RU2802680C2 - Spray-dried sialyllactose - Google Patents

Abstract

FIELD: food industry.

SUBSTANCE: invention relates to solid preparations of breast milk oligosaccharides and to methods for manufacturing solid preparations of breast milk oligosaccharides. A spray-dried powder is proposed for the manufacture of a nutritional composition, where the spray-dried powder contains at least 80 wt% of 3'-sialyllactose (3'-SL) and/or 6'-sialyllactose (6'-SL) obtained by fermentation.

EFFECT: production of a solid preparation of breast milk oligosaccharide without the use of an organic solvent.

16 cl, 6 dwg, 5 tbl, 5 ex

Description

The present invention relates to breast milk oligosaccharide preparations. More particularly, the present invention relates to solid preparations of human milk oligosaccharides and to methods for making said solid preparations of human milk oligosaccharides.

Prior Art

Human breast milk contains a significant amount 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 represent the third most abundant component of human breast milk. More than 150 structurally distinct oligosaccharides are believed to be present in breast milk. Breast milk typically contains 10 to 13 major HMOs, which are present in concentrations ranging from grams to several hundred milligrams per liter (Thurl et al., (2017), Nutrition Reviews 75(11) 920-933). OGMs include neutral OHMs as well as acidic OHMs that contain one or more sialic acid moieties. The most significant HGMs are shown in Table 1. The structural complexity and abundance of these oligosaccharides is specific to human breast milk and is not found in the milk of other mammals, such as, for example, domesticated dairy animals.

Since HGMs are not digested by humans, the physiological role of these saccharides has been studied for several decades. The prebiotic effect of HGM was discovered more than 100 years ago. HMOs have the potential to modulate the human gut microbiome by providing food for beneficial bacteria. Several other functional effects of OGM have been investigated in recent years, especially their effects on neonatal development. OGMs are known to act as decoys to reduce the risk of infections caused by bacterial and viral pathogens that adhere to human cells through binding to cell surface glycoproteins. In addition, various HMOs have anti-inflammatory effects and act as immunomodulators. Therefore, it has been suggested that OGM reduces the risk of developing food allergies.

The positive effect of sialylated HMOs on neonatal brain development has been widely discussed (reviewed in “Prebiotics and Probiotics in Human Milk, Origins and functions of milk-borne oligosaccharides and bacteria”, Academic Press (2017) editors: McGuire M., McGuire M. , and Bode L).

The most common sialylated OGMs are 3'-sialyllactose (NeuAc(α2,3)Gal(β1,4)Glc) and 6'-sialyllactose (NeuAc(α2,6)Gal((β1,4)Glc). In 3'- sialyllactose monosaccharide residue N-acetylneuraminic acid is associated with the galactosyl group of lactose at position 3. 3'-Sialyllactose (3'-SL - from the English 3'-Sialyllactose) binds strongly to several viral strains, including strains of the influenza virus, HIV-1 (virus human immunodeficiency), reovirus and polyomavirus, and may thus alter viral infectivity. 3'-Sialyllactose also affects intestinal colonization by bacteria. In 6'-sialyllactose (6'-SL - from English 6'-Sialyllactose) monosaccharide the N-acetylneuraminic acid residue is linked to the gylactosyl moiety of lactose at position 6. 6'-Sialyl lactose also binds several viral strains.

The first step in reaping the beneficial effects of HMOs in formula-fed infants is to add individual HMOs to the infant formula. However, it would be better to supplement infant formulas with a combination of structurally different HMOs, since the combination of structurally different HMOs will produce actions that are more similar to those of their original source, breast milk, and which cannot be caused by individual HMOs.

The limited addition of individual HMOs to supplement infant formula first led to the development of chemical synthesis of OHMs, followed by the development of biocatalytic approaches using purified enzymes. Today, fermentation of genetically modified bacterial cells is used to produce various HGMs on an industrial scale (WO 2015/150328 A1, WO 2017/043382 A1, WO 2010/070104 A1, WO 2012/097950 A1). HMOs that are synthesized by bacterial cells can be purified from fermentation broth or cell lysate to obtain substantially pure HMO preparations such that they can be used in human nutrition, especially infant nutrition.

During their purification, 3'-SL and/or 6'-SL is/are present in the form of a liquid process stream. Together with purification, the concentration of 3'-SL and/or 6'-SL in a given process stream is increased. However, an aqueous solution of 3'-SL and/or 6'-SL is highly susceptible to bacterial or fungal contamination. Thus, it is preferable to provide the 3'-SL and/or 6'-SL as a dry product having a low water content so that microbial growth is not possible.

Typically, the saccharide is obtained in solid form by crystallization. Crystallization of individual OGMs is described: for 3-fucosyllactose (WO 2014/075680 A), for 2'-fucosyllactose (WO 2011/150939 A), Di-fucosyllactose (WO 2016/086947 A), lacto-N-tetraose (WO 2017/ 101953 A), lacto-N-neotetraose (WO 2014/094783 A). Crystallization of OGM involves the use of organic solvents such as alcohols, mainly ethanol or methanol, or organic acids such as glacial acetic acid. However, the use of organic solvents to crystallize the HMOs as a final step in the process for obtaining the final product in solid form is not appropriate if the HMOs are to be used as food ingredients. In addition, organic solvents are harmful to the environment and to any individual working with them. Thus, the use of organic solvents requires compliance with occupational safety measures and appropriate disposal, which makes the use of organic solvents expensive. Thus, crystallization of OGM to provide OHM in solid form should be considered a disadvantage in the production of OHM on an industrial scale.

Thus, what is desirable is a process that provides the 3'-SL and/or 6'-SL in solid form, which is useful in the industrial production of OGM data and which does not involve the use of an organic solvent at the end of the purification scheme to provide a solid preparation of said OGM.

The problem is solved by a method for producing a powder essentially consisting of 3'-SL and/or 6'-SL, wherein said method comprises spray drying an aqueous solution that contains 3'-SL and/or 6'-SL.

