Spray dried sialyllactose
阅读说明：本技术 喷雾干燥的唾液酸乳糖 (Spray dried sialyllactose ) 是由 S·詹尼温 于 2018-12-07 设计创作，主要内容包括：公开的是一种用于制造基本上由3’-SL和/或6’-SL组成的喷雾干燥的粉末的方法、所述喷雾干燥的粉末、其用于制造营养组合物的用途,和含有该喷雾干燥的粉末的营养组合物。(Disclosed is a process for the manufacture of a spray-dried powder consisting essentially of 3 '-S L and/or 6' -S L, said spray-dried powder, its use for the manufacture of a nutritional composition, and a nutritional composition containing the spray-dried powder.)
1. A spray-dried powder consisting essentially of 3 '-S L and/or 6' -S L that has been produced by microbial fermentation.
2. The spray-dried powder of claim 1, wherein the spray-dried powder comprises at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, or at least 98% by weight of 3 '-S L and/or 6' -S L.
3. The spray-dried powder according to claim 1 or 2, wherein the 3 '-S L and/or 6' -S L is present in amorphous form.
4. The spray-dried powder according to any of claims 1 to 3, wherein the spray-dried powder comprises ≦ 15 wt.% water, preferably ≦ 10 wt.% water, more preferably ≦ 7 wt.% water, most preferably ≦ 5 wt.% water.
5. The spray-dried powder of any one of claims 1 to 4, wherein the spray-dried powder is free of genetically engineered microorganisms and nucleic acid molecules derived from genetically engineered microorganisms.
6. A method for manufacturing a spray-dried powder as defined in any one of claims 1 to 5, the method comprising the steps of:
a) purifying 3 '-S L and/or 6' -S L from the fermentation broth;
b) providing an aqueous solution comprising 3 '-S L and/or 6' -S L of step a), and
c) spray drying the solution of step b).
7. The process of claim 6, wherein the step of purifying 3 '-S L and/or 6' -S L from the fermentation broth (step a)) comprises 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 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 carbon; and/or
vii) subjecting the clarified process stream to at least one crystallization and/or precipitation step.
8. The method of claim 6 or 7, wherein the aqueous solution comprises the 3 '-S L and/or 6' -S L 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. The process according to any one of claims 6 to 8, wherein the aqueous solution comprising 3 '-S L and/or 6' -S L is spray dried at a nozzle temperature of at least 110 ℃, preferably at least 120 ℃, more preferably at least 125 ℃ and less than 150 ℃, preferably less than 140 ℃ and more preferably less than 135 ℃.
10. The process according to any one of claims 6 to 9, wherein the aqueous solution comprising 3 '-S L and/or 6' -S L is spray dried at an outlet temperature of at least 60 ℃, preferably at least 65 ℃, and lower than 80 ℃, preferably lower than 70 ℃.
11. Use of the spray-dried powder according to any one of claims 1 to 5 for the manufacture of a nutritional composition, preferably an infant formula.
12. A nutritional composition containing the spray-dried powder according to any one of claims 1 to 5.
13. The nutritional composition according to claim 12, further comprising at least one additional HMO, wherein the at least one additional HMO is a neutral HMO or a sialylated HMO.
14. Nutritional composition according to claim 12 or 13, wherein the at least one neutral HMO 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 claims 12 to 14, wherein the at least one sialylated HMO is selected from the group consisting of 3 '-sialyllactose, 6' -sialyllactose, sialyl yoghurt-N-tetraose (L ST) -a, L ST-b, L ST-c and disialyllacto-N-tetraose.
16. The nutritional composition according to any one of claims 12 to 15, wherein the nutritional composition comprises at least one probiotic microorganism.
Background
In addition to lactose, one liter of human breast milk also contains oligosaccharides up to 20 g/L, the so-called "Human Milk Oligosaccharides (HMOs)", HMOs represent the third most abundant component in human breast milk it is speculated that there are more than 150 structurally distinct oligosaccharides in human milk typically contains 10 to 13 major HMOs, which are present at concentrations of a few grams to a few hundred milligrams per liter (Thurl et al, (2017), Nutrition Reviews 75 (920) and 933), HMOs include neutral HMOs and acidic HMOs containing one or more sialic acid moieties the structure and complexity of these oligosaccharides in most milk is characteristic of human milk as shown in table 1, while none of the other mammals (e.g. dairy stock) are found.
