PROCESS FOR PURIFICATION OF A SIALIC ACID FROM A FERMENTATION BROTH

Abstract

The present invention describes an efficient way to isolate a sialic acid from a fermentation broth. The sialic acid contained in the fermentation broth is produced by bacterial fermentation. 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 the sialic acid in spray-dried form as well as in form of sialic acid crystals.

Claims

1. A process for purification of a sialic acid from a fermentation broth, comprising: removing biomass from a fermentation broth comprising a sialic acid, wherein a clarified solution is provided, providing a purified solution by subjecting the clarified solution to a cationic ion exchanger treatment with a cationic ion exchanger material, wherein the cationic ion exchanger treatment is performed under conditions in which the sialic acid passes the cationic ion exchanger material and is present in the flowthrough; and an anionic ion exchanger treatment with an anionic ion exchange material, wherein the anionic ion exchanger treatment is performed under conditions in which the sialic acid passes the anionic ion exchanger material and is present in the flowthrough; and removing salt from the purified solution by electrodialysis.

2. The process according to claim 1, wherein the process does not comprise i) a chromatographic separation; and/or ii) a use of ethanol and/or ethyl acetate, optionally does not comprise the use of an organic solvent; and/or iii) a step of eluting the sialic acid from a stationary phase with a solution comprising an organic solvent; and/or iv) a use of a heavy metal; and/or v) a step of heating the fermentation broth, the clarified solution and/or purified solution to a temperature of more than 45 C.

3. The process according to claim 1, wherein the biomass is removed from the fermentation broth by centrifugation and/or filtration.

4. The process according to claim 1, charactcrizcd in that wherein in 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+ or Na+, wherein if the unspecific cations are replaced by H+, the pH of the flowthrough is optionally adjusted to a pH of 6 to 8 before performing a further treatment, optionally by addition of NaOH to the flowthrough.

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 or OH, wherein if the unspecific anions are replaced by Cl, the pH of the flowthrough is optionally adjusted to a pH of 6 to 8 before performing a further treatment, optionally by addition of NaOH to the flowthrough.

7. The process according to claim 1, wherein conditions under which the sialic acid 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 after the cationic ion exchanger treatment and the anionic ion exchanger treatment, the purified solution comprises the sialic acid, one or more colour-giving substances and salt, wherein the salt is optionally NaCl.

9. The process according to claim 1, wherein in 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 a range of 100 to 200 kDa.

12. The process according to claim 1, wherein clarified solution and/or purified solution is concentrated i) up to a concentration of 100 g/L, optionally 200 g/L, optionally 300 g/L, of the sialic acid; 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 to 30 C., optionally 15 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) 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.

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, charactcrizcd in that 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); and/or ii) the conductivity is between 0.2 and 10.0 mS/cm.sup.2, optionally between 0.4 and 5.0 mS/cm.sup.2, optionally between 0.5 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 a step of discolouring, optionally by a treatment with activated charcoal, wherein said removal is optionally performed i) before or after a step of diafiltration and/or concentration of the clarified solution; and/or ii) before or after a step of electrodialysis and/or diafiltration of the clarified solution.

16. The process according to claim 1, wherein the sialic acid comprised in the clarified solution and/or purified solution is converted into sodium form, optionally by treating the purified solution with a strong cationic ion exchanger material in the sodium form.

17. The process according to claim 1, wherein the purified solution is spray-dried, wherein the purified solution optionally comprises the sialic acid in the sodium form.

18. The process according to claim 1, wherein the purified solution is subjected to crystallisation, optionally by addition of acetic acid to the purified solution, wherein particularly preferred optionally, sialic acid is provided in dihydrate crystal form.

19. A composition comprising a) at least 98.00 wt.-% of a sialic acid; b) at most 1.00 wt.-% organic solvent; and c) at most 1.00 wt.-% salt different than a salt form of the sialic acid.

20. The composition according to claim 19, wherein the composition comprises a) 98.50 to 100 wt.-% of the sialic acid, optionally 99.00 to 100.00 wt.-% of the sialic acid, optionally 99.50 to 100.00 wt.-% of the sialic acid, optionally 99.70 to 99.90 wt.-% of the sialic acid; 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.-% salt different than a salt form of the sialic acid, optionally 0.00 to 0.50 wt.-% salt different than a salt form of the sialic acid, optionally 0.00 to 0.40 wt.-% salt different than a salt form of the sialic acid, optionally 0.10 to 0.20 wt.-% salt different than a salt form of the sialic acid.

21. The composition according to claim 19, wherein the sialic acid is present in amorphous form or in crystalline form, optionally in granulate form or in crystalline form, optionally in spray-dried form or in crystalline form.

