Purification of human milk oligosaccharides

Abstract

Process for increasing the concentration of a human milk oligosaccharide (HMO) in a solution, relative to smaller sized saccharides, said process comprising the use of a cascade of at least two membrane filtration stages, including a first membrane filtration stage and a series of one or more membrane filtration stages downstream of said first membrane filtration stage, wherein each HMO-depleted permeate is fed to the next available membrane filtration stage in the series, while the HMO-enriched retentates are recirculated to an upstream membrane filtration stage.

Description

[0001] P1742PC00 PURIFICATION OF HUMAN MILK OLIGOSACCHARIDES

[0002] The invention relates to a process for increasing the concentration of a human milk oligosaccharide (HMO) in a solution, relative to smaller sized saccharides.

[0003] Human milk contains various oligosaccharides (HMOs), which are important for a healthy development of infants. Many HMOs serve an important role in the development of a healthy intestinal microbiome, the establishment of a particular intestinal flora, and stimulation of the immune system. Although HMOs are consumed by infants, it is accepted that the beneficial effects of these oligosaccharides will also apply later in life.

[0004] Numerous HMOs exist and more than 150 HMOs have been structurally characterized. Most HMOs contain a lactose moiety at the reducing end and a lot of them contain a fucose, N-acetyl glucosamine, and / or sialic acid moiety. The monosaccharides from which most HMOs are derived are D-glucose, D-galactose, N-acetylglucosamine, L-fucose, and sialic acid. The HMOs most abundantly present in human milk are fucosylated HMOs such as 2’-fucosyllactose (2’-FL) and 3’-fucosyllactose (3’-FL), galacto-oligosaccharides such as 3’-galactosyllactose (3’-GL) and 6’-galactoseyllactose (6’-GL), sialylated HMOs such as 3’-sialyllactose (3’-SL) and 6’-sialyllactose (6’-SL), and N-acetylated HMOs like lacto-N-tetraose (LNT) and lacto-N-neotetraose (LNnT).

[0005] Galactosyllactoses and fucosylated and N-acetylated HMOs, such as 3’-GL, 6’-GL, 2’-FL, 3-FL, LNT, and LNnT, are neutral, in the sense that they are not charged. Sialylated HMO’s, such as 3’-sialyllactose (3’-SL) and 6’-sialyllactose (6’-SL), are acidic / negatively charged and are generally prepared as a salt, conventionally the sodium salt.

[0006] Many of these oligosaccharides can also be found in milk from other animals. For instance, 3’-SL and 6’-SL can also be found in bovine milk, whereas 2’-FL and 3’-GL can be found in goat milk.

[0007] The commercial production of HMOs and their addition to infant formula has gained increasing interest over the past years. Their synthesis, however, remains a challenge. The most successful synthesis procedures are fermentation processes using2 P1742PC00 recombinant microorganisms. Apart from the challenge of creating an optimal microorganism, also the purification of the HMO from the fermentation broth is a challenging task, since the final fermentation broth generally contains, apart from the target HMO, undesired oligosaccharides (intermediates, by-products, and starting substances), biomass, protein (fragments), DNA (fragments), and minerals. These compounds need to be removed to the highest possible degree.

[0008] A review of fermentative synthesis routes towards HMOs is provided by K. Bych et al., Current Opinions in Biotechnology 56 (2019) 130-137. Generally, recombinant E-coli strains are the microorganisms of choice, together with lactose as the exogeneous sugar acceptor. Sucrose, glucose, and glycerol have been reported as primary carbon and / or energy sources.

[0009] The produced HMO is purified from the fermentation broth by removal of the biomass, followed by various purification techniques, including cation- and anion-exchange chromatography, ultrafiltration, reverse osmosis, electrodialysis, and / or activated carbon treatment.

[0010] Also nanofiltration (NF) has been disclosed as suitable step in the purification of HMOs. Nanofiltration, when applying a membrane with a molecular weight cut-off smaller than the molecular weight of the target HMO, is a technique that is able to concentrate the HMO and at the same time remove smaller molecules and salts. Examples of such smaller molecules are lactose and monosaccharides like glucose, galactose, fucose, etc. A great advantage of nanofiltration is that it is a simple, scalable, and cost-efficient technique that allows continuous processing.

[0011] For instance, WO 2015 / 106943 discloses a nanofiltration step as an alternative or as an addition to electrodialysis in the removal of salts and for the concentration of neutral HMOs obtained by fermentation.

[0012] Luo et al., Bioresource Technology 166 (2014) 9-16, applied nanofiltration for the purification of enzymatically produced 3’-SL. In a first NF step, 3’-SL was retained by the membrane. A second NF step was applied to the permeate of the first NF step, which second NF membrane retained lactose, which lactose was then re-used in the enzymatic process.3 P1742PC00 WO 2022 / 263425 also applied nanofiltration in order to purify an HMO. The HMO was retained in the retentate and this retentate was subsequently subjected to a further NF step in order to further concentrate the HMO.

