CA2163815A1 - Process for the hydrolysis of the triglycerides of polyunsaturated fatty acids - Google Patents
Process for the hydrolysis of the triglycerides of polyunsaturated fatty acidsInfo
- Publication number
- CA2163815A1 CA2163815A1 CA 2163815 CA2163815A CA2163815A1 CA 2163815 A1 CA2163815 A1 CA 2163815A1 CA 2163815 CA2163815 CA 2163815 CA 2163815 A CA2163815 A CA 2163815A CA 2163815 A1 CA2163815 A1 CA 2163815A1
- Authority
- CA
- Canada
- Prior art keywords
- fatty acids
- oil
- process according
- hydrolysis
- lecithin
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
- C12P7/6409—Fatty acids
- C12P7/6427—Polyunsaturated fatty acids [PUFA], i.e. having two or more double bonds in their backbone
Abstract
In order to prepare a mixture of polyunsaturated fatty acids with an optimum quality, an oil rich in polyunsaturated fatty acids is hydrolysed by a non stereospecific lipase from Candida cylindracea in an oil-in-water medium emulsified in the presence of lecithin as an emulsifying agent and under operating conditions preferably at room temperature and in an aqueous medium buffered to a pH close to neutral.
Description
NO 5244/00 216 ~ 815 The invention concerns the total enzymatic hydrolysis of oils rich in polyunsaturated fatty acids.
Fatty acids of the n-6 and n-3 series have nutritional value, in particular as precursors in the biosynthesis of prostagl~n~ns. It can be advantageous to have available fractions enriched in these fatty acids for various nutritional and cosmetic applications. These fatty acids are found naturally mainly in the form of triglycerides.
Free fatty acids are obtained industrially from triglycerides by hydrolysis at high temperatures and pressures. Free fatty acids may also be obtained chemically from triglycerides by saponification with a strong base, followed by liberation of the fatty acids by neutralization of soaps with the aid of a strong acid and finally by extraction of the fatty acids from the medium with the aid of a non polar solvent, for example hexane.
Application of these methods may cause degradation in the case of polyunsaturated fatty acids.
Enzymatic methods represent an alternative to the preceding methods since they enable reactions to be carried out under mild conditions using very little energy and a less stressed system.
Hydrolysis is usually carried out in a two-phase medium while maintaining efficient stirring to ensure the best possible contact between the aqueous phase and the organic phase.
We have discovered a simple enzymatic method for the total hydrolysis of the triglycerides of an oil which enables hydrolysis to be improved in an unexpected manner whilst obtaining a degree of conversion into fatty acids greater than the degree obtained by the enzymatic method in a two-216381~
-phase medium, this being under conditions which cause little degradation and enable an improvement to be obtained in the quality and stability of polyunsaturated fatty acids compared with the chemical method.
The invention concerns an enzymatic process for the preparation of polyunsaturated fatty acids from an oil rich in these polyunsaturated fatty acids, characterized in that the oil is hydrolysed by a non stereospecific lipase from Candida cylindracea in a medium emulsified in the presence of lecithin as an emulsifying agent.
The process according to the invention is applicable in particular to the hydrolysis of oils rich in fatty acids of the n-3 and n-6 series, in particular gammalinolenic acid, in particular oils from blackcurrant, borage and evening primrose seeds.
When putting the process into practice, complete enzymatic hydrolysis is carried out of the triglycerides of oils rich in polyunsaturated fatty acids in an emulsified medium at a temperature of 20C to 45C, preferably at room temperature, at atmospheric pressure and at pH 6 to 8.
These are the control conditions that are advantageous qualitatively and economically.
The enzymatic reaction takes place at the interface between the organic phase and the aqueous phase containing the enzyme and an aqueous buffer solution, at pH 6-8, close to neutral. The emulsion, of the oil-in-water type, is produced by vigorous stirring of the oil in the presence of the aqueous phase and of the emulsifier to produce an emulsion of oil in the form of fine droplets, for example with a mean diameter of about 450 nm, dispersed in the aqueous phase and stabilized by the emulsifier, a lecithin.
