RU2789351C2 - Method for purification of l-fucose from fermentation broth - Google Patents

Abstract

FIELD: food industry.

SUBSTANCE: method for purification of L-fucose from fermentation broth is proposed, according to which biomass is removed by means of centrifugation and/or filtration from fermentation broth containing L-fucose to obtain a clarified solution. A purified solution is obtained by subjecting the clarified solution to treatment on a cation exchanger and treatment on an anion exchanger. Salts are removed from the purified solution by electrodialysis and/or nano-filtration. In this case, biomass contains cells, which produce L-fucose.

EFFECT: invention provides effective isolation of L-fucose from fermentation broth.

17 cl, 7 dwg, 3 tbl, 7 ex

Description

The present invention relates to an efficient method for isolating L-fucose from a fermentation broth. The L-Fucose contained in the fermentation broth is produced by fermentation using microorganisms (bacteria and yeast). The method according to the invention includes the step of removing biomass from the fermentation broth, the step of subjecting the resulting solution to at least one treatment with a cation exchanger and one treatment with an anion exchanger, and the step of removing salts after treatment with an ion exchanger. The use of this method can provide L-fucose in powder form, in granular form, as well as in the form of crystalline L-fucose.

Carbohydrates are involved in all forms of life, playing vital roles in energy storage, structural function, signal transmission, information storage, and so on. To solve these problems, several basic monosaccharides are synthesized in nature, such as glucose, N-acetylglucosamine, mannose, N-acetylmannosamine, fructose, fucose, ribose, L-fucose, xylose, etc., and several auxiliary monosaccharides for more specialized applications, such such as D-allose.

L-Fucose (6-deoxy-L-galactose) is one of the so-called rare monosaccharides. It is found in many natural products in both D- and L-forms: L-fucose (also called rhamnose) and D-fucose (CAS (Chemical Abstracts Service) 3615-37-0). The most common in nature is the L-shape. L-Fucose is a simple molecule for research and development applications and is a characteristic component of the structure of glycans. Scientific research in the field of fucosylated substances has shown, among other things, that they are very important for embryonic development and are involved in the body's immune response.

L-Fucose and fucosylated oligo- and polysaccharides are of particular interest to the chemical, food, cosmetic and pharmaceutical industries because of their great potential for nutritional and biomedical applications (Hauber et al., 2008, Int. J. Med. Sci., 5: 371-376.; Isnard et al., 2005, Ophthalmologics 219: 324-333; Robert et al., 2004, Biomed. Pharmacother., 58: 123-128; Wild et al., 2002, Cells Tissues Organs. 172: 161-173; Adam et al., 1997, Am. J. Respir. Care Med., 155: 2102-2104). In addition to this, fucosylated derivatives are known for their antiallergic and emulsifying properties. It is also known that dietary L-fucose can be used to synthesize fucosylated glycans via the L-fucose recycling pathway found in mammals. Thus, the presence of L-fucose is important for the development of the brain and the immune system. The existence of leukocyte adhesion deficiency syndrome type II, an inborn disorder of fucose metabolism, is also evidence of the importance of this.

In mammalian cells, L-fucose is present in fucosylated glycans such as ABO blood group antigens and human milk oligosaccharides. L-Fucose and fucosylated oligosaccharides or proteins are also of great interest to the chemical, pharmaceutical and nutraceutical industries.

L-Fucose is also found, among other substances, in human breast milk at a concentration of approximately (0.5 g/l). Human breast milk is given an important role in the healthy development of children. The substances it contains, in particular the oligosaccharides (human milk oligosaccharides (HMO)), are one of the main solid components of breast milk and have a core structure that contains a lactose unit at the reducing end and is branched, or the chain ends with an N-acetyllactosamine molecule . Structural variability is enhanced by modifications of the fucose residue at the terminal positions. In terms of health and developmental benefits, the biological function of HMOs has been the subject of much research, but technical purification of these compounds in sufficient quantities is required to elucidate it. It is known in the prior art to obtain L-fucose either by direct extraction from natural sources or by chemical modification of monosaccharides (PT Vanhooren et al., 1999, J. Chem., Technol., Biotechnol., 74, 479).

Although some monosaccharides can be obtained from natural sources in large quantities and at a reasonable cost (for example, glucose, N-acetylglucosamine and fructose), most monosaccharides are quite rare substances and can only be found in nature in small quantities, such such as L-fucose (6-deoxy-L-galactose).

In the case of industrial production of monosaccharides, oligosaccharides obtained from natural sources are almost exclusively used as sources. These oligosaccharides are subjected to acid hydrolysis and individual sugars are isolated from the released monosaccharides. Due to the high chemical similarity of monosaccharides (which differ from each other mainly only in the orientation of the individual hydroxyl groups), the separation of individual monosaccharides in pure form is a rather laborious and expensive process.

Natural sources of L-fucose are polysaccharides from plants such as seaweed (fucoidan, a sulfated polymer based on fucose), potatoes, soybeans, and so on. In plants, fucose is usually included in the composition of polysaccharides having L-fucopyranosyl units at the ends or inside the polysaccharide chain. Another important source of L-fucose is the extracellular polysaccharides of bacteria, fungi and microalgae.

L-Fucose can be obtained from natural sources, such as algae or bacteria, using various extraction methods. These rare naturally occurring substances used to extract L-fucose are usually multi-component mixtures. Currently, L-fucose is obtained through the hydrolysis of these complex oligosaccharides. The isolation of L-fucose having a sufficient degree of purity is an elusive procedure and represents a problem of the state of the art.

To purify individual monosaccharides from complex hydrolysates, toxic chemicals often must be used. For example, US 3240776 discloses a method for extracting L-fucose, in which L-fucose is isolated by the use of lead acetate and excess amounts of organic solvents.

Therefore, the isolation of individual monosaccharides from a complex hydrolyzate of oligosaccharides is problematic (due to the great chemical similarity of these released individual monosaccharides) and harmful to the environment (due to excessive use of toxic chemicals such as lead carbonate).

In addition, in nature, the availability of oligosaccharides rich in a certain sugar can be quite limited and also vary greatly due to seasonal changes. L-Fucose is derived primarily from the polysaccharide fucoidan, fucane monosulphate, present in all common brown seaweeds in the families Fucaceae and Laminariaceae (Black, WAP (1954): The seasonal variation in the combined L-fucose content of the common british Laminariaceae and Fucaceae, J. Sci. Food Agric, 5, 445-448).

To date, L-fucose is obtained in large quantities mainly from the collection of brown seaweeds belonging to the Fucaceae family, which can be found throughout the world, but in large quantities on the European Atlantic coast. The large-scale harvesting of brown seaweed from the sea coast leads to environmental problems and is restricted by environmental laws. L-Fucose and L-fucose-containing substances are the subject of ongoing research.

For example, JP 2000351790 discloses a method for extracting fucoidan and obtaining and isolating a fucose-containing oligosaccharide from the extracted fucoidan.

WO 2012/034996 A1 shows that L-fucose can also be obtained by hydrolysis of naturally occurring bacterial polysaccharides containing L-fucose. This document discloses a strain belonging to the Enterobacteriaceae family, wherein the strain is capable of producing extracellular polysaccharides that contain L-fucose. To obtain L-fucose, the polysaccharides produced by this strain are recovered and subjected to hydrolysis, for example by treatment with sulfuric acid or trifluoroacetic acid.

In addition to extracting L-fucose from poly- or oligosaccharide hydrolysates, various methods have been developed to obtain L-fucose by synthesis. The starting material in such preparations are other monosaccharides such as L-arabinose, D-galactose, L-rhamnose, D-mannose and D-glucose. Using the most efficient synthesis route starting from the rare monosaccharide L-rhamnose, efficient synthesis of L-fucose can be achieved (Defaye et al., 1984, Carbohydr. Res., 126, 165-169). As a rule, these chemical synthesis methods often show rather low yields and involve several chemical transformation steps. To carry out the chemical synthesis of L-fucose, in addition to including several stages of synthesis, a large amount of chemical reactions should be used to introduce protective groups. In general, there is no evidence for the economic feasibility of large-scale chemical synthesis of monosaccharides compared to the extraction of L-fucose from polysaccharides harvested from natural sources.

In general, the recovery of L-fucose requires complex separation techniques such as chromatography using anion or cation exchange resins, dialysis, fractional crystallization, etc., depending on the nature of the accompanying sugars or sugar-related compounds (WO 2005/040430 A1).

JP 63027496 describes the direct extraction of L-fucose from algae belonging to the family Chordariaceae or Spermatochnaceae. These algae were dispersed in water and treated with concentrated sulfuric acid. The resulting solution of hydrolyzed algae was cooled and insoluble matter was removed by filtration. The pH of the filtrate was adjusted to 5, the filtrate was treated with activated carbon and filtered. In order to break down and remove other sugars other than L-fucose, yeast was added to the filtrate. The mixture was treated with activated carbon and filtered. The filtrate was treated with cation and anion exchange resins for deionization and concentrated. The concentrated sugar solution was mixed with ethanol and allowed to crystallize. In this way, L-fucose was obtained with a purity of 98.7%.

One publication describes the isolation of pectins from the ethanol-insoluble residue of beet pulp (Rombouts et al., 1986, Carbohydrate Research, 154: 177-187). The isolated pectins were purified by chromatography on DEAE(diethylaminoethyl)-cellulose or by precipitation using CuSO ₄ . The pectins had a relatively high content of neutral sugars. The main neutral sugars in each pectin were arabinose and galactose; other sugars present were rhamnose, L-fucose, xylose, mannose, and glucose. L-Fucose was not isolated from the sugar/pectin mixture.

Thus, today, to obtain any monosaccharide in pure form, considerable effort must be made to separate the desired monosaccharide from other monosaccharides in mixtures, often involving large volumes of organic solvents and other toxic chemicals. As a consequence, the exclusive accumulation of one desired monosaccharide, such as L-fucose, would be of great help. However, most microorganisms are limited in terms of the types of monosaccharides they are able to use. In addition, they often show a significant preference for certain monosaccharides when several monosaccharides are simultaneously available as a carbon source.

Another possible way to obtain L-fucose is chemical synthesis. As a result of deoxygenation of D-galactose at the carbon atom in the C-6 position, D-fucose is formed. However, this methodology is not practical in the case of L-fucose synthesis because L-galactose is not available in large quantities. L-Fucose can be obtained from L-arabinose through a complex sequence of reactions involving numerous intermediates. Configuration inversion at the C-5 atom and deoxygenation at C-6 in D-glucose results in L-fucose by a multi-step procedure.

L-Fucose can also be obtained by chemical synthesis from L-arabinose (Tanimura, 1961, Chem. Abstr., 55: 12306), from D-glucose (Chiba, T. & Tejima, 1979, Chem. Pharm. Bull., 27: 2838-2840), from methyl-L-rhamnose (Defaye, J., et al., 1984, Carbohydrate Res., 126: 165-169), from D-mannose (Gesson, J-P et al., 1992, Tetrahedron Lett., 33: 3637-3640) and from D-galactose (Dejter-Juszynski, M & Flowers, H-M., 1973, Carbohydrate Res., 28: 144-146). An enzymatic synthesis of L-fucose and its analogues from fuculose-6-phosphate was carried out using a multi-enzyme system (see WO 97/15683 A1).

To obtain L-fucose starting from D-mannose, stereoselective chain elongation at C-1 and cleavage of the terminal glycol-containing moiety are required. In the case of L-rhamnose, as 6-deoxyhexose, OH inversions are necessary to obtain L-fucose, namely, at the C-2 and C-4 atoms. Apparently, D-galactose was the most suitable starting material for obtaining L-fucose, since there is no need to perform an inversion: L-fucose is formed as a result of the reduction of the formyl group at C-1 and the oxidation of the primary hydroxyl at C-6 to formyl. A common characteristic of the above methods is the inevitable temporary protection of hydroxyls, which in this method should not be subjected to the steps of configuration inversion, deoxygenation and reduction and/or oxidation. The repeated protection/deprotection steps, often requiring selective methods, make these methodologies inefficient. In addition, in some cases, laborious chromatographic separations require the separation of intermediates from by-products.

WO 2014/067696 A1 describes for the first time a process for the production of L-fucose using a recombinant microorganism having a glycosyltransferase and a glycosidase that work together to synthesize free form L-fucose. This method requires two enzymes and an acceptor molecule. Glycosyltransferase catalyzes the transfer of fucose from GDP-L-fucose (guanosine diphosphate-L-fucose) to an acceptor such as lactulose to form fucosyl lactulose. The fucosylated acceptor (eg, fucosyllactulose) is then hydrolyzed by glycosidase to the acceptor molecule and L-fucose. The acceptor is then again available for fucosylation by the fucosyl transferase used. L-fucose is then released from the cell by isolation into the medium, after which it can be recovered from the supernatant. Thus, the feedback inhibition of the GDP-fucose pathway can be easily overcome, and significant (several g/l) amounts of free L-fucose can be obtained from fermentation using microorganisms.

L-fucose itself and fucosylated substrates are important starting materials in the chemical and pharmaceutical industries, as well as useful substances for the manufacture of cosmetics and nutraceuticals. Therefore, there is a continuing need for innovative manufacturing methods for the production of L-fucose on an industrial scale.

