Method of making powder blends of oligosaccharides

Abstract

Disclosed is a method for manufacturing a powder blend consisting essentially of at least two human milk oligosaccharides (HMOs) of different structures, the method comprises a) providing at least two spray-dried powders, where each spray-dried powder consists essentially of a structurally different HMO, and b) mixing the at least two spray-dried powders in a vertical convective mixer having a stationary mixing vessel and a motor-driven stirrer, wherein the mixing takes place by rotation of the stirrer within the stationary mixing vessel.

Description

Technical Field

[0001] This invention relates to a method for manufacturing a powder blend composed of human milk oligosaccharides and the powder blend obtained by the method. Background Technology

[0002] Human milk is composed of fat, protein, vitamins, minerals, trace elements, and complex oligosaccharides. Besides lactose, human milk contains a variety of structurally different oligosaccharides, 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). Currently, more than 150 structurally different oligosaccharides have been identified in human milk. With very few exceptions, HMOs are characterized by the presence of lactobiose residues at their reducing ends. Furthermore, many HMOs contain fucose residues, galactose residues, etc., at their non-reducing ends. N -acetylglucosamine or N - Acetylneuraminic acid residues. Furthermore, both straight-chain and branched representatives exist. Typically, the monosaccharide residues of HMOs are D-glucose, D-galactose, N -acetylglucosamine, L-fucose and N - Acetylneuraminic acid (also known as sialic acid or lactamic acid). Studies have reported several health benefits of HMOs, including positive effects on the gut microbiota and immune system. HMOs are also used as beneficial bacteria, such as Bifidobacteria ( Bifidobacteria ) or Lactobacillus ( Lactobacilli The substrate of ).

[0003] Due to the difficulties involved in the chemical synthesis of human milk oligosaccharides, several enzymatic and fermentation methods have been developed. Specifically, fermentation methods require the purification of the desired oligosaccharides from highly complex fermentation broths containing hundreds of different individual compounds. The carbohydrate portion of the fermentation broth alone consists of a complex mixture of monosaccharides and oligosaccharides and their derivatives, including substrates (e.g., lactose, fructose, glucose, sucrose, and other sugars used as carbon sources), biosynthetic intermediates, and individual monosaccharides (such as glucose, galactose, etc.). N -acetylglucosamine, L-fucose and N α-acetylneuraminic acid), metabolic byproducts, and other oligosaccharides and polysaccharides synthesized by microorganisms.

[0004] The resulting HMOs need to be purified and ideally dried. The drying process has specific requirements in principle. The oligosaccharides to be separated and dried typically exhibit chemical reactivity comparable to standard primary or secondary alcohols, amides, α-functionalized carboxylic acids, acetals, and hemiacetals. Furthermore, these structures are redox, biologically active, and temperature-sensitive. Therefore, the drying process must be very gentle, and excessive stress must not be applied to the material through, for example, mechanical stress; additionally, the material must not be exposed to high thermal shock. Furthermore, the materials used in the drying machinery must be inert to carbohydrates and meet food quality standards.

[0005] In principle, different drying methods can be considered for drying HMOs from solution. Spray drying is a common method used for drying and formulating carbohydrates or carbohydrate-containing foods (Woo, MW 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, SP, Chapter 5: Spray Drying, 57-94, In Anandharamakrishnan, C. (Editor-in-Chief) Handbook of Drying for Dairy Products, ISBN: 9781118930526). Spray drying is a method of producing dry powder from a liquid or slurry using a hot air stream. Because of the relatively short heat contact time, heat-sensitive materials, such as food and pharmaceuticals, can be dried in this way, thus ensuring a consistent particle size distribution. In most cases, air is the heating drying medium; however, nitrogen can be used when inert conditions are required. Spray dryers use some type of disc or nozzle to disperse liquid or slurry into a controlled spray of droplet size. Dry powders generally have the advantage of free flow. Some disadvantages of spray drying are the risk of dust explosions and its high energy and air requirements. Spray drying is a purely drying method, not a purification technique, and unlike crystallization, it does not achieve purification. Spray drying can be used for purified solutions obtained from fermented broths, but it can also be used for sugar solutions that have previously been obtained in dried form, such as those obtained from crystallization or from other drying methods, such as belt or strip drying and drum or drum drying.

[0006] Depending on their final application, several HMOs with different structures are mixed and applied as a mixture. For this, the HMOs must be mixed in specific proportions. The proportions of the different HMOs should be uniform throughout the entire product volume.

[0007] Therefore, until now, HMO has been mixed in solution and then dried from the solution by spray drying (WO2019110800 A1). The wet mixing step prior to spray drying has several disadvantages: before mixing, the stability of individual HMOs in solution is not as good as in dry form, which significantly limits the possible storage time. Furthermore, dissolved HMOs occupy more volume in storage or during transportation than dry powder, and thus take up more space. However, the homogeneity of the blends obtained from solutions containing HMO mixtures is very high.

[0008] An alternative to mixing different HMO solutions is to mix dry powders of different HMOs. However, several problems exist when implementing this method: it is difficult to achieve homogeneity similar to that obtained by wet mixing. During dry mixing, temperature rise and mechanical damage are possible due to the grinding of the mixed powders.

