WO1996037586A1 - Production method for fats with long chain polyunsaturated fatty acids - Google Patents

Production method for fats with long chain polyunsaturated fatty acids Download PDF

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Publication number
WO1996037586A1
WO1996037586A1 PCT/EP1996/002132 EP9602132W WO9637586A1 WO 1996037586 A1 WO1996037586 A1 WO 1996037586A1 EP 9602132 W EP9602132 W EP 9602132W WO 9637586 A1 WO9637586 A1 WO 9637586A1
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Prior art keywords
lcpufa
fatty acids
free fatty
product
mixture
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PCT/EP1996/002132
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French (fr)
Inventor
Frederick William Cain
John Bernard Harris
Stephen Raymond Moore
Gerald Patrick Mcneill
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Loders Croklaan B.V.
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Priority to AU58988/96A priority Critical patent/AU5898896A/en
Publication of WO1996037586A1 publication Critical patent/WO1996037586A1/en

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    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12PFERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00Preparation of oxygen-containing organic compounds
    • C12P7/64Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
    • C12P7/6436Fatty acid esters
    • C12P7/6445Glycerides
    • C12P7/6458Glycerides by transesterification, e.g. interesterification, ester interchange, alcoholysis or acidolysis
    • AHUMAN NECESSITIES
    • A23FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23GCOCOA; COCOA PRODUCTS, e.g. CHOCOLATE; SUBSTITUTES FOR COCOA OR COCOA PRODUCTS; CONFECTIONERY; CHEWING GUM; ICE-CREAM; PREPARATION THEREOF
    • A23G3/00Sweetmeats; Confectionery; Marzipan; Coated or filled products
    • A23G3/34Sweetmeats, confectionery or marzipan; Processes for the preparation thereof
    • A23G3/346Finished or semi-finished products in the form of powders, paste or liquids
    • AHUMAN NECESSITIES
    • A23FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23DEDIBLE OILS OR FATS, e.g. MARGARINES, SHORTENINGS, COOKING OILS
    • A23D7/00Edible oil or fat compositions containing an aqueous phase, e.g. margarines
    • A23D7/015Reducing calorie content; Reducing fat content, e.g. "halvarines"
    • AHUMAN NECESSITIES
    • A23FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23DEDIBLE OILS OR FATS, e.g. MARGARINES, SHORTENINGS, COOKING OILS
    • A23D9/00Other edible oils or fats, e.g. shortenings, cooking oils
    • AHUMAN NECESSITIES
    • A23FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23GCOCOA; COCOA PRODUCTS, e.g. CHOCOLATE; SUBSTITUTES FOR COCOA OR COCOA PRODUCTS; CONFECTIONERY; CHEWING GUM; ICE-CREAM; PREPARATION THEREOF
    • A23G9/00Frozen sweets, e.g. ice confectionery, ice-cream; Mixtures therefor
    • A23G9/52Liquid products; Solid products in the form of powders, flakes or granules for making liquid products ; Finished or semi-finished solid products, frozen granules
    • CCHEMISTRY; METALLURGY
    • C11ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11BPRODUCING, e.g. BY PRESSING RAW MATERIALS OR BY EXTRACTION FROM WASTE MATERIALS, REFINING OR PRESERVING FATS, FATTY SUBSTANCES, e.g. LANOLIN, FATTY OILS OR WAXES; ESSENTIAL OILS; PERFUMES
    • C11B7/00Separation of mixtures of fats or fatty oils into their constituents, e.g. saturated oils from unsaturated oils
    • C11B7/0008Separation of mixtures of fats or fatty oils into their constituents, e.g. saturated oils from unsaturated oils by differences of solubilities, e.g. by extraction, by separation from a solution by means of anti-solvents
    • C11B7/0025Separation of mixtures of fats or fatty oils into their constituents, e.g. saturated oils from unsaturated oils by differences of solubilities, e.g. by extraction, by separation from a solution by means of anti-solvents in solvents containing oxygen in their molecule
    • CCHEMISTRY; METALLURGY
    • C11ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11CFATTY ACIDS FROM FATS, OILS OR WAXES; CANDLES; FATS, OILS OR FATTY ACIDS BY CHEMICAL MODIFICATION OF FATS, OILS, OR FATTY ACIDS OBTAINED THEREFROM
    • C11C1/00Preparation of fatty acids from fats, fatty oils, or waxes; Refining the fatty acids
    • C11C1/02Preparation of fatty acids from fats, fatty oils, or waxes; Refining the fatty acids from fats or fatty oils
    • C11C1/04Preparation of fatty acids from fats, fatty oils, or waxes; Refining the fatty acids from fats or fatty oils by hydrolysis
    • C11C1/045Preparation of fatty acids from fats, fatty oils, or waxes; Refining the fatty acids from fats or fatty oils by hydrolysis using enzymes or microorganisms, living or dead
    • CCHEMISTRY; METALLURGY
    • C11ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11CFATTY ACIDS FROM FATS, OILS OR WAXES; CANDLES; FATS, OILS OR FATTY ACIDS BY CHEMICAL MODIFICATION OF FATS, OILS, OR FATTY ACIDS OBTAINED THEREFROM
    • C11C1/00Preparation of fatty acids from fats, fatty oils, or waxes; Refining the fatty acids
    • C11C1/08Refining
    • CCHEMISTRY; METALLURGY
    • C11ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11CFATTY ACIDS FROM FATS, OILS OR WAXES; CANDLES; FATS, OILS OR FATTY ACIDS BY CHEMICAL MODIFICATION OF FATS, OILS, OR FATTY ACIDS OBTAINED THEREFROM
    • C11C3/00Fats, oils, or fatty acids by chemical modification of fats, oils, or fatty acids obtained therefrom
    • C11C3/003Fats, oils, or fatty acids by chemical modification of fats, oils, or fatty acids obtained therefrom by esterification of fatty acids with alcohols
    • CCHEMISTRY; METALLURGY
    • C11ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11CFATTY ACIDS FROM FATS, OILS OR WAXES; CANDLES; FATS, OILS OR FATTY ACIDS BY CHEMICAL MODIFICATION OF FATS, OILS, OR FATTY ACIDS OBTAINED THEREFROM
    • C11C3/00Fats, oils, or fatty acids by chemical modification of fats, oils, or fatty acids obtained therefrom
    • C11C3/02Fats, oils, or fatty acids by chemical modification of fats, oils, or fatty acids obtained therefrom by esterification of fatty acids with glycerol
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12PFERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00Preparation of oxygen-containing organic compounds
