WO1996037587A1

WO1996037587A1 - Production of materials high in long chain polyunsaturated fatty acids

Info

Publication number: WO1996037587A1
Application number: PCT/EP1996/002131
Authority: WO
Inventors: Frederick William Cain; John Bernard Harris; Stephen Raymond Moore; Gerald Patrick Mcneill
Original assignee: Loders Croklaan B.V.
Priority date: 1995-05-24
Filing date: 1996-05-13
Publication date: 1996-11-28
Also published as: AU6610996A

Abstract

Materials, enriched in long chain polyunsaturated fatty acids (=LCPUFA's) are obtained from a material A, containing ≥5 % LCPUFA's and comprising ≥2 LCPUFA's (L1 and L2) in a ratio XA by splitting A into parts B and C, so that B has ratio L1:L2=XB, which is at least 1.5 times XA, while C has ratio L1:L2=XC that is « 0.7 times XA; whereupon component B is split into components D and E: D having a total LCPUFA-content that is ⊃1.2 times its content in B; and/or component C is split into components F and G, F having an LCPUFA-content that is » 1.2 times its content in C.

Description

Production of materials high in long chain polyunsaturated fatty acids.

It is fairly known from the literature, that fats, having a minimum amount of long chain polyunsaturated fatty acids (=LCPUFA ' s) do have a number of health benefits , (cf . e . g . EP 265.699; WO 90/04012; WO 94/00044; European Patent

Application 95302942.8; EP 298.293 etc.) Moreover it is known that these fats can also suitably be applied in infant-food formulations. However for above applications it would be very beneficial if fats could be obtained, that have increased levels of LCPUFA's and/or wherein specific ratios between different LCPUFA's (e.g. L₁ and L₂) present in the fat could be achieved, as different LCPUFA's, such as DHA and EPA have a different health effect. It would be most suitable, if such fats could be obtained from cheap fat sources without having to apply complicated and

expensive chemical and/or physical conversion-methods.

Simultaneously it would be beneficial if methods could be found with which these materials could be obtained in high yields, using high throughputs with good separation- efficiencies. It would also be very advantageous, if fats made according to such methods could be used for the preparation of concentrates, wherein the LCPUFA's would be present in specific levels and ratios, so that these concentrates could be blended with other fats with minimal changes in their functional properties. Shimada discloses in J Am Oil Chem. Soc. 71 (1994) 951-954 a process for the concentration of DHA and EPA in glycerides by hydrolysing triglycerides, containing them with Geotrichum candidum or with Candida cylindracea. The hydrolysis treatment can be repeated with the same enzyme. However from the data mentioned in this paper it can be concluded that the enrichments achieved are too low for our aims. Tanaka in J Am Oil Chem. Soc. 71 (1994) 331-334 discloses a process, wherein a fish oil is subjected to enzymic

hydrolysis, using Candida rugosa, whereupon the total, crude mix obtained is subjected to directed titration. As a result free fatty acids are obtained with an unknown level of DHA and EPA, while also a glyceride-mix, containing mono;di-and tryglycerides, but also including some free fatty acids, is obtained. This total mix is reesterified, resulting in triglycerides with an increased DHA-level compared with the starting fish oil and with a slightly decreased EPA-level, compared with the starting fish oil. So the directed titration is performed on the total crude mix, resulting from the first enzymic conversion; therefore the results of the directed titration are insufficient and the method is uneconomic.

From JP 07/051075 a process is known, wherein a fish oil is subjected to hydrolysis in the presence of Cand.cyl. or Cand. rugosa. The resulting product has an increased DHA- level. This product is further hydrolysed, using a lipase from Penicillium. So by this treatment the diglycerides are removed from the mixture. The oil layer, resulting from the first hydrolysis can also be subjected to a basic ethanolic extraction. According to the data mentioned our aims for enrichment (ratio L₁:L₂ and total L₁+L₂) can not be achieved by this process.

According to JP 05/095792 a three step process is

disclosed, wherein in a first step a hydrolysis of a fish oil is performed, using e.g. Pseudomonas lipase. The resulting product is concentrated in highly unsaturated fatty acids, e.g. by low temperature fractionation, urea adduction or absorption methods. The concentrate obtained is reconverted into triglycerides by reaction with

glycerol, using e.g. genus Candida, while water is removed. However the glycerides and free fatty acids, formed in the first step are not separated and therefore the second step is performed on the crude mixture obtained in the first step. This causes that enrichments obtained are

insufficient.

