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    • 专利摘要:
      A method for purifying sialylated oligosaccharides from a fermentation broth, cell lysate or biocatalytic reaction mixture is disclosed to obtain large amounts of desired high purity sialylated oligosaccharides. The method is particularly suitable for the large-scale economic purification of sialylated oligosaccharides from human milk (such as 3'-sialyl-lactose, 6'-sialyl-lactose or sialyzed lacto-N-tetraose derivatives) from microbial fermentation , with the use of recombinant bacterial cells or yeast cells. The material obtained is of high purity and can be used for medical or food applications, such as medical nutrition products, infant formula, dietary supplements, general nutrition products (for example, milk drinks).
    公开号:BR112020003502A2
    申请号:R112020003502-5
    申请日:2018-08-29
    公开日:2020-09-01
    发明作者:Stefan Jennewein；Markus HELFRICH；Benedikt Engels
    申请人:Jennewein Biotechnologie Gmbh；
    IPC主号:
    专利说明:

    [001] [001] The present invention relates to the purification of sialylated oligosaccharides. More specifically, the present invention relates to the purification of sialylated oligosaccharides, in particular of human milk sialylated oligosaccharides (sHMOs), from a fermentation broth, a clean cell lysate or a reaction mixture. BACKGROUND
    [002] [002] Human milk is a complex mixture of carbohydrates, fats, proteins, vitamins, minerals and trace elements. Carbohydrates are by far the most abundant fraction, which can be further divided into lactose and more complex oligosaccharides, called human milk oligosaccharides (HMOs). While lactose is used as an energy source, complex oligosaccharides are not metabolized by the baby. The fraction of complex oligosaccharides represents up to 10% of the total carbohydrate fraction and probably consists of more than 150 different oligosaccharides. The occurrence and concentration of these complex oligosaccharides are specific to humans and, therefore, cannot be found in large quantities in the milk of other mammals, such as domesticated animals for milk supply.
    [003] [003] The presence of these complex oligosaccharides in human milk has been known for a long time and the physiological functions of these oligosaccharides have been the subject of medical research for many decades (Gura, T. (2014) Science 345: 747-749; Kunz, C . & Egge, H. (2017) In: Prebiotics and Probiotics in Human Milk. Eds. McGuire, MK; McGuire, MA & Bode, L. Elsevier, London pp. 3 to 16). For some of the most abundant HMOs, specific functions have already been identified (Bode, L. (2012) Glycobiology 22: 1147-1162; Bode, L. and Jantscher-Krenn, E. (2012) Adv. Nutr. 3: 383S- 391 S; Morrow et al. (2004) J.
    [004] [004] The limited supply of individual HMOs and the inability to produce sufficient quantities of these molecules led to the development of processes based on chemical synthesis to generate some of these complex molecules. However, the chemical synthesis of HMOs, as well as enzymatic synthesis and fermentation-based production proved to be extremely challenging. Large-scale production of HMOs of sufficient quality for food applications has hardly been achieved until now. In particular, the chemical synthesis of HMOs, such as 2'-fucosyl-lactose (WO 2010/115935 A1) requires a number of harmful chemicals, which can contaminate the final product.
    [005] [005] The disadvantages of HMO chemical synthesis led to the development of several enzymatic and fermentation-based methods (Miyazaki et al., (2010) Methods in Enzymol. 480: 511 to 524; Murata et al., (1999) Glycoconj J. 16: 189-195; Baumgartner et al. (2013) Microb. Cell Fact. 12: 40; Lee et al., (2012) Microb. Cell Fact. 11: 48; US 7,521,212 B1; Albermann et al. , (2001) Carbohydr. Res. 334: 97 to 103; Fierfort, N. and Samain, E. (2008) J. Biotechnol. 134: 216 to 265). However, these processes tend to generate complex mixtures of oligosaccharides, so that the desired product is contaminated with starting material, such as lactose, as well as intermediates, unwanted by-products (for example, by-products that originate from secondary activities of certain glycosyltransferases) and substrates, such as monosaccharides and individual polypeptides.
    [006] [006] State-of-the-art methods for purifying individual oligosaccharides from mixtures of oligosaccharides are technically complex, difficult to perform on a large scale and uneconomical for food applications. Industrial-scale processes have been developed to purify lactose and sucrose disaccharides from whey or molasses respectively, but these methods involve multiple crystallization steps that are elaborated and offer low yield. However, whey and molasses are initial "food grade" products and nowhere near as complex and challenging from the regulatory point of view as fermentation broths obtained from fermentation processes of recombinant bacteria or recombinant yeast.
    [007] [007] Gel filtration chromatography is the best method for purifying complex oligosaccharides, such as HMOs produced by microbial fermentation, but the disadvantages of gel filtration chromatography include its lack of scalability and its incompatibility with continuous processing . Gel filtration chromatography is therefore not economical and cannot be used to produce HMOs, such as 3'-sialyl-lactose or 6'-sialyl-lactose or any other sialylated oligosaccharide of sufficient quality and in sufficient quantity for food particularly for infant and child nutrition products. However, the production of sialylated HMOs (such as 3'-sialyl-lactose (3'-SL), 6'-sialyl-lactose (6'-SL), sialyl-lacto-N-tetraose a (LST-a), sialyl-lacto-N-tetraose b (LST-b), sialyl-lacto-N-tetraose c (LST-c), 3-fucosyl-sialyl-lactose (F-SL), disialyl-lacto-N-tetraose (DS -LNT) and fucosyl-LST b (F-LSTb) is interesting, since sialylated oligosaccharides are - for example - associated with improved neural development.
    [008] [008] The use of recombinant microorganisms (bacteria or yeast) for fermentative production of HMOs is also problematic, since recombinant DNA or proteins can contaminate the final product, and this is not acceptable to consumers and current regulatory authorities. Since the detection limits, in particular, for recombinant DNA molecules are very low (for example, when using detection based on qPCR, which is currently considered as the standard of excellence for detection) even a single DNA molecule in an oligosaccharide product can be detected. In addition, proteins pose the risk of causing allergic reactions and therefore must also be efficiently removed from the desired oligosaccharide product.
    [009] [009] Starting from this previous stage, it was an objective to provide a process to purify sialylated oligosaccharides, in particular, sialylated HMOs, which were produced by means of microbial fermentation, in which the referred process is applicable for commercial or industrial scale manufacture. sialylated oligosaccharides, which can lead to a product that has a purity that makes the product suitable for human consumption. SUMMARY
    [0010] [0010] In a first aspect, a method is provided to purify sialylated oligosaccharides that were produced through microbial fermentation or in-vitro biocatalysis.
    [0011] [0011] In a second aspect, preparations of a sialylated oligosaccharide are provided that have been produced through microbial fermentation or in-vitro biocatalysis.
    [0012] [0012] In a third aspect, the use of sialylated oligosaccharides is provided according to the second aspect.
    [0013] [0013] In a fourth aspect, nutritional compositions comprising at least one sialylated oligosaccharide are provided, wherein said at least one sialylated oligosaccharide has been produced by means of microbial fermentation or in-vitro biocatalysis.
    [0014] [0014] In a fifth aspect, a spray dried GMO free powder is provided that consists essentially of a sialylated oligosaccharide. BRIEF DESCRIPTION OF THE DRAWINGS
    [0015] [0015] Figure 1 shows the chemical structures of 3'-sialyl-lactose and 6'-sialyl-lactose.
    [0016] [0016] Figure 2 shows a diagram illustrating a modality of the process for purifying a sialylated oligosaccharide from fermentation broth.
