Spray-dried human milk oligosaccharide mixtures

Abstract

Disclosed herein are spray-dried powders containing a mixture of structurally distinct human milk oligosaccharides, methods for producing the spray-dried powders, their use for preparing nutritional compositions, and nutritional compositions containing the spray-dried powders.

Description

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

[0002] Human breast milk contains a large amount of carbohydrates. The carbohydrates present in human breast milk include monosaccharides (such as L-fucose and N-acetylneuraminic acid), the disaccharide lactose, and up to 20 g / l of oligosaccharides (so-called "human 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 milk. Table 1 shows selected HMOs. Each of about 10 to 13 of these HMOs is present in human milk at concentrations ranging from several hundred milligrams to several grams per liter (Thurl et al., (2017), Nutrition Reviews 75 (11) 920-933). Among HMOs, neutral HMOs and acidic HMOs containing at least one N-acetylneuraminic acid (NeuAc) moiety are known. The structural complexity and abundance of these oligosaccharides are unique to human milk and have not been found in the milk of other mammals (such as domesticated dairy animals).

[0003] Since humans cannot digest HMOs, the physiological effects of these saccharides have been studied for decades. The prebiotic effects of HMOs were discovered more than 100 years ago. When consumed, HMOs are able to modulate the composition of the human gut microbiome by supporting the growth of beneficial bacteria.

[0004]

[0005]

[0006]

[0007] Table 1: Structures of notable oligosaccharides in human breast milk.

[0008] In recent years, several other functional effects of HMO have been discovered, especially their effects on neonatal development. It is known that HMO can be used as bait to reduce the risk of infection by bacterial and viral pathogens (which adhere to human cells by binding to human cell surface glycoproteins). In addition, various HMOs have anti-inflammatory effects and act as immunomodulators. Therefore, it is proposed that HMOs reduce the risk of food allergies. The positive effects of sialylated HMOs on the development of the central nervous system of neonates have also been widely discussed (reviewed in "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).

[0009] In order to take advantage of the beneficial effects of HMOs, efforts have been made to add individual HMOs to nutritional compositions, particularly infant formulas. However, supplementing nutritional compositions with a combination of different HMOs would be even better because such compositions are more similar to the natural source of HMOs (i.e., human milk) and are more likely to have a better effect on human health and development than compositions containing only a single type of HMO.

[0010] The limited supply of HMOs for supplementing nutritional compositions has led to the development of methods for chemically synthesizing HMOs. The drawbacks of this chemical synthesis have led to biocatalytic methods, in which HMOs are synthesized in vitro using purified enzymes (e.g., glycosyltransferases). Today, individual HMOs are produced on an industrial scale using 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 the fermentation medium and / or cell lysate to obtain a substantially pure HMO preparation.

[0011] During the recovery of HMO from the fermentation broth, the HMO is usually present in the form of a liquid process stream, such as an aqueous solution containing the desired HMO and possibly also containing unwanted HMO, which is a by-product generated during the fermentation production of the desired HMO. As the desired HMO (i.e., the desired HMO) is recovered, its concentration in the process stream and its purity increase. However, aqueous solutions containing HMO are susceptible to bacterial or fungal contamination. Therefore, it is preferred to provide the desired HMO in the form of a dry product or a solid product containing a small amount of water. On / in such a solid product, microbial growth is almost impossible.

[0012] Typically, sugars are obtained in solid form by crystallization. Crystallization of individual HMOs from aqueous solutions has been documented: 3-fucosyllactose (WO 2014 / 075680 A), 2'-fucosyllactose (WO 2011 / 150939 A), difucosyllactose (WO 2016 / 086947 A), lacto-N-tetraose (WO 2017 / 101953 A), lacto-N-neotetraose (WO 2014 / 094783 A). Crystallization of HMOs involves the use of organic solvents such as alcohols (mainly ethanol or methanol), or organic acids (such as glacial acetic acid). However, if the HMOs are to be used for human consumption, it is inappropriate to use alcohols (especially methanol) to crystallize the HMOs at the end of the recovery process. In addition, organic solvents are expensive to purchase and handle. Moreover, organic solvents are also harmful to the environment and the personnel who handle them. Therefore, in the production of HMOs on an industrial scale, crystallization of HMOs is disadvantageous and should be avoided, especially at the end of the recovery process of the desired HMOs.

[0013] Therefore, there is a need for a process for providing a mixture of HMOs in solid form, preferably in combination with at least one monosaccharide in solid form, which process is suitable for industrial scale production and does not involve the use of organic solvents at the end of the purification of one or more HMOs to provide a solid preparation of said HMOs.

[0014] This object has been achieved by a process for the preparation of a spray-dried powder comprising a mixture of structurally different HMOs. Summary of the invention

[0015] In a first aspect, there is provided a spray-dried powder consisting essentially of or containing a mixture of structurally distinct HMOs.

[0016] In a second aspect, a method is provided for preparing a spray-dried powder consisting essentially of or containing a mixture of structurally different HMOs.

[0017] In a third aspect, there is provided the use of a spray-dried powder consisting essentially of or containing a mixture of structurally different HMOs for the preparation of a nutritional composition.

[0018] In a fourth aspect, there is provided a nutritional composition comprising a spray-dried powder consisting essentially of or containing a mixture of structurally distinct HMOs. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Shown is a graph illustrating the results of X-ray powder diffraction of spray-dried 3-fucosyllactose.

[0020] Figure 2 A graph showing the results of X-ray powder diffraction of spray-dried lacto-N-tetraose.

[0021] Figure 3 Shown is a graph illustrating the results of X-ray powder diffraction of spray-dried 6'-sialyllactose.

[0022] Figure 4 Shown is a graph illustrating the results of X-ray powder diffraction of spray-dried 3'-sialyllactose.

[0023] Figure 5 Shown is a graph illustrating the results of X-ray powder diffraction of a spray-dried mixture of 2'-fucosyllactose and lacto-N-tetraose.

[0024] Figure 6 Shown is a graph illustrating the results of spray-dried X-ray powder diffraction of a mixture of 2'-fucosyllactose, 3-fucosyllactose, lacto-N-tetraose, 3'-sialyllactose, and 6'-sialyllactose. DETAILED DESCRIPTION

[0025] According to a first aspect, there is provided a spray-dried powder consisting essentially of or containing a mixture of structurally different HMOs.

[0026] In one embodiment, the spray-dried powder consists essentially of a mixture of structurally distinct HMOs.

[0027] As used herein, the term "consisting essentially of" refers to a composition consisting of the compound specified after the term, and (optionally) inevitable by-products. The inevitable by-products include, for example, compounds produced during microbial fermentation for the production of one or more HMOs, and compounds that are introduced into the process stream (from which the HMOs are recovered) but cannot be removed therefrom. The term "consisting essentially of" as applied to a spray-dried powder includes the spray-dried powder containing at least 80% by weight, at least 85% by weight, at least 90% by weight, at least 93% by weight, at least 95% by weight, or at least 98% by weight of the HMOs relative to the dry matter of the spray-dried powder. The term "consisting essentially of" also applies to spray-dried powders, process streams, and solutions containing HMOs.