A lecithin that can be used according to the invention may be, preferably, selected from the following phospholipids:
- A lecithin containing more than 60 ~ by weight of acetone insoluble matter (hereinafter AIM), - An oil-free lecithin, containing more than 90 ~ of AIM, - A lecithin fraction soluble in alcohol, containing more than 60 ~ by weight of AIM, enriched in phosphatidyl choline (PC), - An alcohol soluble, oil-free lecithin, containing more than 90 ~ by weight of AIM, - A lecithin fraction containing more than 50 ~ by weight of PC and - Lecithins or their preceding fractions modified by phospholipidase A2 f pancreatic or microbial origin, in which 10 ~ to 90 ~ by weight of the PC is converted into lyso-PC (LPC).
The emulsion can be prepared in advance with a view to subsequent use and for example placed in sterilized containers.
The composition of the emulsion is determined so as to give it lasting stability. If the proportion of oil is increased beyond a certain limit, for example beyond about 20 ~ by weight, the emulsion is no longer homogeneous and the degree of hydrolysis obtained is reduced. A lecithin content of less than about 1 ~ by weight also leads to a reduction in the degree of hydrolysis. Preferably, the lecithin content is 0.1 ~ to 5 ~ by weight, advantageously 0.3 ~ to 0.8 ~ by weight and particularly about 0.5 ~ by weight of the composition. Preferably, the lecithin content is fixed so as to correspond to 1.5 ~ to 4 ~ by weight, and particularly about 2.5 ~ by weight of pure PC, based on the oil content of the emulsion.
The non stereospecific lipase is from Candida cylindracea.
The optimum reaction conditions depend on the nature of the 216~815 . .
substrate to be hydrolysed and the intrinsic properties of the various lipases.
The reaction takes place between 20C and 45C, preferably at the lowest temperature, for example about 20C so that hydrolysis is as complete as possible, which enables in particular polyunsaturated fatty acids to be handled which are particularly sensitive to oxidative degradation which increases with temperature.
The pH does not influence the degree of hydrolysis for values between 6 and 8. A neutral buffer solution of a salt is preferably used, such as, for example, a phosphate with a pH of 6.88, so as to preserve reaction conditions leading to constant and reproducible results. Under the optimum conditions described above, complete hydrolysis can be obtained in 2 to 20 hours.
The degree of hydrolysis depends, under the operating conditions previously described, on the concentration of enzyme used, but beyond a certain limit, this parameter no longer takes part, since the enzymatic reaction takes place at the interface between the oil and the aqueous medium and the necessary quantity of enzyme necessary passes through a maximum depending on the surface area of the available interface. A concentration of 10 to 100 mg/g of oil is preferably used.
Once the reaction has finished, the lipase can preferably be recycled and reused. In order to do this, the emulsion is centrifuged at a high speed to break it, and the lipid phase is then recovered by extraction with a solvent, washing with water, drying over sodium sulphate and evaporating the solvent. Fatty acids are preferably stored away from the light, under an inert atmosphere and at a negative temperature (on the Celsius scale) preferably at about -25C. The lipase in solution in the aqueous phase 21~815 with glycerol formed during hydrolysis is recovered by concentrating it, for example by ultrafiltration. The lipase can then be recycled.
The mixture of fatty acids obtained can be used as a raw material in a process for the enrichment of specific polyunsaturated fatty acids or for the preparation of structured triglycerides in which the fatty acids as well as their positions in the glycerol molecule are specific.
The products obtained can in their turn be used in nutritional, cosmetic and pharmaceutical compositions.
The following examples illustrate the invention. In these, the percentages and parts are by weight, unless otherwise stated.