Biotechnological methods for the extraction of L-fucose have been proposed. For example, WO 2012/034996 A1 describes a process for the production of L-fucose using an isolated microorganism from the family Enterobacteriaceae to enzymatically produce L-fucose. However, L-fucose is obtained only after a long fermentation as a crude mixture, so carefully designed additional purification steps are necessary to obtain L-fucose.

US 8642297 B2 postulates a general enzymatic method proposed for the production of L-fucose using recombinant mannitol-1-dehydrogenase from Apium graveolens. However, neither a specific embodiment nor an example is described for this purpose. In addition, no alternative enzyme has been described that could be used for this reaction.

WO 2005/087941 A1 discloses a combined fermentation-enzymatic process for the production of L-fucose. In this case, the formation of L-fuculose from L-fucit first occurs as a result of the use of dehydrogenase from acetobacteria. Then from it in the next stage should be synthesized to obtain L-fucose.

Another method describes the separation of a complex mixture of oligosaccharides by anion exchange chromatography (Derevitskaya et al., 1975, Dokl. Akad. Nauk. SSSR, 223: 1137-1139). In accordance with this description, 2-amino-2-deoxyglucite, glucosamine, galactose and L-fucose were successfully separated from mixtures of oligosaccharides buffered with 0.2 M borate solution by anion exchange chromatography.

JP H11-035591 discloses a process for producing L-fucose from fucoidan obtained from Cladosiphon okamuranus Tokida or an extract containing fucoidan. This method is a multi-step process including, for example, water and/or acid treatments, neutralization, dialysis and electrodialysis treatments, and ion exchanger treatment using an alkaline solution as eluent. Finally, L-fucose is crystallized from alcohol.

Enzymatic and microbiological synthesis was also used to obtain L-fucose. L-Fucose is obtained by enzymatic synthesis from dihydroxyacetone phosphate (DHAP) and DL-lactaldehyde catalyzed by L-fuculose-1-phosphate aldolase followed by reaction involving acid phosphatase and L-fucose isomerase. The L-fucose product was isolated by Dowex® 50W-X8 chromatography (in Ba ²⁺ form), possibly in combination with silica gel separation (Wong et al., 1995; J. Org. Chem., 60: 7360-7363).

EP 0102535 A2 discloses a process for the production of deoxysaccharides selected from fucose and rhamnose by fermentation using Alcaligenes, Klebsiella, Pseudomonas or Enterobacter species that produce extracellular polysaccharides containing more than 10% fucose and/or rhamnose. It is described that fucose and/or rhamnose can be recovered from the hydrolyzate of the fermentation product by chromatography, ion exchange or adsorption (eg using celites), or by further processing by fermentation.

Similarly, it has been found that a bacterial strain of the family Enterobacteriaceae under aerobic fermentation conditions is capable of producing L-fucose-containing polysaccharides, and the fucose has been separated and isolated by acid hydrolysis and numerous purification steps (WO 2012/034996 A1).

However, low-cost methods for producing fucosylated lactoses are needed, such as by fermentation using transformed E. coli (Drouillard et al., 2006, Angew. Chem, Int. Ed., 45, 1778; WO 2010/070104 A1; WO 2010/ 142305 A1, WO 2012/097950 A1, WO 2012/112777 A2, Baumgartner et al., 2013, Microb.Cell Fact., 12:40).

WO 2015/032412 A1 discloses a method for producing a high yield mixture of 2'-fucosyl lactose (2'-FL) and difucosyl lactose (DFL) by culturing a genetically modified cell having a recombinant gene that encodes a single fucosyl transferase in the presence of lactose. To form L-fucose in high yields, the resulting mixture of 2'-FL and DFL can be subjected to acid-initiated or fucosidase-mediated hydrolysis.

WO 2016/150629 A1 shows a process for the biocatalytic and/or enzymatic production of L-fucose using L-fucite as a starting material. To convert L-fucit to L-fucose in a one-step reaction, galactose oxidase is used after isolation in the biocatalysis step or directly in the producing cells in vivo.

WO 2016/120448 A1 describes a process for the enzymatic production of L-fucose. Described is a method for obtaining a monosaccharide, such as L-fucose, in free form using a microbiological fermentation process. The microorganism used exhibits hydrolase activity against intracellularly produced sugars activated by nucleotides and releases the monosaccharide in an unmodified free form. Free form L-Fucose is transported into the culture supernatant.

Existing attempts to obtain purified L-fucose have included only small-scale examples, but to date, there is no way to purify L-fucose from fermentation broth on a large scale with purity and quality suitable for use in the food industry.

The purpose of the present invention was to develop a large-scale (industrial scale) purification process to obtain high purity L-fucose. In particular, the process should be suitable for the production of L-fucose on a scale of a few kg to several tons in a quality suitable for use in the food industry, and should be suitable for continuous operation.

This goal is achieved by developing a method according to claim 1, a composition according to claim 18, a food composition according to claim 21, a liquid, ready-to-use food product for infants or toddlers according to claim 28, a spray-dried product in the form of a mixture for children infants according to claim 29, a nutritional supplement according to claim 30, a premix according to claim 31, and the use of a composition according to claim 33. The dependent claims illustrate preferred embodiments.

According to the invention, a method is provided for purifying L-fucose from a fermentation broth. This method includes the following steps:

removal of biomass from the fermentation broth containing L-fucose, thus obtaining a clarified solution,

obtaining a purified solution by subjecting the clarified solution to treatment on a cation exchanger using a cation exchange carrier, while the treatment on the cation exchanger is carried out under conditions under which L-fucose passes through the cation exchange carrier and is present in the passing stream; And

treatment on an anion exchanger using an anion exchange carrier, while the treatment on an anion exchanger is carried out under conditions under which L-fucose passes through the anion exchange carrier and is present in the passing stream; And

removal of salts from the purified solution by electrodialysis and/or nanofiltration.

The method according to the invention is suitable for obtaining the desired L-fucose in high purity (of food grade quality) and on a large scale (on an industrial scale ranging from one kg to several tons per cycle). In addition, the method according to the invention can be carried out very quickly and without great expense. Therefore, it can be carried out in a very economical manner. In addition, the method can be carried out in a batch or continuous manner. The latter further improves the yield of the resulting product per unit time. At the end of the process, the purity of L-fucose may be above 80%, preferably above 90%, more preferably above 95%, most preferably at least 98%.

The advantage of the proposed L-fucose purification method also lies in the fact that the L-fucose preparation does not contain recombinant DNA and recombinant proteins occurring during fermentation from recombinant microbial strains that are used to obtain L-fucose.

Although the method of the invention was conceived for the purification of a particular L-fucose from a fermentation broth, said method can also be applied to purify L-fucose obtained using an in vitro enzymatic reaction, the so-called in vitro biocatalytic reaction, or using biocatalysis method using permeabilized whole cells. Obviously, when purifying L-fucose from the reaction mixture in the case of an in vitro biocatalytic reaction, there is no need to remove the biomass from the reaction mixture. Thus, the reaction mixture in the case of an in vitro biocatalytic reaction corresponds to a clarified process stream.

Definitions

According to the invention, the term "purity" refers to chemical purity and specifies the degree to which a substance, such as L-fucose, is undiluted or unmixed with foreign matter. Thus, chemical purity is a measure of the relationship between an individual substance and by-products/impurities. Chemical purity is expressed as a percentage (%) and calculated using the following formula:

In a composition containing L-fucose, the purity of L-fucose can be determined by any suitable method known to the person skilled in the art, for example, through the use of high performance liquid chromatography (HPLC). A suitable detector may be a detector selected from the group consisting of an electrochemical detector, a refractive index (RI) detector, a mass spectrometric (MS) detector, a diode array detector (DAD), and a nuclear magnetic resonance (NMR) detector. For example, in the case of HPLC, this is the ratio of the area under the peak representing the amount of L-fucose to the sum of the areas under the peaks representing both the amount of L-fucose and compounds other than this L-fucose in the same chromatogram. However, this implies that all impurities can be analyzed by the chosen method using HPLC. Otherwise, an approach based on the mass balance is needed, i.e. on determining the absolute amount of the desired product (eg L-fucose). In this approach, pure substances are used as a reference substance for purity quantification, and purity is then assessed for the resulting product as a dry matter (desired product with all impurities). This mass balance approach can also be used to determine purity according to the invention.

According to the invention, the term "culture liquid" refers to any liquid after fermentation, which contains L-fucose to be purified. The terms "culture liquid", "fermentation broth" and "culture medium" are used in this description as synonyms. The culture fluid contains L-fucose to be purified, as well as biomass (eg, biological cells and cell debris), media components, salts, and impurities such as other acids and colored compounds. The purity of L-fucose within the culture fluid can be less than 80%. The biological cells contained in the culture fluid are biological cells that produce L-fucose intracellularly and secrete the produced L-fucose into the culture fluid. Biological cells may include or be genetically modified biological cells, such as genetically modified E. coli cells. The genetic modification may include or be a modification to produce L-fucose, in particular during the growth phase of said biological cells.

The term "biomass" as used herein refers to the totality of biological cells present in the fermentation broth at the end of the fermentation step. The biomass includes cells of the microorganism that have produced L-fucose, progeny cells of the microorganism that may lose their ability to produce L-fucose during the fermentation step, and any other cells that happen to be in the fermentation broth at the end of the fermentation step. Therefore, essentially all biological cells present in the fermentation broth at the end of the fermentation step are separated from the fermentation broth, whereby the clarified fermentation broth, i. the process stream is substantially free of cells.

The term "process stream" refers to any solution containing L-fucose that is to be purified.

The term "through stream" refers to a solution containing the L-fucose to be purified, which has just passed through an ion exchange carrier (ie cation and/or anion exchange carrier), i.e. is a mobile phase containing L-fucose after contact with the solid phase of the ion exchange carrier. In other words, the L-fucose present in the passing stream was not adsorbed or absorbed by the stationary phase.

According to the present invention, the difference between a weak cation exchange carrier and a strong cation exchange carrier is that, in the former, the chemical groups suitable for ion exchange have pKa values of at least 1 (for example, a pKa of 2 to 5, similar to a carboxylate group), while the latter has pKa values less than 1 (for example, pKa from -4 to 0, similar to the sulfo group).

Obtaining a clarified solution

The biomass preferably contains biological cells that produce L-fucose, preferably bacterial cells that produce L-fucose, more preferably recombinant bacterial cells that produce L-fucose, most preferably recombinant E. coii cells, recombinant Bacillus sp. and/or recombinant cells of Corynebacterium sp., in particular recombinant cells of Bacillus subtilis and/or recombinant cells of Bacillus megaterium, which produce L-fucose.

Biomass can be removed from the fermentation broth by centrifugation and/or filtration, with the filtration being preferably selected from the group consisting of microfiltration, ultrafiltration, tangential flow filtration, diafiltration, and combinations thereof.

In centrifugation methods suitable for removing biomass from the culture fluid, the biomass is obtained as a centrifugation pellet and the supernatant as a clarified process stream which is subjected to further treatments. In filtration methods suitable for removing biomass from culture fluid, the filtrate becomes a clarified process stream. The preferred filtration method for biomass removal is microfiltration and/or ultrafiltration. Ultrafiltration can remove even finer particles than microfiltration. It is possible that ultrafiltration is ultrafiltration in tangential flow.

Microfiltration is a physical separation process in which a particle-containing liquid is passed through a medium, said medium being either a porous substance containing tortuous channels to retain particles (deep filtration) and/or a membrane with a defined pore size to allow particles/molecules to pass through. , the size of which is less than the specified pore size (membrane filtration or dead-end filtration). The term "microfiltration" as used herein refers to a physical separation process in which biological cells (and cell debris) are removed from the fermentation broth to produce a (clarified) process stream.

Ultrafiltration is a form of membrane filtration that is not radically different from dead-end microfiltration. In ultrafiltration, the forces generated by pressure and concentration gradients result in the removal of particles and large solute molecules by passing a liquid containing such particles and large solute molecules through a semi-permeable membrane, causing the particles and large solute molecules to be held in such a way. called retentate, while water and low molecular weight solutes such as L-fucose pass through the membrane into the permeate (filtrate). Ultrafiltration membranes are characterized according to their molecular weight cut-off value (MWCO), which describes the maximum molecular weight of a solute molecule that can pass through the membrane into the permeate. Any particles, as well as molecules larger than MWCO, are unable to pass through the membrane and remain in the retentate. Ultrafiltration can be applied in tangential flow mode, where the liquid flow is parallel to the membrane surface, or in dead-end mode, where the liquid flow is perpendicular to the membrane surface.

