Method for drying human milk oligosaccharides

Abstract

The present invention relates to a method for drying human milk oligosaccharides (HMO). More specifically, the present invention relates to a simple and economical method for roller drying or drum drying of human milk oligosaccharides.

Description

Field of the Invention

[0001] The present invention relates to a method for drying human milk oligosaccharides (HMO). More specifically, the present invention relates to a method for roller drying or drum drying of human milk oligosaccharides. Background Art

[0002] Human milk is considered to be the best diet for infant development, and it is composed of fat, protein, vitamins, minerals, trace elements and complex oligosaccharides. In addition to lactose, human milk and other mammalian emulsions also contain various oligosaccharides with diverse structures, which are also referred to as human milk oligosaccharides (HMO) (Usashima T et al. 2011, Milk 20 Oligosaccharides, Nova Biomedical Books, New York ISBN 978-1-61122-831-1). It is now believed that there are more than 150 kinds of structurally different oligosaccharides in human milk. Except for very few exceptions, on the one hand, HMO is characterized by the lactose disaccharide residues at their reducing ends. On the other hand, many HMO contain fucose residues, galactose residues, N-acetyllactosamine or N-acetylneuraminic acid residues at their non-reducing ends. In addition, there are also representatives of straight and branched chains. Typically, the monosaccharide residues of HMOs are D-glucose, D-galactose, N-acetyllactosamine, L-fucose, and N-acetylneuraminic acid (the latter also known as sialic acid or lactamic acid). The importance of HMOs for infant nutrition is directly related to their biological activities, including protecting newborns from pathogens, promoting infant immune system development and cognitive abilities. In addition, HMOs can serve as substrates for beneficial bacteria such as Bifidobacteria or Lactobacilli.

[0003] Due to the challenges involved in chemical synthesis of human milk oligosaccharides, some enzyme methods and fermentation methods have been developed. In particular, the fermentation method needs to purify the required oligosaccharides from the highly complex fermentation broth containing hundreds of different individual compounds. Only the carbohydrate part of the fermentation broth is composed of a complex mixture of monosaccharides and oligosaccharides and their derivatives, including substrates (such as lactose, fructose, glucose, sucrose and other sugars used as carbon sources), biosynthetic intermediates, single monosaccharides (such as glucose, galactose, N-acetyllactosamine, L-fucose and N-acetylneuraminic acid), metabolic byproducts and other oligosaccharides and polysaccharides synthesized by microorganisms. In addition, the structures of many oligosaccharides present in the fermentation broth are difficult to identify (for example, oligosaccharides naturally produced by synthetic hosts, such as glycosylation structures on cell surfaces or oligosaccharides produced by organisms due to stress). Therefore, in many cases, the purification and preparation of biotechnology products will be more expensive and time-consuming than its production by fermentation.

[0004] In addition, the HMOs produced in this way need not only to be purified, but also to be separated and, more ideally, to be dried. The drying process is in principle subject to special requirements. The sugars to be separated and dried generally show chemical reactivity comparable to that of standard primary or secondary alcohols, amides, α-functionalized carboxylic acids, acetals and hemiacetals. In addition, these structures are redox and biologically active and, in addition, temperature sensitive. Therefore, the drying process must be very gentle, must not impose excessive stress on the material, for example by mechanical stress, and the material must not be exposed to high thermal shocks. In addition, the materials used in the drying machinery must be inert to carbohydrates and meet food quality standards.

[0005] In particular, the saccharides to be treated in the present invention may be considered as macroscopic materials located at the interface between low molecular weight and high molecular weight materials, macromolecules or even oligomeric and / or polymeric materials. The above-mentioned classes of substances differ in many aspects, but in the present application, it is important that oligosaccharides such as HMOs and polysaccharides such as starch, cellulose or chitin are not chemically damaged by mechanical stress (Liu, W.-C. et al., Macromolecules, 2010, 43 (6), 2855-2864; Davidson, VJ et al. J. Food Sci. 1984, 49 (4), 1154-1157; Ott, RL, J. Polym. Sci. A Pol. Chem. 1964, 2, 973-982; Marx-Figini, M., Macromol. Mater. Eng. 1995, 224, 1, 179-189; Martinez-Camacho, AP, Carbohyd. Polym. 2010, 82, 2, 305-315). Furthermore, dissolved sugar entities are able to react chemically by oxidative or reductive conditions, which requires the use of inert materials and conditions, and the drying of such materials is usually carried out in a vacuum environment or under protective gas, which is attractive. Another aspect to consider that makes the treated sugar unstable is that it undergoes all kinds of intramolecular and intermolecular substitution and hydrolysis reactions, which in extreme cases may even lead to decomposition and coloration of the final product through, for example, the formation of hydroxymethylfurfural (HMF), which is a sugar decomposition product that occurs when applying overly basic, overly acidic and / or overheated conditions ( al., Eur.Food Res.Technol.1999,209(3-4),261-265; Fagerson,IS,J.Agric.Food Chem.1969,17(4),747-750;Wilson,K.et al.,2014,Chapter 19,Bio-based chemicals from biorefining:carbohydrate conversion and utilization, 624-658, In Waldron, K. (Editor), Advances in Biorefineries: Biomass and Waste SupplyChain Exploitation, ISBN: 978-0-85709-521-3; Van der Fels-Klerx, HJetal., FoodRes.Int.2014, 57, 210-217).

[0006] In principle, the different drying methods can be considered in terms of how the material is or should be characterized before drying, how the drying process is carried out and which properties of the material can be derived from the drying method.

[0007] Treated sugars may have been dissolved or present as a suspension or as an emulsion before drying. If the sugar dissolves, the material must be recovered from the solution in an amorphous or crystalline form, wherein the excess solvent must be removed by draining and / or drying. On the other hand, if the sugar exists as a suspension, then only the excess solvent must be removed from the sugar mass. When a crystalline material is obtained, this will usually be formed in a specific hydrate form or even without any crystal water, which is the reason for the presence of a certain water content in the dried material. Then, if the sugar is obtained in an amorphous form, there cannot be any crystal water, but depending on the drying method, it is expected that there will be a certain residual moisture. The hydrate form and the residual water content affect the macroscopic properties of the material, such as solubility and hygroscopicity. In addition to residual moisture, each drying method is accompanied by a specific texture and particle size of the material, which also affects the macroscopic properties of the material, such as hygroscopicity and fluidity.

[0008] Known methods for drying mono-, oligo- and polysaccharides and all other kinds of food products containing carbohydrates, non-food products containing carbohydrates and products not containing carbohydrates are described below.

