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PROCESS FOR THE PURIFICATION OF L-FUCOSE FROM A FERMENTATION BROTH

20200354760 ยท 2020-11-12

    Inventors

    Cpc classification

    International classification

    Abstract

    The present invention concerns an efficient way to isolate L-fucose from a fermentation broth. The L-fucose contained in the fermentation broth is produced by microbial fermentation (bacterial or yeasts). The inventive process comprises a step of removing biomass from the fermentation broth, a step of subjecting the resulting solution to at least one of a cationic ion exchanger treatment and an anionic ion exchanger treatment and a step of removing salts after the ion exchanger treatment. The process can provide L-fucose in powder form, in granulated form as well as in form of L-fucose crystals.

    Claims

    1. A process for the purification of L-fucose from a fermentation broth, comprising: removing biomass from a fermentation broth comprising L-fucose, wherein a clarified solution is provided, providing a purified solution by subjecting the clarified solution to a cationic ion exchanger treatment and an anionic ion exchanger treatment; and removing salt from the purified solution by electrodialysis and/or nanofiltration.

    2. The process according to claim 1, wherein the cationic ion exchanger treatment is performed under conditions in which L-fucose passes the cationic ion exchanger material and is present in the flow-through and wherein the anionic ion exchanger treatment is performed under conditions in which L-fucose passes the anionic exchanger material and is present in the flow-through.

    3. The process according to claim 1, wherein the biomass i) is removed from the fermentation broth by centrifugation and/or filtration, wherein the filtration is optionally selected from the group consisting of microfiltration, ultrafiltration, cross-flow filtration, diafiltration and combinations thereof; and/or ii) comprises cells which produce L-fucose, optionally bacterial cells, optionally recombinant bacterial cells, optionally recombinant E. coli cells, recombinant Bacillus sp. cells and/or recombinant Corynebacterium sp. cells, especially recombinant Bacillus subtilis and/or recombinant Bacillus megaterium.

    4. The process according to claim 1, wherein the cationic ion exchanger treatment, a strong cationic ion exchanger is used and/or in the anionic ion exchanger treatment, a strong anionic exchanger is used.

    5. The process according to claim 1, wherein the cationic ion exchanger treatment is performed to remove unspecific cations and replace them by specific cations, optionally to replace them by the specific cation H.sup.+ or Na.sup.+, wherein if the unspecific cations are replaced by H.sup.+, the pH of the flow-through is optionally adjusted to a pH of 6 to 8 before performing a further treatment, optionally by addition of NaOH to the flow-through.

    6. The process according to claim 1, wherein the anionic exchanger step is performed to remove unspecific anions and replace them by specific anions, optionally by the specific anion Cl.sup. or OH.sup.,wherein if the unspecific anions are replaced by Cl.sup., the pH of the flow-through is optionally adjusted to a pH of 6 to 8 before performing a further treatment, optionally by addition of NaOH to the flow-through.

    7. The process according to claim 1, wherein conditions under which the L-fucose passes the anionic exchanger material and cationic exchanger material are established by adjusting the pH and/or salt concentration of the clarified solution, optionally by adjusting the pH of the clarified solution to a pH in the range of 6 to 8.

    8. The process according to claim 1, wherein the cationic ion exchanger treatment and the anionic ion exchanger treatment, the purified solution comprises the L-fucose, one or more color-giving substances and salt, wherein the salt is optionally NaCl.

    9. The process according to claim 1, wherein the anionic ion exchanger treatment, an anionic exchanger material in the chloride form is used and/or in the cationic ion exchanger treatment, a cationic ion exchanger material in the hydrogen form is used.

    10. The process according to claim 1, wherein the clarified solution is subjected firstly to the cationic ion exchanger treatment and subsequently to the anionic ion exchanger treatment.

    11. The process according to claim 1, wherein the purified solution is concentrated, optionally by nanofiltration and/or reverse osmosis, optionally by nanofiltration, wherein optionally a nanofiltration membrane is used which has a molecular weight cut-off in the range of 100 to 200 kDa.