[0009] The method proposed in this invention is a simple, energy-saving, and effective method for obtaining a homogeneous powder containing HMO mixtures with different structures. Summary of the Invention

[0010] This invention relates to a method for mixing several human milk oligosaccharides with different structures. This method is suitable for mixing sensitive HMOs. HMO mixtures are intended for use in the pharmaceutical or food industries and are therefore subject to strict regulation. High-quality requirements must be met.

[0011] The object of this invention is to provide a method for preparing powder blends that meet these requirements by mixing at least two powders in a vertical convection mixer.

[0012] In some implementations, the vertical convection mixer is a ribbon mixer.

[0013] Another objective of this invention is to provide an industrial-scale method for blending at least two HMOs. Specifically, the method should be suitable for producing powder blends ranging from kilograms to tons.

[0014] Another object of the present invention is to provide a powder blend prepared by the method described above. Attached Figure Description

[0015] Figure 1 A schematic diagram of a conical mixing container is shown.

[0016] Figure 2 A schematic diagram of a cylindrical mixer is shown, and Figure 3 A schematic diagram of a cylindrical mixer with a flat bottom is shown. Detailed Implementation

[0017] This invention provides a method for manufacturing a powder blend substantially composed of at least two structurally different human milk oligosaccharides (HMOs). The method includes at least the following steps: a) Provide at least two spray-dried powders, wherein each spray-dried powder is substantially composed of HMOs with different structures, and b) The at least two spray-dried powders are mixed in a vertical convection mixer having a fixed mixing container and a motor-driven agitator, wherein the mixing occurs by the rotation of the agitator within the fixed mixing container.

[0018] In step a), at least two powders obtained by spray drying are provided. Each spray-dried powder is substantially composed of HMOs with different structures; therefore, each spray-dried powder is substantially composed of HMOs different from those of the other spray-dried powders.

[0019] Each spray-dried powder is obtained by spray drying an HMO solution. The solution preferably contains HMO produced through fermentation. However, it is also possible that one, some, or all of the HMOs are produced through biocatalysis or chemical synthesis.

[0020] In the context of this application, if a powder or powder blend is substantially composed of compounds / groups of compounds, then the content of each compound / group of compounds in the 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% based on dry matter. The most relevant group of compounds in this application is the human milk oligosaccharide group.

[0021] In step b), the spray-dried powder to be mixed is added to the mixing container of a vertical convection mixer. Inside the mixer, a motor-driven agitator is installed, which rotates during operation. Therefore, the agitator is connected to the motor that drives its rotation. The motor is preferably mounted outside the mixing container. Due to the mechanical motion of the rotating agitator, the different powders are reoriented relative to each other. This mixes the single spray-dried powder, resulting in a homogeneous mixture.

[0022] Vertical mixers are preferably filled from the top of the container via a loading valve and emptied via a discharge valve at the bottom of the container. This allows for yields up to 99%, provided the product characteristics permit, as very little residue remains inside the vertical mixer. Spray-dried HMO powders are particularly well-suited for this purpose due to their good flowability. Of course, depending on the mixer's volume, the mixer may include more than one loading valve and more than one discharge valve. A mixer with one loading valve and one valve for emptying the mixer is preferred, as this facilitates cleaning and maintenance.

[0023] The terms top and bottom refer to the orientation of the mixer within its space. The material flows from top to bottom due to gravity. Therefore, the bottom is part of the mixer oriented towards the room floor.

[0024] Furthermore, the vertical mixer can operate in both batch and continuous modes. For the use of spray-dried powder composed of HMO obtained from fermentation, batch mode operation is preferred over continuous mode because the fermentation process is also intermittent.

[0025] The mixing, achieved by a rotary agitator driven by an internal motor, provides a very gentle mixing process. The agitator is made of an inert and rigid material, preferably stainless steel or aluminum. The agitator is preferably mounted only on one side of the container, and more preferably on the top, which facilitates product unloading. The agitator can have different shapes, such as blades, paddles, belts, or screws. Preferably, only one agitator is installed inside the container.

[0026] One advantage of vertical mixing vessels is that the mixer can operate at different fill levels ranging from 5 vol% to 100 vol%, which provides great flexibility in production applications.

[0027] In another embodiment of the method according to the invention, at least two spray-dried powders are agitated in a spiral upward flow around the periphery of the mixing container and a downward flow at the center of the mixing container. Therefore, the agitator is designed to generate a spiral upward flow of material, moving particles from the bottom to the top of the container and from the top to the bottom at the center of the container. These movements contribute to the formation of a homogeneous mixture of materials. In another embodiment, the agitator is a ribbon agitator. Ribbon agitators are particularly suitable for generating a spiral upward flow around the periphery of the mixing container and a downward flow at the center of the mixing container. Preferably, the ribbon agitator has a double-ribbon shape, thus comprising an inner ribbon and an outer ribbon. The outer ribbon generates movement of particles toward the center of the container, while the inner ribbon generates movement toward the outside of the container. A downward flow of material along the axis of rotation is generated by the circumferential velocity difference between the inner and outer ribbons. This effectively distributes the particles.

