Method for manufacturing l-fucose powder by spray-drying and spray-dried l-fucose powder

Abstract

Disclosed is a spray-dried powder consisting essentially of L-fucose and an additive that enables spray-drying of L-fucose, said additive being maltodextrin or skimmed milk powder, a method of spray-drying L-fucose to obtain said spray-dried powder, and the use of the spray-dried powder for manufacturing nutritional compositions or pharmaceutical compositions.

Description

[0001] METHOD FOR MANUFACTURING L-FUCOSE POWDER BY SPRAY-DRYING AND SPRAY-DRIED L-FUCOSE POWDER

[0002] The present invention relates to a preparation of L-fucose. More specifically, the present invention relates of a solid preparation of L-fucose and to a method for manufacturing said solid preparation.

[0003] Background

[0004] L-fucose, also known as 6-deoxy-L-galactose, is a dietary monosaccharide having important roles in cellular biology. L-fucose is used in cells in a process called fucosylation to post-translationally modify proteins and regulate their function. Fucosylation is an essential process in normal organs, in the development of the immune system, and in homeostasis. Fucosylation has been found to be perturbed in a number of pathophysiological contexts such as cancer and Congenital Disorders of Glycosylation (CDGs).

[0005] A CDG affecting fucosylation leads to a developmental delay and to slightly coarse facial features in a patient. In a CDG wherein the amount or activity of GDP-L- Fucose Synthase (GFUS) and GDP-L-fucose levels is reduced, overall hypo- fucosylation in the patient's glycoproteins in serum, leukocytes, platelets and fibroblasts is found. Oral L-fucose supplementation normalized fucosylation of proteins within 4 weeks as measured in serum and leukocytes.

[0006] In another fucosylation-related CDG, clinical-grade L-fucose has been implemented as an experimental treatment of leukocyte adhesion deficiency-ll (LAD-II). Oral administration of L-fucose at least partially reverted symptoms of this pathology by increasing fucosylation, thus illustrating the clinical utility and the safety of L-fucose supplementation.

[0007] L-fucose also exerts various beneficial effects upon topical administration. These effects include skin-whitening, skin-moisturizing, and anti-aging effects. Thus, L- fucose has several potential applications in cosmetics and in medicine.

[0008] Microbial fermentation as for example disclosed in WO 2014 / 067696 A1 is considered an economically versatile manufacturing of L-fucose. In this process, L-fucose is released intracellularly GDP-L-fucose and exported into the fermentation broth. A scalable process for purifying L-fucose from the fermentation broth is provided in WO 2019 / 101629 A1 wherein the L-fucose is crystallized at the end of the purification process using organic solvents, or is subjected to wet granulation. Nevertheless, granulation is known to pose challenges due to high quality requirement of the formed granules in terms of content uniformity and physicochemical properties such as granule size, bulk density, porosity, hardness, moisture, compressibility, etc. together with physical and chemical stability of the compound.

[0009] In contrast to granulation, spray-drying provides good control of the size, shape and morphology of particles. In addition, spray-drying is a fast and energy-efficient technology. Therefore, a method of spray-drying L-fucose is demanded. However, it is known that spray-drying mixtures of low-molecular weight saccharides, such as monosaccharides, is a challenging task. Spray-drying low-molecular weight saccharides suffer from low recovery due to high deposition of these sugars to the walls of the spray-dryer and co-adhesion of dried saccharide particles. This stickiness of low-molecular weight sugars is due to their thermoplastic behavior when heated to a temperature where they change from a glassy state to a liquid-like rubbery state.

[0010] Wrzosek, K. et al. (Acta Chimica Slovaca, Vol. 6, No. 2, 2013, pp. 177-181) investigated optimal conditions of spray-drying of a fructooligosaccharide (FOS)-rich mixture prepared by enzymatic conversion of sucrose. It was revealed that stickiness upon spray-drying of a multi-compound mixture containing a ratio (by weight) of about 1.6 % fructose, 29.7 % glucose, 10.5 % sucrose, and 58.2 % FOS can be overcome when the mixture contains 25 or 50 %-wt., with respect to the dry weight of the mixture, maltodextrin.

[0011] It has surprisingly been found that L-fucose can be spray-dried when blended with a carrier in an aqueous solution prior to spray-drying.

[0012] Summary

[0013] In a first aspect, provided is a spray-dried powder consisting essentially of L-fucose and an additive. In a second aspect, provided is a method for spray-drying L-fucose.

