Recovery of cholesterol from fish oil residues

Abstract

The invention provides a method for separating and recovering cholesterol from fish oil residues or saponified derivatives thereof using a solvent mixture comprising water and an alcohol. Furthermore, a fatty acid enriched subfraction can be obtained, which can be used in other applications such as biofuel production and animal feed.

Description

[0001] Purpose of the invention

[0002] The present invention provides a method for separating and recovering cholesterol from fish oil residues or saponified derivatives thereof using a solvent mixture comprising water and alcohol. In addition, a subfraction rich in fatty acids can be obtained, which can be used in other applications such as biofuel production and animal feed. Technical Field

[0003] The raw materials used in the present invention include saponifiable or saponified residues from the refining of fish oils or derivatives obtained from fish oils, such as acid oils or residues from the fractionation of fatty acids from fish oils. Of particular interest to the present invention is the use of residues derived from the acquisition of EPA / DHA concentrates (eicosapentaenoic acid / docosahexaenoic acid). Background of the Invention

[0005] Cholesterol is a sterol synthesized by animals that performs a series of relevant functions in their physiology, such as the synthesis of bile acids, vitamin D, hormones, maintenance of cell membrane structure, etc. In industry, cholesterol is of interest because it is used in animal nutrition, in the production of vitamin D and as an excipient for cosmetics and pharmaceuticals.

[0006] Currently, most of the cholesterol on the market is obtained as a by-product of lanolin production from sheep and, to a lesser extent, from fish oil residues and by chemical synthesis.

[0007] In the prior art, some techniques for obtaining cholesterol from fish oil-derived residues are reported. For example, document WO 2016 / 096989 A1 describes a method for cholesterol recovery, which comprises: 1) saponification, followed by: 2) extraction with a solvent at least immiscible with water. The product obtained can be further purified by crystallization using at least one alcohol, an alcohol / water mixture or with aromatic or aliphatic hydrocarbons or alcohol / hydrocarbon mixtures. The main problem associated with this method is that multiple extraction steps are required to obtain a high yield of cholesterol recovery, which makes the method complex and expensive.

[0008] Document US10,196,583B1 discloses a method for obtaining cholesterol from fish oil, which comprises the following steps: a) distilling fish oil in a vacuum tower; b) distilling a first distillate in a vacuum tower; c) contacting the residue of the second distillation with an alkali to saponify it; d) extracting the saponified mixture with a non-polar organic solvent or a mixture of non-polar organic solvents; 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 containing cholesterol. The main problem associated with the method disclosed in US10,196,583B1 is that the oil contains a small amount of cholesterol, which makes the method economically unfeasible; especially due to the continuous increase in price caused by the increasing demand for fish oil in recent years. In addition, similar to WO 2016 / 096989 A1, several extraction steps are required before crystallization. In addition, even if a distillate with a cholesterol concentration of up to 9.2% is obtained after the first distillation (Example 1), a second distillation step under high vacuum and high temperature conditions is required, which increases production costs and may also cause cholesterol degradation in the sample.

[0009] Document WO 2019 / 053744 A1 discloses a method for obtaining cholesterol from fish oil residues, which comprises the following steps: a) saponifying the residue in the presence of 4-dimethylaminopyridine as a catalyst and then neutralizing with an acid; b) heating the saponified product in 2-butanone with calcium bromide at high temperature to form a cholesterol adduct; c) isolating the adduct and then recrystallizing it in methanol. Similar to previous patent documents, several process stages and a large amount of solvent are required. In addition, due to the high toxicity of 4-dimethylaminopyridine, its use is problematic.

[0010] Document US10,836,701B2 discloses a method for obtaining cholesterol from fish oil residues, which comprises: a) saponifying the residue with alkali; b) distilling the saponified mixture; c) distilling the residue of the first distillation by vacuum distillation; d) distilling the residue of the second distillation by vacuum distillation. This method produces a product with high purity and high yield. However, the use of special equipment (e.g., high vacuum distillation unit) and high temperature greatly increases investment and production costs. In fact, the feed of fatty acid salts needs to be operated at a temperature above 200°C throughout the process to keep the feed fluid, which complicates the handling of materials in the process and leads to cholesterol decomposition.