    [0017] [0017] Figure 3 shows a diagram that illustrates a modality of the process for purifying a sialylated oligosaccharide from fermentation broth.
    [0018] [0018] Figure 4 shows a diagram illustrating a modality of the process for purifying a sialylated oligosaccharide from fermentation broth.
    [0019] [0019] Figure 5 illustrates the principle of a simulated four-zone moving bed chromatography.
    [0020] [0020] Figure 6 illustrates the principle of an eight-zone simulated moving bed chromatography.
    [0021] [0021] Figure 7 shows a graph illustrating the results of a powdered X-ray powder diffraction of 6'-SL.
    [0022] [0022] Figure 8 shows a graph illustrating the results of a powdered X-ray diffraction of 3'-SL spray-dried. DETAILED DESCRIPTION
    [0023] [0023] According to the first aspect, a method or process is provided to purify sialylated oligosaccharides that were produced by means of microbial fermentation. The method comprises the steps of: i) separating biomass from the fermentation broth; ii) remove cations from the fermentation broth; iii) remove anionic impurities from the fermentation broth; and iv) removing compounds that have a molecular weight less than that of the sialylated oligosaccharide to be purified.
    [0024] [0024] In one embodiment, the desired sialylated oligosaccharides are produced by microbial fermentation. Therefore, cells that have the ability to produce a desired sialylated oligosaccharide are grown under conditions that are permissive for the cells to produce the desired sialylated oligosaccharide. Cells suitable for producing the desired oligosaccharide include bacteria, such as Escherichia coli, Lactobacillus lactis, Corynebacterium glutamicum, Bacillus subtilis, Pseudomonas putita, or yeasts, such as Saccharomyces cerevisiae, Schizosaccharomyces pombe, Pichia pastoris.
    [0025] [0025] The cells can be genetically modified to produce a desired sialylated oligosaccharide that a non-genetically modified precursor cell does not have the capacity to produce, or to improve the production efficiency of the desired oligosaccharide. Escherichia coli, which is a preferred host for metabolic engineering, has already been used for the fermentation of HMOs (neutral HMOs as well as sialylated HMOs). However, other host strains, such as yeasts (such as Saccharomyces cerevisiae), lactic acid bacteria, Corynebacterium glutamicum, Bacillus species, which have GRAS (generally recognized as safe) status, can be equally well modified for production of oligosaccharide HMOs in general, as well as sialylated HMOs in particular.
    [0026] [0026] For the production of a desired sialylated oligosaccharide, the bacterial or yeast host strain typically contains one or more heterologous glycosyltransferases, typically at least one heterologous sialyltransferase, overexpressing genes for sialic acid-CMP synthesis (such as a synthetase sialic acid-CMP plus genes involved in the absorption or de-synthesis of sialic acid), a lactose importer and / or an exporter suitable for the desired sHMO. The expression of a suitable exporter is particularly advantageous when using yeast as production hosts for sHMOs, since it is common knowledge that S. cerevisiae does not secrete the heterologously produced oligosaccharide in economically viable quantities in the fermentation broth without having a suitable exporter .
    [0027] [0027] The term "desired" related to the sialylated oligosaccharide refers to the sialylated oligosaccharide that must be produced by the cell. The desired sialylated oligosaccharide is the oligosaccharide to be purified by the processes disclosed in the present invention. The term "desired" related to the sialylated oligosaccharide, as used in the present invention, also serves to distinguish between the sialylated oligosaccharide to be produced and another sialylated oligosaccharide that can be produced unintentionally by cells.
    [0028] [0028] The term "oligosaccharide", as used in the present invention, refers to linear or branched saccharides that consist of three to 20 monosaccharide residues.
    [0029] [0029] In one embodiment, the sialylated oligosaccharide is a sialylated HMO. The term "sialylated HMO", as used in the present invention, refers to human milk oligosaccharides that comprise one or more sialic acid residues.
    [0030] [0030] The method comprises the step of separating the biomass from the fermentation broth. This step is the first step in the purification process of sialylated oligosaccharides.
    [0031] [0031] The term "biomass", as used in the present invention, refers to the totality of cells present in the fermentation broth at the end of the fermentation stage. The cells that are present in the fermentation broth at the end of the fermentation step comprise the cells that have the ability to produce the desired sialylated oligosaccharide, optionally, auxiliary cells that are present in the fermentation broth to assist in the production of the sialylated oligosaccharide such as - for example - cells that degrade unwanted by-products. Therefore, the cells present in the fermentation broth at the end of the fermentation step are separated from the fermentation broth so that the resulting fermentation broth is substantially cell-free.
    [0032] [0032] Suitable methods for separating the biomass from the fermentation broth include centrifugation in which the biomass is obtained as a pellet and the fermentation broth as a supernatant. In an additional and / or alternative mode, the biomass is separated from the fermentation broth by means of filtration. Suitable filtration methods for separating cells from the fermentation broth include microfiltration and ultrafiltration.
    [0033] [0033] Microfiltration as such is a physical filtration process in which a particle-containing fluid passes through a special pore size membrane to separate particles from the fluid. The term "microfiltration", as used in the present invention, refers to a physical filtration process in which cells are separated from the fermentation broth.
    [0034] [0034] Ultrafiltration is a variety of membrane filtration and is 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 solutes are retained in the so-called retentate, while water and low molecular weight solutes, such as the desired sialylated oligosaccharide, pass through the membrane in the permeate (filtered).
    [0035] [0035] Ultrafiltration membranes are defined by the molar cutoff point (MWCO) of the membrane used. Ultrafiltration is applied in cross-flow or dead-end mode.
    [0036] [0036] The filters suitable for microfiltration or ultrafiltration are SPIRA-CEL® DS MP005 4333 and fiber FS10-FC FUS1582 (Microdyn-Nadir GmbH, Wiesbaden, DE).
    [0037] [0037] Typically, cells synthesize the desired sialylated oligosaccharide intracellularly and secrete it in the fermentation broth. The sialylated oligosaccharide produced in this way ends in the fermentation broth which is then subjected to additional process steps to purify the desired sialylated oligosaccharide, as described later in the present invention.
    [0038] [0038] In modalities, when the desired sialylated oligosaccharide remains intracellularly after its biosynthesis, the biomass is separated from the fermentation broth, and said biomass is used to purify the desired sialylated oligosaccharide. For this purpose, the biomass cells are lysed and the resulting lysate is cleared in which insoluble constituents, nucleic acids, lipids and proteins are removed from the lysate. Methods for lysing cells and for removing insoluble constituents, nucleic acids, lipids and / or proteins from a cell lysate are known. The clean lysate thus obtained that contains the desired sialylated oligosaccharide is then subjected to the same process steps as the cell-free fermentation broth containing the desired sialylated oligosaccharide in order to purify the desired sialylated oligosaccharide.
    [0039] [0039] Although the process to purify sialylated oligosaccharides is used to purify sialylated oligosaccharides that were produced by means of microbial fermentation, the said process can also be employed to purify sialylated oligosaccharides that were produced by an in-vitro enzymatic reaction, called reaction in-vitro biocatalysis. The desired sialylated oligosaccharide is obtained by means of one or more enzymatic reactions in-vitro, and can be purified from the reaction mixture at the end of the biocatalytic reaction to which the said reaction mixture is subjected - instead of the free fermentation broth cell or clean lysate - to the purification process described in the present invention. It is understood that the purification of sialylated oligosaccharides from the in-vitro biocatalysis reaction mixture does not require the removal of biomass from the reaction mixture.