In JP 90/071781 a process is disclosed, wherein a fish oil is split by treatment with Cand. rugosa. The resulting product is separated in free fatty acids and in glycerides. The free fatty acids are converted to esters by reaction with an alcohol, while the esters formed are subjected to urea adduction. The glycerides obtained by the separation on converted into esters by reaction with alcohol, where upon the esters formed are subjected to urea adduction.

According to J. Japan, Oil Chem. Soc. (1993), 35-43 a fish oil is hydrolysed in the presence of Cand. cyl. whereupon the mix obtained is subjected to an enzymic treatment for the removal of diglycerides. The free fatty acids obtained are converted to triglycerides. Although some enrichment in L₁:L₂ will be obtained, our levels of enrichments can not be achieved by this disclosed technology.

Thus so far, our objectives could not be achieved by known preparation-methods. Therefore we studied whether we could find novel methods, with which above objectives could be fulfilled. This study has resulted in our invention.

Basically our invention concerns a novel process for the production of materials, enriched in long chain

polyunsaturated fatty acids (= LCPUFA), wherein a material A, containing at least 5 wt% of total LCPUFA's and

comprising at least two different LCPUFA's, from which L₁ and L₂ are the two most abundant LCPUFA's, while material A contains L₁ and L₂ in a weight ratio L₁: L₂ = X_A, is first split into two parts B and C, such that B has a ratio L₁: L₂ = X_B, that is at least 1.5 times the ratio X_A in A and C has a ratio L₂:L₂ = X_c, that is less than 0.7 times X_A;

component B is optionally converted by esterification to triglycerides B¹ or optionally hydrolysed to a product B¹¹, rich in free fatty acids; component B or component B¹ or component B¹¹ is then split into at least two parts D and E; D having a total LCPUFA-content that is at least 1.2 preferably at least 1.25 times, more preferably at least 1.3 times the total LCPUFA-content of B or B¹ or B¹¹; and/or component C is split into at least two further parts F and G, from which F has a LCPUFA-content that is at least 1.2 times that of C.

Material A thus contains at least 5 wt% of LCPUFA's, however higher levels of LCPUFA's in the end-product are obtained, when material A contains at least 10 wt%,

preferably at least 15 wt%, more preferably at least 20 wt% and most preferably 25-50 wt% of LCPUFA's.

Material A further comprises at least two different

LCPUFA's (L₁ or L₂). The most preferred L₁ and L₂ are selected from linolenic acid (C_18:3) ; arachidonic acid

(C_20:4), eicosapentaenoic acid (C_20:5), docosapentaenoic acid (C_22:5) and docosahexaenoic acid (C_22:6) . However also C_18:4;

C_18:5; C_20:3; C_22:3; C_22:4; C_24:3; C_24:4; C_24:5; and C_24:6 can be applied. The ratio X_a (=L₁,: L₂) in material A is preferably more than 1.0, more preferably more than 2.0, most

preferably more than 3.0.

Very suitable materials A are selected from the group, consisting of at least one of the following oils:

(1) marine oils, in particular Menhaden oil, cod

liver oil; tuna oil; sardine oil; anchovy oil; herring oil; sand eel oil or salmon oil.

(2) oils from microbial fermentation, in particular from a mortierella species; Penicillium; Phytium; Chlorella; Euglena; Porphyridium; Monodus or Nitzchia.

(3) vegetable oils, in particular linseed oil,

evening primrose oil, borage oil or blackcurrent seed oil.

In particular the fish oils are suitable sources, as a number of fish oils are cheap, while they still contain relatively high levels of LCPUFA's, which LCPUFA's consist in general of different LCPUFA's, such as DHA (docosahexa enoic acid: C_22:6) and eicosapentaenoic acid (or EPA: C_20:5) .

The split of material A into parts B and C is performed by an enzymatic hydrolysis. As the enzyme, an enzyme is applied, that can distinguish LCPUFA's of different chain length. A preferred enzyme is Candida rugosa. Product B formed comprises partial glycerides and triglycerides, while product C formed, comprises free fatty acids. However it is also possible that product B comprises free fatty acids and product C comprises the partial glycerides and triglycerides. Products B and C however always will have a different composition. B and C can be separated by a physical separation method. Physical separation methods that can be applied are:

