Process for purifying sialic acid from a fermentation broth

Abstract

An efficient way of isolating sialic acid from a fermentation broth is described. The sialic acid contained in the fermentation broth is produced by bacterial fermentation. The method of the invention comprises the steps of removing biomass from the fermentation broth, subjecting the resulting solution to at least one of a cation exchanger treatment and an anion exchanger treatment, and removing salts after the ion exchanger treatment. The method can provide sialic acid in the form of spray-dried sialic acid and in the form of sialic acid crystals.

Description

[0001] An efficient process for isolating sialic acid from a fermentation broth is described. The sialic acid contained in the fermentation broth is produced by bacterial fermentation. The process of the present invention includes the step of removing biomass from the fermentation broth, the step of subjecting the resulting solution to at least one of a cation exchanger treatment and an anion exchanger treatment, and the step of removing salts after the ion exchanger treatment. The process can provide sialic acid in a spray-dried form as well as in a sialic acid crystal form.

[0002] Sialic acid is a generic term for N- or O-substituted derivatives of neuraminic acid, a monosaccharide with a backbone of nine carbons and with a carboxylic acid function. The carboxylic acid group can impart a negative charge to the sugar at appropriate pH. All known sialic acid structures are derived from four major core structures (a) 2-keto-3-deoxynononic acid (Kdn), (b) neuraminic acid (Neu), (c) N-acetylneuraminic acid (Neu5Ac), and (d) N-glycolylneuraminic acid (Neu5Gc), possibly with substitutions (O-acetyl, O-methyl, O-sulfate, O-lactyl, or phosphate) at the hydroxyl groups at C-4, C-7, C-8, and C-9. A lactone can occur between the C-2 and C-7, C-4, or C-8 amino groups of Neu, or a lactam can occur between the C-2 and C-5 amino groups. In addition, naturally occurring dehydrogenated sialic acids are known, such as 2-deoxy-2,3-didehydro-N-acetylneuraminic acid (Neu2en5Ac, also known as DANA). Dehydrogenated sialic acids such as DANA can have inhibitory properties against sialidases, and thus have potential inhibitory properties against certain viruses (e.g., influenza virus).

[0003] Sialic acids are typically found in the form of a-glycosides, which occupy the non-reducing termini of heterooligosaccharides in glycoconjugates (e.g., glycolipids and glycoproteins) and provide an extreme example of glycan diversity in structure and function. The sialic acid family consists of over 40 derivatives of the nine-carbon neuraminic acid, including N- and O-substituted derivatives. The structural feature of the family members is unique due to the amino group at position 5 and the carboxyl group at position 1, which makes all sialic acids strong organic acids with a negative charge under physiological conditions.

[0004] Sialic acids exist as terminal sugars of glycans present in cell surface glycoconjugates (glycoproteins and glycolipids) of vertebrates and higher invertebrates. Sialic acids are also components of the lipopolysaccharides and capsular polysaccharides of pathogenic bacteria, including Escherichia coli K1, Haemophilus influenzae, Haemophilus ducreyi, Pasteurella multocida, Neisseria gonorrhoeae, Neisseria meningitidis, Campylobacter jejuni, and Streptococcus agalactiae. In mammals, sialic acids are found primarily as terminal residues of glycoconjugates on the cell surface. Two derivatives of Neu are the most common sialic acid structures found on mammalian cells: N-acetylneuraminic acid (Neu5Ac) and its hydroxylated derivative, N-glycolylneuraminic acid (Neu5Gc). In mammals, humans are the known exception, lacking Neu5Gc due to an inactivating mutation of the hydroxylase enzyme that modifies CMP-Neu5Ac to CMP-Neu5Gc.

[0005] Neuraminic acid is not found in nature in its unsubstituted form, the most widely found sialic acid is N-acetylneuraminic acid (also abbreviated as "Neu5Ac" or "NANA"). Neu5Ac is a sialic acid in which the N-atom of the amino group is acetylated. In the literature, the term "sialic acid" is sometimes used as a synonym for the specific sialic acid Neu5Ac, but in the following, the term "sialic acid" is to be understood as the family of all sialic acids, and Neu5Ac is to be understood as a specific member of said family, namely N-acetylneuraminic acid.

[0006] Neu5Ac performs various biological functions by acting as a receptor for microorganisms, viruses, toxins, and hormones. Neu5Ac is a characteristic component of amino sugars that are important for intercellular interactions.

[0007] Another important naturally occurring variant is N-glycolylneuraminic acid (Neu5Gc), which is formed by replacing one of the hydrogen atoms in the methyl moiety of the acetyl group with a hydroxyl group. Neu5Gc is present in a large number of animal species, particularly in porcine tissues, but has not been detected in human tissues, except in individuals with specific types of cancer.

[0008] Furthermore, Neu5Ac is used to protect proteins from protease degradation. Due to the high concentration of Neu5Ac in gangliosides present in the membranes of nerve cells, Neu5Ac plays an important role in neuronal development as well as in the functioning of the nervous system. In addition, it is known that many viral pathogens, such as human influenza virus or avian influenza virus, make use of sialylated compounds in the infection of human organisms. Neu5Ac, compounds comprising Neu5Ac and compounds derived from Neu5Ac have been used in active substances against viral infections. Further research is focused on the use of these compounds for ensuring optimal neuronal development or for preventing degenerative brain diseases.

[0009] Neu5Ac modifications of cell surface molecules play a role in many biological phenomena, such as protein structure stability, regulation of cell adhesion and signal transduction. Neu5Ac deficiency, such as GNE myopathy, which is also known as hereditary inclusion body myopathy (HIBM), distal myopathy with rimmed vacuoles (DMRV) or Nonaka myopathy, is a clinical disease caused by reduced sialic acid production. The production of Neu5Ac is a key reason for the disease caused by mutations. In theory, replacement of metabolites after genetic block in this pathway can alleviate the symptoms of Neu5Ac deficiency. (Jay et al., Gene Reg. and Sys., 2009 Biology 3: 181-190). However, administration of one or more compounds in the Neu5Ac biosynthesis pathway in vivo is a major challenge. These compounds have an ultrafast clearance rate and are excreted in the urine before they can be metabolized.

[0010] In the human body, the highest concentration of Neu5Ac can be found in the brain, where Neu5Ac participates in synapse formation and neurotransmission as a major part of the ganglioside structure (Wang et al., Eur J Clin Nutr. 2003 Nov;57(11): 1351-69). Especially in the first year of life, Neu5Ac appears to play an important role in brain development. The rapid growth of the infant brain places exceptionally high demands on the nutritional supply of the diet, especially for preterm infants. Neu5Ac is an essential component of brain gangliosides and polySia chains, which modify the neural cell adhesion molecule (NCAM). The sialic acid levels in human milk are high, mainly the level of the specific sialic acid Neu5Ac. In contrast, infant formulae contain low levels of Neu5Ac and Neu5Gc (Wang, Annu Rev Nutr. 2009; 29: 177-222). Therefore, Neu5Ac shows a great potential for use in infant formulae to support infant brain development.

[0011] Sialic acids play an important role in many physiological and pathophysiological processes, including embryonic nervous system development, metastasis, modulation of immune responses, and infection by bacteria or viruses. Sialic acids are a fundamental component of brain gangliosides and polymeric sialic acid chains, which modify neural cell adhesion molecules (NCAMs), facilitating intercellular interactions, neurite outgrowth, modification of synaptic connectivity, and memory formation. In piglets, a sialic acid-rich diet increases brain sialic acid levels and increases expression of two learning-related genes. Thus, the diet also enhances learning and memory.

[0012] Infants, particularly preterm infants, have a high demand for nutrients including sialic acid due to the rapid growth of the brain at this developmental stage and their developing immune system. High levels of sialic acid, in particular Neu5Ac (about 0.5 g / L), are also found in human milk. In contrast, infant formulae to date contain little or even negligible amounts of Neu5Ac. In addition to free Neu5Ac, human milk contains a variety of acidic, i.e. sialylated, oligosaccharides, which belong to the family of human milk oligosaccharides (HMOs) (Urashima T. et al., (2011) Nutrition and diet research progress: Milk oligosaccharides, Nova Sciences Publishers Inc, New York, ISBN-978-1-61122-831-1), most notably the sialylated derivatives of 3'-sialyllactose, 6'-sialyllactose and lacto-N-tetraose (LNT), such as LST-a, LST-b, LST-c and disialylated-LNT. While milk from certain mammals such as rodents is particularly rich in sialylated milk oligosaccharides, human milk is particularly rich in neutral oligosaccharides, such as 2'-fucosyllactose, 3-fucosyllactose, lacto-N-tetraose, lacto-N-neotetraose and lacto-N-fucopentaose I.

[0013] Another aspect that is important in drug development is that the active substance should have a stable possible crystal form for use in pharmaceutical quality drug formulations. Those skilled in the art of pharmacy understand that crystallization of active pharmaceutical ingredients provides the best approach to control important physicochemical properties such as stability, solubility, bioavailability, particle size, bulk density, flow properties, polymorphic content and others. Thus, there is a need for crystalline forms of sialic acids such as Neu5Ac, and methods of producing these forms. These crystalline forms should be suitable for pharmaceutical use.

[0014] Another use of Neu5Ac derivatives is as neuraminidase inhibitors to treat viral infections, such as influenza. For example, Neu5Ac is a potential starting material for the synthesis of zanamivir, which can be used to prevent and treat infection with influenza A and B, such as avian influenza virus H5N1 (Kawei et al., Clin Infect Dis 2009, 48:996-997). In addition, Neu5Ac has a wide range of medical applications, such as anticancer, antiadhesion, and anti-inflammatory activities (Varki et al., Lab Invest 2007, 87:851-857).

[0015] The high cost of Neu5Ac is due to its insufficient availability and supply. There are several strategies for preparing Neu5Ac. Traditionally, Neu5Ac is extracted from natural materials such as egg yolk, whey, and edible bird’s nests. However, the Neu5Ac content in natural sources is relatively low, and thus the isolation and purification processes are relatively complex, time-consuming, and tedious, especially considering the complex purification process, impure starting materials, and low yield (Tao et al., Appl Microbiol Biotechnol 2010, 87:1281-1289). Therefore, the conventional method is not cost-effective for large-scale production of Neu5Ac.

[0016] As previously mentioned, Neu5Ac can be isolated from edible bird’s nests. Edible bird’s nests are the highest natural source of Neu5Ac content. The Neu5Ac content in edible bird’s nests is as high as 100 g / kg, while in egg yolk it is 2 g / kg (Koketsu et al., Glycoconj J. 1992 Apr; 9(2): 70-4.). In most cases, Neu5Ac is not fully purified, but only enriched. The product is then sold as a bird’s nest extract. This bird’s nest extract contains only 1.5% of sialic acid (Bird’s Nest Extract, ORYZA OIL & FAT CHEMICAL CO., LTD).

[0017] CN 104072533 A discloses a method for isolating Neu5Ac from bird’s nests. The material is incubated with water, heated to a maximum of 121 °C, and then freeze-dried. Neu5Ac is removed from the so-treated nests by adding ethanol. The ethanol is filtered to remove insolubles and incubated with ethyl acetate to precipitate Neu5Ac. This results in Neu5Ac with a purity of about 79% and a purification yield of 55 to 60%.

[0018] One possible method for the industrial production of Neu5Ac is chemical synthesis. For example, N-acetyl-glucosamine (GlcNAc) and oxaloacetate can be condensed under basic conditions followed by decarboxylation (described in Cornforth et al., Biochem J 1958 68:57-61). Another method is the preparation by asymmetric synthesis from D-mannose and a non-sugar precursor such as 1,2-cis-dihydro-o-benzosemiquinone (Danishefsky et al., J Am Soc 1988, 110:3929-3940). However, in most cases, chemical synthesis of Neu5Ac involves or requires tedious repetitive sequential protection and deprotection steps and can lead to the formation of many reaction intermediates and isomers. These facts can lead to highly complex, difficult and costly separation processes.

[0019] A third option for the industrial production of Neu5Ac is production by biocatalytic processes. The biocatalytic methods include enzyme catalysis, whole-cell biocatalysis and fermentation. Biocatalysis and fermentation methods have become important tools for the production of different substances. These technologies are used for the large-scale production or synthesis of a large number of chemicals, agrochemical intermediates, active pharmaceuticals and food ingredients. Biocatalysis and fermentation offer the possibility of easy and low-cost production of different substances. Most of the methods employ environmentally friendly procedures under mild conditions.

[0020] For the production of Neu5Ac, two different enzymes are described that can be used for biocatalytic or enzyme-based production. These enzymes are Neu5Ac synthase (EC 4.1.3.19) and Neu5Ac aldolase (NAL, formerly known as Neu5Ac lyase, EC 4.1.3.3). Neu5Ac aldolase is more preferred than Neu5Ac synthase because its substrate pyruvate is more readily available than the substrate of Neu5Ac synthase, phosphoenolpyruvate (Tao et al., Appl Microbiol Biotechnol 2010, 87:1281-1289). NAL was first used for Neu5Ac production in 1960, using N-acetyl-D-mannosamine (ManNAc) and pyruvate as starting materials (Comb et al., J Biol Chem 160 235:2529-2537). One key point for the large-scale production of Neu5Ac with this method is that N-acetyl-D-mannosamine is very expensive. Furthermore, N-acetyl-D-mannosamine is not available in large quantities so far.

