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    • 专利摘要:
    Disclosed is a spray-dried powder consisting of a mixture of structurally different human milk oligosaccharides, methods for the production of the spray-dried powder and the use for the preparation of the nutritional compositions and nutritional compositions with this spray-dried powder.
    公开号:AT17129U1
    申请号:TGM50151/2019U
    申请日:2018-12-07
    公开日:2021-06-15
    发明作者:Jennewein Stefan
    申请人:Jennewein Biotechnologie Gmbh；
    IPC主号:
    专利说明:

    description
    NUTRITIONAL COMPOSITION COMPRISING A SPRAY DRIED MIXTURE OF HUMAN MILK OLIGOSACCHARIDES
    The present invention relates to human milk oligosaccharide preparations. In particular, the present invention relates to solid preparations of human milk oligosaccharides.
    GENERAL STATE OF THE ART
    Human breast milk contains significant amounts of carbohydrates. The carbohydrates present in human breast milk include monosaccharides such as L-fucose and N-acetyl-neuraminic acid, the disaccharide lactose and up to 20 g / l oligosaccharides, the so-called "human milk oligosaccharides (HMO)". HMOs represent the third most common constituent of human breast milk. It is assumed that there are more than 150 structurally different oligosaccharides in human milk. Selected HMOs are shown in Table 1. About 10 to 13 of these HMOs are present in human milk at a concentration of several hundred milligrams to grams per liter (Thurl et al., (2017), Nutrition Reviews 75 (11) 920-933). Among the HMOs, neutral HMOs and acidic HMOs are known which contain at least one N-acetylneuraminic acid (NeuAc) unit. The structural complexity and richness of these oligosaccharides is unique to human milk and is not found in the milk of other mammals such as - for example - domesticated dairy animals.
    Since HMOs are not digested by humans, the physiological role of these saccharides has been the subject of research for several decades. The prebiotic effects of HMOs were discovered more than 100 years ago. When consumed, HMOs can modulate the composition of the human gut microbiome by promoting the growth of beneficial bacteria.
    Name chemical structure
    2-FL Fuc (a1,2) Gal (ß1.4) Glc 3-FL Gal (ß1.4) (Fuc (a1,3)) Glc DFL Fuc (a1,2) Gal (ß1,4) Glc (a1 , 2)! Fuc LNT Gal (ß1.3) GICNAc (ß 1.3) Gal (ß 1.4) Glc LNnT Gal (ß1.4) GICNAc (ß1.3) Gal (ß 1.4) GIc LNFP-I Fuc (a1 , 2) Gal (ß1.3) GICcNAc (ß1.3) Gal (ß 1.4) Glc LNFP-II Gal (ß1.3) (Fuc (a1.4)) GICNAc (ß 1.3) Gal (ß 1,4) GIc LNFP-II Gal (ß1,4) (Fuc (a1,3)) GICcNAc (ß1.3) Gal (ß1.4) Glc LNFP-V Gal (ß1.3) GICNAc (ß1.3) Gal (ß 1,4) (Fuc (a1,3)) Glc 3'-SL NeuAc (a2,3) Gal (ß 1,4) Glc 6'-SL NeuAc (a2,6) Gal (ß 1,4 ) Glc LSTa NeuAc (a2.3) Gal (ß 1.3) GICNAc (ß1.3) Gal (ß 1.4) Glc
    LSTb NeuAc
    1 (02.6) Gal (ß1.3) GICNAc (ß 1.3) Gal (ß 1.4) Glc LSTc NeuAc (a2.6) Gal (ß1.4) GICNAc (ß1.3) Gal (ß 1 , 4) Glc LNDFH-I Fuc (a1,2) Gal (ß1,3) GIcNAc (ß1,3) Gal (ß 1,4) Glc ı (a1,4)
    Fuc LNDFH-II Gal (ß1,3) GICNAc (ß 1,3) Gal (ß 1,4) GIc 1 (a1,4) (a1,3)! Fuc Fuc
    Name Chemical structure LNDFH-IIN Gal (ß1,3) GICNAc (ß 1,3) Gal (ß 1,4) GIc 1 (a1,3) (a1,3)! Fuc Fuc F-LSTa NeuAc (a2,3) Gal (ß 1,3) GICcNAc (ßB1,3) Gal (ß1,4) Glc ı (a1,4) Fuc F-LSTb NeuAc 1 (02,6) Fuc ( a1,2) Gal (ß1,3) GICcNAc (ßB1,3) Gal (ß 1,4) Glc F-LSTc NeuAc (a2,6) Gal (ß 1,4) GICcNAc (ß1,3) Gal (ß1, 4) Glc (a1,3)! Fuc FSL NeuAc (a2,3) Gal (ß 1,4) Glc (a1,3)! Fuc DS-LNT NeuAc 1 (a2,6) NeuAc (a2,3) Gal (ß 1,3) GICNAc (ß1,3) Gal (ß 1,4) Gle FDS-LNT-I NeuAc 1 (a2,6) NeuAc (a2,3) Gal (ß 1,4) GICNAc (ßB1,3) Gal (ß 1,4) Gle FDS-LNT-II NeuAc 1 (a2,6) NeuAc (a2,3) Gal (ß 1, 4) GICcNAc (ß1,3) Gal (ß1,4) Glc (a1,3)! Fuc
    Table 1: Structures of noteworthy oligosaccharides in human breast milk.
    In recent years, several other functional effects of HMOs have been uncovered, particularly their effect on neonatal development. It is known that HMOs act as decoys to reduce the risk of infection by bacterial and viral pathogens that attach to the surface glycoproteins of human cells by binding them to them. In addition, various HMOs have anti-inflammatory effects and act as immunomodulators. Therefore, it has been suggested that HMOs reduce the risk of developing food allergies. A positive effect of sialylated HMOs on the development of the central nervous system of a newborn is also intensely discussed (discussed in “Prebiotics and Probiotics in Human Milk, Origins and functions of milk-borne oligosaccharides and bacteria”, Academic Press (2017), editor: McGuire M ., McGuire M. and Bode L.).
    In order to take advantage of the beneficial effects of HMOs, efforts are being made to add individual HMOs to the nutritional composition, in particular the infant formula. However, it would be better to supplement nutritional compositions with a combination of different HMOs, since such compositions more closely resemble the natural origin of the HMOs, namely human milk, and are all the more likely to have better effects on the health and development of a person than compositions that only contain a single type of HMOs contain.
    The limited supply of HMOs to supplement nutritional compositions led to the development of methods for the chemical synthesis of HMOs. The disadvantages of these chemical syntheses lead to biocatalytic approaches, whereby HMOs are synthesized in vitro using purified enzymes such as glycosyltransferases. Today, individual HMOs become genetically modified microbial on an industrial scale through fermentation
    Cells produced (WO 2015/150328 A1, WO 2017/043382 A1, WO 2010/070104 A1, WO 2012/097950 A1). The HMOs are synthesized by genetically modified microbial cells and can be obtained from the fermentation medium and / or cell lysate in order to obtain an essentially pure preparation of the HMO.
    During their recovery from a fermentation broth, HMO are usually in the form of a liquid process stream, for example an aqueous solution which contains the HMO of interest and may also contain undesired HMO which are by-products generated during the fermentative production of the desired HMO were. Along with the recovery of the desired HMO, i.e. the HMO of interest, its concentration in the process stream and its purity are increased. However, an aqueous solution containing HMOs is prone to bacterial or fungal contamination. Therefore, it is preferable to provide the desired HMOs as a dry or solid product containing a small amount of water. The growth of microbial organisms on / in such a solid product is hardly possible.
    Typically, a saccharide is obtained in solid form by crystallization. The crystallization of individual HMO from an aqueous solution was carried out for 3-fucosyllactose (WO 2014/075680 A), for 2'-fucosyllactose (WO 2011/150939 A), difucosyllactose (WO 2016/086947 A), lacto-N-tetraose (WO 22017/101953 A), Lacto-N-neo-tetraose (WO 2014/094783 A). The crystallization of HMO involves the use of organic solvents such as alcohols, mainly ethanol or methanol, or organic acids such as glacial acetic acid. However, the use of alcohols, in particular methanol, to crystallize HMO at the end of the recovery process is unsuitable if the HMO is to be used for human consumption. In addition, organic solvents are expensive to purchase and dispose of. In addition, organic solvents are harmful to the environment and the employees who handle them. Therefore, the crystallization of HMO is a disadvantage in the production of HMO on an industrial scale and should be avoided especially at the end of the recovery process of the desired HMO.
