Process for manufacturing a powder blend of oligosaccharides

Abstract

Disclosed is a process for manufacturing a powder blend essentially consisting of at least two structurally distinct human milk oligosaccharides (HMOs), the process comprising a) providing at least two spray-dried powders, wherein each spray-dried powder essentially consists of a structurally distinct HMO and b) mixing the at least two spray-dried powders in a vertical convective mixer having a fixed mixing vessel and a motor-driven agitator, wherein the mixing occurs by rotation of the agitator inside the fixed mixing vessel.

Description

[0001] PROCESS FOR MANUFACTURING A POWDER BLEND OF OLIGOSACCHARIDES

[0002] The present invention relates to a process for manufacturing a powder blend consisting of human milk oligosaccharides and a powder blend obtained via the process.

[0003] Background

[0004] Human milk is composed of fats, proteins, vitamins, minerals, trace elements and complex oligosaccharides. Besides lactose, human milk contains various structurally diverse oligosaccharides which are also known as human milk oligosaccharides (HMOs) (Urashima T. et al., 2011, Milk Oligosaccharides, Nova Biomedical Books, New York ISBN 978-1-61122-831-1). Today more than 150 structurally different oligosaccharides have been found in human milk. With very few exceptions, HMOs are characterized by a lactose disaccharide residue at their reducing end on the one hand. On the other hand, many HMOs contain a fucose residue, a galactose residue, a / V-acetylglucosamine or a / V-acetylneuraminic acid residue at their nonreducing end. Furthermore, there are linear as well as branched representatives. Generally, the monosaccharide residues of HMOs are D-glucose, D-galactose, / V- acetylglucosamine, L-fucose and / V-acetylneuraminic acid (the latter also known as sialic acid or lactaminic acid). Studies have reported several health benefits of HMOs, amongst others positive effects on the gut microbiota and the immune system. HMOs also serve as a substrate for beneficial bacteria like Bifidobacteria or Lactobacilli.

[0005] Due to the challenges involved in the chemical synthesis of human milk oligosaccharides, several enzymatic methods and fermentative approaches were developed. In particular, the fermentative approach requires purification of the desired oligosaccharide from a highly complex fermentation broth containing several hundreds of different individual compounds. The carbohydrate fraction of the fermentation broth alone is composed of a complex mixture of mono- and oligosaccharides as well as derivatives thereof including substrates (e.g. lactose, fructose, glucose, saccharose and other sugars used as carbon source), biosynthetic intermediates, individual monosaccharides (such as glucose, galactose, / V-acetylglucosamine, L-fucose and / V-acetylneuraminic acid), metabolic side products and other oligo- and polysaccharides synthesized by the microbe.

[0006] The HMOs thus produced, need to be purified and ideally, dried. The drying process is in principle subject to special requirements. The oligosaccharides to be isolated and dried generally show chemical reactivity comparable to standard primary or secondary alcohols, amides, a-functionalized carboxylic acids, acetals and hemiacetals. Furthermore, these structures are redox- and also biologically active and in addition temperature-sensitive. Therefore, the drying process must be very gentle and must not stress the material in excess by, for example, mechanical stress; also the material must not be exposed to a high heat impact. Moreover, materials used in the drying-machinery must be inert to the carbohydrate and meet food quality criteria.

[0007] In principle, different drying methods can be considered for drying an HMO from solution. Spray-drying is a drying process that is often used for drying and for the formulation of carbohydrates or carbohydrate-containing foods (Woo, M. W. et al. 2013, Chapter 2, Spray drying for food powder production, 29-56, In Bhandari, B., Bansal, N., Zhang, M., Schuck, P., (Editors) Handbook of Food Powders, Processes and Properties, ISBN: 978-0-85709-513-8; Ishwarya, S. P., Chapter 5: Spray Drying, 57-94, In Anandharamakrishnan, C. (Editor) Handbook of Drying for Dairy Products, ISBN: 9781118930526). Spray drying is a method of producing a dry powder from a liquid or slurry by means of a hot gas flow. Thermally-sensitive materials such as foods and pharmaceuticals can be dried in this way as heat contact time is rather short, whereby spray drying ensures a consistent particle size distribution. In most cases air is the heated drying medium; however, nitrogen can be used when inert conditions are necessary. Spray dryers use some type of disc or spray nozzle to disperse the liquid or slurry into a controlled drop size spray. The dry powder often has the advantage to be free-flowing. Some of the disadvantages of spray drying are that there is a risk of a dust explosion and it has a high energy requirement as well as a high demand for air. Spray drying is a pure drying method rather than a purification technique by which no purification is achieved in comparison to crystallization. Spray-drying can be used on a purified solution obtained from the fermentation broth but as well for solutions of sugars that have been obtained in dry form before, for example from crystallization or from another drying method such as band or belt drying and roller or drum drying.

[0008] Depending on their final application, several structurally distinct HMOs are mixed and applied as a mixture. For this purpose, the HMOs have to be mixed in a specific ratio. The ratio of different HMOs should be homogeneous over the whole product volume.

[0009] For this purpose, until now, HMOs have been mixed in dissolved form and then dried from this solution by spray-drying (WO 2019110800 A1). The wet blending step before spray-drying has several disadvantages: The stability of the separate HMOs in solution before mixing is not as good as in dried form, which significantly limits the possible storage times. Furthermore, the HMOs in dissolved form take up more volume and therefore space in the warehouse or during transport than the dried powder. However, the homogeneity of blends obtained from a solution containing a mixture of HMOs is very high.