[0021] DE 3937891 A1 discloses the production of Neu5Ac by a biocatalytic process, which can be transferred to larger scale. The isomerization of N-acetylglucosamine to N-acetylmannosamine catalyzed by N-acetylglucosamine-2-epimerase (EC 5.1.3.8) and the subsequent synthesis of Neu5Ac in the presence of N-acetylneuraminic acid pyruvate lyase (EC 4.1.3.3) and pyruvate can be successfully performed in a bioreactor.

[0022] WO 94 / 29476 A1 discloses an in vitro process for the preparation of N-acetyl-D- neuraminic acid from N-acetyl-D-glucosamine (NAG, GlcNAc). In the preparation, NAG is converted to N-acetyl-D-mannosamine (NAM, ManNAc) by an alkali-catalyzed epimerization. Subsequently, NAM is reacted with pyruvate in a reaction catalyzed by Neu5Ac-aldolase to yield Neu5Ac. The Neu5Ac-aldolase is prepared from recombinant E. coli cells expressing said Neu5Ac-aldolase. The aldolase is immobilized by mixing beads with a crude extract of said recombinant E. coli cells. The conversion of NAM to Neu5Ac is started by adding said immobilized enzyme beads to a mixture of NAM and pyruvate. Neu5Ac is obtained from the reaction mixture, wherein the enzyme is removed by filtration and the filtrate is mixed with glacial acetic acid and a small amount of seeds to obtain a "wet cake". The wet cake is subjected to acetone desolvation or rhombohedral crystallization.

[0023] EP 0578825 A1 discloses an in vitro process for the production of Neu5Ac by treating a mixture of N-acetylglucosamine and pyruvate with N-acetylneuraminic acid lyase under alkaline conditions. The reaction products are separated by ion exchange column chromatography using Dowex 1 (Dow Chemical Company) and the isolate is concentrated by crystallization.

[0024] ​To avoid the use of N-acetyl-D-mannosamine for the reaction mixture, an enzymatic process is known in which N-acetyl-D-glucosamine is used as a substrate. In a first step, N-acetyl-D-glucosamine is converted to N-acetyl-D-mannosamine by N-acyl-D-glucosamine-2-epimerase with a conversion rate of 77%. In a second step, the thus produced N-acetyl-D-mannosamine and pyruvate are used as substrates for N-acetylneuraminate lyase to produce Neu5Ac (Maru et al., Carbohydrate Research, 1998; 306: 575-578). By this method, 29 kg of Neu5Ac can be produced from 27 kg of N-acetyl-D-glucosamine. The produced Neu5Ac is recovered by direct crystallization. The solution is heated to 80°C for 5 min to precipitate the contained enzymes. The insoluble fraction is removed by filtration. To recover Neu5Ac from the reaction mixture, 5 volumes of glacial acetic acid are added to the solution. After crystallization, Neu5Ac is recovered by filtration and washed with ethanol to remove residual acetic acid. The material is dried at 40°C until a constant weight is obtained. Using high-performance liquid chromatography (HPLC) and infrared (IR) spectroscopy, the thus produced and isolated Neu5Ac is not distinguishable from natural Neu5Ac.

[0025] Such a production of Neu5Ac can also be learned from another method for large-scale production. In said method, Neu5Ac is synthesized using an aldosaccharide reductase preparation [N-acetylneuraminate lyase; CAS: 9027-60-5; E.C.: 4.1.3.3. aldosaccharide reductase] obtained from a modified strain of E. coli K12 derivative. This enzyme catalyzes the coupling of N-acetylmannosamine and sodium pyruvate to produce N-acetylneuraminic acid. (Gras Notice 602, GRAS Exemption Claim for / V-Acetyl-D-neuraminic acid (NANA), Glycom A / S). The Neu5Ac produced in this way is then purified in several steps. The first step is the removal of proteins from the catalytic production step by filtration. After filtration, anhydrous Neu5Ac is removed from the reaction mixture by crystallization. After the first crystallization step, the material is dissolved again, treated with charcoal to remove color and impurities, and crystallized again to receive Neu5Ac in the form of crystalline dihydrate. However, the biocatalytic production of sialic acid is technically feasible only on a few hundred kilogram scale, not a realistic option for providing sialic acid (e.g. Neu5Ac) on a several ton scale for food applications, e.g. infant and toddler nutrition products. The inclusion of Neu5Ac in infant nutrition products at a natural concentration of about 0.5 g / l would require an amount of about 3.75 g Neu5Ac per 1 kg of infant food formula. Thus, several tons of material would be required even for a "small" infant food formula product. In addition, the biocatalytic production of sialic acid is too expensive for today's food applications.

[0026] In addition to the in vitro biocatalytic production method, Neu5Ac can also be produced by in vivo fermentation with the help of microorganisms. In this case, the enzymes used will be produced in the microorganism, but will not be isolated as described above. Instead, the entire cell is used as a reaction cup, Neu5Ac is produced inside the cell, and during the production process, Neu5Ac is secreted into the surrounding culture medium. After fermentation, Neu5Ac is isolated from the culture broth. For the production of Neu5Ac, bacteria such as E. coli and yeasts are particularly described in the literature.

[0027] EP 1484406 A1 describes a method for producing Neu5Ac using microorganisms which have the ability to produce Neu5Ac, but have a limited or no ability to decompose Neu5Ac compared to wild-type strains, so that Neu5Ac accumulates in the culture medium and can be recovered therefrom. In order to be able to carry out the production of Neu5Ac, the microorganisms have a strong N-acetylneuraminate synthase activity and / or an N-acetylglucosamine 2-epimerase activity.

[0028] CN 106929461 A discloses a method for producing Neu5Ac using Bacillus subtilis cells expressing genes encoding glucosamine-fructose-6-phosphate transaminase, glucosamine-6-phosphate N-acetyltransferase, N-acetylglucosamine isomerase and N-acetylneuraminic acid synthase. The ptsG gene of the cells has been deleted, which gene encodes the glucose-specific component of the phosphotransferase system EIICBA. By cultivating these cells in a glucose-containing medium, 0.66 g·L -1 of Neu5Ac is obtained.

[0029] Zhu, D. and coworkers (Zhu, D. et al. (2017) Biotechnol. Lett. 39: 227-234) report that overexpression of the PEP synthesis related genes pck and ppsA in E. coli using a high copy number co-expression vector enhances Neu5Ac production. More specifically, E. coli cells are subjected to random mutagenesis and transformed with expression plasmids encoding N-acetylneuraminic acid synthase and N-acetylglucosamine-2-epimerase. Cell lines that grow favorably on glucose-containing media but show limited or no growth on Neu5Ac-containing media are selected. After cultivation for a certain period of time, the cells are pelleted by centrifugation, stored as so-called "wet cells" at -20 °C and used after thawing as required. For production of Neu5Ac, a reaction mixture (30 mL) is provided, which contains 90 g·L -1 N-acetylglucosamine, 50 g·L -1 glucose, 10 mL·L -1 xylene and 200 g·L -1 of the wet cells (permeabilized by the presence of 4 g·L -1 of detergent). After completion of the in vitro reaction, the formation of Neu5Ac is assessed by HPLC.

[0030] US 2003 / 0109007 Al discloses a method for producing Neu5Ac by utilizing a permeabilized microorganism. The method comprises preparing a mixture comprising: (i) a culture of a microorganism having N-acetylneuraminic acid aldolase activity or N-acetylneuraminic acid synthase activity, or a treatment derived from said culture; (ii) a culture of a microorganism capable of producing pyruvate (or a treatment derived from said culture), or a culture of a microorganism capable of producing phosphoenolpyruvate (or a treatment derived from said culture); (iii) N-acetylmannosamine; and (iv) an energy source necessary for the formation of pyruvate or phosphoenolpyruvate. The mixture is prepared in an aqueous medium containing a chelating agent or a surfactant, thereby allowing Neu5Ac to form and accumulate in the aqueous medium. The reaction product is quantified using a carbohydrate analysis system. The disadvantage of the above method is that only small scale production can be performed and that excess pyruvate is required to push the reaction equilibrium towards Neu5Ac. Furthermore, N-acetylglucosamine, N-acetylmannosamine and phosphoenolpyruvate are expensive substrates for these reactions.

[0031] WO 2008 / 040717 A2 discloses a method for producing Neu5Ac comprising culturing a microorganism in a culture medium, wherein the microorganism carries heterologous genes encoding sialate synthase (NeuB) and UDP-GlcNAc epimerase (NeuC), wherein the microorganism lacks a gene encoding CMP-Neu5Ac synthase (NeuA), or wherein any gene encoding CMP-Neu5Ac synthase (NeuA) has been inactivated or deleted, and wherein endogenous genes encoding sialate aldolase (NanA), sialate transporter (NanT) and optionally ManNAc kinase (NanK) have been deleted or inactivated. Neu5Ac is purified from the culture supernatant (2 liters) by precipitation using glacial acetic acid.

[0032] WO 2008 / 097366 A2 relates to metabolically engineered E. coli cells producing Neu5Ac. In the cells, the nanT (sialate transporter) and nanA (sialate aldolase) genes are inactivated and the neuC and neuB genes facilitating Neu5Ac biosynthesis in group B of Neisseria meningitidis are introduced and overexpressed in the nanT-nanA E. coli cells using expression plasmids. In addition, the E. coli glucosamine synthase gene (glmS) is co-overexpressed with neuB and neuC. Neu5Ac is purified from the culture broth by ion exchange chromatography.

[0033] CN 106929461 A relates to genetically engineered strains of Bacillus subtilis for improved production of Neu5Ac. The amount of Neu5Ac in the fermentation culture is determined by HPLC. It is therefore an object to provide microorganisms that are able to produce Neu5Ac more efficiently on an industrial scale and that use inexpensive carbon sources as the sole carbon source.

[0034] WO 2012 / 083329 A1 discloses methods and reagents for the production of Neu5Ac by genetically engineered fungal cells of the genus Trichoderma that constitutively express N-acetylglucosamine-2-epimerase and N-acetylneuraminic acid synthase. Such Trichoderma cells are cultivated in the presence of GlcNAc. The presence of Neu5Ac in the mycelium of this Trichoderma strain is analyzed by HPLC-MS.

[0035] EP 0474410 A2 discloses a method for producing Neu5Ac by hydrolytic delipidation of egg yolk. The method comprises the steps of desalting a solution containing Neu5Ac obtainable by hydrolytic delipidation of egg yolk, adsorbing Neu5Ac onto an anion exchange resin, and eluting Neu5Ac. Desalting can be achieved by using a reverse osmosis membrane, an electrodialysis membrane or a dialysis membrane. The adsorption process can comprise passing the desalted hydrolysate through a cation exchange resin and further through an anion exchange resin. The final eluate is dried under reduced pressure to obtain a solid.

[0036] JP 08-119986 A describes a method for purifying Neu5Ac or its analogs, which comprises synthesizing Neu5Ac by condensing N-acetylmannosamine with pyruvic acid in the presence of a sialate aldolase. The solution containing Neu5Ac or its analogs is concentrated using an evaporator. The concentrated solution is mixed with an organic acid containing two or three carbon atoms. Subsequently, the mixture is heated to 50°C and left to stand at 4°C to precipitate white crystals of Neu5Ac.

[0037] Existing attempts to provide purified sialic acids have only involved small-scale demonstrations, but so far no method has been available for purifying sialic acids from large-scale fermentation broths to food-grade purity and food-grade quality.

[0038] It is an object of the present invention to provide a method for purifying sialic acids, such as Neu5Ac, on a large scale (industrial scale) with high purity. In particular, the method should be suitable for providing sialic acids on a kilogram to ton scale with food-grade quality and should be suitable for running in a continuous manner.

[0039] This object is achieved by the method according to claim 1, the composition according to claim 19, the food composition according to claim 22, the liquid ready-to-feed baby or infant nutritional product according to claim 29, the spray-dried baby formula according to claim 30, the dietary supplement according to claim 31, the premix according to claim 32 and the use of the composition according to claim 34. The dependent claims describe advantageous embodiments.

[0040] According to the present application, a method for purifying sialic acid from a fermentation broth is provided. The method comprises the following steps:

[0041] removing biomass from the fermentation broth comprising sialic acid, wherein a clarified solution is provided,

[0042] providing a purified solution by subjecting the clarified solution to:

[0043] a cation exchanger treatment with a cation exchange material, wherein the cation exchanger treatment is performed under conditions in which sialic acid passes through the cation exchange material and is present in a flow-through; and

[0044] an anion exchanger treatment with an anion exchange material, wherein the anion exchanger treatment is performed under conditions in which sialic acid passes through the anion exchange material and is present in a flow-through; and

[0045] removing salts from the purified solution by electrodialysis.

[0046] The method of the present application is suitable for providing the desired sialic acid in high purity (food grade quality) and on a large scale (industrial scale of kilogram to several tons per run). In addition, the method of the present application can be performed very quickly and in an inexpensive manner. Thus, it can be operated very economically. Furthermore, the method can be performed in a batch-wise manner or in a continuous manner. The latter even further increases the obtainable yield per time. At the end of the method, the purity of the sialic acid can be > 80%, preferably > 90%, more preferably > 95%, most preferably at least 98%.

[0047] The proposed purification method of sialic acid is also advantageous in that the sialic acid preparation is free of recombinant DNA and recombinant proteins originating from the recombinant microbial fermentation strain used for producing the desired sialic acid.

[0048] Although the process of the application is envisaged for purifying a specific sialic acid (e.g. Neu5Ac) from a fermentation broth, the process can also be used for purifying a specific sialic acid produced by an in vitro enzymatic reaction (so-called in vitro biocatalytic reaction), or by a permeabilized whole-cell biocatalytic process. It will be appreciated that purifying a sialic acid from a reaction mixture of an in vitro biocatalytic reaction does not require the removal of biomass from the reaction mixture. The reaction mixture of an in vitro biocatalytic reaction thus corresponds to a clarified process stream.