    There is consequently a need to provide a solid preparation of the HMOs mentioned, the disadvantages mentioned above being at least partially improved.
    The object is achieved by a nutritional composition comprising a spray-dried powder which comprises a mixture of structurally different HMOs.
    SHORT REPRESENTATION
    In a first aspect, a nutritional composition is provided comprising a spray-dried powder, which consists essentially of a mixture of structurally different HMOs or contains these.
    DESCRIPTION OF THE FIGURES
    Fig. 1 is a graph illustrating the results of powder X-ray diffraction of spray-dried 3-fucosyllactose.
    Figure 2 is a graph illustrating the results of powder X-ray diffraction of spray-dried lacto-N-tetraose.
    Figure 3 is a graph illustrating the results of powder X-ray diffraction of spray-dried 6'-sialyllactose.
    Figure 4 is a graph illustrating the results of powder X-ray diffraction of spray-dried 3'-sialyllactose.
    Figure 5 is a graph illustrating the results of powder X-ray diffraction of a spray-dried mixture of 2'-fucosyllactose and lacto-N-tetraose.
    Fig. 6 is a graph showing the results of powder X-ray diffraction of a spray-dried mixture of 2'-fucosyllactose, 3-fucosyllactose, lacto-ntetraose, 3'-sialyllactose and 6'-sialyllactose.
    DETAILED DESCRIPTION
    According to one aspect, a spray-dried powder is provided which consists essentially of structurally different HMOs or contains a mixture of structurally different HMOs.
    In one embodiment, the spray-dried powder consists essentially of a mixture of structurally different HMOs.
    The term “consisting essentially of”, as used here, relates to compositions consisting of the compound (s) specified after the expression and - optionally - unavoidable by-products. These inevitable by-products include, for example, compounds generated during microbial fermentation to produce one or more of the HMOs, as well as compounds that were introduced into a process stream from which the HMO (s) are obtained, but which are not removed therefrom could. The expression “essentially consisting of” in relation to spray-dried powder encompasses spray-dried powders which, based on the dry substance of the spray-dried powder, contain 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 contain at least 98% by weight of HMOs. The term “consisting essentially of” is used equally in relation to spray-dried powders, process streams, and solutions containing HMOs.
    In an additional and / or alternative embodiment, the mixture of structurally different HMOs consists of two, three, four, five, six, seven or more than seven structurally different HMOs. The structurally different HMOs include neutral HMOs and sialylated HMOs. The HMO mixture can thus contain at least one neutral HMO and / or at least one acidic HMO.
    The at least one neutral HMO can be selected from the group consisting of 2'-fucosyllactose (2'-FL), 3-fucosyllactose (3-FL), lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT )) and Lacto-N-Fucopentaose | (LNPFI) must be selected.
    The at least one acidic HMO can be selected from the group consisting of sialylated HMO, preferably from the group consisting of 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).
    Therefore, the structurally different HMOs of the mixture of structurally different HMOs can be selected from the group consisting of 2'-FL, 3-FL, LNT, LNnT, LNFPI, 3'-SL, 6'-SL, LST-a , LST-b, LST-c and DSLNT.
    In an additional and / or alternative embodiment, the mixture of structurally different HMOs contains five structurally different HMOs or consists essentially of these. In an additional embodiment, the five structurally different HMOs are 2'-FL, 3-FL, LNT, 3'-SL and 6'-SL. In an exemplary composition of the mixture, the five structurally different HMOs are present in the mixture of structurally different HMOs in amounts as indicated in Table 2.
    HMO |% by weight 2Z-FL 52.2 3-FL 13.0 LNT 26.1 3'-SL 3.5 6 '' - SL 5.2 total 100.0
    Table 2: Composition of an exemplary mixture, consisting of five structurally different HMOs.
    In an additional and / or alternative embodiment, the mixture of structurally different HMOs contains seven structurally different HMOs or consists essentially of these. In an additional embodiment, the seven structurally different HMOs are 2'-FL, 3-FL, LNT, LNnT, LNFPI, 3'-SL and 6'-SL. In an exemplary composition of the mixture, the seven structurally different HMOs are present in the mixture in amounts as indicated in Table 3.
    HMO |% by weight 2Z-FL 39.0 3-FL 12.0 LNT 23.0 LNNT 2.0 LNFPI 16.0 3'-SL 3.0 6 '' - SL 5.0 total 100.0
    Table 3: Composition of an exemplary mixture consisting of seven structurally different HMOs.
    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. The at least one monosaccharide is preferably selected from the group consisting of 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-acetyl neuraminic acid.
    In a particular embodiment, the spray-dried powder consists essentially of the HMOs 2'-FL, 3-FL, LNT, LNnT, LNFPI, 3'-SL and 6'-SL and the monosaccharides L-fucose and N-acetylneuraminic acid .
    In an exemplary composition, the seven structurally different HMOs and the two monosaccharides are present in amounts as indicated in Table 4.
    HMO |% by weight 2Z-FL 33.6 3-FL 10.7 LNT 20.1 LNNT 2.0 LNFPI 13.4 3'-SL 2.7 6 '' - SL 4.0 NeuAc 8.1 L- Fucose 5.4
    100.0
    Table 4: Composition of an exemplary mixture containing HMOs and monosaccharides.
    In an additional and / or alternative embodiment, the spray-dried powder comprises or consists essentially of the composition given in Table 5.
    Saccharide A [% by weight] B [% by weight] 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 LNNT 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
    Table 5: Exemplary compositions of the spray-dried powder
    In an additional and / or alternative embodiment, at least one of the HMOs of the mixture of structurally different HMOs and / or of the spray-dried powder was produced by microbial fermentation. In a particular embodiment, all HMOs of the mixture of structurally different HMOs and / or of the spray-dried powder were produced by microbial fermentation.
    In an additional and / or alternative embodiment, in which the spray-dried powder contains at least one monosaccharide, the at least one monosaccharide was produced by microbial fermentation. In another embodiment, in which the spray-dried powder contains L-fucose and N-acetylneuraminic acid, both monosaccharides were produced by microbial fermentation.
    Therefore, in a particular embodiment, all of the saccharides present in the spray-dried powder, i.e. the HMO or the HMOs and the monosaccharides, were produced by microbial fermentation.
    In an additional and / or alternative embodiment, at least one of the HMOs in the spray-dried powder, preferably all of the HMOs in the spray-dried powder, is in amorphous form. In those embodiments in which the spray-dried powder contains L-fucose and / or N-acetyl-neuraminic acid, the monosaccharide, at least one of the monosaccharides or both monosaccharides is / are in amorphous form.
    In an additional and / or alternative embodiment, the spray-dried powder contains a small amount of water. The expression “small amount of water” relates to an amount of = 15% by weight of water, preferably = 10% by weight of water, more preferably = 7% by weight of water, most preferably = 5% by weight of water.
    In an additional and / or alternative embodiment, the spray-dried powder is free from genetically modified microorganisms and free from nucleic acid molecules which originate from genetically modified microorganisms.
    A spray-dried powder, which consists essentially of a mixture of structurally different HMOs, optionally in combination with at least one monosaccharide, is advantageous over liquid compositions in that the spray-dried powder is less susceptible to microbial contamination. The spray-dried powder is also advantageous over powders with the same composition of ingredients obtained by freeze-drying or lyophilization in that the spray-dried powder is less hygroscopic and remains fluid for much longer.
    In the following, a method for the production of a spray-dried powder is provided which consists essentially of a mixture of structurally different HMOs or comprises these, wherein at least one of the structurally different HMOs, preferably all structurally different HMOs, was (s) produced by microbial fermentation . The procedure consists of the following steps:
    a) recovering at least one of the structurally different HMOs from a fermentation broth;
    b) providing an aqueous solution of the at least one HMO from step a); and
    C) subjecting the solution from step b) to spray drying.
