PROCESS FOR THE PURIFICATION OF AN ACIDIC HUMAN MILK OLIGOSACCHARIDE FROM FERMENTATION BROTH

Abstract

The present invention relates to a process for the purification of an acidic human milk oligosaccharide (HMO) from a fermentation broth using ion exchange methods. This process allows for a reduction of the number and/or extent of desalting operations, such as electrodialysis. It is even possible to refrain from such operations.

Claims

1. Process for the purification of an acidic human milk oligosaccharide (HMO) from a fermentation broth, the process comprising: (i) separating biomass from a fermentation broth to provide a crude solution; (ii) subjecting the crude solution to cation exchange using a cation exchange material in the H+ form, thereby obtaining a solution with a pH in the range 1-3; (iii) subjecting the solution with pH in the range 1-3 to anion exchange using a weakly basic anion exchange material in the Cl— form, thereby obtaining a solution with a pH in the range 1.5-5.5; (iv) subjecting the solution with pH in the range 1.5-5.5 to adsorption using an adsorbent material in order to remove neutral organic compounds; (v) optionally adjusting the pH of the solution having been subjected to adsorption to a value in the range 5-6; (vi) optionally subjecting the solution to anion exchange using an anion exchange resin, thereby binding the acidic human milk oligosaccharide to the resin followed by eluting the acidic human milk oligosaccharide from the resin using a salt solution, this optional step being conducted between steps (iii) and (iv) or steps (iv) and (v); thereby obtaining a purified solution comprising said acidic human milk oligosaccharide.

2. Process according to claim 1, wherein the acidic human milk oligosaccharide is selected from the group consisting of 6′-sialyllactose (6′-SL), 3′-sialyllactose (3′-SL), disialyllacto-N-tetraose, 3′-sialyl-3-fucosyllactose, and sialyllacto-N-tetraose.

3. Process according to claim 1, wherein the cation exchange material in step (ii) is a strong acid cation exchange material.

4. Process according to claim 1, wherein the weakly basic anion exchange material in step (iii) is selected from the group consisting of acrylic gel-type and styrene/divinylbenzene gel-type matrices with tertiary amine.

5. Process according to claim 1, wherein the adsorbent material in step (iv) is a weak cation exchange material.

6. Process according to claim 1, wherein the anion exchange resin in step (vi) is a weakly basic anion exchange resin.

7. Process according to claim 1, wherein step (i) includes the use of a membrane having a molecular weight cut-off of 5 kDa or less.

8. Process according to claim 1, wherein step (i) includes a microfiltration step which is performed at a temperature in the range of 20-75° C.

9. Process according to claim 8, wherein the microfiltration step forms an MF permeate and the MF permeate is subjected to a cooling step before step (ii).

10. Process according to claim 1, wherein the anion exchange resin in step (vi) is in the OH— form.

11. Process according to claim 1, wherein the process further comprises subjecting the purified solution to nanofiltration and/or reverse osmosis.

12. Process according to claim 1, wherein the purified solution is, optionally after one or more further treatment steps, subjected to a drying or crystallisation step.

13. Process according to claim 12, wherein the human milk oligosaccharide is obtained in the form of a powder having a water content of less than 10 wt %.

14. Process according to claim 1, wherein the acidic human milk oligosaccharide is obtained in the form of a syrup.

15. Method for producing an acidic human milk oligosaccharide, the method comprising: producing an acidic human milk oligosaccharide by microbial fermentation in a fermentation broth and purifying the produced acidic human milk oligosaccharide using the process according to claim 1.

Description

EXAMPLE

[0063] A fermentation broth (20 kg) containing more than 15 g/l 3′-SL was microfiltrated in order to remove the bacterial biomass from the liquid. The MF permeate was cross-flow ultrafiltrated with a 5 kDa cut-off ceramic UF membrane (Tami Industries) in order to remove protein and remaining DNA.

[0064] The UF permeate was then further purified by ion exchange chromatography. In the first column, cations were removed on a strong cation exchange resin (styrene-divinyl benzene gel matrix with sulfonate functional groups) in the H.sup.+ form. The solution leaving the column had a pH of 1.7-1.9. In the second chromatographic column, the solution was treated with a weakly basic anion exchange resin (crosslinked acrylic gel matrix with tertiary amines functional groups) in Cl.sup.− form, thereby exchanging anions with Cl.sup.− while keeping the pH between 1.7 and 1.9. The 3′-SL was not bound to the resin under these conditions. A third chromatographic purification step was conducted with adsorber resin in order to remove colorants and other adsorbing impurities, thereby not changing the pH of the solution. The adsorber resin was a styrene divinylbenzene copolymer matrix with sulphonic functional groups in the H.sup.+ form, having a porosity of about 1.0 ml/g, an average pore size of 600-900 Ånstroms, and a surface area ≥700 m.sup.2/g.

[0065] The purification effect of the ion exchange treatment is shown by the following analytical results (all amounts in % on dry matter; “n.d.” stands for “not detected” and means that the concentration was below the detection limit of the respective analytical method).

TABLE-US-00001 Component before IEX after AEX after adsorber 3′-SL 67.79 80.26 80.42 Phosphorus 1.20 n.d. n.d. Sulphate (inorganic) 0.66 n.d. n.d. Chloride n.d. 2.63 2.10 Sodium 0.67 n.d. 0.42 Potassium 3.37 n.d. n.d. Calcium 0.06 n.d. n.d. Magnesium 0.23 n.d. n.d.

[0066] After the chromatography steps, the pH of the solution could be increased to the desired level e.g. by the addition of sodium hydroxide. Still remaining substances such as chloride, organic acids, and small sugars can be further removed by means of crossflow nanofiltration, e.g. by using polymeric polypiperazine or polyamid membranes with a molecular weight cut-off between 150 and 500 Da. By doing so, a 3′-SL purity of 88% on dry matter and significantly higher can be reached.