Fish oil cholesterol

Abstract

Disclosed is a process for producing cholesterol from fish oil, including the following steps: (a) distilling fish oil in a vacuum distillation column to obtain a first residue and a first distillate, (b) distilling the first distillate in a vacuum distillation column to obtain a second distillate and a second residue, (c) contacting the second residue with an alkali to produce a saponified mixture, (d) contacting the saponified mixture with a non-polar organic solvent or a mixture of non-polar organic solvents to produce an organic phase and an aqueous phase, (e) separating the organic phase from the aqueous phase, (f) cooling the organic phase to form a solid phase and a liquid phase, and (g) separating the solid phase from the organic phase, wherein the separated solid phase includes cholesterol.

Claims

1. A process for producing cholesterol from fish oil, comprising the following steps: (a) distilling fish oil in a vacuum distillation column to obtain a first residue and a first distillate, wherein the fish oil is distilled in an admixture with an auxiliary fluid comprising an ethyl ester of a fatty acid, (b) distilling the first distillate in a vacuum distillation column to obtain a second distillate and a second residue, (c) contacting the second residue with an alkali metal hydroxide to produce a saponified mixture, (d) contacting the saponified mixture with a non-polar organic solvent or a mixture of non-polar organic solvents to produce an organic phase and an aqueous phase, (e) separating the organic phase from the aqueous phase, (f) cooling the organic phase to form a solid phase and a liquid phase, and (g) separating the solid phase from the organic phase, wherein the separated solid phase comprises cholesterol comprising at least 95 wt. % of cholesterol and having a lower content of anthropogenic contaminants than the fish oil, wherein the cholesterol is formed in a single extraction-crystallization step.

2. The process according to claim 1, wherein the vacuum distillation column is a short-path distillation column.

3. The process according to claim 1, wherein the fish oil is fed into the vacuum distillation column in step (a) at a rate of 1 to 150 kg/h per m.sup.2 of evaporator area.

4. The process according to claim 1, wherein the weight ratio of the auxiliary fluid to the fish oil in the mixture is about 1:100 to 10:100.

5. The process according to claim 1, wherein the admixture is fed into the vacuum distillation column at a rate of 1 to 150 kg/h per m.sup.2 of evaporator area.

6. The process according to claim 1, wherein step (a) is conducted at an evaporation temperature of 150 to 300 C. and a column pressure of 0.0001 to 0.5 mbar.

7. The process according to claim 1, wherein the first distillate is fed into the vacuum distillation column in step (b) at a rate of 10 to 350 kg/h per m.sup.2 of evaporator area.

8. The process according to claim 1, wherein step (b) is conducted at an evaporation temperature of 100 to 250 C. and a column pressure of 0.0001 to 0.5 mbar.

9. The process according to claim 1, wherein the alkali metal hydroxide of step (c) is NaOH or KOH.

10. The process according to claim 1, wherein the non-polar organic solvent or the mixture of non-polar organic solvents of step (d) comprises aliphatic hydrocarbon solvent.

11. The process according to claim 1, wherein the organic phase and the aqueous phase are separated by decanting or centrifuging.

12. The process according to claim 1, wherein the separated organic phase is kept at less than 30 C. to form a solid phase and a liquid phase.

13. The process according to claim 1, wherein the ethyl ester of a fatty acid are C10 to C22 fatty acids.

Description

COMPARATIVE EXAMPLE

(1) Cholesterol from Anchovy Oil According to the Process of Patent GB 489623.

(2) Anchovy oil was processed according to the process disclosed in patent GB 489623, as embodied in Example 1 of GB 489623 for whale oil.

(3) 100 kg of the anchovy oil having a total cholesterol content of 7.4 mg/g was fed to a VK 83 short path distillation column and distilled at the temperature of 90 C. and the pressure of 0.003 mbar. The condenser temperature was set at 50 C. A distillate D1 in the amount of 1.6 kg was obtained together with a residue R1, the residual anchovy oil of the first distillation. The content of cholesterol of D1 was below 0.1%.

