Separation of human milk oligosaccharides from fermentation broth

Abstract

The present invention relates to a method for purifying neutral or sialylated human milk oligosaccharides (HMOs) from a fermentation broth. Furthermore, the invention also relates to the neutral or sialylated HMO obtained by the method of the invention and to the use thereof in food, feed and medical applications.

Description

Technical Field

[0001] The present invention relates to the separation and isolation of neutral or sialylated human milk oligosaccharides (HMO) from a reaction mixture in which the neutral or sialylated human milk oligosaccharides (HMO) are produced. Background Art

[0002] During the past few decades, people's interest in the preparation and commercialization of human milk oligosaccharides (HMOs) has been steadily increasing. The importance of HMOs is directly related to their unique biological activities. Therefore, HMOs have become important potential products for nutritional and therapeutic uses. Therefore, people have been looking for low-cost ways to produce HMOs on an industrial scale.

[0003] To date, the structures of more than 140 HMOs have been determined, and a considerable number of HMOs may be present in human milk (Urashima et al.: Milk oligosaccharides, Nova Biomedical Books, 2011; Chen Adv. Carbohydr. Chem. Biochem. 72, 113 (2015)). HMOs include a lactose (Galβ1-4Glc) moiety at the reducing end and may be extended with N-acetylglucosamine or one or more N-acetyllactosamine moieties (Galβ1-4GlcNAc) and / or lacto-N-disaccharide moieties (Galβ1-3GlcNAc). Lactose and N-acetyllactosamined or lacto-N-disaccharified lactose derivatives may be further substituted with one or more fucose and / or sialic acid residues, or lactose may be substituted with additional galactose to obtain the HMOs known to date.

[0004] Direct fermentation production of HMOs, especially trisaccharides, has recently become feasible (Han et al., Biotechnol. Adv. 30, 1268 (2012) and references cited therein). This fermentation technology uses a recombinant Escherichia coli (E. coli) system in which one or more glycosyltransferases of viral or bacterial origin are co-expressed to glycosylate exogenously added lactose that has been internalized by the LacY permease of E. coli. However, the use of recombinant glycosyltransferases, especially a series of recombinant glycosyltransferases, to produce oligosaccharides of four or more monosaccharide units always results in the formation of by-products, thus producing a complex mixture of oligosaccharides in the fermentation broth. In addition, the fermentation broth inevitably contains a wide range of non-carbohydrate substances, such as cells, cell debris, proteins, protein fragments, DNA, DNA fragments, endotoxins, caramelization by-products, minerals, salts or other charged molecules.

[0005] For the separation of HMOs from carbohydrate byproducts and other contaminating components, activated carbon treatment combined with gel filtration chromatography has been proposed as an alternative method (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); et al., ChemBioChem 15, 1896 (2014). Although gel filtration chromatography is a convenient laboratory-scale method, it cannot be effectively scaled up to industrial production scale. Instead, more recent methods include ion exchange resin treatment (for removal of charged organic and inorganic species) combined with activated carbon treatment (for decolorization) (e.g., see WO 2015 / 106943; WO 2017 / 182965). However, activated carbon treatment generally has the following disadvantages: the activated carbon may adsorb relatively large amounts of HMO, which can lead to a decrease in HMO yield. In addition, the regeneration of activated carbon is complicated, which makes the reuse of activated carbon less attractive.

[0006] Alternative and / or improved methods for separating and purifying neutral or sialylated HMO from non-carbohydrate components of a fermentation broth producing the neutral or sialylated HMO, in particular methods suitable for industrial scale, are needed to improve the recovery of neutral or sialylated HMO, and / or simplify the methods of the prior art, while at least maintaining, preferably improving, the purity of the neutral or sialylated HMO. Furthermore, such alternative purification methods preferably produce purified neutral or sialylated HMO that is free of proteins and recombinant material originating from the recombinant microbial strain used, and are therefore very suitable for use in food, medical food and feed applications. Summary of the invention

[0007] The present invention relates to a method for purifying neutral or sialylated human milk oligosaccharides (HMO) from a fermentation broth, comprising the following steps:

[0008] I. separating a stream containing HMOs from biomass;

[0009] II. Purifying the HMO-containing stream with an acidic cation exchange resin, then

[0010] III. Treat the resin eluate with an adsorbent resin.

[0011] Preferably, the method comprises:

[0012] a. separating the fermentation broth to form a stream containing HMO and a biomass waste stream;

[0013] b. Purifying the HMO-containing stream by nanofiltration;

[0014] c. purifying the HMO-containing stream with an acidic cation exchange resin;

[0015] d. purifying the HMO-containing stream with an adsorptive resin; and

[0016] e. The purified HMO-containing stream is concentrated and dried to obtain neutral or sialylated HMO in solidified form.

[0017] Preferably, step b) is performed before step c) and / or step d).

[0018] Preferably, step c) is performed immediately after step b).

[0019] Preferably, step d) is performed immediately after step c).

[0020] Even more preferably, the process does not comprise electrodialysis and / or anion exchange resin treatment.

[0021] Even more preferably, an electrodialysis step and / or an anion exchange resin treatment is excluded.

[0022] Even more preferably, a purification / decolorization step using activated carbon is excluded.

[0023] In another aspect, the present invention relates to neutral or sialylated human milk oligosaccharides obtained by the process according to the present invention.

[0024] Another aspect of the present invention relates to the medical use of neutral or sialylated human milk oligosaccharides obtained according to the process of the present invention.

[0025] Another aspect of the present invention relates to the use of neutral or sialylated human milk oligosaccharides obtained according to the process of the present invention for food and / or feed applications.

[0026] Another aspect of the present invention relates to a food or cosmetic product comprising neutral or sialylated human milk oligosaccharides obtained according to the process of the present invention. DETAILED DESCRIPTION

[0027] 1. Terms and Definitions

[0028] As used in this specification, the term "fermentation broth" refers to the product of fermentation of a microbial organism. Thus, the fermentation product includes cells (biomass), fermentation medium, salts, residual substrate materials, and any molecules / byproducts produced during the fermentation process, such as the desired neutral or sialylated HMO. After each step of the purification process, one or more components of the fermentation product are removed, resulting in a more purified neutral or sialylated HMO.

[0029] The term "monosaccharide" refers to sugars of 5 to 9 carbon atoms, which may be aldoses (e.g., D-glucose, D-galactose, D-mannose, D-ribose, D-arabinose, L-arabinose, D-xylose, etc.), ketoses (e.g., D-fructose, D-sorbose, D-tagatose, etc.), deoxysugars (e.g., L-rhamnose, L-fucose, etc.), deoxyaminosugars (e.g., N-acetylglucosamine, N-acetylmannosamine, N-acetylgalactosamine, etc.), sugar acids, keturonic acids (e.g., sialic acid), or their equivalents.

[0030] The term "disaccharide" refers to a carbohydrate composed of two monosaccharide units linked to each other by a glycosidic bond.

[0031] The term "trisaccharide or higher oligosaccharide" refers to a carbohydrate consisting of at least three, preferably three to eight, more preferably three to six monosaccharide units (see above). Oligosaccharides may have a linear or branched structure containing monosaccharide units linked to each other by glycosidic bonds.

[0032] The term "human milk oligosaccharide" or "HMO" refers to a complex carbohydrate found in human milk (Urashima et al.: Milk Oligosaccharides, Nova Medical Books, NY, 2011; Chen Adv. Carbohydr. Chem. Biochem. 72, 113 (2015)). HMO has a lactose unit at the reducing end i) extended by β-N-acetyl-glucosamine or ii) by one or more β-N-acetyl-lactosamine and / or one or more β-lacto-N-diosyl units, and the core structure may be substituted with α-L-pyranosyl and / or α-N-acetylneuraminic acid (sialic acid) moieties. In this regard, non-acidic (or neutral) HMOs have no sialic acid groups, and acidic HMOs have at least one sialic acid residue in their structure. Non-acidic (or neutral) HMOs may be fucosylated or non-salinosylated. Examples of such neutral non-fucosylated HMOs include lacto-N-triose II (LNTri, GlcNAc(β1-3)Gal(β1-4)Glc) lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT), lacto-N-neohexaose (LNnH), p-lacto-N-neohexaose (pLNnH), p-lacto-N-hexaose (pLNH) and lacto-N-hexaose (LNH). Examples of neutral salinaglycosylated HMOs include 2'-fucosyllactose (2'-FL), lacto-N-salina pentasaccharide I (LNFP-I), lacto-N-disalina hexaose I (LNDFH-I), 3-fucosyllactose (3-FL), disalinaglycosyllactose (DFL), lacto-N-salina pentasaccharide II (LNFP-II), lacto-N-salina pentasaccharide III (LNFP-III), lacto-N-disalina hexaose I (LNDFH-I), The present invention relates to lacto-N-hexaose III (LNDFH-III), fucosyl-lacto-N-hexaose II (FLNH-II), lacto-N-salinarum pentasaccharide V (LNFP-V), lacto-N-disalinarum hexaose II (LNDFH-II), fucosyl-lacto-N-hexaose I (FLNH-I), fucosyl-p-lacto-N-hexaose I (FpLNH-I), fucosyl-p-lacto-N-neohexaose II (F-pLNnH II), and fucosyl-lacto-N-neohexaose (FLNnH).Examples of acidic HMOs include 3'-sialyllactose (3'-SL), 6'-sialyllactose (6'-SL), 3-fucosyl-3'-sialyllactose (FSL), LST a, fucosyl-LST a (FLST a), LST b, fucosyl-LST b (FLST b), LST c, fucosyl-LST c (FLST c), sialic acid-LNH (SLNH), sialic acid-lacto-N-hexaose (SLNH), sialic acid-lacto-N-neohexaose I (SLNH-I), sialic acid-lacto-N-neohexaose II (SLNH-II), and disialyl-lacto-N-tetraose (DSLNT).

