WO2014086373A1 - Crystallisation of human milk oligosaccharides (hmo) - Google Patents
Crystallisation of human milk oligosaccharides (hmo) Download PDFInfo
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- HMOs could be isolated in crystalline form from the aqueous enzymatic or fermentation media, in which the HMOs were produced, that would provide a simple and cheap method to separate the HMOs from contaminants in such aqueous media and obtain the HMOs as pure products. Indeed, purification and isolation by crystallization generally makes any process more attractive and cost-effective industrially. See EP-A-1405856. However, there has, to date, been no commercially suitable method for purifying and isolating, by crystallization, HMOs produced enzymatically or by fermentation.
- non-acidic preferably refers to a compound containing no carboxyl group, e.g., no sialic acid group.
- non-acidic human milk oligosaccharide or “non- acidic HMO” preferably means a compound with one of the following core structures or desialylated analogs thereof:
- Particularly preferred non-acidic HMOs include:
- non-acidic precursor of a human milk oligosaccharide or “non-acidic HMO precursor” preferably means fucose, N-acetyl glucosamine, galactose, N-acetyl lactosamine, lacto-N-biose or lacto-N-triose II.
- the dry, preferably protein-free powder is then treated with a hot, preferably boiling, d-C 6 alcohol solvent, preferably a CrC 4 alcohol solvent, especially methanol, to produce an alcohol solution of the non-acidic HMO or HMO precursor.
- a hot, preferably boiling, d-C 6 alcohol solvent preferably a CrC 4 alcohol solvent, especially methanol
- the alcohol solvent can, if desired, be heated, preferably to boiling, only after it is used to dissolve the non-acidic HMO or HMO precursor to produce the alcohol solution.
- non-acidic HMOs and precursors are crystallized in essentially the same manner after fermentation thereof: lacto-N-triose II (US 7521212, Priem et al. Glycobiology 12, 235 [2002]), LNnT( US 7521212, or Priem et al. Glycobiology ⁇ 2, 235 [2002]),
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Abstract
The present invention relates to a method of obtaining a non-acidic human milk oligosaccharide (HMO) or a precursor in crystalline form from an aqueous medium, such as a fermentation broth, by drying the aqueous medium and then treating it with a hot C1-C6 alcohol solvent.
Description
CRYSTALLISATION OF HUMAN MILK OLIGOSACCHARIDES (HMO) FIELD OF THE INVENTION
The present invention relates to a method of crystallizing non-acidic human milk oligosaccharides (HMOs) or precursors thereof from aqueous media, particularly as produced by fermentation.
BACKGROUND OF THE INVENTION
Human milk oligosaccharides (HMOs) have become of great interest in the past few years due to their important functions in human development. To date, the structures of at least 1 15 HMOs have been determined (see Urashima et al.: Milk
Oligosaccharides, Nova Biomedical Books, New York, 201 1 , ISBN: 978-1 -61 122- 831 -1 ), and considerably more are probably present in human milk. The following thirteen core structures have been identified, to date, for the 1 15 HMOs:
Low cost ways have been sought for making industrial quantities of as many as possible of the HMOs, so that their uses in nutritional and therapeutic formulations for infants, as well as as possibly children and adults, could be discovered, developed and exploited by researchers worldwide. A few HMOs have recently been
synthesized in high purity and in high yields by lengthy sequences of chemical process steps, but even lower cost ways of producing them have continued to be sought. As a result, ways have been sought for synthesizing HMOs enzymatically or by fermentation of transformed one-cell organisms, particularly E. coli. See Drouillard et al. Angew. Chem. Int. Ed. 45,1778 (2006) and Han et al. Biotechnol. Adv. 30, 1268 (2012). However, the costs of producing HMOs in such ways have remained stubbornly high because it has been difficult and expensive to purify the HMOs in high yields from the aqueous enzymatic or fermentation media, in which they were produced.
If HMOs could be isolated in crystalline form from the aqueous enzymatic or fermentation media, in which the HMOs were produced, that would provide a simple and cheap method to separate the HMOs from contaminants in such aqueous media and obtain the HMOs as pure products. Indeed, purification and isolation by crystallization generally makes any process more attractive and cost-effective industrially. See EP-A-1405856. However, there has, to date, been no commercially suitable method for purifying and isolating, by crystallization, HMOs produced enzymatically or by fermentation.
