DK181291B1 - Separation of neutral human milk oligosaccharides from a fermentation broth - Google Patents
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Abstract
The invention relates to a method for recovery and purification of (neutral) human milk oligosaccharides (HMOs) 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 separated HMO-containing stream, concentrating the purified HMO-containing stream, and drying the purified HMO-containing stream to obtain solidified HMO. Moreover, the invention also concerns a human milk oligosaccharide obtained by the inventive method, as well as its use in food, feed, and medical application.
Description
DK 181291 B1 1
SEPARATION OF NEUTRAL HUMAN MILK OLIGOSACCHARIDES FROM A
FERMENTATION BROTH
The present invention relates to the separation and isolation of 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 (GalB1-4Glc) 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 resulting in a complex mixture of oligosaccharides in the fermentation broth.
Further, a fermentation broth inevitably contains a wide range of non-oligosaccharide substances such as cells, cell fragments, proteins, protein fragments, DNA, DNA fragments, endotoxins, caramelized by-products, minerals, salts, or other charged molecules.
DK 181291 B1 2
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), Baumgartner 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 A1 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. 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).
EP 3741770 A1 claims a method for the purification of an oligosaccharide of interest, preferably an
HMO, from a fermentation broth, the method comprises: - providing a fermentation broth which contains the oligosaccharide of interest, biomass, microbial — cells and carbohydrates other than the oligosaccharide of interest; - removing the microbial cells from the fermentation broth, thereby providing a process stream; - subjecting the process stream to a first filtration step using a nanofiltration membrane, thereby providing a filtrate which contains the oligosaccharide of interest; - subjecting the filtrate to a second filtration step using a nanofiltration membrane, thereby providing a retentate which contains the oligosaccharide of interest; and
DK 181291 B1 3 - removing salts from the process stream using electrodialysis thereby providing a purified preparation of the oligosaccharide of interest.
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, 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, consisting of 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;
DK 181291 B1 4 c. concentrating the purified HMO-containing stream; and d. drying the purified HMO-containing stream to obtain a solidified HMO, optionally with an active carbon treatment step, wherein the nanofiltration membrane in step b) has a molecular weight cut-off (MWCO) of 500-3000 Da, the active layer of the membrane is composed of polyamide, and its MgSO4 rejection is about 50-90 %.
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 HMO. After each step of the purification method, one or more of the components of the fermentation product is removed, resulting in a more neutral purified HMO.
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.), an 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 B- lacto-N-biosyl units, and which core structures can be substituted by an a-L-fucopyranosyl and/or an o-N-acetyl-neuraminyl (sialyl) moiety. In this regard, the non-acidic (or neutral) HMOs are
DK 181291 B1 devoid of a sialyl residue, and the acidic HMOs have at least one sialyl residue in their structure.
The non-acidic (or neutral) HMOs can be fucosylated or non-fucosylated. Examples of such neutral non-fucosylated HMOs include lacto-N-triose IT (LNTri, GleNAc{BI-3¥Gal{P1-4)Gle), lacto-N- tetraose (LNT), lacto-N-neotetraose (LNnT), lacto-N-neohexaose (I NaH}, para-lacto-N- 5 neohexaose (pLNnH), para-lacto-N-hexaose (pL.NH) and lacto-N-hexaose (LNH). Examples of neutral fucosylated HMOs include 2'-fucosyllactose (2”-FL.), lacto-N-fucopentaose I (LNFP-D), lacto-N-difucohexaose I (LNDFH-T), 3-fucosyllactose (3-FL), difucosyllactose (DFL), lacto-N- fucopentaose IT (LNFP-ID), lacto-N-fucopentaose HI (LNFP-IID), lacto-N-difucohexaose III {LNDFH-I}, fucosyl-lacto-N-bexaose II (FLNH-ID), lacto-N-fucopentaøse V (ENFP-V), lacto-N- difucohexaose II (LNDFH-ID), fucosyl-lacto-N-hexaose I (FLNH-D), fucosyl-para-lacto-N-hexaose I (FOLNH-D, fucosyl-para-lacto-N-neohexaose II (F-pl.NnH IT) and fucosyl-lacto-N-nechexaose {FLNnH). Examples of acidic HMOs melude 3'-siafyllactose (3'-SL), 6 -sialyllactose (6'-SL), 3- fucosyl-3 -sialyllactose (FSL). LST a, fucosyl-LST a (FEST a), LST b, fucosyl-LST b (FLST b},
LST c, fucosyl-LST ¢ (FLST 0), stalyl-LNH (SLNH), sialyl-lacto-N-hexaose (SLNH), sialyl-lacto- 13 N-neohexaose I (SLNH-I), sialfyl-lacto-N-neohexaose II (SLNH-ID and disialyl-lacto-N-tetraose (DSLNT).