[0028] As an alternative, a ribbon agitator is a single ribbon agitator that creates an upward spiral flow around the periphery by moving the powder to the top of the container, while a gravity flow in the center moves the particles downward from the top of the container. A single ribbon generates minimal mechanical and thermal stress during mixing.

[0029] In another embodiment of the method according to the invention, the mixing container has a conical shape, at least towards the bottom of the mixing container. The mixing container is wider at the top and narrower at the bottom. The larger diameter at the top ensures a large volume available for the material. The smaller diameter at the bottom facilitates material discharge, as the material is guided by the contracting walls of the container to the discharge valve. This design enables the recovery of up to 99% or more of the starting material. In another embodiment, the entire mixing container has a conical shape.

[0030] In an alternative embodiment, the mixing container is shaped as a cylinder with a fixed radius at the top and a cone with a variable radius at the bottom. This mixing container has a relatively high mixing volume while providing efficient emptying of the container. Preferably, the ratio c / h of the cone height c to the container height h is between 0.15 and 0.80.

[0031] In another embodiment, the mixing container has a cylindrical shape. This container has a large usable mixing volume. The mixing container has a flat bottom with a valve for emptying the container. In another embodiment, a tool for scraping material from the bottom of the container is integrated into the mixer. This tool may be in the form of a rotating sheet that contacts the flat bottom of the container and ensures no material adheres to it. It collects material from the bottom and directs it to the valve. In the case of emptying the container, the rotating sheet ensures that the material is completely removed from the container. In one embodiment, the rotating sheet is separate from the motor-driven agitator. In an alternative embodiment, the rotating sheet is part of or connected to the motor-driven agitator.

[0032] In another embodiment, the vertical convection mixer has a single mixing chamber, preferably containing one agitator. This design facilitates container emptying and cleaning compared to a two-chamber design with two agitators arranged adjacent to each other. In this case, mixing occurs in the overlapping area between the two moving agitators. This allows for gentle mixing. However, it has been shown that the mixing efficiency of spray-dried HMO powder is high in mixing containers with a single mixing chamber. Material recovery is also more efficient in mixers with a single mixing chamber, which is extremely important because material loss must be minimized in the final stages of the production process.

[0033] In another embodiment of the method according to the invention, mixing is carried out for at least 1 minute, preferably at least 2 minutes, more preferably at least 3 minutes, but not more than 15 minutes, preferably not more than 10 minutes, and more preferably not more than 5 minutes. It has been shown that these mixing times are sufficient to produce a homogeneous blend of spray-dried HMO powder. These relatively short mixing times ensure that the integrity of the sensitive HMO is not compromised. Longer mixing times result in increased mechanical stress.

[0034] In another embodiment of the method according to the invention, the stirrer rotates at a speed of 5 revolutions per minute (R / min) up to 80 R / min, and the volume of the mixing container is between 300 dm³ and 15000 dm³. The rotation frequency is affected by the volume of the mixing container. For larger volumes, a lower rotation frequency is required, while for smaller volumes, a higher rotation frequency is possible. In another embodiment, the stirrer rotates at a speed of 5 R / min to 25 R / min, and the volume of the mixing container is between 5000 dm³ and 15000 dm³. 3 Up to 15000dm 3 Between. Alternatively, the stirrer rotates at 8 R / min to 45 R / min, and the mixing container volume is between 40-50 dm³. 3 Up to 10000dm 3 Between 40 rpm and 75 rpm, and between 200 dm³. Alternatively, the stirrer rotates at 40 rpm to 75 rpm, and the mixing container volume is between 200 dm³. 3 Up to 950dm 3 between.

[0035] In another embodiment, the mixing container volume is filled with at least two spray-dried powders at a minimum of 15%, 20%, 30%, or 40% and a maximum of 70%, 80%, 90%, or 95%. The advantage of a vertical convection mixer is that this wide range of fill levels leads to satisfactory results. A higher fill level between 50% and 95% is more preferred, as this results in a higher percentage of recovered material. Spray-dried HMO powders can adhere to the walls of the mixing container. Therefore, the smaller the dead volume in the mixer, the higher the percentage of recovered material, which is particularly relevant for mixed HMOs.

[0036] In another embodiment of the method according to the invention, mixing is carried out at a temperature between 14°C and 45°C, preferably between 15°C and 35°C, and more preferably between 17°C and 25°C. Preferably, mixing is carried out without active cooling of the mixing container and / or without active cooling of the agitator. It has been shown that HMO powder blends are stable at these temperatures. The absence of active cooling of the container walls or agitator improves the energy efficiency of this method compared to methods requiring cooling.

[0037] In another embodiment of the method according to the invention, three, four, five, six, seven, eight, or more spray-dried powders are mixed. Therefore, in those cases, the resulting powder blends are essentially composed of three, four, five, six, seven, eight, or more structurally different human milk oligosaccharides. These blends containing more than two HMOs more closely resemble naturally occurring phenomena, where several HMOs are typically present at different concentrations.