[0028] In an additional and / or alternative embodiment, the mixture of structurally different HMOs consists of two, three, four, five, six, seven or more structurally different HMOs. The structurally different HMOs include neutral HMOs and sialylated HMOs. Thus, the mixture of HMOs may contain at least one neutral HMO and / or at least one acidic HMO.

[0029] The at least one neutral HMO may be selected from 2'-fucosyllactose (2'-FL), 3-fucosyllactose (3-FL), lacto-N-tetraose (LNT), lacto-N-tetraose (LNnT) and lacto-N-fucose I (LNPFI).

[0030] The at least one acidic HMO may be selected from sialylated HMOs, preferably 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).

[0031] Thus, the structurally different HMO in the mixture of structurally different HMOs may be selected from 2'-FL, 3-FL, LNT, LNnT, LNFPI, 3'-SL, 6'-SL, LST-a, LST-b, LST-c and DSLNT.

[0032] In an additional and / or alternative embodiment, the mixture of structurally distinct HMOs contains or consists essentially of five structurally distinct HMOs. In another embodiment, the five structurally distinct HMOs are 2'-FL, 3-FL, LNT, 3'-SL, and 6'-SL. In an exemplary composition of the mixture, the five structurally distinct HMOs are present in the mixture of structurally distinct HMOs in amounts as shown in Table 2.

[0033] HMO weight% 2'-FL 52.2 3-FL 13.0 LNT 26.1 3'-SL 3.5 6'-SL 5.2 total 100.0

[0034] Table 2: Composition of an exemplary mixture of five structurally distinct HMOs

[0035] In an additional and / or alternative embodiment, the mixture of structurally distinct HMOs contains or consists essentially of seven structurally distinct HMOs. In another embodiment, the seven structurally distinct HMOs are 2'-FL, 3-FL, LNT, LNnT, LNFPI, 3'-SL, and 6'-SL. In an exemplary composition of the mixture, the seven structurally distinct HMOs are present in the mixture of structurally distinct HMOs in amounts as shown in Table 3.

[0036]

[0037]

[0038] Table 3: Composition of an exemplary mixture of seven structurally distinct HMOs

[0039] In an additional and / or alternative embodiment, the spray-dried powder contains or consists essentially of a mixture of structurally different HMOs and at least one monosaccharide. Preferably, the at least one monosaccharide is selected from L-fucose and N-acetylneuraminic acid (NeuAc). In an additional and / or alternative embodiment, the spray-dried powder contains the monosaccharides L-fucose and N-acetylneuraminic acid.

[0040] In a specific embodiment, the spray-dried powder consists essentially of HMO 2'-FL, 3-FL, LNT, LNnT, LNFPI, 3'-SL, and 6'-SL, and the monosaccharides L-fucose and N-acetylneuraminic acid.

[0041] In one exemplary composition, seven structurally distinct HMOs and two monosaccharides are present in the amounts shown in Table 4.

[0042]

[0043]

[0044] Table 4: Composition of exemplary mixtures containing HMOs and monosaccharides

[0045] In an additional and / or alternative embodiment, the spray-dried powder comprises or consists essentially of the composition provided in Table 5.

[0046] sugar A [wt%] B [wt%] 2'-FL 30.0-55.0 33.6-52.2 3-FL 10.0-15.0 10.7-13.0 LNT 20.0-30.0 20.1-26.1 LqCy 0.0-5.0 0.0-2.0 LNFPI 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-fucose 0.0-6.0 0.0-5.4 total 100.0 100.0

[0047] Table 5: Exemplary composition of spray dried powders

[0048] In an additional and / or alternative embodiment, at least one HMO in the mixture of structurally different HMOs and / or in the spray-dried powder has been produced by microbial fermentation. In a specific embodiment, all HMOs in the mixture of structurally different HMOs and / or in the spray-dried powder have been produced by microbial fermentation.

[0049] In an additional and / or alternative embodiment, the spray-dried powder contains at least one monosaccharide that has been produced by microbial fermentation. In another embodiment, the spray-dried powder contains L-fucose and N-acetylneuraminic acid, both of which are produced by microbial fermentation.

[0050] Thus, in a particular embodiment, all sugars present in the spray-dried powder, ie HMOs or HMOs and monosaccharides, have been produced by microbial fermentation.

[0051] In an additional and / or alternative embodiment, at least one HMO in the spray-dried powder, preferably all HMOs in the spray-dried powder, are present in amorphous form. In those embodiments where the spray-dried powder contains L-fucose and / or N-acetylneuraminic acid, monosaccharides, at least one of the monosaccharides or both of the monosaccharides are present in amorphous form.

[0052] In an additional and / or alternative embodiment, the spray-dried powder contains a small amount of water. The term "small amount of water" means that the amount of water is ≤15 wt %, preferably the amount of water is ≤10 wt %, more preferably the amount of water is ≤7 wt %, and most preferably the amount of water is ≤5 wt %.

[0053] In an additional and / or alternative embodiment, the spray-dried powder is free of genetically engineered microorganisms and is free of nucleic acid molecules derived from genetically engineered microorganisms.

[0054] A spray-dried powder consisting essentially of a mixture of structurally different HMOs, optionally in combination with at least one monosaccharide, has advantages over liquid compositions in that it is less susceptible to microbial contamination. It also has advantages over powders of the same composition (obtained by freeze-drying or lyophilization) in that it is less hygroscopic and remains flowable for a longer period of time.

[0055] According to a second aspect, there is provided a process for preparing a spray-dried powder consisting essentially of or comprising a mixture of structurally different HMOs, wherein at least one of the structurally different HMOs, preferably all of the structurally different HMOs, are produced by microbial fermentation. The process comprises the following steps:

[0056] a) recovering at least one structurally distinct HMO from the fermentation broth;

[0057] b) providing an aqueous solution of at least one HMO of step a); and

[0058] c) spray drying the solution of step b).

[0059] In an additional and / or alternative embodiment, purifying at least one HMO from the fermentation broth (step a) comprises one or more of the following steps:

[0060] i) removing microbial cells from the fermentation broth to obtain a process stream;

[0061] ii) subjecting the process stream to at least one ultrafiltration;

[0062] iii) treating the process stream at least once with a cation exchange resin and / or at least once with an anion exchange resin;

[0063] iv) subjecting the process stream to at least one nanofiltration;

[0064] v) subjecting the process stream to at least one electrodialysis;

[0065] vi) treating the process stream at least once with activated carbon; and / or

[0066] vii) subjecting the process stream to at least one crystallization and / or precipitation step.