    [0040] [0040] The cell-free fermentation broth, clean lysate or reaction mixture contains the desired sialylated oligosaccharide as well as a substantial amount of unwanted impurities and constituents including other oligosaccharides in addition to the desired sialylated oligosaccharide, monovalent salts, divalent salts, amino acids, polypeptides , proteins, organic acids, nucleic acids and monosaccharides.
    [0041] [0041] The process for purifying sialylated oligosaccharides comprises the step of a cation exchange chromatography to remove positively charged compounds from the cell-free fermentation broth, the clean lysate or the reaction mixture.
    [0042] [0042] Suitable cation exchange resins for removing positively charged compounds include Lewatit S2568 (H +) (Lanxess AG, Cologne, DE).
    [0043] [0043] The process for purifying sialylated oligosaccharides comprises the step of an anion exchange chromatography to remove unwanted, negatively charged compounds from the cell-free fermentation broth, clean lysate or reaction mixture.
    [0044] [0044] Suitable anion exchange resins include Lewatit S6368 A, Lewatit S4268, Lewatit S5528, Lewatit S6368A (Lanxess AG. Cologne, DE), Dowex
    [0045] [0045] The process for purifying sialylated oligosaccharide comprises the step of removing compounds that have a lower molecular weight than that of the sialylated oligosaccharide to be purified. Suitable methods for removing compounds that have a molecular weight less than that of the sialylated oligosaccharide to be purified include nanofiltration and diafiltration.
    [0046] [0046] Diafiltration involves adding fresh water to a solution in order to remove (by washing) permeable membrane components. Diafiltration can be used to separate components based on their molecular size and charge using appropriate membranes, where one or more species are efficiently retained and other species are membrane permeable. In particular, diafiltration using a nanofiltration membrane is effective for separating low molecular weight compounds from salts. Nanofiltration membranes usually have a molar cutoff in the range of 150 to 300 Daltons. Nanofiltration is widely used in the dairy industry for the concentration and demineralization of whey.
    [0047] [0047] Suitable membranes for nanofiltration and / or diafiltration include Dow Filmtec NF270-4040, Trisep 4040-XN45-TSF (Microdyn-Nadir GmbH, Wiesbaden, DE), GE4040F30 and GH4040F50 (GE Water & Process Technologies, Ratingen, DE) .
    [0048] [0048] Diafiltration using nanofiltration membranes has been found to be efficient as a pre-treatment to remove significant amounts of contaminants before electrodialysis treatment of the solution containing the HMO. However, nanofiltration has been found to be efficient for the removal of low molecular weight contaminants after an ultrafiltration step, in which said removal is beneficial for concentrating and demineralizing the HMO solution before treatment with an ion exchanger. The use of nanofiltration membranes for concentration and diafiltration during HMO purification results in less energy and processing costs, and better product quality due to reduced thermal exposure, which leads to Maillard reactions and reduced aldolic reactions.
    [0049] [0049] The purification process provides the desired sialylated oligosaccharide in a preparation in which the purity of said desired sialylated oligosaccharide is ≥ 80%, ≥85%, ≥90%, ≥95%. The process provides a preparation of the sialylated oligosaccharide in which the purity of the sialylated oligosaccharide is suitable for food and feed applications.
    [0050] [0050] In addition, the process is economically profitable and easy to be produced on a large scale, making it suitable as a basis for a multi-ton scale manufacturing process.
    [0051] [0051] The process for purifying a sialylated oligosaccharide is also advantageous in that the desired sialylated oligosaccharides are obtained free of recombinant DNA and recombinant proteins derived from recombinant microbial fermentation strains that can be used as processing aids.
    [0052] [0052] In an additional and / or alternative modality, the process additionally comprises a nanofiltration step to increase the concentration of saccharides in the solution.
    [0053] [0053] In an additional and / or alternative modality, the process comprises an electrodialysis stage.
    [0054] [0054] Electrodialysis combines dialysis and electrolysis, and can be used to separate ion concentration in solutions based on their selective electromigration through a semipermeable membrane.
    [0055] [0055] Electrodialysis (ED) combines dialysis and electrolysis and can be used for the separation or concentration of ions in solutions based on their selective electromigration through semipermeable membranes. Industrial electrodialysis applications date back to the early 1960s, when this method was used for the demineralization of cheese whey for inclusion in infant formula. Additional electrodialysis applications include adjusting the pH of beverages such as wine, must, apple juice and orange juice.
    [0056] [0056] The desalination of brackish water for the production of drinking water and the demineralization of whey for the production of infant food are the most widespread applications of electrodialysis today (Tanaka, Y. (2010) Ion Exchange Membrane Electrodialysis. Nova Science Publishers, Inc. New York).
    [0057] [0057] The basic principle of electrodialysis consists of an electrolytic cell that comprises a pair of electrodes submerged in an electrolyte for conducting ions, connected to a direct current generator. The electrode connected to the positive pole of the direct current generator is the anode, and the electrode connected to the negative pole is the cathode. The electrolyte solution then supports the current flow, which results from the movement of negative and positive ions towards the anode and cathode, respectively. The membranes used for electrodialysis are essentially sheets of porous ion exchange resins with negative or positive charge groups and are therefore described as cationic or anionic membranes, respectively. Ion exchange membranes are usually produced from polystyrene that carries a suitable functional group (such as sulfonic acid for cationic membranes or a quaternary ammonium group for anionic membranes) cross-linked with divinylbenzene. The electrolyte can be, for example, sodium chloride, sodium acetate, sodium propionate or sulfamic acid. The electrodialysis stack is then mounted in such a way that the anionic and cationic membranes are parallel as in a filter press between two electrode blocks, so that the flow subjected to ion depletion is well separated from the flow subjected to enrichment. ion (the two solutions are also called the diluted (subjected to ion depletion) and concentrated (subjected to ion enrichment). The core of the electrodialysis process is the membrane stack, which consists of several anion exchange membranes and membranes of cationic exchange separated by spacers, installed between two electrodes.Applying a direct electric current, anions and cations will migrate through the membranes towards the electrodes generating a diluted (desalinated) and a concentrated flow.
    [0058] [0058] Applying an acidic condition during electrodialysis, sHMOs can be protonated so that they appear uncharged (protonation of the carbonyl group of the sialic acid part of the oligosaccharide). As an alternative, electrodialysis can be performed under neutral conditions with the use of bipolar membranes. In this case, the sialylated oligosaccharides can even be concentrated in a separate electrodialysis concentrate circuit. In this way, the sialylated oligosaccharide can even be enriched during electrodialysis.
    [0059] [0059] The pore size of the ion exchange membranes is small enough to avoid diffusion of the product from the diluted flow to the concentrated flow, motivated by large differences in concentration between the two flows. After the separation of biomass, proteins and, in particular, recombinant DNA molecules (in size in the range of fragments to whole genomes) need to be quantitatively removed from the desired product. If possible in any way, the electrodialysis of such large molecules (when compared to the molecular size of HMOs) will take a long time, certainly accompanied by significant losses of the desired product from the diluted to the concentrate.
    [0060] [0060] In an additional and / or alternative modality, the process for purifying sialylated oligosaccharides additionally comprises a step of simulated moving bed chromatography (SMB).
    [0061] [0061] Simulated moving bed chromatography (SMB) originated in the petrochemical and mineral industries. Currently, SMB chromatography is used by the pharmaceutical industry to isolate enantiomers from racemic mixtures. Large-scale SMB chromatography has already been used for the separation of fructose monosaccharide from fructose-glucose solutions and for the separation of sucrose disaccharide from sugar beet or sugar cane syrups. However, SMB chromatography has not yet been used for the purification of sialylated oligosaccharides from human milk, such as sialylated lacto-N-tetraose, 3'-sialyl-lactose or 6'-sialyl-lactose during chemical, enzymatic or based synthesis fermentation. SMB was used to purify sialyl-lactoses from bovine milk, but bovine milk is a completely different matrix when compared to microbial fermentation broth, which is used in the present invention as a source of HMOs.