Accordingly, it is an object of the present invention to provide a method for obtaining, in crystalline form, HMOs, as well as their precursors such as fucose, N-acetyl glucosamine, galactose, N-acetyl lactosamine, lacto-N-biose and lacto-N-triose II from aqueous mixtures thereof as produced by fermentation, particularly as produced extracellularly, as well as from aqueous solutions thereof as produced enzymatically.
SUMMARY OF THE INVENTION
A first aspect of this invention relates to a method of obtaining, in crystalline form, a non-acidic human milk oligosaccharide (or HMO) or a non-acidic precursor of an HMO produced in an aqueous media, enzymatically or by fermentation, particularly
produced intracellularly in an aqueous fermentation broth, quite particularly produced by E. coli, said method comprising the steps of: a) drying, preferably freeze-drying, the aqueous media, preferably after removing proteins therefrom, to produce a dry powder that contains the non- acidic HMO or HMO precursor, preferably at a concentration of at least about
15 wt%, preferably at least about 25 wt%, especially at least about 30-40%, particularly at least about 40-60%, more particularly at least 95 wt%, even more particularly at least 97 wt%, and that preferably also contains no more than about 5% water, especially no more than about 3-4% water; b) then treating the dry powder with a hot, preferably boiling, C C6 alcohol solvent, preferably a CrC4 alcohol solvent, optionally containing up to 50 v/v%, preferably up to about 35 v/v% water, especially up to about 15 v/v%, particularly up to about 5 v/v%, to produce an alcohol solution of the non-acidic HMO or HMO precursor; and c) then crystallizing the non-acidic HMO or HMO precursor from the alcohol solution.
Advantageously, the method also comprises, between steps b) and c), one or more, preferably all, of the following intermediate steps: i) the alcohol solution is filtered to separate insolubles; ii) then the alcohol solution is concentrated down to at least about 60-70% of its original volume, and iii) then, the alcohol solution is seeded with crystals of the non- acidic HMO or HMO precursor.
A second aspect of this invention relates to a method of obtaining a non-acidic HMO or HMO precursor from a powder, preferably a protein-free powder, produced by drying, preferably freeze drying, an aqueous media, in which the non-acidic HMO or HMO precursor was produced enzymatically or by fermentation, particularly produced intracellularly in an aqueous fermentation broth, quite particularly produced
by E. coli, comprising steps b) and c) above, and advantageously also comprising intermediate steps i) to iii).
DETAILED DESCRIPTION OF THE INVENTION
In accordance with this invention, the term "d-C6 alcohol" preferably means a hydroxy- or dihydroxy-alkanol having 1 to 6 carbon atoms, such as methanol, ethanol, n-propanol, /-propanol, ethylene glycol, 1 ,2-propanediol, 1 ,3-propanediol, butanol and an i-hexanol, especially a "CrC4 alcohol", such as methanol, ethanol, 1 ,2-propanediol, and butanol, particularly methanol.
Also in this invention, the term "non-acidic" preferably refers to a compound containing no carboxyl group, e.g., no sialic acid group.
Also in this invention, the term "non-acidic human milk oligosaccharide" or "non- acidic HMO" preferably means a compound with one of the following core structures or desialylated analogs thereof:
Abbreviation Chemical Structure
2'-FL Fuc(ecl-2)Gal tfl-4)Glc
3-FL Gal #l-4)Glc
Fuc(al-3)
DFL Fuc(al-2)Gal tfl-4)Glc
Fuc(al-3)
3'-SL Neu5Ac(a2-3)Gal #l-4)Glc
FSL Neu5Ac(a2-3)Gal #l-4)Glc
1 1
Fuc(al-3)
LNT Gal #l-3)GlcNAc tfl-3)Gal tfl-4)Glc
LNFP I Fuc( l-2)Gal 51-3)GlcNAc 51-3)Gal 51-4)Glc
LNFP II Gal #l-3)GlcNAc tfl-3)Gal tfl-4)Glc
Fuc(ecl-4)
LNFP III Gal #l-4)GlcNAc tfl-3)Gal tfl-4)Glc
Fuc(al-3)
LNFP V Gal 51-3)GlcNAc 51-3)Gal 51-4)Glc
1
Fuc(al-3)
LNDFH I Fuc( l-2)Gal 51-3)GlcNAc 51-3)Gal 51-4)Glc
1
Fuc(al-4)
LNDFH II Gal 51-3)GlcNAc 51-3)Gal 51-4)Glc
1 1