Examples 1-7 An oil-in-water emulsion was prepared containing 20~ of blackcurrant seed oil and 1~2% of soya lecithin (Asol 100 (R), Lucas Meyer) dissolved in 78.8~ of an aqueous solution of a 0.0 5 M phosphate buffer of pH 6.88 and was passed 5 times through a microfluidizer (110 T, Microfluidics Corporation, Newton) which led to a mean oil droplet diameter of about 450 nm.
Lipase was solubilized in the phosphate buffer, and was then centrifuged at 4000 g for 20 min to remove insoluble residues. The supernatant was used for experiments. 10 ml of the preceding emulsion (containing 2g of blackcurrant seed oil) were placed in a stoppered 25ml Erlenmeyer flask, in a bath thermostatically maintained at the selected temperature with magnetic stirring at 250 rpm, and the enzyme solution was added, corresponding to 0.2g of lipase.
After the reaction, the medium was centrifuged at 4000 g to break the emulsion and the lipid phase was recovered by extraction with ether. It was washed with water and dried 215381~
over sodium sulfate and the solvent was then removed by evaporation. The fatty acids obtained were stored at -25C
away from the light under nitrogen.
The lipase in solution in the aqueous phase as well as the glycerol formed were recovered by ultrafiltration (YM10 module, cut-off threshold 10,000, Amicon, Denver, USA) which gave a concentrated solution of lipase which could be reused.
The degree of hydrolysis, corresponding to the percentage of free fatty acids liberated during the reaction was determined by acid-base titration using a Metrohm 692 titroprocessor. The sample analysed, dissolved in 25 ml of an equivolume mixture of ethanol and ethyl ether was titrated with an alcoholic solution of KOH at a concentration of 0.1 N.
The hydrolysis conditions are shown in table I below:
Fatty acids of the n-6 and n-3 series have nutritional value, in particular as precursors in the biosynthesis of prostagl~n~ns. It can be advantageous to have available fractions enriched in these fatty acids for various nutritional and cosmetic applications. These fatty acids are found naturally mainly in the form of triglycerides.
Free fatty acids are obtained industrially from triglycerides by hydrolysis at high temperatures and pressures. Free fatty acids may also be obtained chemically from triglycerides by saponification with a strong base, followed by liberation of the fatty acids by neutralization of soaps with the aid of a strong acid and finally by extraction of the fatty acids from the medium with the aid of a non polar solvent, for example hexane.
Application of these methods may cause degradation in the case of polyunsaturated fatty acids.
Enzymatic methods represent an alternative to the preceding methods since they enable reactions to be carried out under mild conditions using very little energy and a less stressed system.
Hydrolysis is usually carried out in a two-phase medium while maintaining efficient stirring to ensure the best possible contact between the aqueous phase and the organic phase.
We have discovered a simple enzymatic method for the total hydrolysis of the triglycerides of an oil which enables hydrolysis to be improved in an unexpected manner whilst obtaining a degree of conversion into fatty acids greater than the degree obtained by the enzymatic method in a two-216381~
-phase medium, this being under conditions which cause little degradation and enable an improvement to be obtained in the quality and stability of polyunsaturated fatty acids compared with the chemical method.
The invention concerns an enzymatic process for the preparation of polyunsaturated fatty acids from an oil rich in these polyunsaturated fatty acids, characterized in that the oil is hydrolysed by a non stereospecific lipase from Candida cylindracea in a medium emulsified in the presence of lecithin as an emulsifying agent.
The process according to the invention is applicable in particular to the hydrolysis of oils rich in fatty acids of the n-3 and n-6 series, in particular gammalinolenic acid, in particular oils from blackcurrant, borage and evening primrose seeds.
When putting the process into practice, complete enzymatic hydrolysis is carried out of the triglycerides of oils rich in polyunsaturated fatty acids in an emulsified medium at a temperature of 20C to 45C, preferably at room temperature, at atmospheric pressure and at pH 6 to 8.
These are the control conditions that are advantageous qualitatively and economically.