Non-limiting examples and suitable microfiltration and/or ultrafiltration filters for removing biomass from fermentation broth include SPIRA-CEL® DS MP005 4333, which is a module containing a helically wound polyethersulfone membrane for compact design and better performance and having a nominal pore size 0.05 µm for ultrafiltration applications. Suitable membranes for biomass removal by microfiltration may have a pore size of at least 0.2 µm. Alternatively, biomass removal can be accomplished by microfiltration using membranes having an MWCO of 100 to 1000 kDa, preferably 150 kDa to 500 kDa, to remove biomass and additional cellular debris such as larger proteins. For example, FS10-FC FUS1582 (Microdyn-Nadir GmbH, Wiesbaden, DE), a hollow fiber ultrafiltration module using a polyethersulfone membrane (5 m ² ) with an MWCO of 150,000 daltons (150 kDa) can be used as an alternative.

In a further and/or alternative embodiment, smaller particles and large solute molecules are removed from the clarified process stream using tangential flow ultrafiltration. Here, the clarified process stream may be subjected to an ultrafiltration step using a filter having a MWCO of 10 kDa, such as Spira-Cel® DSUP010 (Microdyn-Nadir GmbH, Wiesbaden, DE), a helical wound ultrafiltration module using a polyethersulfone membrane (5, 7 m ² ) with MWCO 10,000 daltons (10 kDa).

In summary, the following possibilities can be used in the present invention to remove biomass from the fermentation broth:

1) collection by centrifugation. Undissolved parts are removed from the culture fluid in one step. Benefit: fast removal of undissolved parts;

2) collection by microfiltration. Undissolved parts and large molecules above a certain size are removed from the culture fluid in one step. When performing microfiltration, you can use spiral wound membranes or a hollow fiber filter device in a tangential flow mode. You can use microfiltration membranes that have a molecular weight cut-off ≥500 kDa, more preferably ≥150 kDa. Advantage: fast removal of undissolved parts and large molecules above a certain size;

3) collection by ultrafiltration. Undissolved parts, large molecules and small molecules above a certain size are removed from the culture liquid in one step. When performing ultrafiltration, you can use spiral wound membranes or a hollow fiber filter device in a tangential flow mode. You can use ultrafiltration membranes that have a molecular weight cut-off ≦100 kDa, more preferably ≦10 kDa. Advantage: fast removal of undissolved parts, large molecules and small molecules above a certain size;

4) collection by centrifugation combined with microfiltration. Undissolved parts and large molecules above a certain size are removed from the culture liquid in two stages. When performing microfiltration, you can use spiral wound membranes or a hollow fiber filter device in a tangential flow mode. You can use microfiltration membranes that have a molecular weight cut-off ≥500 kDa, more preferably ≥150 kDa. Benefit: fast removal of undissolved parts and large molecules above a certain size without clogging the pores in membranes or filters used in microfiltration;

5) collection by centrifugation combined with ultrafiltration. Undissolved parts, large molecules and small molecules above a certain size are removed from the culture liquid in two stages. When performing ultrafiltration, you can use spiral wound membranes or a hollow fiber filter device in a tangential flow mode. You can use ultrafiltration membranes that have a molecular weight cut-off value of ≦100 kDa, more preferably ≦10 kDa. Advantage: fast removal of undissolved parts, large molecules and small molecules above a certain size without clogging the pores in membranes or filters used in ultrafiltration;

6) collection by microfiltration combined with an ultrafiltration step. Undissolved parts, large molecules and small molecules above a certain size are removed from the culture liquid in two stages. When performing microfiltration and/or ultrafiltration, spiral wound membranes or a hollow fiber filter device in tangential flow mode can be used. You can use microfiltration membranes that have a molecular weight cut-off ≥500 kDa, more preferably ≥150 kDa. You can use ultrafiltration membranes that have a molecular weight cut-off value of ≦100 kDa, more preferably ≦10 kDa. Benefit: Most rapid removal of undissolved particles, large molecules and small molecules above a certain size with reduced risk of plugging pores in membranes or filters used in ultrafiltration.

The clarified process stream containing L-fucose can typically contain a significant amount of undesirable impurities, including (but not limited to) monovalent ions, divalent ions, amino acids, polypeptides, proteins, organic acids, nucleic acids, monosaccharides and/or oligosaccharides.

Steps which are preferably absent in the method of the invention

The process of the invention may include one or more chromatographic separation steps. However, in preferred embodiments, this method may be characterized in that it does not require or involve chromatographic separation (eg, there is no need for chromatographic fractionation). The advantage of not having a chromatographic separation is that the process can be done faster and at a lower cost because there is no time and expense involved in preparing the elution solution, using it to elute L-fucose from the solid phase, and collecting fractions after elution.

In addition, the method of the invention may differ in that it does not include the use of methanol, n-propanol, isopropanol, 2-butanone and/or ethyl acetate, possibly does not include the use of any organic solvent. The advantage of not using an organic solvent is that it can guarantee the absence of methanol, ethanol, n-propanol, isopropanol, 2-butanone and/or ethyl acetate in the final product, the final L-fucose or composition containing L-fucose, possibly the absence of any or an organic solvent. In addition, this method becomes less costly, environmentally friendly and safer for workers.

In addition, the method according to the invention may differ in that it does not include the steps of eluting L-fucose from the stationary phase using a solution containing an organic solvent. The advantage is that the purified L-fucose can be stored without any organic solvent. In addition, this method becomes less costly, environmentally friendly and safer for workers.

Moreover, the method according to the invention may be characterized in that it does not require the use of a heavy metal. The advantage is that it can guarantee the absence of heavy metals in purified L-fucose.

The method according to the invention may include one or more stages of heating the process stream, preferably to a temperature above 45°C. However, in a preferred embodiment, the method of the invention does not include the step of heating the fermentation broth, the clarified solution and/or the purified solution to a temperature above 45°C. The advantage is that, compared to prior art methods using such a heat treatment step, energy is saved to heat the respective solutions, making the process more economical and more environmentally friendly.

Processing on a cation exchanger

Cations can be removed from the clarified process stream, among other impurities, in the cation exchanger treatment step of the process of the invention, i.e. by applying at least one cation exchanger treatment of the clarified process stream. Specifically, the cations are exchanged for other cations that are bound to the cation exchange carrier (stationary phase) before the clarified process stream is fed to the cation exchanger treatment step. Significantly, it was found that the cation exchanger treatment removed ammonia and some of the protein contaminants from the clarified process stream.

In the cation exchanger treatment step, positively charged substances can be removed from the cell-free culture fluid as they bind to the resin. The aqueous solution of L-fucose is brought into contact in any suitable manner which allows the positively charged species to be adsorbed onto the cation exchange carrier while the L-fucose passes through it. The liquid obtained after contacting the cation exchange resin contains water, certain cations (those that were immobilized on the cation exchange carrier prior to this step), anions, colorants and L-fucose.

The treatment on the cation exchanger can be carried out using a weak cation exchange carrier or using a strong cation exchange carrier, preferably it is carried out using a strong cation exchange carrier.

When processing on a cation exchanger according to the method according to the invention, a strong cation exchanger can be used. Cation exchange resins suitable for removing positively charged compounds are strongly acidic cation exchange resins, such as resins that contain a carboxyl group (weak cation exchanger) or a sulfo group (strong cation exchanger) attached to a solid backbone (eg, a polystyrene backbone). Suitable resins include (but are not limited to) ^Lewatit® S2568 (H ⁺ ) (Lanxess AG, Cologne, DE), ^Dowex® 50WX2 (Merck KGaA, Darmstadt, DE); ^Amberlite® IR-116 (Japan Organo Co., Ltd.) and Diaion™ SK-102 (Mitsubishi Chemical Industries, Ltd.).

The cation exchanger treatment step may be the first step after removal of the biomass from the culture medium, i. e. the first stage, which is subjected to a clarified culture medium.

Non-specific cations are preferably replaced by a specific H ⁺ or Na ⁺ cation. If non-specific cations are replaced by H ⁺ , then the pH value in the passing stream is preferably adjusted to pH 6-8 before carrying out the subsequent processing step (eg, processing on an anion exchanger), most preferably by adding NaOH to the passing stream.

In a preferred embodiment, before the clarified process stream enters the cation exchanger, the cation exchange carrier is in the H ⁺ form, i. the ion-exchanging ligand on the cation-exchange support is protonated. This leads to the fact that during the cation exchange, the cations in the clarified process stream are replaced by H ⁺ . Therefore, the pH value of the solution after completion of this stage (in the passing stream) is corresponding to more acidic environments than the pH value of the solution before the said stage. Preferably, the pH of the through stream is raised to a neutral or near neutral pH (eg, pH ≧6.5 and pH ≦7.5) before carrying out subsequent purification steps. Raising the pH value can be achieved by adding NaOH to the process stream.

Any alkali metal ion (Li ⁺ , Na ⁺ , K ⁺ ), alkaline earth metal ion (such as Ca ²⁺ , Mg ²⁺ ), ammonium ion or carbonate ion can be used as the counterion in the cation exchanger. Preferably, the counterion of the cation exchanger is sodium ion (Na ⁴ ). More preferably, the counterion is a hydrogen ion (H ⁴ ). The advantage of the hydrogen ion as a counterion is that upon contact with the cation exchange carrier, the protonated form of the anions is formed in the process stream and these protonated anions are found in the passing stream, ie. in solution passed through a cation-exchange carrier. The protonated form of said anions can then be neutralized by adding a base (eg NaOH) to the product containing stream, whereby the protonated form of said anions is converted to a specific salt form (eg the sodium salt form). The presence of anions in the sodium salt form is preferred because sodium ions are relatively small cations and can be more easily removed by nanofiltration and/or electrodialysis than larger cations.

Preferably, the cation exchange step is carried out before the anion exchange step. However, in principle, the cation exchange treatment can also be carried out after the anion exchange treatment step.

The particle size of the cation exchange resin is preferably in the range of 0.1 to 1 mm. This range of particle sizes allows efficient passage of the cell-free culture fluid used, while still effectively removing charged substances on the cation exchange resin. In order to allow efficient ion exchange to take place, the volumetric flow rate should preferably be from 0.5 to 2.5 times the volume of the chromatography layer, more preferably from 1.0 to 2.0 times the volume of the chromatography layer. The treatment on the ion exchanger can be carried out in a conventional manner, for example in batches or continuously, preferably continuously.

The conditions under which L-fucose passes through the cation exchange carrier can be adjusted 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 value in the range of 6-8 and/or by adjusting the salt concentration if necessary.

After treatment with a cation exchanger and treatment with an anion exchanger, the purified solution may contain L-fucose, coloring agents and a salt, the salt being preferably NaCl. According to a preferred embodiment of the invention, in the anion exchanger treatment, the anion exchange carrier is used in the chloride form.

According to a further preferred embodiment of the invention, the cation exchange carrier is used in hydrogen form in the cation exchange treatment. It has been found that the use of a cation exchanger in the hydrogen form (the counterion is H ⁺ ) is preferred because a much better binding of salts and contaminant proteins is achieved compared to a cation exchange carrier in any other form (the counterion is not H ⁺ ). The use of the hydrogen form results in the passing stream having a pH corresponding to more acidic media than the solution that was applied for the cation exchanger treatment. However, this pH value can easily be raised by adding a base, preferably NaOH, to a neutral pH, preferably to a pH value in the range of 6-8. The implementation of neutralization using NaOH is preferred because the introduced sodium ions are relatively small and can be easily removed by nanofiltration and/or electrodialysis.

The particle size of the cation exchange resin should be chosen to facilitate efficient passage of the cell-free culture fluid used, while still effectively removing charged substances on the cation exchange resin. In order to allow efficient ion exchange to take place, the volumetric flow rate should preferably be from 0.2 to 2.0 chromatographic layer volume, more preferably 0.5 to 1.5 chromatographic layer volume. The treatment on the ion exchanger can be carried out in a conventional manner, for example in batches or continuously, preferably continuously.

First, the clarified solution can be treated with a cation exchanger and then treated with an anion exchanger. The advantage of this sequence in the method is that the cation exchange carrier can be in the H ⁺ form to obtain strong binding of contaminant proteins, and after neutralization using a base (for example, NaOH), a solution containing a certain salt form of anions can be passed through the anion exchange carrier in the process stream (for example, anions in the Na ⁺ -form).

Processing on an anion exchanger

Preferably, the anion exchange step is carried out after the cation exchange step. However, in principle, it can also be carried out before the stage of treatment on the cation exchanger, ie. before the process stream is processed by the cation exchanger.

The anion exchange step is carried out to remove non-specific anions and replace them with specific anions, preferably a specific Cl ^- or OH ^- anion. If non-specific anions are replaced by Cl ^- , then the pH value in the passing stream is preferably adjusted to pH 6-8 before carrying out the subsequent processing step (for example, removal of salts from the purified solution by electrodialysis), most preferably by adding NaOH to the passing stream.

Anions can be removed from the clarified process stream in the anion exchanger treatment step of the process of the invention, i.e. by applying at least one anion exchanger treatment to the clarified process stream. The anion exchange treatment step may be the first step after removal of the biomass from the culture medium, ie. the first stage, which is subjected to a clarified culture medium. Preferably, the anion exchanger treatment is carried out in a step carried out after the cation exchanger treatment step.

