Spray-dried human breast milk oligosaccharide mixture

Abstract

A spray-dried powder containing a mixture of structurally different human breast milk oligosaccharides, a method for preparing said spray-dried powder, the use thereof for preparing a nutritional composition, and a nutritional composition containing said spray-dried powder are disclosed.

Description

Technology Field

[0001] The present invention relates to a preparation of human breast milk oligosaccharides. More specifically, the present invention relates to a solid preparation of human breast milk oligosaccharides and a method for preparing said solid preparation. Background Technology

[0002] Human breast milk contains a significant amount of carbohydrates. The carbohydrates present in human breast milk are L-fucose and N - It includes monosaccharides such as acetylneuraminic acid, the disaccharide lactose, and up to 20 g / L oligosaccharides, so-called “human breast milk oligosaccharides (HMOs).” HMOs represent the third most abundant component in human breast milk. It is estimated that more than 150 structurally different oligosaccharides are present in human breast milk. Selected HMOs are shown in Table 1. Each of these approximately 10 to 13 types of HMOs is present in human breast milk at concentrations of several hundred milligrams to grams per liter (Thurl et al., (2017), Nutrition Reviews 75(11) 920-933). Among the HMOs, as well as neutral HMOs, at least one N Acidic HMOs containing the acetylneuraminic acid (NeuAc) moiety are known. The structural complexity and abundance of these oligosaccharides are unique to human breast milk and are not found in the breast milk of other mammals, such as dairy cattle, for example.

[0003] Because HMOs are not digested by humans, the physiological roles of these sugars have been under study for decades. The prebiotic effects of HMOs were discovered over 100 years ago. When consumed, HMOs can regulate the composition of the human gut microbiome by supporting the growth of beneficial bacteria.

[0004]

[0005]

[0006] Several different functional effects of HMOs, particularly their impact on neonatal development, have been identified over the past few years. HMOs are known to act as baits to reduce the risk of infection by bacterial and viral pathogens attached to human cells by binding to the cell surface glycoproteins. Additionally, various HMOs possess anti-inflammatory effects and act as immunomodulators. Consequently, HMOs have been suggested to reduce the risk of food allergies. The positive effects of sialylated HMOs on the central nervous system development of newborns have also been discussed in depth (discussed below: “Prebiotics and Probiotics in Human Milk, Origins and functions of milk-borne oligosaccharides and bacteria”, Academic Press (2017) editors: McGuire M., McGuire M., and Bode L).

[0007] Efforts have been made to add individual HMOs to nutritional compositions, particularly infant formula, in order to take advantage of the beneficial effects of HMOs. However, supplementing nutritional compositions with a combination of different HMOs is better because such compositions are more similar to HMOs from natural sources, namely human breast milk, and are more likely to have better effects on human health and development than compositions containing only a single type of HMO.

[0008] The limited supply of HMOs for supplementing nutritional compositions led to the development of methods for the chemical synthesis of HMOs. Due to the drawbacks of such chemical synthesis, biocatalytic approaches emerged, in which HMOs are synthesized in vitro using purified enzymes such as glycosyltransferase. Today, individual HMOs are produced on a commercial scale using the fermentation of genetically engineered microbial cells (WO 2015 / 150328 A1, WO 2017 / 043382 A1, WO 2010 / 070104 A1, WO 2012 / 097950 A1). HMOs are synthesized by genetically engineered microbial cells and can be recovered from fermentation media and / or cell lysates to obtain substantially pure HMO preparations.

[0009] In the recovery process from the fermentation liquid, HMOs generally exist in the form of a liquid process stream, for example, an aqueous solution containing the HMO of interest and potentially also containing undesirable HMOs, which are byproducts generated during the fermentation production of the desired HMO. With the recovery of the desired HMO, i.e., the target HMO, its concentration and purity in the process stream increase. However, aqueous solutions containing HMOs are susceptible to bacterial or fungal contamination. Therefore, it is desirable to provide the desired HMO as a dry or solid product containing a small amount of water. The growth of microbial organisms is virtually impossible on or within such solid products.

[0010] Typically, sugars are obtained in solid form by crystallization. 3-Fucosyllactose (WO 2014 / 075680 A), 2'-Fucosyllactose (WO 2011 / 150939 A), D-Fucosyllactose (WO 2016 / 086947 A), Lacto- N- Tetraose (WO 2017 / 101953 A), lacto- N-NeoThe crystallization of individual HMOs from aqueous solutions is described in Tetraose (WO 2014 / 094783 A). The crystallization of HMOs involves the use of alcohols, organic solvents such as ethanol or methanol, or organic acids such as glacial acetic acid. However, when HMOs are used for human consumption, using alcohols, particularly methanol, for HMO crystallization at the end of the recovery process is inappropriate. Furthermore, organic solvents are expensive to purchase and dispose of. In addition, organic solvents are harmful to the environment and to personnel handling them. Therefore, the crystallization of HMOs is a problem in industrial-scale HMO production and must be avoided, especially at the end of the process for recovering the desired HMOs.

[0011] Therefore, preferably, a process is required to provide a mixture of HMOs combined with at least one monosaccharide in solid form, wherein the process is applicable to industrial-scale production and does not involve the use of organic solvents when the purification of one or more HMOs is completed to provide the solid formulation of said HMO.

[0012] The above objective was achieved by a method for producing a spray-dried powder containing a mixture of structurally different HMOs.

[0013] In a first embodiment, a spray-dried powder is provided that essentially consists of or contains a mixture of structurally different HMOs.

[0014] In a second embodiment, a method for producing a spray-dried powder is provided, which essentially consists of or contains a mixture of structurally different HMOs.

[0015] In a third embodiment, a use is provided for the preparation of a nutritional composition, which is essentially composed of or contains a mixture of structurally different HMOs, of spray-dried powder.

[0016] In a fourth embodiment, a nutritional composition is provided comprising a spray-dried powder that is essentially composed of or contains a mixture of structurally different HMOs. Brief explanation of the drawing

[0017] Figure 1 shows a graph illustrating the X-ray powder diffraction results of spray-dried 3-fucosilactose. Figure 2 shows spray-dried lacto- N- A graph showing the X-ray powder diffraction results of tetraose is illustrated. Figure 3 shows a graph illustrating the X-ray powder diffraction results of spray-dried 6'-sialyllactose. Figure 4 shows a graph illustrating the X-ray powder diffraction results of spray-dried 3'-sialyllactose. Fig. 5 shows 2'-fucosyllactose and lacto- N- A graph showing the X-ray powder diffraction results of a spray-dried mixture of tetraose is shown. Fig. 6 shows 2'-fucosyllactose, 3-fucosyllactose, lacto- N- A graph showing the X-ray powder diffraction results of a spray-dried mixture of tetraose, 3'-siallyllactose, and 6'-siallyllactose is shown. Specific details for implementing the invention

[0018] According to the first embodiment, a spray-dried powder is provided that essentially consists of or contains a mixture of structurally different HMOs.

[0019] In an embodiment, the spray-dried powder essentially consists of a mixture of structurally different HMOs.

[0020] As used herein, the term “essentially constitutes” means a composition comprising the compound(s) specified after the term and—optionally—an inevitable byproduct. The inevitable byproduct includes—e.g.—compounds generated during microbial fermentation for the production of one or more HMOs, as well as compounds introduced into the process stream where the HMO(s) are recovered but could not be removed therefrom. With respect to spray-dried powder, the term “essentially constitutes” comprises spray-dried powder containing at least 80 wt%, at least 85 wt%, at least 90 wt%, at least 93 wt%, at least 95 wt%, or at least 98 wt% of HMO with respect to the dry material of the spray-dried powder. The term “essentially constitutes” is likewise used with respect to spray-dried powder, process stream, and solution containing HMO.

[0021] In additional and / or alternative embodiments, a mixture of structurally different HMOs consists of 2, 3, 4, 5, 6, 7, or 7 or more structurally different HMOs. The structurally different HMOs include neutral HMOs and sialylated HMOs. Thus, the HMO mixture may contain at least one neutral HMO and / or at least one acidic HMO.

[0022] At least one neutral HMO is 2'-fucosyllactose (2'-FL), 3-fucosyllactose (3-FL), lacto- N -Tetraose (L N T), lacto- N-Neo Tetraose (L N nT) and lacto- N -Foucopentaos I (L N It can be selected from a group consisting of FPI).