The term "sialyl” or “sialyl moiety” means the glycosyl residue of sialic acid (N-acetyl-neuraminic acid, NeuSAc), preferably linked with ¢-linkage:
HO pr COOH
HO ud 4
The tern “fucosyl” means an L-fucopyranosyl group, preferably linked with a-mterglycosidie
Linkage: ane
HC oF os -
OH
"N-acetyl-slucosanunvi” means an N-aceiyl-D-amino-2-deoxy-D-elucopyranosyl (GicNAc) group, preferably linked with B-linkage:
DK 181291 B1 8
OH ga %
HO- \
NHAC
SN-acetyl-lactosamimyl” means the glycosyl residue of N-acetyl-lactosamine {LacNAc, Galp1-
AGIeNAcp), preferably linked with B-linkage: ox OH
OH
HO... 3 må % oH HO \ —
NHAC
S Furthermore, the term "lacto-N-biosy]” means the glycosyl residue of lacio-N-biose (LNB, GalpB1- 3GIeNAcp), preferably linked with B-linkage:
OH OH OH am
HO G
OH NHAc
The term "neutral human milk oligosaccharide” means a non-sialviated (therefore neutral) complex carbohydrate found in human breast milk (Urasluma et al.: AX oligosaccharides, Nova Biomedical
Books, 2011; Chen ddv. Carbohydr. Chem. Biochem. 72, 113 (20155) comprisine a core structure being a lactose unit at the reducing end that is a) substituted with one or two &-L-fucopyranosyl moieties, b) substituted with a galactosyl residue, or c) elongated, via ifs 3'-OH group, by an N- acetylglucosamine, a lacto-N-biose (GalBI-3GIcNAc) or an N-acetyllactosamine (Gal 1-4GIcNAc) moiety. The N-acetyllactosamine containing derivatives can be further substituted with N- — sctyllactosanune and/or lacto-N-biose (lacto-N-biose 1s always a non-reducing terminal). The N- acetyHactosamine and the lacto-N-biose contamine derivatives can optionally be substituted by one or more 4-L-fucopyranosyl moieties. Examples of neutral trisaccharide HMOs melude 2°-0- fucosyllactose (2°-FL., Fucoal-2Galf1-4G10), 3-O-fucosyllactose {3-FL, Galft1-4(Fucal-3)Gle) or lacto-N-triose IT {GleNAcB1-3GalB1-4Gle); examples of neutral tefrasaccharide HMOs include 273-di-O-fucosyllactose (DFL. Fucai-2GalB1-4(Fucal-3)Gc), lacto-N-tetraose (LNT, Galfil- 3GHKNAcCB1-30a181-4G1c) or lacto-N-neotetraose (LNT, Gal 1-4GIcNAcf1-3GalB1-4CGH10); examples of neutral pentasacchande HMOs include lacto-N-fucopentaose I (LNFP I, Fucal- 2Galfl-3GIeNAcPI-3Galf1-4Gle), lacto-N-fucopentaose II (LNFP II, Gal 1-3(Fucal-
AGINAcf1-3Ga1B1-4GH0), lacto-N-fucopentacse TTT (LNFP IH, Galfil-4(Fuca1-3)GRN AcB1-
DK 181291 B1 7 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, Fuca1-2GalB1- 3(Fucal-4)GlcNAcP1-3Galp1-4Glc), lacto-N-difucohexaose II (LNDFH II, GalB1-3(Fucal- 4)GIcNAcB1-3GalpP1-4(Fuca1-3)GIc), lacto-N-difucohexaose III (LNDFH III, Galp1-4(Fucal- 3)GIcNAcB1-3GalpP1-4(Fuca1-3)GIc), lacto-N-hexaose (LNH, GalB1-3GIcNAcB1-3(Galp1- 4GIcNAcP1-6)GalB1-4Glc), para-lacto-N-hexaose (pLNH, Galp1-3GIcNAcB1-3Galp1- 4GlIcNAcfp1-3GalB1-4G1c), 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 a 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 providing a neutral HMO, 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 broth or enzymatic process mixture.