Negatively charged substances can be removed from the process stream as they bind to the anion exchange resin. The aqueous solution containing L-fucose is brought into contact in any suitable manner which allows the negatively charged species to be adsorbed onto the anion exchange carrier while the L-fucose passes through it. The liquid obtained after contact with the anion exchange resin contains water, certain cations, certain anions, colorants and L-fucose. The conditions of the anion exchange treatment step are such that L-fucose does not bind to the anion exchange carrier, i.e. is in the passing stream after contact with the carrier.

At the start of the anion exchanger treatment, the pH of the product-containing stream is preferably adjusted to pH 6-8 (most preferably pH 7). The passing stream (containing the product stream passing through the anion exchange carrier) may have a lower pH value, for example, a pH in the range from 4.5 to 6. At this pH value, the anions in the passing stream may be in protonated form. The pH value in the passing stream can be increased by adding a base, such as NaOH.

During anion exchange, anions in the clarified process stream may be exchanged for Cl ^- . Therefore, the pH value in the process stream becomes corresponding to slightly more acidic environments. Preferably, the pH is raised prior to subsequent purification steps, preferably until a neutral or nearly neutral pH (ie pH ≥6.5 and pH ≤7.5) is reached in the process stream. More preferably, this increase in pH in the process stream is achieved by adding NaOH to the process stream.

The anion exchanger may be a weak anion exchanger or a strong anion exchanger, preferably a strong anion exchanger.

Suitable strong base anion exchange resins are resins that contain a trimethylammonium group or a hydroxyethyl group attached to a solid backbone (eg a polystyrene backbone). Suitable resins include (but are not limited to) ^Lewatit® S6368A, ^{Lewatit® S4268} , Lewatit® S5528, Lewatit® ^S6368A (Lanxess AG, Cologne, DE), ^Dowex® AG 1×2, ^Dowex® 1×8, ^Purolite® Chromalite CGA100x4 (Purolite GmbH, Ratingen, DE), Amberlite® FPA51 (Dow Chemicals, MI, USA). The anion exchange resin is preferably in the chloride form. Suitable weak anion exchange resins are resins which contain an amino group attached to a solid backbone (eg a polystyrene backbone).

The anion exchanger can be used with any base as the counterion, eg HCO ₃ ^- , I ^- , Br ^- , NO ₃ ^- . Preferably, the counterion is hydroxide ion (OH ^- ). More preferably, the counterion is chloride ion (Cl ^- ). The advantage of using the chloride ion as the counterion is that the Cl anion ^is smaller than many other anions that are originally present in the fermentation broth. This results in the chloride anion being more easily removed from the process stream by electrodialysis and/or nanofiltration (eg diafiltration). The advantage of using the OH anion ^as the counterion is that when the pH is adjusted to neutral using HCl in the post-anion exchange step, then a small chlorine anion is introduced into the process stream, which can be removed more easily than other anions.

The particle size of the anion exchange resin is preferably in the range of 0.1 to 1 mm, which allows efficient passage of the process stream used while still effectively removing charged substances on the anion exchange resin. In order to allow efficient ion exchange to take place, the volumetric flow rate should preferably be from 0.5 to 2.5 times the volume of the chromatography layer, more preferably from 1.0 to 2.0 times the volume of the chromatography layer. The treatment on the ion exchanger can be carried out in a conventional manner, for example in batches or continuously, preferably continuously.

The conditions under which L-fucose passes through the anion exchange carrier can be adjusted by adjusting the pH and/or salt concentration of the clarified solution, preferably by adjusting the clarified solution to a pH value in the range of 6-8 and/or by adjusting the salt concentration if necessary. For example, it is noted that if a solution containing L-fucose, which is obtained by treating a passing stream on a cation exchanger with H ⁺ as a counterion and in which the pH value is adjusted to 6-8 by adding NaOH, is treated on an anion exchanger, then the concentration salts in the passing stream is high enough to prevent the binding of L-fucose with the anion exchange resin. However, it is noted that undesirable impurities (eg, some proteins, DNA and RNA molecules (in particular, recombinant DNA and RNA molecules) and colored substances) still bind to the anion exchange resin under these conditions.

In a preferred embodiment, the purification process comprises treatment using a cation exchange resin in hydrogen (H ⁺ ) form and an anion exchange resin in chloride (Cl ^- ) form. The cation exchange resin is preferably a strong cation exchanger. The anion exchanger may be a weak or strong anion exchanger, preferably it is a strong anion exchanger. In addition, the anion exchange carrier may also be an absorbent carrier. Treatment using both the cation exchange resin and the anion exchange resin removes all non-specific ions from the product-containing stream. Thus, non-specific ions can be replaced by specific ions, preferably a sodium cation (eg, introduced by neutralization of a product-containing stream using NaOH after contact with a cation exchange carrier with H ⁺ as a counterion) and a chlorine anion. The sodium cation and chloride anion are both relatively small ions and can be removed by electrodialysis and/or nanofiltration (diafiltration) in a faster and more economical manner than larger ions.

Concentration of the purified solution

Особенно предпочтительной является нанофильтрация и/или обратный осмос.Преимущество нанофильтрации по сравнению с обратным осмосом заключается в том, что нанофильтрация обеспечивает более быстрое концентрирование, а также позволяет частично удалять ионы солей. Преимущество нанофильтрации и/или обратного осмоса по сравнению с упариванием в вакууме заключается в том, что никаких реакций карамелизации с L-фукозой не происходит, т.е. в процессе концентрирования не образуется никаких окрашенных "карамельных" продуктов.In a preferred embodiment, the ion exchanger (throughstream) solution that contains L-fucose 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 the preferred methods (for example, nanofiltration using a nanofiltration membrane having a size exclusion limit

Particularly preferred is nanofiltration and/or reverse osmosis. An advantage of nanofiltration compared to reverse osmosis is that nanofiltration provides faster concentration and also allows partial removal of salt ions. The advantage of nanofiltration and/or reverse osmosis over vacuum evaporation is that no caramelization reactions occur with L-fucose, i.e. no colored "caramel" products are formed during the concentration process.

It is most preferred to concentrate the purified solution by nanofiltration, with the most preferred nanofiltration membrane used being a membrane that has a molecular weight cut-off value in the range of 100-200 kDa. The advantage of this molecular weight cutoff is that L-fucose is retained while salt ions can pass through the membrane, i.e. in addition to concentration, desalination is carried out.

Before concentrating the solution, the concentration of L-fucose in the solution may be ≦20% (w/w), ≦10% (w/w), or ≦5% (w/w).

During this process, the clarified solution and/or the purified solution can be concentrated to an L-fucose concentration of ≧100 g/l, preferably ≧200 g/l, more preferably ≧300 g/l.

In addition, the clarified solution and/or the purified solution can be concentrated by nanofiltration at temperatures below 80°C, preferably below 50°C, more preferably 4°C to 45°C, more preferably 10°C to 40°C, even more preferably 15°C to 30°C, most preferably 15°C to 20°C.

In addition, 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.

Moreover, the clarified solution and/or the purified solution can be concentrated by nanofiltration at pressures ranging from above 5 bar (0.5 MPa) to below 50 bar (5 MPa), preferably at pressures from above 10 bar (1 MPa) to below 40 bar (4 MPa), more preferably at pressures above 15 to below 30 bar (3 MPa).

In addition, the clarified solution and/or the purified solution can be concentrated by reverse osmosis at a pressure in the range from above 5 bar (0.5 MPa) to below 100 bar (10 MPa), preferably at a pressure from above 10 bar (1 MPa) to below 80 bar (8 MPa), more preferably at pressures from above 15 to below 70 bar (7 MPa).

Reverse osmosis is a membrane filtration method that removes particles larger than 0.1 nm from a solution (process stream). Only water will be removed from the process stream, while all other molecules such as ions, sugars, etc. will be concentrated inside the retentate. When using reverse osmosis, only an increase in the concentration of L-fucose can be carried out, but without its desalting.

In a further and/or alternative embodiment, the concentration step is characterized by at least one of the following, possibly all of the following:

1) concentration is carried out to achieve a concentration of L-fucose ≥100 g/l, preferably ≥200 g/l, more preferably ≥300 g/l;

2) the concentration is carried out at a temperature below 80°C, preferably ≤50°C, more preferably from 4°C to 45°C; most preferably from 4°C to 40°C if the concentration is carried out by nanofiltration;

3) concentration is carried out at a temperature below 50°C, preferably from 20°C to 45°C; most preferably from 30°C to 45°C, more preferably from 35°C to 45°C, if the concentration is carried out by reverse osmosis and/or evaporation in vacuum; And

4) concentration is carried out at a pressure in the range from above 5 bar (0.5 MPa) to below 50 bar (5 MPa), preferably at a pressure from above 10 bar (1 MPa) to below 40 bar (4 MPa), more preferably at pressure from above 15 to below 30 bar (3 MPa).

предпочтительно с использованием нанофильтрации или обратного осмоса, более предпочтительно с использованием нанофильтрации.In a preferred embodiment of the invention, the solution containing L-fucose is concentrated prior to the electrodialysis step using vacuum evaporation (for example, using a falling film evaporator or plate evaporator), reverse osmosis, or nanofiltration (for example, nanofiltration using a nanofiltration membrane having an exclusion limit to size

preferably using nanofiltration or reverse osmosis, more preferably using nanofiltration.

предпочтительно с использованием нанофильтрации или обратного осмоса, более предпочтительно с использованием нанофильтрации.In another preferred embodiment of the invention, the solution containing L-fucose is concentrated after an electrodialysis step using vacuum evaporation (for example, using a falling film evaporator or plate evaporator), reverse osmosis or nanofiltration (for example, nanofiltration using a nanofiltration membrane having an exclusion limit to size

preferably using nanofiltration or reverse osmosis, more preferably using nanofiltration.

Preferably, the solution containing L-fucose is concentrated prior to isolation of L-fucose in solid form (eg, crystalline form or granular form).

Salt removal

The method for purifying L-fucose includes a step in which salts are removed from the solution, preferably removed from the clarified solution and/or the purified solution. Salt removal can be achieved by subjecting the solution to desalting using nanofiltration and/or electrodialysis.

Nanofiltration is a membrane filtration method in which the membrane contains nanometer-sized pores. Nanofiltration membranes have pore sizes ranging from 1 to 10 nanometers. The pore size of nanofiltration membranes is smaller than the pore sizes used in microfiltration and even smaller than the pore sizes of ultrafiltration membranes, but usually larger than the pore sizes of membranes used for reverse osmosis. Membranes for nanofiltration applications are predominantly made from thin polymer films. Commonly used materials include polyethylene terephthalate or metals such as aluminium. The pore density can range from 1 to 10 ⁶ pores per cm ² . Nanofiltration is used in the L-fucose purification process to increase the concentration of L-fucose in a solution, such as a clarified solution and/or a purified solution. In addition, the solution (process stream) is desalted.

Membranes suitable for nanofiltration include those made of polyamide or polypiperazine, a thin film composite membrane material providing size exclusion in the range of 150 to 300 Da, such as Dow Filmtec™ NF270 (Dow Chemical Company, USA). Such membranes allow you to work with a large flow. In particular, nanofiltration membranes with a molecular weight cutoff in the range of 100 to 300 kDa are advantageous for increasing the concentration of L-fucose in the process stream. Membranes with this molecular weight cut-off prevent L-fucose from passing through the membrane and have the advantage that they also perform desalting since the salt (e.g. sodium chloride) that is present in the process stream after treatment on the ion exchange carrier(s) ), passes through the membrane and is separated from L-fucose. Additional examples of membranes suitable for nanofiltration include ^Trisep® 4040-XN45-TSF (Microdyn-Nadir GmbH, Wiesbaden, DE), GE4040F30 and GH4040F50 (GE Water & Process Technologies, Ratingen, DE).

It has been found that significant amounts of impurities (eg, salt ions) are effectively removed using nanofiltration prior to electrodialysis of the solution containing L-fucose. Nanofiltration has also been found to be effective in removing low molecular weight contaminants from the clarified fermentation broth after removal of the biomass from the fermentation broth (eg, in an ultrafiltration step). Removal of low molecular weight components is useful for concentrating and demineralizing a solution containing L-fucose before processing on an ion exchanger. The use of nanofiltration to increase the concentration of L-fucose leads to a reduction in energy and processing costs, as well as an improvement in product quality due to a decrease in thermal effects.

Electrodialysis combines dialysis and electrolysis and can be used to separate or concentrate ions in solutions based on their selective electromigration across a semipermeable membrane. The first industrial applications of electrodialysis date back to the early 1960s, when this method was used to demineralize cheese whey for inclusion in infant formula. Other uses for electrodialysis include its use to adjust the pH of beverages such as wines, grape must, apple juice, and orange juice.

The desalination of brackish water for drinking water and the demineralization of whey for the production of baby food are the most common applications of electrodialysis today. The basic principle of electrodialysis is the use of an electrolytic cell containing a pair of electrodes immersed in an electrolyte to provide ionic conductivity and connected to a direct current source. The electrode connected to the positive pole of the DC source is the anode, and the electrode connected to the negative pole is the cathode. Thus, the electrolyte solution maintains an electric current, which is caused by the movement of negative and positive ions towards the anode and cathode, respectively. The membranes used for electrodialysis are essentially sheets of porous ion exchange resins with negatively or positively charged groups and are therefore described as cationic or anionic membranes, respectively. Ion exchange membranes are typically made from polystyrene bearing a suitable functional group (such as a sulfo group for cationic membranes or a quaternary ammonium group for anionic membranes) crosslinked using divinylbenzene.

