Method for obtaining crystalline 2′-fucosyllactose

Abstract

The present invention relates to a method for obtaining crystalline 2'-fucosyllactose from a 2'-FL starting material, the 2'-FL starting material containing 2'-FL as the main component and at least 0.5% by weight, typically at least 1% by weight, particularly at least 2% by weight, more particularly at least 5% by weight, and especially at least 8% by weight of one or more monosaccharides or oligosaccharides different from 2'-FL, based on the total amount of monosaccharides and oligosaccharides in the starting material, wherein the method comprises: a) providing an aqueous solution of the 2'-FL starting material, the solution comprising not more than 10% by weight, preferably not more than 7% by weight, more preferably not more than 5% by weight, of an organic solvent, based on the total amount of water; b) achieving crystallization of 2'-FL from the solution provided in step a) by inducing controlled supersaturation conditions in the solution; and c) separating crystalline 2'-FL from the mother liquor, wherein during the controlled supersaturation in step b), not more than 10% by weight, preferably not more than 7% by weight, more preferably not more than 5% by weight, of an organic solvent, based on the total amount of water present during step b) is present.

Description

[0001] The present invention relates to a method for obtaining crystalline 2'-fucosyllactose from a 2'-fucosyllactose raw material, and particularly to a method for selectively obtaining the hydrate form A or the anhydrate form II of 2'-fucosyllactose. Background technology:

[0002] 2′-Fucosyllactose (CAS No. 41263-94-9: α-L-fucopyranosyl-(1→2)-O-β-D-galactopyranosyl-(1→4)-D-glucopyranose, hereinafter referred to as 2′-FL) is an oligosaccharide found in relatively large amounts in breast milk. It has been reported in many sources that the presence of 2′-FL in breast milk causally reduces the risk of infection in breastfed newborns (see, for example, Weichert et al., Nutrition Research, 33 (2013), Volume 10, 831-838; Jantscher-Krenn et al., Minerva Pediatr. 2012, 64 (1) 83-99; Morrow et al., J. Pediatr. 145 (2004) 297-303). Therefore, 2′-FL is of particular interest as a component of food supplements, in particular as an additive to humanized milk products, especially infant nutrition.

[0003] The preparation of 2′-O-fucosyllactose by classical chemical or biochemical means has been described variously in the literature (for classical chemical means, see for example US 5,438,124, WO 2010 / 070616, WO 2010 / 115934, WO 2010 / 115935, WO 2016 / 038192 and WO 2017 / 153452; for biochemical means, see for example Drouillard et al. Angew. Chem. Int. Ed. 45, 1778 (2006), WO 2010 / 070104, WO 2012 / 007481, WO 2012 / 097950, WO 2012 / 112777, WO 2013 / 139344, WO 2014 / 086373, WO 2015 / 188834 and WO 2016 / 095924).

[0004] In principle, the production of 2'-FL by fermentation processes using transformed microorganisms such as transformed Escherichia coli (E. coli) is promising for economic and environmental reasons. However, the isolation of 2'-FL is tedious and usually requires:

[0005] - separating the supernatant containing the product by centrifugation,

[0006] - adsorption of the product onto an activated carbon bed, which is washed with water to remove water-soluble contaminants such as salts, amino acids and protein fragments,

[0007] - eluting the product with alcohol or aqueous alcohol, and

[0008] - Last but not least, 2'-FL is separated from other carbohydrates such as lactose and fucose by gel permeation chromatography on a bed of charcoal diatomaceous earth or by flash chromatography.

[0009] The main disadvantage of this separation method is that a chromatographic separation is required in order to obtain a pure substance or at least a mixture enriched in the target compound but still containing undesirable derivatives. Although repeated chromatographic separations can lead to an increase in purity, the high costs and relatively long technical times for processing the feed solution and the column packing for separation and optionally regenerating the packing (especially on a large or industrial scale) can be disadvantageous and / or cumbersome.

[0010] Crystallization or recrystallization is, in principle, a simple and inexpensive method for isolating a product from a reaction mixture and separating it from contaminants to obtain a purified substance. Therefore, separation or purification using crystallization can make the entire technical process robust and cost-effective, and is therefore, in principle, advantageous and attractive compared to other operations. Although crystallization of 2′-FL prepared by classical organic synthesis is an effective means of isolating or purifying 2′-FL, crystallization methods cannot be easily applied to 2′-FL prepared by non-classical organic synthesis because the products obtained by fermentative production of 2′-FL contain a large number of by-products, in particular, oligosaccharides other than 2′-FL, but also monosaccharides. Since these monosaccharides and oligosaccharides have considerable polarity and therefore considerable solubility, they are difficult to separate by crystallization processes.

[0011] Kuhn et al. (Chem. Ber. 1956, p. 2513) reported that 2′-FL purified by repeated chromatography did not readily crystallize, remaining in a syrupy state. The authors noted that when aqueous solutions of 2′-FL were allowed to stand for extended periods, virtually no 2′-FL crystals formed. Crystalline 2′-FL could only be obtained in large quantities from solutions containing mixtures of water and large amounts of organic solvents.

[0012] WO 2014 / 086373 describes a method for obtaining oligosaccharides such as 2′-FL from a fermentation broth, which comprises freeze-drying the fermentation broth (preferably after removing proteins therefrom) to produce a dry powder, treating the dry powder with an aliphatic alcohol (such as methanol) to dissolve the oligosaccharide, and then crystallizing the oligosaccharide from the alcoholic solution. This method is cumbersome because it requires prior freeze-drying of the fermentation broth and the use of an organic solvent.

[0013] WO 2015 / 188834 describes a method for crystallizing 2′-FL from an aqueous solution containing 2′-FL and fucosylated oligosaccharides (such as difucosyllactose), which comprises fermenting 2′-FL using genetically modified cells having a recombinant gene encoding 1,2-fucosyltransferase, separating the supernatant from non-carbohydrate solids and contaminants, and adding C1-C4-alkanol to achieve crystallization of 2′-FL.

[0014] WO 2016 / 095924 describes a method for crystallizing 2′-FL from an aqueous solution containing 2′-FL and fucosylated oligosaccharides (such as difucosyllactose), which method comprises providing an aqueous solution of 2′-FL and fucosylated oligosaccharides as described in WO 2015 / 188834, and adding acetic acid to the aqueous solution to achieve crystallization of 2′-FL.

[0015] WO 2014 / 009921 describes different polymorphic forms of 2'-FL. Although polymorph B can be obtained by recrystallizing pure 2'-FL (e.g., pure polymorph A) from water, it does not describe the purification of 2'-FL-containing starting materials (which also contain a large amount of mono- or oligosaccharides other than 2'-FL).

[0016] WO 2018 / 164937 describes a method for crystallizing 2′-FL from aqueous solution, which requires precipitating 2′-FL from a supersaturated aqueous solution at a temperature above 60° C. By this method, a crystalline anhydrate of 2-FL, Form II of 2′-FL, is obtained, which is described in WO 2011 / 150939.

[0017] Methods for obtaining crystals still require large amounts of organic solvents during crystallization, which are often difficult to remove because they are often trapped in the crystalline material. Especially for infants, the use of organic solvents is unacceptable because it always carries the risk of not being able to completely remove the organic solvents. Summary of the invention:

[0019] There remains a need for an efficient method for obtaining crystalline 2′-FL from a 2′-FL starting material containing a large amount of monosaccharides and oligosaccharides other than 2′-FL, such as lactose, acetylated 2′-FL, fucosylated lactose other than 2′-FL, and fucosylated lactulose, from an aqueous solution of such starting material, in particular from an aqueous solution obtained by a fermentation process. Such a method would particularly allow the avoidance of organic solvents and provide 2′-FL in high purity and yield.

[0020] It has been discovered that 2′-FL can be efficiently and reliably crystallized from aqueous solutions of 2′-FL starting materials containing significant amounts of monosaccharides and oligosaccharides other than 2′-FL by inducing controlled supersaturation conditions in the aqueous solution, thereby achieving selective crystallization of 2′-FL. Inducing controlled supersaturation conditions in the aqueous solution of the 2′-FL starting material enables efficient crystallization without the use of significant amounts of organic solvents during crystallization. This is particularly surprising, as 2′-FL is highly soluble in water, even pure 2′-FL is difficult to crystallize from water, and the significant amounts of monosaccharides and oligosaccharides contained in the 2′-FL starting material should further hinder 2′-FL crystallization.

[0021] Therefore, the present invention relates to a method for obtaining crystalline 2′-fucosyllactose from a 2′-FL raw material containing 2′-FL as a main component and at least 0.5% by weight, typically at least 1% by weight, particularly at least 2% by weight, more particularly at least 5% by weight and especially at least 8% by weight of one or more monosaccharides or oligosaccharides different from 2′-FL, based on the total amount of monosaccharides and oligosaccharides in the raw material, the method comprising:

[0022] a) providing an aqueous solution of the 2′-FL raw material, wherein the solution comprises no more than 10 wt %, preferably no more than 7 wt %, more preferably no more than 5 wt % of an organic solvent based on the total amount of water;

[0023] b) crystallizing 2'-FL from the solution provided in step a) by inducing controlled supersaturation conditions in said solution at a temperature preferably of at most 60°C; and

[0024] c) separating crystalline 2′-FL from the mother liquor,

[0025] And wherein during the controlled supersaturation in step b), no more than 10 wt.-%, preferably no more than 7 wt.-%, more preferably no more than 5 wt.-% of organic solvent is present, based on the total amount of water present during step b).

[0026] The method of the present invention is associated with several advantages. It enables efficient separation of 2′-FL from other oligosaccharides, resulting in high yields and a high purity (based on the organic matter in the crystalline 2′-FL), typically greater than 93%, particularly greater than 95%, and especially at least 97% or at least 98%). In particular, the method does not require the use of organic solvents during crystallization, thereby minimizing the risk of crystalline 2′-FL containing large amounts of entrapped organic solvents. The process yields a mother liquor that is colorless or nearly colorless and can therefore be subjected to further crystallization steps or reintroduced into solution for crystallization prior to achieving crystallization.

[0027] By the process of the present invention, pure crystalline 2'-FL is obtained in the form of dense crystals.

[0028] Surprisingly, this method enables the selective crystallization of the three polymorphic forms of 2′-FL in a reliable manner, namely

[0029] anhydrate form II, which is described in WO 2011 / 150939 and can be identified, for example, by its characteristic reflections in the X-ray powder diffraction pattern, in particular the following reflections expressed in 2θ values: 16.98±0.2°, 13.65±0.2° and 18.32±0.2° (at 25°C and Cu-Kα radiation);

[0030] - hydrate form A, which is described in WO 2014 / 009921 and can be identified, for example, by its characteristic reflections in the X-ray powder diffraction pattern, in particular the following reflections expressed in 2θ values: 18.86±0.2°, 17.05±0.2° and 9.89±0.2° (at 25°C and Cu-Kα radiation); or

[0031] -hydrate form B, which is described in WO 2014 / 009921 and can be identified, for example, by its characteristic reflections in the X-ray powder diffraction pattern, in particular the following reflections expressed in 2θ values: 20.48±0.2°, 11.90±0.2° and 9.96±0.2° (at 25°C and Cu-Kα radiation).

[0032] This is particularly important for the registration of 2'-FL, which may require the reliable production of a specific polymorphic form. The method enables the selective preparation of crystalline anhydrate Form II or crystalline hydrate Forms A or B of 2'-FL, depending on the temperature at which crystallization of 2'-FL is achieved. In particular, if 2'-FL is crystallized at a temperature of at most 52°C, in particular at most 50°C, more particularly at most 48°C, and in particular at most 45°C, for example at a temperature of 0 to 52°C, in particular at 0 to 50°C, more particularly at 0 to 48°C, and in particular at 0 to 45°C, hydrate Forms A or B are obtained, while if 2'-FL is crystallized at a temperature above 52°C, in particular at a temperature above 53°C, anhydrate Form II is obtained. It should be noted that when crystallization is achieved at a temperature of at most 52°C, in particular at most 50°C, more particularly at most 48°C, and in particular at most 45°C, crystalline hydrate Form B is initially formed. However, crystalline hydrate Form B converts to crystalline hydrate Form A upon drying.