[0009] When processing pure isolated fractions of monosaccharides or oligosaccharides in single forms (e.g. glucose, galactose, mannose, fructose, ribose, rhamnose, fucose, N-acetylneuraminic acid, sucrose, lactose, maltose and N-acetylated variants of the mentioned saccharides or all kinds of oligomeric human milk oligosaccharides), the mother liquor is removed after crystallization and the crystals obtained are then dried. Spray drying is another drying method commonly used for drying and for 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., (Editor) 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) Handbook of Drying for Dairy Products, ISBN: 9781118930526). Freeze drying is used for drying carbohydrates or carbohydrate-containing foods (Ratti, C. 2013, Chapter 3, Freeze drying for food powder production, 57-84, In Bhandari,B.,Bansal,N.,Zhang,M.,Schuck,P.,(Editors)Handbook of Food Powders, Processes and Properties,ISBN:978-0-85709-513-8;Bhushani,A.,Chapter 6: Freeze Drying,95-121,In Anandharamakrishnan,C.(Editor)Handbook of Drying for Dairy Products, ISBN: 9781118930526).Spray drying or freeze drying can also be used as a method for drying carbohydrate-containing foods (Ishwarya, SP, Chapter 7: Spray Freeze Drying, 123-148, In Anandharamakrishnan, C. (Editor) Handbook of Drying for Dairy Products, ISBN: 9781118930526). Band drying and / or belt drying and vacuum band drying or vacuum belt drying. Finally, roller drying, also known as drum drying or vacuum roller drying or vacuum drum drying (Courtois, F. 2013, Chapter 4, Roller and drum drying for food powder production, 85-104, In Bhandari, B., Bansal, N., Zhang, M., Schuck, P., (Editors) Handbook of Food Powders, Processes and Properties, ISBN: 978-0-85709-513-8; Karthik, P. et al., Chapter 4: Drum Drying, 43-56, In Anandharamakrishnan, C. (Editor) Handbook of Drying for Dairy Products, ISBN: 9781118930526). It is important to consider that when a vacuum environment is applied to belt drying and roller or drum drying, it significantly changes the process as well as the product parameters, which is why these methods should be considered separately.

[0010] Sugars cannot be simply dried from their solutions, but must undergo special drying steps. The reason is that sugars have water-like alcohol functional groups and may usually interact with water. This is why sugar solubility is usually high in water and aqueous mixtures. When attempting to separate carbohydrates from solutions by simple drying, sugars tend to form gels or hydrogels, and crystallization is hindered due to the reduced mobility and limited freedom of sugar molecules. However, almost all homologous sugars can crystallize, and therefore, crystallization, drainage and subsequent drying of crystal masses (mass), followed by washing and removal of uncrystallized sugar molecules and impurities are long-established methods for processing sugars in industry.

[0011] However, it should be considered that due to the extreme affinity of carbohydrates to water, carbohydrates as described above cannot be crystallized anhydrous in all cases. Sugar molecules, monosaccharides and oligosaccharides often cocrystallize with water molecules. In this article, conversely, for each monosaccharide, more than one crystal hydrate form is possible. For example, the known crystallized glucose is anhydrous form and monohydrate form, sialic acid is crystallized in anhydrous form and dihydrate form, the known crystallized lactose is anhydrous form and monohydrate form, and known maltose is anhydrous form and monohydrate form. Sugar exists or sells in which crystal form or in which polymorph, which will affect its material properties, and what is mentioned here is its stability, hygroscopicity and solubility. In addition, when there are other components in the solution, crystallization usually fails, and the other components are chemically similar to the material to be crystallized, and these components prevent the three-dimensional orientation of the target material from being stable during the crystallization process. This is a common phenomenon, especially in the case of a sugar mixture derived from fermentation. In addition, crystallization is a fairly slow process, and it sometimes (if not continuously running) each batch will take a few days, so a faster solution is preferred.

[0012] After crystallization, the residual mother liquor needs to be separated batchwise or continuously without compromising the integrity and yield of the crystals. In addition to water and water vapor, water-miscible organic solvents, such as all kinds of alcohols and mixtures of water and water-miscible organic solvents, are used for separation in combination with mechanical separation using suction filters or centrifuges.

[0013] In short, while crystallization provides an excellent method for isolating sugars prior to drying, crystallization parameters have not been determined for any conceivable sugar, nor has its industrial applicability been demonstrated. In addition, the crystal mass must be washed, which requires additional stress and contamination of the sugar mass, and often reduces yield before the crystal mass is finally dried.

[0014] The second drying method is spray drying. Spray drying is a method of producing dry powders from liquids or slurries by means of a stream of hot gas. Because the heat contact time is fairly short, spray drying can ensure a consistent particle size distribution, so heat-sensitive materials such as foods and pharmaceuticals can be dried in this way. In most cases, air is the heated drying medium; however, nitrogen can be used when inert conditions are required. Spray dryers use some type of atomizer or spray nozzle to disperse the liquid or slurry into a controlled droplet size spray. The dried powders generally have the advantage of being free-flowing. Some disadvantages of spray drying are that there is a risk of dust explosions and that it has high energy requirements as well as a high demand for air. In contrast to crystallization, spray drying is a pure drying method rather than a purification technique and no purification is achieved.

[0015] Another drying method is freeze drying. Freeze drying, also known as lyophilization or low temperature dehydration, is a low temperature dehydration method that involves freezing the product, reducing the pressure, and then removing the ice by sublimation. Due to the low temperatures used in this method, freeze drying results in high quality products. The original shape of the product is maintained, and in most cases, the quality of the rehydrated product is excellent. The main applications of freeze drying include biological, biopharmaceutical and food processing and preservation. Like spray drying, freeze drying does not result in product purification, but only produces its anhydrous preparation. Some disadvantages of this method are that it requires high energy input and requires a lot of time compared to other types of drying and large surfaces. The finished product produced by freeze drying has an enhanced sensitivity to water absorption.

[0016] A special method of drying is spray freeze drying or freeze spray drying, in which the material to be dried is sprayed into, for example, liquid nitrogen to form small ice particles, which are subsequently dried by sublimation in a freeze drying process. This ultimately results in freeze-dried spherical particles. However, due to the high energy requirements, this method is difficult to apply on an industrial scale.

[0017] Belt drying is the next method to be discussed. Belt dryers are thermal process devices for continuous drying and cooling of all possible suspensions as well as food agglomerates with the aid of air, inert gases or flue gases. Belt dryers or belt coolers are devices for particularly gentle product treatment. The wet material is fed continuously and evenly in the inlet chamber and usually passes through the drying chamber on a horizontally arranged perforated drying belt. It is passed through a hot gas flow. The drying chamber is divided into several chambers, each of which can be equipped with a circulating air fan and a heating coil. Each dryer unit can be controlled individually. The product flow rate of the drying / cooling air can be varied in each unit. In addition, the conveying speed of the material to be dried can be varied, thus providing an additional parameter for adjusting the drying time of the material. The heating or cooling of the unit can be carried out directly or indirectly with all heating media, such as oil, steam, hot water or hot gas. The disadvantages of this method are the difficulty of miniaturization and uneven product discharge. It can usually be accelerated by additionally applying a vacuum to the process and made to operate under milder conditions. This expands the potential product portfolio to materials that are too sensitive for standard vacuum belt drying processes. Therefore, vacuum belt drying is declared as a separate process. The disadvantage is that it is difficult to construct an airtight and vacuum-compatible belt chamber with additional engineering.

