DK202100635A1 - Separation of neutral human milk oligosaccharides from a fermentation broth - Google Patents
Separation of neutral human milk oligosaccharides from a fermentation broth Download PDFInfo
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- DK202100635A1 DK202100635A1 DKPA202100635A DKPA202100635A DK202100635A1 DK 202100635 A1 DK202100635 A1 DK 202100635A1 DK PA202100635 A DKPA202100635 A DK PA202100635A DK PA202100635 A DKPA202100635 A DK PA202100635A DK 202100635 A1 DK202100635 A1 DK 202100635A1
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- Prior art keywords
- hmo
- lacto
- neutral
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- 230000007935 neutral effect Effects 0.000 title claims abstract description 126
- 238000000855 fermentation Methods 0.000 title claims abstract description 60
- 230000004151 fermentation Effects 0.000 title claims abstract description 60
- 229920001542 oligosaccharide Polymers 0.000 title claims abstract description 47
- 150000002482 oligosaccharides Chemical class 0.000 title claims abstract description 47
- 235000020256 human milk Nutrition 0.000 title claims abstract description 41
- 210000004251 human milk Anatomy 0.000 title claims abstract description 37
- 238000000926 separation method Methods 0.000 title description 11
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- 150000002597 lactoses Chemical class 0.000 description 1
- JCQLYHFGKNRPGE-FCVZTGTOSA-N lactulose Chemical compound OC[C@H]1O[C@](O)(CO)[C@@H](O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 JCQLYHFGKNRPGE-FCVZTGTOSA-N 0.000 description 1
- 229960000511 lactulose Drugs 0.000 description 1
- PFCRQPBOOFTZGQ-UHFFFAOYSA-N lactulose keto form Natural products OCC(=O)C(O)C(C(O)CO)OC1OC(CO)C(O)C(O)C1O PFCRQPBOOFTZGQ-UHFFFAOYSA-N 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 229910001629 magnesium chloride Inorganic materials 0.000 description 1
- 235000011147 magnesium chloride Nutrition 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 230000003278 mimic effect Effects 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 125000001483 monosaccharide substituent group Chemical group 0.000 description 1
- 229920005615 natural polymer Polymers 0.000 description 1
- 230000035764 nutrition Effects 0.000 description 1
- ZQPPMHVWECSIRJ-KTKRTIGZSA-N oleic acid group Chemical group C(CCCCCCC\C=C/CCCCCCCC)(=O)O ZQPPMHVWECSIRJ-KTKRTIGZSA-N 0.000 description 1
- 230000036961 partial effect Effects 0.000 description 1
- 230000000737 periodic effect Effects 0.000 description 1
- 239000012071 phase Substances 0.000 description 1
- IVXQBCUBSIPQGU-UHFFFAOYSA-N piperazine-1-carboxamide Chemical compound NC(=O)N1CCNCC1 IVXQBCUBSIPQGU-UHFFFAOYSA-N 0.000 description 1
- 229920000747 poly(lactic acid) Polymers 0.000 description 1
- 229920001467 poly(styrenesulfonates) Polymers 0.000 description 1
- 229920002492 poly(sulfone) Polymers 0.000 description 1
- 229920002239 polyacrylonitrile Polymers 0.000 description 1
- 239000004626 polylactic acid Substances 0.000 description 1
- 229920001155 polypropylene Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 229920002981 polyvinylidene fluoride Polymers 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- 239000001508 potassium citrate Substances 0.000 description 1
- 229960002635 potassium citrate Drugs 0.000 description 1
- QEEAPRPFLLJWCF-UHFFFAOYSA-K potassium citrate (anhydrous) Chemical compound [K+].[K+].[K+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O QEEAPRPFLLJWCF-UHFFFAOYSA-K 0.000 description 1
- 235000011082 potassium citrates Nutrition 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 238000011027 product recovery Methods 0.000 description 1
- 239000008213 purified water Substances 0.000 description 1
- 239000011541 reaction mixture Substances 0.000 description 1
- 238000004366 reverse phase liquid chromatography Methods 0.000 description 1
- 238000002390 rotary evaporation Methods 0.000 description 1
- 238000010079 rubber tapping Methods 0.000 description 1
- 239000003813 safflower oil Substances 0.000 description 1
- 235000005713 safflower oil Nutrition 0.000 description 1
- 238000004062 sedimentation Methods 0.000 description 1
- 235000020183 skimmed milk Nutrition 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000001509 sodium citrate Substances 0.000 description 1
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 description 1
- 235000011083 sodium citrates Nutrition 0.000 description 1
- 229910052938 sodium sulfate Inorganic materials 0.000 description 1
- 235000011152 sodium sulphate Nutrition 0.000 description 1
- 239000008247 solid mixture Substances 0.000 description 1
- 239000007790 solid phase Substances 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 238000010561 standard procedure Methods 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 125000001273 sulfonato group Chemical group [O-]S(*)(=O)=O 0.000 description 1
- 239000001117 sulphuric acid Substances 0.000 description 1
- 235000011149 sulphuric acid Nutrition 0.000 description 1
- 239000002600 sunflower oil Substances 0.000 description 1
- 230000002195 synergetic effect Effects 0.000 description 1
- 229920001059 synthetic polymer Polymers 0.000 description 1
- 150000004044 tetrasaccharides Chemical class 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- QORWJWZARLRLPR-UHFFFAOYSA-H tricalcium bis(phosphate) Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 description 1
- 235000013337 tricalcium citrate Nutrition 0.000 description 1
- 238000013060 ultrafiltration and diafiltration Methods 0.000 description 1
- 235000015112 vegetable and seed oil Nutrition 0.000 description 1
- 239000008158 vegetable oil Substances 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
- 229940088594 vitamin Drugs 0.000 description 1
- 229930003231 vitamin Natural products 0.000 description 1
- 235000019155 vitamin A Nutrition 0.000 description 1
- 239000011719 vitamin A Substances 0.000 description 1
- 235000019156 vitamin B Nutrition 0.000 description 1
- 239000011720 vitamin B Substances 0.000 description 1
- 235000019164 vitamin B2 Nutrition 0.000 description 1
- 239000011716 vitamin B2 Substances 0.000 description 1
- 235000019154 vitamin C Nutrition 0.000 description 1
- 239000011718 vitamin C Substances 0.000 description 1
- 235000019166 vitamin D Nutrition 0.000 description 1
- 239000011710 vitamin D Substances 0.000 description 1
- 239000012610 weak anion exchange resin Substances 0.000 description 1
- 235000021119 whey protein Nutrition 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H1/00—Processes for the preparation of sugar derivatives
- C07H1/06—Separation; Purification
- C07H1/08—Separation; Purification from natural products
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D61/00—Processes of separation using semi-permeable membranes, e.g. dialysis, osmosis or ultrafiltration; Apparatus, accessories or auxiliary operations specially adapted therefor
- B01D61/02—Reverse osmosis; Hyperfiltration ; Nanofiltration
- B01D61/027—Nanofiltration
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D61/00—Processes of separation using semi-permeable membranes, e.g. dialysis, osmosis or ultrafiltration; Apparatus, accessories or auxiliary operations specially adapted therefor
- B01D61/14—Ultrafiltration; Microfiltration
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D61/00—Processes of separation using semi-permeable membranes, e.g. dialysis, osmosis or ultrafiltration; Apparatus, accessories or auxiliary operations specially adapted therefor
- B01D61/58—Multistep processes
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H3/00—Compounds containing only hydrogen atoms and saccharide radicals having only carbon, hydrogen, and oxygen atoms
- C07H3/06—Oligosaccharides, i.e. having three to five saccharide radicals attached to each other by glycosidic linkages
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
Abstract
The invention relates to a method for recovery and purification of a neutral human milk oligosaccharide (HMO) from a fermentation broth, comprising the steps of separating the fermentation broth to form a separated HMO-containing stream and a biomass waste stream, purifying the HMO-containing stream by nanofiltration, purifying the HMO-containing stream with an acidic cation exchange resin, concentrating the purified HMO-containing stream, and drying the purified HMO-containing stream to obtain a solidified neutral HMO. Moreover, the invention also concerns a neutral human milk oligosaccharide obtained by the inventive method, as well as its use in food, feed, and medical application.
Description
DK 2021 00635 A1
SEPARATION OF NEUTRAL HUMAN MILK OLIGOSACCHARIDES FROM
A FERMENTATION BROTH
The present invention relates to the separation and isolation of neutral human milk oligosaccharides (HMOs) from a reaction mixture in which they are produced.
During the past decades, the interest in the preparation and commercialisation of human milk oligosaccharides (HMOs) has been increasing steadily. The importance of HMOs is directly linked to their unique biological activities. Therefore, HMOs have become important potential products for nutrition and therapeutic uses. As a result, low cost ways of producing industrially HMOs have been sought.
To date, the structures of more than 140 HMOs have been determined, and considerably more are probably present in human milk (Urashima et al.: Milk oligosaccharides, Nova Biomedical Books, 2011; Chen Adv. Carbohydr. Chem. Biochem. 72, 113 (2015)). The HMOs comprise a lactose (Galp1-4Glc) moiety at the reducing end and may be elongated with an N-acetylglucosamine, or one or more N-acetyllactosamine moiety/moieties (Galf1-4GlcNAc) and/or a lacto-N-biose moiety (Galp1-3GlcNAc). Lactose and the N-acetyllactosaminylated or lacto-N-biosylated lactose derivatives may further be substituted with one or more fucose and/or sialic acid residue(s), or lactose may be substituted with an additional galactose, to produce HMOs known so far.
Direct fermentative production of HMOs, especially of those being a trisaccharide, has recently become practical (Han et al. Biotechnol. Adv. 30, 1268 (2012) and references cited therein). Such fermentation technology has used a recombinant E. coli system wherein one or more types of glycosyl transferases originating from viruses or bacteria have been co-expressed to glycosylate exogenously added lactose, which has been internalized by the LacY permease of the E. coli.