[0033] Therefore, the present invention also relates to a method for selectively obtaining crystalline hydrate form A or B of 2′-FL or crystalline anhydrate form II of 2′-FL from a 2′-FL starting material as defined herein, which method comprises carrying out the method for obtaining crystalline 2′-FL as described herein, provided that:

[0034] - crystallization of 2′-fucosyllactose is carried out at a temperature of 0° C. to 52° C., particularly 0° C. to 50° C., more particularly 0° C. to 48° C., especially 0° C. to 45° C. to obtain crystalline form A or form B of 2′-fucosyllactose;

[0035] - Alternatively, the crystallization of 2'-fucosyllactose is effected at a temperature above 52°C, in particular at least or above 53°C, preferably at most 60°C to obtain form II of 2'-fucosyllactose. Detailed Description of the Invention

[0037] Here and hereinafter, the terms 2′-FL and 2′-fucosyllactose are used synonymously and refer to α-L-fucopyranosyl-(1→2)-O-β-D-galactopyranosyl-(1→4)-D-glucopyranose, including the α- and β-anomers and mixtures thereof.

[0038] As used herein, the term "2'-FL starting material" refers to an oligosaccharide composition containing 2'-FL as the main component, in particular in an amount of at least 70 wt.-%, and a considerable amount, i.e., at least 0.5 wt.-%, typically at least 1 wt.-%, particularly at least 2 wt.-%, more particularly at least 5 wt.-%, and especially at least 8 wt.-%, of one or more monosaccharides or oligosaccharides other than 2'-fucosyllactose, based on the total amount of monosaccharides and oligosaccharides in the starting material. In particular, the 2'-FL starting material from which crystalline 2'-FL is obtained by the process of the present invention comprises:

[0039] - 70 to 98% by weight, in particular 75 to 95% by weight, especially 78 to 92% by weight, of 2′-FL, based on the total amount of monosaccharides and oligosaccharides in the starting material, and

[0040] 2 to 30% by weight, in particular 5 to 25% by weight, especially 8 to 22% by weight, of one or more monosaccharides and oligosaccharides other than 2′-fucosyllactose, based on the total amount of monosaccharides and oligosaccharides in the starting material.

[0041] Typical monosaccharides and oligosaccharides other than 2′-fucosyllactose contained in the 2′-FL raw material include, but are not limited to, lactose, fucosylated lactose other than 2′-FL, fucose, galactose, glucose, lactulose, and fucosylated lactulose. These monosaccharides and oligosaccharides are hereinafter referred to as “carbohydrate impurities or by-products.”

[0042] As used herein, the term "fucosylated lactose other than 2'-FL" includes any monofucosylated lactose other than 2'-FL. The term "fucosylated lactose other than 2'-FL" also includes any polyfucosylated lactose, in particular difucosylated lactose also known as "difucosyllactose", such as 2,2'-O-difucosyllactose or 2',3-O-difucosyllactose.

[0043] Likewise, the term "fucosylated lactulose" as used herein includes any monofucosylated lactulose and polyfucosylated lactulose, i.e. lactulose fucosylated with one or more, e.g. one or two, fucose moieties on the galactose moiety of lactulose.

[0044] The aforementioned carbohydrate impurities or byproducts may be formed during fermentation or under post-fermentation conditions. For example, fucosylated lactose other than 2'-FL may be formed due to insufficient, defective, or impaired fucosylation other than α-1,2-fucosylation on the galactose moiety of lactose, or due to fucose migration from 2'-FL under fermentation or post-fermentation conditions, or due to fucose hydrolysis from polyfucosylated lactose. Other carbohydrate impurities or byproducts may be formed by rearrangement, such as lactulose and fucosylated lactulose, or by hydrolysis, such as fucose, glucose, galactose, and lactose, or may be unconsumed raw materials, such as glucose or lactose.

[0045] In particular, the 2′-FL raw material comprises at least one fucosylated lactose other than 2′-FL, in particular difucosyllactose. In particular, the amount of fucosylated lactose is 0.3 to 10% by weight, in particular 0.5 to 10% by weight, and especially 1 to 10% by weight, based on the weight of the monosaccharides and oligosaccharides contained in the 2′-FL raw material. In particular, the 2′-FL raw material further comprises at least one of lactulose and fucosylated lactulose, or a mixture of both. In particular, the total amount of lactulose and fucosylated lactose is 0.2 to 10% by weight, in particular 0.5 to 10% by weight, and especially 1 to 10% by weight, based on the weight of the monosaccharides and oligosaccharides contained in the 2′-FL raw material.

[0046] In the first step a) of the process according to the present invention, an aqueous solution of the 2′-FL starting material is provided, which is then crystallized in the second step b) under controlled supersaturation conditions. In principle, any aqueous solution of the 2′-FL starting material can be used in the process according to the present invention, which solution contains no more than 10% by weight, preferably no more than 7% by weight, and more preferably no more than 5% by weight, of an organic solvent, based on the amount of water present therein.

[0047] It is essential to the present invention that the aqueous solution of the 2′-FL starting material provided in step a) and crystallized in step b) and the water present during step b) do not contain significant amounts of organic solvents. According to the present invention, the concentration of organic solvents in the solution provided in step a) does not exceed 5% by weight, particularly 2% by weight, and especially 1% by weight, based on the water present in the solution provided in step a). Furthermore, the concentration of organic solvents in the water present during step b) does not exceed 10% by weight, preferably 7% by weight, more preferably 5% by weight, particularly 2% by weight, and especially 1% by weight, based on the water present in step b). In this context, the term "organic solvent" encompasses any organic compound having a boiling point of 30 to 250° C. at atmospheric pressure, and includes, for example, organic alcohols, particularly C1-C4-alkanols and C1-C4-alkanoic acids, as well as any other organic compound commonly used in organic chemistry, particularly in carbohydrate chemistry.

[0048] The aqueous solution may be one obtained by a biochemical method or by a conventional method (ie, a chemical method).

[0049] The aqueous solution of 2′FL starting material used in the process of the present invention is preferably obtained by a biochemical process, such as a process in which 2′-FL is obtained by enzymatic biocatalytic fucosylation of lactose or by fermentation, as described, for example, in Drouillard et al. Angew. Chem. Int. Ed. 45, 1778 (2006), WO 2010 / 070104, WO 2012 / 007481, WO 2012 / 097950, WO 2012 / 112777, WO 2013 / 139344, WO 2014 / 086373, WO 2015 / 188834 and WO 2016 / 095924.

[0050] The fermentation broth typically contains at least 25 g / L of 2′-FL in the supernatant of the culture medium and may contain up to 120 g / L or even more than 120 g / L of 2′-FL. In addition, the supernatant may also contain DFL, typically in an amount of about 1.5 to 20% by weight relative to 2′-FL. The 2′-FL / DFL mixture optionally contains fucosylated lactulose produced in the culture medium and / or lactose or other monosaccharides or oligosaccharides as an acceptor for unconsumed lactulose.

[0051] If the aqueous solution of the starting material used in the process according to the invention is obtained by a biochemical process, in particular by fermentation, the aqueous solution obtained is usually worked up before crystallization.

[0052] Such post-treatment may include a conventional demineralization step, during which minerals, salts and other charged molecules are extracted from the aqueous solution prior to crystallization. Desalination can be performed using conventional ion exchange resins, i.e., by passing the aqueous solution through H + The cation exchange resin is preferably a strong exchanger and the anion exchange resin is preferably a weak exchanger. In addition to removing salts and charged molecules from the solution, the ion exchange resin can also physically adsorb proteins, DNA, and coloring / caramel bodies that are optionally left in the solution after the previous purification step. Alternatively, desalination can be performed by conventional electrodialysis. In addition, an adsorbent such as activated carbon can be optionally used to remove colored compounds from the aqueous solution.

[0053] In some cases, it may be desirable to selectively remove certain components of the aqueous solution of 2'-FL before crystallization. This can be achieved using different types of chromatography, such as elution chromatography with or without a recycle loop or with a continuous chromatography process, such as simulated moving bed chromatography (SMB), including variants thereof with asynchronous switching of the inlet and outlet and / or variations in the flow rate and / or feed concentration during the switching interval. For example, methods for purifying aqueous solutions of oligosaccharides (such as 2'-FL) obtained by fermentation using SMB have been described by T. Eiwegger et al., Pediatric Research, Vol. 56 (2004), pp. 536-540, CN 102676604 and EP 2857410, which can be used by analogy to remove certain components of the aqueous solution of 2'-FL before crystallization. A review of suitable methods for performing SMB can be found in M. Ottens et al., “Advances in process chromatography and application”, Chapter 4.4.3, pp. 132–135, Woodhead Publishing Limited 2010, and references cited therein.

[0054] The solution obtained by any of the above methods can then be concentrated by conventional evaporation steps or conventional nanofiltration steps (including ultrafiltration and diafiltration). Similarly, microfiltration can be incorporated to remove proteins and macromolecules. A further final ("sterile") filtration can be included before crystallization to remove microbial contaminants.

[0055] It has been found to be advantageous if the aqueous solution of the 2′-FL starting material provided in step a) is substantially free of water-insoluble solid matter (i.e., the amount of water-insoluble matter is less than 5000 ppm, particularly less than 1000 ppm, based on the 2′-FL contained therein, or at most 3000 ppm, particularly at most 1000 ppm, based on the weight of the aqueous solution of the 2′-FL starting material). Therefore, post-treatment will preferably include a conventional clarification step. By this clarification step, post-fermentation cell fragments (debris) and proteins are removed. Clarification is preferably performed before the carbon treatment described below. Clarification can be performed in a conventional manner, for example by centrifugal sedimentation to produce a clear or partially clear supernatant. Alternatively, the fermentation broth can be subjected to a filtration step, such as microfiltration or ultrafiltration, before being subjected to the crystallization in step b). For example, ultrafiltration is performed in a conventional manner to remove high molecular weight components. The semipermeable membrane used for ultrafiltration of the 2′-FL fermentation broth can suitably have a cut-off value of 5-50 kDa, preferably 10-25 kDa, and more preferably about 15 kDa. Depending on the characteristics of the fermentation broth to be clarified, a combination of higher and lower cut-off membranes (in this order) within the ranges given above can be used. Optionally, centrifugation or ultrafiltration can be followed by nanofiltration, during which the aqueous solution containing 2′-FL and carbohydrate byproducts is concentrated in a conventional manner before treatment with charcoal. In this nanofiltration step, the membrane can have a pore size that ensures retention of 2′-FL with a molecular weight of 488; therefore, a cut-off membrane of 200-300 Da can typically be used.

[0056] In addition, post-treatment may further include conventional carbon treatment, preferably prior to the desalting step, to remove color bodies and any residual water-soluble bio-junk from previous purification steps. Carbohydrate compounds have a strong affinity for adsorbing on carbon in aqueous media; therefore, water-soluble contaminants can be easily washed away with water (distilled, preferably food grade). The carbohydrates can then be eluted from the carbon bed with alcohol or aqueous alcohol.

[0057] In step b), crystallization of 2'-FL is achieved by inducing controlled supersaturation conditions in the solution of the 2'-FL starting material.

[0058] Depending on the temperature of the aqueous solution, the concentration of 2′-FL in the aqueous solution subjected to crystallization in step b) may typically be 400 to 750 g / L or 500 to 750 g / L, in particular 500 to 700 g / L. Typically, the total concentration of carbohydrates (i.e., 2′-FL and monosaccharides and oligosaccharides other than 2′-FL) ranges from 510 to 950 g / L, in particular 510 to 850 g / L.