    [0062] [0062] SMB chromatography is used as a continuous separation process analogous to continuous chemical separation processes, such as rectification. In rectification, a countercurrent is established between the liquid and gaseous phases that allows the continuous application of feed and the continuous removal of product. Countercurrent chromatography should, in theory, achieve superior separation when compared to conventional cross current systems, but the mobile and stationary phases in countercurrent systems will need to move in opposite directions. SMB chromatography was developed as a solution to the practical difficulties encountered when trying to move a solid chromatography material in a continuous chromatographic separation process.
    [0063] [0063] The classic SMB concept involves four different zones with four external flows: the feed flow containing the components to be separated, the desorption or mobile phase flow, the extract, and the raffinate flow (the latter being the last represents the components retained less efficiently). These liquid flows divide the SMB system into four different zones (each zone or section can comprise one or more columns) with the following functions: zone I is necessary for the regeneration of the solid phase, the purpose of zone II is desorption of the less strongly adsorbed material, the function of zone III is the adsorption of the strongly adsorbed material, and finally, the function of zone IV is the adsorption of the less adsorbed material (Figure 5). In this way, stronger adsorbent components establish a concentration wave in zone II and are transported to the extract port while less strong adsorbent components migrate to the raffinate port.
    [0064] [0064] In principle, zones I and IV serve for the regeneration of the solid phase (regeneration zones) while zones II and III can be considered as the real separation zones of the system (separation zones). In addition to the resulting four liquid flows and zones, the system contains (for closed loop operation) a recycling pump for the mobile phase (desorbent), which passes the mobile phase through the fixed zones in one direction. The countercurrent flow is then achieved by periodic switching and continuous supply or removal of power, desorbed, and products sequentially from one column to the next in the system.
    [0065] [0065] In addition to the classic four-zone closed circuit SMB 4 system, three-zone open circuit systems are available, and can be more economically cost-effective if fresh solvent is not expensive, for example, when water or mixtures of water / ethanol are used as the mobile phase. In the three-zone circuit configuration, regeneration of the liquid phase is no longer necessary, making zone IV obsolete.
    [0066] [0066] In addition to the classic SMB systems for separating mixtures of two components, closed loop systems of eight zones (Figure 6) and open circuit systems of five zones have been developed for the separation of more than 2 components.
    [0067] [0067] Given the continuous mode of operation, the recycling of the mobile phase and also the potential for using large column sizes, SMB systems can, in principle, be increased to reach production volumes of hundreds of tons.
    [0068] [0068] The simulated moving bed chromatography process step is advantageous in that this process step allows for additional removal of oligosaccharides that are structurally closely related to the desired sialylated oligosaccharide.
    [0069] [0069] In an additional and / or alternative modality, the process additionally comprises a step of removing dyes.
    [0070] [0070] Suitable process steps for removing dyes include treating cell-free fermentation broth or clean lysate with activated carbon, such as activated carbon.
    [0071] [0071] The broth treatment with activated carbon removes any unwanted dyes and provides a preparation of the desired oligosaccharide that has an acceptable appearance.
    [0072] [0072] In an additional and / or alternative embodiment, the sialylated oligosaccharide purification process comprises at least one step of increasing the concentration of the desired sialylated oligosaccharide.
    [0073] [0073] In additional and / or alternative process, the solution containing the desired sialylated oligosaccharide is concentrated after at least one of the purification steps i) to iv), preferably after the purification step iv), with the use of vacuum evaporation (for example, using a descending film evaporator or a plate evaporator) or reverse osmosis or nanofiltration (for example, nanofiltration with a nanofiltration membrane that has a size exclusion limit of ≤ 20 Å (2 nm) a) at a concentration of ≥ 100 g / L, preferably ≥ 200 g / L, more preferably ≥ 300 g / L; and / or b) at a temperature of <80 ° C, preferably <50 ° C, more preferably, 20 ° C to 50 ° C, even more preferably, 30 ° C to 45 ° C, with maximum preference, 35 ° C at 45 ° C (specifically relevant for vacuum evaporation or reverse osmosis); and / or c) at a temperature of <80 ° C, preferably <50 ° C, more preferably 4 ° C to 40 ° C (specifically relevant for nanofiltration).
    [0074] [0074] Suitable methods for increasing the concentration of the desired sialylated oligosaccharide include nanofiltration and solvent evaporation.
    [0075] [0075] In an additional and / or alternative method of the process, according to the invention, the purified solution is sterile filtered and / or subjected to the removal of endotoxin, preferably through filtration of the purified solution through a filter 3 kDa or 6 kDa filter.
    [0076] [0076] The process to purify the desired oligosaccharide from a fermentation broth, cell lysate or reaction mixture from a biocatalytic reaction provides an aqueous solution of the desired oligosaccharide. In a further and / or alternative embodiment, the process additionally comprises a step of removing the solvent from the sialylated oligosaccharide so that either a solution of the desired oligosaccharide is provided that includes a high concentration of the desired oligosaccharide, or so that a preparation solid of the desired oligosaccharide is obtained.
    [0077] [0077] Suitable methods of removing solvent from the liquid preparation of the sialylated oligosaccharide to obtain a solid preparation of the desired oligosaccharide include crystallization and lyophilization (freeze drying - a process in which the aqueous solution containing sHMO is frozen and then by reducing the surrounding pressure, the frozen water in the sublime material is then allowed directly from the solid to the gas phase - this usually leads to a hygroscopic sHMO powder.
    [0078] [0078] In an alternative embodiment, the solvent can be removed from the liquid preparation of the sialylated oligosaccharide by means of spray drying. The inventors have surprisingly found that a liquid preparation containing the desired sialylated oligosaccharide can be spray-dried to obtain a powder that essentially consists of the desired sialylated oligosaccharide, although it is generally known that carbohydrates are not typically drying. sprinkling, this also includes lactose, sialic acid, fucose, etc.
    [0079] [0079] The separation of biomass from the fermentation broth is typically the first stage of the purification process. Since the process comprises the step of removing the solvent from the preparation, that step is typically the final step of purifying the desired oligosaccharide. The order of the additional process steps is not particularly limited.
    [0080] [0080] Referring to Figure 2, a modality of the process for purifying a sialylated oligosaccharide is shown schematically, in which the sialylated oligosaccharide is an sHMO produced by means of microbial fermentation. At the end of fermentation, the biomass is separated from the fermentation broth by means of cross-flow filtration. The filtrate (cell-free fermentation broth) is subjected to cation exchange chromatography and anion exchange chromatography. Subsequently, the eluate is concentrated and treated with activated carbon. The resulting solution is subjected to SMB chromatography and the concentration of the sialylated oligosaccharide in the resulting solution is increased. Finally, filtration with a 3 kDa cut is followed by sterile filtration to obtain a solution that contains a high concentration of the desired sHMO.
    [0081] [0081] Figure 3 illustrates another embodiment of the process, which differs from the embodiment shown in Figure 2 in that the solution containing a high concentration of the desired sHMO is spray-dried to obtain the desired powdered sHMO. Unexpectedly, it was possible to identify conditions in which 3'-sialyl-lactose and 6'-sialyl-lactose could be spray dried. In contrast to lactose or sialic acid, which cannot be spray-dried, conditions were found that allowed these sHMOs to be obtained as a spray-dried powder. The spray-dried powder also appeared to be just a little hygroscopic. The use of spray drying has several advantages over other processes, such as crystallization or freeze drying, as it is highly economical, compatible with large-scale manufacturing of sHMOs, it avoids organic solvents as in the case of crystallization.