However, the use of a recombinant glycosyl transferase, especially series of recombinant glycosyl transferases to produce oligosaccharides of four or more monosaccharide units, has always led to by-product formation hence resulting in a complex mixture of oligosaccharides in the fermentation broth. Further, a fermentation broth inevitably contains a wide range of non-oligosaccharide 1
DK 2021 00635 A1 substances such as cells, cell fragments, proteins, protein fragments, DNA, DNA fragments, endotoxins, caramelized by-products, minerals, salts, or other charged molecules.
For separating HMOs from carbohydrate by-products and other contaminating components, active carbon treatment combined with gel filtration chromatography has been proposed as a method of — choice (WO 01/04341, EP-A-2479263, Dumon et al. Glycoconj. J. 18, 465 (2001), Priem et al.
Glycobiology 12, 235 (2002), Drouillard et al. Angew. Chem. Int. Ed. 45, 1778 (2006), Gebus et al.
Carbohydr. Res. 361, 83 (2012), Baumgårtner et al. ChemBioChem 15, 1896 (2014)). Although gel filtration chromatography is a convenient lab scale method, it cannot be efficiently scaled up for industrial production.
EP 2896628 Al describes a process for purification of 2’-FL from a fermentation broth obtained by microbial fermentation comprising the following steps: ultrafiltration, strong cation exchange resin chromatography (H”-form), neutralization, strong anion exchange resin chromatography (acetate- form), neutralization, active carbon treatment, electrodialysis, second strong cation exchange resin chromatography (H'- or Na"-form), second strong anion exchange resin chromatography (CI”- form), second active carbon treatment, optional second electrodialysis and sterile filtration.
However, such a purification process is intrinsically limited to neutral human milk oligosaccharides.
WO 2017/182965 and WO 2017/221208 disclose a process for purification of LNT or LNnT from fermentation broth comprising ultrafiltration, nanofiltration, active carbon treatment and treatment with strong cation exchange resin (H -form) followed by weak anion exchange resin (base form).
WO 2015/188834 and WO 2016/095924 disclose the crystallization of 2'-FL from a purified fermentation broth, the purification comprising ultrafiltration, nanofiltration, active carbon treatment and treatment with strong cation exchange resin (H”-form) followed by weakly basic resin (base form).
Other prior art documents have disclosed purification methods elaborated for low lactose or no- lactose fermentation broths. According to these procedures, lactose added in excess during the fermentative production of a neutral HMO has been hydrolysed in situ after completion of the fermentation by the action of a B-galactosidase, resulting in a broth that substantially does not contain residual lactose. Accordingly, WO 2012/112777 discloses a series of steps to purify 2’-FL comprising centrifugation, capturing the oligosaccharide on carbon followed by elution and flash chromatography on ion exchange media. WO 2015/106943 discloses purification of 2'-FL comprising ultrafiltration, strong cation exchange resin chromatography (H'-form), neutralization, 2
DK 2021 00635 A1 strong anion exchange resin chromatography (Cl -form), neutralization, nanofiltration/diafiltration, active carbon treatment, electrodialysis, optional second strong cation exchange resin chromatography (Na”-form), second strong anion exchange resin chromatography (CI'-form), second active carbon treatment, optional second electrodialysis and sterile filtration. WO 2019/063757 discloses a process for purification of a neutral HMO comprising separating biomass from fermentation broth and treatment with a cation exchange material, an anion exchange material, and a cation exchange adsorbent resin.
However, alternative and/or improved procedures for isolating and purifying neutral HMOs from non-carbohydrate components of the fermentation broth in which they have been produced, — especially those suitable for industrial scale, are needed to improve the recovery yield of the neutral
HMO and/or to simplify prior art methods while the purity of the neutral HMO is at least maintained, and preferably, improved. Moreover, such alternative purification procedures preferably lead to purified neutral HMOs that are free of proteins and recombinant materials originating from the used recombinant microbial strains, which are thus well suited for use in food, medical food, and feed applications.
The invention relates to a method for recovery and purification of a neutral human milk oligosaccharide (HMO) from a fermentation broth, comprising the steps of: a. separating the fermentation broth to form a separated HMO-containing stream and a biomass waste stream; b. purifying the separated HMO-containing stream: by nanofiltration/diafiltration followed by purifying with an acidic cation exchange resin, or with an acidic cation exchange resin followed by purifying by nanofiltration/diafiltration; c. concentrating the purified HMO-containing stream; and d. drying the purified HMO-containing stream to obtain a solidified neutral HMO.
In another aspect, the invention relates to a neutral human milk oligosaccharide obtained by the method according to the invention.
Another aspect of the invention relates to a neutral human milk oligosaccharide obtained by the method according to the invention for use in medicine. 3
DK 2021 00635 A1
Another aspect of the invention relates to the use of a neutral human milk oligosaccharide obtained by the method according to the invention for food and/or feed applications.
Another aspect of the invention relates to a food or cosmetic product comprising the neutral human milk oligosaccharide obtained by the method according to the invention.
1. Terms and definitions
The term "fermentation broth", as used in this specification, refers to a product obtained from fermentation of the microbial organism. Thus, the fermentation product comprises cells (biomass), the fermentation medium, salts, residual substrate material, and any molecules/by-products produced during fermentation, such as the desired neutral HMOs. After each step of the purification method, one or more of the components of the fermentation product is removed, resulting in more purified neutral HMOs.
The term “monosaccharide” means a sugar of 5-9 carbon atoms that is an aldose (e.g., D-glucose,
D-galactose, D-mannose, D-ribose, D-arabinose, L-arabinose, D-xylose, etc.), a ketose (e.g., D- fructose, D-sorbose, D-tagatose, etc.), a deoxysugar (e.g., L-rhamnose, L-fucose, etc.), a deoxy- aminosugar (e.g., N-acetylglucosamine, N-acetylmannosamine, N-acetylgalactosamine, etc.), a uronic acid, a ketoaldonic acid (e.g., sialic acid) or equivalents.
The term “disaccharide” means a carbohydrate consisting of two monosaccharide units linked to each other by an interglycosidic linkage. — The term “tri- or higher oligosaccharide” means a sugar polymer consisting of at least three, preferably from three to eight, more preferably from three to six, monosaccharide units (vide supra).
The oligosaccharide can have a linear or branched structure containing monosaccharide units that are linked to each other by interglycosidic linkages.
The term "human milk oligosaccharide" or "HMO" means a complex carbohydrate found in human breast milk (Urashima et al.: Milk Oligosaccharides, Nova Medical Books, NY, 2011; Chen Adv.
Carbohydr. Chem. Biochem. 72, 113 (2015)). The HMOs have a core structure being a lactose unit at the reducing end that is elongated by one or more B-N-acetyl-lactosaminyl and/or one or more 3- lacto-N-biosyl units, and which core structures can be substituted by an a-L-fucopyranosyl and/or an a-N-acetyl-neuraminyl (sialyl) moiety. In this regard, the non-acidic (or neutral) HMOs are devoid of a sialyl residue, and the acidic HMOs have at least one sialyl residue in their structure. 4
DK 2021 00635 A1
The non-acidie for neutral} HMOs can be fucosylated or non-fucosylated. Examples of such neutral non-fucosylated HMOs imelude lacto-N-triose II (LNTri, GleNAc(B1-3}Gal{B1-4)Gle}, lacto-N- tetraose (LNT), lacto-N-neotetraose (LNNT), lacto-N-nechexaose (LNnH), para-lacto-N- neohexaose (pLNnH), para-lacto-N-hexaose {(pLNH} and lacto-N-hexaose (L NH). Examples of neutral fucosylated HMOs include ?'-fucosyllactose (2'-FL), lacto-N-fucopentaose I (LNFP-D), lacto-N-difucohexnose I (LNDFH-D), 3-fucosyllaciose (3-FL). difucosyllactose (DFL), lacto-N- fucopentacse I {LNFP-II}, lacto-N-fucopentaose HI (LNFP-III), lacto-N-difucohexaose HI (LNDFH-ID), fucosyl-lacto-N-hexaose I (FLNH-ID), lacto-N-fucopentaose V (LNFP-V}, lacto-N- difucohexaose II (LNDFH-ID), fucosyl-lacto-N-hexaose I (FLNH-I}, fucosyl-para-lacto-N-hexaose I — (FPpLNH-D, fucosyl-para-lactio-N-neohexaose II {F-pLNnH ID) and fucosyl-lacto-N-neohexaose (FLNAH). Examples of acidic HMOs melude 3 -s1alyliactose (37-SL), 6'-sialyllactose (6'-SL), 3- fucosyl-3'-stalvllactose (FSL), LST a, fucosyl-LST a (FLST a}, LST b, fucosyl-LST b (FLST b),
LST c, fucosyl-LST ¢ (FLST 0), ualyl-LNH (SLNH), sialyl-lacto-N-hexaose (SLNH), sialyl-lacio-
N-neohexavse I (SLNH-D), sialyl-lacto-N-neohexaose II (SLNH-II} and disialyvl-lacto-N-tetraose 18 (DSLNT).
