WO2016096989A1

WO2016096989A1 - Method of extracting cholesterol from fish oil residue

Info

Publication number: WO2016096989A1
Application number: PCT/EP2015/079997
Authority: WO
Inventors: Frank Eisele; Paul MUGFORD; Alfred ROLLE
Original assignee: Dsm Ip Assets B.V.
Priority date: 2014-12-19
Filing date: 2015-12-16
Publication date: 2016-06-23

Abstract

The present invention relates to method of extracting cholesterol from fish oil, especially from fish oil waste residue, which is a waste stream in the fish oil production.

Description

METHOD OF EXTRACTING CHOLESTEROL FROM FISH OIL RESIDUE

The present invention relates to a method of extracting cholesterol from fish oil, especially from fish oil waste residue, which is a waste stream in the fish oil production. The so isolated crystalline cholesterol is obtained in excellent yield and purity.

Cholesterol, which lUPAC name is (33)-cholest-5-en-3-ol and which systematic name is 2,15-dimethyl-14-(1 ,5-dimethylhexyl)tetracyclo[8.7.0.0²'⁷.0¹¹'¹⁵]heptacos-7-en-5-ol can be found in cheese, egg yolks, beef, pork, poultry, fish, and shrimp. These sources usually contain cholesterol in its free form as well as in the esterified form.

The most important source is lanolin, which is obtained from the wool of sheep.

Cholesterol, is a sterol (or modified steroid), a lipid molecule and is biosynthesized by all animal cells because it is an essential structural component of animal cell membranes that is required to maintain both membrane structural integrity and fluidity.

In addition to its importance within cells, cholesterol also serves as a precursor for the biosynthesis of steroid hormones, bile acids, and vitamin D.

Due to the importance of cholesterol as intermediate in the production of vitamin D and vitamin D derivatives, there is always a need to provide improved as well as alternative isolation methods for providing cholesterol.

Surprisingly it was found that cholesterol can be extracted in high yields and good quality from fish oil. In fact it is also possible to extract cholesterol not only from fish oil, but also from fish oil residue (from which for example the polyunsaturated fatty acids (PUFAs) have been removed), and which is usually burnt.

From this residue, which is a waste stream in the fish oil industry, a valuable product (cholesterol) can be extracted and afterwards the remaining residue (freed from cholesterol in its free as well as esterified form) can still be burnt. The fish oil residue (waste stream of the fish oil industry) which is used for the extraction of the cholesterol is more or less the residue, wherein such compounds as the PUFA oils have been removed by commonly known methods (extractions, distillations, etc.). The residue which is mainly used for this process usually contains up to 15 wt-%, based on the total amount of the residue, of cholesterol equivalents, may it be in free form or es- terified with fatty acids. The content of cholesterol (and the derivatives) can vary. The fish oil residue from which the cholesterol is extracted contains the high boiling fraction of the fish oil process (boiling point more than 400°C at atm. pressure).

Now it was found that the cholesterol can be extracted in high amounts (more than 70% of the overall cholesterol (free and esterified) content) from the fish oil residue. The cho- lesterol is isolated from the fish oil residue in its crystalline form in excellent yield and quality (purity).

The extraction process comprises the following two steps:

(i) saponification followed by

(ii) an extraction with at least one non-water miscible solvent, and

wherein the extraction process (step (ii)) is carried out at elevated temperature, which is above 30°C.

Preferably the temperature used in step (ii) is between 30°C to 80°C, more preferably 40 to 70°C.

Therefore the present invention relates to a process (P) for extracting cholesterol from fish oil residue by

(i) saponification followed by

(ii) an extraction with at least one non-water miscible solvent, and

wherein the extraction process (step (ii)) is carried out at a temperature of above

30°C.

Therefore the present invention relates to a process (P1 ) for extracting cholesterol from fish oil residue by

(i) saponification followed by

(ii) an extraction with at least one non-water miscible solvent, and wherein the extraction process (step (ii)) is carried out at a temperature between 30°C to 80°C.

Therefore the present invention relates to a process (P2) for extracting cholesterol from fish oil residue by

(i) saponification followed by

(ii) an extraction with at least one non-water miscible solvent, and

wherein the extraction process (step (ii)) is carried out at a temperature between 40 to 70°C.

