Purification of oligosaccharides from a fermentation broth by using filtration
Disclosed is method for the purification of an oligosaccharide of interest from a fermentation broth, the method comprises providing a cell-free fermentation broth to a first filtration step using a nanofiltration membrane, thereby providing a filtrate which contains the oligosaccharide of interest; subjecting the filtrate to a second filtration step using a nanofiltration membrane, thereby providing a retentate which contains the oligosaccharide of interest; and removing salts from the retentate thereby providing a purified preparation of the oligosaccharide of interest.
1 . A method for purification of an oligosaccharide of interest from a fermentation broth, wherein the method comprises:
(i) providing a fermentation broth which comprises the oligosaccharide of interest, biomass, microbial cells and carbohydrates other than the oligosaccharide of interest;
(ii) removing the microbial cells from the fermentation broth, thereby providing a process stream;
(iii) subjecting the process stream to a first filtration using a nanofiltration membrane, thereby providing a filtrate which contains the oligosaccharide of interest, wherein the nanofiltration membrane used in the first filtration has a molecular weight cut-off of between about 700 Dalton and about 3,000 Dalton;
(iv) subjecting the filtrate to a second filtration using a nanofiltration membrane, thereby providing a retentate which contains the oligosaccharide of interest, wherein the nanofiltration membrane used in the second filtration has a molecular weight cut-off between 100 Dalton and 1,000 Dalton; and
(v) removing one or more salts from the process stream using electrodialysis thereby providing a purified preparation of the oligosaccharide of interest
wherein the method does not comprise an ion exchange step.
2 . The method according to claim 1 , wherein the microbial cells are removed from the fermentation broth by subjecting the fermentation broth to at least one centrifugation and/or to at least one filtration.
3 . The method according to claim 2 , wherein the at least one filtration is a microfiltration.
4 . The method according to claim 3 , wherein the process stream is subjected to at least one ultrafiltration, using a membrane having a molecular weight cut-off of about 50 kDa.
5 . The method according to claim 1 , wherein the nanofiltration membrane used in the first filtration has a molecular weight cut-off of about 1,000 and about 2,000 Dalton.
6 . The method according to claim 1 , wherein the nanofiltration membrane used in the second filtration has a molecular weight cut-off of about 150 Dalton and about 500 Dalton.
7 . The method according to claim 1 , wherein the oligosaccharide of interest is a HMO selected from the group consisting of 2′-fucosyllactose, 3-fucosyllactose, 2′, 3-difucosyllactose, lacto-N-triose II, lacto-N-tetraose, lacto-N-neotetraose, lacto-N-fucopentaose I, lacto-N-neofucopentaose, lacto-N-fucopentaose II, lacto-N-fucopentaose III, lacto-N-fucopentaose V, lacto-N-neofucopentaose V, lacto-N-difucohexaose I, lacto-N-difucohexaose II, 6′-galactosyllactose, 3′-galactosyllactose, lacto-N-hexaose and lacto-N-neohexaose 3′-sialyllactose, 6′-sialyllactose, sialyllacto-N-tetraose a, sialyllacto-N-tetraose b, sialyllacto-N-tetraose c, 3-fucosyl-sialyllactose, disialyl-lacto-N-tetraose and fucosyl-LST b, or any other HMO as listed in Table 1.
8 . The method according to claim 1 , wherein the process stream is subjected to the second filtration such that
i) the amount of salt in the retentate is <10%-wt., and/or
ii) the conductivity is between 0.5 and 10.0 mS/cm 2 .
9 . The method according to claim 1 , wherein the process stream is subjected to a concentration.
10 . The method according to claim 9 , wherein the concentration is performed
by nanofiltration at a temperature of <80° C.; and/or
by reverse osmosis at a temperature of 20° C. to 50° C.; and/or
at a pressure between >5 bar and <50 bar.
11 . The method according to claim 1 , wherein the process stream is subjected to a concentration such that the concentration of the oligosaccharide of interest is ≥100 g/L.
12 . The method according to claim 1 , wherein the process stream is subjected to removal of one or more colorants.
13 . The method according to claim 12 , wherein the removing of colorants is performed
i) before or after diafiltration;
ii) before or after concentrating the process stream; and/or
iii) before or after electrodialysis.
14 . The method according to claim 1 , wherein the electrodialysis is an electrodialysis under neutral conditions or an electrodialysis under acidic conditions.
15 . The method according to claim 1 , wherein the process stream after removal of one or more salts contained therein
i) comprises an amount of salt that is <1%-wt.; and/or
ii) has a conductivity of between 0.05 and 1.0 mS/cm 2 .
16 . The method according to claim 1 , wherein the process stream is spray-dried, preferably optionally spray dried
i) at an inlet temperature in the range of 110-150° C. and an outlet temperature in the range of 60-80° C.; and/or
ii) at a concentration of the oligosaccharide of interest in the process stream of 5-60%-wt.
17 . The method according to claim 1 , wherein the purity of the oligosaccharide of interest in the purified preparation is ≥80%-wt., with respect to dry matter of the purified preparation.
18 . The method according to claim 3 , wherein the microfiltration comprises using a membrane which has a molecular weight cut-off of about 500 kDa.
19 . The method according to claim 3 , wherein the microfiltration comprises using a membrane which has a molecular weight cut-off of about 150 kDa.
20 . The method according to claim 3 , wherein the process stream is subjected to at least one ultrafiltration, using a membrane having a molecular weight cut-off of about 30 kDa.