[0059] Typically, a dilute solution having a 2'-FL concentration of at most 500 g / L, in particular at most 450 g / L, especially at most 400 g / L, e.g. 25 to 450 g / L or 50 to 400 g / L, is provided in step a) and is then subjected to a concentration step, e.g. by evaporation of water to a concentration of 2'-FL at which crystallization can occur, in particular in the range of 400 to 750 g / L or 500 to 750 g / L, in particular 420 to 720 g / L or 510 to 720 g / L.

[0060] The dilute solution can be concentrated to the concentration range required for crystallization and crystallized in a single step (i.e., in a crystallization device). It is also possible to first perform a pre-concentration step in which water is removed by evaporation until a concentration of 2'-FL is reached that is still lower than the solubility of 2'-FL under equilibrium conditions. The solution is then introduced into the crystallization device and controlled supersaturation conditions are induced in the solution thus concentrated. The concentration of 2'-FL corresponding to the solubility under equilibrium conditions is also referred to as the equilibrium concentration or equilibrium solubility c* under given conditions. As described above, the 2'-FL concentration range in the solution in which controlled supersaturation conditions are induced is typically 400 to 750 g / L, in particular 410 to 700 g / L or 410 to 650 g / L.

[0061] For the purposes of the present invention, it has been found to be advantageous if the concentration of 2′-FL in the aqueous solution of the 2′-FL starting material subjected to crystallization in step b) does not exceed 650 g / L, in particular 630 g / L, for example 400 to 650 g / L or 500 to 650 g / L, in particular 410 to 630 g / L or 500 to 630 g / L, especially 510 to 630 g / L.

[0062] However, it is also possible to carry out the crystallization in step b) on an aqueous solution of the 2'-FL starting material having a concentration of 2'-FL higher than 630 g / L, in particular higher than 650 g / L.

[0063] Inducing controlled supersaturation conditions under given conditions ensures that the desired polymorph can be selectively crystallized from an aqueous solution of the 2'-FL starting material.

[0064] Controlled supersaturation means that during crystallization, the supersaturation does not exceed a value at which uncontrolled (i.e., spontaneous) crystallization occurs. Supersaturation is understood to be the ratio of the actual concentration c of 2′-FL dissolved during crystallization to the equilibrium solubility c* of 2′-FL in water under given conditions, i.e., the ratio c:c*. In particular, the ratio c:c* does not exceed a value of 1.5:1, in particular a value of 1.3:1, more particularly a value of 1.2:1, and especially a value of 1.15:1. Obviously, supersaturation requires that the ratio c:c* exceeds the thermodynamic equilibrium state (i.e., the state where the ratio c:c* is 1), i.e., c:c* has a value greater than 1:1. A value greater than 1:1 refers, for example, to values of 1.00001:1, 1.0005:1, 1.0001:1, 1.0005:1, 1.001:1, or 1.0002:1, in particular values of 1.00001 to 1.002:1. The equilibrium concentration, c*, of 2′-FL in water at a given temperature or pressure is known or can be determined by routine experimentation. The actual concentration of 2′-FL dissolved in water can be calculated using the concentration of 2′-FL in the aqueous solution, the amount of 2′-FL fed to the crystallization apparatus, the amount of water removed, and the amount of 2′-FL crystallized. The actual concentration of a solution or suspension can also be determined experimentally, for example, by ATR-FTIR (attenuated total reflectance Fourier transform infrared spectroscopy) or by density measurement.

[0065] The concentration of dissolved 2′-FL, and thus the degree of supersaturation, is typically adjusted by removing water from the aqueous solution of the 2′-FL starting material (i.e., by increasing the concentration of 2′-FL under the crystallization conditions), and / or by cooling (i.e., by reducing the solubility of 2′-FL under the crystallization conditions), and in particular by evaporation, or by a combination of both.

[0066] To achieve or maintain supersaturated conditions, water is preferably removed by evaporation. In particular, supersaturated conditions are induced and maintained by evaporation of water or by combined evaporation / cooling. In other words, crystallization is preferably performed as evaporative crystallization, i.e., the concentration of 2′-FL in the reaction vessel is increased by evaporation of water under crystallization conditions. This can of course be accompanied by cooling, or, after evaporation of water, the initially obtained aqueous suspension of crystalline 2′-FL is cooled to increase the yield of crystallized 2′-FL.

[0067] Preferably, the water is removed by evaporation under reduced pressure. Preferably, the water is evaporated at a pressure of 10 to 900 mbar, in particular at a pressure of 50 to 800 mbar.

[0068] Preferably, the evaporation is carried out at a temperature of at least 20°C, particularly at least 25°C, more particularly at least 30°C, and especially at least 35°C. Typically, the temperature will not exceed 105°C, particularly not more than 100°C or 95°C. In particular, the evaporation temperature will not exceed 62°C or 60°C. The temperature at which the evaporation is carried out will also depend on the type of polymorph produced. If it is desired to obtain polymorph A or polymorph B, the aqueous solution is typically concentrated at a temperature of 20 to below 52°C, particularly 25 to 50°C, more particularly 30 to 48°C, and especially 35 to 45°C, while to obtain the anhydrate form II, the aqueous solution of the 2′-FL starting material is typically concentrated at a temperature of above 52 to 105°C, typically 52 to 100°C or 52 to 95°C, particularly 52 to 65°C or 52 to 60°C.

[0069] The evaporation of water can be achieved by conventional means using any equipment that enables the removal of water by distillation. The type of equipment will depend in a known manner on whether the water is to be removed during preconcentration or in order to induce conditions of controlled supersaturation, and whether the crystallization is to be carried out discontinuously (i.e. batchwise or semi-batchwise) or continuously.

[0070] To induce supersaturated conditions by evaporating water during crystallization in batch or semi-batch operation, a simple vessel can be used, wherein the necessary heat is transferred by heating means (e.g., by a double jacket, by a heating element in the vessel, by an external pumping circuit with a heat exchanger, or by a combination of these means). If the crystallization is carried out in a continuous manner, supersaturated conditions are induced by evaporating water using a continuously operated crystallization apparatus, such as a stirred tank vessel, a stirred tank vessel with a guide tube, a forced circulation crystallizer (FC), a draft tube baffle crystallizer (DTB), or an Oslo crystallizer. The evaporator can be heated with a conventional heating medium, such as heating oil or heating steam, including steam from a steam network or steam provided by steam recompression in the process of the present invention.

[0071] The evaporation of water in the pre-concentration step can be accomplished by conventional means using any equipment capable of removing water by distillation, such as stirred tank vessels, thin-film evaporators, falling-film evaporators, and spiral tube evaporators. Preferably, the evaporation of water in the pre-concentration step is accomplished with the aid of a falling-film evaporator, preferably using heated steam obtained through mechanical vapor recompression. Mechanical vapor recompression reduces the amount of fresh steam required, thereby lowering overall costs. Vapor recompression is preferably accomplished using one or more rotary compressors. Due to the moderate compression stroke of vapor recompression and the resulting limited temperature rise in the heating section, falling-film evaporators are preferred because they can operate with small temperature gradients. Falling-film evaporators enable high evaporation rates at low circulation rates and low pressure drops. Consequently, falling-film evaporators enable short residence times for temperature-sensitive 2′-FL. Furthermore, the low pressure drop of a falling-film evaporator facilitates vapor recompression and, therefore, heat recovery. Connecting several evaporators in series is advantageous because it allows for a high temperature difference between the heating and process sides, thereby allowing for a small surface area in the heat exchanger.

[0072] The amount of water removed is generally selected so that, at least at the beginning of crystallization, the concentration of dissolved 2'-FL in the aqueous medium present during crystallization is within the ranges given above and, depending on the temperature during crystallization, can therefore vary from 400 to 750 g / L or 500 to 750 g / L, in particular from 410 to 720 g / L or 510 to 720 g / L. As mentioned above, a concentration of dissolved 2'-FL in the aqueous medium present during crystallization of up to 650 g / L, in particular up to 630 g / L, for example from 400 to 650 g / L, especially from 410 to 630 g / L, can be advantageous. However, a 2'-FL concentration in the aqueous medium present during crystallization of greater than 630 g / L, in particular greater than 650 g / L, is also possible. It is also obvious that, in a continuously operated crystallization, the concentration of dissolved 2'-FL in the water present during crystallization remains within the ranges given herein throughout the crystallization.

[0073] To achieve crystallization, controlled supersaturation is generally induced at a temperature of at least 0° C., in particular at least 10° C. or at least 20° C. The temperature at which controlled supersaturation is induced will generally not exceed 105° C., in particular not exceed 100° C., more in particular not exceed 95° C. or 90° C., especially not exceed 85° C. To avoid discoloration, the temperature is preferably not more than 70° C., in particular 65° C. or 60° C., and in particular below 60° C.

[0074] If controlled supersaturation is induced by a process involving evaporation of water (hereinafter referred to as evaporative crystallization), the temperature at which supersaturation is induced is generally at least 20°C, particularly at least 25°C, especially at least 30°C or at least 35°C. Typically, the temperature will not exceed 105°C, particularly not exceed 100°C, more particularly not exceed 95°C or 90°C, and especially not exceed 85°C. To avoid discoloration of the mother liquor, the crystallization temperature is preferably not more than 70°C, particularly 65°C or 60°C, and especially below 60°C. In particular, supersaturation is induced at a temperature of 0 to 95°C or 0 to 60°C, more particularly 0 to 90°C or 0 to below 60°C, and especially 0 to 85°C or 0 to 58°C. If controlled supersaturation is induced by evaporative crystallization, supersaturation is preferably induced at a temperature of 25 to 95°C or 25 to 60°C, more preferably 30 to 90°C or 30 to below 60°C, and especially 35 to 85°C or 35 to 58°C.

[0075] If the polymorph B or A, respectively, is to be prepared by evaporative crystallization, supersaturation is generally induced at a temperature of 20 to 52° C., in particular 25 to 50° C., more particularly 30 to 48° C., especially 35 to 45° C. For the preparation of the anhydrate form II, supersaturation is generally induced at a temperature of above 52 to 105° C., in particular 52 to 100° C., especially 52 to 95° C. or 52 to 90° C. or 52 to 85° C. or 52 to 60° C. or 52 to below 60° C. or 52 to 58° C.

[0076] If controlled supersaturation is induced by a method that does not involve evaporation of water, for example in the case of a temperature at which supersaturation is induced by cooling, the temperature may be below the range given above and may be as low as 0° C. In this case, the temperature at which crystallization is induced is generally from 0 to 60° C., in particular from 0 to below 60° C., or from 0 to 58° C. The temperature will of course depend on the desired polymorphic form of 2′-FL.

[0077] Crystallization of 2'-FL is typically carried out at ambient pressure or under reduced pressure, for example, at a pressure of 10 to 1020 mbar. The pressure will, of course, depend on the temperature and concentration of the aqueous solution of the 2'-FL starting material. It may be advantageous to carry out crystallization of 2'-FL under reduced pressure to facilitate the removal of water by evaporation during crystallization. Crystallization of 2'-FL is preferably carried out at a pressure range of 10 to 900 mbar, particularly 20 to 800 mbar, and especially 30 to 700 mbar.

[0078] During crystallization, the temperature may be further reduced and / or water may be further evaporated to drive the crystallization to completion, particularly if the crystallization is performed in a batch or semi-batch operation. Of course, if the crystallization of 2-FL' is performed continuously, the temperature will be within the above range.

[0079] To achieve controlled supersaturation, measures are taken to favor crystallization and prevent kinetic inhibition of crystallization and, therefore, excessive supersaturation. Such measures are, in particular, to carry out the crystallization in the presence of a solid, such as amorphous 2′-FL or, in particular, crystalline 2′-FL. Mixtures of amorphous and crystalline 2′-FL can also be used. If crystalline 2′-FL is used for this purpose, any crystal form can be used. Other solids, including solid CO₂, can also be used. Ultrasound can also be applied to prevent kinetic inhibition of crystallization. Measures favoring crystallization are, of course, taken when the ratio c:c* does not exceed a value of 1.5:1, in particular a value of 1.3:1, more particularly a value of 1.2:1, and especially a value of 1.15:1.