    [0082] [0082] The method of the process illustrated schematically in Figure 4 differs from the method shown in Figure 3 in that the process does not comprise SMB chromatography to obtain a high purity sHMO preparation.
    [0083] [0083] According to the second aspect, sialylated oligosaccharide preparations are provided, in which the sialylated oligosaccharides were produced by means of microbial fermentation or in-vitro biocatalysis.
    [0084] [0084] The sialylated oligosaccharide of the preparation was purified from the fermentation broth, cells or reaction mixture by the process described in the present invention.
    [0085] [0085] In an additional and / or alternative modality, the sialylated oligosaccharide is a sialylated HMO. In an additional and / or alternative modality, the sialylated HMO is selected from the group consisting of 3'-SL, 6'-SL, LST- a, LST-b, LST-c, F-SL, F-LST -b and DS-LNT.
    [0086] [0086] The preparation of the sialylated oligosaccharide can be present in liquid form as a solution of the sialylated oligosaccharide in a solvent, preferably in water. In an alternative embodiment, the preparation of the sialylated oligosaccharide is present in solid form, preferably in the form of a powder. The powder comprises the sialylated oligosaccharide in the form of particles in which the sialylated oligosaccharide is present in the form of amorphous particles or in the form of crystalline particles. More preferably, the sialylated oligosaccharide is obtained as a spray-dried powder with a water content of less than 10%. Name Structure 3'-sialyl-lactose Neu5Ac (α2-3) Gal (βb1-4) Glc 6'-sialyl-lactose Neu5Ac (α2-6) Gal (β1-4) Glc F-SL Neu5Ac (α2-3) Gal (β1-4) Glc | Fuc (α1-3) LSTa Neu5Ac (α2-3) Gal (β1-3) GlcNAc (β1-3) Gal (β1-4) Glc LSTb Neu5Ac (α2-6) | Gal (β1-3) GlcNAc (β1-3) Gal (β1-4) Glc LSTc Neu5Ac (α2-6) Gal (β1-4) GlcNAc (β1-3) Gal (β1-4) Glc DS-LNT Neu5Ac ( α2-6) | Neu5Ac (α2-3) Gal (β1-3) GlcNAc (β1-3) Gal (β1-4) Glc F-LSTb Neu5Ac (α2-6) | Fuc (α1-2) Gal (β1-3) GlcNAc (β1-3) Gal (β1-4) Glc
    [0087] [0087] The preparation of sialylated oligosaccharides obtainable by a process, as described in the present invention, comprises the desired sialylated oligosaccharide in a purity of ≥ 80% by weight.
    [0088] [0088] According to the third aspect, the use of a sialylated oligosaccharide, as previously described in the present invention, especially of a sialylated HMO, is provided for the preparation of a nutritional composition, preferably of an infant formula.
    [0089] [0089] The purification process of sialylated oligosaccharides provides preparations of sialylated oligosaccharides in which the oligosaccharide is present in a purity sufficient for human consumption.
    [0090] [0090] Said nutritional composition contains at least one sialylated oligosaccharide which was produced by a method, as previously disclosed in the present invention.
    [0091] Thus, according to the fourth aspect, nutritional compositions are provided which contain at least one sialylated oligosaccharide, preferably at least one sHMO, which was produced by means of a method, as previously disclosed in the present invention. The at least one sHMO in the nutritional composition is selected from the group consisting of 3'-SL, 6'-SL, LST-a, LST-b, LST-c, F-SL, DS-LNT and F-LSTb . Said at least one sialylated oligosaccharide was produced by means of microbial fermentation or in-vitro biocatalysis.
    [0092] [0092] In an additional modality, the nutritional composition is selected from the group consisting of medicinal formulations, infant formula, milk drinks and dietary supplements. The nutritional composition additionally comprises micro and / or macronutrients, such as,
    [0093] [0093] As a medicinal formulation, the nutritional composition can be used to improve the symptoms of diabetes, as siallylactoses improve insulin secretion and thereby increase blood glucose level to prevent or relieve diabetes mellitus. In addition, a nutritional composition that contains 3'-SL can be effective against osteoarthritis.
    [0094] [0094] As an infant formula, the nutritional composition meets the compositional requirements established in Regulation (EU) 2016/127. Exemplary infant formula compositions are specified in Table 2 and Table 3. Infant formula: Skimmed milk Vegetable oils (palm oil, rapeseed oil, sunflower oil) Human milk oligosaccharides Skimmed milk powder Mortierella alpine oil Fish oil Calcium carbonate Potassium chloride Vitamin C Sodium chloride Vitamin E Iron acetate Zinc sulphate Niacin Calcium D-pantothenate
    [0095] [0095] In an additional and / or alternative modality, the nutritional composition additionally comprises microorganisms, preferably probiotic microorganisms. In the case where used for infant food applications, preferred microorganisms are derived from or can be found in a healthy human microbiome. Preferably, but without limitation, the microorganisms are selected from the genus Bifidobacterium, Lactobacillus, Enterococcus, Streptococcus, Staphylococcus, Peptostreptococcus, Leuconostoc, Clostridium, Eubacterium, Veilonella, Fusobacterium, Bacterioidesichia, Propacterla, Esoteribcherichia, Propacterla, Esteribla, In an additional and / or alternative modality, the microorganism is selected from the group consisting of Bifidobacterium breve, Bifidobacterium longum, Bifidobacterium lactis, Bifidobacterium animalis, Bifidobacterium bifidum, Bifidobacterium infantil, Bifidobacterium aldolescentis, Lactobacillus, lactobacillus, bacterium Lactobacillus casei, Lactobacillus gasseri, Lactobacillus reuteri, Lactobacillus rhamnosus, Lactobacillus plantarum, Lactobacillus salivarius, Lactococcus lactis, Lactobacillus paracasei, Lactobacillus bulgaricusus, helactobacillus, Lactobacillus; Escherichia coli, Enterococcus faecium and Streptococcus thermophilus (VSL # 3).
    [0096] [0096] In addition to combining sHMOs with living organisms, these oligosaccharides can also be used in combination with dead cultures. In the field of probiotics, exterminated cultures are sometimes used (for example, industrialized bacteria). It is added that these exterminated cultures provide proteins, peptides, oligosaccharides, fragments of the outer cell wall, natural products, which can lead to a short-term stimulation of the immune system.
    [0097] [0097] The combination of at least one sialylated oligosaccharide, in particular, at least one sHMO, and probiotic microorganisms in the nutritional composition is particularly advantageous in that it establishes or restores an appropriate microbiome in the intestine, and the health benefits associated with it are facilitated.
    [0098] [0098] Even more advantageous is the combination of at least one sialylated oligosaccharide, in particular, a sHMO, with established prebiotics, such as GOS (Galacto-oligosaccharides) and / or FOS (Fructo-oligosaccharides, Inulin).
    [0099] [0099] The nutritional composition can be present in liquid or solid form including, but not limited to, powders, granules, flakes and pellets.
    [00100] Thus, a nutritional composition is also provided which comprises at least 5 HMOs, in which said at least 5 HMOs are selected from the group consisting of 2'-fucosyl-lactose, 3-fucosillactose, Lacto-N -tetraose, Lacto-N-neotetraose, Lacto-N-fucopentaose I, 3'-sialyl-lactose and 6'-sialyl-lactose.
