in the diet improves learning ability, whereas deficiencies of DHA are associated with deficits in learning. DHA is taken up by the brain in preference to other fatty acids. The turnover of DHA in the brain is very fast, more so than is generally realized. The visual acuity of healthy, full-term, formula-fed infants is increased when their formula in- eludes DHA. During the last 50 years, many infants have been fed formula diets lacking DHA and other omega-3 fatty acids. DHA deficiencies are associated with foetal alcohol syndrome, attention deficit hyperactivity disorder, cystic fibrosis, phenyl-ketonuria, unipolar depression, aggressive hostility, and adrenoleukodystrophy. Decreases in DHA in the brain are associated with cognitive decline during aging and with onset of sporadic Alzheimer disease.

The leading cause of death in western nations is cardiovascular disease. Epidemiological studies have shown a strong correlation between fish consumption and reduction in sudden death from myocardial infarction. The reduction is approximately 50% with 200 mg/day of DHA from fish. DHA is the active component in fish. Not only does fish oil reduce triglycerides in the blood and decrease thrombosis, but it also prevents cardiac arrhythmias. The association of DHA deficiency with depression is the reason for the robust positive correlation between depression and myocardial infarction.

It is thus reported that fish oil decreases the proliferation of tumour cells. It is also been reported that DHA has a positive effect on diseases such as hyper- tension, arthritis, atherosclerosis, depression, adult-onset diabetes mellitus, myocardial infarction, thrombosis, and some cancers.

EPA and DPA are further n-3 polyunsaturated fatty acids existing in most fish oils. They are usually converted into DHA in the metabolic system. This con- verting process is much faster than other shorter chain n-3 fatty acids such as linolenic acid. Therefore, EPA and DPA are also used as nutritional fatty acids as DHA. EPA itself is the precursor of the series 3 prostaglandins, which are potent anti -inflammatory and anticlotting agents. It has the following functions: reducing arthritic inflammation and pain, elevating HDL levels, reducing abnormal platelet aggregation, and lowering triglyceride levels. It is reported that EPA has significant beneficial effects on diabetic neuropathy and serum lipids as well as other diabetic complications such as nephropathy and macroangiopathy. It also seems that EPA, and not DHA, is the fatty acid primarily responsible for the triglyceride-lowering effect of fish oil in rats.

The enrichment of n-3 polyunsaturated fatty acids including EPA, DPA, and DHA from marine oils such as fish oils or oils produced by bacteria have long been a target for academia and industry as well. Many products have been produced in the markets. The methods for the production include chroma- tographic methods, distillation methods, low temperature crystallization methods, supercritical fluids extraction methods, urea complexation methods, salt solubility method, extraction with aqueous silver nitrate solutions, io- dolactonization method, low temperature acetone extraction methods, and enzyme methods. A general review may be found in Shahidi and Wa- nasundara, Omega-3 fatty acid concentrates: nutritional aspects and production technologies, Trends in Food Science and Technology 9:230-240, 1998. and Medina et al. Downstream processing of algal polyunsaturated fatty acids, Biotechnology Advances 16: 517-580, 1998.

GB2218984 discloses a process essentially based on the use of molecular distillation for the preparation of EPA and DHA fatty acids and of their ethyl esters. By this process, complexes constituted by EPA and DHA or by their ethyl esters with a total concentration of not lower than 90% can be obtained. EP0292846 discloses a process for the extraction of EPA and DHA ethyl esters from fish oils by means of transesterification with ethanol and H₂SO₄ and two-step molecular distillation. These processes can only produce the mix- ture of polyunsaturated fatty acids in the form of free acid or their ethyl esters. To produce their glycerides, especially triglycerides, chemical esterifica- tion or interesterification has been employed under high temperature (up to 175 °C) with acidic or base catalysts (US5149851). Under these conditions, fatty acids like EPA and DHA will undergo an isomerisation of the double bonds, which leads to the loss of nutritional functions and other anti- nutritional effects. Due to these reasons, chemical synthesis of triglycerides containing high content of polyunsaturated fatty acids such as EPA and DHA has gradually lost its relevance and applications.