It is surprising that the extraction process is improved significantly by the use of elevated temperature.

Furthermore, the cholesterol obtained by this process can then be recrystallized and it is then obtained in even higher purity. As stated above the obtained cholesterol can then be used in further chemical reactions. The quality (as well as the yield) of the isolated cholesterol is already sufficient without the recrystallization, but if it is needed the quality can be improved.

The first essential step of the process is a saponification. Saponification is a well-known process. The saponification step (step (i)) of the present invention is carried out using the following reaction conditions.

At least one strong base is used in the saponification step. Suitable strong bases are NaOH and KOH.

Usually an aqueous solution of at least one base is used.

The saponification is usually carried out at elevated temperatures. Usual reaction temperatures (internal temperatures) are between 30°C and 150°C.

The saponification according to the present invention is usually carried in a solvent or a mixture of solvent (for example alcohol/water mixture). The reaction time of the saponification step can vary. Usually the saponification step is carried out for a few hours. Therefore the present invention also relates to a process (P3), which is process (P), (P1 ) or (P2), wherein the saponification step (step (i)) is carried out with NaOH and/or KOH.

Therefore the present invention also relates to a process (P4), which is process (P), (P1 ), (P2) or (P3), wherein the saponification step (step (i)) is carried out at elevated tempera- tures, preferably between 30°C and 150°C.

After the saponification step (step (i)), an extraction step (step (ii)) with at least one non- water miscible solvent is carried out.

Step (ii) is carried out by using at least one non-water miscible solvent. Suitable non- water miscible solvents are aromatic as well as aliphatic hydrocarbons.

Preferred are aliphatic hydrocarbons.

Suitable solvents are heptane, hexane, octane, cyclohexane, methyl cyclohexane and nonane. Therefore the present invention also relates to a process (P5), which is process (P), (P1 ), (P2), (P3) or (P4), wherein the extraction step (step (ii)) is carried out with at least one aromatic hydrocarbon and/or aliphatic hydrocarbon.

Therefore the present invention also relates to a process (P6), which is process (P), (P1 ), (P2), (P3) or (P4), wherein the extraction step (step (ii) is carried out with at least one aliphatic hydrocarbon.

Therefore the present invention also relates to a process (Ρ6'), which is process (P6), wherein the extraction step (step (ii)) is carried out with at least one aliphatic hydrocarbon chosen from the group consisting of heptane, hexane, octane, cyclohexane, methyl cyclohexane and nonane.

The non-water miscible solvent (or the mixture of such solvents) is added to the reaction mixture after the saponification step (step (i)). The extraction process (step (ii)) is carried out at elevated temperature. The temperature is usually not higher than the boiling point of the solvent (or mixture of solvents). It is also possible to add NaCI to the reaction mixture. This can be done before or after the addition of the non-water miscible solvent (or the mixture of such solvents) or at the same time.

Then this reaction mixture is mixed for a sufficient time. After the two phases have been separated, the organic phase is set aside. This procedure is usually repeated at least once. At the end all the organic phases are usually combined and the solvent is removed usually by evaporation and the cholesterol is obtained in excellent yields and purity.

The several steps of step (ii) can be carried in any suitable and usual sequence. Step (ii) can be carried our batch-wise as well as continuously. It is possible to evaporate each or- ganic phase separately or to evaporate after all the phases are combined at the end.

As stated above the cholesterol is obtained in crystalline form in good yield as well as good quality (purity).

The purity of the cholesterol can be further improved by commonly known purification processes, such as for example crystallization.

Crystallization is a preferred process for purification of the cholesterol obtained by the extraction process according to the present invention. The crystallization process is usually carried out with at least one alcohol (methanol, ethanol), alcohol/water-mixture or with ar- omatic or aliphatic hydrocarbons or alcohol/hydrocarbon mixtures.

The extraction can be carried out with or without backwash. Backwash is a process wherein the organic phase (non-water miscible solvent containing the cholesterol) is washed with water-miscible solvents to remove any remaining water-soluble impurities. For the backwash water or mixtures of EtOH, NaOH, NaCI and water can be used.

The obtained waste stream of this extraction process according to the present invention can be used as boiler fuel after appropriate treatment (as in example 13). It is still useful as fuel. The following Examples illustrate the invention further without limiting it. All percentages and parts, which are given, are related to the weight and the temperatures are given in °C, when not otherwise stated.