The term “HMO-containing stream” preferably 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
DK 181291 B1 8 metabolites and debris that could interfere with the eventual purification of the 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 “molecular weight cut-off or MWCO” refers to membrane descriptor provided by a manufacturer of the membrane in the list of membrane specifications as a way to characterize the membrane pore size. According to the definition, MWCO implies rejection factor of 90 % under given conditions (such as temperature, cross-flow, trans-membrane pressure TMP, etc.) for the one of the standard solutes used by the manufacturer with molecular weight equal to MWCO. It is well - — known to the skilled person that MWCO is one of many membrane descriptors which could provide only a rough estimation of particular solute rejection behaviour.
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 c) of the method according to the invention refers to the removal of liquid, mostly water, thus resulting in a higher concentration of a neutral HMO in the purified neutral HMO-containing product stream. 2. Method for the purification of 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, consisting of the steps of’
DK 181291 B1 9 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. concentrating the purified HMO-containing stream; and d. drying the purified HMO-containing stream to obtain a solidified HMO, optionally with an active carbon treatment step, wherein the nanofiltration membrane in step b) has a molecular weight cut-off (MWCO) of 500-3000 Da, the active layer of the membrane is composed of polyamide, and its MgSO4 rejection is about 50-90 %.
In a preferred embodiment. the method steps a)-d) are performed in the consecutive order a)-d) as — given above.
The fermentation broth:
In an embodiment, the neutral human milk oligosaccharide 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. The microbial organism is a genetically modified bacterium or yeast such, as an E. coli strain, a Bacillus strain, 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. 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,
Zygosaccharomyces bailii; and the Bacillus is Bacillus amyloliquefaciens, Bacillus licheniformis or
Bacillus subtilis.
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 weight%, preferably <60 weight%, more preferably <50 weight%, most preferably <40 weight%.
DK 181291 B1 10
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 neutral HMO in the fermentation broth is a single HMO.
In an embodiment, the HMO in the fermentation broth is a mixture of various individual HMOs. — In an embodiment, the HMO is a mixture of two individual HMOs. In another embodiment, the
HMO is a mixture of three individual HMOs. In another embodiment, the HMO is a mixture of four individual HMOs. In another embodiment, the HMO is a mixture of five individual HMOs.
In an embodiment, the neutral 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 neutral 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 neutral 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 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 microfiltration. The microfiltration 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 microfiltration membrane in the permeate. This microfiltration permeate is an aqueous solution containing the neutral human milk oligosaccharide — also referred to as the HMO-containing stream, whereas the microfiltration retentate comprises the
DK 181291 B1 11 biomass waste stream. Any conventional microfiltration membrane can be used having a pore size ranging from 0.1 to 10 um. Step a) of the method according to the invention may comprise more than one microfiltration step using membranes with different pore size, e.g., applying two microfiltration separations, wherein the first membrane has a bigger pore size 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 the neutral human milk oligosaccharides of interest.
In another 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. The 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) higher than 10 kDa and lower than 500 kDa, such as 10-50 kDa, 50-100 kDa, 100-250 kDa, 300- 400 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 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 — stream, while the remaining material, i.e., the “biomass waste stream” can be separated out. By
DK 181291 B1 12 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.
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.
DK 181291 B1 13
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 L. In some embodiments, the working volume can be less than 1, 5, 10, 15, 20, 50, 100, 300, or SOOL. In some embodiments, the working volume can be greater than 1, 5, 10, 15, 20, 50, 100, 300, or SOOL.