The beneficial effects of sialylated HMOs on neonatal brain development have therefore been discussed extensively (reviewed in "cosmetics and Probiotics in Human man, organics and carbohydrates of the same microorganisms and bacteria, and research on the physiological effects of these sugars, Academic: Guirea, and Guirea, edited by McMcMcMcJ, 2017, and Guirea, L).
The most abundant sialylated HMOs are 3 ' -sialyllactose (NeuAc (a2, 3) Gal (β 1, 4) Glc) and 6 ' -sialyllactose (NeuAc (α 2, 6) Gal (β 1, 4) Glc). in 3 ' -sialyllactose, the monosaccharide residue N-acetylneuraminic acid is attached at position 3 to the galactosyl moiety of lactose 3 ' -sialyllactose (3 ' -S L) binds avidly (avidly) to several viral strains, including influenza, HIV-1, reovirus and polyomavirus, and can thus alter the viral infectivity.3 ' -sialyllactose also affects the bacterial colonization of the gut.in 6 ' -sialyllactose (6 ' -S L), the residue N-acetylneuraminic acid is attached at position 6 to the galactosyl moiety of lactose 6 ' -sialyllactose.
The first step in the beneficial effects of HMOs on artificially fed infants is the addition of HMOs alone to the infant formula. However, it would be better to supplement infant formulas with a combination of structurally different HMOs, as the combination of structurally different HMOs would have an effect that is more similar to its original source (human milk) and cannot be produced by HMOs alone.
The limited supply of HMOs alone for supplementation of infant formula has led first to the development of chemical synthesis of HMOs, followed by the development of biocatalytic methods using purified enzymes. Fermentation of genetically engineered bacterial cells is now used to produce different HMOs on a commercial scale (WO 2015/150328 a1, WO 2017/043382 a1, WO 2010/070104 a1, WO 2012/097950 a 1). HMOs synthesized by bacterial cells can be purified from fermentation broths or cell lysates to produce substantially pure HMO preparations, making them useful in human foods, especially infant foods.
During its purification, 3 '-S L and/or 6' -S L are typically present in the form of a liquid process stream, with purification, the concentration of 3 '-S L and/or 6' -S L in the process stream increases, however, aqueous 3 '-S L and/or 6' -S L solutions are very susceptible to bacterial or fungal contamination.
Typically, the sugar is obtained in solid form by crystallization. The crystallization of HMO alone has been described as follows: 3-fucosyllactose (WO 2014/075680A), 2' -fucosyllactose (WO 2011/150939A), difucosyllactose (WO 2016/086947A), lacto-N-tetraose (WO 2017/101953A), lacto-N-neotetraose (WO 2014/094783A). Crystallization of HMOs involves the use of organic solvents, such as alcohols, primarily ethanol or methanol; or an organic acid such as glacial acetic acid. However, if HMO is to be used as a food ingredient, the use of an organic solvent to crystallize HMO is not suitable as a final step in the process to obtain the final product in solid form. In addition, organic solvents are harmful to the environment and to any individual handling them. Therefore, the use of organic solvents requires occupational safety measures and proper disposal, which makes the use of organic solvents costly. Therefore, crystallizing HMO to provide HMO in solid form must be considered a disadvantage in the production of HMO on an industrial scale.
Therefore, there is a need for a process to provide 3 '-S L and/or 6' -S L in solid form, which is suitable for use in the industrial scale production of these HMOs and does not involve the use of organic solvents at the end of the purification scheme for providing the solid preparation of said HMOs.
This problem is solved by a process for providing a powder consisting essentially of 3 '-S L and/or 6' -S L, wherein the process comprises spray drying an aqueous solution comprising 3 '-S L and/or 6' -S L.
Disclosure of Invention
In a first aspect, a method is provided that provides a spray-dried powder consisting essentially of 3 '-S L and/or 6' -S L.
In a second aspect, a method is provided for making a spray-dried powder consisting essentially of 3 '-S L and/or 6' -S L.