    In an additional and / or alternative embodiment, the purification of the at least one HMO from the fermentation broth (step a) comprises one or more of the steps for
    i) removing microbial cells from the fermentation broth to obtain a process stream;
    il) subjecting the process stream to at least one ultrafiltration;
    ill) treating the process stream at least once with a cation exchange resin and / or at least once with an anion exchange resin;
    iv) subjecting the process stream to at least one nanofiltration;
    V) subjecting the process stream to at least one electrodialysis;
    Vi) treating the process stream at least once with activated carbon; and or
    vii) subjecting the process stream to a crystallization and / or precipitation step at least once.
    The at least one HMO or one of the structurally different HMOs of the mixture can be produced by microbial fermentation, a genetically modified microorganism which is able to synthesize an HMO, cultivated in a culture medium (fermentation broth) and under such conditions that are permitted for the synthesis of the HMO by the genetically modified microorganism. The purification of the HMO, which was synthesized by the cells of the genetically modified microorganism, comprises the step of separating the microbial cells from the fermentation broth in order to obtain a process stream. The process stream is essentially cell-free and contains the HMO (s). This step is the first step in the process of purifying the desired HMO.
    Suitable methods for separating the microbial cells from the fermentation broth include centrifugation, the microbial cells being obtained as a pellet and the fermentation broth as a supernatant. In an additional and / or alternative embodiment, the microbial cells are separated from the fermentation broth by means of filtration. Suitable filtration methods for separating the cells from the fermentation broth include microfiltration and ultrafiltration.
    Microfiltration is per se a physical filtration process in which a liquid containing particles is passed through a membrane with a special pore size in order to separate the particles from the liquid. As used herein, the term “microfiltration” refers to a physical filtration process that separates cells from the fermentation broth.
    Ultrafiltration is a membrane filtration variant and not fundamentally different. In ultrafiltration, forces such as pressure or concentration gradients lead to separation through a semipermeable membrane. Cells, suspended solids and high molecular weight dissolved substances are retained in the so-called retentate, while water and low molecular weight dissolved substances such as the desired sialylated oligosaccharide pass through the membrane in the permeate (filtrate).
    Ultrafiltration membranes are defined by the molecular exclusion limit (MWCO) of the membrane used. Dead-end and crossflow processes are used in ultrafiltration.
    71727
    Typically, the microbial cells synthesize the HMO intracellularly. Depending on the structure of the HMO, the HMO is either released into the fermentation broth or remains in the microbial cell. In the first case, the HMO produced in this way is present in the fermentation broth at the end of the fermentation and can be obtained from the fermentation broth, which thus becomes the process stream. In the latter case, the microbial cells carrying the HMO are separated from the fermentation broth and lyzed to release the HMO. Thus, the cell lysate contains the HMO and becomes the process stream for the purification of HMO, as described below.
    Even when the method is used to purify HMOs produced by microbial fermentation, the method can also be used to purify HMOs produced by enzymatic catalysis in vitro. The HMO can then be purified from the reaction mixture at the end of the biocatalytic reaction. This reaction mixture is subjected to the purification process as a process stream.
    The process stream contains the desired HMO (s) as well as by-products and undesired impurities such as - for example - monosaccharides, disaccharides, unwanted oligosaccharide by-products, ions, amino acids, polypeptides, proteins and / or nucleic acid molecules.
    In an additional and / or alternative embodiment, the method for purifying the HMO comprises the step of at least one cation exchange treatment in order to remove positively charged compounds from the clarified process stream.
    Suitable cation exchange resins for removing positively charged compounds from the process stream are Lewatit® S 6368 A (Lanxess AG, Cologne, DE) in H * form; Lewatit® S 2568 (H *) (Lanxess AG, Cologne, DE).
    In an additional and / or alternative embodiment, the method for purifying the HMO comprises the step of an anion exchange treatment in order to remove undesired negatively charged compounds from the clarified process stream.
    Suitable anion exchange resins include Lewatit® S 2568 (CI) (Lanxess AG, Cologne, DE) Lewatit® ”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 100200), Purolite® Chromalite® CGA100x4 (Purolite GmbH, Ratingen, DE), Dow® Amberlite'M FPA51 (Dow Chemicals, MI, USA).
    In an additional and / or alternative embodiment, the method for purifying the HMO comprises a nanofiltration and / or a diafiltration step in order to remove low molecular weight impurities and to concentrate the desired HMO.
    In diafiltration, fresh water is added to a solution in order to remove (wash out) membrane-permeable components. Diafiltration can be used to separate components based on their molecular size and charge using suitable membranes, with one or more species being effectively retained and other species being membrane permeable. In particular, diafiltration using a nanofiltration membrane is effective for separating low molecular compounds such as small molecules and salts. Nanofiltration membranes usually have a molecular exclusion limit in the range of 150-1000 Daltons. Nanofiltration is often used in the dairy industry to concentrate and demineralize whey. Suitable membranes for nanofiltration and / or diafiltration include Dow® Filmtec'M NF270-4040, Trisep® 4040-XN45TSF (Microdyn-Nadir GmbH, Wiesbaden, DE), GE4040F30 and GH4040F50 (GE Water & Process Technologies, Ratingen, DE).
    Diafiltration using nanofiltration membranes has been found to be an effective pretreatment for removing significant amounts of contaminants prior to electrodialysis treatment of the solution containing the oligosaccharide. The use of nanofiltration membranes for concentration and diafiltration during the purification of
    HMO leads to lower energy and processing costs and better product quality due to a lower thermal load, which leads to lower Maillard and aldol reactions.
    In an additional and / or alternative embodiment, the method for purifying the HMO comprises at least one electrodialysis step.
    Electrodialysis (ED) combines dialysis and electrolysis and can be used to separate or concentrate ions in solutions due to their selective electromigration through semipermeable membranes.
    The basic principle of electrodialysis consists of an electrolysis cell which comprises two electrodes which are immersed in an electrolyte for conducting ions and which are connected to a direct current 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 solution then supports the flow of current that results from the movement of the negative and positive ions to the anode and the cathode, respectively. The membranes used for electrodialysis are essentially foils made of porous ion exchange resins with negatively or positively charged groups and are therefore referred to as cationic or anionic membranes. The ion exchange membranes usually consist of polystyrene which carries a suitable functional group (such as sulfonic acid for cationic membranes or a quaternary ammonium group for anionic membranes) which is cross-linked with divinylbenzene. The electrolyte can be, for example, sodium chloride, sodium acetate, sodium propionate or sulfamic acid. The electrodialysis stack cell is then assembled in such a way that the anionic and cationic membranes are arranged in parallel, as in a filter press between two electrode blocks, so that the current that is subject to ion depletion is well separated from the current that is subject to ion enrichment ( The two solutions are also referred to as diluate (underlying ion depletion) and concentrate (underlying ion enrichment). The heart of the electrodialysis process is the membrane stack, which consists of several anion exchange membranes and cation exchange membranes, which are separated from one another by spacers and installed between two electrodes an electrical direct current migrate anions and cations across the membranes to the electrodes.
    In an additional and / or alternative embodiment, the method for purifying the HMO further comprises a step of continuous chromatography such as simulated countercurrent chromatography (SMB).
    The simulated moving bed chromatography (SMB) has its origin in the petrochemical and mineral industries. Today, SMB chromatography is used in the pharmaceutical industry to isolate enantiomers from racemic mixtures. Large-scale SMB chromatography has already been used to separate the monosaccharide fructose from fructose-glucose solutions and to separate the disaccharide sucrose from sugar beet or sugar cane syrups.
    SMB chromatography processes that are used to separate saccharides use, for example, calcium-charged, cross-linked polystyrene resins, anion resins in the bisulfite form (Bechthold M., et al., Chemie Ingenieur Technik, 2010, 82, 65-75), or polystyrene -Gel, strongly acidic cation exchanger in hydrogen form (Purolite® PCR833H) (Purolite, Bala Cynwyd, USA).
    Due to the continuous operation, the recycling of the mobile phase and also the potential to use large column sizes, SMB chromatography systems can in principle be scaled to reach production volumes of hundreds of tons.
    The process step of simulated countercurrent chromatography is advantageous insofar as this process step enables further removal of oligosaccharides which are structurally closely related to the desired oligosaccharide.