[0011] Based on the above, the present patent application solves the technical problems associated with subjecting the raw material to high temperatures (more than 100° C.) to reclaim and purify cholesterol, which results in lower cholesterol decomposition and lower degradation product generation. In addition, a solid product having a cholesterol concentration of at least 30%, preferably at least 50%, more preferably at least 70% on a dry basis is obtained by using only one precipitation stage, which reduces investment and production costs. Finally, the method does not require the use of highly toxic compounds such as 4-dimethylaminopyridine.

[0012] Description of the invention

[0013] The present invention discloses a method for producing cholesterol or a cholesterol-enriched fraction having a cholesterol concentration of at least 30%, preferably at least 50%, and more preferably at least 70% on a dry basis from a fish oil residue containing at least 5% free or esterified cholesterol and at least 50% free or esterified fatty acids.

[0014] The present invention preferably uses, but is not limited to, residues from the production of EPA+DHA concentrates from fish oil due to the high cholesterol concentration in these residues. Preferably, but not limited to, vacuum distillation bottoms can be used, which can exhibit a cholesterol concentration of 5% to 50%, depending on the method from which they are obtained as shown in US10196583 B1, US10836701 B2 and WO 2019 / 053744A1.

[0015] According to the present invention, this method has avoided relating to high thermal stress and causes the prior art process of cholesterol decomposition, reduces the generation of unwanted by product and increases the advantageous aspect of cholesterol recovery.In addition, proposed method needs still less unit operation than other methods of employing a series of distillations and crystallization described in the prior art.Less unit operation causes lower investment and production cost.

[0016] The invention provides a method for separating and recovering cholesterol from fish oil residues or saponified derivatives thereof using a solvent mixture comprising water and alcohol. A subfraction rich in fatty acids is obtained from the cholesterol recovery process. The subfraction can be used for other applications, such as biofuel production and animal feeding.

[0017] The method of the present invention comprises the following stages:

[0018] a) saponifying the fish oil residue using an alkali in water or an alkali in a solution of water and alcohol;

[0019] b) adding a solvent selected from the group consisting of water or an alcohol or a solution of water and an alcohol to the mixture obtained in stage a) until the ratio of solvent to residue is between 15 w / w and 30 w / w;

[0020] c) maintaining the mixture obtained in stage b) at a temperature lower than the temperature of stage b) to precipitate a cholesterol-rich solid;

[0021] d) recovering a cholesterol-rich solid from the mixture after stage c); and

[0022] e) washing the cholesterol-enriched solid with a washing solution comprising water and alcohol.

[0023] Optionally, after the saponification reaction in stage a) and before stage c), part of the base used can be neutralized with an acid.

[0024] Optionally, stage b) can be carried out in a series of stages until a solvent to residue ratio of 15 w / w to 30 w / w is reached.

[0025] In another object of the present invention, a method is provided in which phase a) and phase b) can be changed by the following sequence of operations:

[0026] a.1) saponification of fish oil residues using alkali in water or alkali in a solution of water and alcohol;

[0027] a.2) adding an acid to the solution obtained in a.1) until the pH value is between 2 and 6;

[0028] a.3) recovering the oil phase after addition of the acid in a.2) and removing the aqueous phase and the precipitated salt obtained;

[0029] a.4) mixing the oil phase obtained in a.3) with a solvent comprising a base and a solution of water and an alcohol until the ratio of solvent to residue is 15 w / w to 30 w / w;

[0030] a.5) maintaining the mixture obtained in stage a.4) at a temperature lower than the temperature of stage a.4) in order to precipitate a cholesterol-rich solid;

[0031] a.6) recovering a cholesterol-rich solid from the mixture after stage a.5); and

[0032] a.7) washing the cholesterol-enriched solid with a washing solution comprising water or a solution of water and alcohol.

[0033] In the context of the present invention, without limiting its scope, the term "aqueous-alcoholic solution" means a mixture comprising water and at least one alcohol.