[0067] At least one HMO or any of the structurally different HMOs of the mixture can be produced by microbial fermentation, wherein a genetically engineered microorganism capable of synthesizing the HMO is cultured in a culture medium (fermentation broth) and under conditions that allow synthesis of the HMO by the genetically engineered microorganism. Purification of the HMO synthesized by cells of the genetically engineered microorganism comprises the step of separating the microbial cells from the fermentation broth to obtain a process stream. The process stream is substantially free of cells and contains the HMO. This step is the first step in the process of purifying the desired HMO.

[0068] Suitable methods for separating microbial cells from fermented liquid include centrifugation, wherein the microbial cells obtained are precipitation and the fermented liquid is a supernatant. In an additional and / or alternative embodiment, microbial cells are separated from the fermented liquid by filtration. Suitable filtration methods for separating cells from the fermented liquid include microfiltration and ultrafiltration.

[0069] Such microfiltration is a physical filtration process in which a fluid containing particles is passed through a membrane having a specific pore size to separate the particles from the fluid. As used herein, the term "microfiltration" refers to a physical filtration process for separating cells from a fermentation broth.

[0070] Ultrafiltration is a variation of membrane filtration and is not fundamentally different. In ultrafiltration, forces such as pressure or concentration gradients cause separation across 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 sialylated oligosaccharides) pass through the membrane into the permeate (filtrate).

[0071] Ultrafiltration membranes are defined by the molecular weight cutoff (MWCO) of the membrane used. Ultrafiltration is applied in cross-flow or dead-end mode.

[0072] Typically, microbial cells synthesize HMOs intracellularly. Depending on the structure of the HMO, the HMO is delivered to the fermentation broth or retained within the microbial cells. In the former case, the HMOs so produced are present in the fermentation broth at the end of the fermentation and can be recovered from the fermentation broth, which thus becomes a process stream. In the latter case, the microbial cells carrying the HMOs are separated from the fermentation broth and lysed to release the HMOs. Thus, the cell lysate contains the HMOs and becomes a process stream for purifying the HMOs, as described below.

[0073] 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 then be purified from the reaction mixture at the end of the biocatalytic reaction. The reaction mixture is subjected to the purification process as a process stream.

[0074] The process stream contains the desired HMOs as well as byproducts and undesirable impurities such as, for example, monosaccharides, disaccharides, undesirable oligosaccharide byproducts, ions, amino acids, polypeptides, proteins and / or nucleic acid molecules.

[0075] In an additional and / or alternative embodiment, the method for purifying HMO comprises at least one step of cation exchange treatment to remove positively charged compounds from the clarified process stream.

[0076] Suitable cation exchange resins for removing positively charged compounds from process streams include H + Formal S 6368A (Lanxess AG, Cologne, DE); S2568(H + )(Lanxess AG, Cologne, DE).

[0077] In an additional and / or alternative embodiment, the method for purifying HMO comprises a step of anion exchange treatment to remove unwanted negatively charged compounds from the clarified process stream.

[0078] Suitable anion exchange resins include S2568 (Cl) (Lanxess AG, Cologne, DE), S 6368A (Lanxess AG, Cologne, DE), S 4268 (Lanxess AG, Cologne, DE), S 5528 (Lanxess AG, Cologne, DE), AG 1x2(Mesh 200-400), 1x8(Mesh 100-200), CGA100x4 (Purolite GmbH, Ratingen, DE), Amberlite TM FPA51 (Dow Chemicals, MI, USA).

[0079] In an additional and / or alternative embodiment, the method for purifying HMOs comprises a nanofiltration and / or diafiltration step to remove impurities with lower molecular weight and to concentrate the desired HMOs.

[0080] Diafiltration involves adding fresh water to a solution to remove (wash out) membrane permeable components. Diafiltration can be used to separate components based on their molecular size and charge using a suitable membrane, where one or more substances are effectively retained, while other substances are membrane permeable. In particular, diafiltration using nanofiltration membranes can effectively separate low molecular weight compounds (such as small molecules) and salts. The molecular weight cutoff of nanofiltration membranes is typically 150-1000 Daltons. Nanofiltration is widely used in the dairy industry for the concentration and demineralization of whey.

[0081] Suitable membranes for nanofiltration and / or diafiltration include Filmtec TM NF270-4040, 4040-XN45-TSF (Microdyn-Nadir GmbH, Wiesbaden, DE), GE4040F30 and GH4040F50 (GE Water & Process Technologies, Ratingen, DE).

[0082] Diafiltration using nanofiltration membranes has been found to be effective as a pretreatment to remove a large number of contaminants prior to subjecting a solution containing oligosaccharides to electrodialysis. In the purification of HMOs, concentration and diafiltration using nanofiltration membranes can achieve lower energy and processing costs, as well as better product quality due to reduced heat exposure, resulting in reduced Maillard and aldol reactions.

[0083] In an additional and / or alternative embodiment, the method for purifying HMO comprises at least one electrodialysis step.

[0084] Electrodialysis (ED) combines dialysis and electrolysis and can be used to separate or concentrate ions in solution based on the selective electrical migration of ions across a semipermeable membrane.

[0085] The basic principle of electrodialysis is composed of an electrolytic cell, which includes a pair of electrodes immersed in an electrolyte for conducting ions, which are connected to a DC generator. The electrode connected to the positive pole of the DC generator is the anode, and the electrode connected to the negative pole is the cathode. The electrolyte then supports the flow of electric current, which is generated due to 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 typically composed 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. The electrolyte can be, for example, sodium chloride, sodium acetate, sodium propionate and / or sulfamic acid. The electrodialysis stack is then assembled in such a way that the anion membrane and the cation membrane are parallel, as between two electrode blocks in a filter press, so that the stream undergoing ion depletion is fully separated from the stream undergoing ion enrichment (the two solutions are also referred to as the diluent (for ion depletion) and the concentrate (for ion enrichment)). The core of the electrodialysis process is the 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 across the membranes toward the electrodes.

[0086] In an additional and / or alternative embodiment, the method for purifying HMO further comprises a step of continuous chromatography, such as simulated moving bed (SMB) chromatography.

[0087] Simulated Moving Bed (SMB) chromatography has its origins in the petrochemical and mineral 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 cane syrups.

[0088] SMB chromatography for separation of sugars uses, for example, calcium-containing, cross-linked polystyrene resins, anionic resins in the bisulfite form (Bechthold M., et al., Chemie Ingenieur Technik, 2010, 82, 65-75), or polystyrene gel strong acid cationic resins in the hydrogen form ( PCR833H) (Purolite, Bala Cynwyd, USA).

[0089] Taking into account the continuous mode of operation, the recycling of mobile phase and the potential for using large columns, the SMB chromatography system can in principle be scaled up to achieve a production volume of hundreds of tons.

[0090] An advantage of the process step of simulating moving bed chromatography is that this process step allows for further removal of oligosaccharides that are structurally closely related to the desired oligosaccharide.

[0091] In an additional and / or alternative embodiment, a method for purifying at least one HMO comprises treating a process stream with activated carbon to remove contaminants, such as pigments, from the process stream.