    12. The process according to claim 1, wherein the clarified solution and/or the purified solution is concentrated i) up to a concentration of 100 g/L, optionally 200 g/L, optionally 300 g/L, of L-fucose; and/or ii) by nanofiltration at a temperature of <80 C., optionally <50 C., optionally 4 C. to 45 C., optionally 10 C. to 40 C., optionally 15 C. to 30 C., optionally 15 C. to 20 C.; and/or iii) by reverse osmosis at a temperature of 20 C. to 50 C., optionally 30 C. to 45 C., optionally 35 C. to 45 C.; and/or iv) by nanofiltration at a pressure between >5 bar and <50 bar, optionally at a pressure between >10 bar and <40 bar, optionally at a pressure between >15 and <30 bar; and/or v) by reverse osmosis at a pressure between >5 bar and <100 bar, optionally at a pressure between >10 bar and <80 bar, optionally at a pressure between >15 and <70 bar.

    13. The process according to claim 1, wherein the electrodialysis is an electrodialysis under neutral conditions or an electrodialysis under acidic conditions.

    14. The process according to claim 1, wherein after removing salt from the purified solution, i) the amount of salt in the purified solution is <10% (w/w), optionally <5% (w/w), optionally 1% (w/w), optionally 0.5% (w/w), optionally 0.4% (w/w), especially 0.2% (w/w); and/or ii) the conductivity is between 0.2 mS/cm.sup.2 and 10.0 mS/cm.sup.2, optionally between 0.4 mS/cm.sup.2 and 5.0 mS/cm.sup.2, optionally between 0.5 mS/cm.sup.2 and 1.0 mS/cm.sup.2.

    15. The process according to claim 1, wherein the clarified solution and/or purified solution is subjected to discolouring, optionally by a treatment with activated charcoal and/or a treatment with a cationic ion exchanger and an anionic ion exchanger which are coupled in series, wherein said removal is optionally performed i) before or after diafiltration and/or concentration of the clarified solution; and/or ii) before or after electrodialysis and/or diafiltration of the clarified solution.

    16. The process according to claim 1, wherein the purified solution is subjected to i) granulation; ii) spray-drying; iii) roller-drying; or iv) lyophilization.

    17. The process according to claim 1, wherein the purified solution is subjected to crystallisation, optionally by addition of butanol to the purified solution or without addition of any organic solvent to the purified solution.

    18. A composition comprising 1. at least 95.00 wt.-% of L-fucose; 2. at most 1.00 wt.-% organic solvent; and 3. at most 1.00 wt.-% salt.

    19. The composition according to claim 18, wherein the composition comprises a) 95.50 to 100 wt.-% of L-fucose, optionally 98.00 to 100.00 wt.-% of L-fucose, optionally 98.50 to 100.00 wt.-% of L-fucose, optionally 98.70 to 99.90 wt.-% of L-fucose; and/or b) 0.00 to 1.00 wt.-% organic solvent, optionally 0.00 to 0.50 wt.-% organic solvent, optionally 0.00 to 0.40 wt.-% organic solvent, optionally 0.10 to 0.200 wt.-% organic solvent; and/or c) 0.00 to 1.00 wt.-% salts, optionally 0.00 to 0.50 wt.-% salts, optionally 0.00 to 0.40 wt.-% salt, optionally 0.10 to 0.20 wt.-% salt.

    20. The composition according claim 18, wherein L-fucose is present in amorphous form or in crystalline form, optionally in granulate form or in crystalline form.

    21. A food composition, optionally an infant food formula, a toddler food formula or a medical nutrition product, comprising L-fucose and at least one other carbohydrate selected from galactooligosaccharides (GOS), fructooliogsoaccharides (FOS), inulin, lactulose, isomaltose, maltodextrin, lactose, and human milk oligosaccharides.

    22. The food composition according to claim 21, wherein the food composition comprises at least one human milk oligosaccharide selected from the group consisting of 2-fucosyllactose, 3-fucosyllactose, lacto-N-tetraose, lacto-N-triose II, lacto-N-fucopentaose I, lacto-N-fucopentaose Ill, lacto-N-fucopentaose V, difucosyllactose, lacto-N-neotetraose, 3-sialyllactose, 6-sialyllactose and sialylated lacto-N-neotetraose and lacto-N-tetraose derivatives.

    23. The food composition according to claim 21, wherein the food composition is i) a liquid food composition and comprises the L-fucose at a concentration of 1 mg/l to 2 g/l, optionally at a concentration of 5 mg/l to 1.5 g/l, optionally at a concentration of 20 mg/l to 1 g/l, optionally at a concentration of 50 mg/l to 0.7 g/l; or ii) a solid food composition and comprises the L-fucose at a concentration of 5 mg/kg to 15 g/kg, optionally at a concentration of 25 mg/kg to 10 g/kg, optionally at a concentration of 100 mg/kg to 10 g/kg, optionally at a concentration of 375 mg/kg to 5.25 g/kg.

