Spray-dried 3-fucosyllactose

Abstract

To provide solid preparations of human milk oligo saccharides and methods for manufacturing of solid preparations of human milk oligosaccharides.SOLUTION: The present invention provides spray-dried powder consisting essentially of 3-fucosyllactose which has been produced by microbial fermentation, and a nutritional composition containing the spray-dried powder. A method for manufacture of the spray-dried powder comprises the steps of: a) purifying the 3-fucosyllactose from a fermentation broth; b) providing an aqueous solution containing the 3-fucosyllactose of the step a); and c) subjecting the solution of the step b) to spray-drying.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to preparations of human milk oligosaccharides. More particularly, the present invention relates to solid preparations of human milk oligosaccharides and methods for producing said solid preparations of human milk oligosaccharides. [Background technology]

[0002] Human breast milk contains significant amounts of carbohydrates. These carbohydrates include monosaccharides such as L-fucose and N-acetylneuraminic acid (Neu5Ac). The disaccharide lactose is also present in human breast milk. In addition to lactose, one liter of human breast milk contains up to 20 g / L of oligosaccharides, so-called "human milk oligosaccharides (HMOs)." HMOs are the third most abundant component of human breast milk. It is estimated that there are over 150 structurally distinct oligosaccharides in human milk. Human milk typically contains between 10 and 13 major HMOs, which are present at concentrations ranging from several hundred milligrams to several grams per liter (Thurl et al. (2017), Nutrition Reviews 75(11) 920-933). HMOs include neutral and acidic HMOs containing one or more sialic acid moieties. The most well-known HMOs are listed in Table 1. The structural complexity and abundance of these oligosaccharides is unique to human milk and is not found in the milk of other mammals, such as dairy animals.

[0003] Because HMOs are not digested by humans, the physiological role of these sugars has been investigated for decades. The prebiotic effect of HMOs was discovered over 100 years ago. HMOs can modulate the human gut microbiota by providing beneficial bacteria. Several other functional effects of HMOs, particularly their effects on neonatal development, have been investigated in recent years. HMOs are known to function as decoys to reduce the risk of infection by bacterial and viral pathogens, attaching to human cells by binding to cell surface glycoproteins. Furthermore, various HMOs have anti-inflammatory effects and function as immunomodulators. Therefore, it has been proposed that HMOs reduce the risk of developing food allergies. The beneficial effects of sialylated HMOs on neonatal brain development are hotly debated (reviewed in "Prebiotics and Probiotics in Human Milk, Origins and Functions of Milk-Borne Oligosaccharides and Bacteria," Academic Press (2017), editors: McGuire M., McGuire M., and Bode L.).

[0004] One HMO is 3-fucosyllactose (Gal(β1-4)[Fuc(β1-3)]Glc). 3-Fucosyllactose (3-FL) was first isolated from milk in 1958 and from the urine of non-secretors with blood type O during pregnancy and lactation in 1977. 3-Fucosyllactose is resistant to enzymatic hydrolysis in the infant's gastrointestinal tract. It is hypothesized that 3-fucosyllactose reaches the large intestine, where it is absorbed and metabolized by bacteria.

[0005] The first step to take advantage of the beneficial effects of HMOs on milk-fed infants is to add individual HMOs to infant formula. However, it is better to supplement infant formula with structurally different HMO combinations, because these have effects that are more similar to their original source, human milk, and this cannot be achieved by individual HMOs.

[0006] The limited supply of individual HMOs for supplementing infant formulas led to the development of chemical synthesis of HMOs, followed by biocatalytic approaches using purified enzymes. Today, a variety of HMOs are produced on a commercial scale using fermentation of genetically engineered bacterial cells (WO2015 / 150328A1, WO2017 / 043382A1, WO2010 / 070104A1, WO2012 / 097950A1). HMOs synthesized by bacterial cells can be purified from fermentation broths or cell lysates to obtain substantially pure preparations of HMOs that can be used in human foods, particularly infant foods.

[0007] During its purification, 3-fucosyllactose is usually present in the form of liquid process stream.With purification, the concentration of 3-fucosyllactose in process stream increases.However, the aqueous solution of 3-fucosyllactose is very vulnerable to bacterial or fungal contamination.Therefore, it is preferred to provide 3-fucosyllactose as a dry product with low moisture content, so that microbial growth is impossible.

[0008] Typically, sugars are obtained in solid form by crystallization. Crystallization of individual HMOs has been described for 3-fucosyllactose (WO 2014 / 075680A), 2'-fucosyllactose (WO 2011 / 150939A), di-fucosyllactose (WO 2016 / 086947A), lacto-N-tetraose (WO 2017 / 101953A), and lacto-N-neotetraose (WO 2014 / 094783A). Crystallization of HMOs involves the use of organic solvents such as alcohols, primarily ethanol or methanol, or organic acids such as glacial acetic acid. However, using organic solvents to crystallize HMOs as the final step in a process to obtain a solid final product is not appropriate when the HMO is used as a food ingredient. Furthermore, organic solvents are harmful to the environment and to anyone who handles them. Therefore, the use of organic solvents requires occupational safety measures and proper disposal, which makes their use expensive. Therefore, the crystallization of HMOs to provide solid forms of HMOs should be considered a drawback in the production of HMOs on an industrial scale.

[0009] Therefore, a method for providing HMOs, particularly 3-fucosyllactose, in solid form is desirable that is applicable to industrial-scale production of HMOs and does not involve the use of organic solvents at the end of the purification scheme to provide a solid preparation of said HMO.

[0010] This problem is solved by a method for providing a powder consisting essentially of purified HMO, said method comprising spray drying an aqueous solution containing HMO. Summary of the Invention

[0011] In a first aspect, a spray-dried powder consisting essentially of 3-fucosyllactose is provided and a method is provided. In a second aspect, a method is provided for producing a spray-dried powder consisting essentially of 3-fucosyllactose.