Fuc(ecl-4) Fuc(al-3)
LNDFH III Gal 51-4)GlcNAc 51-3)Gal 51-4)Glc
1 1
Fuc(al-3) Fuc(al-3)
LSTa Neu5Ac( 2-3)Gal 51-3)GlcNAc 51-3)Gal 51-4)Glc
LSTb Neu5Ac(ec2-6)
1
Gal 51-3)GlcNAc 51-3)Gal 51-4)Glc
LSTc Neu5Ac( 2-6)Gal 51-4)GlcNAc 51-3)Gal 51-4)Glc
F-LSTa Neu5Ac( 2-3)Gal 51-3)GlcNAc 51-3)Gal 51-4)Glc
1
Fuc(ecl-4)
F-LSTb Neu5Ac(ec2-6)
1
Fuc( l-2)Gal 51-3)GlcNAc 51-3)Gal 51-4)Glc
F-LSTc Neu5Ac( 2-6)Gal 51-4)GlcNAc 51-3)Gal 51-4)Glc
1
Fuc(al-3)
DS-LNT Neu5Ac(a2-6)
1
Neu5Ac( 2-3)Gal 51-3)GlcNAc 51-3)Gal 51-4)Glc
FDS-LNT I Neu5Ac(ec2-6)
1
Neu5Ac( 2-3)Gal 51-3)GlcNAc 51-3)Gal 51-4)Glc
1
Fuc(ecl-4)
FDS-LNT II Neu5Ac(ec2-6)
1
Neu5Ac( 2-3)Gal 51-3)GlcNAc 51-3)Gal 51-4)Glc
1
Fuc(al-3)
LNnT Gal 51-4)GlcNAc 51-3)Gal 51-4)Glc especially:
LNnT and LNT,
the monofucosyl LNTs, LNFP I, LNFP II and LNFP V, the difucosyl LNTs, LNDFH I and LNDFH II,
the fucosyl lactose derivatives 2'-FL, 2',3-DFL, 3-FL, and - the mono- and difucosyl LNnTs, LNFP III and LNDFH III.
Particularly preferred non-acidic HMOs include:
- a compound of formula 1 :
wherein R independently of each other is fucosyl or H, provided that one R is fucosyl, a compound of formula 2 :
2
a compound of formula 3:
Further in this invention, the term "fucosyl" preferably means an L-fucopyranosyl group of formula 4 attached to the core compound with a-interglycosidic linkage:
Still further in this invention, the term "non-acidic precursor of a human milk oligosaccharide" or "non-acidic HMO precursor" preferably means fucose, N-acetyl glucosamine, galactose, N-acetyl lactosamine, lacto-N-biose or lacto-N-triose II.
Still further in this invention, the term "dry" or "dried" preferably means a water content of no more than about 10%, preferably 1 -5%, particularly no more than 3-4%.
Still further in this invention, the term "hot" with regard to the alcohol solvent used in the method preferably means a temperature range from about 40-50 °C to the boiling point of that alcohol.
HMOs and their precursors can be produced in high yields, in aqueous media enzymatically of by fermentation of genetically transformed E. coli. See, for example, EP-A-1405856 and EP-A-1 194584, Drouillard et al. Agnew. Chem. Int. Ed. 45,1778 (2006) and Han et al. Biotechnol. Adv. 30, 1268 (2012).
To separate a non-acidic HMO or HMO precursor in high yield as a pure crystalline material from an aqueous solution, in which it was made enzymatically, the aqueous media is preferably contacted with cationic and/or anionic ion exchange resins to remove proteins. To separate a non-acidic HMO or HMO precursor, preferably a non-acidic HMO, in high yield as a pure crystalline material from an aqueous fermentation broth, in which it was made, the aqueous fermentation broth is preferably separated, preferably by centrifugation, from the fermented, preferably E. coli, cells and then proteins and salts are removed from the fermentation broth, preferably by contacting it with cationic and anionic ion exchange resins.
The resulting aqueous medium, produced enzymatically or by fermentation, is then dried to remove at least most of its water content by evaporation, azeotropic distillation, spray drying, freeze drying or a combination thereof, preferably by freeze drying. The dry powder, so-produced, contains no more than about 8-10 wt% water, preferably no more than about 5 wt% water, especially no more than 3-4 wt% water. For best results in carrying out the method of this invention, the aqueous medium, produced enzymatically or by fermentation, preferably contains at least about 10 g/l, especially at least 15-20 g/l, particularly at least about 30-40 g/l, quite particularly at least at least about 50 g/l, of the non-acidic HMO or HMO precursor, and the dry powder therefrom preferably contains the non-acidic HMO or HMO precursor at a concentration of at least about 15 wt%, preferably at least about 25 wt%, especially at least 30-40 wt%, particularly at least at least 50-60 wt%, more particularly at least 95 wt%, even more particularly at least 97 wt%.