[0023] At least one acidic HMO is a group consisting of sialylated HMOs, preferably 3'-sialyllactose (3'-SL), 6'-sialyllactose (6'-SL), sialyllactose- N-tetraose a (LST-a), sialyllacto- N -tetraose b (LST-b), sialyllacto- N -tetraose c (LST-c) and disialyllacto- N It can be selected from the group consisting of -tetraose (DSLNT).

[0024] Therefore, the structurally different HMOs in the mixture of structurally different HMOs are 2'-FL, 3-FL, L N T, L N nT, L N It can be selected from the group consisting of FPI, 3'-SL, 6'-SL, LST-a, LST-b, LST-c, and DSLNT.

[0025] In additional and / or alternative embodiments, the mixture of structurally different HMOs contains or essentially consists of five structurally different HMOs. In additional embodiments, the five structurally different HMOs are 2'-FL, 3-FL, L N They are T, 3'-SL and 6'-SL. In an exemplary composition of the mixture, five structurally different HMOs exist as a mixture of structurally different HMOs in amounts as specified in Table 2.

[0026] Composition of an exemplary mixture consisting of five structurally different HMOs HMO weight% 2'-FL 52.2 3-FL 13.0 L N T 26.1 3'-SL 3.5 6'-SL 5.2 gun 100.0

[0027] In additional and / or alternative embodiments, a mixture of structurally different HMOs contains or essentially consists of seven structurally different HMOs. In an additional embodiment, the seven structurally different HMOs are 2'-FL, 3-FL, L N T, L N nT, L N They are FPI, 3'-SL, and 6'-SL. In an exemplary composition of the mixture, seven structurally different HMOs are present in the mixture in amounts as specified in Table 3.

[0028] Composition of an exemplary mixture consisting of seven structurally different HMOs HMO weight% 2'-FL 39.0 3-FL 12.0 L N T 23.0 L N nT 2.0 L N FPI 16.0 3'-SL 3.0 6'-SL 5.0 gun 100.0

[0029] In additional and / or alternative embodiments, the spray-dried powder contains a mixture of structurally different HMOs and at least one monosaccharide, or is essentially composed of these. Preferably, the at least one monosaccharide is L-fucose and N - Selected from the group consisting of acetylneuraminic acid (NeuAc). In additional and / or alternative embodiments, the spray-dried powder is the monosaccharide L-fucose and N - Contains acetylneuraminic acid.

[0030] In a specific embodiment, the spray-dried powder is an HMO, 2'-FL, 3-FL, L N T, L N nT, L N FPI, 3'-SL and 6'-SL, and the monosaccharide L-fucose and N - It is essentially composed of acetylneuraminic acid.

[0031] In an exemplary composition, seven structurally different HMOs and two monosaccharides are present in amounts as specified in Table 4.

[0032] Composition of an exemplary mixture containing HMOs and monosaccharides HMO weight% 2'-FL 33.6 3-FL 10.7 L N T 20.1 L N nT 2.0 L N FPI 13.4 3'-SL 2.7 6'-SL 4.0 NeuAc 8.1 L-Foucos 5.4 gun 100.0

[0033] In additional and / or alternative embodiments, the spray-dried powder comprises the compositions provided in Table 5 or essentially consists of these.

[0034] Exemplary composition of spray-dried powder Sugars A [Weight %] B [Weight %] 2'-FL 30.0 - 55.0 33.6 - 52.2 3-FL 10.0 - 15.0 10.7 - 13.0 L N T 20.0 - 30.0 20.1 - 26.1 L N nT 0.0 - 5.0 0.0 - 2.0 L N FPI 0.0 - 20.0 0.0 - 16.0 3'-SL 2.0 - 4.0 2.7 - 3.5 6'-SL 4.0 - 6.0 4.0 - 5.2 NeuAc 0.0 - 10.0 0.0 - 8.1 L-Foucos 0.0 - 6.0 0.0 - 5.4 gun 100.0 100.0

[0035] In additional and / or alternative embodiments, at least one HMO of the mixture of structurally different HMOs and / or the spray-dried powder was produced by microbial fermentation. In a specific embodiment, all HMOs of the mixture of structurally different HMOs and / or the spray-dried powder were produced by microbial fermentation.

[0036] In additional and / or alternative embodiments in which the spray-dried powder contains at least one monosaccharide, said at least one monosaccharide was produced by microbial fermentation. The spray-dried powder is L-fucose and N In another embodiment containing acetylneuraminic acid, the two monosaccharides were produced by microbial fermentation.

[0037] Accordingly, in a specific embodiment, all sugars present in the spray-dried powder, namely HMO or HMO and monosaccharides, were produced by microbial fermentation.

[0038] In additional and / or alternative embodiments, at least one HMO in the spray-dried powder, preferably all HMOs in the spray-dried powder, exists amorphously. The spray-dried powder is L-fucose and / or N - In this embodiment containing acetylneuraminic acid, the monosaccharides, at least one monosaccharide or two monosaccharides, exist in an amorphous form.

[0039] In additional and / or alternative embodiments, the spray-dried powder contains a small amount of water. The term “small amount of water” refers to 15 weight percent or less of water, preferably 10 weight percent or less of water, more preferably 7 weight percent or less of water, and most preferably 5 weight percent or less of water.

[0040] In additional and / or alternative embodiments, the spray-dried powder is free of genetically engineered microorganisms and does not contain nucleic acid molecules derived from genetically engineered microorganisms.

[0041] A spray-dried powder, essentially composed of a mixture of structurally different HMOs, optionally combined with at least one monosaccharide, has an advantage over a liquid composition in that it is less susceptible to microbial contamination. The spray-dried powder also has an advantage over a powder having components of the same composition obtained by freeze-drying or lyophilization in that it has lower hygroscopicity and can maintain fluidity for a much longer period.

[0042] According to a second embodiment, a method for producing a spray-dried powder is provided, which essentially consists of or comprises a mixture of structurally different HMOs, wherein at least one structurally different HMO, preferably all structurally different HMOs, is produced by microbial fermentation. The method comprises the following steps:

[0043] a) a step of recovering at least one structurally different HMO from the fermentation broth;

[0044] b) a step of providing an aqueous solution of at least one HMO of step a); and

[0045] c) A step of spray-drying the solution from step b).

[0046] In additional and / or alternative embodiments, the step of purifying at least one HMO from the fermentation liquid (step a) comprises one or more of the following steps:

[0047] i) a step of removing microbial cells from the fermentation liquid to obtain a process stream;

[0048] ii) a step of applying the process stream to at least one ultrafiltration process;

[0049] iii) a step of treating the process stream with a cation exchange resin at least once and / or an anion exchange resin at least once;

[0050] iv) a step of applying the process stream to at least one nanofiltration;

[0051] v) a step of applying a process stream to at least one electrodialysis;

[0052] vi) a step of treating the process stream with activated carbon at least once; and / or

[0053] vii) A step of applying the process stream to at least one crystallization and / or precipitation step.

[0054] At least one of the structurally different HMOs of the mixture or at least one of the HMOs may be produced by microbial fermentation, wherein a genetically engineered microorganism capable of synthesizing the HMO is cultured in a culture medium (fermentation liquid) under conditions that allow the synthesis of the HMO by said genetically engineered microorganism. The purification of the HMO synthesized by the cells of the genetically engineered microorganism includes the step of separating the microbial cells from the fermentation liquid to obtain a process stream. The process stream is essentially cell-free and contains said HMO(s). This step is the first step in the process of purifying the desired HMO.

[0055] A suitable method for separating microbial cells from the fermentation liquid includes centrifugation, wherein the microbial cells are obtained as a pellet and the fermentation liquid as a supernatant. In additional and / or alternative embodiments, the microbial cells are separated from the fermentation liquid by filtration. A suitable filtration method for separating cells from the fermentation liquid includes microfiltration and ultrafiltration.

[0056] Such microfiltration is a physical filtration process in which particles are separated from a fluid by passing a particle-containing fluid through a membrane of a specific pore size. As used herein, the term “microfiltration” refers to a physical filtration process in which cells are separated from a fermentation liquid.