22. A food composition, optionally an infant food formula, a toddler food formula or a medical nutrition product, comprising a sialic acid and at least one human milk oligosaccharide.

23. The food composition according to claim 22, wherein the food composition comprises at least one sugar selected from the group consisting of 2-fucosyllactose, 3-fucosyllactose, lacto-N-tetraose, 3-sialyllactose, 6-sialyllactose and lacto-N-neotetraose.

24. The food composition according claim 22, wherein the food composition is i) a liquid food composition and comprises the sialic acid at a concentration of 1 mg/I to 2 g/I, optionally at a concentration of 5 mg/I to 1.5 g/I, optionally at a concentration of 20 mg/I to 1 g/I, optionally at a concentration of 50 mg/I to 0.7 g/I; or ii) a solid food composition and comprises the sialic acid 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, at a concentration of 375 mg/kg to 5.25 g/kg.

25. The food composition according to claims 22, characterized in that wherein the food composition comprises i) at least one neutral human milk oligosaccharide, prcfcrably 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) L-fucose.

26. The food composition according to claim 22, 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.

27. The food composition according to claim 22, 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.

28. The food composition according to claim 22, 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.

29. A liquid, ready-to-use infant or toddler nutrition product comprising a sialic acid at a concentration of 1 mg/I to 2 g/I, L-fucose and i) at least one neutral human milk oligosaccharide selected form the group consisting of 2-fucosyllactose, 3-fucosyllactose, difucosyl-lactose, 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.

30. A spray-dried infant formula product comprising a sialic acid at a concentration of 5 mg/kg to 15g/kg, L-fucose and i) at least one neutral human milk oligosaccharides selected form the group consisting of 2-fucosyllactose, 3-fucosyllactose, difuco-syllactose, 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.

31. A dietary supplement comprising a sialic acid and at least one neutral HMO selected from the group consisting of 2-fucosyllactose, 3-fucosyllactose, difucosyllactose, lacto-N-tetraose and lacto-N-neotetraose.

32. A premix, optionally in the form of a spray-dried, granulated or liquid product, comprising a sialic acid 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), L-fucose, a further sialic acid, lactose and combinations thereof; and/or vii) the probiotic strain is selected from the group consisting of (encapsulated) Lactobacillus, Bifidobacterium strain and combinations thereof.

33. A pharmaceutical composition, optionally a pharmaceutical composition for use in preventing and/or treating at least one of a viral infection, a bacterial infection, a memory loss and dysbiosis, comprising a composition according to claim 19 and optionally comprising at least one sugar different than a sialic acid 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.

34. A product comprising the composition according to claim 19 for manufacture of a food composition and/or pharmaceutical composition.

Description

[0277] 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.

[0278] FIG. 1 shows an example of an inventive process for purification of the sialic acid N-acetylneuraminic acid. After fermentation, the fermentation broth is clarified by centrifugation and/or filtration. The clarified fermentation broth is subjected to an ionic exchanger treatment. The flow-through of the ionic exchanger treatment is then subjected to diafiltration and/or concentration. After said step, the solution comprising the sialic acid is passed over activated charcoal. Then, the solution comprising the sialic acid is subjected to electrodialysis and/or diafiltration. Subsequently, the solution comprising the sialic acid is subjected to spray-drying or crystallization.

[0279] FIG. 2 shows an example of a second inventive process for purification of the sialic acid N-acetylneuraminic acid. After fermentation, the fermentation broth is clarified by centrifugation and/or filtration. The clarified fermentation broth is subjected to an ionic exchanger treatment. The flow-through of the ionic exchanger treatment is then subjected to electrodialysis and/or diafiltration. After said step, the solution comprising the sialic acid is passed over activated charcoal. Then, the solution comprising the sialic acid is subjected to diafiltration and/or concentration. Subsequently, the solution comprising the sialic acid is subjected to spray-drying or crystallization.

[0280] FIG. 3 shows an example of a third inventive process for purification of the sialic acid N-acetylneuraminic acid. After fermentation, the fermentation broth is clarified by centrifugation and/or filtration. The clarified fermentation broth is subjected to an ionic exchanger treatment. The flow-through of the ionic exchanger treatment is then passed over activated charcoal. After said step, the solution comprising the sialic acid is subjected to diafiltration and/or concentration. Then, the solution comprising the sialic acid is subjected to electrodialysis and/or diafiltration. Subsequently, the solution comprising the sialic acid is subjected to spray-drying or crystallization.