[0033] The term "sialyl" or "sialic acid moiety" refers to the sugar portion of sialic acid (N-acetyl-neuraminic acid, Neu5Ac), preferably linked with an α-linkage:

[0034]

[0035] The term "fucosyl" refers to L-fucopyranosyl, preferably linked with an α-glycosidic linkage:

[0036]

[0037] "N-acetyl-aminoglucosyl" refers to an N-acetyl-2-amino-2-deoxy-D-glucopyranosyl (GlcNAc) group, preferably linked by a β-linkage:

[0038]

[0039] "N-acetyl-lactosyl" refers to a glycosyl residue of N-acetyl-lactosamine (LacNAc, Galpβ1-4GlcNAc), preferably linked by a β-linkage:

[0040]

[0041] Furthermore, the term "lacto-N-biosyl" refers to the glycosyl residue of lacto-N-biose (LNB, Galpβ1-3GlcNAc), preferably linked with a β-bond:

[0042]

[0043] The term "biomass" in the context of fermentation refers to suspended, precipitated or insoluble material derived from fermenting cells, such as whole cells, disrupted cells, cell debris, proteins, protein fragments, polysaccharides.

[0044] The term "Brix" refers to the Brix degree, i.e. the sugar content of an aqueous solution (the number of grams of sugar in 100 g of solution). In this regard, the Brix of the human milk oligosaccharide solution of the present application refers to the total carbohydrate content of the solution including human milk oligosaccharides and their accompanying carbohydrates. Brix is ​​measured with a calibrated refractometer.

[0045] "Demineralization" preferably refers to the process of removing minerals or mineral salts from a liquid. In the context of the present invention, demineralization may take place in a nanofiltration step, especially when it is combined with diafiltration, or using cation and anion exchange resins, if applicable.

[0046] The term "protein-free aqueous medium" preferably refers to an aqueous medium or broth from a fermentation or enzymatic process which has been treated to remove from the mixture of the fermentation or enzymatic process substantially all proteins as well as peptides, peptide fragments, RNA and DNA as well as endotoxins and glycolipids which could interfere with the final purification of one or more neutral or sialylated HMOs and / or one or more of their components, in particular mixtures thereof.

[0047] The term "HMO-containing stream" preferably refers to an aqueous medium containing neutral or sialylated HMOs obtained from a fermentation process, which has been treated to remove suspended particulate matter and contaminants from the process, in particular cells, cell components, insoluble metabolites and debris, which could interfere with the final purification of one or more hydrophilic oligosaccharides, in particular one or more neutral or sialylated HMOs and / or one or more HMO components, in particular mixtures thereof.

[0048] The term "biomass waste stream" preferably refers to suspended particulate matter and contaminants resulting from a fermentation process, in particular cells, cellular components, insoluble metabolites and debris.

[0049] The rejection factor of salt (in percent) was calculated as (1-κ p / κ r )·100, where κ p is the conductivity of the salt in the permeate, κ r is the conductivity of the salt in the retentate.

[0050] The carbohydrate retention rate (in percent) was calculated as (1-C p / C r )·100, where C p is the concentration of carbohydrates in the permeate, C r is the carbohydrate concentration in the retentate.

[0051] The term "diafiltration" refers to the addition of solvent (water) during membrane filtration. If diafiltration is applied during ultrafiltration, it will increase the yield of the desired HMO in the permeate. If diafiltration is applied during nanofiltration, it will improve the separation of small molecule impurities and salts into the permeate. The solute yield and therefore the product enrichment can be calculated based on the rejection and the relative amount of water added according to formulas known to those skilled in the art.

[0052] The term "concentrating" refers to the removal of liquids, primarily water, resulting in a higher concentration of neutral or sialylated HMOs in the purified HMO-containing product stream.

[0053] The term "decolorization" refers to the removal of a color body from a solution to the extent required by the product specification. Decolorization of solutions containing carbohydrates is primarily based on van der Waals type interactions between the color body and the adsorbent. In the context of the present invention, the color of the solution is quantified by the visible light absorbance at 400 nm (Abs_400) and normalized by concentration and path length. Therefore, the color index CI_400 is defined as 1000×Abs_400 / Brix, path length = 1 cm. Typically, if CI_400 is less than 5, the solid product separated from its solution is in the form of a colorless (white) solid. The crude supernatant containing the HMO product typically has a color index CI_400 in the range of 100-400 after fermentation.

[0054] 2. Method for purifying neutral or sialylated HMO from fermentation broth:

[0055] The present invention relates to a method for purifying neutral or sialylated human milk oligosaccharides (HMO) from a fermentation broth, comprising the following steps:

[0056] I. separating a stream containing HMOs from biomass;

[0057] II. Purifying the HMO-containing stream with an acidic cation exchange resin, then

[0058] III. Treat the resin eluate with an adsorbent resin.

[0059] Preferably, the method comprises:

[0060] a. separating the fermentation broth to form a stream containing HMO and a biomass waste stream;

[0061] b. Purifying the HMO-containing stream by nanofiltration;

[0062] c. purifying the HMO-containing stream with an acidic cation exchange resin;

[0063] d. purifying the HMO-containing stream with an adsorptive resin; and

[0064] e. The purified HMO-containing stream is concentrated and dried to obtain neutral or sialylated HMO in solidified form.

[0065] In one embodiment, the method according to the invention consists of steps a) to e).

[0066] In a preferred embodiment, the method does not include an anion exchange resin treatment step.

[0067] In a preferred embodiment, the anion exchange resin treatment step is excluded from the process according to the invention.

[0068] In a preferred embodiment, method steps a) to e) are carried out in the consecutive order a) to e) as given above.

[0069] Fermentation broth:

[0070] The neutral or sialylated HMOs present in the fermentation broth are obtained by culturing a genetically modified microorganism capable of producing neutral or sialylated human milk oligosaccharides from internalized carbohydrate precursors. 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 strain, 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; the bacterium is Bacillus amyloliquefaciens. amyloliquefaciens), Bacillus licheniformis or Bacillus subtilis.

[0071] In one embodiment, at least one neutral or sialylated human milk oligosaccharide present in the fermentation broth is not obtained by microbial fermentation but is, for example, added to the fermentation broth after production by non-microbial methods such as chemical and / or enzymatic synthesis.

[0072] In one embodiment, the purity of neutral or sialylated HMO in the fermentation broth is ≤70%, preferably ≤60%, more preferably ≤50%, and most preferably ≤40%.

[0073] Preferably, the HMO is a neutral HMO. In one embodiment, the neutral HMO is preferably selected from 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 (also known as: lacto-N-fucopentaose VI), lacto-N-difucohexaose I, lacto-N-difucohexaose II, lacto-N-difucohexaose III, 6'-galactosyl lactose, 3'-galactosyl lactose, lacto-N-hexaose, lacto-N-neohexaose and any mixture thereof. More preferably, the HMO is 2'-fucosyllactose, 3-fucosyllactose, 2',3-difucosyllactose, lacto-N-triose II, lacto-N-tetraose, lacto-N-neotetraose or lacto-N-fucopentaose, more preferably 2'-fucosyllactose, LNT, LNnT or lacto-N-fucopentaose.

[0074] In one embodiment, the sialylated HMO is selected from 3'-sialyllactose (3'-SL) and 6'-sialyllactose (6'-SL).

[0075] In one embodiment, the HMO in the fermentation broth is a single neutral or sialylated HMO.

[0076] In one embodiment, the HMO in the fermentation broth is a mixture of different individual neutral or sialylated HMOs.

[0077] In one embodiment, the HMO in the fermentation broth is a mixture of two individual neutral or sialylated HMOs. In another embodiment, the HMO in the fermentation broth is a mixture of three individual neutral or sialylated HMOs. In another embodiment, the HMO in the fermentation broth is a mixture of four individual neutral or sialylated HMOs. In another embodiment, the HMO in the fermentation broth is a mixture of five individual neutral or sialylated HMOs.