In a third aspect, there is provided the use of a spray dried powder consisting essentially of 3 '-S L and/or 6' -S L for the manufacture of a nutritional composition.
In a fourth aspect, a nutritional composition is provided comprising a spray-dried powder consisting essentially of 3 '-S L and/or 6' -S L.
Drawings
Figure 1 shows a graph illustrating the X-ray powder diffraction results of spray dried 3-fucosyllactose.
Figure 2 shows a graph illustrating the X-ray powder diffraction results for spray dried lacto-N-tetraose.
FIG. 3 shows a graph illustrating the X-ray powder diffraction results of spray dried 6' -sialyllactose.
Figure 4 shows a graph illustrating the X-ray powder diffraction results of spray dried 3' -sialyllactose.
Figure 5 shows a graph illustrating the X-ray powder diffraction results for a spray-dried mixture of 2' -fucosyllactose and lacto-N-tetraose.
Figure 6 shows a graph illustrating the X-ray powder 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, there is provided a spray-dried powder consisting essentially of 3 '-S L and/or 6' -S L produced by microbial fermentation.
As described herein below, the term "consisting essentially of.. means that the spray-dried powder consists of 3 '-S L and/or 6' -S L and, optionally, byproducts produced during fermentation of a microorganism used to produce 3 '-S L and/or 6' -S L but cannot be removed from the process stream resulting from fermentation of the microorganism.
In an additional and/or alternative embodiment, the 3 '-S L and/or 6' -S L is present in the spray-dried powder in amorphous form.
In an additional and/or alternative embodiment, the spray-dried powder comprises ≦ 15 wt.% water, preferably ≦ 10 wt.% water, more preferably ≦ 7 wt.% water, most preferably ≦ 5 wt.% water.
In an additional and/or alternative embodiment, 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 process for the manufacture of a spray-dried powder consisting essentially of 3 '-S L and/or 6' -S L, said 3 '-S L and/or 6' -S L having been produced by microbial fermentation, the process comprising the steps of:
a) purifying 3 '-S L and/or 6' -S L from the fermentation broth;
b) providing an aqueous solution of 3 '-S L and/or 6' -S L of step a), and
c) spray drying the solution of step b).
In an additional and/or alternative embodiment, the purification of 3 '-S L and/or 6' -S L from the fermentation broth comprises 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 to at least one crystallization and/or precipitation step.
3 '-S L and/or 6' -S L can be produced by fermentation of a microorganism wherein a genetically engineered microorganism capable of synthesizing 3 '-S L and/or 6' -S L is cultured in a culture medium (fermentation broth) and under conditions permitting synthesis of 3 '-S L and/or 6' -S L by said genetically engineered microorganism purification of 3 '-S L and/or 6' -S L produced by fermentation of a microorganism comprises the step of separating microbial cells from the fermentation broth to obtain a clarified process stream substantially free of cells and containing 3 '-S L and/or 6' -S L, which step is the first step in the process for purifying the desired oligosaccharide.
Suitable methods for isolating microbial cells from a fermentation broth comprise centrifugation, wherein the microbial cells are obtained in pellet form and the fermentation broth is obtained in supernatant form. In an additional and/or alternative embodiment, the microbial cells are isolated from the fermentation broth by filtration. Suitable filtration methods for separating microbial cells from the fermentation broth include microfiltration and ultrafiltration.
Microfiltration is itself a physical filtration process in which a fluid containing particles is passed through a membrane of a particular pore size to separate the particles from the fluid. As used herein, the term "microfiltration" refers to a physical filtration process in which cells are separated from a fermentation broth.
Ultrafiltration is a series of membrane filtrations and is not fundamentally different. In ultrafiltration, forces such as pressure or concentration gradients cause separation across a semi-permeable membrane. Cells, suspended solids and high molecular weight solutes remain 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).
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 (dead-end mode).
Suitable filters for microfiltration or ultrafiltration areDS MP0054333 and fiber FS10-FC FUS1582(Microdyn-Nadir GmbH, Wiesbaden, Germany).