(i) evaparation

and or (ii) extraction with an aqueous organic solvent, preferably methanol

and or (iii) treatment with an inorganic or organic

absorbent, preferably basic alumina

The invention furhter concerns, a process wherein product B or its converted triglyceride-product B¹ or its hydrolysed product B¹¹ are split in other products, that have an increased level of total LCPUFA's, compared to the starting products B, B¹ or B¹¹. So our invention also concerns a process, wherein product B is optionally esterified to a triglyceride product B¹, or optionally randomly hydrolysed to free fatty acids B¹¹ or a product B¹¹ rich in free fatty acids, using a non-specific lipase or a base, while product B or product B¹ or product B¹¹ is split into products D and E by one of the following processes:

(i) by solvent fractionation involving

filtration to remove a stearin fraction (ii) by directed interesterification, both

chemically and enzymically, which

interesterification is followed by removal of precipitated triglycerides rich in saturated fatty acids by filtration, either dry or in solvent.

(iii) by glycerolysis, both chemically and

enzymicly, which glycerolysis is followed by removal of precipitated saturated partial glycerides by filtration, either dry or in solvent.

(iv) by hydrolysis, followed by evaporation, or extraction with aqueous alcohol, preferably methanol, or treatment with an inorganic or organic absorbent, preferably being basic alumina.

The solvent fractionation preferably is a low temperature fractionation (i), performed at temperatures between 0 and -70°C, in particular between -25 and -60°C. Although a dry- fractionation is possible, we found that better results are obtained, if a wet-fractionation is performed. Solvents that can be applied for such a wet-fractionation are e.g. hexane, petroleum ether and acetone. However other solvents known for the wet-fractionation of fats can also be used. Suitable weight-ratios fats: solvent are 1:4 to 4:1, preferable: 1:3 to 3:1. The oleine-fraction is normally The directed interesterification (ii) can be performed by adding a base, such as Na-methylate to the mixture. The temperature applied will range from -10 to 50°C, in

particular -5 to 20°C. Because of the presence of the base an interesterification of fatty acid moieties, bonded at the glycerol backbone will occur. This will result in the formation of all kinds of triglycerides and partial

glycerides, including glycerides rich in saturated fatty acids (such as trisaturated triglycerides). These

glycerides, rich in saturated fatty acids will precipitate in the crude reaction mixture and will therefore direct the interesterification. At the end of the conversion the precipitate is separated from the other (liquid)

glycerides. This separation can be performed by any known suitable separation-technique for separating a liquid and a solid phase. The liquid phase is the product, enriched in LCPUFA's.

The interesterification can also be performed as an enzymic interesterification. In that instance we prefer to use a lipase selected from Chromobacterium; Pseudomonas;

Rhizomucor; Humicola; Rhizopus or Candida. The enzymic interesterification (ii) is performed in the presence of a limited amount of water (i.e. up to 2 wt%)). The conditions that can be applied are set our in e.g. GB 1,577,933.

Again the reaction can be directed by precipitation of the glycerides, rich in saturated fatty acid moieties.

The glycerolysis (iii) also can be performed by using a base (e.g. Na-methylate) or by using an enzyme. Enzymes, that are known for glycerolysis-purposes, are disclosed in our earlier patent-application EP 94302325.9 The crude reaction product is a mixture of triglycerides and partial glycerides (most diglycerides), with a whole spectrum of fatty acid moieties in it. However the triglycerides and partial glycerides rich in saturated fatty acid moieties will precipitate in the crude reaction-mixture. This precipitation will direct the course of the glycerolysis, so that a product D, enriched in LCPUFA's can be separated from a product E, enriched in saturated fatty acids. The hydrolysis (iv) is performed by using a lipase, that is selective against LCPUFA's over other fatty acids. Example of such lipase are: Geotrichum candidum, Lipase G and Mucor Miehei. The techniques to separate D and E in this instance are well-known physical separation techniques.

The products D and E, which are formed by our process can either be both triglycerides and/or partial glycerides, or product D can be a mixture of partial glycerides and triglycerides, while product E is a mixture of free fatty acids or D and E are both free fatty acids, however having a different composition.

A very beneficial process is obtained if part of product D or E is hydrolysed, resulting in a mixture comprising free fatty acids and glycerol; removing the glycerol from this mixture and reconverting the remaining free fatty acids with another part of D or E, or the original non-hydrolysed material, preferably using stoichiometric amounts of reactants. The results of this process are triglycerides with an increased total level of LCPUFA's, wherein L₁ and L₂ are present in a ratio, different from its ratio in starting material A.