[0281] FIG. 4 shows an example of a fourth inventive process for purification of the sialic acid N-acetylneuraminic acid. After fermentation, the fermentation broth is clarified by centrifugation and/or filtration. The clarified fermentation broth is subjected to an ionic exchanger treatment. The flow-through of the ionic exchanger treatment is then subjected to electrodialysis and/or diafiltration. After said step, the solution comprising the sialic acid is subjected to diafiltration and/or concentration. Then, the solution comprising the sialic acid is passed over activated charcoal. Subsequently, the solution comprising the sialic acid is subjected to spray-drying or crystallization.

[0282] FIG. 5 shows a microscopic picture of crystals of N-acetylneuraminic acid. It can be seen that N-acetylneuraminic acid forms small needles with a length of 2 to 8 m.

[0283] FIG. 6 shows an HPLC diagram which was recorded for N-acetylneuraminic acid that has been crystallized as dihydrate. The purity of the crystallized N-acetylneuraminic acid is detected as 98.7%.

[0284] FIG. 7 shows an HPLC diagram which was recorded for N-acetylneuraminic acid that has been crystallized with acetic acid. The purity of the crystallized N-acetylneuraminic acid is detected as 98.2% in comparison to other sugars. The amount of acetic acid after drying was 2.8% in comparison to total mass of material.

[0285] FIG. 8 shows an HPLC diagram which was recorded for N-acetylneuraminic acid that has been spray-dried. The purity of the spray-dried N-acetylneuraminic acid is detected as 99.1%.

EXAMPLE 1

Purification of the Sialic Acid N-Acetylneuraminic Acid from Bacterial Fermentation

[0286] The sialic acid N-acetylneuraminic acid was produced by a bacterial fermentation and harvested by filtration wherein a clear particle-free sialic acid solution (37 g/1) was obtained.

[0287] The clear particle-free solution was firstly treated with a strong cationic ion exchanger (Lewatit 52568 in proton form, Lanxess).

[0288] After neutralization with sodium hydroxide (NaOH), the solution was additionally processed with a strong anionic ion exchanger (Lewatit S 6368A in chloride form).

[0289] For concentration and desalting of N-acetylneuraminic acid in the solution after ionic exchanger treatment, the solution was further processed by a nanofiltration step. An Emrich EMRO 1.8 reverse osmosis system (Emrich Edelstahlbau) was equipped with a Trisep XN 45 nano filtration module. The inlet pressure was set to 30 bar and the solution was concentrated until the flow rate drops under 100 liter per hour. The solution was diafiltrated three times with an equal amount of reverse osmosis water. To reduce the amount of salt, the solution was diafiltrated three times with equal volume of reverse osmosis water.

[0290] For removal of color, the N-acetylneuraminic acid containing solution was treated with activated carbon powder. The solution was incubated for 2 hours under stirring with Norit SA2 activated charcoal. After incubation the activated carbon was removed by filtration and the pH was adjusted to pH 7.0 with sodium hydroxide.

[0291] To remove remaining color and unspecific ions 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). After said treatments, the pH was adjusted to pH 7.0.

[0292] For removal of sodium chloride, the solution was electrodialysed using a PCCell P15 electrodialysis system (PCell, Heusweiler, Germany) equipped with an 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 solution was electrodialysed until a stable conductivity was reached. During electrodialysis, the pH was stabilized at pH 7.0 by addition of NaOH.

[0293] After electrodialysis, the solution was concentrated to 20% (w/v) dry matter by reverse osmosis using an Emrich EMRO 1.8 reverse osmosis system (Emrich Edelstahlbau) which was equipped with a CSM RE8040BE reverse osmosis module.

[0294] Subsequently, the solution was filtrated to remove endotoxins and sterile filtration by passing the solution through a 5 kDa ultrafiltration membrane (Spira-Cell WY UP005 2440 C module). The sterile solution was then stored at room temperature (20 C.) and used as a sterile liquid concentrate product or the sterile solution was spray-dried.

EXAMPLE 2

Conversion of the Sodium Salt of the Sialic Acid N-Acetylneuraminic Acid to its Free Acid

[0295] To isolate the sialic acid in its free acid form, 30% hydrochloric acid was added after the activated charcoal treatment mentioned in Example 1 until the pH was between 1.7 and 2.3.

[0296] After the pH adjustment, the solution was treated again with a strong cationic exchanger (Lewatit 52568 in proton form, Lanxess). After ionic exchanger treatment, the solution was electrodialysed again using a PCCell P15 electrodialysis system (PCell, Heusweiler, Germany) equipped with an 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 solution was electrodialysed until a stable conductivity was reached. During electrodialysis the pH was stabilized at pH 2.0 by addition of hydrochloric acid.