[0013] WO 2019 / 229118 discloses the purification of sialyllactose from other carbohydrates using two different membranes in the nanofiltration range; a first one (300-550 Da) for removing smaller molecules and leaving the sialyllactose in the retentate; a second one (600-800 Da) for retaining larger molecules and passing the sialyllactose through the membrane into the permeate. Hence, the sialyllactose-enriched retentate resulting from the first filtration was subjected to the second filtration; the sialyllactose ending up in the permeate of the second filtration.

[0014] As indicated above, a starting material in the fermentative production of HMOs is lactose. Not all the lactose will generally be converted, meaning that lactose is generally one of the components that has to be removed during HMO purification. The conventional HMO purification steps as listed above, except for nanofiltration, are not able to remove lactose from neutral HMOs.

[0015] Theoretically, nanofiltration should be able to remove lactose from an HMO, but especially for neutral trisaccharides like 2’-FL and 3-FL this is a challenge, as it requires a membrane that permeates lactose (MW 342 g / mol) but retards 2’-FL or 3-FL (MW488 g / mol).

[0016] Similar problems arise with the removal of intermediate oligosaccharides from larger target HMOs, such as the removal of LNT-II from LNT.

[0017] The removal, with nanofiltration, of saccharides with molecular weights close to that of the target HMO requires a compromise in terms of membrane selection and filtration conditions: optimized permeation of impurities on the one hand and optimized retention of the HMO on the other. The effect of such compromise is that a significant amount of the target HMO will still permeate the membrane, thereby negatively affecting the HMO yield, and / or a significant amount of lactose will remain in the retentate, thereby negatively affecting the purity of the HMO.

[0018] The present invention provides a solution to this problem by using a cascade of membrane filtration stages in which the HMO-depleted permeate(s) is / are subjected to one or more downstream membrane filtration stages and wherein the HMO-4 P1742PC00 enriched retentate(s) of the downstream membrane filtration stage(s) is / are recirculated to an upstream membrane filtration stage. The HMO-enriched retentate of the first membrane filtration stage is collected and optionally submitted to further membrane filtration, purification, concentration, and / or drying steps.

[0019] In this way, HMO initially permeating the membrane is captured in the downstream membrane filtration stages, thereby improving the purification yield.

[0020] The invention therefore relates to a process for increasing the concentration of a human milk oligosaccharide (HMO) in a solution, relative to smaller sized saccharides, said process comprising the use of a cascade of at least two membrane filtration stages, including a first membrane filtration stage and a series of one or more membrane filtration stages downstream of said first membrane filtration stage, the process comprising the following steps:

[0021] (i) feeding the solution to the first membrane filtration stage in order to obtain a first HMO-enriched retentate and a first HMO-depleted permeate,

[0022] (ii) feeding the first HMO-depleted permeate to said series of one or more membrane filtration stages, each stage producing an HMO-enriched retentate and an HMO-depleted permeate, each HMO-depleted permeate being fed to the next available membrane filtration stage in the series,

[0023] (iii) recirculating the HMO-enriched retentates resulting from step (ii) by feeding said HMO-enriched retentates to an upstream membrane filtration stage within said cascade of at least two membrane filtration stages,

[0024] (iv) optionally feeding the HMO-enriched retentate from the first membrane filtration stage to a second series of one or more membrane filtration stages, each stage producing an HMO-enriched retentate and a HMO-depleted permeate, each HMO-enriched retentate being fed to the next available membrane filtration stage in the series, and

[0025] (v) collecting the HMO-enriched retentate of either the first membrane filtration stage or, if step (iv) is conducted, the last membrane filtration stage of the second series of one or more membrane filtration stages.

[0026] The process involves the purification / concentration of a human milk oligosaccharide (HMO) relative to smaller sized saccharides. The term “human milk oligosaccharide” refers to specific saccharides, irrespective of their actual origin. That is: the HMO to5 P1742PC00 be purified or concentrated does not have to originate from human milk, but may also originate from milk from other animals or be made synthetically. The reference to human milk only serves to indicate that the specific saccharide is known to be present in human milk.

[0027] For the avoidance of doubt, the term “oligosaccharide” implies a polymerization degree (DP) of at least three, meaning that mono- and di-saccharides are not encompassed by the term (human milk) oligosaccharides.

[0028] HMOs that can be purified / concentrated via the process of the present invention include neutral and acidic HMOs, preferably being selected from galactosyllactoses and oligosaccharidess containing fucosyl, N-acetyl glucosamine, and / or sialyl groups. Examples of neutral HMOs are 3’-galactosyllactose (3’GL), 4’-galactosyllase (4’-GL), 6’-galactosyllactose (6’-GL), N-acetyllactosamine (LacNAc), lacto-N-biose (LNB), lacto-N-triose II (LNT II), lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT), lacto-N-hexaose (LNH), lacto-N-neohexaose (LNnH), para-lacto-N-hexaose (para-LNH), para-lacto-N-neohexaose (para-LNnH), 2'-fucosyllactose (2'-FL), 3-fucosyllactose (3-FL), 2',3-difucosyllactose (DFL), lacto-N-fucopentaose I (LNFP I), lacto-N-neofucopentaose I (LnNFP I), lacto-N-fucopentaose II (LNFP II), lacto-N-fucopentaose III (LNFP III), lacto-N-fucopentaose V (LNFP V), lacto-N-neofucopentaose V (LnNFP V), F-LNnH, lacto-N-difucohexaose I (DF-LNH I), lacto-N-difucohexaose II (DF-LNH II), para-lacto-N-difucohexaose (DF-para-LNH) and para-lacto-N-difuconeohexaose (DF-para-LNnH).