The electrolyte may be, for example, an aqueous solution containing sodium chloride, sodium acetate, sodium propionate and/or sulfamic acid. The electrolyte surrounds the cathode and anode and serves to ensure the flow of electrical current within the cell. The electrodialysis stack is then assembled in such a way that the anionic and cationic membranes are arranged in parallel, as in a filter press between two electrode blocks, so that the ion-depleted stream is well separated from the ion-rich stream (these two solutions are also called the dilute solution). (solution subjected to depletion of ions) and concentrate (solution subjected to enrichment in ions)).

The basis of the electrodialysis process is the use of a membrane stack, which consists of several spaced anion exchange membranes and cation exchange membranes installed between two electrodes. When a direct current source is connected, anions and cations will migrate through the membranes towards the electrodes, forming a (desalinated) dilute solution stream and a concentrate stream.

The pore size of ion exchange membranes for electrodialysis applications is small enough to prevent diffusion of product from the dilute solution stream into the concentrate stream due to the large concentration difference between the two streams. After separation of the biomass and/or exchange of cations and/or anions, quantitative removal of proteins and especially recombinant DNA molecules (ranging in size from fragments to whole genomes) from the desired product must occur.

Electrodialysis is used to remove ions from an aqueous solution while the L-fucose remains inside the process stream. An important advantage of electrodialysis is that recombinant DNA molecules can be completely removed from a solution containing L-fucose. In addition, it has been found that by means of electrodialysis it is possible to significantly reduce the amount of salt in the process stream. In fact, it has been discovered that sodium chloride can be completely removed from the product-containing stream. This has the advantage that it is possible to obtain L-fucose free of salt such as sodium chloride, which prevents any negative effect that the presence of salt (eg sodium chloride) in the final product, for example for infant nutrition, can have.

Removal of salts (for example, by electrodialysis) can be carried out until a stable value of electrical conductivity (mS/cm ² ) is reached in the range from 0.2 to 10.0 mS/cm ² , preferably from 0.4 to 5, 0 mS/cm ² , more preferably 0.5 to 1.0 mS/cm ² . In addition, electrodialysis can be carried out until the salt concentration (g/l) is below 10.0 g/l, preferably below 5.0 g/l, more preferably below 1.0 g/l, even more preferably ≦0.5 g/l, most preferably ≦0.4 g/l, in particular ≦0.2 g/l.

The electrodialysis can be carried out under neutral conditions or under acidic conditions. The difference between these two options lies in the form of the anions present in the process stream.

In the case of electrodialysis under neutral conditions, before the start of electrodialysis, the pH of the process stream can be adjusted using an acid, preferably hydrochloric acid (HCl), or a base, preferably sodium hydroxide (NaOH), until a pH of 5.0 to 9 is reached, 0, preferably 6.0 to 8.0, more preferably 6.5 to 7.5.

In the case of electrodialysis under acidic conditions, before the start of electrodialysis, the process stream is acidified using an acid, preferably hydrochloric acid (HCl), until a pH of 1.0 to 3.0 is reached, preferably 1.5 to 2.5, more preferably from 1.8 to 2.2. Electrodialysis is preferably carried out until stable values of electrical conductivity (mS/cm ² ) and pH are reached.

Electrodialysis can be carried out until a stable value of electrical conductivity (mS/cm ² ) is reached in the range from 1.0 to 10.0 mS/cm ² , preferably from 1.5 to 10.0 mS/cm ² , more preferably from 2.0 to 8.0 mS/cm ² . During electrodialysis, the pH must be adjusted and adjusted using a base, preferably sodium hydroxide. Under neutral conditions, electrodialysis can be carried out using bipolar membranes. In this case, L-fucose can be concentrated in a separate circuit for electrodialysis concentrate. Thus, during electrodialysis, L-fucose can be enriched.

In this method, after removing salts from the purified solution

1) the amount of salt in the purified solution may be less than 10% (w/w), preferably less than 5% (w/w), more preferably less than 1% (w/w), even more preferably ≤0 .5% (w/w), most preferably ≦0.4% (w/w), especially ≦0.2% (w/w); and/or

2) The conductivity value may be in the range of 0.2 to 10.0 mS/cm ² , preferably in the range of 0.4 to 5.0 mS/cm ² , more preferably in the range of 0.5 to 1.0 mS /cm ² .

Decolorization of the clarified and/or purified solution The clarified solution and/or purified solution can be subjected to a decolorization step, preferably by treatment with activated carbon and/or treatment using a cation exchanger and an anion exchanger which are connected in series.

The bleaching step can be carried out

1) before or after the stage of diafiltration and/or concentration of the clarified solution; and/or

2) before or after the stage of electrodialysis and/or diafiltration of the clarified solution.

Preferably, the bleaching step is carried out after the electrodialysis step, especially when said step is carried out by treatment using a cation exchanger and an anion exchanger (which are connected in series). The advantage is that the cation exchanger and anion exchanger can be operated using a process stream that contains very low salt concentration and this results in many electrically charged substances (e.g. electrically charged peptides and/or DNA molecules) being bound to ion-exchange carrier, while L-fucose remains unbound and is in the passing stream. Thus, at this stage, the purity of L-fucose in terms of colored substances and uncolored electrically charged substances can be significantly improved. It is possible that said step is carried out after the nanofiltration step and/or before the electrodialysis step.

The advantage of removing coloring substances by treatment with activated carbon compared to treatment using a cation exchanger and an anion exchanger (which are connected in series) is that both electrically charged and electrically uncharged (neutral) coloring substances can be removed and can be removed electrically. uncharged (neutral) carbohydrates (for example, oligosaccharides, monosaccharides).

Activated carbon, also called activated carbon, is a form of carbon that has been processed to produce small, low volume pores that increase the surface area available for adsorption. Typically, only one gram of activated carbon, due to its high degree of microporosity, has a surface area in excess of 3000 m ² as determined from gas adsorption data.

A carbohydrate like a monosaccharide or an oligosaccharide tends to bind to the surface of activated carbon particles from an aqueous solution. The interaction of oligosaccharides with activated carbon is much stronger than the interaction of monosaccharides. This behavior is due to their structure and leads to the fact that L-fucose binds weaker, i.e. in a smaller amount, with activated carbon than the impurity oligosaccharide. In addition to oligosaccharides, colored substances are adsorbed on activated carbon. Other water-soluble compounds, such as salts, bind weaker and elute from the activated charcoal along with L-fucose by washing the activated charcoal with water (after incubation). After eluting with water, the adsorbed oligosaccharides and colored substances still remain bound to the activated charcoal. Therefore, at the stage of treatment with activated carbon, it is possible to remove impurity oligosaccharides and colored impurities. Finally, the activated carbon step removes colorants, other impurities, and reduces water-soluble contaminants such as salt.

Suitable activated carbons for removing coloring compounds, oligosaccharides, or contaminants include, but are not limited to, granular activated carbons such as ^Norit® GAC830EN (Carbot Cooperation) and ^Epibon® Y 12x40 spezial (Donaucarbon) or powdered activated carbon such as Norit ^® DX1, Norit ^® SA2 (Carbot Cooperation) and Carbopal ^® MB 4 (Donaucarbon).

The removal of color-imparting substances by treatment with a cation exchanger and an anion exchanger (which are connected in series) has the advantage over activated carbon treatment that not only color-imparting substances can be removed from the solution, but non-specific salt ions can also be replaced in the solution with specific salt ions such as, for example, Na ⁺ and Cl ^- and electrically charged peptides can be removed. The advantage of this ion exchange is that the desalting process can become more efficient (Na ⁺ and Cl ^- are small ions compared to other possible ions and can therefore be more easily removed in the desalting treatment step). An additional advantage is that the negative influence of some non-specific salts on the crystallization of L-fucose can be avoided.

The treatment on the ion exchanger is carried out in such a way that charged substances and coloring substances are adsorbed on the appropriate ion exchange carrier, while L-fucose passes through the appropriate ion exchange carrier, i.e. is in the flow. The resulting liquid (the passing stream) contains water, a small amount of certain ions, a lower amount of coloring agents and L-fucose.

The cation exchanger may be a weak cation exchanger or a strong cation exchanger, preferably a strong cation exchanger.

Suitable cation exchange resins are strongly acid cation exchange resins such as (but not limited to) ^Lewatit® S2568(H ⁺ ) (Lanxess AG, Cologne, DE), ^Dowex® 50WX2 (Merck KGaA, Darmstadt, DE); ^Amberlite® IR-116 (Japan Organo Co., Ltd.) and Diaion™ SK-102 (Mitsubishi Chemical Industries, Ltd.).

The cation exchanger can be used with any alkali metal ion (Li ⁺ , Na ⁺ , K ⁺ ), alkaline earth metal ion (such as Ca ²⁺ , Mg ²⁺ ), ammonium ion or carbonate ion as the counter ion. Preferably, the counterion is a sodium ion (Na ⁺ ).

The anion exchanger may be a weak anion exchanger or a strong anion exchanger, preferably a strong anion exchanger.

Suitable anion exchange resins are strongly basic (type I) anion exchange resins such as, but not limited to, ^Lewatit® S6368 A, Lewatit® ^S4268 , ^Lewatit® S5528, ^Lewatit® S6368A (Lanxess AG, Cologne, DE), ^Dowex® AG 1× 2, ^Dowex® 1×8, ^Purolite® Chromalite CGA100×4 (Purolite GmbH, Ratingen, DE), ^Amberlite® FPA51 (Dow Chemicals, MI, USA). Preferably, the anion exchange resin is in the chloride form.

The anion exchanger can be used with any base as the counterion, eg HCO ₃ ^- , I ^- , Br ^- , NO ₃ ^- . Preferably, hydroxide ion (OH) is used as the counterion. More preferably, chloride ion (Cl ^- ) is used as the counterion.

In the case of a serial connection of the cation exchanger and the 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 case of a series connection. Thus, a solution passing through such a serial connection first passes through a cation exchanger and then passes through an anion exchanger.

The pH value of the process stream that has passed through the cation exchanger and/or anion exchanger (i.e. the respective passing stream) is preferably above 2.0 and below 10.0, more preferably above 3.0 and below 9.0, most preferably above 4 .0 and below 8.0.

The particle size of the ion exchange resin should be chosen to facilitate efficient flow of the process stream while effectively removing charged and coloring agents on the ion exchange resin. To ensure efficient ion exchange, the volumetric flow rate should preferably be from 0.2 to 2.0 chromatographic layer volume, more preferably 0.5 to 1.5 chromatographic layer volume, and most preferably 0.75 to 1.2 chromatographic layer volume. layer. The treatment on the ion exchanger can be carried out in a conventional manner, for example in batches or continuously, preferably continuously.

However, if an endotoxin-free strain is used (eg, a Bacillus strain and/or a genetically modified endotoxin-free strain), the endotoxin removal step may not be required.

Sterilizing Filtration and/or Endotoxin Removal In a preferred embodiment of the invention, the purified solution is sterile filtered and/or endotoxin removed, preferably by filtering the purified solution through a filter module (≤10 kDa). As an example, the purified solution containing L-fucose is sterilized by filtration and/or subjected to an endotoxin removal step by filtering the purified solution through a filter (3 kDa) or through a filter (6 kDa). Removal of endotoxins is necessary if the L-fucose is intended for human consumption.

Obtaining the granular form of L-fucose

Agglomeration of powdered food concentrate is commonly used to improve the fast cooking properties of granular (eg spray dried) products. This method is used when it is desirable to increase the particle size of the product in order to improve its flowability and/or appearance.

In general, the basic properties of granulates or agglomerates, such as size, porosity, solubility, wettability, shape and/or density, depend on the type of agglomeration (granulation) process and on the process conditions in the agglomeration process. Fluidized bed agglomeration is one of the methods suitable for obtaining agglomerates with high porosity and good mechanical strength during transportation and packaging.

The principles of fluidized bed agglomeration are to fluidize the particles with a stream of hot air and wet the surface of the particles by spraying a liquid binder. As a result of collisions between wet particles in a fluidized bed, liquid bridges are formed and the particles merge. After drying of these particles, the bridges harden, which leads to coarsening of the agglomerates.

According to the invention, the purified solution can be granulated, in particular granulated at a L-fucose concentration of 5-50% (w/w), preferably 10-40% (w/w), more preferably 15-35 % (w/w).

The inlet temperature may be in the range of 50°C-120°C, preferably 65°C-110°C, more preferably 80°C-100°C.

The outlet temperature (product temperature) may be in the range of 30°C-80°C, preferably 35°C-75°C, more preferably 40°C-75°C.

The resulting granulate should have a loss on drying, preferably from 0.1 to 5% by weight of the substance, more preferably from 0.5 to 2.5% by weight of the substance, most preferably from 0.7 to 1.5% by weight of the substance.