Component C, or a conversion product of component C, can be split into parts F and G by physical separation methods. Examples of such physical separation methods are:

(i) molecular distillation

(ii) solvent or dry fractionation

(iii) urea adduction

(iv) solvent fractionation of metal salts of the free fatty acids, followed by filtration to remove stearin fraction, and reconversion to free fatty acids by adding an acid (= directed titration) (v) treatment with an inorganic or organic absorbent preferably being basic alumina

The products F and G resulting from the above split are enriched in total LCPUFA's , respectively depleted in

LCPUFA's compared to C.

The solvent fractionation (ii) is carried out, using hexane, petroleum either or acetone as solvent. The solvent/oil-ratio is 4:1 to 1:4, preferably 3:1 to 1:3. The temperature applied is -20 to -60 C°, preferably -25 to -35 C°.

The free fatty acid products obtained by our novel process can be used for the esterification of glycerol or partial glycerides, preferably in stoichiometric amounts for the production of triglycerides.

Component C comprising free fatty acids can also be split into two parts by reaction with alcohols, using a lipase selective against LCPUFA. The reaction forms esters depleted in LCPUFA, leaving free fatty acid, enriched in

LCPUFA.

As mentioned before the products, as obtainable by the different processes have many health-benefits. So it is possible to use these products per se in a number of consumer products. However the products often suffer from oxygen-sensitivity. In order to improve this oxygen- sensitivity blends of materials are made, comprising a mixture of the products, as obtainable by the process of claims 1-15 and anti-oxidants, selected from the group of natural or synthetic tocopherols or other anti-oxidants, enzymes with anti-oxidant properties, such as glucose oxidase and/or catalase, BHA, BHT, TBHQ, ascorbyl

palmitate; propyl gallate; Lecithin; catechins or

flavenols. According to another embodiment of our invention the triglycerides, partial glycerides, or free fatty acids, as obtainable by the process according to the invention or its blends with anti-oxidants can also be mixed with other lipid materials that have a solid fat index at 5°C (N₅:

NMR-pulse, not stabilised) that is at least 5 units

different from the N₅ of the fatty products, obtainable by the process of claims 1-16. In this way fatblends can be obtained, that are appropriate for specific applications.

Part of our invention are also consumer products, such as food products, in particular spreads, cream alternatives, infant food, ice cream, mayonnaise, dressings, toppings etcetera, pharmaceutical products, skin-care products, such as lotions or skin-creams comprising a fatty component or a free fatty acid, wherein the fatty component or the free fatty acid comprises a product as obtainable by the process according to claims 1-16, or wherein the fatty component or free fatty acid comprises a blend according to claims 17- 18.

According to a last embodiment our invention also concerns the use of materials, enriched in LCPUFA's, wherein the products, as obtainable by the process of claims 1-16 or wherein the blends according to claims 17-18 are used to improve the health benefits of consumer goods, such as food products or personal products. EXAMPLES : EXAMPLES 1

Two batches each consisting of 10Kg of refined Chilean fish oil containing 100 ppm of TBHQ as antioxidant were mixed with 4g of Candida rugosa lipase dissolved in 10Kg of pH7 phosphate buffer and stirred at 25°C for 26 hours under a nitrogen blanket until 60% of the oils had been hydrolysed to free fatty acid. The mixtures were rapidly heated to 90°C to destroy enzyme activity, washed with water then dried under vacuum. The free fatty acids were removed by evaporation at 190°C at a pressure of 0.02 to 0.04 mBars and a flow rate of 30 to 35 ml/min.

A triglyceride/ partial glyceride mixture was thus obtained with a composition as given in table 1. Fatty acid compositions were determined by fatty acid methyl ester gas chromatography (FAME GC) using the method given in AOCS Ce 1b-89, free fatty acid (FFA) contents were determined by titration against standard sodium hydroxide solution and are expressed as % oleic acid. Partial glyceride contents were determined by silica gel high performance liquid chomatography (HPLC ) using an

evaporative light scattering detector with 12, hydroxy iso-octane as an internal standard.

6Kg of the combined triglyceride/ partial glyceride mixture were vigorously stirred with an equal volume of water and with 180g of Rhizomucor miehei immobilised onto Duolite. The mixture was stirred under a nitrogen blanket at 35 °C for 3 hours until the free fatty acid content was 27 %. The enzyme was removed by filtration and the free glycerol removed by water washing. 100 ppm of TBHQ were added. The partial glycerides and free fatty acid were reesterified to triglyceride using Rhizomucor miehei

immobilised onto Duolite. 5.9Kg of the partial glyceride mixture were mixed with 295g of Rhizomucor miehei at 55°C for 44 hours with a vacuum of 50mBars.The enzyme was removed by filtration and any remaining free fatty acids in the triglyceride rich product were removed by

neutralisation with sodium hydroxide.