Enzymatic processing of fish oils or other related oils has also been ad- dressed in the patent literature. JP09238693-A discloses a method for purification of polyunsaturated fatty acids by esterifying the mixture of fatty acids produced from oils and fats containing unsaturated fatty acids with linear higher alcohols with lipases. This method uses the specificity of lipases having less reactivity towards some polyunsaturated fatty acids such as EPA and DHA. Mainly other common fatty acids are esterified. WO9524459 discloses a method using enzymatic alcoholysis to purify EPA and DHA. The oils and fats containing EPA and DHA are subjected to a reaction with alcohols under lipase catalysis. After separation, the residual fraction containing glycehdes are recovered to contain higher content of EPA and DHA. JP07203979-A discloses a method using a Geotrichum sp. Lipase to concentrate the highly unsaturated fatty acids by hydrolysis. After hydrolysis, a glyceride fraction can be recovered to contain high content of EPA and DHA. JP06287594-A discloses a method for producing triglycerides containing DHA at the sn-2 position by reaction between an oil and fat and a fatty acid or its ethyl esters with the catalysis of sn-1 ,3 lipases. The triglycerides formed contain DHA primarily at the sn-2 position. JP06116585-A discloses a method for concentrating DHA by alcoholysis in the presence of lipases to remove other fatty acids as lower alkyl esters. This method also uses the specificity of lipases towards DHA. The resulting glycerides, which contain higher content of DHA, can be recovered and further modified to ethyl esters by chemical reactions with ethanol. JP05331105-A discloses a method for preparing DHA triglyceride by esterification between DHA and glycerol under the catalysis of a lipase. JP03019694-A discloses a method for producing a concentrate of DHA glycerides by hydrolysing fat and oil containing long chain unsaturated fatty acids with a lipase derived from Candida rugosa. The resulting glycerides can be obtained by alkaline deoxidation or steam distillation. This glyceride fraction is claimed to contain a higher content of DHA. JP03019693-A dis- closes a method for producing a concentrate of DHA glycerides by hydrolysing fat and oil containing long chain unsaturated fatty acids in the presence of an immobilized lipase from Candida cylindracea. The resulting glycerides can be obtained by alkaline deoxidation or steam distillation. This glyceride frac- tion is claimed to contain a higher content of DHA. JP02025447-A discloses a method for preparing highly unsaturated fatty acids by hydrolysis of fish oil using a lipase, separating the fatty acids and glyceride components, performing an esterification, forming an urea adduct followed by purification. JP58165796-A discloses a method for producing a concentrate of EPA and DHA glycerides by hydrolysing fat and oil containing long chain unsaturated fatty acids with Candida cylindracea lipase. WO98/18952 discloses a process for producing fats containing highly unsaturated fatty acids comprising selectively concentrated DHA. Lipases having an elevated capability of selectively concentrating DHA are used. All these disclosures teach how to obtain free PUFA or their methyl or ethyl esters or partial glycerides for use for nutritional purposes.

However, experimental work has shown that EPA and DHA in the form of triglycerides is better absorbed (Lawson and Hughes, Human absorption of fish oil fatty acids as triacylglycerols, free acids, and ethyl esters, Biochem. Biophys. Acta 152: 328-335, 1988). Therefore, for nutritional purposes, it would be an advantage if the beneficial n-3 products were made in the glyceride form instead of EPA and DHA ethyl esters.

WO 97/19601, EP 0 862 369 and US6020020 disclose compositions based on fish oil. The fish oil concentrates have no less than 40% of long chain polyunsaturated fatty acids, less than 20% of saturated fatty acids, less than 15% oleic acid and with a weight ratio DHA/EPA=0.5-3.0. The process for the production involves the following steps: subjecting the fish oil to an en- zymatic conversion (hydrolysis or alcoholysis), removing free fatty acids or esters by distillation from the conversion product, subjecting the product of the above to enzymatic hydrolysis to free fatty acids, using 1 ,3 specific lipase or partial glyceride-specific lipase, washing the glycerol and drying the product, and re-esterifying the dried product into triglycerides. This is the only process known by the inventor starting from fish oil and ending up with a different oil containing higher amount of polyunsaturated fatty acids.