[0080] According to one embodiment of the present invention, seed crystals of 2'-FL are added, preferably, but not necessarily, those having the desired polymorphic form. This measure is particularly taken if the crystallization is carried out discontinuously. The amount of seed crystals is then typically 0.01 to 5% by weight, in particular 0.02 to 3% or 0.02 to 1% by weight, relative to the pure 2'-FL in the aqueous solution from which the crystallization is carried out in step b).

[0081] To carry out crystallization in the presence of crystalline 2′-FL, an aqueous solution can also be fed to a suspension of crystalline 2′-FL in water under controlled supersaturation conditions. The solids content in the aqueous suspension is preferably in the range of 5 to 60 wt %, particularly 10 to 45 wt %, and especially 20 to 40 wt %, based on the total weight of the suspension. Preferably, depending on the temperature during crystallization and the desired polymorphic form, the concentration of dissolved 2′-FL in the aqueous phase of the 2′-FL suspension under supersaturation conditions is preferably in the range of 400 to 750 g / L or 500 to 750 g / L, particularly 410 to 720 g / L or 510 to 720 g / L, more particularly 400 to 650 g / L, and especially 410 to 630 g / L.

[0082] In a very preferred group of embodiments, the crystallization of step b) is carried out at a temperature of 20 to 52°C, particularly 25 to 50°C, more particularly 30 to 48°C, especially 35 to 45°C, in the presence of solid 2′-fucosyllactose, particularly crystalline 2′-fucosyllactose (any known polymorph of 2′-FL may be used), and wherein the crystallization is achieved from a supersaturated aqueous solution under conditions of controlled supersaturation as described herein, wherein the supersaturated aqueous solution has a concentration of dissolved 2′-fucosyllactose, at least initially, of 410 to 630 g / L.

[0083] The crystallization of 2'-FL can be carried out in any type of crystallization apparatus that can be used for crystallizing organic compounds from aqueous solutions. Suitable crystallization apparatus include, but are not limited to, stirred tank crystallizers, stirred tank crystallizers with guide tubes, stirred tank crystallizers with guide tubes and optionally with a device for crystal classification, so-called draft tube crystallizers or draft tube baffle (DTB) crystallizers, forced circulation crystallizers (such as Oslo-type crystallizers) optionally with a device for crystal classification, induced forced circulation crystallizers optionally with a device for crystal classification, and cooling plate crystallizers. Preferred crystallizers are selected from forced circulation crystallizers, draft tube crystallizers, draft tube baffle crystallizers, Oslo-type crystallizers, and induced forced circulation crystallizers, with draft tube baffle crystallizers and induced forced circulation crystallizers being particularly preferred.

[0084] As mentioned above, the process of the present invention can be carried out discontinuously (ie, batchwise, semi-batchwise) or continuously.

[0085] Batch processing involves adding an aqueous solution of the 2′-FL starting material to a crystallization vessel and inducing controlled supersaturation conditions therein to achieve crystallization of 2′-FL. This depletes the 2′-FL in solution, and the concentration of 2′-FL decreases. To prevent kinetic inhibition of crystallization, it is preferred to add a solid substance, particularly amorphous or crystalline 2′-fucosyllactose, especially seed crystals of 2′-FL. To maintain controlled supersaturation conditions, water can be evaporated during crystallization, or the temperature can be lowered, or both. In particular, a solid substance, particularly amorphous or crystalline 2′-fucosyllactose (any known polymorph of 2′-FL can be used), or a mixture of crystalline and amorphous 2′-FL, especially seed crystals of 2′-FL, is added when the ratio c:c* does not exceed a value of 1.5:1, particularly a value of 1.3:1, more particularly a value of 1.2:1, and especially a value of 1.15:1. Typically, when the desired amount of 2′-fucosyllactose has been crystallized from the solution, the aqueous suspension of crystalline 2′-fucosyllactose obtained is discharged from the crystallization vessel and subjected to a solid-liquid separation step. Typically, batch crystallization is carried out so that the suspension ultimately contains solid crystalline 2′-FL in an amount of 5 to 55% by weight, in particular 10 to 45% by weight, and especially 20 to 40% by weight, based on the weight of the suspension.

[0086] Semi-batch means that a portion of the aqueous solution of the 2′-FL raw material is added to the crystallization vessel and controlled supersaturation conditions are induced therein to achieve crystallization of 2′-FL. In order to prevent kinetic inhibition of crystallization, it is preferred to add a solid substance, in particular amorphous or crystalline 2′-fucosyllactose (any known polymorph of 2′-FL or a mixture of amorphous and crystalline 2′-FL can be used), especially seeds of 2′-FL. Then, the other amount of the aqueous solution of the 2′-FL raw material is fed into the crystallization device and thus fed into the aqueous suspension of partially or completely crystallized 2′-FL. In order to maintain controlled supersaturation conditions, water can be evaporated during crystallization, or the temperature can be lowered during crystallization, or both measures can be taken. Typically, when the desired amount of 2′-fucosyllactose has been crystallized from the solution, the aqueous suspension of crystalline 2′-fucosyllactose obtained is discharged from the crystallization vessel and subjected to a solid-liquid separation step. The semibatch crystallization is generally carried out so that the suspension finally contains 5 to 55% by weight, in particular 10 to 45% by weight and especially 20 to 40% by weight of solid crystalline 2′-FL, based on the weight of the suspension.

[0087] In another embodiment, crystallization is performed continuously. To this end, the aqueous solution containing the 2'-FL feedstock provided in step a) is fed to a continuously operated crystallization apparatus containing an aqueous suspension of 2'-fucosyllactose crystals. In other words, the aqueous solution of 2'-FL is continuously fed to the continuously operated crystallization apparatus, and crystallized 2'-FL is continuously discharged from the crystallization apparatus.

[0088] In a continuously operated crystallization apparatus, conditions of controlled supersaturation are maintained throughout the crystallization process. Preferably, conditions of controlled supersaturation are maintained by continuously removing a defined amount of water, preferably by evaporation, or by cooling, or by a combination of these measures.

[0089] Typically, continuously operated crystallization apparatuses are operated in such a way that the conditions of controlled supersaturation are quasi-static or almost quasi-static, in particular with temperature variations of less than 5 K and / or pressure variations of less than 60 mbar.

[0090] Typically, the continuously operated crystallization apparatus contains an aqueous suspension of 2′-FL crystals. Preferably, the solids content of the aqueous suspension contained in the continuously operated crystallization apparatus (i.e., the amount of 2′-FL crystals) is from 5 to 60% by weight, in particular from 10 to 45% by weight, and especially from 20 to 40% by weight, based on the total weight of the continuously operated crystallization apparatus or the suspension contained in the active volume of the continuously operated crystallization apparatus. The active volume is understood to be that part of the crystallization apparatus where crystallization takes place, e.g., that part of the free-flowing aqueous suspension containing 2′-FL crystals.

[0091] Typically, step b) of a continuously operated crystallization apparatus comprises the following sub-steps:

[0092] b1) continuously feeding an aqueous solution of 2′-FL starting material into a continuously operated crystallization apparatus comprising an aqueous suspension of crystalline 2′-FL, the aqueous suspension preferably containing crystalline 2′-FL in an amount of 5 to 60% by weight, particularly 10 to 45% by weight, especially 20 to 40% by weight, based on the weight of the suspension;

[0093] b2) continuously removing water from the aqueous suspension of 2′-FL contained in the crystallization apparatus, preferably by evaporation, in particular by evaporation under reduced pressure;

[0094] b3) The aqueous suspension of 2'-FL is continuously removed from the crystallization apparatus.

[0095] It has been found to be advantageous if the stream of the aqueous 2′-FL suspension removed from the crystallizer in step b3) is divided into two streams: the first stream is subjected to the separation of 2′-FL crystals, while the remaining portion is partially returned to the crystallization apparatus together with the fresh aqueous solution of the 2′-FL starting material provided in step b1). To this end, a portion of the aqueous 2′-FL suspension removed in step b3) is mixed with the aqueous solution of the 2′-FL starting material from step b1) before being fed to the crystallization apparatus. The resulting mixture is then returned to the crystallization apparatus. The volume ratio of the total stream removed from the crystallizer in step b3) to the first stream subjected to the separation of 2′-FL crystals is at least 4:1, particularly at least 7:1, more particularly at least 10:1, for example, from 4:1 to 200:1, or from 7:1 to 80:1, or from 10:1 to 60:1.

[0096] In order to remove water by evaporation, the energy required for evaporation must be introduced into the crystallizer. This can be achieved by conventional heating elements. Preferably, the heat of evaporation is introduced into the crystallizer by feeding a heated aqueous solution stream of 2′-FL raw material to the reactor. The heated aqueous solution stream of 2′-FL raw material fed to the reactor can be heated by any conventional heat exchanger. The heat exchanger can be operated with a conventional heating medium, such as heating oil or heating steam, including steam from a steam network, or steam provided by vapor recompression of water evaporated during crystallization or concentration of the aqueous solution of 2′-FL raw material in the process of the present invention. Preferably, the heated solution of 2′-FL raw material fed to the crystallizer is heated by using a forced circulation reduced pressure evaporator, which is preferably heated by steam from vapor recompression of water evaporated during crystallization or concentration of the aqueous solution of 2′-FL raw material. The use of a forced circulation reduced pressure evaporator minimizes fouling on the surfaces of the heat exchanger.

[0097] The continuously operated crystallization apparatus is preferably a forced circulation crystallizer.

[0098] The crystallization of step b) is generally carried out such that at least 30%, in particular at least 40%, for example 30 to 95%, in particular 40 to 90% of the 2′-fucosyllactose initially contained in the aqueous solution subjected to crystallization in step b) has been crystallized. A person skilled in the art will immediately appreciate that a low percentage of crystallized 2′-fucosyllactose leads to a higher purity, whereas a high percentage of crystallized 2′-fucosyllactose leads to a lower purity of the obtained crystalline 2′-fucosyllactose.

[0099] In step b), a suspension of crystalline 2′-fucosyllactose in an aqueous mother liquor is obtained. In step c), the crystalline 2′-FL is separated from the aqueous mother liquor. To this end, the suspension of crystalline 2′-FL in the aqueous mother liquor is subjected to solid / liquid separation. Suitable measures for separating solids from liquids include centrifugation, filtration or washing towers. The apparatus for centrifugation may include, but is not limited to, a pusher centrifuge, a worm screen centrifuge, a scraper centrifuge and a decanter. The apparatus for filtration may include, but is not limited to, a rotary pressure filter, a belt filter, a suction filter, a chamber filter and a chamber filter press. Suitable washing towers may include, but are not limited to, gravity wash columns, mechanical wash columns, hydraulic wash columns and piston wash columns. Preferably, solid / liquid separation is performed by centrifugation, in particular by utilizing a pusher centrifuge or a worm screen centrifuge, because low residual moisture in the obtained solid can be achieved, which is typically less than 10% by weight, for example 1 to 8% by weight.

[0100] The solid / liquid separation can be performed stepwise or continuously.

[0101] The obtained solid can be washed to remove the adhering mother liquor, for example, by washing with a cold solvent (such as water) or a saturated aqueous solution of pure 2′-FL. Suitable solvents for washing solid 2′-FL can also be mixtures of water and a non-solvent for 2′-FL. Typical non-solvents are C1-C4-alkanols such as methanol, ethanol, n-propanol or n-butanol, and acetic acid. If crystallization is carried out in more than one crystallization stage, a suitable solvent for washing solid 2′-FL can also be the mother liquor of a subsequent crystallization step. If crystallization is carried out in more than one crystallization stage, a suitable solvent for washing solid 2′-FL can also be a mixture of water and a non-solvent for 2′-FL, that is, a mixture of a non-solvent and a mother liquor of a subsequent crystallization step. Washing can be carried out, for example, by spraying solid crystalline 2′-FL with a cold solvent followed by further liquid / solid separation, or by suspending solid crystalline 2′-FL in a cold solvent followed by further liquid / solid separation. Washing can be carried out in a single step or by a plurality of washing steps, for example by 2, 3 or more steps. If washing is carried out by a plurality of washing steps, the washing steps can be operated in co-current or preferably in counter-current.