[0010] The alternative to mixing solutions of different HMOs would be mixing of dried powders of different HMOs. However, there are several problems when implementing this process: It is difficult to obtain a homogeneity similar to the one obtained by wet blending. During dry blending, an increase of temperature and mechanical destruction due to grinding of the mixed powders are possible.

[0011] The process suggested in the present invention is a simple, energy efficient and effective method for obtaining a homogeneous powder containing a mixture of structurally different HMOs.

[0012] Summary

[0013] The present invention concerns a method for mixing several structurally distinct human milk oligosaccharides. The method is suitable for the mixing of the sensitive HMOs. The HMO mixtures are intended for the pharmaceutical or food industry and are therefore heavily regulated. High quality demands have to be fulfilled. It is an object to provide a method for preparing a powder blend that fulfills these requirements by mixing at least two powders in a vertical convective mixer.

[0014] In some embodiment the vertical convective mixer is a helical-ribbon mixer.

[0015] It is another object of the present invention to provide an industrial scale method of mixing at least two HMOs. Specifically, the method should be suitable for providing a powder blend ranging from kg scale to tons scale.

[0016] It is another object of the invention to provide a powder blend that has been prepared by the described process.

[0017] Brief description of the drawings

[0018] Fig. 1 shows a schematical drawing of a conical mixing vessel,

[0019] Fig. 2 shows a schematical drawing of a cylindrical mixer and

[0020] Fig. 3 shows a schematical drawing of a cylindrical mixer with flat bottom.

[0021] Detailed description

[0022] The present invention provides a method for manufacturing a powder blend essentially consisting of at least two structurally distinct human milk oligosaccharides (HMOs). The process comprises at least the following steps: a) providing at least two spray-dried powders, wherein each spray-dried powder essentially consists of a structurally distinct HMO and b) mixing the at least two spray-dried powders in a vertical convective mixer having a fixed mixing vessel and a motor-driven agitator, wherein the mixing occurs by rotation of the agitator inside the fixed mixing vessel.

[0023] In step a) at least two powders, that have been obtained by spray-drying are provided. Each spray-dried powder essentially consists of a structurally distinct HMO; thus each spray-dried powder essentially consists of a different HMO than the other spray-dried powder. Each spray-dried powder is obtained by spray-drying a solution of an HMO. The solutions preferably contain HMOs produced by fermentation. However, it is also possible that one, some or all HMOs have been produced by biocatalysis or chemical synthesis.

[0024] In the context of this application, if a powder or a powder blend essentially consists of a compound I group of compounds, the content of the respective compound I group of compounds in this powder or powder blend is at least 75 wt.-%, at least 80 wt.-%, at least 85 wt.-%, at least 90 wt.-%, at least 95 wt.-% or at least 98 wt.-% on a dry matter basis. The group of compounds most relevant in this application is the group of human milk oligosaccharides.

[0025] In step b), the spray-dried powders to be mixed are added into the mixing vessel of the vertical convective mixer. Inside the mixer, a motor-driven agitator is mounted, which rotates during operation. Thus, the agitator is connected to a motor, which drives the rotation of the agitator. The motor is preferably mounted on the outside of the mixing vessel. The different powders are reoriented in relation to one another because of the mechanical movement by the rotating agitator. Thereby the single spray-dried powders are mixed, thereby resulting in a homogeneous mixture.

[0026] The vertical mixer is preferably loaded from the top of the vessel through a charge valve and emptied through a discharge valve at the bottom of the vessel. Thereby yields up to 99% can be obtained as only little residue stays inside the vertical mixer if the characteristics of the product allow it. The spray-dried powders of HMOs are very suitable in this respect as they have a good flowability. The mixer can of course comprise more than one loading valve and more than one discharge valve depending on the volume of the mixer. Preferred is a mixer with one loading valve and one valve to empty the mixer since this facilitates cleaning and maintenance of the mixer.

[0027] The terms top and bottom refer to the orientation of the mixer in the room where it is located. The direction wherein a material flows due to gravity is from top to bottom. The bottom is thus the part of the mixer oriented towards the floor of the room.

[0028] Furthermore, the operation of a vertical mixer in batch mode and continuous mode is possible. For the use with spray-dried powders consisting of HMOs obtained from fermentation, the operation in batch mode is preferred over the continuous mode, since the fermentation process is working in batches as well.

[0029] The mixing by the motor-driven rotating agitator inside the vessel provides for very gentle mixing. The agitator is made from an inert and stiff material, preferably stainless steel or aluminum. The agitator is preferably only mounted at one side of the vessel, preferably at the top, which facilitates the unloading of the product. The agitator can have different shapes, such as a blade, paddle, ribbon or screw. It is preferred that only one agitator is mounted inside the vessel.

[0030] One advantage of the vertical mixing vessel is that the mixer can be operated at different filling levels ranging from 5 vol.-% up to 100 vol-%, which provides for a very flexible use in production.

[0031] In another embodiment of the process according to the invention, the at least two spray-dried powders are agitated in a helical upward flow at the periphery of the mixing vessel and a downward flow in the center of the mixing vessel. Thus, the agitator is designed in such a way that it creates a helical upward material flow moving particles from the bottom to the top of the vessel, from where the particles move to the bottom in the center of the vessel. These movements facilitate the formation of a homogeneous mix of the material. In another embodiment, the agitator is a helical ribbon agitator. A helical ribbon agitator is especially suitable for creating a helical upward flow at the periphery of the mixing vessel and a downward flow in the center of the mixing vessel. Preferably the helical ribbon agitator has the shape of a double helical ribbon, thus comprising an inner and an outer helical ribbon. The outer helical ribbon creates a movement of the particles towards the center of the vessel whereas the inner helical ribbon creates a movement towards the outside of the vessel. The downward material flow along the rotation axis is created through the difference in peripheral speed between the inner and the outer helical ribbon. Thereby the particles are efficiently distributed.