The above-mentioned processes are primarily for the concentration of DHA. The reason for this is that the lipases used in all the above applications have not the same specificity towards both EPA and DHA. Usually lipases have higher specificity (less selectivity) towards EPA than towards DHA (Shahidi and Wanasundara, Omega-3 fatty acid concentrates: nutritional aspects and production technologies, Trends in Food Science and Technology 9:230-240, 1998; Haraldsson and Kristinsson, J. Am. Oil Chem. Soc. 75, 1551-1556; Pedersen and Hølmer, ibid. 72, 239-243, 1995; Hill et al., ibid. 67, 561-564, 1990; Shimada et al., ibid. 74, 1441-1446, 1997. Tanaka et al., ibid. 69, 1210-1214, 1992). Therefore, in any type of the above-discussed reactions, DHA is taking part in the transesterification to a lesser degree than EPA. As a consequence, EPA in the original oils and fats are largely lost or not recovered, either on purpose or as the result of the enzymatic process. Since EPA is also a n-3 fatty acid and highly nutritional as discussed above, it would be advantageous if EPA could be recovered into the final products as well. This will definitely increase the efficiency and outcome of the processes and reduce the loss of nutritional parts of the oils. The same applies to DPA because it is highly nutritional as well even though the amount of DPA is usually low in fish oils.

Moreover, in order to supply a more balanced nutritional product, it would be advantageous if other nutritional fatty acids such as gamma-linolenic acid, conjugated linoleic acid, or arachidonic acid could be incorporated into the fish oil product, if desired. OBJECTS OF THE INVENTION

One object of the invention is to increase the content of nutritional valuable long chain polyunsaturated fatty acids in fish oil products by recovering the content of EPA, DHA, DPA as well as other essential fatty acids from the original fish oils into the final fish oil products.

A further object is to provide an enzymatic process for increasing the content of nutritional valuable polyunsaturated fatty acids in fish oil products.

SUMMARY OF THE INVENTION

The present invention provides an enzymatic process for increasing the content of long chain polyunsaturated fatty acids (PUFA), the omega-3 fatty acids, including eicosapentaenoic acid (EPA), docosapentaenoic acid (DPA), and docosahexaenoic acid (DHA) and other essential or functional fatty acids in fish oil production in the form of triglycerides.

In summary, the process involves the following steps. Firstly, the acylglycerols of fish oils are hydrolysed or alcoholysized employing a sn-1 ,3 specific lipase.

Secondly other fatty acids than DHA, DPA and EPA are removed through short path distillation or membrane separation after separation of water and enzyme.

Thirdly, esterification will take place in a reaction step between partial glycerides and the free DHA, DPA, EPA and other fatty acids not removed in the above step employing a non-specific enzyme.

The final content of PUFA in the final product is more than 55% by weight and the final content of triglycerides is no less than 70% by weight. A recovery of 94 % of the valuable PUFA shows that it is a very efficient method. Moreover, it is possible with this method to increase the content of EPA in triglycerides of fish oil. EPA is normally lost or not recovered in the existing processes.

FIGURES Figure 1 shows a process scheme for the production of a fish oil product enriched in nutritional valuable PUFA.

PUFA: polyunsaturated fatty acids such as EPA, DHA, DPA, gamma- linolenic, conjugated linoleic acid, arachidonic acid

FA: free fatty acids

AE: Alky I esters

DEFINITIONS

By "PUFA" is meant polyunsaturated fatty acids such as the omega-3 fatty acids, including eicosapentaenoic acid (EPA), docosapentaenoic acid (DPA), and docosahexaenoic acid (DHA) as well as gamma-linolenic acid, conjugated linoleic acid, or arachidonic acid.

By "triglycerides enriched in polyunsaturated fatty acids (PUFA)" is understood the overall content of PUFA, which is present in the form of triglycerides.

By "FA" is meant free fatty acids and by "AE" alkyl esters.

By "partial glycerides" is meant mono- and di-glycerides.

By "fish oil" is meant an oil originating from fish or any other oil containing EPA and/or DHA and/or DPA. By "marine oils" is meant fish oils or oils produced from other marine animal and plants or oils produced by microoganisms such as bacteria or algae, which contain significant amount (higher than 10%) of polyunsaturated fatty acids (including EPA, DHA, or DPA)

DETAILED DESCRIPTION OF THE INVENTION

Due to a still increasing need for fish oil products or similar oil products comprising triglycerides enriched in polyunsaturated fatty acids a new enzymatic process has been developed. Non-limiting examples of other marine oils con- taining EPA, DHA or DPA are for example oils from algae or microbial oils.

Surprisingly, it has been found that with this process it is possible to increase the polyunsaturated fatty acids content in triglycerides of fish oil. Moreover, it is also possible to increase the content of EPA in triglycerides of fish oil, which is normally lost or not recovered in the existing processes.