[0078] In one embodiment, the HMO in the fermentation broth is a mixture of neutral or sialylated HMO obtained by microbial fermentation and HMO not obtained by microbial fermentation but obtained, for example, by chemical and / or enzymatic synthesis.

[0079] In step i) or step a) of the process according to the invention, the fermentation broth is separated to form a stream containing HMOs and a stream containing Material waste stream

[0080] In step a) of the process according to the invention, a stream containing HMOs is separated from a biomass waste stream.

[0081] In addition to the desired neutral or sialylated HMOs, the fermentation broth typically contains the cell biomass of the microorganisms used together with proteins, protein fragments, peptides, DNA, RNA, endotoxins, biogenic amines, amino acids, organic acids, inorganic salts, unreacted carbohydrate acceptors such as lactose, sugar by-products, monosaccharides, pigments, etc. In step a) of the process according to the invention, the biomass is separated from the neutral or sialylated HMOs.

[0082] In a preferred embodiment, the biomass is separated from the neutral or sialylated HMO in step a) by ultrafiltration. The ultrafiltration step is to separate the biomass and preferably also the high molecular weight components and suspended solids from the low molecular weight soluble components of the fermentation broth, which low molecular weight soluble components will pass through the ultrafiltration membrane into the permeate. This ultrafiltration permeate is an aqueous solution containing neutral or sialylated human milk oligosaccharides, also referred to as the HMO-containing stream, while the ultrafiltration retentate comprises the biomass waste stream.

[0083] Any conventional ultrafiltration membrane with a molecular weight cut-off (MWCO) in the following range can be used: about 1 to about 500 kDa, such as 10-250 kDa, 50-100 kDa, 200-500 kDa, 100-250 kDa, 1-100 kDa, 1-50 kDa, 10-25 kDa, 1-5 kDa or any other suitable sub-range. The membrane material can be ceramic, or made of synthetic or natural polymers, such as polysulfone, polyvinylidene fluoride, polyacrylonitrile, polypropylene, cellulose, cellulose acetate or polylactic acid. The ultrafiltration step can be carried out in a dead-end or tangential flow mode. The step a) of the method according to the invention can include using a membrane with different MWCO as defined above to carry out more than one ultrafiltration step, such as, applying two ultrafiltration separations, wherein the MWCO of the first membrane is higher than that of the second membrane. This arrangement can provide a better separation efficiency of high molecular weight components in the fermentation broth. After this separation step, the permeate contains materials with molecular weights less than the MWCO of the second membrane, including neutral or sialylated human milk oligosaccharides of interest.

[0084] In one embodiment, the fermentation broth is ultrafiltered using a membrane having a MWCO of 5-30 kDa, such as 10-25 kDa, 15 kDa or 20 kDa.

[0085] In preferred embodiments, the yield of the desired neutral or sialylated 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%.

[0086] In another embodiment, in step a) of the process according to the invention, the fermentation broth obtained from the fermentation is centrifuged to separate the biomass from the neutral or sialylated HMO (HMO-containing stream). In said embodiment, the supernatant represents the HMO-containing stream, while the residual material, i.e. the "biomass waste stream", can be separated. By centrifugation, a clarified supernatant comprising neutral or sialylated HMO can be obtained, which represents the HMO-containing stream.

[0087] Centrifugation can be at laboratory scale or, advantageously relative to previous centrifugation methods, at commercial scale (eg, industrial scale, full production scale).

[0088] In some embodiments, multiple steps of 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, centrifugation can be a single step. Centrifugation provides rapid biomass removal.

[0089] In certain embodiments, a Flottweg-designed and manufactured Centrifuge.

[0090] The specific type of centrifuge is not limited, and various types of centrifuges can be used. Centrifugation can be a continuous process. In some embodiments, centrifugation can add feed. For example, centrifugation can continuously add feed. In certain embodiments, centrifugation can include solid removal, such as wet solid removal. Wet solid removal can be continuous in certain embodiments, and can be periodic in other embodiments.

[0091] For example, a conical plate centrifuge (e.g., a disk bowl centrifuge or a disc stack separator) may be used. A conical plate centrifuge can be used to remove solids (usually impurities) from a liquid, or to separate two liquid phases from each other by high centrifugal forces. The denser solids or liquids subjected to these forces move outward toward the walls of the rotating bowl, while the less dense liquid moves toward the center. The special plates (called a disc stack) increase the surface settling area, thereby speeding up the separation process. Different disc stack designs, arrangements, and shapes are used for different processes, depending on the type of feed present. The concentrated, denser solids or liquids can then be removed continuously, manually, or intermittently, depending on the design of the conical plate centrifuge. This type of centrifuge is well suited for clarifying liquids that contain small amounts of suspended solids.

[0092] The centrifuge works on the principle of inclined plate setters. A set of parallel plates are mounted at an angle θ relative to the horizontal plane to reduce the distance over which particles settle. The reason for the angle of inclination is to allow the settled solids on the plates to slide downwards under the action of centrifugal force so that they do not accumulate and block the channels formed between adjacent plates.

[0093] This type of centrifuge can have different designs, such as nozzle type, manual cleaning, self-cleaning, and sealing. The specific centrifuge is not limited.

[0094] Factors that affect a centrifuge include disc angle, gravity effects, disc spacing, feed solids, discharge cone angle, discharge frequency, and liquid discharge.

[0095] Alternatively, a solid bowl centrifuge (e.g., a decanter centrifuge) can be used. This is a centrifuge that uses the principle of sedimentation. A centrifuge uses the centrifugal force generated by continuous rotation to separate a mixture consisting of two substances of different densities. It is commonly used to separate solid-liquid, liquid-liquid, and solid-solid mixtures. Compared with other types of centrifuges, an advantage of industrial solid bowl centrifuges is that they are easy to install. There are three design types of solid bowl centrifuges, namely conical, cylindrical, and conical-cylindrical.

[0096] Solid drum centrifuges can have many different designs, any of which can be used for the disclosed methods. For example, conical solid drum centrifuges, cylindrical solid drum centrifuges, and conical-cylindrical solid drum centrifuges can be used.

[0097] Centrifugation can be carried out at various speeds and residence times. For example, centrifugation can be carried out at a relative centrifugal force (RCF) of 20000g, 15000g, 10000g or 5000g. In some embodiments, centrifugation can be carried out at a relative centrifugal force (RCF) of less than 20000g, 15000g, 10000g or 5000g. In some embodiments, centrifugation can be carried out at a relative centrifugal force (RCF) of greater than 20000g, 15000g, 10000g or 5000g.

[0098] In some embodiments, centrifugation 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 1. In some embodiments, the working volume can be greater than 1, 5, 10, 15, 20, 50, 100, 300, or 500 1.

[0099] In some embodiments, centrifugation 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.

[0100] The time (e.g., residence time) spent by centrifugation can also be different. 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.

[0101] Any of the above-mentioned supernatant characteristics can be obtained by a single centrifugation. Alternatively, they can also be obtained by multiple centrifugations.

[0102] In view of the above, step a) of the process according to the invention can be carried out by ultrafiltration or centrifugation as defined above, or by a combination of ultrafiltration and centrifugation. Preferably, process step a) is carried out by ultrafiltration as defined above to obtain a stream containing HMOs separated from a biomass waste stream.

[0103] Before ultrafiltration and / or centrifugation steps, the fermented liquid can be subjected to a pretreatment step. The pretreatment of the fermented liquid can include pH adjustment and / or dilution and / or heat treatment. In certain embodiments, pH adjustment, dilution and heat treatment all three can be performed. In other optional embodiments, pH adjustment and dilution can be performed. In other optional embodiments, pH adjustment and heat treatment can be performed. In other optional embodiments, heat treatment and dilution can be performed. Advantageously, the combination of multiple pretreatment methods can provide an improved synergistic effect not found in a single pretreatment.

[0104] In certain embodiments, during the biomass removal by centrifugation and / or ultrafiltration in step a), as described above, one or more of the aforementioned pretreatment steps may occur. For example, the centrifugation container may be capable of heating the fermentation broth between the steps of multiple centrifugation steps, or during the centrifugation process.

[0105] Advantageously, the pretreatment can increase the sedimentation velocity of solid particles (biomass) in the fermentation broth by 100-20,000 times, making the separation of biomass by centrifugation more efficient and thus applicable on an industrial scale. In addition to the sedimentation velocity, at least three other parameters are significantly improved due to the pretreatment: the yield of neutral or sialylated HMOs in the HMO-containing stream is increased; the protein and DNA content in the supernatant is reduced; and further, the content of residual suspended solids can be greatly reduced.