The enzymatic reaction takes place at the interface between the organic phase and the aqueous phase containing the enzyme and an aqueous buffer solution, at pH 6-8, close to neutral. The emulsion, of the oil-in-water type, is produced by vigorous stirring of the oil in the presence of the aqueous phase and of the emulsifier to produce an emulsion of oil in the form of fine droplets, for example with a mean diameter of about 450 nm, dispersed in the aqueous phase and stabilized by the emulsifier, a lecithin.
A lecithin that can be used according to the invention may be, preferably, selected from the following phospholipids:
- A lecithin containing more than 60 ~ by weight of acetone insoluble matter (hereinafter AIM), - An oil-free lecithin, containing more than 90 ~ of AIM, - A lecithin fraction soluble in alcohol, containing more than 60 ~ by weight of AIM, enriched in phosphatidyl choline (PC), - An alcohol soluble, oil-free lecithin, containing more than 90 ~ by weight of AIM, - A lecithin fraction containing more than 50 ~ by weight of PC and - Lecithins or their preceding fractions modified by phospholipidase A2 f pancreatic or microbial origin, in which 10 ~ to 90 ~ by weight of the PC is converted into lyso-PC (LPC).
The emulsion can be prepared in advance with a view to subsequent use and for example placed in sterilized containers.
The composition of the emulsion is determined so as to give it lasting stability. If the proportion of oil is increased beyond a certain limit, for example beyond about 20 ~ by weight, the emulsion is no longer homogeneous and the degree of hydrolysis obtained is reduced. A lecithin content of less than about 1 ~ by weight also leads to a reduction in the degree of hydrolysis. Preferably, the lecithin content is 0.1 ~ to 5 ~ by weight, advantageously 0.3 ~ to 0.8 ~ by weight and particularly about 0.5 ~ by weight of the composition. Preferably, the lecithin content is fixed so as to correspond to 1.5 ~ to 4 ~ by weight, and particularly about 2.5 ~ by weight of pure PC, based on the oil content of the emulsion.
The non stereospecific lipase is from Candida cylindracea.
The optimum reaction conditions depend on the nature of the 216~815 . .
substrate to be hydrolysed and the intrinsic properties of the various lipases.
The reaction takes place between 20C and 45C, preferably at the lowest temperature, for example about 20C so that hydrolysis is as complete as possible, which enables in particular polyunsaturated fatty acids to be handled which are particularly sensitive to oxidative degradation which increases with temperature.
The pH does not influence the degree of hydrolysis for values between 6 and 8. A neutral buffer solution of a salt is preferably used, such as, for example, a phosphate with a pH of 6.88, so as to preserve reaction conditions leading to constant and reproducible results. Under the optimum conditions described above, complete hydrolysis can be obtained in 2 to 20 hours.
The degree of hydrolysis depends, under the operating conditions previously described, on the concentration of enzyme used, but beyond a certain limit, this parameter no longer takes part, since the enzymatic reaction takes place at the interface between the oil and the aqueous medium and the necessary quantity of enzyme necessary passes through a maximum depending on the surface area of the available interface. A concentration of 10 to 100 mg/g of oil is preferably used.
Once the reaction has finished, the lipase can preferably be recycled and reused. In order to do this, the emulsion is centrifuged at a high speed to break it, and the lipid phase is then recovered by extraction with a solvent, washing with water, drying over sodium sulphate and evaporating the solvent. Fatty acids are preferably stored away from the light, under an inert atmosphere and at a negative temperature (on the Celsius scale) preferably at about -25C. The lipase in solution in the aqueous phase 21~815 with glycerol formed during hydrolysis is recovered by concentrating it, for example by ultrafiltration. The lipase can then be recycled.
The mixture of fatty acids obtained can be used as a raw material in a process for the enrichment of specific polyunsaturated fatty acids or for the preparation of structured triglycerides in which the fatty acids as well as their positions in the glycerol molecule are specific.
The products obtained can in their turn be used in nutritional, cosmetic and pharmaceutical compositions.
The following examples illustrate the invention. In these, the percentages and parts are by weight, unless otherwise stated.