[0057] Ultrafiltration is a type of membrane filtration and is fundamentally no different. In ultrafiltration, forces such as pressure or a concentration gradient lead to separation through a semipermeable membrane. High molecular weight cells, suspended solids, and solutes are retained in the so-called residue, while low molecular weight solutes, such as water and the desired sialylated oligosaccharides, pass through the membrane in the permeate (filtrate).

[0058] Ultrafiltration membranes are defined by the molecular weight cutoff (MWCO) of the membrane used. Ultrafiltration is applied in cross-flow or dead-end modes.

[0059] Typically, microbial cells synthesize HMOs intracellularly. Depending on the structure of the HMO, the HMO is either delivered to the fermentation broth or remains within the microbial cells. In the former case, the HMO thus produced is present in the fermentation broth at the end of fermentation and can be recovered from the fermentation broth, thereby becoming a process stream. In the latter case, the microbial cells containing the HMO are separated from the fermentation broth and lysed to release the HMO. Thus, the cell lysate contains HMO and subsequently becomes a process stream for the purification of the HMO as described herein.

[0060] Although the process is used for the purification of HMOs produced by microbial fermentation, the process can also be used to purify HMOs produced by in vitro enzyme catalysis. The HMOs can then be purified from the reaction mixture upon completion of the biocatalytic reaction. The reaction mixture is applied to the purification process as a process stream.

[0061] The process stream contains not only the desired HMO(s) but also by-products and unwanted impurities such as monosaccharides, disaccharides, unintended oligosaccharide by-products, ions, amino acids, polypeptides, proteins, and / or nucleic acid molecules.

[0062] In additional and / or alternative embodiments, the purification process of HMO comprises at least one cation exchange treatment step for removing positively charged compounds from the purified process stream.

[0063] Cation exchange resins suitable for removing positively charged compounds from process streams are H + Lewatit of the form ® S 6368 A (Lanxess AG, Cologne, DE); Lewatit ® S 2568 (H + Includes ) (Lanxess AG, Cologne, DE).

[0064] In additional and / or alternative embodiments, the purification process of HMO includes an anion exchange treatment step to remove unwanted negatively charged compounds from the cleared process stream.

[0065] Suitable anion exchange resin is Lewatit ® S 2568 (Cl - ) (Lanxess AG, Cologne, DE) ® S 6368 A (Lanxess AG, Cologne, DE), Lewatit ® S 4268 (Lanxess AG, Cologne, DE), Lewatit ® S 5528 (Lanxess AG, Cologne, DE), Dowex ® AG 1x2 (Mesh 200-400), Dowex ® 1x8 (Mesh 100-200), Purolite ® Chromalite ® CGA100x4 (Purolite GmbH, Ratingen, DE), Dow ® Amberlite TM Includes FPA51 (Dow Chemicals, ML, USA).

[0066] In additional and / or alternative embodiments, the purification process of the HMO includes a nanofiltration and / or dialysis filtration step to remove low molecular weight impurities and concentrate the desired HMO.

[0067] Dialysis involves adding fresh water to a solution to remove (wash) membrane-permeable components. Dialysis can be used to separate components based on molecular size and charge using appropriate membranes, where one or more species are efficiently retained and others are membrane-permeable. In particular, dialysis using nanofiltration membranes is effective for the separation of low molecular weight compounds, such as low molecules and salts. Nanofiltration membranes generally have molecular weight cutoffs in the range of 150 to 1,000 daltons. Nanofiltration is widely used in the dairy industry for the concentration and demineralization of whey.

[0068] Membranes suitable for nanofiltration and / or dialysis filtration are Dow ® Filmtec TM NF270-4040, Trisep ® Includes 4040-XN45-TSF (Microdyn-Nadir GmbH, Wiesbaden, DE), GE4040F30 and GH4040F50 (GE Water & Process Technologies, Ratingen, DE).

[0069] Dialysis filtration using nanofiltration membranes has been found to be effective as a pretreatment for removing significant amounts of contaminants prior to electrodialysis of oligosaccharide-containing solutions. The use of nanofiltration membranes for concentration and dialysis filtration during HMO purification lowers energy and processing costs, improves product quality due to reduced heat exposure, and decreases Maillard and Aldol reactions.

[0070] In additional and / or alternative embodiments, the purification process of HMO includes at least one electrodialysis step.

[0071] Electrodialysis (ED) combines dialysis and electrolysis and can be used for the separation or concentration of ions in solution based on selective electrophoresis through a semipermeable membrane.

[0072] 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 electrode of the DC generator is the anode, and the electrode connected to the negative electrode is the cathode. The electrolyte solution then supports the flow of current, which causes anions and cations to move to the positive and negative electrodes, respectively. The membranes used in electrodialysis are essentially sheets of porous ion-exchange resin having negative or positive charge groups, and are therefore described as cation or anion membranes, respectively. Ion-exchange membranes are generally made of polystyrene having suitable functional groups (such as sulfonic acid for cation membranes or quaternary ammonium groups for anion membranes) cross-linked with divinylbenzene. The electrolyte can be, for example, sodium chloride, sodium acetate, sodium propionate, or sulfamic acid. The electrodialysis stack is subsequently assembled with anion and cation membranes in parallel, as in a filter press between two electrode blocks, to ensure that the stream undergoing ion depletion is effectively separated from the stream becoming ion-rich (the two solutions are also referred to as the dilute (where ion depletion occurs) and the concentrate (where ion enrichment occurs). The core of the electrodialysis process is the membrane stack, which consists of several anion-exchange and cation-exchange membranes separated by spacers and installed between two electrodes. By applying a direct current, anions and cations move across the membrane toward the electrodes.

[0073] In additional and / or alternative embodiments, the purification process of HMO further includes a continuous chromatography step, such as simulated bed moving (SMB) chromatography.

[0074] Semi-moving bed (SMB) chromatography originated in the petrochemical and mineral industries. Today, SMB chromatography is used in the pharmaceutical industry to separate enantiomers from racemic mixtures. Large-scale SMB chromatography has already been used to separate the monosaccharide fructose from fructose-glucose solutions and the disaccharide sucrose from sugar beet or sugarcane syrup.

[0075] The SMB chromatography process used for separating sugars utilizes, for example, calcium-charged, cross-linked polystyrene resins, bisulfite-type anionic resins (Bechthold M., et al., Chemie Ingenieur Technik, 2010, 82, 65-75), or hydrogen-type polystyrene-based gel strong acid cation resins (Purolite). ® We use PCR833H (Purolite, Bala Cynwyd, USA).

[0076] Considering the continuous operation mode, mobile phase recycling, and the possibility of using large column sizes, the SMB chromatography system can be sized to theoretically achieve production capacities of hundreds of tons.

[0077] The process step of pseudo-moving bed chromatography has an advantage in that it allows for the additional removal of oligosaccharides that are structurally closely related to the desired oligosaccharide.

[0078] In additional and / or alternative embodiments, at least one HMO purification process includes treating the process stream with activated carbon to remove contaminants, such as coloring agents, from the process stream.

[0079] In additional and / or alternative embodiments, the purification process of at least one HMO comprises at least one step of crystallizing or precipitating the HMO from the process stream. The crystallization or precipitation of at least one HMO from the process stream is miscible with water in the process stream containing at least one HMO. This can be performed by adding an appropriate amount of organic solvent. The organic solvent may be selected from the group consisting of C1 to C6-alcohols and C1 to C4-carboxylic acids. At least one crystallization or precipitation step of HMO is not performed at the end of the recovery process so that any residual amount of organic solvent or carboxylic acid can be removed by a subsequent process step.

[0080] An additional and / or alternative embodiment of at least one HMO purification process comprises a sterile filtration and / or endotoxin removal step, preferably by filtration of the process stream through a 3 kDa filter or a 6 kDa filter.

[0081] In additional and / or alternative embodiments, the purification process of at least one HMO includes a step of increasing the concentration of at least one HMO in a process stream. The concentration of at least one HMO in the process stream may be increased by applying the process stream to vacuum evaporation, reverse osmosis, or nanofiltration (e.g., nanofiltration using a nanofiltration membrane with a size exclusion limit of 20 Å or less). Alternatively, to obtain at least one HMO aqueous solution, crystallized or precipitated HMO is dissolved in water, wherein the aqueous solution has said at least one HMO at a desired concentration.