[0034] By applying the proposed method, a cholesterol-enriched solid having a cholesterol concentration of at least 30%, preferably at least 50%, more preferably at least 70%, based on dry matter, can be obtained with only one precipitation stage in stage e) and stage a.7).

[0035] The method for obtaining cholesterol concentrate results in the production of a liquid stream comprising a solvent and a residue that is not precipitated in stage c) or a.5). In addition, another liquid stream or waste washing solution is produced in stage e) or a.7), which is composed of a washing solution and a portion of the residue that is not precipitated. Both liquid streams can be evaporated or distilled to recover the solvent, but a large amount of the solvent used will result in the high operating cost associated with the energy consumption. Therefore, the present invention proposes a solvent recovery method based on the following stages of washing (stage e) and a.7):

[0036] f) adding acid to the liquid stream obtained in step d) or a.6) after removal of the cholesterol-rich solids and to the spent washing solution of step e) or a.7) until a pH between 2 and 6 is obtained; and

[0037] g) recovering the oil phase obtained after the addition of the acid in step f) and removing the non-oily liquid phase and the salts obtained.

[0038] Optionally, non-oily liquid phase can be maintained at low temperature to precipitate solid residue, then the solid residue formed can be removed.These stages produce the non-oily liquid phase that is rich in water and alcohol, this liquid phase can be reused in any step of step a) and / or step b) and / or step e) and / or step a.1) and / or step a.4) and / or step a.7), and does not need to use evaporation and / or distillation to reclaim water and alcohol after each operation.On the other hand, the oil phase obtained in this process is rich in fatty acid, so this oil phase can be used for other applications, such as biofuel and animal breeding.

[0039] For the saponification of the residue in stage a) or a.1), any method known in the prior art can be used without limiting the scope of the present invention. Preferably, for example, aqueous and alcoholic solutions with sodium hydroxide and / or potassium hydroxide as bases are recommended for saponification because of their high efficiency and low cost.

[0040] Preferably, saponification of the fish oil residue using alkali in water or alkali in a solution of water and alcohol is carried out at a temperature of at least above 65°C.

[0041] Preferably, the process is characterized in that the mixture or dispersion obtained in stage b) and / or a.4) has a pH of more than 9.

[0042] Preferably, the process is characterized in that after stage b) or a.4), the mixture has an alcohol / water ratio of at least between 0.7 w / w and 5 w / w. Preferably, if ethanol is the only alcohol used, the ethanol / water ratio is at least between 0.8 w / w and 4 w / w.

[0043] Preferably, the process comprises maintaining the mixture obtained in step c) or a.5) at a temperature below 30°C to precipitate a cholesterol-rich solid.

[0044] Preferably, the mixture obtained in step c) or a.5) is kept at a temperature below 30°C for at least 15 minutes, preferably at least 30 minutes, more preferably at least 60 minutes.

[0045] Preferably, the method is characterized in that the alcohol to be used in the water / alcohol solution can be but is not limited to methanol, ethanol, n-propyl alcohol, isopropyl alcohol, n-butyl alcohol, sec-butyl alcohol, tert-butyl alcohol, isobutyl alcohol, ethylene glycol, diethylene glycol, diacetone alcohol or a mixture thereof. Methanol and ethanol are preferred because their boiling points are lower, which makes them easier to remove from the cholesterol-rich solid. Preferably, ethanol is used when the by-product from the ethyl ester production of fish oil is used as a raw material.

[0046] Preferably, the method is characterized in that in the acidification or neutralization of the base, any organic or inorganic acid can be used, such as hydrochloric acid, sulfuric acid, boric acid, citric acid, lactic acid, phosphoric acid, nitric acid, acetic acid, propionic acid, etc.

[0047] The cholesterol-rich solids may be recovered in stage d) or a.6) by using any method known in the art for separating solids from liquid streams, such as filtration and centrifugation, without limiting the scope of the present application.

[0048] Preferably, the process is characterized in that the precipitation and separation in stages c) and d) or a.5) and a.6) can be carried out in 2 or more consecutive steps at different temperatures, wherein these temperatures are below 30°C.