[0012] In a third aspect, there is provided the use of a spray-dried powder consisting essentially of 3-fucosyllactose for the manufacture of a nutritional composition. In a fourth aspect, there is provided a nutritional composition comprising a spray-dried powder consisting essentially of 3-fucosyllactose. [Brief explanation of the drawings]

[0013] [Figure 1] Graph showing the results of powder X-ray diffraction of spray-dried 3-fucosyllactose. [Figure 2] 1 is a graph showing the results of powder X-ray diffraction of spray-dried lacto-N-tetraose. [Figure 3] 1 is a graph showing the results of powder X-ray diffraction of spray-dried 6'-sialyllactose. [Figure 4] 1 is a graph showing the results of powder X-ray diffraction of spray-dried 3'-sialyllactose. [Figure 5] 1 is a graph showing powder X-ray diffraction results of a spray-dried mixture of 2'-fucosyllactose and lacto-N-tetraose. [Figure 6] 1 is a graph showing powder X-ray diffraction results of a spray-dried mixture of 2'-fucosyllactose, 3-fucosyllactose, lacto-N-tetraose, 3'-sialyllactose, and 6'-sialyllactose. DETAILED DESCRIPTION OF THE INVENTION

[0014] According to a first aspect, there is provided a spray-dried powder consisting essentially of 3-fucosyllactose produced by microbial fermentation. 3-fucosyllactose is produced by microbial fermentation as described herein below.The term "consisting essentially of" as used herein means that the spray-dried powder is composed of 3-fucosyllactose and optionally by-products that are produced during the microbial fermentation for the production of 3-fucosyllactose, but may not be removed from the process stream obtained from microbial fermentation.The term "consisting essentially of" includes the spray-dried powder that is composed of at least 80wt%, at least 85wt%, at least 90wt%, at least 93wt%, at least 95wt%, or at least 98wt% of 3-fucosyllactose.

[0015] In further and / or alternative embodiments, the 3-fucosyllactose is present in the spray-dried powder in amorphous form. In further and / or alternative embodiments, the spray-dried powder contains no more than 15 wt% water, preferably no more than 10 wt% water, more preferably no more than 7 wt% water, and most preferably no more than 5 wt% water.

[0016] In further and / or alternative embodiments, the spray-dried powder is free of genetically engineered microorganisms and nucleic acid molecules derived from genetically engineered microorganisms. According to a second aspect, there is provided a method for producing a spray-dried powder consisting essentially of 3-fucosyllactose produced by microbial fermentation, the method comprising: a) purifying 3-fucosyllactose from the fermentation broth; b) providing an aqueous solution of 3-fucosyllactose of step a); c) subjecting the solution of step b) to spray drying; Includes:

[0017] In further and / or alternative embodiments, the step of purifying 3-fucosyllactose from the fermentation broth comprises: i) removing microbial cells from the fermentation broth to obtain a clarified process stream; ii) subjecting the clarified process stream to at least one ultrafiltration; iii) treating the clarified process stream at least once with a cation exchange resin and / or at least once with an anion exchange resin; iv) subjecting the clarified process stream to at least one nanofiltration; v) subjecting the clarified process stream to at least one electrodialysis; vi) treating the clarified process stream at least once with activated charcoal; and / or vii) subjecting the clarified process stream to at least one crystallization and / or precipitation step. Contains one or more of:

[0018] 3-fucosyllactose can be produced by microbial fermentation, and a genetically engineered microorganism capable of synthesizing 3-fucosyllactose is cultivated in a culture medium (fermentation broth) and under conditions that allow the genetically engineered microorganism to synthesize 3-fucosyllactose.The purification of the 3-fucosyllactose produced by microbial fermentation comprises separating microbial cells from the fermentation broth to obtain a clarified process stream that is essentially free of cells and contains 3-fucosyllactose.This step is the first step in the method for purifying desired oligosaccharides.

[0019] Suitable methods for separating microbial cells from the fermentation broth include centrifugation, with the microbial cells obtained as a pellet and the fermentation broth obtained as a supernatant. In additional and / or alternative embodiments, the microbial cells are separated from the fermentation broth by using filtration. Suitable filtration methods for separating cells from the fermentation broth include microfiltration and ultrafiltration.

[0020] Such microfiltration is a physical filtration process in which a particle-containing fluid is forced through a membrane of a specific pore size to separate the particles from the fluid. The term "microfiltration," as used herein, refers to a physical filtration process in which cells are separated from a fermentation broth.

[0021] Ultrafiltration is a type of membrane filtration and is not fundamentally different. In ultrafiltration, forces such as pressure or a concentration gradient effect separation across a semipermeable membrane. Cells, suspended solids, and high molecular weight solutes are retained in the so-called retentate, while water and low molecular weight solutes, such as the desired sialylated oligosaccharides, pass through the membrane into the permeate (filtrate).

[0022] Ultrafiltration membranes are defined by the molecular weight cut-off (MWCO) of the membrane used. Ultrafiltration is applied in cross-flow or dead-end mode. Typically, microbial cells synthesize 3-fucosyllactose intracellularly and secrete it into fermentation broth.The 3-fucosyllactose thus produced ultimately ends up in the fermentation broth, which is then subjected to further process steps for purifying 3-fucosyllactose as described hereinafter.

[0023] The method is used to purify 3-fucosyllactose produced by microbial fermentation, but it can also be used to purify 3-fucosyllactose produced by in vitro enzyme catalysis. 3-fucosyllactose can be purified from the reaction mixture at the end of the biocatalytic reaction. This reaction mixture is subjected to the purification process as a clarified process stream.

[0024] The clarified process stream contains 3-fucosyllactose, as well as by-products and unwanted impurities, such as monosaccharides, disaccharides, unwanted oligosaccharide by-products, ions, amino acids, polypeptides, proteins and / or nucleic acids.

[0025] In further and / or alternative embodiments, the method for purifying 3-fucosyllactose comprises at least one step of cation exchange treatment to remove positively charged compounds from the clarified process stream.

[0026] Suitable cation exchange resins for removing positively charged compounds include Lewatit S2568 (H+) (Lanxess AG, Cologne, DE). In further and / or alternative embodiments, the method for purifying 3-fucosyllactose includes a step of anion exchange treatment to remove undesirable negatively charged compounds from the clarified process stream.

[0027] Suitable anion exchange resins include Lewatit S6368A, Lewatit S4268, Lewatit S5528, Lewatit S6368A (Lanxess AG, Cologne, DE), Dowex AG 1x2 (mesh 200-400), Dowex 1x8 (mesh 100-200), Purolite Chromalite CGA 100x4 (Purolite GmbH, Ratingen, DE), Dow Amberlite FPA51 (Dow Chemicals, MI, USA).