[0106] In step b), the HMO-containing stream is purified by nanofiltration:

[0107] In step b) of the process according to the invention, the stream containing neutral or sialylated HMOs is purified by nanofiltration.

[0108] Nanofiltration (NF) can be used to remove low molecular weight molecules that are smaller than the desired neutral or sialylated HMOs, such as mono- and disaccharides, short peptides, small organic acids, water, and salts.

[0109] The product stream, ie the stream containing neutral or sialylated HMOs, is the NF retentate. Therefore, the MWCO or pore size of the nanofiltration membrane is to ensure the retention of the neutral or sialylated human milk oligosaccharides of interest, ie the MWCO of the nanofiltration membrane is adjusted accordingly.

[0110] Typically, the nanofiltration membrane has a pore size of 0.5 nm to 2 nm and / or a molecular weight cut-off (MWCO) of 150 Daltons (Da) to MWCO 3500 Da.

[0111] In one embodiment, the MWCO of the membrane is in the range of 150-300 Da, which is defined as a "tight" NF membrane.

[0112] In a preferred embodiment, the MWCO of the membrane is above 300 Da, and preferably the MWCO is not above 3500 Da. In said embodiment, the membrane is considered to be a "loose" NF membrane.

[0113] In another preferred embodiment, the "loose" nanofiltration membrane has a molecular weight cut-off (MWCO) of 500-3500 Da, and the active (top) layer of the membrane is preferably composed of a polyamide, more preferably a piperazine-based polyamide. Thus, the retention of trisaccharides or higher oligosaccharides is ensured, and at least a portion of disaccharides are allowed to pass through the membrane. In this embodiment, the nanofiltration membrane used should be tight for trisaccharides or higher oligosaccharides to be effectively retained. At the same time, the membrane should be relatively loose for MgSO4, that is, its rejection rate (rejection) is about 50-90%, so that disaccharides can pass through the membrane. In this way, it is possible to separate, for example, lactose (which is a precursor for producing human milk oligosaccharides, for example, by fermentation) from neutral or sialylated human milk oligosaccharides with good efficiency, and additionally, a considerable portion of divalent ions also pass into 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 MgSO4 rejection factor on the membrane is 80-90%. Preferably, the NaCl rejection factor of the membrane is lower than the MgSO4 rejection factor. In one embodiment, the NaCl rejection factor is no greater than 50%. In another embodiment, the NaCl rejection factor is no greater than 40%. In another embodiment, the NaCl rejection factor is no greater than 30%. In this latter embodiment, a significant reduction in total monovalent salts in the retentate can also be achieved. In the embodiment, the membrane is a thin film composite (TFC) membrane. Examples of suitable piperazine-based polyamide TFC membranes are Other examples of suitable NF membranes include Synder NFG (600-800 Da), Synder NDX (500-700 Da), and XN-45 (500Da).

[0114] Preferably, the yield of the desired neutral or sialylated 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%.

[0115] In a preferred embodiment, step b) comprises a diafiltration step, ie a nanofiltration step performed in diafiltration mode. Preferably, the diafiltration step is performed after the aforementioned (conventionally performed) nanofiltration step.

[0116] The diafiltration process involves adding pure water to a solution during membrane filtration to more effectively remove membrane permeable components. Thus, by using an appropriate membrane, diafiltration can be used to separate multiple components based on the properties of the components, particularly molecular size, charge or polarity, where one or more substances are effectively retained while other substances are permeable to the membrane.

[0117] In a preferred embodiment, diafiltration and nanofiltration are combined in one step (referred to as nanofiltration / diafiltration or NF / DF), wherein diafiltration is performed while using a nanofiltration membrane, which is effective for separating low molecular weight compounds and / or salts from neutral or sialylated HMOs. Diafiltration using a "loose" NF membrane as defined above is particularly effective for the removal of monovalent and divalent salts and the separation of disaccharides from neutral or sialylated HMOs.

[0118] In a preferred embodiment, the DF step or the NF / DF step is carried out so that the pH is set to less than 5.0, preferably less than 4.5, advantageously less than 4.0, but preferably not less than 3.0. Under such conditions, salts consisting of monovalent cations, such as sodium salts (i.e. sodium ions together with the corresponding anions), are effectively removed, resulting in a low-salt or practically salt-free purified solution containing neutral or sialylated HMOs in the retentate.

[0119] In one embodiment, a second nanofiltration step is performed in the process according to the invention so that it is included in step b). In the second nanofiltration step, the nanofiltration membrane is a "loose" NF membrane or a "tight" NF membrane. The optional second nanofiltration step is performed after the first nanofiltration step (step b), but preferably before step c) of the process according to the invention.

[0120] Similarly, a second diafiltration may be performed in the process according to the invention. This optional second diafiltration step may also be combined with a second nanofiltration step. When a "loose" NF membrane is applied as disclosed above, this second NF / DF step is performed so that the pH is set to less than 5.0, preferably less than 4.5, advantageously less than 4.0, but preferably not less than 3.0.

[0121] In one embodiment, the purified solution obtained after step b) of the process according to the invention contains neutral or sialylated HMOs in a purity of ≥ 80%, preferably ≥ 85%, more preferably ≥ 90%.

[0122] In one embodiment, the purified solution obtained after step b) of the method according to the invention is free of proteins and / or recombinant genetic material.

[0123] Step c) Purification of the HMO-containing stream with an acidic cation exchange resin:

[0124] The process according to the invention comprises purifying the HMO-containing stream with an acidic cation exchange resin (step c)).

[0125] In a cation exchange treatment step, positively charged species can be effectively removed from the HMO-containing stream before or after nanofiltration, since they bind to the resin, whereas HMO are not retained by the acidic cation exchange resin. Thus, the amount of salts and / or colorants and / or proteins can also be further reduced.

[0126] In a preferred embodiment, the stationary phase (resin) comprises sulfonate groups which are negatively charged in aqueous solution and bind tightly to cationic compounds.

[0127] In a preferred embodiment, the acidic cation exchange resin is a strongly acidic cation exchange resin, preferably a polystyrene-divinylbenzene cation exchange resin having sulfonic acid functional groups.

[0128] In a further preferred embodiment, the acidic cation exchange resin is H + -Formal.

[0129] The binding capacity of acidic cation exchange resins is usually 1.2-2.2 eq / l.

[0130] When using ion exchange resins, the degree of crosslinking can be selected according to the operating conditions of the ion exchange column. Highly crosslinked resins have the advantages of durability and high mechanical integrity, but have the disadvantages of reduced porosity and decreased mass transfer. Low crosslinked resins are more fragile and tend to swell due to absorption of mobile phase. The particle size of the ion exchange resin is selected to allow the eluent to flow effectively while the charged species are still effectively removed. Suitable flow rates can also be obtained by applying negative pressure to the elution end of the column or applying positive pressure to the loading end of the column and collecting the eluent. A combination of positive and negative pressures can also be used. The ion exchange treatment can be carried out in a conventional manner, such as intermittently or continuously.

[0131] Non-limiting examples of suitable acidic cation exchange resins may be, for example, Amberlite IR100, Amberlite IR120, Amberlite FPC22, Dowex 50WX, Finex CS16GC, Finex CS13GC, Finex CS12GC, Finex CS11GC, Lewatit S, Diaion SK, Diaion UBK, Amberjet 1000, Amberjet1200.

[0132] Preferably, the cation exchange resin treatment step is performed after the nanofiltration step.

[0133] In a preferred embodiment, the purified solution produced in step c) contains neutral or sialylated HMOs in a purity of ≥ 80%, preferably ≥ 85%, more preferably ≥ 90%.

[0134] In a preferred embodiment, the purified solution produced in step c) is free of proteins and / or recombinant genetic material.

[0135] In a particularly preferred embodiment, after the HMO-containing stream obtained in step a) has been purified by NF or NF / DF, the retentate from step b) of NF or NF / DF is subjected to a strong cation exchange resin (H + -form).

[0136] In step d), the HMO-containing stream is purified using an adsorptive resin:

[0137] "Adsorbent" is a material with a high specific surface area, i.e. >400m 2 / g (determined using nitrogen adsorption BET technique) of insoluble solid porous material, which is capable of removing certain compounds from solution by physical adsorption and / or chemical adsorption, while adsorption occurs on the pore surface of the adsorbent. Physical adsorption involves non-stoichiometric processes and non-covalent interactions, including, for example, van der Waals forces, polar or ionic interactions without ion exchange. Chemical adsorption or chemisorption occurs due to chemical reactions, i.e., involving electron exchange and chemical bond formation on the adsorbent surface. Examples of adsorbents include activated carbon (activated charcoal or AC) and adsorbent resins.