    24. The food composition according to claim 21, wherein the food composition comprises i) at least one neutral human milk oligosaccharide, optionally at least one neutral human milk oligosaccharide selected from the group consisting of 2-fucosyllactose, 3-fucosyllactose, lacto-N-tetraose and lacto-N-neotetraose, optionally all of said neutral human milk oligosaccharides; and ii) at least one acidic human milk oligosaccharide, optionally at least one acidic human milk oligosaccharide selected from the group consisting of 3-sialyllactose and 6-sialyllactose, optionally all of said acidic human milk oligosaccharides; and iii) a sialic acid, optionally N-acetylneuraminic acid.

    25. The food composition according to claim 21, wherein the food composition comprises at least one substance, optionally all substances, selected from the group consisting of lactose, whey protein, biotin, skimmed milk, vegetable oil, skimmed milk powder, oil of Mortierella alpine, fish oil, calcium carbonate, potassium chloride, vitamin C, sodium chloride, vitamin E, iron acetate, zinc sulfate, niacin, calcium-D-panthothenate, copper sulfate, vitamin A, vitamin B1, vitamin B6, magnesium sulphate, potassium iodate, folic acid, vitamin K, sodium selenite and vitamin D.

    26. The food composition according to claim 21, wherein the food composition comprises at least one substance selected from the group consisting of a protein source, a vitamin, an oil, a mineral, an enzyme, a further carbohydrate and a probiotic strain.

    27. The food composition according to claim 21, wherein the food composition is a composition selected from the group consisting of a medical food composition, a dietary supplement, a sachet product, a liquid ready-to-use infant nutrition product, a liquid ready-to-use toddler nutrition product, a granulated product, a spray-dried infant formula product and combinations thereof.

    28. A liquid, ready-to-use infant or toddler nutrition product comprising L-fucose at a concentration of 1 mg/l to 2 g/l, a sialic acid and i) at least one neutral human milk oligosaccharide selected form the group consisting of 2-fucosyllactose, 3-fucosyllactose, difucosyllactose, lacto-N-tetraose and lacto-N-neotetraose; and/or ii) at least one acidic human milk oligosaccharide selected from the group consisting of 3-sialylactose or 6-sialyllactose.

    29. A spray-dried infant formula product comprising L-fucose at a concentration of 5 mg/kg to 15 g/kg, a sialic acid and i) at least one neutral human milk oligosaccharides selected form the group consisting of 2-fucosyllactose, 3-fucosyllactose, difucosyllactose, lacto-N-tetraose and lacto-N-neotetraose; and/or ii) at least one acidic human milk oligosaccharide selected from the group consisting of 3-sialyllactose and 6-sialyllactose.

    30. A dietary supplement comprising L-fucose and at least one neutral HMO selected from the group consisting of 2-fucosyllactose, 3-fucosyllactose, difucosyllactose, lacto-N-triose II, lacto-N-tetraose and lacto-N-neotetraose, lacto-N-fucopentaose I.

    31. A premix, optionally in the form of a spray-dried, granulated or liquid product, comprising a L-fucose and at least one substance selected from the group consisting of a protein source, a vitamin, an oil, a mineral, an enzyme, a further carbohydrate and a probiotic strain, wherein optionally the i) protein source is selected from the group consisting of whey, corn soya blend, protein hydrolysates and combinations thereof; and/or ii) vitamin is selected from the group consisting of vitamin A, thiamine, riboflavin, vitamin B12, folate and combinations thereof; and/or iii) oil is selected from the group consisting of palm oil, DHA, arachidonic acid and combinations thereof; and/or iv) mineral is selected from the group consisting of potassium chloride, potassium iodate, zinc oxide and combinations thereof; and/or v) enzyme is selected from the group consisting of amylase, amyloglucosidase and combinations thereof; and/or vi) carbohydrate is selected from the group consisting of a human milk oligosaccharide (HMO), a galactooligosaccharide (GOS), inulin (FOS), lactose, isomaltose, sialic acid, and combinations thereof; and/or vii) the probiotic strain is selected from the group consisting of (encapsulated) Lactobacillus, Bifidobacterium, Bacillus, yeast and combinations thereof.