In accordance with this invention, the dry, preferably protein-free powder is then treated with a hot, preferably boiling, d-C6 alcohol solvent, preferably a CrC4 alcohol solvent, especially methanol, to produce an alcohol solution of the non-acidic HMO or HMO precursor. In this regard, the alcohol solvent can, if desired, be heated, preferably to boiling, only after it is used to dissolve the non-acidic HMO or HMO precursor to produce the alcohol solution. The alcohol solvent preferably contains no water, but where water enhances the solubility differential between the desired product and fermentation by-products, the alcohol solvent can suitably contain up to about 50 v/v% water, preferably up to 30 v/v% water, especially no more than 15 v/v%, particularly no more than 5 v/v%. In this regard, the dry powder can be dissolved in water or aqueous alcohol before treating it with the alcohol solvent.
Optionally, the alcohol solvent contains a filtration aid, such as activated carbon or diatomaceous earth, to be removed in a subsequent filtration step. The amount of alcohol solvent used is not critical, but preferably at least about 4-10 ml, especially about 5-7 ml, of alcohol are used per gram of the dry powder. Depending on the choice of solvent and its water content, if any, at least about 33 wt%, preferably at least 40-60 wt%, of the solids content of the dry powder become dissolved in the alcohol solution.
Preferably for certain non-acidic HMO or HMO precursors, the alcohol solution is then filtered, preferably while still hot, especially boiling hot, to remove insoluble fermentation by-products. Depending on the choice of solvent and its water content, if any, at least about 30 wt%, preferably about 40-60 wt%, especially about about 45- 55 wt%, of the solids content of the dry powder are filtered from the alcohol solution. The hot alcohol solution, preferably after filtration, is then concentrated by partial evaporation down to at least about 60-70 % of its original volume, preferably down to about 40-60 %, especially down to about 45-55 %, of its original volume. The concentrated alcohol solution is then allowed to cool somewhat, preferably down to about 30-50 °C, especially about 35-45 °C. Seed crptals of the desired non-acidic HMO or HMO precursor can be added to the concentrated alcohol solution while it is still warm, preferably at about 30-50 °C, especialy at about 35-45 °C. The
concentrated alcohol solution is then allowed to cool, for example to room
temperature, and precipitated crystals of the desired non-acidic HMO or HMO precursor can then be filtered from the solution. If desired, the precipitated crystals can then be recrystallized using a hot CrC6 alcohol solvent, preferably the same alcohol solvent as was used for the original crystallization, optionally containing up to about 50 v/v% water.
In a preferred embodiment, the non-acidic HMO to be obtained by crystallization is a fucosylated HMO and is preferably a compound of formula 1 , above (2'-FL, 3-FL or 2',3-DFL). Its aqueous medium, produced enzymatically or by fermentation (see e.g. Drouillard et al. Angew. Chem. Int. Ed. 45, 1778 (2006), WO 2010/070104, WO 2010/142305, WO 2012/1 12777) is dried, by removing at least most of its water content by evaporation, azeotropic distillation, spray drying, freeze drying or a combination thereof, preferably by freeze drying, to form a dry powder that contains no more than about 8-10 wt% water, preferably no more than about 5 wt% water, especially no more than 3-4 wt% water. For best results, the aqueous medium, preferably contains at least about 15-20 g/l, particularly at least about 30-40 g/l, quite particularly at least at least about 50 g/l, of a compound of formula 1 , and the dry powder therefrom preferably contains the compound of formula 1 at a concentration of at least about 30-40 wt%, preferably at least at least 50-60 wt%, more particularly at least 95 wt%, even more particularly at least 97 wt%. The dry, preferably protein-
free powder is then treated with a hot, C C4 alcohol solvent, preferably boiling methanol, to produce an alcohol solution of a compound of formula 1. In this regard, the alcohol solvent can, if desired, be heated, preferably to boiling, only after it is used to dissolve a compound of formula 1 to produce the alcohol solution. The alcohol solvent preferably contains no water. Preferably, the alcohol solution is then filtered, preferably while still hot, especially boiling hot, to remove insoluble
fermentation by-products. The hot alcohol solution, preferably after filtration, is then concentrated by partial evaporation down to at least about 60-70 % of its original volume, preferably down to about 40-60 %, especially down to about 45-55 %, of its original volume. The concentrated alcohol solution is then allowed to cool somewhat, preferably down to about 30-50 °C, especially about35-45 °C, seed crystals are then preferably added to the concentrated alcohol solution while it is still warm, preferably at about 30-50 °C, especially at about 35-45 °C. Tte concentrated alcohol solution is then allowed to cool, for example to room temperature, and precipitated crystals of a compound of formula 1 can then be filtered from the solution. If desired, the precipitated crystals can then be recrystallized using a hot C C4 alcohol solvent, preferably the same alcohol solvent as was used for the original crystallization, which can contain water.