258g of the refined triglyceride rich fraction were

dissolved in 1800mls of acetone and cooled to -60°C . A stearine fraction was removed by filtration and washed with a further 1800mls of acetone. The wash was combined with the oleine fraction to produce a long chain polyunsaturated (LCPUFA) enriched product , the compositions are given in table 1.1.

A SPREAD was prepared using the LCPUFA enriched oleine fraction which was compared to a reference spread made with sunflower oil. The spreads were made with the following formulation:

Fat Phase

The fat blend for the reference was 13% InEs, 87% SF.

For the LCPUFA product, the fat blend used was:- InEs 13%

Sunflower 78%

Fish Blend 9%

In Es = Interesterified mix of fully hardened palm oil and fully hardened palmkernel olein.

2 kg of material was prepared and processed.

A micro-votator processing lines was set up as follows:-

Premix conditions - Stirrer Speed 60 rpm

- Temperature 50°C pump - Proportioning pump set at 60% (30 g/min.).

A₁ conditiocns - Shaft speed 1000 rpm A₁ conditions - Shaft speed 1000 rpm

- Temperature set at 8°C

C₁ conditions - Shaft speed 1000 rpm

- Temperature set to 10°C

A₂ conditions - Shaft Speed 1000 rpm

- Temperature set to 10°C C₂ conditions - Shaft speed 1000 rpm

- Temperature set to 13°C

The aqueous phase was prepared by heating the required amount of water to approximately 80°C and then, using a silverson mixer, slowly mixing in the ingredients. The pH of the system was adjusted to 5.1 by adding 20% Lactic acid solution as required.

A premix was prepared by stirring the fat phase in the premix tank and then slowly adding in the aqueous phase. When addition was complete, the mix was stirred for a further 5 minutes before pumping through the line. When the process had stabilised (around 20 minutes), product was collected for storage and evaluation.

Very good oil continuous low fat spreads were produced using this system for both the reference and the LCPUFA product. The spreads were evaluated, after 5 days storage at 5°C and 20°C, for hardness using a cone penetrometer, electrical conductivity and for the plasticity of the product by formation of a collar. The results are given in table 1.3.

(Collar formation is scored on a scale of 1 to 6. A collar of 1 shows that the product has little structure a score of 6 has a lot of structure and is butterlike.)

Both samples spread very easily on grease-proof paper, with no obvious signs of water loss.

A "RANCH STYLE" DRESSING was prepared using the LCPUFA enriched oleine which was compared to a reference dressing made with sunflower oil. The formulation for the dressing is given in table 1.4 A "RANCH STYLE" DRESSING was prepared using the LCPUFA enriched oleine which was compared to a reference dressing made with sunflower oil. The formulation for the dressing is given in table 1.4

The liquid oil for the reference was sunflower and for the LCPUFA containing product was 90/10 sunflower oil /

enriched oleine. The water and maltodextrin were first blended using a homogeniser. The egg yolk, xanthum gum and vinegar were sequentially added whilst continuing to stir until complete mixing had occurred. At this stage the pH =3.25 .

The liquid oils were slowly added to the aqueous phase whilst homogenising. Mixing was continued until all the oil appeared to have been dispersed. The dressings were then transferred to sterile bottles.

The dressings were evaluated after 24 hours storage at ambient temperature. The viscosities of the samples were determined using a Brookfield Viscometer fitted with a number 4 spindle rotating at 10 rpm. The samples were contained in identical 200ml plastic bottles hence the viscosities are directly comparable with each other. For each sample the average of three measurements was taken with the sample being allowed to relax for 1 minute between each 1 minute of shear.

The oil droplet size distribution was determined using a Malvern Mastersizer fitted with a a 45mm lens.

EXAMPLE 2

A Chilean fish oil was hydrolysed using Candida rugosa lipase to a free fatty acid content of 60% and the acids removed by evaporation as described in example 1. A triglyceride/ partial glyceride mixture was thus obtained with a composition as given in table 2.1. Analytical procedures were as described in example 1. To 5g of the triglyceride/ partial glyceride mixture were added 0.2g of Geotrichum Candida lipase dissolved in 5g of pH6.5 phosphate buffer. The mixture was stirred with a

EXAMPLE 3

Chilean fish oil was hydrolysed using Candida rugosa lipase to a free fatty acid content of 60% and the acids removed by evaporation as described in example 1. A triglyceride/ partial glyceride mixture was thus obtained with a

composition as given in table 3. Analytical procedures were as described in example 1. 100g of the partial glyceride fraction were hydrolysed to free fatty acids by refluxing with 23g of potassium

hydroxide in 130 mis of ethanol and 44mls of water for 1 hour. The potassium salts were converted to free fatty acids by addition of hydrochloric acid and then extracted into hexane.

