Method for obtaining crystalline 2′-fucosyllactose

Abstract

The present invention relates to a method for obtaining crystalline 2′-fucosyllactose from a 2′-FL raw material, which contains 2′-FL as a main constituent and at least 0.5% by weight, frequently at least 1% by weight, in particular at least 2% by weight, more particularly at least 5% by weight, and especially at least 8% by weight,based on the total amount of mono-and oligosaccharides in the raw material, of one or more mono- or oligosaccharides different from 2′-FL, where the method comprises a)providing a solution of the 2′-FL raw material in water, which does not contain more than 10% by weight, preferably not more than 7% by weight, more preferably not more than 5% by weight of organic solvents, based on the total amount of water; b) effecting the crystallization of 2′-FL from the solution provided in step a) by inducing conditions of a controlled super saturation in the solution; and c) separating crystalline 2′-FL from the mother liquor, and where during 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 organic solvents are present, based on the total amount of water present during step b).

Claims

1. Method for selectively obtaining crystalline form A or form B of 2′-fucosyllactose from a 2′-fucosyllactose raw material, which contains 2′-FL as a main constituent and at least 0.5% by weight, based on the total amount of mono-and oligosaccharides in the raw material, of one or more mono-or oligosaccharides different from 2′-fucosyllactose, where the method comprises a) providing a solution of the 2′-fucosyllactose raw material in water, which does not contain more than 10% by weight of organic solvents, based on the total amount of water; b) effecting the crystallization of 2′-fucosyllactose at a temperature in the range from 0 to 52° C. by inducing conditions of a controlled supersaturation in the solution, where form A or form B of 2′-fucosyllactose is obtained and a water miscible organic solvent is added to the suspension when the crystallization is almost complete; and c) separating crystalline form A or form B of 2′-fucosyllactose from the mother liquor; and where during controlled supersaturation in step b) not more than 10% by weight of organic solvents are present, based on the total amount of water present during step b).

2. The method of claim 1, where the conditions of a controlled supersaturation are induced in a manner such that the ratio c:c* of the concentration c of dissolved 2′-fucosyllactose to the equilibrium solubility c* of 2′-fucosyllactose under the conditions of controlled supersaturation is from more than 1:1 to 1.5:1, thereby effecting the crystallization of 2′-fucosyllactose.

3. The method of claim 1, where the controlled supersaturation is induced by removing water and/or by cooling.

4. The method of claim 1, where the crystallization is effected in the presence of solid 2′-fucosyllactose and is carried out as an evaporation crystallization.

5. The method of claim 1, where the concentration of dissolved 2′-fucosyllactose under the conditions of controlled supersaturation is in the range from 400 to 750 g/L.

6. The method of claim 1, where the 2′-fucosyllactose raw material contains at least one oligosaccharide selected from lactose, difucosyllactose, lactulose and fucosylated lactulose.

7. The method of claim 1, where the solution of 2′-fucosyllactose provided in step a) does not contain more than 5000 ppm of solid insoluble material, based on the total weight of the solution.

8. The method of claim 1, where the aqueous solution of 2′-fucosyllactose provided in step a) is obtained by a fermentation process.

9. The method of claim 1, where the aqueous solution of 2′-fucosyllactose provided in step a) is fed to a continuously operated crystallization apparatus, which contains an aqueous suspension of 2′-fucosyllactose crystals.

10. The method of claim 9, where step b) comprises b1) continuously feeding the aqueous solution of 2′-fucosyllactose to a crystallization apparatus containing an aqueous suspension of 2′-fucosyllactose; b2) continuously removing water from the aqueous suspension of 2′-fucosyllactose contained in the crystallization apparatus to maintain conditions of controlled supersaturation; b3) continuously removing the aqueous suspension of 2′-fucosyllactose from the crystallization apparatus.

11. The method of claim 9, where a portion of the aqueous suspension of 2′-fucosyllactose removed in step b3) is mixed with the aqueous solution of 2′-fucosyllactose of step b1) and the mixture is fed back it into the crystallization apparatus.

12. The method of claim 1, where the solution of the 2′-fucosyllactose raw material is subjected to a crystallization in batch- or fed-batch operated crystallization apparatus.

13. The method of claim 1, where the crystallization is carried out at a temperature in the range from 20 to 52° C. in the presence of solid 2′-fucosyllactose and where the crystallization is effected from an aqueous supersaturated solution under the conditions of controlled supersaturation, where the aqueous supersaturated solution has a concentration of dissolved 2′-fucosyllactose in the range from 410 to 630 g/L.

14. The method of claim 1, where at least a portion of the mother liquor obtained in step c) is subjected to a crystallization of 2′-fucosyllactose by inducing conditions of a controlled supersaturation in the mother liquor.

15. The method of claim 14, where at least a portion of the mother liquor is mixed with the solution of the 2′-fucosyllactose raw material prior to carrying out step b).