Examples 1-7 An oil-in-water emulsion was prepared containing 20~ of blackcurrant seed oil and 1~2% of soya lecithin (Asol 100 (R), Lucas Meyer) dissolved in 78.8~ of an aqueous solution of a 0.0 5 M phosphate buffer of pH 6.88 and was passed 5 times through a microfluidizer (110 T, Microfluidics Corporation, Newton) which led to a mean oil droplet diameter of about 450 nm.
Lipase was solubilized in the phosphate buffer, and was then centrifuged at 4000 g for 20 min to remove insoluble residues. The supernatant was used for experiments. 10 ml of the preceding emulsion (containing 2g of blackcurrant seed oil) were placed in a stoppered 25ml Erlenmeyer flask, in a bath thermostatically maintained at the selected temperature with magnetic stirring at 250 rpm, and the enzyme solution was added, corresponding to 0.2g of lipase.
After the reaction, the medium was centrifuged at 4000 g to break the emulsion and the lipid phase was recovered by extraction with ether. It was washed with water and dried 215381~
over sodium sulfate and the solvent was then removed by evaporation. The fatty acids obtained were stored at -25C
away from the light under nitrogen.
The lipase in solution in the aqueous phase as well as the glycerol formed were recovered by ultrafiltration (YM10 module, cut-off threshold 10,000, Amicon, Denver, USA) which gave a concentrated solution of lipase which could be reused.
The degree of hydrolysis, corresponding to the percentage of free fatty acids liberated during the reaction was determined by acid-base titration using a Metrohm 692 titroprocessor. The sample analysed, dissolved in 25 ml of an equivolume mixture of ethanol and ethyl ether was titrated with an alcoholic solution of KOH at a concentration of 0.1 N.
The hydrolysis conditions are shown in table I below:
2~63815 Table I
Example Candida cylindracea Temperature Duration %
lipase C h hydrolysis Type B, Biocatalysts Ltd 37 4 99.9 Cardiff, Fngl~n~
2 Type VII, Sigma Chemical 20 20 91.6 Co., St Louis, USA
I0 3 Lot 189, Biogenzia Lemania 37 20 96 SA, T~ nne, Switzerland 4 Type OF, Meito Sangyo Co. 20 8 99.2 Ltd., Tokyo, Japan Type OF, Meito Sangyo Co. 30 8 96.3 Ltd., Tokyo, Japan 6 Type MY, Meito Sangyo Co. 37 20 91.1 Ltd., Tokyo, Japan 7 Type F5, Enzymatix, 37 20 97.6 Cambridge, Fngl~n~l As a comparison, hydrolysis in a two-phase system using the enzymes, temperature conditions and durations of examples 1 to 4, resulted in the following results:
Lipase of example % Hydrolysis in a two-phase system 4 81.1 Examples 8-13 By varying the concentration of enzyme, the proportions of oil and lecithin and the number of successive cycles with recycled lipase, whilst using the same other conditions as in example 4, the degrees of hydrolysis shown below in table II were obtained:
216381~
Table II
Example Composition Concentration Number of % hydrolysis of emulsion of enzyme 20 h (æ) mg/g oil hydrolysis cycles 8 Oil, 30 - - 78 Lecithin, 1.2 Buffer, 68.8 0 9 Oil, 20 - - 89 Lecithin, 1 Buffer, 79 - 20 - 88.6 11 - 50 - 92.7 13 - - 3 94.6 -: Same conditions as in example 4.
Example 14 The quality and stability of the fatty acids obtained by the enzymatic method were evaluated in comparison with those obtained by chemical saponification of blackcurrant seed oil under the following conditions:
100 g of blackcurrant seed oil were mixed with 213 g of an alkaline solution composed of 47.7~ water, 14.3~ sodium hydroxide, 37.8~ ethanol and 0.15~ disodium ethylenediaminetetra-acetate and the mixture was refluxed at 80-85C for 120 min. After addition of 40 g of water, and then 80 ml of fuming hydrochloric acid, the mixture was vigorously stirred for 60 min. The fatty acids formed were extracted with hexane and, the organic phase was washed with water and dried over sodium sulphate and the solvent removed by evaporation.