In another preferred embodiment, the non-acidic HMO to be crystallized has no fucosyl residue and is preferably LNT or LNnT. Its aqueous medium, produced enzymatically or by fermentation (see e.g. WO 01 /04341 , Priem et al. Glycobiology 12, 235 (2002)) is dried, by removing at least most of its water content, by means of evaporation, azeotropic distillation, spray drying, freeze drying or combination thereof, preferably by freeze drying, to form a dry powder that contains no more than about 8-10 wt% water, preferably no more than about 5 wt% water, especially no more than 3-4 wt% water and preferably contains LNT or LNnT at a concentration of at least about 30-40 wt%, preferably at least at least 50-60 wt%, more particularly at least 95 wt%, even more particularly at least 97 wt%. The dry, preferably protein-free powder is then treated with a hot Ci-C4 alcohol solvent, preferably boiling methanol, which can contain up to 30-40 % of water, to produce an alcohol solution of LNT or LNnT. In this regard, the alcohol solvent, water or a mixture thereof can, if desired, be heated, preferably to around 60 °C, only after t is used to dissolve LNT or LNnT
to produce the alcohol solution. Optionally, the alcohol solution can be filtered, preferably while still hot, to remove insoluble fermentation by-products. The hot alcohol solution is then allowed to cool somewhat, preferably down to room
temperature or 0-4 °C, and precipitated crystals of LNT or LNnT can then be filtered from the solution. It is possible to assist crystallization by adding seed crystals. If desired, the precipitated crystals can then be recrystallized using a hot C C4 alcohol solvent, preferably the same alcohol solvent as was used for the original
crystallization.
EXAMPLES Example 1
2'-0-fucosyllactose (a-L-fucopyranosyl-(1→2)-p-D-galactopyranosyl-(1→4)-D- glucose ["2'-FL"]) was expressed by an E. coli strain K12 that had been transformed with a glycosyl transferase gene and that had been cultivated in an aqueous medium containing glycerol (as a carbon and energy source) and lactose (as an acceptor for glycosylation with fucose, produced internally in the E. coli). The fermentation was carried out as described in Drouillard et al. Angew. Chem. Int. Ed. 45, 1778 (2006).
When fermentation ended, the aqueous fermentation broth, containing about 30 g/l of 2'-FL, was centrifuged to remove the E. coli cells from the broth. The remaining liquid was treated with an anionic ion exchange resin and with a cationic ion exchange resin to remove the cell proteins from the liquid. The cell- and protein-free broth was then freeze dried, so that it contained about 50-55 wt% of 2'-FL and no more than about 4 wt% water.
The dried powder (1 g) was then treated with boiling anhydrous methanol (6 ml). About 50 wt% of the solids content of the dry powder dissolved in the methanol. The methanol solution was then filtered while still boiling hot to remove insoluble fermentation by-products (amounting to about 50 wt% of the solids content of the dried powder). The filtered methanol solution was then kept hot to partially evaporate it to about 50 % of its original volume and then allowed to cool somewhat. Seed crystals of 2'-FL were then added to the still warm (40 °C), concentrated methanol solution.
The concentrated methanol solution was then allowed to cool to room temperature (22 °C), and precipitated crystals (0.45 g) of 2'-FL were filtered from the solution with high purity.
Example 2 9.17 g of LNT (approximately 80%, obtained after fermentation with genetically modified E. coll according to the teaching of WO 01 /04341 , treatment with resins and freeze-drying) was dissolved in water (30 ml) and MeOH (60 ml) by heating to 60 °C, and the solution was stirred at room temperature for 2 h followed by 4 °C over night. Filtration, washing with MeOH-H2O 3:1 and drying (50 °C and vacuum overnight) gave lacto-N-tetraose (5.89 g). Purity 95% (HPLC).