[0029] Examples of acidic HMOs are 3’-sialyllactose (3'-SL), 6’-sialyllactose (6'-SL), fucosylsialyllactose (FSL), sialyllacto-N-tetraose a (LST-a), sialyllacto-N-tetraose b (LST-b), sialyllacto-N-tetraose c (LST-c) and disyalyllacto-N-tetraose (DS-LNT), fuco-sialyllacto-N-tetraose a (F-LST-a), fuco-sialyllacto-N-tetraose b (F-LST-b), and fuco-sialyllacto-N-tetraose c (F-LST-c).

[0030] Preferably, the HMO is selected from the group consisting of 2’-fucosyllactose (2’-FL), 3-fucosyllactose (3-FL), 3’-sialyllactose (3’-SL), 6’-sialyllactose (6’-SL), lacto-N-tetraose (LNT), and lacto-N-neotetraose (LNnT), even more preferably it is selected from 2’-fucosyllactose (2’-FL) and 3-fucosyllactose (3-FL), and most preferably it is 2’-fucosy I lactose (2’-FL).6 P1742PC00 The HMO is purified / concentrated from a solution comprising, apart from said HMO, smaller sized impurities, in particular smaller sized saccharides. This solution and the HMO may be of natural origin or of synthetic origin.

[0031] Examples of solutions containing HMO from natural origin are milk, e.g. bovine or goat milk, and milk fractions, e.g. whey, whey ultrafiltration permeate, or delactosed whey ultrafiltration permeate.

[0032] Examples of solutions containing HMO of synthetic origin are fermentation broths resulting from the fermentative production of an HMO, solutions resulting from the enzymatic production of an HMO, and solutions resulting from the chemical synthesis of an HMO.

[0033] In a preferred embodiment, the solution originates from an HMO-containing fermentation broth from which at least biomass, and preferably also protein (fragments), DNA (fragments), and at least part of the minerals, has / have been removed.

[0034] As already explained above, HMO-containing fermentation broths can be obtained by methods disclosed in the prior art using recombinant microorganisms, such as recombinant bacteria, yeasts, or fungi, more preferably recombinant bacteria or yeasts, even more preferably recombinant bacteria, most preferably recombinant E. coli. Lactose is generally used as exogeneous sugar acceptor, while sucrose, glucose, and glycerol are conventionally used as primary carbon and / or energy sources.

[0035] The resulting HMO-containing fermentation broth will contain, apart from the target HMO, various types of impurities, including undesired oligosaccharides (intermediates, by-products, and starting substances), biomass, protein (fragments), DNA (fragments), and minerals.

[0036] The biomass can be removed from the fermentation broth by conventional methods, such as centrifugation, microfiltration (MF), and / or ultrafiltration (UF), as extensively described in WO 2022 / 26425. MF is a preferred method for the removal of biomass and is preferably carried out with a membrane having a pore size of less than 1 pm, preferably of about 0.1 to about 0.2 pm. MF is particularly suitable to remove cell material (complete cells, fragments thereof) and other supramolecular debris. MF can be performed at a temperature in the range of 20-75°C, more preferably 30-75°C, most preferably 35-70°C, such as 40-50°C or 60-70°C. A temperature in the range 60-70°C is advantageous for achieving a higher concentration factor of cell material during7 P1742PC00 microfiltration, which has a positive effect on the yield of the HMO. Prior to or during biomass removal, the fermentation broth can be subjected to a degassing step. Degassing is advantageous in that it reduces the risk of the formation of gas bubbles during subsequent purification steps.

[0037] Proteins, DNA (fragments), and / or endotoxins can be conveniently removed from the clarified fermentation broth by ultrafiltration (UF), e.g., using a membrane with a molecular weight cut-off of 10 kDa or less, more preferably about 1-5 kDa, most preferably 1 -3 kDa.

[0038] The majority of the minerals may be conveniently removed by applying nanofiltration and / or ion exchange steps. Said ion exchange steps may include both anion and cation exchange treatments. Ion-exchange steps may be combined with electrodialysis and / or nanofiltration steps.

[0039] If desired, also additional purification steps may be conducted, such as crystallization, chromatography steps, optionally in the form of simulated moving bed chromatography, activated carbon treatment (to remove, e.g., colouring bodies), depth filtration with charged media (to reduce negatively charged DNA, endotoxins and other host cell proteins), sterile filtration, and / or pasteurization or heat sterilization (to remove, e.g. microorganisms).

[0040] The resulting solution, optionally after adjustment of the pH to below 8, preferably below 7, and most preferably to a value in the range 3-5, is fed to the cascade of membrane filtration stages according to the process of the present invention. This cascade serves to remove remaining impurities with a smaller molecular size than the target HMO, more in particular monosaccharides, disaccharides (incl. lactose), small organic acids, and salts.