[0082] In additional and / or alternative embodiments, the resulting process stream is an aqueous solution containing at least one HMO at a concentration of 20 g / L or more, 25 g / L or more, 30 g / L or more, 40 g / L or more, 60 g / L or more, 100 g / L or more, 200 g / L or even 300 g / L or more.

[0083] In additional and / or alternative embodiments, the aqueous solution contains at least one HMO with a purity of at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, or at least 98% with respect to the weight of the dry material / solute in the solution.

[0084] As used herein, the term "purity" refers to chemical purity, that is, the degree to which a substance is not diluted or mixed with external substances. Accordingly, chemical purity is an indicator of the relationship between at least one HMO and byproducts / impurities. Chemical purity is expressed as a percentage (%) and is calculated using the following formula:

[0085]

[0086] The purity of HMO in the formulation can be determined by using any suitable method known to those skilled in the art, e.g., HPLC, and calculating the ratio of the area under the peak(s) representing the amount of HMO(s) to the sum of the areas under the peaks representing HMO(s) and all other compounds other than said HMO(s) in the same chromatogram.

[0087] An aqueous solution containing at least one HMO can be stored under appropriate conditions, for example, said aqueous solution can be frozen.

[0088] At least one HMO purification process is cost-effective and easy to scale up, making it suitable as the basis for multi-ton scale manufacturing processes.

[0089] At least one HMO purification process also has the advantage of being free of genetically engineered microorganisms and nucleic acid molecules derived from genetically engineered microorganisms in the aqueous solution. Furthermore, the aqueous solution is free of proteins. Completely removing proteins eliminates the risk of causing allergies to potential consumers.

[0090] A method for preparing a spray-dried powder comprises the step of providing an aqueous solution containing at least one HMO or a mixture of structurally different HMOs, and - optionally - at least one monosaccharide.

[0091] In additional and / or alternative embodiments, at least one HMO is 2'-FL, 3-FL, L N T, L N nT, L N It is selected from the group consisting of FPI, 3'-SL, 6'-SL, LST-a, LST-b, LST-c, and DSLNT.

[0092] In additional and / or alternative embodiments, the mixture of structurally different HMOs comprises five structurally different HMOs, preferably 2'-FL, 3-FL, L N It consists of T, 3'-SL, and 6'-SL. In another embodiment, the mixture of structurally different HMOs consists of seven types of structurally different HMOs, preferably 2'-FL, 3-FL, L N T, L N nT, L N It consists of FPI, 3'-SL and 6'-SL.

[0093] In additional and / or alternative embodiments, the aqueous solution is preferably L-fucose and N - It further contains at least one monosaccharide selected from the group consisting of acetylneuraminic acid. In another embodiment, the aqueous solution contains L-fucose and N - Contains more acetylneuraminic acid.

[0094] In additional and / or alternative embodiments, the aqueous solution contains at least one HMO or a mixture of HMOs, and / or at least one monosaccharide in an amount of 20% (w / v), 30% (w / v), 35% (w / v), and up to 45% (w / v), 50% (w / v), 60% (w / v) of total sugars.

[0095] In additional and / or alternative embodiments, the aqueous solution contains at least one HMO or a mixture of HMOs with a purity of at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, or at least 98% with respect to the weight of the dry material / solute in the solution.

[0096] In additional and / or alternative embodiments, the aqueous solution does not contain genetically engineered microorganisms, nucleic acid molecules derived from genetically engineered microorganisms, or proteins.

[0097] In a method for manufacturing a spray-dried powder, an aqueous solution containing at least one HMO or a mixture of structurally different HMOs is spray-dried.

[0098] Spray drying is a method for obtaining a dry powder, in which a solution containing a substance of interest is first sprayed into small droplets that are rapidly dried by hot air.

[0099] Spray drying is very fast, and the exposure of the material to high temperature is very short.

[0100] In additional and / or alternative embodiments, an aqueous solution containing at least one HMO or a structurally different HMO mixture 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 150°C, preferably less than 140°C, and more preferably less than 135°C.

[0101] In additional and / or alternative embodiments, an aqueous solution containing at least one HMO or a mixture of structurally different HMOs is spray-dried at an outlet temperature of at least 60°C, preferably at least 65°C, and less than 80°C, preferably less than 70°C. In a particularly preferred embodiment, an aqueous solution containing HMO(s) is spray-dried at a nozzle temperature of about 68°C to about 70°C.

[0102] It should be understood that, optionally combined with at least one monosaccharide, each species of structurally different HMOs can be purified and individually spray-dried, and the resulting spray-dried powders can be mixed in any desired proportion. In an alternative embodiment, an aqueous solution contains all structurally different HMOs, and a resulting aqueous solution containing a mixture of structurally different HMOs in a desired proportion is spray-dried.

[0103] In additional and / or alternative embodiments, an aqueous solution containing at least one HMO or a mixture of structurally different HMOs is—e.g.—L-fusose and / or N - It further contains at least one monosaccharide such as acetylneuraminic acid. An aqueous solution containing at least one HMO or a mixture of structurally different HMOs and at least one monosaccharide is subsequently spray-dried to obtain a spray-dried powder essentially composed of at least one HMO, at least one monosaccharide or a mixture of structurally different HMOs and at least one monosaccharide.

[0104] The proportion of sugars (HMO and monosaccharide(s)) in the derived spray-dried powder corresponds to the proportion of these sugars in the aqueous solution.

[0105] The latter process has the advantage that monosaccharides that cannot be spray-dried individually can be spray-dried in the presence of one or more HMOs.

[0106] Spray drying of an aqueous solution containing at least one HMO or a mixture of structurally different HMOs provides a powder with low hygroscopicity, wherein the HMO exists amorphously and the particle size is homogeneous. A spray-dried powder essentially composed of at least one HMO or a mixture of structurally different HMOs has lower hygroscopicity than a powder of the same composition obtained by freeze-drying. Accordingly, a spray-dried powder as described herein has advantages in terms of further use and processing.

[0107] According to a third embodiment, the mixture essentially consists of a mixture of structurally different HMOs or a mixture of structurally different HMOs and at least one monosaccharide, preferably L-fusose and / or N - A use is provided for the preparation of a nutritional composition of a spray-dried powder containing acetylneuraminic acid. A mixture of structurally different HMOs - and optionally, preferably L-fucose and N A spray-dried powder essentially composed of at least one monosaccharide selected from the group consisting of acetylneuraminic acid is suitable for human consumption and can therefore be included in formulations for human consumption such as pharmaceutical formulations, infant formula, dairy beverages or dietary supplements.

[0108] According to a fourth embodiment, a nutritional composition is provided that contains a spray-dried powder such as that described in the first embodiment and / or prepared according to the second embodiment.

[0109] In additional and / or alternative embodiments, the nutritional composition is Neu5Ac, 2'-FL, 3-FL, L N T, L N nT, L N It includes a mixture essentially composed of FPI, 3'-SL, 6'-SL, and L-fucose. A composition containing a preferred amount of each of the above compounds is provided in Table 6.

[0110] The composition according to column 2 of Table 6 has a particular advantage in that it supplements infant formula so that the final infant formula consumed directly contains the compounds of the mixture at the concentrations specified in column 3 of Table 6.

[0111] Composition of a representative mixture containing Neu5AC suitable for infant formula. compound Proportion in the mixture (weight percentage) Final concentration (g / L) in infant formula 2'-FL 34 2.5 3-FL 11 0.8 L N T 20 1.5 L N nT 2 0.15 L N FPI 13 1.0 3'-SL 3 0.2 6'-SL 4 0.3 Neu5Ac 8 0.6 L-Foucos 5 0.4 gun 100 7.45

[0112] In additional and / or alternative embodiments, the nutritional composition contains one or more additional ingredients. The one or more additional ingredients are selected from the group consisting of oils, fats and fatty acids (e.g., olive oil, sunflower oil, coconut oil, nut oil, rapeseed oil, palm oil, flaxseed oil, fish oil, linolenic acid, soybean oil, etc.), carbohydrates (e.g., glucose, fructose, lactose, maltodextrin, starch, sucrose, inositol, etc.), proteins (skimmed milk, whey, casein (derived from any dairy animal), or derived from soybeans), vitamins (A, B1, B2, B5, B6, B12, C, D, E, K, biotin, folic acid, niacin, choline), minerals and trace elements (sodium, potassium, chloride, calcium, phosphorus, magnesium, iron, zinc, manganese, fluoride, selenium, iodine, copper).