Summary of the invention

In a first aspect, a spray-dried powder is provided substantially consisting of 3'-SL and/or 6'-SL.

A second aspect provides a method for making a spray-dried powder substantially consisting of 3'-SL and/or 6'-SL.

A third aspect provides the use of a spray-dried powder substantially consisting of 3'-SL and/or 6'-SL for the manufacture of a nutritional composition.

In a fourth aspect, a nutritional composition is provided comprising a spray-dried powder substantially consisting of 3'-SL and/or 6'-SL.

Description of graphic materials

In FIG. 1 is a graph illustrating powder X-ray diffraction results of 3-fucosyllactose dried by spray drying.

In FIG. 2 is a graph illustrating powder X-ray diffraction results of lacto-N-tetraose dried by spray drying.

In FIG. 3 is a graph illustrating powder X-ray diffraction results of 6'-sialyllactose dried by spray drying.

In FIG. 4 is a graph illustrating powder X-ray diffraction results of 3'-sialyllactose dried by spray drying.

In FIG. 5 is a graph illustrating powder X-ray diffraction results of a spray-dried mixture of 2'-fucosyllactose and lacto-N-tetraose.

In FIG. 6 is a graph illustrating 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

According to a first aspect, a spray-dried powder is provided substantially consisting of 3'-SL and/or 6'-SL produced by microbial fermentation.

3'-SL and/or 6'-SL are produced by microbial fermentation as described below herein. The term "essentially consisting of", as used herein, means that the spray-dried powder consists of 3'-SL and/or 6'-SL and - possibly - by-products that are formed during microbial fermentation to produce 3'-SL and/or 6'-SL, but which could not be removed from the process stream resulting from microbial fermentation. The term "essentially consisting of" includes spray-dried powders consisting of 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'-SL and/or 6'-SL.

In an additional and/or alternative embodiment, the 3'-SL and/or 6'-SL is present in the spray-dried powder in amorphous form.

In an additional and/or alternative embodiment, the spray-dried powder contains 15 wt. % water or less, preferably 10 wt. % water or less, more preferably 7 wt. % water or less, most preferably 5 wt. % water or less.

In a further and/or alternative embodiment, the spray-dried powder does not contain genetically modified microorganisms and nucleic acid molecules derived from genetically modified microorganisms.

According to a second aspect, there is provided a method of making a spray-dried powder substantially consisting of 3'-SL and/or 6'-SL that has been/are produced by microbial fermentation. The method includes the following stages:

a) purification of 3'-SL and/or 6'-SL from the fermentation broth;

b) providing an aqueous solution of the 3'-SL and/or 6'-SL of step a); And

c) subjecting the solution to step b) to spray drying.

In an additional and/or alternative embodiment, purification of 3'-SL and/or 6'-SL from the fermentation broth includes one or more of the following steps:

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 carbon; and/or

vii) subjecting the clarified process stream at least once to crystallization and/or precipitation steps.

3'-SL and/or 6'-SL may be produced by microbial fermentation in which a genetically modified microorganism that is capable of synthesizing 3'-SL and/or 6'-SL is cultured in a culture medium ( fermentation broth) and under conditions that are permissive for the synthesis of 3'-SL and/or 6'-SL by the specified genetically modified microorganism. Purification of 3'-SL and/or 6'-SL produced by microbial fermentation includes the step of separating microbial cells from the fermentation broth to produce a clarified process stream that is substantially cell-free and that contains 3'-SL and/or or 6'-SL. This step represents the first step in the process for purifying the desired oligosaccharides.

Suitable methods for separating microbial cells from the fermentation broth include centrifugation, in which the microbial cells are obtained as a sediment and the fermentation broth as a supernatant. In a further and/or alternative embodiment, microbial cells are separated from the fermentation broth by filtration. Suitable filtration-based methods for separating cells from the fermentation broth include microfiltration and ultrafiltration.

Microfiltration itself is a physical filtration method where a liquid containing particles is passed through a membrane with a special pore size to separate the particles from the liquid. The term "microfiltration", as used herein, refers to a physical filtration method where cells are separated from the fermentation broth.

Ultrafiltration is a type of membrane filtration and is not fundamentally different. In ultrafiltration, forces, like pressure or concentration gradients, drive separation across a semipermeable membrane. Cells, suspended solids, and high molecular weight solutes are retained in what is called the retentate, while water and low molecular weight solutes, such as the desired sialylated oligosaccharide, pass through the membrane into the permeate (filtrate).

Ultrafiltration membranes are determined by the nominal molecular weight cut-off (MWCO) of the membrane used. Ultrafiltration is used in cross-flow or blunt-end mode.

Suitable filters for microfiltration or ultrafiltration are SPIRA-CEL® DS MP005 4333 and FS10-FC FUS1582 fiber (Microdyn-Nadir GmbH, Wiesbaden, DE).

Typically, microbial cells synthesize 3'-SL and/or 6'-SL intracellularly and secrete it(here) into the fermentation broth. The 3'-SL and/or 6'-SL thus obtained is passed into the fermentation broth, which is then subjected to further processing steps to purify the 3'-SL and/or 6'-SL, as described below in this document. Provided that the 3'-SL and/or 6'-SL are present in the fermentation broth at the end of the fermentation, said fermentation broth becomes a clarified process stream after cell removal. Provided that all or some of the 3'-SL and/or 6'-SL remains within the cells, the cells can be removed from the fermentation broth and lysed. Insoluble components can be removed from the cell lysate, which then becomes a clarified process stream containing 3'-SL and/or 6'-SL.

Although the method is used to purify 3'-SL and/or 6'-SL that has been produced by microbial fermentation, the method can also be used to purify 3'-SL and/or 6'-SL that has been obtained as a result of enzymatic catalysis in-vitro. The 3'-SL and/or 6'-SL can be purified from the reaction mixture at the end of the biocatalytic reaction. The said reaction mixture is subjected to a purification process in the form of a clarified process stream.