[0028] In further and / or alternative embodiments, the method for purifying 3-fucosyllactose includes nanofiltration and / or diafiltration steps to remove lower molecular weight impurities and concentrate the desired oligosaccharides.

[0029] Diafiltration involves adding fresh water to a solution to remove (wash) membrane-permeable components. Diafiltration can be used to separate components based on their molecular size and charge by using an appropriate membrane that efficiently retains one or more species while allowing others to permeate the membrane. Diafiltration using nanofiltration membranes is particularly effective for separating low-molecular-weight compounds, such as small molecules and salts. Nanofiltration membranes typically have a molecular weight cutoff in the range of 150 to 1000 daltons. Nanofiltration is widely used in the dairy industry for the concentration and demineralization of whey.

[0030] 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).

[0031] Diafiltration using nanofiltration membranes has been found to be an efficient pretreatment method for removing significant amounts of contaminants prior to electrodialysis of oligosaccharide-containing solutions. The use of nanofiltration membranes for concentration and diafiltration during HMO purification results in lower energy and processing costs, as well as better product quality due to reduced heat exposure, leading to reduced Maillard and aldol reactions.

[0032] In further and / or alternative embodiments, the method for purifying 3-fucosyllactose comprises at least one electrodialysis step. Electrodialysis (ED) is a combination of dialysis and electrolysis that can be used to separate or concentrate ions in a solution based on their selective electromigration through a semipermeable membrane.

[0033] The basic principle of electrodialysis consists of an electrolytic cell equipped with a pair of electrodes immersed in an electrolyte for ion conduction, 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 then carries the current flow, which results from the migration of anions and cations toward the anode and cathode, respectively. The membranes used in electrodialysis are essentially sheets of porous ion-exchange resins with negatively or positively charged groups, and are therefore described as cationic or anionic membranes, respectively. Ion-exchange membranes are typically made of polystyrene bearing suitable functional groups (e.g., sulfonic acid groups for cationic membranes, or quaternary ammonium groups for anionic membranes) cross-linked with divinylbenzene. The electrolyte can be, for example, sodium chloride, sodium acetate, sodium propionate, or sulfamic acid. The ion-depleted stream is then thoroughly separated from the ion-enriched stream (the two solutions are also referred to as dilution). The electrodialysis stack is assembled in a manner where the anion and cation membranes are parallel, like a filter press, between two electrode blocks, resulting in two separate electrodes called the concentrate (ion-depleted) and concentrate (ion-enriched). The heart of the electrodialysis process is the membrane stack, which consists of several anion and cation exchange membranes separated by spacers that are placed between the two electrodes. By applying a direct current, the anions and cations migrate through the membranes to the electrodes.

[0034] In further and / or alternative embodiments, the method for purifying 3-fucosyllactose further comprises a step of continuous chromatography, such as simulated moving bed (SMB) chromatography.

[0035] Simulated moving bed (SMB) chromatography originated in the petrochemical and mining industries. Today, SMB chromatography is used by the pharmaceutical industry to separate enantiomers from racemic mixtures. Large-scale SMB chromatography has already been used to separate the monosaccharide fructose from fructose-glucose solutions and the disaccharide sucrose from sugar beet or sugarcane syrup.

[0036] SMB processes used to separate sugars use, for example, calcium-loaded cross-linked polystyrene resin, an anionic resin in the bisulfite form (Bechthold M. et al., Chemie Ingenieur Technik, 2010, 82, 65-75), or a polystyrene gel strong acid cation resin in the hydrogen form (Purolite PCR833H) (Purolite, Bala Cynwyd, USA).

[0037] Considering the possibility of using a continuous mode of operation, mobile phase recycling, and large column sizes, SMB systems can in principle be scaled to achieve production volumes of several hundred tonnes.

[0038] The simulated moving bed chromatography process step is advantageous in that it allows for the further removal of oligosaccharides that are closely structurally related to the desired oligosaccharide.

[0039] In further and / or alternative embodiments, the method for purifying 3-fucosyllactose comprises treating the process stream with activated charcoal to remove contaminants, such as colorants, from the process stream.

[0040] In additional and / or alternative embodiments, the method for purifying 3-fucosyllactose comprises at least one step of crystallizing or precipitating 3-fucosyllactose from a clarified process stream.Crystallizing or precipitating 3-fucosyllactose from a process stream can be carried out by adding a suitable amount of water-miscible organic solvent to the process stream containing 3-fucosyllactose.The organic solvent can be selected from the group consisting of C1-C6-alcohols and C1-C4-carbon acids.

[0041] In further and / or alternative embodiments, the method for purifying 3-fucosyllactose includes a step of sterile filtration and / or endotoxin removal, preferably by filtration of the process stream through a 3 kDa filter or a 6 kDa filter.

[0042] In additional and / or alternative embodiments, the method for purifying 3-fucosyllactose includes increasing the concentration of 3-fucosyllactose in a process stream. The concentration of 3-fucosyllactose in the process stream can be increased by subjecting the process stream to vacuum evaporation, reverse osmosis, or nanofiltration (e.g., a process having a size exclusion limit of 20 Å or less). Alternatively, the crystallized or precipitated 3-fucosyllactose can be dissolved in water to obtain a solution of 3-fucosyllactose having the desired concentration.

[0043] In further and / or alternative embodiments, the resulting process stream is an aqueous solution containing 3-fucosyllactose at a concentration of 20 g / L or more, 25 g / L or more, 30 g / L or more, 40 g / L or more, 60 g / L or more, 100 g / L or more, 200 g / L or more, or even 300 g / L or more.

[0044] In further and / or alternative embodiments, the aqueous solution comprises 3-fucosyllactose that is at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, or at least 98% pure by weight of dry matter / solutes in the solution.

[0045] The resulting concentrate containing purified 3-fucosyllactose can be stored under appropriate conditions. The method for purifying 3-fucosyllactose is cost-effective, easy to scale up and suitable as the basis for a multi-tonne scale manufacturing process.

[0046] The method for purifying 3-fucosyllactose is also advantageous in that the aqueous solution does not contain genetically engineered microorganisms and nucleic acid molecules derived from genetically engineered microorganisms.In addition, the aqueous solution does not contain protein.Completely removing protein eliminates the risk of causing allergies to potential consumers.