(4) Next, R1 was fed to a VK 83 short path distillation column and distilled at the temperature of 130 C. and the pressure of 0.002 mbar. The condenser temperature was set at 50 C. A distillate D2 in the amount of 1.1 kg was obtained together with a residue R2, the residual anchovy oil of the second distillation. The content of cholesterol of D2 was 0.8%.

(5) As the D2 split was low, around 1%, the following distillations were done using an auxiliary fluid of the composition shown in Table 1 below. It should be noted, that in similar circumstances, use of an auxiliary fluid may help prevent the clogging of the condenser, as disclosed in U.S. Pat. No. 2,126,467.

(6) R2 was admixed with 5 kg of auxiliary fluid of the composition shown in Table 1 and the mixture was fed to a VK 83 short path distillation column and distilled at the temperature of 180 C. and the pressure of 0.002 mbar. The condenser temperature was set to 20 C. A distillate D3 in the amount of 5.8 kg was obtained together with a residue R3, the residual anchovy oil of the third distillation. The content of cholesterol in D3 was 6.6%.

(7) Next, R3 was admixed with 5 kg of auxiliary fluid of the composition shown in Table 1 and the mixture was fed to a VK 83 short path distillation column and distilled at the temperature of 220 C. and the pressure of 0.002 mbar. The condenser temperature was set to 20 C. A distillate D4 in the amount of 5.3 kg was obtained together with a residue R4, the residual anchovy oil of the fourth distillation. The content of cholesterol in D3 was 6.2%.

(8) TABLE-US-00001 TABLE 1 Auxiliary fluid composition in Comparative Example. Fatty acid ethyl ester Composition concentration, % Myristic acid (C14:0) ethyl ester. 6.6 Palmitic acid (C16:0) ethyl ester 8.2 Palmitoleic acid (C16:1) ethyl ester 46.4 Stearic acid (C18:0) ethyl ester 1.9 Oleic acid (C18:1) ethyl ester 29.3 Linoleic acid (C18:2) ethyl ester 4.1 Alpha-linolenic acid (18:3) ethyl ester 3.5

(9) Next, 580 g of D3 and 530 g of D4 were combined and saponified. Extraction was performed two times with 5 kg of ethyl ether. The ethyl ether extract was evaporated recovering 94 g of residue. The residue was dissolved with 250 g of ethyl acetate and cooled in a refrigerator overnight. Solid crystals were formed. The solids were separated by filtration and then dried in a vacuum oven to obtain 62 g of dry solids having a cholesterol concentration of 90.4%. Total poly aromatic hydrocarbons (PAH) of the solids was 31.5 ppb, higher than in the original anchovy oil.

(10) Concerning the residual fish oil of each distillation, Table 2 shows the combined EPA and DHA content, trans fatty acid content and acid number of each residue.

(11) TABLE-US-00002 TABLE 2 Combined EPA and DHA content, trans fatty acid content an acid number Anchovy Oil R1 R2 R3 R4 EPA + DHA, % 26.7 26.8 26.2 25.1 24.3 Trans Fatty Acids, % 0.3% 0.3% 0.4% 0.6% 0.7% Acid Number, mg KOH/g 6.3 0.3 0.1 <0.05 <0.05

(12) As can be observed in Table 2, the obtaining of cholesterol fractions by fractionation of the fish oil as disclosed in patent GB 489623, leads to an increase of the trans fatty content and a loss of (EPA+DHA), presumably due to polymerization in the residual fish oil. After four successive distillations, the trans fatty acid content increased by about 130% and the EPA+DHA content decreased by about 9%.

(13) Comparative Example shows that cholesterol obtained according to the process of GB 489623 is not a pharmaceutical grade cholesterol, contains a detectable amount of impurities coming from fish oil, and thus the residual or processed fish oil is not suitable for human or animal consumption.

EXAMPLE 1

(14) Cholesterol from Neutralized Anchovy Oil.

(15) Anchovy oil (same raw material as in Example 1) was neutralized with caustic soda and washed with hot water to yield a neutralized anchovy oil with an acid number of 0.2 mg KOH/g.

(16) 250 kg of the neutralized anchovy oil was admixed with 15 kg of the auxiliary fluid set forth in Table 1.