[0038] In another embodiment of the method according to the present invention, the at least two structurally different HMOs are selected from 2'-fucosylated lactose (2'-FL), 3-fucosylated lactose (3-FL), lactose- N -Tetrasaccharide (L N T), lact-N-neotetrasaccharide (L) N nT), milk- N - Fucopentose I (LNPFI), lacto-N-fucopentose II (LNPFII), lacto-N-fucopentose III (LNPFIII), 3'-sialyllactose (3'-SL), 6'-sialyllactose (6'-SL), sialyl-N-tetrasaccharide a (LST-a), sialyl-N-tetrasaccharide b (LST-b), sialyl-N-tetrasaccharide c (LST-c), and disialyl-sialyl-N-tetrasaccharide (DSLNT).

[0039] In another embodiment of the method according to the invention, the powder blend consists of five HMOs with different structures, preferably 2'-FL, 3-FL, LNT, 3'-SL, and 6'-SL. In another embodiment, the powder blend consists of seven HMOs with different structures, preferably 2'-FL, 3-FL, LNT, LNnT, LNFPI, 3'-SL, and 6'-SL.

[0040] In another embodiment of the method according to the invention, at least two spray-dried powders each consist substantially of HMOs with different structures, wherein the HMOs are present in the powders with a purity greater than 85%, preferably greater than 90%, and more preferably greater than 95%. The HMOs provided as spray-dried powders with such high purity benefit from the method of the invention, which provides gentle mixing and low thermal stress.

[0041] The term purity, as used in this application, refers to chemical purity, that is, the degree to which a substance is undiluted or unmixed with foreign materials. Therefore, chemical purity is an indicator of the relationship between at least one HMO and byproducts / impurities. Chemical purity is expressed as a percentage (%) and calculated using the following formula: Purity percentage = 100 × (mass of desired compound in the sample) / (total mass of sample) Purity can be determined by any suitable method known to those skilled in the art. One suitable method is HPLC (High Performance Liquid Chromatography). In the obtained chromatogram, the ratio of the peak area representing the amount of HMO to the sum of the peak areas representing HMO and all other compounds besides HMO is calculated.

[0042] In another embodiment of the method according to the invention, at least two spray-dried powders composed of HMOs with different free structures contain less than 15 wt%, preferably less than 10 wt%, and more preferably 4 wt% to 8 wt% water. At these water contents, no agglomeration was observed after mixing.

[0043] In another embodiment of the method, the method is carried out intermittently. Preferably, 200 kg to 10,000 kg, more preferably 2,000 kg to 7,000 kg of powder blends are produced in batches.

[0044] In another embodiment of the method, the obtained powder blend has a density of 0.30 kg / dm³. 3 Up to 0.50 kg / dm 3 Preferably 0.40 kg / dm 3 Up to 0.05 kg / dm 3 The bulk density ensures excellent flowability, which is important for product handling. The bulk density is determined according to the international standard DIN / ISO 679.

[0045] In another embodiment of the method according to the invention, in step a), HMOs with different structures are obtained by microbial fermentation. The solution obtained after purification of the fermented broth is spray-dried to obtain a spray-dried powder consisting primarily of HMOs. These spray-dried powders are mixed according to the method to produce a powder blend with high homogeneity in a short time, thereby requiring minimal energy input.

[0046] HMOs are preferably obtained by microbial fermentation, wherein genetically engineered microorganisms capable of synthesizing each desired HMO are cultured in a culture medium (fermented broth) and under conditions that allow the genetically engineered microorganisms to synthesize the desired HMO.

[0047] Purification of HMOs produced by microbial fermentation involves the steps of isolating microbial cells from the fermentation broth to obtain a clear process stream that is substantially cell-free and contains the desired HMOs. This step is the first step in the process of purifying the desired oligosaccharide.

[0048] Purification of HMOs from the fermented broths mentioned above includes one or more of the following: i) Remove microbial cells from the fermented broth to obtain a clarified process stream; ii) Perform ultrafiltration on the clarified process stream at least once; iii) Treat the clarified process stream with cation exchange resin at least once and / or with anion exchange resin at least once; iv) Perform nanofiltration on the clarified process stream at least once; v) subject the clarified process stream to at least one electrodialysis step; vi) Treat the clarified process stream with activated carbon at least once; and / or vii) subject the clarified process stream to at least one crystallization and / or precipitation step.

[0049] Suitable methods for removing microbial cells from fermented broth include centrifugation, wherein the microbial cells are obtained as granules and the fermented broth is obtained as a supernatant. In another and / or alternative embodiment, microbial cells are removed from the fermented broth by filtration. Suitable filtration methods for removing cells from fermented broth include microfiltration and ultrafiltration.

[0050] Microfiltration is a physical filtration method in which a fluid containing particles is passed through a membrane with specially designed pore size to separate the particles from the fluid. As used herein, the term "microfiltration" refers to a physical filtration method for separating cells from fermented broth.

[0051] Ultrafiltration is a type of membrane filtration and is not fundamentally different. In ultrafiltration, forces such as pressure or concentration gradients cause separation through a semipermeable membrane. Cells, suspended solids, and high molecular weight solutes are retained in the so-called retentate, while water and low molecular weight solutes, such as the desired HMO, pass through the membrane and are in the permeate (filtrate). Ultrafiltration membranes are defined by the molecular weight cutoff (MWCO) of the membrane used. Ultrafiltration is applied in cross-flow or dead-end mode.