[0092] In additional and / or alternative embodiments, the method for purifying at least one HMO comprises at least one step of crystallizing or precipitating the HMO from a process stream. Crystallizing or precipitating the at least one HMO from the process stream can be performed by adding an appropriate amount of a water-miscible organic solvent to the process stream containing the at least one HMO. The organic solvent can be selected from C1- to C6-alcohols and C1- to C4-carboxylic acids. The step of crystallizing or precipitating the at least one HMO is not performed at the end of the recovery process, so that residual amounts of organic solvent or carboxylic acid can be removed by subsequent process steps.

[0093] In additional and / or alternative embodiments of the method for purifying at least one HMO, a step of sterile filtration and / or endotoxin removal is comprised, preferably by filtering the process stream through a 3 kDa filter or a 6 kDa filter.

[0094] In an additional and / or alternative embodiment, the method for purifying at least one HMO comprises the step of increasing the concentration of at least one HMO in a process stream. The concentration of at least one HMO can be increased by subjecting the process stream to vacuum evaporation, reverse osmosis, or nanofiltration (e.g., a process stream having a size exclusion limit of ≤ Alternatively, the crystallized or precipitated HMO is dissolved in water to obtain an aqueous solution of the at least one HMO, wherein the aqueous solution has the desired concentration of the at least one HMO.

[0095] In an additional and / or alternative embodiment, the resulting process stream is an aqueous solution containing at least one HMO in a concentration of ≥20 g / l, ≥25 g / l, ≥30 g / l, ≥40 g / l, ≥60 g / l, ≥100 g / l, ≥200 g / l or even ≥300 g / l.

[0096] In an additional and / or alternative embodiment, the aqueous solution contains at least one HMO having a purity of at least 80%, at least 85%, at least 90%, at least 93%, at least 95% or at least 98% relative to the weight of dry matter / solutes in the solution.

[0097] As used herein, the term "purity" refers to chemical purity, i.e., the degree to which a substance is not diluted or mixed with foreign substances. Thus, 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:

[0098]

[0099] The purity of HMO in the preparation can be determined by any suitable method known to those skilled in the art, for example by using HPLC and calculating the ratio of the area under the peak representing the amount of HMO to the sum of the areas under the peaks representing HMO and all other compounds except HMO in the same chromatogram.

[0100] The aqueous solution containing at least one HMO may be stored under appropriate conditions, for example the aqueous solution may be frozen.

[0101] The method for purifying at least one HMO is cost-effective and easily scalable, making it suitable as the basis for a multi-ton scale production process.

[0102] The method for purifying at least one HMO also has the advantage that the aqueous solution is free of genetically engineered microorganisms and nucleic acid molecules derived from genetically engineered microorganisms. In addition, the aqueous solution is free of proteins. The complete removal of proteins eliminates the risk of allergies to potential consumers.

[0103] The method for preparing the spray-dried powder comprises the steps of providing an aqueous solution containing at least one HMO or a mixture of structurally different HMOs, and (optionally) at least one monosaccharide.

[0104] In an additional and / or alternative embodiment, the at least one HMO is selected from 2'-FL, 3-FL, LNT, LNnT, LNFPI, 3'-SL, 6'-SL, LST-a, LST-b, LST-c and DSLNT.

[0105] In an additional and / or alternative embodiment, the mixture of structurally different HMOs consists of five structurally different HMOs, preferably consisting of 2'-FL, 3-FL, LNT, 3'-SL and 6'-SL. In another embodiment, the mixture of structurally different HMOs consists of seven structurally different HMOs, preferably consisting of 2'-FL, 3-FL, LNT, LNnT, LNFPI, 3'-SL and 6'-SL.

[0106] In an additional and / or alternative embodiment, the aqueous solution further contains at least one monosaccharide, preferably selected from L-fucose and N-acetylneuraminic acid. In another embodiment, the aqueous solution further contains L-fucose and N-acetylneuraminic acid.

[0107] In an additional and / or alternative embodiment, the aqueous solution contains at least one HMO or a mixture of HMOs, and / or at least one monosaccharide in a total amount of at least 20% (w / v), 30% (w / v), 35% (w / v), and at most 45% (w / v), 50% (w / v), 60% (w / v).

[0108] In an additional and / or alternative embodiment, the aqueous solution contains at least one HMO or mixture of HMOs having a purity of at least 80%, at least 85%, at least 90%, at least 93%, at least 95% or at least 98% relative to the weight of dry matter / solutes in the solution.

[0109] In an additional and / or alternative embodiment, the aqueous solution is free of genetically engineered microorganisms, nucleic acid molecules and proteins derived from genetically engineered microorganisms.

[0110] In the process for preparing the spray-dried powder, an aqueous solution containing at least one HMO or a mixture of structurally different HMOs is spray-dried.

[0111] Spray drying is a method of obtaining dry powders in which a solution containing the substance of interest is first sprayed into droplets, which are rapidly dried using hot air. Spray drying is very fast and the substance to be dried is exposed to high temperatures for a very short time.

[0112] In an additional and / or alternative embodiment, the aqueous solution containing at least one HMO or a mixture of structurally different HMOs is spray dried at a nozzle temperature of at least 110°C, preferably at least 120°C, more preferably at least 125°C and below 150°C, preferably below 140°C and more preferably below 135°C.

[0113] In an additional and / or alternative embodiment, the 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 below 80° C., preferably below 70° C. In a particularly preferred embodiment, the aqueous solution containing HMO is spray dried at a nozzle temperature of about 68° C. to about 70° C.

[0114] It will be appreciated that each structurally distinct HMO (optionally in combination with at least one monosaccharide) can be purified and spray dried separately, and the resulting spray dried powders can be mixed in any desired ratio. In an alternative embodiment, the aqueous solution contains all of the structurally distinct HMOs, and the resulting aqueous solution containing a mixture of the structurally distinct HMOs in the desired ratio is spray dried.

[0115] In an additional and / or alternative embodiment, the aqueous solution containing at least one HMO or a mixture of structurally different HMOs further contains at least one monosaccharide such as, for example, L-fucose and / or N-acetylneuraminic acid. The aqueous solution containing at least one HMO or a mixture of structurally different HMOs and at least one monosaccharide is then spray dried to obtain a spray-dried powder consisting essentially of at least one HMO and at least one monosaccharide or a mixture of structurally different HMOs and at least one monosaccharide.

[0116] The ratios of sugars (HMO and monosaccharides) in the resulting spray-dried powder correspond to the ratios of these sugars in the aqueous solution.

[0117] The advantage of the latter method is that monosaccharides that are not spray-dryable alone become spray-dryable in the presence of one or more HMOs.

[0118] Spray drying of an aqueous solution containing at least one HMO or a mixture of structurally different HMOs provides a powder of low hygroscopicity, wherein the HMO is present in amorphous form and wherein the particle size is uniform. Spray-dried powders consisting essentially of at least one HMO or a mixture of structurally different HMOs have a lower hygroscopicity than powders of the same composition obtained by freeze drying. Therefore, the spray-dried powders described herein are advantageous with regard to their further use and processing.