~16~815 Analysis by gas chromatography of the fatty acid of the two hydrolysates in the form of their methyl esters showed the perfect agreement of their composition, which enabled it to be affirmed that the soya lecithin was not hydrolysed.
Analysis by W spectroscopy of the fatty acids in iso-octane solution showed that the values for the adsorption maxima of the fatty acids obtained by enzymatic hydrolysis were lower than those of fatty acids obtained chemically.
Moreover, the values of the molar extinction coefficients, calculated for several wavelengths and for the two types of fatty acid, were higher in the case of fatty acids obtained chemically than in the case of fatty acids obtained enzymatically.
Finally, a determination of the peroxide index of the fatty acids enabled the two hydrolysis methods and the quality of the fatty acids obtained to be compared. The values found by converting ferric ions into ferric thiocyanate with a red colour measured at a wavelength of 510 nm on a Hewlett Packard 845A spectrophotometer, enabled the peroxide index to be determined expressed in meq of O2/kg. The results were as follows:
Fatty acids obtained by saponification 7.9 Fatty acids obtained enzymatically 1.4 The preceding analytical results confirmed that the biocatalysed hydrolysis reaction led to a mixture of fatty acids having a quality appreciably greater than did chemical hydrolysis.
Example Candida cylindracea Temperature Duration %
lipase C h hydrolysis Type B, Biocatalysts Ltd 37 4 99.9 Cardiff, Fngl~n~
2 Type VII, Sigma Chemical 20 20 91.6 Co., St Louis, USA
I0 3 Lot 189, Biogenzia Lemania 37 20 96 SA, T~ nne, Switzerland 4 Type OF, Meito Sangyo Co. 20 8 99.2 Ltd., Tokyo, Japan Type OF, Meito Sangyo Co. 30 8 96.3 Ltd., Tokyo, Japan 6 Type MY, Meito Sangyo Co. 37 20 91.1 Ltd., Tokyo, Japan 7 Type F5, Enzymatix, 37 20 97.6 Cambridge, Fngl~n~l As a comparison, hydrolysis in a two-phase system using the enzymes, temperature conditions and durations of examples 1 to 4, resulted in the following results:
Lipase of example % Hydrolysis in a two-phase system 4 81.1 Examples 8-13 By varying the concentration of enzyme, the proportions of oil and lecithin and the number of successive cycles with recycled lipase, whilst using the same other conditions as in example 4, the degrees of hydrolysis shown below in table II were obtained:
216381~
Table II
Example Composition Concentration Number of % hydrolysis of emulsion of enzyme 20 h (æ) mg/g oil hydrolysis cycles 8 Oil, 30 - - 78 Lecithin, 1.2 Buffer, 68.8 0 9 Oil, 20 - - 89 Lecithin, 1 Buffer, 79 - 20 - 88.6 11 - 50 - 92.7 13 - - 3 94.6 -: Same conditions as in example 4.
Example 14 The quality and stability of the fatty acids obtained by the enzymatic method were evaluated in comparison with those obtained by chemical saponification of blackcurrant seed oil under the following conditions:
100 g of blackcurrant seed oil were mixed with 213 g of an alkaline solution composed of 47.7~ water, 14.3~ sodium hydroxide, 37.8~ ethanol and 0.15~ disodium ethylenediaminetetra-acetate and the mixture was refluxed at 80-85C for 120 min. After addition of 40 g of water, and then 80 ml of fuming hydrochloric acid, the mixture was vigorously stirred for 60 min. The fatty acids formed were extracted with hexane and, the organic phase was washed with water and dried over sodium sulphate and the solvent removed by evaporation.