The term “sialyl” or “sialyl moiety” means the glveosyl residue of sialic acid (N-acetyl-neuraminie acid, Neu%4c), preferably linked with o-linkage:
OH
HO AL COOH
AcH NZ oF
The term "fucosyl” means an L-fucopyranosyl group, preferably linked with a-interelycosidie — linkage:
SVN
OH
HO
OH
SN-acetyl-elucosaminyl” means an N-acetyl-2-amino-2-deoxv-D-glucopyranosyl (GicNAc) group, preferably linked with fi-linkage: 5
DK 2021 00635 A1
OH al
HØ…—
NHAC
SN-acetyl-lactosaminyl” means the glycosyl residue of N-acetyl-lactosamine {LacNAc, GalpPi- 4GleNAcp}, preferably linked with B-linkage: on OH
OH
J 2 0
HO~ oH as 3
NHAC
Furthermore, the term “lacto-N-biosvl” means the glveosyl residue of lacto-N-liose (LNB. Galp1- 3GleNAep), preferably linked with B-linkage: on OH OH amba
HON, 0 -
OR NHAC
The term "neutral human milk oligosaccharide” means a non-sialylated (therefore neutral) complex carbohydrate found in buman breast milk (Urashima et al: MIF oligosaccharides, Nova Biomedical
Books, 2011; Chen Adv. Carbohyvdr. Chen, Bfochem. 72, 113 (2015) comprising a core structure being a lactose unit at the reducing end that 1s a) substituted with one or two o-L-fucopyranosyl moieties, b) substituted with a galactosyl residue, or ¢) elongated, via its 3"-OH group, by an N- acetylglucosamine, a lacto-N-biose (GalfiI-3GIcN Ao) or an N-acetyllactosamine {Gal 1-4GleNAc) moiety. The N-acetyllactosamine containing derivatives can be further substituted with N- 18 — sctyllaciosamine and/or lacio-N-biose (lacto-N-biose 1s always a non-reducing terminal). The N- acetyHactosamine and the lacto-N-biose contaumne derivatives can optionally be substituted by one or more u-L-fucopyranosyl moieties. Examples of neutral trisaccharide HMOs melude 27-0- fucosyllactose (2°-FL. Fucol-2GaiB1-4G10), 3-0-fucosyllactose (3-FL, Galf"i-4(Fucoal-3)Gle) or lacto-N-triose I {GIcNAcPB1-3GalBi-4Gle); examples of neutral tetrasaccharide HMOs include 2 3-di-O-fucosylactose (DFL. Fucai-2GalB1-4(Fucal-3)YG10), lacto-N-tetraose (LNT, Gaifi1- 3GRONACB1-3Ga11-4G1c) or lacto-N-neotetraose (LNAT, Galfl-4GLNAcf1-3Ga1B 1-4GH10); examples of neutral pentasaccharide HMOs inelude lacto-N-fucopentaose I (LNFP I, Fucal- 2Galfi-3GIeNAcBI-3Galf1-4Gle), lacto-N-fucopentaose IT (LNFP II, Gal 1-3(Fucal- 4jGINAeP1-3Ga191-4G10), lacto-N-fucopentaose IIT (LNFP IT. GalB1-4iFuco1-3)GIoNAcD1- 6
DK 2021 00635 A1 3GalB1-4Glc), lacto-N-fucopentaose V (LNFP V, Gal1-3GlcNAcB1-3GalB1-4(Fucal-3)Glc), lacto-N-fucopentaose VI (LNFP VI, Galp1-4GlcNAcB1-3Galp1-4(Fucal-3)Glc); examples of neutral hexasaccharide HMOs include lacto-N-difucohexaose I (LNDFH I, Fucal-2Galp1- 3(Fucal-4)GlcNAcB1-3Galp1-4Glce), lacto-N-difucohexaose II (LNDFH II, Galp1-3(Fucal- 4)GIcNAcB1-3Galp1-4(Fucal-3)Glc), lacto-N-difucohexaose III (LNDFH III, GalB1-4(Fucal- 3)GIcNAcB1-3GalpP1-4(Fuca1-3)GIc), lacto-N-hexaose (LNH, GalB1-3GIcNAcB1-3(Galp1- 4GIcNACcB1-6)Galp1-4Glc), para-lacto-N-hexaose (pLNH, GalB1-3GIcNAcB1-3GalP1- 4GIcNACcB1-3GalB1-4Glc), lacto-N-neohexaose (LNnH, GalB1-4GIcNAcB1-3(GalB1-4GIcNAcP1- 6)GalB1-4Glc) or para-lacto-N-neohexaose (pLNnH, Gal 1-4GIcNAcB1-3GalB1-4GIcNAcB1- 3Galp1-4Glc).
The term "biomass”, in the context of fermentation, refers to the suspended, precipitated, or insoluble materials originating from fermentation cells, like intact cells, disrupted cells, cell fragments, proteins, protein fragments, polysaccharides.
The term “Brix” refers to degrees Brix, that is the sugar content of an aqueous solution (g of sugar — in 100 g of solution). In this regard, Brix of the human milk oligosaccharide solution of this application refers to the overall carbohydrate content of the solution including the human milk oligosaccharides and its accompanying carbohydrates. Brix is measured by a calibrated refractometer. “Demineralization” preferably means a process of removing minerals or mineral salts from a liquid.
In the context of the present invention, demineralization can occur in the nanofiltration step, especially when it is combined with diafiltration.
The term “protein-free aqueous medium” preferably means an aqueous medium or broth from a fermentation or enzymatic process, which has been treated to remove substantially all the proteins, as well as peptides, peptide fragments, RNAs and DNAs, as well as endotoxins and glycolipids that — could interfere with the eventual purification of the one or more neutral HMOs and/or one or more of their components, especially the mixture thereof, from the fermentation or enzymatic process mixture.
The term "HMO-containing stream” means an aqueous medium containing neutral HMOs obtained from a fermentation process, which has been treated to remove suspended particulates and contaminants from the process, particularly cells, cell components, insoluble metabolites and debris that could interfere with the eventual purification of the one or more hydrophilic oligosaccharides, 7
DK 2021 00635 A1 especially one or more neutral HMOs and/or one or more HMO components, especially mixtures thereof.
The term “biomass waste stream” preferably means suspended particulates and contaminants from the fermentation process, particularly cells, cell components, insoluble metabolites, and debris.
Rejection factor of a salt (in percent) is calculated as (1-kp/kr): 100, wherein «p is the conductivity of the salt in the permeate and kr is the conductivity of the salt in the retentate.
Rejection factor of a carbohydrate (in percent) is calculated as (1-Cp/C;): 100, wherein Cp is the concentration of the carbohydrate in the permeate and Cy is the concentration of the carbohydrate in the retentate.
The term “diafiltration” refers to solvent addition (water) during the membrane filtration process. If diafiltration is applied during ultrafiltration, it improves the yield of the desired HMO in the permeate. If diafiltration is applied during nanofiltration, it improves the separation of small size impurities and salts to the permeate. The solute yield and therefore the product enrichment could be calculated based on the formulas known to the skilled person based on rejection factors and relative — amount of water added.
The term "concentrating” as used in step d) of the method according to the invention refers to the removal of liquid, mostly water, thus resulting in a higher concentration of the neutral HMO in the purified HMO-containing product stream. 2. Method for the purification of a neutral human milk oligosaccharides from a fermentation broth
The invention relates to a method for recovery and purification of a neutral human milk oligosaccharide (HMO) from a fermentation broth, comprising the steps of’ a. separating the fermentation broth to form a separated HMO-containing stream and a biomass waste stream; b. purifying the separated HMO-containing stream by nanofiltration/diafiltration (NF/DF) followed by purifying with an acidic cation exchange resin, or with an acidic cation exchange resin followed by purifying by NF/DF; c. concentrating the purified HMO-containing stream; and 8
DK 2021 00635 A1 d. drying the purified HMO-containing stream to obtain a solidified neutral HMO.
In a preferred embodiment, the NF/DF is conducted at a pH of less than 5.0, preferably less than 4.5, advantageously less than 4.0, but not less than 3.0.
In a preferred embodiment, step b) comprises two NF/DF steps, more preferably the second NF/DF — step is conducted at a pH of less than 5.0, preferably less than 4.5, advantageously less than 4.0, but not less than 3.0.
In a preferred embodiment, step b) comprises two NF/DF steps, wherein an acidic cation exchange resin purification step is performed between the NF/DF steps, more preferably wherein the second
NF/DF step is conducted at a pH of less than 5.0, preferably less than 4.5, advantageously less than — 4.0, but not less than 3.0.
In one embodiment, the method according to the invention consists of steps a)-d).
In a preferred embodiment, the method does not contain a basic anion exchanger treatment step.
In a preferred embodiment, a basic anion exchanger treatment step is excluded from the method according to the invention.
In a preferred embodiment, method steps a)-d) are performed in the consecutive order a)-d) as given above.
The fermentation broth:
In an embodiment, the neutral being present in the fermentation broth has been obtained by culturing a genetically modified microorganism capable of producing said neutral human milk oligosaccharide from an internalized carbohydrate precursor. Preferably, the microbial organism is a genetically modified bacterium or yeast such as a Saccharomyces strain, a Candida strain, a
Hansenula strain, a Kluyveromyces strain, a Pichia strain, a Schizosaccharomyces stain, a
Schwanniomyces strain, a Torulaspora strain, a Yarrowia strain, or a Zygosaccharomyces strain.
More preferably, the yeast is Saccharomyces cerevisiae, Hansenula polymorpha, Kluyveromyces lactis, Kluyveromyces marxianus, Pichia pastoris, Pichia methanolica, Pichia stipites, Candida boidinii, Schizosaccharomyces pombe, Schwanniomyces occidentalis, Torulaspora delbrueckii,
Yarrowia lipolytica, Zygosaccharomyces rouxii, or Zygosaccharomyces bailii; and the Bacillus is
Bacillus amyloliquefaciens, Bacillus licheniformis or Bacillus subtilis. 9
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In an embodiment, at least one neutral human milk oligosaccharide being present in the fermentation broth has not been obtained by microbial fermentation, but has been e.g., added to the fermentation broth after it has been produced by a non-microbial method, e.g., chemical and/or enzymatic synthesis.
In an embodiment, the purity of the neutral HMO in the fermentation broth is <70%, preferably <60%, more preferably <50%, most preferably <40%.
In a preferred embodiment, the neutral HMO is preferably 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-fucopentaose II, lacto-N-fucopentaose III, lacto-N- fucopentaose V, lacto-N-neofucopentaose V (alternative name: lacto-N-fucopentaose VI), lacto-N- difucohexaose I, lacto-N-difucohexaose II, lacto-N-difucohexaose III, 6'-galactosyllactose, 3'- galactosyllactose, lacto-N-hexaose, lacto-N-neohexaose, or any mixture thereof. Preferably, the
HMO is 2'-fucosyllactose, LNT or LNnT.