In a preferred embodiment, the granular L-fucose is preferably at least 50% (w/w) a substance with a particle size of 150 µm to 1400 µm, more preferably at least higher than 60% (w/w). ), with a particle size of 150 µm to 1400 µm, most preferably greater than 70% (w/w), with a particle size of 150 µm to 1400 µm.

Particle size can be determined by sieving the material through sieves having different pore sizes. For example, in order to obtain L-fucose as a substance with a particle size of 150 µm to 1400 µm, a 1400 µm sieve and a 150 µm sieve can be used sequentially (in any order) to select a substance having desired particle size.

Obtaining the form of L-fucose dried on a roller dryer

L-Fucose can be obtained in roll-dried form. To this end, the purified solution can be dried on a roller dryer, i.e. subjected to a drying procedure on a roller dryer.

Obtaining the crystalline form of L-fucose

L-Fucose can be obtained in crystalline form. The method of crystallization of L-fucose from aqueous solutions will be described in this description.

It has been found that L-fucose can be selectively crystallized after applying a biocatalytic approach using enzymes or from a fermentation broth (culture fluid).

In a preferred embodiment, the aqueous L-fucose solution to be crystallized has a given carbohydrate content of greater than 50% (w/w), preferably greater than 60% (w/w), preferably greater than 70% (w/w) , in particular, above 80% (wt./mass.).

Alternatively, the concentration of L-fucose in the aqueous solution to be crystallized is 650-950 g/l, preferably 800-880 g/l.

The concentration ranges and ratios given above can be achieved in a conventional manner by concentrating an aqueous process stream from the culture fluid, preferably after removal of e.g. coloring cells, salts and/or charged molecules from the culture fluid using the previously described methods.

Concentration of the solution containing L-fucose can be carried out by removal of water using nanofiltration and/or vacuum evaporation (for example, using a rotary evaporator or plate evaporator), preferably nanofiltration in combination with vacuum evaporation.

Seed crystals can be added to said concentrated L-fucose solution to start crystallization at a temperature in the range of 20°C to 50°C, preferably at 25°C.

The concentrated solution containing L-fucose preferably has a concentration of this carbohydrate, in particular above 75% (w/w).

The solution containing L-fucose can be incubated under vacuum, preferably below 200 mbar (20 kPa), more preferably below 100 mbar (10 kPa), in particular below 50 mbar (5 kPa).

In addition, the solution containing L-fucose can be incubated at a temperature in the range from 15°C to 60°C, preferably from 20°C to 45°C, more preferably from 25°C to 40°C, in particular from 30°C to 35°C.

In addition, a solution containing L-fucose can be incubated until the concentration of this carbohydrate reaches a value above 85%, preferably above 90%, or until a viscous crystallization slurry is formed.

The solution (crystallizable mixture) containing L-fucose can then be placed in a suitable crystallization vessel and incubated at room temperature (25° C.) for 12-96 hours until a solid crystallized mass is formed.

The crystallized mass can be separated mechanically and an organic solvent such as methanol, acetone, isopropanol and/or ethanol, preferably ethanol, can be added to the crystal solution. In order to separate the L-fucose crystals, 1 kg of the crystallized substance solution is mixed with 0.5-3 L of ethanol, preferably 0.7-2.0 L of ethanol, more preferably 1.0-1.5 L of ethanol.

L-fucose crystals can be recovered 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, for example methanol, acetone, isopropanol and/or ethanol, preferably ethanol. To remove the remaining mother liquor, 1 kg of crystallized L-fucose is preferably washed with 0.5-3 L, preferably 0.7-2.0 L, more preferably 1.0-1.5 L of ethanol.

The resulting crystals can be dried for 4-48 hours, preferably 8-36 hours, more preferably 12-24 hours, or until no change in weight occurs. The drying temperature is chosen in the range 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° to 50°C.

For crystallizing L-fucose from an aqueous solution, a second alternative method for crystallizing L-fucose from aqueous solutions using organic solvents will be described herein.

In a preferred embodiment, the aqueous L-fucose solution to be crystallized has a given carbohydrate content of greater than 40% (w/w), preferably greater than 50% (w/w), more preferably greater than 60% (w/w). ), in particular above 70% (w/w).

Alternatively, the concentration of L-fucose in the aqueous solution to be crystallized may be 650-850 g/l, preferably 700-780 g/l.

The above concentration ranges and ratios can be achieved in a conventional manner by concentrating an aqueous process stream from the culture fluid, preferably after removal of, for example, cells, colorants, salts and/or charged molecules from the culture fluid using methods previously described.

The concentration of the solution containing L-fucose can be carried out by removal of water using nanofiltration and/or evaporation in vacuum (for example, using a rotary evaporator or plate evaporator). Preferably, nanofiltration is used in combination with vacuum evaporation.

After concentrating a solution containing L-fucose, this solution has an L-fucose carbohydrate concentration of more than 70% (w/w) in particular. In order to remove the remaining water from the mixture to be crystallized, 0.5 volumes of butanol can be added to this solution. The mixture to be crystallized can then be incubated under vacuum until an equivalent amount of added liquid has been removed by vacuum distillation.

Seed crystals can be added to said concentrated L-fucose solution to start crystallization at a temperature in the range of 20°C to 50°C, preferably at 25°C.

The crystallization can preferably be carried out at a pressure below 200 mbar (20 kPa), more preferably below 100 mbar (10 kPa), in particular below 50 mbar (5 kPa).

In addition, the crystallization can be carried out at a temperature of the crystallizing solution in the range of 15°C to 60°C, preferably 20°C to 45°C, more preferably 25°C to 40°C, in particular 30°C. up to 35°С.

Moreover, the crystallizable solution can be incubated until the concentration of this carbohydrate reaches a value above 85%, preferably above 90%, or until a viscous crystallization slurry is formed.

The crystallizable mixture can then be placed in a suitable crystallization vessel and incubated at room temperature (25° C.) for 12-96 hours until a solid crystallized mass is formed.

The crystallized mass can be separated mechanically and an organic solvent such as methanol, acetone, isopropanol and/or ethanol, preferably ethanol, can be added to the crystallized mass. In order to separate the L-fucose crystals, 1 kg of the crystallized substance solution is preferably mixed with 0.5-3 L of ethanol, preferably with 0.7-2.0 L of ethanol, more preferably with 1.0-1.5 L of ethanol.

L-fucose crystals can be recovered 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, for example methanol, acetone, isopropanol and/or ethanol, preferably ethanol. To remove the remaining mother liquor, 1 kg of crystallized L-fucose is preferably washed with 0.5-3 L, preferably 0.7-2.0 L, more preferably 1.0-1.5 L of ethanol.

The resulting crystals can be dried for 4-48 hours, preferably 8-36 hours, more preferably 12-24 hours, or until no change in weight occurs. The drying temperature can be selected in the range of 10°C to 80°C, preferably 20°C to 70°C, more preferably 30°C to 60°C, in particular 40° to 50°C.

Obtaining a lyophilized form of L-fucose

The purified solution containing L-fucose can also be lyophilized. In said lyophilization, the purified solution containing L-fucose is frozen and then the pressure is reduced, whereby the frozen water sublimes directly from the solid phase to the gas phase. This method usually results in a hygroscopic L-fucose powder.

Compositions and products according to the invention

The present invention provides a composition which contains

a) at least 98 wt. % L-fucose;

b) not more than 1 wt. % organic solvent; And

c) not more than 1 wt. % salts.

In a preferred embodiment, the composition contains

a) from 95.50 to 100.00 wt. % L-fucose, preferably from 98.00 to 100.00 wt. % L-fucose, more preferably from 98.50 to 100.00 wt. % L-fucose, possibly from 98.70 to 99.90 wt. % L-fucose; and/or

b) from 0.00 to 1.00 wt. % organic solvent, preferably from 0.00 to 0.50 wt. % organic solvent, more preferably from 0.00 to 0.40 wt. % organic solvent, possibly from 0.10 to 0.20 wt. % organic solvent; and/or

c) from 0.00 to 1.00 wt. % salts, preferably from 0.00 to 0.50 wt. % salts, more preferably from 0.00 to 0.40 wt. % salts, possibly from 0.10 to 0.20 wt. % salts.

Preferably, the composition contains (only)

a) from 0.00 to 0.50 wt. % proteins, more preferably from 0.00 to 0.40 wt. % proteins, possibly from 0.10 to 0.20 wt. % proteins; and/or

b) from 0.00 to 0.50 wt. % DNA, more preferably from 0.00 to 0.40 wt. % DNA, possibly from 0.10 to 0.20 wt. % DNA.

The composition may be characterized in that the L-fucose is in amorphous form or in crystalline form, preferably in granular form or in crystalline form. The crystalline form is preferably the dihydrate crystalline form.

Breast milk is the best source of nutrition for an infant. Breast milk contains free L-fucose. It is known that fucose can be taken up by humans and activated via the fucose recycling pathway to form GDP-fucose. GDP-fucose is then used to synthesize fucosylated glycans, which play an important role in cell-to-cell communication processes such as immune recognition and in brain development.

A food composition is provided, preferably an infant formula, a toddler formula, or a health food product, wherein the food composition contains L-fucose and a probiotic carbohydrate (e.g., galactooligosaccharide (GOS), fructooligosaccharide (FOS), inulin, maltodextrin, isomaltose, lactulose, etc.) or another monosaccharide (for example, sialic acid such as N-acetylneuraminic acid).

The nutritional composition, preferably an infant formula, a toddler formula, or a health food product, may contain L-fucose and at least one human milk oligosaccharide.

The food composition may contain at least one sugar selected from the group consisting of 2'-fucosyllactose, 3-fucosyllactose, lacto-N-tetraose, lacto-N-triose II, lacto-N-fucopentaose I, lacto-N-fucopentaose III, lacto-N-fucopentose V, difucosyllactose, lacto-N-neotetraose, 3'-sialyllactose, 6'-sialyllactose and sialylated derivatives of lacto-N-neotetraose and lacto-N-tetraose.

The food composition may be

1) liquid food composition and contain L-fucose at a concentration of 1 mg/l to 2 g/l, more preferably at a concentration of 5 mg/l to 1.5 g/l, even more preferably at a concentration of 20 mg/l up to 1 g/l, most preferably at a concentration of 50 mg/l to 0.7 g/l; or

2) a solid food composition and contain L-fucose at a concentration of 5 mg/kg to 15 g/kg, more preferably at a concentration of 25 mg/kg to 10 g/kg, even more preferably at a concentration of 100 mg/kg to 10 g/kg, most preferably at a concentration of 375 mg/kg to 5.25 g/kg.

The food composition may contain sialic acid (eg, N-acetylneuraminic acid) in the same concentration (same range) as for L-fucose (see above).

The food composition may contain

1) at least one neutral human milk oligosaccharide, preferably at least one neutral human milk oligosaccharide selected from the group consisting of 2'-fucosyl lactose, 3-fucosyl lactose, lacto-N-tetraose and lacto-N-neotetraose, most preferably all from said neutral breast milk oligosaccharides; And

2) at least one human milk acid oligosaccharide, preferably at least one human milk acid oligosaccharide selected from the group consisting of 3'-sialyllactose and 6'-sialyllactose, most preferably all of said human milk acid oligosaccharides; And

3) sialic acid (eg N-acetylneuraminic acid).

The composition may further comprise at least one, possibly all, selected from the group consisting of lactose, whey protein, biotin, skimmed milk, vegetable oil, skimmed milk powder, Mortierella alpine oil, fish oil, carbonate calcium, potassium chloride, vitamin C, sodium chloride, vitamin E, iron acetate, zinc sulfate, niacin, calcium D-pantothenate, copper sulfate, vitamin A, vitamin B1, vitamin B6, magnesium sulfate, potassium iodate, folic acid, vitamin K , sodium selenite and vitamin D.

The food composition may contain at least one substance selected from the group consisting of a protein source, a vitamin, an oil, a mineral, an enzyme, an additional carbohydrate, and a probiotic strain.

The nutritional composition may be a composition selected from the group consisting of a health food composition, a dietary supplement, a sachet product, a liquid ready-to-use infant food, a liquid ready-to-use toddler food, a granular product, dried spraying the product as a mixture for infants and combinations thereof.

The following is a liquid ready-to-use food product for infants or toddlers containing L-fucose at a concentration of 1 mg/l to 2 g/l, sialic acid (for example, N-acetylneuraminic acid) (preferably at a concentration of 1 mg/l l up to 2 g/l) and

1) at least one neutral human milk oligosaccharide selected from the group consisting of 2'-fucosyl lactose, 3-fucosyl lactose, difucosyl lactose, lacto-N-tetraose and lacto-N-neotetraose; and/or

2) at least one human milk acid oligosaccharide selected from the group consisting of 3'-sialyllactose or 6'-sialyllactose.

The invention also provides a spray-dried infant formula containing L-fucose at a concentration of 5 mg/kg to 15 g/kg, sialic acid (for example, N-acetylneuraminic acid) (preferably at a concentration of 5 mg/kg/kg). kg up to 15 g/kg) and

1) at least one neutral human milk oligosaccharide selected from the group consisting of 2'-fucosyl lactose, 3-fucosyl lactose, difucosyl lactose, lacto-N-tetraose and lacto-N-neotetraose; and/or

2) at least one human milk acid oligosaccharide selected from the group consisting of 3'-sialyllactose and 6'-sialyllactose.

