Resumen
This invention relates to a procedure for isolating exosomes from buttermilk that comprises: (a) selecting the buttermilk as a byproduct of the dairy industry, (b) subjecting it to a first centrifugation, under conditions between 10,000 g and 15,000 g, 1°C and 8°C, and 15 and 30 min, obtaining a fatty upper layer, an aqueous fraction, and a precipitate; (c) subjecting the aqueous fraction to a second centrifugation, under conditions between 30,000 g and 35,000 g, 1 and 10°C, and between 50 min and 1 hour and 20 min, obtaining a precipitate and a supernatant; (d) collecting the upper 75% of the supernatant; (e) concentrating or separating the exosomes from the supernatant fraction; and (f) filtering the fraction obtained to obtain a fraction enriched in exosomes. The invention also relates to the exosomes obtained by the procedure of the invention and their applications in medicine, cosmetics and nutraceuticals.
Reivindicaciones
1. - Procedure for isolating exosomes from a byproduct of the dairy industry characterized in that it comprises the following stages: (a) selecting buttermilk as a byproduct of the dairy industry, (b) carrying out a first centrifugation of the buttermilk obtained in (a), under conditions of centrifugal force between 10,000 g and 15,000 g, at a temperature between 1°C and 8°C and for a time between 15 and 30 min, to obtain a fatty upper layer, an aqueous fraction, and a precipitate; (c) subjecting the aqueous fraction from stage (b) to a second centrifugation, under centrifugal force conditions between 30,000 g and 35,000 g, at a temperature between 1 and 10°C and for a time between 50 min and 1 hour and 20 min; to obtain a precipitate and a supernatant; (d) collecting the upper 75% of the supernatant obtained in c); (e) concentrating or separating the exosomes from the supernatant fraction collected in d); and (f) filtering the fraction obtained in e) to obtain a fraction enriched in exosomes. 2. Procedure for isolating exosomes according to claim 1 characterized in that the buttermilk selected in a) can be freshly obtained buttermilk as a byproduct of the industrial process of butter production or b) frozen buttermilk. 3. Procedure for isolating exosomes according to claim 1 characterized in that stage e) is carried out by means of differential ultracentrifugation, under conditions of centrifugal force between 75,000 and 125,000 g, for a time between 1 and 2 h, and at a temperature between 1 and 10°C, to obtain a gel-shaped pellet; a dense exosome-rich phase on the gel, an aqueous supernatant, and a thin upper layer rich in phospholipids. 4. Procedure for isolating exosomes according to claim 1 characterized in that stage e) is carried out by means of a combination of two tangential filtration operations comprising microfiltration followed by ultrafiltration. 5. Procedure for isolating exosomes according to claim 4 characterized in that the microfiltration comprises the filtration of the supernatant obtained in d) through the use of a polypropylene membrane, with a pore size between 800 and 1,200 nm, with a pressure between 0.5 and 3 bar and a flow between 100 and 200 L/m<2> h, until reaching a concentration of 90%. 6. Procedure for isolating exosomes according to claim 5 wherein the filtration is carried out with a polypropylene spiral membrane, with a pore size of 1 µm, at 1.5 bar and a flow of 150 L/m<2> h until a concentration of 90% is reached. 7. Procedure for isolating exosomes according to claim 5 or 6, characterized in that the ultrafiltration is carried out in a hollow fiber membrane, with a MWCO (Molecular Weight Cut-Off) between 200 and 700 kD, at a pressure between 1.5 and 20 bar and with a flow between 20 and 150 L/m<2> h. 8. Procedure for isolating exosomes according to claim 7 wherein the hollow fiber membrane is a hollow fiber polyethersulfone membrane, with a MWCO (Molecular Weight Cut-Off) of 500 kD, at a pressure of 17 bar and a flow of 50 L/m<2> h. 9. Procedure for isolating exosomes according to claim 1 wherein stage e) comprises a precipitation with one or more hydrophilic polymers, followed by centrifugation. 