    • C12P7/64Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
    • C12P7/6409Fatty acids
    • C12P7/6427Polyunsaturated fatty acids [PUFA], i.e. having two or more double bonds in their backbone
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12PFERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00Preparation of oxygen-containing organic compounds
    • C12P7/64Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
    • C12P7/6436Fatty acid esters
    • C12P7/6445Glycerides
    • C12P7/6472Glycerides containing polyunsaturated fatty acid [PUFA] residues, i.e. having two or more double bonds in their backbone
    • AHUMAN NECESSITIES
    • A23FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23GCOCOA; COCOA PRODUCTS, e.g. CHOCOLATE; SUBSTITUTES FOR COCOA OR COCOA PRODUCTS; CONFECTIONERY; CHEWING GUM; ICE-CREAM; PREPARATION THEREOF
    • A23G2200/00COCOA; COCOA PRODUCTS, e.g. CHOCOLATE; SUBSTITUTES FOR COCOA OR COCOA PRODUCTS; CONFECTIONERY; CHEWING GUM; ICE-CREAM; PREPARATION THEREOF containing organic compounds, e.g. synthetic flavouring agents
    • A23G2200/08COCOA; COCOA PRODUCTS, e.g. CHOCOLATE; SUBSTITUTES FOR COCOA OR COCOA PRODUCTS; CONFECTIONERY; CHEWING GUM; ICE-CREAM; PREPARATION THEREOF containing organic compounds, e.g. synthetic flavouring agents containing cocoa fat if specifically mentioned or containing products of cocoa fat or containing other fats, e.g. fatty acid, fatty alcohol, their esters, lecithin, paraffins

Definitions

  • LCPUFA's such as DHA and EPA have a different health effect .
  • fats could be made with high levels of EPA, as it is very difficult to make these fats by conventional one step processes. 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. 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.
  • a three step process 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.
  • 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.
  • 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 are converted into esters by reaction with alcohol, where upon the esters formed are subjected to urea adduction.
  • LCPUFA long chain poly- unsaturated fatty acids
  • 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.
  • Very suitable materials A are selected from the group, consisting of at least one of the following oils :
  • marine oils in particular Menhaden oil; cod liver oil; tuna oil; sardine oil; anchovy oil; herring oil; sand eel oil; or salmon oil.
  • oils from microbial fermentation in particular from a Mortierella species; Penicillium; Phytium; Chlorella; Euglena; Porphyridium; Monodus or Nitzchia.
  • vegetable oils in particular linseed oil, evening primrose oil, borage oil or blackcurrant seed oil .
  • 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 (docosahexaeonic acid: C 22:6 ) and eicosapentaenoic acid (or
  • Beneficial fats are obtained, when the long chain polyunsaturated fatty acids L x and L 2 are selected from fatty acids with at least 18 carbon atoms and at least 3 unsaturations, preferably C 18:3 , C 20:4 , C 20:5 C 22:5 , and C 22:6 . however also C 18:4 , C 18:5 , C 20:3 , C 22:3 , C 24:3 , C 24:4 , C 24:5 and C 24:6 can be applied.
  • material A can be subjected to the split, it is also possible to hydrolyse A first, preferably using randomising methods, such as by applying a non-specific lipase or a base, such as ethanolic potassium hydroxide.
  • the product A 1 obtained is rich in free fatty acids.
  • the split of material A or A 1 into parts B and C can be performed in a number of ways. Very suitably this split is performed either by: (i) low temperature fractionation, in particular solvent fractionation, followed by filtration to remove stearin-fraction, or
  • glycerolysis both chemically, using a base and enzymically, which glycerolysis is followed by removal of preciptitated saturated partial glycerides by filtration, either dry or in solvent, or
  • hydrolysis using a lipase that is selective against CPUFA's over other fatty acids, followed by evaporation, or by extraction with aqueous alcohol, preferably methanol, or by treatment with an inorganic or organic absorbent, preferably basic alumina or : (v) urea adduction, followed by filtration to remove stearin fraction, or
  • the low temperature fractionation (i) is performed at temperatures between -20 and -65°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 1:8 to 1:2, preferably: 1:6 to 1:4.
  • the oleine-fraction is normally obtained in a yield of 10-50 wt%.
  • the oleine-fraction is the fraction enriched in LCPUFA's (so this is part B) .
  • the directed interesterification (ii) can be performed by adding a base, such as Na-methylate to the mixture.
  • a base such as Na-methylate
  • the temperature applied will range from -5 to 80°C, in particular 10-50°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, including triglycerides rich in saturated fatty acids (such as trisaturated triglycerides) . These triglycerides, rich in saturated triglycerides 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) triglycerides.
  • This separation can be performed by any known suitable separation-technique for separating a liquid and a solid phase.
  • the liquid phase is our part B
  • the solid phase is our part C.
  • 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 out in e.g. GB 1,577,933. This assures that high levels of triglycerides are obtained, while the formation of extensive quantities of diglycerides is avoided.
  • reaction can be directed by precipitation of the triglycerides, 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 B, enriched in LCPUFA's can be separated from a product C, enriched in saturated fatty acids.
  • the hydrolysis (iv) is performed by using a lipase, that is selective against LCPUFA's over other fatty acids.
  • lipases are: Geotrichum candidum, Lipase G and Mucor Miehei.
  • the products B and C obtained in this way are a mixture of triglycerides and partial glycerides (as product B) and a mixture of free fatty acids (as product C) . Because of the use of a lipase that is selective against LCPUFA's over other fatty acids, product B is enriched in LCPUFA, while the fatty acids from product C are enriched in the non- LCPUFA's.