    In an additional and / or alternative embodiment, the method comprises
    Purification of the at least one HMO a treatment of the process stream with activated carbon in order to remove contaminating substances such as dyes from the process stream.
    In an additional and / or alternative embodiment, the method for purifying the at least one HMO comprises at least one step of crystallization or precipitation of the HMO from the process stream. The crystallization or precipitation of the at least one HMO from the process stream can be carried out by adding a suitable amount of a water-miscible organic solvent to the process stream which contains the at least one HMO. The organic solvent can be selected from the group consisting of C + - to Ce alcohols and C + - to C4-carboxylic acids. The step of crystallizing or precipitating the at least one HMO is not carried out at the end of the recovery process, so that residual amounts of the organic solvent or the carboxylic acid can be removed by subsequent process steps.
    In an additional and / or alternative embodiment of the method for purifying the at least one HMO, step-by-step sterile filtration and / or endotoxin removal takes place, preferably by filtering the process stream through a 3 kDa filter or a 6 kDa filter.
    In an additional and / or alternative embodiment, the method for purifying the at least one HMO comprises a step of increasing the concentration of the at least one HMO in the process stream. The concentration of the at least one HMO in the process stream can be increased by subjecting the process stream to vacuum evaporation, reverse osmosis or nanofiltration (e.g. nanofiltration with a nanofiltration membrane with a size exclusion limit of = 20 A). Alternatively, crystallized or precipitated HMOs are dissolved in water in order to obtain an aqueous solution of the at least one HMO, the aqueous solution having the desired concentration of the at least one HMO.
    In an additional and / or alternative embodiment, the resulting process stream is an aqueous solution which contains the at least one HMO in a concentration of = 20 g / l, = 25 g / l, = 30 g / l, = 40 g / contains l, = 60 g / l, = 100 g / l, = 200 g / l or even = 300 g / l.
    In an additional and / or alternative embodiment, the aqueous solution contains the at least one HMO in a purity of at least 80%, at least 85%, at least 90%, at least 93%, at least 95% or at least 98% by weight the dry matter / solutes in the solution.
    As used herein, the term "purity" refers to chemical purity, i.e. the degree to which a substance is undiluted or unmixed with foreign matter. The chemical purity is therefore an indicator of the relationship between the at least one HMO and by-products / impurities. Chemical purity is expressed as a percentage (%) and calculated using the following formula:
    Mass of the desired compound in the sample Percent Purity = 100 x
    Total mass of the sample
    The purity of an HMO in a preparation can be determined by any suitable method known to those skilled in the art, for example using HPLC and calculating the ratio of the area under the peak (s) that represent the amount of HMO , to the sum of the areas below the peaks that represent the HMO (the HMO) and all compounds other than the HMO (the HMO) in the same chromatogram.
    The aqueous solution containing the at least one HMO can be stored under suitable conditions, for example the aqueous solution can be frozen.
    The process for purifying the at least one HMO is inexpensive and easy to scale, making it suitable as a basis for a production process on a multi-ton scale.
    The method for purifying the at least one HMO is also advantageous in this respect
    when the aqueous solution is free from genetically modified microorganisms and from nucleic acid molecules derived from genetically modified microorganisms. In addition, the aqueous solution is free of proteins. The complete removal of proteins eliminates the risk of causing allergies in a potential consumer.
    The method for producing the spray-dried powder comprises the step of providing an aqueous solution which contains the at least one HMO or the mixture of structurally different HMO and - optionally - the at least one monosaccharide.
    In an additional and / or alternative embodiment, the at least one HMO is selected from the group consisting of 2'-FL, 3-FL, LNT, LNnT, LNFPI, 3'-SL, 6'-SL, LST-a , LST-b, LST-c and DSLNT.
    In an additional and / or alternative embodiment, the mixture of structurally different HMOs consists of five structurally different HMOs, preferably 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 of 2'-FL, 3FL, LNT, LNNT, LNFPI, 3'-SL and 6 '' SL.
    In an additional and / or alternative embodiment, the aqueous solution also contains at least one monosaccharide, preferably selected from the group consisting of L-fucose and N-acetyl neuraminic acid. In another embodiment, the aqueous solution also contains L-fucose and N-acetyl neuraminic acid.
    In an additional and / or alternative embodiment, the aqueous solution contains the at least one HMO or the mixture of HMOs and / or the at least one monosaccharide in the amount of a total saccharide amount of at least 20% (w / v), 30% (w / v), 35% (w / v) and up to 45% (w / v), 50% (w / v), 60% (w / vV).
    In an additional and / or alternative embodiment, the aqueous solution contains the at least one HMO or the mixture of HMO in a purity of at least 80%, at least 85%, at least 90%, at least 93%, at least 95% or at least 98 %, based on the weight of the dry substance / dissolved substances in the solution.
    In an additional and / or alternative embodiment, the aqueous solution does not contain any genetically modified microorganisms, nucleic acid molecules which originate from genetically modified microorganisms, and proteins.
    In the process for producing the spray-dried powder, the aqueous solution which contains the at least one HMO or the mixture of structurally different HMOs is subjected to spray drying.
    Spray drying is a method of obtaining dry powders, whereby the solution containing the substance of interest is first sprayed into droplets which are quickly dried by hot air. Spray drying is very quick and exposure of the substance to be dried to high temperatures is quite short.
    In an additional and / or alternative embodiment, the aqueous solution containing the at least one HMO or the mixture of structurally different HMO, at a nozzle temperature of at least 110 ° C, preferably at least 120 ° C, more preferably at least 125 ° C and spray-dried at less than 150 ° C, preferably less than 140 ° C and more preferably less than 135 ° C.
    In an additional and / or alternative embodiment, the aqueous solution containing the at least one HMO or the mixture of structurally different HMOs is at an outlet temperature of at least 60 ° C, preferably at least 65 ° C and less than 80 ° C , preferably less than 70.degree. C., spray-dried. In a particularly preferred embodiment, the aqueous solution containing the HMO (the HMO) is spray-dried at a nozzle temperature of about 68.degree. C. to about 70.degree.
    It goes without saying that each species of the structurally different HMOs - optionally in
    Combination with the at least one monosaccharide - can be cleaned and spray dried individually and that the resulting spray dried powders can be mixed in any desired ratio. In an alternative embodiment, the aqueous solutions all contain structurally different HMOs, and the resulting aqueous solution, which contains the mixture of structurally different HMOs in the desired ratio, is subjected to spray drying.
    In an additional and / or alternative embodiment, the aqueous solution containing at least one HMO or a mixture of structurally different HMOs also contains at least one monosaccharide such as - for example - L-fucose and / or N-acetyl neuraminic acid. The aqueous solution containing the at least one HMO or the mixture of structurally different HMOs and the at least one monosaccharide is then subjected to spray drying in order to obtain a spray-dried powder, which consists essentially of the at least one HMO and the at least one monosaccharide or the Mixture of structurally different HMOs and the at least one monosaccharide.
    The ratio of the saccharides (HMO and monosaccharide (s) in the resulting spray-dried powder corresponds to the ratio of these saccharides in the aqueous solution.
    The latter process is advantageous in that monosaccharides which cannot be spray dried individually can be spray dried in the presence of one or more HMOs.
    The spray drying of the aqueous solution containing the at least one HMO or the mixture of structurally different HMOs provides a powder with low hygroscopicity, the HMO being in amorphous form and the particle size being homogeneous. The spray-dried powder, which consists essentially of at least one HMO or a mixture of structurally different HMOs, is less hygroscopic than a powder with an identical composition that was obtained by freeze-drying. The spray-dried powders described here are therefore advantageous with regard to their further use and processing.
    The 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, is used to produce a nutritional composition. The spray-dried powder, which consists essentially of a mixture of structurally different HMOs and optionally at least one monosaccharide, preferably selected from the group consisting of L-fucose and NacetyIneuraminic acid, is suitable for human consumption and can therefore be used in preparations for human consumption such as medical formulations, infant formula, milk beverages or dietary supplements.
    At least one nutritional composition is provided which comprises a spray-dried powder as described above and / or as prepared according to the method described above.