16. The method of claim 1, which comprises a first crystallization step and a second crystallization step, where the aqueous solution of the 2′-fucosyllactose raw material provided in step a) is subjected to a crystallization of the second crystallization step, where in the second crystallization step the crystallization of 2′-fucosyllactose is effected by inducing conditions of a controlled supersaturation in the solution according to step b), where the aqueous suspension of the crystalline 2′-fucosyllactose obtained in the second crystallization step is subjected to a solid-liquid separation to obtain a crystalline 2′-fucosyllactose and a mother liquor, where the mother liquor obtained in the solid-liquid separation is introduced into the first crystallization step.

17. The method of claim 16, where the crystalline 2′-fucosyllactose obtained in the first crystallization step is dissolved in the aqueous solution of the 2′-fucosyllactose raw material prior to carrying out the second crystallization step.

Description

DESCRIPTION OF THE FIGURES

(1) FIG. 1 shows a basic flow chart of the process according to the invention.

(2) FIG. 2 shows one embodiment of a forced circulation crystallizer.

(3) FIG. 3 shows another embodiment of a forced circulation crystallizer, in this case a draft baffle crystallizer.

(4) FIG. 4 shows an embodiment of induced forced circulation crystallizer.

(5) FIG. 5 shows a block diagram of an embodiment of a multi-stage process according to the invention.

(6) FIG. 6 shows a block diagram of a second group of embodiments of a multi-stage process according to the invention.

(7) FIG. 7 shows schematically one crystallization stage according to the invention.

(8) FIG. 8 shows schematically a two stage crystallization process according to the first group of embodiments of the invention.

(9) FIG. 9 shows schematically a two stage crystallization process according to the second group of embodiments of the invention.

(10) In the figures, the following reference symbols are used:

(11) C crystalline phase/crystals

(12) CR Crystallization

(13) D Discharge

(14) DU Dilution unit

(15) F Feed

(16) L Liquor

(17) ML mother liquor

(18) MLR recycled mother liquor

(19) P Product

(20) R recycled suspension

(21) RL residual liquor

(22) S fresh solution

(23) SLS solid/liquid separation

(24) V Vapor

(25) W condensed vapor (liquid water)

(26) WL wash liquid

(27) i index for the stage

(28) 1 Crystallizer

(29) 2 heat exchanger

(30) 3 Separator

(31) 4 circulation pump

(32) 5 concentrate pump

(33) 6 compressor for vapor

(34) 10 Inlet

(35) 11 slurry withdrawal

(36) 12 suspension outlet

(37) 13 liquid withdrawal/overflow

(38) 14 draft tube

(39) 15 Demister

(40) 16 vapor outlet

(41) 17 settling zone

(42) 18 Agitator

(43) 19 Inducer

(44) 20 vapor separation zone

(45) 21 active volume

(46) As illustrated in FIG. 1, a fresh stream S containing an aqueous solution of the 2′-FL raw material is combined with a recycle stream R and heated in a heat exchanger 2 to a temperature of at least 40° C., for example in the range of from 40° C. to 95° C., to give an aqueous solution of the 2′-FL raw material as feed stream F. The heat exchanger 2 can be arranged either horizontally or vertically depending on the specific requirements. The feed F is then fed to a continuously operated crystallizer 1. The crystallizer 1 contains as active volume an aqueous supersaturated suspension of 2′-FL with a content of solid 2′-FL of 5% to 50% by weight, for example from 20% to 40% by weight, based on the weight of the suspension. Feeding the under-saturated aqueous solution of 2′-FL raw material F into the active volume and removing water at the same time, the concentration of 2′-FL in the over-saturated suspension, i.e. in the active volume of the crystallizer 1 is levelled off. The controlled supersaturation of 2′-FL in the aqueous suspension is effected at a temperature of at least 25° C., for example in the range of from 30° C. to 95° C., depending from the desired polymorph of 2′-FL, and at reduced pressure, for example in the range of from 20 mbar to 800 mbar.

(47) Water is removed from the aqueous suspension of 2′-FL by evaporation, the water vapor V being withdrawn at the head from the crystallizer 1. The vapor V can be further conveyed via a compressor 6 to heat the heat exchanger 2, conducted for example in countercurrent to the feed F to be heated, and leaving the heat exchanger 2 as condensate W.

(48) A discharge D of the slurry containing crystalline 2′-FL is removed at the lower end from the crystallizer 1. From the discharge D, a partial stream is taken as recycle stream R and conveyed via a recycling pump 4 to be mixed with the fresh stream S before, on or after entry into the heat exchanger 2. The discharge D will be portioned 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 in the range of from 40:1 to 60:1.

(49) The other part of the discharge D is routed by means of a concentrate pump 5 to a separator 3. In the separator 3, the slurry D is separated to obtain mother liquor ML and crystalline 2′-FL as product P. If desired, the mother liquor ML can be recycled to the inventive process or a preceding stage.