[0014] In a third aspect, provided is the use of spray-dried powder consisting essentially of L-fucose and an additive for manufacturing a nutritional composition and / or a pharmaceutical composition.

[0015] In a fourth aspect, provided is a nutritional composition and / or a pharmaceutical composition containing a spray-dried powder which consists essentially of L-fucose and an additive.

[0016] Detailed description

[0017] According to the first aspect, provided is a spray-dried powder that consists essentially of L-fucose and an additive. The additive is a compound or component that enables spray-drying of L-fucose in that the additive inhibits or prevents stickiness of the L-fucose during spray-drying, at least in the amount or concentration of the L- fucose in an aqueous solution before being subjected to the spray-drying. Hence, it is understood that L-fucose and the additive are mixed prior to subjecting the L- fucose to spray-drying, and that the additive is present during spray-drying of the L- fucose, but not added to spray-dried L-fucose after said L-fucose has been spray- dried.

[0018] The term “consisting essentially of’ as used herein with respect to the composition of the spray-dried powder is understood such that the spray-dried powder does not contain any other compound besides L-fucose and the additive than those compounds constituting inevitable by-products originating from the manufacturing of the L-fucose and the additive, and which by-products are typically present in commercially available preparations of L-fucose and / or the additive. Thus, in certain embodiments, the spray-dried powder does not contain fructo-oligosaccharides (FOS) and / or galacto-oligosaccharides (GOS).

[0019] In some embodiments, the additive for enabling spray-drying of L-fucose is maltodextrin. The term “maltodextrin” designates two different families of glucose polymers, namely digestible maltodextrins and digestion-resistant maltodextrins. Maltodextrins are classified by a dextrose equivalent (DE) a number between 3 and 20 that corresponds to the number of free chain ends in a given maltodextrin sample. A lower DE value designates chains containing more glucose monomers whereas a higher DE value designates shorter glucose chain lenths.

[0020] In some embodiments, the maltodextrin is a digestible maltodextrin. Digestible maltodextrins are manufactured as white solids by chemical processing of plant starches such as corn starch, potatoe starch, rice starch, wheat starch, or cassava starch. They consist of D-glucose monomers that are connected primarily via a(1 ,4) glycosidic bonds to chains of variable length. Digestible maltodextrins are digested rapidly and provide glucose as energy source to the human body. Digestible maltodextrins are generally recognized as safe. In additional and / or alternative embodiments, the maltodextrin has a DE value of 19, 12 or 6.

[0021] In some embodiments, the additive for enabling spray-drying of L-fucose is skimmed milk powder. Skimmed milk powder is obtained by removing water from pasteurized skim milk, i.e. when almost all the milkfat has been removed from whole milk, by spray-drying. Most skimmed milk is created by spinning whole milk in a centrifuge so that the fat droplets separate out. Skimmed milk tends to contain about 0.1 % fat. Skimmed milk powder is low in fat, a source of protein, calcium, and vitamins. Aditional health benefits provided by consumption of skimmed milk includes better weight loss management, improved control of blood sugar levels, improved bone health, and reduced risk of diseases. Skimmed milk powder is classified according to the heat treatment used in its manufacture (high heat, medium heat, and low heat).

[0022] In certain embodiments, the spray-dried powder contains at least 10 %-wt. of L- fucose, at least 20 %-wt. of L-fucose, at least 30 %-wt. of L-fucose, or at least 35 %- wt. L-fucose. In additional and / or alternative embodiments, the spray-dried powder contains less than 40 %-wt. of L-fucose, less than 39 %-wt. of L-fucose, less than 38 %-wt. of L-fucose, less than 37 %-wt. of L-fucose, or less than 36 %-wt. of L-fucose. Although, generally speaking, a higher amount of L-fucose in the spray-dried powder would be desirable, it appered from the trials made by the inventors that the upper limit of an L-fucose content in the spray-dried powder is below 40 %-wt. In additional and / or alternative embodiments, the ratio of L-fucose : additive by %-wt. is in the range of from 10 : 90 to less than 40 : 60. In certain embodiments the ratio of L-fucose : additive by %-wt. is about 10 : 90, about 20 : 80, about 30 : 70, or about 35 : 65.

[0023] In certain embodiments, the L-fucose is present in the spray-dried powder in amorphous form, but not as crystalline material.