[0119] According to a third aspect, there is provided the use of a spray-dried powder consisting essentially of a mixture of structurally different HMOs, or containing a mixture of structurally different HMOs and at least one monosaccharide, preferably L-fucose and / or N-acetylneuraminic acid, for the preparation of a nutritional composition. The spray-dried powder consisting essentially of a mixture of structurally different HMOs, and optionally at least one monosaccharide, preferably selected from L-fucose and N-acetylneuraminic acid, is suitable for human consumption and can therefore be included in a product for human consumption, such as a pharmaceutical preparation, an infant formula, a dairy beverage or a dietary supplement.

[0120] According to a fourth aspect, there is provided a nutritional composition comprising the spray-dried powder according to the first aspect and / or the spray-dried powder prepared according to the second aspect.

[0121] In an additional and / or alternative embodiment, the nutritional composition comprises a mixture consisting essentially of Neu5Ac, 2'-FL, 3-FL, LNT, LNnT, LNFPI, 3'-SL, 6'-SL and L-fucose. Compositions comprising preferred amounts of each of the compounds are provided in Table 6.

[0122] The composition according to the second column of Table 6 is particularly advantageous for supplementing infant formulas, so that the final infant formula for direct consumption may contain the compounds of the mixture in the concentrations specified in the third column of Table 6.

[0123]

[0124]

[0125] Table 6: Composition of representative Neu5AC-containing mixtures suitable for use in infant formula.

[0126] In an additional and / or alternative embodiment, the nutritional composition comprises one or more other ingredients. The one or more other ingredients are selected from oils, fats and fatty acids (e.g., olive oil, sunflower oil, coconut oil, nut oil, rapeseed oil, palm oil, linseed oil, fish oil, linolenic acid, soybean oil, etc.), carbohydrates (e.g., glucose, fructose, lactose, maltodextrin, starch, sucrose, inositol, etc.), proteins (from skim milk, whey, casein (derived from any domesticated dairy animal) or soy), 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).

[0127] In a preferred embodiment, the nutritional composition containing the spray-dried powder is an infant formula that complies with the composition requirements specified in Regulation (EU) 2016 / 127 and / or Code of Federal Regulations (USA) Title 21 107.100 (nutrition specifications), wherein the spray-dried powder contains / essentially consists of: at least one human milk oligosaccharide, or a mixture of human milk oligosaccharides, or a mixture of at least one human milk oligosaccharide and at least one monosaccharide, or a mixture of structurally different human milk oligosaccharides and other fibers. Representative compositions of infant formulas are listed in Tables 7 and 8.

[0128]

[0129]

[0130] Table 7: Composition of representative infant formulas.

[0131]

[0132]

[0133]

[0134] Table 8: Composition of representative infant formula. Final concentration is based on preparation of 13.5 g powder in 90 ml water.

[0135] In an additional and / or alternative embodiment, the nutritional composition further contains a microorganism, preferably a probiotic microorganism. For the application of baby food, the preferred microorganism is derived from the microbial group of a healthy human being or can be found in the microbial group of a healthy human being. Preferably, but not limited to, the microorganism is selected from the genus Bifidobacterium, Lactobacillus, Enterococcus, Streptococcus, Staphylococcus, Peptostreptococcus, Leuconostoc, Clostridium, Eubacterium, Veilonella, Fusobacterium, Bacterioides, Prevotella, Escherichia, Propionibacterium and Saccharomyces.In an additional and / or alternative embodiment, the microorganism is selected from Bifidobacterium adolescentis, Bifidobacterium animalis, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium infantis, Bifidobacterium lactis, Bifidobacterium longum; Enterococcus faecium; Escherichia coli; Klyveromyces marxianus; Lactobacillus acidophilus. acidophilus), Lactobacillus bulgaricus, Lactobacillus casei, Lactobacillus crispatus, Lactobacillus fermentum, Lactobacillus gasseri, Lactobacillus helveticus, Lactobacillus johnsonii, Lactobacillus paracasei, Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus rhamnosus, Lactobacillus salivarius, Lactobacillus sakei; Lactococcus lactis lactis (including but not limited to subspecies lactis, cremoris and diacetylactis); Leuconostoc mesenteroides (including but not limited to subspecies mesenteroides); Pedicoccus acidilactici, P. pentosaceus; Propionibacterium acidipropionici, P. freudenreichii ssp. shermanii; Staphylococcus carnosus; and Streptococcus thermophilus.

[0136] In addition to combining living organisms, nutritional compositions can also include dead cell cultures. In the field of probiotics, inactivated cell cultures (e.g., tyndalized bacteria) are sometimes used. These inactivated cultures can provide proteins, peptides, oligosaccharides, cell wall fragments, and natural products, resulting in short-term stimulation of the immune system.

[0137] Including probiotic microorganisms in the nutritional composition is particularly advantageous, especially in the presence of HMOs, as it also promotes the establishment of a healthy intestinal microbiome.

[0138] In an additional and / or alternative embodiment, the nutritional composition further comprises a prebiotic such as galacto-oligosaccharides (GOS), fructo-oligosaccharides (FOS), inulin, or a combination thereof.

[0139] The nutritional composition may be in liquid form or solid form, including but not limited to powders, granules, flakes and pellets.

[0140] In an additional embodiment, the nutritional composition is selected from the group consisting of a pharmaceutical formulation, an infant formula, and a dietary supplement.

[0141] The present invention will be described with reference to specific embodiments and with reference to the accompanying drawings, but the 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 for the purpose of describing a sequence in time, space, ranking or any other aspect. It should be understood that the terms so used are interchangeable where appropriate, and that the embodiments of the present invention described herein can be operated in other sequences than those described or shown herein.

[0142] 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 understood as specifying 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 groups 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 with respect to the present invention, the only relevant components of the device are A and B.

[0143] Throughout this specification, references to "one embodiment" or "an embodiment" mean that a particular feature, structure, or characteristic described in conjunction with that embodiment is included in at least one embodiment of the present invention. Thus, the phrases "in one embodiment" or "in one embodiment" appearing throughout this specification do not necessarily always 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 in light of this disclosure.

[0144] Similarly, it should be understood that in the description of representative embodiments of the invention, various features of the invention are sometimes combined in a single embodiment, figure, or description thereof to simplify the disclosure and facilitate 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. Rather, as reflected in the following claims, aspects of the present invention may require less than all the features of any of the aforementioned disclosed embodiments. Therefore, the claims following the detailed description are hereby expressly incorporated into the detailed description, with each claim independently serving as a separate embodiment of the present invention.

[0145] In addition, although some embodiments described herein include some features included in other embodiments but do not include other features, the combination of features of different embodiments is intended to fall within the scope of the present invention and form different embodiments, as will be understood by those skilled in the art. For example, in the claims below, any claimed embodiment can be used in any combination.