[0018] In order to overcome all the disadvantages of known drying methods, the present invention provides a roller drying or drum drying method for drying human milk oligosaccharides which is simple, cost-effective and scalable.

[0019] The roller drying or drum drying method of the invention for drying human milk oligosaccharides represents a simpler, safer and more energy-efficient method. And it is particularly suitable for the drying of HMOs - which, due to their nature, require special attention in terms of drying. The method does not require a heated air flow and, due to the circulating material flow, there is no risk of dust explosion or electrical charges. Summary of the invention

[0020] The present invention relates to a simple and economical method for drying HMO. The drying of sugars, oligosaccharides and polysaccharides in particular, and the drying of HMO in particular, is a very special task in which many parameters must be taken into account. HMO are very sensitive substances which are intended for use in the pharmaceutical or food industry and are therefore intensively regulated and have high quality requirements.

[0021] The object of the present invention is to provide a roll drying or drum drying method for drying human milk oligosaccharides so that they can be obtained as solids.

[0022] As a further embodiment, the present invention is directed to vacuum roll drying or vacuum drum drying applied to the HMO.

[0023] Another object of the present invention is to provide a process for roller drying or drum drying of HMO, wherein the quality of such HMO is obtained with maintained purity due to the mild drying conditions of such process. Wherein HMO with a specific water content, a specific surface texture and a certain particle size is obtained, which gives such HMO unique product properties for use in pharmaceutical and food industries.

[0024] Another object of the present invention is to provide an industrial scale process for roller drying or drum drying of HMO. In particular, the process should be suitable for providing dried HMO from kilogram scale to ton scale. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The HILIC-CAD (Hydrophilic Interaction Liquid Chromatography-Charged Aerosol Detector) spectrum of the sodium salt of N-acetylneuraminic acid used as a starting material in Examples 1 and 2 is shown.

[0026] Figure 2 The HILIC-CAD (hydrophilic interaction liquid chromatography-charged aerosol detector) spectrum of the sodium salt of N-acetylneuraminic acid obtained as the product in Example 1 is shown.

[0027] Figure 3 The HILIC-CAD (hydrophilic interaction liquid chromatography-charged aerosol detector) spectrum of the sodium salt of N-acetylneuraminic acid obtained as the product in Example 2 is shown.

[0028] Figure 4The HILIC-CAD (Hydrophilic Interaction Liquid Chromatography-Charged Aerosol Detector) spectrum of N-acetylneuraminic acid used as a starting material in Example 3 is shown.

[0029] Figure 5 The HILIC-CAD (hydrophilic interaction liquid chromatography-charged aerosol detector) spectrum of N-acetylneuraminic acid obtained as the product in Example 3 is shown.

[0030] Figure 6 The HILIC-CAD (Hydrophilic Interaction Liquid Chromatography-Charged Aerosol Detector) spectrum of 6′-sialyllactose sodium salt used as a starting material in Example 5 is shown.

[0031] Figure 7 The HILIC-CAD (hydrophilic interaction liquid chromatography-charged aerosol detector) spectrum of 6′-sialyllactose sodium salt obtained as the product in Example 5 is shown.

[0032] Figure 8 The HILIC-CAD (Hydrophilic Interaction Liquid Chromatography-Charged Aerosol Detector) spectrum of 6′-sialyllactose sodium salt used as a starting material in Example 6 is shown.

[0033] Fig. 9 The HILIC-CAD (hydrophilic interaction liquid chromatography-charged aerosol detector) spectrum of 6′-sialyllactose sodium salt obtained as the product in Example 6 is shown.

[0034] Fig.10 The HILIC-CAD (Hydrophilic Interaction Liquid Chromatography-Charged Aerosol Detector) spectrum of lacto-N-neotetraose used as a starting material in Example 7 is shown.

[0035] Fig.11 The HILIC-CAD (hydrophilic interaction liquid chromatography-charged aerosol detector) spectrum of lacto-N-neotetraose obtained as the product in Example 7 is shown.

[0036] Fig.12 The HILIC-CAD (Hydrophilic Interaction Liquid Chromatography-Charged Aerosol Detector) spectrum of lacto-N-neotetraose used as a starting material in Example 8 is shown.

[0037] Fig.13 The HILIC-CAD (hydrophilic interaction liquid chromatography-charged aerosol detector) spectrum of lacto-N-neotetraose obtained as the product in Example 8 is shown.

[0038] Fig.14The HILIC-CAD (Hydrophilic Interaction Liquid Chromatography-Charged Aerosol Detector) spectrum of lacto-N-tetraose used as a starting material in Example 9 is shown.

[0039] Fig.15 The HILIC-CAD (hydrophilic interaction liquid chromatography-charged aerosol detector) spectrum of lacto-N-tetraose obtained as the product in Example 9 is shown.

[0040] Fig.16 The residual water content is shown as a function of the drying parameters for selected examples.

[0041] Fig.17 The HPAEC-PAD (High Performance Anion Exchange Chromatography-Pulsed Amperometric Detection) spectrum of water is shown.

[0042] Fig.18 Shown is the HPAEC-PAD (High Performance Anion Exchange Chromatography-Pulsed Amperometric Detection) spectrum of an aqueous solution of ethanol.

[0043] Fig.19 The HPAEC-PAD (High Performance Anion Exchange Chromatography-Pulsed Amperometric Detection) spectrum of the sodium salt of N-acetylneuraminic acid used as a starting material in Example 2 is shown.

[0044] Fig. 20 The HPAEC-PAD (high performance anion exchange chromatography-pulsed amperometric detection) spectrum of the sodium salt of N-acetylneuraminic acid obtained as the product in Example 2 is shown.

[0045] Fig.21 The HPAEC-PAD (High Performance Anion Exchange Chromatography-Pulsed Amperometric Detection) spectrum of 6′-sialyllactose sodium salt used as a starting material in Example 6 is shown.

[0046] Fig. 22 The HPAEC-PAD (High Performance Anion Exchange Chromatography-Pulsed Amperometric Detection) spectrum of 6′-sialyllactose sodium salt obtained as the product in Example 6 is shown.

[0047] Fig.23 The HPAEC-PAD (High Performance Anion Exchange Chromatography-Pulsed Amperometric Detection) spectrum of lacto-N-neotetraose used as a starting material in Example 9 is shown.