[0047] The method for producing the spray-dried powder comprises providing an aqueous solution comprising 3-fucosyllactose. In further and / or alternative embodiments, the aqueous solution contains 3-fucosyllactose in an amount of at least 20% (w / v), 30% (w / v), 35% (w / v), and up to 45% (w / v), 50% (w / v), 60% (w / v).

[0048] In further and / or alternative embodiments, the aqueous solution contains 3-fucosyllactose that is at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, or at least 98% pure by weight of dry matter / solutes in the solution.

[0049] In additional and / or alternative embodiments, the aqueous solution is free of genetically engineered microorganisms, nucleic acid molecules derived from genetically engineered microorganisms, and proteins. In the method for producing the spray-dried powder, the aqueous solution containing 3-fucosyllactose is subjected to spray drying.

[0050] Spray drying is a method for obtaining dry powders in which a solution containing the substance of interest (i.e., 3-fucosyllactose) is first sprayed into droplets, which are rapidly dried by hot air. Spray drying is very fast, and the substance to be dried is only exposed to high temperatures for a very short period of time.

[0051] In further and / or alternative embodiments, the 3-fucosyllactose-containing aqueous solution purified from the fermentation broth or process stream is spray-dried at a nozzle temperature of at least 110°C, preferably at least 120°C, more preferably at least 125°C, and less than 150°C, preferably less than 140°C, more preferably less than 135°C.

[0052] In additional and / or alternative embodiments, the aqueous solution containing 3-fucosyllactose purified from the fermentation broth or process stream is spray dried at an outlet temperature of at least 60° C., preferably at least 65° C., and less than 80° C., preferably less than 70° C. In a particularly preferred embodiment, the aqueous solution containing 3-fucosyllactose is spray dried at a nozzle temperature of about 68° C. to about 70° C.

[0053] Spray drying of an aqueous solution containing 3-fucosyllactose provides a powder with low hygroscopicity, in which the 3-fucosyllactose is present in amorphous form and the particle size is uniform.

[0054] According to a third aspect, there is provided the use of the spray-dried powder containing 3-fucosyllactose purified from process stream for producing nutritional compositions.The spray-dried powder essentially consisting of 3-fucosyllactose is suitable for human consumption, and can therefore be included in preparations for human consumption, such as pharmaceutical preparations, infant formula, milk drinks or dietary supplements.

[0055] According to a fourth aspect, there is provided a nutritional composition comprising the spray-dried powder according to the first aspect produced according to the second aspect. In additional and / or alternative embodiments, the nutritional composition contains at least one additional HMO that is not 3-fucosyllactose. The at least one additional HMO may be a neutral HMO, preferably selected from the group consisting of 2'-fucosyllactose (2'-FL), lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT), and lacto-N-fucopentaose I (LNFPI). In additional and / or alternative embodiments, the at least one additional HMO may be a sialylated HMO, preferably selected from the group consisting of 3'-sialyllactose (3'-SL), 6'-sialyllactose (6'-SL), sialyllact-N-tetraose (LST)-a, LST-b, LST-c, and disialyllact-N-tetraose (DSLNT).

[0056] In further and / or alternative embodiments, the nutritional composition comprises a mixture consisting essentially of Neu5Ac, 2'-FL, 3-FL, LNT, LNnT, LNFPI, 3'-SL, 6'-SL, sialic acid, and L-fucose. Nutritional compositions containing preferred amounts of each of the foregoing compounds are shown in Table 1.

[0057] [Table 1]

[0058] The composition according to column 2 of Table 1 may be used to supplement infant formula so that the final infant formula for direct consumption may contain the compounds of the mixture in the concentrations specified in column 3 of Table 1. This is particularly advantageous for

[0059] In further and / or alternative embodiments, the nutritional composition contains one or more additional ingredients selected from the group consisting of oils, fats and fatty acids (e.g., olive oil, sunflower oil, coconut oil, nut oils, rapeseed oil, palm oil, flaxseed oil, fish oil, linoleic acid, soybean oil, etc.), carbohydrates (e.g., glucose, fructose, lactose, maltodextrin, starch, sucrose, inositol, etc.), proteins (from nonfat milk, whey, casein (from any dairy animal), or soy), vitamins (A, B1, B2, B5, B6, B12, C, D, E, K, biotin, folic acid, niacin, choline), minerals and trace elements (sodium, potassium, chloride, calcium, phosphorus, magnesium, iron, zinc, manganese, fluoride, selenium, iodine, copper).

[0060] In a preferred embodiment, the nutritional composition comprising spray-dried human milk oligosaccharides or a mixture of human milk oligosaccharides or a mixture of human milk oligosaccharides and functional monosaccharides or a mixture of human milk oligosaccharides and other fibers is an infant formula that meets the compositional requirements set forth in Regulation (EU) 2016 / 127 and / or Code of Federal Regulations (USA) Title 21 107.100 (Nutrient Specifications). Representative compositions of infant formulas are shown in Tables 2 and 3.

[0061] [Table 2]

[0062] [Table 3-1]

[0063] [Table 3-2]

[0064] In additional and / or alternative embodiments, the nutritional composition also comprises microorganisms, preferably probiotic microorganisms.For infant food applications, preferred microorganisms can be obtained from or found in healthy human microflora.Preferably, but not limited to, the microorganisms are selected from the genera Bifidobacterium, Lactobacillus, Enterococcus, Streptococcus, Staphylococcus, Peptostreptococcus, Leuconostoc, Clostridium, Eubacterium, Veillonella, Fusobacterium, Bacteroides, Prevotella, Escherichia, Propionibacterium and Saccharomyces. In further and / or alternative embodiments, 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; Kluyveromyces 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, Selected from the group consisting of 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); Pediococcus acidilactici, P. pentosus; Propionibacterium acidipropionici, P. freudenreichii ssp. shermannii; Staphylococcus carnosus; and Streptococcus thermophilus.

[0065] In addition to the combination of living organisms, the nutritional composition may also contain dead cell cultures. In the field of probiotics, dead cell cultures may be used (for example, tyndalized bacteria). These dead cultures provide proteins, peptides, oligosaccharides, cell wall fragments and natural products, and can provide short-term stimulation of the immune system.