(17) The admixture was fed to a VK 83 short path distillation column and distilled at the temperature of 253 C. and the pressure of 0.008 mbar. The condenser temperature was set at 20 C. A distillate D1 in the amount of 18 kg was obtained together with a residue of anchovy oil R1.

(18) Next, 15 kg of distillate D1 was fed to a VK 83 short path distillation column at the temperature of 155 C. and the pressure of 0.007 mbar. The condenser temperature was set at 20 C. A residue R2 in the amount of 3.5 kg was obtained.

(19) Next, 1 kg of R2 was contacted in a stirred reactor with 2 kg of water, 1 kg of ethanol (190 proof) and 110 g of NaOH (99%) to form a first mixture, stirred and heated to 77 C. for a period of time of 2 hours. Then the mixture was cooled to 35 C. and contacted in the same reactor with 5 kg of hexane to form a second mixture which was stirred for 5 minutes then let to stand until two immiscible phases were formed: a first aqueous phase and a first organic phase. After separating the two phases, the first aqueous phase was contacted with 5 kg of fresh hexane to form a second mixture which was agitated for 5 minutes and then let to settle to form a second organic phase and a second aqueous phase. After separating these phases, the first and second organic phases were combined and the combination was contacted with 500 g of water and 500 g of ethanol, agitated and then let to stand until a third organic phase and a third aqueous phase was formed. The third organic phase was separated from the third aqueous phase and then partially evaporated to obtain 1752 g of a residue R3.

(20) Next, residue R3 was cooled overnight in a refrigerator at 5 C. Solid crystals were formed. The solids were separated by filtration and then dried in a vacuum oven to obtain 261 g of dry solids.

(21) Table 3 below presents the analytical results for the Example 1.

(22) TABLE-US-00003 TABLE 3 Analytical results for the Example 1. Neutralized Distillate Dry Anchovy oil D1 Solid Free cholesterol, mg/g 7.0 92.7 971.5 Total cholesterol, mg/g 7.4 93.0 971.5 Cholesterol ester.sup.1, mg/g 0.8 0.5 <LOQ Non-saponifiable matter, % 1.38 13.40 100 Acid number, mg KOH/g 0.2 2.6 <LOQ Dioxins, Furans and Dioxin like 1.41 12.83 <LOQ PCBs, TEQ ppt (lower bound) PCB 209, ppb (lower bound) 18.53 225.27 <LOQ Total PAHs, ppb 14.11 133.6 <LOQ Pesticides, ppb 18.4 241.3 <LOQ .sup.1As mg of cholesteryl oleate/g of sample. LOQ: Limit of Quantification

(23) In R1, there was no difference between the trans fatty acid and EPA+DHA content with respect to the anchovy oil, and the concentrations of toxic and/or harmful anthropogenic contaminants were below the quantification limits.

EXAMPLE 2

(24) Cholesterol from Sardine Oil

(25) 240 kg of sardine oil was fed to a VK 83 short path distillation column and distilled at the temperature of 253 C. and the pressure of 0.03 mbar. The condenser temperature was set to 50 C. A distillate D1 in the amount of 18.6 kg together with a residue of sardine oil R1 was obtained.

(26) Next, 10 kg of distillate D1 was fed to a VK 83 short path distillation column at the temperature of 170 C. and the pressure of 0.01 mbar. The condenser temperature was set to 40 C. A residue R2 in the amount of 3.3 kg was obtained.

(27) Next, 1 kg of R2 was contacted in a stirred reactor with 1.5 kg of water, 1 kg of ethanol (190 proof) and 105 g of NaOH (99%) to form a first mixture, stirred and heated to 78 C. for a period of time of 2 hours. Then the mixture was cooled to 35 C. and contacted in the same reactor with 5 kg of petroleum ether to form a second mixture which was stirred for 5 minutes then let to stand until two immiscible phases were formed: a first aqueous phase and a first organic phase. After separating the two phases, the first aqueous phase was contacted with 5 kg of fresh hexane to form a second mixture which was agitated for 5 minutes and then let to settle to form a second organic phase and a second aqueous phase. After separating these phases, the first and second organic phases were combined and the combination was contacted with 500 g of water and 500 g of ethanol, agitated and then let to stand until a third organic and a third aqueous phase was formed. The third organic phase was separated from the third aqueous phase and then partially evaporated to obtain 1600 g of a residue R3.