In an embodiment, the HMO in the fermentation broth is a single neutral HMO.
In an embodiment, the HMO in the fermentation broth is a mixture of various individual neutral
HMOs.
In an embodiment, the HMO is a mixture of two individual neutral HMOs. In another embodiment, the HMO is a mixture of three individual neutral HMOs. In another embodiment, the HMO is a mixture of four individual neutral HMOs. In another embodiment, the HMO is a mixture of five — individual neutral HMOs.
In an embodiment, the HMO in the fermentation broth is a mixture of a neutral HMO obtained by microbial fermentation and a HMO that has not been obtained by microbial fermentation, but e.g., by chemical and/or enzymatic synthesis.
Separating the fermentation broth to form a separated HMO-containing stream and a biomass waste stream in step a) of the method according to the invention:
In step a) of the method according to the invention, the HMO-containing stream is separated from the biomass waste stream.
The fermentation broth typically contains, besides the desired neutral HMO, the biomass of the cells of the used microorganism together with proteins, protein fragments, peptides, DNAs, RNAs, endotoxins, biogenic amines, amino acids, organic acids, inorganic salts, unreacted carbohydrate 10
DK 2021 00635 A1 acceptors such as lactose, sugar-like by-products, monosaccharides, colorizing bodies, etc. In step a) of the method according to the invention, the biomass is separated from the neutral HMO.
In a preferred embodiment, the biomass is separated from the neutral HMO in step a) by ultrafiltration. The ultrafiltration step is to separate the biomass and, preferably, also high molecular — weight components and suspended solids from the lower molecular weight soluble components of the broth, which pass through the ultrafiltration membrane in the permeate. This ultrafiltration permeate is an aqueous solution containing the neutral human milk oligosaccharide also referred to as the HMO-containing stream, whereas the ultrafiltration retentate comprises the biomass waste stream.
Any conventional ultrafiltration membrane can be used having a molecular weight cut-off (MWCO) range between about 1 and about 500 kDa, such as 10-250, 50-100, 200-500, 100-250, 1-100, 1-50, 10-25, 1-5 kDa, or any other suitable sub-range. The membrane material can be a ceramic or made of a synthetic or natural polymer, e.g., polysulfone, polyvinylidene fluoride, polyacrylonitrile, polypropylene, cellulose, cellulose acetate or polylactic acid. The ultrafiltration step can be applied in dead-end or cross-flow mode. Step a) of the method according to the invention may comprise more than one ultrafiltration step using membranes with different MWCO as defined above, e.g, applying two ultrafiltration separations, wherein the first membrane has a higher MWCO than that of the second membrane. This arrangement may provide a better separation efficacy of the higher molecular weight components of the broth. After this separation step, the permeate contains — materials that have a molecular weight lower than the MWCO of the second membrane, including the neutral human milk oligosaccharides of interest.
In one embodiment, the fermentation broth is ultrafiltered using a membrane having a MWCO of 5 to 30 kDa, such as 10-25, 15 or 20 kDa.
In a preferred embodiment, the yield of the desired neutral HMO in the permeate after the ultrafiltration step performed in step a) is greater than 50%, greater than 60%, greater than 70%, greater than 80%, greater than 90%, greater than 91%, greater than 92%, greater than 93%, greater than 94%, greater than 95%, greater than 96%, greater than 97%, greater than 98%, or greater than 99%.
In another embodiment, the broth obtained from fermentation is subjected to centrifugation to — separate the biomass from the neutral HMO (HMO-containing stream) in step a) of the method according to the invention. In said embodiment, the supernatant represents the HMO-containing 11
DK 2021 00635 A1 stream, while the remaining material, i.e., the “biomass waste stream” can be separated out. By centrifugation, a clear supernatant comprising the neutral HMO can be obtained, which represents the HMO-containing stream.
The centrifuging can be lab scale or, advantageously over previous centrifuging methods, commercial scale (e.g.,, industrial scale, full production scale).
In some embodiments, a multi-step centrifugation can be used. For example, a series of 2, 3, 4, 5, 6, 7, 8, 9, or 10 centrifugation steps can be performed. In other embodiments, the centrifugation may be a single step. Centrifugation provides for a quick biomass-removal.
In certain embodiments, Sedicanter® centrifuge designed and manufactured by Flottweg can be used.
The particular type of centrifuge is not limiting, and many types of centrifuges can be used. The centrifuging can be a continuous process. In some embodiments, the centrifuging can have feed addition. For example, the centrifuging can have a continuous feed addition. In certain embodiments, the centrifuging can include a solid removal, such as a wet solid removal. The wet — solid removal can be continuous in some implementations, and periodic in other implementations.
For example, a conical plate centrifuge (e.g.,, disk bowl centrifuge or disc stack separator) can be used. The conical plate centrifuge can be used to remove solids (usually impurities) from liquids, or to separate two liquid phases from each other by means of a high centrifugal force. The denser solids or liquids which are subjected to these forces move outwards towards the rotating bowl wall while the less dense fluids move towards the centre. The special plates (known as disc stacks) increase the surface settling area which speeds up the separation process. Different stack designs, arrangements and shapes are used for different processes depending on the type of feed present. The concentrated denser solid or liquid can then be removed continuously, manually, or intermittently, depending on the design of the conical plate centrifuge. This centrifuge is very suitable for clarifying liquids that have small proportion of suspended solids.
The centrifuge works by using the inclined plate setter principle. A set of parallel plates with a tilt angle 0 with respect to horizontal plane is installed to reduce the distance of the particle settling.
The reason for the tilted angle is to allow the settled solids on the plates to slide down by centrifugal force so they do not accumulate and clog the channel formed between adjacent plates. 12
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This type of centrifuge can come in different designs, such as nozzle-type, manual-cleaning, self- cleaning, and hermetic. The particular centrifuge is not limiting.
Factors affecting the centrifuge include disk angle, effect of g-force, disk spacing, feed solids, cone angle for discharge, discharge frequency, and liquid discharge.
Alternatively, a solid bowl centrifuge (e.g.,, a decanter centrifuge) can be used. This is a type of centrifuge that uses the principle of sedimentation. A centrifuge is used to separate a mixture that consists of two substances with different densities by using the centrifugal force resulting from continuous rotation. It is normally used to separate solid-liquid, liquid-liquid, and solid-solid mixtures. One advantage of solid bowl centrifuges for industrial uses is the simplicity of installation — compared to other types of centrifuge. There are three design types of solid bowl centrifuge, which are conical, cylindrical, and conical-cylindrical.
Solid bowl centrifuges can have a number of different designs, any of which can be used for the disclosed method. For example, conical solid bowl centrifuges, cylindrical solid bowl centrifuges, and conical-cylindrical bowl centrifuges can be used. — The centrifuging can be performed at a number of speeds and residence times. For example, the centrifuging can be performed with a relative centrifugal force (RCF) of 20000g, 15000g, 10000g, or 5000g. In some embodiments, the centrifuging can be performed with a relative centrifugal force (RCF) of less than 20000g, 15000g, 10000g or 5000g. In some embodiments, the centrifuging can be performed with a relative centrifugal force (RCF) of greater than 20000g, 15000g, 10000g or 5000g.
In some embodiments, the centrifuging can be characterized by working volume. In some embodiments, the working volume can be 1, 5, 10, 15, 20, 50, 100, 300, or 500 1. In some embodiments, the working volume can be less than 1, 5, 10, 15, 20, 50, 100, 300, or 500 I. In some embodiments, the working volume can be greater than 1, 5, 10, 15, 20, 50, 100, 300, or 500 I.
In some embodiments, the centrifuging can be characterized by feed flow rate. In some embodiments, the feed flow rate can be 100, 500, 1000, 1500, 2000, 5000, 10000, 20000, 40000, or 100000 ]/hr. In some embodiments, the feed flow rate can be greater than 100, 500, 1000, 1500, 2000, 5000, 10000, 20000, 40000, or 100000 1/hr. In some embodiments, the feed flow rate can be less than 100, 500, 1000, 1500, 2000, 5000, 10000, 20000, 40000, or 100000 1/hr. 13
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The amount of time spent centrifuging (e.g.,, residence time) can vary as well. For example, the residence time can be 0.1, 0.2, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 minutes. In some embodiments, the residence time can be greater than 0.1, 0.2, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 minutes. In some embodiments, the residence time can be less than 0.1, 0.2, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 minutes.
Any of the above supernatant properties can be produced through a single instance of centrifuging.
Alternatively, it can be produced through multiple instances of centrifuging.
In view of the above, step a) of the method according to the invention can be performed via ultrafiltration as defined above or centrifugation, or via a combination of ultrafiltration and centrifugation. Preferably, method step a) is carried out by ultrafiltration as defined above to obtain — the HMO-containing stream separate from the biomass waste stream.
Before the ultrafiltration and/or centrifugation step, the fermentation broth can be subjected to a pre-treatment step. Pre-treatment of the fermentation broth can include pH adjustment, and/or dilution, and/or heat treatment. In certain implementations, all three of pH adjustment, dilution, and heat treatment can be performed. In alternative embodiments, pH adjustment and dilution can be performed. In alternative embodiments, pH adjustment and heat treating can be performed. In alternative embodiments, heat treating and dilution can be performed. Advantageously, a combination of a plurality of pre-treatment methods can provide an improved synergistic effect not found in individual pre-treatments.
In certain embodiments, one or more of the aforementioned pre-treatment steps can occur during the biomass removal in step a) by centrifuging and/or ultrafiltration as defined above. For example, between steps in a multi-step centrifuging, or the centrifuging vessel may be able to heat the fermentation broth during centrifuging.