[0066] The inclusion of probiotic microorganisms in nutritional compositions, especially in the presence of HMOs, is particularly advantageous in that it also promotes the establishment of a healthy intestinal microflora. In further and / or alternative embodiments, the nutritional compositions also include prebiotics such as galacto-oligosaccharides (GOS), fructooligosaccharides (FOS), inulin, or combinations thereof.

[0067] The nutritional composition is in a solid form including, but not limited to, a powder, granules, flakes, pellets, or combinations thereof. In additional embodiments, the nutritional composition is selected from the group consisting of a pharmaceutical formulation, an infant formula, a dairy drink, and a dietary supplement.

[0068] As a pharmaceutical preparation, the nutritional composition may be used to improve cognitive performance, in particular to improve attention, learning and / or memory. Aspects of the present invention also include the following. Aspect 1 A spray-dried powder consisting essentially of 3-fucosyllactose produced by microbial fermentation. Aspect 2 2. The spray-dried powder of embodiment 1, wherein the spray-dried powder contains at least 80 wt%, at least 85 wt%, at least 90 wt%, at least 93 wt%, at least 95 wt%, or at least 98 wt% 3-fucosyllactose. Aspect 3 3. The spray-dried powder of embodiment 1 or 2, wherein the 3-fucosyllactose is present in amorphous form. Aspect 4 4. The spray-dried powder according to any of the preceding aspects, comprising no more than 15 wt% water, preferably no more than 10 wt% water, more preferably no more than 7 wt% water, and most preferably no more than 5 wt% water. Aspect 5 Aspect 5. The spray-dried powder of any of aspects 1 to 4, which is free of genetically engineered microorganisms and nucleic acid molecules derived from genetically engineered microorganisms. Aspect 6 6. A method for producing a spray-dried powder according to any of aspects 1 to 5, comprising the steps of: a) purifying 3-fucosyllactose from the fermentation broth; b) providing an aqueous solution containing 3-fucosyllactose of step a); c) subjecting the solution of step b) to spray drying; A method comprising: Aspect 7 A step of purifying 3-fucosyllactose from the fermentation broth (step a) i) removing microbial cells from the fermentation broth to obtain a clarified process stream; ii) subjecting the clarified process stream to at least one ultrafiltration; iii) treating the clarified process stream at least once with a cation exchange resin and / or at least once with an anion exchange resin; iv) subjecting the clarified process stream to at least one round of nanofiltration and / or diafiltration; v) subjecting the clarified process stream to at least one electrodialysis; vi) treating the clarified process stream at least once with activated charcoal; and / or vii) subjecting the clarified process stream to at least one crystallization and / or precipitation step. 7. The method of embodiment 6, comprising one or more of: Aspect 8 The method of claim 6 or 7, wherein the aqueous solution comprises 3-fucosyllactose in an amount of at least 20% (w / v), 30% (w / v), 35% (w / v), and up to 45% (w / v), 50% (w / v), 60% (w / v). Aspect 9 A method according to any one of aspects 6 to 8, wherein the 3-fucosyllactose-containing aqueous solution is spray-dried at a nozzle temperature of at least 110°C, preferably at least 120°C, more preferably at least 125°C, and less than 150°C, preferably less than 140°C, more preferably less than 135°C. Aspect 10 10. A method according to any one of aspects 6 to 9, wherein the 3-fucosyllactose-containing aqueous solution is spray-dried at an outlet temperature of at least 60°C, preferably at least 65°C, and less than 80°C, preferably less than 70°C. Aspect 11 6. Use of the spray-dried powder according to any of aspects 1 to 5 for the manufacture of a nutritional composition, preferably an infant formula. Aspect 12 6. A nutritional composition comprising the spray-dried powder of any of aspects 1 to 5. Aspect 13 13. The nutritional composition of embodiment 12, further comprising at least one additional HMO, wherein said at least one additional HMO is a neutral HMO or a sialylated HMO. Aspect 14 14. The nutritional composition according to embodiment 12 or 13, wherein the at least one neutral HMO is selected from the group consisting of 2'-fucosyllactose, lacto-N-tetraose, lacto-N-neotetraose and lacto-N-fucopentaose I. Aspect 15 15. The nutritional composition of any of aspects 12 to 14, wherein the at least one sialylated HMO is selected from the group consisting of 3'-sialyllactose, 6'-sialyllactose, sialyllact-N-tetraose (LST)-a, LST-b, LST-c and disialyllacto-N-tetraose. Aspect 16 16. A nutritional composition according to any of aspects 12 to 15, comprising at least one probiotic microorganism. The present invention will be described with respect to specific embodiments and with reference to the drawings, but the invention is not limited thereto, but only by the claims. Moreover, terms such as first, second, etc. in the specification and claims are used to distinguish between similar elements and do not necessarily describe an order in time, space, ranking, or any other manner. It should be understood that terms so used are interchangeable under appropriate circumstances, and that the embodiments of the invention described herein may function in orders other than those described or illustrated herein.

[0069] It should be noted that the term "comprises", when used in the claims, should not be interpreted as being limited to the means recited thereafter, and does not exclude other elements or steps. It should therefore be interpreted as specifying the presence of the stated feature, integer, step, or component as referenced, but not as excluding the presence or addition of one or more other features, integers, steps, or components, or groups thereof. Thus, the scope of the expression "a device comprising means A and B" should not be limited to a device consisting only of components A and B. This means that, in the context of the present invention, the only relevant components of the device are A and B.

[0070] References throughout this specification to "one embodiment" or "an embodiment" mean that a particular feature, structure, or characteristic described in connection with an embodiment is included in at least one embodiment of the present invention. Thus, the appearances of the phrases "in one embodiment" or "in an embodiment" in various places throughout this specification are not necessarily always referring to the same embodiment. Furthermore, as will be apparent to one of ordinary skill in the art from this disclosure, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0071] Similarly, in describing representative embodiments of the invention, it should be understood that various features of the invention may be grouped together in a single embodiment, figure, or description thereof to simplify the disclosure and facilitate understanding of one or more of the various inventive aspects. This method of disclosure is not to be interpreted as reflecting an intention that the claimed invention requires more features than are expressly recited in each claim. Rather, as the following claims reflect, aspects of the invention may require fewer than all features of any foregoing disclosed embodiment. Thus, the claims following the detailed description are expressly incorporated into this detailed description, with each claim standing on its own as a separate embodiment of the invention.