[0041] The cascade of at least two membrane filtration stages according to the present invention provides a first membrane filtration stage and a series of one or more, preferably one or two, most preferably one membrane filtration stage(s) downstream of said first membrane filtration stage.

[0042] A membrane filtration stage is defined as a series of one or more membrane filtration loops, each loop resulting in a retentate bleed stream that is fed to the subsequent loop in the series, if any. A membrane filtration stage therefore results in a single retentate - i.e. the retentate of the last loop in the series - and a collected permeate for all loops.8 P1742PC00 A membrane filtration loop is defined as a system in which a feed stream is continuously circulated - e.g., by using a recirculation pump - through one or more pressure vessels each containing one or more membranes. Within a loop, the pressure vessels may be connected in parallel or in series.

[0043] During the entire process, it is preferred that the fluid flow is tangential to the membrane surfaces. This creates a constant disruption of accumulated mass due to constant shear and thereby limits the buildup of a fouling layer on the membrane surface. The opposite model, called dead end filtration, is not preferred. It results in fluid being pressed against a membrane, which over time results in a thick cake being formed due to the constant removal of permeate. This cake needs to be removed periodically, e.g. via backwashing, mechanical cleaning, and / or air scouring, resulting in lost process time and product loss with discharge of the cake residue.

[0044] The HMO-containing solution is fed to the first membrane filtration stage in the cascade.

[0045] The type of membrane(s) and filtration conditions to be used in this first membrane filtration stage should be such that it enables the retention of a significant part of the target HMO, while permeating a significant part of the smaller sized saccharides. In other words, the first membrane filtration stage should result in an HMO-enriched retentate - i.e. a retentate that has a higher concentration of the target HMO, relative to smaller sized saccharides, than the solution that was fed to the first membrane filtration stage - and an HMO-depleted permeate - i.e. a permeate that has a lower concentration of the target HMO relative to smaller sized saccharides, than the solution that was fed to the first membrane filtration stage.

[0046] The membranes to be used have a molecular weight cut-off in the range 200-2500 Da and preferably a MgSO4 rejection of at least 10%. Such membranes are generally called nanofiltration membranes, but may also be referred to as tight ultrafiltration membranes. The specific membrane to be used will depend on the HMO to be purified. In one embodiment, the membranes have a MgSO4 rejection of at least 50%, preferably 70-99%, more preferably 90-98.5%, and most preferably 94-98%. Membranes with this MgSO4 rejection are generally tight for tri- and higher oligosaccharides and loose for mono- and disaccharides, thereby facilitating the9 P1742PC00 separation of HMO from smaller sized impurities. Such membranes are particularly preferred for the purification of neutral trisaccharides, such as 2’-FL and 3’-FL. The membranes of this embodiment preferably have an molecular weight cut-off (MWCO) in the range of 200-1500 Da, more preferably 250-700 Da, and most preferably 300-500 Da.

[0047] In another embodiment, the membranes have a MgSO4 rejection in the range of 10-90%, preferably 20-80%, more preferably 30-70%, and most preferably 40-60%. Such membranes are particularly preferred for the purification of acidic trisaccharides, such as 3’-SL and 6’-SL. The membranes of this embodiment preferably have a MWCO in the range of 500-2500 Da, more preferably 800-1500 Da, and most preferably 1000-1200 Da.

[0048] The MgSO4 rejection of a membrane can be determined by circulating an aqueous 2000 ppm MgSO4 solution across the selected membrane while circulating the permeate stream back into a feed tank. The system is equilibrated for 10 minutes at 10 bars trans membrane pressure (TMP) and 20°C before taking samples from the permeate and retentate. The rejection factor is calculated from the measured conductivity of the samples: (1-κp / κr)·100, wherein κp is the conductivity of MgSO4 in the permeate and κr is the conductivity of MgSO4 in the retentate.

[0049] The top layer of the membrane, i.e. the membrane surface that comes into contact with the HMO, can be made of various materials; either inorganic, polymeric, or hybrid. Examples of polymeric materials are polysulfone (PS), (modified) polyethersulfone (PES), sulfonated polyethersulfone (S-PES), polyvinylidene difluoride (PVDF), polyamide (PA), polyelectrolyte multilayer (PEM), polyacrylonitrile (PAN), cellulose acetate (CA), and polypropylene (PP), while preferred polymeric materials are polysulfone (PS), polyethersulfone (PES), sulfonated polyethersulfone (S-PES), polyamide (PA), polyelectrolyte multilayer (PEM), and polyvinylidene difluoride (PVDF). In a preferred embodiment, the membrane is a thin-film composite with a top layer made of a polyamide (PA), more preferably polypiperazine, or a polyelectrolyte multilayer membrane.