The clarified process stream contains 3'-SL and/or 6'-SL, as well as by-products and unwanted impurities such as - for example - monosaccharides, disaccharides, unwanted oligosaccharide by-products, ions, amino acids, polypeptides, proteins and/or nucleic acids .

In an additional and/or alternative embodiment, the method for purifying 3'-SL and/or 6'-SL includes the step of at least one treatment with a cation exchange resin to remove positively charged compounds from the clarified process stream.

Suitable cation exchange resins for removing positively charged compounds include Lewatit S2568 (H ⁺ ) (Lanxess AG, Cologne, DE).

In an additional and/or alternative embodiment, the 3'-SL and/or 6'-SL purification method includes the step of treating with an anion exchange resin to remove unwanted negatively charged compounds from the clarified process stream.

Suitable anion exchange resins include Lewatit S6368 A, Lewatit S4268, Lewatit S5528, Lewatit S6368A (Lanxess AG. Cologne, DE), Dowex AG 1x2 (Mesh 200-400), Dowex 1x8 (Mesh 100-200), Purolite Chromalite CGA100×4 (Purolite GmbH, Ratingen, DE), DowAmberlite FPA51 (Dow Chemicals, Ml, USA).

In an additional and/or alternative embodiment, the 3'-SL and/or 6'-SL purification method includes a nanofiltration and/or diafiltration step to remove low molecular weight impurities and concentrate the desired oligosaccharides.

Diafiltration involves adding fresh water to a solution to remove (wash out) membrane-permeable components. Diafiltration can be used to separate components based on the size and charge of their molecules through the use of appropriate membranes where one or more compounds are effectively retained and the other compounds are membrane permeable. In particular, diafiltration using a nanofiltration membrane is effective in separating low molecular weight compounds, like small molecule, and salts. Nanofiltration membranes typically have a nominal molecular weight cutoff in the range of 150 - 1000 Daltons. Nanofiltration is widely used in the dairy industry to concentrate and demineralize whey.

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).

Diafiltration using nanofiltration membranes has been found to be effective as a pre-treatment to remove significant amounts of contaminants prior to electrodialysis-based treatment of the oligosaccharide-containing solution. The use of nanofiltration membranes for concentration and diafiltration during OGM purification results in lower energy and processing costs and better product quality due to reduced thermal stress, resulting in reduced levels of Maillard and aldol reactions.

In an additional and/or alternative embodiment, the method for purifying 3'-SL and/or 6'-SL includes at least one electrodialysis step.

Electrodialysis (ED - from the English electrodialysis) combines dialysis and electrolysis and can be used to separate or concentrate ions in solutions based on their selective electromigration through semi-permeable membranes.

The basic principle of electrodialysis is an electrolytic cell containing a pair of electrodes immersed in an electrolyte to conduct ions, connected to a direct current generator. The electrode connected to the positive pole of the DC generator is the anode, and the electrode connected to the negative pole is the cathode. In this case, the electrolyte solution supports the passage of electric current, which is formed as a result of the movement of negatively charged and positively charged ions towards the anode and cathode, respectively. Membranes used for electrodialysis are essentially sheets of porous ion exchange resins with negatively charged or positively charged groups, and are thus described as cationic or anionic membranes, respectively. Ion exchange membranes are typically made from polystyrene bearing a suitable functional group (such as a sulfonic acid in the case of cationic membranes or a quaternary ammonium group in the case of anionic membranes) cross-linked with divinylbenzene. The electrolyte may, for example, be sodium chloride, sodium acetate, sodium propionate or aminosulfonic acid. In such a case, the electrodialysis package is assembled in such a way that the anion and cation membranes are parallel, as in a filter press between two electrode blocks, so that the ion-depleted stream is well separated from the ion-rich stream (these two solutions are also called dilute (ion-depleted) and concentrate (ion-enriched). The basis of the electrodialysis method is a membrane package, which consists of several anion-exchange membranes and cation-exchange membranes separated by spacers installed between two electrodes. By applying a constant electric current, anions and cations will migrate through the membranes towards the electrodes.

In an additional and/or alternative embodiment, the method for purifying 3'-SL and/or 6'-SL further includes a continuous chromatography step, such as simulated bed moving chromatography (SMB).

Smooth moving bed (SMB) chromatography has its origins in the petrochemical and minerals industries. Currently, SMB chromatography is used in the pharmaceutical industry to isolate enantiomers from racemic mixtures. Large-scale SMB chromatography has already been used to isolate the monosaccharide fructose from fructose-glucose solutions and to isolate the disaccharide sucrose from sugar beet or sugar cane syrups.

SMB processes used for separating saccharides use, for example, calcium-charged crosslinked polystyrene resins, anion exchange resins in bisulfite form (Bechthold M., et al., Chemie Ingenieur Technik, 2010, 82, 65-75) or based on a strong acid cation exchanger in hydrogen form and polystyrene gel (Purolite PCR833H) (Purolite, Bala Cynwyd, USA).

With continuous operation, reuse of the mobile phase and also the ability to use larger column sizes, SMB systems can in principle be scaled up to achieve production volumes of hundreds of tons.

The pseudo-moving bed chromatography step of the method is advantageous in that the step allows further removal of oligosaccharides that are structurally related to the desired oligosaccharide.

In an additional and/or alternative embodiment, the 3'-SL and/or 6'-SL purification method includes treating the process stream with activated carbon to remove contaminants, such as dyes, from the process stream.

In an additional and/or alternative embodiment, the method for purifying 3'-SL and/or 6'-SL includes at least one step of crystallizing or precipitating 3'-SL and/or 6'-SL from the clarified process stream. Crystallization or precipitation of 3'-SL and/or 6'-SL from the process stream can be accomplished by adding a suitable amount of an organic solvent that is miscible with water to the process stream containing 3'-SL and/or 6'-SL. The organic solvent may be selected from the group consisting of C ₁ -C ₆ alcohols and C ₁ -C ₄ carboxylic acids.

