Resumen
In the preparation of 4-O-beta -D-galactopyranosyl-D-xylose (I) by reacting D-xylose, a beta -galactopyranoside substrate and beta -D-galactosidase (BDG) in aqueous buffer, (I) is isolated (after deactivating or removing the BDG and removing the substrate aglycone) by adding celite, carrying out solid-liquid extraction and eluting from a column, and (I)-containing fractions are treated by crystallization. Preparation of 4-O-beta -D-galactopyranosyl-D-xylose (I) involves: (i) treating a mixture of 2-20 wt.% D-xylose, 0.5-5 wt.% of a beta -galactopyranoside (as substrate) and 75-97.5 wt.% aqueous buffer of pH 5.0-9.0 (as reaction medium) with 10-1000 U (per g of substrate) of beta -D-galactosidase (BDG); (ii) carrying out disaccharide-forming reaction at a temperature between the freezing point of the mixture and 45[deg]C for 2-48 hours; (iii) deactivating the BDG by freezing at -20 to -170[deg]C or by heating at 95-115[deg]C or removing the BDG from the mixture by ultrafiltration; (iv) removing the aglycone of the substrate by extraction or filtration, and (v) isolating the (I)-containing fractions. Step (v) is carried out by adding celite, carrying out solid-liquid extraction with a solvent and eluting from a column with a first eluant, or by directly adding activated carbon, filtering and eluting with a second solvent. The (I)-containing fractions are further treated by crystallization from a mixture of acetone and methanol (or water) in a ratio of 5-20:1. An independent claim is included for (I) obtained by the process.
Reivindicaciones
CLAIMS 1. 1.- An enzymatic process to obtain 4-O-β-D- galactopyranosyl-D-xylose that comprises: a first step of preparation of a first reaction mixture of 2-20% by weight of D-xylose 0-5-5% by weight of a β-D-galactopyranoside substrate 75-97.5% by weight of a reaction medium that comprises buffered water at a pH between 5.0 and 9.0; adding 10 to 1,000 units of a β-D-galactosidase enzyme, per gram of β-D-galactopyranoside, to the first reaction mixture; and obtaining a second reaction mixture a second step wherein the second reaction mixture is subjected to a reaction at a temperature comprised between a temperature higher than the freezing point of the second reaction mixture and 45°C, for 2 to 48 hours, in order to form disaccharides in the second reaction mixture; a third step wherein the reaction is stopped when the disaccharides have been formed in the desired amount, by means of a treatment chosen between deactivation of β- D-galactosidase by freezing the second reaction mixture at a temperature between 20°C and -170°C, deactivation of β-D-galactosidase by heating the second reaction mixture at a temperature between 95 and 110°C, and separation of β-D-galactosidase from the second reaction mixture by ultrafiltration; obtaining a third reaction mixture; a fourth step wherein an aglyconic fragment of the β-D-galactopyranoside substrate used in the first step is separated from the third reaction mixture by extraction or filtration; obtaining a fourth reaction mixture; a fifth step comprising isolation of fractions that contain 4-O-β-D-galactopyranosyl-D-xylose, characterized in that, the fifth step is selected between addition of celite to the fourth reaction mixture, followed by solid-liquid extraction with a solvent and elution with a first eluent in a column; and directly adding active carbon to the fourth reaction mixture followed by filtration and elution with a second eluent, and in that, in a sixth step, the fractions that contain 4-O-β-D- galactopyranosyl-D-xylose, are crystallized in a crystallization mixture selected among mixtures of acetone/methanol in a ratio between 5/1 to 20/1 and mixtures of acetone/water in a ratio between 5/1 to 20/1. 2. 2.- Process according to claim 1, characterized in that the fourth reaction mixture is concentrated before being subjected to elution in the column. 3. 3.- Process according to claim 1, characterized in that the mixture of acetone/methanol has a ratio of 10/1. 4. 4.- Process according to claim 1, characterized in that the mixture of acetone/water has a ratio of 10/1. 5. 5.- Process according to claim 1, characterized in that the first eluent is a mixture of water/isopropanol that contains 1 to 10% (v/v) of isopropanol. 6. 6.- Process according to claim 1, characterized in that the mixture of water/isopropanol contains 20 (v/v) of isopropanol. 7. 7.- Process according to claim 1, characterized in that the fifth step consists of adding celite to the fourth reaction mixture and concentrating to dryness, followed by solid-liquid extraction with an organic solvent in a Soxhlet extractor that has a cartridge made out of a material compatible with said solvent, and eluting with a first eluent in a column selected among filtration columns with cross-linked dextrane polymer fillers, filtration columns with acrylamide polymer fillers, filtration columns of active carbon or active carbon-celite columns. 8. 8.- Process according to claim 7, characterized in that the solvent is ethyl acetate. 9. 9.- Process according to claim 7, characterized in that the solvent is used in an amount comprised between ml and 25 ml per gram of initial xylose. 10. 10.- Process according to claim 7, characterized in that the celite is used in an amount comprised between 1 g and 2 g per gram of initial xylose. 11. 11.- Process according to claim 7, characterized in that the column is of active carbon-celite wherein the carbon is deactivated by adding 35% hydrochloric acid. 12. 12.- Process according to claim 11, characterized in that the celite is used in an amount comprised between 0.5 g and 2 g of celite per gram of initial xylose. 