[0049] By applying the proposed method, a cholesterol-enriched solid having a cholesterol concentration higher than 30% on a dry basis can be obtained in stage e) or a.7). More preferably, in the case of only one precipitation, the cholesterol concentration in the solid is at least 50%, more preferably at least 70% on a dry basis.

[0050] Advantageously, the process is characterized in that the cholesterol recovery in stage e) or a.7) is at least 25%, preferably at least 50%, of the cholesterol in the fish oil residue.

[0051] The cholesterol-enriched solid obtained in stage e) may be dried using any drying technique known in the art, such as spray drying, drums, trays, tapes, etc., to obtain a water-free or low-moisture solid.

[0052] The cholesterol-enriched solid of stage e) can optionally be purified by crystallization to increase the cholesterol concentration. Preferably, a mixture of water, alcohol and hydrocarbon is used in the crystallization.

[0053] Examples of Applications

[0054] Example 1. Recovery of cholesterol from a residue containing 11.1% cholesterol

[0055] 50 g of fish oil residue from the production process of EPA and DHA concentrate was saponified at 85° C. using 15.3 g of NaOH in a solution containing 26.3 g of water and 101 g of ethanol for 5 hours. The solution was then neutralized to a pH between 3.3 and 3.5 using a 50% H2SO4 ethanolic solution. The oil phase formed was recovered and dried in a rotary evaporator (100-200 mbar) at 85° C. The oil phase contained 11.1% of cholesterol (analyzed using gas chromatography) and an acid value of 102 mg of NaOH / g.

[0056] A series of precipitation experiments using ethanol or methanol were carried out, and the results are summarized in Table 1. In order to conduct the experiment, 4g of sample was mixed with ethanol or methanol, water and 514.8mg of NaOH in the amount shown in the table at 60°C for 1 hour. Then, the mixture was cooled to the precipitation temperature (Tp) and kept at this temperature for 3 hours. After 3 hours, the sample was centrifuged at 1,000rpm for 10 minutes, and the supernatant was removed. The solid was washed with a solution of 4g water and alcohol, the composition of which was the same as the solution used in the precipitation. Finally, the solid was recovered by dissolving it in hexane, and the solution was transferred to a round-bottomed flask. Hexane was removed using a rotary evaporator (100-200mbar) at 85°C. The moisture content of the solid was measured by drying the solid to constant weight at 105°C in an oven, and the cholesterol content was measured by gas chromatography.

[0057] Table 1. Precipitation conditions and results obtained for samples used in Example 1

[0058]

[0059] Tp: Precipitation temperature.

[0060] alcohol: E (ethanol) or M (methanol).

[0061] Recovery rate: Recovery of cholesterol relative to the cholesterol present in the feed

[0062] The results in Table 1 show that different alcohols can be used to recover cholesterol from fish oil residues. From these results, it is clear that good recovery and purity of cholesterol obtained from fish oil can be achieved according to the operating conditions disclosed in this application. In addition, our results show that NaOH can be used as a base.

[0063] Example 2. Recovery of cholesterol from a residue containing 12.5% ​​cholesterol

[0064] Fish oil residue from the production process of EPA / DHA concentrate was treated using the same procedure used in Example 1. After this procedure, the oil phase contained 12.5% ​​cholesterol and an acid value of 113 mg NaOH / g. For the experiments, 4 g of the sample were mixed with the amounts of alcohol, water and 700 mg KOH shown in Table 2 for 1 hour at 60°C. Other precipitation conditions were evaluated and the results of the precipitation are shown in Table 2. The precipitation procedure was the same as that followed in Example 1. The solid was washed with 16 g of a solution of water and alcohol, the composition of which was the same as that used in the precipitation.

[0065] Table 2. Precipitation conditions for samples of Example 2 and results obtained.

[0066] M: methanol; E: ethanol.

[0067]

[0068] Tp: Precipitation temperature.

[0069] alcohol: E (ethanol) or M (methanol).