Advantageously, the pre-treatment can increase the settling velocity of the solid particles (biomass) in the fermentation broth by a factor of 100 to 20000, making the biomass separation by centrifugation much more efficient and thus applicable in industrial scale. In addition to settling velocity, at least three other parameters are substantially improved due to pre-treatment that are, improved neutral HMO yield in the HMO-containing stream, reduced protein and DNA content in the supernatant, and further residual suspended solid content can be substantially reduced. 14
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Purifying the HMO-containing stream in step b):
In step b) of the method according to the invention, the HMO-containing stream is purified by nanofiltration followed by purifying with an acidic cation exchange resin, or purified with an acidic cation exchange resin followed by purifying by nanofiltration. — Nanofiltration (NF) can be used to remove low molecular weight molecules smaller than the desired neutral HMOs, such as mono- and disaccharides, short peptides, small organic acids, water, and salts.
The product stream, 1.e., the HMO-containing steam, is the NF retentate. The nanofiltration membrane thus has a MWCO that ensures the retention of the neutral of interest, i.e., the MWCO of — the nanofiltration membrane is adjusted accordingly.
Typically, the pore size of the nanofiltration membrane is from 0.5 nm to 2 nm and/or from 150 dalton (Da) molecular weight cut-off (MWCO) to 3000 Da MWCO.
In an embodiment, the membranes are in the range of 150-300 Da MWCO, which are defined as “tight” NF membranes.
In a preferred embodiment, the membranes are above 300 Da MWCO, and preferably not higher than 3000 Da MWCO. In said embodiment, the membranes are considered “loose” NF membranes.
In another preferred embodiment, the “loose” nanofiltration membrane has a molecular weight cut- off (MWCO) of 500-3000 Da and the active (top) layer of the membrane is preferably composed of polyamide. Thereby, the retention of tri- or higher oligosaccharides is ensured and at least part of — the disaccharides is allowed to pass the membrane. In this embodiment, the applied nanofiltration membrane shall be tight for tri- and higher oligosaccharides for them to be efficiently retained. At the same time, the membrane shall be relatively loose for MgSOu, that its rejection is about 50-90 %, as well as for disaccharides. This way, it is possible to separate e.g., lactose, which is a precursor in making human milk oligosaccharides e.g., by fermentation, from the neutral human milk oligosaccharides product with a good efficacy, and additionally a substantial part of divalent ions also passes to the permeate. In some embodiments, the MgSO4 rejection factor is 60-90 %, 70-90 %, 50-80 %, 50-70 %, 60-70 % or 70-80 %. Preferably, the MgSQOs rejection factor on said membrane is 80-90 %. Preferably, the membrane has a rejection factor for NaCl that is lower than that for MgSO4. In one embodiment, the rejection factor for NaCl is not more than 50 %. In another embodiment, the rejection factor for NaCl is not more than 40 %. In another embodiment, the 15
DK 2021 00635 A1 rejection factor for NaCl is not more than 30 %. In this latter embodiment, a substantial reduction of all monovalent salts in the retentate is also achievable. In said embodiment, the membrane is a thin- film composite (TFC) membrane. An example of a suitable piperazine based polyamide TFC membrane is TriSep” UA60.
Preferably, the yield of the desired neutral HMO in the retentate after the nanofiltration step is greater than 50%, greater than 60%, greater than 70%, greater than 80%, greater than 90%, greater than 91%, greater than 92%, greater than 93%, greater than 94%, greater than 95%, greater than 96%, greater than 97%, greater than 98%, or greater than 99%.
In a preferred embodiment, the method according to the invention further comprises a diafiltration step.
In an embodiment, the further diafiltration step follows the aforementioned nanofiltration step.
Diafiltration is a process that involves the addition of purified water to a solution during membrane filtration process in order to remove membrane permeable components more efficiently. Thus, diafiltration can be used to separate components on the basis of their properties, in particular molecular size, charge or polarity by using appropriate membranes, wherein one or more species are efficiently retained and other species are membrane permeable.
In a preferred embodiment, diafiltration and nanofiltration can be combined within one step (referred to as nanofiltration/diafiltration or NF/DF) in which diafiltration is executed while using a nanofiltration membrane that is effective for the separation of low molecular weight compounds and/or salts from the neutral HMOs. Diafiltration with “loose” NF membrane as defined above, is particularly efficient for both mono- and divalent salts removal and disaccharides removal from neutral HMOs.
In a preferred embodiment, the DF step or the NF/DF step is performed so that the pH is set below 5.0, preferably, below 4.5, advantageously below 4.0, but preferably not less than 3.0. Under this — condition, salts comprised of monovalent cations such as sodium salts (that is sodium ion together with the co-anion(s)) are effectively removed, giving rise to a low-salt or a practically salt-free purified solution containing a neutral HMO in the retentate.
The method according to the invention comprises further purification of the HMO-containing stream with an acidic cation exchange resin in step b). 16
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In the cation exchanger treatment step, positively charged materials can be efficiently removed from the HMO-containing stream, either before or after nanofiltration, as they bind to the resin, while the neutral HMOs will not be retained by the acidic cation exchange resin. Thereby, also the amounts of salts and/or colorizing agents and/or proteins can be further reduced.
In a preferred embodiment, the stationary phase (resin) comprises sulfonate groups that are negatively charged in aqueous solution and that tightly bind cationic compounds.
In a preferred embodiment, the acidic cation exchange resin is a strongly acidic cation exchange resin, preferably a styrene-divinylbenzene cation exchange resin.
In a further preferred embodiment, the acidic cation exchange resin is in H form.
The binding capacity of an acidic cation exchange resin is generally from 1.2 to 2.2 eq/l.
When using a cationic ion exchange resin, its degree of crosslinking can be chosen depending on the operating conditions of the ion exchange column. A highly crosslinked resin offers the advantage of durability and a high degree of mechanical integrity, however, suffers from a decreased porosity and a drop off in mass-transfer. A low-crosslinked resin is more fragile and tends to swell by absorption of mobile phase. The particle size of the ion exchange resin is selected to allow an efficient flow of the eluent, while the charged materials are still effectively removed. A suitable flow rate may also be obtained by applying a negative pressure to the eluting end of the column or a positive pressure to the loading end of the column, and collecting the eluent. A combination of both positive and negative pressure may also be used. The cationic ion exchange treatment can be carried out in a conventional manner, e.g., batch-wise or continuously.
Non-limiting examples of a suitable acidic cation exchange resin can be e.g., Amberlite IR100,
Amberlite IR120, Amberlite FPC22, Dowex S0WX, Finex CS16GC, Finex CS13GC, Finex
CS12GC, Finex CS11GC, Lewatit S, Diaion SK, Diaion UBK, Amberjet 1000, Amberjet 1200.
Preferably, the cation exchange step is performed after the nanofiltration step. However, said cation exchange step can also be conducted after a further optional step making use of active carbon as further described below.
In a preferred embodiment, step b) results in a purified solution containing the neutral HMO at a purity of > 80%, preferably > 85%, more preferably > 90%.
In a preferred embodiment, step b) results in a purified solution that is free of proteins and/or recombinant genetic material. 17
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In an even preferred embodiment, a second nanofiltration/diafiltration step is carried out in the method according to the invention. In said second nanofiltration step, the nanofiltration membrane is a “loose” NF membrane, see above. The second optional NF/DF step is performed after the first nanofiltration step, but is preferably performed before step c) of the method according to the invention. The second nanofiltration is performed in diafiltration mode. This second NF/DF step is performed so that the pH is set below 5.0, preferably below 4.5, advantageously below 4.0, but preferably not less than 3.0.
In a particularly preferred embodiment, step b) comprises a first NF or NF/DF purification of the
HMO-containing stream obtained in step a) followed by an strong cation exchange resin treatment (H -form) of the retentate from the first NF or NF/DF step, setting the pH of the resin eluate with
NaOH-solution between 5.0-7.0, then performing a second NF/DF step wherein the pH is set below 5.0, preferably below 4.5, advantageously below 4.0, but preferably not less than 3.0. As cation exchanger removes effectively cations, only sodium ion is reintroduced after neutralization. The present inventors surprisingly found that the rejection of sodium salts at pH below 5.0, preferably below 4.5 is low when “loose” NF membrane was used in NF/DF. In this regard, the sodium ions bring the anions to the permeate, making it possible that an aqueous solution of the neutral HMO is collected in the retentate that is practically salt-free, but at least has a very low salt content.
Therefore, the use of basic anion exchangers is avoidable in the purification of neutral HMOs.
Concentrating the purified HMO-containing stream in step c) of the method according to the invention:
A concentration step is used to economically remove significant quantities of liquid, mostly water, from the neutral HMO-containing stream using e.g., evaporation, nanofiltration, or reverse-osmosis filtration. Evaporation processes can include, e.g., falling film evaporation, climbing film evaporation and rotary evaporation. The evaporation can also be conducted under vacuum. The incoming solids concentration to the process is preferably approximately 5 to 30 wt.%. The exit solids concentration from such a process is typically greater than 30 wt.%., preferably greater than 50 wt.%. More preferably, the solids concentration exiting the dewatering operation is 60 to 80 wt.%. The solids portion of the recovered material is preferably greater than 80 wt.% of neutral
HMO.
In an embodiment, the purified neutral HMO-containing stream is concentrated to a concentration of > 100 g/L of neutral HMO, preferably of > 200 g/L, more preferably of > 300 g/L. 18
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When the purified neutral HMO-containing stream is concentrated by evaporation, the evaporation is preferably carried out at a temperature of from about 20 to about 80 °C. In some embodiments, the evaporation is carried out at a temperature of from 25 to 75 °C. In some embodiments, the evaporation is carried out at a temperature of from 30 to 70 °C. In some embodiments, the evaporation is carried out at a temperature of from 30 to 65 °C. Preferably, the evaporation is carried out under vacuum.
When the purified neutral HMO-containing stream is concentrated by membrane filtration, any membrane, typically nanofiltration membrane, is suitable that sufficiently rejects the neutral HMO.
Concentration by membrane filtration usually provides an HMO-solution of around 30-35 wt%. — This concentration may be suitable for conducting the subsequent drying-solidification step, e.g. freeze-drying. However, other drying methods may require more concentrated solutions, e.g. spray- drying or crystallization. In this case, concentration by evaporation, preferably under vacuum, is the preferred embodiment. Alternatively, the neutral HMO-containing stream obtained in the previous step is concentrated to around 30-35 wt% using a nanofiltration membrane, and the solution is — further concentrated by evaporation.