  • product B is split into products D and E by performing an enzymic hydrolysis, using a lipase that can distinguish LCPUFA's of different chain length, preferably by using Candida rugosa.
  • Products D and E are separated by physical separation methods. Suitable methods are : evaporation, extraction with an aqueous alcohol preferably methanol and treatment with an inorganic or organic absorbent, preferably basic alumina.
  • product B mainly comprises free fatty acids.
  • This product B can be split into D and E by an enzymic conversion with glycerol, using an enzyme selective against LCPUFA.
  • the resulting mixture comprises triglycerides and partial glycerides, depleted in LCPUFA and free fatty acids, enriched in LCPUFA.
  • This mixture can be separated by physical separation methods. These methods include: evaporatio ; - extraction with an aqueous alcohol, preferably methanol or treatment with an inorganic or organic absorbent, preferably basic alumina.
  • product D and/or E as obtained by the enzymic hydrolysis or esterification are used for different purposes.
  • part of product D or E being a mixture of triglycerides and partial glycerides enriched in L x or L 2 , is hydrolysed, resulting in a mixture comprising different free fatty acids and glycerol; the glycerol is removed from the mixture and remaining free fatty acids are reconverted with another part of product D or E, preferably in such a way that the reaction mixture has a stoichiometric composition.
  • product D or E comprising mainly free fatty acids, enriched in L x or L 2 can also be converted to triglycerides by esterification with glycerol or with partial glycerides, preferably in ratios corresponding with stoichiometric compositions.
  • reaction product D or E comprising partial glycerides and optionally also triglycerides is converted with a free fatty acid or mixture of free fatty acids, in particular comprising saturated and mono-unsaturated free fatty acids, to a triglyceride mixture.
  • the products 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.
  • anti-oxidants selected from the group of natural or synthetic tocopherols or other anti-oxidants, enzymes with anti-oxidant properties, such as Glucose oxidase, catalase, BHA, BHT, TBHQ, ascorbyl palmitate, propyl gallate, lecithin, catechins or flavenols.
  • the fats 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 (N 5 : NMR-pulse, not stabilised) that is at least 5 units different from the N 5 of the fatty products, obtainable by the process of claims 1-14 or of the blend of claim 15.
  • N 5 NMR-pulse, not stabilised
  • 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 1-14, or wherein the fatty component or free fatty acid comprises a blend according to claims 15- 16.
  • our invention also concerns the use of materials, enriched in LCPUFA's, wherein the products, as obtainable by the process of claims 1-14 or wherein the blends according to claims 15-16 are used to improve the health benefits of consumer goods, such as food products or personal products .
  • 450g of the oleine fraction were mixed with 3.2g of Candida rugosa lipase dissolved in 650g of water and stirred at 25°C for 68 hours under a nitrogen blanket until 60% of the triglycerides had been hydrolysed to free fatty acid. The mixture was rapidly heated to 80°C to destroy the enzyme activity then the lipid layer was decanted off.
  • the hydrolysed reaction mixture was deacidified by extraction into aqueous methanol .
  • 446g of hydrolysed products were extracted with 3L of methanol containing 2.5 % water at 55°C.
  • a triglyceride/ partial glyceride rich fraction and a free fatty acid rich fraction were thus obtained with compositions as given in table 1.1.
  • the free fatty acid rich fraction from the methanol extraction was esterified with glycerol to produce a triglyceride rich fat.
  • 10 g of the fatty acids were mixed with 1 g of glycerol and 0.6g of Rhizomucor miehei immobilised onto Duolite. The mixture was stirred, in an open glass vial at 55°C for 136 hours with nitrogen blowing across the surface.
  • the composition of the triglyceride rich fat is given in table 1.1. TABLE 1.1 ANALYTICAL DATA EXAMPLE 1
  • a "RANCH STYLE" DRESSING was prepared using the LCPUFA enriched recombined partial glyceride fraction which was compared to a reference dressing made with sunflower oil
  • the formulation for the dressing is given in table 1.2
  • the liquid oil for the reference was sunflower and for the LCPUFA containing product was 90/10 sunflower oil/ enriched product.
  • 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.
  • the pH 3.25 .
  • 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 45mm lens.
  • AN ICECREAM was prepared using the LCPUFA enriched recombined partial glyceride fraction which was compared to a reference ice-cream 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.
  • 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 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 m Bars and a flow rate of 40 to 50 ml/ min.
  • 2.2Kg of the partial glyceride fraction were mixed with 13.2g of Candida rugosa lipase dissolved in 2.2Kg of pH7 phosphate buffer and stirred at 25°C for 70 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 m Bars and a flow rate of 30 to 35 ml/ min.
  • a second triglyceride/ partial glyceride mixture was thus obtained with a composition as given in table 2.1. 252g of the second partial glyceride fraction were vigorously stirred with an equal volume of water and with 5.3g of Rhizomucor miehei immobilised onto Duolite. The mixture was stirred under a nitrogen blanket at 35°C for 48 hours until the free fatty acid content was approximately 55 %. The enzyme was removed by filtration and the free glycerol extracted by water washing. 100 ppm of BHT were added. This hydrolysed partial glyceride fraction was re- esterified to triglyceride using Rhizomucor miehei immobilised onto Duolite as catalyst.
  • the second partial glyceride fraction (before partial hydrolysis) was re-esterified with a mixture of fatty acids produced by the random hydrolysis of sunflower oil. 9.8g of the partial glyceride fraction were mixed with 1.2 g of sunflower oil acids and 0.5g 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 2.1.
  • the free fatty acid rich fraction from the Candida rugosa hydrolysis was esterified with glycerol to form a triglyceride rich product.
  • 9.7 g of the fatty acids were mixed with 1.1 g of glycerol and 0.5g of Rhizomucor miehei immobilised onto Duolite. The mixture was stirred in an open glass vial at 55°C for 212 hours with nitrogen blowing across the surface.
  • the composition of the triglyceride rich fat is given in table 2.1. TABLE 2.1 ANALYTICAL DATA EXAMPLE 2
  • a SPREAD was prepared using the LCPUFA enriched triglyceride 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.