EXAMPLE 3

Obtaining the Sialic Acid N-Acetylneuraminic Acid in Solid Form by Spray Drying

[0297] The sialic acid obtained by isolation process in sodium form or in free acid form as mentioned above in Examples 1 and 2 was concentrated to 20% (w/w) and the solution was filtrated to remove endotoxins and sterile filtration by passing the solution through a 5 kDa ultrafiltration membrane (Spira-Cell WY UP005 2440 C module, Microdyn Nadir, Wiesbaden, Germany). The so obtained sterile solution comprising N-acetylneuraminic acid was then spray-dried using a NUBILOSA LTC-GMP spray-dryer (NUBILOSA, Konstanz, Germany). For the spray-drying of the N-acetylneuraminic acid solution, the solution was passed under pressure with 3.5 bar through the spray-dryer nozzles set to 130C and flow was adjusted to maintain an exhaust temperature between 66 C. to 67 C. Using these settings, a spray-dried powder with less than 5% moisture could be obtained. The moisture contents were determined by Karl-Fischer titration.

EXAMPLE 4

Crystallization of the Sialic Acid N-Acetylneuraminic Acid as Dihydrate Form

[0298] The sialic acid N-acetylneuraminic acid in free acid form was concentrated to 20% (w/w) dry matter by reverse osmosis using an Emrich EMRO 1.8 reverse osmosis system (Emrich Edelstahlbau) which was equipped with a CSM RE8040BE reverse osmosis module.

[0299] For crystallization, 5 liters of this solution was concentrated by a Hei-VAP industrial evaporator (Heidolph Instruments GmbH, Schwabach Germany) to 50% (w/w) dry matter. The solution was inoculated with seed crystals and concentrated under vacuum until crystals were formed (Dry mater of the solution: 75% w/w).

[0300] The crystal solution was mixed 1:1 with isopropanol and stirred for 5 min. The solution was incubated for at least 12 hours at 4 C. Crystallized sialic acid was removed from the mother liquor by vacuum filtration and the crystals were washed with isopropanol.

[0301] The yield of precipitated N-acetylneuraminic acid was between 75 and 85%.

EXAMPLE 5

Crystallization of the Sialic Acid N-Acetylneuraminic Acid with Acetic Acid

[0302] The sialic acid N-acetylneuraminic acid in free acid form was concentrated to 30% (w/w) dry matter by reverse osmosis using an Emrich EMRO 1.8 reverse osmosis system (Emrich Edelstahlbau) which was equipped with a CSM RE8040BE reverse osmosis module.

[0303] For crystallization, 5 Liter of glacial acetic acid was added under stirring in portions to 1 liter of the N-acetylneuraminic solution. The solution was cooled down from room temperature (20 C.) to 4 C. in 3 hours. After the final temperature (4.5C) was reached the crystallization approach was incubated for 12 to 36 hours at this temperature.

[0304] The crystal solution was mixed 1:1 with isopropanol and stirred for 5 min. The crystallized N-acetylneuraminic acid was removed from the mother liquor by vacuum filtration and the crystals were washed twice with the equal amount of isopropanol.

[0305] The yield of precipitated N-acetylneuraminic acid was between 74 and 81%.

[0306] The purity degree in an exemplary purification of N-acetylneuraminic acid from culture broth is shown in Table 1.

TABLE-US-00001 TABLE 1 Purity is given as the desired mass of N-acetylneuraminic acid in comparison to the total mass. Purification Concentration sialic acid Dry Matter Purity step sialic acid (g/l) (kg) (kg) (%) Harvest 37 259 399 64.9 Cationic 37 256 382 67.02 exchanger Anionic 36 248 378 65.5 exchanger Concentration/ 152 244 316.6 77.06 Diafiltration Activated 145 232.3 298.4 77.9 carbon Electrodialysis 135 181 184 98.3 Concentration 203 180 183 98.3

EXAMPLE 6

Composition of a Representative Infant Formula Product

[0307] In the following, a composition of a representative infant formula product is presented (see Table 2 below).

[0308] The composition comprises the sialic acid Neu5Ac 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 L-fucose.

[0309] 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 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 L/VT g 1.11 0.15 L/VnT mg 0-111.11 0-15 L/VPF-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 Neu5Ac and the monosaccharide L-fucose.

[0310] In the following, a composition of a representative premix for an infant formula product is presented (see Table 3 below).

[0311] The composition comprises the sialic acid Neu5Ac 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)), as well as the monosaccharide free L-fucose.

[0312] 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 L/VT 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