[0138] "Adsorbent resins" or "synthetic adsorbents" are specially designed macroporous polymers, usually supplied in the form of spherical beds, with high specific surface areas, e.g. >400 m 2 / g (determined by nitrogen adsorption BET technique) and a defined pore structure for selectively removing specific substances from aqueous solutions. In the context of the present application, the adsorptive resin suitable for step d) is partially functionalized with hydrophilic groups to make it easier to regenerate. Therefore, the adsorptive resin is an acid adsorbent derived from a crosslinked polystyrene or polyacrylic acid polymer and partially functionalized with a primary, secondary or tertiary amine as a weak base. In a preferred embodiment, the adsorptive resin used in step d) is a polystyrene-divinylbenzene copolymer with tertiary amine functional groups.

[0139] In the context of the present application, an adsorbent resin is distinguished from an ion exchanger or ion exchange resin by the fact that the non-stoichiometric physical adsorption process, or chemical adsorption such as acid adsorption by amines on a polymer support, in both cases the molecule is removed from the feed solution as a whole without releasing ions or other substances from the adsorbent. In addition, an adsorbent resin functionalized with basic amino groups in the free base form does not contain any exchangeable ions and therefore cannot be an ion exchanger if used in the free base form. In addition, these resins usually have a low basic capacity, for example 0.1-0.3 eq / l (or about 0.6-1.0 eq / kg on a dry weight basis), which is much lower than the typical capacity of ion exchange resins, i.e. 1-2 eq / l. The resin capacity is a measure of the total capacity, which is determined by tests carried out in the laboratory by titration. A determined amount of alkaline (anionic) resin (including the polystyrene-divinylbenzene copolymer adsorbent resin with tertiary amine functional groups used in step d) is completely converted to an alkaline form with an excess of a strong base such as a NaOH solution, and then rinsed thoroughly. Then, a determined amount of a strong inorganic acid such as HCl or a sulfuric acid solution is passed through the resin to completely deplete the resin. The effluent is collected. The acid that passes through the column represents the amount that is not captured by the resin. The solution is then titrated with a base to neutralize it, and the amount of base required is expressed in equivalents. The difference between the total acid equivalents that pass through the column and the acid equivalents that flow out of the column represents the total acid equivalents captured by the resin. The capacity of the resin is then determined.

[0140] In a preferred embodiment, the surface area of ​​the adsorptive resin is >400 m 2 In one embodiment, the surface area of ​​the adsorptive resin is 400-1200m 2 / g. More preferably, the alkaline capacity of the adsorptive resin is 0.1-0.3 eq / l (or about 0.6-1.0 eq / kg by weight). In addition, preferably, the adsorptive resin used in step d) is a polyacrylic acid polymer, more preferably a methacrylic acid polymer adsorptive resin. Advantageously, the polymer is a functionalized polystyrene (PS), more preferably a cross-linked PS with divinylbenzene (DVB).

[0141] Suitable adsorptive resins are Dowex Optipore SD-2, Purolite MN100 or MN102.

[0142] In the context of the present application, the substances to be removed are a wide variety of colored compounds, hydrophobic impurities, large biomolecules such as proteins, DNA, RNA, polysaccharides and fragments thereof.

[0143] In a preferred embodiment, purification with an adsorptive resin results in a HMO-containing stream with a CI_400<5, preferably, a CI_400<4.

[0144] In a preferred embodiment, step d) results in a purified HMO-containing solution having a purity of ≥ 80%, preferably ≥ 85%, more preferably ≥ 90%.

[0145] In the process according to the invention, the adsorbent resin defined above can be used as a replacement for the activated carbon commonly used in known processes for purifying HMO streams. The adsorbent resin is as effective as activated carbon in removing pigments, large biomolecules (proteins, DNA, etc.) and hydrophobic compounds and has the following advantages over the use of activated carbon:

[0146] - It does not adsorb HMO like carbon. Activated carbon can adsorb up to 30-35wt% HMO relative to the weight of carbon used, which can reduce the recovery yield of HMO;

[0147] - It can be easily regenerated and reused many times, unlike activated carbon, which requires harsh conditions or the use of organic solvents;

[0148] - Due to the regeneration and non-adsorption of products, unlike activated carbon, the amount of adsorbent resin is not limited, that is, it can be used in any amount as long as it is sufficient to remove impurities;

[0149] - it does not contain trace amounts of toxic elements such as nickel, lead and arsenic that may be present in activated carbon and that may be released into the product stream; and

[0150] - Due to the presence of weakly basic sites, it is used as an insoluble base with acid adsorption properties, replacing the activated carbon and basic anion exchange resin treatment steps that characterize the prior art processes.

[0151] In step e), the purified HMO-containing stream is concentrated and dried to obtain neutral or salivary fluid in solidified form. Acidified HMO:

[0152] Preferably, after all separation / purification steps have been applied, in particular after process steps a) to d), the neutral or sialylated HMO of interest is provided in its solid form by a concentration and drying step (step e)).

[0153] The concentration step is used to economically remove a large amount of liquid, mainly water, from a stream containing neutral or sialylated HMO using, for example, evaporation, nanofiltration or reverse osmosis filtration. The evaporation process can include, for example, falling film evaporation, rising film evaporation and rotary evaporation. The evaporation can also be carried out under vacuum. The solid concentration fed to the process is preferably about 5-30 wt.%. The solid concentration at the outlet of this process is generally greater than 30 wt.%, more preferably greater than 50 wt.%. More preferably, the solid concentration at the outlet of the dehydration operation is 60-80 wt.%. The solid portion of the recovered material is preferably greater than 80 wt.% neutral or sialylated HMO.

[0154] In one embodiment, the purified neutral or sialylated HMO-containing stream is concentrated to a neutral or sialylated HMO concentration of ≥ 100 g / l, preferably ≥ 200 g / l, more preferably ≥ 300 g / l.

[0155] When the purified neutral or sialylated HMO-containing stream is concentrated by evaporation, the concentration is preferably carried out at a temperature of about 20 to about 80° C. In some embodiments, the concentration is carried out at a temperature of 25-75° C. In some embodiments, the concentration is carried out at a temperature of 30-70° C. In some embodiments, the concentration is carried out at a temperature of 30-65° C. Preferably, the concentration is carried out under vacuum.

[0156] When the purified stream containing neutral or sialylated HMO is concentrated by membrane filtration, any membrane, typically a nanofiltration membrane, is suitable that can sufficiently reject neutral or sialylated HMO. Concentration by membrane filtration typically provides a solution of about 30-35 wt.%. This concentration may be suitable for subsequent drying and solidification steps, such as freeze drying. However, other drying methods may require a more concentrated solution, such as spray drying or crystallization. In this case, drying by evaporation (preferably under vacuum) is a preferred embodiment. Alternatively, the stream containing neutral or sialylated HMO obtained in the previous step is concentrated to about 30-35 wt.% using nanofiltration, and the solution is further dried by evaporation.

[0157] In one embodiment of concentration by membrane filtration, the membrane of choice is a "tight" NF membrane with a MWCO of 150-300 Da.

[0158] In other embodiments of concentration by membrane filtration, the membrane selected is a nanofiltration membrane ("loose" NF membrane) with a molecular weight cutoff (MWCO) of 500-3500 Da and an active (top) layer of polyamide; the concentration step is carried out so that the pH is set to less than 5.0, preferably less than 4.5, advantageously less than 4.0, but preferably not less than 3.0. In this latter embodiment, a significant reduction in all monovalent salts in the retentate can also be achieved. In said embodiment, the membrane is preferably a thin film composite (TFC) membrane, which is a piperazine-based polyamide membrane, more preferably having a MgSO4 rejection of about 50-90%, and even more preferably having a NaCl rejection of no more than 50%. Examples of such membranes are UA60. Under this condition, residual salt is also effectively removed to obtain a purified neutral or sialylated HMO concentrate with low or virtually no salt. In this embodiment, after the concentration step is completed, the pH of the neutral or sialylated HMO concentrate is advantageously set to 4-6 before the next step (e.g., evaporation, drying and solidification, sterile filtration).

[0159] When the drying step is freeze drying, the concentration step may be optional.

[0160] In one embodiment, the method according to the invention further comprises the following steps: wherein preferably after the concentration step, the HMO-containing solution is sterile filtered and / or subjected to endotoxin removal, preferably by filtering the purified solution through a 3 kDa filter. Said optional steps are preferably performed before the drying step. The sterile filtration step does not affect the purity of the HMO-containing solution.

[0161] In a preferred embodiment, the drying step comprises, preferably consists of, spray drying of the stream containing neutral or sialylated HMOs.

[0162] Preferably, spray drying results in a solidified neutral or sialylated HMO having an amorphous structure, ie an amorphous powder is obtained.

[0163] In one embodiment, spray drying is performed under the following conditions: the concentration of neutral or sialylated HMO is 20-60% (w / v), preferably 30-50% (w / v), more preferably 35-45% (w / v), the inlet temperature is 110-150°C, preferably 120-140°C, more preferably 125-135°C, and / or the outlet temperature is 60-80°C, preferably 65-70°C.

