Solvent free process for extraction of cholesterol from milk fat

Abstract

The present invention discloses solvent free process for extracting cholesterol free of impurities from milk fat. The so isolated cholesterol is useful for the further preparation of vitamin D3. The present invention further provides pharmaceutical grade cholesterol from milk fat of high purity.

Claims

1. A process for extraction of cholesterol from milk fat, comprising: i. a solvent-free step of heating un-saponified milk fat with anhydrous calcium chloride to obtain a CaCl.sub.2-cholesterol adduct; and ii. separating cholesterol from the CaCl.sub.2-cholesterol adduct by refluxing the CaCl.sub.2-cholesterol adduct in an organic solvent, followed by re-crystallization of cholesterol.

2. The process as claimed in claim 1, wherein the solvent-free step includes heating un-saponified milk fat and anhydrous calcium chloride in a molar ratio ranging from 1:1 to 1:12.

3. The process as claimed in claim 1, wherein the solvent-free step includes heating at a temperature ranging from 40° C. to 90° C.

4. The process as claimed in claim 3, wherein the solvent-free step includes heating at a temperature ranging from 75° C. to 85° C.

5. The process as claimed in claim 1, wherein the solvent in the step of separating cholesterol is selected from the group consisting of a lower alcohol, a ketone, and a mixture thereof.

6. The process as claimed in claim 1, wherein the solvent in the step of separating cholesterol is selected from the group consisting of methanol, ethanol, n-propanol, isopropyl alcohol, n-butanol, isobutanol, tert-butanol, and a mixture thereof.

7. The process as claimed in claim 1, wherein the solvent in the step of separating cholesterol is selected from the group consisting of acetone, 2-Butanone, methyl isobutyl ketone, and a mixture thereof.

8. The process as claimed in claim 1, wherein the step of separating cholesterol produces cholesterol having a purity in ranging from 95% to 98%, based on HPLC.

9. A process for synthesis of vitamin D3, comprising: extracting cholesterol from milk fat by the process of claim 1; converting cholesterol to 7-dehydrocholesterol (7-DHC); and converting 7-DHC to vitamin D3 by irradiation.

10. A process for synthesis of vitamin D3, comprising: extracting cholesterol from milk fat by: heating un-saponified milk fat with anhydrous calcium chloride in a molar ratio ranging from 1:1 to 1:12 to obtain a CaCl.sub.2-cholesterol adduct; and separating cholesterol from the CaCl.sub.2-cholesterol adduct by refluxing the CaCl.sub.2-cholesterol adduct in a solvent; converting the separated cholesterol to 7-dehydrocholesterol (7-DHC); and converting 7-DHC to vitamin D3 by irradiation.

11. The process as claimed in claim 10, wherein the heating step includes heating un-saponified milk fat and anhydrous calcium chloride.

12. The process as claimed in claim 10, wherein the step of heating un-saponified milk fat is a solvent-free step, and wherein the solvent-free step includes heating at a temperature ranging from 40° C. to 90° C.

13. The process as claimed in claim 10, wherein the solvent in the step of separating cholesterol is selected from the group consisting of: a lower alcohol selected from the group consisting of methanol, ethanol, n-propanol, isopropyl alcohol, n-butanol, isobutanol, tert-butanol, and a mixture thereof; and a ketone selected from the group consisting of acetone, 2-Butanone, methyl isobutyl ketone, and a mixture thereof.

Description

DESCRIPTION OF FIGURES

(1) FIG. 1 depicts the chromatogram of Standard (STD) cholesterol.

(2) FIG. 2 depicts the chromatogram of cholesterol obtained by the present process.

DETAILED DESCRIPTION OF THE INVENTION

(3) The present invention relates to solvent free process for extraction of cholesterol of high purity from milk fat. The present process avoids the contact of milk fat with potentially harmful solvents in the initial stages that can affect the functionality, flavour or other properties of the milk fat. Further, there is no saponification thereby preventing the use of base, which may lead to losses of the neutral fat resulting in low yields of the desired product. The isolated milk fat free of cholesterol is washed with water to remove traces of inorganic impurities. The residual Milk fat free of cholesterol can be used by the milk industry for their own applications.

(4) In an embodiment, the present invention relates to cost effective, solvent free process for extraction of cholesterol of high purity from milk fat characterized by the process steps which comprises; i. heating un-saponified milk fat with anhydrous calcium chloride in the ratio ranging between about 1:2 to about 1:20 to obtain CaCl.sub.2-cholesterol adduct; and ii. separating cholesterol from the adduct by refluxing in the solvent followed by re-crystallization to yield pure cholesterol.

(5) The milk fat of the present invention includes skim milk such as fluid skim milk, ultra-filtered skim milk, condensed skim milk and various derivatives of skim milk, whey products such as reconstituted fluid whey, whey powder, whey protein isolates, whole milk, reduced fat, reconstituted non-fat dry milk and the like that contribute significant amounts of cholesterol in these foods. The content of cholesterol typically ranges from 2-5 gms per gm of the milk fat.