Examples

Example 1 : Saponification with NaOH and heptane Extraction

To reaction vessel, was added warmed fish oil residue (300.6 g), ethanol/water (95/5) mixture (300.3 g) and caustic 7 M NaOH solution (192.2 g). The mixture was stirred under reflux for about 2 hours at bath temperature of 80°C.

After 3 hours, 483.4 g of an ethanol/water (95/5)-mixture was added followed by 10% brine solution (645.3 g).

This mixture was heated to internal temperature of 65°C. Afterwards heptane (817.5 g) was added and the mixture was stirred vigorously at an internal temperature of 65°C for about 15 min. The mixture was allowed to separate for 10 min with the organic phase set aside and to the polar phased was added again heptane (817.5 g) for a second extraction. This was done for a total of 5 heptane extractions of equal volume. The yield of cholesterol was 86.3%.

Example 2: Saponification with NaOH and hexane Extraction

To reaction vessel, was added warmed fish oil residue (300.2 g), ethanol/water (95/5) mixture (300.3 g) and caustic 7 M NaOH solution (192.2 g). The mixture was stirred under reflux for about 2 hours at bath temperature of 80°C.

After 3 hours, 483.4 g of an ethanol/water (95/5)-mixture was added followed by 10% brine solution (642.9 g).

This mixture was heated to internal temperature 52°C. Afterwards hexane (817.4 g) was added and the mixture was stirred vigorously at an internal temperature of for 15 min. The mixture was allowed to separate for 10 min with the organic phase set aside and to the polar phased was added again heptane (817.4 g) for a second extraction. This was done for a total of 5 hexane extractions of equal volume.

The yield of cholesterol was 80.0% Example 3: Crystallization from Methanol

4043.4 g of the combined heptane layers from an heptane extraction (as Example 1 ) was evaporated to dryness to give 37.1 g crude Cholesterol (Purity 71.3w%).

36.6g of the crude Cholesterol was mixed with 769.5g methanol. The mixture was heated to reflux to dissolve the crude Cholesterol. Then, the mixture was cooled at a cooling rate of 20°C/h and the mixture was allowed to slowly stir at 4°C for no less than 1 hour. Crystals filtered cold, and rinsed with 15 g methanol. Solid was dried under reduced pressure 3mbar at 40°C to afford 24.5g of Cholesterol (Purity 92.2w%, residual solvent <0.1w%, fatty acid salts < 1 %)

Crystallization yield: 90.0%

Example 4: Crystallization from Heptane

973.3 g of the combined heptane layers from a heptane extraction (as Example 1 ) was evaporated to dryness to give 9.0g crude cholesterol (Purity 67.4w%).

8.8g of the crude Cholesterol was mixed with 106.0g heptane. The mixture was heated to an internal temperature of 65°C to dissolve the crude cholesterol. Then, the mixture was cooled for 1 h at a cooling rate of 5°C/h, for 1 h at a cooling rate of 10°C/h and at a cooling rate of 20°C/h to IT = 4°C. The mixture was allowed to slowly stir at 4°C for no less than 1 hour. Crystals filtered cold, and rinsed with 5.2 g heptane. Solid was dried under reduced pressure 3mbar at 40°C to afford 4.5g of Cholesterol (Purity 91.3w%, residual solvent <0.1w%, fatty acid salts < 1 %) Crystallization yield: 70.2%

Example 5: Crystallization from Ethanol with solvent exchange

1699.3 g of the combined heptane layers from a heptane extraction (as Example 1 ) was evaporated partially to give 74.4g of a suspension of crude Cholesterol in heptane.

To this suspension 300.2 g of ethanol was added and the distillation was continued to give 76.9 g of a solution of cholesterol in ethanol at an internal temperature of 50°C.

The solution was heated to an internal temperature of 65°C. Then, the mixture was cooled for 1 h at a cooling rate of 5°C/h, for 1 h at a cooling rate of 10°C/h and at a cooling rate of 20°C/h to IT = 4°C. The mixture was allowed to slowly stir at 4°C for no less than 1 hour. Crystals filtered cold, and rinsed with 3.1 g ethanol. Solid was dried under reduced pressure 3mbar at 40°C to afford 1 1.1 g of Cholesterol (Purity 85.7w%, residual solvent <0.1w%)

Crystallization yield: 85.8%

Example 6: Crystallization from an Ethanol/Water (95/5) mixture

3901.3 g of the combined heptane layers from a heptane extraction (as Example 1 ) was evaporated to dryness to give 37.5g crude Cholesterol (Purity 67.6w%).