    32. Pharmaceutical composition, a pharmaceutical composition for use in preventing or treating at least one of a viral infection, a bacterial infection, a memory loss and dysbiosis, comprising a composition according to claim 18 and optionally comprising at least one sugar different than a L-fucose and/or at least one probiotic bacterial strain, wherein the at least one sugar is optionally at least one sugar selected from the group consisting of lactose, lactulose, inulin and sucrose.

    33. Use of A product comprising the composition according to claim 18 for manufacture of a food composition and/or pharmaceutical composition.

    Description

    [0291] With reference to the following figures and examples, the subject according to the invention is intended to be explained in more detail without wishing to restrict said subject to the special embodiments shown here.

    [0292] FIG. 1 shows an example of an inventive process for purification of L-fucose. After fermentation, the fermentation broth is clarified by centrifugation and/or filtration. The clarified fermentation broth is subjected to an ionic exchanger treatment for removal of electrically charged contaminations and for exchange of unspecific ions to specific ions. The flow-through of the ionic exchanger treatment is then subjected to diafiltration and/or concentration. After said step, the solution comprising L-fucose is subjected to electrodialysis and/or diafiltration. Then, the solution comprising L-fucose is subjected to a further ionic exchanger treatment for removal of electrically charged colored substances and peptides. Subsequently, the solution comprising L-fucose is subjected to an activated charcoal treatment. Then, the solution is either subjected to crystallization of L-fucose or subjected to a further electrodialysis and/or diafiltration step with subsequent granulation and/or spray-drying.

    [0293] FIG. 2 shows an example of a second inventive process for purification of L-fucose. After fermentation, the fermentation broth is clarified by centrifugation and/or filtration. The clarified fermentation broth is subjected to an ionic exchanger treatment for removal of electrically charged contaminations and for exchange of unspecific ions to specific ions. The flow-through of the ionic exchanger treatment is then subjected to diafiltration and/or concentration. After said step, the solution comprising L-fucose is subjected to electrodialysis and/or diafiltration. Then, the solution comprising L-fucose is subjected to an activated charcoal treatment. Subsequently, the solution comprising L-fucose is subjected to a further ionic exchanger treatment for a removal of electrically charged colored substances and peptides. Then, the solution is either subjected to crystallization of L-fucose or subjected to a further electrodialysis and/or diafiltration step with subsequent granulation and/or spray-drying.

    [0294] FIG. 3 shows an example of a third inventive process for purification of L-fucose. After fermentation, the fermentation broth is clarified by centrifugation and/or filtration. The clarified fermentation broth is subjected to diafiltration and/or concentration. Then, the solution is subjected to an ionic exchanger treatment for removal of electrically charged contaminations and for exchange of unspecific ions to specific ions. After said step, the solution comprising L-fucose is subjected to electrodialysis and/or diafiltration. Then, the solution comprising L-fucose is subjected to an activated charcoal treatment. Subsequently, the solution comprising L-fucose is subjected to a further ionic exchanger treatment for a removal of electrically charged colored substances and peptides. Then, the solution is either subjected to crystallization of L-fucose or subjected to a further electrodialysis and/or diafiltration step with subsequent granulation and/or spray-drying.

    [0295] FIG. 4 shows an example of a fourth inventive process for purification of L-fucose. After fermentation, the fermentation broth is clarified by centrifugation and/or filtration. The clarified fermentation broth is subjected to diafiltration and/or concentration. Then, the solution is subjected to an activated charcoal treatment. After said step, the solution comprising L-fucose is subjected to an ionic exchanger treatment for removal of electrically charged contaminations and for exchange of unspecific ions to specific ions. Subsequently, the solution comprising L-fucose is subjected to electrodialysis and/or diafiltration. Afterwards, the solution is subjected to a further ion exchanger treatment for a removal of electrically charged colored substances and peptides. Then, the solution is either subjected to crystallization of L-fucose or subjected to a further electrodialysis and/or diafiltration step with subsequent granulation and/or spray-drying.

    [0296] FIG. 5 shows a microscopic picture of crystals of L-fucose formed by crystallization from an aqueous solution. It can be seen that L-fucose forms needle-like crystals with a length of 50 m to 150 m.

    [0297] FIG. 6 shows an HPLC diagram which was recorded for L-fucose that has been crystallized from an aqueous solution. The purity of the crystallized L-fucose is detected as 99.8%. The water content is 0.1% (w/w).