(28) Next, R3 was contacted with 200 g of ethanol (190 proof) and cooled in a refrigerator at 5 C. for 8 hours. Solid crystals were formed. The solids were separated by filtration and then dried in a vacuum oven to obtain 252 g of dry solids.

(29) Table 4 below presents the analytical results for the Example 2.

(30) TABLE-US-00004 TABLE 4 Analytical results for Example 2. Sardine oil Distillate D1 Dry Solid Free cholesterol, mg/g 9.3 118.6 964.9 Total cholesterol, mg/g 9.6 118.9 964.9 Cholesterol ester.sup.1, mg/g 0.5 0.5 <LOQ Unsaponifiable matter, % 1.47 16.01 100 Acid number, mg KOH/g 14.4 171.3 <LOQ Free (EPA + DHA) % 0.5 6.8 <LOQ Dioxins, Furans and Dioxin like 1.61 16.96 <LOQ PCBs, TEQ ppt (lower bound) PCB 209, ppb (lower bound) 17.21 218.12 <LOQ Total PAHs, ppb 28.15 328.63 <LOQ Pesticides, ppb 21.0 248.5 <LOQ Inorganic As, ppm 2.2 <LOQ <LOQ Heavy metals, ppm 0.06 <LOQ <LOQ .sup.1As mg of cholesteryl oleate/g of sample. LOQ: Limit of Quantification

(31) In R1, there was no difference between the trans fatty acid and EPA+DHA content with respect to the anchovy oil, and the concentrations of toxic and/or harmful anthropogenic contaminants were below the quantification limits.

EXAMPLE 3

(32) Cholesterol from Bleached Sardine Oil

(33) 250 kg of sardine oil with 2 kg of bleaching clay was heated at 70 C. and at a vacuum of 50 mbar in a stirred vessel for 30 minutes. After separating the clay by filtration, 245 kg of bleached sardine oil was obtained.

(34) 240 kg of bleached sardine oil was admixed with 10 kg of auxiliary fluid of the composition shown in Table 2 above and the admixture was fed to a VK 83 short path distillation column and distilled at the temperature of 245 C. and the pressure of 0.008 mbar. The condenser temperature was set to 20 C. A distillate D1 in the amount of 21.3 kg and a residue bleached sardine oil R1 were obtained.

(35) Next, 15 kg of distillate D1 was fed to a VK 83 short path distillation column at the temperature of 167 C. and the pressure of 0.004 mbar. The condenser temperature was set to 20 C. A residue R2 in the amount of 4.4 kg was obtained.

(36) Next, 1 kg of R2 was contacted in a stirred reactor with 2 kg of water, 2 kg of ethanol (190 proof) and 130 g of NaOH (99%) to form a first mixture, stirred and heated to 78 C. for a period of time of 1 hour. Then the mixture was cooled to 40 C. and contacted in the same reactor with 5 kg of cyclohexane to form a second mixture which was stirred for 5 minutes then let to stand until two immiscible phases were formed: a first aqueous phase and a first organic phase. After separating the two phases, the first aqueous phase was contacted with 5 kg of fresh cyclohexane to form a second mixture which was agitated for 5 minutes and then let to settle to form a second organic phase and a second aqueous phase. After separating these phases, the first and second organic phases were combined and the combination was contacted with 500 g of water and 500 g of ethanol, agitated and then let to stand until a third organic phase and a third aqueous phase was formed. The third organic phase was separated from the third aqueous phase, and then evaporated to dryness to obtain 251 g of a residue R3.

(37) Next, the residue R3 was dissolved with 1.2 kg of acetone at 55 C. cooled in a refrigerator at 5 C. for 24 hours. Solid crystals were formed. The solids were separated by filtration and then dried in a vacuum oven to obtain 189 g of dry solids. Table 5 presents the analytical results for Example 3.