  • the fat blend used was: - InEs 13% Sunflower 78% LCPUFA 9%
  • Es interesterified mix of hardened palm oil and hardened palm kernel olein. 2 kg of material was prepared and processed.
  • a micro-votator processing lines was set up as follows :-
  • 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.
  • LCPUFA 150 10 "*5 I 130 lO "5 I produc t (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.)
  • 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 recombined partial glyceride fraction which was compared to a reference ice-cream made with sunflower oil.
  • the formulation and method of production was as described in example 1.
  • the fatty acids were esterified with glycerol to form a triglyceride rich fat .
  • 2.7g of the fatty acids were mixed with 0.4g of glycerol , 0.3g of water and 0.25g of
  • Candida rugosa lipase immobilised onto Accurel The mixture was stirred in an open glass vial at 35°C for 120 hours with nitrogen blowing across the surface. The resulting glyceride species were separated by thin layer chromatography and the fatty acid compositions determined by FAME GC.
  • the fatty acids were esterified with glycerol to form a triglyceride rich fat.
  • 5.0 g of the fatty acids were mixed with 0.5g of glycerol, 0.3g of water and 0.lg of Candida rugosa lipase immobilised onto Accurel.
  • the mixture was stirred in an open glass vial at 35°C for 40 hours with nitrogen blowing across the surface.
  • the resulting glyceride species were separated by thin layer chromatography and the fatty acid compositions determined by FAME GC. Analytical procedures were as described in example 1.

Abstract

Materials, enriched in long chain poly-unsaturated fatty acids (= LCPUFA) can be obtained from a material A, containing at least 5 % LCPUFA's by splitting A in materials B and C, B having to different LCPUFA's, while its total content LCPUFA's is » 1.5 times the total LCPUFA content of A; B is split further in components D and E, D being enriched in a particular LCPUFA (L1 or L2) by a factor of » 1.5, compared to B and E being depleted in the same LCPUFA.

Description

Production method for fats with 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. x and L2) present in the fat could be achieved, as different LCPUFA's, such as DHA and EPA have a different health effect . In particular it would be very beneficial if fats could be made with high levels of EPA, as it is very difficult to make these fats by conventional one step processes. 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. 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
Figure imgf000004_0001
and total Li+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 are 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 Lx plus L2 will be obtained, our levels of enrichments can not be achieved by this disclosed technology.
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 poly- unsaturated fatty acids (= LCPUFA) , wherein a material A, containing at least 5 wt% of total LCPUFA's is first split into two parts B and C; B having at least two different LCPUFA's, from which Li and L2 are the two most abundant LCPUFA's, while B has a total LCPUFA-content that is at least 1.5 times greater than that of A; B is split into at least two components D and E, wherein D compared to B is enriched by a factor of at least 1.5 in one of the LCPUFA's Li or L2 and E simultaneously, compared to B, is depleted in the same LCPUFA Lx or L2.
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.
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 blackcurrant 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 (docosahexaeonic acid: C22:6) and eicosapentaenoic acid (or
Figure imgf000006_0001
Beneficial fats are obtained, when the long chain polyunsaturated fatty acids Lx and L2 are selected from fatty acids with at least 18 carbon atoms and at least 3 unsaturations, preferably C18:3, C20:4, C20:5 C22:5, and C22:6. however also C18:4, C18:5, C20:3, C22:3, C24:3, C24:4, C24:5 and C24:6 can be applied. Although material A can be subjected to the split, it is also possible to hydrolyse A first, preferably using randomising methods, such as by applying a non-specific lipase or a base, such as ethanolic potassium hydroxide. The product A1 obtained is rich in free fatty acids.
The split of material A or A1 into parts B and C can be performed in a number of ways. Very suitably this split is performed either by: (i) low temperature fractionation, in particular solvent fractionation, followed by filtration to remove stearin-fraction, or
(ii) directed interesterification, both chemically using a base and enzymically, which interesterification is followed by removal of precipitated saturated triglycerides by filtration, either dry or in solvent, or
(iii)glycerolysis, both chemically, using a base and enzymically, which glycerolysis is followed by removal of preciptitated saturated partial glycerides by filtration, either dry or in solvent, or (iv) hydrolysis, using a lipase that is selective against CPUFA's over other fatty acids, followed by evaporation, or by extraction with aqueous alcohol, preferably methanol, or by treatment with an inorganic or organic absorbent, preferably basic alumina or : (v) urea adduction, followed by filtration to remove stearin fraction, or
(vi) directed titration, i.e. a solvent fractionation of metal salts of the free fatty acids, followed by filtration to remove stearin fraction and reconversion back to free fatty acids by addition of a strong acid.
The low temperature fractionation (i) is performed at temperatures between -20 and -65°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 1:8 to 1:2, preferably: 1:6 to 1:4. The oleine-fraction is normally obtained in a yield of 10-50 wt%. The oleine-fraction is the fraction enriched in LCPUFA's (so this is part B) .
The directed interesterification (ii) can be performed by adding a base, such as Na-methylate to the mixture. The temperature applied will range from -5 to 80°C, in particular 10-50°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, including triglycerides rich in saturated fatty acids (such as trisaturated triglycerides) . These triglycerides, rich in saturated triglycerides 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) triglycerides. This separation can be performed by any known suitable separation-technique for separating a liquid and a solid phase. The liquid phase is our part B, the solid phase is our part C. 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 out in e.g. GB 1,577,933. This assures that high levels of triglycerides are obtained, while the formation of extensive quantities of diglycerides is avoided.
Again the reaction can be directed by precipitation of the triglycerides, 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 B, enriched in LCPUFA's can be separated from a product C, enriched in saturated fatty acids.
The hydrolysis (iv) is performed by using a lipase, that is selective against LCPUFA's over other fatty acids. Examples of such lipases are: Geotrichum candidum, Lipase G and Mucor Miehei.