    [0298] FIG. 7 shows an HPLC diagram which was recorded for L-fucose that has been granulated from an aqueous solution. The purity of the granulated L-fucose is detected as 97.8%. The water content is 1.7% (w/w).

    EXAMPLE 1: PURIFICATION OF L-FUCOSE FROM BACTERIAL FERMENTATION

    [0299] L-fucose was produced by a bacterial fermentation and harvested by filtration. The resulting clear particle-free L-fucose solution (34 g/l) was treated with a strong cationic ion exchanger (Lewatit 52568 in proton form, Lanxess). After neutralization with sodium hydroxide, the solution was additionally processed with a strong anionic ion exchanger (Lewatit S 6368A in chloride form).

    [0300] For concentration of L-fucose after ionic exchanger treatment, the solution was further processed by a reverse osmosis step. An Emrich EMRO 1.8 reverse osmosis system (Emrich Edelstahlbau) was equipped with a Trisep TS80 (80-40-TS80-TSA) nanofiltration module. The inlet pressure was set to 25 bar and the solution was concentrated until the L-fucose concentration has reached 100 g/l. Alternatively, the L-fucose solution could also concentrated by vacuum evaporation, nevertheless filtration techniques reduces the production of Maillard reactions during concentration.

    [0301] To remove the sodium chloride from the ion exchanger step and other ions the solution was electrodialysed using a PCCell P15 electrodialysis system (PCell, Heusweiler, Germany) equipped with a PCCell ED 1000A membrane stack. Said stack comprised the following membranes: cation exchange membrane CEM: PC SK and the anion exchange membrane CEM:PcAcid60 having a size exclusion limit of 60 Da. The conductivity of the starting solutions was between 15 and 25 mS/cm.sup.2 and the solution was electrodialysed until the conductivity was 1.5 to 2 mS/cm.sup.2.

    [0302] After electrodialysis, the solution was treated again with a strong cationic ion exchanger (Lewatit 52568 in sodium form, Lanxess) and a strong anionic ion exchanger (Lewatit S 6368A in chloride form) to remove a colored substances and possibly remaining unspecific ions (like e.g. peptides).

    [0303] For a more rigorous removal of colored substances, the L-fucose containing solution was further treated with activated carbon powder. The solution was incubated for 2 hours under stirring with Norit DX1 activated charcoal. After incubation, the activated carbon was removed by filtration.

    [0304] After activated carbon treatment, the L-fucose containing solution was concentrated by reverse osmosis filtration using an Emrich EMRO 1.8 reverse osmosis system (Emrich Edelstahlbau) which was equipped with a CSM RE8040BE reverse osmosis module. The solution was concentrated until the flow rate of the filtration system drops below 50 liter/hours. The dry matter after concentration was between 30 and 35% (w/w).

    [0305] Alternatively, the L-fucose could be also concentrated by vacuum evaporation. This technique leads to a higher concentration (50 to 60% (w/w) of L-fucose. The disadvantage of concentration by vacuum evaporation is that L-fucose will partly undergo caramelization which is evidenced by the solution turning into a distinct brown color. In short, product quality, product purity and product yield are decreased by this concentration method.

    EXAMPLE 2: CRYSTALLIZATION OF L-FUCOSE BY USING ORGANIC SOLVENTS

    [0306] For crystallization of L-fucose, 15 liters of a 32% (w/w) solution were concentrated to a final concentration of 80 to 85% (w/w) dry matter by vacuum evaporation using an Hei-VAP industrial evaporator (Heidolph Instruments GmbH, Schwabach Germany) to obtain a mother liquor for crystallization of L-fucose.

    [0307] To start the crystallization process, the mother liquor was inoculated with seed crystals. Additional 2.5 liters (ratio Butanol to fucose 1:2) of n-butanol were added and the solution was concentrated under vacuum until the solution was saturated with crystals.

    [0308] The crystallization mass was removed from the piston and incubated for at least 24 hours until a solid crystallization mass was obtained.

    [0309] The solid crystals were mixed with ethanol in a ratio of 1 to 1 (1 liter ethanol with 1 kg crystallization mass). To remove the mother liquor and the ethanol from the crystals, the solution was centrifuged.