In addition, a nutritional supplement is provided containing L-fucose and at least one neutral HMO selected from the group consisting of 2'-fucosyl lactose, 3-fucosyl lactose, difucosyl lactose, lacto-N-triose II, lacto-N-tetraose and lacto -N-neotetraose, lacto-N-fucopentaose I.

The invention provides a premix for a food product (for example, a premix for infant or toddler formula) which contains L-fucose and at least one substance selected from the group consisting of a protein source, a vitamin, an oil, a mineral. , an enzyme, an additional carbohydrate (eg, a carbohydrate other than the L-fucose already present in the premix), and a probiotic strain. More preferably, the premix contains all of these substances.

The protein source may be selected from the group consisting of whey, soybean grain blend (CSB), protein hydrolysates, and combinations thereof.

The vitamin may be selected from the group consisting of vitamin A, thiamine, riboflavin, vitamin B12, folate, and combinations thereof.

The oil may be selected from the group consisting of palm oil, docosahexaenoic acid (DHA), arachidonic acid, and combinations thereof.

The mineral may be selected from the group consisting of potassium chloride, potassium iodate, zinc oxide, and combinations thereof.

The enzyme may be selected from the group consisting of amylase, amyloglucosidase, and combinations thereof.

The carbohydrate may be selected from the group consisting of human milk oligosaccharide (HMO), galactooligosaccharide (GOS), inulin, fructooligosaccharide (FOS), lactose, isomaltose, sialic acid, and combinations thereof.

The probiotic strain may be selected from the group consisting of strains of Lactobacillus, Bifidobacterium, Bacillus, yeast, and combinations thereof.

The premix may be in the form of a spray-dried, granular or liquid (syrup) product.

The present invention provides a pharmaceutical composition, preferably a pharmaceutical composition, for use in the prevention or treatment of at least one of the following: viral infection, bacterial infection (to improve immune function), memory loss, brain development disorder, and dysbiosis. Such a pharmaceutical composition contains a composition according to the invention and optionally contains at least one sugar other than L-fucose and/or at least one probiotic bacterial strain, wherein at least one sugar is preferably at least one sugar selected from the group consisting of lactose, lactulose, inulin and sucrose.

All compositions of the present invention (e.g., food composition, liquid ready-to-use infant or toddler food, spray-dried infant formula, dietary supplement, and premix) may have at least one of the following: .

The composition preferably contains lactose.

In the case of a solid composition, the lactose concentration may be from 50 to 800 g/kg, preferably from 100 to 750 g/kg, more preferably from 200 to 700 g/kg, even more preferably from 300 to 650 g/kg, most preferably from 400 up to 600, in particular from 440 to 560 g/kg.

In the case of a liquid composition, the lactose concentration may be from 10 to 95 g/l, preferably from 20 to 90 g/l, more preferably from 30 to 85 g/l, even more preferably from 40 to 80 g/l, most preferably from 50 up to 75 g/l, in particular from 60 to 74 g/l.

It has been found that the presence of lactose in the composition has a positive effect on the well-being of the subject who consumes such a composition as food. This effect is due to the combinatorial action of lactose along with other ingredients that are present in the composition of the invention (eg fucose, sialic acid, neutral HMO and acidic HMO). It is believed that lactose, along with other ingredients, work together to protect the body from pathogenic bacteria and reduce the intensity of inflammatory reactions in the gastrointestinal tract of subjects consuming this composition.

The composition preferably contains HMOs (acidic and neutral HMOs) in the maximum amount.

In the case of a solid composition, the maximum HMO concentration may be in the range of 20 to 70 g/kg, preferably 25 to 65 g/kg, more preferably 30 to 60 g/kg, even more preferably 35 to 55 g/kg, most preferably 40 to 50, in particular 42.2 to 48.1 g/kg.

In the case of a liquid composition, the maximum concentration of HMO may be from 2.0 to 9.0 g/l, preferably from 3.0 to 8.0 g/l, more preferably from 4.0 to 7.0 g/l, even more preferably 4.5 to 6.5 g/l, most preferably 5.0 to 7.0 g/l, especially 5.7 to 6.5 g/l.

The composition preferably contains HMO in a certain concentration.

In the case of a solid composition, the concentration of 2'-fucosyllactose (2'-FL) may be from 5 to 40 g/kg, preferably from 10 to 35 g/kg, more preferably from 12.5 to 30 g/kg, most preferably from 15 up to 25 g/kg, in particular from 18.5 to 22.2 g/kg.

In the case of a liquid composition, the concentration of 2'-FL may be from 0.5 to 5.5 g/l, preferably from 1.0 to 4.5 g/l, more preferably from 1.5 to 4.0 g/l, most preferably 2.0 to 3.5 g/l, especially 2.5 to 3.0 g/l.

In the case of a solid composition, the concentration of 3'-FL may be from 3.5 to 7.5 g/kg, preferably from 4.0 to 7.0 g/kg, more preferably from 4.5 to 6.5 g/kg, most preferably 5.0 to 6.0 g/kg, especially 5.5 to 5.9 g/kg.

In the case of a liquid composition, the concentration of 3'-FL may be from 500 to 1000 mg/l, preferably from 600 to 950 mg/l, more preferably from 650 to 900 mg/l, most preferably from 700 to 850 mg/l, in particular from 750 to 800 mg/l.

In the case of a solid composition, the concentration of lacto-N-tetraose (LWT) may be from 7 to 15 g/kg, preferably from 8 to 14 g/kg, more preferably from 9 to 13 g/kg, most preferably from 10 to 12 g/kg. kg, especially 11.1 g/kg.

In the case of a liquid composition, the concentration of LNT may be from 0.2 to 3.5 g/l, preferably from 0.4 to 3.0 g/l, more preferably from 0.8 to 2.5 g/l, most preferably from 1.0 to 2.0 g/l, in particular 1.5 g/l.

In the case of a solid composition, the concentration of 3'-sialyllactose (3'-SL) may be from 0.4 to 3.5 g/kg, preferably from 0.8 to 3.0 g/kg, more preferably from 1.0 to 2 .5 g/kg, most preferably 1.4 to 2.0 g/kg, in particular 1.48 to 1.7 g/kg.

In the case of a liquid composition, the concentration of 3'-SL may be from 50 to 400 mg/l, preferably from 100 to 350 mg/l, more preferably from 150 to 300 mg/l, most preferably from 180 to 250 mg/l, in particular from 200 to 230 mg/l.

In the case of a solid composition, the concentration of 6'-SL may be from 0.5 to 3.5 g/kg, preferably from 1.0 to 3.0 g/kg, more preferably from 1.5 to 2.5 g/kg, most preferably 2.0 to 2.3 g/kg, especially 2.07 to 2.22 g/kg.

In the case of a liquid composition, the concentration of 6'-SL may be from 100 to 500 mg/l, preferably from 150 to 450 mg/l, more preferably from 200 to 400 mg/l, most preferably from 250 to 350 mg/l, in particular from 280 to 300 mg/l.

In the case of a solid composition, the concentration of lacto-N-neotetraose (LNnT) may be from 0.2 to 3.5 g/kg, preferably from 0.4 to 1.8 g/kg, more preferably from 0.8 to 1.4 g/kg, most preferably 1.0 to 1.2 g/kg, in particular 1.11 g/kg.

In the case of a liquid composition, the concentration of LNnT may be from 10 to 350 mg/l, preferably from 20 to 300 mg/l, more preferably from 50 to 250 mg/l, most preferably from 100 to 200 mg/l, in particular 150 mg/l l.

In the case of a solid composition, the concentration of lacto-N-fucopentose I (LNFP-I) may be from 1.0 to 10.0 g/kg, preferably from 3.0 to 9.0 g/kg, more preferably from 6.0 to 8.0 g/kg, most preferably 7.0 to 7.5 g/kg, in particular 7.4 g/kg.

In the case of a liquid composition, the concentration of LNFP-I may be from 0.1 to 2.0 g/l, preferably from 0.3 to 1.5 g/l, more preferably from 0.6 to 1.2 g/l, most preferably 0.8 to 1.1 g/l, in particular 1.0 g/l.

When the composition is a premix, the above concentrations may be at least 2 times higher, preferably at least 4 times higher, more preferably at least 8 times higher, even more preferably at least 16 times higher, most preferably at least 20 times, in particular at least 22 times.

Typical food composition

The method of the invention provides the desired L-fucose with a purity sufficient to be useful in foods or feeds, in particular suitable for inclusion in foods for infants and toddlers. The resulting L-fucose can be used to prepare infant formula by mixing it with the components of the base infant formula. The infant formula may be a dry formula or a ready-to-use liquid formula for infants.

The base mixture may have at least one, possibly all, of the following components.

The use of the composition according to the invention

The proposed use of the composition according to the invention for the manufacture of food compositions and/or pharmaceutical compositions.

It is intended that on the basis of the following figures and examples, the subject matter of the invention will be explained in more detail without limiting said subject matter to the specific embodiments shown in this specification.

On FIG. 1 shows an example of the L-fucose purification process according to the invention. After fermentation, the fermentation medium is clarified by centrifugation and/or filtration. The clarified fermentation medium is treated with an ion exchanger to remove electrically charged impurities and to exchange non-specific ions for specific ions. The passing stream after treatment on the ion exchanger is then subjected to diafiltration and/or concentration. After this step, the solution containing L-fucose is subjected to electrodialysis and/or diafiltration. Then the solution containing L-fucose is subjected to additional processing on an ion exchanger to remove electrically charged colored substances and peptides. Thereafter, the solution containing L-fucose is treated with activated charcoal. The solution is then either subjected to L-fucose crystallization or subjected to an additional electrodialysis and/or diafiltration step followed by granulation and/or spray drying.

On FIG. 2 shows an example of a second L-fucose purification process according to the invention. After fermentation, the fermentation medium is clarified by centrifugation and/or filtration. The clarified fermentation medium is treated with an ion exchanger to remove electrically charged impurities and to exchange non-specific ions for specific ions. The passing stream after treatment on the ion exchanger is then subjected to diafiltration and/or concentration. After this step, the solution containing L-fucose is subjected to electrodialysis and/or diafiltration. The solution containing L-fucose is then treated with activated charcoal. After that, the solution containing L-fucose is subjected to additional processing on an ion exchanger to remove electrically charged colored substances and peptides. The solution is then either subjected to L-fucose crystallization or subjected to an additional electrodialysis and/or diafiltration step followed by granulation and/or spray drying.

On FIG. 3 shows an example of a third L-fucose purification process according to the invention. After fermentation, the fermentation medium is clarified by centrifugation and/or filtration. The clarified fermentation medium is subjected to diafiltration and/or concentration. The solution is then treated with an ion exchanger to remove electrically charged impurities and to exchange non-specific ions for specific ions. After this step, the solution containing L-fucose is subjected to electrodialysis and/or diafiltration. The solution containing L-fucose is then treated with activated charcoal. After that, the solution containing L-fucose is subjected to additional processing on an ion exchanger to remove electrically charged colored substances and peptides. The solution is then either subjected to L-fucose crystallization or subjected to an additional electrodialysis and/or diafiltration step followed by granulation and/or spray drying.

On FIG. 4 shows an example of a fourth L-fucose purification process according to the invention. After fermentation, the fermentation medium is clarified by centrifugation and/or filtration. The clarified fermentation medium is subjected to diafiltration and/or concentration. The solution is then treated with activated carbon. After completing this step, the solution containing L-fucose is treated with an ion exchanger to remove electrically charged impurities and to exchange non-specific ions for specific ions. After that, the solution containing L-fucose is subjected to electrodialysis and/or diafiltration. After that, the solution is subjected to additional processing on an ion exchanger to remove electrically charged colored substances and peptides. The solution is then either subjected to L-fucose crystallization or subjected to an additional electrodialysis and/or diafiltration step followed by granulation and/or spray drying.

On FIG. 5 shows a microscope image of L-fucose crystals formed by crystallization from an aqueous solution. It can be seen that L-fucose forms needle-like crystals with a length of 50 µm to 150 µm.

On FIG. 6 shows the HPLC diagram that was recorded for L-fucose crystallized from an aqueous solution. The purity of crystallized L-fucose was found to be 99.8%. The water content is 0.1% (w/w).

On FIG. 7 shows the HPLC chart that was recorded for L-fucose granulated from an aqueous solution. Granulated L-fucose was found to be 97.8% pure. The water content is 1.7% (w/w).

Example 1 Purification of L-fucose after bacterial fermentation

L-Fucose was formed by bacterial fermentation and was collected by filtration. The resulting clear, particle-free solution of L-fucose (34 g/l) was ^treated using a strong cation exchanger (Lewatit® S2568 in protonated form, Lanxess). After neutralization with sodium hydroxide, the solution was further treated with a strong anion exchanger ( ^Lewatit® S 6368A in chloride form).