[0049] Definitions

[0050] According to the present application, the term "purity" refers to chemical purity and designates the extent to which a substance such as a specific individual sialic acid is not diluted or mixed with extraneous substances. Thus, chemical purity is an indicator of the relationship between a single substance and by-products / impurities. Chemical purity is expressed in percentage (%) and calculated using the following formula:

[0051]

[0052] In a composition comprising sialic acids, the purity of the sialic acids can be determined by any suitable method known to the person skilled in the art, for example by using HPLC. A suitable detector can be a detector selected from the group consisting of an electrochemical detector, a refractive index (RI) detector, a mass spectrometer (MS), a diode array detector (DAD) and an NMR detector. For example, in HPLC, the ratio of the area under the peak representing the amount of a specific sialic acid (e.g. Neu5Ac) to the sum of the areas under the peaks representing the amount of the specific sialic acid and the amount of compounds other than the specific sialic acid in the same chromatogram. However, this means that all impurities can be analysed by the selected HPLC method. Otherwise, a mass balance approach has to be taken, i.e. an absolute quantification of the desired product (e.g. Neu5Ac) is performed. In said method, a pure substance is used as a reference for quantifying purity, and then the purity is judged according to the dry matter obtained from the product (desired product plus all impurities). The mass balance approach can also be used to determine purity according to the present application.

[0053] According to the present application, the term "sialic acid" refers to sialic acid within the family of all sialic acids. Thus, according to the present application, the sialic acid to be purified is preferably an N- or O-substituted derivative of neuraminic acid, more preferably an N-substituted derivative of neuraminic acid, even more preferably N-acetylneuraminic acid (2-keto-5-acetamido-3,5-dideoxy-D-glycero-D-galactononulopyranos-1 - onic acid, abbreviated as "Neu5Ac" or "NANA"), N-glycolylneuraminic acid (abbreviated as "Neu5Gc") or 2-deoxy-2,3-didehydro-N-acetylneuraminic acid (abbreviated as "Neu2en5Ac" or "DANA"). In a particularly preferred embodiment of the present application, the sialic acid is N-acetylneuraminic acid ("Neu5Ac" or "NANA").

[0054] According to the present application, "culture broth" refers to any liquid comprising the sialic acid to be purified after fermentation. The terms "culture broth", "fermentation broth" and "culture medium" are used synonymously herein. The culture broth comprises the sialic acid to be purified as well as biomass (e.g. biological cells and cell debris), medium components, salts and contaminants (such as other acids and colored compounds). The purity of the sialic acid to be purified in the culture broth can be < 80%. The biological cells comprised in the culture broth are biological cells that produce the desired sialic acid (e.g. Neu5Ac) within the cells and secrete the produced sialic acid into the liquid culture medium. The biological cells can comprise or consist of genetically modified biological cells, e.g. genetically modified E. coli cells. The genetic modification can comprise or consist of a modification that results in the production of a single (desired) sialic acid (e.g. Neu5Ac), in particular during the growth phase of the biological cells.

[0055] As used herein, the term "biomass" refers to all biological cells present in the fermentation broth at the end of the fermentation step. The biomass includes cells of the microorganism that produces the desired sialic acid (e.g. Neu5Ac), progeny cells of the microorganism that can have lost their ability to produce the desired sialic acid (e.g. Neu5Ac) during the fermentation step, and any other cells that are inadvertently present in the fermentation broth at the end of the fermentation step. Thus, substantially all biological cells present in the fermentation broth at the end of the fermentation step are separated from the fermentation broth, so that the clarified fermentation broth (i.e. the process stream) is substantially free of cells.

[0056] The term "process stream" refers to any solution comprising a specific sialic acid to be purified.

[0057] The term "flow-through" refers to a solution comprising sialic acid to be purified, which has just passed through an ion exchanger material (i.e. a cation and / or anion exchanger material), i.e. a mobile phase comprising sialic acid after contact with a solid phase ion exchanger. In other words, the sialic acid present in the flow-through has not yet been adsorbed to the stationary phase or has not yet been adsorbed by the stationary phase.

[0058] According to the present application, the difference between a weak cation exchanger material and a strong cation exchanger material is that the chemical groups suitable for ion exchange of the former have a pKa of at least 1 (e.g. a pKa of 2 to 5, such as the pKa of a carboxylic acid group), whereas the latter have a pKa of less than 1 (e.g. a pKa of -4 to 0, such as the pKa of a sulfonic acid group).

[0059] Producing a clear solution

[0060] The biomass can be removed from the fermentation broth by centrifugation and / or filtration.

[0061] In a suitable centrifugation process for removing biomass from the culture broth, the biomass is obtained in the form of a pellet, and the supernatant obtained is a clear process stream for further processing. In a suitable filtration process for removing biomass from the culture broth, the filtrate becomes the clear process stream. Preferred filtration processes for removing biomass are microfiltration and / or ultrafiltration. In ultrafiltration, even smaller particles can be removed than in microfiltration. Optionally, the ultrafiltration is cross-flow ultrafiltration.

[0062] Microfiltration is a physical separation process in which a particle-containing fluid is passed through a medium comprising a porous substance containing tortuous channels to retain particles (depth filtration) and / or a membrane having a specific pore size that allows particles / molecules smaller than the pore size to pass through (membrane filtration or dead-end filtration). As used herein, the term "microfiltration" refers to a physical separation process in which biological cells (and cell debris) are removed from the fermentation broth, leaving a (clear) process stream.

[0063] Ultrafiltration is a form of membrane filtration that is not essentially different from dead-end microfiltration. In ultrafiltration, the forces generated by pressure and concentration gradients remove particles and large soluble molecules by passing a liquid containing these particles and large soluble molecules through a semipermeable membrane, resulting in the particles and large soluble molecules being retained in the so-called retentate, while water and low-molecular-weight solutes (such as the sialic acid to be purified) pass through the membrane into the permeate (filtrate). The membrane used for ultrafiltration is defined by its molecular weight cut-off (MWCO), which describes the maximum molecular weight of soluble molecules that can pass through the membrane into the permeate. Any particles that cannot pass through the membrane, as well as molecules larger than the MWCO, will be retained in the retentate. Ultrafiltration can be applied in a cross-flow mode in which the flow of the liquid is parallel to the membrane surface, or in a dead-end mode in which the flow of the liquid is perpendicular to the membrane surface.

[0064] Non-limiting examples of suitable filters for microfiltration and / or ultrafiltration to remove biomass from the fermentation broth include DS MP005 4333, which is an assembly comprising polyethersulfone membranes, the assembly is spiral wound to provide a compact design and better performance, the nominal pore size for ultrafiltration applications is 0.05 pm. The pore size of a suitable membrane for removing biomass by microfiltration can be at least 0.2 pm. Alternatively, biomass removal can be performed by microfiltration using a membrane with a MWCO of 100 to 1000 KDa, preferably 150 kDa to 500 kDa, to remove biomass and additional cell debris, such as larger proteins. For example, FS10-FC FUS1582 (Microdyn-Nadir GmbH, Wiesbaden, DE) can be used as an alternative, which is a hollow fiber ultrafiltration assembly using polyethersulfone membranes (5 m 2 ) with a MWCO of 150,000 Dalton (150 kDa).

[0065] In additional and / or alternative embodiments, smaller particles and large soluble molecules are removed from the clarified process stream by cross-flow ultrafiltration. In this context, an ultrafiltration step can be performed on the clarified process stream using a filter with a MWCO of 10 kDa, such as SpiraCel DSUP010 (Microdyn-Nadir GmbH, Wiesbaden, DE), which is a spiral wound ultrafiltration assembly using polyethersulfone membranes (5.7 m 2 ) with a MWCO of 10,000 Dalton (10 kDa).

[0066] In summary, the following possible ways to remove biomass from the fermentation broth can be employed in the present application:

[0067] 1) By centrifugation The insoluble fraction is removed from the culture broth in one step. Advantages: fast removal of the insoluble fraction;

[0068] 2) By microfiltration The insoluble fraction and macromolecules above a certain size are removed from the culture broth in one step. A spiral wound membrane or a hollow fiber cross-flow filter can be used for microfiltration. A microfiltration membrane with a molecular weight cut-off > 500 kDa, more preferably > 150 KDa, can be used. Advantage: fast removal of the insoluble fraction and macromolecules above a certain size;

[0069] 3) By ultrafiltrationStep 1 : Removal of insoluble parts, macromolecules above a certain size and small molecules from the culture broth. Spiral wound membranes or hollow fiber cross flow filters can be used for ultrafiltration. Ultrafiltration membranes with a molecular weight cut-off of < 100 kDa, more preferably < 10 KDa can be used. Advantage: fast removal of insoluble parts, macromolecules above a certain size and small molecules;

[0070] 4) By centrifugation combined with microfiltration Step 1 : Removal of insoluble parts and macromolecules above a certain size from the culture broth. Spiral wound membranes or hollow fiber cross flow filters can be used for microfiltration. Microfiltration membranes with a molecular weight cut-off of > 500 kDa, more preferably > 150 KDa can be used. Advantage: fast removal of insoluble parts and macromolecules above a certain size without clogging the membranes or filters used in microfiltration;

[0071] 5) By centrifugation combined with ultrafiltration Step 1 : Removal of insoluble parts, macromolecules above a certain size and small molecules from the culture broth. Spiral wound membranes or hollow fiber cross flow filters can be used for ultrafiltration. Ultrafiltration membranes with a molecular weight cut-off of < 100 kDa, more preferably < 10 KDa can be used. Advantage: fast removal of insoluble parts, macromolecules above a certain size and small molecules without clogging the membranes or filters used in ultrafiltration;

[0072] 6) By microfiltration combined with ultrafiltration steps Step 1 : Removal of insoluble parts, macromolecules above a certain size and small molecules from the culture broth. Spiral wound membranes or hollow fiber cross flow filters can be used for microfiltration and / or ultrafiltration. Microfiltration membranes with a molecular weight cut-off of > 500 kDa, more preferably > 150 KDa can be used. Ultrafiltration membranes with a molecular weight cut-off of < 100 kDa, more preferably < 10 KDa can be used. Advantage: fastest removal of insoluble parts, macromolecules above a certain size and small molecules, thereby reducing the risk of clogging the membranes or filters used in ultrafiltration.

[0073] The clarified process stream comprising sialic acid can typically contain a large amount of unwanted impurities including (but not limited to) monovalent ions, divalent ions, amino acids, polypeptides, proteins, organic acids, nucleic acids, monosaccharides and / or oligosaccharides.

[0074] Steps that are preferably not present in the process of the application

[0075] The process of the application is characterized in that the process does not comprise a chromatographic separation. The advantage is that the process can be performed faster and cheaper, since the time and costs for preparing an eluent and using it for eluting the sialic acid from a solid phase are saved.

[0076] Furthermore, the method of the present invention is characterized in that it does not comprise the use of ethanol and / or ethyl acetate, optionally does not comprise the use of organic solvents. The advantage is that it can be ensured that the final product, the final sialic acid or the sialic acid containing composition is free of ethanol and / or ethyl acetate, optionally free of any organic solvent. In addition, the method becomes cheaper and safer for the workers.

[0077] Furthermore, the method of the present invention is characterized in that it does not comprise the use of heavy metals. The advantage is that it can be ensured that the purified sialic acid is free of heavy metals.

[0078] Furthermore, the method of the present invention is characterized in that it does not comprise the use of heavy metals. The advantage is that it can be ensured that the purified sialic acid is free of heavy metals.

[0079] In addition, the method of the present invention is characterized in that it does not comprise the step of heating the fermentation broth, the clarified solution and / or the purification solution to a temperature higher than 45°C. The advantage is that, compared to prior art methods employing such a heat treatment step, energy for heating the respective solutions is saved, which makes the method more economical and more ecological.

[0080] Cation exchanger treatment

[0081] Among other impurities, cations can be removed from the clarified process stream by the cation exchanger treatment step of the method of the present invention, i.e. by subjecting the clarified process stream to at least one cation exchange treatment. Specifically, cations are exchanged to other cations that bind to the cation exchanger material (stationary phase) before the clarified process stream is applied to the cation exchanger treatment step. Importantly, it has been found that the cation exchanger treatment removes ammonia and a portion of the contaminating proteins from the clarified process stream.

[0082] In the cation exchanger treatment step, the positively charged species are bound to the resin after which they can be removed from the cell-free culture broth. The aqueous solution of sialic acid is contacted in any suitable manner that allows the positively charged material to be adsorbed to the cation exchange material while the sialic acid passes through. After contact with the cation exchange resin, the resulting liquid contains water, defined cations (those cations that were fixed on the cation exchanger material prior to this step), anions, coloring substances and the desired sialic acid.

[0083] The cation exchanger treatment can be performed with a weak cation exchange material or a strong cation exchange material, preferably, it is performed with a strong cation exchange material.

[0084] In the cation exchanger treatment of the method of the present application, a strong cation exchanger can be used. Suitable cation exchange resins for removing positively charged compounds are strong acid cation exchange resins, such as resins comprising carboxylic acid groups (weak cation exchanger) or sulfonic acid groups (strong cation exchanger) attached to a solid backbone, such as a polystyrene backbone. Suitable resins include, but are not limited to S 2568 (H + ) (Lanxess AG, Cologne, DE), 50W X2 (Merck KGaA, Darmstadt, DE), IR-116 (Japan Organo Co., Ltd.) and Diaion TM SK-102 (Mitsubishi Chemical Corporation).