    [0310] The crystals were washed again with ethanol (2.5 liters for 5 kg L-fucose crystals) and centrifuged again. The solid L-fucose crystals were removed from the centrifuge and dried at 40 C. until no ethanol was left. The L-fucose was sifted through a riddle with 0.2 mm diameters.

    [0311] At the end, 3.3 kg L-fucose was obtained from 5 kg L-fucose crystallization approach (yield: 66%).

    EXAMPLE 3: CRYSTALLIZATION OF L-FUCOSE FROM AQUEOUS SOLUTIONS

    [0312] For crystallization of L-fucose from aqueous solutions, 15 liters of a 32% (w/w) solution were concentrated to a final concentration of 80% to 85% (w/w) dry matter by vacuum evaporation using an Hei-VAP industrial evaporator (Heidolph Instruments GmbH, Schwabach Germany).

    [0313] To start the crystallization process, the mother liquor was inoculated with seed crystals. The solution was concentrated under vacuum until a clear crystal formation was observed.

    [0314] The crystallization mass was removed from the piston and incubated for at least 72 hours until a solid crystallization mass was obtained. The solid crystals were mixed with ethanol in a ratio of 1 to 1 (1 liter ethanol with 1 kg crystallization mass).

    [0315] To remove the mother liquor and the ethanol from the crystals, the solution was centrifuged. The crystals were washed with ethanol (2.5 Liter for 5 kg L-fucose crystals) and centrifuged again. The solid L-fucose crystals were removed from the centrifuge and dried at 40 C. until no ethanol was left. The L-fucose was sifted through a riddle with 0.2 mm diameters.

    [0316] At the end, 3.0 kg L-fucose was obtained from 5 kg L-fucose crystallization approach (yield: 60%).

    EXAMPLE 4: ISOLATION OF L-FUCOSE BY GRANULATION

    [0317] For granulation of L-fucose, a fluid bed system from Glatt (Glatt GmbH, Germany) was used. From a crystallization approach of L-fucose, a 40% (w/w) L-fucose solution was provided and used for granulation in the fluid bed system.

    [0318] The fluid bed system was equipped with a total of 500 g of solid L-fucose and the system was started by adding the feed solution. The system was stabilized by a product temperature between 45 C. and 50 C. After 300 g L-fucose, the first agglomerates were formed. After addition of 1000 g 40% (w/w) L-fucose solution (400 g L-fucose), the system was stopped and analysed.

    [0319] After granulation and a recovery rate of 80%, 720 g L-fucose could be recovered with a bulk density of 570 g/l and a dry loss of 0.9%. The size of the granulated L-fucose was analysed by sieving. This test shows that 70% of the material has a size between 150 m and 1400 m.

    EXAMPLE 5: PURITY DEGREES IN AN EXEMPLARY PURIFICATION OF L-FUCOSE

    [0320] The purity degrees in an exemplary purification of L-fucose from culture broth are shown in Table 1.

    TABLE-US-00001 TABLE 1 Purification of L-fucose from culture broth. Purity is declared as the mass of L-fucose in comparison to the total mass. Concentra- Dry Purification tion L-fucose L-fucose Matter Purity step (g/l) (kg) (kg) (%) Harvest 33 204 298 68 Cationic 31 199 281 70.8 exchanger Anionic 29 189 302 62.5 exchanger Concentration 88 186 269 69.1 Electrodialysis 86 156 161 96.9 Activated 81 149 154 96.7 carbon Concentration 310 150 154 97.4 Crystallization 86.3 98.8

    EXAMPLE 6: COMPOSITION OF A REPRESENTATIVE INFANT FORMULA PRODUCT

    [0321] In the following, a composition of a representative infant formula product is presented (see Table 2 below).

    [0322] The composition comprises L-fucose in combination with the abundant neutral HMOs 2-fucosyllactose (2-FL), 3-fucosyllactose (3-FL), lacto-N-tetraose (LNT) and optionally lacto-N-neotetraose (LNnT) and lacto-N-fucopentaose I (LNFP-I), acidic HMOs (6-sialyllactose (6-SL) and 3-sialyllactose (3-SL)), and the sialic acid N-acetylneuraminic acid (Neu5Ac).

    [0323] One or more probiotic strains can be present in the product. The final concentration of each ingredient is based on a preparation of 13.5 g of the powder in 90 ml of water.