[0164] In some embodiments, the Brix value of the stream containing neutral or sialylated HMO fed to the spray dryer is about 8% to about 75% of Brix. In some embodiments, the Brix value is about 30% to about 65% of Brix. In some embodiments, the Brix value is about 50% to about 60% of Brix. In some embodiments, the feed entering the spray dryer is at a temperature of about 2°C to about 70°C in the spray dryer just before being dispersed into droplets. In some embodiments, the feed entering the spray dryer is at a temperature of about 30°C to about 60°C in the spray dryer just before being dispersed into droplets. In some embodiments, the feed entering the spray dryer is at a temperature of about 2°C to about 30°C in the spray dryer just before being dispersed into droplets. In some embodiments, spray drying uses air with an air inlet temperature of 120-280°C. In some embodiments, the air inlet temperature is 120-210°C. In some embodiments, the air inlet temperature is about 130°C to about 190°C. In some embodiments, the air inlet temperature is about 135°C to about 160°C. In some embodiments, spray drying uses air with an air outlet temperature of 80-110°C. In some embodiments, the air outlet temperature is about 100°C to about 110°C. In some embodiments, spray drying is carried out at a temperature of about 20°C 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 fluidized bed. In some embodiments, the spray dryer includes a rotating disk, a high pressure nozzle, or a two-fluid nozzle. In some embodiments, the spray dryer includes an atomizer wheel. In some embodiments, spray drying is the final purification step for the desired neutral or sialylated HMO.

[0165] Alternatively, the drying and curing step comprises an indirect drying method. For the purposes of this specification, indirect dryers are 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 through the walls of the dryer, such as through the walls of the housing in the case of a drum dryer, or alternatively, through the walls of hollow paddles in the case of a paddle dryer, the paddles rotating in the solid while a heat transfer medium circulates in the hollow interior of the paddles. Other examples of indirect dryers include contact dryers and vacuum drum dryers.

[0166] Alternatively, the drying and solidification step comprises freeze drying.

[0167] Alternatively, the drying and solidification step comprises crystallization (provided that the HMO is obtainable in crystalline form).

[0168] Other optional method steps:

[0169] In one embodiment, after the treatment with the acidic cation exchange resin (step c)), the HMO-containing stream is neutralized to a pH of about 7 by adding sufficient sodium hydroxide. In this case, the concentration step is advantageously a membrane filtration, the membrane selected being a nanofiltration membrane ("loose" NF membrane) with a molecular weight cutoff (MWCO) of 500-3500 Da and an active (top) layer of polyamide; the concentration step is carried out so that the pH is set to less than 5.0, preferably less than 4.5, advantageously less than 4.0, but not less than 3.0. Under these conditions, the removal of all monovalent salts (such as sodium salts) in the retentate can be achieved.

[0170] In another embodiment, the method according to the invention, including its preferred and more preferred embodiments, further comprises the step wherein preferably after the concentration and before the drying step, the purified solution containing neutral or sialylated HMO is sterile filtered and / or subjected to endotoxin removal, preferably by filtering the purified solution through a 3 kDa filter, or using a membrane with a pore size of less than 0.5 μm, less than 0.4 μm, less than 0.3 μm or less than 0.2 μm. It is to be noted that although the sterile filtration step does not constitute a solution to the technical problem, i.e., purifying the HMO from the fermentation broth in which the HMO is produced and separating it from biomass, proteins, protein fragments, fragments of genetic material originating from genetically modified microorganisms, salts, fermentation additives, metabolic byproducts, in particular non-carbohydrate byproducts, chromatosomes, etc., so as to make it suitable for human administration. When the method disclosed above is carried out under appropriate sterile conditions, it is not necessary to perform sterile filtration. However, according to one embodiment, the sterile filtration step disclosed above can be part of the method of the invention.

[0171] Specific implementation methods of the invention:

[0172] In a preferred embodiment, the process according to the invention does not comprise an anion exchange resin treatment step.

[0173] In a preferred embodiment, the process according to the invention does not comprise a purification / decolorization step using activated carbon.

[0174] In a further preferred embodiment, the method according to the invention comprises or consists of the following steps (preferably in consecutive order):

[0175] i. separating the fermentation broth to form a stream containing HMO and a biomass waste stream;

[0176] ii. purifying the HMO-containing stream by nanofiltration, wherein the molecular weight cut-off (MWCO) of the nanofiltration membrane is 500-3500 Da;

[0177] iii. The nanofiltration retentate was treated with H + - Purification by acidic cation exchange resins;

[0178] iv. purifying the cation exchange resin eluate with an adsorptive resin;

[0179] v. The adsorption resin eluate is concentrated and the purified HMO concentrate is dried to obtain neutral or sialylated HMO in a solidified form.

[0180] In a further preferred embodiment, the method according to the invention comprises or consists of the following steps (preferably in consecutive order):

[0181] i. separating the fermentation broth to form a stream containing HMO and a biomass waste stream;

[0182] ii. purifying the HMO-containing stream by nanofiltration, wherein the molecular weight cut-off (MWCO) of the nanofiltration membrane is 500-3500 Da;

[0183] iii. The nanofiltration retentate was treated with H + - Purification by strong acidic cation exchange resin;

[0184] iv. purifying the cation exchange resin eluate with an adsorptive resin;

[0185] v. concentrating the adsorbent resin eluate and drying the purified HMO concentrate to obtain neutral or sialylated HMO in a solidified form,

[0186] The cation exchange resin eluate is neutralized to a pH of about 7 before performing step iv).

[0187] More preferably, the active (top) layer of the nanofiltration membrane consists of a polyamide, more preferably a piperazine-based polyamide, with a MgSO4 rejection of about 50-90%, preferably a NaCl rejection of not more than 50%. More preferably, the nanofiltration step is carried out so that the pH is set to less than 5.0, preferably less than 4.5, advantageously less than 4.0, but preferably not less than 3.0, ensuring the rejection of the neutral or sialylated HMOs to be purified, allowing monovalent and divalent salts to pass and accumulate in the permeate, and allowing at least a portion of the lactose to pass and accumulate in the permeate.

[0188] In addition, preferably, including the preferred and more preferred embodiments disclosed above, the adsorptive resin is an acid adsorbent derived from a cross-linked polystyrene or polyacrylic acid polymer and partially functionalized with tertiary amine functional groups. More preferably, the surface area of ​​the adsorptive resin is >400 m 2 / g, for example, 400-1200m 2Still more preferably, the capacity of the adsorptive resin is 0.1-0.3 eq / l.

[0189] 3. Neutral or sialylated human milk oligosaccharides produced by the method according to the present invention

[0190] In another aspect, the present invention relates to neutral or sialylated human milk oligosaccharides obtained by the process according to the present invention.

[0191] The neutral or sialylated HMO recovered and purified according to the methods described in this specification may be amorphous or crystalline, preferably amorphous.

[0192] In a preferred embodiment, the purity of the neutral or sialylated HMO on a dry basis is greater than 80 wt.% on a dry matter basis for a single neutral or sialylated HMO and greater than 70% on a dry matter basis for a mixture of HMOs. More preferably, the purity of a single neutral or sialylated HMO is greater than 90 wt.%.

[0193] In preferred embodiments, the neutral or sialylated HMO has at least one of the following characteristics (by weight): lactose <2%, fucose <3%, galactose <1%, or glucose <3%.

[0194] In one embodiment, the fine particle fraction (less than or equal to 10 μm) of neutral or sialylated HMO is less than 10%, preferably less than 5%, more preferably less than 1%, and most preferably less than 0.1%. In addition, the average particle size (d50) of neutral or sialylated HMO is preferably greater than 100 μm, more preferably greater than 150 μm, and even more preferably greater than 200 μm.

[0195] Neutral or sialylated HMO produced according to the method of the present invention exhibit good flowability. Preferably, the Carr index of the HMO is less than 30, wherein the Carr index (C) is determined by the formula C = 100 (1-ρB / ρT), wherein ρB is the freely settled bulk density of the powder and ρT is the tapped bulk density after "tappingdown". For free-flowing solids, the values ​​of bulk density and tapped bulk density will be close, so the index is small. For solids with poor flowability, the difference between these values ​​will be larger, so the Carr index will be larger.

[0196] In preferred embodiments, the neutral or sialylated 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.%. To optimize product recovery, the pH of the neutral or sialylated HMO is preferably greater than 3.0 in at least 5% of the 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 pH of the neutral or sialylated HMO is between 4 and 7, more preferably between 4.5 and 5.5.