In one embodiment of the concentration by membrane filtration, the membrane of choice is a “tight” NF with 150-300 Da MWCO.
In other embodiment of the concentration by membrane filtration, the membrane of choice is a nanofiltration membrane that has a molecular weight cut-off (MWCO) of 500-3500 Da and an — active (top) layer of polyamide (“loose” NF membrane); and the concentration step is performed so that the pH is set below 5.0, preferably below 4.5, advantageously below 4.0, but not less than 3.0.
In this latter embodiment, a substantial reduction of all monovalent salts in the retentate is also achievable. In said embodiment, the membrane is preferably a thin-film composite (TFC) membrane. An example of a suitable TFC membrane is the piperazine based polyamide membrane
TriSep® UA60. Under this condition, remaining salts are also effectively removed, giving rise to a low-salt or a practically salt-free purified neutral HMO-concentrate. In this embodiment, after completing the concentration step, the pH of the neutral HMO-concentrate is advantageously set between 4-6 before performing the next step (e.g. evaporation, drying-solidification, sterile filtration). 19
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Drying the purified HMO-containing stream to obtain a solidified neutral HMO in step d):
Preferably after the separation/purification/concentration steps according to method steps a)-c) and any of the undermentioned optional method steps, respectively, the neutral HMO of interest is provided in its solid form via a drying step (step d)).
In a preferred embodiment, drying step d) comprises spray drying of the neutral HMO-containing stream, preferably consists of spray drying of the neutral HMO-containing stream.
Preferably, spray-drying leads to solidified neutral HMO having an amorphous structure, i.e., an amorphous powder is obtained.
In an embodiment, spray-drying is performed at a concentration of the neutral HMO of 20-60 % (w/v), preferably 30-50 % (w/v), more preferably 35-45 % (w/v), and an inlet temperature of 110- 150 °C, preferably 120-140 °C, more preferably 125-135 °C and/or an outlet temperature of 60-80 °C, preferably 65-70 °C.
In some embodiment, the neutral HMO-containing stream fed into the spray dryer has a Brix value of from about 8 to about 75% Brix. In some embodiments, the Brix value is from about 30 to about 65% Brix. In some embodiments, the Brix value is from about 50 to about 60% Brix. In some embodiments, the feed into the spray dryer is at a temperature of from about 2 to about 70 °C immediately before being dispersed into droplets in the spray dryer. In some embodiments, the feed into the spray dryer is at a temperature of from about 30 to about 60 °C immediately before being dispersed into droplets in the spray dryer. In some embodiments, the feed into the spray dryer is at a temperature of from about 2 to about 30 °C immediately before being dispersed into droplets in the spray dryer. In some embodiments, the spray drying uses air having an air inlet temperature of from 120 to 280 °C. In some embodiments, the air inlet temperature is from 120 to 210 °C. In some embodiments, the air inlet temperature is from about 130 to about 190 °C. In some embodiments, the air inlet temperature is from about 135 to about 160 °C. In some embodiments, the spray drying — uses air having an air outlet temperature of from about 80 to about 110 °C. In some embodiments, the air outlet temperature is from about 100 to about 110 °C. In some embodiments, the spray drying is carried out at a temperature of from about 20 to about 90 °C. In some embodiments, the spray dryer is a co-current spray dryer. In some embodiments, the spray dryer is attached to an external fluid bed. In some embodiments, the spray dryer comprises a rotary disk, a high-pressure nozzle, or a two-fluid nozzle. In some embodiments, the spray dryer comprises an atomizer wheel.
In some embodiments, spray-drying is the final purification step for the desired neutral HMO. 20
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Alternatively, the drying-solidification step comprises an indirect drying method. For the purposes of this specification, indirect dryers include those devices that do not utilize direct contact of the material to be dried with a heated process gas for drying, but instead rely on heat transfer either through walls of the dryer, e.g., through the shell walls in the case of a drum dryer, or alternately — through the walls of hollow paddles of a paddle dryer, as they rotate through the solids while the heat transfer medium circulates in the hollow interior of the paddles. Other examples of indirect dryers include contact dryers and vacuum drum dryers.
Alternatively, the drying-solidification step comprises freeze-drying.
Alternatively, the drying-solidification step comprises crystallization (provided that the HMO is obtainable in crystalline form).
Optional steps
In a preferred embodiment, the method according to the invention further comprises purification of by an active carbon treatment.
The treatment with active carbon represents a decolorization step (removing colorizing components) and/or a chromatographic step on a neutral solid phase, preferably reversed-phase chromatography to remove hydrophobic contaminants. Preferably, active carbon, such as Norit CA1 activated carbon can be used.
The active carbon treatment may serve to remove colorizing agents and may further reduce the amounts of water-soluble contaminants, such as salts. Moreover, the active carbon treatment may serve to remove proteins, DNAs, RNAs, or endotoxin that may be present in the HMO-containing stream.
Hence, the active carbon treatment leads to a reduction of colorizing agents and/or salts and/or proteins and/or DNAs and/or RNAs and/or endotoxin in the HMO-containing stream.
Under certain conditions, the neutral human milk oligosaccharides do not, or at least not substantially, adsorb to the carbon particles and elution with water gives rise to an aqueous solution of the neutral human milk oligosaccharides without a significant loss in their amounts, while colorizing agents, proteins, DNAs, RNAs, endotoxin, etc. remain adsorbed. It is merely a matter of routine skills to determine the conditions under which the neutral human milk oligosaccharides would bind to the carbon from its aqueous solution. 21
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Hence, the optional active charcoal treatment step is performed so that the neutral HMO is not or at least not substantially adsorbed by the active carbon. Under “not substantially adsorbed”, it is understood that less than 10%, preferably less than 5%, and more preferably less than 1% of the neutral HMO is adsorbed by the active carbon. The amount of active carbon used in this aspect is <100% by weight relative to the neutral HMO being present in the HMO-containing stream, preferably <10%. This can allow most of the neutral HMO to pass while residual biomolecules, coloured compounds, and other hydrophobic molecules, are retaining by the active carbon. In an embodiment, the amount of the applied active carbon is around 2-6 wt.%. This is economical, because all the benefits disclosed above can be conveniently achieved with a very low amount of — carbon. In other embodiment, the active carbon is added in an amount in the range of 0.25 wt.% to 3 wt.%, preferably in the range of 0.5 wt.% to 2.5 w.t%, and more preferably in the range of 0.75 wt.% to 2.2 wt.%, and even more preferably in the range of 1.0 wt.% to 2.0 wt.%, wherein the percentage values are based on the total weight of the HMO-containing stream that is subjected to the active carbon treatment step. This rather small amount of active carbon allows for significant reduction of active carbon consumption as well as for a significant reduction of product losses (neutral HMO).
In one aspect, the active carbon treatment can be conducted by adding carbon powder to the HMO- containing steam under stirring and filtering off the carbon.
In other aspect, for higher scale purification, the aqueous solution containing the neutral human — milk oligosaccharide (HMO-containing stream) is preferably loaded to a column packed with carbon, which may be a granulated carbon or may optionally be mixed with inert filter aid, then the column is washed with the required eluent. The fractions containing the neutral human milk oligosaccharide are collected.
In one embodiment, the active carbon used is granulated. This ensures a convenient flow-rate — without applying high pressure.
In one embodiment, the active carbon treatment, preferably comprising active carbon chromatography is conducted at elevated temperature. At elevated temperature, the binding of colorizing agents, residual proteins, etc. to the carbon particles takes place in a shorter contact time, therefore the flow-rate can be conveniently raised. Moreover, the active carbon treatment conducted atelevated temperature substantially reduces the total number of viable microorganisms (total 22
DK 2021 00635 A1 microbial count) in the HMO-containing stream. The elevated temperature may be at least 30-35 °C, such as at least 40 °C, at least 50 °C, around 40-50 °C, or around 60 °C.
In one embodiment, the active carbon is added as a powder having a particle size distribution with a diameter d50 in the range of 2 pm to 25 pm, preferably in the range of 3 pm to 20 pm, and more preferably in the range of 3 pm to 7 pm, and even more preferably in the range of 5 pm to 7 pm.
The d50 value is determined with standard procedures.
In one embodiment, the pH of the HMO-containing stream is adjusted before the active carbon treatment is carried out to improve the reduction of colorizing agents and/or proteins during step b) of the method according to the invention. Preferably, the pH is adjusted to 5.5, more preferably to 5.0 and even more preferably to 4.5 by the addition of a suitable acid.
The optional active carbon treatment may follow the cation exchange step b) and is preferably conducted before step c) of the method according to the invention. In case an optional second nanofiltration step is performed as described above, the optional active carbon treatment can be performed before or after said optional second nanofiltration step, but preferably before.
In another embodiment, the method according to the invention further comprises a step, wherein the
HMO-containing solution, preferably after concentration according to step c), is sterile filtered and/or subjected to endotoxin removal, preferably by filtration of the purified solution through a 3 kDa filter. Said optional step is preferably conducted after step b) and any of the aforementioned optional purification steps and before the drying step according to step d). — According to one embodiment, both the active charcoal treatment and the sterile filtration step, disclosed above, are part of the method of the invention.
Particular embodiments of the invention
In preferred embodiments, the method according to the present invention does not include a basic anion exchange step.
In preferred embodiments, the method according to the present invention does not include an electrodialysis step.