10g of the fatty acids were mixed with 50mls of 0.5M sodium hydroxide and 100mls of acetone The mixture was stirred in a jacketed vessel with a scrape surface stirrer at 45°C for 30minutes then cooled at 1°C/min to 4°C at which

temperature it was stirred for 1 hour. The crystalline stearine fraction was removed by filtration and washed with a further 50 mis of acetone. The sodium salts in the stearine and oleine fractions were converted back to free fatty acids by addition of hydrochloric acid and then extracted into hexane. The compositions of the fractions are given in table 3.

EXAMPLE 4

composition as given in table 4. Analytical procedures were as described in example 1. 100g of the partial glyceride fraction were hydrolysed to free fatty acids by refluxing with 23g of potassium hydroxide in 130mls of ethanol and 44mls of water for 1 hour. The potassium salts were converted to free fatty acids by addition of hydrochloric acid and then extracted into hexane.

35g of the fatty acids were added to 200g of urea mixed with 600mls of ethanol at 65°C in a jacketed vessel fitted with a scape surface stirrer. The mixture was stirred for 1 hour at 65°C then cooled at 1°C/min to 4°C at which

temperature it was held for 16 hours. The solid fraction was removed by filtration. The ethanol was removed from the oleine fraction and the urea salts converted back to free fatty acids by addition of hydrochloric acid and then extracted into hexane.

The fatty acids were esterified with glycerol to form a triglyceride rich fat. 1.6 g of the fatty acids were mixed with 0.2g of glycerol and 0.1g of Rhizomucor miehei

immobilised onto Duolite. The mixture was stirred in an open glass vial at 55°C for 168 hours with nitrogen blowing across the surface. The composition of the triglyceride rich fat is given in table 4

EXAMPLE 5

composition as given in table 5.

Analytical procedures were as described in example 1. 10g of the partial glyceride fraction were further

hydrolysed with 0.06g of Candida rugosa lipase dissolved in 10mls of water. The mixture was stirred with a magnetic flea at 35°C in a sealed vial with a nitrogen blanket in a magnetic stirrer /hotblock for 72 hours. The resulting glyceride species were separated by thin layer

chromatography and the fatty acid compositions determined by FAME GC.

EXAMPLE 6

Chilean fish oil was hydrolysed using Candida rugosa lipase to a free fatty acid content of 60% and the acids removed by evaporation as described in example 1. A free fatty acid fraction was thus obtained with a composition as given in table 6. Analytical procedures were as described in example 1.

56g of the free fatty acids were added to 210g of urea mixed with 750mls of ethanol at 65°C in a jacketed vessel fitted with a scape surface stirrer. The mixture was stirred for 1 hour at 65°C then cooled at 1°C/min to 4°C at which temperature it was held for 16 hours. The solid fraction was removed by filtration. The ethanol was removed from the oleine fraction and the urea salts converted back to free fatty acids by addition of hydrochloric acid and then extracted into hexane.

The fatty acids were esterified with glycerol to form a triglyceride rich fat. 5.1 g of the fatty acids were mixed with 0.6g of glycerol and 0.3g of Rhizomucor miehei immobilised onto Duolite. The mixture was stirred in an open glass vial at 55°C for 144 hours with nitrogen blowing across the surface. The composition of the triglyceride rich fat is given in table 6

T

EXAMPLE 7

Chilean fish oil was hydrolysed using Candida rugosa lipase to a free fatty acid content of 60% and the acids removed by evaporation as described in example 1. A free fatty acid fraction was thus obtained with a composition as given in table 7. Analytical procedures were as described in example 1.

14g of the fatty acids were mixed with 50mls of 0.5M sodium hydroxide and 100mls of acetone The mixture was stirred in a jacketed vessel with a scrape surface stirrer at 45°C for 30minutes then cooled at 1°C/min to -5°C at which

temperature it was stirred for 1 hour. The crystalline stearine fraction was removed by filtration and washed with a further 50 mis of acetone. The sodium salts in the stearine and oleine fractions were converted back to free fatty acids by addition of hydrochloric acid and then extracted into hexane. The compositions of the fractions are given in table 7.