A two-step enzyme process is used for the modification of fish oils and related oils containing polyunsaturated fatty acids. The process starts from an oil and ends up with a different oil containing a higher content of polyunsatu- rated fatty acids. The loss of EPA, DPA and DHA from the original oil is minimal during the processing. With the special applied process, the EPA can be efficiently recovered together with DHA and DPA into the final product. A process scheme is depicted in Figure 1.

* The process is in summary conducted in the following steps:

1. The oil, which comprises EPA, DPA and DHA, is subjected to a lipase- catalysed hydrolysis or alcoholysis

2. The product of Step 1 is subjected to a treatment for the removal of free fatty acids of less than C₂o carbons or their alkyl esters, either using a separate step of distillation (for example short path distillation) or using an integrated membrane separation during reaction (for example membrane extraction during the enzymatic reaction).

3. The product of Step 2 is subjected to a lipase-catalysed esterification or interesterifi cation optionally with added PUFA under a vacuum system or a pervaporation system to remove water or alcohol.

Advantageously the process results in that up to 94% of the nutritional valuable PUFA in the original oil can be recovered into the final two products [oil product and FA (free fatty acids)/AE (alkyl esters)- product], and the final oil product contains more than 55 % PUFA.

According to the first aspect, the present invention concerns a fish oil product comprising glycerides enriched in polyunsaturated fatty acids wherein the total content of polyunsaturated fatty acids is more than 55 wt%, preferably more than 60 wt%, more preferably more than 65 wt% and most preferably more than 70 wt%. The fish oil product contains at least 70 wt% triglycerides and more preferably at least 80 wt% triglycerides and other glycerides are in the form of partial glycerides. PUFA in the form of triglycerides are provided in order to improve the absorption of PUFA as discussed earlier

The preferred polyunsaturated fatty acids are n-3 polyunsaturated fatty acids, most preferably the n-3 polyunsaturated fatty acids are eicosapentaenoic acid (EPA), docosapentaenoic acid (DPA), and docosahexaenoic acid (DHA).

In yet a preferred embodiment other nutritional valuable PUFA are added to supply a more balanced nutritional product with different nutritional fatty acids. The preferred additional nutritional valuable fatty acids are gamma- linolenic acid, conjugated linoleic acid and arachidonic acid. It may be necessary to add stabilizeres in order to prevent the fish oil from going rancid. In a preferred embodiment of the present invention the fish oil product is therefore stabilized with an effective amount of an oxidation stabilizer known in the art, for example, selected from the group consisting of natural or synthetic tocopherols, propylgallate, TBHQ, BHT, BHA, free radical scavengers, enzymes with anti-oxidant properties and ascorbylesters of fatty acids, and the like.

In another preferred embodiment of the present invention the fish oil product may be used in an food products comprising a fat phase, such as spreads, margarine, cream alternative, infant food, chocolate, confectionery bakery products, sauces, ice-creams, ice-creams coatings, cheese, soups, mayonnaise, dressings, enteral or parenteral products, wherein the fat phase contains fish oil product according to the present invention.

In yet another preferred embodiment the fish oil is used in capsules comprising a filling, encapsulated in an edible coating, wherein the filling consists of a fish oil product according to the present invention alone other together with other oils or ingredients. The capsules may either be hard or soft capsules containing from 0.01 gram to 5 gram, more preferably 0.1 gram - 3 gram and most preferable 0.5 gram to 2.5 gram. The capsules are used as a nutraceu- tical with a daily intake of 1-5 gram depending on the population groups. The capsules may be made from gelatine, hydroxypropyl methylcellulose or the like known to the man skilled in the art.

In a further preferred embodiment the fish oil product according to the present invention is administered as a liquid for nutraceutical purposes with a daily intake of 1-5 mL

According to a second aspect there is provided a process for preparing a fish oil product comprising a) subjecting a fish oil to a enzyme-catalysed hydrolysis or alcoholysis;

b) removing free fatty acids containing less than 20 carbon atoms or their alkyl esters from the product of step a), and

c) subjecting the product of step b) to a enzyme-catalysed esterification or interesterification.