[0102] To drive crystallization to completion and increase the yield of crystalline 2′-fucosyllactose, a water-miscible organic solvent can be added to the suspension of 2′-fucosyllactose in the mother liquor before step b) when crystallization is nearly complete. In this context, nearly complete is preferably understood to mean that at least 80%, in particular at least 90%, of the 2′-fucosyllactose, calculated based on the amount of 2′-fucosyllactose, has crystallized, which theoretically can be crystallized from the solution in step b) under the crystallization conditions selected in step b). Typically, the organic solvent is only added when at least 30%, in particular at least 40%, for example 30 to 95%, in particular 40 to 90%, of the 2′-fucosyllactose initially contained in the aqueous solution subjected to crystallization in step b) has crystallized. Suitable water-miscible organic solvents are completely miscible with deionized water at 20°C and 1 bar. Examples of suitable organic solvents include C1-C4-alkanols and C1-C4-alkanoic acids, in particular ethanol, acetic acid and / or propionic acid, and mixtures thereof. The amount of organic solvent is typically chosen such that the weight ratio of organic solvent to water is at least 1 :1, such as 1 :1 to 10: 1. Surprisingly, the addition of an organic solvent leads to a higher purity of the obtained 2'-fucosyllactose.

[0103] The crystallization of 2′-FL typically involves a single crystallization step, as a single crystallization typically ensures a purity of 2′-FL that is sufficient for most purposes. However, the crystallization of 2′-FL may include two or more crystallization steps, 2 or 3 subsequent crystallization steps or stages. A further crystallization stage may involve recrystallization of the crystalline material obtained in the first crystallization stage. In this case, the further crystallization stage may be carried out according to the above-mentioned method involving crystallization under conditions of controlled supersaturation, and is useful for further improving the desired purity of 2′FL. The mother liquor obtained in the first crystallization stage may also be subjected to a second crystallization stage to increase the yield of 2′-fucosyllactose. In this case, the mother liquor may be mixed with a portion of the aqueous solution of the 2′-fucosyllactose raw material, and the mixture may be crystallized.

[0104] To increase the yield of crystalline 2′-fucosyllactose, part or all of the mother liquor obtained in step c) can be subjected to crystallization of 2′-fucosyllactose by inducing controlled supersaturation conditions in the mother liquor. For this purpose, the mother liquor can preferably be further crystallized according to the methods described herein. However, it is also possible to mix at least a portion of the mother liquor with a solution of the 2′-fucosyllactose starting material before proceeding to step b), and then subject the mixture to further crystallization of 2′-fucosyllactose according to the methods described herein.

[0105] According to a first set of preferred embodiments, the multi-stage crystallization process comprises a first crystallization step and a second crystallization step, and optionally one or more, for example, one or two, further crystallization steps, wherein at least in the second crystallization step, and preferably also in the first crystallization step, the crystallization of 2′-fucosyllactose is achieved by inducing controlled supersaturation conditions in the solution by the method described herein. In this set of preferred embodiments, the aqueous solution of 2′-fucosyllactose provided in step a) is subjected to crystallization in the second crystallization step. From this second crystallization step, an aqueous suspension of crystalline 2′-fucosyllactose in a mother liquor is obtained, which is then subjected to solid-liquid separation according to step c) to obtain crystalline 2′-fucosyllactose and a mother liquor. This mother liquor is then fed to the first crystallization step. The first crystallization step can be performed as described herein for step b) or according to prior art crystallization. Preferably, the first crystallization step is performed according to step b) described herein. The first crystallization step produces an additional amount of crystalline 2′-fucosyllactose. Typically, the purity of the crystalline 2′-fucosyllactose obtained in the first crystallization step is slightly lower than that of the crystalline 2′-fucosyllactose obtained in the second crystallization step. The crystalline 2′-fucosyllactose obtained in the first crystallization step can be used as is. However, it can also be dissolved in the aqueous solution of the 2′-fucosyllactose raw material provided in step a), and the resulting solution can be subjected to crystallization in the second crystallization step.

[0106] According to a second set of embodiments of the multi-stage crystallization process, the aqueous solution of the 2′-FL raw material provided in step a) is fed to a crystallization stage (1), which is operated batchwise or continuously as described above. The crystalline 2′-FL obtained in this stage (1) is then dissolved in water, and the solution obtained is subjected to a subsequent crystallization step (2), wherein purified crystalline 2′-FL and further mother liquor are obtained. The mother liquor of the subsequent crystallization step (2) can be mixed with water, and the mixture is then used to dissolve the crystalline 2′-FL obtained in the crystallization step (1). The crystalline 2′-FL obtained in stage (2) can be subjected to one or more, for example 1 or 2, further crystallization stages (3) and (4), respectively. For example, the mother liquor of the subsequent crystallization step (n+1) is mixed with water, and the mixture is used to dissolve the crystalline 2′-FL obtained in the crystallization step (n), where n represents the corresponding crystallization step. The mother liquor of the first crystallization stage can be discarded.

[0107] According to a combination of the first and second groups of embodiments, the mother liquor of the first crystallization stage is subjected to a further crystallization stage (also called a stripping stage) to obtain a residual mother liquor (which is discarded) and crystalline 2′-FL of lower purity. The crystalline 2′-FL of lower purity obtained in the crystallization stage can be dissolved, for example, in the aqueous 2′-FL solution provided in step a) to obtain a more concentrated solution, which is fed to the crystallization step (1). The crystalline 2′-FL obtained from the mother liquor of step (1) in the crystallization can also be dissolved in a mixture of water and the mother liquor obtained in the crystallization step (1) and combined with the aqueous 2′-FL solution provided in step a) to obtain a more concentrated solution, which is fed to the crystallization step (1).

[0108] According to the present invention, at least the crystallization stage (1) of the second group of embodiments and the combination of the second and first groups of embodiments is carried out according to the above-described method, which comprises crystallization under conditions of controlled supersaturation. If the crystallization stage is followed by a crystallization stage (2), it is also preferred that the crystallization stage (2) is carried out according to the above-described method, which comprises crystallization under conditions of controlled supersaturation.

[0109] Reference below Figures 1 to 9 The method according to the present invention is described in detail. The drawings shown are for illustration only and are not intended to limit the present invention thereto. Description of the drawings:

[0110] Figure 1 A basic flow diagram of the process according to the present invention is shown.

[0111] Figure 2 One embodiment of a forced circulation crystallizer is shown.

[0112] Figure 3 Another embodiment of a forced circulation crystallizer is shown, in this case a draft baffle crystallizer.

[0113] Figure 4 An embodiment of an induced forced circulation crystallizer is shown.

[0114] Figure 5 A block diagram of an embodiment of a multi-stage process according to the present invention is shown.

[0115] Figure 6 A block diagram is shown of a second set of embodiments of a multi-stage process according to the present invention.

[0116] Figure 7 One crystallization stage according to the invention is schematically shown.

[0117] Figure 8 A two-stage crystallization process according to a first group of embodiments of the present invention is schematically illustrated.

[0118] Figure 9 A two-stage crystallization process according to a second set of embodiments of the present invention is schematically illustrated.

[0119] In the drawings, the following reference symbols are used:

[0120] C crystalline phase / crystal

[0121] CR crystal

[0122] D discharge

[0123] DU Dilution Unit

[0124] F Feed

[0125] L liquid (liquor)

[0126] ML stock solution

[0127] MLR recycle mother liquor

[0128] P product

[0129] R Recirculating suspension

[0130] RL Residual Liquid

[0131] S fresh solution

[0132] SLS solid / liquid separation

[0133] V Steam

[0134] W Condensed steam (liquid water)

[0135] WL washing liquid

[0136] i index for the stage

[0137] 1 Crystallizer

[0138] 2 Heat exchanger

[0139] 3 separators

[0140] 4 Circulation pump

[0141] 5. Concentrate pump

[0142] 6 Steam compressor

[0143] 10 Entrance

[0144] 11 Slurry withdrawal

[0145] 12 Suspension outlet

[0146] 13 Liquid withdrawal / overflow

[0147] 14. Diversion tube

[0148] 15 Defoamer

[0149] 16 Steam outlet

[0150] 17 Subsidence Area

[0151] 18. Blender

[0152] 19 Inducer

[0153] 20 Steam separation zone

[0154] 21 Effective volume

[0155] like Figure 1As shown, a fresh stream S comprising an aqueous solution of a 2′-FL feedstock is combined with a recycle stream R and heated to a temperature of at least 40°C (e.g., 40°C to 95°C) in a heat exchanger 2 to produce an aqueous solution of the 2′-FL feedstock as a feed stream F. Heat exchanger 2 can be arranged horizontally or vertically, depending on specific requirements. Feed F is then fed into a continuously operating crystallizer 1. Crystallizer 1 contains a supersaturated aqueous suspension of 2′-FL as its effective volume, having a solid 2′-FL content of 5% to 50% by weight, e.g., 20% to 40% by weight, based on the weight of the suspension. An undersaturated aqueous solution F of the 2′-FL feedstock is fed into the effective volume while water is removed to maintain a steady 2′-FL concentration in the supersaturated suspension (i.e., the effective volume of crystallizer 1). Depending on the desired polymorph of 2'-FL, controlled supersaturation of 2'-FL in aqueous suspension is achieved under reduced pressure (eg, 20 to 800 mbar) at a temperature of at least 25°C (eg, 30 to 95°C).

[0156] Water is removed from the aqueous suspension of 2′-FL by evaporation and water vapor V is discharged from the top of the crystallizer 1. The vapor V can be further conveyed via a compressor 6 to heat the heat exchanger 2, for example flowing countercurrently to the feed F to be heated, and leaves the heat exchanger 2 as condensate W.

[0157] A discharge D containing a slurry of crystals 2′-FL is removed from the lower end of the crystallizer 1. A portion of the discharge D is used as a recycle stream R and is conveyed via a recycle pump 4 to be mixed with a fresh stream S before, during, or after entering the heat exchanger 2. The discharge D is distributed in such a way that the mass ratio of the recycle stream R to the fresh stream S is preferably greater than 5, in particular greater than 10, greater than 20, for example, 40:1 to 60:1.

[0158] Another part of the discharge D is sent to the separator 3 by means of a concentration pump 5. In the separator 3, the slurry D is separated to obtain mother liquor ML and crystals 2'-FL as product P. If necessary, the mother liquor ML can be recycled to the process of the present invention or a previous stage.

[0159] Alternatively, a discharge D containing a slurry of crystals 2′-FL is removed from one side of the lower end of the crystallizer 1. The discharge D is conveyed to a separator 3 by means of a thickening pump 5. In the separator 3, the slurry D is separated to obtain a mother liquor ML and crystals 2′-FL as a product P. If desired, the mother liquor ML can be recycled to the process of the present invention or to a previous stage. A second discharge is removed as a recycle stream R in the central portion of the lower end of the crystallizer 1. The recycle stream R is conveyed via a recycle pump 4 to be mixed with the fresh stream S before, during, or after entering the heat exchanger 2. The mass ratio of the recycle stream R to the fresh stream S is greater than 5, particularly greater than 10, greater than 20, for example, 40:1 to 60:1. Alternating the discharge of the two different slurries can prove particularly advantageous if the slurry D withdrawn from one side of the crystallizer is thicker than the slurry R withdrawn from the bottom of the crystallizer 1 or contains crystals with a different size distribution.

[0160] Crystallization can preferably be effected in a continuously operated crystallizer, for example a forced circulation crystallizer, a draft tube crystallizer or a draft tube baffle crystallizer, or in particular in an induced forced circulation crystallizer.

[0161] Figure 2 A superheated aqueous solution F of 2′-FL feedstock is fed into the crystallizer 1 via inlet 10 , flows upward through the draft tube 14 , and returns downward along the outside of the draft tube 14 .