To concentrate the L-fucose solution after treatment with an ion exchanger, the solution was further processed using a reverse osmosis step. An Emrich EMRO 1.8 reverse osmosis system (Emrich Edelstahlbau) was equipped with a ^Trisep® TS80 nanofiltration module (80-40-TS80-TSA). The inlet pressure was set to 25 bar (2.5 MPa) and the solution was concentrated until the concentration of L-fucose reached 100 g/l. Alternatively, concentration of the L-fucose solution can also be carried out by evaporation in vacuo, however, filtration techniques reduce the possibility of formation of Maillard products during the concentration process.

To remove sodium chloride from the ion exchanger step and other ions, the solution was subjected to electrodialysis using a PCCell P15 electrodialysis system (manufactured by PCCell, Heusweiler, Germany) equipped with a PCCell ED 1000A membrane package. This package contained the following membranes: cation exchange membrane CEM: PC SK and anion exchange membrane CEM: PcAcid60 having a size exclusion limit of 60 Da. The value of the electrical conductivity of the original solutions ranged from 15 to 25 mS/cm ² and the solution was subjected to electrodialysis to achieve a value of electrical conductivity of 1.5-2 mS/cm ² .

After electrodialysis, the solution was once again treated with a strong cation exchanger ( ^Lewatit® S2568 in sodium form, Lanxess) and a strong anion exchanger ( ^Lewatit® S 6368A in chloride form) to remove colored substances and possibly remaining non-specific ions (such as, for example, charged peptides).

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

After activated carbon treatment, the solution containing L-fucose was concentrated by reverse osmosis filtration using an Emrich EMRO 1.8 reverse osmosis system (Emrich Edelstahlbau) which was equipped with a CSM RE8040BE reverse osmosis module. The solution was concentrated until the flow rate dropped below 50 liters per hour. The dry matter content after concentration was 30 to 35% (w/w).

Alternatively, concentration of the L-fucose solution can also be carried out by evaporation in vacuo. This method results in higher concentrations (50 to 60% (w/w) of L-fucose. The disadvantage of concentration by vacuum evaporation is that the L-fucose will partially caramelize, as evidenced by the color change of the solution to a distinct brown color Briefly, this concentration method reduces product quality, product purity, and product yield.

Example 2 Crystallization of L-Fucose Using Organic Solvents

To carry out the crystallization of L-fucose, 15 liters of a 32% (w/w) solution was concentrated to a final dry matter concentration of 80-85% (w/w) by evaporation in vacuo using an industrial Hei-VAP evaporator ( Heidolph Instruments GmbH, Schwabach, Germany) in order to obtain a mother liquor for the crystallization of L-fucose.

To start the crystallization process, seed crystals were introduced into the mother liquor. An additional 2.5 liters of n-butanol (the ratio of butanol and fucose was 1:2) was added and the solution was concentrated under vacuum until the solution was saturated with crystals.

The crystallized mass was removed from the cap of the reservoir (piston) and incubated for at least 24 hours until a solid crystallized mass was obtained.

Solid crystals were mixed with ethanol in a ratio of 1:1 (1 liter of ethanol with 1 kg of crystallized mass). The solution was centrifuged to separate the mother liquor and ethanol from the crystals.

The crystals were washed again with ethanol (2.5 liters per 5 kg of L-fucose crystals) and centrifuged again. Solid crystals of L-fucose were removed from the centrifuge and dried at 40°C until the complete absence of ethanol. L-Fucose was sieved through a coarse sieve with a mesh diameter of 0.2 mm.

As a result, 3.3 kg of L-fucose was obtained from 5 kg of L-fucose-containing crystallizable mixture (yield: 66%).

Example 3 Crystallization of L-Fucose from Aqueous Solutions

To carry out the crystallization of L-fucose from aqueous solutions, 15 liters of a 32% (w/w) solution was concentrated to a final dry matter concentration of 80-85% (w/w) by evaporation under vacuum using an industrial Hei evaporator. -VAP (Heidolph Instruments GmbH, Schwabach, Germany).

To start the crystallization process, seed crystals were introduced into the mother liquor. The solution was concentrated in vacuo until clear crystals began to form.

The crystallized mass was removed from the lid of the reservoir (piston) and incubated for at least 72 hours until a solid crystallized mass was obtained. Solid crystals were mixed with ethanol in a ratio of 1:1 (1 liter of ethanol with 1 kg of crystallized mass).

The solution was centrifuged to separate the mother liquor and ethanol from the crystals. The crystals were washed with ethanol (2.5 liters per 5 kg L-fucose crystals) and centrifuged again. Solid crystals of L-fucose were removed from the centrifuge and dried at 40°C until the complete absence of ethanol. L-Fucose was sieved through a coarse sieve with a mesh diameter of 0.2 mm.

As a result, 3.0 kg of L-fucose was obtained from 5 kg of L-fucose-containing crystallizable mixture (yield: 60%).

Example 4 Isolation of L-Fucose by Granulation

For granulation of L-fucose, a fluid bed system from Glatt (Glatt GmbH, Germany) was used. A 40% (w/w) L-fucose solution was prepared from the L-fucose crystallizable mixture and used for granulation in a fluid bed system.

A total of 500 g of solid L-fucose was charged to the fluidized bed system, and the system was started by adding stock solution. The system constantly maintained the temperature of the product from 45°C to 50°C. After adding 300 g of L-fucose, the first agglomerates were formed. After adding 1000 g of a 40% (w/w) solution of L-fucose (400 g of L-fucose), the system was stopped and analyzed.

After granulation had been carried out and a recovery of 80% had been achieved, 720 g of L-fucose could be obtained with a bulk density of 570 g/l and a loss on drying of 0.9%. The size of the granulated L-fucose was analyzed by sieving. This test shows that 70% of a given substance has a size between 150 µm and 1400 µm.

Example 5 Purity After Typical Purification of L-Fucose

The degree of purity after a typical purification of L-fucose from the culture fluid is shown in Table 1.

Example 6 Composition of a Typical Infant Formula Product

The following is the composition of a typical infant formula product (see Table 2 below).

This composition includes L-fucose in combination with neutral HMOs present in excess, namely: 2'-fucosyllactose (2-FL), 3-fucosyllactose (3-FL), lacto-N-tetraose (LWT) and possibly lacto- N-neotetraose (LNnT) and lacto-N-fucopentaose I (LNFP-I), acid HMOs (6'-sialyllactose (6'-SL) and 3'-sialyllactose (3'-SL)) and N-acetylneuraminic acid sialic acid acid (Neu5Ac).

One or more of the probiotic strains may be present in the product. The value of the final concentration of each ingredient is based on the preparation of the composition at the rate of 13.5 g of powder in 90 ml of water.

Example 7 Composition of a typical premix for an infant formula product containing human milk oligosaccharides, the monosaccharide L-fucose and the monosaccharide N-acetylneuraminic acid (Neu5Ac)

The following is the composition of a typical premix for an infant formula product (see Table 3 below).

This composition includes the monosaccharide L-fucose in combination with the neutral HMOs present in excess, namely: lacto-N-neotetraose (LNnT) and lacto-N-fucopentaose I (LNFP-I), acidic HMOs (such as 6'-sialyllactose (6'-SL), 3'-sialyllactose (3'-SL) and sialylated derivatives LNT) and sialic acid N-acetylneuraminic acid (Neu5Ac).

This premix can be used to reconstitute infant formula by adding said premix to other foods that are needed to reconstitute infant formula, such as whey, lactose, lipids (saturated and unsaturated fatty acids) and minerals. . It is assumed that the premix shown in Table 3 will be used to prepare 1 kg of the final product as an infant formula.

Claims (36)

1. A method for purifying L-fucose from a fermentation broth, comprising the following steps: removal of biomass by centrifugation and/or filtration from the fermentation broth containing L-fucose to obtain a clarified solution, obtaining a purified solution by subjecting the clarified solution to a cation exchanger and an anion exchanger; And removal of salts from the purified solution by electrodialysis and/or nanofiltration, where the biomass contains cells that produce L-fucose. 2. The method of claim 1, wherein the cation exchanger treatment is carried out under conditions under which L-fucose passes through the cation exchange material and is present in the passing stream, and where the anion exchanger treatment is carried out under conditions under which L-fucose passes through the anion exchange material and present in the passing stream. 3. The method according to claim 1 or 2, in which 1) the biomass is removed from the fermentation broth by centrifugation and/or filtration, the filtration being preferably selected from the group consisting of microfiltration, ultrafiltration, tangential flow filtration, diafiltration and combinations thereof; and/or 2) the biomass contains bacterial cells that produce L-fucose, preferably recombinant bacterial cells, most preferably recombinant E. coli cells, recombinant Bacillus sp. and/or recombinant cells of Corynebacterium sp., in particular recombinant cells of Bacillus subtiils and/or recombinant cells of Bacillus megaterium. 4. The method according to any one of paragraphs. 1-3, wherein the cation exchange treatment uses a strong cation exchanger and/or the anion exchange treatment uses a strong anion exchanger. 5. The method according to any one of paragraphs. 1-4, where the treatment on the cation exchanger is performed to remove non-specific cations and to replace them with specific cations, preferably to replace them with a specific H ⁺ or Na ⁺ cation, while if non-specific cations are replaced with H ⁺ , then the pH value in the passing stream preferably adjusted to pH 6-8 before carrying out the subsequent processing step, most preferably by adding NaOH to the passing stream. 6. The method according to any one of paragraphs. 1-5, where the stage of treatment on an anion exchanger is carried out to remove non-specific anions and replace them with specific anions, preferably with a specific Cl ^- or OH ^- anion, while if non-specific anions are replaced with Cl ^- , then the pH value in the passing stream is preferably adjusted to pH 6-8 before carrying out the subsequent stage of processing, most preferably by adding NaOH to the passing stream. 7. The method according to any one of paragraphs. 1-6, wherein the conditions under which L-fucose passes through the anion exchange material and the cation exchange material are set by adjusting the pH and/or salt concentration of the clarified solution, preferably by adjusting the pH of the clarified solution to a pH value in the range of 6-8. 8. The method according to any one of paragraphs. 1-7, where after treatment with a cation exchanger and treatment with an anion exchanger, the purified solution contains L-fucose, coloring agents and a salt, the salt being preferably NaCl. 9. The method according to any one of paragraphs. 1-8 wherein the anion exchanger treatment uses an anion exchange material in chloride form and/or the cation exchanger treatment uses a cation exchange material in hydrogen form. 10. The method according to any one of paragraphs. 1-9, where the clarified solution is first treated with a cation exchanger and then treated with an anion exchanger. 11. The method according to any one of paragraphs. 1-10, where the purified solution is concentrated, preferably by nanofiltration and/or reverse osmosis, more preferably by nanofiltration, most preferably using a nanofiltration membrane that has a molecular weight cut-off in the range of 100-200 kDa. 12. The method according to any one of paragraphs. 1-11 where the clarified solution and/or the purified solution is concentrated 1) until the L-fucose concentration is greater than or equal to 100 g/l, preferably greater than or equal to 200 g/l, more preferably greater than or equal to 300 g/l; and/or 2) by nanofiltration below 80°C, preferably below 50°C, more preferably 4 to 45°C, more preferably 10 to 40°C, even more preferably 15 to 30°C, most preferably 15 to 20°C; and/or 3) by reverse osmosis at 20 to 50°C, more preferably 30 to 45°C, most preferably 35 to 45°C; and/or 4) by nanofiltration at a pressure in the range from above 5 bar (0.5 MPa) to below 50 bar (5 MPa), preferably at a pressure from above 10 bar (1 MPa) to below 40 bar (4 MPa), more preferably at pressure from above 15 bar (1.5 MPa) to below 30 bar (3 MPa); and/or 5) by reverse osmosis at a pressure in the range from above 5 bar (0.5 MPa) to below 100 bar (10 MPa), preferably at a pressure from above 10 bar (1 MPa) to below 80 bar (8 MPa), more preferably at pressures from above 15 bar (1.5 MPa) to below 70 bar (7 MPa). 13. The method according to any one of paragraphs. 1-12, where the electrodialysis is electrodialysis under neutral conditions or electrodialysis under acidic conditions. 14. The method according to any one of paragraphs. 1-13, where after removal of salts from the purified solution 1) the amount of salt in the purified solution is less than 10% (w/w), preferably less than 5% (w/w), more preferably 1% (w/w) or less, even more preferably 0.5% (w/w) or less, most preferably 0.4% (w/w) or less, especially 0.2% (w/w) or less; and/or 2) the conductivity value is in the range of 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 . 15. The method according to any one of paragraphs. 1-14, where the clarified solution and/or the purified solution is subjected to a decolorization step, preferably by treatment with activated carbon and/or treatment using a cation exchanger and an anion exchanger, which are connected in series, and said removal may be carried out 1) before or after the stage of diafiltration and/or concentration of the clarified solution; and/or 2) before or after the stage of electrodialysis and/or diafiltration of the clarified solution. 16. The method according to any one of paragraphs. 1-15, where the purified solution is subjected to 1) granulation; 2) spray drying; 3) drying on a roller dryer; or 4) lyophilization. 17. The method according to any one of paragraphs. 1-16 where the purified solution is subjected to crystallization, possibly by adding butanol to the purified solution or without adding any organic solvent to the purified solution.

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