    In an additional and / or alternative embodiment, the nutritional composition comprises a mixture consisting essentially of Neub5Ac, 2'-FL, 3-FL, LNT, LNnT, LNFPI, 3'-SL, 6'-SL and L- Fucose consists. A composition containing preferred amounts of each of these compounds is shown in Table 6.
    The composition according to the second column in Table 6 is particularly advantageous for supplementing baby food, so that the final baby food for direct consumption can contain the compounds of the mixture in the concentrations given in the third column of Table 6 are.
    Compound Proportion of the mixture Final concentration in the (percentage by weight) baby food (g / l)
    2Z-FL 34 2.5 3-FL 11 0.8 LNT 20 1.5 LNNT 2 0.15 LNFPI 13 1.0 3'-SL 3 0.2 6'-SL 4 0.3 NeubAc 8 0.6 L-fucose 5 0.4 total 100 7.45
    Table 6: Composition of a representative mixture containing Neu5AC suitable for infant formula.
    In an additional and / or alternative embodiment, the nutritional composition contains one or more additional ingredients. The one or more additional ingredients are from the group consisting of oil, fat and fatty acids (such as olive oil, sunflower oil, coconut oil, nut oil, rapeseed oil, palm oil, flaxseed oil, fish oil, linolenic acid, soybean oil, etc.), carbohydrates (such as glucose, fructose, Lactose, maltodextrin, starch, sucrose, inositol etc.), proteins (from skimmed milk, whey, casein (from domesticated dairy animals) or 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) are selected.
    In a preferred embodiment, the nutritional composition with the contained spray-dried powder, which contains at least one human milk oligosaccharide or the mixture of human milk oligosaccharides or the mixture of at least one human milk oligosaccharide with at least one monosaccharide or the mixture of structurally different human milk oligosaccharides with other fibers / consists essentially of it , an infant formula that meets the composition requirements according to Regulation (EU) 2016/127 and / or the Code of Federal Regulations (USA), Title 21, 107.100 (nutrient specifications). Representative baby formula compositions are given in Tables 7 and 8.
    Baby food: skimmed milk vegetable oils (palm oil, rapeseed oil, sunflower oil) human milk oligosaccharides 3-fucosyllactose skimmed milk powder oil from Mortierella alpina fish oil calcium carbonate potassium chloride vitamin C sodium chloride vitamin E iron acetate zinc sulfate niacin calcium D-panthothenate
    Copper sulfate Vitamin A Vitamin B1 Vitamin B6 Magnesium sulfate Potassium iodate Folic acid Vitamin K Sodium selenite Vitamin D.
    Table 7: Components of a representative baby food.
    per 100 g powder | per 100 ml baby food
    Energy kJ 1 | 2094-2145 283
    kcal | 500-512 67-68 fats g 24.2-26.2 3.3-3.5 of which: 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 g 56-58 7.4-7.9 of which: sugars g 44-56 6-7.4 of which: lactose g 44-56 6-7.4 Neub5Ac mg | 440 60 L-Fucose mg 1 | 300 40 HMO g 4.22-4.81 0.57-0.65 of which 2-FL g 1.85-2.22 0.25- 0.30 3-FL mg | 555.56-592.6 | 75-80 LNT g 1.11 0.15 LNNT mg | 0-111.11 0-15 LNPF-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 Vitamins Vitamin A uUg 1357-358 47.3-48.2 Vitamin D Wg 17.8 1.05 Vitamin E mg 18.15 1.1 Vitamin K WUg 1 | 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 | 1.3-1.7 0.18-0.23
    Niacin mg 13.63 0.49 Vitamin B6 Wg | 526-600 71-81 Folic acid Ug | 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 1 | 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 phosphorus mg 1 | 335-352 45.2-46.5 magnesium mg | 49.3-56.3 6.66-7.43 iron mg 14.15 0.56
    Zinc mg | 3.7-3.8 0.49-0.51 copper wg 1274 37 manganese wg 196.3 13 fluoride wg 1 | 30.4-32.6 4.1-4.4 selenium wg 1 | 11 , 1-12.3 1.5-1.6 iodine uUg | 101.5-103.7 13.7-14
    Table 8: Composition of a representative baby food. The final concentration is based on a preparation of 13.5 g of the powder in 90 ml of water.
    In an additional and / or alternative embodiment, the nutritional composition also contains microorganisms, preferably probiotic microorganisms. For infant nutrition applications, the preferred microorganisms are derived from or can be found in the microbiome of a healthy human. Preferably, but without limitation, the microorganisms are selected from the genera Bifidobacterium, Lactobacillus, Enterococcus, Streptococcus, Staphylococcus, Peptostreptococcus, Leuconostoc, Clostridium, Eubacterium, Veilonella, Fusobacterium, Bacterioides, Prevotella, and Saccharomyibacterium, Eubotella. In an additional and / or alternative embodiment, the microorganism is selected from the group consisting of Bifidobacterium adolescentis, B. animalis, B. bifidum, B. breve, B. infantis, B. lactis, B. longum; Enterococcus faecium; Escherichia coli; Klyveromyces marxianus; Lactobacillus acidophilus, L. bulgaricus, L. casei, L. crispatus, L. fermentum, L. gasseri, L. helveticus, L. johnsonii, L. paracasei, L. plantarum, L. reuteri, L. rhamnosus, L. salivarius , L. sakei; Lactococcus lactis (including, but not limited to, the subspecies lactis, cremoris and diacetylactis); Leuconostoc mesenteroides (including but not limited to the subspecies mesenteroides); Pedicoccus acidilactici, P. pentosaceus; Propionibacterium acidipropionici, P. freudenreichii ssp. shermanii; Staphylococcus carnosus; and Streptococcus thermophi / us.
    In addition to the combined living organisms, the nutritional composition can also contain dead cell cultures. In the probiotics field, killed cell cultures are sometimes used (e.g., tyndalized bacteria). These killed cultures can provide proteins, peptides, oligosaccharides, cell outer wall fragments and natural products, which leads to short-term stimulation of the immune system.
    The inclusion of probiotic microorganisms in the nutritional composition, especially in the presence of HMO, is particularly advantageous insofar as it also promotes the establishment of a healthy intestinal microbiome.
    In an additional and / or alternative embodiment, the nutritional supplement comprises
    composition also includes prebiotics such as galactooligosaccharides (GOS), fructooligosaccharides (FOS), inulin or combinations thereof.
    The nutritional composition may be in liquid or solid form, including but not limited to powders, granules, flakes, and pellets.
    In an additional embodiment, the nutritional composition is selected from the group consisting of medicinal formulations, baby foods and nutritional supplements.
    The present invention will be described with respect to certain embodiments and with reference to the drawings, but the invention is not limited in this regard, but only by the claims. Furthermore, the terms first, second, and the like are used in the description and in the claims to distinguish between similar elements and not necessarily to describe a sequence in terms of time, space, rank or otherwise. It will be understood that the terms so used are interchangeable in appropriate circumstances and that the embodiments of the invention described herein may be employed in other sequences than those described or illustrated herein.
    It should be noted that the term "comprising" as used in the claims should not be interpreted as being limited to the means set out below; it does not exclude other elements or steps. It is therefore to be interpreted as an indication of the presence of the named features, whole numbers, steps or components as mentioned, but without excluding the presence or addition of one or more other features, whole numbers, steps or components or groups thereof. Therefore, the scope of the phrase “a device comprising means A and B” should not be limited to devices consisting only of components A and B. It means that in relation to the present invention the only relevant components of the device are A and B.
    Reference in this specification to "an embodiment" or "an embodiment" means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention is. The appearances of the expressions “in an embodiment” or “in an embodiment” in different places in this description therefore do not necessarily all refer to the same embodiment. Furthermore, the particular features, structures, or properties in one or more embodiments can be combined in any suitable manner, as would be apparent to one of ordinary skill in the art from this disclosure.
    Similarly, it will be understood that in describing representative embodiments of the invention, various features of the invention are sometimes combined into a single embodiment, figure, or description thereof for the purpose of simplifying the disclosure and facilitating understanding of one or more of the various inventive elements Aspects are summarized. This method of disclosure is not to be construed as reflecting the intent that the claimed invention require more features than are expressly set out in each claim. Rather, as the following claims reflect, inventive aspects may require less than all features of any preceding disclosed embodiment. Thus, the claims that follow the detailed description are hereby expressly incorporated into this detailed description, with each claim standing on its own as a separate embodiment of this invention.