    [00101] [00101] In an additional and / or alternative embodiment, a nutritional composition, as previously disclosed in the present invention, contains at least one sialylated HMO and at least one neutral HMO and a probiotic microorganism.
    [00102] [00102] According to the fifth aspect, a spray dried GMO free powder is provided which essentially consists of a sialylated oligosaccharide with a purity of> 80% dry weight and has less than 10% water by weight .
    [00103] [00103] In one embodiment, the spray-dried powder is a powder that essentially consists of a sialylated oligosaccharide selected from the group consisting of 3'-sialyl-lactose, 6'-sialyl-lactose, and a mixture of 3 ' -sialyl-lactose and 6'-sialyl-lactose.
    [00104] [00104] In an additional and / or alternative embodiment, the spray dried powder essentially consists of a mixture of 3'-sialyl-lactose, 6'-sialyl-lactose and one or more neutral HMOs, in which said one or more Neutral HMOs are selected from the group consisting of 2'-fucosyl lactose, 3-fucosillactose, lacto-N-tetraose, lacto-N-neotetraose, lacto-N-fucopentaose I.
    [00105] [00105] The present invention will be described in relation to the particular modalities and with reference to the drawings, but the invention is not limited to them, but only by the claims. In addition, the terms first, second and similar, in the description and in the claims, are used to distinguish between similar elements and not necessarily to describe a sequence, either temporally, spatially, in order or in any other way. It is to be understood that the terms used in this way are interchangeable under appropriate circumstances and that the modalities of the invention described in the present invention have the ability to operate in sequences other than those described or illustrated in the present invention.
    [00106] [00106] It should be noted that the term "comprising", used in the claims, should not be interpreted as being restricted to the means listed later; it does not exclude other elements or steps. It should therefore be interpreted as specifying the presence of the resources, whole numbers, steps or components established as mentioned, but does not exclude the presence or addition of one or more other resources, whole numbers, steps or components, or groups of themselves. Therefore, the scope of the expression "a device comprising means A and B" should not be limited to devices consisting only of components A and B. It means, in relation to the present invention, that the only relevant components of the device are A and B.
    [00107] [00107] The reference throughout this specification to "a modality" or "a modality" means that a particular resource, structure or characteristic, described in conjunction with the modality, is included in at least one embodiment of the present invention. In this way, occurrences of the expressions "in a modality" or "in a modality", in various places throughout this specification, are not necessarily referring to the same modality, but they may be. In addition, particular resources, structures or characteristics can be combined in any appropriate way, as will be apparent to a person skilled in the art from this disclosure, in one or more modalities.
    [00108] [00108] Similarly, it should be noted that, in the description of exemplary modalities of the invention, several features of the invention are sometimes grouped together in a single modality, figure or description of the same in order to simplify the disclosure and assist in understanding one or more of the various aspects of the invention. This method of disclosure, however, should not be interpreted as reflecting an intention that the claimed invention requires more resources than are expressly cited in each claim. Instead, as the following claims reflect, aspects of the invention are found in less than the totality of the resources of a single embodiment previously revealed. Accordingly, claims that follow the detailed description are expressly incorporated into this detailed description, each claim being self-sufficient as a separate embodiment of this invention.
    [00109] [00109] Furthermore, although some modalities described in the present invention include some, but not other resources included in other modalities, combinations of resources of different modalities are intended to be within the scope of the invention, and form different modalities, as will be understood by technicians on the subject. For example, in the following claims, any of the claimed modalities can be used in any combination.
    [00110] [00110] In addition, some of the modalities are described in the present invention as a method or combination of elements of a method that can be implanted by a processor of a computer system or by other means of performing the function. Thus, a processor with the instructions necessary to execute such a method or element of a method forms a means to execute the method or element of a method. In addition, an element described in the present invention of an apparatus embodiment is an example of a means for performing the function performed by the element for the purpose of carrying out the invention.
    [00111] [00111] In the description and drawings provided in the present invention, numerous specific details are established. However, it is understood that the modalities of the invention can be practiced without these specific details. In other cases, well-known methods, structures and techniques have not been shown in detail in order not to obscure an understanding of this description.
    [00112] [00112] The invention will be described in the following by means of a detailed description of the various modalities of the invention. It is clear 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 teaching of the invention, the invention is limited only by the terms of the appended claims. Example 1: Fermentation of 3'-sialyl-lactose with the use of a recombinant microorganism.
    [00113] [00113] A batch fermentation fed with 3'-sialyl-lactose that employs a strain of E. coli that synthesizes recombinant 3'-sialyl-lactose (E. coli BL21 (DE3) ∆lacZ), which contains a genomic integration of a 3'-sialyltransferase gene from Vibrio sp. JT-FAJ-16. To enhance the biosynthesis of sialic acid-CMP genes that encode E. coli's GlucS-6-phosphate synthase, N-acetylglucosamin2-epimerase Slr1975 from Synechocystis sp., Glucosamine 6-phosphate N-acetyltransferase Gna1 from Saccharomyces cerev , E. coli phosphoenolpyruvate synthase, NeuB N-acetylneuraminate synthase and sialic acid-CMP NeuA synthase, the latter both of Campylobacter jejuni, were chromosomally integrated to the E. coli BL21 (DE3) host. In addition, the gene encoding the lactose permease LacY from E. coli, and the genes cscB (sucrose permease), cscK (fructose kinase), cscA (sucrose hydrolase) and cscR (transcriptional regulator) from E. coli W have been integrated to the BL21 genome. Transcription of integrated genes is initiated from constitutive promoters, either the tetracycline Ptet promoter or the PT5 promoter. A functional gal-operon, consisting of the galE (UDP-glucose-4-epimerase), galT (galactose-1-phosphate uridylyltransferase), galK (galactokinase) and galM (galactose-1-epimerase) genes was transferred from E. coli K12 for the genome of the BL21 strain. To avoid the degradation of N-acetylglucosamine 6-phosphate genes that code for N-acetylglucosamine-6-phosphate deacetylase (NagA), glucosamine-6-phosphate deaminase (NagB) and the specific PTS protein of N-acetylglucosamine IIABC ( NagE) were deleted from the chromosome. Additionally, the manXYZ operon, which encodes a sugar transporter from the E. coli PTS system for mannose, glucose, glucosamine and N-acetylglucosamine was deleted, as well as the nanA, nanK, nanE and nanT genes, which encode N-acetylneuraminate lyase, N-acetylmannosamine kinase, N-acetylmannosamine-6-phosphate epimerase and the sialic acid transporter, respectively. The gene encoding N-acetylgalactosamine-6-phosphate deacetylase (AgaA) has also been deleted.
    [00114] [00114] The 3'-sialyl-lactose production strain was grown in a defined mineral salt medium, comprising 7 g L-1 of NH4H2PO4, 7 g L-1 of K2HPO4, 2 g L-1 of KOH, 0.3g L-1 citric acid, 5 g L-1 NH4CI, 1 ml L-1 antifoam (Struktol J673, Schill + Seilacher), 0.1 mM CaCI2, 8 mM MgSO4, trace elements and 2% sucrose as a carbon source.
    [00115] [00115] The trace elements consisted of 0.101 g L-1 of nitrilotriacetic acid, pH 6.5, 0.056 g L-1 of iron and ammonium citrate, 0.01 g L-1 of MnCI2 x 4 H2O, 0.002 g L-1 of CoCI2 x 6 H2O, 0.001 g L-1 of CuCI2 x 2 H2O, 0.002 g L-1 of boric acid, 0.009 g L-1 of ZnSO4 x 7 H2O, 0.001 g L 1 of Na2MoO4 x 2 H2O, 0.002 g L-1 of Na2SeO3, 0.002 g L-1 of NiSO4 x 6 H2O.