[0070] Recovery rate: Recovery of phytosterols relative to phytosterols present in the feedstock

[0071] The results in Table 2 show that different alcohols can be used to recover cholesterol from fish oil residues. From these results, it is clear that good recovery and purity of cholesterol obtained from fish oil can be achieved according to the operating conditions disclosed in this application. Our results also show that KOH can be used as a base.

[0072] Example 3. Recovery of cholesterol from a residue containing 28.7% cholesterol

[0073] The fish oil residue from the production process of EPA / DHA concentrate was treated using the same procedure used in Example 1. In this case, the oil phase contained 28.7% cholesterol and had an acid value of 85.3 mg of NaOH / g. For the experiments, 3 g of the sample were mixed with the amounts of alcohol, water and 418 mg of NaOH shown in Table 3 for 1 hour at 60°C. The precipitation procedure was the same as that followed in Example 1. The results obtained from the precipitation are shown in Table 3.

[0074] Table 3. Precipitation conditions for samples of Example 3 and results obtained.

[0075]

[0076] The results in Table 3 show that methanol and ethanol can be used to recover cholesterol from fish oil residues. From these results, it is clear that good recovery and purity of cholesterol from fish oil can be achieved according to the operating conditions disclosed in this application.

[0077] Example 4. Recovery of cholesterol directly from samples after saponification

[0078] 50.0 g of fish oil residue from the EPA and DHA concentrate production process (Example 3) was mixed with 15.2 g of NaOH and 125 g of an aqueous solution containing 58.8% ethanol. In this case, ethanol is preferred because ethanol is formed after the hydrolysis of fatty acid ethyl esters. The mixture was refluxed and stirred at 1,000 rpm for 4 hours to saponify the sample. The mixture was then cooled. Ethanol and water were added to a solvent / sample ratio of 20 and an ethanol / water ratio of 1.42. The mixture was then cooled to 15 ° C and kept for 60 minutes. The solid formed was centrifuged and then washed with 200 g of an aqueous solution containing 58.8% ethanol. A cholesterol-rich solid was recovered, which had 82.5% cholesterol on a dry basis (cholesterol recovery = 88.1%).

[0079] 50.1 g of fish oil residue from the EPA and DHA concentrate production process (Example 3) was saponified using NaOH and 125 g of an aqueous solution containing 79% ethanol. The mixture was then cooled. Ethanol and water were added to a solvent / sample ratio of 17.5 and an ethanol / water ratio of 3.83. The mixture was then cooled to 15 ° C and kept for 60 minutes. The solid formed was centrifuged and then washed with 200 g of an aqueous solution containing 79% ethanol. A cholesterol-rich solid was recovered, which had 90.0% cholesterol on a dry basis (cholesterol recovery = 84.6%).

[0080] 50.0 g of fish oil residue from the EPA and DHA concentrate production process (Example 1) was mixed with 15.0 g of NaOH and 125 g of an aqueous solution containing 59.7% ethanol. The mixture was refluxed and stirred at 1,000 rpm for 4 hours to saponify the sample. The mixture was then cooled. Ethanol and water were added to a solvent / sample ratio of 19 and an ethanol / water ratio of 1.5. The mixture was then cooled to 5 ° C and kept for 60 minutes. The formed solid was centrifuged and then washed with 100 g of an aqueous solution containing 59.7% ethanol. A cholesterol-rich solid was recovered, which had 64.2% cholesterol on a dry basis (cholesterol recovery = 71.7%).

[0081] 50.0 g of fish oil residue from the EPA and DHA concentrate production process (Example 2) was mixed with 15.0 g of NaOH and 125 g of an aqueous solution containing 59.7% ethanol. The mixture was refluxed and stirred at 1,000 rpm for 4 hours to saponify the sample. The mixture was then cooled. Ethanol and water were added to reach a solvent / sample ratio of 19 and an ethanol / water ratio of 1.5. The mixture was then cooled to 5°C and kept for 60 minutes. The formed solid was centrifuged and then washed with 200 g of an aqueous solution containing 59.7% ethanol. A cholesterol-rich solid was recovered, which had 77.2% cholesterol on a dry basis (cholesterol recovery = 86.4%).