    While some embodiments described herein incorporate some, but not different, features that are incorporated in other embodiments, combinations of features of different embodiments are intended to be within the scope of the invention and constitute different embodiments, as will be understood by those skilled in the art. For example, in the following claims, each of the claimed embodiments
    men can be used in any combination.
    Furthermore, some of the 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 for performing the function. A processor with the necessary instructions for carrying out such a method or element of a method thus forms a means for carrying out the method or element of a method. Additionally, an element of an apparatus embodiment described herein is an example of a means for performing the function performed by the element for the purpose of practicing the invention.
    In the description and drawings provided herein, numerous specific details are set forth. It should be understood, however, that embodiments of the invention can be practiced without these specific details. In other instances, known methods, structures, and techniques are not shown in detail in order to facilitate understanding of the description and drawings.
    The invention will now be described through a detailed description of several embodiments of the invention. It will be understood that other embodiments of the invention can be configured according to the knowledge of those skilled in the art without departing from the true spirit or technical value of the invention, the invention being limited only by the terms of the appended claims.
    Example 1: Purification of 2'-Fucosyllactose from Fermentation Broth
    The production of 2'-fucosyllactose by fermentation using a genetically modified E. coli strain was carried out as described in European patent application No. 16 196 486.1. The 2'-fucosyllactose was purified from the fermentation broth by filtration, ion exchange chromatography, nanofiltration, diafiltration or electrodialysis and treatment with charcoal as described in WO 2015/106943 A1. The resulting solution containing 2'-fucosyl lactose was subjected to spray drying to obtain a stable solid product.
    Example 2: Purification of 3-Fucosyllactose from Fermentation Broth
    3-Fucosyllactose was produced by fermentation using a genetically modified E. coli strain as described in European Patent Application No. 16 196 486.1.
    The cells were separated from the culture medium by ultrafiltration (0.05 μm cut-off) (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, which contained about 30 g / l 3-fucosyllactose, was passed over a strong cationic ion exchanger (Lewatit S 2568 (Lanxess, Cologne, Germany) in H * form in order to remove positively charged impurities adjusted to pH 7.0 with sodium hydroxide solution and applied in chloride form to an anionic ion exchanger (Lewatit S6368 A, Lanxess). Both ion exchangers were used in volumes of 200 |. After 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. The concentrated solution with a conductivity of about 15 mS cm “was filtered (10 kDa ; Microdyn-Nadir, Wiesbaden, Germany), clarified with activated carbon (CAS: 7440-44-0, Carl Roth, Karlsruhe, Germany) and deionized by electrodialysis de a PC-Cell BED 1-3 electrodialysis machine (PCCell, Heusweiler, Germany) with a PC-Cell E200 membrane stack that contained the following membranes: Cation exchange membrane CEM: PC SK and anion membrane AEM: PCAcid60. Sulfamic acid 0.25 M was used as the electrolyte. To reduce the brownish coloration caused by Maillard reactions and the aldol products from the fermentation process, a second round of ion exchange chromatography was carried out.
    graphie carried out using the same ion exchange material as mentioned above in Na * - and CI 'form, but in a volume of 50 I. After concentrating the sugar solution by evaporation, the conductivity was again carried out by electrodialysis using the above-mentioned PC-Cell BED 1-3 decreased from 4 mS cm ”to 0.4 mS cm” or less. For further decolorization, activated carbon (CAS: 7440-44-0, Carl Roth, Karlsruhe, Germany) was added to the solution and a virtually colorless solution was obtained by filtration.
    Example 3: Purification of Lacto-N-tetraose from fermentation broth
    The fermentative production of lacto-N-tetraose was carried out using a genetically modified E. coli BL21 (DE3) A / acZ strain with genomically integrated genes that are necessary for the in vivo synthesis of lacto-N-tetraose are essential, namely an N-acetylglucosamine glycosyltransferase (IgtA from Neisseria meningitidis MC58), a βB-1,3-galactosyltransferase (wbdO from Salmonella enterica subsp. salamae serovar Greenside), lacY from E. co K12, the UDP-glucose-4- Epimerase galE, and the UTP-glucose-1-phosphate uridyltransferase galU, both from E. coli K12. For the fermentative production of Lacto-N-tetraose, the strain was grown in a defined mineral salt medium containing 7 g I "NH4H-PO-", 7 g I "K" HPO "s, 2 g I KOH, 0.3 g I" citric acid , 5 g I NH4Cl, 0.1 mM CaCl, 8 mM MgSO ", trace elements (0.101 g I nitrilotriacetic acid, pH 6.5, 0.056 g I ammonium iron citrate, 0.01 g I MnCl2 x 4 H2O, 0.002 g I" CoCl " x 6 H2O, 0.001g I "CuCIz x 2 H2O, 0.002 g I" boric acid, 0.009 g I ZnSO4 x 7 H2O, 0.001 g I "Na2MoO" x 2 H; O, 0.002 g I "Na2SeOs, 0.002 g I NiSO4 x 6 H: O) and 2% glucose as the carbon source. If necessary, antifoam agents (Struktol J673, Schill + Seilacher) were added. The pH was controlled using a 25% ammonia solution. Lactose was added gradually up to a final concentration of 15 mM from a 216 g I-lactose stock solution, the lactose concentration in the culture medium being kept constant during the fermentation process. Residual lactose and Lacto-N-Triose II, which were by-produced during the process, were hydrolyzed by a second E. coli strain which was added to the fermenter. This strain expressed a functional beta-lactamase, a beta-N-acetylhexosaminidase (bbh / from Bifidobacterium bifidum JCM1254) and a functional Gal operon for the degradation of monosaccharides (EP 2 845 905 A).
    The cells were separated from the fermentation broth and the liquid containing lacto-N-tetraose was purified to a purity of 75-80%, determined by mass balance according to the method described in Example 2.
    Contaminating carbohydrate by-products resulting from inefficient enzymatic degradation and inefficient metabolism were removed by chromatography using simulated countercurrent chromatography (SMB chromatography) according to WO 2015/049331. Alternatively, the lacto-N-tetraose was purified by crystallization with isopropanol. For crystallization, the solution containing lacto-N-tetraose 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 the spray drying nozzles with an inlet temperature of 130 ° C under a stream of nitrogen, while the product flow was controlled so that an outlet temperature of 67 ° C to 68 ° C ° C was maintained.
    The solid material was a mixture of isopropanol and water (3: 1 (v / v)) in a ratio of 1 kg of powder in 12 | Isopropanol / water given. The suspension was stirred vigorously, then the insoluble lacto-N-tetraose was filtered and dried at 40.degree. Starting from a material with a purity of 73-89%, the crystallized lacto-N-tetraose was purified to about 95% with a yield of 85%. The sugar was dissolved in water to a concentration of 25% and passed successively through a 6 kDa filter (Pall Microza ultrafiltration module SIP-2013, Pall Corporation, Dreieich, Germany) and a 0.2 μm sterile filter. Solid material was obtained by spray drying the sterile material under the conditions described above.
    Example 4: Purification of 3'- and 6'-Sialyllactose from fermentation broth
    Recombinant E. coli BL21 (DE3) AlacZ strains were used to produce 3'- and 6'-sialyllactose. The strains shared genetic modifications: chromosomal, constitutive expression of the glucosamine-6-phosphate synthase GImS from E. coli, the N-acetylglucosamine-2-epimerase SIr1975 from Synechocystis sp., The glucosamine-6-phosphate-N-acetyltransferase Gna1 from Saccharomyces cerevisiae, the phosphoenolpyruvate synthase PpsA from E. col, the N-acetyl neuraminic acid synthase NeuB and the CMP sialic acid synthetase NeuA, the latter both from Campylo-bacterjejuni. In addition, the genes encoding the lactose permease LacY from E. col, cscB (sucrose permease), cscK (fructokinase), cscA (sucrose hydrolase) and cscR (transcription regulator) from E. col / W, and a functional gal operon consisting of the genes ga / E (UDP-glucose-4-epimerase), galT (galactose-1-phosphate-uridylyl transferase), galk (galactokinase) and ga / M (galactose-1-epimerase) from E. coli K12 in the genome of the BL21- Strain integrated and constitutively expressed.