In a preferred embodiment the enzyme in step a) is a lipase. The lipase is preferably selected from Lipozyme RM IM, Pseudomonas sp. lipase, Candida cylindracea lipase, Aspergillus niger lipase, and Geotrichum candidum lipase and the lipase is most preferably Lipozyme RM IM.

In another preferred embodiment the free fatty acids with less than 20 carbon atoms or their alkyl esters are removed by using either a separate step of distillation or using integrated membrane separation. The separate step of distillation is most preferably short path distillation, whereas the integrated membrane separation is the most preferred extraction method during the enzymatic reaction of step a). Short path distillation is an operation with high vacuum and certain temperature based on the boiling property of the separated compounds. Fatty acids with fewer carbons are more volatile at certain conditions. The process may be conducted under the following conditions: evaporator temperature 60-160°C, vacuum 0.0005-0.1 mbar, roller speed 300-500 rpm; preferably evaporator temperature 130-150°C, vacuum 0.0005- 0.005 mbar, roller speed 400 rpm. Membrane separation is based on the molecule size and other related properties. Fatty acids with fewer carbons are also easier to be separated. For a practical process, a membrane inter- phase is installed with the reaction system on one side and the extraction system on the other side. The extraction phase is the same alcohol as used in the reaction system. On the reaction side, 0.5-2 bar pressure, preferably 1 bar, is applied. In yet another preferred embodiment, the enzyme in step c) is a lipase. The lipase is preferably selected from Novozyme 435, Lipozyme TL IM, Rhizopus delemar lipase, Chromobacterium viscosum lipase, Candida rugosa lipase, Pseudomonas sp. lipase, and Lipozyme RM IM and the lipase is most preferably Novozyme 435, from Novozymes A S, Bagsvaerd, Denmark. The optimal conditions is substrate weight ratio 1:0.5-3 (glycerides residual from step b: PUFA), reaction time 10-40 h, temperature 40-70 °C, stirring 300- 1000 rpm, vacuum 5-100 mbar, and enzyme amount 3-15 wt%; preferably 1 :1-2, 20-30 h, 50-60 °C, 400-700 rpm , 10-80 m bar, and 5-10 wt%, respectively.

In a further embodiment polyunsaturated fatty acids may be added in step c). The added polyunsaturated fatty acids are preferably n-3 polyunsaturated fatty acids and are most preferably eicosapentaenoic acid (EPA), and/or docosapentaenoic acid (DPA), and/or docosahexaenoic acid (DHA) and/or most preferably docosapentaenoic acid (DPA). Free polyunsaturated fatty acids are added in order to increase the content of glyceride bound polyunsaturated fatty acids.

In yet a preferred embodiment other nutritional valuable PUFA may be added to supply a more balanced nutritional product with different nutritional fatty acids. The preferred additional nutritional valuable PUFA are gamma- linolenic acid, conjugated linoleic acid and arachidonic acid.

In another embodiment of the invention water or alcohol is removed in step c). Water or alcohol is preferably removed by employing either a vacuum system or a pervaporation system. The removal of water or alcohol results in higher yield of the esterification via the shifting of equilibrium to the product side, indicating higher content of triglycerides will be formed. With reference to Figure 1, typical non-limiting examples of process schemes within the present invention could be as in the following, where letters designate starting material (A), intermediate product (B-F) and final product (G)

A. Any oils and fats containing EPA, DPA and/or DHA, preferably EPA content no less than 5 wt% and DHA content no less than 5 wt%.

B. In the alcohol phase, EPA-FA AE content is no more than 5 wt% among other FA/AEs. C. In the distillate mixture, EPA-FA/AE content is no more than 5 wt% among other FA/AEs.

D. The residual contains 50-90 wt% partial glycerides and 10-50 wt% FA/AE.

E. External PUFA-FA/AE is added to improve the afterwards esterifica- tion or interesterification. It preferably contains no less than 70 wt%

PUFA. PUFA can be EPA, DHA, DPA, gamma-linolenic acid, conjugated linoleic acid, or arachidonic acid.

F. The PUFA-FA/AE product contains no less than 55 wt% PUFA.

G. The product oil contains no less than 55 wt% PUFA and no less than 70 wt% triglycerides.

Three alternative processes and the conditions under which the individual steps are carried out are described in the following. Numerals l-VI describe one process, where the first reaction step is hydrolysis with a li- pase. Letters (i-iii) describes an alternative process to steps l-ll where the initial step is an alcoholysis with a lipase, whereas letters (a-c) describes an alternative process to steps I-III where the first reaction step is alcoholysis with a lipase in a membrane reactor.