    [00116] [00116] The sucrose feed (500 g L-1) was supplemented with 8 mM MgSO, 0.1 mM CaCl2, trace elements and 5 g L-1 of NH4CI. For the formation of 3'-sialyl-lactose, a lactose feed of 216 g L-1 was employed. The pH was controlled using ammonia solution (25% v / v). The fed batch fermentation was conducted at 30 ° C under constant aeration and stirring for 72 hours by applying a sucrose feed rate of 5.5 to 7 ml L-1 h-1, referring to the initial volume. 72 hours after the start of fermentation, most of the added lactose was converted to 3'-sialyl-lactose. In order to remove residual lactose in the fermentation supernatant, β-galactosidase was added to the fermentation vessel. The resulting monosaccharides were metabolized by the production strain. Example 2: Fermentation of 6'-sialyl-lactose an sHMO with the use of a recombinant microorganism.
    [00117] [00117] The strain that synthesizes 6'-sialyl-lactose comprises the same genetic resources as the strain producing 3'-sialyl-lactose, despite the sialyltransferase. For the production of 6'-sialylactose, the photobacterium leiognathi plsT6 gene JT-SHIZ-119 that encodes an alpha-2,6-sialyltransferase was integrated into the E. coli BL21 (DE3) genome.
    [00118] [00118] For the fermentative production of 6'-sialyl-lactose, the strain was grown in a defined mineral salt medium, comprising 7 g L-1 of NH4H2PO4, 7 g L-1 of K2HPO4, 2 g L-1 KOH, 0.3g L-1 citric acid, 5 g L-1 NH4CI, 1 ml L-1 antifoam (Struktol J673, Schill + Seilacher), 0.1 mM CaCI2, 8 mM MgSO4, trace (0.101 g L-1 of nitrilotriacetic acid, pH 6.5, 0.056 g L-1 of iron and ammonium citrate, 0.01 g of MnCI2 x 4 H2O, 0.002 g L-1 of CoCI2 x 6 H2O, 0.001 g L-1 of CuCl2 x 2 H2O, 0.002 g L-1 of boric acid, 0.009 g L-1 of ZnSO4 x 7 H2O, 0.001 g L-1 of Na2MoO4 x 2 H2O, 0.002 g L-1 of Na2SeO3, 0.002 g NiSO4 L-1 x 6 H2O) and 2% sucrose as a carbon source.
    [00119] [00119] The sucrose feed (500 g L-1) was supplemented with 8 mM MgSO, 0.1 mM CaCl2, trace elements, and 5 g L-1 of NH4CI. For the formation of 6'-sialyl-lactose, a lactose feed of 216 g L-1 was employed. The pH was controlled using ammonia solution (25% v / v). The fed batch fermentation was conducted at 30 ° C under constant aeration and stirring for 72 hours by applying a sucrose feed rate of 5.5 to 7 ml L-1 h-1, referring to the initial volume. Lactose that was not converted to 6'-sialyl-lactose at the end of the production process was degraded by the addition of β-galactosidase and monosaccharides from lactose hydrolysis were metabolized by the production strain. Example 3: Purification of 6'-sialyl-lactose and 3'-sialyl-lactose from fermentation broth
    [00120] [00120] The biomass was separated from the fermentation medium by means of ultrafiltration followed by the sequential use of a winding module filter (0.05 μm cut) (CUT membrane technology, Erkrath, Germany), and a cross flow filter (150 kDa of cut) (Microdyn-Nadir, Wiesbaden, Germany). A cell-free fermentation medium of approximately 1 m3 was obtained which contains more than 20 g L-1 of sialylated oligosaccharides.
    [00121] [00121] The cell-free liquid was then deionized by means of ion exchange chromatography. First, cationic contaminants were removed in a strong cationic exchanger in a volume of 200 l (Lewatit S 2568 (Lanxess, Cologne, Germany) in the form of H +. Using NaOH, the pH of the solution obtained was set to 7.0 In a second step, anionic ions and unwanted dyes were removed from the solution using the Lewatit S 6368 S strong anion exchanger (Lanxess, Cologne, Germany) in the form of chloride.The ion exchanger had a bed volume of 200 L With the use of a second filtration step in the cross flow filter (150 kDa cut) (Microdyn-Nadir, Wiesbaden, Germany), precipitates originating from acidification of the solution were removed. For the sugar concentration, the solution was nanofiltered on a Dow Filmtec NF270-4040 (Inaqua, Monchengladbach, Germany) or alternatively on a Trisep 4040-XN45-TSF membrane (0.5 kDa cut) (Microdyn-Nadir, Wiesbaden, Germany). of the latter, the monosaccharide N-acetylglucosamine, originating from the fermentation and which contaminates the sialylactose solution, was separated from the product. The concentrated sialyl-lactose solution was then treated with activated carbon (CAS: 7440-44-0, Carl Roth, Karlsruhe, Germany) to remove dyes, such as Maillard reaction products and aldolic reaction products. In order to separate the sialyl-lactose from by-products that originate from the fermentation process such as sialic acid and N-acetylglucosamine, the solution was filtered with a 1 kDa cut membrane GE4040F30 (GE water & process technologies, Ratingen, Germany), and diafiltered at a conductivity of 0.6 to 0.8 mS. The diluted solution was concentrated on a rotary evaporator at a concentration of about 300 g L-1. In a final chromatographic separation, other contaminating sugars, such as di-sialylactose, were removed. Therefore, the concentrated solution was applied to a weak anion ion exchange resin in the form of acetate (Amberlite FPA51, Dow Chemical, Michigan, USA). While sialyl-lactose is hardly bound to resin, di-sialyl-lactose is adsorbed. In this way, sialyl-lactose is eluted with 10 mM ammonium acetate, while di-sialyl-lactose is eluted with 1 M ammonium acetate. For the removal of ammonium acetate, sialyl-lactose was precipitated with a 10-fold excess of ethanol. The solid fraction was filtered and dried.
    [00122] [00122] The product was finished by passing a 20% solution of sialylactose sequentially through a 6 kDa filter (Pall Microza ultrafiltration module SIP-2013, Pall Corporation, Dreieich, Germany) and a sterile 0 filter , 2 μm.
    [00123] [00123] Part of the solution was spray-dried using a Büchi spray dryer (Büchi Mini Spray Dryer B-290) (Büchi, Essen, Germany), applying the following parameters: Inlet temperature: 130 ° C, Outlet temperature 67 ° C to 71 ° C, gas flow 670 L / h, vacuum cleaner 100%.
    [00124] [00124] The spray-dried 6'-sialyl-lactose had a purity of 91%, while the 3'-sialyl-lactose material had a purity of 93%. Example 4: Analysis of spray-dried sialyl-lactoses by wide-angle powder X-ray diffraction (XDR)
    [00125] [00125] Wide angle powder X-ray diffraction (XRD) was used to study the morphology of lyophilized products. The Empyrean X-ray diffractometer (Panalytical, Almelo, Netherlands) equipped with a copper anode (45 kV, 40 mA, Κα1 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 of 5 to 45 ° 2Θ, with a step size of 0.04 ° 2Θ and a counting time of 100 seconds per step.
