Simple method for the purification of lacto-N-neotetraose (LNnT) from carbohydrates obtained by microbial fermentation

1. A method for purification of lacto-N-neotetraose from other carbohydrates, wherein the method comprises subjecting an aqueous solution containing lacto-N- neotetraose (LNnT) to membrane filtration using a membrane having a molecular weight cut-off of about 300 to about 500 Dalton to remove carbohydrates having a molecular weight that is smaller than that of LNnT, followed by, subjecting the retentate of the membrane filtration to a continuous chromatography to remove carbohydrates having a molecular weight larger than that of LNnT from the aqueous solution after the membrane filtration.

2. The method according to claim 1 , wherein the aqueous solution is obtained from a fermentation or enzymatic process for production of LNnT.

3. The method according to claim 1 , wherein the aqueous solution is obtained from a fermentation by separating biomass from fermentation broth.

4. The method according to claim 3 , wherein separating biomass from fermentation broth comprises a step of at least one ultrafiltration, of at least two ultrafiltrations, or of at least one of a first ultrafiltration using a membrane having a molecular weight cut-off of about 500 kDa and a second ultrafiltration using a membrane having a molecular weight cut-off of about 150 kDa.

5. The method according to claim 1 , wherein the aqueous solution is further treated with a cation exchange resin and an anion exchange resin before subjecting aqueous solution which has been treated with ion exchange resins to membrane filtration.

6. The method according to claim 1 , wherein the method further comprises dialysis or electrodialysis.

7. The method according to claim 1 , wherein the purity of lacto-N- neotetraose in the aqueous solution is ≤70%, ≤60%, ≤50%, ≤40%, ≤30%, ≤20%, ≤ 10 %, or ≤5% with respect to areas of peaks of a HPLC chromatogram prior to purification and/or the purified aqueous solution contains lacto-N-neotetraose at a purity of ≥80%, ≥85%, or ≥90% with respect to areas of peaks of a HPLC chromatogram after purification.

8. The method according to claim 3 , wherein the method further comprises using at least one glycosidase for hydrolyzing one or more carbohydrates other than LNnT in the fermentation broth.

9. The method according to claim 3 , wherein a second microorganism is added to the fermentation broth after fermentative production of LNnT, wherein said second microorganism expresses a β-galactosidase and/or a N-acetylhexosaminidase.

10. The method according to claim 9 , wherein the β-galactosidase is encoded by E. coli lacZ, and/or the N-acetylhexosaminidase is encoded by bbhl of B. bifidum.

11. The method according to claim 5 , wherein the cation exchange resin is in H+ form, and the anion exchange resin is in Cl-form.