8.1 g of the crude Cholesterol was mixed with 96.9g ethanol/water 95/5. The mixture was heated to reflux to dissolve the crude Cholesterol. Then, the mixture was cooled for 1 h at a cooling rate of 5°C/h, for 1 h at a cooling rate of 10°C/h and at a cooling rate of 20°C/h to IT = 4°C. The mixture was allowed to slowly stir at 4°C for no less than 1 hour. Crystals filtered cold, and rinsed with 3.0 g ethanol/water 95/5. Solid was dried under reduced pressure 3mbar at 40°C to afford 5.8g of Cholesterol (Purity 92.0w%, residual solvent <0.1w%, fatty acid salts < 1 %) Crystallization yield: 83.1 %

Example 7: Back-Wash of heptane layer

1948.4 g of the combined heptane layers from an heptane extraction (as Example 1 ) was extracted at IT = 65°C with 487.0 g of a mixture of 4 mole/l aq. NaOH, 10% brine and 95% ethanol in a ratio of 1 :1 :1 .4. The bottom layer was removed and the organic layer was extracted twice with 487.5 g water each at IT = 65°C. 1884.6 g of back-washed organic layer was obtained. Back-wash yield: 99.3% Example 8: Crystallization from Heptane after back-wash

442.3 g of the back-washed heptane layer (as from Example 7) was evaporated to dryness to give 3.8g crude Cholesterol (Purity 78.3w%).

3.5g of the crude Cholesterol was mixed with 42.2g heptane. The mixture was heated to an internal temperature of 65°C to dissolve the crude Cholesterol. Then, the mixture was cooled for 1 h at a cooling rate of 5°C/h, for 1 h at a cooling rate of 10°C/h and at a cooling rate of 20°C/h to IT = 4°C. The mixture was allowed to slowly stir at 4°C for no less than 1 hour. Crystals filtered cold, and rinsed with 2.9 g heptane. Solid was dried under reduced pressure 3mbar at 40°C to afford 2.0g of Cholesterol (Purity 93.4w%, residual solvent <0.1w%, fatty acid salts < 0.5%)

Crystallization yield: 81.2%

Example 9: Crystallization from Ethanol after back-wash with solvent exchange

1864.9 g of the back-washed heptane layer (as from Example 7) was evaporated partially to give 61 .4g of a suspension of crude Cholesterol in heptane.

To this suspension 300.1 g of ethanol was added and the distillation was continued to give 75.3 g of a solution of cholesterol in ethanol at an internal temperature of 50°C.

The solution was heated to an internal temperature of 65°C. Then, the mixture was cooled for 1 h at a cooling rate of 5°C/h, for 1 h at a cooling rate of 10°C/h and at a cooling rate of 20°C/h to IT = 4°C. The mixture was allowed to slowly stir at 4°C for no less than 1 hour. Crystals filtered cold, and rinsed with 3.1 g ethanol. Solid was dried under reduced pressure 3mbar at 40°C to afford 1 1.6g of Cholesterol (Purity 90.2w%, residual solvent <0.1w%, fatty acid salts < 0.5%)

Crystallization yield: 86.0%

Example 10: Crystallization from an Ethanol/Water (95/5) mixture after back-wash

1874.5 g of the back-washed heptane layer (as from Example 7) was evaporated to dryness to give 17.7g crude Cholesterol (Purity 79.4w%).

8.0g of the crude Cholesterol was mixed with 96.2g ethanol/water 95/5. The mixture was heated to reflux to dissolve the crude Cholesterol. Then, the mixture was cooled for 1 h at a cooling rate of 5°C/h, for 1 h at a cooling rate of 10°C/h and at a cooling rate of 20°C/h to IT = 4°C. The mixture was allowed to slowly stir at 4°C for no less than 1 hour. Crystals filtered cold, and rinsed with 3.0 g ethanol/water 95/5. Solid was dried under reduced pressure 3mbar at 40°C to afford 5.8g of Cholesterol (Purity 91.6w%, residual solvent <0.1w%, fatty acid salts < 0.3%)

Crystallization yield: 84.6%

Example 11 : Crystallization from a Heptane/Ethanol (9/1) mixture after back-wash with solvent exchange

917.5 g of the back-washed heptane layer (as from Example 7) was evaporated partially to give 35. Og of a suspension of crude Cholesterol in heptane.