[0085] The cation exchanger treatment step can be the first step after removal of the biomass from the culture medium, i.e. the first step of treating the clarified culture medium.

[0086] The non-specific cations are preferably replaced by the specific cation H + or Na + . If the non-specific cations are replaced by H + , it is preferred to adjust the pH of the flow-through to a pH of 6 to 8, most preferably by adding NaOH to the flow-through, before performing a further treatment step, such as an anion exchanger treatment.

[0087] In a preferred embodiment, the cation exchange material is present in the form of H + , i.e. the ion exchange ligands of the cation exchange material are protonated before applying the clarified process stream to the cation exchanger. This allows for the replacement of the cations in the clarified process stream by H + during the cation exchange. Thus, the pH of the solution after the described step (flow-through) is more acidic than the pH of the solution before the described step. Preferably, the pH of the flow-through is increased to a neutral or almost neutral pH value (e.g. > pH 6.5 and < pH 7.5) before a subsequent purification step. The increase of the pH can be achieved by adding NaOH to the process stream. By this measure, the sialic acid in the protonated form (H + form) is converted to the sialate salt. In case NaOH is added, the sodium salt form (Na + form) of the sialic acid is obtained.

[0088] Cation exchangers can be used and in any alkali metal (Li + , Na + , K +), an alkali earth metal (e.g. Ca 2+ , Mg 2+ ), an ammonium ion or a carbonate ion as counter ion. Preferably, sodium (Na + ) is the counter ion of the cation exchanger. More preferably, hydrogen (H + ) is the counter ion. The advantage of hydrogen as counter ion is that upon contact with the cation exchanger, a protonated form of the sialic acid is generated, which is present in the flow-through, i.e. in the solution that passes through the cation exchange material. Subsequently, the protonated form of the sialic acid can be neutralized by adding a base (e.g. NaOH) to the product stream, which converts the sialic acid into its salt form (e.g. sodium form). The sodium form of the sialic acid is beneficial, since sodium ions are relatively small cations and can be more easily removed by nanofiltration and / or electrodialysis compared to larger cations.

[0089] Preferably, the cation exchanger treatment step is performed prior to the anion exchanger treatment step. The advantage of this sequence is that the salt concentration of the process stream that has passed through the anion exchanger material and comprises the desired sialic acid is low, since the sialic acid does not bind to the cation exchanger material (i.e. is in the flow-through). The flow-through can then be subjected to anion exchanger treatment without the need for a desalting step. However, in principle, the cation exchanger treatment can also be performed after the anion exchanger treatment step.

[0090] The particle size of the cation exchange resin is preferably between 0.1 and 1 mm. This particle size range allows for an efficient flow of the used cell-free culture fluid, while the charged material is still efficiently removed by the cation exchange resin. To ensure that an efficient ion exchange can take place, the flow rate should preferably be between >0.5 and <2.5 times the bed volume, more preferably between >1.0 and <2.0 times the bed volume. The ion exchange treatment can be performed in a conventional manner, e.g. batch-wise or continuously, preferably continuously.

[0091] The conditions for the passage of the sialic acid through the cation exchange material can be established by adjusting the pH and / or the salt concentration of the clarified solution, preferably by adjusting the pH of the clarified solution to a pH between 6 and 8, and / or, if necessary, adjusting the salt concentration.

[0092] After the cation exchanger treatment and the anion exchanger treatment, the purified solution can comprise sialic acid, coloring substances and salts, wherein the salts are preferably NaCl. According to a preferred embodiment of the present application, in the anion exchanger treatment, an anion exchange material in the form of a chloride is used.

[0093] According to another preferred embodiment of the present application, in the cation exchanger treatment, a cation exchange material in the form of hydrogen is used. It has been found that the use of a cation exchange material in the form of hydrogen (H +Cation exchangers in the H + form are beneficial because the binding of salts and contaminating proteins is much better compared to any other form of cation exchanger (counterion ≠ H The use of the H form results in a flow-through having a more acidic pH than the solution used for cation exchanger treatment. However, the pH can easily be increased to a neutral pH, preferably pH 6 to 8, by addition of a base, preferably NaOH. Neutralization with NaOH is advantageous because the introduced sodium ions are relatively small and can easily be removed by nanofiltration and / or electrodialysis.

[0094] The particle size of the cation exchange resin should be chosen to allow efficient flow of the used cell-free culture fluid, while the charged material can still be efficiently removed by the cation exchange resin. To ensure that efficient ion exchange can take place, the flow rate should preferably be > 0.2 to < 2.0 times the bed volume, more preferably > 0.5 to < 1.5 times the bed volume. The ion exchange treatment can be performed in a conventional manner, for example batch-wise or continuously, preferably continuously.

[0095] The clarified solution can first be subjected to cation exchanger treatment and then to anion exchanger treatment. The advantage of this method order is that the cation exchanger material can be in the H + form to obtain strong binding of contaminating proteins, and that after neutralization with a base, for example NaOH, the anion exchanger material can be run with a solution comprising a defined salt form of sialic acid, for example the Na + form of sialic acid.

[0096] Anion exchanger treatment

[0097] Preferably, the anion exchanger treatment step is performed after the cation exchanger treatment step. However, in principle, it can also be performed before the cation exchanger treatment step, i.e. after the process stream is treated with a cation exchanger.

[0098] The anion exchanger step is performed to remove non-specific anions and replace them with specific anions, preferably the specific anions CI - or OH - If the non-specific anions are replaced by CI - , the pH of the flow-through is preferably adjusted to pH 6 to 8, most preferably by adding NaOH to the flow-through, before a further treatment step is performed, for example removal of salts from the purified solution by electrodialysis.

[0099] Anions can be removed from the clarified process stream by an anion exchanger treatment step of the process of the application, i.e. by applying at least one anion exchanger treatment to the clarified process stream. The anion exchanger treatment step can be the first step after the removal of the biomass from the culture medium, i.e. the first step in which the clarified culture medium is treated. Preferably, the anion exchanger treatment is performed in a step subsequent to the cation exchanger treatment step.

[0100] When the negatively charged material is bound to the anion exchange resin, it can be removed from the process stream. The aqueous solution comprising the desired sialic acid is contacted in any suitable manner which allows the negatively charged material to be adsorbed onto the anion exchange material, while the sialic acid passes through. After contact with the anion exchange resin, the resulting liquid contains water, defined cations, defined anions, colouring substances and the desired sialic acid. The conditions of the anion exchanger step are such that the sialic acid does not bind to the anion exchanger material, i.e. it is present in the flow-through after contact with the material.

[0101] At the beginning of the anion exchanger treatment, the pH of the product stream is preferably adjusted to a pH of 6 to 8 (most preferably a pH of 7). The flow-through (product stream which has passed through the anion exchanger material) can have a lower pH, for example a pH of 4.5 to 6. At this pH, the sialic acid is present in the sodium form. The pH of the flow-through can be increased by the addition of a base, for example NaOH.

[0102] During the anion exchange process, the anions in the clarified process stream can be replaced by CI - The pH of the process stream thus becomes slightly more acidic. Preferably, the pH is increased prior to the subsequent purification steps, preferably to a neutral or almost neutral pH value (i.e. > pH 6.5 and < pH 7.5) of the process stream. More preferably, the increase in the pH of the process stream is achieved by the addition of NaOH to the process stream.

[0103] The anion exchanger can be a weak anion exchanger or a strong anion exchanger, preferably a strong anion exchanger.

[0104] Suitable strong basic anion exchange resins are resins comprising trimethylammonium groups or hydroxyethyl groups attached to a solid backbone, for example a polystyrene backbone. Suitable resins include (but are not limited to) S6368 A, S4268, S5528, S6368 A (Lanxess AG, Cologne, DE), AG 1 x 2 (200-400 mesh), 1 x 8 (100-200 mesh), Chromalite CGA100x4 (Purolite GmbH, Ratingen, DE), FPA51 (Dow Chemicals, Ml, USA). Preferably, the anion exchange resin is present in chloride form. Suitable weak anion exchange resins are resins comprising amino groups attached to a solid backbone, such as a polystyrene backbone.

[0105] An anion exchanger can be used, with any base as counterion, such as HC03 - , I - , Br - , N03 - . Preferably, the counterion is hydroxide (OH - ). More preferably, the counterion is chloride (CI - ). An advantage of using chloride as counterion is that CI - anions are smaller than many other anions originally present in the fermentation broth. This enables that the chloride anions can be more easily removed from the process stream by electrodialysis. An advantage of using OH - anions as counterion is that, in a step after the anion exchanger treatment, small anions chloride are introduced into the process stream which are more easily removed compared to other anions when neutralizing the pH with HC1.

[0106] The particle size of the anion exchange resin is preferably 0.1 to 1 mm to allow efficient flow of the process stream used, while still effectively removing the charged material by the anion exchange resin. To ensure that an efficient ion exchange can take place, the flow rate should preferably be > 0.5 to < 2.5 times the bed volume, more preferably > 1.0 to < 2.0 times the bed volume. The ion exchange treatment can be carried out in a conventional manner, such as batchwise or continuously, preferably continuously.

[0107] If necessary, conditions for sialic acid passage through the anion exchanger material can be established by adjusting the pH and / or salt concentration of the clarified solution, preferably by adjusting the pH of the clarified solution to 6-8 and / or by adjusting the salt concentration. For example, it has been observed that if a solution comprising sialic acid, which originates from a flow-through of a cation exchanger treatment with H+as counterion and has been adjusted to a pH of 6 to 8 by addition of NaOH, is subjected to anion exchanger treatment, the salt concentration of the flow-through is sufficiently high to avoid binding of sialic acid to the anion exchange resin. However, it has been observed that under said conditions, contaminants, such as certain proteins, DNA molecules and colored substances, still bind to the anion exchanger resin.

[0108] In a preferred embodiment, the purification method comprises treating the clarified solution with hydrogen (H +) form of cation exchange resins and chlorides (Cl) - Treatment is performed using anion exchange resins in the form of cation exchange resins. The cation exchange resin is preferably a strong cation exchange resin. The anion exchange resin can be a weak or strong anion exchange resin, preferably a strong anion exchange resin. Alternatively, the anion exchange material can also be an absorbent material. Treatment using both cation and anion exchange resins allows for the removal of all non-specific ions from the product stream. Therefore, non-specific ions can be replaced by specific anions, preferably sodium cations (e.g., in the presence of H+). + After contact with the cation exchanger material acting as a counterion (introduced by neutralizing the product stream with NaOH), sodium cations and chloride anions are introduced. Both sodium cations and chloride anions are relatively small ions and can be removed more quickly and economically by electrodialysis compared to larger ions.

[0109] Concentration of purified solution

[0110] In a preferred embodiment, the solution (flowthrough) treated with an ion exchanger containing the desired sialic acid (e.g., N-acetylneuraminic acid) is concentrated, preferably by nanofiltration, reverse osmosis, and / or vacuum evaporation (e.g., using a falling film evaporator, rotary evaporator, or plate evaporator). Reverse osmosis and / or nanofiltration are preferred methods (e.g., using a size exclusion limit ≥ ). Nanofiltration (nanofiltration membranes) is particularly preferred. The advantages of nanofiltration over reverse osmosis are that it achieves faster concentration and also partially removes salt ions. Compared to vacuum evaporation, nanofiltration does not undergo caramelization with sialic acid, meaning that colored caramel bodies are not produced during the concentration process.

[0111] The solution is most preferably concentrated and purified by nanofiltration, wherein a nanofiltration membrane with a molecular weight cutoff of 100 to 200 kDa is most preferably used. This molecular weight cutoff has the advantage of retaining the sialic acid to be purified, while salt ions can pass through the membrane, thus achieving desalination in addition to concentration.

[0112] Before concentrating the solution, the concentration of sialic acid in the solution can be ≤20% (w / w), ≤10% (w / w), or ≤5% (w / w).

[0113] In this method, the clarified solution and / or purified solution can be concentrated to a sialic acid concentration of up to ≥100 g / L, preferably ≥200 g / L, and more preferably ≥300 g / L.

[0114] Furthermore, the clarified and / or purified solutions can be concentrated by nanofiltration at a temperature of <80°C, preferably <50°C, more preferably 4°C to 45°C, even more preferably 10°C to 40°C, and even more preferably 15°C to 30°C, most preferably 15°C to 20°C.

[0115] Furthermore, the clarified solution and / or the purified solution can be concentrated by reverse osmosis at a temperature of 20°C to 50°C, more preferably 30°C to 45°C, most preferably 35°C to 45°C.

[0116] Furthermore, the clarified solution and / or the purified solution can be concentrated at a pressure of > 5 bar to < 50 bar, preferably at a pressure of > 10 bar to < 40 bar, more preferably at a pressure of > 15 to < 30 bar.

[0117] Reverse osmosis is a membrane filtration method that removes particles larger than 0.1 nm from a solution (process stream). Only water is removed from the process stream, while all other molecules (such as ions, sugars, etc.) will be concentrated in the retentate. By using reverse osmosis, only an increase in sialic acid concentration can be achieved, not desalination.

[0118] In additional and / or alternative embodiments, the concentration step comprises at least one, optionally all, of the following parameters:

[0119] i) concentration is performed to a concentration of sialic acid of up to > 100 g / L, preferably > 200 g / L, more preferably > 300 g / L;

[0120] ii) concentration is performed at a temperature of < 80°C, preferably < 50°C, more preferably 4°C to 45°C; most preferably 4°C to 40°C if concentration is performed by nanofiltration.