[0113] In a preferred embodiment, a nutritional composition containing a spray-dried powder containing at least one human breast milk oligosaccharide or a mixture of human breast milk oligosaccharides or a mixture of at least one human breast milk oligosaccharide having at least one monosaccharide or a mixture of other fibers of structurally different human breast milk oligosaccharides, or essentially composed of such a mixture, is an infant formula that satisfies the compositional requirements specified in Regulation (EU) 2016 / 127 and / or the U.S. Federal Regulations (USA) Title 21 107.100 (nutrient specification). Representative compositions of infant formula are specified in Tables 7 and 8.

[0114] Typical ingredients of infant formula. Infant Formula: Skim milk vegetable oil (palm oil, rapeseed oil, sunflower oil) human breast milk oligosaccharide 3-fucosyllactose skim milk powder Mortierella alpina ( Mortierella alpine ) Oilfish oil, calcium carbonate, potassium chloride, vitamin C, sodium chloride, vitamin E, iron acetate, zinc, sulfate, niacin, calcium-D-pantothenate, copper, sulfate, vitamin A, vitamin B1, vitamin B6, magnesium, potassium sulfate, iodate, folic acid, vitamin K, sodium selenite, vitamin D

[0115] Composition of a typical infant formula. The final concentration is based on a powder formulation of 13.5 g in 90 ml of water. Per 100g of powder Per 100ml of infant formula energy kJ 2094-2145 283 kcal 500-512 67-68 Fat (of this specification): g 24.2-26.2 3.3-3.5 saturated fatty acids g 8.7-9.4 1.2-1.3 monounsaturated fatty acids g 10.4 1.4 polyunsaturated fatty acids g 5.5-5.9 0.7-0.8 Carbohydrates (of this specification): g 56-58 7.4-7.9 Sugars g 44-56 6-7.4 (of the present specification): Lactose g 44-56 6-7.4 Neu5Ac mg 440 60 L-Foucos mg 300 40 HMO (of this specification) g 4.22-4.81 0.57-0.65 2'-FL g 1.85-2.22 0.25-0.30 3-FL mg 555.56-592.6 75-80 L N T g 1.11 0.15 L N nT mg 0-111.11 0-15 L N PF-I mg 0-740.74 0-100 3'-SL mg 148.15-170.37 20-23 6'-SL mg 207.4-222.22 28-30 protein g 11.11-11.85 1.5-1.6 salt g 0.47-0.59 0.06-0.08 vitamin Vitamin A μg 357-358 47.3-48.2 Vitamin D μg 7.8 1.05 Vitamin E mg 8.15 1.1 Vitamin K μg 43.7-44.4 5.9-6.0 Vitamin C mg 115-118 15-16 Vitamin B1 mg 0.51-0.60 0.068-0.079 Vitamin B2 mg 1.3-1.7 0.18-0.23 Niacin mg 3.63 0.49 Vitamin B6 μg 526-600 71-81 folic acid μg 160-164 21.6-21.7 Vitamin B12 μg 1.7-1.9 0.23-0.25 biotin μg 22-30 3.0-3.9 Pantothenic acid mg 4.6-5.4 0.62-0.72 minerals sodium mg 187-236 25.3-31.2 potassium mg 673-675 88.8-91.2 chloride mg 327-333 43.1-44.9 calcium mg 460-504 62.1-66.5 person mg 335-352 45.2-46.5 magnesium mg 49.3-56.3 6.66-7.43 steel mg 4.15 0.56 zinc mg 3.7-3.8 0.49-0.51 copper μg 274 37 manganese μg 96.3 13 Fluoride μg 30.4-32.6 4.1-4.4 Selenium μg 11.1-12.3 1.5-1.6 iodine μg 101.5-103.7 13.7-14

[0116] In additional and / or alternative embodiments, the nutritional composition also contains microorganisms, preferably probiotic microorganisms. In application to infant food, the preferred microorganisms may be derived from or found in the healthy human microbial community. Preferably, without limitation, the microorganisms are Bifidobacterium ( Bifidobacterium ), Lactobacillus ( Lactobacillus ), Enterococcus ( Enterococcus ), Streptococcus ( Streptococcus ), Staphylococcus ( Staphylococcus ), Peptostreptococcus ( Peptostreptococcus ), Leuconostoc ( Leuconostoc ), Clostridium ( Clostridium ), Eubacterium ( Eubacterium ), Veilonella ( Veilonella ), Fusobacterium ( Fusobacterium ), Bacteroides ( Bacterioides ), Prevotella ( Prevotella ), Essericia ( Escherichia ), Propionibacterium ( Propionibacterium ), and Saccharomyces ( Saccharomyces It is selected from the genus ). In additional and / or alternative embodiments, the microorganism is Bifidobacterium adolesentis ( Bifidobacterium adolescentis ), Bifidobacterium animalis ( B. animalis ), Bifidobacterium bifidum ( B. bifidum ), Bifidobacterium breve ( B. breve ), Bifidobacterium infantis ( B. infantis ), Bifidobacterium lactis ( B. lactis ), Bifidobacterium longum ( B. longum ); Enterococcus faecium ( Enterococcus faecium ); Esserikia Colai ( Escherichia coli Cluiberomyces Marcianus ( Klyveromyces marxianus Lactobacillus acidophilus ( Lactobacillus acidophilus ), Lactobacillus bulgaricus ( L. bulgaricus ), Lactobacillus casei ( L. casei ), Lactobacillus crispatus ( L. crispatus), Lactobacillus fermentum ( L. fermentum ), Lactobacillus gasseri ( L. gasseri ), Lactobacillus helveticus ( L. helveticus ), Lactobacillus johnsonii ( L. johnsonii ), Lactobacillus paracasei ( L. paracasei ), Lactobacillus plantarum ( L. plantarum ), Lactobacillus reuteri ( L. reuteri ), Lactobacillus rhamnosus ( L. rhamnosus ), Lactobacillus salivarius ( L. salivarius ), Lactobacillus sakei ( L. sakei ); Lactococcus lactis ( Lactococcus lactis ) (Subspecies Lactis ( lactis ), Cremoris ( cremoris ) and diacetyllactis ( diacetylactis Includes, but not limited to)); Leuconostoc mesenteroides ( Leuconostoc mesenteroides ) (Subspecies mesenteroides ( mesenteroides Includes, but not limited to)); Pediococcus acidiractis ( Pedicoccus acidilactici ), Pediococcus Pentosaeus ( P. pentosaceus ); Propionibacterium acidipropionis ( Propionibacterium acidipropionici ), Propionibacterium freudenreich schermanii ( P. freudenreichii ssp. shermanii Staphilococcus carnosus ( Staphylococcus carnosus ); and Streptococcus thermophilus ( Streptococcus thermophilus It is selected from a group consisting of ).

[0117] In addition to a combination of living organisms, the nutritional composition also includes dead cell cultures. In the field of probiotics, dead cell cultures (e.g., tyndalized bacteria) are often used. These dead cultures can induce short-term stimulation of the immune system by providing proteins, peptides, oligosaccharides, cell wall fragments, and natural products.

[0118] In particular, including probiotic microorganisms in the nutritional composition in the presence of HMOs has a particular advantage in that it also promotes the establishment of a healthy gut microbiome.

[0119] In additional and / or alternative embodiments, the nutritional composition also includes prebiotics such as galacto-oligosaccharides (GOS), fructo-oligosaccharides (FOS), inulin, or combinations thereof.

[0120] The nutritional composition may exist in liquid form or in solid form, including but not limited to powder, granules, flakes, and pellets.

[0121] In additional embodiments, the nutritional composition is selected from the group consisting of pharmaceutical formulations, infant formula, and dietary supplements.

[0122] The present invention will be described with reference to specific embodiments and drawings, but is not limited thereto and is defined only by the claims. Additionally, terms such as "first," "second," etc., in the description and claims are used to distinguish similar elements and are not necessarily intended to describe an order in chronological, spatial, sequential, or any other manner. It should be understood that the terms used in this manner are interchangeable in appropriate circumstances, and that embodiments of the invention described herein may operate in a different order than those described or illustrated herein.