(50) Alternatively, a discharge D of the slurry containing crystalline 2′-FL is removed on the side of the lower end from the crystallizer 1. The discharge D is routed by means of a concentrate pump 5 to a separator 3. In the separator 3, the slurry D is separated to obtain mother liquor ML and crystalline 2′-FL as product P. If desired, the mother liquor ML can be recycled to the inventive process or a preceding stage. A second discharge is removed as recycle stream R in the center part of the lower end from the crystallizer 1. The recycle stream R is conveyed via a recycling pump 4 to be mixed with the fresh stream S before, on or after entry into the heat exchanger 2. The mass ratio of the recycle stream R to the fresh stream S is greater than 5, in particular greater than 10, greater than 20, for example in the range of from 40:1 to 60:1. This alternative withdrawal of two different slurries can prove in particular advantageous if the slurry D taken at the side of the crystallizer is thicker or contains crystals of a different size distribution than the slurry R taken at the bottom of the crystallizer 1.

(51) The crystallization may be preferably effected in a continuously operated crystallizer, for example a forced circulation crystallizer, a draft tube crystallizer or a draft tube baffled crystallizer, or in particular in an induced forced circulation crystallizer.

(52) FIG. 2 shows a draft tube crystallizer. Superheated aqueous solution of the 2′-FL raw material F is fed to the crystallizer 1 via an inlet 10, flows upward through a draft tube 14 and returns downward along the outer side of the draft tube 14.

(53) Water evaporated from the suspension in the active volume 21 rises as vapor V to the head of the crystallizer 1. The vapor V passes a vapor separation zone 20 and a demister 15 to remove liquid droplets and leaves the crystallizer 1 via a vapor outlet 16. The vapor V is further conveyed via a compressor 6 to heat the heat exchanger 2, conducted for example in countercurrent to the feed F to be heated, and leaving the heat exchanger 2 as condensate W. Around the active volume 21, a settling zone 17 may be arranged. Via a suspension outlet 12 in the lower region of the active volume 21, suspension R is removed and combined with the fresh solution S. The combined stream of R and S is recycled via a circulation pump 4 through a heat exchanger 2 as feed F into the crystallizer. The circulation pump 4 provides for the necessary agitation of the suspension mixed with the incoming solution F and effects the circulation of the suspension within the active volume 21.

(54) Via a slurry withdrawal 11 situated at the bottom of the crystallizer 1 below the active volume 21, slurry D is removed from the crystallizer 1. The withdrawn slurry D contains the desired crystalline 2′-FL.

(55) FIG. 3 shows a draft tube baffled crystallizer with forced circulation. Superheated aqueous solution of 2′-FL raw material F is fed to the crystallizer 1 via an inlet 10, flows upward through a draft tube 14 and returns downward along the outer side of the draft tube 14. A bottom entry agitator 18 provides for the necessary agitation of the suspension mixed with the incoming solution F at moderate energy consumption and effects the circulation of the suspension within the active volume 21.

(56) Water evaporated from the suspension in the active volume 21 rises as vapor V to the head of the crystallizer 1. The vapor V passes a vapor separation zone 20 and a demister 15 to remove liquid droplets and leaves the crystallizer 1 via a vapor outlet 16.

(57) Peripheral to the active volume 21, a settling zone 17 is arranged by means of baffles. In the settling zone 17, excess mother liquor L and/or fines can be withdrawn for further processing at an overflow 13 in the upper region of the settling zone 17. This basically clear liquor L can be recycled to the process to regulate the temperature and/or the concentration of the solution of 2′-FL at any stage.

(58) Via a suspension outlet 12 in the lower region of the settling zone 12, suspension R is removed and recycled to be mixed with the fresh feed stream S.

(59) Via a slurry withdrawal 11 situated below the settling zone 12, slurry D is removed from the crystallizer 1. The withdrawn slurry D contains the desired crystalline 2′-FL as product P.

(60) The induced forced circulation crystallizer shown in FIG. 4 operates similarly to the forced circulation crystallizers shown in FIGS. 2 and 3 as explained above. Different to the embodiment shown in FIG. 3, the induced forced circulation crystallizer works without any internal agitation device.

(61) Superheated aqueous solution of 2′-FL raw material F is fed to the crystallizer 1 via an inlet 10, flows upward through a draft tube 14 and returns downward along the outer side of the draft tube 14. Water evaporated from the suspension in the active volume 21 rises as vapor V to the head of the crystallizer 1. The vapor V passes a vapor separation zone 20 and a demister 15 to remove liquid droplets and leaves the crystallizer 1 via a vapor outlet 16.

(62) Peripherical to the active volume 21, a settling zone 17 is arranged. Liquor L is withdrawn at a liquid withdrawal 13 in the upper region of the settling zone 17. This basically clear liquor L is recycled via the circulation pump 4. Via a suspension outlet 12 below the settling zone 12, suspension R is removed and combined with the clear liquor L in an external circuit. Fresh solution S is fed to the recycled stream L before, simultaneously or after combination with stream R. The combined recycled stream is heated in a heat exchanger (not shown in the figure) and fed to the crystallizer 1 as feed F. Analogously to the embodiment shown in FIG. 2, the vapor V may be used to heat the heat exchanger 2.