[0162] The water evaporated from the suspension in the effective volume 21 rises as steam V to the top of the crystallizer 1. The steam V passes through the steam separation zone 20 and the demister 15 to remove droplets and leaves the crystallizer 1 via the steam outlet 16. The steam V is further conveyed via the compressor 6 to heat the heat exchanger 2, for example, flowing countercurrently with the feed F to be heated, and leaves the heat exchanger 2 as condensate W.

[0163] Around the working volume 21, a settling zone 17 may be provided. Via the suspension outlet 12 in the lower region of the working volume 21, the suspension R is removed and combined with the fresh solution S. The combined stream of R and S is recirculated through the heat exchanger 2 via the circulation pump 4 as feed F to the crystallizer. The circulation pump 4 provides the necessary stirring of the suspension mixed with the incoming solution F and enables the circulation of the suspension within the working volume 21.

[0164] The slurry D is removed from the crystallizer 1 through the slurry discharge port 11 located at the bottom of the crystallizer 1 below the effective volume 21. The discharged slurry D contains the desired crystals 2'-FL.

[0165] Figure 3A draft tube baffle crystallizer with forced circulation is shown. A superheated aqueous solution F of the 2′-FL feedstock is fed into the crystallizer 1 via inlet 10, flows upward through draft tube 14, and returns downward along the outside of draft tube 14. A bottom-entry agitator 18 provides the necessary agitation of the suspension, which mixes with the incoming solution F, with moderate energy consumption and circulates the suspension within the active volume 21.

[0166] Water evaporated from the suspension in the active volume 21 rises to the top of the crystallizer 1 as steam V. The steam V passes through the steam separation zone 20 and the demister 15 to remove droplets and leaves the crystallizer 1 through the steam outlet 16.

[0167] A settling zone 17 is provided with the aid of baffles at the periphery of the effective volume 21. In the settling zone 17, excess mother liquor L and / or fines can be discharged for further treatment at an overflow 13 in the upper region of the settling zone 17. The substantially clarified liquor L can be recycled into the process at any stage to adjust the temperature and / or concentration of the 2′-FL solution.

[0168] Via the suspension outlet 12 in the lower region of the settling zone 12 , the suspension R is removed and recirculated to be mixed with the fresh feed stream S.

[0169] The slurry D is removed from the crystallizer 1 through the slurry discharge port 11 located below the settling zone 12. The discharged slurry D contains the desired crystals 2'-FL as the product P.

[0170] As explained above, Figure 4 The induced forced circulation crystallizer shown is Figure 2 and 3 The forced circulation crystallizer shown operates similarly. Figure 3 Unlike the embodiment shown, the induced forced circulation crystallizer can be operated without any internal stirring device.

[0171] The superheated aqueous solution F of the 2′-FL feedstock is fed into the crystallizer 1 via the inlet 10, flows upward through the draft tube 14, and returns downward along the outside of the draft tube 14. Water evaporated from the suspension in the effective volume 21 rises to the top of the crystallizer 1 as steam V. The steam V passes through the steam separation zone 20 and the demister 15 to remove droplets, and leaves the crystallizer 1 via the steam outlet 16.

[0172] A settling zone 17 is provided on the periphery of the effective volume 21. The liquid L is discharged at the liquid discharge port 13 in the upper region of the settling zone 17. The substantially clarified liquid L is recirculated via the circulation pump 4. Via the suspension outlet 12 below the settling zone 12, the suspension R is removed and combined with the clarified liquid L in an external loop. Before, simultaneously with, or after being combined with the stream R, fresh solution S is fed into the recirculating stream L. The combined recirculating stream is heated in a heat exchanger (not shown in the figure) and fed into the crystallizer 1 as feed F. Figure 2 Similar to the embodiment shown, the steam V can be used to heat the heat exchanger 2 .

[0173] The throughput of the circulation pump 4 provides the siphoning of the recirculating suspension R and the necessary stirring of the suspension in the effective volume 21. No other stirring means are required, so that the crystals in the suspension are handled with the least possible strain.

[0174] The slurry D is removed from the crystallizer 1 through the slurry discharge port 11 located at the bottom of the crystallizer 1 below the effective volume 21 and below the settling zone 12. The discharged slurry D contains the desired crystals 2'-FL as product P.

[0175] In accordance with Figure 5 In a multi-stage process, crystallization is carried out in n stages. It should be noted that stage 3 to stage n are optional stages. Feed F is introduced into the first crystallization stage (i=1). The solvent is removed from the first crystallization, for example, by evaporation. The suspension is separated into a residual liquid RL and a first crystalline phase C1. The first crystalline phase C1 enters the second crystallization stage (i=2). The mother liquor from the second crystallization stage (i=2) is recycled to the first crystallization stage (i=1), for example by mixing it with water and using the mixture to dissolve the crystalline phase C1 obtained in the first crystallization stage. In each crystallization stage (i=2 to n), water is removed (for example, by discharging it in the form of solvent vapor V), and the suspension is separated into mother liquor ML and crystalline phase C. The crystalline phase from each crystallization stage (i) enters the subsequent crystallization stage (i+1). The mother liquor from each crystallization stage (i) is recycled to the previous crystallization stage (i-1), for example by mixing it with water and using the mixture to dissolve the crystals 2′-FL from the previous crystallization stage. From the last stage n, a crystalline phase containing the desired 2'-FL crystals is discharged. The number of stages n depends on the desired quality of the crystals in terms of form, purity, flow properties and storage performance.

[0176] In accordance with Figure 6 In the multi-stage process, crystallization is carried out in n stages, the first stage (i=1) being the stripping stage. It should be noted that stages 3 to n are optional stages. This process is similar to Figure 5, but the feed F is introduced between the stripping stage (i=1) and the second crystallization stage (i=2). Figure 6 The process gave higher yields of the desired product.

[0177] according to Figure 7 The crystallization stage (i) comprises two steps for crystallizing CR i and SLS for solid / liquid separation i Typically, the equipment used for crystallization of CR i The apparatus is a crystallizer suitable for crystal suspensions, such as a stirred tank reactor (e.g., a Swenson crystallizer), a forced circulation crystallizer (e.g., an Oslo reactor), a draft tube reactor, a draft tube baffle crystallizer (see Figure 3 ) or induced forced circulation crystallizer (see Figure 4 ). Used for solid / liquid separation SLS i The equipment used is usually a centrifuge, decanter, filter, filter press or washing tower.

[0178] Feed F for each stage (i) i Each contains the crystalline phase C from the previous stage (i-1) i-1 Suspension and / or fresh feed F, and recycled mother liquor MLR i . With solvent vapor V i The form of CR i The distillate is then removed from the suspension in a solid / liquid separation SLS. i Separate into mother liquor ML i and crystalline phase C i The crystalline phase C from each crystallization stage (i) i Can be used as feed F i+1 Transfer to the subsequent crystallization stage (i+1) or discharge as product. A portion of the mother liquor ML from each crystallization stage (i) i Recirculation to MLR i The remaining mother liquor MLi from each crystallization stage (i) can be recycled to the previous crystallization stage (i-1) or discharged. In order to improve the purity of the product 2'-FL, a solid / liquid separation SLS can be used. i In addition, washing liquid WL i As washing liquid WL i , preferably using cold water or the cold mother liquor of the subsequent crystallization stage (i+1).

[0179] exist Figure 8 In the Figure 6A two-stage process. The feed F is introduced into a dilution unit DU between the stripping stage (i=1) and the second crystallization stage (i=2), in which the crystalline 2′-fucosyllactose C1 obtained in the first crystallization stage is dissolved in the feed, i.e., the aqueous solution of the 2′-fucosyllactose raw material. Water is removed from the second crystallization stage, for example by evaporation in the form of steam V. A suspension of 2′-fucosyllactose in the mother liquor is thereby obtained, which is subjected to a solid-liquid separation SLS2 to obtain a mother liquor ML and purified crystalline 2′-fucosyllactose C2. The mother liquor from the second crystallization stage (i=2) is recycled to the first crystallization stage (i=1). Water is removed from the first crystallization stage, for example by evaporation in the form of steam V. A suspension of 2′-fucosyllactose in the mother liquor is thereby obtained, which is subjected to a solid-liquid separation SLS1 to obtain a residual liquid RL (which is discarded) and crystalline 2′-fucosyllactose C1.

[0180] exist Figure 9 In the Figure 5 A two-stage process. Feed F (i.e. an aqueous solution of 2′-fucosyllactose raw material) is introduced into the first crystallization stage (i=1). Water is removed from the first crystallization stage, for example by evaporation in the form of steam V. Thus, a suspension of 2′-fucosyllactose in the mother liquor is obtained, which is subjected to solid-liquid separation SLS1 to obtain residual liquid RL (which is discarded) and purified crystalline 2′-fucosyllactose C1. Crystalline 2′-fucosyllactose C1 is dissolved in solvent S (water or other feed) in a dilution unit DU. The solution thus obtained is passed to the second crystallization stage (i=2). Water is removed from the second crystallization stage, for example by evaporation in the form of steam V. Thus, a suspension of 2′-fucosyllactose in the mother liquor is obtained, which is subjected to solid-liquid separation SLS2 to obtain mother liquor ML and purified crystalline 2′-fucosyllactose C2. The mother liquor from the second crystallization stage (i=2) is recycled to the first crystallization stage (i=1), for example by mixing it with feed F.

[0181] abbreviation:

[0182] 2'-FL: 2'-O-fucosyllactose

[0183] DiFL: difucosyllactose

[0184] bw: by weight

[0185] rpm: revolutions per minute

[0186] RT: room temperature, i.e., about 22°C

[0187] analyze:

[0188] HPLC:

[0189] Column: Spherisorb NH2 column (amine-modified silica: particle size 3 μm, pore size ), length 250 mm, inner diameter 4.5 mm (Waters Corporation)

[0190] Eluent: acetonitrile / water 82.5 / 17.5 v / v

[0191] Detection: RID

[0192] Parameters: flow rate 1.3 ml / min, T = 35 ° C, pressure 112 bar, 5 μl injection volume

[0193] Water Determination: The water concentration was determined by Karl-Fischer titration.

[0194] The dry matter content was determined by drying 2 g of sample at 130°C for 2 hours.

[0195] The filter cake resistance is calculated based on the measured volume flow of the filtrate in the pressure filter, the applied pressure and the filter area.

[0196] Determination of crystal form: powder X-ray diffraction (PXRD)

[0197] Using Cu-K radiation at 25°C X-ray diffraction patterns were recorded using a Panalytical X'Pert Pro diffractometer (manufacturer: Panalytical) in reflection geometry (Bragg-Brantano) with, for example, an increment of 0.017° and a measurement time of 20 s / step in the range 2θ = 3°-40°. The tube voltage was 45 kV and the current was 40 mA. The sample was placed in a silicon single crystal sample holder at a depth of 0.2 mm and flattened. Crystallization Example:

[0198] In Examples 1 to 3, an aqueous solution of 2'-FL feedstock obtained by fermentation and subsequent downstream processing, including passing the fermentation broth through an ion exchange resin bed, and concentration of the fermentation broth to a solids content of 61.1% by weight was used. This aqueous solution contained 52.5% by weight of 2'-FL and 8.6% by weight of monosaccharides and oligosaccharides, including lactose, DiFL, and fucosyllactulose.

[0199] Example 1:

[0200] In a reaction flask equipped with a distillation bridge and a stirrer, an aqueous solution of 100 g of 2′-FL starting material is heated to 50° C. (bath temperature) with the aid of a water bath. At a pressure of 30 mbar, 19.42 g of water are distilled off to give a syrup containing 65% by weight of 2′-FL. The weight ratio of product (2′-FL) to water in the resulting syrup is 2.74:1. The container is expanded to ambient pressure, and the resulting viscous solution is cooled to 45° C. (bath temperature) and seeded with 0.05 g of form II of 2′-FL obtained from a previous operation. The mixture is stirred at 45° C. (bath temperature) for a further 4 hours, allowed to cool to room temperature and stirred for a further 16 hours. The thick suspension thus obtained is warmed to 35° C. (bath temperature) and stirred at 35° C. for 2 hours at ambient pressure. The hot suspension was filtered through a heated suction filter (35° C.), and the filter cake was washed four times with 10 ml of ethanol / water (80 / 20 w / w) and then dried at 40° C. and 0.8 mbar for 12 hours. 38.9 g of crystalline material with the following composition were obtained (yield 70.5%):

[0201] Composition (HPLC): 95.1% 2'-FL, 0.2% lactose, 0.3% fucosyllactulose, 0.9% DiFL. The crystalline material obtained contained 3.3% by weight of water as determined by Karl-Fischer titration.