Typically, microbial cells synthesize 3 '-S L and/or 6' -S L intracellularly and secrete it into the fermentation broth 3 '-S L and/or 6' -S L thus produced is finally in the fermentation broth, which is then subjected to further process steps for purification of 3 '-S L and/or 6' -S L, as described later herein, provided that 3 '-S L and/or 6' -S L is present in the fermentation broth at the end of the fermentation, the fermentation broth becomes a clarified process stream after the cells have been removed, provided that all or some of the 3 '-S L and/or 6' -S L remain intracellularly, the cells can be removed from the fermentation broth and lysed.
Although this method is used for the purification of 3 '-S L and/or 6' S L that has been produced by microbial fermentation, the method may also be employed to purify 3 '-S L and/or 6' -S L produced by in vitro enzymatic catalysis, 3 '-S L and/or 6' -S L may be purified from the reaction mixture at the end of the biocatalytic reaction, the reaction mixture being subjected to the purification method as a clarified process stream.
The clarified process stream contains 3 '-S L and/or 6' -S L as well as byproducts and unwanted impurities such as, for example, monosaccharides, disaccharides, unwanted oligosaccharide byproducts, ions, amino acids, polypeptides, proteins, and/or nucleic acids.
In an additional and/or alternative embodiment, the method for purifying 3 '-S L and/or 6' -S L comprises at least one cation exchange treatment step.
Suitable cation exchange resins for removing positively charged compounds include L ewatit S2568(H +) (L anxess AG, Cologne, DE).
In an additional and/or alternative embodiment, the method for purifying 3 '-S L and/or 6' -S L includes the step of anion exchange treatment to remove unwanted negatively charged compounds from the clarified process stream.
Suitable anion exchange resins include L ewatit S6368A, L ewatit S4268, L ewatit S5528, L ewatit S6368A (L anxess AG. Cologne, DE), Dowex AG 1x2(200-400 mesh), Dowex 1x8(100-200 mesh), Purolite Chromalite CGA 100x4(Purolite GmbH, Ratinogen, DE), Dow Amberlite FPA51(Dow Chemicals, Ml, USA).
In an additional and/or alternative embodiment, the process for purifying 3 '-S L and/or 6' -S L comprises a nanofiltration and/or diafiltration step to remove impurities with lower molecular weight and concentrate the desired oligosaccharides.
Diafiltration involves the addition of fresh water to the solution to remove (wash out) membrane-permeable components. Diafiltration may be used to separate components by using a suitable membrane, based on the molecular size and charge of the components, in which one or more substances are effectively retained, while other substances are permeable by the membrane. In particular, diafiltration using nanofiltration membranes is effective for separating low molecular weight compounds such as small molecules and salts. The molecular weight cut-off of the nanofiltration membrane is typically 150-. Nanofiltration is widely used in the dairy industry for the concentration and demineralization of whey.
Suitable membranes for nanofiltration and/or diafiltration include Dow Filmtec NF270-4040, Trisep4040-XN45-TSF (Microdyn-Nadir GmbH, Wiesbaden, DE), GE4040F30, and GH4040F50(GE Water & Process Technologies, Ratingen, DE).
It has been found that diafiltration using nanofiltration membranes is effective as a pretreatment to remove large amounts of contaminants prior to electrodialysis treatment of the oligosaccharide containing solution. The use of nanofiltration membranes for concentration and diafiltration during HMO purification results in lower energy consumption and processing costs and better product quality due to reduced thermal exposure, which results in reduced Maillard reactions and aldol condensation reactions.
In an additional and/or alternative embodiment, the process for purifying 3 '-S L and/or 6' -S L comprises at least one electrodialysis step.
Electrodialysis (ED) combines dialysis and electrolysis and can be used for separation or concentration of ions in solution based on the selective electromigration of ions through a semi-permeable membrane.