In an additional and/or alternative embodiment, the 3'-SL and/or 6'-SL purification method includes the step of sterile filtration and/or endotoxin removal, preferably by filtering the process stream through a 3 kDa filter or a 6 kDa filter.

In an additional and/or alternative embodiment, the method for purifying 3'-SL and/or 6'-SL includes the step of increasing the concentration of 3'-SL and/or 6'-SL in the process stream. The concentration of 3'-SL and/or 6'-SL in the process stream can be increased by subjecting the process stream to vacuum evaporation, reverse osmosis, or nanofiltration (eg, nonofiltration using a nanofiltration membrane having an exclusion limit of 20 A or less). Alternatively, the crystallized or precipitated 3'-SL and/or 6'-SL is dissolved in water to obtain a solution of 3'-SL and/or 6'-SL having the desired concentration of 3'-SL and/or 6'-SL .

In a further and/or alternative embodiment, the resulting process stream is an aqueous solution that contains 3'-SL and/or 6'-SL at a concentration of 20 g/L or greater, 25 g/L or greater, or 30 g /L or more, equal to 40 g/L or more, 60 g/L or more, equal to 100 g/L or more, equal to 200 g/L or more, or even equal to 300 g/L or more.

In an additional and/or alternative embodiment, the aqueous solution contains 3'-SL and/or 6'-SL with a purity of at least 80%, at least 85%, at least 90%, at least 93%, at least 95% or at least 98% based on the weight of dry matter/solutes in solution.

The resulting concentrate containing purified 3'-SL and/or 6'-SL can be stored under appropriate conditions.

The 3'-SL and/or 6'-SL purification process is cost-effective and easily scaled up, making it suitable as the basis for a multi-ton scale production process.

The 3'-SL and/or 6'-SL purification method is also advantageous in that the aqueous solution does not contain genetically modified microorganisms and nucleic acid molecules derived from genetically modified microorganisms. In addition, the aqueous solution does not contain proteins. The general removal of proteins eliminates the risk of causing allergies in potential consumers.

A method for producing a spray-dried powder includes the step of providing an aqueous solution containing 3'-SL and/or 6'-SL.

In an additional and/or alternative embodiment, the aqueous solution contains 3'-SL and/or 6'-SL 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).

In an additional and/or alternative embodiment, the aqueous solution contains 3'-SL and/or 6'-SL with a purity of at least 80%, at least 85%, at least 90%, at least 93%, at least 95% or at least 98% based on the weight of dry matter/solutes in solution.

In a further and/or alternative embodiment, the aqueous solution does not contain genetically modified microorganisms, nucleic acid molecules derived from genetically modified microorganisms, and proteins.

In a method for producing a spray-dried powder, an aqueous solution containing 3'-SL and/or 6'-SL is spray-dried.

Spray drying is a method of producing dry powders where a solution containing the test substance (namely 3'-SL and/or 6'-SL) is first sprayed into droplets, which are quickly dried by hot air. Spray drying is very fast and the exposure of the substance to be dried to high temperatures is quite short.

In an additional and/or alternative embodiment, the aqueous solution containing 3'-SL and/or 6'-SL, which has been 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 and more preferably less than 135°C.

In an additional and/or alternative embodiment, the aqueous solution containing 3'-SL and/or 6'-SL, which has been purified from the fermentation broth or process stream, is spray dried at an outlet temperature of at least 60° C., preferably at least at least 65°C and less than 80°C, preferably less than 70°C. In a particularly preferred embodiment, the aqueous solution containing 3'-SL and/or 6'-SL is spray dried at a nozzle temperature of from about 68°C to about 70°C.

It is understood that the 3'-SL and/or 6'-SL can be purified and spray dried separately, and that the resulting spray dried powders can be mixed in any desired ratio. In a further and/or alternative embodiment, separate aqueous solutions containing 3'-SL and/or 6'-SL, respectively, can be mixed in any desired ratio, and the resulting aqueous solution containing 3'-SL and/or 6'- SL in the desired ratio, can be spray dried. The ratio of 3'-SL and/or 6'-SL in the powder obtained by spray drying corresponds to the ratio of 3'-SL and/or 6'-SL in aqueous solution.

Spray drying of an aqueous solution containing 3'-SL and/or 6'-SL provides a powder of low hygroscopicity, where the 3'-SL and/or 6'-SL is in amorphous form, and where the particle size is uniform.

A third aspect provides the use of a spray-dried powder containing 3'-SL and/or 6'-SL that has been purified from the process stream to make a nutritional composition. The spray-dried powder essentially consisting of 3'-SL and/or 6'-SL is suitable for human consumption and can thus be included in preparations for human consumption such as medical compositions, infant formulas, milk drinks or biologically active additives.

According to a fourth aspect, nutritional compositions are provided that comprise a spray-dried powder as described in the first aspect or as prepared according to the second aspect.

In an additional and/or alternative embodiment, the nutritional composition contains at least one additional HMO that is neither a 3'-SL nor a 6'-SL. The at least one additional HMO may be a neutral HMO, preferably selected from the group consisting of 2'-fucosyllactose (2'-FL - from English 2'-fucosyllactose), 3-fucosyllactose (3-FL - from English 3 -fucosyllactose), lacto-N-tetraose (LNT - from the English lacto-N-tetraose), lacto-N-neotetraose (LNnT - from the English lacto-N-neotetraose) and lacto-N-fucopentaose I (LNFPI). In an additional and/or alternative embodiment, the at least one additional HGM may be a sialylated HGM, preferably selected from the group consisting of 3'-sialyllactose (3'-SL - 3'-sialyllactose), 6'-sialyllactose ( 6'-SL - from the English 6'-sialyllactose), sialyllacto-N-tetraose (LST - from the English sialyllacto-N-tetraose)-a, LST-b, LST-c and disialyllacto-N-tetraose (DSLNT - from the English disialyllacto-N-tetraose).