[0050] The HMO-depleted permeate resulting from the first membrane filtration stage is then fed to the series of one or more downstream membrane filtration stages. Each of these downstream stages produces an HMO-enriched retentate (i.e. a retentate that has a10 P1742PC00 higher concentration of the target HMO, relative to smaller sized saccharides, than the solution that was fed to the downstream stage) and an HMO-depleted permeate (i.e. a permeate that has a lower concentration of the target HMO, relative to smaller sized saccharides, than the solution that was fed to the downstream stage). The permeate is then fed to the next downstream membrane filtration stage, if any. The permeate resulting from the final membrane filtration stage in the series, which will hardly contain any residual HMO, can be discarded (optionally after a reverse osmosis treatment), while the HMO-enriched retentates of the downstream membrane filtration stages are fed back to an upstream membrane filtration stage within the cascade. This means that the HMO-enriched retentate resulting from the second membrane filtration stage in the cascade (i.e. the first stage downstream of the first membrane filtration stage) will be fed to the first membrane filtration stage. The HMO-enriched retentate resulting from the third and higher membrane filtration stages in the cascade (i.e. the second and higher stages downstream of the first membrane filtration stage) can be fed directly to the first membrane filtration stage or to any other upstream stage. Preferably, the HMO-enriched retentate resulting from the nthstage in the cascade is fed to the (n-1 )thstage of the cascade.

[0051] The HMO-enriched retentate resulting from the first membrane filtration stage may optionally be fed to a second series of one or more membrane filtration stages, each stage producing an HMO-enriched retentate and an HMO-depleted permeate, each HMO-enriched retentate being fed to the next available membrane filtration stage in the series.

[0052] The retentate of this first membrane filtration stage or, if said retentate is fed to said second series of membrane filtration stages, the retentate of the last stage in said second series, is collected.

[0053] In one embodiment, the same membrane is used in all membrane filtration stages. The advantage of this embodiment is that it simplifies the process.

[0054] In another embodiment, the membranes in different stages differ. For instance, the membranes in the first stage may have a lower retention for the HMO (and, thus, a lower MgSO4 rejection) than the membrane(s) in the downstream stage(s). Such embodiment increases lactose permeation and, thus, purification in the first stage,11 P1742PC00 while reducing HMO loss in the subsequent stage(s). When more than one downstream stage is applied, the membranes in these downstream stages may be the same or may increase in HMO retention along the series. The term “retention” in this respect relates to 1-(mass fraction of a specific compound in the permeate relative to the mass fraction of that compound in the retentate).

[0055] In a particularly preferred embodiment, the membrane in the first stage has an HMO / lactose separation factor of at least 1, more preferably at least 2, most preferably at least 5; the separation factor being defined as:

[0056]

[0057] wherein the concentration (c) is in g / kg.

[0058] In a preferred embodiment, one or more of the membrane filtration stages is / are combined with diafiltration. Diafiltration involves the addition of fresh water in order to remove membrane permeable components, thereby improving the HMO purity and yield.

[0059] In one embodiment, the HMO-enriched retentate collected from the first membrane filtration stage or, if step(iv) is applied, the last membrane filtration stage of the second series of one or more membrane filtration stages, preferably has a purity of at least 90 wt% on dry matter, more preferably at least 94 wt% on dry matter, and most preferably at least 96 wt% on dry matter.

[0060] If desired, also additional purification steps may be conducted, such as crystallization, chromatography steps, optionally in the form of simulated moving bed chromatography, activated carbon treatment (to remove, e.g., colouring bodies), depth filtration with charged media (to reduce negatively charged DNA, endotoxins and other host cell proteins), sterile filtration, and / or pasteurization or heat sterilization (to remove, e.g. microorganisms). It is also possible to conduct a demineralisation step at this stage, e.g. by electrodialysis, ion-exchange, or a combination thereof. This is especially preferred if no such demineralisation has been conducted to the solution before the membrane filtration treatment. The advantage of performing such demineralisation after, instead of before the membrane filtration is that smaller equipment is required12 P1742PC00 for said demineralisation step (as part of the minerals has been removed with the membrane filtration), thereby making the process more economical.

[0061] The resulting HMO-containing product may subsequently be dried to form a powder, for instance by spray-drying, freeze-drying, or spray-cooling, most preferably by spraydrying. Before drying, the HMO may first be concentrated by, e.g., evaporation or reverse osmosis.

[0062] It is also possible to enzymatically convert any remaining lactose in the product into galacto-oligosaccharides (GOS), thereby producing a composition comprising both HMO and GOS. This is particularly advantageous for HMO-enriched retentates obtained by applying the process of the present invention to milk, whey, whey ultrafiltration permeate, or de-lactosed whey ultrafiltration permeate as the solution fed to the first membrane filtration stage.

[0063] The purified / concentrated HMO-containing product resulting from the process of the present invention can be used in nutritional compositions. This can be nutritional compositions for pregnant women (MUM compositions), infants or young children (e.g. formula milk), adolescents (13-20 years of age), or adults (>20 years of age). The nutritional composition can be used as a regular food composition, as nutritional therapy, as nutritional support, as medical food, as a food for special medical purposes, or as a nutritional supplement.

[0064] An example of a nutritional composition is formula milk. Formula milk includes infant formulas, follow-up formulas and growing-up formulas (also called young child formulas). Other examples of nutritional compositions are compositions for adults, such as patients or frail elderly or anyone else desiring to boost the immune system or gut health.