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 L/hr. In some embodiments, the feed flow rate can be greater than 100, 500, 1000, 1500, 2000, 5000, 10000, 20000, 40000, or 100000 L/hr. In some embodiments, the feed flow rate can be less than 100, 500, 1000, 1500, 2000, 5000, 10000, 20000, 40000, or 100000 L/hr.
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.
DK 181291 B1 14
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 microfiltration as defined above, ultrafiltration as defined above or centrifugation, or via a — combination of: ultrafiltration and centrifugation, microfiltration and ultrafiltration, microfiltration and centrifugation, microfiltration and 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.
In a preferred embodiment, the yield of the desired neutral HMO in the permeate/supernatant after the microfiltration, ultrafiltration or centrifugation step, or any combination thereof, 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%.
Before the ultrafiltration and/or microfiltration 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 and/or microfiltration 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.
DK 181291 B1 15
Purifying the separated neutral HMO-containing stream in step b) by nanofiltration:
In step b) of the method according to the invention, the neutral HMO-containing stream is purified 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, i.e., the neutral HMO-containing steam, is the NF retentate. The nanofiltration membrane thus has a MWCO or a pore size that ensures the retention of the neutral human milk oligosaccharide of interest, i.e., the MWCO of the nanofiltration membrane is adjusted accordingly.
The nanofiltration membrane has a molecular weight cut-off (MWCO) of 500-3000 Da and the active (top) layer of the membrane is 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 MgSQy, 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 MgSO, 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 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. Other examples of suitable NF membranes include Synder NFG (600-800 Da), Synder NDX (500-700
Da), and TriSep” XN-45 (500 Da).
Preferably, the yield of the desired neutral HMO in the retentate after a nanofiltration step is greater than 50%, greater than 60%, greater than 70%, greater than 80%, greater than 90%, greater than
DK 181291 B1 16 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%.
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 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.
In an embodiment, a second nanofiltration step is carried out in the method according to the invention. In said second nanofiltration step, the nanofiltration membrane is either a “loose” NF membrane or a “tight” NF membrane.
Likewise, a second diafiltration can be performed in the method according to the invention if combined with the second nanofiltration step. This second NF/DF step, when “loose” NF membrane is applied, 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 an embodiment, the purified solution obtained after step b) of the method according to the invention, contains the neutral HMO at a purity of >80 weight%, preferably >85 weight%, more preferably >90 weight%.
In an embodiment, the purified solution obtained after step b) of the method according to the invention is free proteins and/or recombinant genetic material.
DK 181291 B1 17
Concentrating the purified neutral 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.
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
DK 181291 B1 18 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 preferably 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).
Drying the purified neutral HMO-containing stream to obtain solidified 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, 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
DK 181291 B1 19 120 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.
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 step
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
DK 181291 B1 20 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.
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 wt%, 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
DK 181291 B1 21 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 at elevated temperature substantially reduces the total number of viable microorganisms (total 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 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.
According to one embodiment, the active charcoal treatment, disclosed above, is part of the method of the invention.
Particular embodiments of the invention
In one embodiment, the method according to the invention consists of the following steps (in consecutive order):
DK 181291 B1 22 i. separating the fermentation broth to form a separated neutral HMO-containing stream and a biomass waste stream by ultrafiltration; ii. purifying the separated neutral HMO-containing stream by combined nanofiltration and diafiltration, wherein the nanofiltration membrane has a molecular weight cut-off (MWCO) of 500-3000 Da, the active layer of the membrane is composed of polyamide, and its MgSOy rejection is about 50-90 %; iii. purifying the neutral HMO-containing stream by active carbon treatment; iv. purifying the separated neutral 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; v. concentrating the purified neutral HMO-containing stream by evaporation; and vi. — spray drying the purified neutral HMO-containing stream to obtain 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
One embodiment relates to a neutral human milk oligosaccharide obtained by the method according tothe invention.
The neutral HMO recovered and purified according to the method described in this specification can be amorphous or crystalline, preferably amorphous.