[0082] Example 5. Crystallization of a cholesterol-rich solid

[0083] Make 0.5g of cholesterol purity obtained as described in this patent application be 84.8% cholesterol-rich solid crystallization.At 60 ℃, sample is dissolved in the mixture containing 2.5g heptane, 0.25g methanol and 0.5g water.Then, mixture is cooled at 15 ℃, and kept 2 hours at 15 ℃.Finally, filter the solid of crystallization and wash with 5g methanol.The final product (cholesterol recovery=69.2%) with 92.8% cholesterol.Use the identical solid that is rich in cholesterol to carry out similar experiment, but use 5g heptane to replace methanol in the washing step.Recovered the product (cholesterol recovery=85.2%) with 91.7% cholesterol.

[0084] Similarly, 0.5 g of a cholesterol-enriched solid with a cholesterol content of 78.9% was crystallized using 2.5 g of heptane, 0.25 g of methanol and 0.5 g of water at 15° C. for 2 hours. A product with 93.2% cholesterol was recovered (cholesterol recovery = 75.6%).

Claims

1. A method for separating and recovering cholesterol from fish oil residues or saponified derivatives thereof, characterized in that The following phases are included: a) saponifying the fish oil residue using an alkali in water or an alkali in a solution of water and alcohol; b) adding a solvent selected from the group consisting of water or an alcohol or a solution of water and an alcohol to the mixture obtained in stage a) until the ratio of solvent to residue is between 15 w / w and 30 w / w; c) maintaining the mixture obtained in stage b) at a temperature lower than the temperature of stage b) to precipitate a cholesterol-rich solid; d) recovering said cholesterol-enriched solid from said mixture after stage c); and e) washing the cholesterol-enriched solid with a washing solution comprising water and alcohol.

2. The method for separating and recovering cholesterol according to claim 1, characterized in that The saponification is carried out using sodium hydroxide, potassium hydroxide or a mixture thereof as the base.

3. The method for separating and recovering cholesterol according to claims 1 and 2, characterized in that The solution of water and alcohol in stage b) has an alcohol / water ratio of at least between 0.7 w / w and 5 w / w.

4. The method for separating and recovering cholesterol according to claim 3, characterized in that: The alcohol is ethanol, and the ethanol / water ratio is at least between 0.8 w / w and 4 w / w.

5. The method for separating and recovering cholesterol according to claim 1, characterized in that After the saponification reaction in stage a) and before stage c), part of the base used can be partially neutralized using an acid.

6. The method for separating and recovering cholesterol according to claim 5, characterized in that The acid used is any usable organic or inorganic acid selected from the group consisting of hydrochloric acid, sulfuric acid, boric acid, citric acid, lactic acid, phosphoric acid, nitric acid, acetic acid, propionic acid.

7. The method for separating and recovering cholesterol according to claim 1, characterized in that: The alcohol used is selected from the group consisting of methanol, ethanol, n-propanol, isopropanol, n-butanol, sec-butanol, tert-butanol, isobutanol, ethylene glycol, diethylene glycol, diacetone alcohol or mixtures thereof.

8. The method for separating and recovering cholesterol according to claim 1, characterized in that Step b) is performed in at least two stages to obtain a solvent to residue ratio of 15 w / w to 30 w / w.

9. The method for separating and recovering cholesterol according to claim 1, characterized in that: The solution or dispersion of stage b) has a pH above 9.

10. The method for separating and recovering cholesterol according to claim 1, characterized in that: Stage c) and stage d) may be carried out in 2 or more consecutive steps at different temperatures.

11. The method for separating and recovering cholesterol according to claim 1, characterized in that: Recovery of the cholesterol-enriched solids may be performed by any method useful for separating solids from liquid streams, such as filtration and centrifugation.

12. The method for separating and recovering cholesterol according to claim 1, characterized in that: The solvent of the liquid stream of stage c) and the washing solution of stage e) is recovered by: f) adding acid to the liquid stream obtained in d) after removal of the cholesterol-rich solids and to the spent wash solution in e) until a pH between 2 and 6 is obtained; as well as g) recovering the oil phase obtained after the addition of the acid in step f) and removing the non-oily liquid phase and the salt obtained therefrom.