[0121] iii) concentration is performed at a temperature of < 50°C, preferably 20°C to 45°C; most preferably 30°C to 45°C, more preferably 35°C to 45°C if concentration is performed by reverse osmosis and / or vacuum evaporation; and

[0122] iv) concentration is performed at a pressure of > 5 bar to < 50 bar, preferably at a pressure of > 10 bar to < 40 bar, more preferably at a pressure of > 15 to < 30 bar.

[0123] In another preferred embodiment of the present application, after the electrodialysis step, the solution comprising sialic acid is concentrated using vacuum evaporation (e.g. by using a falling film evaporator or a plate evaporator), reverse osmosis or nanofiltration (e.g. nanofiltration using a nanofiltration membrane with a size exclusion limit of > 0.1 nm to < 10 nm, preferably > 0.1 nm to < 5 nm, more preferably > 0.1 nm to < 2 nm, most preferably > 0.1 nm to < 1 nm), preferably using nanofiltration or reverse osmosis, more preferably using nanofiltration.

[0124] Removal of salts

[0125] The method of purifying sialic acid comprises a step of removing salts from the solution, preferably from the clarified solution and / or the purified solution. The salts can be removed by nanofiltration and / or electrodialysis of the solution to be desalted. ​

[0126] Nanofiltration is a membrane filtration process in which the membrane contains pores of nanometer size. Nanofiltration membranes have a pore size of 1 to 10 nanometers. The pore size of nanofiltration membranes is smaller than that of microfiltration membranes and even smaller than that of ultrafiltration membranes, but in fact larger than that of membranes used for reverse osmosis. Membranes for nanofiltration are mainly made of polymeric thin films. Commonly used materials include polyethylene terephthalate or metals such as aluminum. The pore density can be 1 to 10 6 pores / cm 2 In this process nanofiltration is used to purify the desired sialic acid to increase the concentration of sialic acid in the solution (e.g. in the clarified solution and / or in the purified solution). In addition, desalination of the solution (process stream) will also occur.

[0127] Suitable membranes for nanofiltration include polyamide or polyazipyrrolidine thin film composite membrane materials providing a size exclusion of 150 to 300 Da, such as Dow Filmtec™ NF270 (Dow Chemical Company, USA). Such membranes allow for high flux. In particular, nanofiltration membranes with a molecular cut-off of 100 to 300 KDa are advantageous to increase the concentration of the desired sialic acid (e.g. Neu5Ac) in the process stream. Membranes with this molecular cut-off prevent the passage of sialic acid through the membrane and have the advantage that they also achieve desalination, since salts (e.g. sodium chloride) present in the process stream after treatment with ion exchanger material pass through the membrane and are separated from the sialic acid. Other examples of suitable membranes for nanofiltration include Trisep 4040-XN45-TSF (Microdyn-Nadir GmbH, Wiesbaden, DE), GE4040F30 and GH4040F50 (GE Water & Process Technologies, Ratingen, DE).

[0128] It was found that nanofiltration effectively removes a large amount of contaminants (e.g. salt ions) prior to the electrodialysis treatment of a solution containing sialic acid. It was also found that nanofiltration effectively removes low molecular weight contaminants from the clarified fermentation broth after removal of the biomass (e.g. by an ultrafiltration step) from the fermentation broth. Removal of low molecular weight components is advantageous for concentration and demineralization of the solution comprising sialic acid prior to ion exchange treatment. The use of nanofiltration to increase the concentration of sialic acid results in lower energy and processing costs and better product quality due to reduced heat exposure.

[0129] Electrodialysis combines dialysis and electrolysis and can be used for the separation and concentration of ions in a solution based on the selective electromigration of ions in the solution through a semipermeable membrane. The application of industrial electrodialysis can be traced back to the early 1960s when the method was used for the demineralization of cheese whey for inclusion in infant formula. Other applications of electrodialysis include adjusting the pH of beverages (e.g. wine, grape juice, apple juice and orange juice).

[0130] Desalination of salt and fresh water for drinking water production and demineralization of whey for infant food production are the most widespread applications of electrodialysis today. The basic principle of electrodialysis consists of an electrolytic cell comprising a pair of electrodes for conducting ions immersed in an electrolyte solution connected to a direct current generator. The electrode connected to the positive pole of the direct current generator is the anode and the electrode connected to the negative pole is the cathode. The electrolyte solution then supports an electric current which is generated by the movement of negative and positive ions towards the anode and cathode respectively. The membranes used in electrodialysis are essentially porous ion exchange resin sheets with negative or positive charge groups and are therefore described as cationic or anionic membranes respectively. Ion exchange membranes are usually composed of a polystyrene matrix with suitable functional groups cross-linked with divinylbenzene, such as sulfonic acid for cationic membranes or quaternary ammonium groups for anionic membranes.

[0131] The electrolyte solution can be, for example, an aqueous solution comprising sodium chloride, sodium acetate, sodium propionate and / or sulfamic acid. The electrolyte solution surrounds the cathode and anode and serves to allow the flow of electric current within the cell. The electrodialysis stack is then assembled in such a way that the anionic and cationic membranes are parallel, as between two electrode blocks in a filter press, to separate the ion-depleted stream from the ion-enriched stream sufficiently (both solutions are also known as diluate (ion-depleted) and concentrate (ion-enriched)).

[0132] The core of the electrodialysis process is the membrane stack, which consists of several anion and cation exchange membranes mounted between two electrodes separated by spacers. By applying a direct current, anions and cations will migrate across the membranes towards the electrodes, resulting in a diluate stream and a concentrate stream.

[0133] The pore size of the ion exchange membranes used in electrodialysis is small enough to prevent the diffusion of products from the diluate stream into the concentrate stream driven by the high concentration difference between the two streams. After separation from the biomass and / or exchange of cations and / or anions, proteins, and especially recombinant DNA molecules (ranging in size from fragments to entire genomes), must be quantitatively removed from the desired product.

[0134] Electrodialysis is used to remove ions from an aqueous solution, whereas sialic acid will remain in the process stream. An important advantage of electrodialysis is that it can completely remove recombinant DNA molecules from a solution comprising sialic acid to be purified. In addition, it has been found that electrodialysis can significantly reduce the amount of salts in the process stream. In fact, it has been found that sodium chloride can be completely removed from the product stream. Such an advantage is that it is possible to provide sialic acid free of salts such as sodium chloride, preventing any negative effects of the presence of salts (e.g. sodium chloride) in the final product, such as infant food.

[0135] Electrodialysis can be performed until a concentration of 0.2 to 10.0 mS / cm is achieved. 2 Stable electrical conductivity (mS / cm) 2 Preferably, the concentration is 0.4 to 5.0 mS / cm. 2 More preferably 0.5 to 1.0 mS / cm 2 Furthermore, electrodialysis can be performed until the amount of salt (g / l) is <10.0 g / l, preferably <5.0 g / l, more preferably <1.0 g / l, and most preferably <0.5 g / l.

[0136] Electrodialysis can be performed under neutral or acidic conditions. The difference between the two lies in the form of sialic acid. At neutral pH, sialic acid exists as a sodium salt. In this case, the pH must be controlled during electrodialysis. At acidic pH, sialic acid exists in its free form (hydrogen form). The advantage of using electrodialysis under acidic conditions is that the electrodialysis is performed on sialic acid in its sodium form, leaving sialic acid in its free (hydrogen) form after electrodialysis. In short, electrodialysis under acidic conditions not only removes contaminating salts from sialates (e.g., the sodium salt of sialic acid) but also converts sialates into the free (hydrogen) form of sialic acid.

[0137] By applying neutral conditions during electrodialysis, the sodium form of sialic acid to be purified exhibits non-charge (due to the interaction between the negatively charged carboxyl group of sialic acid and the positively charged sodium ion). Before starting electrodialysis, the pH of the process stream can be adjusted with an acid (preferably hydrochloric acid (HCl)) or a base (preferably sodium hydroxide (NaOH)) until a pH of 5.0 to 9.0 is reached, preferably 6.0 to 8.0, and more preferably 6.5 to 7.5.

[0138] By applying acidic conditions during electrodialysis, the protonated form of sialic acid becomes uncharged (the negatively charged carboxyl group of sialic acid interacts with the positively charged proton). Before initiating electrodialysis, the process flow is acidified with acid (preferably hydrochloric acid (HCl)) until a pH of 1.0 to 3.0 is reached, preferably 1.5 to 2.5, more preferably 1.8 to 2.2. Electrodialysis is preferably carried out until a stable conductivity (mS / cm) is achieved. 2 ) and pH.

[0139] Electrodialysis can be performed up to 1.0 to 10.0 mS / cm. 2 Stable electrical conductivity (mS / cm) 2 Preferably, the concentration is 1.5 to 10.0 mS / cm. 2 More preferably 2.0 to 8.0 mS / cm 2. In the electrodialysis process, base (preferably sodium hydroxide) has to be used to control and adjust the pH value. Under the neutral conditions, the electrodialysis can be performed using bipolar membranes. In this case, the sialic acid can be concentrated in a separate electrodialysis concentrate circuit. Thus, the sialic acid can be enriched in the electrodialysis process.

[0140] In the method, the salt is removed from the purified solution,

[0141] i) the amount of salt in the purified solution can be < 10% (w / w), preferably < 5% (w / w), more preferably < 1% (w / w); and / or

[0142] ii) the conductivity can be 0.2 to 10.0 mS / cm 2 , preferably 0.4 to 5.0 mS / cm 2 , more preferably 0.5 to 1.0 mS / cm 2 .

[0143] Decolorization of the clarified and / or purified solution

[0144] The clarified solution and / or the purified solution can be subjected to a decolorization step, preferably by treatment with activated carbon and / or treatment with a cation exchanger and an anion exchanger coupled in series.

[0145] The decolorization step can be performed:

[0146] i) before or after the diafiltration and / or concentration step of the clarified solution; and / or

[0147] ii) before or after the electrodialysis and / or diafiltration step of the clarified solution.

[0148] Optionally, the step is performed after the nanofiltration step and / or before the electrodialysis step.

[0149] The advantage of the treatment with activated carbon compared to the treatment with cation exchanger and anion exchanger (coupled in series) is that both charged and uncharged (neutral) colorants can be removed and uncharged (neutral) oligosaccharides can be removed.

[0150] Activated carbon (also known as activated charcoal) is a form of carbon that has been treated to have small, small-volume pores that increase the surface area available for adsorption. Typically, due to its high microporosity, only one gram of activated carbon has a surface area greater than 3000 m 2 of surface area, as determined by gas adsorption.

[0151] Carbohydrate species, such as monosaccharides or oligosaccharides, tend to bind from aqueous solution to the surface of charcoal particles. Oligosaccharides interact much more strongly with activated carbon than monosaccharides. This behaviour is due to their structure and results in a weaker binding of sialic acids, such as Neu5Ac, to activated carbon, i.e. in smaller amounts. In addition to oligosaccharides, coloured substances are also adsorbed on activated carbon. Other water-soluble materials, such as salts, bind in a weaker manner and are washed out of the activated carbon together with the desired sialic acids by rinsing the activated carbon with water (after incubation). After washing out with water, the adsorbed oligosaccharides and coloured substances remain bound to the activated carbon. Thus, the treatment step with activated carbon removes the contaminating oligosaccharides and coloured contaminants. In summary, the activated carbon step removes colouring agents, other impurities and reduces the amount of water-soluble contaminants such as salts.

[0152] Suitable activated carbons for removing colouring compounds, oligosaccharides or contaminants are, but not limited to, granulated activated carbons, such as Norit GAC830EN (Carbot Cooperation) and Epibon Y 12 x 40 spezial (Donaucarbon), or powdered activated carbons, such as Norit DX1, Norit SA2 (Carbot Cooperation) and Carbopal MB 4 (Donaucarbon).

[0153] The advantage of removing colouring substances by treatment with cation exchangers and anion exchangers (coupled in series) compared to treatment with activated carbon is that not only colouring substances can be removed from the solution, but also non-specific salt ions in the solution can be exchanged for specific salt ions, such as Na + and CI". The advantage of the ion exchange is that the desalination process can become more efficient (Na + and CI" are small ions and thus can be removed more easily by the desalination treatment step). Another advantage is that the adverse effects of certain non-specific salts on sialic acid crystallisation can be avoided.

[0154] The ion exchanger treatment is performed so that the charged material and the colouring substances are adsorbed to the respective ion exchange material, while the sialic acids pass through the respective ion exchange material, i.e. are in the flow-through. The resulting liquid (flow-through) contains water, small amounts of the defined ions, small amounts of colouring substances and sialic acids.

[0155] The cation exchanger can be a weak cation exchanger or a strong cation exchanger, preferably a strong cation exchanger.

[0156] Suitable cation exchange resins are strong acid cation exchange resins, such as, but not limited to S2568 (H + ) (Lanxess AG, Cologne, DE), 50W X2 (Merck KGaA, Darmstadt, DE), IR-116 (Japan Organo Co., Ltd.) and Diaion TM SK-102 (Mitsubishi Chemical Corporation).

[0157] Cation exchangers can be used with any alkali metal (Li + , Na + , K + ), alkaline earth metal (e.g. Ca 2+ , Mg 2 + ), ammonium ion or carbonate ion as counter ion. Preferably, sodium ion (Na + ) is the counter ion.

[0158] Anion exchangers can be weak anion exchangers or strong anion exchangers, preferably it is a strong anion exchanger.