I. The hydrolysis is conducted under 1 :0.2-1.2 wt ratio with water (oil: water), 30-60°C, 5-15% enzyme dosage, 300-700 rpm, and with the addition of 1-3% emulsifiers, preferably lecithin. The time is con- trolled until the free fatty acid content reaches 30-70%. The lipase may be Lipozyme RM IM, Pseudomonas sp. lipase, Candida cylindracea lipase, Aspergillus niger lipase, or Geotrichum candidum lipase, and is preferably Lipozyme RM IM. II. Filtration/centrifugation is conducted to remove lipase for further reuses.

III. Short path distillation is conducted to remove non-PUFA (hydrolysis process) or their alkyl esters (alcoholysis process). The process is preferably conducted under the following conditions: evaporator tem- perature 60-180°C, vacuum 0.0005-0.1 mbar, roller speed 300-500 rpm. Distillate yield is 15-55%.

IV. Esterification (for hydrolysis process) or interesterification (for alcoholysis process) is conducted in a vacuum system under 1 :0.5-3 wt substrate ratio [residual (D): external PUFA-FA/AE (E), as indicated in Figure 1], 40-70°C temperature, 1-100 mbar vacuum, 300-700 rpm stirring, and 5-15% enzyme dosage. The time is controlled until triglycerides content reach no less than 70%. The reaction can also be conducted in a pervaporation system using a nanofiltration membrane or reverse osmosis membrane with the material of polysulfone or cellulose. The same conditions can be used as above. Vacuum is conducted through the membrane phase during the reaction. Lipases for the reaction may be Novozyme 435, Lipozyme TL IM, Rhizopus delemar lipase, Chromobacterium viscosum lipase, Candida rugosa lipase, Pseudomonas sp. lipase, or Lipozyme RM IM. It is preferable to use Novozyme 435 or Lipozyme TL IM.

V. Filtration is conducted to remove lipase for further reuses.

VI. Short path distillation is conducted to remove FA/AE in the mixture under evaporator temperature 100-180°C and vacuum 0.0005-0.01 mbar. i. Alcoholysis is conducted under 1:0.3-2.0 wt ratio with alcohols (oil: alcohol), 30-60°C, 5-15% enzyme dosage, 300-700 rpm. Alcohols are lower carbon alcohols with the carbons from 2 to 5. The time is controlled until the fatty acid alkyl ester content reach 30-70%. The lipases may be Lipozyme RM IM, Pseudomonas sp. lipase, Candida cylindracea lipase, Aspergillus niger lipase, or Geotrichum candidum lipase, and is preferably Lipozyme RM IM. ii. Filtration is conducted to remove lipase for further reuses.

,iii. Vacuum is conducted to remove alcohol.

a. Alcoholysis reaction is conducted in the same way as Step i. A membrane inter-phase is installed with the reaction system in one side and the extraction system in the other side. The membranes may be polysulfone-, ceramic, polymer- based nanofiltration membrane, ul- trafiltration membrane, or reverse osmosis membrane with the Cutoff of no more than 30000. The extraction phase is the same alcohol as for the reaction. In the reaction side, 0.5-2 bar pressure is applied. b. Filtration is conducted to remove lipase for further reuses. c. Vacuum is conducted to remove alcohol.

The fish oil product is preferably obtained by the present advantageous process.

The present invention will be further illustrated in the following non-limiting examples.

Example 1:

491 g refined and deodorised salmon oil (A1), with the fatty acid composition in Table 1, was mixed with 0.17 g GP117 and 0.33 g Toco 50, 5 g soybean lecithin, 300 g distilled water, and 39.28 g Lipozyme RM IM (Novozymes A/S). The reaction was conducted under N₂ protection. The conditions were 40°C, 600rpm stirring. After 24 hours of reaction, the mixture was filtrated with a filter to remove the enzyme. Another two batches were manufactured with the same lipase under the same conditions and the same operation procedure. The batches were pooled and the total liquid was then centrifuged (2800 rpm). The lipid phase 1442 g was recovered with free fatty acid content around 66%. 10OOg of the mixture were used for short path distillation under 140°C and 0.005 mbar. The residual was further distilled under the same conditions twice. Finally 400 g residual (D1 ) was obtained with the free fatty acid content around 30%. The distillate (C1 ) was also sampled for GC analysis. The glycerides are prepared for GC analysis by converting the glycerides into methyl esters of the fatty acids by methylation.