10. Procedure for isolating exosomes according to claim 9 wherein the precipitation is carried out using polyethylene glycol. 11. Procedure for isolating exosomes according to claim 10 wherein: - the supernatant obtained in d) is mixed with polyethylene glycol with a molecular weight between 3,000 and 10,000 D, in ratios between 5 and 15% (w/w), - the mixture obtained in the previous stage is kept under gentle stirring for a period between 1 and 10 min and is subsequently allowed to rest for between 1 and 15 h, and - after resting, the mixture is subjected to gentle centrifugation under conditions between 6,000 and 8,000 g, at a temperature between 1 and 8°C, and for a time between 10 and 40 min, to obtain a highly concentrated precipitate enriched in exosomes. 12. Procedure for isolating exosomes according to claim 11 wherein: - the supernatant obtained in d) is mixed with polyethylene glycol 6000, in a ratio of 10% (w/w), - the mixture obtained in the previous stage is kept gently stirring for 3 min and then allowed to rest for 8 h, and - after resting, the mixture is subjected to gentle centrifugation at 8,000 g, at a temperature of 4°C, and for 30 min. 13. Procedure for isolating exosomes according to claim 9 wherein the precipitation is carried out using a combination of two food-grade hydrophilic polymers with a percentage of the mixture for the first of them between 95 and 80% (w/w) and for the second of them between 5% and 20% (p/p). 14. Procedure for isolating exosomes according to claim 1 wherein stage e) is carried out by reverse dialysis. 15. Procedure for isolating exosomes according to claim 14 wherein the reverse dialysis is carried out with polyethylene glycol in a ratio of 8 to 12%. 16. Procedure for isolating exosomes according to any of the preceding claims, characterized in that a stage d'), prior to stage e), is optionally carried out, which comprises a chemical precipitation followed by centrifugation to obtain a precipitate that mainly comprises caseins and a supernatant comprising the exosomes. 17. Procedure for isolating exosomes according to claim 16 characterized in that stage d') comprises: - subjecting the supernatant fraction obtained in d) to a chemical precipitation by adding an acid until reaching a pH of between 4.0 and 4.5 and keeping it under gentle stirring or resting for a period of time between 10 and 30 min and at a temperature between 20°C and 30°C, and - subjecting the liquid fraction from the previous stage to a centrifugation operation under conditions between 2,000 g and 8,000 g, at a temperature between 1°C and 8°C, and for a time between 10 and 30 min. 18. Procedure for isolating exosomes according to claim 17 wherein the acid used is a food-grade acid. 19. Procedure for isolating exosomes according to claim 18 wherein the chemical precipitation is carried out by adding hydrochloric acid until a pH of 4.5 is reached. 20. Procedure for isolating exosomes according to any of the preceding claims wherein the filtration of stage f) is carried out using a filter with a pore size of 0.22 µm. 21. Procedure for isolating exosomes according to any of the preceding claims wherein, optionally, in an additional stage g) therapeutic, cosmetic or nutritional agents are incorporated into the exosomes obtained in f). 22. Procedure for isolating exosomes according to claim 21 wherein stage g) is carried out by incubating the exosomes obtained in f) with a solution of lipofectant agent and a nucleic acid molecule, for a time between 30 and 60 min, at room temperature, or by direct contact. 23. Procedure for isolating exosomes according to any of the preceding claims wherein the fraction enriched in exosomes obtained in stage f) or g) is subjected to an additional purification stage h) by using molecular exclusion chromatography. 24. Exosomes isolated from buttermilk by the procedure of any of claims 1-23. 25. Use of the isolated exosomes according to claim 24 as nanocarriers of compounds. 26. Use of isolated exosomes, according to claim 25, wherein the exosomes comprise therapeutic, cosmetic or nutritional compounds incorporated therein. 27. Exosomes according to claim 24 for their use in medicine.