[0123] It should be noted that the term “comprising” as used in the claims should not be interpreted as being limited to the means enumerated below; it should not be interpreted as excluding other elements or steps. Accordingly, it should be interpreted as specifying the presence of the mentioned features, integers, steps, or components as described, and not excluding the presence or addition of one or more other features, integers, steps, or components, or groups thereof. Accordingly, the scope of the expression “device comprising means A and B” should not be limited to a device composed solely of components A and B. This means that, in relation to the present invention, the only relevant components of the device are A and B.

[0124] Throughout this specification, references to “one embodiment” or “an embodiment” mean that a specific feature, structure, or characteristic described in relation to an embodiment is included in at least one embodiment of the invention. Accordingly, phrases such as “in one embodiment” or “in an embodiment” appearing at various locations throughout this specification do not necessarily always refer to the same embodiment. Furthermore, a specific feature, structure, or characteristic may be combined in any suitable manner in one or more embodiments, as is apparent to those skilled in the art from this disclosure.

[0125] Likewise, in the description of representative embodiments of the invention, it should be understood that various features of the invention are sometimes grouped together within a single embodiment, drawing, or description for the purpose of simplifying the disclosure and facilitating understanding of one or more different embodiments of the invention. This method of disclosure should not be interpreted as reflecting an intention that the claimed invention requires more features than explicitly enumerated in each claim. Rather, as reflected by the following claims, an embodiment of the invention may require fewer features than all of the embodiments disclosed above. Accordingly, the claims following the detailed description are hereby explicitly incorporated into the detailed description, and each claim is presented as a separate embodiment of the invention in itself.

[0126] Furthermore, as is understood by those skilled in the art, some embodiments described herein include some features included in other embodiments but not others, whereas combinations of features of different embodiments are intended to be within the scope of the invention and form different embodiments. For example, in the following claims, any claimed embodiment may be used in any combination.

[0127] Additionally, some embodiments are described herein as a method or a combination of elements of a method that may be implemented by a processor of a computer system or by other means performing a function. Accordingly, a processor having instructions necessary to perform such a method or elements of a method forms the means for performing the method or elements of a method. Additionally, the elements of the device embodiments described herein are examples of means for performing a function performed by the elements for the purpose of carrying out the present invention.

[0128] In the detailed description and drawings provided herein, many specific details are described. However, it is understood that embodiments of the invention may be practiced without these specific details. In other cases, well-known methods, structures, and techniques have not been described in detail to aid in understanding the description and drawings.

[0129] The present invention will now be described by the detailed description of various embodiments of the invention. It is evident that other embodiments of the invention may be constructed according to the knowledge of those skilled in the art without departing from the true spirit or technical merit of the invention, and that the invention is limited only by the terms of the appended claims.

[0130] Example 1: Purification of 2'-Fucosyllactose from Fermentation Broth

[0131] As described in European patent application no. 16 196 486.1, genetically modified E. coli The production of 2'-fucosyllactose by fermentation using a strain was performed. 2'-fucosyllactose was purified from the fermentation broth by filtration, ion exchange chromatography, nanofiltration, dialysis filtration or electrodialysis, and treatment with charcoal as described in WO 2015 / 106943 A1. The resulting solution containing 2'-fucosyllactose was spray-dried to obtain a stable solid product.

[0132] Example 2: Purification of 3-Fucosyllactose from Fermentation Broth

[0133] As described in European patent application no. 16 196 486.1, genetically modified E. coli 3-fucosyllactose was produced by fermentation using a strain.

[0134] Cells were separated from the culture medium by ultrafiltration (0.05 μm cutoff) (CUT membrane technology, Erkrath, Germany) followed by a 150 kDa MWCO cross-flow filter (Microdyn-Nadir, Wiesbaden, Germany). H+ Positively charged contaminants were removed by passing a cell-free fermentation medium containing approximately 30 g / L of 3-fucosyllactose through a strong cation exchanger (Lewatit S 2568 (Lanxess, Cologne, Germany)). The solution was then adjusted to pH 7.0 using sodium hydroxide and transferred to a chloride-type anion exchanger (Lewatit S6368 A, Lanxess). Both exchangers were used at a volume of 200 L. Following a second filtration (150 kDa; Microdyn-Nadir, Wiesbaden, Germany), the particle-free solution was concentrated 5-fold by nanofiltration using a Filmtech NF270 membrane (Dow, Midland, USA) and 2.5-fold by vacuum evaporation. Conductivity approximately 15 mS cm⁻¹. -1 The concentrated solution was filtered (10 kDa; Microdyn-Nadir, Wiesbaden, Germany), purified with activated carbon (CAS:7440-44-0, Carl Roth, Karlsruhe, Germany), and deionized by electrodialysis. Accordingly, a PC-Cell BED 1-3 electrodialysis unit (PC-Cell, Heusweiler, Germany) equipped with a PC-Cell E200 membrane stack was used with the following membranes: cation exchange membrane CEM:PC SK and anion membrane AEM:PC Acid 60. 0.25 M sulfamic acid was used as the electrolyte in the process. To reduce brown discoloration caused by the Maillard reaction and aldol products derived from the fermentation process, the second ion exchange chromatography was performed using Na as described above. + and Cl - The procedure was performed using an ion exchange material of the same type but with a volume of 50 L. After concentrating the sugar solution by evaporation, the conductivity was again reduced to 4 mS cm⁻¹ by electrodialysis using the aforementioned PC-Cell BED 1-3. -1 to 0.4 mS cm -1It was reduced to or below that level. For further decolorization, the solution was mixed with activated carbon (CAS: 7440-44-0, Carl Roth, Karlsruhe, Germany), and a nearly colorless solution was obtained by filtration.

[0135] Example 3: Lacto- from fermentation liquid N - Purification of Tetraose

[0136] Lacto- N- Genome-integrated genes essential for the in vivo synthesis of tetraose, namely, N- Acetylglucosamine glucosyltransferase (Neisseria meningitidis ( Neisseria meningitidis ) IgtA from MC58), β-1,3-galactosyltransferase (Salmonella Enterica (Salmonella enterica ) subsp. salamae serovar From Greenside wbdO ), E. coli (E. coli ) lacY from K12, E. coli UDP-glucose-4-epimerase galE from K12, and E. coli Genetically modified having UTP-glucose-1-phosphate uridyltransferase galU from K12 E. coli BL21 (DE3) △ lacZ Using strain t, lacto- N- Fermentation production of tetraose was carried out. Lacto- N- 7 gl for the fermentation production of tetraose -1 NH4H2PO4, 7 gl -1 K2HPO4, 2 gl -1 KOH, 0.3gl -1 Citric acid, 5 gl -1 NH4Cl, 0.1 mM CaCl2, 8 mM MgSO4, trace elements (0.101 gl -1 Nitrilotriacetic acid, pH 6.5, 0.056 gl -1 Ammonium ferric citrate, 0.01 gl -1MnCl2x₄ H₂O, 0.002 gl -1 CoCl2x 6 H2O, 0.001 gl -1 CuCl2x₂ 2 H₂O, 0.002 gl -1 Boric acid, 0.009 gl -1 ZnS04 x 7 H2O, 0.001 gl -1 Na2Mo04 x 2 H2O, 0.002 gl -1 Na2Se03, 0.002 gl -1 The strain was grown in a mineral salt-restricted medium containing NiSO₄ (6 H₂O) and 2% glucose as a carbon source. An antifoaming agent (Struktol J673, Schill + Seilacher) was added as needed. pH was adjusted using a 25% ammonia solution. Lactose was 216 g -1 Lactose was added stepwise from the lactose stock to a final concentration of 15 mM, and the lactose concentration in the culture medium was maintained constant throughout the fermentation process. Residual lactose and lacto-, which is an accumulation during the process as a byproduct, were N- Trios II is the second one added to the fermenter E. coli It was hydrolyzed by a strain. The strain contains functional beta-lactamase, beta- for the breakdown of monosaccharides. N- Acetylhexosaminidase (Bifidobacterium bifidum ( Bifidobacterium bifidum ) from JCM1254 bbhl ), and expressed a functional gal-operon (EP 2 845 905 A).

[0137] According to the process described in Example 2, cells were separated from the fermentation broth, and lacto- N - The tetraose-containing fluid was purified to a purity of 75-80% as determined by mass balance.