~16~815 Analysis by gas chromatography of the fatty acid of the two hydrolysates in the form of their methyl esters showed the perfect agreement of their composition, which enabled it to be affirmed that the soya lecithin was not hydrolysed.
Analysis by W spectroscopy of the fatty acids in iso-octane solution showed that the values for the adsorption maxima of the fatty acids obtained by enzymatic hydrolysis were lower than those of fatty acids obtained chemically.
Moreover, the values of the molar extinction coefficients, calculated for several wavelengths and for the two types of fatty acid, were higher in the case of fatty acids obtained chemically than in the case of fatty acids obtained enzymatically.
Finally, a determination of the peroxide index of the fatty acids enabled the two hydrolysis methods and the quality of the fatty acids obtained to be compared. The values found by converting ferric ions into ferric thiocyanate with a red colour measured at a wavelength of 510 nm on a Hewlett Packard 845A spectrophotometer, enabled the peroxide index to be determined expressed in meq of O2/kg. The results were as follows:
Fatty acids obtained by saponification 7.9 Fatty acids obtained enzymatically 1.4 The preceding analytical results confirmed that the biocatalysed hydrolysis reaction led to a mixture of fatty acids having a quality appreciably greater than did chemical hydrolysis.
Claims (10)
1. Enzymatic process for the preparation of polyunsaturated fatty acids from an oil rich in these polyunsaturated fatty acids, characterized in that the oil is hydrolysed by a non stereospecific lipase from Candida cylindracea in a medium emulsified in the presence of lecithin as an emulsifying agent.
2. Process according to claim 1, characterized in that the emulsion is of the oil-in-water type and contains up to 30 % by weight of oil and 0.1 % to 5 %, and in particular 0.3 % to 0.8 % by weight of lecithin.
3. Process according to claim 1, characterized in that the reaction takes place at room temperature.
4. Process according to claim 1, characterized in that the aqueous phase contains a buffer salt maintaining the pH at about neutral.
5. Process according to claim 1, characterized in that a quantity of enzyme is used corresponding to 10 to 100 mg/g of oil.
6. Process according to claim 1, characterized in that the reaction takes 2 to 20 hours.
7. Process according to claim 1, characterized in that, once the reaction is complete, the emulsion is centrifuged at a high speed to break it, and the lipid phase is then recovered by extracting it with a solvent, washing it with water, drying it and evaporating the solvent, the fatty acids obtained being stored out of the light, under an inert atmosphere and at a negative temperature whilst awaiting use.
8. Process according to claim 1, characterized in that the lipase in solution in the aqueous phase with glycerol formed during hydrolysis is recovered by concentrating it by ultrafiltration and by recycling for re-use in a new hydrolysis.
9. Process according to claim 1, characterized in that the oil used is an oil rich in fatty acids of the n-3 and n-6 series, particularly in gammalinolenic acid, particularly selected from the oil of blackcurrant, borage and evening primrose seeds.