[0202] In the crystalline material obtained, 2'-FL was present essentially in Form A as determined by PXRD.

[0203] Example 2:

[0204] In a reaction flask equipped with a distillation bridge and a stirrer, an aqueous solution of 200 g of 2'-FL starting material was heated to 55°C (bath temperature) with the aid of a water bath. At a pressure of 30 mbar, 31.65 g of water was distilled off to obtain a syrup containing 62.4% by weight of 2'-FL. The weight ratio of product (2'-FL) to water in the resulting syrup was 2.3:1. The resulting viscous solution was expanded to ambient pressure, cooled to 45°C (bath temperature), and seeded with 0.05 g of crystalline 2'-FL (Form A) obtained in Example 1. The mixture was stirred at 45°C (bath temperature) and ambient pressure for another 4 hours and then cooled to 10°C. A sample showing that Form A of 2'-FL had formed was taken. 136.2 ml of acetic acid was then added over 30 minutes while maintaining the temperature at 10°C, resulting in a volume ratio of acetic acid to water of 3:1 v / v. The resulting suspension was stirred at 10°C under ambient pressure for 0.5 hours. The suspension thus obtained was filtered through a suction filter, and the filter cake was washed three times with 10 ml of acetic acid / water (80 / 20 w / w) and then dried at 40° C. and 0.8 mbar for 12 hours. 53.6 g (yield 49.1%) of a crystalline substance having the following composition were obtained:

[0205] Composition (HPLC): 0.5% DiFL and 96.2% 2'-FL (no detectable amounts of lactose and fucosyllactulose). The crystalline material obtained contained 3.9% by weight of water as determined by Karl-Fischer titration.

[0206] In the crystalline material obtained, 2'-FL was present essentially in Form A as determined by PXRD.

[0207] Example 3:

[0208] In a reaction flask equipped with a distillation bridge and a stirrer, an aqueous solution of 200 g of the 2′-FL starting material was heated to 65° C. (internal temperature; 80° C. bath temperature) with the aid of a water bath. At a pressure of 250 mbar, 38 g of water were distilled off to give a syrup containing 64.8% by weight of 2′-FL. The weight ratio of product (2′-FL) to water in the resulting syrup was 2.5:1. The resulting syrup was expanded to ambient pressure and cooled to 50° C. (bath temperature). The suspension was stirred at 50° C. under ambient pressure (bath temperature) for a further 3 hours, then cooled to 20° C. and stirred at 20° C. for a further 1 hour. 117 ml of acetic acid were then added over 30 minutes, while maintaining the temperature at 20° C., so that the volume ratio of acetic acid to water was 3:1 v / v. The resulting suspension was stirred at 10° C. for 0.5 hours. The suspension thus obtained was filtered through a suction filter, and the filter cake was washed three times with 15 ml of acetic acid / water (80 / 20 w / w) and then dried at 40° C. and 0.8 mbar for 12 hours. 80.3 g (yield 75.6%) of a crystalline substance having the following composition were obtained:

[0209] Composition (HPLC): 0.2% lactose, 0.3% fucosyllactulose and 98.8% 2'-FL (no detectable amount of DiFL). The crystalline material obtained contained 0.015% by weight of water as determined by Karl-Fischer titration.

[0210] In the crystalline material obtained, 2'-FL was present essentially in Form II as determined by PXRD.

[0211] As a variation of Example 3 above, the following is possible:

[0212] a. Instead of increasing the 2′-FL concentration in the syrup to above 60 wt %, as detailed in Example 3, syrups with lower 2′-FL concentrations (2′-FL concentrations up to only 50 wt %) can also be used. The results of experiments starting with these lower concentration solutions were essentially the same as those in Example 3.

[0213] b. As another possibility, the amount of acetic acid used in Example 3 can be increased up to 3 times its amount and still obtain substantially the same results as in Example 3.

[0214] c. As another possibility, the solution can be seeded with any polymorphic form of 2'-FL, even amorphous 2'-FL, with essentially the same results.

[0215] “Substantially the same results” means that the purity of 2′-FL and the absolute amount of by-product content vary to a very small extent, ie, differ from the experimental results of Example 3 by less than about 5%.

[0216] In Example 4, an aqueous solution of 2'-FL feedstock was used, obtained by fermentation and subsequent downstream processing, including passing the fermentation broth through an ion exchange resin bed, and concentrating the fermentation broth to a solids content of 61.5% by weight. This aqueous solution contained 49.4% by weight of 2'-FL and 12.1% by weight of monosaccharides and oligosaccharides, including 0.8% lactose, 0.4% fucosyllactulose, and 2.5% DiFL.

[0217] Example 4:

[0218] In a reaction flask equipped with a distillation bridge and a stirrer, an aqueous solution of 200 g of 2′-FL raw material was heated to 45° C. (bath temperature) with the aid of a water bath. At a pressure of 20–50 mbar, about 30 g of water was distilled off to obtain a syrup containing about 58% by weight of 2′-FL. The weight ratio of product (2′-FL) to water in the resulting syrup was 2.1:1. The resulting viscous syrup was expanded to ambient pressure and seeded with 0.05 g of crystalline 2′-FL (Form II) obtained in Example 3 at 45° C. (bath temperature). The mixture was stirred for another 16 hours at ambient pressure and 45° C. (bath temperature). The thick suspension thus obtained was discharged from the reactor and filtered through a heated suction filter (40° C.), and the filter cake was dried under an inert gas flow at 40° C. and 10 mbar for 16 hours. 78.1 g of a crystalline substance with the following composition was thus obtained (yield 69.3%):

[0219] Composition (HPLC): 0.72% lactose, 0.57% fucosyllactulose, 2.61% DiFL and 87.64% 2'-FL. The crystalline material obtained contained 3.0% by weight of water as determined by Karl-Fischer titration.

[0220] In the crystalline material obtained, 2'-FL was present essentially in Form A as determined by PXRD.

[0221] In Examples 5 to 8 and Comparative Examples C1 and C2 below, an aqueous solution of 2'-FL feedstock obtained by fermentation and subsequent downstream processing, including decolorization, microfiltration, ultrafiltration, desalination, and reverse osmosis, was used. This aqueous solution had a dry matter content of 25% by weight and contained 21.0% by weight of 2'-FL and 4% by weight of monosaccharides and oligosaccharides, including lactose and DiFL.

[0222] Example 5:

[0223] An aqueous solution of the 2′-FL starting material was evaporated on a rotary evaporator at 60°C under reduced pressure to a dry matter content of approximately 73% by weight and a 2′-FL concentration of approximately 59% by weight. 1517 g of the resulting solution were filled into a baffled tank and stirred at 60°C (internal temperature). 12 g of amorphous 2′-FL were then added to this solution, and solid formation was observed. The resulting suspension was stirred at 60°C (internal temperature) for 20 hours. The resulting suspension was then cooled to 25°C over 1.5 hours while stirring. The resulting suspension was then filtered without washing.

[0224] 467 g of a wet crystalline mass having the following composition are thus obtained:

[0225] The filter cake had a composition of 83 wt% 2'-FL, 1.2 wt% lactose, 0.9 wt% fucosyllactulose, 2.1 wt% DiFL, and 9 wt% water. In the filter cake, 2'-FL was primarily present in Form II, as determined by PXRD. The calculated yield, based on the fucosyllactulose content in the concentrated solution, was 42%.

[0226] The filtrate had the following composition: 49 wt% 2'-FL, 1.4 wt% fucosyllactulose, 2 wt% lactose, 3.7 wt% DiFL and 36 wt% water.

[0227] The filter cake resistance is 5x10 12 Pas / m 2 .

[0228] The same variables a and c mentioned in the context of Example 3 also apply here, leading to essentially the same results as Example 5.

[0229] Example 6:

[0230] In a rotary evaporator, the aqueous solution of the 2′-FL raw material was evaporated at 40° C. under reduced pressure to a dry matter content of about 62% by weight and a concentration of about 50% by weight of 2′-FL. 1532 g of the solution thus obtained were filled into a baffle and stirred at 40° C. (internal temperature). 5 g of crystalline 2′-FL (Form II) were then added to the solution, and the formation of a solid was observed. The suspension thus formed was stirred at 40° C. (internal temperature) for 19 hours. The suspension thus formed was then evaporated at 40° C. under reduced pressure to a 2′-FL concentration of 61% by weight and then cooled to 25° C. under stirring within 1.0 hour. The suspension was stirred at 25° C. for another 22 hours. The suspension thus obtained was then filtered without washing. 772 g of a wet crystalline material having the following composition was thereby obtained:

[0231] The filter cake had a composition of 77 wt% 2'-FL, 1.1 wt% lactose, 0.8 wt% fucosyllactulose, 1.9 wt% DiFL, and 15 wt% water. The calculated yield, based on the 2'-FL content in the concentrated solution, was 77%. In the filter cake, 2'-FL was primarily present in Form B, as determined by PXRD.

[0232] The filtrate had the following composition: 36 wt% 2'-FL, 2 wt% fucosyllactulose, 3.1 wt% lactose, 5 wt% DiFL and 40 wt% water.

[0233] The filter cake resistance is 4x10 11 Pas / m 2 .

[0234] Example 7:

[0235] In a rotary evaporator, an aqueous solution of the 2′-FL starting material was evaporated at 40°C under reduced pressure to a dry matter content of approximately 65% by weight and a 2′-FL concentration of approximately 52% by weight. 1572 g of the solution thus obtained was filled into a baffled trough and stirred at 40°C (internal temperature). 26 g of crystalline 2′-FL (Form A) was then added to this solution, and the formation of a solid was observed. The suspension thus formed was stirred at 40°C (internal temperature) for 1 hour. The suspension thus formed was then evaporated at 40°C under reduced pressure to a 2′-FL concentration of 55% by weight, followed by stirring at 40°C for 12 hours and then cooled to 25°C with stirring over 1.0 hour. The suspension was stirred at 25°C for an additional 7 hours. The suspension thus obtained was then filtered without washing. This yielded a wet crystalline material containing 2′-FL in its Form B, as determined by PXRD.

[0236] After drying the filter cake at 60° C. and 100 mbar for 2 days, a crystalline material with the following composition was obtained:

[0237] 90 wt% 2'-FL, 0.5 wt% lactose, 0.3 wt% fucosyllactulose, 1.1 wt% DiFL, and 5.9 wt% water. The calculated yield, based on the 2'-FL content in the concentrated solution, is 41%. In the dried crystalline material, 2'-FL is primarily in Form A, as determined by PXRD.

[0238] The filtrate had the following composition: 45 wt% 2'-FL, 1.3 wt% fucosyllactulose, 1.9 wt% lactose, 3.7 wt% DiFL and 39 wt% water.

[0239] The filter cake resistance is 5x10 11 Pas / m 2 .

[0240] Example 8:

[0241] A total of 1099 g of the filtrate and water obtained in Examples 5, 6 and 7 were placed in a baffled trough and stirred at 40° C. The concentration of 2'-FL in the solution was 36% by weight. Stirring was continued while water was evaporated from the solution at 40° C. under reduced pressure until the concentration of 2'-FL was 43% by weight (dry matter content was 67% by weight). 8 g of crystalline 2'-FL (Form A) was then added to the solution, and the formation of a solid was observed. The suspension thus formed was stirred at 40° C. (internal temperature) for 2 hours. The suspension thus formed was then evaporated at 40° C. under reduced pressure to a concentration of 47% by weight of 2'-FL, followed by stirring at 40° C. for 1 hour, and then cooled to 25° C. under stirring within 1.0 hour. The suspension was stirred at 25° C. for another 72 hours. The suspension thus obtained was then filtered without washing. Thus, a wet crystalline material was obtained, which contained 2'-FL in its Form B.