(63) The throughput of the circulation pump 4 provides for the syphoning of the recycled suspension R and the necessary agitation of the suspension within the active volume 21. No further agitation devices are required, so that the crystals in the suspension are treated with the least possible strain.

(64) Via a slurry withdrawal 11 situated at the bottom of the crystallizer 1 below the active volume 21 and below the settling zone 12, slurry D is removed from the crystallizer 1. The withdrawn slurry D contains the desired crystalline 2′-FL as product P.

(65) In the multi-stage process according to FIG. 5, the crystallization is performed in n stages. It should be noted that stages 3 to n are optional stages. A feed F is introduced into a first crystallization stage (i=1). Solvent is removed from the first crystallization e.g. by way of evaporation. The suspension is separated into residual liquor RL and a first crystalline phase C.sub.1. The first crystalline phase C.sub.1 is passed into a second crystallization stage (i=2). Mother liquor from the second crystallization stage (i=2) is recycled into the first crystallization stage (i=1), e.g. by mixing it with water and using the mixture for dissolving the crystalline phase C.sub.1 obtained in the first crystallization stage. In each crystallization stage (i=2 to n), water is removed, e.g. by withdrawing it in the form of solvent vapor V and the suspension is separated into mother liquor ML and a crystalline phase C. The crystalline phase from each crystallization stage (i) is passed into the following crystallization stage (i+1). Mother liquor from each crystallization stage (i) is recycled into the previous crystallization stage (i−1), for example by mixing it with water and utilizing the mixture for dissolving the crystalline 2′-FL from the previous crystallization stage. A crystalline phase containing the desired 2′-FL crystals is withdrawn from the last stage n. The number of stages n depends on the desired quality of the crystals in respect of form, purity, flow characteristics and storage properties.

(66) In the multi-stage process according to FIG. 6, the crystallization is performed in n stages, the first stage (i=1) being a stripping section. It should be noted that stages 3 to n are optional stages. The flow is similar to the flow described in FIG. 5, but feed F is introduced between the stripping stage (i=1) and the second crystallization stage (i=2). In general, the process according to FIG. 6 gives higher yields of the desired product.

(67) A crystallization stage (i) according to FIG. 7 comprises an apparatus each for crystallization CR.sub.i and for solid/liquid separation SLS.sub.i. Apparatuses employed for the crystallization CR.sub.i are in general crystallizers suitable for crystalline suspensions such as stirred tank reactors, e.g. Swenson type crystallizers, forced circulation crystallizers, e.g. Oslo type reactors, draft tube reactors, draft tube baffled crystallizer (see FIG. 3), or induced forced circulation crystallizer (see FIG. 4). Apparatuses employed for the solid/liquid separation SLS.sub.i are in general centrifuges, decanters, filters, filter presses, or washing towers.

(68) The feed F.sub.i for each stage (i) comprises suspension containing the crystalline phase C.sub.i-1 from the previous stage (i-1) and/or fresh feed F, respectively, as well as recycled mother liquor MLR.sub.i. Distillate is withdrawn from the crystallization CR.sub.i in the form of solvent vapor V.sub.i. Subsequently, the suspension is separated in the solid/liquid separation SLS.sub.i into mother liquor ML.sub.i and a crystalline phase C.sub.i. The crystalline phase C.sub.i from each crystallization stage (i) can be passed as feed F.sub.i+1 into the following crystallization stage (i+1) or be withdrawn as product, respectively. One portion of mother liquor ML.sub.i from each crystallization stage (i) is recycled into the same stage as MLR.sub.i. The rest of mother liquor ML.sub.i from each crystallization stage (i) can be recycled into the previous crystallization stage (i−1) or be withdrawn, respectively.

(69) To enhance the purity of the product 2′-FL, washing liquid WL.sub.i can additionally be employed in the solid/liquid separation SLS.sub.i. As washing liquid WL.sub.i cold water or cold mother liquor of a subsequent crystallization stage (i+1) is preferably used.

(70) In FIG. 8, a two stage process similar to FIG. 6 is depicted. The feed F is introduced between the stripping stage (i=1) and the second crystallization stage (i=2) into a dilution unit DU, where the crystalline 2′-fucosyllactose C1 obtained in the first crystallization stage is dissolved in the feed, i.e. in the aqueous solution of the 2′-fucosyllactose raw material. Water is removed from the second crystallization stage e.g. as vapor V by way of evaporation. Thereby a suspension of 2′-fucosyllactose in the mother liquor is obtained, which is subjected to a solid-liquid separation SLS2 to obtain a mother liquor ML and the purified crystalline 2′-fucosyllactose C2. The mother liquor from the second crystallization stage (i=2) is recycled into the first crystallization stage (i=1). Water is removed from the first crystallization stage e.g. as vapor V by way of evaporation. Thereby a suspension of 2′-fucosyllactose in a mother liquor is obtained, which is subjected to a solid-liquid separation SLS1 to obtain a residual liquor RL, which is discarded, and the crystalline 2′-fucosyllactose C1.