10. Mixture of fatty acids obtained by working out the process according to one of claims 1 to 9.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| EP94118663A EP0713917A1 (en) | 1994-11-28 | 1994-11-28 | Process for hydrolysis of polyunsaturated fatty acid triglycerides |
| EP94118663.7 | 1994-11-28 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2163815A1 true CA2163815A1 (en) | 1996-05-29 |
Family
ID=8216490
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA 2163815 Abandoned CA2163815A1 (en) | 1994-11-28 | 1995-11-27 | Process for the hydrolysis of the triglycerides of polyunsaturated fatty acids |
Country Status (6)
| Country | Link |
|---|---|
| EP (1) | EP0713917A1 (en) |
| JP (1) | JPH08205879A (en) |
| CN (1) | CN1131697A (en) |
| AU (1) | AU3776595A (en) |
| BR (1) | BR9505529A (en) |
| CA (1) | CA2163815A1 (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2016187415A1 (en) * | 2015-05-20 | 2016-11-24 | Cargill, Incorporated | Modified lecithin for asphalt applications |
| US20220098628A1 (en) * | 2020-09-30 | 2022-03-31 | Chant Oil Co., Ltd. | METHOD FOR PRODUCING CIS-UNSATURATED FATTY ACID BY RECOMBINANT CANDIDA RUGOSA LIPASE 1 (rCRL1) |
| US11352537B2 (en) | 2014-09-29 | 2022-06-07 | Cargill, Incorporated | Lecithin drying using fatty acids |
Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DK1411129T3 (en) * | 2001-07-02 | 2010-04-19 | Suntory Holdings Ltd | Process for the production of fat comprising triglyceride containing high unsaturated fatty acid |
| US8153391B2 (en) * | 2008-08-29 | 2012-04-10 | Bunge Oils, Inc. | Hydrolases, nucleic acids encoding them and methods for making and using them |
| WO2014166421A1 (en) * | 2013-04-12 | 2014-10-16 | Rhodia Operations | Hydrolysis of an ester compound |
| CN103725716B (en) * | 2013-12-25 | 2016-04-13 | 无锡新和源发酵技术研究院有限公司 | A kind of take neutral fat as the method for substrate biosynthesizing cis-3-hexenol |
| CN114317625B (en) * | 2020-09-30 | 2024-04-12 | 承德油脂股份有限公司 | Method for producing cis-unsaturated fatty acid by recombinant candida rugosa lipase |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4865978A (en) * | 1986-07-03 | 1989-09-12 | The United States Of America As Represented By The Secretary Of Agriculture | Lipolytic splitting of fats and oils |
| IN171359B (en) * | 1986-09-17 | 1992-09-19 | Colgate Palmolive Co |
-
1994
- 1994-11-28 EP EP94118663A patent/EP0713917A1/en not_active Withdrawn
-
1995
- 1995-11-09 AU AU37765/95A patent/AU3776595A/en not_active Abandoned
- 1995-11-27 BR BR9505529A patent/BR9505529A/en active Search and Examination
- 1995-11-27 JP JP7307018A patent/JPH08205879A/en not_active Withdrawn
- 1995-11-27 CA CA 2163815 patent/CA2163815A1/en not_active Abandoned
- 1995-11-27 CN CN 95121741 patent/CN1131697A/en active Pending
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US11352537B2 (en) | 2014-09-29 | 2022-06-07 | Cargill, Incorporated | Lecithin drying using fatty acids |
| US11713408B2 (en) | 2014-09-29 | 2023-08-01 | Cargill, Incorporated | Lecithin drying using fatty acids |
| WO2016187415A1 (en) * | 2015-05-20 | 2016-11-24 | Cargill, Incorporated | Modified lecithin for asphalt applications |
| US10689406B2 (en) | 2015-05-20 | 2020-06-23 | Cargill, Incorporated | Modified lecithin for asphalt applications |
| US11345718B2 (en) | 2015-05-20 | 2022-05-31 | Cargill, Incorporated | Modified lecithin for asphalt applications |
| US11820786B2 (en) | 2015-05-20 | 2023-11-21 | Cargill, Incorporated | Modified lecithin for asphalt applications |
| US20220098628A1 (en) * | 2020-09-30 | 2022-03-31 | Chant Oil Co., Ltd. | METHOD FOR PRODUCING CIS-UNSATURATED FATTY ACID BY RECOMBINANT CANDIDA RUGOSA LIPASE 1 (rCRL1) |
| US11685939B2 (en) * | 2020-09-30 | 2023-06-27 | Chant Oil Co., Ltd. | Method for producing cis-unsaturated fatty acid by recombinant Candida rugosa lipase 1 (rCRL1) |
Also Published As
| Publication number | Publication date |
|---|---|
| JPH08205879A (en) | 1996-08-13 |
| EP0713917A1 (en) | 1996-05-29 |
| BR9505529A (en) | 1997-11-04 |
| AU3776595A (en) | 1996-06-06 |
| CN1131697A (en) | 1996-09-25 |
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