[0072] Furthermore, although some embodiments described herein include some features and not other features included in other embodiments, combinations of features from different embodiments are intended to be within the scope of the present invention and form different embodiments, as would be understood by one skilled in the art. For example, in the following claims, any of the claimed embodiments may be used in any combination.

[0073] Furthermore, some of the embodiments are described herein as methods or combinations of elements of methods that may be implemented by a processor of a computer system or other means for performing a function. Thus, a processor with the necessary instructions for carrying out such a method or element of a method forms a means for carrying out the method or element of a method. Furthermore, the elements described herein of apparatus embodiments are examples of means for carrying out the functions performed by the elements for carrying out the invention.

[0074] In the description and drawings set forth herein, numerous specific details are set forth. However, embodiments of the present invention may be practiced without these specific details. In other instances, well-known methods, structures and techniques have not been shown in detail to facilitate understanding of the description and drawings.

[0075] The present invention will now be described by detailed descriptions of several embodiments of the present invention. Although other embodiments of the present invention may be constructed according to the knowledge of those skilled in the art without departing from the spirit or technical advantages of the present invention, it is apparent that the present invention is limited only by the terms of the appended claims. [Example]

[0076] Example 1 Purification of 2'-fucosyllactose from fermentation broth As described in European Patent Application No. 16196486.1, 2'-fucosyllactose was produced by fermentation using a genetically modified Escherichia coli strain. As described in WO2015 / 106943A1, 2'-fucosyllactose was purified from the fermentation broth by filtration, ion exchange chromatography, nanofiltration, diafiltration or electrodialysis, and treatment with charcoal. The resulting 2'-fucosyllactose-containing solution was subjected to spray drying to obtain a stable solid product.

[0077] Example 2 Purification of 3-fucosyllactose from fermentation broth 3-Fucosyllactose was produced by fermentation using a genetically modified Escherichia coli strain as described in European Patent Application No. 16196486.1.

[0078] Cells were separated from the culture medium by ultrafiltration (0.05 μm cutoff) (CUT membrane technology, Erkrath, Germany) followed by filtration through a cross-flow filter with a MWCO of 150 kDa (Microdyn-Nadir, Wiesbaden, Germany). The cell-free fermentation medium containing approximately 30 g / L of 3-fucosyllactose was collected by H + The solution was then passed through a strong cationic ion exchanger (Lewatit S2568, Lanxess, Cologne, Germany) in the chloride form to remove positively charged contaminants. The solution was then set to pH 7.0 using sodium hydroxide and applied to an anionic ion exchanger (Lewatit S6368A, Lanxess) in the chloride form. Both ion exchangers were used in a volume of 200 L. After a second filtration (150 kDa; Microdyn-Nadir, Wiesbaden, Germany), the particle-free solution was concentrated 5-fold by nanofiltration using a Filmtech NF270 membrane (Dow, Midland, USA) and 2.5-fold by vacuum evaporation. The concentration was approximately 15 mS cm. -1The concentrated solution, with a conductivity of 10 kDa (Microdyn-Nadir, Wiesbaden, Germany), was filtered, purified with activated carbon charcoal (CAS: 7440-44-0, Carl Roth, Karlsruhe, Germany), and deionized by electrodialysis. For this purpose, a PC-Cell BED 1-3 electrodialysis device (PC-Cell, Heusweiler, Germany) was used, which had a PC-Cell E200 membrane stack containing the following membranes: cation exchange membrane CEM: PC SK and anion exchange membrane AEM: PC Acid 60. 0.25 M sulfamic acid was used as the process electrolyte. To reduce brown discoloration caused by the Maillard reaction and aldol products from the fermentation process, Na was added. + and Cl - A second ion exchange chromatography was performed using the same ion exchange material as above, but in a volume of 50 L. After concentrating the sugar solution by evaporation, the conductivity was again reduced to 4 mScm by electrodialysis using the PC-Cell BED 1-3 described above. -1 to 0.4 mScm -1 For further decolorization, the solution was mixed with activated charcoal (CAS: 7440-44-0, Carl Roth, Karlsruhe, Germany) and filtered to obtain an almost colorless solution.

[0079] Example 3 Purification of lacto-N-tetraose from fermentation broth Fermentative production of lacto-N-tetraose was carried out using a genetically modified E. coli BL21(DE3)ΔlacZ strain containing the essential genes for in vivo synthesis of lacto-N-tetraose, namely, N-acetylglucosamine glycosyltransferase (lgtA from Neisseria meningitidis MC58), β-1,3-galactosyltransferase (wbdO from Salmonella enterica subsp. salame serovar Greenside), and lacY from E. coli K12. The UDP-glucose-4-epimerase galE and UTP-glucose-1-phosphate uridyltransferase galU, both from E. coli K12, were also overexpressed. For lacto-N-tetraose fermentative production, the strains were grown in defined mineral salts medium containing 2% glucose as the carbon source. Antifoam was added as needed. pH was controlled using 25% ammonia solution. 216gl -1 Lactose was added stepwise from a lactose stock solution to a final concentration of 15 mM, and the lactose concentration in the culture medium was kept constant throughout the fermentation process. Residual lactose and lacto-N-triose II accumulated during the process as by-products and were hydrolyzed by a second E. coli strain added to the fermenter. This strain expressed functional beta-lactamase, beta-N-acetylhexosaminidase (bbhI from Bifidobacterium bifidum JCM1254), and a functional gal operon for the degradation of monosaccharides (EP2845905A).

[0080] The cells were separated from the fermentation broth and the lacto-N-tetraose-containing fluid was purified according to the procedure described in Example 2 to a purity of 75-80% as determined by mass balance. Contaminating carbohydrate by-products resulting from inefficient enzymatic degradation and metabolism were removed by simulated moving bed (SMB) chromatography according to WO 2015 / 049331. Alternatively, lacto-N-tetraose was purified by crystallization using isopropanol. For crystallization, the lacto-N-tetraose-containing solution was concentrated to a concentration of 20% by evaporation and then spray-dried. Using a NUBILOSA LTC-GMP spray dryer (NUBILOSA, Konstanz, Germany), the solution was passed under a nitrogen flow through a spray dryer nozzle with an inlet temperature of 130 °C, while the product stream was controlled to maintain an outlet temperature of 67 °C to 68 °C.