[0032] Alternatively, the helical ribbon agitator is a single helical ribbon agitator, creating the helical upward flow at the periphery by moving the powders to the top of the vessel, from where gravity flow moves the particles downwards in the center. The single helical ribbon creates minimal mechanical and thermal stress during the mixing process.

[0033] In another embodiment of the process according to the invention, the mixing vessel has a conical shape at least towards the bottom of the mixing vessel. The mixing vessel is wider at the top of the vessel and thinner at the bottom of the vessel. The larger diameter at the top of the vessel ensures a large volume available for material. The smaller diameter at the bottom of the vessel facilitates the discharge of the material, since the material is directed by the converging walls of the vessel towards the discharge valve. This design enables recovering of up to more than 99% of the starting material. In another embodiment, the entire mixing vessel has a conical shape.

[0034] In an alternative embodiment, the mixing vessel has the shape of a cylinder with a fixed radius at the top of the vessel and a cone with a variable radius at the bottom of the vessel. Such a mixing vessel has a relatively high mixing volume and at the same time provides for efficient emptying of the vessel. Preferably, the ratio c / h of the height of the conical part c to the height of the vessel h, is between 0.15 and 0.80.

[0035] In another embodiment, the mixing vessel has the shape of a cylinder. Such a vessel has a large mixing volume available. The mixing vessel has a flat bottom with a valve for emptying the vessel. In another embodiment, a tool for scraping off material from the bottom of the vessel is integrated into the mixer. Such a tool can have the form of a rotating sheet that touches the flat bottom of the vessel and ensures no material sticks to the vessel. It collects the material from the bottom and directs it towards the valve. In the case of emptying the vessel, the rotating sheet ensures that the material is entirely removed from the vessel. In one embodiment the rotating sheet is separate from the motor-driven agitator. In an alternative embodiment, the rotating sheet is part of the motor-driven agitator or connected to it.

[0036] In another embodiment, the vertical convective mixer has a single mixing chamber with preferably one agitator in the single mixing chamber. This design facilitates the emptying and cleaning of the vessel compared to designs with two chambers with two agitators arranged next to each other. The mixing in this case takes place in a superimposed zone between the two moving agitators. This allows for gentle mixing. However, it has been shown that the efficiency of the mixing of spray-dried HMO powders is high in mixing vessels having a single mixing chamber. The recovery of material is more efficient in mixers having a single mixing chamber, which is extremely important since the material loss at the last stage of the production process has to be minimized.

[0037] In another embodiment of the process according to the invention, the mixing is performed for at least 1 minute, preferably for at least 2 minutes, more preferably for at least 3 minutes, but no longer than 15 minutes, preferably no longer than 10 minutes, more preferably no longer than 5 minutes. It has been shown that these mixing times suffice to produce homogeneous blends of spray-dried HMO powders. These rather short mixing times ensure that the integrity of the sensitive HMOs is not compromised. Longer mixing times lead to an increase of mechanical stress.

[0038] In another embodiment of the process according to the invention, the agitator rotates with 5 revolutions per minute (R / min) up to 80 R / min and the volume of the mixing vessel is between 300 dm3and 15 000 dm3. The rotational frequency is influenced by the volume of the mixing vessel. For higher volumes, a lower rotational frequency is required, whereas for lower volumes, a higher rotational frequency is possible. In another embodiment, the agitator rotates with 5 R / min to 25 R / min and the volume of the mixing vessel is between 5 000 dm3and 15 000 dm3. Alternatively, the agitator rotates with 8 R / min to 45 R / min and the volume of the mixing vessel is between 4 050 dm3and 10 000 dm3. Alternatively, the agitator rotates with 40 R / min and 75 R / min and the volume of the mixing vessel is between 200 dm3and 950 dm3.

[0039] In another embodiment, a minimum of 15%, 20%, 30% or 40% and a maximum of 70%, 80%, 90% or 95% of the volume of the mixing vessel is filled with the at least two spray-dried powders. The advantage of the vertical convective mixer is that such a large range of filling levels leads to satisfying results. More preferred are high filling levels between 50 % and 95%, since this results in a higher percentage of recovered material. The spray-dried HMO powders can stick to the wall of the mixing vessel. Thus, the lower the dead volume in the mixer, the higher the percentage of recovered material, which is especially relevant for the mixed HMOs.

[0040] In another embodiment of the process according to the invention, the mixing is performed at a temperature between 14°C and 45°C, preferably between 15°C and 35°C, more preferably between 17°C and 25°C. Preferably, the mixing is performed without active cooling of the mixing vessel and I or without active cooling of the agitator. It has been shown that the HMO powder blends are stable at these temperatures. Not applying active cooling of the walls of the vessel or of the agitator increases the engery-efficiency of the process compared to a process where cooling is required.

[0041] In another embodiment of the process according to the invention, three, four, five, six, seven, eight or more spray-dried powders are mixed. Thus, the resulting powder blend in those cases essentially consists of three, four, five, six, seven, eight or more structurally distinct human milk oligosaccharides. These mixes containing more than two HMOs come closer to the natural occurance in nature, where usually several HMOs are present in different concentrations.