(71) In FIG. 9, a two stage process similar to FIG. 5 is depicted. The feed F, i.e. the aqueous solution of the 2′-fucosyllactose raw material, is introduced into the first crystallization stage (i=1). Water is removed from the first crystallization stage e.g. as vapor V by way of evaporation. Thereby a suspension of 2′-fucosyllactose in the mother liquor is obtained, which is subjected to a solid-liquid separation SLS1 to obtain a residual liquor RL, which is discarded, and a purified crystalline 2′-fucosyllactose C1. The crystalline 2′-fucosyllactose C1 is dissolved in solvent S (water or in further feed) in a dilution unit DU. The thus obtained solution is passed into a second crystallization stage (i=2). Water is removed from the second crystallization stage e.g. as vapor V by way of evaporation. Thereby a suspension of 2′-fucosyllactose in the mother liquor is obtained, which is subjected to a solid-liquid separation SLS2 to obtain a mother liquor ML and the purified crystalline 2′-fucosyllactose C2. Mother liquor ML from the second crystallization stage (i=2) is recycled into the first crystallization stage (i=1), e.g. by mixing it with the feed F.

(72) Abbreviations:

(73) 2′-FL: 2′-O-fucosyllactose

(74) DiFL: difucosyllactose

(75) b.w.: by weight

(76) rpm: rotations per minute

(77) RT: Room temperature, i.e. about 22° C.

(78) Analytics:

(79) HPLC:

(80) Column: Spherisorb NH2 column (amine modified silica: particle size 3 μm, pore size 80 Å) length 250 mm, internal diameter 4.5 mm (Waters Corporation)

(81) Eluent: acetonitrile/water 82.5/17.5 v/v

(82) Detection: RID

(83) Parameters: flow rate 1.3 ml/min, T=35° C., pressure 112 bar, 5 μl injection volume

(84) Determination of water: The concentration of water was determined by Karl-Fischer titration.

(85) Dry matter content was determined by drying 2 g of the sample at 130° C. for 2 hours

(86) Filter cake resistance was calculated based on the measured volume flow of the filtrate in the pressure nutsche, the applied pressure and the filter area.

(87) Determination of crystalline form: Powder X-Ray Diffraction (PXRD)

(88) X-ray diffraction patterns were recorded with a Panalytical X′Pert Pro diffractometer (manufacturer: Panalytical) in reflection geometry (Bragg-Brantano) in the range from 2θ=3°-40° with increments of e.g. 0.017° and measurement time of 20 s/step using Cu-Kα radiation (1.54178 Å) at 25° C. The tube voltage was 45 kV and current 40 mA. The sample was placed in a silicon single crystal sample holder of 0.2 mm depth and flattened.

CRYSTALLIZATION EXAMPLES

(89) In examples 1 to 3 an aqueous solution of a 2′-FL raw material was used, which was obtained by fermentation and subsequent downstream processing including passing the fermentation broth through a bed of an ion exchange resin and concentration of the thus treated broth to a solids content of 61.1% by weight was used. The aqueous solution contained 52.5% by weight of 2′-FL and 8.6% by weight of mono- and oligosaccharides including lactose, DiFL and fucosyllactulose.

Example 1

(90) In a reaction flask equipped with a distillation bridge and a stirrer 100 g of the aqueous solution of the 2′-FL raw material was heated by means of a water bath to 50° C. (bath temperature). At a pressure of 30 mbar 19.42 g of water were distilled off resulting in a syrup containing 65% by weight of 2′-FL. The weight ratio of product (2′-FL) to water in the obtained syrup was 2.74:1. The vessel was expanded to ambient pressure and the resulting viscous solution was allowed to cool to 45° C. (bath temperature) and seeded with 0.05 g of crystalline Form II of 2′-FL obtained from a previous run. The mixture was stirred at 45° C. (bath temperature) for further 4 h, allowed to cool to RT and stirred for further 16 h. The thus obtained thick suspension was warmed to 35° C. (bath temperature) and stirred for 2 h at 35° C. at ambient pressure. The warm suspension was filtered through a heated suction filter (35° C.) and the filter cake was washed 4 times with each 10 ml of ethanol/water (80/20 w/w) and thereafter dried at 40° C. and 0.8 mbar for 12 h. Thereby, 38.9 g of crystalline material (yield 70.5%) having the following composition was obtained:

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

(92) In the obtained crystalline material 2′-FL was present essentially as form A, as determined by PXRD.