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
DK 181291 B1 23 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 (d 50), 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 neutral HMO has a Carr index of less than 30, where the Carr index (C) is determined by the formula C = 100(1-ps/pr), where pg is the freely settled bulk density of the powder, and pr is the tapped bulk density of the powder after "tapping down”. For free-flowing solids, the values of 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%, less than 10 wt%, less than 9% less than 8 wt%, less than 7 wt%, and less than 6 wt%. In order to optimize product recovery, preferably, the HMO has a pH greater than 3.0 in at least 5% solution, more preferably the neutral HMO has a pH greater than 4.0. Typically, this is achieved by adjusting the — pH of the neutral 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 an embodiment, the HMO obtained by the method according to the invention is a dried neutral
HMO, preferably having a water content of less than 6 wt%.
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.
DK 181291 B1 24
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 be roughly mimic human breast milk. In some embodiments, a neutral HMO purified by a process 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 neutral HMO 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 embodiment relates to a neutral human milk oligosaccharide obtained by the method according to the invention for use in medicine.
Hence, another embodiment 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, embodiment relates to a food or cosmetic product comprising the neutral human milk oligosaccharide obtained by the method according to the invention for use in medicine.
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: LNT was produced by microbial fermentation using a genetically modified E.coli strain. The fermentation was performed by culturing the strain in the presence of exogenously added lactose and a suitable carbon source, thereby producing LNT which was accompanied with
DK 181291 B1 25 trisaccharide LNT-II, hexasaccharide p-LNH-2 and unreacted lactose as major carbohydrate impurities in the fermentation broth. In the end of fermentation, the pH was adjusted to 4 by adding 25% sulfuric acid.
Broth pre-treatment and biomass removal by centrifugation:
A portion of the obtained broth (4.3 kg, pH 4.45 after storage) was diluted to total of 11.1 kg with distilled water followed by heating to 80 °C within 40 min and allowed to cool immediately to ambient temperature. The pH was adjusted to 3.2 and the broth was centrifuged. The obtained supernatant (9.0 kg) was carefully pumped out to separate from the sediment (2130 g).
The obtained clarified supernatant had the following parameters: Brix=8.1, conductivity 7.50 mS/cm, pH 3.27.
Membrane filtration:
Part of the obtained supernatant (4.5 kg) was subjected to membrane filtration in a MMS SW 18 system equipped with Synder-XT2B-1812F membrane element (UF PES membrane, MWCO 1000
Da, area 0.32 m?) at TMP=3.0 bar, T=23-25 °C and cross-flow of 400 L/h. 3.5 kg of membrane permeate was collected in 35 min. Then the remaining retentate (Brix 9.7, conductivity 7.28 mS/cm) was diafiltrated by continuous water addition (3.0 L at 5.4 L/h flow rate) under the same conditions to give a final membrane permeate (6.9 kg, Brix 5.4) and a retentate which was mostly depleted from HMOs (Brix 1.0) and enriched with proteins and residual suspended solids.
Loose NF: — The product-containing membrane permeate obtained above (6.8 kg, conductivity 5.84 mS/cm, pH 3.40) was first concentrated in the same SW 18 system equipped with Trisep UA60-1812 membrane element (piperazine-amide, MWCO 1000-3500 Da, area 0.23 m?, LNT rejection factor >99 %) by removing 5.44 kg of the permeate in 26 min (TMP=30 bar, T=40-42°C, cross-flow 400 L/h) followed by diafiltration under the same conditions with 4 L of water added at 5.0 L/h flow rate to — give total of 9.4 kg of NF permeate (Brix 0.6, conductivity 4.28 mS/cm, pH 3.21). The obtained retentate (Brix 20.2, conductivity 1.67 mS/cm, pH 3.54) was further diafiltrated with additional 10
L of water to give a second NF permeate (10.3 kg, Brix 0.1, conductivity 0.458 mS/cm, pH 3.71) and the final NF retentate which was practically free of salts (ca. 1500 g, Brix 18.2, conductivity 0.48 mS/cm, pH 4.28).