The following non-acidic HMOs and precursors are crystallized in essentially the same manner after fermentation thereof: lacto-N-triose II (US 7521212, Priem et al. Glycobiology 12, 235 [2002]), LNnT( US 7521212, or Priem et al. Glycobiology Λ2, 235 [2002]),
LNDFH III (Dumon et al. Biotechnol. Prog. 20, 412 [2004]),
LNFP V Gal(jS1 -4)GlcNAc(jS1 -3)Gal(jS1 -4)[Fuc(a1 -3)]Glc (Dumon et al.
Glycoconj. J. 18, 465 [2001 ]).
Claims
1 . A method of obtaining, in crystalline form, a non-acidic HMO or a non-acidic precursor of an HMO produced in an aqueous media, enzymatically or by
fermentation, particularly produced intracellularly in an aqueous fermentation broth, quite particularly produced by E. coli, said method comprising the steps of: a) drying, preferably freeze-drying, the aqueous media, preferably after removing proteins therefrom, to produce a dry powder that contains the non- acidic HMO or HMO precursor; b) then treating the dry powder with a hot CrC6 alcohol solvent, preferably a C C4 alcohol solvent, especially methanol, optionally containing up to 50 v/v%, preferably up to 35 v/v%, especially no more than 15 v/v%, particularly no more than 5 v/v%, water, to produce an alcohol solution of the non-acidic HMO or HMO precursor; and c) then crystallizing the non-acidic HMO or HMO precursor from the alcohol solution.
2. The method of claim 1 , wherein the dry powder is at least about 15 wt%, preferably at least about 25 wt%, especially at least 30-40 wt%, particularly at least 50-60 wt%, more particularly at least 95 wt%, even more particularly at least 97 wt% non-acidic HMO or HMO precursor.
3. The method of any one of the claims 1 -2, wherein the dry powder contains no more than 8-10 wt% water, preferably no more than about 5 wt% water, especially no more than 3-4 wt% water.
4. The method of any one of the claims 1 -3, which also comprises, between steps b) and c), one or more, preferably all, of the following intermediate steps: i) the alcohol solution is filtered to separate insolubles; ii) then the alcohol solution is concentrated down to at least
about 60-70 %, preferably down to about 40-60 %, especially
down to about 45-55 %, of its original volume, and
iii) then, the alcohol solution is seeded with crystals of the non- acidic HMO or HMO precursor.
5. The method of any one of the claims 1 -4, wherein the non-acidic HMO is a fucosylated HMO, preferably a compound of formula 1
wherein R independently of each other is fucosyl or H, provided that at least one R is fucosyl.
6. The method of claim 5, comprising the steps of: a) drying, preferably freeze-drying, the aqueous media, preferably after removing proteins therefrom, to produce a dry powder that contains a
compound of formula 1 ; b) then treating the dry powder with a hot C C4 alcohol solvent, preferably methanol, that preferably contains no water, to produce an alcohol solution of compound of formula 1 ; and c) then crystallizing the compound of formula 1 from the alcohol solution.
7. The method of claim 6, which also comprises, between steps b) and c), an intermediate step wherein the alcohol solution is concentrated down to 40-60 %, preferably down to 45-55 %, of its original volume, preferably by keeping it hot to partially evaporate it.
8. The method of claim 7, wherein the hot alcohol solution is cooled to 30-50 °C, preferably 35-45 °C, after being concentrated in the intermediate step above.
9. The method of any one of the claims 5-8, wherein the compound of formula 1 is 2'-FL, and the alcohol is methanol.
10. The method of any one of the claims 1 -4, wherein the non-acidic HMO is a HMO having no fucosyl residue, preferably LNT or LNnT.
1 1 . The method of claim 10, comprising the steps of: a) drying, preferably freeze-drying, the aqueous media, preferably after removing proteins therefrom, to produce a dry powder that contains LNT or LNnT; b) then treating the dry powder with a hot CrC4 alcohol solvent, preferably methanol, that preferably contains up to 30-40 % of water, to produce an alcohol solution LNT or LNnT; and c) then crystallizing LNT or LNnT from the alcohol solution.
12. The method of any one of the claims 10-1 1 , wherein the non-acidic HMO is LNT, and the alcohol is methanol.
13. A method of obtaining a non-acidic HMO or HMO precursor from a powder, preferably a protein-free powder, produced by drying, preferably freeze drying, an aqueous media, in which the non-acidic HMO or HMO precursor was produced enzymatically or by fermentation, particularly produced intracellularly in an aqueous fermentation broth, quite particularly produced by E. coli, comprising carrying out the method of any one of the claims 1 -12.
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