[0048] Fig.24 The HPAEC-PAD (High Performance Anion Exchange Chromatography-Pulsed Amperometric Detection) spectrum of lacto-N-neotetraose obtained as the product in Example 9 is shown.

[0049] Fig.25 The relative ethanol content after drying, as determined by HPAEC-PAD (High Performance Anion Exchange Chromatography coupled with Pulsed Amperometric Detection), is shown relative to the ethanol content before drying of selected examples. Specific implementation plan

[0050] Human milk oligosaccharides are provided from the microbial fermentation liquid to roll drying or drum drying, thereby obtaining human milk oligosaccharides in a powder form.

[0051] The present invention provides a method for drying human milk oligosaccharides and / or obtaining human milk oligosaccharides in powder form, the method comprising the following steps:

[0052] a) providing an aqueous solution containing purified human milk oligosaccharides;

[0053] b) applying the aqueous solution to a roll dryer or a drum dryer.

[0054] The HMO used in this drying method can be produced by microbial fermentation, wherein a genetically engineered microorganism capable of synthesizing the desired HMO is cultured in a culture medium (fermentation broth) and under conditions that allow the genetically engineered microorganism to synthesize the desired HMO.

[0055] Purification of HMO produced by microbial fermentation includes the step of separating microbial cells from the fermentation broth to obtain a clarified process stream that is substantially free of cells and contains the desired HMO. This step is the first step in the process of purifying the desired oligosaccharides.

[0056] Purification of HMO from the fermentation broth may include one or more of the following steps:

[0057] i) removing microbial cells from the fermentation broth to obtain a clarified process stream;

[0058] ii) subjecting the clarified process stream to at least one ultrafiltration;

[0059] iii) treating the clarified process stream at least once with a cation exchange resin and / or with an anion

[0060] treating the clarified process stream at least once with an ion exchange resin;

[0061] iv) subjecting the clarified process stream to at least one nanofiltration;

[0062] v) subjecting the clarified process stream to at least one electrodialysis;

[0063] vi) treating the clarified process stream at least once with activated carbon; and / or

[0064] vii) subjecting the clarified process stream to at least one crystallization and / or precipitation step.

[0065] Suitable methods for removing microbial cells from fermented broth include centrifugation, wherein the microbial cells are obtained as pellets and the fermented broth is obtained as supernatant. In additional and / or alternative embodiments, microbial cells are removed from the fermented broth by filtration. Suitable filtration methods for removing cells from the fermented broth include microfiltration and ultrafiltration.

[0066] Such microfiltration is a physical filtration process in which a fluid containing particles is passed through a membrane of a specific pore size thereby separating the particles from the fluid. As used herein, the term "microfiltration" refers to a physical filtration process in which cells are separated from a fermentation broth.

[0067] Ultrafiltration is a variation of membrane filtration and is not fundamentally different. In ultrafiltration, forces like pressure or concentration gradients lead to 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 into the permeate (filtrate). Ultrafiltration membranes are defined by the molecular weight cut-off (MWCO) of the membrane used. Ultrafiltration is applied as cross-flow filtration or dead-end filtration.

[0068] Typically, microbial cells synthesize the desired HMO intracellularly and secrete it into the fermentation broth. The HMO so produced ends up in the fermentation broth, which is then subjected to further processing steps to purify the HMO, as described below.

[0069] Although the method is used to purify HMOs that have been produced by microbial fermentation, the method can also be used to purify HMOs produced by in vitro enzymatic catalysis. The HMOs can be purified from the reaction mixture at the end of the biocatalytic reaction. The reaction mixture is subjected to the purification method as a clarified process stream.

[0070] The clarified process stream contains the HMOs as well as by-products and undesirable impurities, such as monosaccharides, disaccharides, undesirable oligosaccharide by-products, ions, amino acids, polypeptides, proteins and / or nucleic acids.

[0071] In additional and / or alternative embodiments, the method for purifying the desired HMO includes at least one step of 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, Logne, DE).

[0072] In additional and / or alternative embodiments, the method for purifying the desired HMO includes an anion exchange treatment to remove undesirable 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 1x2 (Mesh 200-400), Dowex 1x8 (Mesh 100-200), Purolite Chromalite CGA 100x4 (Purolite GmbH, Ratingen, DE), Dow Amberlite FPA51 (Dow Chemicals, Ml, USA).

[0073] In additional and / or alternative embodiments, the method for purifying HMO includes a nanofiltration and / or diafiltration step to remove impurities with lower molecular weight and concentrate the desired oligosaccharides. Diafiltration involves adding fresh water to the solution to remove (wash off) membrane permeable components. Diafiltration can be used to separate components based on the molecular size and charge of the separated components by using appropriate membranes, wherein one or more substances are effectively retained and other substances are membrane permeable. Specifically, diafiltration using nanofiltration membranes can effectively separate low molecular weight compounds, such as small molecules and salts. Nanofiltration membranes typically have a molecular weight cutoff of 150-1000 Daltons. Nanofiltration is widely used in the dairy industry for whey concentration and desalination.

[0074] 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).

[0075] Diafiltration using nanofiltration membranes has been found to be effective as a pretreatment to remove bulk contaminants prior to electrodialysis treatment of solutions containing oligosaccharides. During the purification of HMOs, the use of nanofiltration membranes for concentration and diafiltration results in low energy consumption and processing costs, as well as better product quality due to reduced heat exposure, resulting in reduced Maillard and aldol reactions.

[0076] In additional and / or alternative embodiments, the method for purifying HMO comprises at least one electrodialysis step. Electrodialysis (ED) transports salt ions from one solution to another through an ion exchange membrane under the influence of an applied potential difference and can be used for the separation or concentration of ions in solution based on selective electrical migration through a semipermeable membrane.

[0077] The basic principle of electrodialysis is composed of an electrolytic cell connected to a DC generator, and the electrolytic cell includes a pair of electrodes immersed in an electrolyte for conducting ions. The electrode connected to the positive pole of the DC generator is an anode, and the electrode connected to the negative pole is a cathode. Then, the electrolyte solution supports the electric current generated by the movement of negative ions and positive ions toward the anode and cathode respectively. The membrane used for electrodialysis is basically a porous ion exchange resin sheet with negative or positive charge groups, and is therefore described as a cationic membrane or an anionic membrane, respectively. Ion exchange membranes are usually made of polystyrene with a suitable functional group (such as sulfonic acid for cationic membranes or quaternary ammonium groups for anionic membranes) cross-linked with divinylbenzene. Electrolyte can be, for example, sodium chloride, sodium acetate, sodium propionate or aminosulfonic acid. The electrodialysis stack is then assembled in a parallel manner with anion and cation membranes in a filter press between two electrode blocks, so that the stream undergoing ion depletion is well separated from the stream undergoing ion enrichment (the two solutions are also referred to as dilute (experiencing ion depletion) and concentrate (experiencing ion enrichment). The core of the electrodialysis process is a membrane stack, which consists of several anion exchange membranes and cation exchange membranes separated by spacers, mounted between two electrodes. By applying a direct current, anions and cations will migrate through the membranes towards the electrodes.