The basic principle of electrodialysis consists of an electrolytic cell comprising a pair of electrodes immersed in an electrolyte for conducting ions and connected to a direct current generator. The electrode connected to the positive pole of the dc generator is the anode and the electrode connected to the negative pole is the cathode. The electrolyte solution then supports the current flow, which is generated as negative and positive ions move toward the anode and cathode, respectively. The membranes used for electrodialysis are essentially porous ion-exchange resin sheets with negative or positive charged groups and are therefore described as cationic or anionic membranes, respectively. Ion exchange membranes are typically made from polystyrene with suitable functional groups (such as sulfonic acid for cationic membranes, or quaternary ammonium groups for anionic membranes) crosslinked with divinylbenzene. The electrolyte may be, for example, sodium chloride, sodium acetate, sodium propionate, or sulfamic acid. The electrodialysis stack (electrodialysis stack) is then assembled in such a way that the anion and cation membranes are parallel in the filter press between two electrode blocks, so that the stream undergoing ion depletion is well separated from the stream undergoing ion enrichment (the two solutions are also called diluate (undergoing ion depletion) and concentrate (undergoing ion enrichment)). The core of the electrodialysis process is a membrane stack consisting of several anion and cation exchange membranes separated by spacers and mounted between two electrodes. By applying a direct current, anions and cations will migrate across the membrane towards the electrodes.
In an additional and/or alternative embodiment, the process for purifying 3 '-S L and/or 6' -S L further comprises the step of continuous chromatography, such as Simulated Moving Bed (SMB) chromatography.
Simulated Moving Bed (SMB) chromatography originates from the petrochemical and mineral industries. Today, the pharmaceutical industry uses SMB chromatography to separate enantiomers from racemic mixtures. Large scale SMB chromatography has been used to separate the monosaccharide fructose from fructose-glucose solutions, as well as the disaccharide sucrose from sugar beet or cane sugar slurries.
The SMB process for the isolation of sugars uses, for example, crosslinked polystyrene resins of the calcium ion type, anionic resins in the bisulfite form (bechtold m. et al, Chemie ingnieur Technik, 2010, 82, 65-75), or polystyrene gel strong acid cationic resins in the hydrogen form (Purolite PCR833H) (Purolite, Bala Cynwyd, USA).
SMB systems are in principle scalable to achieve throughput of several hundred tons, given the continuous mode of operation, recovery of mobile phase and the potential to use large size chromatography columns.
An advantage of the process step of simulated moving bed chromatography is that it allows for further removal of oligosaccharides structurally closely related to the desired oligosaccharide.
In an additional and/or alternative embodiment, the process for purifying 3 '-S L and/or 6' -S L includes treating the process stream with activated carbon to remove contaminant materials, such as pigments, from the process stream.
In an additional and/or alternative embodiment, the process for purifying 3 '-S L and/or 6' -S L includes at least one step of crystallizing or precipitating 3 '-S L and/or 6' -S L from a clarified process stream crystallization or precipitation of 3 '-S L and/or 6' -S L from a process stream may be carried out by adding an appropriate amount of a water-miscible organic solvent to the process stream containing 3 '-S L and/or 6' -S L1To C6Alcohol and C1To C4An acid of carbon.
In an additional and/or alternative embodiment, the method for purifying 3 '-S L and/or 6' -S L comprises a step of sterile filtration and/or endotoxin removal, which preferably filters the process stream through a 3kDa filter or a 6kDa filter.
In an additional and/or alternative embodiment, the method for purifying 3 '-S L and/or 6' -S L includes the step of increasing the concentration of 3 '-S L and/or 6' -S L in the process stream the concentration of 3 '-S L and/or 6' -S L in the process stream can be increased by subjecting the process stream to vacuum evaporation, reverse osmosis, or nanofiltration (e.g., with a membrane having one or more pores that are filled with a solvent, such as water, ethanol, or a combination thereof)Alternatively, the crystallized or precipitated 3 '-S L and/or 6' -S L is dissolved in water to obtain a solution of 3 '-S L and/or 6' -S L with the desired concentration of 3 '-S L and/or 6' -S L.
In an additional and/or alternative embodiment, the resulting process stream is an aqueous solution comprising 3 '-S L and/or 6' -S L at a concentration of ≥ 20 g/L, ≥ 25 g/L, ≥ 30 g/L, ≥ 40 g/L, ≥ 60 g/L, ≥ 100 g/L, ≥ 200 g/L or even ≥ 300 g/L.
In an additional and/or alternative embodiment, the aqueous solution comprises 3 '-S L and/or 6' S L in a purity of at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, or at least 98% relative to the weight of dry matter/solute in the solution.
The resulting concentrate containing purified 3 '-S L and/or 6' -S L can be stored under appropriate conditions.