In an additional and/or alternative embodiment, the nutritional composition includes a mixture consisting essentially of Neu5Ac, 2'-FL, 3-FL, LNT, LNnT, LNFPI, 3'-SL, 6'-SL, sialic acid and L-fucose. A nutritional composition comprising preferred amounts of each of these components is provided in Table 1.

The composition according to the second column of Table 1 is particularly advantageous for supplementing infant formula such that the final infant formula for direct consumption may contain the formula compounds in concentrations as indicated in the third column of Table 1.

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

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 with functional monosaccharides or a mixture of human milk oligosaccharides with other fibers is an infant formula that meets the composition requirements set forth in Regulation (EU) 2016/127 and/or US Code of Federal Regulations section 21 107.100 (nutrient requirements). Typical compositions of infant formulas are shown in Tables 2 and 3.

Infant formula: Skim milk

Vegetable oils (palm oil, rapeseed oil, sunflower oil)

Breast milk oligosaccharides

3'-SL and/or 6'-SL

Skim milk powder

Mortierella alpine oil

Fish fat

Calcium carbonate

Potassium chloride

Vitamin C

Sodium chloride

Vitamin E

Iron acetate

Zinc sulfate

Niacin

Calcium d-pantothenate

Copper sulfate

Vitamin A

Vitamin B1

Vitamin B6

Magnesium sulfate

Potassium iodate

Folic acid

Vitamin K

Sodium Selenite

Vitamin D

In an additional and/or alternative embodiment, the nutritional composition also contains microorganisms, preferably probiotic microorganisms. For infant formula applications, the preferred microorganisms originate from or can be found in the microbiome of a healthy individual. Preferably, but without limitation, the microorganisms are selected from species of Bifidobacterium, Lactobacillus, Enterococcus, Streptococcus, Staphylococcus, Peptostreptococcus, Leuconostoc, Clostridium, Eubacterium, Veilonella, Fusobacterium, Bacterioides, Prevotella, Escherichia, Propionibacterium and Saccharomyces. In an additional and/or alternative embodiment, the microorganism is selected from the group consisting of Bifidobacterium adolescentis, B. animalis, B. bifidum, B. breve, B. infantis, B. lactis, B. longum; Enterococcus faecium; Escherichia coli; Klyveromyces marxianus; Lactobacillus acidophilus, L. bulgaricus, L. casei, L. crispatus, L, fermentum, L, gasseri, L, helveticus, L, johnsonii, L, paracasei, L. plantarum, L. reuteri, L. rhamnosus, L, salivarius , L. sakei; Lactococcus lactis (including but not limited to subspecies lactis, cremoris and diacetyiactis); Leuconostoc mesenteroides (including but not limited to mesenteroides subspecies); Pedicoccus acidilactici, P. pentosaceus; Propionibacterium acidipropionici, P. freudenreichii ssp.shermanii; Staphylococcus carnosus and Streptococcus thermophiius.

In addition to the combination of living organisms, the nutritional composition may also include cultures of non-living cells. In the probiotic field, killed cell cultures (eg, tindalized bacteria) are sometimes used. These killed cultures may provide proteins, peptides, oligosaccharides, outer cell wall fragments and natural products, resulting in short-term stimulation of the immune system.

The inclusion of probiotic microorganisms in a nutritional composition, especially in the presence of HGM, is of particular interest in that it also stimulates the establishment of a healthy intestinal microbiome.

In an additional and/or alternative embodiment, the nutritional composition also includes prebiotics such as galacto-oligosaccharides (GOS), fructo-oligosaccharide (FOS), inulin, or combinations thereof.

The nutritional composition is in solid form, including, but not limited to, powders, granules, flakes, pellets, or combinations thereof.

In a further embodiment, the nutritional composition is selected from the group consisting of medicinal products, infant formulas, dairy beverages and dietary supplements.

As a medicinal product, the nutritional composition can be used to improve cognitive performance, especially to improve attention, learning and/or memory.

The present invention will be described with respect to specific embodiments and with reference to drawings, but the invention is not limited thereto, but only by the claims. In addition, the terms first, second, and the like in the specification and claims are used to distinguish between like elements and not necessarily to describe sequence, or in time, or in space, or in arrangement, or in any other manner. It should be understood that the terms used in this manner are interchangeable in appropriate circumstances, and that the embodiments of the invention described herein are capable of operating in sequences other than those described or illustrated herein.

It should be noted that the term “comprising” as used in the claims should not be considered limited to the means listed below; it does not exclude other elements or stages. Thus, it is to be understood as specifying precisely the presence of the stated features, integers, steps or components referred to, but does not exclude the presence or addition of one or more other features, integers, steps or components or groups thereof. Thus, the scope of the expression “device comprising means A and B” should not be limited to devices consisting only of components A and B. This means that for the purposes of the present invention, A and B are only relevant components of the device.

Reference throughout this specification to “one embodiment” or “embodiment” means that the particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, appearances of the phrases “in one embodiment” or “in an embodiment” at different places throughout this specification do not necessarily always refer to the same embodiment. Moreover, specific features, structures, or characteristics may be combined in any suitable manner, as will be apparent to one of ordinary skill in the art based on this disclosure, in one or more embodiments.

Likewise, it should be understood that in the description of illustrative embodiments of the invention, various features of the invention are sometimes grouped together in one single embodiment, figure, or description thereof for the purpose of simplifying the disclosure and assisting in understanding one or more different aspects of the invention. This manner of disclosure should not be taken as indicating an intention that the claimed invention requires more features than are expressly listed in each claim. On the contrary, as evidenced by the claims below, aspects of the invention may require less than all of the features of any of the above disclosed embodiments. Thus, the claims following the detailed description are hereby expressly incorporated into this detailed description, with each claim standing by itself as a separate embodiment of the invention.

