US 11,685,758 B2
Simple method for the purification of lacto-N-neotetraose (LNnT) from carbohydrates obtained by microbial fermentation
Stefan Jennewein, Bad Honnef (DE); and Markus Helfrich, Bad Hoenningen (DE)
Assigned to Chr. Hansen HMO GmbH, Rheinbreitbach (DE)
Appl. No. 17/53,317
Filed by Chr. Hansen HMO GmbH, Rheinbreitbach (DE)
PCT Filed May 6, 2019, PCT No. PCT/EP2019/061530
§ 371(c)(1), (2) Date Nov. 5, 2020,
PCT Pub. No. WO2019/215073, PCT Pub. Date Nov. 14, 2019.
Claims priority of application No. 18171114 (EP), filed on May 7, 2018.
Prior Publication US 2021/0230199 A1, Jul. 29, 2021
Int. Cl. C07H 1/08 (2006.01); C07H 3/06 (2006.01); C12N 9/10 (2006.01)
CPC C07H 1/08 (2013.01) [C07H 3/06 (2013.01); C12N 9/1051 (2013.01)] 8 Claims
 
1. A method for purification of lacto-N-neotetraose from other carbohydrates, wherein the lacto-N-neotetraose is produced by microbial fermentation and wherein the method comprises:
a) separating biomass from a microbial fermentation broth comprising lacto-N-neotetraose to obtain a cell-free fermentation broth comprising lacto-N-neotetraose;
b) subjecting the cell-free fermentation broth to ion-exchange resin treatment to remove charged material, wherein the cell-free fermentation broth is treated with a cation exchange resin and an anion exchange resin to obtain an aqueous solution comprising lacto-N-neotetraose;
c) subjecting the aqueous solution comprising lacto-N-neotetraose obtained in step b) to:
i) a first membrane filtration step to remove carbohydrates having a molecular weight smaller than that of lacto-N-neotetraose, wherein the first membrane is selected from a molecular weight cut-off of between about 300 and about 500 Dalton;
ii) a second membrane filtration step to remove carbohydrates having a molecular weight larger than that of lacto-N-neotetraose, wherein the second membrane is selected from a molecular weight cut-off of between about 600 and about 800 Dalton, and
d) a step for increasing the concentration of the lacto-N-neotetraose present in the filtrate of step c).