    [00126] [00126] It was found that the spray-dried 6'-sialyl-lactose had a completely amorphous structure (Figure 7), while the sample of the 3'-sialyl-lactose shows two diffraction peaks, at 7 ° and 31 °, which indicate a partially crystalline structure, however, the 3'-sialyl-lactose sample also predominantly provides an amorphous signal (Figure 8). Example 5: Analysis of spray dried sialyl lactoses by Differential Scanning Calorimetry (DSC)
    [00127] [00127] Differential scanning calorimetry (DSC) in a Mettler Toledo 821e (Mettler Toledo, Giessen, Germany) was used to determine thermal events of spray-dried products (glass transition temperature (Tg), exo and endothermic events).
    [00128] [00128] Approximately 25 mg of the spray dried product was analyzed in corrugated Al-crucibles (Mettler Toledo, Giessen, Germany). The samples were cooled to 0 ° C with 10 K / min and reheated to 100 ° C with a scan rate of 10 K / min. After cooling the samples to 0 ° C in a second heating cycle, the samples were heated to 150 ° C. The midpoint of the baseline endothermic change during the heating analysis was obtained as Tg. Exothermic and endothermic peaks are reported using the peak temperature and normalized energy of the event.
    [00129] [00129] The spray dried 6'-sialyl lactose sample exhibited a Tg value of 48 ° C in the first heating analysis, and a Tg of 50 ° C in the second heating analysis with an endothermic relaxation peak after first Tg. When compared to 6'-sialyl-lactose, it showed the Tg values of 3'-sialyl-lactose of 22 ° C in both heating analyzes, indicating a greater stability of 6'-sialyl-lactose at higher temperatures.
    权利要求:
    Claims (23)
    [1]
    1. A method for purifying sialylated oligosaccharides that have been produced by microbial fermentation or in-vitro biocatalysis, the method comprising the steps of i) separating biomass from the fermentation broth; ii) removing cations from the fermentation broth or reaction mixture; iii) remove anionic impurities from the fermentation broth or reaction mixture; and iv) removing compounds that have a molecular weight less than that of the sialylated oligosaccharide to be purified from the fermentation broth or reaction mixture.
    [2]
    The method according to claim 1, further comprising one or more steps selected from the group consisting of v) increasing the concentration of the sialylated oligosaccharide; vi) removing unwanted oligosaccharides; vii) remove dyes; viii) removing endotoxins; ix) sterilize; and x) spray drying or crystallizing the sialylated oligosaccharide.
    [3]
    The method according to claim 1 or 2, wherein the sialylated oligosaccharide is a human milk sialylated oligosaccharide, preferably selected from the group consisting of 3'-SL, 6'-SL, LST-a, LST -b, LST-c, 3-F-SL, DS-LNT and F-LST-b.
    [4]
    The method according to any one of claims 1 to 3, wherein the removal of biomass from the fermentation broth is carried out by subjecting the fermentation broth to an ultrafiltration, preferably an ultrafiltration which removes the biomass and compounds that have a weight molecular weight ≥ 500 kDa of the fermentation broth, more preferably to an ultrafiltration that removes biomass and compounds that have a molecular weight of ≥ 150 kDa from the fermentation broth, and even more preferably to an ultrafiltration that removes biomass and compounds that have a molecular weight ≥ 100 kDa of the fermentation broth.
    [5]
    The method according to any of claims 1 to 4, wherein the removal of cations from the fermentation broth is carried out by cation exchange chromatography.
    [6]
    The method according to any one of claims 1 to 5, in which the removal of anionic impurities from the fermentation broth is carried out by anionic exchange chromatography.
    [7]
    The method according to any one of claims 1 to 6, wherein the removal of compounds that have a molecular weight less than that of the sialylated oligosaccharide to be purified is carried out by cross-flow filtration.
    [8]
    The method according to any one of claims 1 to 7, wherein the concentration of the sialylated oligosaccharide to be purified is increased by nanofiltration or evaporation of the solvent.
    [9]
    The method according to any one of claims 1 to 87, wherein the removal of dyes is carried out by treating the fermentation broth / solution containing the desired sialylated oligosaccharide with activated carbon.
    [10]
    The method according to any one of claims 1 to 9, wherein the removal of endotoxins is carried out by filtering the solution containing the desired oligosaccharide through a 6 kDa filter or a 3kDa filter.
    [11]
    The method according to any one of claims 1 to 10, wherein the solution is sterilized by filtering the solution through a 0.2 μm filter.
    [12]
    The method according to any of claims 1 to 11 wherein it additionally comprises an SMB chromatography step.
    [13]
    The method according to any one of claims 1 to 12 wherein it further comprises an electrodialysis step.
    [14]
    14. A preparation of a sialylated oligosaccharide, wherein said sialylated oligosaccharide has been purified by a method according to any one of claims 1 to 13.
    [15]
    The preparation according to claim 14, wherein the sialylated oligosaccharide is present in the preparation in a purity of ≥ 80% by weight.
    [16]
    The preparation according to claim 14 or 15, wherein the preparation is a fluid or a powder, preferably a powder in which the particles are amorphous or crystalline particles.
    [17]
    17. Use of the preparation according to any one of claims 14 to 16 to produce a nutritional composition, preferably an infant formula.
    [18]
    18. A nutritional composition comprising at least one sialylated oligosaccharide, in which said at least one sialylated oligosaccharide has been produced by microbial fermentation or biocatalysis, and in which said nutritional composition is a medicinal formulation, a dietary supplement, a milk drink or a infant formula.
    [19]
    19. A nutritional composition containing at least 5 HMOs, in which said at least 5 HMOs are selected from the group consisting of 2'-fucosyl-lactose, 3-fucosyl-lactose, lacto-N-tetraose, lacto- N-neotetraose, lacto-N-fucopentaose I, 3'-sialyl-lactose and 6'-sialyl-lactose.
    [20]
    20. A nutritional composition according to claim 18 or 19,
    wherein the nutritional composition contains at least one sialylated HMO and at least one neutral HMO and a probiotic microorganism.
    [21]
    21. A spray-dried GMO-free powder consisting essentially of a sialylated oligosaccharide with a purity of> 80% dry weight and having less than 10% water by weight.
    [22]
    The spray-dried powder product according to claim 21, wherein the product is selected from the group consisting of 3'-sialyl-lactose, 6'-sialyl-lactose, and a mixture of 3'- sialyl-lactose and 6'-sialyl-lactose.
    [23]
    The spray dried powder product according to claim 22, wherein the spray dried powder essentially consists of a mixture of 3'-sialyl-lactose, 6'-sialyl-lactose and one or more neutral HMOs, the referred to one or more neutral HMOs being selected from the group consisting of 2'-fucosyl-lactose, 3-fucosyl-lactose, lacto-N-tetraose, lacto-N-neotetraose, lacto-N-fucopentaose I.
    类似技术:
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    同族专利:
    公开号 | 公开日
    JP2020531039A|2020-11-05|
    RU2020108730A|2021-09-30|
    EP3676274A1|2020-07-08|
    US20200308211A1|2020-10-01|
    AU2018322785A1|2020-02-27|
    CN111094311A|2020-05-01|
    KR20200041370A|2020-04-21|
    PH12020550069A1|2021-02-15|
    WO2019043029A1|2019-03-07|
    EP3450443A1|2019-03-06|
    SG11202001296UA|2020-03-30|
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    法律状态:
    2021-11-23| B350| Update of information on the portal [chapter 15.35 patent gazette]|
    优先权:
    申请号 | 申请日 | 专利标题
    EP17188280.6A|EP3450443A1|2017-08-29|2017-08-29|Process for purifying sialylated oligosaccharides|
    EP17188280.6|2017-08-29|
    PCT/EP2018/073178|WO2019043029A1|2017-08-29|2018-08-29|Process for purifying sialylated oligosaccharides|
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