[0065] Infant formula, baby formula or just formula (American English) or baby milk, infant milk or first milk (British English), is a manufactured food designed and marketed for feeding to babies and infants under 12 months of age, usually prepared for bottlefeeding or cup-feeding from powder (mixed with water) or liquid (with or without additional water). The U. S. Federal Food, Drug, and Cosmetic Act (FFDCA) defines infant formula as "a food which purports to be or is represented for special dietary use solely as a food for infants by reason of its simulation of human milk or its suitability13 P1742PC00 as a complete or partial substitute for human milk". Similarly, the Codex Alimentarius international food standards (WHO and FAO) defines infant formula as a breast-milk substitute specially manufactured to satisfy, by itself, the nutritional requirements of infants during the first months of life up to the introduction of appropriate complementary feeding. The Codex Alimentarius describes the essential composition of an infant formula with amounts and specifications for the lipid source, protein source, carbohydrate source, vitamins and minerals.

[0066] In order to constitute the nutritional composition, in particular the formula milk, the purified / concentrated HMO-containing product obtained by the invention - either as aqueous composition or a (spray)dried powder obtained from it - is blended with the further ingredients of the nutritional composition. In case of formula milk, these ingredients include at least one protein source, at least one lipid source, vitamins and minerals. Preferably, the aqueous composition is added to a liquid blend of said ingredients.

[0067] The lipid source for use in formula milk may be any lipid or fat suitable for use in formula milk. Preferred fat sources include milk fat, safflower oil, egg yolk lipid, canola oil, olive oil, coconut oil, palm kernel oil, soybean oil, fish oil, palm oleic, high oleic sunflower oil and high oleic safflower oil, and microbial fermentation oil containing long-chain, polyunsaturated fatty acids. In one embodiment, anhydrous milk fat is used. The lipid source may also be in the form of fractions derived from these oils such as palm olein, medium chain triglycerides, and esters of fatty acids such as arachidonic acid, linoleic acid, palmitic acid, stearic acid, docosahexaenoic acid, linolenic acid, oleic acid, lauric acid, capric acid, caprylic acid, caproic acid, and the like. Small amounts of oils containing high quantities of preformed arachidonic acid and docosahexaenoic acid such as fish oils or microbial oils may be added. The fat source preferably has a ratio of n-6 to n-3 fatty acids of about 5:1 to about 15:1; for example about 8:1 to about 10:1. In a specific aspect, the infant formula comprises an oil mix comprising palmitic acid esterified to triacylglycerols, for example wherein the palmitic acid esterified in the sn-2 position of triacylglycerol is in the amount from 10% to 60% by weight of total palmitic acid and palmitic acid esterified in the sn-1 / sn-3 position of triacylglycerol is in the amount of from 30% to 80% by weight of total palmitic acid.14 P1742PC00 Examples of protein sources include milk, preferably bovine milk, whey protein sources like whey protein concentrate and serum protein concentrate, and various plant proteins. The proteins may be hydrolysed or unhydrolysed.

[0068] Examples of vitamins and minerals that are preferably present in formula milk are vitamin A, vitamin B1, vitamin B2, vitamin B6, vitamin B12, vitamin E, vitamin K, vitamin C, vitamin D, folic acid, inositol, niacin, biotin, pantothenic acid, choline, calcium, phosphorous, iodine, iron, magnesium, copper, zinc, manganese, chloride, potassium, sodium, selenium, chromium, molybdenum, taurine, and L-carnitine. Minerals are usually added in salt form.

[0069] If necessary, the nutritional composition may contain emulsifiers and stabilisers such as soy lecithin, citric acid esters of mono- and di-glycerides, and the like. It may also contain other substances which may have a beneficial effect such as lactoferrin, nucleotides, nucleosides, probiotics, and the like. Suitable probiotics include Lactobacteria, Bifidobacterium lactis such as Bifidobacterium lactis Bb12, Streptococcus thermophilus, Lactobacillus johnsonii La1, Bifidobacterium longum BL999, Lactobacillus rhamnosus LPR, L rhamnosus GG, Lactobacillus reuteri, Lactobacillus salivarius. Such prebiotics are commercially available.

[0070] FIGURE

[0071] Figure 1 displays a flow chart of a single stage process (left) and a two-stage cascade (right) HMO purification process.

[0072] EXAMPLES

[0073] Example 1

[0074] A Design of Experiments (DOE RSM design) was performed with the total component concentration (dry matter, dm; wt%), the temperature (T; °C), the membrane cross flow velocity (u; m / s), and the transmembrane pressure (TMP; bar) as factors. The total flux and the retention of the different components were selected as response variables. The experiments were conducted on a lab scale unit using a flat sheet of a Trisep® XN45 membrane (Mann+Hummel), with a membrane surface area of 0.025 m2A spacer of 34 mil was used.15 P1742PC00 The HMO liquid feed material contained a mixture of 2’-FL, monosaccharides (DP1 ), disaccharides (DP2), and other carbohydrates, as analysed by HPLC. Glucose and lactose were used to represent the DP1 and DP2 groups.