The content of PUFA is calculated as follows: Methyl esters of PUFA in the products 00/Methyl esters of all FAs in the product

The method error range is less than 0.1 %, compared to if the following equation was used:

PUFA in the products 00/all FAs in the product

The method error range is less than 0.2%, compared to if the following equa- tion was use:

PUFA in the products 00/total product Therefore, for all practical purposes, the first equation is used in the present context.

All fatty acid compositions are included in Table 1.

200 g residual (D1 ) is mixed with 400 g PUFA-FA concentrate (E1 ) and 0.10 g GP117 and 0.25 g Toco 50. 30 g Novozyme 435 was added to the mixture under the conditions 60°C, 600 rpm, and 80 mbar. The reaction was finished after 30 hours. The mixture was cooled down to 30 °C and filtration was conducted to remove the lipase. The liquid is subjected to short path distillation to remove all free fatty acids. The operation conditions were 170°C and 0.001 mbar. 360 g residual (G1) was obtained with the free fatty acid content around 3%. Consequently, ca. 220 g distillate (F1) was obtained. The fatty acid compositions are given in Table 1 where PUFA = (20:5 + 22:5 + 22:6).

Example 2

An example of process steps i-iii-lll is given in the following:

200 g refined and deodorised salmon oil (A2), with the fatty acid composition in Table 2, was mixed with 0.1 g GP117 and 0.1 g Toco 50, 100 g ethanol, and 10 g Lipozyme RM IM to form the reaction phase. The extraction phase was pure ethanol. In between an ETNA01 A flat membrane was installed. One bar pressure was applied to the reaction side to allow some fatty acid ethyl esters into the extraction side. The reaction was conducted under N₂ protection. The conditions were 40°C, 600rpm stirring. After 30 hours of reac- tion, the extraction phase was removed and the reaction mixture was filtrated with a filter. The liquid mixture was vacuumized under 100 mbar for 1 hour at 40 °C. The de-ethanolized lipid phase 188 g was recovered. The mixture (188 g) was used for short path distillation under 80°C and 0.005 mbar. The residual was further distilled under the same conditions for two hours. Finally ca. 90 g residual (D2) was obtained. All the fatty acid compositions are included in Table 2. After obtaining residual D2 the process may be continued as outlined in Example 1

Table 2. Fatty acid compositions (wt%)

Claims

1. Fish oil product comprising triglycerides enriched in polyunsaturated fatty acids wherein the total content of polyunsaturated fatty acids is more than 55 wt%.

2. Fish oil product according to claim 1 wherein the total content of polyunsaturated fatty acids is preferably more than 60 wt%, more preferably more than 65 wt% and most preferably more than 70 wt%.

3. Fish oil product according to claim 1 to 2 having a total content of triglycerides of at least 70 wt% and more preferably at least 80 wt%.

4. Fish oil product according to claim 1 to 3, wherein the polyunsaturated fatty acids are n-3 polyunsaturated fatty acids.

5. Fish oil product according to claim 4, wherein the n-3 polyunsaturated fatty acids are eicosapentaenoic acid (EPA)and/or docosapentaenoic acid (DPA), and/or docosahexaenoic acid (DHA)

6. Fish oil product according to claim 1 to 5 comprising other nutritional valuable fatty acids in the triglycerides.

7. Fish oil product according to claim 6, wherein the other nutritional valuable fatty acids are gamma-linolenic acid, conjugated linoleic acid, or arachidonic acid.

8. Fish oil product according to any of claims 1 to 7, wherein the enriched fish oil contain an effective amount of an oxidation stabilizer, selected from the group consisting of natural or synthetic tocopherols, propylgallate, TBHQ,

BHT, BHA, free radical scavengers, enzymes with anti-oxidant properties and ascorbylesters of fatty acids.

9. Food products, comprising a fat phase, such as spreads, margarine, cream alternative, infant food, chocolate, confectionery bakery products, sauces, ice-creams, ice-creams coatings, cheese, soups, mayonnaise, dressings, enteral or parenteral products, wherein the fat phase contains fish oil product according to any of claims 1 to 8.

10. Capsules comprising a filling, encapsulated in an edible coating, wherein the filling consists of fish oil product according to any of claims 1 to 8.