[0138] Contamination by carbohydrate byproducts resulting from inefficient enzymatic degradation and metabolism was removed by chromatography using pseudo-moving bed (SMB) chromatography according to WO 2015 / 049331. Alternatively, lacto- N- Tetraose was purified by crystallization using isopropanol. For crystallization, lacto- N - A tetraose-containing solution was concentrated to a concentration of 20% by evaporation and spray-dried. Using a NUBILOSA LTC-GMP spray dryer (NUBILOSA, Konstanz, Germany), the solution was passed through a spray dryer nozzle with an inlet temperature of 130°C under a nitrogen flow, while the product flow was controlled to maintain an outlet temperature of 67°C to 68°C.

[0139] The solid material was added to a mixture of isopropanol and water (3:1 (vol / vol)) at a ratio of 1 kg of powder in 12 L of isopropanol / water. After vigorously stirring the suspension, the insoluble lacto- N- The tetraose was filtered and dried at 40 °C. Starting from 73-89% pure material, crystallized lacto- N- Tetraose was purified to approximately 95% and recovered to 85%. The sugar was dissolved in water at a concentration of 25% and passed sequentially through a 6 kDa filter (Pall Microza ultrafiltration module SIP-2013, Pall Corporation, Dreieich, Germany) and a 0.2 μm sterile filter. Under the aforementioned conditions, the sterile material was spray-dried to obtain a solid material.

[0140] Example 4: Purification of 3'- and 6'-sialyllactose from fermentation broth

[0141] Recombinant for the production of 3'- and 6'-sialyllactose E. coli The BL21 (DE3) △lacZ strain was used. This strain has common genetic variations: E. coliGlucosamine-6-phosphate synthase from GlmS, Synechocytis species ( Synechocystis from sp.) N- Acetylglucosamine 2-epimerase Slr1975, glucosamine 6-phosphate from Saccharomyces cerevisiae N- Acetyltransferase Gna1, E. coli Phosphoenolpyruvate synthase PpsA from, Campylobacter jejuni ( Campylobacter jejuni from ) N- It had chromosomal constitutive expression of acetylneuraminate synthase NeuB and CMP-sialic acid synthase NeuA from Campylobacter jejuni. Additionally, E. coli Lactose permia LacY from E. coli from W cscB (sucrose permia), cscK (fructokinase), cscA (sucrose hydrolase), and cscR (Warrior Regulator), and E. coli Gene from K12 galE (UDP-glucose-4-epimerase), galT (galactose-1-phosphate uridyltransferase), galK (galactokinase), and galM Functional components consisting of (galactose-1-epimerase) gal - Genes encoding the operon were integrated into the genome of the BL21 strain and constitutively expressed.

[0142] Strains that synthesize 3'-sialyllactose are Vibrio species ( Vibrio While possessing the 3'-sialyltransferase gene from sp.) JT-FAJ-16, the 6'-sialyllactose-producing strain is Photobacterium rayognathi ( Photobacterium leiognathi It contains alpha-2,6-sialyltransferase plsT6 from JT-SHIZ-119.

[0143] The sialyllactose-producing strain is 7 gl -1NH4H2PO4, 7 gl -1 K2HPO4, 2 gl -1 KOH, 0.3gl -1 Citric acid, 5 gl -1 NH4Cl, 1 ml l -1 They were grown in a mineral salt-restricted medium containing an antifoaming agent (Struktol J673, Schill + Seilacher), 0.1 mM CaCl2, 8 mM MgSO4, trace elements, and 2% sucrose as a carbon source. Sucrose feed supplied during the fed-batch period (500 gl -1 ) in 8 mM MgSO4, 0.1 mM CaCl2, trace elements, and 5 gl -1 NH4Cl was added.

[0144] Trace elements are 0.101 gl -1 Nitrilotriacetic acid, pH 6.5, 0.056 gl -1 Ammonium ferric citrate, 0.01 gl -1 MnCl2x₄ H₂O, 0.002 gl -1 CoCl2x⁶ H₂O, 0.001 gl -1 CuCl2x₂ 2 H₂O, 0.002 gl -1 Boric acid, 0.009 gl -1 ZnS04 x 7 H2O, 0.001 gl -1 Na2Mo04 x 2 H2O, 0.002 gl -1 Na2Se03, 0.002 gl -1 It is composed of NiS04 x 6 H2O.

[0145] For sialyllactose formation, 216 gl -1 A lactose feedstock was used. The pH was adjusted using an ammonia solution (25% v / v). Fed-batch fermentation was carried out at 30°C under constant aeration and stirring. To remove residual lactose at the end of fermentation, β-galactosidase was added to the fermentation vessel. The produced monosaccharides were metabolized by the production strain.

[0146] Subsequently, the cell-free liquid was deionized by ion exchange chromatography. First, cationic contaminants H + It was removed in a 200 L volume strong cation exchanger (Lewatit® S 2568 (Lanxess, Cologne, Germany)). The pH of the obtained solution was set to 7.0 using NaOH. In the second step, anionic ions and unwanted colorants were removed from the solution using a chloride-type strong anion exchanger, Lewatit® S 6368 S (Lanxess, Cologne, Germany). The bed volume of the ion exchanger was 200 L. Precipitates resulting from the acidification of the solution were removed using a second filtration step in a cross-flow filter (150 kDa cutoff) (Microdyn-Nadir, Wiesbaden, Germany). For sugar concentration, the solution was nanofiltered on a Dow FILMTECH NF270-4040 (Inaqua, Monchengladbach, Germany), or alternatively, a Trisep 4040-XN45-TSF membrane (0.5 kDa cutoff) (Microdyn-Nadir, Wiesbaden, Germany). Using the latter, monosaccharides generated during the fermentation process that contaminate the sialyllactose solution N- Acetylglucosamine was separated from the product. The concentrated sialyllactose solution was subsequently treated with activated carbon (CAS: 7440-44-0, Carl Roth, Karlsruhe, Germany) to remove coloring agents such as Maillard reaction products and aldol reaction products. Sialyllactose was separated into sialic acid and N- To separate from by-products from the fermentation process, such as acetylglucosamine, the solution was filtered through a 1 kDa cutoff membrane GE4040F30 (GE water & process technologies, Ratingen, Germany) at 0.6 to 0.8 mS cm⁻¹ -1It was dialysis-filtered with a conductivity of . The diluted solution was evaporated in a rotary evaporator at a rate of approximately 300 g L -1 It was concentrated to a specific concentration. Other contaminating sugars, such as di-sialyllactose, were removed during the final chromatographic separation. For this purpose, the concentrated solution was added to a weak anion exchange resin in acetate form (Amberlite FPA51, Dow Chemical, Michigan, USA). Sialyllactose hardly binds to the resin, while di-sialyllactose is adsorbed. Therefore, sialyllactose was eluted with 10 mM ammonium acetate, whereas di-sialyllactose was eluted with 1 M ammonium acetate. To remove the ammonium acetate, sialyllactose was precipitated with a 10-fold excess of ethanol. The solid fraction was filtered and dried.

[0147] A 20% sialyllactose solution was passed sequentially through a 6 kDa filter (Pall Microza ultrafiltration module SIP-2013, Pall Corporation, Dreieich, Germany) and a 0.2 μm sterile filter to obtain the final product.

[0148] Apply the following parameters to a portion of the solution B chi spray dryer (B chi Mini Spray Dryer B-290) (B Spray drying was performed using chi, Essen, Germany): inlet temperature: 130℃, outlet temperature: 67℃-71℃, gas flow: 670 L / h, suction: 100%.

[0149] Spray-dried 6'-sialyllactose had a purity of 91%, while the 3'-sialyllactose material had a purity of 93%.

[0150] Example 5: Preparation of HMO Mixture

[0151] Mixtures of different HMOs were prepared from solid products. Accordingly, individual HMOs were spray-dried, and the resulting powders were mixed. HMO-Mix I consists of 2'-fucosyllactose and lacto- N- It contains tetraose in a ratio of 70 wt% to 30 wt%; HMO-Mix II contains 2'-fucosyllactose (52 wt%), 3-fucosyllactose (13 wt%), lacto- N- It contained tetraose (26 wt%), 3'-sialyllactose (4 wt%), and 6'-sialyllactose (5 wt%). The mixed powder was dissolved in water in a solution containing 20 wt% HMO, and the resulting solution was B as described in Example 4. It was spray-dried again using a chi spray dryer.