[0081] The solid material was added to a mixture of isopropanol and water (3:1 (vol / vol)) at a ratio of 1 kg of powder to 12 L of isopropanol / water. The suspension was vigorously stirred, and then the insoluble lacto-N-tetraose was filtered and dried at 40 °C. Starting with material of 73-89% purity, the crystallized lacto-N-tetraose was purified to approximately 95% with an 85% recovery. The sugar was dissolved in water to a concentration of 25% and passed sequentially through a 6 kDa filter (Pall Microza Ultrafiltration Module SIP-2013, Pall Corporation, Dreieich, Germany) and a 0.2 μm sterile filter. The solid material was obtained by spray-drying the sterile material under the conditions described above.

[0082] Example 4 Purification of 3'- and 6'-sialyllactose from fermentation broth For the production of 3'-sialyllactose and 6'-sialyllactose, we used the recombinant Escherichia coli BL21(DE3)ΔlacZ strain. The strains shared the following genetic modifications: glucosamine-6-phosphate synthase GlmS from E. coli, N-acetylglucosamine 2-epimerase Slr1975 from Synechocystis sp., glucosamine 6-phosphate N-acetyltransferase Gna1 from Saccharomyces cerevisiae, phosphoenolpyruvate synthase PpsA from E. coli, and chromosomal constitutive expression of N-acetylneuraminate synthase NeuB and CMP-sialic acid synthetase NeuA, both from Campylobacter jejuni. Furthermore, a functional gal operon consisting of the genes encoding lactose permease LacY from E. coli, cscB (sucrose permease), cscK (fructokinase), cscA (sucrose hydrolase), and cscR (transcription regulator) from E. coli W, and the genes galE (UDP-glucose-4-epimerase), galT (galactose-1-phosphate uridylyltransferase), galK (galactokinase), and galM (galactose-1-epimerase) from E. coli K12, was integrated into the genome of the BL21 strain and constitutively expressed.

[0083] The strain synthesizing 3'-sialyllactose harbors the alpha-2,3-sialyltransferase gene from Vibrio sp. JT-FAJ-16, while the 6'-sialyllactose-producing strain contains the alpha-2,6-sialyltransferase plsT6 from Photobacterium leiognatii JT-SHIZ-119.

[0084] The sialyllactose-producing strain was grown in defined mineral salts medium containing 2% sucrose as the carbon source. The sucrose feed (500 g l) provided in the fed-batch phase was -1 ) with 8 mM MgSO4, 0.1 mM CaCl2, trace elements, and 5 g -1 of NH4Cl was added.

[0085] For sialyllactose formation, 216gl -1 A lactose feed of 1000 kJ / ml was used. The pH was controlled by using an ammonia solution (25% v / v). Fed-batch fermentation was carried out at 30°C under constant aeration and agitation. β-galactosidase was added to the fermenter to remove residual lactose at the end of the fermentation. The resulting monosaccharides were metabolized by the production strain.

[0086] The cell-free fluid was then deionized by ion exchange chromatography. +The cationic impurities were removed with a 200 L volume of a strong cation exchanger (Lewatit S2568 (Lanxess, Cologne, Germany) in chloride form. The pH of the resulting solution was set to 7.0 using NaOH. In a second step, anionic ions and undesired colorants were removed from the solution using a strong anion exchanger (Lewatit S6368S (Lanxess, Cologne, Germany) in chloride form. The ion exchanger had a bed volume of 200 L. A second filtration step with a cross-flow filter (150 kDa cutoff) (Microdyn-Nadir, Wiesbaden, Germany) was used to remove precipitate resulting from acidification of the solution. To concentrate the sugars, the solution was nanofiltered using a Dow FILMTECH NF270-4040 (Inaqua, Mönchengladbach, Germany) or alternatively, a Trisep 4040-XN45-TSF Membrane (0.5 kDa cutoff) (Microdyn-Nadir, Wiesbaden, Germany). The latter was used to separate the monosaccharide N-acetylglucosamine, which originates from the fermentation process and contaminates the sialyllactose solution, from the product. The concentrated sialyllactose solution was then treated with activated charcoal (CAS: 7440-44-0, Carl Roth, Karlsruhe, Germany) to remove colorants such as Maillard and aldol reaction products. To separate sialyllactose from by-products derived from the fermentation process, such as sialic acid and N-acetylglucosmine, the solution was filtered through a 1 kDa cut-off membrane GE4040F30 (GE water & process technologies, Ratingen, Germany) at 0.6–0.8 mScm -1The diluted solution was diafiltered to a conductivity of approximately 300 g / L. The diluted solution was concentrated on a rotary evaporator to a concentration of approximately 300 g / L. In a final chromatographic separation, other contaminating sugars, such as di-sialyllactose, were removed. To do so, the concentrated solution was applied to a weak anionic ion-exchange resin in acetate form (Amberlite FPA51, Dow Chemical, Michigan, USA). Sialyllactose rarely binds to the resin, whereas di-sialyllactose is adsorbed. Therefore, sialyllactose is eluted with 10 mM ammonium acetate, and di-sialyllactose with 1 M ammonium acetate. To remove the ammonium acetate, sialyllactose was precipitated with a 10-fold excess of ethanol. The solid fraction was filtered and dried.

[0087] The product was finalized by passing the 20% sialyllactose solution sequentially through a 6 kDa filter (Pall Microza Ultrafiltration Module SIP-2013, Pall Corporation, Dreieich, Germany) and a 0.2 μm sterile filter.

[0088] A portion of the solution was spray dried using a Buchi spray dryer (Buchi Mini Spray Dryer B-290) (Buchi, Essen, Germany) applying the following parameters: inlet temperature 130 °C, outlet temperature 67 °C–71 °C, gas flow rate 670 L / h, aspirator 100%.

[0089] 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 mixture Mixtures of HMOs were prepared from the solid products. To do this, single HMOs were spray-dried and the powders were mixed. HMO Mixture I contained 2'-fucosyllactose and lacto-N-tetraose in a ratio of 70% to 30%, while HMO Mixture II contained 2'-fucosyllactose (52%), 3-fucosyllactose (13%), lacto-N-tetraose (26%), 3'-sialyllactose (4%), and 6'-sialyllactose (5%). The mixed powders were dissolved in water to a 20% sugar solution and again spray-dried using a Buchi spray dryer as described in Example 4.