    TABLE-US-00002 TABLE 2 Comparison of a representative infant formula product per 100 g per 100 ml powder infant formula Energy kJ 2094-2145 283 kcal 500-512 67-68 Fats, g 24.2-26.2 3.3-3.5 hereof: saturated fatty acids g 8.7-9.4 1.2-1.3 monounsaturated fatty acids g 10.4 1.4 polyunsaturated fatty acids g 5.5-5.9 0.7-0.8 Carbohydrates g 56-58 7.4-7.9 Sugars g 44-56 6-7.4 hereof: Lactose g 44-56 6-7.4 Sialic acid (Neu5Ac) mg 300-450 40-60 L-fucose mg 300-450 40-60 HMOs g 4.22-4.81 0.57-0.65 Hereof 2-FL g 1.85-2.22 0.25-0.30 3-FL mg 555.56-592.6 75-80 LNT g 1.11 0.15 LNnT mg 0-111.11 0-15 LNFP-I mg 0-740.74 0-100 3-SL mg 148.15-170.37 20-23 6-SL mg 207.4-222.22 28-30 Protein g 11.11-11.85 1.5-1.6 Salt g 0.47-0.59 0.06-0.08 Vitamins Vitamin A g 357-358 47.3-48.2 Vitamin D g 7.8 1.05 Vitamin E mg 8.15 1.1 Vitamin K g 43.7-44.4 5.9-6.0 Vitamin C mg 115-118 15-16 Vitamin B1 mg 0.51-0.60 0.068-0.079 Vitamin B2 mg 1.3-1.7 0.18-0.23 Niacin mg 3.63 0.49 Vitamin B6 g 526-600 71-81 Folic acid g 160-164 21.6-21.7 Vitamin B12 g 1.7-1.9 0.23-0.25 Biotin g 22-30 3.0-3.9 Panthothenic acid mg 4.6-5.4 0.62-0.72 Minerals Sodium mg 187-236 25.3-31.2 Potassium mg 673-675 88.8-91.2 Chloride mg 327-333 43.1-44.9 Calcium mg 460-504 62.1-66.5 Phosphorous mg 335-352 45.2-46.5 Magnesium mg 49.3-56.3 6.66-7.43 Iron mg 4.15 0.56 Zinc mg 3.7-3.8 0.49-0.51 Copper g 274 37 Manganese g 96.3 13 Fluoride g 30.4-32.6 4.1-4.4 Selenium g 11.1-12.3 1.5-1.6 Iodine g 101.5-103.7 13.7-14

    EXAMPLE 7: COMPOSITION OF A REPRESENTATIVE PREMIX FOR AN INFANT FORMULA PRODUCT COMPRISING HUMAN MILK OLIGOSACCHARIDES, THE MONOSACCHARIDE L-FUCOSE AND THE MONOSACCHARIDE N-ACETYLNEURAMINIC ACID (NEU5AC)

    [0324] In the following, a composition of a representative premix for an infant formula product is presented (see Table 3 below).

    [0325] The composition comprises the monosaccharide L-fucose in combination with the abundant neutral HMOs 2-fucosyllactose (2-FL), 3-fucosyllactose (3-FL), lacto-N-tetraose (LNT) and optionally lacto-N-neotetraose (LNnT) and lacto-N-fucopentaose I (LNFP-I) and acidic HMOs (such as 6-sialyllactose (6-SL), 3-sialyllactose (3-SL) and sialylated LNT derivatives), and the sialic acid N-acetylneuraminic acid (Neu5Ac).

    [0326] The premix can be used to reconstitute an infant formula by adding said premix to other nutritional products which are necessary to reconstitute an infant food formula, such as whey, lactose, lipids (saturated and unsaturated fatty acids) and minerals. The premix shown in Table 3 is intended to be used for 1 kg of final infant formula product.

    TABLE-US-00003 TABLE 3 HMO/Sialic acid/Fucose Vitamin premix Sialic acid (Neu5Ac) g 3.0 L-fucose g 3.0 2-FL g 18.5 3-FL g 5.5 LNT g 10.0 3-SL g 1.5 6-SL g 2.0 Vitamins Vitamin A mg 3.5 Vitamin D g 78.0 Vitamin E mg 81.5 Vitamin K g 437.0 Vitamin C g 1.1 Vitamin B1 mg 5.1 Vitamin B2 mg 13.0 Niacin mg 36.3 Vitamin B6 mg 5.2 Folic acid mg 1.6 Vitamin B12 g 17.0 Biotin g 220.0 Panthothenic acid mg 46.0