In preferred embodiments, the method according to the present invention does not include an electrodialysis step and a basic anion exchange step. 23
DK 2021 00635 A1
In a preferred embodiment, the method according to the invention comprises or consists of the following steps (in consecutive order): i. separating the fermentation broth to form a separated HMO-containing stream and a biomass waste stream by ultrafiltration; ii. purifying the separated HMO-containing stream by combined nanofiltration and diafiltration, wherein the nanofiltration membrane is preferably in the range of 500-3000
Da MWCO; ii. purifying the HMO-containing stream by a strongly acidic cation exchange resin; iv. purifying the separated HMO-containing stream by an active carbon treatment; v. optionally, purifying the HMO-containing stream by a second nanofiltration step, preferably combined with diafiltration, wherein the nanofiltration membrane is preferably in the range of 150-300 Da MWCO; vi. concentrating the purified HMO-containing stream by evaporation; and vii. — spray drying the purified HMO-containing stream to obtain a solidified neutral HMO. — In one aspect, the method according to the invention, including the preferred and more preferred embodiments disclosed above, comprises at least one nanofiltration step wherein the nanofiltration membrane has a molecular weight cut-off (MWCO) of 500-3000 Da, the active (top) layer of the membrane is composed of polyamide, the membrane has a MgSO rejection factor of about 50-90 % and a NaCl rejection factor of not more than 50 %, and the nanofiltration step is performed so that the pH is set below 5.0, preferably below 4.5, advantageously below 4.0, but preferably not less than 3.0, ensuring the retention of the neutral HMO to be purified and allowing the mono-and divalent salts to pass and accumulate in the permeate, and also allowing at least a part of lactose to pass and accumulate in the permeate. 3. Neutral human milk oligosaccharide produced by the method according to the invention
In another aspect, the invention relates to a neutral human milk oligosaccharide obtained by the method according to the invention.
The neutral HMO recovered and purified according to the method described in this specification can be amorphous or crystalline, preferably amorphous. 24
DK 2021 00635 A1
In a preferred embodiment, the purity of the neutral HMO on a dry basis is greater than 80 wt.% for a single neutral HMO based on dry matter; or for mixtures of HMOs, greater than 70% purity based on dry matter, for the combination. More preferably, the purity of a single neutral HMO is greater than 90 wt.%.
In a preferred embodiment, the neutral HMO has at least one of the following characteristics (by weight): <2% lactulose, < 3% fucose, < 1% galactose, or < 3% glucose.
In an embodiment, the neutral HMO has a fines fraction (less than or equal to 10 um), of less than 10%, preferably less than 5%, more preferably less than 1%, most preferably less than 0.1%. The neutral HMO also preferably has an average particle size (d50), of greater than 100 um, more preferably greater than 150 um, even more preferably greater than 200 um.
The neutral HMO produced by the method according to the invention, demonstrates good flowability. Preferably, the HMO has a Carr index of less than 30, where the Carr index (C) is determined by the formula C = 100(1-p B/ p T), where p B is the freely settled bulk density of the powder, and p T is the tapped bulk density of the powder after "tapping down”. For free-flowing solids, the values bulk and tapped density would be similar, so the value is small. For poorer flowing solids, the differences between these values would be larger, so that the Carr index would be larger.
In a preferred embodiment, the neutral HMO has a water content of less than 15 wt.%, less than 10 wt.%, less than 9 wt.%, less than 8 wt.%, less than 7 wt.%, or less than 6 wt.%. In order to optimize product recovery, preferably, the neutral HMO has a pH greater than 3.0 in at least 5% solution.
Typically, this is achieved by adjusting the pH of the HMO-containing stream to greater than 3.0 prior to the drying step. Preferably, the neutral HMO has a pH of from 4 to 7, more preferably from 4.5 to 5.5.
In a preferred embodiment, the neutral HMO is preferably 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-fucopentaose II, lacto-N-fucopentaose III, lacto-N- fucopentaose V, lacto-N-neofucopentaose V (alternative name: lacto-N-fucopentaose VI), lacto-N- difucohexaose I, lacto-N-difucohexaose II, lacto-N-difucohexaose III, 6'-galactosyllactose, 3'- galactosyllactose, lacto-N-hexaose, lacto-N-neohexaose, or any mixture thereof. Preferably, the
HMO is 2'-fucosyllactose, LNT or LNnT. 25
DK 2021 00635 A1
In an embodiment, the neutral HMO obtained by the method according to the invention, is incorporated into a food product (e.g., human or pet food), dietary supplement or medicine product.
In some embodiments, the neutral HMO is incorporated into a human baby food (e.g., infant formula). Infant formula is generally a manufactured food for feeding to infants as a complete or partial substitute for human breast milk. In some embodiments, infant formula is sold as a powder and prepared for bottle- or cup-feeding to an infant by mixing with water. The composition of infant formula is typically designed to roughly mimic human breast milk. In some embodiments, a neutral
HMO purified by a method in this specification is included in infant formula to provide nutritional benefits similar to those provided by one or more neutral HMOs in human breast milk. In some embodiments, the H neutral MO is mixed with one or more ingredients of the infant formula.
Examples of infant formula ingredients include skimmed milk, carbohydrate sources (e.g., lactose), protein sources (e.g., whey protein concentrate and casein), fat sources (e.g., vegetable oils - such as palm, high oleic safflower oil, rapeseed, coconut and/or sunflower oil; and fish oils), vitamins (such as vitamins A, B , B2, C and D), minerals (such as potassium citrate, calcium citrate, magnesium chloride, sodium chloride, sodium citrate and calcium phosphate).
Hence, another aspect of the invention relates to a neutral human milk oligosaccharide obtained by the method according to the invention for use in medicine.
Hence, another aspect of the invention relates to the use of a neutral human milk oligosaccharide obtained by the method according to the invention for food and/or feed applications.
Hence, another aspect of the invention relates to a food or cosmetic product comprising the neutral human milk oligosaccharide obtained by the method according to the invention.
Example 1
General: Carbohydrate and impurity content were quantified by calibrated HPLC and/or HPAEC.
Soluble proteins were quantified by Bradford assay. Colour was quantified by UV-absorption measurement at 400 nm in a cuvette with 1 cm path. The colour index CI 400 is calculated according to the following formula: CI 400 = 1000%Abs 400/Brix.
Fermentation: 2'-FL was produced by microbial fermentation using a genetically modified E. coli strain comprising a recombinant gene encoding an a-1,2-fucosyltransferase. The fermentation was performed by culturing the strain in the presence of exogenously added lactose and a suitable 26
DK 2021 00635 A1 carbon source, thereby producing 2’-FL which was accompanied with DFL and unreacted lactose as major carbohydrate impurities in the fermentation broth.
Purification: 1. UF/DF: The obtained broth was acidified to pH=3.8 with sulphuric acid followed by ultrafiltration-diafiltration through 15 kDa ceramic membrane elements at T= +60 °C in industrial continuous UF system with the DF water flow of approximately 2-times the feed flow and UF retentate flow of ca. half of the feed flow. 2. NF/DF: The UF permeate stream containing the product (Brix ca. 5) was immediately processed by loose nanofiltration with diafiltration in industrial continuous NF system equipped with Trisep UA60 membrane (piperazine-amide, MWCO 1000-3500 Da) elements (TMP=30 bar, T= 15 °C) and with a DF water flow approximately twice as the feed (UF permeate) flow. 3. Strong cation exchange resin treatment: A sample of the obtained NF retentate (4.7 kg, Brix 23.8, conductivity: 2.20 mS/cm, pH= 3.9, CI 400: 134) was passed through a column packed with 800 ml of Dowex-88 resin in H”-form (capacity 1.8 eq/l) at 2 bed volumes per hour flow rate followed by elution with water (730 ml). 400 ml fractions were collected. Each fraction was titrated with 1M NaOH to pH=4.5 and analysed for sugar, colour and protein content. The pH- adjusted fractions #2-13 were combined to give 5.4 kg of yellow solution (Brix 20.2, conductivity: 3.48 mS/cm, pH= 4.55, CI 400: 67.3). 4. Active charcoal decolorization: Part of the obtained solution (3.5 kg) was passed through granulated active charcoal CPG LF (75 g, 150 ml) packed in a column (ID=16 mm) at +60 °C and at 2 bed volumes per hour flow rate followed by water elution (300 ml). 150 ml fractions were collected. Fractions #2-24 were combined to give 3.5 kg of nearly colourless solution (Brix 19.7, conductivity 3.42 mS/cm, pH= 5.19, CI 400: 1.23). 5. NF/DF: The obtained solution (3.4 kg) was subjected to constant volume diafiltration in MMS
SW18 membrane filtration system equipped with 1812-size spiral wound Trisep UA60 membrane under the following conditions: cross-flow= 400 1/h, TMP= 30 bar, T= 30-35 °C and
DF water flow 4.0 I/h. First, the DF was performed at a pH of around 5 (10 1), then at 3.8 (extra 10 1). Finally, the retentate was further concentrated at TMP= 39 bar followed by a pH adjustment to 4.5 with 4 % NaOH solution and pumped out from the system to give 1.8 kg of a final solution (Brix 30.6, conductivity: 0.216 mS/cm, pH= 4.49, CI 400: 1.35). 27
DK 2021 00635 A1 6. Microfiltration and freeze-drying: The above obtained NF retentate was passed through a
PES 0.2 um micro-filter to give 1.6 kg of solution (Brix 30.6) and freeze-dried to give 492 g of a white powder.
Example 2: comparison of MgSO, and Na2SO rejection 2.01 of 0.2 % MgSO solution were loaded into a MMS SW18 system equipped with 1812-size spiral wound Trisep UA60 element (membrane area 0.23 m?). The system was run at 400 1/h cross- flow with permeate circulating back to the feed tank. It was equilibrated for at least 5 min or until constant conductivity in the permeate under each condition. The pH was adjusted by adding a small amount of 25 % H>SO4 solution. The conductivity of the permeate and the retentate are disclosed in the table below.
MgSO4 rejection vs pH 25%
TMP | T |HSO4| pH | Flow | Flux oe oo Rejection fe] 2 (bar) | (°C) added (retentate) | (1/h) | (I/m”h) (mS/cm) (mS/cm) factor 10 [247] 0 | 606 [211 [ 916 | 2100 0.785 62.61 % 2.100 0.587 72.05 % 2.130 0.624 70.70 % 10 [251] 80 | 393 [205 [ 892 | 2170 0.520 76.04 % 2.260 0.538 76.19 %
The same experiment was performed with 0.2 % Na&>SO4 solution.