[0146] In addition, some embodiments are described herein as a combination of methods or method elements that can be applied by a processor of a computer system or by other means of performing the function. Therefore, a processor with necessary instructions for performing such a method or method element constitutes a means for performing the method or method element. In addition, the elements of the device embodiments described herein are examples of means for implementing the functions performed by the element to implement the present invention.

[0147] 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 implemented without these specific details. In other cases, well-known methods, structures and techniques are not shown in detail to facilitate understanding of the description and drawings.

[0148] 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 can be configured according to the knowledge of those skilled in the art without departing from the true spirit or technical advantages of the present invention, and the present invention is limited only by the terms of the appended claims.

[0149] Example 1: Purification of 2'-fucosyllactose from fermentation broth

[0150] As described in European patent application 16 196 486.1, 2'-fucosyllactose is produced by fermentation using a genetically modified strain of Escherichia coli (E. coli). As described in WO 2015 / 106943 A1, 2'-fucosyllactose is purified from the fermentation broth by filtration, ion exchange chromatography, nanofiltration, diafiltration or electrodialysis and treatment with activated carbon. The resulting solution containing 2'-fucosyllactose is spray dried to obtain a stable solid product.

[0151] Example 2: Purification of 3-fucosyllactose from fermentation broth

[0152] As described in European patent application 16 196 486.1, 3-fucosyllactose is produced by fermentation using a genetically modified strain of Escherichia coli.

[0153] Cells were separated from the culture broth by ultrafiltration (cut-off 0.05 pm) (CUT Membrane Technology, Erkrath, Germany) followed by a cross-flow filter with a MWCO of 150 kDa (Microdyn-Nadir, Wiesbaden, Germany). The cell-free fermentation medium containing about 30 g / L 3-fucosyllactose was passed through H + The solution was then adjusted to pH 7.0 using sodium hydroxide and applied to an anion exchanger in the form of chloride (Lewatit S 2568 A, Lanxess). Both ion exchangers were used in a volume of 200 L. After a second filtration (150 kDa; Microdyn-Nadir, Wiesbaden, Germany), the particle-free solution was concentrated 5 times by nanofiltration using a Filmtech NF270 membrane (Dow, Midland, USA) and concentrated 2.5 times by vacuum evaporation. The conductivity of the concentrated solution was about 15 mS cm -1, filtered (10 kDa; Microdyn-Nadir, Wiesbaden, Germany), clarified by activated carbon (CAS: 7440-44-0, Carl Roth, Karlsruhe, Germany), and deionized by electrodialysis. For this purpose, a PC-Cell BED 1-3 electrodialysis unit (PC-Cell, Heusweiler, Germany) with a PC-Cell E200 membrane stack was used, which contained the following membranes: a cation exchange membrane CEM: PC SK and an anion membrane AEM: PCAcid60. 0.25 M aminosulfonic acid was used as an electrolyte in the process. In order to reduce the brown color caused by the Maillard reaction and the aldol products from the fermentation process, the same Na + and Cl - A second round of ion exchange chromatography was performed using the ion exchange material in the form of 50 L, but in a volume of 50 L. After concentrating the sugar solution by evaporation, the conductivity was increased from 4 mS cm to 1.5 mS cm by electrodialysis using the PC-Cell BED 1-3 described above. -1 Reduced to 0.4 mS cm -1 For further decolorization, the solution was mixed with activated carbon (CAS: 7440-44-0, Carl Roth, Karlsruhe, Germany) and an almost colorless solution was obtained by filtration.

[0154] Example 3: Purification of lacto-N-tetraose from fermentation broth

[0155] The fermentative production of lacto-N-tetraose was carried out using a genetically modified Escherichia coli BL21 (DE3) ΔlacZ strain, which has genomic integration of genes essential for the in vivo synthesis of lacto-N-tetraose, namely N-acetylglucosaminyltransferase (IgtA from Neisseria meningitidis MC58), β-1,3-galactosyltransferase (wbdO from Salmonella enterica subsp.salamae serovarGreenside), lacY from Escherichia coli K12, UDP-glucose-4-epimerase galE and UTP-glucose-1-phosphate uridyltransferase galU (both from Escherichia coli K12). For the fermentative production of lacto-N-tetraose, the strain was grown in a defined mineral salt medium containing 7 g l -1 NH4H2PO4, 7g l -1 K2HPO4, 2g l - 1KOH, 0.3gl -1 Citric acid, 5 g l -1 NH4Cl, 0.1mM CaCl2, 8mM MgSO4, trace elements (0.101gl -1 Nitrilotriacetic acid, pH 6.5, 0.056g l -1 Ammonium ferric citrate, 0.01gl -1 MnCl2 x 4H2O, 0.002gl -1 CoCl2 x 6H2O, 0.001gl -1 CuCl2 x 2H2O, 0.002gl -1 Boric acid, 0.009gl -1 ZnSO4 x 7H2O, 0.001g Na2MoO4 x2H2O, 0.002gl -1 Na2SeO3, 0.002gl -1 NiSO4 x 6H2O) and 2% glucose as carbon source. Defoamer (Struktol J673, Schill+Seilacher) was added if necessary. 25% ammonia solution was used to control pH. -1 It is 15mM to progressively add lactose to a final concentration in the lactose stock solution, which keeps the lactose concentration in the substratum constant during the fermentation. During this process, the residual lactose and milk-N-triose II accumulated as byproducts are added to the second Escherichia coli strain hydrolysis in the fermentor tank. This strain expresses functional beta-lactamase, beta-N-acetylhexosaminidase (bbhl from bifidobacterium bifidum (Bifidobacterium bifidum) JCM1254) and a functional gal-operon (EP 2845 905 A) for the degradation of monosaccharides.

[0156] The cells were separated from the fermentation broth according to the method described in Example 2 and the lacto-N-tetraose containing fluid was purified to a purity of 75-80% by mass balance.

[0157] According to WO 2015 / 049331, contaminating carbohydrate byproducts produced by ineffective enzymatic degradation and metabolism are removed by chromatography using simulated moving bed (SMB) chromatography. Alternatively, lacto-N-tetraose is purified by crystallization using isopropanol. For crystallization, a solution containing lacto-N-tetraose is concentrated by evaporation to a concentration of 20% and spray dried. A NUBILOSA LTC-GMP spray dryer (NUBILOSA, Konstanz, Germany) is used to pass the solution through a spray dryer nozzle under a nitrogen stream with an inlet temperature of 130°C while controlling the product flow to maintain an outlet temperature of 67°C to 68°C.