[0159] Suitable anion exchange resins are strong basic (Type I) anion exchange resins, such as (but not limited to) S6368A, S4268, S5528, S6368A (Lanxess AG, Cologne, DE), AG 1 x 2 (200-400 mesh), 1 x 8 (100-200 mesh), Chromalite CGA100 x 4 (Purolite GmbH, Ratingen, DE), FPA51 (Dow Chemicals, Ml, USA). Preferably, the anion exchange resin is present in chloride form.

[0160] Anion exchangers can be used with any alkali as counter ion, such as HC03 - , I - , Br - , N03 - . Preferably, hydroxide (OH - ) is used as counter ion. More preferably, chloride (CI - ) is used as counter ion.

[0161] In the tandem coupling of a cation exchanger and an anion exchanger, the anion exchanger can be located upstream or downstream of the cation exchanger. Preferably, the anion exchanger is located downstream of the cation exchanger in the tandem coupling. Thus, the solution passing through this tandem first passes through the cation exchanger and then through the anion exchanger.

[0162] The pH of the process stream (i.e. the respective flow-through) through the cation exchanger and / or the anion exchanger is preferably > 2.0 and < 10.0, more preferably > 3.0 and < 9.0, most preferably > 4.0 and < 8.0.

[0163] The particle size of the ion exchange resin should be chosen to allow efficient flow of the process stream, while still allowing efficient removal of the charged material and colouring substances by the ion exchange resin. To ensure that efficient ion exchange can take place, the flow rate should preferably be > 0.2 to < 2.0 times the bed volume, more preferably > 0.5 to < 1.5 times the bed volume, most preferably > 0.75 to < 1.2 times the bed volume. The ion exchange treatment can be carried out in a conventional manner, e.g. batchwise or continuously, preferably continuously.

[0164] Sterile filtration and / or endotoxin removal

[0165] In a preferred embodiment of the present application, the purified solution is sterile filtered and / or subjected to endotoxin removal, preferably by filtering the purified solution through a < 10 kDa filter module. For example, the purified solution is filtered through a 3 kDa filter or through a 6 kDa filter, the purified solution containing sialic acid is filtered sterile and / or subjected to an endotoxin removal step. If the sialic acid is to be consumed by humans, endotoxins must be removed.

[0166] Conversion of free sialic acid (hydrogen form) into its salt form

[0167] To convert sialic acid in free acid form or sialic acid in sodium salt form into a different salt form, any cation exchanger of an alkali metal (Li + , Na + , K + ), an alkaline earth metal (e.g. Ca 2+ , Mg 2+ ), an ammonium ion or a carbonate ion can be used to form the respective salt. Sialic acid in salt form, in particular in sodium form, is significantly more stable than sialic acid in free acid form, since, unlike the free acid form, no discoloration reactions have been found to occur in aqueous media.

[0168] The sialic acids comprised in the clarified solution and / or the purified solution are preferably converted into their sodium form, preferably by treating the purified solution with a strong cation exchanger material in the sodium form. The advantage of the sodium form is that it is a pH neutral substance, i.e. when dissolved in water, the pH of the water remains neutral. In contrast, when sialic acids in the free form (hydrogen form) are dissolved in water, the pH of the water becomes acidic. It has been observed that under acidic conditions and at temperatures above 10 °C, sialic acids, in particular Neu5Ac, are prone to develop a color change, i.e. are prone to form colored compounds. Since the sodium form of sialic acids does not exhibit a pH lowering behavior, the sodium form is more stable and prevents the formation of colored compounds.

[0169] Conversion of the sodium form of sialic acids into their free form (hydrogen form)

[0170] The sodium salt of sialic acids can be converted into the free sialic acid (protonated form) by another cation exchange step. In this step, the sodium ions (Na + ) associated with the sialic acids or the sodium ions (Na + ) in the clarified process stream are replaced by protons (H + ). This exchange results in a more acidic pH of the process stream.

[0171] By treating a solution comprising sodium sialic acid salts with a cation exchanger in the hydrogen (H + ) form, the positively charged sodium ions can be removed from the sialic acids. The cation exchanger can be a weak cation exchanger or a strong cation exchanger, preferably a strong cation exchanger. In this step, the positively charged sodium ions from the sialic acids can be removed from the deprotonated sialic acid molecules and bound to the resin, wherein H + ions are released from the resin and can protonate the deprotonated sialic acids. The aqueous solution of sialic acids is contacted in any suitable manner that allows the exchange of sodium ions for hydrogen ions and the adsorption of sodium ions to the cation exchange material, while the sialic acids pass through. After contact with the cation exchange resin, the resulting liquid contains protonated sialic acids.

[0172] Suitable cation exchange resins for removing the positively charged sodium from the sialic acid solution are strong acidic cation exchange resins, such as (but not limited to) S2568 (H + ) (Lanxess AG, Cologne, DE), 50WX2 (Merck KGaA, Darmstadt, DE), IR-116 (Japan Organo Co., Ltd.) and Diaion TM SK-102 (Mitsubishi Chemical Corporation).

[0173] Instead of converting the salt of sialic acid to the free acid or to a different salt form by treatment with a cation exchanger, an electrodialysis step can be implemented. Electrodialysis can be used to remove sodium from the process stream under acidic conditions, while sialic acid will remain in the process stream in protonated form.

[0174] By applying acidic conditions during the electrodialysis, the sialic acid can be protonated, making it behave uncharged (protonation of the carbonyl group). Prior to starting the electrodialysis, the process stream is acidified with an acid, preferably hydrochloric acid (HC1), until a pH of 1.0 to 3.0, preferably 1.5 to 2.5, more preferably a pH of 1.8 to 2.2 is reached. The electrodialysis is preferably performed until a stable conductivity (mS / cm 2 ) and pH is reached.

[0175] Providing sialic acid in spray-dried form

[0176] In a preferred embodiment, the purified solution is spray-dried. The purified solution subjected to spray-drying preferably comprises sialic acid in sodium form.

[0177] The purified solution can be spray-dried at a sialic acid concentration of 5-35% (w / w), preferably 10-30% (w / w), more preferably 15-25% (w / w). The inlet temperature can be 110-150°C, preferably 120-140°C, more preferably 125-135°C. The outlet temperature can be 60-80°C, preferably 65-70°C.

[0178] Providing sialic acid in crystalline form

[0179] The purified solution can be subjected to crystallization, preferably by adding acetic acid to the purified solution. According to this embodiment, sialic acid is preferably provided in dihydrate crystal form.

[0180] For the purpose of following the embodiments of the present application, a method for crystallizing sialic acid from an aqueous solution will be described herein. It has been found that sialic acid (e.g. Neu5Ac) can be selectively crystallized in dihydrate form from a biocatalytic process using enzymes or fermentation broth (culture broth).

[0181] In a preferred embodiment, the purified sialic acid (aqueous) solution used for crystallization has a carbohydrate content of more than 20% (w / w), preferably more than 30% (w / w), preferably more than 40% (w / w), in particular more than 50% (w / w). Alternatively, the sialic acid concentration in the purified (aqueous) solution to be used for crystallization is 450-600 g / l, preferably 500-550 g / l.

[0182] The above concentration ranges and ratios can be achieved in a conventional manner by concentrating the aqueous process stream from the culture broth, preferably after removal of e.g. cells, coloring substances, salts and / or charged molecules from the culture broth by using the aforementioned methods.

[0183] Concentration of the sialic acid containing solution can be achieved by vacuum evaporation, e.g. by using a rotary evaporator or a plate evaporator, or by nanofiltration. For the initiation of crystallization, seed crystals are added to the concentrated sialic acid solution at a temperature of 20°C to 50°C, preferably at 25°C.

[0184] The carbohydrate concentration of the sialic acid containing solution can be > 50% (w / w). The crystallization process can be performed under vacuum, preferably under at least one of the following conditions:

[0185] i) a pressure of less than 200 mbar, more preferably at a pressure of less than 100 mbar, especially at a pressure of less than 50 mbar;

[0186] ii) a product temperature of 15°C to 60°C, preferably of 20°C to 45°C, more preferably of 25°C to 40°C, in particular of 30°C to 35°C; and

[0187] iii) an incubation time until the carbohydrate concentration reaches > 60%, preferably < 70%, or a sticky crystalline slurry occurs.

[0188] An organic solvent, preferably isopropanol, can be added to the crystallization solution. To enhance the crystallization process, 1 L of the crystalline material is mixed with 0.5 to 3 L of isopropanol, preferably with 0.7 to 2.0 L, more preferably with 1.0 to 1.5 L. The resulting crystallization process with an initial temperature of 20°C to 30°C is cooled down to a temperature of 0 to 15°C, preferably to 2 to 12°C, more preferably to 4 to 8°C, without stirring or with stirring. After reaching the preferred temperature, the crystallization process is incubated at the chosen temperature for 2 to 48 h, preferably for 6 to 36 h, more preferably for 12 to 24 h, without stirring or with stirring.

[0189] The sialic acid crystals can be removed from the liquid phase by filtration, with or without vacuum, or by centrifugation. To remove the remaining mother liquor, the crystals can be washed with an organic solvent, such as ethanol, methanol and / or isopropanol, preferably isopropanol. To remove the remaining mother liquor, 1 kg of the crystallized sialic acid, e.g. N-acetylneuraminic acid, is washed with 0.5 to 3 L of isopropanol, preferably with 0.7 to 2.0 L, more preferably with 1.0 to 1.5 L.

[0190] The obtained crystals are dried for 4 to 48 h, preferably 8 to 36 h, more preferably 12 to 24 h, or until no weight change is observed. The drying temperature can be from 10 °C to 80 °C, preferably from 20 °C to 70 °C, more preferably from 30 °C to 60 °C, in particular from 40 °C to 50 °C.

[0191] For the crystallization of sialic acid (e.g. Neu5Ac) from aqueous solution in the form of dihydrate, a second alternative method for the crystallization of sialic acid from aqueous solution in the form of dihydrate will be described herein.

[0192] For the start of the crystallization, seed crystals are added to the concentrated sialic acid at a temperature of 20 °C to 50 °C, preferably at 25 °C. The carbohydrate concentration of the sialic acid solution can be > 50 % (w / w). The crystallization method is incubated under stirring until crystallization occurs, preferably for 0.5 to 2 h, more preferably for 1 to 1.5 h.

[0193] An organic solvent, preferably isopropanol, is added to the crystallization solution. To enhance the crystallization process, 1 L of the crystallization material is mixed with 0.5 to 6.0 L of isopropanol, preferably with 1.0 to 4.0 L, more preferably with 1.5 to 2.5 L. The obtained crystallization method with a starting temperature of 20 °C to 30 °C is cooled to a temperature of 0 to 15 °C, preferably to 2 to 12 °C, more preferably to 4 to 8 °C, without stirring or with stirring. After reaching the preferred temperature, the crystallization method is incubated at the chosen temperature for 2 to 48 h, preferably for 6 to 36 h, more preferably for 12 to 24 h, without stirring or with stirring.

[0194] The sialic acid crystals can be removed from the liquid phase by filtration or centrifugation under vacuum or without vacuum. To remove the remaining mother liquor, the crystals can be washed with an organic solvent, such as ethanol, methanol and / or isopropanol, preferably isopropanol. To remove the remaining mother liquor, 1 kg of the crystallized sialic acid (e.g. N-acetylneuraminic acid) is washed with 0.5 to 3 L of isopropanol, preferably with 0.7 to 2.0 L, more preferably with 1.0 to 1.5 L.

[0195] The obtained crystals are dried for 4 to 48 h, preferably 8 to 36 h, more preferably 12 to 24 h, or until no weight change is observed. The drying temperature can be from 10 °C to 80 °C, preferably from 20 °C to 70 °C, more preferably from 30 °C to 60 °C, in particular from 40 °C to 50 °C.

[0196] An alternative method for the selective crystallization of sialic acid (e.g. Neu5Ac) from aqueous solution will be described herein. It has been found that by adding glacial acetic acid, sialic acid can be selectively crystallized from a biocatalytic process using enzymes or fermentation broth (culture broth).

[0197] In a preferred embodiment, the purified (aqueous) solution of sialic acid to be treated with acetic acid has a carbohydrate content of more than 10% (w / w), preferably more than 20% (w / w), more preferably more than 30% (w / w), in particular 35 to 40% (w / w). Alternatively, the sialic acid concentration in the purified (aqueous) solution to be treated with acetic acid is 250-350 g / l, preferably 290-330 g / l.

[0198] The above-mentioned concentration ranges and ratios can be achieved in a conventional manner by concentrating the aqueous process stream from the culture broth, preferably after removal of, e.g., cells, coloring substances, salts and / or charged molecules from the culture broth by using the aforementioned methods.

[0199] The concentration of the sialic acid-containing solution can be achieved by vacuum evaporation, e.g., by using a rotary evaporator or a plate evaporator, or by nanofiltration.

[0200] At a temperature of 20°C to 50°C, preferably at 25°C, acetic acid is added to the resulting aqueous solution. The acetic acid is added completely in one step or in portions over a period of 0.5 to 2 h, preferably 1 to 1.5 h. Preferably, the aqueous solution is continuously stirred at the same temperature, preferably also after the time point, while crystallization occurs, while the acetic acid is being added.

[0201] Preferably, about 5-25 L glacial acetic acid is used per kg of the aqueous sialic acid solution. Thus, for an aqueous sialic acid solution of 250-350 g / L, preferably 290-330 g / L, with a carbohydrate content of more than 30% (w / w), at least about 5-25 L acetic acid, preferably 7-20 L, more preferably 10-18 L, in particular 12-16 L, is used per kg of the aqueous sialic acid solution.