[0052] Typically, microbial cells synthesize the desired HMOs intracellularly and secrete them into the fermentation broth. The resulting HMOs are then placed in the fermentation broth, which is then subjected to further processing steps for HMO purification as described below.

[0053] Although this method is used to purify HMOs produced by microbial fermentation, it can also be used to purify HMOs produced by in vitro enzymatic catalysis. HMOs can be purified from the reaction mixture at the end of the biocatalytic reaction. The reaction mixture is then used as a clarified process stream for purification.

[0054] The clarified process stream contains HMOs as well as byproducts and undesirable impurities, such as monosaccharides, disaccharides, undesirable oligosaccharide byproducts, ions, amino acids, peptides, proteins and / or nucleic acids.

[0055] In other and / or alternative embodiments, the method for purifying the desired HMO includes performing at least one cation exchange treatment to remove positively charged compounds from the clarified process stream. Suitable cation exchange resins for removing positively charged compounds include Lewatit S2568 (H+) (Lanxess AG, Cologne, DE).

[0056] In other and / or alternative embodiments, the method for purifying the desired HMO includes an anion exchange treatment to remove unwanted negatively charged compounds from the clarified 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 1 x8 (Mesh 100-200), PuroliteChromalite CGA100x4 (Purolite GmbH, Ratingen, DE), and Dow Amberlite FPA51 (DowChemicals, MI, USA).

[0057] In other / or alternative embodiments, methods for purifying HMOs include nanofiltration and / or percolation steps to remove impurities with lower molecular weights and concentrate the desired oligosaccharides. Percolation involves adding water to the solution to remove (wash) membrane-permeable components. By using appropriate membranes, percolation can be used to separate components based on their molecular size and charge, where one or more substances are effectively retained while others permeate through the membrane. Specifically, percolation using nanofiltration membranes is effective for the separation of low molecular weight compounds, such as small molecules and salts. Nanofiltration membranes typically have a molecular weight cutoff in the range of 150–1000 Daltons. Nanofiltration is widely used in the dairy industry for whey concentration and desalting.

[0058] Membranes suitable for nanofiltration and / or percolation include Dow Filmtec NF270-4040, Trisep 4040-XN45-TSF (Microdyn-Nadir GmbH, Wiesbaden, DE), GE4040F30 and GH4040F50 (GE Water & Process Technologies, Ratingen, DE).

[0059] It has been found that percolation using nanofiltration membranes is an effective pretreatment for removing significant contaminants from solutions containing oligosaccharides prior to electrodialysis. The use of nanofiltration membranes for concentration and percolation during HMO purification reduces heat exposure, leading to fewer Maillard and aldol condensation reactions, thus lowering energy and processing costs and resulting in better product quality.

[0060] In other and / or alternative embodiments, the method for purifying HMO includes at least one electrodialysis step. Electrodialysis (ED) is used to transfer salt ions from one solution to another through an ion-exchange membrane under the influence of an applied potential difference, and based on the selective electromigration of ions through a semipermeable membrane, it can be used for the separation or concentration of ions in solution.

[0061] The basic principle of electrodialysis consists of an electrolytic cell comprising a pair of electrodes immersed in an electrolyte for ion conduction, connected to a DC generator. The electrode connected to the positive terminal of the DC generator is the anode, and the electrode connected to the negative terminal is the cathode. An electrolyte solution then supports the flow of current generated by the movement of negative and positive ions toward the anode and cathode, respectively. The membranes used in electrodialysis are essentially porous ion-exchange resin sheets with negatively or positively charged groups, and are therefore described as cation or anion membranes, respectively. Ion-exchange membranes are typically made of polystyrene with suitable functional groups (such as sulfonic acid for cation membranes or quaternary ammonium groups for anion membranes) crosslinked with divinylbenzene. The electrolyte can be, for example, sodium chloride, sodium acetate, sodium propionate, or aminosulfonic acid. The electrodialysis stack is then assembled such that the anion and cation membranes are parallel, as in a filter press between two electrode blocks, thereby effectively separating the stream undergoing ion consumption from the stream undergoing ion enrichment (these two solutions are also referred to as the diluent (undergoing ion consumption) and the concentrate (undergoing ion enrichment)). At the heart of the electrodialysis process is a membrane stack positioned between two electrodes, consisting of several anion-exchange and cation-exchange membranes separated by septa. By applying a direct current, anions and cations migrate across the membranes toward the electrodes.

[0062] In additional and / or alternative embodiments, the method for purifying HMOs also includes a continuous chromatographic step, such as simulated moving bed (SMB) chromatography. Simulated moving bed (SMB) chromatography originated in the petrochemical and mineral industries. Currently, the pharmaceutical industry uses SMB chromatography to separate enantiomers from racemic mixtures. Large-scale SMB chromatography has been used to separate monosaccharides from fructose-glucose solutions, and disaccharides from beet or cane syrup. SMB methods for separating sugars use, for example, calcium-crosslinked polystyrene resins, anion exchange resins in the form of bisulfite (Bechthold M. et al., Chemie Ingenieur Technik, 2010, 82, 65-75), or hydrogen-form polystyrene gel strong acid cationic resin (Purolite PCR833H) (Purolite, Bala Cynwyd, USA).