The process for purifying 3 '-S L and/or 6' -S L is cost-effective and easily scalable, making it suitable as a basis for a multi-ton scale manufacturing process.
The method for purifying 3 '-S L and/or 6' -S L is also advantageous because the aqueous solution is free of genetically engineered microorganisms and nucleic acid molecules derived from genetically engineered microorganisms.
The method for manufacturing a spray-dried powder comprises the step of providing an aqueous solution comprising 3 '-S L and/or 6' -S L.
In an additional and/or alternative embodiment, the aqueous solution comprises 3 '-S L and/or 6' -S L 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 comprises 3 '-S L and/or 6' S L in a purity of at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, or at least 98% relative to the weight of dry matter/solute in the solution.
In an additional and/or alternative embodiment, the aqueous solution does not comprise genetically engineered microorganisms, nucleic acid molecules derived from genetically engineered microorganisms, and proteins.
In a method for producing a spray-dried powder, an aqueous solution containing 3 '-S L and/or 6' -S L is spray-dried.
Spray drying is a method of obtaining a dry powder, in which a solution containing the target substance (i.e. 3 '-S L and/or 6' -S L) is first sprayed into droplets, which are rapidly dried by hot air.
In an additional and/or alternative embodiment, the aqueous solution containing 3 '-S L and/or 6' -S L that has been purified from the fermentation broth or process stream is spray dried at a nozzle temperature of at least 110 ℃, preferably at least 120 ℃, more preferably at least 125 ℃, and less than 150 ℃, preferably less than 140 ℃ and more preferably less than 135 ℃.
In an additional and/or alternative embodiment, the aqueous solution containing 3 '-S L and/or 6' -S L that has been purified from the fermentation broth or process stream is spray dried at an outlet temperature of at least 60 ℃, preferably at least 65 ℃, and less than 80 ℃, preferably less than 70 ℃.
In an additional and/or alternative embodiment, the individual aqueous solutions containing 3 '-S L and/or 6' -S L, respectively, can be mixed in any desired ratio and the resulting aqueous solution containing 3 '-S L and/or 6' -S L in the desired ratio can be dry sprayed.
Spray drying of an aqueous solution containing 3 '-S L and/or 6' -S L provides a low hygroscopic (hygroscopy) powder in which 3 '-S L and/or 6' -S L are present in amorphous form and in which the particle size is uniform.
Spray-dried powders consisting essentially of 3 '-S L and/or 6' -S L are suitable for human consumption and may therefore be included in preparations for human consumption, such as pharmaceutical preparations, infant formulas, milk drinks or dietary supplements.
According to a fourth aspect, there is provided a nutritional composition containing a spray-dried powder as described in the first aspect or manufactured according to the second aspect.
In an additional and/or alternative embodiment, the nutritional composition contains at least one further HMO, said further HMO not being 3 '-S L and/or 6' -S L the at least one further HMO may be a neutral HMO, preferably selected from the group consisting of 2 '-fucosyllactose (2' -F L0), 3-fucosyllactose (3-F L1), lacto-N-tetraose (L2 NT), lacto-N-neotetraose (L NnT), and lacto-N-fucopentaose I (L NFPI). in an additional and/or alternative embodiment the at least one further HMO may be a sialylated HMO, preferably selected from the group consisting of 3 '-sialyllactose (3' -S L), 6 '-sialyllactose (6' -S L), sialylyogurt-N-tetraose (L ST) -a, L ST-b, sialyl L ST-c and disialyllactose-N-tetraose (DS L).
Table 1: compositions containing exemplary mixtures suitable as supplements for infant formula
In an additional and/or alternative embodiment, the nutritional composition includes a mixture consisting essentially of Neu5Ac, 2 ' -F L, 3-F L, L NT, L NnT, L NFPI, 3 ' -S L, 6 ' -S L, sialic acid, and L-fucose the nutritional composition including preferred amounts of each of the compounds is provided in table 1.
The composition of the second column of table 1 is particularly advantageous for supplementing infant formulas so that the final infant formula for direct consumption may contain the compounds of the mixture at the concentrations specified in the third column of table 1.