The products B and C obtained in this way are a mixture of triglycerides and partial glycerides (as product B) and a mixture of free fatty acids (as product C) . Because of the use of a lipase that is selective against LCPUFA's over other fatty acids, product B is enriched in LCPUFA, while the fatty acids from product C are enriched in the non- LCPUFA's. So summarizing the above the products B and C obtained after completing the splits (i) to (iv) are respectively; different triglycerides for B and C (reactions i and ii) - a mixture of triglycerides and partial glycerides, both for B and C, which mixture is different for B compared with the mixture for C. (reaction iii) a mixture of triglycerides and partial glycerides (for B) and a mixture of free fatty acids (for C) (reaction iv) .
In a following step product B is split into products D and E by performing an enzymic hydrolysis, using a lipase that can distinguish LCPUFA's of different chain length, preferably by using Candida rugosa. Products D and E are separated by physical separation methods. Suitable methods are : evaporation, extraction with an aqueous alcohol preferably methanol and treatment with an inorganic or organic absorbent, preferably basic alumina. As a result of the use of a lipase, that can distinguish LCPUFA's of different chain length, so e.g. C22:6 over C20:5, a glyceride product D is obtained, wherein the two long chain polyunsaturated fatty acids Lx and L2, originally present in product B in ratio XB are now present in another ratio XD. E.g. when using Candida rugosa product D will have a ratio:
Figure imgf000010_0001
which is higher than the ratio of these fatty acids in product B. The opposite will acount for product E.
It is also possible to perform our process in such away that product B mainly comprises free fatty acids. This product B can be split into D and E by an enzymic conversion with glycerol, using an enzyme selective against LCPUFA. The resulting mixture comprises triglycerides and partial glycerides, depleted in LCPUFA and free fatty acids, enriched in LCPUFA. This mixture can be separated by physical separation methods. These methods include: evaporatio ; - extraction with an aqueous alcohol, preferably methanol or treatment with an inorganic or organic absorbent, preferably basic alumina.
The products D and/or E as obtained by the enzymic hydrolysis or esterification are used for different purposes. E.g. part of product D or E, being a mixture of triglycerides and partial glycerides enriched in Lx or L2, is hydrolysed, resulting in a mixture comprising different free fatty acids and glycerol; the glycerol is removed from the mixture and remaining free fatty acids are reconverted with another part of product D or E, preferably in such a way that the reaction mixture has a stoichiometric composition.
However product D or E, comprising mainly free fatty acids, enriched in Lx or L2 can also be converted to triglycerides by esterification with glycerol or with partial glycerides, preferably in ratios corresponding with stoichiometric compositions.
It is however also possible that reaction product D or E, comprising partial glycerides and optionally also triglycerides is converted with a free fatty acid or mixture of free fatty acids, in particular comprising saturated and mono-unsaturated free fatty acids, to a triglyceride mixture.
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 1-14 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, catalase, BHA, BHT, TBHQ, ascorbyl palmitate, propyl gallate, lecithin, catechins or flavenols.
According to another embodiment of our invention the fats 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 1-14 or of the blend of claim 15. 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 1-14, or wherein the fatty component or free fatty acid comprises a blend according to claims 15- 16.
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 1-14 or wherein the blends according to claims 15-16 are used to improve the health benefits of consumer goods, such as food products or personal products . EXAMPLES:
EXAMPLE 1
Three batches each consisting of 800g of refined Chilean fish oil containing 100 ppm of TBHQ as antioxidant were each dissolved in 3200g of acetone and cooled to -65°C. Stearin fractions were removed by filtration and washed with another 3200g of acetone. All the washes and oleine fractions were combined to produce a long chain poly¬ unsaturated (LCPUFA) enriched product, the composition of which is given in table 1.1. Fatty acid compositions were determined by fatty acid methyl ester gas chromatography (FAME GC) using the method given in AOCS Ce lb-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.
450g of the oleine fraction were mixed with 3.2g of Candida rugosa lipase dissolved in 650g of water and stirred at 25°C for 68 hours under a nitrogen blanket until 60% of the triglycerides had been hydrolysed to free fatty acid. The mixture was rapidly heated to 80°C to destroy the enzyme activity then the lipid layer was decanted off.
The hydrolysed reaction mixture was deacidified by extraction into aqueous methanol . 446g of hydrolysed products were extracted with 3L of methanol containing 2.5 % water at 55°C. A triglyceride/ partial glyceride rich fraction and a free fatty acid rich fraction were thus obtained with compositions as given in table 1.1.
90g of the triglyceride/ partial glyceride mixture were vigorously stirred with an equal volume of water and with lg of Rhizomucor miehei immobilised onto Duolite. The mixture was stirred under a nitrogen blanket at 35°C for 140 hours until the free fatty acid content was approximately 20 %. The enzyme was removed by filtration and the free glycerol removed by water washing. 100 ppm of BHT were added. This hydrolysed partial glyceride fraction was re-esterified to triglyceride using Rhizomucor miehei immobilised onto Duolite as catalyst. 90g of the partial glyceride mixture were mixed with
5g of Rhizomucor miehei at 55°C for 96 hours under vacuum. The enzyme was removed by filtration. The remaining free fatty acid in the triglyceride rich product was removed by treatment with basic alumina in hexane. 100 ppm of BHT were added. The composition of the triglyceride rich fat is given in table 1.1.