[0078] In additional and / or alternative embodiments, the method for purifying HMOs further comprises a step of continuous chromatography such as simulated moving bed (SMB) chromatography. Simulated moving bed (SMB) chromatography originates from the petrochemical and mining industries. Today, the pharmaceutical industry uses SMB chromatography to separate enantiomers from racemic mixtures. Large-scale SMB chromatography has been used to separate the monosaccharide fructose from fructose-glucose solutions, and to separate the disaccharide sucrose from beet or sugar cane syrups.

[0079] The SMB method for separating sugars uses, for example, calcium charged cross-linked polystyrene resin, anionic resin in the bisulfite form (Bechthold M., et al., Chemie Ingenieur Technik, 2010, 82, 65-75) or hydrogen form polystyrene gel strong acid cation resin (Purolite PCR833H) (Purolite, BalaCynwyd, USA).

[0080] Taking into account the continuous mode of operation, the recirculation of the mobile phase and the potential to use large column sizes, the SMB system can in principle be scaled up to achieve production volumes of hundreds of tons.

[0081] The process step of simulating moving bed chromatography is advantageous because it allows further removal of oligosaccharides that are structurally closely related to the desired oligosaccharide.

[0082] In additional and / or alternative embodiments, the method for purifying HMO comprises treating the process stream with activated carbon to remove contaminants such as colorants from the process stream.

[0083] In additional and / or alternative embodiments, the method for purifying HMO comprises at least one step of crystallizing or precipitating HMO from a clarified process stream. Crystallizing or precipitating HMO from a process stream can be performed by adding a suitable amount of a water-miscible organic solvent to the process stream containing HMO. The organic solvent can be selected from C1-C6 alcohols and C1-C4 carbonic acids.

[0084] In additional and / or alternative embodiments, the method for purifying the target HMO comprises a step of sterile filtration and / or endotoxin removal, preferably by filtering the process stream through a 3 kDa filter or a 6 kDa filter.

[0085] In additional and / or alternative embodiments, the method for purifying HMO includes a step of increasing the concentration of HMO in the process stream. The process stream may be subjected to vacuum evaporation, reverse osmosis, or nanofiltration (e.g., with a concentration of ≤ Alternatively, the crystallized or precipitated HMO is dissolved in water to obtain an HMO solution having the desired HMO concentration.

[0086] In additional and / or alternative embodiments, the resulting treated stream is an aqueous solution comprising the desired HMO at 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.

[0087] In additional and / or alternative embodiments, the aqueous solution comprises HMO in 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 / solutes in the aqueous solution.

[0088] The method of obtaining human milk oligosaccharides in the form of powder by using a vacuum roll dryer or a vacuum drum dryer includes the step of providing an aqueous solution containing the desired purified HMO.

[0089] Such aqueous solutions contain at least 1% (w / v), 10% (w / v), 20% (w / v), 30% (w / v), 35% (w / v) and up to 45% (w / v), 50% (w / v) or at least 60% (w / v) of the desired HMO.

[0090] Such an aqueous solution does not contain genetically engineered microorganisms, nucleic acid molecules derived from genetically engineered microorganisms and proteins.

[0091] In the method of obtaining HMO dry powder, an aqueous solution containing HMO is subjected to a roll drying or drum drying method.

[0092] Roller drying or drum drying is a method of drying food, etc. Before the substance is dried on the rollers, a solution, suspension or emulsion or a mixture thereof is applied to previously heated rollers through a nozzle or gap, the obtained substance is usually amorphous - depending on the material, it can also be in crystalline form, which is then peeled off from the roller surface with a blade. In this method, one or two rollers, in some cases four rollers are installed in the device, and if at least two rollers are installed, they rotate in opposite directions to achieve an increase in the effective surface. The ready-made material is mainly amorphous, or sometimes in the form of crystals and flakes. Usually, the material is distinguished from similar materials with a specific dry form, obtained, for example, from crystallization, spray drying or freeze drying processes, by its water content, hygroscopicity, flowability and other physico-chemical properties (such as porosity or density).

[0093] In certain embodiments, applying additional vacuum to the process can generally speed it up, allowing it to be carried out under milder conditions and to achieve a target dried material with a lower water content. This expands the potential product portfolio to materials that are too sensitive to standard vacuum roll or drum drying methods, such as human milk oligosaccharides.

[0094] Roller drying or drum drying is a method for obtaining dry powders, in which a solution containing the substance of interest (e.g. any kind of human milk oligosaccharides) is applied dropwise into the gap formed between heated drum cylinders. The drum cylinders are usually heated with steam or hot oil. The aqueous solution containing the HMO is evenly distributed on the heated drum cylinders, and the moisture content evaporates during the partial rotation of such drum cylinders. After drying the aqueous solution, it is removed from the drum cylinders by a scraper and falls as a powder onto a tray.

[0095] There are many types of roller dryers or drum dryers, depending on the configuration of the drum cylinder. A drum dryer comprises at least one drum cylinder. The drum body of the drum or drums of the drum dryer is heated. Typically, the drum body is heated from the inside by steam.

[0096] In one embodiment, the roller dryer or drum dryer has a single drum dryer cylinder. The feed type for the single drum dryer is selected from splash feed or spray feed, dip feed, below roll feed, top roll feed, side roll feed and multi roll feed. When the tank is placed below the drum to feed liquid, splash feed or spray feed is performed. The liquid is splashed to stick to the drum with a rotating wing installed in the liquid tank. When a shallow bowl-shaped liquid tank is provided below the drum, dip feed is performed. Liquid is fed into the tank. The drum is immersed in a fluid, which sticks to the drum surface. In the liquid tank, an agitator is provided to prevent solid precipitation and keep the liquid density constant. For roll feeding, liquid is fed between the drum and the feeding roller to form a film on the drying drum surface, which can be selected from under-roll feeding, top-roll feeding, side-roll feeding and multi-roll feeding depending on where the feeding part is located.

[0097] In a particular embodiment, the roller dryer or drum dryer is a twin-drum dryer. In a twin-drum dryer (two drum dryer cylinders), in which two heated drum cylinders are provided, the gap between the cylinders can be adjusted even during rotation of the cylinders.

[0098] In a specific embodiment, roll or drum drying is operated under vacuum. The advantage of vacuum roll drying or vacuum drum drying is that it is very fast and the exposure of the material to be dried using high temperatures is very short.