In addition, while some embodiments described herein include some, but not other, features included in other embodiments, it is understood that combinations of features of different embodiments are within the scope of the invention and form different embodiments, as will be appreciated by those skilled in the art. this area. For example, in the following claims, any of the claimed embodiments may be used in any combination.

In addition, certain embodiments are described herein as a method or combination of elements of a method that may be performed by a computer system processor or other means of performing a function. Thus, a processor with the necessary instructions for implementing such method or method element constitutes means for implementing the method or method element. In addition, the device embodiment described herein is an example of a means of implementing the function performed by the element for the purpose of carrying out the invention.

Many specific details are set forth in the descriptions and graphics provided herein. However, it will be understood that embodiments of the invention may be practiced without these specific details. In other examples, well-known methods, structures and techniques are not shown in detail in order to facilitate understanding of the specification and drawings.

The invention will now be described by way of detailed description of several embodiments of the invention. It is clear that other embodiments of the invention may be configured in accordance with the knowledge of those skilled in the art without departing from the true spirit or technical concept of the invention, the invention being limited only by the terms of the appended claims.

Example 1: Purification of 2'-fucosyllactose from fermentation broth The production of 2'-fucosyllactose by fermentation using a genetically modified E coli strain was carried out as described in European Patent Application No. 16 196 486.1. 2'-Fucosyllactose was purified from the fermentation broth by filtration, ion exchange chromatography, nanofiltration, diafiltration or electrodialysis and charcoal treatment as described in WO 2015/106943 A1. The resulting solution containing 2'-fucosyllactose was spray dried to obtain a stable solid product.

Example 2: Purification of 3-fucosyllactose from fermentation broth 3-Fucosyllactose was produced by fermentation using a genetically modified E. coli strain as described in European Patent Application No. 16 196 486.1.

Cells were separated from the culture medium by ultrafiltration (0.05 μm cutoff) (CUT membrane technology, Erkrath, Germany) followed by a 150 kDa MWCO cross-flow filter (Microdyn-Nadir, Wiesbaden, Germany). Cell-free fermentation medium containing approximately 30 g/L 3-fucosyllactose was passed through a strong cation exchanger (Lewatit S 2568 (Lanxess, Cologne, Germany) in the H ⁺ form to remove positively charged contaminants. The solution was then adjusted to pH 7 ,0, using sodium hydroxide, and it was applied to an anion exchanger (Lewatit S6368 A, Lanxess) in the form of chloride. Both ion exchangers were used in a volume of 200 L. After a second filtration (150 kDa; Microdyn-Nadir, Wiesbaden, Germany), the solution did not contain particles were concentrated 5-fold by nanofiltration using a Filmtech NF270 membrane (Dow, Midland, USA) and 2.5-fold by vacuum evaporation.The concentrated solution with a conductivity of approximately 15 mS/cm ^-1 was filtered (10 kDa; Microdyn- Nadir, Wiesbaden, Germany), purified by activated carbon (CAS:7440-44-0, Carl Roth, Karlsruhe, Germany) and deionized by electrodialysis using a PC-Cell BED 1-3 electrodialyzer (PC-Cell, Heusweiler, Germany) with a PC-Cell E200 membrane package containing the following membranes: cation exchange membrane CEM:PC SK and anion exchange membrane AEM:PCAcid60. 0.25 M aminosulfonic acid was used as an electrolyte in this method. To reduce the brownish coloration due to Maillard reactions and aldol products originating from the fermentation process, a second round of ion exchange chromatography was performed using the same ion exchange material as mentioned above in the form of Na ⁺ and Cl ^- , but in a volume of 50 L. After concentrating the Sugar solution by evaporation, the conductivity was again reduced from 4 mS/cm ^-1 to 0.4 mS/cm ^-1 or less by electrodialysis using the PC-Cell BED 1-3 mentioned earlier. For additional decolorization, the solution was mixed with activated carbon (CAS:7440-44-0, Carl Roth, Karlsruhe, Germany) and an almost colorless solution was obtained by filtration.

Example 3: Purification of lacto-N-tetraose from fermentation broth Fermentation production of lacto-N-tetraose was carried out using a strain of genetically modified E. coli BL21 (DE3) ΔlacZ with genes integrated into the genome necessary for the in vivo synthesis of lacto-N-tetraose, namely, the genes of N-acetylglucosamine glycosyltransferase (IgtA from Neisseria meningitidis MC58), (β-1,3-galactosyltransferase (wbdO from Salmonella enterica subspecies salamae serovar Greenside), lacY from E, coli K12, UDP (uridine diphosphate)-glucose-4-epimerase galE and UTP (uridine triphosphate)-glucose-1-phosphate uridyl transferase galU, both from E, coli K12. In addition, the galS gene encoding glucosamine-6-phosphate synthase was overexpressed. For fermentation production of lacto-N-tetraose, the strain was grown in a defined medium with mineral salts containing 2% glucose as a carbon source. Antifoaming agent was added if necessary. The pH was controlled using 25% ammonium solution. Lactose was added gradually to a final concentration of 15 mM from 216 g l ^-1 lactose stock, lactose concentration in The culture medium remained constant throughout the fermentation process. Residual lactose and lacto-N-triose II accumulated throughout the process as a by-product were hydrolyzed by a second strain of E. coli, which was added to the fermenter. This strain expressed a functional beta-lactamase, beta-N-acetylhexosaminidase (bbhl from Bifidobacterium bifidum JCM1254) and a functional gal operon for monosaccharide degradation (EP 2,845,905 A).

The cells were separated from the fermentation broth and the liquid containing lacto-N-tetraose was purified to 75-80% purity, determined by mass balance, according to the method described in Example 2.

Contaminating carbohydrate by-products resulting from inefficient enzymatic degradation and metabolization were removed by pseudo-moving bed (SMB) chromatography according to WO 2015/049331. Alternatively, lacto-N-tetraose was purified by isopropanol crystallization. For crystallization, the solution containing lacto-N-tetraose was concentrated by evaporation to a concentration of 20% and spray dried. Using a NUBILOSA LTC-GMP spray dryer (NUBILOSA, Konstanz, Germany), the solution was passed in a nitrogen stream through the spray dryer nozzles with an inlet temperature of 130°C, while controlling that the product stream maintained an outlet temperature of 67°C to 68°C .