EXAMPLE 8

Chilean fish oil was hydrolysed using Candida rugosa lipase to a free fatty acid content of 60% and the acids removed by evaporation as described in example 1. A free fatty acid fraction was thus obtained with a composition as given in table 8.1. Analytical procedures were as described in example 1. 400g of the fatty acids were dissolved in 1600g of acetone and cooled to -60°C. A stearine fraction was removed by filtration and washed with another 1600g of acetone. The oleine fraction fatty acids were esterified with glycerol to form a triglyceride rich fat. 166g of the fatty acids were mixed with 14.9g of glycerol and 7.9g of

Rhizomucor miehei immobilised onto Duolite. The mixture was stirred in a round bottom flask at 55°C for 144 hours under vacuum. The enzyme was removed by filtration. The remaining free fatty acid was removed by treatment with basic alumina in hexane. The composition of the triglyceride rich fat is given in table 8.1.

A SPREAD was prepared using the LCPUFA enriched

triglyceride fraction which was compared to a reference spread made with sunflower oil using the formulation and method given in example 1.

Very good oil continuous low fat spreads were produced using this system for both the reference and the LCPUFA product.

The spreads were evaluated as described in example 1. .3

(Collar formation is scored on a scale of 1 to 6 . A collar of 1 shows that the product has little structure a score of 6 has a lot of structure and is butterlike.) Both samples spread very easily on grease-proof paper, with no obvious signs of water loss.

A "RANCH STYLE" DRESSING was prepared using the LCPUFA enriched triglyceride fraction which was compared to a reference dressing made with sunflower oil. The formulation and method of production was as described in example 1.

AN ICE-CREAM was prepared using the LCPUFA enriched

triglyceride fraction which was compared to a reference spread made with sunflower oil. The Ice-creams were made according to the following recipe:

Sherex IC 9330^® is a product from Quest International and comprises mono- and diglycerides admixed with different stabilizers.

The fat blend for the reference was PO / Sunflower oil 90/10 and the fat blend according to the invention was:

90/10 PO/LCPUFA product.

All ingredients except the water and the fat were mixed. Then the cold water was added to this mixture. This mixture was heated in a water bath till a temperature of 70°C. Then the fully liquid palm oil was added to the mixture while "stirred" in the ultra-turrax. This emulsion was cooled in a water bath of 20°C. The emulsion was stirred in the ultra-turrax again. The batch ice cream machine was held for 24 hours at -28°C prior to use. The emulsion was placed in the batch ice cream machine and stirred for 15 minutes. The resulting ice cream was stored at -20°C for 24 hours and then evaluated. The viscosity of the ice cream emulsion, prior to freezing was measured. The overrun and hardness were determined. The viscosity was measured by using the Haake viscometer.

Hardness was measured by using a Stevens texture analyser with a 45° cone at a speed of 0.5 mm/second till a deepness of 2 mm.

The viscosities of the emulsions were similar.

EXAMPLE 9

Chilean fish oil was hydrolysed using Candida rugosa lipase to a free fatty acid content of 60% and the acids removed by evaporation as described in example 1. A free fatty acid fraction was thus obtained with a composition as given in table 9. Analytical procedures were as described in example 1. 356g of the fatty acids were cooled to 30°C and held for 24 hours at which time 10% of the mixture had solidified. This solid fraction was removed by filtration under a pressure of 0-24 bar for 2 hours then 24 bar for a further 2 hours.

Claims

1. Process for the production of materials, enriched in long chain polyunsaturated fatty acids (= LCPUFA), wherein a material A, containing at least 5 wt% of total LCPUFA's and comprising at least two different LCPUFA's, from which L₁ and L₂ are the two most abundant LCPUFA's, while material A contains L₁ and L₂ in a weight ratio L₂:L₂ = X_A, is first split into two parts B and C, such that B has a ratio L₁: L₂ = X_B, that is at least 1.5 times the ratio X_A in A and C has a ratio L₁:L₂ = X_c, that is less than 0.7 times X_A;

component B is optionally converted by esterification to triglycerides B¹ or optionally hydrolysed to a product B¹¹, rich in free fatty acids; component B or component B¹ or component B¹¹ is then split into at least two parts D and E; D having a total LCPUFA- content that is at least 1.2 times, preferably at least 1.25 times, more preferably at least 1.3 times the total LCPUFA-content of B or B¹ or B¹¹; and/or component C is split into at least two further parts F and G, from which F has a LCPUFA-content that is at least 1.2 times that of C.