[0099] Another embodiment of the vacuum roll drying or vacuum drum drying process uses an aqueous solution containing HMO, which is provided to a heated, rotating drum cylinder in a vacuum chamber. After the solvent is removed, the product is stripped from the drum cylinder in a continuous or batch manner and obtained as an amorphous or crystalline solid material. Due to the mild drying conditions, high quality materials are obtained with maintained purity. In addition, materials with specific water content, specific surface texture and certain particle size are obtained, which provide unique product properties for the pharmaceutical and food industries.

[0100] The aqueous solution containing the HMO of interest which has been purified from a fermentation broth or a process stream is dried at a drum wall temperature of at least 25°C, preferably at least 30°C, preferably at least 35°C, preferably at least 40°C, preferably at least 45°C, preferably at least 50°C, preferably at least 55°C, preferably at least 60°C, preferably at least 65°C, preferably at least 70°C, more preferably at least 75°C, and less than 200°C, less than 195°C, less than 190°C, less than 185°C, less than 180°C, preferably less than 175°C, less than 170°C, less than 165°C, less than 160°C, less than 155°C, more preferably less than 150°C.

[0101] The aqueous solution containing HMOs that has been purified from a fermentation broth or a process stream is dried at a shell temperature of at least 25° C., preferably at least 30° C., preferably at least 35° C., preferably at least 40° C., preferably at least 45° C., preferably at least 50° C., and less than 150° C., preferably less than 145° C., preferably less than 140° C., preferably less than 135° C., preferably less than 130° C., more preferably less than 145° C. In a particularly preferred embodiment, the aqueous solution containing the target HMOs is vacuum roll dried or vacuum drum dried at a shell temperature of about 80° C. to about 90° C.

[0102] The aqueous solution containing HMOs which has been purified from the fermentation broth or process stream is dried at a rotation speed of the drum drum of at least 0.1 rpm, preferably at least 1.0 rpm and less than 20 rpm, more preferably less than 30 rpm. In a particularly preferred embodiment, the aqueous solution containing the target HMOs is vacuum roll dried or vacuum drum dried at a rotation speed of the drum drum of 1.5 to 6.5 rpm.

[0103] The aqueous solution containing HMOs which has been purified from a fermentation broth or a process stream is dried in a gap between drum cylinders of at least 0.02 mm, preferably at least 0.2 mm, less than 20 mm, more preferably less than 10 mm. In a particularly preferred embodiment, the aqueous solution containing the target HMOs is vacuum roll dried or vacuum drum dried at a gap width of 0.02 to 2 mm.

[0104] The aqueous solution containing HMOs which has been purified from the fermentation broth or process stream is dried at a pressure of at least 0.55 mbar, preferably at least 5.5 mbar, and less than 1013 mbar, more preferably less than 550 mbar. In a particularly preferred embodiment, the aqueous solution containing the target HMOs is vacuum rolled or vacuum drum dried at a pressure of 55 mbar.

[0105] In additional and / or alternative embodiments, the aqueous solution containing HMOs that has been purified from a fermentation broth or process stream is dried from a solution having an ethanol content of at least 0.1% (v / v), preferably at least 1.0% (v / v), and less than 100% (v / v), more preferably less than 50% (v / v). In a particularly preferred embodiment, the aqueous solution containing the target HMOs is vacuum roll dried or vacuum drum dried from a solution having an ethanol content of 10-50% (v / v).

[0106] In additional and / or alternative embodiments, the aqueous solution containing HMOs that has been purified from a fermentation broth or process stream is dried from a solution having a relative ethanol content of at least 0.01%, preferably at least 0.1%, and less than 100%, more preferably less than 10%, as determined by HPAEC-PAD. In a particularly preferred embodiment, the aqueous solution containing the target HMOs is vacuum roll dried or vacuum drum dried from a solution having a relative ethanol content of 0.5 to 5.0%, as determined by HPAEC-PAD.

[0107] In additional and / or alternative embodiments, the aqueous solution containing HMOs that has been purified from a fermentation broth or process stream is dried to obtain a product having a relative ethanol content of at least 0.01%, preferably at least 0.1%, and less than 100%, more preferably less than 10%, as determined by HPAEC-PAD. In a particularly preferred embodiment, the aqueous solution containing the target HMOs is vacuum roll dried or vacuum drum dried to obtain a dry HMO having a relative ethanol content of 0.5-1.0%, as determined by HPAEC-PAD.

[0108] Vacuum roll drying or vacuum drum drying of an aqueous solution containing HMO provides a low hygroscopic powder in which the HMO is present in an amorphous form and in which the particle size is uniform.

[0109] The vacuum roll dried or vacuum drum dried powder containing HMO which has been purified from the process stream is used to make a nutritional composition. The vacuum roll dried or vacuum drum dried powder consisting of HMO is suitable for human consumption and can therefore be included in a formulation for human consumption, such as a pharmaceutical formulation, infant formula, dairy beverage or dietary supplement.

[0110] The HMO may be a neutral HMO, preferably selected from 2'-fucosyllactose (2'-FL), 3-fucosyllactose (3-FL), 2',3-difucosyllactose (DiFL), lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT) and lacto-N-fucopentaose I (LNFPI). In further and / or alternative embodiments, the at least one additional HMO may be a sialylated HMO, preferably selected from 3'-sialyllactose (3'-SL), 6'-sialyllactose (6'-SL), sialyllacto-N-tetraose (LST)-a, LST-b, LST-c and disialyllacto-N-tetraose (DSLNT).

[0111] The present invention will be described by specific embodiments and with reference to the accompanying drawings, but the present invention is not limited thereto, but only by the claims. In addition, the terms "first", "second", etc. in the specification and claims are used to distinguish similar elements, and are not necessarily used to describe the order of time, space, level or any other aspects. It should be understood that the terms used in this way are interchangeable where appropriate, and the embodiments of the present invention described herein can be operated in other orders than those described or illustrated herein.

[0112] It should be noted that the term "comprising" used in the claims should not be interpreted as being limited to the means listed thereafter; it does not exclude other elements or steps. It should therefore be interpreted as specifically indicating the presence of the features, integers, steps or components mentioned, but does not exclude the presence or addition of one or more other features, integers, steps or components or combinations thereof. Thus, the scope of the expression "a device comprising components A and B" should not be limited to devices consisting only of components A and B. This means that for the purposes of the present invention, the only relevant components of the device are A and B.

[0113] Throughout this specification, reference to "one or a) embodiment means that a particular feature, structure, or characteristic described with respect to that embodiment is included in at least one embodiment of the present invention. Thus, the phrase "in one embodiment" appearing in various places throughout this specification does not necessarily always refer to the same embodiment, but may refer to the same embodiment. Furthermore, in one or more embodiments, the particular features, structures, or characteristics may be combined in any suitable manner, as will be apparent to one of ordinary skill in the art from this disclosure.