(6) Accordingly, the process comprises heating the sterol containing un-saponified milk fat with anhydrous calcium chloride in molar ratio ranging between about 1:2 to about 1:20, in solvent free medium, to obtain addition product of free cholesterol. The heating is performed at temperature in the range of 40−90° C., preferably 40-80° C., most preferably 75-85° C. for a period of 2-10 hours.

(7) The reaction mass is then cooled to 40-50° C. and maintained at same temperature for 10-36 hours and filtered. The cholesterol adduct is suspended in organic solvent and refluxed for 1-10 hours, preferably 1-5 hours, more preferably 2-4 hours. The reaction mass is cooled to 0-50° C., preferably at 15-45° C., more preferably at 20-30° C., stirred at same temperature for about 2-3 hours, filtered, washed further with organic solvent and dried under vacuum to obtain cholesterol.

(8) The isolated crude cholesterol is further purified by crystallization in solvent selected from lower alcohols such as methanol, ethanol, n-propanol, IPA, n-butanol, isobutanol or tert-butanol, preferably methanol or ketones selected from acetone, 2-Butanone, methyl isobutyl ketone, preferably acetone.

(9) The residual milk fat free of cholesterol obtained after filtration of cholesterol-calcium chloride adduct satisfies the quality standard required by the Milk industry and can be used by Milk industry for their own applications.

(10) In an embodiment, the cholesterol obtained by the present process is substantially free of impurities and is characterized by HPLC purity in the range of 95-99% as depicted in the comparative tables 1 and 2 below. The pharmaceutical grade cholesterol can be used as precursor for further preparation of vitamin D3.

(11) TABLE-US-00001 TABLE 1 HPLC data of STD cholesterol (w.r.t FIG. 1) Retention Peak No. time Area Height Area % 1. 10.895 4714241 1020260 99.335 2. 11.140 14800 4917 0.312 3. 11.258 12567 4208 0.265 4. 11.616 2640 739 0.056 5. 11.738 1544 400 0.033 Total 4745792 1030524 100.000

(12) TABLE-US-00002 TABLE 2 HPLC data of cholesterol obtained by the present process (w.r.t FIG. 1) Retention Peak No. time Area Height Area % 1. 10.893 5311138 1083092 99.337 2. 11.128 4877 1305 0.091 3. 11.246 16260 5626 0.304 4. 11.711 14315 2456 0.268 Total 5346590 1092479 100.000

(13) In another embodiment, the process for preparation of vitamin D3 from pharmaceutical grade cholesterol obtained from milk fat of the present invention which comprises (a) converting cholesterol to 7-dehydrocholesterol (7-DHC; pro-vitamin D3) as per International Patent Publication No. WO 2015/170341, filed by the present Applicant, and (ii) converting 7-DHC to vitamin D3 by irradiation.

(14) In the advantageous embodiment of the present invention, the milk fat used for extraction of cholesterol is not exposed to solvents in the initial stages due to which the functionality, flavour or other properties of the milk fat are not disturbed; there is no saponification thereby preventing the use of base, which may lead to losses of the neutral fat resulting in low yields of the desired product; the residual milk fat free of cholesterol of good quality can be used by the Milk Industry for further applications according to their requirement. The cholesterol obtained by the present process is of pharmaceutical grade and can be used as precursor for further preparation of vitamin D3. The process is simple and industrially feasible with minimum work up steps.

(15) The example herein is provided to illustrate particular aspect of the disclosure and do not limit the scope of the present invention.

Example 1: Cholesterol from Milk Fat

(16) Milk fat (100 gms) and anhydrous calcium chloride (8.5 gms) were heated at 80° C. for 2-4 hours. The reaction mass was cooled to 45° C. and maintained at same temperature for 15 hours. The reaction mass was filtered at 45° C., and the solid residue of cholesterol-adduct was suspended in methanol (100 ml). The reaction mass was refluxed for 4-6 hours, cooled to 25-30° C., stirred at same temperature for 2-3 hours, filtered and washed with methanol (10 ml) to obtain crude cholesterol. The crude cholesterol was recrystallized by dissolving in methanol (100 ml), filtered, washed with 20-50 ml of cold Methanol, dried under vacuum at 45° C. to obtain pure cholesterol.

(17) Yield: 35 gms

(18) HPLC Purity: 96-99%.

(19) It is to be understood that the above description is intended to be illustrative and not restrictive. Many embodiments will be apparent to a person skilled in the art upon reviewing the description. The scope of the invention should therefore, be determined not with reference to the above description, but instead should be determined with reference to the appended claims, along with the full scope of equivalents to which such claims are entitled.