Example 2

(93) In a reaction flask equipped with a distillation bridge and a stirrer 200 g of the aqueous solution of the 2′-FL raw material was heated by means of a water bath to 55° C. (bath temperature). At a pressure of 30 mbar 31.65 g of water were distilled off resulting in a syrup containing 62.4% by weight of 2′-FL. The weight ratio of product (2′-FL) to water in the obtained syrup was 2.3:1. The resulting viscous solution was expanded to ambient pressure and allowed to cool to 45° C. (bath temperature) and seeded with 0.05 g of crystalline 2′-FL obtained from example 1 (Form A). The mixture was stirred at 45° C. (bath temperature) and ambient pressure for further 4 h and then cooled to 10°. A sample was taken which showed that form A of 2′-FL had been formed. Then 136.2 ml of acetic acid was added within 30 min while keeping the temperature at 10° C., such that the volume ratio of acetic acid and water was 3:1 v/v. The obtained suspension was stirred for 0.5 h at 10° C. at ambient pressure. The thus obtained suspension filtered through a suction filter and the filter cake was washed 3 times with each 10 ml of acetic acid/water (80/20 w/w) and thereafter dried at 40° C. and 0.8 mbar for 12 h. Thereby, 53.6 g (yield 49.1%) of crystalline material having the following composition was obtained:

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

(95) In the obtained crystalline material 2′-FL was present essentially as form A, as determined by PXRD.

Example 3

(96) In a reaction flask equipped with a distillation bridge and a stirrer 200 g of the aqueous solution of the 2′-FL raw material was heated by means of a water bath to 65° C. (internal temperature; 80° C. bath temperature). At a pressure of 250 mbar 38 g of water were distilled off resulting in a syrup containing 64.8% by weight of 2′-FL. The weight ratio of product (2′-FL) to water in the obtained syrup was 2.5:1. The resulting syrup was expanded to ambient pressure and allowed to cool to 50° C. (bath temperature). The suspension was stirred at 50° C. at ambient pressure (bath temperature) for further 3 h and then cooled to 20° C. and stirred for further 1 h at 20° C. Then 117 ml of acetic acid was added within 30 min while keeping the temperature at 20° C., such that the volume ratio of acetic acid and water was 3:1 v/v. The obtained suspension was stirred for 0.5 h at 10° C. The thus obtained suspension filtered through a suction filter and the filter cake was washed 3 times with each 15 ml of acetic acid/water (80/20 w/w) and thereafter dried at 40° C. and 0.8 mbar for 12 h. Thereby, 80.3 g (yield 75.6%) of crystalline material having the following composition was obtained:

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

(98) In the obtained crystalline material 2′-FL was present essentially as form II, as determined by PXRD

(99) As variations to example 3 above the following are possible: a. Instead of increasing the concentration of 2′-FL in the syrup above 60% by weight as detailed in example 3, it is also possible to use less concentrated syrups of 2′-FL with concentrations of only up to 50% by weight of 2′-FL. The results of the experiment performed starting from those less concentrated solutions is basically the same as in example 3. b. As a further possibility, the amount of acetic acid as used in example 3 can be increased in its amounts up to 3-fold, still leading to the basically same results as in example 3. c. As a further possibility, the solution could be seeded with any polymorphic form of 2′-FL and even with amorphous 2′-FL also yielding the basically same results.

(100) “Basically the same results” means that the absolute amounts of the purity of 2′-FL and the contents of the by-products vary to a very small extent, i.e. about less than 5% deviation from the experimental results of example 3.

(101) In example 4 an aqueous solution of a 2′-FL raw material was used, which was obtained by fermentation and subsequent downstream processing including passing the fermentation broth through a bed of an ion exchange resin and concentration of the thus treated broth to a solids content of 61.5% by weight was used. The aqueous solution contained 49.4% by weight of 2′-FL and 12.1% by weight of mono- and oligosaccharides including 0.8% of lactose, 0.4% of fucosyllactulose and 2.5% of DiFL.

Example 4

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

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

(104) In the obtained crystalline material 2′-FL was present essentially as form A, as determined by PXRD.

(105) In the following examples 5 to 8 and comparative examples C1 and C2 an aqueous solution of a 2′-FL raw material was used, which was obtained by fermentation and subsequent downstream processing including decolorization, microfiltration, ultrafiltration, demineralization and reverse osmosis. The 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 mono- and oligosaccharides including lactose and DiFL.

Example 5

(106) In a rotary evaporator the aqueous solution of a 2′-FL raw material was evaporated at 60° C. under reduced pressure to a dry matter content of about 73% by weight and a concentration of 2′-FL of about 59% by weight. 1517 g of the thus obtained solution were filled into a baffled tank and stirred at 60° C. (internal temperature). Then, 12 g of amorphous 2′-FL were added to this solution and formation of solids was observed. The thus formed suspension was stirred for 20 h at 60° C. (internal temperature).Then, the thus formed suspension was cooled within 1.5 h to 25° C. with stirring. The thus obtained suspension was then filtered without washing. Thereby, 467 g of a wet crystalline material having the following composition was obtained:

(107) Composition of the filter cake: 83% b.w. 2′-FL, 1.2% b.w. lactose, 0.9% b.w. fucosyllactulose, 2.1% b.w. DiFL and 9% b.w. water. In filter cake 2′-FL was present essentially as form II, as determined by PXRD. The calculated yield was 42% based on the fucosyllactose contained in the concentrated solution.

(108) The filtrate had the following composition: 49% b.w. 2′-FL, 1.4% b.w. fucosyllactulose, 2% b.w. lactose, 3.7% b.w. DiFL and 36% b.w. water.

(109) The filter cake resistance was 5×10.sup.12 Pas/m.sup.2.