[0114] Similarly, it should be understood that in the description of representative embodiments of the present invention, the various features of the present invention are sometimes combined together in a single embodiment, its drawings or description to simplify the disclosure and facilitate the understanding of one or more of the various inventive aspects. The method of the present disclosure should not be interpreted as reflecting the following intention: the claimed invention requires more features than the features explicitly listed in each claim. Instead, as reflected in the following claims, the inventive aspects may require fewer features than all the features of any previously disclosed embodiment. Therefore, the claims following the specification are hereby expressly incorporated into this detailed description, with each claim independently serving as an independent embodiment of the present invention.

[0115] In addition, although some embodiments described herein include some features and do not include other features included in other embodiments, the combination of features of different embodiments is intended to fall within the scope of the present invention and form different embodiments, as understood by those skilled in the art. For example, in the claims below, any of the claimed embodiments can be used in any combination.

[0116] In addition, herein, some embodiments are described as a combination of methods or method elements that can be performed by a processor of a computer system or by other tools that perform functions. Therefore, a processor with necessary instructions for performing the method or method elements forms a tool for performing the method or method elements. In addition, the elements of the device embodiments described herein are examples of tools for performing such functions, which are performed by elements for achieving the purpose of the present invention.

[0117] In the description and drawings provided herein, many specific details are set forth. However, it should be understood that embodiments of the present invention can be practiced without these specific details. In other cases, well-known methods, structures, and techniques are not shown in detail to facilitate understanding of the specification and drawings.

[0118] The present invention will now be described by a detailed description of several embodiments of the present invention. Obviously, other embodiments of the present invention may be provided according to the knowledge of those skilled in the art without departing from the true spirit or technical teachings of the present invention, and the present invention is limited only by the appended claims.

[0119] Example

[0120] Example 1: Drying of N-acetylneuraminic acid sodium salt in aqueous solution

[0121] The drum cylinder wall temperature of the vacuum drum dryer was set to 105°C, and the shell temperature was set to 85°C. The vacuum was set to 55mbar, the roller speed was 2.5rpm, and the gap width was 2.0mm. An aqueous solution of N-acetylneuraminic acid sodium salt with a DSC of 25.0% was prepared. The solution was applied dropwise to the gap of a heated drum cylinder that was set to rotate in opposite directions. The material immediately began to dry on the drum cylinder and was removed by means of a blade. After the addition was complete, the vacuum was released to atmospheric pressure, and the drum cylinder wall temperature was set to room temperature. A white fine powder with a residual water content of 7.46% determined by Karl-Fisher-Titration was obtained.

[0122] Example 2: Drying of N-acetylneuraminic acid sodium salt in ethanol solution

[0123] The drum cylinder wall temperature of the vacuum drum dryer is set to 120 ° C, and the shell temperature is set to 85 ° C. The vacuum is set to 55mbar, the roller speed is 3.5rpm, and the gap width is 2.0mm. An aqueous solution of N-acetylneuraminic acid sodium salt with an EtOH content of 50% is prepared so that its DSC is 6.9%. The HPAEC-PAD of the starting material shows that the relative ethanol content is 4.06%. The solution is applied dropwise to the gap of the heated drum cylinder that is set to rotate in the opposite direction. The material immediately begins to dry on the drum cylinder and is removed by means of a blade. After the addition is completed, the vacuum is released to atmospheric pressure, and the drum cylinder wall temperature is set to room temperature. A white fine powder with a residual water content of 5.71% determined by Karl-Fischer titration is obtained. The HPAEC-PAD of the dried product shows that the relative ethanol content is 0.64%.

[0124] Example 3: Drying of N-acetylneuraminic acid in aqueous solution

[0125] The drum wall temperature of the vacuum drum dryer was set to 85°C and the shell temperature was set to 85°C. The vacuum was set to 55mbar, the roller speed was set to 3.0rpm and the gap width was set to 2.0mm. An aqueous solution of the free acid form of N-acetylneuraminic acid with a DSC of 3.6% was prepared. The solution was applied dropwise to the gap of a heated drum cylinder that was set to rotate in the opposite direction. The material immediately began to dry on the drum cylinder and was removed with the aid of a blade. After the addition was complete, the vacuum was released to atmospheric pressure and the drum wall temperature was set to room temperature. A slightly pink fine powder with a residual water content of 2.05% determined by Karl-Fischer titration was obtained.

[0126] Example 4: Drying of 2'-fucosyllactose in aqueous solution

[0127] The drum cylinder wall temperature of the vacuum drum dryer was set to 100°C and the shell temperature was set to 85°C. The vacuum was set to 55mbar, the roller speed was 2.5rpm and the gap width was 2.0mm. An aqueous solution of 2'-fucosyllactose with a DSC of 45.0% was prepared. The solution was applied dropwise into the gap of a heated drum cylinder that was set to rotate in opposite directions. The material immediately began to dry on the drum cylinder and was removed with the aid of a blade. After the addition was complete, the vacuum was released to atmospheric pressure and the drum cylinder wall temperature was set to room temperature. An ivory-colored gritty powder with a residual water content of 4.53% as determined by Karl-Fischer titration was obtained.

[0128] Example 5: Drying of 6'-sialyllactose sodium salt in aqueous solution

[0129] The drum cylinder wall temperature of the vacuum drum dryer was set to 105°C, and the shell temperature was set to 85°C. The vacuum was set to 55mbar, the roller speed was 3.5rpm, and the gap width was 2.0mm. An aqueous solution of 6'-sialyllactose sodium salt with a DSC of 26.2% was prepared. The solution was applied dropwise to the gap of a heated drum cylinder that was set to rotate in the opposite direction. The material immediately began to dry on the drum cylinder and was removed by means of a blade. After the addition was completed, the vacuum was released to atmospheric pressure, and the drum cylinder wall temperature was set to room temperature. A slightly yellow fine powder with a residual water content of 5.23% determined by Karl-Fischer titration was obtained.

[0130] Example 6: Drying of 6'-sialyllactose sodium salt in ethanol solution

[0131] The drum cylinder wall temperature of the vacuum drum dryer is set to 105 ° C, and the shell temperature is set to 85 ° C. The vacuum is set to 55mbar, the roller speed is set to 4.5rpm, and the gap width is set to 2.0mm. An aqueous solution of 6'-sialyllactose sodium salt with an EtOH content of 10% is prepared, and its DSC is 26.4%. The HPAEC-PAD of the starting material shows a relative ethanol content of 1.10%. The solution is applied dropwise to the gap of the heated drum cylinder that is set to rotate in the opposite direction. The material immediately begins to dry on the drum cylinder and is removed by means of a blade. After the addition is completed, the vacuum is released to atmospheric pressure, and the drum cylinder wall temperature is set to room temperature. A slightly yellow powder with a residual water content of 4.61% determined by Karl-Fischer titration is obtained. The HPAEC-PAD of the dried product shows a relative ethanol content of 0.62%.