In an additional and/or alternative embodiment, the nutritional composition contains one or more additional ingredients. The one or more other ingredients are selected from oils, fats and fatty acids (e.g. olive oil, sunflower oil, coconut oil, nut oil, rapeseed oil, palm oil, linseed oil, fish oil, linolenic acid, soybean oil, etc.), carbohydrates (e.g. glucose, fructose, lactose, maltodextrin, starch, sucrose, inositol, etc.), proteins (from skimmed milk, whey, casein (from any domestic 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, fluorine, selenium, iodine, copper).
In a preferred embodiment, the nutritional composition comprising spray dried human milk oligosaccharides, or a mixture of human milk oligosaccharides and functional monosaccharides, or a mixture of human milk oligosaccharides and other fibres is an infant formula meeting the compositional requirements specified in regulation (eu)2016/127 and/or Code of Federal Regulations (USA) Title 21107.100 (nutritional specifications). Table 2 and table 3 specifically indicate representative compositions of infant formula.
Infant formula: skimmed milk
Vegetable oil (palm oil, rapeseed oil, sunflower oil)
Human milk oligosaccharides
LNFPI
Defatted milk powder
Mortierella alpina (Mortierella alpina) oil
Fish oil
Calcium carbonate
Potassium chloride
Vitamin C
Sodium chloride
Vitamin E
Ferric acetate
Zinc sulfate
Nicotinic acid
D-calcium pantothenate
Copper sulfate
Vitamin A
Vitamin B1
Vitamin B6
Magnesium sulfate
Potassium iodate
Folic acid
Vitamin K
Sodium selenite
Vitamin D
Table 2: components of exemplary infant formulas.
Table 3: composition of exemplary infant formula. The final concentration is based on a formulation of 13.5g of powder in 90ml of water.
In an additional and/or alternative embodiment the nutritional composition further comprises a microorganism, preferably a probiotic microorganism, preferably a microorganism from the group of microorganisms of healthy people or may be found in the group of microorganisms of healthy people, preferably but not limited to, the microorganisms are selected from the group consisting of Bifidobacterium (Bifidobacterium), Lactobacillus (L bacteria), Enterococcus (Enterococcus), Streptococcus (Streptococcus), Staphylococcus (Staphylococcus), Streptococcus digestions (Peptostreptococcus), Leuconostoc (L gluconostoc), Clostridium (Clostridium), Eubacterium (Eubacterium), Veilonella), Clostridium (Fusobacterium), Anaerobacterium anaerobacterium (Bacillus), Propionibacterium (Lactobacillus), Propionibacterium (Lactobacillus), Lactobacillus plantarum (Lactobacillus), Lactobacillus paracasei), Lactobacillus (Lactobacillus), Lactobacillus paracasei (Lactobacillus), Lactobacillus paracasei (Lactobacillus paracasei), Lactobacillus paracasei (Streptococcus faecalis), Lactobacillus paracasei (Lactobacillus paracasei), Lactobacillus (Streptococcus faecalis, Lactobacillus), Lactobacillus (Streptococcus, Lactobacillus), Lactobacillus strain (Streptococcus, Lactobacillus strain (Streptococcus, Lactobacillus), Lactobacillus strain (Streptococcus faecalis, Lactobacillus), Lactobacillus strain (Streptococcus strain, Lactobacillus), Lactobacillus strain (Streptococcus strain, Lactobacillus strain (Streptococcus strain), Lactobacillus strain (Streptococcus strain), Lactobacillus strain (Streptococcus strain), Lactobacillus strain (Streptococcus strain), Lactobacillus strain (Streptococcus strain), Lactobacillus strain (Streptococcus strain), Lactobacillus strain (Streptococcus strain), Lactobacillus strain (Streptococcus strain), Lactobacillus strain (Streptococcus strain), Lactobacillus strain (Streptococcus strain), Lactobacillus strain (368. strain), Lactobacillus strain (Streptococcus strain), Lactobacillus strain (Streptococcus strain), Lactobacillus strain (Streptococcus), Lactobacillus strain (Streptococcus), Lactobacillus strain (Streptococcus strain (367. strain (Streptococcus), Lactobacillus strain (Corynebacterium), Lactobacillus strain.