    The strain that synthesizes 3'-sialyllactose harbors the 3'-sialyltransferase gene from Vibrio sp. JT-FAJ-16, while the 6'-sialyllactose producing strain contains the alpha-2,6-sialyltransferase plsT6 from Photobacterium leiognathi JT-SHIZ-119.
    The sialyllactose-producing strains were grown in a defined mineral salt medium that contained 7 g I "NHıH2-PO-., 7 g I K2HPOs, 2 g I" KOH, 0.3 g I "citric acid, 5 g I NH4Cl, 1 ml 1 ”antifoam agent (Struktol J673, Schill + Seilacher), 0.1 mM CaCl2, 8 mM MgSO-, trace elements and 2% sucrose as carbon source. The sucrose feed (500 g I ") added in the fed batch phase was supplemented with 8 mM MgSO-«, 0.1 mM CaCl, trace elements and 5 g I NH4Cl.
    The trace elements consisted of 0.101 g I "nitrilotriacetic acid, pH 6.5, 0.056 g I ammonium iron (III) citrate, 0.01 g I MnCl2 x 4 H2: O, 0.002 g I" CoCl »x 6 H2: O, 0.001g I ”CuCl, x 2 H2O, 0.002 g I” boric acid, 0.009 g I ”ZnSO4 x 7 H2O, 0.001 g I NazMoO« x 2 H2: O, 0.002 g I ”Na2SeOs, 0.002 g I” NiSO: x 6 H2: O.
    For sialyllactose formation, a lactose feed of 216 g I "was used. The pH value was controlled using ammonia solution (25% v / v). The FedBatch fermentation was carried out at 30 ° C below constant aeration and agitation carried out In order to remove residual lactose at the end of the fermentation, βB-galactosidase was added to the fermentation vessel and the resulting monosaccharides were metabolized by the production strain.
    The cell-free liquid was then deionized by ion exchange chromatography. First, cationic impurities were removed in H * form on a strong cation exchanger in a volume of 200 l (Lewatit® S 2568 (Lanxess, Cologne, Germany). Using NaOH, the pH of the resulting solution was adjusted to 7.0 In a second step, anionic ions and unwanted dyes were removed from the solution in chloride form with the strong anion exchanger Lewatit® S 6368 S (Lanxess, Cologne, Germany) .The ion exchanger had a bed volume of 200 I. Using a second filtration step on the Cross-flow filters (150 kDa cut-off) (Microdyn-Nadir, Wiesbaden, Germany) were removed from the acidification of the solution. To concentrate the sugar, the solution was filtered through a Dow FILMTECH NF270-4040 (Inaqua, Mönchengladbach, Germany) or alternatively nanofiltered using a Trisep 4040-XN45-TSF membrane (0.5 kDa cutoff) (Microdyn-Nadir, Wiesbaden, Germany) The monosaccharide N-acetylglucosamine which originates from the fermentation process and which contaminates the sialyllactose solution is separated from the product. The concentrated sialyllactose solution was then treated with activated charcoal (CAS: 7440-44-0, Carl Roth, Karlsruhe, Germany) in order to remove dyes such as Maillard reaction products and aldol reaction products. In order to separate the sialyllactose from by-products that originate from the fermentation process, such as sialic acid and N-acetylglucosamine, the solution was filtered with a 1 kDa exclusion membrane GE4040F30 (GE Water & Process Technologies, Ratingen, Germany) and tested to a conductivity of 0, 6 to 0.8 mS cm diafiltered. The diluted solution was rotated
    evaporator concentrated to a concentration of approx. 300 g / l. In a final chromatographic separation, other contaminating sugars such as disialyl lactose were removed. For this purpose, the concentrated solution was applied to a weak anion exchange resin in acetate form (Amberlite FPA51, Dow Chemical, Michigan, USA). While the sialyllactose rarely binds to the resin, the disialyllactose is adsorbed. Thus, the sialyl lactose is eluted with 10 mM ammonium acetate, while the disialyl lactose is eluted with 1 M ammonium acetate. To remove the ammonium acetate, the sialyllactose was precipitated with a 10-fold excess of ethanol. The solid fraction was filtered and dried.
    The product was completed by passing a 20% strength sialyllactose solution successively through a 6 kDa filter (Pall Microza ultrafiltration module SIP-2013, Pall Corporation, Dreieich, Germany) and a 0.2 µm sterile filter.
    Part of the solution was spray-dried using a Büchi spray dryer (Büchi Mini Spray Dryer B-290) (Büchi, Essen, Germany) using the following parameters: inlet temperature: 130 ° C, outlet temperature 67 ° C to 71 ° C, gas flow 670 l / h, aspirator 100%.
    The spray-dried 6'-sialyllactose had a purity of 91%, while the 3'Sialyllactose material had a purity of 93%.
    Example 5: Preparation of HMO Mixtures
    Mixtures of various HMOs were made from solid products. For this purpose, the individual HMOs were spray-dried and the powders obtained were mixed. HMO mixture | contained 2’-fucosyllactose and lacto-N-tetraose in a ratio of 70% by weight to 30% by weight; HMO mixture II contained 2'-fucosyllactose (52% by weight), 3-fucosyllactose (13% by weight), lacto-N-tetraose (26% by weight, 3'-sialyllactose (4% by weight), and 6 '-Sialyllactose (5 wt.%) The mixed powders were dissolved in water to form a solution containing 20 wt.% HMO, and the resulting solution was spray dried again using the Buchi spray dryer described in Example 4.
    Analysis of the resulting spray-dried powder revealed that, in terms of the ratio of the various HMOs, it had the same composition as the solution that was spray-dried.
    Example 6: Preparation of saccharide mixtures
    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 as described in Example 4 using the Büchi spray dryer. A spray-dried powder was obtained which essentially consisted of 2'-FL and L-fucose, the ratio of 2'-FL and L-fucose in the spray-dried powder being the ratio of 2'-FL and L-fucose in the solution, which was spray dried was identical. Thus, L-fucose can be spray dried in the presence of an HMO.
    Example 7: Characterization of spray-dried human milk oligosaccharides 1. Dynamic differential calorimetry (DSC)
    Using the dynamic differential calorimetry (DSC) on a Mettler Toledo 821e (Mettler Toledo, Giessen, Germany), thermal events of the spray-dried human milk oligosaccharides, namely 3-fucosyllactose, 6'-sialyllactose, 3'-sialyllactose, lacto-N- tetraose and the spray-dried mixtures of human milk oligosaccharides, a mixture (HMO mixture |) of 2'-fucosyllactose / lacto-N-tetraose or a mixture (HMO mixture II) of 2'-fucosyllactose, 3-fucosyllactose, lacto-N -tetraose, 6'-sialyllactose, 3'-sialyllactose.
    With a Mettler Toledo 821e (Mettler Toledo, Giessen, Germany) thermal events of the spray-dried products (glass transition temperature (Tg), other ex and endothermic events) were determined.
    About 25 mg of the spray-dried human milk oligosaccharides were analyzed in crimped aluminum pans (Mettler Toledo, Giessen, Germany). The samples were cooled to 0 ° C at 10 K / min and heated again to 100 ° C at a sampling rate of 10 K / min. After cooling the samples to 0 ° C in a second heating cycle, the samples were 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). Exothermic and endothermic peaks are reported using the peak temperature and the normalized energy of the event.
    The first heating scan in all samples showed a major glass transition throughout the heat flow, as evidenced by a major step transition in the range of approximately 48-58 ° C in most samples, with the important glass transition event observed in the first heating scan recurring in the second heating scan. The results of the DSC analyzes are summarized in Table 9.