[0202] The resulting crystallization process at a starting temperature of 20°C to 50°C is cooled to a temperature of 0 to 15°C, preferably to 2 to 12°C, more preferably to 4 to 8°C, with or without stirring. After the preferred temperature has been reached, the crystallization process is incubated at the chosen temperature for 2 to 48 h, preferably 6 to 36 h, more preferably 12 to 24 h, with or without stirring.

[0203] The sialic acid crystals are removed from the liquid phase by filtration or centrifugation under vacuum or without vacuum. To remove the remaining sialic acid, the crystals are washed with an organic solvent, such as ethanol, methanol and / or isopropanol, preferably with isopropanol. To remove the remaining acetic acid, 1 kg of the crystallized sialic acid is washed with 0.5 to 3 L isopropanol, preferably 0.7 to 2.0 L, more preferably 1.0 to 1.5 L.

[0204] The obtained crystals are dried for 4 to 48 h, preferably 8 to 36 h, more preferably 12 to 24 h, or until no weight change is observed. The drying temperature is chosen between 10 °C and 80 °C, preferably between 20 °C and 70 °C, more preferably between 30 °C and 60 °C, in particular between 40 °C and 50 °C.

[0205] Providing sialic acid in lyophilized form

[0206] The purified solution comprising sialic acid can also be lyophilized. In said lyophilization, the purified solution comprising sialic acid is frozen and then the pressure is reduced so that the frozen water sublimates directly from the solid phase to the gas phase. This method usually results in a hygroscopic sialic acid powder.

[0207] Compositions and products provided by the present application

[0208] According to the present application, a composition is provided, comprising:

[0209] a) at least 98 wt.% sialic acid; and

[0210] b) at most 1 wt.% organic solvent; and

[0211] c) at most 1 wt.% of a salt different from the salt form of sialic acid.

[0212] In a preferred embodiment, the composition comprises:

[0213] a) 98.50 to 100.00 wt.% sialic acid, preferably 99.00 to 100.00 wt.% sialic acid, more preferably 99.50 to 100.00 wt.% sialic acid, optionally 99.70 to 99.90 wt.% sialic acid; and / or

[0214] b) 0.00 to 1.00 wt.% organic solvent, preferably 0.00 to 0.50 wt.% organic solvent, more preferably 0.00 to 0.40 wt.% organic solvent, optionally 0.10 to 0.20 wt.% organic solvent; and / or

[0215] c) 0.00 to 1.00 wt.% of a salt different from the salt form of sialic acid, preferably 0.00 to 0.50 wt.% of a salt different from the salt form of sialic acid, more preferably 0.00 to 0.40 wt.% of a salt different from the salt form of sialic acid, optionally 0.10 to 0.20 wt.% of a salt different from the salt form of sialic acid.

[0216] Preferably, the composition comprises (only):

[0217] a) 0.00 to 0.50 wt.% protein, more preferably 0.00 to 0.40 wt.% protein, optionally 0.10 to 0.20 wt.% protein; and / or

[0218] b) 0.00 to 0.50 wt.% DNA, more preferably 0.00 to 0.40 wt.% DNA, optionally 0.10 to 0.20 wt.% DNA.

[0219] The composition is characterized in that the sialic acid is present in amorphous form or in crystalline form, preferably in particulate form or in crystalline form, more preferably in spray-dried form or in crystalline form. The crystalline form is preferably a dihydrate crystalline form.

[0220] The present invention provides a food composition, preferably an infant food formula, a follow-on food formula or a medical nutrition product, wherein the food composition comprises sialic acid and at least one human milk oligosaccharide.

[0221] The food composition can comprise 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.

[0222] The food composition can be:

[0223] i) a liquid food composition comprising sialic acid in a concentration of 1 mg / l to 2 g / l, more preferably in a concentration of 5 mg / l to 1.5 g / l, even more preferably in a concentration of 20 mg / l to 1 g / l, most preferably in a concentration of 50 mg / l to 0.7 g / l; or

[0224] ii) a solid food composition comprising sialic acid in a concentration of 5 mg / kg to 15 g / kg, more preferably in a concentration of 25 mg / kg to 10 g / kg, even more preferably in a concentration of 100 mg / kg to 10 g / kg, most preferably in a concentration of 375 mg / kg to 5.25 g / kg.

[0225] The food composition can comprise:

[0226] i) at least one neutral human milk oligosaccharide, preferably at least one neutral human milk oligosaccharide selected from the group consisting of 2'-fucosyllactose, 3-fucosyllactose, lacto-N-tetraose and lacto-N-neotetraose, most preferably all of said neutral human milk oligosaccharides; and

[0227] ii) at least one acidic human milk oligosaccharide, preferably at least one acidic human milk oligosaccharide selected from the group consisting of 3'-sialyllactose and 6'-sialyllactose, most preferably all of said acidic human milk oligosaccharides; and

[0228] iii) L-fucose.

[0229] The composition can further comprise at least one, optionally all, of the 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, ferrous acetate, zinc sulphate, niacin, D-calcium pantothenate, copper sulphate, vitamin A, vitamin B1, vitamin B6, magnesium sulphate, potassium iodate, folic acid, vitamin K, sodium selenite and vitamin D.

[0230] The food composition can comprise at least one substance selected from the group consisting of a protein source, a vitamin, an oil, a mineral, an enzyme, another carbohydrate and a probiotic bacterial strain.

[0231] The food composition can be a composition selected from the group consisting of a medical food composition, a dietary supplement, a sachet product, a liquid ready-to-feed infant nutritional product, a liquid ready-to-feed toddler nutritional product, a granule product, a spray-dried infant formula product and combinations thereof.

[0232] The present application also provides a liquid ready-to-feed infant or toddler nutritional product comprising sialic acid, L-fucose and

[0233] i) at least one neutral human milk oligosaccharide selected from the group consisting of 2'-fucosyllactose, 3-fucosyllactose, difucosyllactose, lacto-N-tetraose and lacto-N-neotetraose; and / or

[0234] ii) at least one acidic human milk oligosaccharide selected from the group consisting of 3'-sialyllactose and 6'-sialyllactose.

[0235] The present application also provides a spray-dried infant formula product comprising sialic acid, L-fucose and

[0236] i) at least one neutral human milk oligosaccharide selected from the group consisting of 2'-fucosyllactose, 3-fucosyllactose, difucosyllactose, lacto-N-tetraose and lacto-N-neotetraose; and / or

[0237] ii) at least one acidic human milk oligosaccharide selected from the group consisting of 3'-sialyllactose and 6'-sialyllactose.

[0238] Furthermore, the present application also provides a dietary supplement comprising 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.

[0239] According to the present application, the present application provides a premix for a food product, such as a premix for an infant food formula, comprising sialic acid, such as Neu5Ac, and at least one substance selected from the group consisting of a protein source, a vitamin, an oil, a mineral, an enzyme, another carbohydrate (such as a carbohydrate different from the sialic acid already present in the premix), and a probiotic bacterial strain. More preferably, the premix comprises all of said substances.

[0240] The protein source can be selected from the group consisting of whey, corn soy blend (CSB), protein hydrolysate, and combinations thereof.

[0241] The vitamin can be selected from the group consisting of vitamin A, thiamine, riboflavin, vitamin B12, folic acid, and combinations thereof.

[0242] The oil can be selected from the group consisting of palm oil, DHA, arachidonic acid, and combinations thereof.

[0243] The mineral can be selected from the group consisting of potassium chloride, potassium iodate, zinc oxide, and combinations thereof.

[0244] The enzyme can be selected from the group consisting of amylase, amyloglucosidase, and combinations thereof.

[0245] The carbohydrate can be selected from the group consisting of human milk oligosaccharides (HMOs), galacto-oligosaccharides (GOS), inulin (FOS), L-fucose, other sialic acids, lactose, and combinations thereof.

[0246] The probiotic bacterial strain can be selected from the group consisting of (capsulated) Lactobacillus, Bifidobacterium strains, and combinations thereof.

[0247] The premix can be in the form of a spray-dried, granulated, or liquid (syrup) product.

[0248] According to the present application, a pharmaceutical composition, preferably for the prevention or treatment of at least one of a viral infection, a bacterial infection, memory loss, and dysbiosis, is provided. The pharmaceutical composition comprises a composition according to the present application, and optionally at least one sugar different from sialic acid and / or at least one probiotic bacterial strain, wherein the at least one sugar is preferably at least one sugar selected from the group consisting of lactose, lactulose, inulin, and sucrose.

[0249] Release formulations of sialic acid or pharmaceutically acceptable salts, solvates, or esters thereof are known in the prior art and described, for example, in WO 2012 / 009474 and WO 2013 / 109906, the contents of which are incorporated herein by reference.

[0250] Exemplary food composition

[0251] The method of this invention provides sialic acid of sufficient purity for use in food or feed applications, particularly for inclusion in infant and toddler nutritional products. The obtained sialic acid, especially Neu5Ac, can be used to prepare infant formula by mixing it with components of an infant formula base. The infant formula can be a powder formula or a ready-to-eat liquid infant formula.

[0252] The base formulation may contain at least one of the following components, and optionally all of the following components:

[0253] Basic formulation ingredients: Components of representative basic formulation ingredients:

[0254] Skim milk

[0255] Vegetable oils (palm oil, rapeseed oil, sunflower oil)

[0256] skim milk powder

[0257] Oil of Alpine Mosporum

[0258] fish oil

[0259] Calcium carbonate

[0260] Potassium chloride

[0261] Vitamin C

[0262] Sodium chloride

[0263] Vitamin E

[0264] Ferrous acetate

[0265] Zinc sulfate

[0266] niacin

[0267] D-Calcium pantothenate

[0268] Copper sulfate

[0269] Vitamin A

[0270] Vitamin B1

[0271] Vitamin B6

[0272] Magnesium sulfate

[0273] Potassium iodate

[0274] folic acid

[0275] Vitamin K

[0276] Sodium selenite

[0277] Vitamin D

[0278] Use of the compositions of the present invention

[0279] The present application suggests the use of the composition of the present application for the preparation of a food composition and / or a pharmaceutical composition.

[0280] The present application is intended to explain the subject matter of the present application in more detail with reference to the following figures and examples, without wishing to limit the subject matter to the specific embodiments shown herein.

[0281] Figure 1 An example of a method of the present application for purifying the sialic acid N-acetylneuraminic acid is shown. After fermentation, the fermentation broth is clarified by centrifugation and / or filtration. The clarified fermentation broth is subjected to ion exchanger treatment. The flow-through of the ion exchanger treatment is then subjected to diafiltration and / or concentration. After this step, the solution comprising sialic acid is passed through activated carbon. Then, the solution comprising sialic acid is subjected to electrodialysis and / or diafiltration. Subsequently, the solution comprising sialic acid is subjected to spray drying or crystallization.

[0282] Figure 2 An example of a second method of the present application for purifying the sialic acid N-acetylneuraminic acid is shown. After fermentation, the fermentation broth is clarified by centrifugation and / or filtration. The clarified fermentation broth is subjected to ion exchanger treatment. The flow-through of the ion exchanger treatment is then subjected to electrodialysis and / or diafiltration. After this step, the solution comprising sialic acid is passed through activated carbon. Then, the solution comprising sialic acid is subjected to diafiltration and / or concentration. Subsequently, the solution comprising sialic acid is subjected to spray drying or crystallization.

[0283] Figure 3 An example of a third method of the present application for purifying the sialic acid N-acetylneuraminic acid is shown. After fermentation, the fermentation broth is clarified by centrifugation and / or filtration. The clarified fermentation broth is subjected to ion exchanger treatment. The flow-through of the ion exchanger treatment is then passed through activated carbon. After this step, the solution comprising sialic acid is subjected to diafiltration and / or concentration. Then, the solution comprising sialic acid is subjected to electrodialysis and / or diafiltration. Subsequently, the solution comprising sialic acid is subjected to spray drying or crystallization.

[0284] Figure 4 An example of a fourth method of the present application for purifying the sialic acid N-acetylneuraminic acid is shown. After fermentation, the fermentation broth is clarified by centrifugation and / or filtration. The clarified fermentation broth is subjected to ion exchanger treatment. The flow-through of the ion exchanger treatment is then subjected to electrodialysis and / or diafiltration. After this step, the solution comprising sialic acid is subjected to diafiltration and / or concentration. Then, the solution comprising sialic acid is passed through activated carbon. Subsequently, the solution comprising sialic acid is subjected to spray drying or crystallization.

[0285] Figure 5A micrograph of crystals of N-acetylneuraminic acid is shown. It can be seen that N-acetylneuraminic acid forms small needles with a length of 2 to 8 pm.

[0286] Figure 6 An HPLC diagram of the recorded N-acetylneuraminic acid, which has been crystallized as dihydrate, is shown. A purity of 98.7% of the crystallized N-acetylneuraminic acid was detected.

[0287] Figure 7 An HPLC diagram of the recorded N-acetylneuraminic acid, which has been crystallized with acetic acid, is shown. A purity of 98.2% of the crystallized N-acetylneuraminic acid was detected compared to other sugars. The amount of acetic acid after drying was 2.8% compared to the total mass of the material.

[0288] Figure 8 An HPLC diagram of the recorded N-acetylneuraminic acid, which has been spray-dried, is shown. A purity of 99.1% of the spray-dried N-acetylneuraminic acid was detected.

[0289] Example 1 : Purification of the sialic acid N-acetylneuraminic acid from bacterial fermentation

[0290] The sialic acid N-acetylneuraminic acid was produced by bacterial fermentation and collected by filtration, wherein a clear, particle-free sialic acid solution (37 g / l) was obtained.