[0152] Analysis of the resulting spray-dried powder showed that it had the same composition as the spray-dried solution for different HMO ratios.

[0153] Example 6: Preparation of a sugar mixture

[0154] 8 g of 2'-FL and 1 g of L-fucose were dissolved in 50 ml of distilled water. The resulting solution was B as described in Example 4. Spray drying was performed using a chi spray dryer. A spray-dried powder essentially composed of 2'-FL and L-fucose was obtained, wherein the ratio of 2'-FL and L-fucose in the spray-dried powder was the same as the ratio of 2'-FL and L-fucose in the spray-dried solution. Therefore, L-fucose can be spray-dried in the presence of HMO.

[0155] Example 7: Characteristics of spray-dried human breast milk oligosaccharides

[0156] 1. Differential Scanning Calorimetry (DSC)

[0157] Spray-dried human breast milk oligosaccharides, namely 3-fucosyllactose, 6'-sialyllactose, 3'-sialyllactose, lacto- N- Spray-dried mixture of tetraose, and human breast milk oligosaccharides, 2'-fucosyllactose / lacto- N- Mixture of tetraose (HMO-Mix I), and 2'-fucosyllactose, 3-fucosyllactose, lacto- N- The thermal events of each mixture of tetraose, 6'-siallyllactose, and 3'-siallyllactose (HMO Mix II) were determined.

[0158] A Mettler Toledo 821e (Mettler Toledo, Giessen, Germany) was used to determine the thermal events (glass transition temperature (Tg), additional exothermic and endothermic events) of the spray-dried product.

[0159] Approximately 25 mg of spray-dried human breast milk oligosaccharides were analyzed in a corrugated Al crucible (Mettler Toledo, Giessen, Germany). The sample was cooled to 0 °C at a rate of 10 K / min and reheated to 100 °C at a scan rate of 10 K / min. In the second heating cycle, the sample was cooled to 0 °C and then reheated to 150 °C. The midpoint of the endothermic shift of the baseline during the heating scan was defined as the glass transition temperature (Tg). Exothermic and endothermic peaks were reported based on the peak temperature and normalized energy of the event.

[0160] In all samples, the first heating scan showed major glass transition events within the entire heat flow, as evidenced by major phase transitions in the range of approximately 48–58 °C, and in most samples, the major glass transition events observed in the first heating scan reappeared in the second heating scan. The results of the DSC analysis are summarized in Table 9.

[0161] Thermal events of HMOs determined by differential scanning calorimetry Sample 1st heating scan 2nd heating scan Tg [℃] Tg [℃] 3-Foucosylactos 57,6 59,9 Lacto- N- Tetraos 49,9 79,4 6'-sialyllactose 47,6 49,6 3'-sialyllactose 48,8 54,3 2'-Fucosyllactos / lacto- N- Tetraos 56,3 59 HMO mix 54,2 55,6

[0162] In the case of 3-fucosyllactose, an endothermic relaxation peak after Tg was detected in the first heating scan. Lacto- N- In the case of tetraose, a much higher Tg was detected at approximately 79 °C during the second heating scan compared to other samples. This may be due to an endothermic event during the first heating scan at approximately 89 °C (-6.04 J / g). As with 3-fucosyllactose, an endothermic relaxation peak after Tg was also detected for 6'-sialyllactose, but an additional endothermic event occurred at 77 °C in this sample (-0.22 J / g). No endothermic events were detected for 3'-sialyllactose and HMO-Mix I, while for HMO-Mix II, the endothermic event during the first heating scan was at 79 °C (0.34 J / g).

[0163] 2. X-ray Powder Diffraction (XRD)

[0164] The morphology of the freeze-dried product was studied using wide-angle X-ray powder diffraction (XRD). Copper anode (K at 45 kV, 40 mA, wavelength of 0.154 nm) α1 An X-ray diffractometer Empyrean (Panalytical, Almelo, The Netherlands) equipped with an emission and PIXcel3D detector was used. Approximately 100 mg of spray-dried samples were analyzed in reflection mode within an angle range of 5–45° 2θ, with a step size of 0.04° 2θ and a counting time of 100 seconds per step.

[0165] All single oligosaccharides, as well as HMO mixes I and II, exhibited a completely amorphous state (Figs. 1 to 6). Lacto- N- In the case of tetrasole, a second (amorphous) signal was detected at approximately 9-10°.

[0166] 3. Laser diffraction

[0167] Powder particle size was evaluated by laser diffraction. The system detects light scattered and diffracted by an array of concentric sensor elements. Subsequently, a software algorithm approximates the particle count by calculating the z-values ​​of the light intensity reaching different sensor elements. The analysis was performed using a SALD-7500 Aggregate Sizer (Shimadzu Corporation, Kyoto, Japan) quantitative laser diffraction system (qLD).

[0168] A small amount (spatula tip) of each sample was dispersed in 2 ml of isooctane and homogenized by sonication for 5 minutes. The dispersion was transferred to a batch cell filled with isooctane and analyzed in manual mode.

[0169] The data acquisition settings are as follows: average signal count per measurement: 128, accumulated signal count: 3, and interval: 2 seconds.

[0170] Before measurement, the system was emptied with isooctane. Each sample dispersion was measured three times, and the mean and standard deviations were reported. The data were evaluated using the software WING SALD II version V3.1. Since the refractive index of the samples was unknown, the refractive index (1.530) of the sugar (disaccharide) particles was used to determine the size distribution profile. Size values ​​for the mean and median diameters were reported.

[0171] The average particle sizes of all samples were very similar, with slightly lower values ​​measured for HMO-Mix II. Particle size characteristics are summarized in Table 10. Furthermore, the particle size distribution showed the presence of a single major size group for all samples.

[0172] Particle size of HMO determined by laser diffraction size 3-Foucosylactos Lacto- N- Tetraos 6'-sialyllactose 3'-sialyllactose HMO Mix I HMO Mix II Average [nm] 119.2± 0.5 117.3± 0.7 113.8± 1.5 115.4± 0.6 113.1± 0.3 97.3± 5.3 Median [nm] 141.3± 0.0 141.3± 0.0 141.3± 0.0 121.9± 16.7 141.3± 0.0 112.2± 0.0

Claims

Claim 1 A nutritional composition comprising at least one probiotic microorganism, tyndalized probiotic bacteria, and a spray-dried powder, wherein the spray-dried powder contains a mixture of structurally different human breast milk oligosaccharides and at least one monosaccharide selected from the group consisting of L-fucose and N-acetylneuraminic acid, wherein all human breast milk oligosaccharides in the mixture are produced by microbial fermentation, and said spray-dried powder contains at least 80 weight% of human breast milk oligosaccharides, and comprising the following: . Claim 2 In paragraph 1, the nutritional composition is a formula milk for infants. Claim 3 A nutritional composition according to claim 1, wherein the spray-dried powder contains at least 85 weight% of human breast milk oligosaccharides. Claim 4 A nutritional composition according to claim 1, wherein the spray-dried powder contains at least 90 weight% of human breast milk oligosaccharides. Claim 5 A nutritional composition according to claim 1, wherein the spray-dried powder contains at least 93 weight% of human breast milk oligosaccharides. Claim 6 A nutritional composition according to claim 1, wherein the spray-dried powder contains at least 95 weight% of human breast milk oligosaccharides. Claim 7 A nutritional composition according to claim 1, wherein the spray-dried powder contains at least 98 weight% of human breast milk oligosaccharides. Claim 8 A nutritional composition according to claim 1, wherein the spray-dried powder contains 15% by weight or less of water. Claim 9 A nutritional composition according to claim 1, wherein the spray-dried powder contains 10% by weight or less of water. Claim 10 A nutritional composition according to claim 1, wherein the spray-dried powder contains 7% by weight or less of water. Claim 11 A nutritional composition according to claim 1, wherein the spray-dried powder contains 5% by weight or less of water. Claim 12 A nutritional composition according to claim 1, wherein at least one monosaccharide is produced by microbial fermentation. Claim 13 A nutritional composition according to claim 1, wherein the monosaccharide is produced by microbial fermentation. Claim 14 delete Claim 15 delete Claim 16 delete Claim 17 delete Claim 18 delete

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