    Sample 1. 2. Heating scan Heating scan Tg [° C] Tg [° C]
    3-Fucosyllactose 57.6 59.9 Lacto-N-tetraose 49.9 79.4 6 & '- Sialyllactose 47.6 49.6 3'-Sialyllactose 48.8 54.3 Z'-Fucosyllactose / Lacto-N-tetraose 56.3 59 HMO blend 54.2 55.6
    Table 9: Thermal events of HMOs as determined by dynamic differential calorimetry
    For 3-fucosyllactose, an endothermic relaxation peak after Tg was found in the first heating scan. A much higher Tg of about 79 ° C was found for Lacto-N-tetraose in the 2nd heating scan compared to the other samples. This can be caused by an endothermic event during the first heating scan at about 89 ° C (-6.04 J / g). As for 3-fucosyllactose, an endothermic relaxation peak after Tg was also determined for 6′-sialyllactose. However, an endothermic event at 77 ° C. (0.22 J / g) also occurred in this sample. For the 3'-sialyllactose and the HMO mixture | no endothermic events were found, for the HMO mixture II the endothermic event was during the first heating scan at 79 ° C. (0.34 J / g).
    2, powder x-ray diffraction (XRD)
    Wide angle powder x-ray diffraction (XRD) was used to study the morphology of lyophilized products. The X-ray diffractometer Empyrean (Panalytical, Almelo, Netherlands) with a copper anode (45 kV, 40 mA, Ka1 emission at a wavelength of 0.154 nm) and a PIXcel3D detector was used. Approximately 100 mg of the spray dried samples were analyzed in reflection mode in the angular range 5-45 ° 268, with a step size of 0.04 ° 26 and a counting time of 100 seconds per step.
    All singular oligosaccharides and the HMO mixtures | and II showed a completely amorphous state (Figures 1 to 6). A second (amorphous) signal around 9-10 ° was detected for lacto-N-tetraose.
    3 laser diffraction
    The powder particle size was evaluated by laser diffraction. The system detects scattered and diffracted light through an arrangement of concentrically arranged sensor elements. The software algorithm then approximates the particle numbers by calculating the z-values of the light intensity values that reach the various sensor elements. Analysis was performed using a quantitative laser diffraction system (gqLD) SALD-7500 aggregate
    gate Sizer (Shimadzu Corporation, Kyoto, Japan).
    A small amount (spatula tip) of each sample was dispersed in 2 ml of isooctane and homogenized by ultrasonic treatment for five minutes. The dispersion was transferred to a batch cell filled with isooctane and analyzed in manual mode.
    The settings for the data acquisition were as follows: number of signal averages per measurement: 128, number of signal accumulations: 3, and interval: 2 seconds.
    Before the measurement, the system was zeroed with isooctane. Each sample dispersion was measured three times and the mean values and standard deviation are reported. The data were evaluated with the software WING SALD II Version V3.1. Since the refractive index of the sample was unknown, the refractive index of sugar particles (disaccharide particles) (1.530) was used to determine the size distribution profiles. The size values for the mean and median diameter are given.
    The mean particle sizes for all samples were very similar; somewhat lower values were measured for HMO mixture II. The particle size properties are summarized in Table 10. In addition, the particle size distribution for all samples indicated the presence of a predominantly size population.
    [nm] +0.7 | +1.5 + 0.6 median [141.3 141.3 [| 141.3 + * 0.0 [121.9 [141.3 + 0.0 [112.2 [nm] + 0.0 + 0.0 + 16.7 +0.0
    Table 10: Particle size of the HMO, determined by laser diffraction
    权利要求:
    Claims (13)
    [1]
    1. Nutritional composition comprising a spray-dried powder containing a mixture of structurally different human milk oligosaccharides, said mixture containing structurally different human milk oligosaccharides 2'-FL, 3-FL, 3'-SL, 6'-SL, and LNT and / or LNnT.
    [2]
    2. The nutritional composition of claim 1, wherein said spray-dried powder further comprises LNFP | contains.
    [3]
    3. The nutritional composition of claim 1 or 2, wherein the spray-dried powder further contains at least one monosaccharide selected from the group consisting of L-fucose and N-acetyl neuraminic acid.
    [4]
    4. A nutritional composition according to any one of claims 1 to 3, wherein the spray-dried powder contains or consists of:
    Saccharide A [% by weight] B [% by weight] 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 LNNT 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
    [5]
    5. Nutritional composition according to claim 1, wherein the mixture of the structurally different HMOs consists of 52.2% wt. 2'-FL, 13.0% weight 3-FL, 26.1% wt. LNT, 3.5% wt. 3'-SL and 5.2% weight 6'-SL.
    [6]
    6. Nutritional composition according to claim 1, wherein the mixture of the structurally different HMOs consists of 39.0% wt. 2'-FL, 12.0% weight 3-FL, 23.0% wt. LNT, 2.0% wt. LNnT, 16.0% wt. LNFP I, 3.0% wt. 3'-SL and 5.0% weight 6'-SL.
    [7]
    7. Nutritional composition according to claim 3, wherein the spray-dried powder consists of 33.6% wt. 2'-FL, 10.7% - wt. 3-FL, 20.1% - wt. LNT, 2.0% - wt. LNnT, 13.4% - wt. LNFP I, 2.7% - wt. 3'-SL, 4.0% - wt 6'-SL, 8.1% by weight of NeuAc, and 5.4% by weight of L-fucose.
    [8]
    8. Nutritional composition according to one of claims 1 to 7, wherein preferably the whole of the spray-dried powder is 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% wt. Contains human milk oligosaccharides.
    [9]
    9. Nutritional composition according to one of claims 3 to 8, wherein at least one human milk oligosaccharide, preferably all human milk oligosaccharides, and the at least one monosaccharide are contained in amorphous form.
    [10]
    10. The nutritional composition according to any one of claims 1 to 9, wherein the nutritional composition further contains at least one probiotic microorganism.
    [11]
    11. Nutritional composition according to one of claims 1 to 10, wherein the nutritional composition contains one or more additional ingredients selected from the group consisting of oil, fat, fatty acids, carbohydrates, proteins, vitamins, minerals and trace elements.
    [12]
    12. The nutritional composition of claim 11, wherein the proteins are selected from the group of proteins derived from skimmed milk, whey, casein and soybeans.
    [13]
    13. Nutritional composition according to one of claims 1 to 13, wherein the nutritional composition contains at least one prebiotic selected from the group consisting of galacto-oligosaccharides, fructo-oligosaccharides and inulin.
    In addition 3 sheets of drawings
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    JP2021505174A|2021-02-18|
    WO2019110801A1|2019-06-13|
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    KR20200096793A|2020-08-13|
    DK201900113U8|2020-11-05|
    SG11202004650SA|2020-06-29|
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    AU2018380957A1|2020-06-11|
    BR112020011300A2|2020-11-17|
    PH12020550849A1|2021-05-17|
    KR20200096770A|2020-08-13|
    AU2018380959A1|2020-06-18|
    BR112020011202A2|2020-11-17|
    US20210161192A1|2021-06-03|
    RU2020119946A|2022-01-10|
    PH12020550848A1|2021-05-17|
    SG11202004414PA|2020-06-29|
    KR20200096771A|2020-08-13|
    AU2018380960A1|2020-06-04|
    JP2021505171A|2021-02-18|
    RU2020119953A|2022-01-10|
    KR20200096779A|2020-08-13|
    DE202018006272U1|2019-12-20|
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    RU2020119944A|2022-01-10|
    JP2021505177A|2021-02-18|
    BR112020011236A2|2020-11-17|
    BR112020011242A2|2020-11-17|
    WO2019110800A1|2019-06-13|
    DK201900113U1|2020-01-07|
    US20210171992A1|2021-06-10|
    PH12020550843A1|2021-05-17|
    US20200383367A1|2020-12-10|
    JP2021505170A|2021-02-18|
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    法律状态:
    2021-10-15| HC| Change of the firm name or firm address|Owner name: CHR. HANSEN HMO GMBH, DE Effective date: 20210901 |
    优先权:
    申请号 | 申请日 | 专利标题
    EP17206159.0A|EP3494805A1|2017-12-08|2017-12-08|Spray-dried tetrasaccharides|
    EP17206223.4A|EP3494806A1|2017-12-08|2017-12-08|Spray-dried lacto-n-fucopentaose|
    EP17206124.4A|EP3494804A1|2017-12-08|2017-12-08|Spray-dried 3-fucosyllactose|
    EP17206414.9A|EP3494807A1|2017-12-11|2017-12-11|Spray-dried sialyllactose|
    EP18155669.7A|EP3524067A1|2018-02-08|2018-02-08|Spray-dried mixture of human milk oligosaccharides|
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