13. 13.- Process according to claim 11, characterized in that the active carbon is used in an amount comprised between 0.5 g and 2 g of active carbon per gram of initial xylose. 14. 14.- Process according to claim 7, characterized in that said first eluent is used in an amount comprised between 5 ml and 25 ml per gram of initial xylose. 15. 15.- Process according to claim 11, characterized in that the hydrochloric acid is used in an amount comprised between 0.5 ml and 1.5 ml per gram of initial xylose. 16. 16.- Process according to claim 1, characterized in that in the fifth step, the fourth reaction mixture is subjected to direct addition of at least a second eluent on the active carbon wherein the 4-O-β-D- galactopyranosyl-D-xylose is adsorbed on the active carbon and the second eluent is water followed by diluted isopropanol with a growing proportion in volume of isopropanol in successive steps. 17. 17.- Process according to claim 16, characterized in that the proportion in volume of isopropanol is comprised between 1% and 3% in a first step, between 3% and 5% in a second step and between 5% and 7% in a third step. 18. 18.- Process according to claim 16, characterized in that the active carbon is used in an amount comprised between 2 g and 4 g of active carbon per gram of initial xylose. 19. 19.- Process according to claim 16, characterized in that the second eluent is used in a total amount comprised between 30 ml and 50 ml of second eluent per gram of initial xylose. 20. 20.- Process according to claim 1 or 16, characterized in that the reaction is stopped by cooling the second reaction mixture at 0°C. 21. 21.- Process according to claim 1, 16 and 20, characterized in that the fourth reaction mixture is obtained by separating the aglyconic fragment from the β- D-galactopyranoside substrate by means of filtration. 22. 22.- Process according to claim 1, characterized in that the proportion of D-xylose in the second reaction mixture is 7.5% by weight. 23. 23.- Process according to claim 1, characterized in that the proportion of β-D-galactopyranoside in the second reaction mixture is 1.5% by weight. 24. 24.- Process according to claim 1, characterized in that 20 units of β-D-galactosidase per gram of β-D- galactopyranoside are added. 25. 25.- Process according to claim 1, characterized in that the reaction medium also comprises at least a cosolvent medium selected among dimethylsulfoxide, dimethylformamide, dioxane and mixtures thereof. 26. 26.- Process according to claim 25, characterized in that the reaction medium comprises 20% by weight of the cosolvent medium. 27. 27.- Process according to claim 1, characterized in that the reaction is carried out at a constant temperature. 28. 28.- Process according to claim 1 or 27, characterized in that the reaction temperature is from -5°C to 40°C. 29. 29.- Process according to claim 1 or 27, characterized in that the reaction temperature is higher than the freezing temperature of the second mixture and lower than 0°C. 30. 30.- Process according to claim 1, 28 or 29, characterized in that the reaction temperature is -5°C. 31. 31.- Process according to claim 1 or 28, characterized in that the reaction temperature is room temperature. 32. 32.- Process according to claim 1, 26 or 27, characterized in that the reaction medium is buffered to a pH of 7. 33. 33.- Process according to claim 1, characterized in that, in the third step, the reaction is stopped by freezing the second reaction mixture at a temperature of -78°C. 34. 34.- Process according to claim 1, characterized in that, in the third step, the reaction is stopped by heating the second reaction mixture up to a temperature of 100°C. 35. 35.- Process according to claim 1, characterized in that, in the third step, the reaction is stopped by separating the β-D-galactosidase by ultrafiltration. 36. 36.- Process according to claim 1, characterized in that the β-D-galactopiranoside substrate is selected between o-nitrophenyl β-D-galactopiranoside and lactose. 37. 37.- Process according to claim 1, characterized in that the β-D-galactosidase enzyme is E. coli β-D- galactosidase. 38. 38.- Process according to claim 1, characterized in that the β-D-galactosidase enzyme is Kluyveramyces lactis β-D-galactosidase. 39. 39.- A 4-O-β-D-galactopyranosyl-D-xylose characterized in that it has been obtained by means of the process defined in any of the claims 1 to 38. 40. 40.- A composition for in vivo evaluation of intestinal lactase in humans, characterized in that it comprises a 4-O-β-D-galactopyranosyl-D-xylose obtained by means of the process defined in any of the claims 1 to 38. 41. A solution for the in vivo evaluation of intestinal lactase in humans, characterized in that it comprises a solution selected between aqueous solutions and saline solutions of a 4-O-β-D-galactopyranosyl-D- xylose obtained by means of the process defined in any of the claims 1 to 38. 42. 42.- Use of 4-O-β-D-galactopyranosyl-D-xylose prepared according to any of the claims 1 to 38, in the preparation of a composition for in vivo evaluation of intestinal lactase in humans. 43. 43.- Use of 4-O-β-D-galactopyranosyl-D-xylose prepared according to any of the claims 1 to 38, in the preparation of a solution selected between saline solutions and aqueous solutions for in vivo evaluation of intestinal lactase in humans. 44. 44.- Use according to claim 42 or 43, characterized in that the 4-O-β-D-galactopyranosyl-D-xylose is combined with pharmaceutically acceptable amounts of at least one additive selected from among stabilizers, protecting agents, flavoring agents, lactose, gelling agents, fluidizing agents and preservatives.