(38) TABLE-US-00005 TABLE 5 Analytical results for Example 3 Sardine oil Distillate D1 Dry Solid Free cholesterol, mg/g 7.5 82.5 975.1 Total cholesterol, mg/g 8.2 83.9 975.1 Cholesterol ester.sup.1, mg/g 1.2 2.4 <LOQ Unsaponifiable matter, % 1.66 13.04 100 Acid number, mg KOH/g 7.3 171.0 <LOQ Free (EPA + DHA) % 0.7 7.6 <LOQ Dioxins, Furans and Dioxin like 3.61 17.08 <LOQ PCBs, TEQ ppt(lower bound) PCB 209, ppb(lower bound) 17.45 247.49 <LOQ Total PAHs, ppb 22.1 256.4 <LOQ Pesticides, ppb 12.9 142.7 <LOQ Inorganic As, ppm 5.2 <LOQ <LOQ Heavy metals, ppm 0.04 <LOQ <LOQ .sup.1As mg of cholesteryl oleate/g of sample. LOQ: Limit of Quantification

(39) In R1, there was no difference between the trans fatty acid and EPA+DHA content with respect to the anchovy oil, and the concentrations of toxic and/or harmful anthropogenic contaminants were below the quantification limits.

EXAMPLE 4

(40) Contaminant Analysis of the Dry Solids from Example 2

(41) A comprehensive contaminant analysis was performed on the cholesterol product of Example 2. The results are shown in Table 6:

(42) TABLE-US-00006 TABLE 6 Dried solids of Example 2 Dioxins and Furans WHO(2005)-PCDD/F <LOQ (17 PCDD/F) TEQ (lower-bound), pg/g Polychlorinated WHO(2005)-PCB TEQ <LOQ biphenyls (12 WHO (lower-bound), pg/g PCB) Polychlorinated Total 6 ndl-PCB (lower- <LOQ biphenyls (6 ICES bound), ng/g PCB) TEQ-Totals WHO- WHO(2005)-PCDD/F + <LOQ PCDD/F and PCB PCB TEQ (lower-bound), pg/g PCB 209, Total Mono- to DecaCB <LOQ polychlorinated (lower bound), ng/g biphenyls 209 in total Polybrominated sum of 24 BDEs (excl. <LOQ biphenyls ethers (24 LOQ), ng/g PBDE) Ester-bound 2- Total 2-MCPD (free and <LOQ chloropropane-1,3- bound), g/kg diol (2-MCPD ester) Ester-bound 3- Total 3-MCPD (free and <LOQ chloropropane-1,2- bound), g/kg diol (3-MCPD ester) Ester-bound 3- Total 3-MCPD (free and <LOQ chloropropane-1,2- bound), g/kg diol (3-MCPD ester) and glycidol (glycidyl ester) Arsenic (As) Arsenic (As), mg/kg <LOQ 13 PAH (EPA).sup.1 Sum of all positive <LOQ identified PAH, g/kg Benzo(a)pyrene Benzo(a)pyrene, g/kg <LOQ Organochlorine DDT (total), mg/kg <LOQ Pesticides and Pyrethroides Organochlorine DDE, p,p-, mg/kg <LOQ Pesticides and Pyrethroides Methoxylated (MeO-) 2-MeO-PBDE-68, ng/g <LOQ PBDEs Methoxylated (MeO-) 2-MeO-PBDE-47, ng/g <LOQ PBDEs .sup.1Environmental Protection Agency LOQ: Limit of Quantification

(43) 2-MeO-PBDE-68 and 2-MeO-PBDE-47 are naturally occurring methoxylated PBDEs which accumulate in fish oil via the marine food web but may also originate by bio-transformation of PBDEs.

(44) Examples show that cholesterol obtained according to the present invention is a pharmaceutical grade cholesterol, does not contain a detectable amount of impurities coming from fish oil, and thus the residual or processed fish is suitable for human or animal consumption.

(45) All references cited and/or discussed in this specification are incorporated herein by reference in their entireties and to the same extent as if each reference was individually incorporated by reference.