The present invention relates to a method of extracting cholesterol from milk fat. 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[8.7.0.0 ² ' ⁷ .0 ¹¹ ' 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 method for providing cholesterol.

Surprisingly it was found that crystalline cholesterol can be extracted in high yields from milk fat.

Milk fat (also named butterfat), especially cow milk fat, is the fatty portion of milk. The milk fat can be removed from the milk by commonly known processes. Milk fat can be bought commercially.

The content of cholesterol (and the derivatives) in milk fat can vary. It is depending on the feed of the cows as well as for example the cow species. 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 milk fat. The cholesterol is isolated from the milk fat 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 milk fat 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 milk fat 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 milk fat 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°C to 70°C. It is surprising that the extraction process is improved significantly by the elevated temperature.

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 or KOH, preferably NaOH.

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, preferably with NaOH. 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 temperatures, 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 ali- phatic 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 cyclo- hexane and nonane.

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). Usually the non-water miscible solvent (or the mixture of such solvents) is added to the reaction mixture after the saponification step (step (i)).

It is also possible and also preferred 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.

This 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 organic 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 impurities.

For the backwash water or mixtures of EtOH, NaOH, NaCI and water can be used.

The following Example illustrates 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 and Extraction of Cholesterol

Milk fat (1.0 g) was suspended in ethanol (40 ml) and treated with 50% aqueous KOH (8 ml). The white suspension was heated to 60 °C for 1 h. After cooling to room temperature, n-hexane (60 ml) and water (40 ml) were added at once. The resulting two layers were separated and the aqueous layer washed with n-hexane (2 x 60 ml). The combined organic layers were extracted with water (3 x 40 ml, slow layer separation, foam was formed). The organic layer was filtered over sodium sulfate and evaporated at 50 °C/ 300 to <30 mbar to give 352 mg pale yellow, oily solid which contained 82.4% cholesterol (HPLC w w%; 85.1 % NMR assay; 80.2% yield).