[0242] After drying the filter cake at 60° C. and 100 mbar for 2 days, a crystalline material with the following composition was obtained:

[0243] 88 wt% 2'-FL, 0.9 wt% lactose, 0.5 wt% fucosyllactulose, and 1.7 wt% DiFL. The calculated yield, based on the 2'-FL content in the concentrated solution, was 26%. In the dried crystalline material, 2'-FL was primarily present in Form A, as determined by PXRD.

[0244] The filtrate had the following composition: 40 wt% 2'-FL, 2.1 wt% fucosyllactulose, 3.2 wt% lactose, 5.7 wt% DiFL and 35 wt% water.

[0245] The filter cake resistance is 6x10 11 Pas / m 2 .

[0246] Comparative Example C1:

[0247] In a distillation apparatus, an aqueous solution of the 2′-FL starting material was evaporated under reduced pressure at 60°C to a 2′-FL concentration of approximately 42% by weight. 1663 g of the resulting solution was filled into a baffled trough and evaporated under reduced pressure at 60°C to a dry matter content of 85% by weight and a 2′-FL concentration of 69%. Spontaneous crystallization then occurred, and the viscosity of the suspension was too high to drain from the baffled trough. PXRD analysis of the suspension indicated that 2′-FL was present primarily in Form II.

[0248] Comparative Example C2:

[0249] In a distillation apparatus, an aqueous solution of the 2′-FL starting material was evaporated under reduced pressure at 50°C to a 2′-FL concentration of approximately 50% by weight. 1607 g of the resulting solution was filled into a baffled trough and evaporated under reduced pressure at 40°C to a dry matter content of 80% by weight and a 2′-FL concentration of 65%. Spontaneous crystallization then occurred, and the viscosity of the suspension was too high to drain from the baffled trough. PXRD analysis of the suspension indicated that the 2′-FL was primarily present in Form B.

[0250] In Examples 9 and 10 below, an aqueous solution of 2'-FL feedstock was used, obtained by fermentation and subsequent downstream processing, including decolorization, microfiltration, ultrafiltration, desalination, and reverse osmosis. This aqueous solution had a dry matter content of 29% by weight and contained 24.5% by weight of 2'-FL and 4% by weight of monosaccharides and oligosaccharides, including lactose and DiFL.

[0251] The aqueous solution of 2'-FL starting material was concentrated on a rotary evaporator under reduced pressure to a 2'-FL concentration of approximately 52.1 wt %. This solution was referred to as the "pre-evaporation feed" and was used in Examples 9 and 10 below.

[0252] Example 9:

[0253] 1612 g of pre-evaporation feed was filled into a baffled trough and stirred at 60° C. (internal temperature). Water was evaporated under reduced pressure at 60° C. with stirring to a 2′-FL concentration of 62% by weight. 12 g of crystalline 2′-FL (Form II) suspended in a small volume of pre-evaporation feed was then added to the solution, and solid formation was observed. The suspension thus formed was stirred at 60° C. (internal temperature) for 19 hours while stirring at 450 rpm. The suspension thus formed was then filtered without washing using a heated pressure suction filter (60° C., pressure difference 0.5 bar, 230 s). Thus, a wet crystalline material was obtained, which, as demonstrated by PXRD, contained 2′-FL in its Form II.

[0254] The filter cake was dried at 60° C. and 100 mbar for 1 day. The resulting crystalline material had the following composition: 94.6% by weight of 2′-FL, 0.8% by weight of lactose, 2.1% by weight of DiFL. The calculated yield, based on the 2′-FL content of the concentrated solution, was 45%.

[0255] The same variables a and c mentioned in Example 3 also apply here, resulting in essentially the same results as Example 9.

[0256] Example 10:

[0257] 1628 g of pre-evaporation feed was filled into a baffled trough and stirred at 40° C. (internal temperature). Water was evaporated under reduced pressure at 40° C. with stirring to a 2′-FL concentration of 63% by weight. 13 g of crystalline 2′-FL (Form A) suspended in a small volume of pre-evaporation feed was then added to the solution, and solid formation was observed. The suspension thus formed was stirred at 40° C. (internal temperature) for 21 hours while stirring at 450 rpm. The suspension thus formed was then filtered without washing using a heated pressure suction filter (40° C., pressure difference 0.5 bar, 403 s). Thus, a wet crystalline material was obtained, which, as demonstrated by PXRD, contained 2′-FL in its Form B.

[0258] The filter cake was dried at 60°C and 100 mbar for one day. The resulting crystalline material had the following composition: 88 wt% 2'-FL, 1.4 wt% lactose, and 4.3 wt% DiFL. The calculated yield, based on the 2'-FL content in the concentrated solution, was 80%. PXRD analysis of the dried crystalline material indicated that 2'-FL was primarily present in Form A, as determined by PXRD.

[0259] Example 11:

[0260] An aqueous solution of 2'-FL starting material was used, obtained by fermentation and subsequent downstream processing, including passing the fermentation broth through an ion exchange resin bed and concentrating the thus-treated broth to a 2'-FL content of 50.4% by weight. This aqueous solution also contained 0.9% by weight of lactose, 2.9% by weight of DiFL, and 1.6% by weight of fucosyllactulose.

[0261] In a reaction flask equipped with a distillation bridge and a stirrer, an aqueous solution of 150 g of 2′-FL raw material is heated to 50° C. (bath temperature) with the aid of a water bath. At a pressure of 30-100 mbar, water is distilled off to obtain a syrup containing 65% by weight of 2′-FL. The container is expanded to ambient pressure, and the resulting viscous solution is cooled to 45° C. (bath temperature) and seeded with 0.15 g of crystalline 2′-FL obtained from the previous operation. The mixture is stirred at 45° C. (bath temperature) for another 4 hours, allowed to cool to room temperature and stirred at room temperature for another 30 hours. 84 g of glacial acetic acid are added to the thick suspension thus obtained, and the mixture is stirred for another 3 hours to complete the crystallization. The suspension is filtered through a suction filter, and the filter cake is washed 3 times with glacial acetic acid and then dried at 40° C. and 1.0 mbar for 12 hours. 61 g of a crystalline material with the following composition is thus obtained:

[0262] Composition (HPLC): 92.9% 2'-FL, 0.1% lactose, 0.1% fucosyllactulose, 0.4% DiFL and 0.6% acetic acid. The crystalline material obtained contained 3.6% by weight of water as determined by Karl-Fischer titration.

[0263] In the crystalline material obtained, 2'-FL was present essentially in Form A as determined by PXRD.

Claims

1. A method for obtaining crystalline form A and / or crystalline form B of 2'-fucosyllactose from a 2'-fucosyllactose raw material, wherein the 2'-fucosyllactose raw material contains 2'-FL as a main component and at least 0.5 wt. % of one or more monosaccharides or oligosaccharides other than 2'-fucosyllactose, based on the total amount of monosaccharides and oligosaccharides in the raw material, The method comprises: a) providing an aqueous solution of the 2'-fucosyllactose raw material, wherein the solution comprises an organic solvent in an amount not exceeding 10 wt % based on the total amount of water; b) achieving crystallization of 2'-fucosyllactose by inducing conditions of controlled supersaturation in said solution at a temperature of 20 to 52°C; and c) separating crystalline 2'-fucosyllactose from the mother liquor, and wherein during the controlled supersaturation in step b), not more than 10 wt.-% of organic solvent is present, based on the total amount of water present during step b), wherein controlled supersaturation means that during crystallization, the supersaturation does not exceed a value where uncontrolled crystallization occurs, wherein the controlled supersaturated condition is induced in such a manner that the ratio c:c* of the concentration c of 2'-fucosyllactose dissolved under the controlled supersaturated condition to the equilibrium solubility c* of 2'-fucosyllactose is greater than 1:1 to 1.5:1, thereby achieving crystallization of the 2'-fucosyllactose.

2. The method according to claim 1, wherein the 2'-fucosyllactose raw material contains 2'-FL as a main component and at least 1 wt% of one or more monosaccharides or oligosaccharides other than 2'-fucosyllactose based on the total amount of monosaccharides and oligosaccharides in the raw material.

3. The method according to claim 1, wherein the 2'-fucosyllactose raw material contains 2'-FL as a main component and at least 2 wt% of one or more monosaccharides or oligosaccharides other than 2'-fucosyllactose based on the total amount of monosaccharides and oligosaccharides in the raw material.

4. The process according to claim 1, wherein the solution in step a) comprises not more than 7% by weight of organic solvent based on the total amount of water.

5. The method according to claim 1, wherein the solution in step a) contains no more than 5% by weight of organic solvent based on the total amount of water.

6. The process of claim 1 , wherein during the controlled supersaturation of step b), no more than 7 wt.% of organic solvent is present, based on the total amount of water present during step b).

7. The process of claim 1 , wherein during the controlled supersaturation of step b), no more than 5 wt.% of organic solvent is present, based on the total amount of water present during step b).

8. The process according to any one of claims 1 to 7, wherein the controlled supersaturation is induced by removing water and / or by cooling.

9. The process according to any one of claims 1 to 7, wherein controlled crystallization is achieved in the presence of solid 2'-fucosyllactose.

10. The method of claim 9, wherein the controlled crystallization is achieved in the presence of crystalline 2'-fucosyllactose.

11. The process according to any one of claims 1 to 7, wherein the crystallization is carried out as evaporative crystallization.

12. The method according to any one of claims 1 to 7, wherein the 2'-fucosyllactose starting material comprises at least one oligosaccharide selected from the group consisting of lactose, difucosyllactose, lactulose and fucosylated lactulose.

13. The method according to any one of claims 1 to 7, wherein the solution of 2'-fucosyllactose provided in step a) contains not more than 5000 ppm of solid insoluble matter, based on the total weight of the solution.

14. The method according to any one of claims 1 to 7, wherein the aqueous solution of 2'-fucosyllactose provided in step a) is obtained by a fermentation process.

15. The process according to any one of claims 1 to 7, wherein the aqueous solution of 2'-fucosyllactose provided in step a) is fed to a continuously operated crystallization apparatus comprising an aqueous suspension of 2'-fucosyllactose crystals.

16. The method according to claim 15, wherein step b) comprises: b1) continuously feeding the aqueous solution of 2′-fucosyllactose into a crystallization device containing an aqueous suspension of 2′-fucosyllactose; b2) continuously removing water from the aqueous suspension of 2'-fucosyllactose contained in the crystallization apparatus in order to maintain conditions of controlled supersaturation; b3) continuously removing the aqueous suspension of 2'-fucosyllactose from the crystallization apparatus.

17. The process according to claim 16, wherein a portion of the aqueous suspension of 2'-fucosyllactose removed in step b3) is mixed with the aqueous solution of 2'-fucosyllactose in step b1), and the mixture is returned to the crystallization apparatus.

18. The process according to any one of claims 1 to 7, wherein the solution of 2'-fucosyllactose starting material is crystallized in a crystallization apparatus operated in batch or fed-batch mode.

19. The process according to any one of claims 1 to 7, wherein at least a portion of the mother liquor obtained in step c) is subjected to crystallization of 2'-fucosyllactose by inducing conditions of controlled supersaturation in the mother liquor.

20. The method of claim 19, wherein at least a portion of the mother liquor is mixed with the solution of the 2'-fucosyllactose starting material before performing step b).

21. The process according to any one of claims 1 to 7, wherein before step c), a water-miscible organic solvent is added to the suspension obtained in step b) when the crystallization is almost complete.

22. The method according to claim 1, wherein the conditions are such that the crystallization of 2'-fucosyllactose is achieved at a temperature of 20 to 50°C to obtain the crystalline form A or form B of 2'-fucosyllactose.

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