[0075] The HMO liquid feed material composition was (on dry matter):

[0076] 2’-FL: 918.0 g / kg

[0077] Monosaccharides:17.6 g / kg

[0078] Disaccharides: 37.4 g / kg

[0079] Other carbohydrates: 27.0 g / kg

[0080] To simulate both the cascade process according to the invention as well as a single stage process, mathematic modelling (JMP 18.0.1) was performed to create models for the flux and transmembrane pressure at 10°C and 50°C for different concentrations of the HMO component retentions as a function of temperature, TMP-Π, dry matter, and crossflow velocity; where TMP-Π is the transmembrane pressure (TMP) minusthe osmotic pressure (Π), the driving force for the filtration.

[0081] The osmotic pressure was measured by varying the feed material and recording the obtained flux through the membrane. Linear regression was applied to the observed flux as a function of transmembrane pressure for each dry matter / temperature combination. The osmotic pressure for the given concentration is the value for which the transmembrane pressure equals zero flux. The results are listed in the Table below:

[0082]

[0083] 16 P1742PC00 The osmotic pressure results were fitted with the following equation:

[0084]

[0085] where:

[0086] Xdm = total solids mass fraction (kg / kg),

[0087] T = Temperature (°C)

[0088] D0, D1, D2, D3 = Fitted parameters

[0089] The values of the fitted parameters are shown in the following table:

[0090]

[0091] The flux and retention modelling was performed using the software’s fit model function. The stepwise fit function was used to determine which parameters should be included in the model and which should not. To avoid overfitting, the model with the lowest residuals but also the lower corrected Akaike Information Criterion value (AICc) was selected. As fitted models, a response surface including the third order polynomials of each operating parameter were obtained.17 P1742PC00 The equation giving both flux and retentions is:

[0092] xdm- 0.1885 T - 30.8 (TMP - 11) - 27.566 Flux or rt= Ao i+ Al i-n__r- 1-2t — - 1- A3 t- „„ „ „ „ -10.1775 ' 21.1 ' 27.414 u — 0.4825 -I- A. - 1- A..

[0093]

[0094] .34'10.28955'1\ 0.1775 J6'1\ 0.1775 J

[0095]

[0096] / T - 30.8\2 / T - 30.8\3((TMP - 11) - 27.566+ i47'i -\ 21.1 J+ A8’i -\ 21.1 J+ A9'i -^ 27.414

[0097]

[0098] ((TMP - n) - 27.566^, (u - 0.4825^,

[0099] + A10'z’ \ 27.414 J+ A1' \ 0.2895 )+ A12'Z

[0100] (u — 0.4825\3ixim— 0.1885\ / T — 30.8\

[0101] . _ ] -I- A Iam_ l. l _ 1 -1- 4 \ 0.2895 J13'1\ 0.1775 J \ 21.1 / 14'1

[0102] / xdm~ 0.1885\ / (TMP — 11) — 27.566\ / xdm~ 0.1885\ ’ \ 0.1775 J ’ \ 27.414 J+ A15’1' \ 0.1775 J / u - 0.4825^, / T - 30.8\ ( TMP - 11) - 27.566^,

[0103] ’ \ 0.2895 J+ A16' 21.1 J ' \ 27.414 J+ A17’1

[0104]

[0105] ' \ 21.1 J \ 0.2895 J1Sli\ 27.414 ) / u — 0.4825\

[0106] ' \ 0.2895 J

[0107] wherein:

[0108] Xdm = Solution total solids mass fraction (kg / kg),

[0109] T = Temperature (°C),

[0110] TMP-Π = Transmembrane Pressure – Osmotic pressure (bar)

[0111] u = Crossflow velocity (m / s)

[0112] A0-A18 = Fitted parameters18 P1742PC00 The fitted parameters for the flux and each component retention are shown in the following table:

[0113]

[0114] The flux and retention models described above were integrated in flowsheet models in gPROMS representing the single stage process and the two-stage cascade. These flowsheet models were then used to compare the performance of the single stage process to the two-stage cascade. The following input settings were used for both cases: feed flowrate 10 m3 / h, feed pressure 10 bar in loops 1, 2 and 4 and 30 bar in loop 3, pressure drop 1.2 bar in all membrane housings, and temperature 50°C in all loops. The dry matter of the feed could vary between 0.01 and 0.05 kg / kg. The retentate dry matter content was always between 0.28 and 0.32 kg / kg.

[0115] The tables below list the yield at different purity levels for the single stage process and the two-stage cascade, and the average flux and retention values in each loop for the19 P1742PC00 single stage case and the two-stage cascade case, respectively. The results show that significantly higher yield values can be obtained at the same purity level by using a cascade instead of a single stage process.

[0116]

[0117] Average flux and retention values in each loop for the single stage process (retentions of all other carbohydrates equal to 1):

[0118]

[0119] 20 P1742PC00 Average flux and retention values in each loop fora two-stage cascade process (retentions of all other carbohydrates are equal to 1):

[0120]

[0121] Example 2

[0122] Example 1 was repeated, except for using a 3’-SL-containing solution. This solution had a composition in line with that disclosed by Nordvang et al., Separation and Purification Technology 138 (2014) 77-83:

[0123]

[0124] 21 P1742PC00 Three scenarios were applied, based on the results for three different membranes disclosed by Nordvang et al., using the following flux and retention values:

[0125]

[0126] The same single stage process and two stage cascade that were used for the 2’-FL example above are also used here. The maximum achievable yield at different purity levels for the three scenarios are displayed below. As in the 2’-FL case, also with 3’-SL a significant increase in yield can be obtained by using a two stage cascade instead of a single stage process.