The free fatty acid rich fraction from the methanol extraction was esterified with glycerol to produce a triglyceride rich fat. 10 g of the fatty acids were mixed with 1 g of glycerol and 0.6g of Rhizomucor miehei immobilised onto Duolite. The mixture was stirred, in an open glass vial at 55°C for 136 hours with nitrogen blowing across the surface. The composition of the triglyceride rich fat is given in table 1.1. TABLE 1.1 ANALYTICAL DATA EXAMPLE 1
% TG %DG %FFA 20:5 22:6 TOTAL YIELD LCPUFA
FISH OIL 98 1.7 0 15.9 12.3 33.5 A
LOW TEMPERATURE
SOLVENT
FRACTIONATION
STEARIN 12.8 11.2 30.2 77% C
OLEINE 27 15.9 53.2 23% B
60* HYDROLYSIS OF OLEINE FRACTION USING C. RUGOSA LIPASE
60% HYDROLYSIS 30 9 61 27.6 15.9 53.4 PRODUCT
DEACIDIFICATION BY METHANOL EXTRACTION
PARTIAL GLYCERIDE 83 8 8 24.8 28.9 65.9 D FRACTION
FREE FATTY ACID 0 9 91 28.7 11.2 49.1 E FRACTION
RECOMBINATION TO TRIGLYCERIDES USING M. MIEHEI LIPASE
PARTIALLY HYDROLYSE 89 2 9 24.9 29.6 66.1 PARTIAL GLYCERIDE FRACTION THEN RECOMBINE TO TG
RECOMBINED FFA 78 18 2 28.2 10.5 48.7 FRACTION
A "RANCH STYLE" DRESSING was prepared using the LCPUFA enriched recombined partial glyceride fraction which was compared to a reference dressing made with sunflower oil The formulation for the dressing is given in table 1.2
TABLE 1.2 RANCH STYLE DRESSING FORMULATION
Weight percent
Liquid oil 25
Maltodextrin 20
Dried Egg yolk 0.8
Xanthum gum 0.4
Vinegar 5
Water 48.8
The liquid oil for the reference was sunflower and for the LCPUFA containing product was 90/10 sunflower oil/ enriched product.
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 45mm lens.
TABLE 1.3 RANCH DRESSINGS EVALUATION RESULTS EXAMPLE 1
VISCOSITY CP SAUTER MEAN PARTICLE
OIL DIAMETER μm
REFERENCE 4387 2.46
LCPUFA PRODUCT 4527 2.52
AN ICECREAM was prepared using the LCPUFA enriched recombined partial glyceride fraction which was compared to a reference ice-cream made with sunflower oil. The ice creams were made according to the following recipe:
wt%
Fat blend 10.0
Skimmed milk powder 10.0
Icing sugar 12.0 Corn syrup solids 4.0
Dextrose monohydrate 2.0
Sherex IC 9330® 0.6
Water 61.4
Total 100.0
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 (= PO) 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.
TABLE 1.4 ICE-CREAM EVALUATION EXAMPLE 1
Sample Overrun Hardness (%) (gram)
Reference 16.3 458
LCPUFA 62.7 293 PRODUCT
The viscosities of the emulsions were similar. EXAMPLE 2
5Kg of refined Chilean fish oil containing 100 ppm of TBHQ as antioxidant were mixed with 250g of Geotrichum Candida lipase dissolved in 5kg of pH7 phosphate buffer and stirred at 30°C for 48 hours under a nitrogen blanket. At this stage the free fatty acid content was 34 % .
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 m Bars and a flow rate of 40 to 50 ml/ min.
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.
2.2Kg of the partial glyceride fraction were mixed with 13.2g of Candida rugosa lipase dissolved in 2.2Kg of pH7 phosphate buffer and stirred at 25°C for 70 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 m Bars and a flow rate of 30 to 35 ml/ min.
A second triglyceride/ partial glyceride mixture was thus obtained with a composition as given in table 2.1. 252g of the second partial glyceride fraction were vigorously stirred with an equal volume of water and with 5.3g of Rhizomucor miehei immobilised onto Duolite. The mixture was stirred under a nitrogen blanket at 35°C for 48 hours until the free fatty acid content was approximately 55 %. The enzyme was removed by filtration and the free glycerol extracted by water washing. 100 ppm of BHT were added. This hydrolysed partial glyceride fraction was re- esterified to triglyceride using Rhizomucor miehei immobilised onto Duolite as catalyst. 153g of the partial glyceride mixture were mixed with 7.8 g of Rhizomucor miehei and 7.7g of glycerol at 55°C for 184 hours under vacuum .-The enzyme was removed by filtration. The remaining free fatty acid and partial glycerides in the triglyceride rich product were removed by treatment with silica gel in hexane. 100 ppm of BHT were added. The composition of the triglyceride rich fat is given in table 2.1.
The second partial glyceride fraction (before partial hydrolysis) was re-esterified with a mixture of fatty acids produced by the random hydrolysis of sunflower oil. 9.8g of the partial glyceride fraction were mixed with 1.2 g of sunflower oil acids and 0.5g 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 2.1.
The free fatty acid rich fraction from the Candida rugosa hydrolysis was esterified with glycerol to form a triglyceride rich product. 9.7 g of the fatty acids were mixed with 1.1 g of glycerol and 0.5g of Rhizomucor miehei immobilised onto Duolite. The mixture was stirred in an open glass vial at 55°C for 212 hours with nitrogen blowing across the surface. The composition of the triglyceride rich fat is given in table 2.1. TABLE 2.1 ANALYTICAL DATA EXAMPLE 2
%TG %DG %FFA 20:5 22:6 TOTAL LCPUFA
FISH OIL 97.2 1.8 0 15.4 11.5 33.5 A
PARTIAL HYDROLYSIS USING G. CANDIDA LIPASE
34% HYDROLYSIS PRODUCT 34
DEACIDIFICATION BY EVAPORATION
PARTIAL GLYCERIDE FRACTION 68 28.9 1 22.9 18.4 50.3 B
FREE FATTY ACID FRACTION 0 0 100 4 0.6 7.5 C
HYDROLYSIS OF PARTIAL GLYCERIDE FRACTION USING C. RUGOSA LIPASE
HYDROLYSIS PRODUCT
DEACIDIFICATION BY EVAPORATION
PARTIAL GLYCERIDE FRACTION 72 21.5 6.1 20.3 32.7 63.8 D
FREE FATTY ACID FRACTION 100 23.7 10.4 41.8 E
RECOMBINATION TO TRIGLYCERIDE USING M. MIEHEI LIPASE
PARTIALLY HYDROLYSE PARTIAL 91 5.2 3.8 18.7 33.7 62.9 GLYCERIDES THEN RECOMBINE TO TG
PARTIAL GLYCERIDES RECOMBINE 82 6 11 17 30 56 WITH SUNFLOWER ACIDS
FFA FRACTION RECOMBINE TO 82 10 7 23.9 10.4 42.1 TRIGLYCERIDE
A SPREAD was prepared using the LCPUFA enriched triglyceride fraction which was compared to a reference spread made with sunflower oil . The spreads were made with the following formulation: Fat Phase
Fat Blend 40 %
Hymono 7804 0.3 %
Colour (2% β-carotene) 0.02 %
Total 40.32 %
Aqueous Phase (to pH 5.1)
Water 56.44 .