13. The method for separating and recovering cholesterol according to claim 12, characterized in that: The non-oil liquid phase is kept at a low temperature to precipitate solid residues, and the formed solid residues are removed.

14. The method for separating and recovering cholesterol according to claim 12, characterized in that: The obtained oil phase is rich in fatty acids and can be used for other applications such as biofuel or animal feeding.

15. A method for separating and recovering cholesterol from fish oil residues or saponified derivatives thereof, characterized in that The following phases are included: a.1) saponification of fish oil residues using alkali in water or alkali in a solution of water and alcohol; a.2) adding an acid to the solution obtained in a.1) until the pH value is between 2 and 6; a.3) recovering the oil phase after adding the acid in a.2) and removing the aqueous phase and the precipitated salt obtained; a.4) mixing the oil phase obtained in a.3) with a solvent comprising a base and a solution of water and an alcohol until the ratio of solvent to residue is 15 w / w to 30 w / w; a.5) maintaining the mixture obtained in stage a.4) at a temperature lower than the temperature of stage a.4) in order to precipitate a cholesterol-rich solid; a.6) recovering the cholesterol-enriched solid from the mixture after stage a.5); and a.7) washing the cholesterol-enriched solid with a washing solution comprising water or a solution of water and alcohol.

16. The method for separating and recovering cholesterol according to claim 15, characterized in that The saponification is carried out using sodium hydroxide, potassium hydroxide or a mixture thereof as the base.

17. The method for separating and recovering cholesterol according to claims 15 and 16, characterized in that The water-alcohol solution of stage a.4) has an alcohol / water ratio of at least between 0.7 w / w and 5 w / w.

18. The method for separating and recovering cholesterol according to claim 17, characterized in that: The alcohol is ethanol, and the ethanol / water ratio is at least between 0.8 w / w and 4 w / w.

19. The method for separating and recovering cholesterol according to claim 15, characterized in that: The obtained oil phase is rich in fatty acids and can be used for other applications such as biofuel or animal feeding.

20. The method for separating and recovering cholesterol according to claim 15, characterized in that: The acid used is any usable inorganic or organic acid selected from the group consisting of hydrochloric acid, sulfuric acid, boric acid, citric acid, lactic acid, phosphoric acid, nitric acid, acetic acid, propionic acid.

21. The method for separating and recovering cholesterol according to claim 15, characterized in that: The alcohol used is selected from the group consisting of methanol, ethanol, n-propanol, isopropanol, n-butanol, sec-butanol, tert-butanol, isobutanol, ethylene glycol, diethylene glycol, diacetone alcohol or mixtures thereof.

22. The method for separating and recovering cholesterol according to claim 15, characterized in that: The recovery of the cholesterol-enriched solids may be performed by any method for separating solids from liquid streams, such as filtration and centrifugation.

23. The method for separating and recovering cholesterol according to claim 15, characterized in that The solvent of the liquid stream of stage a.5) and of the washing solution of stage a.7) is recovered by: f) adding acid to the liquid stream obtained in a.6) after removing the cholesterol-rich solids and to the spent washing solution in a.7) until a pH between 2 and 6 is obtained; g) recovering the oil phase obtained after the addition of the acid in step f) and removing the non-oily liquid phase and the salt obtained therefrom.

24. The method for separating and recovering cholesterol according to claim 23, characterized in that The non-oil liquid phase is kept at a low temperature to precipitate solid residues, and the formed solid residues are removed.

25. The method for separating and recovering cholesterol according to claim 23, characterized in that The obtained oil phase is rich in fatty acids and can be used for other applications such as biofuel or animal feeding.

26. The method for separating and recovering cholesterol according to claim 15, characterized in that Stage a.5) and stage a.6) can be carried out in 2 or more consecutive steps at different temperatures.

27. The method for separating and recovering cholesterol according to the preceding claims, characterized in that The cholesterol-enriched solid may optionally be purified by crystallization using a mixture of water, alcohol and hydrocarbons.

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