[0090] Example 6 Characterization of spray-dried human milk oligosaccharides 6.1 Differential Scanning Calorimetry (DSC) Differential scanning calorimetry (DSC) was used on a Mettler Toledo 821e (Mettler Toledo, Giessen, Germany) to determine the thermal events of spray-dried human milk oligosaccharides, namely, 3-fucosyllactose, 6'-sialyllactose, 3'-sialyllactose, lacto-N-tetraose, and a spray-dried mixture of human milk oligosaccharides, a mixture of 2'-fucosyllactose / lacto-N-tetraose (HMO mixture I), and a mixture of 2'-fucosyllactose, 3-fucosyllactose, lacto-N-tetraose, 6'-sialyllactose, and 3'-sialyllactose (HMO mixture II).

[0091] A Mettler Toledo 821e (Mettler Toledo, Giessen, Germany) was used to determine the thermal events (glass transition temperature (Tg), further exothermic and endothermic events) of the spray-dried products.

[0092] Approximately 25 mg of spray-dried human milk oligosaccharides were analyzed in crimped Al crucibles (Mettler Toledo, Giessen, Germany). The sample was cooled to 0°C at 10 K / min and reheated to 100°C at a scan rate of 10 K / min. After cooling the sample to 0°C in a second heating cycle, the sample was reheated to 150°C. The midpoint of the endothermic shift in the baseline during the heating scan was considered the glass transition temperature (Tg). Exothermic and endothermic peaks are reported using the peak temperature and normalized energy of the event.

[0093] The first heating scan of all samples showed a major glass transition event at the total heat flow rate, evident by a major stepwise transition in the range of approximately 48–58 °C, and in most of the samples, the major glass transition event observed in the first heating scan occurred again in the second heating scan. The results of the DSC analysis are summarized in Figure 4.

[0094] [Table 4]

[0095] In the case of 3-fucosyllactose, an endothermic relaxation peak was detected after the Tg in the first heating scan. In the case of lacto-N-tetraose, a much higher Tg of approximately 79 °C was detected in the second heating scan compared to the other samples. This may be caused by an endothermic event at approximately 89 °C (-6.04 J / g) during the first heating scan. Similar to 3-fucosyllactose, an endothermic relaxation peak was also detected after the Tg in 6'-sialyllactose, but this sample also had an endothermic event at 77 °C (-0.22 J / g). No endothermic event was detected in 3'-sialyllactose and HMO mixture I, but in the case of HMO mixture II, the endothermic event during the first heating scan was 79 °C (0.34 J / g).

[0096] 6.2 Powder X-ray diffraction (XRD) Wide-angle powder X-ray diffraction (XRD) was used to probe the morphology of the freeze-dried products. A copper anode (45 kV, 40 mA, K at a wavelength of 0.154 nm) was used.α1 An Empyrean X-ray diffractometer (Panalytical, Almelo, The Netherlands) equipped with a luminescence and PIXcel3D detector was used. Approximately 100 mg of spray-dried sample was analyzed in reflectance mode within a 2θ angle range of 5–45°, with a 2θ step size of 0.04° and a counting time of 100 s per step.

[0097] All single oligosaccharides and HMO mixtures I and II exhibited a completely amorphous state (Figures 1-6). In the case of lacto-N-tetraose, a second (amorphous) signal was detected around 9-10°.

[0098] 6.3 Laser diffraction Powder particle size was assessed by laser diffraction. The system detects scattered and diffracted light using an array of concentrically arranged sensor elements. A software algorithm then approximates particle counts by calculating z values ​​of the light intensity values ​​reaching different sensor elements. Analysis was performed using a SALD-7500 Aggregate Sizer (Shimadzu Corporation, Kyoto, Japan) quantitative laser diffraction system (qLD). .

[0099] A small amount (spatula tip) of each sample was dispersed in 2 ml of isooctane and homogenized by sonication for 5 minutes, and the dispersion was transferred into a batch cell filled with isooctane and analyzed in manual mode.

[0100] The data acquisition settings were as follows: signal averaging count per measurement: 128, signal accumulation count: 3, and interval: 2 seconds. Prior to the measurements, the system was zeroed with isooctane. Each sample dispersion was measured three times, and the mean and standard deviation are reported. The data were evaluated using the software WING SALD II, version V3.1. Since the refractive index of the samples was unknown, the refractive index of sugar (disaccharide) particles (1.530) was used to determine the size distribution profile. The mean and median diameter size values ​​are reported.

[0101] The mean particle size of all samples was very similar, with a slightly lower value measured for HMO Mix II. The particle size characteristics are summarized in Table 5. Furthermore, the particle size distributions indicated the presence of a single major size population for all of the samples.

[0102] [Table 5]

Claims

1. A spray-dried powder, the spray-dried powder containing at least 80 wt% 3-fucosyllactose produced by microbial fermentation, free of genetically engineered microorganisms and nucleic acid molecules derived from genetically engineered microorganisms, and containing 15 wt% or less water; at least one probiotic microorganism; and intermittently sterilized probiotic bacteria. Nutritional composition.

2. The nutritional composition described in claim 1, further containing at least one additional HMO, wherein the at least one additional HMO is a neutral HMO or a sialylated HMO.

3. The nutritional composition described in claim 2, wherein at least one neutral HMO is selected from the group consisting of 2'-fucosyllactose, lacto-N-tetraose, lacto-N-neotetraose and lacto-N-fucopentaose I.

4. A nutritional composition described in any one of claims 2 to 3, wherein at least one sialylated HMO is selected from the group consisting of 3'-sialyllactose, 6'-sialyllactose, sialyllact-N-tetraose (LST)-a, LST-b, LST-c and disialyllact-N-tetraose.

5. A nutritional composition described in any one of claims 1 to 4, wherein the spray-dried powder contains at least 85 wt%, at least 90 wt%, at least 93 wt%, at least 95 wt%, or at least 98 wt% 3-fucosyllactose.

6. A nutritional composition according to any one of claims 1 to 5, wherein the 3-fucosyllactose is present in amorphous form.

7. A nutritional composition described in any one of claims 1 to 6, wherein the spray-dried powder contains less than 10 wt% water, preferably less than 7 wt% water, more preferably less than 5 wt% water.

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