[0158] The solid material was added to a mixture of isopropanol and water (3:1 (volume / volume)) in a ratio of 1 kg powder in 12 L isopropanol / water. The suspension was stirred vigorously, and then the insoluble lacto-N-tetraose was filtered and dried at 40°C. Starting from pure raw materials with a purity of 73-89%, the crystallized lacto-N-tetraose was purified to about 95% with a recovery of 85%. The sugar was dissolved in water to a concentration of 25%, and then passed through a 6kDa filter (Pall Microza ultrafiltration module SIP-2013, Pall Corporation, Dreieich, Germany) and a 0.2 pm sterile filter. The solid material was obtained by spray drying the sterile material under the above conditions.

[0159] Example 4: Purification of 3'- and 6'-sialyllactose from fermentation broth

[0160] For the production of 3'- and 6'-sialyllactose, recombinant E. coli BL21 (DE3) ΔlacZ strains were used. These strains have common genetic modifications: chromosomal constitutive expression of glucosamine-6-phosphate synthase GlmS from E. coli, N-acetylglucosamine 2-epimerase Slr1975 from Synechocystis sp., glucosamine 6-phosphate N-acetyltransferase Gna1 from Saccharomyces cerevisiae, phosphoenolpyruvate synthase PpsA from E. coli, N-acetylneuraminic acid synthase NeuB and CMP-sialic acid synthase NeuA, the latter two both from Campylobacter jejuni. In addition, genes encoding the lactose permease LacY from E. coli, cscB (sucrose permease), cscK (fructokinase), cscA (sucrose hydrolase) and cscR (transcription regulator) from E. coli W, and a functional gal-operon (composed of the genes galE (UDP-glucose-4-epimerase), galT (galactose-1-phosphate uridyltransferase), galK (galactokinase) and galM (galactose-1-epimerase) from E. coli K12) were integrated into the genome of the BL21 strain and constitutively expressed.

[0161] The strain synthesizing 3'-sialyllactose carries the 3'-sialyltransferase gene from Vibrio sp. JT-FAJ-16, while the strain producing 6'-sialyllactose contains the α-2,6-sialyltransferase plsT6 from Photobacterium leiognathi JT-SHIZ-119.

[0162] Sialyl lactose-producing strains were grown in a defined mineral salts medium containing 7 g l - 1 NH4H2PO4, 7g l -1 K2HPO4, 2g l -1 KOH, 0.3gl -1 Citric acid, 5 g l -1 NH4Cl, 1ml l -1 Antifoam (Struktol J673, Schill + Seilacher), 0.1 mM CaCl2, 8 mM MgSO4, trace elements and 2% glucose as carbon source. Sucrose feed (500 g l -1 ) supplemented with 8 mM MgSO4, 0.1 mM CaCl2, trace elements, and 5 g l -1 NH4Cl.

[0163] Trace elements are composed of: 0.101gl -1 Nitrilotriacetic acid, pH 6.5, 0.056g l -1 Ammonium ferric citrate, 0.01gl -1 MnCl2 x 4H2O, 0.002gl -1 CoCl2 x 6H2O, 0.001gl -1 CuCl2 x 2H2O, 0.002gl -1 Boric acid, 0.009gl -1 ZnSO4 x 7H2O, 0.001gl -1 Na2MoO4 x 2H2O, 0.002gl -1 Na2SeO3, 0.002gl - 1 NiSO4 x 6H2O.

[0164] To form sialyllactose, 216 g l -1 The lactose was fed with 1% lactose. The pH was controlled by 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 the fermentation, β-galactosidase was added to the fermentation vessel. The resulting monosaccharides were metabolized by the production strain.

[0165] The cell-free liquid was then deionized by ion exchange chromatography. First, cationic contaminants were removed on a strong cation exchanger in the H+ form (Lewatit S2568 (Lanxess, Cologne, Germany) with a volume of 200 L. The pH of the resulting solution was set to 7.0 using NaOH. In a second step, anions and unwanted pigments were removed from the solution using a strong anion exchanger in the chloride form, Lewatit S6368S (Lanxess, Cologne, Germany). The bed volume of the ion exchanger was 200 L. A second filtration step on a cross-flow filter (cutoff 150 kDa) (Microdyn-Nadir, Wiesbaden, Germany) was used to remove the precipitate originating from the acidified solution. To concentrate the sugars, the solution was filtered through a Dow FILMTECH NF270-4040 (Inaqua, Germany) or alternatively on a Trisep 4040-XN45-TSFMembrane (cutoff 0.5 kDa) (Microdyn-Nadir, Wiesbaden, Germany). When the latter is used, the monosaccharide N-acetylglucosamine is separated from the product, which originates from the fermentation process and contaminates the sialyllactose solution. The concentrated sialyllactose solution is then treated with activated carbon (CAS: 7440-44-0, Carl Roth, Karlsruhe, Germany) to remove pigments, such as Maillard reaction products and aldol reaction products. In order to separate sialyllactose from by-products derived from the fermentation process (such as sialic acid and N-acetylglucosamine), the solution is filtered with a membrane GE4040F30 with a cutoff of 1 kDa (GE water & process technologies, Ratingen, Germany) and diafiltered to a conductivity of 0.6 to 0.8 mS cm -1 . The diluted solution was concentrated on a rotary evaporator to a concentration of about 300 g / L. In the final chromatographic separation, other contaminating sugars, such as disialyl lactose, were removed. For this reason, the concentrated solution was applied to a weak anion exchange resin in the acetate form (Amberlite FPA51, Dow Chemical, Michigan, USA). Although sialyllactose hardly binds to the resin, disialyl lactose is adsorbed. Therefore, sialyllactose is eluted with 10 mM ammonium acetate, while disialyl lactose is eluted with 1 M ammonium acetate. In order to remove ammonium acetate, sialyllactose is precipitated with 10 times excess ethanol. The solid fraction is filtered and dried.

[0166] The product was finalized by passing the 20% sialyllactose solution through a 6 kDa filter (Pall Microza ultrafiltration module SIP-2013, Pall Corporation, Dreieich, Germany) and then a 0.2 μm sterile filter.

[0167] Part of the solution was spray dried using a Büchi Mini Spray Dryer B-290 (Büchi, Essen, Germany) with the following parameters: inlet temperature: 130° C., outlet temperature 67° C.-71° C., air flow 670 L / h, aspirator 100%.

[0168] The purity of the spray-dried 6'-sialyllactose was 91%, while the purity of the 3'-sialyllactose was 93%.

[0169] Example 5: Preparation of HMO mixture

[0170] Mixtures of different HMOs were prepared from the solid product. Therefore, the individual HMOs were spray dried and the powders obtained were mixed. HMO-Mix I contained 2'-fucosyllactose and lacto-N-tetraose in a ratio of 70 wt % to 30 wt %; HMO-MixII contained 2'-fucosyllactose (52 wt %), 3-fucosyllactose (13 wt %), lacto-N-tetraose (26 wt %), 3'-sialyllactose (4 wt %) and 6'-sialyllactose (5 wt %). The mixed powders were dissolved in water to prepare a solution containing 20 wt % HMO, and the resulting solution was spray dried again using a Büchi spray dryer as described in Example 4.