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

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

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

The 3'-sialyllactose-producing strain carries 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 leiognathi JT-SHIZ-119.

Sialyl lactose-producing strains were grown in a defined mineral salt medium containing 2% sucrose as a carbon source. The sucrose feed (500 g L ^-1 ) supplied to the batch feeding phase was supplemented with 8 mM MgSO ₄ , 0.1 mM CaCl ₂ , trace elements and 5 g L ^-1 NH ₄ Cl .

To form sialyllactose, a lactose feed of 216 g l ^-1 was used. The pH was controlled using ammonium solution (25% v/v). Batch fermentation with feeding was carried out at 30°C with constant aeration and stirring. To remove residual lactose at the end of fermentation, β-galactosidase was added to the fermentation tank. The resulting monosaccharides were metabolized by the production strain.

The cell-free liquid was then deionized by ion exchange chromatography. First, cationic contaminants were removed in a 200 L strong cation exchanger (Lewatit S 2568 (Lanxess, Cologne, Germany) in the form of H ⁺ . Using NaOH, the pH of the resulting solution was adjusted to 7.0. In the second stage, anions and unwanted dyes were removed from solution using a strong anion exchanger Lewatit S 6368 S (Lanxess, Cologne, Germany) in chloride form. The ion exchanger had a bed volume of 200 L. Using a second filtration stage on a cross-flow filter (150 kDa cutoff) (Microdyn-Nadir, Wiesbaden, Germany ), precipitates resulting from acidification of the solution were removed. To concentrate the sugar, the solution was nanofiltered on a Dow FILMTECH NF270-4040 (Inaqua, Monchengladbach, Germany) or alternatively on a Trisep 4040-XN45-TSF membrane (0.5 kDa cutoff) ( Microdyn-Nadir, Wiesbaden, Germany) Using the latter, the monosaccharide N-acetylglucosamine resulting from the fermentation process and the contaminating sialyllactose solution were separated from the product. The concentrated sialyllactose solution was then treated with activated carbon (CAS:7440-44-0, Carl Roth, Karlsruhe, Germany) to remove dyes such as Maillard reaction products and aldol reaction products. To separate sialyllactose from by-products that arise from the fermentation process, like sialic acid and N-acetylglucosamine, the solution was filtered on a GE4040F30 membrane (GE water & process technologies, Ratingen, Germany) with a cut-off of 1 kDa, and diafiltrated to a conductivity of 0. 6 - 0.8 mS/cm ^-1 . The diluted solution was concentrated on a rotary evaporator to a concentration of approximately 300 g/L. The final chromatographic separation removed other contaminating sugars, like di-sialyllactose. To do this, the concentrated solution was applied to a weak anion exchange resin in the form of acetate (Amberlite FPA51, Dow Chemical, Michigan, USA). While sialyllactose rarely binds to the resin, di-sialyllactose is adsorbed. Thus, sialyllactose is eluted with 10 mM ammonium acetate, while di-sialyllactose is eluted with 1 M ammonium acetate. To remove ammonium acetate, sialyllactose was precipitated with a 10-fold excess of ethanol. The solid fraction was filtered and dried.

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

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

The spray-dried 6'-sialyllactose material was 91% pure, while the 3'-sialyllactose material was 93% pure.

Example 5: Preparation of OGM mixtures

OGM mixtures were obtained from solid products. To do this, spray drying of individual OGMs was carried out, and the powder was mixed. OGM-Mixture I contained 2'-fucosyllactose and lacto-N-tetraose in a ratio of 70% - 30%; OGM-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 form a 20% sugar solution and spray dried again using a Buchi spray dryer as described in Example 4.

Example 6: Characterization of Spray Dried Breast Milk Oligosaccharides

6.1 Differential scanning calorimetry (DSC)

Using differential scanning calorimetry (DSC) on a Mettler Toledo 821e (Mettler Toledo, Giessen, Germany), the thermal events of spray-dried human milk oligosaccharides, namely 3-fucosyllactose, 6'-sialyllactose, 3'-sialyllactose, lacto-N were determined -tetraose, and spray-dried mixtures of human milk oligosaccharides, mixture (OGM-Mixture I) of 2'-fucosyllactose/lacto-N-tetraose and mixture (OGM-Mixture II) of 2'-fucosyllactose, 3-fucosyllactose, lacto-N -tetraose, 6'-sialyllactose, 3'-sialyllactose, respectively.

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

Approximately 25 mg of spray-dried human milk oligosaccharides were analyzed in Al-fluted crucibles (Mettler Toledo, Giessen, Germany). The samples were cooled to 0°C at a rate of 10 K/min and reheated to 100°C at a scan rate of 10 K/min. After cooling the samples to 0°C in the second heating cycle, the samples were reheated to 150°C. The midpoint of the endothermic shift of the initial level during the heating scan was taken as the glass transition temperature (Tg). Exothermic and endothermic peaks are reported by the maximum temperature and normalized energy of a given event.

The first heating scan in all samples demonstrated a major glass transition event in the overall heat flux, as evidenced by a major step transition in the range of approximately 48-58°C for most samples, with the major glass transition event observed in the first heating scan occurring again in the second heating scan. The results of DSC analyzes are summarized in Table 4.

In the case of 3-fucosyllactose, an endothermic relaxation peak was detected after Tg in the first heating scan. In the case of lacto-N-tetraose, the ^2nd heating scan revealed a much higher Tg of approximately 79°C, compared to the Tg of other samples. This may have been caused by an endothermic event during the first heating scan at approximately 89°C (−6.04 J/g). As in the case of 3-fucosyllactose, also in the case of 6'-sialyllactose an endothermic relaxation peak was detected after Tg, however, in this sample an additional endothermic event occurred at 77°C (-0.22 J/g). No endothermic events were detected in the case of 3'-sialyllactose and OGM-Mixture I; in the case of OGM-Mixture II, the endothermic event during the ^1st heating scan occurred at 79°C (0.34 J/g).