(110) The same variations a and c as mentioned in the context of example 3 are here applicable as well, leading to the basically same results as in example 5.

Example 6

(111) In a rotary evaporator the aqueous solution of a 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 2′-FL of about 50% by weight. 1532 g of the thus obtained solution were filled into a baffled tank and stirred at 40° C. (internal temperature). Then, 5 g of crystalline 2′-FL (form II) were added to this solution and formation of solids was observed. The thus formed suspension was stirred for 19 h at 40° C. (internal temperature).Then, the thus formed suspension was evaporated at 40° C. under reduced pressure to a concentration of 2′-FL of 61% by weight and thereafter cooled within 1.0 h to 25° C. with stirring. The suspension was stirred for further 22 h at 25° C. The thus obtained suspension was then filtered without washing. Thereby, 772 g of wet crystalline material having the following composition was obtained:

(112) Composition of the filter cake: 77% b.w. 2′-FL, 1.1% b.w. lactose, 0.8% b.w. fucosyllactulose, 1.9% b.w. DiFL and 15% b.w. water. The calculated yield was 77% based on the 2′-FL contained in the concentrated solution. In filter cake 2′-FL was present essentially as form B, as determined by PXRD.

(113) The filtrate had the following composition: 36% b.w. 2′-FL, 2% b.w. fucosyllactulose, 3.1% b.w. lactose, 5% b.w. DiFL and 40% b.w. water.

(114) The filter cake resistance was 4×10.sup.11 Pas/m.sup.2.

Example 7

(115) In a rotary evaporator the aqueous solution of a 2′-FL raw material was evaporated at 40° C. under reduced pressure to a dry matter content of about 65% by weight and a concentration of 2′-FL of about 52% by weight. 1572 g of the thus obtained solution were filled into a baffled tank and stirred at 40° C. (internal temperature). Then, 26 g of crystalline 2′-FL (form A) were added to this solution and formation of solids was observed. The thus formed suspension was stirred for 1 h at 40° C. (internal temperature).Then, the thus formed suspension was evaporated at 40° C. under reduced pressure to a concentration of 2′-FL of 55% by weight followed by stirring for 12 h at 40° C. and subsequent cooling within 1.0 h to 25° C. with stirring. The suspension was stirred for further 7 h at 25° C. The thus obtained suspension was then filtered without washing. Thereby, a wet crystalline material was obtained, which contained 2′-FL as its form B, as determined by PXRD.

(116) After drying the filter cake for 2 days at 60° C. and 100 mbar crystalline material having the following composition was obtained:

(117) 90% b.w. 2′-FL, 0.5% b.w. lactose, 0.3% b.w. fucosyllactulose, 1.1% b.w. DiFL and 5.9% b.w. water. The calculated yield was 41% based on the 2′-FL contained in the concentrated solution. In the dry crystalline material 2′-FL was present essentially as form A, as determined by PXRD.

(118) The filtrate had the following composition: 45% b.w. 2′-FL, 1.3% b.w. fucosyllactulose, 1.9% b.w. lactose, 3.7% b.w. DiFL and 39% b.w. water.

(119) The filter cake resistance was 5×10.sup.11 Pas/m.sup.2.

Example 8

(120) The filtrates obtained in examples 5, 6 and 7 and water to a total of 1099 g were filled into a baffled tank and stirred at 40° C. The concentration of 2′-FL in this 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). Then, 8 g of crystalline 2′-FL (form A) were added to this solution and formation of solids was observed. The thus formed suspension was stirred for 2 h at 40° C. (internal temperature).Then, the thus formed suspension was evaporated at 40° C. under reduced pressure to a concentration of 2′-FL of 47% by weight followed by stirring for 1 h at 40° C. and subsequent cooling within 1.0 h to 25° C. with stirring. The suspension was stirred for further 72 h at 25° C. The thus obtained suspension was then filtered without washing. Thereby, a wet crystalline material was obtained, which contained 2′-FL as its form B.

(121) After drying the filter cake for 2 days at 60° C. and 100 mbar crystalline material having the following composition was obtained:

(122) 88% 2′-FL, 0.9% b.w. lactose, 0.5% b.w. fucosyllactulose and 1.7% b.w. DiFL. The calculated yield was 26% based on the 2′-FL contained in the concentrated solution. In the dry crystalline material 2′-FL was present essentially as form A, as determined by PXRD.

(123) The filtrate had the following composition: 40% b.w. 2′-FL, 2.1% b.w. fucosyllactulose, 3.2% b.w. lactose, 5.7% b.w. DiFL and 35% b.w. water.

(124) The filter cake resistance was 6×10.sup.11 Pas/m.sup.2.

Comparative Example C1

(125) In a distillation apparatus the aqueous solution of a 2′-FL raw material was evaporated at 60° C. under reduced pressure to a concentration of 2′-FL of about 42% by weight. 1663 g of the thus obtained solution were filled into a baffled tank and evaporated at 60° C. under reduced pressure to a dry matter content of 85% by weight and concentration of 2′-FL of 69%. Then spontaneous crystallization occurred and the viscosity of the suspension was too high to drain it off the baffled tank. A PXRD of the suspension showed that the 2′-FL was present essentially as form II.