[0042] In another embodiment of the process according to the invention, the at least two structurally distinct HMOs are selected from the group consisting of 2’-fucosyllactose (2’-FL), 3-fucosyl lactose (3-FL), lacto-ZV-tetraose (LA / T), lacto-ZV-neotetraose (L / VnT), lacto-ZV-fucopentaose I (L / VPFI), lacto- / V-fucopentaose II (L / VPFII), lacto-A / - fucopentaose III (L / VPFI II), 3’-sialyllactose (3’-SL), 6’-sialyllactose (6’-SL), sialyllacto-N-tetraose a (LST-a), sialyllacto-N-tetraose b (LST-b), sialyllacto-N- tetraose c (LST-c) and disialyllacto-N-tetraose (DSLNT).

[0043] In another embodiment of the process according to the invention, the powder blend consists of five structurally distinct HMOs, preferably of 2’-FL, 3-FL, L / VT, 3’-SL and 6’-SL. In another embodiment, the powder blend consists of seven structurally distinct HMOs, preferably of 2’-FL, 3-FL, L / VT, L / VnT, L / VFPI, 3’-SL and 6’-SL.

[0044] In another embodiment of the process according to the invention, the at least two spray-dried powders each essentially consist of a structurally distinct HMO, wherein the HMO is present in the powder at a purity of more than 85%, preferably a purity of more than 90%, more preferably a purity of more than 95%. HMOs provided as spray-dried powders in such a high purity benefit from the inventive process, which provides for gentle mixing and low thermal stress.

[0045] The term purity used in this application refers to chemical purity, thus the degree to which a substance is undiluted or unmixed with extraneous material. Hence, the chemical purity is an indicator of the relationship between the at least one HMO and by-products / impurities. Chemical purity is expressed as a percentage (%) and is calculated using the following formula:

[0046] Percent purity= 100x (mass of desired compound in sample) / (total mass of sample)

[0047] The purity can be determined by any suitable method known to the person skilled in the art. One suitable method is HPLC (high-performance liquid chromatography). In the obtained chromatogram, the ratio of the area underneath the peak(s) representing the amount of HMO(s) to the sum of areas underneath the peaks representing the HMO(s) and all other compounds than said HMO(s) in the chromatogram is calculated.

[0048] In another embodiment of the process according to the invention, the at least two spray-dried powders each consisting of a structurally distinct HMO contain less than 15 wt.-%, preferably less than 10 wt.-% of water, more preferably from 4 wt.-% of water up to 8 wt.-% of water. With these water contents, no lumps have been observerd after the mixing.

[0049] In another embodiment of the process, the process is performed in batches. Preferably 200 kg to 10 000 kg, more preferably 2 000 kg to 7 000 kg of the powder blend are produced in one batch.

[0050] In another embodiment of the process, the obtained powder blend has a bulk density from 0.30 kg / dm3to 0.50 kg / dm3, preferably from 0.40 kg / dm3to 0.05 kg / dm3. This bulk density ensures a very good flowability, which is important for handling of the product. The bulk density is determined according to the International Standard DIN / ISO 679.

[0051] In another embodiment of the process according to the invention, in step a) the structurally distinct HMOs have been obtained by microbial fermentation. A solution obtained after a purification of the fermentation broth has been spray-dried to yield the spray-dried powder essentially consisting of an HMO. The mixing of such spray- dried powders according to the described process, yields powder blends with high homogeneity in a short time, requiring minimial energy input.

[0052] The HMOs are preferably obtained by microbial fermentation, wherein a genetically- engineered microorganism that is able to synthesize each desired HMO is cultivated in a culture medium (fermentation broth) and under conditions that are permissive for the synthesis of the desired HMO by said genetically-engineered microorganism.

[0053] The purification of the HMO produced by microbial fermentation comprises the step of separating the microbial cells from the fermentation broth to obtain a cleared process stream which is essentially free of cells and which contains the desired HMO. This step is the first step in the process of purifying the desired oligosaccharides.

[0054] The purification of the HMO from a fermentation broth mentioned above, includes one or more of the following: i) removing the microbial cells from the fermentation broth to obtain a cleared process stream; ii) subjecting the cleared process stream to at least one ultrafiltration; iii) treating the cleared process stream at least one time with a cation exchange resin and / or at least one time with an anion exchange resin; iv) subjecting the cleared process stream to at least one nanofiltration; v) subjecting the cleared process stream to at least one electrodialysis; vi) treating the cleared process stream at least one time with activated charcoal; and / or vii) subjecting the cleared process stream at least one time to a crystallization and / or precipitation step. Suitable methods for removing the microbial cells from the fermentation broth include centrifugation wherein the microbial cells are obtained as a pellet and the fermentation broth as a supernatant. In an additional and / or alternative embodiment, the microbial cells are removed from the fermentation broth by means of filtration. Suitable filtration methods for removing the cells from the fermentation broth include microfiltration and ultrafiltration.

[0055] Microfiltration as such is a physical filtration process where a particle-containing fluid is passed through a special pore-sized membrane to separate the particles from the fluid. The term "microfiltration" as used herein refers to a physical filtration process where cells are separated from the fermentation broth.

[0056] Ultrafiltration is a variety of membrane filtration and is not fundamentally different. In ultrafiltration, forces like pressure or concentration gradients lead to a separation through a semipermeable membrane. Cells, suspended solids and solutes of high molecular weight are retained in the so-called retentate, while water and low molecular weight solutes such as the desired HMO pass through the membrane in the permeate (filtrate). Ultrafiltration membranes are defined by the molecular weight cut-off (MWCO) of the membrane used. Ultrafiltration is applied in cross-flow or dead-end mode.

[0057] Typically, the microbial cells synthesize the desired HMO intracellularly and secrete it into the fermentation broth. The thus produced HMO ends up in the fermentation broth which is then subjected to further process steps for the purification of the HMO as described herein after.