[0063] Considering the continuous operation mode, mobile phase recirculation, and the potential for using large column sizes, the SMB system can, in principle, be scaled up to achieve a production capacity of hundreds of tons.

[0064] The process step of simulated moving bed chromatography is advantageous because it allows for the further removal of oligosaccharides that are structurally closely related to the desired oligosaccharide.

[0065] In other and / or alternative embodiments, methods for purifying HMOs include treating the process stream with activated carbon to remove contaminants, such as colorants, from the process stream.

[0066] In other and / or alternative embodiments, the method for purifying HMO includes at least one step of crystallizing or precipitating HMO from a clarified process stream. Crystallization or precipitation of HMO from the process stream can be performed by adding an appropriate amount of a water-miscible organic solvent to the HMO-containing process stream. The organic solvent can be selected from C1- to C6-alcohols and C1- to C4-carbon acids.

[0067] Additional and / or alternative embodiments of the method for purifying the target HMO include sterile filtration and / or endotoxin removal steps, preferably by filtering the process stream through a 3kDa or 6kDa filter.

[0068] In other and / or alternative embodiments, the method for purifying HMO includes the step of increasing the HMO concentration in the process stream. This can be achieved by subjecting the process stream to vacuum evaporation, reverse osmosis, or nanofiltration (e.g., with a size exclusion limit ≤ 20). Nanofiltration (using a nanofiltration membrane) can increase the concentration of HMO in the process stream. Alternatively, crystallized or precipitated HMO can be dissolved in water to obtain an HMO solution with the desired HMO concentration.

[0069] In other and / or alternative embodiments, the resulting process stream is an aqueous solution containing the desired HMO at the following concentrations: ≥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.

[0070] In other and / or alternative embodiments, the aqueous solution contains HMO with a purity of at least 50%, at least 65%, at least 80%, at least 90%, at least 95%, or at least 98% relative to the weight of dry matter / solute in the aqueous solution.

[0071] This aqueous solution does not contain genetically engineered microorganisms, nor does it contain nucleic acid molecules or proteins derived from genetically engineered microorganisms.

[0072] In a method for obtaining spray-dried HMO powder, an aqueous solution containing HMO is subjected to spray drying.

[0073] Spray drying is a method for obtaining dry powder in which a solution containing a target substance is sprayed into droplets, and the droplets are rapidly dried by hot air. Spray drying is very fast, and the time the substance to be dried is exposed to high temperatures is very short.

[0074] In other and / or alternative embodiments, the aqueous solution containing 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.

[0075] In other and / or alternative embodiments, the aqueous solution containing 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.

[0076] Spray drying of an aqueous solution containing HMO yields a powder with low hygroscopicity, where HMO exists in an amorphous form. The hygroscopicity of a spray-dried powder composed primarily of HMO is lower than that of a powder of the same composition obtained by freeze drying. This is advantageous for the mixing process because the water content of the dried powder does not increase significantly during the applied mixing time.

[0077] Another objective of the present invention is to provide a powder blend obtained by the method described above.

[0078] This powder blend is preferably used in the manufacture of nutritional compositions. The method according to the invention provides a powder blend with a uniformly distributed HMO concentration, making the blend suitable for human consumption, such as pharmaceutical preparations, infant formula, dairy beverages, or dietary supplements.

[0079] The invention will be described with reference to specific embodiments and the accompanying drawings, but is not limited thereto; rather, it is limited only by the claims. Furthermore, the terms first, second, etc., in the specification and claims are used to distinguish similar elements and are not necessarily used to describe an order in time, space, sequence, or any other manner. It should be understood that the terminology used is interchangeable where appropriate, and the embodiments of the invention described herein can operate in orders other than those described or shown herein.

[0080] It should be noted that the term "comprising" as used in the claims should not be construed as limited to the manner listed below; it does not exclude other elements or steps. Therefore, it is to be understood as indicating the presence of the specified feature, integer, step, or component mentioned, but not excluding the presence or addition of one or more other features, integers, steps, or components, or groups thereof. Thus, the scope of the expression "apparatus comprising devices A and B" should not be limited to an apparatus consisting solely of components A and B. This means that, with respect to the invention, the only relevant components of the apparatus are A and B.

[0081] Throughout this specification, references to "one embodiment" or "an embodiment" indicate that at least one embodiment of the invention includes a specific feature, structure, or characteristic described in connection with that embodiment. Therefore, the appearance of the phrase "in one embodiment" or "in one embodiment" in multiple places throughout the specification does not necessarily refer to all of the same embodiment, but may refer to the same embodiment. Furthermore, in one or more embodiments, specific features, structures, or characteristics may be combined in any suitable manner, as will be apparent to those skilled in the art based on this disclosure.

[0082] Similarly, it should be understood that in the description of exemplary embodiments of the invention, various features of the invention are sometimes combined together in a single embodiment, drawing, or description therein in order to simplify the disclosure and aid in understanding one or more of the various inventive aspects. However, this approach of the disclosure should not be construed as reflecting an intention that the claimed invention requires more features than expressly listed in each claim. Rather, as reflected in the following claims, inventive aspects lie in fewer than all the features of a single foregoing disclosed embodiment. Therefore, the claims following the detailed description are thus expressly incorporated into that detailed description, wherein each claim is an independent embodiment of the invention.