Na2SO4 rejection vs pH
TMP T 25% pH Flow | Flux Retentate Permeate Rejection (bar) | (°C) | H2SOs | (retentate) | (I/h) | (/m?h) | conductivity | conductivity factor added (mS/cm) (mS/cm) 1 10 [220] 0 | 570 [167 | 726 | 2840 0.0755 97.34 % 2.840 0.1706 | 93.99% 2.840 0.824 70.99 % 2.850 1.706 40.14 % 2.880 1.939 32.67 % 2.970 2.090 28.67 % 3.060 2.230 27.12 % 3.280 2.500 23.78 % 1310 3.730 2.870 23.06 % 28
DK 2021 00635 A1
It was demonstrated that the sodium salt rejection with divalent counter-ion such as sulfate is strongly pH dependent in case of NF with polyamide membrane. Because a substantial amount of sodium salt is present in the collected fractions after the strong cation exchange resin treatment due to neutralization with NaOH solution (see step #3 in Example 1), these salts can be effectively removed in a second NF/DF (see step #5 in Example 1) when the DF is conducted at a pH of less than 4.5, advantageously less than 4.0, resulting in a practically salt-free solution (as assessed from conductivity). In this regard, no basic anionic resins are necessary to use to obtain a salt-free solution. 29
Claims (15)
1. A method for recovery and purification of a neutral human milk oligosaccharide (HMO) from a fermentation broth, comprising the steps of:
a. separating the fermentation broth to form a separated HMO-containing stream and a biomass waste stream;
b. purifying the separated HMO-containing stream by nanofiltration;
c. purifying the HMO-containing stream with an acidic cation exchange resin;
d. concentrating the purified HMO-containing stream; and e. drying the purified HMO-containing stream to obtain a solidified neutral HMO.
2. The method according to claim 1, wherein the purity of the HMO in the fermentation broth is <70%, preferably <60%, more preferably <50%, most preferably <40%.
3. The method according to claim 1 or claim 2, wherein the HMO is 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-fucopentaose II, lacto- N-fucopentaose III, lacto-N-fucopentaose V, lacto-N-fucopentaose VI, lacto-N- difucohexaose I, lacto-/V-difucohexaose II, lacto-N-difucohexaose III, 6'-galactosyllactose, 3'-galactosyllactose, lacto-N-hexaose, lacto-N-neohexaose, or any mixture thereof.
4. The method according to any of the preceding claims, wherein step a) comprises at least one of ultrafiltration, microfiltration, and centrifugation, preferably consists of ultrafiltration.
5. The method according to any of the preceding claims, wherein the nanofiltration membrane in step b) has a molecular weight cut-off (MWCO) of 500-3000 Da, and the active layer of the membrane is preferably composed of polyamide.
6. The method according to claim 5, wherein step b) is performed so that the pH is set below
5.0, preferably below 4.5, advantageously below 4.0, but preferably not less than 3.0.
7. The method according to any of the preceding claims, wherein the acidic cation exchange resin in step c) is a strongly acidic cation exchange resin, preferably a styrene- divinylbenzene cation exchange resin.
DK 2021 00635 A1
8. The method according to any of the preceding claims, wherein the method comprises further purification of the HMO-containing stream by an active carbon treatment.
9. The method according to any of the preceding claims, wherein the method further comprises at least one diafiltration step, preferably said at least one diafiltration step is combined with nanofiltration in step b).
10. The method according to any of the preceding claims, wherein step b) results in a purified solution containing the HMO at a purity of >50%, preferably >60%, more preferably >70%.
11. The method according to any of the preceding claims, wherein step c) results in a purified solution containing the HMO at a purity of >80%, preferably >85%, more preferably >90%.
12. The method according to any of the preceding claims, wherein step c) results in a purified solution that is free of proteins and/or recombinant genetic material.
13. The method according to any of the preceding claims, wherein step d) comprises evaporation and/or reverse-osmosis filtration, preferably consists of evaporation.
14. The method according to any of the preceding claims, wherein step e) comprises, preferably consists of, spray-drying to obtain a solidified neutral HMO.
15. The method according to claim 1, wherein the method consists of steps a)-e) and/or is performed in the consecutive order a)-e). 31
SEARCH REPORT - PATENT Application No. PA 2021 00635
1.U] Certain claims were found unsearchable (See Box No. I).
2.[] Unity of invention is lacking prior to search (See Box No. ID.
A. CLASSIFICATION OF SUBJECT MATTER CO7H 1/08 (2006.01), BOID 61/02 (2006.01), BO1D 61/14 (2006.01), BO1D 61/58 (2006.01), CO7H 3/06 (2006.01), C12P 19/00 (2006.01) According to International Patent Classification (IPC)
B. FIELDS SEARCHED PCT-minimum documentation searched (classification system followed by classification symbols) IPC/CPC: A23C, A23L, BO1D, CO7H, C12P Documentation searched other than minimum documentation to the extent that such documents are included in the fields searched Electronic database consulted during the search (name of database and, where practicable, search terms used) EPODOC, WPI, English Full text databases
C. DOCUMENTS CONSIDERED TO BE RELEVANT Category* Citation of document, with indication, where appropriate, of the relevant passages Relevant for claim No. X WO 2019/226431 A1 (DSM IP ASSETS B.V.) 28 November 2019, see sections 1-15
[0023]-[0031], [0045]-[0051], [0071], [0077], figure 1 and claims. X WO 2025064679 Al (GLYCOM A/S) 8 April 2021, see page 21, line 25 — page 1-15 22, line 11, page 23, line 9-24, line page 28, line 11-30 and page 38, line 21 — page 39, line 16. A WO 2019/0631757 Al (FRIESLANDCAMPINA NEDERLAND B.V.) 4 April 1-15 2019, see page 10, line 19 — page 12, line 20 and claims. A EP 3741770 A1 JENNEWEIN BIOTECHNOLOGIE GMBH) 25 November 1-15 2020, see sections [0042], [0058]-[0063], [0078], [0092], [0093], [0097]-[0099], claims and figure 1. X Further documents are sted in the continuation of Box C. + Special categories of cited documents: "pr Document published prior to the filing date but later than the "A" — Document defining the general state of the art which is not priority date claimed. considered to be of particular relevance. "TT" Document not in conflict with the application but cited to nym Document cited in the application. understand the principle or theory underlying the invention. "E" Earlier application or patent but published on or after the filing date. | x Document of particular relevance; the claimed invention cannot be . . Co considered novel or cannot be considered to involve an inventive "" Document which may throw doubt on priority claim(s) or which is step when the document is taken alone cited to establish the publication date of another citation or other won . Co . . special reason (as specified). Y Document of particular relevance; the claimed invention cannot be . . oo considered to involve an inventive step when the document is "O" Document referring to an oral disclosure, use, exhibition or other combined with one or more other such documents, such means. combination being obvious to a person skilled in the art. "&" Document member of the same patent family. Danish Patent and Trademark Office Date of completion of the search report Helgeshøj Allé 81 17 December 2021 DK-2630 Taastrup Denmark Authorized officer Verner Holm
Tel.: +45 4350 8000
Tel.: +45 43 50 83 54 October 2021 1/4
Application No. SEARCH REPORT - PATENT PP ono PA 2021 00635 C (Continuation). DOCUMENTS CONSIDERED TO BE RELEVANT Citation of document, with indication, where appropriate, of the relevant passages Relevant for claim No. A US 2616/0237104 A1 (JENNEWEIN et al.) 18 August 2016, see sections [0137], 1-15
[0138] and claims. A WO 2015/106943 A1 (JENNEWEIN BIOTECHNOLOGIE GMBH) 23 July 1-15 2015, see example 1, claims and figure 1. A WO 2019/110800 Al (JENNEWEIN BIOTECHNOLOGIE GMBH) 13 June 1-15 2019, see example 2 and claims. October 2021 2/4
SEARCH REPORT - PATENT Application No. PA 2021 00635 Box No. I Observations where certain claims were found unsearchable This search report has not been established in respect of certain claims for the following reasons:
1.[] Claims Nos.: because they relate to subject matter not required to be searched, namely:
2. U] Claims Nos.: because they relate to parts of the patent application that do not comply with the prescribed requirements to such an extent that no meaningful search can be carried out, specifically:
3. I Claims Nos. because of other matters, Box No. II Observations where unity of invention is lacking prior to the search The Danish Patent and Trademark Office found multiple inventions in this patent application, as follows: Application No. SEARCH REPORT - PATENT PA 2021 00635 October 2021 3/4
SUPPLEMENTAL BOX Continuation of Box [.] October 2021 4/4
Priority Applications (4)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DKPA202100635A DK202100635A1 (en) | 2021-06-15 | 2021-06-15 | Separation of neutral human milk oligosaccharides from a fermentation broth |
| BE20225468A BE1029437B1 (en) | 2021-06-15 | 2022-06-14 | SEPARATION OF BREAST MILK OLIGOSACCHARIDES FROM A FERMENTATION BROTH |
| PCT/EP2022/066133 WO2022263426A1 (en) | 2021-06-15 | 2022-06-14 | Separation of human milk oligosaccharides from a fermentation broth |
| EP22734562.6A EP4355465A1 (en) | 2021-06-15 | 2022-06-14 | Separation of human milk oligosaccharides from a fermentation broth |
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| DKPA202100635A DK202100635A1 (en) | 2021-06-15 | 2021-06-15 | Separation of neutral human milk oligosaccharides from a fermentation broth |
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| DK202100635A1 true DK202100635A1 (en) | 2023-06-06 |
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| DKPA202100635A DK202100635A1 (en) | 2021-06-15 | 2021-06-15 | Separation of neutral human milk oligosaccharides from a fermentation broth |
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| DK (1) | DK202100635A1 (en) |
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