[0132] Example 7: Drying of lacto-N-neotetraose in aqueous solution

[0133] The drum cylinder wall temperature of the vacuum drum dryer was set to 105°C and the shell temperature was set to 85°C. The vacuum was set to 55mbar, the roller speed was 2.5rpm and the gap width was 2.0mm. An aqueous solution of lacto-N-neotetraose with a DSC of 21.8% was prepared. The solution was applied dropwise to the gap of a heated drum cylinder that was set to rotate in opposite directions. The material immediately began to dry on the drum cylinder and was removed with the aid of a blade. After the addition was complete, the vacuum was released to atmospheric pressure and the drum cylinder wall temperature was set to room temperature. A fine white powder with a residual water content of 6.07% determined by Karl-Fischer titration was obtained.

[0134] Example 8: Drying of lacto-N-neotetraose from ethanol solution

[0135] The drum cylinder wall temperature of the vacuum drum dryer is set to 105°C, and the shell temperature is set to 85°C. The vacuum is set to 55mbar, the roller speed is 5.5rpm, and the gap width is 2.0mm. The lacto-N-neotetraose aqueous solution with an EtOH content of 10% is prepared, and its DSC is 27.3%. The solution is applied dropwise to the gap of the heated drum cylinder that is set to rotate in the opposite direction. The material immediately begins to dry on the drum cylinder and is removed by means of a blade. After the addition is completed, the vacuum is released to atmospheric pressure, and the drum cylinder wall temperature is set to room temperature. A white fine powder with a residual water content of 4.03% determined by Karl-Fischer titration is obtained.

[0136] Example 9: Drying of lacto-N-tetraose from ethanol solution

[0137] The drum cylinder wall temperature of the vacuum drum dryer was set to 105°C, and the shell temperature was set to 85°C. The vacuum was set to 55mbar, the roller speed was set to 6.5rpm, and the gap width was set to 2.0mm. An aqueous solution of lacto-N-tetraose with an EtOH content of 10% was prepared, and its DSC was 17.6%. The HPAEC-PAD of the starting material showed a relative ethanol content of 0.81%. The solution was applied dropwise to the gap of a heated drum cylinder set to rotate in opposite directions. The material immediately began to dry on the drum cylinder and was removed by means of a blade. After the addition was completed, the vacuum was released to atmospheric pressure, and the drum cylinder wall temperature was set to room temperature. A slightly yellow powder with a residual water content of 5.26% determined by Karl-Fischer titration was obtained. The HPAEC-PAD of the dried product showed a relative ethanol content of 0.77%.

[0138]

[0139]

Claims

1. A method for drying human milk oligosaccharides and / or obtaining human milk oligosaccharides in powder form, the method comprising the following steps: a) providing an aqueous solution containing purified human milk oligosaccharides; b) applying the aqueous solution to a roll dryer or a drum dryer; wherein the roller dryer or the drum dryer is a vacuum roller dryer or a vacuum drum dryer, the aqueous solution is dried at a pressure of at least 0.55 mbar and less than 550 mbar, wherein the aqueous solution is dried at a drum cylinder wall temperature of 85°C to 120°C, and wherein the aqueous solution is dried at a drum cylinder rotation speed of at least 0.1 rpm and less than 30 rpm.

2. The method of claim 1, wherein the aqueous solution comprises the purified human milk oligosaccharides at 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.

3. The method of claim 1 or 2, wherein the aqueous solution comprises human milk oligosaccharides with a total 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 / solutes in the aqueous solution.

4. The method according to claim 1 or 2, wherein the roller dryer or the drum dryer is selected from a single-drum dryer or a double-drum dryer.

5. The method of claim 1 or 2, wherein the aqueous solution is dried at a shell temperature of at least 25°C and less than 150°C.

6. The method of claim 5, wherein the aqueous solution is dried at a shell temperature of at least 30°C.

7. The method of claim 5, wherein the aqueous solution is dried at a shell temperature of at least 35°C.

8. The method of claim 5, wherein the aqueous solution is dried at a shell temperature of at least 40°C.

9. The method of claim 5, wherein the aqueous solution is dried at a shell temperature of at least 45°C.

10. The method of claim 5, wherein the aqueous solution is dried at a shell temperature of at least 50°C.

11. The method of claim 5, wherein the aqueous solution is dried at a shell temperature of less than 145°C.

12. The method of claim 5, wherein the aqueous solution is dried at a shell temperature of less than 140°C.

13. The method of claim 5, wherein the aqueous solution is dried at a shell temperature of less than 135°C.

14. The method of claim 5, wherein the aqueous solution is dried at a shell temperature of less than 130°C.

15. The method of claim 5, wherein the aqueous solution containing the target HMO is vacuum roll dried or vacuum drum dried at a shell temperature of about 80°C to about 90°C.

16. The method of claim 1 or 2, wherein the aqueous solution is dried at a rotation speed of the drum cylinder of at least 1.0 rpm and less than 20 rpm.

17. The method of claim 1 or 2, wherein the aqueous solution is dried in a gap between drum cylinders of at least 0.02 mm and less than 20 mm.

18. The method of claim 17, wherein the aqueous solution is dried in a gap between drum cylinders of at least 0.2 mm.

19. The method of claim 17, wherein the aqueous solution is dried in a gap between drum cylinders of less than 10 mm.

20. The method of claim 1 or 2, wherein the aqueous solution is dried from a solution having an ethanol content of at least 0.1% (v / v) and less than 100% (v / v).

21. The method of claim 20, wherein the aqueous solution is dried from a solution having an ethanol content of at least 1.0% (v / v).

22. The method of claim 20, wherein the aqueous solution is dried from a solution having an ethanol content of less than 50% (v / v).

23. The method of claim 20, wherein the aqueous solution is dried from a solution having a relative ethanol content of at least 0.01% and less than 10%.

24. The method of claim 1 or 2, wherein the human milk oligosaccharide is selected from N-acetyllactosamine, lacto-N-biose I, sialylated oligosaccharides and fucosylated oligosaccharides.

25. The method according to claim 24, wherein the N-acetyllactosamine or lacto-N-biose I is selected from one or more of lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT) and lacto-N-fucopentaose I (LNFPI).

26. The method of claim 24, wherein the sialylated oligosaccharide is selected from 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).

27. The method of claim 24, wherein the fucosylated oligosaccharide is selected from 2'-fucosyllactose (2'-FL), 3-fucosyllactose (3-FL), 2',3-difucosyllactose (DiFL) and lacto-N-fucopentaose I (LNFPI).

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