Comparative Example C2

(126) In a distillation apparatus the aqueous solution of a 2′-FL raw material was evaporated at 50° C. under reduced pressure to a concentration of 2′-FL of about 50% by weight. 1607 g of the thus obtained solution were filled into a baffled tank and evaporated at 40° C. under reduced pressure to a dry matter content of 80% by weight and concentration of 2′-FL of 65%. Then spontaneous crystallization occurred and the viscosity of the suspension was too high to drain it off the baffled tank. A PXRD of the suspension showed that the 2′-FL was present essentially as form B.

(127) In the following examples 9 to 10 an aqueous solution of a 2′-FL raw material was used, which was obtained by fermentation and subsequent downstream processing including decolorization, microfiltration, ultrafiltration, demineralization and reverse osmosis. The 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 mono- and oligosaccharides including lactose and DiFL.

(128) The aqueous solution of a 2′-FL raw material was concentrated in a rotary evaporator under reduced pressure to a concentration of 2′-FL of about 52.1% by weight. The solution is termed “pre-evaporated feed” and was used in the following examples 9 and 10.

Example 9

(129) 1612 g of pre-evaporated feed were filled into a baffled tank and stirred at 60° C. (internal temperature). Water was evaporated under reduced pressure with stirring at 60° C. to a concentration of 62% be weight of 2′-FL. Then, 12 g of crystalline 2′-FL (form II), suspended in a small volume of the pre-evaporated feed, were added to this solution and formation of solids was observed. The thus formed suspension was stirred for 19 h at 60° C. (internal temperature) with stirring at 450 rpm. Then, the thus formed suspension was filtered without washing by using a heated pressure nutsche (60° C., pressure difference 0.5 bar, 230 s). Thereby, a wet crystalline material was obtained, which contained 2′-FL as its form II as evidenced by PXRD.

(130) The filter cake was dried for 1 day at 60° C. and 100 mbar. The obtained crystalline material had the following composition: 94.6% b.w. 2′-FL, 0.8% b.w. lactose, 2.1% b.w. DiFL. The calculated yield was 45% based on the 2′-FL contained in the concentrated solution.

(131) The same variations a and c as mentioned in example 3 are here applicable as well, leading to the basically same results as in example 9.

Example 10

(132) 1628 g of pre-evaporated feed were filled into a baffled tank and stirred at 40° C. (internal temperature). Water was evaporated under reduced pressure with stirring at 40° C. to a concentration of 63% be weight of 2′-FL. Then, 13 g of crystalline 2′-FL (form A), suspended in a small volume of the pre-evaporated feed, were added to this solution and formation of solids was observed. The thus formed suspension was stirred for 21 h at 40° C. (internal temperature) with stirring at 450 rpm. Then, the thus formed suspension was filtered without washing by using a heated pressure nutsche (40° C., pressure difference 0.5 bar, 403 s). Thereby, a wet crystalline material was obtained, which contained 2′-FL as its form B as evidenced by PXRD.

(133) The filter cake was dried for 1 day at 60° C. and 100 mbar. The obtained crystalline material had the following composition: 88% b.w. 2′-FL, 1.4% b.w. lactose, 4.3% b.w. DiFL. The calculated yield was 80% based on the 2′-FL contained in the concentrated solution. PXRD of the dried crystalline material showed that 2′-FL was essentially present as form A, as determined by PXRD.

Example 11

(134) An aqueous solution of a 2′-FL raw material was used, which was obtained by fermentation and subsequent downstream processing including passing the fermentation broth through a bed of an ion exchange resin and concentration of the thus treated broth to a 2′-FL content of 50.4% by weight. The aqueous solution additionally contained 0.9% by weight of lactose, 2.9 by weight of DiFL and 1.6% by weight of fucosyllactulose.

(135) In a reaction flask equipped with a distillation bridge and a stirrer 150 g of the aqueous solution of the 2′-FL raw material was heated by means of a water bath to 50° C. (bath temperature). At a pressure of 30-100 mbar water was distilled off resulting in a syrup containing 65% by weight of 2′-FL. The vessel was expanded to ambient pressure and the resulting viscous solution was allowed to cool to 45° C. (bath temperature) and seeded with 0.15 g of crystalline 2′-FL obtained from a previous run. The mixture was stirred at 45° C. (bath temperature) for further 4 h, allowed to cool to RT and stirred for further 30 h at RT. To the thus obtained thick suspension 84 g of glacial acetic acid were added and the mixture was stirred for further 3 h to complete crystallization. The suspension was filtered through a suction filter and the filter cake was washed 3 times with glacial acetic acid and thereafter dried at 40° C. and 1.0 mbar for 12 h. Thereby, 61 g of crystalline material having the following composition was obtained:

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

(137) In the obtained crystalline material 2′-FL was present essentially as form A, as determined by PXRD.