[0058] Notwithstanding that the process is used for the purification of an HMO that has been produced by microbial fermentation, said process may also be employed to purify an HMO that was produced by enzymatic catalysis in-vitro. The HMO can be purified from the reaction mixture at the end of the biocatalytic reaction. Said reaction mixture is subjected to the process for the purification as a cleared process stream. The cleared process stream contains the HMO as well as by-products and undesired impurities such as, for example, monosaccharides, disaccharides, undesired oligosaccharide by-products, ions, amino acids, polypeptides, proteins and / or nucleic acids.

[0059] In an additional and / or alternative embodiment, the process for the purification of the desired HMO comprises the step of at least one cation exchange treatment to remove positively charged compounds from the cleared process stream. Suitable cation exchange resins for removing positively charged compounds include Lewatit S2568 (H+) (Lanxess AG, Cologne, DE).

[0060] In an additional and / or alternative embodiment, the process for the purification of the desired HMO comprises the step of an anion exchange treatment to remove undesired negatively charged compounds from the cleared process stream. Suitable anion exchange resins include Lewatit S6368 A, Lewatit S4268, Lewatit S5528, Lewatit S6368A (Lanxess AG. Cologne, DE), Dowex AG 1 x2 (Mesh 200-400), Dowex 1x8 (Mesh 100-200), Purolite Chromalite CGA100x4 (Purolite GmbH, Ratingen, DE), Dow Amberlite FPA51 (Dow Chemicals, Ml, USA).

[0061] In an additional / or alternative embodiment, the process for the purification of the HMO comprises a nanofiltration and / or a diafiltration step to remove impurities having a lower molecular weight, and to concentrate the desired oligosaccharides. Diafiltration involves the addition of fresh water to a solution to remove (wash out) membrane-permeable components. Diafiltration can be used to separate components on the basis of their molecular size and charge by using appropriate membranes, wherein one or more species are efficiently retained, and other species are membrane permeable. In particular, diafiltration using a nanofiltration membrane is effective for the separation of low molecular weight compounds like small molecules and salts. Nanofiltration membranes usually have a molecular weight cutoff in the range 150 - 1000 Daltons. Nanofiltration is widely used in the dairy industry for the concentration and demineralization of whey. Suitable membranes for nanofiltration and / or diafiltration include Dow Filmtec NF270-4040, Trisep 4040-XN45-TSF (Microdyn-Nadir GmbH, Wiesbaden, DE), GE4040F30 and GH4040F50 (GE Water & Process Technologies, Ratingen, DE).

[0062] Diafiltration using nanofiltration membranes was found to be efficient as a pretreatment to remove significant amounts of contaminants prior to electrodialysis treatment of the solution containing the oligosaccharide. The use of nanofiltration membranes for concentration and diafiltration during the purification of HMOs results in lower energy and processing costs, and better product quality due to reduced thermal exposure, leading to reduced Maillard reactions and aldol reactions.

[0063] In an additional and / or alternative embodiment, the process for the purification of the HMO comprises at least one electrodialysis step. Electrodialysis (ED) is used to transport salt ions from one solution through ion-exchange membranes to another solution under the influence of an applied electric potential difference and it can be used for the separation or concentration of ions in solutions based on their selective electromigration through semipermeable membranes.

[0064] The basic principle of electrodialysis consists of an electrolytic cell comprising a pair of electrodes submerged into an electrolyte for the conduction of ions, connected to a direct current generator. The electrode connected to the positive pole of the direct current generator is the anode, and the electrode connected to the negative pole is the cathode. The electrolyte solution then supports the current flow, which results from the movement of negative and positive ions towards the anode and cathode, respectively. The membranes used for electrodialysis are essentially sheets of porous ion-exchange resins with negative or positive charge groups, and are therefore described as cationic or anionic membranes, respectively. The ionexchange membranes are usually made of polystyrene carrying a suitable functional group (such as sulfonic acid for cationic membranes or a quaternary ammonium group for anionic membranes) cross-linked with divinylbenzene. The electrolyte can be, for example, sodium chloride, sodium acetate, sodium propionate or sulfamic acid. The electrodialysis stack is then assembled in such a way that the anionic and cationic membranes are parallel as in a filter press between two electrode blocks, such that the stream undergoing ion depletion is well separated from the stream undergoing ion enrichment (the two solutions are also referred to as the dilute (undergoing ion depletion) and concentrate (undergoing ion enrichment)). The heart of the electrodialysis process is the membrane stack, which consists of several anion-exchange membranes and cation-exchange membranes separated by spacers, installed between two electrodes. By applying a direct electric current, anions and cations will migrate across the membranes towards the electrodes.

[0065] In an additional and / or alternative embodiment, the process for the purification of the HMO further comprises a step of continuous chromatography like simulated moving bed (SMB) chromatography. Simulated moving bed (SMB) chromatography originated in the petrochemical and mineral industries. Today, SMB chromatography is used by the pharmaceutical industry to isolate enantiomers from racemic mixtures. Large-scale SMB chromatography has already been used for the separation of the monosaccharide fructose from fructose-glucose solutions and for the separation of the disaccharide sucrose from sugar beet or sugar cane syrups.

[0066] SMB processes used to separate saccharides use e.g. calcium charged, crosslinked polystyrene resins, anion resins in the bisulfite form (Bechthold M., et al., Chemie Ingenieur Technik, 2010, 82, 65-75), or polystyrenic gel strong acid cation resin in the hydrogen form (Purolite PCR833H) (Purolite, Bala Cynwyd, USA).