Skimmed Milk Powder 1.5 %
Gelatin (270 bloom) 1.5 %
Potassium Sorbate 0.15 % Citric Acid Powder 0.07 %
All percentages on product basis,
The fat blend for the reference was 13% InEs, 87% SF.
For the LCPUFA product, the fat blend used was: - InEs 13% Sunflower 78% LCPUFA 9%
PRODUCT
In Es = interesterified mix of hardened palm oil and hardened palm kernel 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. )
x conditions Shaft speed 1000 rpm Temperature set at 8°C
C-L 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. TABLE 2.2 TYPICAL PROCESS CONDITIONS FOR SPREAD PRODUCTION
Sample i Exit ^-1 Exit "2 Exit C2 Exit Line
(°C) (°C) (°C) (°C) Pressure
(bar)
Referenc 13.2 18.7 13.6 15.6 0.5 to 2 e
LCPUFA 13.0 18.9 12.2 16.8 1.3 to product 2.2
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 2.3
TABLE 2.3 SPREAD EVALUATIONS EXAMPLE 2
5°C 20°C
Sample C- Conductiv Collar C- Conductiv Collar
Value ity value ity g/cm2 μScrrr1 g/cm2 μSciϊT1
Contro 170 lO-5 I 190 10"5 I 1
LCPUFA 150 10"*5 I 130 lO"5 I produc t (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.
TABLE 2. 4 RANCH DRESSINGS EVALUATION RESULTS EXAMPLE 2
VISCOSITY cP SAUTER MEAN PARTICLE
OIL DIAMETER μm
Reference 4387 2.46
LCPUFA Product 4613 2.31
AN ICE-CREAM was prepared using the LCPUFA enriched recombined partial glyceride fraction which was compared to a reference ice-cream made with sunflower oil. The formulation and method of production was as described in example 1.
TABLE 2.5 TCE-CRF.AM EVALUATION EXAMPLE 2
Sample Overrun Hardness (%) (gram)
Reference 16.3 458
LCPUFA 57.1 342 PRODUCT
The viscosities of the emulsions were similar. EXAMPLE 3
100g of Chilean fish oil were hydrolysed to free fatty acids by refluxing with 23g of potassium hydroxide in 130mls of ethanol and 44mls of water for lhour. The potassium salts were converted to free fatty acids by addition of hydrochloric acid and then extracted into hexane.
21g of the fatty acids were mixed with lOOmls of 0.5M sodium hydroxide and 200mls of acetone The mixture was stirred in a jacketed vessel with a scrape surface stirrer at 40°C for 30minutes then cooled at l°C/min to 4°C at which temperature it was stirred for 1 hour. The crystalline stearin fraction was removed by filtration and washed with a further 75mls of acetone. The sodium salts in the stearin 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. Analytical procedures were as described in example 1.
The fatty acids were esterified with glycerol to form a triglyceride rich fat . 2.7g of the fatty acids were mixed with 0.4g of glycerol , 0.3g of water and 0.25g of
Candida rugosa lipase immobilised onto Accurel. The mixture was stirred in an open glass vial at 35°C for 120 hours with nitrogen blowing across the surface. The resulting glyceride species were separated by thin layer chromatography and the fatty acid compositions determined by FAME GC.
TABLE 3 ANA YTTCAT, DATA EXAMPLE 3
Figure imgf000029_0001
EXAMPLE 4
lOOg of Chilean fish oil were hydrolysed to free fatty acids by refluxing with 23g of potassium hydroxide in 130mls of ethanol and 44mls of water for lhour. The potassium salts were converted to free fatty acids by addition of hydrochloric acid and then extracted into hexane.
41g of the fatty acids were added to 200g 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 2 hours at 70°C then cooled at l°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. 5.0 g of the fatty acids were mixed with 0.5g of glycerol, 0.3g of water and 0.lg of Candida rugosa lipase immobilised onto Accurel. The mixture was stirred in an open glass vial at 35°C for 40 hours with nitrogen blowing across the surface. The resulting glyceride species were separated by thin layer chromatography and the fatty acid compositions determined by FAME GC. Analytical procedures were as described in example 1.
TABLE 4 ANALYTICAL DATA EXAMPLE 4
%TG %DG %FFA 20:5 22:6 TOTAL YIELD LCPUF A
FISH OIL 98 2 0 15.4 11.5 33.9 A
RANDOM HYDROLYSIS USING KOH CATALYST
RANDOM HYDROLYSIS 0 0 100 15.4 11.5 33.9 PRODUCT
UREA FRACTIONATION
STEARIN
OLEINE 0 0 100 36.0 23.4 69.9 41% B
RECOMBINATION OF OLEINE ACIDS TO TRIGLYCERIDE USING C RUGOSA LIPASE
15 _ 14 57 36.0 23.4 69.9
RECOMBINATION PRODUCT
SEPARATION
TRIGLYCERIDE 34.3 8.7 58.1 FRACTION
DIGLYCERIDE 42.3 5.9 60.9 IE FRACTION
EMONOGLYCERIDE 45.4 9.6 67 FRACTION
FREE FATTY ACID 30.6 36.2 78.9 D FRACTION

Claims

Claims
1. Process for the production of materials, enriched in long chain poly-unsaturated fatty acids (= LCPUFA) , wherein a material A, containing at least 5 wt% of total LCPUFA's is first split into two parts B and C; B having at least two different LCPUFA's, from which L-L and L2 are the two most abundant LCPUFA's, while B has a total LCPUFA-content that is at least 1.5 times greater than that of A; B is split into at least two components D and E, wherein D compared to B is enriched by a factor of at least 1.5 in one of the LCPUFA's Lx or L2 and E simultaneously, compared to B, is depleted in the same LCPUFA L-L or L2.
2. Process according to claim 1, wherein 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.
3. Process according to claim 1 or 2, wherein material A is 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 and anchovy 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 blackcurrant seed oil .