[0197] Preferably, the HMO is a neutral HMO. In one embodiment, the neutral HMO is preferably selected from 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 (also known as: lacto-N-fucopentaose VI), lacto-N-difucohexaose I, lacto-N-difucohexaose II, lacto-N-difucohexaose III, 6'-galactosyl lactose, 3'-galactosyl lactose, lacto-N-hexaose, lacto-N-neohexaose and any mixture thereof. More preferably, the HMO is 2'-fucosyllactose, 3-fucosyllactose, 2',3-difucosyllactose, lacto-N-triose II, lacto-N-tetraose, lacto-N-neotetraose or lacto-N-fucopentaose, more preferably 2'-fucosyllactose, LNT, LNnT or lacto-N-fucopentaose.

[0198] In one embodiment, the sialylated HMO is selected from 3'-sialyllactose (3'-SL) and 6'-sialyllactose (6'-SL).

[0199] In one embodiment, the neutral or sialylated HMO obtained by the method according to the invention is incorporated into a food (eg, human or pet food), a nutritional supplement or a pharmaceutical product.

[0200] In some embodiments, neutral or sialylated HMOs are incorporated into human infant foods (e.g., infant formula). Infant formula is typically a manufactured food for feeding infants as a complete or partial substitute for human breast milk. In some embodiments, infant formula is sold in powder form and is prepared for feeding infants in bottles or cups by mixing with water. The composition of infant formula is typically designed to roughly mimic human breast milk. In some embodiments, neutral or sialylated HMOs purified by the methods of this specification are included in infant formula to provide similar nutritional benefits to those provided by one or more neutral or sialylated HMOs in human breast milk. In some embodiments, neutral or sialylated HMOs are mixed with one or more infant formula ingredients. Examples of ingredients of infant formula include skim milk, a carbohydrate source (e.g. lactose), a protein source (e.g. whey protein concentrate and casein), a fat source (e.g. vegetable oils, such as palm oil, high oleic safflower oil, rapeseed oil, coconut oil and / or sunflower oil; and fish oil), vitamins (e.g. vitamins A, B, B2, C and D), minerals (e.g. potassium citrate, calcium citrate, magnesium chloride, sodium chloride, sodium citrate and calcium phosphate).

[0201] In this context, another aspect of the present invention relates to the use of neutral or sialylated human milk oligosaccharides obtained by the process according to the invention in medicine.

[0202] In this context, a further aspect of the present invention relates to the use of neutral or sialylated human milk oligosaccharides obtained by the process according to the invention for food and / or feed applications.

[0203] In this regard, another aspect of the present invention relates to a food or cosmetic product comprising a neutral or sialylated human milk oligosaccharide obtained by the process according to the invention.

[0204] Example

[0205] Example 1: Production and purification of lacto-N-salina pentasaccharide I (LNFP-I) and 2'-fucosyllactose (2'-FL)

[0206] Fermentation: Using LacZ - ,LacY +A genetically modified Escherichia coli (E. coli) strain of a genotype that produces a fermentation broth containing LNFP-I at a 20 L scale by fermentation, wherein the strain includes: a recombinant gene encoding a β-1,3-N-acetyl-glucosaminyltransferase (the enzyme is capable of transferring the GlcNAc of UDP-GlcNAc to internalized lactose); a recombinant gene encoding a β-1,3-galactosyltransferase (the above enzyme is capable of transferring the galactosyl residue of UDP-Gal to N-acetyl-glucosaminylated lactose (lacto-N-triose II or LNT-2) to form LNT (lacto-N-tetraose)) and an α-1,2-fucosyltransferase (the enzyme is capable of transferring the fucose of GDP-fucose to LNT); and genes encoding the synthesis pathways of UDP-GlcNAc, UDP-Gal and GDP-fucose. Fermentation was performed by culturing the strain in the presence of exogenously added lactose and a suitable carbon source, thereby producing LNFP-I in the fermentation broth, accompanied by 2'-FL, trace amounts of LNT and unreacted lactose as major carbohydrate impurities.

[0207] Through ultrafiltration, nanofiltration and strong acid cation exchange (UF / NF / SAC (H + )) Purification: The obtained fermentation broth was acidified to pH = 4.0 with H2SO4. A portion of the fermentation broth (12 kg of the 4x3 kg batch) was purified by Kerasep 15 kDa membrane (BE-8 channel, size 1178x25 mm, area 0.21 m 2 ) was treated by ultrafiltration in batch mode under the following conditions: temperature 60°C, transmembrane pressure (TMP) 2-2.5 bar, tangential flow (cross-flow) 600 l / h in two steps: first, the volume was reduced by removing the permeate to a concentration factor CF=2, and then diafiltration was continued with 6 kg of deionized water, each time producing 7.00 kg of ultrafiltration permeate (4x7 kg batches, a total of 28 kg).

[0208] The combined permeate (27.7 kg) was filtered through a Trisep UA60 membrane (spiral wound size 1812, area 0.23 m 2, pore size / MWCO 1000-3500Da) was treated by tangential flow loose nanofiltration at 30-40°C and TMP 38: 23kg of permeate was first collected to obtain a concentrated retentate (Brix 19.4, conductivity 17.3mS / cm), which was then continuously diafiltered with 25kg of deionized water to obtain an additional 27kg of diafiltration permeate. The obtained retentate (2.680kg, Brix 22.0) was collected from the system and subjected to additional washing (deionized water, 500ml) to obtain a final nanofiltration / diafiltration retentate (3.17kg, Brix 18.8, conductivity 1.30mS / cm, pH 4.11) with greatly reduced conductivity.

[0209] A portion of the obtained nanofiltration retentate (2.67 kg) was passed through a strongly acidic cation exchange resin Dowex-88 (H + -form, bed volume (BV) = 800 ml), followed by water (600 ml) to give 3.21 kg of a tan solution (Brix 15.2, conductivity 4.22 mS / cm, pH 1.85).

[0210] Purification by adsorption resin (AR): A small amount of sample (119 g, pH 1.85) treated with Dowex-88 was passed through a PDMS filter filled with Dowex Optipore SD-2 adsorption resin (15.6 g, 58% water content, surface area: about 800 m 2 / g, dry weight capacity: 0.8eq / kg, BV=24ml) and eluted with water to obtain 167g of solution (Brix 10.0, conductivity 0.110mS / cm, pH 3.64). After each step, the column was regenerated by washing with an aqueous wash solution (water wash) of 4% HCl, 4% NaOH and 4% NaCl. The obtained solution was reprocessed through the regenerated resin at 75ml / h (3BV / h) to obtain 215g of a completely colorless solution (Brix 7.5, conductivity 0.024mS / cm, pH 5.0, Abs_400=0.0000, protein<0.5mg / l), which was then freeze-dried.

[0211] Analysis of the data showed that AR treatment not only completely removed the color but also reduced the protein concentration by an order of magnitude.

[0212] Example 2: Production and purification of 2'-fucosyllactose (2'-FL) and difucosyllactose (DFL):

[0213] Fermentation: Using LacZ - ,LacY +A genetically modified Escherichia coli strain of a genotype produces a fermentation broth containing 2'-FL, wherein the strain includes a recombinant gene encoding an α-1,2-fucosyltransferase (an enzyme capable of transferring fucose of GDP-fucose to internalized lactose) and a gene encoding a GDP-fucose synthesis pathway. Fermentation is performed by culturing the strain in the presence of exogenously added lactose and a suitable carbon source, thereby producing 2'-FL in the fermentation broth, which is accompanied by unreacted lactose as a major carbohydrate impurity.

[0214] Purification by ultrafiltration, nanofiltration and strong acid cation exchange (UF / NF): The fermentation broth obtained was treated by UF / NF as described in Example 1, i.e., by pH adjustment, tangential flow UF and diafiltration NF to obtain a NF retentate with the following parameters: Brix 23.3, conductivity 3.80 mS / cm, pH 3.65, Abs_400 2.899, color index CI_400 124.

[0215] The strong acid cation exchange and adsorption resin (SAC (H + ) / AR) purification: A portion of the NF retentate (1.11 kg, 1020 ml, 6 BV) was passed through a Dowex-88 (H + -form, BV=170 ml), then immediately passed through Dowex Optipore SD-2 adsorbent resin (120 g wet resin, 58% water content, BV=172 ml) packed in another XK16 / 100 column, with a flow rate of about 500 ml / h (3 BV / h), followed by water (2 BV, 340 ml). 35 ml (0.2 BV) fractions were collected and analyzed for Brix, conductivity, pH, Abs-400 and protein content.

[0216] Fraction #6-23 (630 ml, 3.66 BV, Brix 18.3, CI_400 1.78) was combined, pH was adjusted to 4.8 with 4% NaOH solution, and freeze-dried (protein 17 mg / kg). The remaining fractions #24-40 were also combined and freeze-dried.