[0067] Given the continuous mode of operation, the recycling of the mobile phase and also the potential to use large column sizes, SMB systems can in principle be scaled to achieve production volumes of hundreds of tons.

[0068] The process step of simulated moving bed chromatography is advantageous in that this process step allows further removal of oligosaccharides being structurally closely related to the desired oligosaccharide.

[0069] In an additional and / or alternative embodiment, the process for the purification of the HMO comprises a treatment of the process stream with activated charcoal to remove contaminating substances such as colorants from the process stream.

[0070] In an additional and / or alternative embodiment, the process for the purification of the HMO comprises at least one step of crystallization or precipitation of the HMO from the cleared process stream. Crystallization or precipitation of the HMO from the process stream may be performed by adding a suitable amount of an organic solvent that is miscible with water to the process stream containing the HMO. The organic solvent may be selected from the group consisting of C1- to C6-alcohols and C1- to C4-carbon acids.

[0071] An additional and / or alternative embodiment of the process for the purification of the aimed HMO comprises a step of sterile filtration and / or endotoxin removal, preferably by filtration of the process stream through a 3 kDa filter or 6 kDa filter.

[0072] In an additional and / or alternative embodiment, the process for the purification of the HMO comprises a step of increasing the concentration of the HMO in the process stream. The concentration of the HMO in the process stream can be increased by subjecting the process stream to vacuum evaporation, reverse osmosis or nanofiltration (e.g. nanofiltration with a nanofiltration membrane having a size exclusion limit of < 20 A). Alternatively, crystallized or precipitated HMO is dissolved in water, to obtain a solution of the HMO possessing the desired concentration of HMO.

[0073] In an additional and / or alternative embodiment, the resulting process stream is an aqueous solution which contains the desired HMO in a concentration of > 1 g / L, > 10 g / L, > 20 g / L, > 25 g / L, > 30 g / L, > 40 g / L, > 60 g / L, > 100 g / L, > 200 g / L, > 300 g / L or even > 400 g / L.

[0074] In an additional and / or alternative embodiment, the aqueous solution contains the HMO in a purity of at least 50 %, at least 65 %, at least 80 %, at least 90 %, at least 95 % or at least 98 % with respect to the weight of dry matter / solutes within such aqueous solution.

[0075] Such aqueous solution does not contain genetically-engineered microorganisms, nucleic acid molecules derived from genetically-engineered microorganisms and proteins. In the process for obtaining a spray-dried powder of an HMO, the aqueous solution containing the HMO is subjected to a spray-drying method.

[0076] Spray-drying is a method to obtain dry powders, wherein the solution containing the substance of interest is sprayed into droplets which are rapidly dried by hot air. Spray-drying is very fast and exposure of the substance to be dried to high temperatures is quite short.

[0077] In an additional and / or alternative embodiment, the aqueous solution containing the HMO is spray-dried at a nozzle temperature of at least 110 °C, preferably at least 120 °C, more preferably at least 125 °C, and less than 190 °C, preferably less than 180 °C and more preferably less than 160 °C.

[0078] In an additional and / or alternative embodiment, the aqueous solution containing the HMO is spray-dried at an outlet temperature of at least 60 °C, preferably at least 65 °C, and less than 90 °C, preferably less than 80 °C.

[0079] The spray-drying of the aqueous solution containing the HMO provides a powder of low hygroscopy, wherein the HMO is present in amorphous form. The spray-dried powder consisting essentially of the HMO is less hygroscopic than a powder of identical composition which was obtained by freeze-drying. This is beneficial for the mixing process, as the water content of the powder after drying is not increasing significantly during the mixing times applied.

[0080] It is another object of the invention to provide a powder blend that has been obtained with the process described above.

[0081] The powder blend is preferably being used for the manufacture of a nutritional composition. The process according to the invention provides powder blends with a defined concentration of HMOs, in a homogeneous distribution, so that the blend is suitable for human consumption such as medicinal formulations, infant formula, dairy drinks or dietary supplements.

[0082] The present invention will be described with respect to particular embodiments and with reference to drawings, but the invention is not limited thereto but only by the claims. Furthermore, the terms first, second and the like in the description and in the claims, are used for distinguishing between similar elements and not necessarily for describing a sequence, either temporally, spatially, in ranking or in any other manner. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments of the invention described herein are capable of operation in other sequences than described or illustrated herein.

[0083] It is to be noticed that the term “comprising”, used in the claims, should not be interpreted as being restricted to the means listed thereafter; it does not exclude other elements or steps. It is thus to be interpreted as specifying the presence of the stated features, integers, steps or components as referred to, but does not preclude the presence or addition of one or more other features, integers, steps or components, or groups thereof. Thus, the scope of the expression “a device comprising means A and B” should not be limited to devices consisting only of components A and B. It means that with respect to the present invention, the only relevant components of the device are A and B.

[0084] Reference throughout this specification to “one embodiment” or “an embodiment” means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, appearances of the phrases “in one embodiment” or “in an embodiment” in various places throughout this specification are not necessarily all referring to the same embodiment, but may. Furthermore, the particular features, structures or characteristics may be combined in any suitable manner, as would be apparent to one of ordinary skill in the art from this disclosure, in one or more embodiments.

[0085] Similarly it should be appreciated that in the description of exemplary embodiments of the invention, various features of the invention are sometimes grouped together in a single embodiment, figure, or description thereof for the purpose of streamlining the disclosure and aiding in the understanding of one or more of the various inventive aspects. This method of disclosure, however, is not to be interpreted as reflecting an intention that the claimed invention requires more features than are expressly recited in each claim. Rather, as the following claims reflect, inventive aspects lie in less than all features of a single foregoing disclosed embodiment. Thus, the claims following the detailed description are hereby expressly incorporated into this detailed description, with each claim standing on its own as a separate embodiment of this invention.