To this suspension 2.9 g of ethanol was added and the solution was heated to IT = 65°C. Then, the mixture was cooled for 1 h at a cooling rate of 5°C/h, for 1 h at a cooling rate of 10°C/h and at a cooling rate of 20°C/h to IT = 4°C. The mixture was allowed to slowly stir at 4°C for no less than 1 hour. Crystals filtered cold, and rinsed with 2.5 g ethanol. Solid was dried under reduced pressure 3mbar at 40°C to afford 4.5g of Cholesterol (Purity 91.9w%, residual solvent <0.1w%, fatty acid salts < 0.5%)

Crystallization yield: 69.2%

Example 12: Crystallization from Heptane/Ethanol 1/9 after back-wash

921 .1 g of the back-washed heptane layer (as from Example 7) was evaporated to dryness to give 7.9g crude Cholesterol (Purity 80.4w%).

7.0g of the crude Cholesterol was mixed with 28.3g heptane/ethanol 1/9. The mixture was heated to reflux to dissolve the crude Cholesterol. Then, the mixture was cooled for 1 h at a cooling rate of 5°C/h, for 1 h at a cooling rate of 10°C/h and at a cooling rate of 20°C/h to IT = 4°C. The mixture was allowed to slowly stir at 4°C for no less than 1 hour. Crystals filtered cold, and rinsed with 2.0 g heptane/ethanol 1/9. Solid was dried under reduced pressure 3mbar at 40°C to afford 5.2g of Cholesterol (Purity 90.3w%, residual solvent <0.1w%, fatty acid salts < 0.5%) Crystallization yield: 82.7%

Example 13: Recycle of boiler fuel for incineration

475.6 g of the bottom layer from heptane extraction (as in Example 1 ), 144.3 g distillate from solvent exchange (as in Examples 5, 9 or 1 1 ), 90.0 g mother liqueur of a crystallization from ethanol/water 95/5 (as in example 10) and a total 790.3g of all three aqueous layers from back-extraction (as from Example 7) were mixed, heated to 40°C and acidified to pH = 2 with 57.1 g 35% aq. HCI. The before homogeneous mixture gave two phases upon acidification, which were separate at an internal temperature of 40°C.

132.0 g upper organic layer and 1403.8 g lower aqueous layer were obtained. The lower layer was submitted to stripping for ethanol/water azeotrope recovery.

130.0 g of the upper layer was evaporated to give two layers of distillate, 46.4 g of an upper heptane rich layer and 17.3 g of a lower ethanol rich layer, which were recycled. The sump consisted of 58.6 g solvent free and cholesterol reduced boiler fuel ready for incin- eration (78.4% of the original boiler fuel).

Claims

1. A process for extracting cholesterol from fish oil residue by

(i) saponification followed by

(ii) an extraction with at least one non-water miscible solvent, and

wherein the extraction process (step (ii)) is carried out at a temperature of above 30°C.

2. Process according to claim 1 , wherein the extraction process (step (ii)) is carried out at a temperature between 30 and 80°C.

.

3. Process according to any one of the preceding claims, wherein the saponification step (step (i)) is carried out with NaOH and/or KOH.

4. Process according to any one of the preceding claims, wherein the saponification step (step (i)) is carried out at elevated temperatures, preferably between 30°C and 150°C.

5. Process according to any one of the preceding claims, wherein the extraction step (step (ii)) is carried out with at least one aromatic hydrocarbon and/or aliphatic hydro- carbon.

6. Process according to any one of the preceding claims, wherein the extraction step (step (ii)) is carried out with at least one aliphatic hydrocarbon.

7. Process according to any one of the preceding claims, wherein the extraction step (step (ii)) is carried out with at least one aliphatic hydrocarbon chosen from the group consisting of heptane, hexane, octane, cyclohexane, methyl cyclohexane and nonane.

8. Process according to any one of the preceding claims, wherein the extracted choles- terol is purified by crystallization.