[0291] The clear, particle-free solution was first treated with a strong cation exchanger (Lewatit S 2568 in proton form, Lanxess).

[0292] After neutralization with sodium hydroxide (NaOH), the solution was additionally treated with a strong anion exchanger (Lewatit S 6368 A in chloride form).

[0293] To concentrate and desalt the N-acetylneuraminic acid in the solution after the ion exchanger treatment, the solution was further treated by a nanofiltration step. An Emrich EMRO 1.8 reverse osmosis system (Emrich Edelstahlbau) was equipped with XN 45 nanofiltration modules. The inlet pressure was set to 30 bar and the solution was concentrated until the flow rate fell below 100 L / h. The solution was diafiltered three times with an equal amount of reverse osmosis water. To reduce the amount of salts, the solution was diafiltered three times with an equal volume of reverse osmosis water.

[0294] To remove color, the N-acetylneuraminic acid-containing solution was treated with activated carbon powder. The solution was incubated with Norit SA2 activated carbon under stirring for 2 h. After incubation, the activated carbon was removed by filtration and the pH was adjusted to pH 7.0 with sodium hydroxide.

[0295] To remove residual color and non-specific ions, the solution was again treated with a strong cation exchanger (Lewatit S2568 in sodium form, Lanxess) and a strong anion exchanger (Lewatit S6368A in chloride form). After the treatment, the pH was adjusted to pH 7.0.

[0296] To remove sodium chloride, the solution was electrodialyzed using a PCCell P15 electrodialysis system (PCell, Heusweiler, Germany) equipped with a PCCell ED 1000A membrane stack. The stack comprised the following membranes: a cation exchange membrane CEM:PC SK and an anion exchange membrane CEM:PcAcid60 with a size exclusion limit of 60 Da. The solution was electrodialyzed until a stable conductivity was reached. During the electrodialysis, the pH was stabilized at pH 7.0 by addition of NaOH.

[0297] 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) equipped with a CSM RE8040BE reverse osmosis module.

[0298] Subsequently, the solution was filtered to remove endotoxins and the solution was sterile filtered through a 5 kDa ultrafiltration membrane (Spira-Cell WYUP005 2440C 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.

[0299] Example 2: Conversion of the sodium salt of sialic acid N-acetylneuraminic acid to its free acid

[0300] To isolate the sialic acid in its free acid form, after the active carbon treatment mentioned in example 1, 30% hydrochloric acid was added until a pH of 1.7 to 2.3.

[0301] After adjusting the pH, the solution was again treated with a strong cation exchanger (Lewatit S2568 in proton form, Lanxess). After the ion exchanger treatment, the solution was again electrodialyzed using a PCCell P15 electrodialysis system (PCell, Heusweiler, Germany) equipped with a PCCell ED 1000A membrane stack. The stack comprised the following membranes: a cation exchange membrane CEM:PC SK and an anion exchange membrane CEM:PcAcid60 with a size exclusion limit of 60 Da. The solution was electrodialyzed until a stable conductivity was reached. During the electrodialysis, the pH was stabilized at pH 2.0 by addition of hydrochloric acid.

[0302] Example 3: Obtaining sialic acid N-acetylneuraminic acid in solid form by spray drying

[0303] The sialic acid obtained in sodium form or in free acid form by the isolation process in Examples 1 and 2 above was concentrated to 20% (w / w), the solution was filtered to remove endotoxins and sterile filtered by passing the solution through a 5 kDa ultrafiltration membrane (Spira-Cell WYUP005 2440C module, Microdyn Nadir, Wiesbaden, Germany). The sterile solution thus obtained containing N-acetylneuraminic acid was then spray dried using a NUBILOSA LTC-GMP spray dryer (NUBILOSA, Konstanz, Germany). For spray drying of the N-acetylneuraminic acid solution, the solution was passed through the spray dryer nozzle set to 130 °C at a pressure of 3.5 bar and the flow was adjusted to maintain the exhaust temperature at 66 °C to 67 °C. Using these settings, a spray-dried powder with a moisture content of less than 5% could be obtained. The moisture content was determined by Karl-Fischer titration.

[0304] Example 4: Crystallization of sialic acid N-acetylneuraminic acid in dihydrate form

[0305] 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) equipped with a CSM RE8040BE reverse osmosis module.

[0306] For crystallization, 5 liters of this solution were concentrated to 50% (w / w) dry matter by a Hei-VAP industrial evaporator (Heidolph Instruments GmbH, Schwabach Germany). The solution was seeded with crystals and concentrated under vacuum until crystals formed (dry matter of the solution: about 75% w / w).

[0307] The crystal solution was mixed 1 : 1 with isopropanol and stirred for 5 min. The solution was incubated at 4 °C for at least 12 h. The crystallized sialic acid was removed from the mother liquor by vacuum filtration and the crystals were washed with isopropanol.

[0308] The yield of precipitated N-acetylneuraminic acid was 75 to 85%.

[0309] Example 5: Crystallization of sialic acid N-acetylneuraminic acid with acetic acid

[0310] 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) equipped with a CSM RE8040BE reverse osmosis module.

[0311] For crystallization, 5 liters of glacial acetic acid were added portion-wise under stirring to 1 liter of N-acetylneuraminic acid solution. The solution was cooled from room temperature (20°C) to 4°C within 3 h. After reaching the final temperature (4.5°C), the crystallization process was incubated at this temperature for 12 to 36 h.

[0312] 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 an equal amount of isopropanol.

[0313] The yield of precipitated N-acetylneuraminic acid was 74 to 81%.

[0314] The purity of the exemplarily purified N-acetylneuraminic acid from the culture broth is shown in Table 1.

[0315] Purification step Sialic acid concentration (g / l) Sialic acid (kg) Dry matter (kg) Purity (%) Collection 37 259 399 64,9 Cation exchanger 37 256 382 67,02 Anion exchanger 36 248 378 65,5 Concentration / diafiltration 152 244 316,6 77,06 Activated carbon 145 232,3 298,4 77,9 Electrodialysis 135 181 184 98,3 Concentration 203 180 183 98,3

[0316] The purity in Table 1 is expressed as the mass of the desired N-acetylneuraminic acid compared to the total mass.

[0317] Example 6: Composition of a representative infant formula

[0318] In the following, a composition of a representative infant formula is given (see Table 2 below).

[0319] The composition comprises the sialic acid Neu5Ac 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), the acidic HMOs (6'-sialyllactose (6'-SL) and 3'-sialyllactose (3'-SL)) and L-fucose.

[0320] One or more probiotic bacterial strains can be present in the product. The final concentration of each ingredient is based on a preparation of 13.5 g powder in 90 ml water.

[0321]

[0322]

[0323]

[0324] Table 2

[0325] Example 7: Composition of a representative premix of an infant formula comprising human milk oligosaccharides, the monosaccharide Neu5Ac and the monosaccharide L-fucose.

[0326] In the following, the composition of a representative premix of an infant formula is given (see Table 3 below).

[0327] The composition comprises the sialic acid Neu5Ac with the rich 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), the acidic HMOs (6'-sialyllactose (6'-SL) and 3'-sialyllactose (3'-SL)) and the monosaccharide free L-fucose.

[0328] The premix can be used to reconstitute an infant formula by adding the premix to the other nutritional products (e.g. whey, lactose, lipids (saturated and unsaturated fatty acids) and minerals) necessary for the reconstitution of the infant formula. The premix shown in Table 3 is intended for 1 kg of final infant formula.

[0329]

[0330]

[0331] Table 3

Claims

1. A method for purifying sialic acid from fermentation broth, comprising the following steps: Biomass is removed from the fermentation broth containing sialic acid, providing a clarified solution. A purified solution is provided by treating the clarified solution as follows: Cation exchanger treatment with cation exchanger material, wherein the cation exchanger treatment is carried out under conditions in which sialic acid passes through the cation exchanger material and is present in the flow-through liquid, and the conditions for sialic acid to pass through the cation exchanger material are established by adjusting the pH of the clarified solution to 6 to 8; and Anion exchanger treatment using anion exchanger material, wherein the anion exchanger treatment is carried out under conditions in which sialic acid passes through the anion exchanger material and is present in the flow-through liquid, sialic acid being present in the flow-through liquid after contact with the anion exchanger material, and the conditions for sialic acid to pass through the anion exchanger material are established by adjusting the pH of the clarified solution to 6 to 8. and Salts were removed from the purified solution by electrodialysis. The ion exchange treatment is performed before the electrodialysis step. First, the clarified solution is treated with a cation exchanger, followed by an anion exchanger treatment. Furthermore, in the cation exchanger treatment, a strong cation exchanger is used, and in the anion exchanger treatment, a strong anion exchanger is used, wherein the cation exchanger treatment is performed to remove non-specific cations and replace them with specific cations H+, and wherein the anion exchanger step is performed to remove non-specific anions and replace them with specific anions Cl-.

2. The method according to claim 1, characterized in that, The method does not include: i) Chromatographic separation; and / or ii) Use ethanol and / or ethyl acetate; and / or iii) The step of eluting sialic acid from the stationary phase with a solution containing an organic solvent; and / or iv) Use of heavy metals; and / or v) The step of heating the fermentation broth, clarified solution, and / or purified solution to a temperature above 45°C.

3. The method according to claim 2, characterized in that, The method does not include the use of organic solvents.

4. The method according to any one of claims 1 to 3, characterized in that, Biomass is removed from the fermentation broth by centrifugation and / or filtration.

5. The method according to claim 1, characterized in that, Adjustments were made by adding NaOH to the flow-through solution.

6. The method according to any one of claims 1 to 3, characterized in that, After treatment with cation exchangers and anion exchangers, the purified solution contains sialic acid, coloring substances, and salts.

7. The method according to claim 6, characterized in that, The salt mentioned is NaCl.

8. The method according to any one of claims 1 to 3, characterized in that, The solution is concentrated and purified by nanofiltration and / or reverse osmosis.

9. The method according to claim 8, characterized in that, Concentration is achieved through nanofiltration.

10. The method according to claim 9, characterized in that, Nanofiltration membranes with a molecular weight cutoff of 100 to 200 kDa are used.

11. The method according to claim 1, characterized in that, Concentrate the clarified solution and / or purify the solution. i) Until the concentration of sialic acid is ≥100 g / L; and / or ii) It is carried out by nanofiltration at a temperature <80°C; and / or iii) It is carried out by reverse osmosis at a temperature of 20°C to 50°C; and / or iv) It is carried out under pressures of >5 bar and <50 bar.

12. The method according to claim 11, characterized in that, Until the concentration of sialic acid is ≥200g / L.

13. The method according to claim 11, characterized in that, Until the concentration of sialic acid is ≥300g / L.

14. The method according to claim 11, characterized in that, It is carried out by nanofiltration at a temperature of <50°C.

15. The method according to claim 11, characterized in that, It is carried out by nanofiltration at temperatures ranging from 4°C to 45°C.

16. The method according to claim 11, characterized in that, It is carried out by nanofiltration at temperatures ranging from 10°C to 40°C.

17. The method according to claim 11, characterized in that, It is carried out by nanofiltration at a temperature of 15 to 30°C.

18. The method according to claim 11, characterized in that, It is carried out by nanofiltration at a temperature of 15 to 20°C.

19. The method according to claim 11, characterized in that, It is carried out through reverse osmosis at a temperature of 30°C to 45°C.

20. The method according to claim 11, characterized in that, It is carried out through reverse osmosis at a temperature of 35°C to 45°C.

21. The method according to claim 11, characterized in that, It is carried out under pressures of >10 bar and <40 bar.

22. The method according to claim 11, characterized in that, It is carried out under pressures of >15 bar and <30 bar.

23. The method according to any one of claims 1 to 3, characterized in that, Electrodialysis is electrodialysis under neutral conditions or electrodialysis under acidic conditions.

24. The method according to any one of claims 1 to 3, characterized in that, After removing the salt from the purified solution, i) The amount of salt in the purified solution is <10% w / w; and / or ii) The electrical conductivity is 0.2 to 10.0 mS / cm².

25. The method according to claim 24, characterized in that, The amount of salt in the purified solution is <5% w / w.

26. The method according to claim 24, characterized in that, The amount of salt in the purified solution is <1% w / w.

27. The method according to claim 24, characterized in that, The electrical conductivity ranges from 0.4 to 5.0 mS / cm².

28. The method according to claim 24, characterized in that, Electrical conductivity ranges from 0.5 to 1.0 mS / cm 2 .

29. The method according to any one of claims 1 to 3, characterized in that, A decolorization step is performed on the clarified solution and / or purified solution, wherein the salt is removed. i) Performed before or after the percolation step of clarifying the solution; and / or ii) Performed before or after the electrodialysis step of clarifying the solution; and / or iii) Performed before or after the concentration step of the clarified solution.

30. The method according to claim 29, characterized in that, Decolorization is achieved through treatment using activated carbon.

31. The method according to any one of claims 1 to 3, characterized in that, The sialic acid contained in the clarified and / or purified solutions is converted into its sodium form.

32. The method according to claim 31, characterized in that, The conversion is achieved by treating the purified solution with a strong cation exchanger material in the form of sodium.

33. The method according to any one of claims 1 to 3, characterized in that, The purified solution is spray-dried, wherein the purified solution contains sialic acid in the form of sodium.

34. The method according to any one of claims 1 to 3, characterized in that, The purified solution is then crystallized.

35. The method according to claim 34, characterized in that, Crystallization was carried out by adding acetic acid to the purified solution, wherein sialic acid was provided in the form of dihydrate crystals.

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