[0171] Analysis of the resulting spray-dried powder showed that it had the same composition as the solution subjected to spray drying, with regard to the ratios of the different HMOs.

[0172] Example 6: Preparation of sugar mixture

[0173] 8 g of 2'-FL and 1 g of L-fucose were dissolved in 50 ml of distilled water. The resulting solution was spray dried using a Büchi spray dryer as described in Example 4. A spray-dried powder consisting essentially of 2'-FL and L-fucose was obtained, wherein the ratio of 2'-FL to L-fucose in the spray-dried powder was the same as the ratio of 2'-FL to L-fucose in the solution subjected to spray drying. Thus, L-fucose can be spray dried in the presence of HMO.

[0174] Example 7: Characterization of spray-dried human milk oligosaccharides

[0175] 1. Differential Scanning Calorimetry (DSC)

[0176] The thermal events of spray-dried human milk oligosaccharides (i.e., 3-fucosyllactose, 6'-sialyllactose, 3'-sialyllactose, lacto-N-tetraose and human milk oligosaccharides) and spray-dried mixtures of human milk oligosaccharides (a mixture of 2'-fucosyllactose / lacto-N-tetraose (HMO-Mix I) and a mixture of 2'-fucose, 3-fucosyllactose, lacto-N-tetraose, 6'-sialyllactose, 3'-sialyllactose (HMO Mix II)) were measured using differential scanning calorimetry (DSC) on a Mettler Toledo 821e (Mettler Toledo, Giessen, Germany).

[0177] The thermal events (glass transition temperature (Tg), as well as exothermic and endothermic events) of the spray-dried products were determined using a Mettler Toledo 821e (Mettler Toledo, Giessen, Germany).

[0178] Approximately 25 mg of spray-dried human milk oligosaccharides were analyzed in crimped aluminum crucibles (Mettler Toledo, Giessen, Germany). The sample was cooled to 0°C at a rate of 10 K / min and then reheated to 100°C at a scan rate of 10 K / min. After cooling the sample to 0°C in the second heating cycle, the sample was reheated to 150°C. The midpoint of the endothermic shift of the baseline during the heating scan was taken as the glass transition temperature (Tg).

[0179] Exothermic and endothermic peaks are reported by the peak temperature and normalized energy of the event.

[0180] The first heating scan in all samples showed a major glass transition event in the total heat flow, which was evidenced by a major step transition in the range of about 48-58°C. In most samples, the major glass transition event observed in the first heating scan occurred again in the second heating scan. The results of the DSC analysis are summarized in Table 9.

[0181]

[0182]

[0183] Table 9: Thermal events of HMOs determined by differential scanning calorimetry

[0184] For 3-fucosyllactose, an endothermic relaxation peak was detected after Tg in the first heating scan. For lacto-N-tetraose, a much higher Tg of about 79°C was detected in the second heating scan than for other samples. This may be due to the endothermic event that occurred during the first heating scan at about 89°C (-6.04 J / g). Like 3-fucosyllactose, an endothermic relaxation peak was also detected after Tg for 6'-sialyllactose, but in this sample, an endothermic event additionally occurred at 77°C (-0.22 J / g). For 3'-sialyllactose and HMO-Mix I, no endothermic event was detected, and for HMO-Mix II, the endothermic event during the first heating scan was at 79°C (0.34 J / g).

[0185] 2. X-ray powder diffraction (XRD)

[0186] Wide-angle X-ray powder diffraction (XRD) was used to study the morphology of the freeze-dried product. An X-ray diffractometer Empyrean (Panalytical, Almelo, The Netherlands) equipped with a copper anode (45 kV, 40 mA, Kα1 emission at a wavelength of 0.154 nm) and a PIXcel3D detector was used. About 100 mg of the spray-dried sample was analyzed in reflection mode with an angle range of 5-45° 2θ, a step size of 0.04° 2θ, and a counting time of 100 seconds per step.

[0187] All single oligosaccharides as well as HMO mixtures I and II showed a completely amorphous state ( Figure 1-6 ). For lacto-N-tetraose, a second (amorphous) signal was detected around 9-10°.

[0188] 3. Laser diffraction

[0189] Powder particle size was assessed by laser diffraction. The system detects scattered and diffracted light through an array of concentrically arranged sensor elements. The software algorithm then approximates the particle count by calculating the z-values ​​of the light intensity values ​​reaching the different sensor elements. The SALD-7500 Aggregate Sizer (Shimadzu Corporation, Kyoto, Japan) quantitative laser diffraction system (qLD) was used for analysis.

[0190] A small amount (spatula tip) of each sample was dispersed in 2 ml isooctane and homogenized by sonication for 5 minutes. The dispersion was transferred to a batch cell filled with isooctane and analyzed in manual mode.

[0191] The data acquisition settings are as follows: Signal Averaging Count per Measurement: 128, Signal Accumulation Count: 3, Interval: 2 seconds.

[0192] Before measuring, the system was blanked with isooctane. The dispersion of each sample was measured 3 times, and the average value and standard deviation were reported. The data were evaluated using software WING SALD II version V3.1. Since the refractive index of the sample was unknown, the refractive index (1.530) of the sugar (disaccharide) particles was used to determine the size distribution curve. The size values ​​of the mean and median diameter were reported.

[0193] The average particle size for all samples was very similar, with HMO-Mix II measuring slightly lower. The particle size characteristics are summarized in Table 10. In addition, the particle size distribution showed that for all samples there was one major population of particle sizes.

[0194]

[0195] Table 10: Particle size of HMO measured by laser diffraction

Claims

1. A spray-dried powder containing a mixture of structurally different human milk oligosaccharides and at least one monosaccharide selected from L-fucose and N-acetylneuraminic acid, wherein the spray-dried powder comprises the following composition: The spray-dried powder contains at least 85% by weight human milk oligosaccharides and ≤15% by weight water, and all of the milk oligosaccharides in the mixture have been produced by microbial fermentation.

2. The spray-dried powder of claim 1, wherein the spray-dried powder contains at least 90 wt%, at least 93 wt%, at least 95 wt%, or at least 98 wt% human milk oligosaccharides.

3. The spray-dried powder according to any one of claims 1 to 2, wherein all HMO in the mixture is present in amorphous form.

4. The spray-dried powder according to any one of claims 1 to 3, wherein all monosaccharides selected from L-fucose and N-acetylneuraminic acid are present in an amorphous form.

5. The spray-dried powder according to any one of claims 1 to 4, wherein the spray-dried powder contains ≤ 10 wt% water, preferably ≤ 7 wt% water, more preferably ≤ 5 wt% water.

6. Use of the spray-dried powder according to any one of claims 1 to 5 for the preparation of a nutritional composition, preferably an infant formula.

7. A nutritional composition comprising the spray-dried powder according to any one of claims 1 to 5.

8. The nutritional composition according to claim 7, wherein the nutritional composition additionally contains at least one probiotic microorganism.

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