[0086] Furthermore, while some embodiments described herein include some but not other features included in other embodiments, combinations of features of different embodiments are meant to be within the scope of the invention, and form different embodiments, as would be understood by those in the art. For example, in the following claims, any of the claimed embodiments can be used in any combination.

[0087] Furthermore, some of the embodiments are described herein as a method or combination of elements of a method that can be implemented by a processor of a computer system or by other means of carrying out the function. Thus, a processor with the necessary instructions for carrying out such a method or element of a method forms a means for carrying out the method or element of a method. Furthermore, an element described herein of an apparatus embodiment is an example of a means for carrying out the function performed by the element for the purpose of carrying out the invention.

[0088] In the description and drawings provided herein, numerous specific details are set forth. However, it is understood that embodiments of the invention may be practiced without these specific details. In other instances, well-known methods, structures and techniques have not been shown in detail in order not to obscure an understanding of this description.

[0089] The invention will now be described by a detailed description of several embodiments of the invention. Other embodiments of the invention can be configured according to the knowledge of persons skilled in the art without departing from the true spirit or technical teaching of the invention, the invention being limited only by the terms of the appended claims.

[0090] Examples: Examples 1 and 2: Preparing a powder blend of five structurally distinct HMOs

[0091] Five spray-dried powders each essentially consisting of a structurally distinct HMO were provided. The five HMOs were obtained by microbial fermentation followed by purification and spray-drying as described in WO 2019 110800 A1.

[0092] The spray-dried powders consisted of two fucosylated HMOs (HMO1 and HMO2), one neutral HMO (HMO3) and two sialylated HMOs (HMO4 and HMO5). The purities of the separate spray-dried powders ranged from 94% to 99%. The water content of the separate powders ranged from 4 wt.-% to 7 wt.-%.

[0093] These powders were filled into a vertical convective mixer with a volume of 10000 dm3. In total 2252 kg of spray-dried powders were filled into the mixer. The filling level of the mixer was about 50%. The mixer had a fixed mixing vessel with a loading valve on top and a discharge valve at the bottom of the mixing vessel. The mixer had a motor-driven agitator. The agitator and the mixing vessel were made of stainless steel, which is an inert material. This choice of material ensures that no chemical interaction between the powders and the material of the vessel or the agitator took place. The agitator had the shape of a single helical ribbon. Upon rotation of the agitator, the powders inside the vessel are moved upwards at the periphery of the mixing vessel and moved downwards in the center of the mixing vessel. The mixer contained a single mixing chamber with a single agitator.

[0094] The powders were then mixed for in total 5 min using a rotation frequency of 1844 R / min. The mixing was performed starting at room temperature of 18 °C without active cooling. After 1 min, 2 min, 3 min, 4 min, and at the end of the mixing, samples were taken. For each HMO of the final powder blend, a tolerance interval was set in advance. Each sample was analyzed by HPLC.

[0095] Table 1 shows the results of two different mixing processes performed independently. For each HMO, the final obtained value achieved after 3 min of mixing is given in % for the final blend. The % value is a wt.-% value referring to the dry matter of the blend. For each HMO, the time until the target value was reached is listed. This is the time after which the observed percentage fell within the tolerance interval of the target value. As can be seen in Table 1, a mixing time of 3 min is enough to get a blend that meets the specification given by the target values. The deviations were comparable to results obtained by mixing HMO solutions (wet blending) and spray-drying from the mixed solution. The short mixing duration is extremely important, since the mixed materials are sensitive to humidity, temperature, and mechanical stress. Furthermore, the shorter the mixing times the lower is the energy required for the mixing.

[0096] The integrity of the HMOs did not suffer during the mixing. It was not necessary to apply active cooling during the process. This is another advantage, especially when the scale of the mixing is in the tons-range.

[0097] After the mixing process, the powder blend was emptied via the discharge valve at the bottom of the mixer. The mixer had a cylindrical shape, and a removal device was used to ensure that no material sticks to the bottom of the mixer, which facilitated the discharging. 99% of the material used in the mixer were recovered, which is very important since the production and isolation is costly.

[0098] The resulting 5 HMO Mix had a bulk density of 0.48 kg / dm3and 0.49 kg / dm3in Example 1 and 2, respectively. This bulk density results in a good flowability, which is important for the handling of the final mix. The bulk density was measured according to the DIN / ISO 697 standard.

[0099] The final water content of the resulting 5-HMO Mix was determined by Karl-Fischer titration 6.2 wt.% and 6.1 wt.% for Example 1 and 2, respectively.

[0100] Table 1: Results from mixing examples 1 and 2

[0101] After the mixing was completed, the obtained blend was filled into six big bags. To confirm that the mixing resulted in a homogeneous blend, the mixtures obtained after filling into big bags were analyzed again by HPLC. From six different big bags, samples were taken. Table 2 shows the average composition and standard deviation for the different HMOs of the composition. The low standard deviation confirms that a homogeneous mixture was obtained.

[0102] Table 2: Composition of mixtures obtained from Examples 1 and 2 after filling into big bags Figures 1 and Figures 2 and 3 show a schematical view of a conical mixing vessel and two mixers, respectively. The drawings herein are not to scale.