AC treatment:
DK 181291 B1 26
A portion of the obtained NF retentate (379 g) was passed at ca. 6 mL/min feed rate through a short column packed with Silcarbon CW20 (6.00g, ID=2.1 cm, bed height=5.3 cm pre-washed with 200 mL of de-ionised water) followed by 50 mL water elution to give 407 g of colourless solution (Brix 16.2, conductivity 0.455 mS/cm, pH 4.17, Abs 400 0.0130 corresponding to CI 400 = 0.80) — Solid product isolation by freeze-drying:
The obtained solution was freeze-dried to give 62 g of a white solid.
Reference Example 2
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 — 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-
DK 181291 B1 27 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
SW 18 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 1/h. First, the DF was performed at a pH of around 5 (10 I), 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). 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 3: comparison of MgSO4 and Na2SO4 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.
MgSO, rejection vs pH 25%
TMP |... | H2504 pH Flow | Flux | Retentate | Permeate | pejcction (bar) TC added | (retentate) | (I/h) | (I/m?h) conductivity | conductivity factor (ul) (mS/cm) (mS/cm) 10 [247] o | 606 [211] 916 | 2.100 0.785 62.61% 2.100 0.587 72.05 % 2.130 0.624 70.70 %
DK 181291 B1 28 uw [25] 3 | 393 [05 [wa | 200 | 05 | 7600% 2.260 0538 | 76.19%
The same experiment was performed with 0.2 % Na2SO4 solution.
NaxS04 rejection vs pH
TMP | T(°C)| 25% pH Flow Flux Retentate Permeate Rejection (bar) H>2S5O1 | (retentate) | (I/h) | (I/m?h) | conductivity | conductivity factor added (mS/cm) (mS/cm) (ul) i [m0] o | 570 |17| 726 | 23% | 00 | 730% 2.840 0.1706 | 93.99% 2.840 0.824 70.99 % 2.850 1.706 40.14 % 2880 | 1989 | 3267% 2.970 2.090 28.67 % 3.060 2230 | 27.12% 3.280 2.500 23.78 % 1310 3.730 2.870 23.06 %
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 2), these salts can be effectively removed in a second NF/DF (see step #5 in Example 2) 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.
Example 4 — Determination of a substance rejection factor on a membrane
The NaCl and MgSO4 rejection on a membrane is determined as follows: in a membrane filtration — system, a NaCl (0.1 %) or a MgSO4 (0.2 %) solution is circulated across the selected membrane sheet (for Tami: tubular module) while the permeate stream is circulated back into the feed tank.
The system is equilibrated at 10 bars and 25 °C for 10 minutes before taking samples from the permeate and retentate. The rejection factor is calculated from the measured conductivity of the samples: (1-kp/kr): 100, wherein kp is the conductivity of NaCl or MgSO; in the permeate and kr is the conductivity of NaCl or MgSOs in the retentate.
DK 181291 B1 29
NaCl rej. factor MgSO; rej. factor membrane | active layer | MWCO | supplier lab. supplier lab. spec. measurement spec. measurement
Trisep piperazine- | 1000- 0 0 0
UA60 PA 3500 10 % 80 % 81-89 % 1000 | - [| 1 0%
A carbohydrate rejection factor is determined in a similar way with the difference that the rejection factor is calculated from the concentration of the samples (determined by HPLC): (1-CpC;): 100, wherein Cy is the concentration of the carbohydrate in the permeate and Cy is the concentration of the carbohydrate in the retentate.
Claims (13)
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| DKPA202100626A DK181291B1 (en) | 2021-06-15 | 2021-06-15 | Separation of neutral human milk oligosaccharides from a fermentation broth |
| EP22734558.4A EP4355464A1 (en) | 2021-06-15 | 2022-06-14 | Separation of human milk oligosaccharides from a fermentation broth |
| PCT/EP2022/066100 WO2022263406A1 (en) | 2021-06-15 | 2022-06-14 | Separation of human milk oligosaccharides from a fermentation broth |
| BE20225464A BE1029434B1 (en) | 2021-06-15 | 2022-06-14 | SEPARATION OF BREAST MILK OLIGOSACCHARIDES FROM A FERMENTATION BROTH |
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