[0083] Furthermore, although some embodiments described herein include features that are included in other embodiments but are not otherwise, combinations of features from different embodiments are intended to be included within the scope of the invention and form different embodiments, as will be understood by those skilled in the art. For example, any claimed embodiments may be used in any combination in the following claims.

[0084] Furthermore, in this document, some embodiments are described as methods or combinations of method elements that can be implemented by a processor of a computer system or by other means of performing the function. Thus, a processor having the necessary instructions for performing this method or method element forms a manner for performing the method or method element. Furthermore, the elements of the device embodiments described herein are examples of manners for performing the functions performed by these elements for the purposes of this invention.

[0085] Several specific details are set forth in the description and accompanying drawings provided herein. However, it should be understood that embodiments of the invention can be practiced without these specific details. In other instances, well-known methods, structures, and techniques have not been shown in detail so as not to obscure the understanding of this description.

[0086] The present invention will now be described in detail through several embodiments thereof. Other embodiments of the invention may be configured based on the knowledge of those skilled in the art without departing from the true spirit or technical teachings of the invention, which is limited only by the terms of the appended claims.

[0087] Example: Examples 1 and 2: Preparation of powder blends of five HMOs with different structures Five spray-dried powders were provided, each consisting essentially of HMOs with different structures. As described in WO 2019110800 A1, the five HMOs were obtained through microbial fermentation, followed by purification and spray drying.

[0088] The spray-dried powder consists of two fucoidylated HMOs (HMO1 and HMO2), one neutral HMO (HMO3), and two sialylated HMOs (HMO4 and HMO5). The purity of the separated spray-dried powder ranges from 94% to 99%. The water content of the separated powder ranges from 4 wt% to 7 wt%.

[0089] These powders were filled to a volume of 10,000 dm³ 3A vertical convection mixer was used. A total of 2252 kg of spray-dried powder was filled into the mixer. The fill level of the mixer was approximately 50%. The mixer has a fixed mixing container with a loading valve at the top and a discharge valve at the bottom. The mixer has a motor-driven agitator. The agitator and mixing container are made of stainless steel as an inert material. This choice of material ensures that no chemical interaction occurs between the powder and the material of the container or agitator. The agitator has a single spiral ribbon shape. When the agitator rotates, the powder inside the container moves upward around the periphery of the mixing container and downward at the center of the mixing container. The mixer contains a single mixing chamber with a single agitator.

[0090] The powders were then mixed for a total of 5 min using a rotational frequency of 1844 R / min. Mixing was initiated at room temperature (18 °C) without active cooling. Samples were collected at 1 min, 2 min, 3 min, 4 min, and after mixing. Pre-defined tolerance ranges were established for each HMO in the final powder blend. Each sample was analyzed by HPLC.

[0091] Table 1 shows the results of two independently performed mixing processes. For each HMO, the final value achieved after 3 minutes of mixing is given as a percentage of the final blend. The % value represents the wt% value of the dry matter in the blend. For each HMO, the time to reach the target value is listed. This is the time after the observed percentage falls within the acceptable range of the target value.

[0092] As shown in Table 1, a mixing time of 3 minutes is sufficient to obtain blends that meet the specifications given by the target values. The deviation is comparable to that obtained by mixing an HMO solution (wet mixing) and then spray-drying the mixture. A short mixing duration is extremely important because the mixed materials are sensitive to humidity, temperature, and mechanical stress. Furthermore, the shorter the mixing time, the lower the energy required for mixing.

[0093] The integrity of the HMO was not affected during blending. No active cooling was required during the process. This is another advantage, especially when the blending scale is in the tonne range.

[0094] After the mixing process, the powder blend is emptied through a discharge valve at the bottom of the mixer. The mixer has a cylindrical shape and uses a removal device to ensure that no material adheres to the bottom of the mixer, which facilitates discharge. It is crucial to recover 99% of the material used in the mixer, as production and separation are costly.

[0095] In Examples 1 and 2, the five HMO mixtures obtained had a concentration of 0.48 kg / dm³. 3 and 0.49 kg / dm 3The bulk density is [value missing]. This bulk density results in good flowability, which is important for the handling of the final mixture. The bulk density is measured according to DIN / ISO 697 standard.

[0096] For Examples 1 and 2, the final water content of the obtained 5-HMO mixtures was determined to be 6.2 wt% and 6.1 wt%, respectively, by Karl Fischer titration.

[0097]

[0098] After mixing, the resulting blend was packed into six large bags. To confirm that the mixing produced a homogeneous blend, the mixtures obtained after packing into the large bags were analyzed again by HPLC. Samples were collected from the six different large bags. Table 2 shows the average composition and standard deviation of the different HMOs in the composition. The low standard deviation confirms the acquisition of a homogeneous mixture.

[0099]

[0100] Figure 1 as well as Figure 2 and 3 Schematic diagrams of the conical mixing container and two mixers are shown. The accompanying drawings are not to scale.