2. Process according to claim 1, wherein material A

contains at least 10 wt%, preferably at least 15 wt%, more preferably at lest 20 wt% and most preferably 25- 50 wt% of LCPUFA's.

3. Process according to claim 1 or 2, wherein material A comprises at least two different LCPUFA's L₁ and L₂, selected from C_18:3, C_20:4, C_20:5, C_22:5 and C_22:6.

4. Process according to claims 1-3, wherein material A contains L₁ and L₂ in a ratio X_A of more than 1.0, preferably more than 2.0, most preferably more than 3.0. 5. Process according to claims 1-4, wherein material A is selected from the group consisting of at least one of the following oils:

(1) marine oils, in particular sardine oil, anchovy oil, menhaden oil, cod liver oil and tuna oil

(2) oils from microbial fermentation, in particular from a Mortierella species

(3) vegetable oils, in particular linseed oil,

evening primrose oil, borage oil or black currant seed oil 6. Process according to claims 1-5, wherein the split of A into B and C is performed by an enzymic hydrolysis, using a lipase, that can distinguish LCPUFA's of different chain length, preferably by using Candida rugosa followed by physical separation from B and C. 7. Process according to claim 6, wherein B and C are

separated by either

(i) evaporation, or

(ii) extraction with an aqueous organic solvent,

preferably being methanol, or

(iii) treatment with an inorganic or organic

preferably being basic alumina, or

(iv) by combinations of (i) - (iii) 8. Process according to claims 1-7, wherein product B

formed comprises free fatty acids, partial glycerides and triglycerides, while product C formed, comprises free fatty acids, partial glycerides and

triglycerides, with the prerequisite that B is different from C.

9. Process according to claims 1-8, wherein product B is optionally esterified to a triglyceride product B¹, or optionally randomly hydrolysed to free fatty acids or a product B¹¹, rich in free fatty acids, using a nonspecific lipase or a base, while product B or product B¹ or product B¹¹ is split into products D and E by one of the following processes:

(i) by solvent fractionation involving filtration to remove a stearin fraction.

(ii) by directed interesterification, both

chemically and enzymically, which

interesterification is followed by removal of precipitated triglycerides rich in saturated fatty acids, by filtration, either dry or in solvent.

(iii) by glycerolysis, both chemically and

enzymacally, which glycerolysis is followed by removal of precipitated saturated partial glycerides by filtration either dry or in solvent.

(iv) by hydrolysis, followed by evaporation, or

extraction with aqueous alcohol, preferably methanol, or treatment with an inorganic or organic absorbent, preferably basic alumina.

10. Process according to claim 9, wherein the products D and E are both triglycerides and/or partial

glycerides, or wherein product D is a mixture of partial glycerides and triglycerides and E is a mixture of free fatty acids, or D and E are both free fatty acids, however having a different composition.

11. Process according to claims 1-10, wherein part of

product D or E is hydrolysed, resulting in a mixture comprising free fatty acidas and glycerol; removing the glycerol from this mixture and reconverting the NOT TAKEN INTO CONSIDERATION

FOR THE PURPOSES OF INTERNATIONAL PROCESSING

17. Blends of materials, comprising a mixture of products, as obtainable by the process according to claims 1-16, and anti-oxidants, selected from the group consisting of natural or synthetic tocopherols, or other anti- oxidants; enzymes with anti-oxidant properties, such as glucose oxidase and/or catalase; BHA; BHT.

18. Blends of materials, comprising the triglyceride,

partial glycerides or free fatty acids, as obtainable by the process according to claims 1-16, and other lipid materials, that have a solid fat index at 5°C (N₅: NMR-pulse, not-stab) that is at least 5 units different from the N₅ of the triglycerides or partial glycerides, obtainable according to the process of claims 1-16.

19. Consumer-products, such as food products, in

particular spreads, cream alternatives, infant foods, ice cream, mayonnaise, dressings, toppings etc., pharmaceutical products, skin-care products, such as lotions or skin-creams, comprising a fatty component or a free fatty acid, wherein the fatty component or the free fatty acid comprises a product as obtainable by the process according to claims 1-16 or wherein the free fatty acid or the fatty component comprises a blend, according to claims 17-18.

20. Use of materials, enriched in LCPUFA's, wherein the products, obtainable by the process according to claims 1-16 or wherein the blends, according to claims 17-18 are used to improve the health benefits of consumer goods, in particular of food products and personal products.