[0217] Example 3: Lacto-N-tetraose (LNT), lacto-N-triose II (LNTri II) and p-lacto-N-hexaose II (pLNHII) Production and purification of

[0218] Fermentation: Using LacZ - ,LacY +A genetically modified Escherichia coli strain of a genotype, producing a fermentation broth containing LNT by fermentation at a scale of 20 l, wherein the strain comprises: a recombinant gene encoding a β-1,3-N-acetyl-glucosaminyltransferase (the enzyme is capable of transferring the GlcNAc of UDP-GlcNAc to internalized lactose); a recombinant gene encoding a β-1,3-galactosyltransferase (the enzyme is capable of transferring the galactosyl residue of UDP-Gal to N-acetyl-glucosaminylated lactose (lacto-N-triose II) to form LNT (lacto-N-tetraose)); and genes encoding the biosynthetic pathways of UDP-GlcNAc and UDP-Gal. The strain is fermented by culturing the strain in the presence of exogenously added lactose and a suitable carbon source, thereby producing LNT in the fermentation broth, which is accompanied by lacto-N-triose II, pLNH II and unreacted lactose as the main carbohydrate impurities.

[0219] Ultrafiltration (UF): The fermentation broth obtained was acidified to pH = 3.7 with H2SO4. A portion of the fermentation broth (12.86 kg) was filtered through a Tami 15 kDa membrane (39 channels, size 1178 x 25 mm, area 0.5 m 2 ) was treated by UF in batch mode under the following conditions: temperature 60°C, transmembrane pressure 6-7 bar, tangential flow 600 l / h in two steps: 1) 9190 g of permeate was collected, and then 2) diafiltration was continued with 14.55 kg of water at a feed rate of about 5-6 l / h. A total of 24.52 kg of permeate was collected and the retentate (about 2.85 kg) was discarded.

[0220] Nanofiltration (NF): The obtained UF permeate (24.43 kg, Brix 9.9, conductivity 9.10 mS / cm, pH 4.58, Abs_400 0.9465) was filtered through a Trisep UA60 membrane (spiral wound size 1812, area 0.23 m 2, MWCO 1000-3500Da) was processed by tangential flow loose nanofiltration at 30-48°C, TMP=30-31bar, and 14.85kg of permeate and concentrated retentate (Brix 23.0, conductivity 10.10mS / cm, pH 4.30, Abs_400 2.0552) were collected. The pH of the retentate was adjusted to 5.6 with 50% NaOH solution, and then diafiltered with 30l of water added continuously at a flow rate of 5.1l / h to obtain 46kg of combined permeate and intermediate retentate (about 8.38kg, Brix 21.1, conductivity 3.05mS / cm, pH 5.45, Abs_400 1.6358). Then, the pH of the intermediate retentate was adjusted to 3.71 with 25% H2SO4 solution, and then additional diafiltration was performed with 10l of water. The obtained retentate (Brix 20.4, conductivity 1.77 mS / cm, pH 3.83, Abs_400 1.3853) was further concentrated under the condition of TMP=39 bar to obtain the final retentate (5.48 kg, Brix 30.4, conductivity 1.35 mS / cm, pH 4.06, Abs_400 2.0307).

[0221] Strong acid cation exchange (SAC (H + )) The obtained NF retentate (5.41 kg) was passed through Dowex-88 (H + -form, BV=800 ml), eluted with water to obtain a yellow-brown solution (6.1 kg, Brix 26.6, conductivity 5.21 mS / cm, pH 1.62, Abs_400 0.8269).

[0222] Adsorbent resin (AR) treatment: The SAC (H + ) was passed through a column filled with Dowex Optipore SD-2 adsorptive resin (170 ml) at a flow rate of 510 ml / h and eluted with water to obtain 415 g of solution (Brix 15.5, conductivity 0.023 mS / cm, pH 8.4, Abs_400 0.0450), which was adjusted to pH 4.4 with 25% H2SO4 solution and freeze-dried to obtain 60.9 g of white solid.

[0223] Example 4: Determination of the retention rate of a substance on a membrane

[0224] The retention of NaCl and MgSO4 on the membrane was determined as follows: In the membrane filtration system, a NaCl (0.1%) or MgSO4 (0.2%) solution was circulated across the selected membrane (for Tami: tubular module), while the permeate stream was circulated back to the feed tank. The system was equilibrated at 10 bar and 25°C for 10 minutes, and then samples were taken from the permeate and retentate. The retention was calculated from the conductivity measured by the samples: (1-κ p / κ r )·100, where κ p is the conductivity of NaCl or MgSO4 in the permeate, κ r It is the conductivity of NaCl or MgSO4 in the retentate.

[0225]

[0226] The carbohydrate retention was determined in a similar manner, except that the retention was calculated from the concentration of the sample (determined by HPLC): (1-C p / C r )·100, where C p is the carbohydrate concentration in the permeate, C r is the carbohydrate concentration in the retentate.

[0227] Example 5: Comparison of MgSO4 and Na2SO4 Retention Rates

[0228] 2.0 1 of 0.2% MgSO4 solution was loaded onto a Trisep UA60 element (piperazine-amide, MWCO 1000-3500 Da, membrane area 0.23 m2) equipped with a 1812-size spiral wound 2 ) in an MMS SW18 system. The system was operated at 400 l / h tangential flow with the permeate recycled back to the feed tank. The system was equilibrated for at least 5 minutes at each condition or until the conductivity in the permeate was constant. The pH was adjusted by adding a small amount of 25% H2SO4 solution. The conductivity of the permeate and retentate are disclosed in the table below.

[0229]

[0230] The same experiment was performed using 0.2% Na2SO4 solution.

[0231]

[0232] It has been demonstrated that in the case of polyamide membranes NF, the rejection of sodium salts with divalent counterions, such as sulfate, is highly pH dependent. Thus, when DF is performed at a pH of less than 4.5, advantageously less than 4.0, inorganic anions can be effectively removed in the NF / DF step together with sodium cations, resulting in a virtually salt-free solution (as assessed by conductivity). In this regard, it is not necessary to use a basic anion resin in order to obtain a salt-free solution.

Claims

1. A method for purifying neutral or sialylated human milk oligosaccharides (HMO) from a fermentation broth, comprising the following steps: a. separating the fermentation broth to form a stream containing HMO and a biomass waste stream; b. Purifying the HMO-containing stream by nanofiltration; c. purifying the HMO-containing stream with an acidic cation exchange resin; d. Purifying the HMO-containing stream with an adsorptive resin; and e. concentrating and drying the purified HMO-containing stream to obtain neutral or sialylated HMO in solidified form, Provided that, when the drying step is freeze-drying, the concentration step is optional.

2. The method according to claim 1, wherein the molecular weight cut-off (MWCO) of the nanofiltration membrane in step b) is 500-3500 Da.

3. The method according to claim 2, wherein the active layer of the membrane consists of a polyamide, more preferably a piperazine-based polyamide, and the membrane has a MgSO4 rejection of about 50-90%.

4. The process according to claim 3, wherein step b) is carried out so that the pH is set to less than 5.0, preferably less than 4.5, advantageously less than 4.0, but preferably not less than 3.

0.

5. The method according to any one of claims 1 to 4, wherein the acidic cation exchange resin in step c) is a strongly acidic cation exchange resin.

6. The method according to claim 5, wherein the strongly acidic cation exchange resin is a polystyrene-divinylbenzene cation exchange resin having a sulfonic acid functional group, more preferably H + -form.

7. The process according to any one of the preceding claims, wherein the concentration step prior to drying is evaporation or nanofiltration.

8. The method according to any one of the preceding claims, wherein the drying step is spray drying or freeze drying.

9. The method according to any one of the preceding claims, wherein the adsorptive resin is an acid adsorbent derived from a cross-linked polystyrene or polyacrylic polymer and the adsorptive resin is partially functionalized with tertiary amine functional groups.

10. The method of claim 9, wherein the surface area of ​​the adsorptive resin is greater than 400 m 2 / g.

11. The method according to claim 10, wherein the acid adsorption capacity of the adsorbent resin is 0.6-1.0 eq / kg on a dry weight basis.

12. The method according to any one of the preceding claims, wherein the HMO is a neutral HMO.

13. The method according to claim 12, wherein the neutral 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-N-difucopentaose II, lacto-N-neotetra ... Algaecohexose II, lacto-N-difucohexaose III, 6'-galactosyl lactose, 3'-galactosyl lactose, lacto-N-hexaose and lacto-N-neohexose, more preferably 2'-fucosyllactose, 3-fucosyllactose, 2',3-difucosyllactose, lacto-N-triose II, lacto-N-tetraose, lacto-N-neotetraose and lacto-N-fucopentaose, and even more preferably 2'-fucosyllactose, LNT, LNnT and lacto-N-fucopentaose.

14. The method of any one of claims 1-11, wherein the HMO is a sialylated HMO.

15. The method of claim 14, wherein the sialylated HMO is 3'-sialyllactose (3'-SL) or 6'-sialyllactose (6'-SL).

16. A process according to any one of the preceding claims, wherein no purification / decolorisation step using activated carbon is involved.

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