[0103] Referring now to Figure 1 , a schematical drawing of a conical mixing vessel 2 is shown. The fixed mixing vessel 2 has a cylindrical shape with a constant diameter d in the upper part and a conical shape in the bottom part. The mixing vessel 2 is wider at the top of the vessel and thinner at the bottom. This larger diameter at the top ensures a large volume available for mixing. The conical shape at the bottom directs all the material towards the discharge valve 6, thereby increasing the efficiency of the emptying process. The material to be mixed can be loaded via the charge valve into the mixing vessel. The ratio c / h of the height of the conical part c to the height of the vessel h, is in this example approximately 0.45.

[0104] Referring now to Figure 2, a cross-section of a cylindrical mixer 1 with a perspective view of the motor-driven agitator 3 inside the mixer 1 is depicted. The motor-driven agitator 3 rotates during operation. The motor 4 is mounted on top of the vessel on the outside of the mixing vessel 2. This facilitates maintenance of the motor 4. The mixing vessel 2 has a cylindrical shape with an about constant diameter over the whole height of the vessel 2. Thus, the mixing vessel 2 has about the same diameter at the top as at the bottom of the vessel 2. Thereby the mixing volume is maximized. Only at the lowest part of the vessel in the area of the discharge valve 6, the walls of the vessel are tilted towards the discharge valve 6 in order to facilitate the emptying of the vessel 2 through the discharge valve 6.

[0105] The motor-driven agitator 3 has the shape of a single helical ribbon. The helical ribbon creates an helical upward flow at the periphery by moving the powders to the top of the vessel (indicated by the dashed arrow directed upwards), from where gravity flow moves the particles downwards in the center (indicated by the dashed arrow directed downwards). The dashed arrows in the drawing only schematically indicate the material flow. The actual material flow is more complex. The advantage of the single helical ribbon is that only minimal mechanical and thermal stress are created during the mixing process.

[0106] Referring now to Figure 3, the difference to the mixer depicted in Figure 2 is the shape of the vessel 2. The vessel 2 has a flat bottom and the agitator 3 is acting as a device for removing powder sticking to the bottom of the vessel and directing it towards the discharge valve 6. This ensures an almost complete emptying of the vessel.

[0107] List of reference characters

[0108] 1 mixer

[0109] 2 mixing vessel 3 agitator

[0110] 4 motor

[0111] 5 charge valve, loading valve

[0112] 6 discharge valve

Claims

CLAIMS1. A process for manufacturing a powder blend essentially consisting of at least two structurally distinct human milk oligosaccharides (HMOs), the process comprising a) providing at least two spray-dried powders, wherein each spray-dried powder essentially consists of a structurally distinct HMO and b) mixing the at least two spray-dried powders in a vertical convective mixer (1) having a fixed mixing vessel (2) and a motor-driven agitator (3), wherein the mixing occurs by rotation of the agitator (3) inside the fixed mixing vessel (2).

2. The process according to claim 1 , wherein the at least two spray-dried powders are agitated in a helical upward flow at the periphery of the mixing vessel and a downward flow in the center of the mixing vessel.

3. The process according to claim 2, wherein the agitator (3) is a helical ribbon agitator, preferably a double helical ribbon agitator, or more preferably a single helical ribbon agitator.

4. The process according to any one of claims 1 to 3, wherein the mixing vessel (2) has the shape of a cylinder.

5. The process according to any one of claims 1 to 3, wherein the mixing vessel (2) has a conical shape at least at its bottom.

6. The process according to any one of claims 1 to 5, wherein the mixing is performed for at least 1 minute, preferably for at least 2 minutes, more preferably for at least 3 minutes, but no longer than 15 minutes, preferably no longer than 10 minutes, more preferably no longer than 5 minutes.

7. The process according to any one of claims 1 to 6, wherein the agitator (3) rotates with 5 revolutions per minute (R / min) up to 80 R / min and the volume of the mixing vessel (2) is between 300 dm3and 15 000 dm3.

8. The process according to any one of claims 1 to 7, wherein between 15% and 95%, preferably between 20% and 90% of the volume of the mixing vessel (2) is filled with the at least two spray-dried powders.

9. The process according to any one of claims 1 to 8, wherein the mixing is performed at a temperature between 14 °C and 45 °C, preferably without active cooling of the mixing vessel (2) and I or of the agitator (3).

10. The process according to any one of claims 1 to 9, wherein three, four, five, six, seven or eight spray-dried powders are mixed.

11. The process according to any one of claims 1 to 10, wherein the at least two structurally distinct HMOs are selected from the group consisting of 2’- fucosy I lactose (2’-FL), 3-fucosyllactose (3-FL), lacto-ZV-tetraose (LA / T), lacto-A / - neotetraose (L / VnT), lacto-ZV-fucopentaose I (L / VPFI), lacto-ZV-fucopentaose II (L / VPFII), lacto-ZV-fucopentaose III (L / VPFIII), 3’-sialyllactose (3’-SL), 6’- sialyllactose (6’-SL), sialyllacto-N-tetraose a (LST-a), sialyllacto-N-tetraose b (LST-b), sialyllacto-N-tetraose c (LST-c) and disialyllacto-N-tetraose (DSLNT).

12. The process according to any one of claims 1 to 11 , wherein the at least two spray-dried powders each essentially consist of a structurally distinct HMO, wherein the HMO is present in the powder at a purity of at least 85%, preferably a purity of at least 90%, more preferably a purity of at least 95%.

13. The process according to any one of claims 1 to 12, wherein the at least two spray-dried powders each consisting of a structurally distinct HMO each contain less than 15 wt.-%, preferably less than 10 wt.-% of water.

14. The process according to any one of claims 1 to 13, wherein in step a) the structurally distinct HMOs are provided from a microbial fermentation.

15. A powder blend obtained via a process according to any one of claims 1 to 14.

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