11. A process for preparing a fish oil product according to claims 1 to 7, which comprises

a) subjecting a fish oil to a enzyme-catalysed hydrolysis or alcoholysis;

b) removing free fatty acids with less than 20 carbon atoms or their alkyl ester from the product of step a)

12. A process according to claim 11 , wherein the enzyme in step a) is a lipase.

13. A process according to claim 12, wherein the lipase is selected from Li- pozyme RM IM, Pseudomonas sp. lipase, Candida cylindracea lipase, As- pergillus niger lipase, and Geotrichum candidum lipase.

14. A process according to claim 13, wherein the lipase is Lipozyme RM IM.

15. A process according to any of claims 11 to 14, wherein the free fatty acids with less than 20 carbon atoms or their alkyl esters are removed by using either a separate step of distillation or using integrated membrane separation.

16. A process according to claim 15, wherein the separate step of distillation is short path distillation.

17. A process according to claim 15, wherein the integrated membrane separation is extraction during the enzymatic reaction of step a)

18. A process according to any of claim 11 to 17, wherein the enzyme in step c) is a lipase.

19. A process according to claim 18, wherein the lipase is selected from Novozyme 435, Lipozyme TL IM, Rhizopus delemar lipase, Chromobacterium viscosum lipase, Candida rugosa lipase, Pseudomonas sp. lipase, and Lipozyme RM IM.

20. A process according to claim 19, wherein the lipase is Novozyme 435.

21. A process according to any of claim 11 to 20, wherein polyunsaturated fatty acids are added in step c).

22. A process according to claim 21 , wherein the added polyunsaturated fatty acids are n-3 polyunsaturated fatty acids

23. A process according to claim 22, wherein the n-3 polyunsaturated fatty acids are eicosapentaenoic acid (EPA), docosapentaenoic acid (DPA), or docosahexaenoic acid (DHA).

24. A process according to any of claims 11 to 23, wherein other nutritional valuable fatty acids are added in step c).

25. A process according to claim 24, wherein the other nutritional valuable fatty acids are gamma-linolenic acid, conjugated linoleic acid, or arachidonic acid.

26. A process according to any of claims 11 to 25, wherein water or alcohol is removed in step c).

27. A process according to claim 26, wherein water or alcohol is removed by employing either a vacuum system or a pervaporation system.

28. A fish oil product obtained by the method of claims 11 to 27.

29. A fish oil product according to claim 28 comprising triglycerides enriched in polyunsaturated fatty acids wherein the total content of polyunsaturated fatty acids is more than 55 wt%.

30. A fish oil product according to claim 29 wherein the total content of polyunsaturated fatty acids is preferably more than 60 wt%, more preferably more than 65 wt% and most preferably more than 70 wt%.

31. Fish oil product according to claim 29 to 30 having a total content of triglycerides of at least 70 wt% and more preferably at least 80 wt%.

32. Fish oil product according to claim 29 to 31, wherein the polyunsaturated fatty acids are n-3 polyunsaturated fatty acids.

33. Fish oil product according to claim 32, wherein the n-3 polyunsaturated fatty acids are eicosapentaenoic acid (EPA)and/or docosapentaenoic acid (DPA), and/or docosahexaenoic acid (DHA)

34. Fish oil product according to claim 28 to 33 comprising other nutritional valuable fatty acids in the triglycerides.

35. Fish oil product according to claim 34, wherein the other nutritional valuable fatty acids are gamma-linolenic acid, conjugated linoleic acid, or arachidonic acid.

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36. Fish oil product according to any of claims 28 to 35, wherein the enriched fish oil contain an effective amount of an oxidation stabilizer, selected from the group consisting of natural or synthetic tocopherols, propylgallate, TBHQ, BHT, BHA, free radical scavengers, enzymes with anti-oxidant properties and l o ascorbylesters of fatty acids.

37. Food products, comprising a fat phase, such as spreads, margarine, cream alternative, infant food, chocolate, confectionery bakery products, sauces, ice-creams, ice-creams coatings, cheese, soups, mayonnaise,

15 dressings, enteral or parenteral products, wherein the fat phase contains fish oil product according to any of claims 28 to 36.

38. Capsules comprising a filling, encapsulated in an edible coating, wherein the filling consists of fish oil product according to any of claims 28 to 36.

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