In addition to combining living organisms, the nutritional composition may also include dead cell cultures. In the field of probiotics, killed cell cultures (e.g. intermittently killed (tyndalized) bacteria) are sometimes used. These killed cultures can provide proteins, peptides, oligosaccharides, extracellular wall fragments and natural products, resulting in short term stimulation of the immune system.
The inclusion of probiotic micro-organisms in the nutritional composition is particularly advantageous, especially in the presence of HMOs, as it also promotes the establishment of a healthy gut microbiome.
In an additional and/or alternative embodiment, the nutritional composition further comprises prebiotics, such as galacto-oligosaccharides (GOS), fructo-oligosaccharides (FOS), inulin, or combinations thereof.
The nutritional composition is in a solid form including, but not limited to, a powder, granules, flakes, pellets, or a combination thereof.
In another embodiment, the nutritional composition is selected from the group consisting of a pharmaceutical formulation, an infant formula, a milk drink, and a dietary supplement.
As pharmaceutical preparations, the nutritional compositions may be used for improving cognitive abilities, in particular for improving attention, learning and/or memory.
The present invention will be described with respect to particular embodiments and with reference to certain drawings but the invention is not limited thereto but only by the claims. Furthermore, the terms "first," "second," and the like in the description and in the claims, are used for distinguishing between similar elements and not necessarily for describing a sequential or chronological order. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments of the invention described herein are capable of operation in other sequences than described or illustrated herein.
It is to be noticed that the term 'comprising', used in the claims, should not be interpreted as being limitative to the means listed thereafter; it does not exclude other elements or steps. It is thus to be interpreted as specifying the presence of the stated features, integers, steps or components as referred to, but does not preclude 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 the devices a and B" should not be limited to devices consisting of only the components a and B. This means that, in the sense of the present invention, the only relevant components of the device are a and B.
Reference throughout this specification to "one or a (a) embodiment" means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, the appearances of the phrase "in one embodiment" appearing in various places throughout the specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner, as would be apparent to one of ordinary skill in the art from this disclosure, in one or more embodiments.
Similarly, it should be appreciated that in the description of representative embodiments of the invention, various features of the invention are sometimes grouped together in a single embodiment, figure, or description thereof for the purpose of streamlining the disclosure and aiding in the understanding of one or more of the various inventive aspects. The methods of the present disclosure should not be construed as reflecting the intent: the claimed invention requires more features than are expressly recited in each claim. Rather, as the following claims reflect, inventive aspects may lie in less than all features of any foregoing disclosed embodiment. Thus, the claims following the detailed description are hereby expressly incorporated into this detailed description, with each claim standing on its own as a separate embodiment of this invention.
Furthermore, although some embodiments described herein include some features but not other features included in other embodiments, combinations of features of different embodiments are intended to fall within the scope of the invention and form different embodiments, as will be understood by those familiar with the art. For example, in the following claims, any of the claimed embodiments may be used in any combination.
Furthermore, some embodiments are described herein as a method or combination of elements of a method that can be performed by a processor of a computer system or by other means of performing functions. Thus, a processor with the necessary instructions for performing the method or elements of the method forms a tool for performing the method or elements of the method. Further, the elements of the apparatus embodiments described herein are examples of means for performing the functions performed by the elements for carrying out the objects of the invention.
In the description and drawings provided herein, numerous specific details are set forth. However, it is understood that embodiments of the invention may be practiced without these specific details. In other instances, well-known methods, structures and techniques have not been shown in detail in order not to obscure the understanding of this description and the drawings.
The present invention will now be described by a detailed description of several embodiments of the invention. It will be apparent that other embodiments of the invention can be set forth according to the knowledge of a person skilled in the art without departing from the true spirit or technical advantages of the invention, which is limited only by the claims appended hereto.
Example 1: purification of 2' -fucosyllactose from fermentation broths
2' -fucosyllactose is produced by fermentation with a genetically modified strain of Escherichia coli (E.coli) as described in European patent application 16196486.1. The 2' -fucosyllactose is purified from the fermentation broth by filtration, ion exchange chromatography, nanofiltration, diafiltration or electrodialysis, and treatment with charcoal (charpore), as described in WO 2015/106943 a 1. The resulting solution containing 2' -fucosyllactose was spray dried to obtain a stable solid product.