6.2 X-ray powder diffraction (XRD)

Wide-angle X-ray powder diffraction (XRD) was used to study the morphology of lyophilized products. An Empyrean X-ray diffractometer (Panalytical, Almelo, the Netherlands) equipped with a copper anode (45 kV, 40 mA, K _α1 emission at 0.154 nm) and a PIXcel3D detector was used. Approximately 100 mg of spray dried samples were analyzed in reflective mode over an angular range of 5 to 45° 26 with a step size of 0.04° 29 and a counting time of 100 seconds/step.

All individual oligosaccharides, as well as OGM-Mixes I and II, showed a completely amorphous state (Fig. 1 - 6). In the case of lacto-N-tetraose, the second (amorphous) signal was detected at about 9-10°.

6.3 Laser diffraction

Powder particle size was assessed by laser diffraction. The system detects scattered and diffracted light through a series of concentrically arranged sensor elements. A software algorithm then approximates the number of particles by calculating z-scores of light intensity values that arrive at different sensor elements. The analysis was performed using the quantitative laser diffraction (qLD) system of the SALD-7500 aggregate calibration machine (Shimadzu Corporation, Kyoto, Japan).

A small amount (at the tip of a spatula) of each sample was dispersed in 2 ml of isooctane and homogenized by sonication for five minutes. The dispersion was transferred to a batch cell filled with isooctane and analyzed manually.

The data acquisition settings were as follows: number of signal averages per measurement: 128, number of signal rises: 3, and interval: 2 seconds.

Before measurements, the system was quenched with isooctane. Measurement on each sample dispersion was carried out 3 times, and the average values and standard deviation are presented. Data were assessed using WING SALD II software, version V3.1. Since the refractive index of the sample was unknown, the refractive index of the sugar (disaccharide) particles (1.530) was used to determine the particle size distribution profiles. Size values for mean and median diameters are presented.

The average particle sizes for all samples were very similar, with slightly lower values measured for OGM-Mixture II. The particle size characteristics are summarized in Table 5. In addition, the particle size distribution demonstrated the presence of one main size population for all samples.

Claims (27)

1. A spray-dried powder for the manufacture of a nutritional composition, wherein the spray-dried powder contains at least 80 wt.% 3'-sialyllactose (3'-SL) and/or 6'-sialyllactose (6'-SL), which was obtained by microbial fermentation. 2. The spray-dried powder according to claim 1, wherein said spray-dried powder contains at least 85 wt%, at least 90 wt%, at least 93 wt%, at least 95 wt%, or at least at least 98 wt.% 3'-SL and/or 6'-SL. 3. Spray-dried powder according to claim 1 or 2, wherein the 3'-SL and/or 6'-SL is in amorphous form. 4. Spray-dried powder according to any one of claims. 1-3, wherein said spray-dried powder contains 15 wt% water or less, preferably 10 wt% water or less, more preferably 7 wt% water or less, most preferably 5 wt% water or less. 5. Spray-dried powder according to any one of claims. 1-4, wherein said spray-dried powder does not contain genetically modified microorganisms and nucleic acid molecules derived from genetically modified microorganisms. 6. A method for producing spray-dried powder according to any one of claims. 1-5, including the following stages: a) purification of 3'-SL and/or 6'-SL from the fermentation broth; b) providing an aqueous solution containing 3'-SL and/or 6'-SL stages A); And c) subjecting the solution to step b) to spray drying. 7. The method according to claim 6, in which step a) purification of 3'-SL and/or 6'-SL from the fermentation broth includes one or more of the following steps: i) removing microbial cells from the fermentation broth to obtain a clarified process stream; ii) subjecting said clarified process stream to at least one ultrafiltration; iii) treating said clarified process stream at least once with a cation exchange resin and/or at least once with an anion exchange resin; iv) subjecting said clarified process stream to at least one nanofiltration and/or diafiltration; v) subjecting said clarified process stream to at least one electrodialysis; vi) treating said clarified process stream at least once with activated carbon; and/or vii) subjecting said clarified process stream at least once to crystallization and/or precipitation steps. 8. The method according to claim 6 or 7, in which the aqueous solution contains 3'-SL and/or 6'-SL 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). 9. Method according to any one of paragraphs. 6-8, wherein the aqueous solution containing 3'-SL and/or 6'-SL is spray dried at a nozzle temperature of at least 110°C, preferably at least 120°C, more preferably at least 125° C and less than 150°C, preferably less than 140°C and more preferably less than 135°C. 10. Method according to any one of paragraphs. 6-9, wherein the aqueous solution containing 3'-SL and/or 6'-SL 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. 11. Use of the spray-dried powder according to any one of claims. 1-5 for the manufacture of a nutritional composition, where the nutritional composition is an infant formula. 12. A nutritional composition comprising a spray-dried powder according to any one of claims. 1-5. 13. The nutritional composition of claim 12, further comprising at least one additional human milk oligosaccharide (HMO), wherein said at least one additional HMO is a neutral HMO or a sialylated HMO. 14. The nutritional composition according to claim 12 or 13, wherein said at least one neutral HGM is selected from the group consisting of 2'-fucosyllactose, 3-fucosyllactose, lacto- N -tetraose, lacto- N -neotetraose and lacto- N -fucopentaose I. 15. Nutritional composition according to any one of paragraphs. 12-14, wherein said at least one sialylated HGM is selected from the group consisting of 3'-sialyllactose, 6'-sialyllactose, sialyllacto -N -tetraose (LST)-a, LST-b, LST-c and disialyllacto- N -tetraose. 16. Nutritional composition according to any one of paragraphs. 12-15, wherein said nutritional composition contains at least one probiotic microorganism.