4. Process according to claims 1-3, wherein the long chain poly-unsaturated fatty acids L-L and L2 are fatty acids with at least 18 carbon atoms and at least 3 unsaturations, preferably C18:3, C20:4, C20:S, C22.5 and C22:6.
5. Process according to claims 1-4, wherein material A is optionally hydrolyzed, using an enzyme or a base, to a product A1, rich in free fatty acids, whereupon A or
A1 is split into B and C by:
(i) low temperature fractionation, in particular solvent fractionation, followed by filtration to remove the stearin fraction. or
(ii) directed interesterification, both chemically using a base and enzymically, which interesterification is followed by removal of the precipitated saturated triglycerides by filtration either dry or in solvent. or
(iii) glycerolysis, both chemically, using a base and enzymically, which glycerolysis is followed by removal of the precipitated saturated partial glycerides by filtration, either dry or in solvent. or
(iv) hydrolysis, using a lipase that is selective against LCPUFA's over other fatty acids, followed by evaporation, or extraction with aqueous alcohol, preferably methanol, or by treatment with an absorbing inorganic or organic material, preferably basic alumina.
6. Process according to claims 1-5, wherein the split of A or A1 into B and C is performed by:
(i) urea adduction, followed by filtration to remove stearin-fraction, or (ii) directed titration.
7. Process according to claim 5 (iv) , wherein the lipase is selected from: Geotrichum candidum, Lipase G and Mucor Miehei.
8. Process according to claim 5, wherein the products B and C, obtained according to claim 5 (i) and 5 (ii) are both triglycerides; according to claim 5 (iii) are both triglycerides and/or partial glycerides and according to claim 5 (iv) are triglycerides and/or partial glycerides (= product B) and free fatty acids
(= product C) .
9. Process according to claims 1-8, wherein product B is split into products D and E by performing an enzymic hydrolysis, using a lipase that can distinguish LCPUFA's of different chain length, preferably by using Candida rugosa, followed by a physical separation of D from E by either evaporation, or extraction with aqueous alcohol, preferably methanol or treatment with an inorganic absorbent, preferably basic alumina.
10. Process according to claim 1, wherein a product B, comprising mainly free fatty acids is split into D and E by an enzymic conversion with glycerol, using an enzyme selective against long chain PUFA, resulting in a mixture of triglyceride, and particle glycerides depleted in long chain PUFA and free fatty acids enriched in LCPUFA, which mixture is separated by physical methods.
11. Process according to claim 11, wherein the physical separation method is selected from :
- evaporation;
or - extraction with aqueous alcohol, preferably methanol
- or treatment with an inorganic or organic absorbent, preferably basic alumina.
12. Process according to claim 1, wherein part of product D or E, being a mixture of triglycerides and partial glycerides enriched in Lx or L2, is hydrolysed, resulting in a mixture comprising different free fatty acids and glycerol; removing the glycerol from the mixture and reconverting the remaining free fatty acids with another part of product D or E, preferably in such a way that the reaction mixture has a stoichiometric composition.
13. Process according to claim 1, wherein product D or E comprising mainly free fatty acids, enriched in Lx or L2 is converted to triglycerides by esterification with glycerol or with partial glycerides, preferably in ratios corresponding with stoichiometric compositions.
14. Process according to claim 1, wherein the reaction product D or E, comprising partial glycerides and optionally also triglycerides is converted with a free fatty acid or mixture of free fatty acids, in particular comprising saturated and mono-unsaturated free fatty acids, to a triglyceride mixture.
15. Blends of materials, comprising a mixture of the products, as obtainable by the process of claims 1-14 and anti-oxidants, selected from the group of natural or synthetic tocopherols or other anti-oxidants, enzymes with anti-oxidant properties, in particular Glucose oxidase and/or catalase, BHA, BHT or TBHQ
16. Blends of materials, comprising a mixture of the products, as obtainable by the process of claims 1-14 or the blends of claim 15 and 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 1-14 or of the blend of claim 15.
17. 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 1-14, or wherein the fatty component or free fatty acid comprises a blend according to claims 15-16.
18. Use of materials, enriched in LCPUFA's, wherein the products, as obtainable by the process of claims 1-14 or wherein the blends according to claims 15-16 are used to improve the health benefits of consumer goods, such as food products or personal products.
PCT/EP1996/002132 1995-05-24 1996-05-13 Production method for fats with long chain polyunsaturated fatty acids WO1996037586A1 (en)

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EP0964058A1 (en) * 1998-05-12 1999-12-15 Loders Croklaan B.V. Process for the enrichment of compounds in trans-10 isomers
US6127562A (en) * 1998-05-12 2000-10-03 Loders Croklaan B.V. Process for the enrichment of compounds in trans-10 isomers
JP2002537442A (en) * 1999-02-17 2002-11-05 ノルスク ハイドロ アーエスアー Lipase-catalyzed esterification of marine oil
US6762203B2 (en) 1999-08-03 2004-07-13 Kao Corporation Oil composition
EP1211305A4 (en) * 1999-08-03 2002-11-13 Kao Corp Fat compositions
US6852758B2 (en) 1999-08-03 2005-02-08 Kao Corporation Oil composition
WO2003094625A1 (en) * 2002-05-08 2003-11-20 Danmarks Tekniske Uni Technica A facile two-step enzyme process for increasing the content of polyunsaturated fatty acids in fish oil
EP1978102A1 (en) 2007-04-02 2008-10-08 Cognis IP Management GmbH A mixture containing fatty acid glycerides
EP1978101A1 (en) 2007-04-02 2008-10-08 Cognis IP Management GmbH Method for enriching polyunsaturated fatty acids
US7737289B2 (en) 2007-04-02 2010-06-15 Cognis Ip Management Gmbh Process for enriching polyunsaturated fatty acids
US11872201B2 (en) 2018-06-21 2024-01-16 Nuseed Nutritional Us Inc. DHA enriched polyunsaturated fatty acid compositions
CN109182403A (en) * 2018-09-18 2019-01-11 宁波大学 A kind of preparation method of the glyceride rich in DHA

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