[0101] Now for reference Figure 1 A schematic diagram of a conical mixing container 2 is shown. The fixed mixing container 2 is a cylindrical shape with a constant diameter d at the top and a conical shape at the bottom. The mixing container 2 is wider at the top and narrower at the bottom. This larger diameter at the top ensures a large volume available for mixing. The conical shape at the bottom directs all material to the discharge valve 6, thereby improving the efficiency of the emptying process. The material to be mixed can be loaded into the mixing container through the loading valve. In this embodiment, the ratio of the height c of the conical portion to the container height h, c / h, is approximately 0.45.

[0102] Now refer to Figure 2 The diagram shows a cross-section of a cylindrical mixer 1 and a perspective view of a motor-driven agitator 3 inside the mixer 1. The motor-driven agitator 3 rotates during operation. A motor 4 is mounted on top of the container, located outside the mixing container 2. This facilitates the maintenance of the motor 4. The mixing container 2 has a cylindrical shape with a substantially constant diameter over its entire height. Therefore, the diameter of the top of the mixing container 2 is approximately the same as the diameter of the bottom of the container 2. This maximizes the mixing volume. Only at the lowest part of the container, in the area of ​​the discharge valve 6, is the container wall inclined towards the discharge valve 6 to facilitate the emptying of the container 2 through the discharge valve 6.

[0103] The motor-driven stirrer 3 has a single helical ribbon shape. The ribbon creates an upward spiral flow around the periphery by moving the powder to the top of the container (indicated by the upward-pointing dashed arrow), while a gravitational flow at the center moves the particles downwards from the top of the container (indicated by the downward-pointing dashed arrow). The dashed arrows in the attached diagram are only schematic representations of the material flow. The actual material flow is more complex. The advantage of the single helical ribbon is that it generates minimal mechanical and thermal stress during mixing.

[0104] Now for reference Figure 3 ,and Figure 2 The difference in the mixer shown is the shape of container 2. Container 2 has a flat bottom, and the agitator 3 acts as a device to remove powder adhering to the bottom of the container and guide it to the discharge valve 6. This ensures that the container is almost completely emptied.

[0105] Reference character list 1 Mixer 2 mixing containers 3 mixers 4 motors 5. Loading valve, load valve 6 Discharge valve

Claims

1. A method for manufacturing a powder blend substantially composed of at least two structurally different human milk oligosaccharides (HMOs), the method comprising: a) Provide at least two types of spray-dried powders, wherein, Each spray-dried powder is essentially composed of HMOs with different structures, and b) The at least two spray-dried powders are mixed in a vertical convection mixer (1) having a fixed mixing container (2) and a motor-driven agitator (3), wherein the mixing occurs by the rotation of the agitator (3) within the fixed mixing container (2).

2. The method according to claim 1, wherein, The at least two spray-dried powders are stirred in a spiral upward flow around the periphery of the mixing container and in a downward flow at the center of the mixing container.

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

4. The method according to any one of claims 1 to 3, wherein, The mixing container (2) has a cylindrical shape.

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

6. The method according to any one of claims 1 to 5, wherein, The mixing process shall be carried out for at least 1 minute, preferably at least 2 minutes, more preferably at least 3 minutes, but not more than 15 minutes, preferably not more than 10 minutes, and more preferably not more than 5 minutes.

7. The method according to any one of claims 1 to 6, wherein, The stirrer (3) rotates at 5 revolutions per minute (R / min) up to 80 R / min, and the volume of the mixing container (2) is 300 dm³. 3 Up to 15000dm 3 between.

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

9. The method according to any one of claims 1 to 8, wherein, The mixing is carried out at a temperature between 14°C and 45°C, and preferably the mixing container (2) and / or the stirrer (3) are not actively cooled.

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

11. The method according to any one of claims 1 to 10, wherein, The at least two structurally distinct HMOs are selected from 2'-fucosylated lactose (2'-FL), 3-fucosylated lactose (3-FL), lact- N -Tetrasaccharide (L N T), lact-N-neotetrasaccharide (L) N nT), lact-N-fucopentose I (L N PFI), lact-N-fucopentose II (L N PFII), lacto-N-fucopentose III (L N PFIII), 3'-sialyl lactose (3'-SL), 6'-sialyl lactose (6'-SL), sialyl lacto-N-tetrasaccharide a (LST-a), sialyl lacto-N-tetrasaccharide b (LST-b), sialyl lacto-N-tetrasaccharide c (LST-c) and disialial lacto-N-tetrasaccharide (DSLNT).

12. The method according to any one of claims 1 to 11, wherein, The at least two spray-dried powders are each substantially composed of HMOs with different structures, wherein the HMOs are present in the powders with a purity of at least 85%, preferably at least 90%, and more preferably at least 95%.

13. The method according to any one of claims 1 to 12, wherein, The at least two spray-dried powders composed of HMOs with different free structures each contain less than 15 wt%, preferably less than 10 wt% water.

14. The method according to any one of claims 1 to 13, wherein, In step a), the structurally different HMOs are provided by microbial fermentation.

15. A powder blend obtained by the method according to any one of claims 1 to 14.

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