[0127]

Claims

22 P1742PC00 CLAIMS1. Process for increasing the concentration of a human milk oligosaccharide (HMO) in a solution, relative to smaller sized saccharides, said process comprising the use of a cascade of at least two membrane filtration stages, including a first membrane filtration stage and a series of one or more membrane filtration stages downstream of said first membrane filtration stage, the process comprising the following steps:(i) feeding the solution to the first membrane filtration stage in order to obtain a first HMO-enriched retentate and a first HMO-depleted permeate, (ii) feeding the first HMO-depleted permeate to said series of one or more membrane filtration stages, each stage producing an HMO-enriched retentate and an HMO-depleted permeate, each HMO-depleted permeate being fed to the next available membrane filtration stage in the series, (iii) recirculating the HMO-enriched retentates resulting from step (ii) by feeding said them to an upstream membrane filtration stage, (iv) optionally feeding the HMO-enriched retentate from the first membrane filtration stage to a second series of one or more membrane filtration stages, each stage producing an HMO-enriched retentate and an HMO- depleted permeate, each HMO-enriched retentate being fed to the next available membrane filtration stage in the series, and(v) collecting the HMO-enriched retentate of either the first membrane filtration stage or, if step (iv) is conducted, the last membrane filtration stage of the second series of one or more membrane filtration stages.

2. The process according to claim 1 wherein the HMO is selected from the group consisting of neutral and acidic HMOs containing fucosyl, N-acetyl glucosamine, and / or sialyl groups, preferably selected from the group consisting of 2’- fucosy I lactose (2’-FL), 3-fucosy I lactose (3-FL), 3’-sialyllactose (3’-SL), 6’- sialyllactose (6’-SL), lacto-N-tetraose (LNT), and lacto-N-neotetraose (LNnT), even more preferably selected from 2’-fucosyllactose (2’-FL) and 3- fucosy I lactose (3-FL), and most preferably is 2’-fucosyllactose (2’-FL).23 P1742PC00 3. The process according to claim 1 or 2 wherein the solution is obtained by fermentative production of the HMO to obtain a fermentation broth, followed by removal of biomass, protein (fragments), DNA (fragments), and minerals from said fermentation broth.

4. The process according to claim 3 wherein the fermentation broth has been subjected to microfiltration, ultrafiltration, and ion-exchange steps.

5. The process according to claim 1 or 2 wherein the solution is selected from bovine milk, whey, whey ultrafiltration permeate, and de-lactosed whey ultrafiltration permeate.

6. The process according to any one of the preceding claims wherein the collected HMO-enriched retentate of the first membrane filtration stage or, if step (iv) is conducted, the last membrane filtration stage of the second series of one or more membrane filtration stages, is subjected to one or more demineralization steps, preferably selected from the group consisting of ion exchange and electrodialysis7. The process according to any one of the preceding claims wherein the collected HMO-enriched retentate of the first membrane filtration stage or, if step (iv) is conducted, the last membrane filtration stage of the second series of one or more membrane filtration stages, is dried to form a powder.

8. The process according to any one of the preceding claims wherein the process is conducted using membranes with a MgSO4rejection of at least 50%, preferably in the range of 70-99%, more preferably 90-98.5%, and most preferably 94-98%.

9. The process according to any one of claims 1 -7 wherein the process is conducted using membranes with a MgSO4rejection in the range of 10-90%, preferably in the range of 20-80%, more preferably 30-70%, and most preferably 40-60%.24 P1742PC00 10. The process according to any one of the preceding claims wherein the process is conducted using membranes with a molecular weight cut-off (MWCO) in the range of 200-1500 Da, preferably 250-700 Da, most preferably 300-500 Da.

11. The process according to any one of claims 1 -9 wherein the process is conducted using membranes with a molecular weight cut-off (MWCO) in the range of 500- 2500 Da, preferably 800-1500 Da, most preferably 1000-1200 Da.

12. The process according to any one of the preceding claims wherein the membranes in the first membrane filtration stage have a lower MgSO4retention and / or a lower molecular weight cut-off (MWCO) than the membranes in downstream membrane filtration stages.

13. The process according to any one of the preceding claims wherein membranes are polyelectrolyte multilayer membranes or have a polypiperazine top layer.

14. Process for the preparation of a composition comprising a human milk oligosaccharide (HMO) and galacto-oligosaccharides (GOS) comprising the steps of(i) performing the process of any one of claims 1 -12,(ii) enzymatically converting lactose that is present in the HMO-enriched retentate of the first membrane filtration stage or, if step (iv) is conducted, the last membrane filtration stage of the second series of one or more membrane filtration stages, into galacto-oligosaccharides (GOS).

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