[0024] In additional and / or alternative embodiments, the spray-dried powder comprises water, wherein the content of water in the spray-dried powder is less than 4 %-wt. of the spray-dried powder, less than 3.5 %-wt., less than 3.3 %-wt., less that 3.0 %-wt., less than 2.5 %-wt., or even less than 2.2 %-wt. The water content of the spray- dried powder can be determined pursuant to various methods known to the skilled person in the art, but should be determined by Karl-Fischer titration as was done in the examples herein below.

[0025] According to the second aspect, provided is a method for manufacturing a spray- dried powder that consists essentially of L-fucose and an additive for enabling spray-drying of L-fucose. The method comprises the step of providing an aqueous solution containing a mixture of L-fucose and the additive. In certain embodiments, the additive is selected from the group consisting of maltodextrin and skimmed milk powder.

[0026] In additional embodiments, the L-fucose is obtained by microbial fermentation. For the fermentative production of L-fucose, a population of microbial cells that were metabolically engineered to synthesize L-fucose are cultured in a medium and under conditions that are permissive for the microbial cell to synthesize L-fucose, thereby producing L-fucose. Microbial fermentation for producing L-fucose is a biotechnological method that can be scaled up for industrial production of L-fucose.

[0027] In additional and / or alternative embodiments, the L-fucose is recovered from the culture medium to provide L-fucose that was synthesized by the genetically engineered microbial cells during fermentation in the desired and / or prescribed purity. The recovery of L-fucose comprises removal of the microbial cells from the culture medium by means of centrifugation and / or filtration to obtain a clarified culture medium. The clarified culture medium is then subjected to a series of purification steps to remove undesired impurities such as proteins, peptides, nucleic acid molecules, nucleotides, lipids, secondary metabolites, educts, metabolic byproducts and the like from the clarified culture medium thus becoming a process stream, and to provide an aqueous solution containing L-fucose in a desired and / or prescribed purity. Said aqueous solution typically contains the L-fucose in a concentration that is higher than the L-fucose concentration in the culture medium at the end of the fermentation step. The purification steps may include the steps of subjecting the clarified culture medium and / or the process stream to at least one step of cation exchange treatment, at least one step of anion exchange treatment, at least one step of electrodialysis, at least one step of treatment with activated carbon, at least one step of diafiltration, of microfiltration, of ultrafiltration and / or of nanofiltration, at least one concentration step, and / or at least one evaporation step.

[0028] Hence, in certain embodiments the method for manufacturing the spray-dried powder that consisting essentially of L-fucose and an additive enabling spray-drying of L-fucose comprises the step of culturing a population of microbial cells that were metabolically engineered to synthesize L-fucose are cultured in a medium and under conditions that are permissive for the microbial cells to synthesize L-fucose; and the step of recovering the thus synthesized L-fucose from the culture medium to provide an aqueous solution containing L-fucose.

[0029] In some embodiments, the aqueous solution contains at least 4 %-wt., at least 5 %- wt., at least 10 %-wt., at least 15 %-wt., at least 20 %-wt., at least 25 %-wt., at least 30 %-wt., 32 %-wt., 33 %-wt. or at least 35 %-wt., but equal or less than 50 %-wt., equal or less than 45 %-wt., equal or less 40 %-wt. L-fucose.

[0030] In additional and / or alternative embodiments, the aqueous solution containing the L- fucose before the additive is added contains the L-fucose in a purity of at least 80 %, at least 85 %, at least 90 %, at least 91 %, at least 92 %, at least 93 %, at least 94 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, or at least 99 %. Purity is declared as the mass of L-fucose in comparison to the total dry matter in the aqueous solution.

[0031] In an additional embodiment, the additive enabling spray-drying of L-fucose is added to the aqueoaus solution containing the recovered L-fucose. The additive is added to the aqueous solution in an amount that is necessary to provide an aqueous solution that contains L-fucose and the additive enabling spray-drying of L-fucose in the desired ratio. Thus, the method for manufacturing a spray-dried powder that consists essentially of L-fucose and an additive enabling spray-drying of L-fucose may also comprise a step of determining the cocentration of L-fucose in the aqueous solution containing the recovered L-fucose.

[0032] In some embodiments, the additive enabling spray-drying of L-fucose is added in an amount such that the ratio of L-fucose : additive in the aqueous solution by %-wt. of dry matter is from 10 : 90 to less than 40 : 60, e.g. 39 : 61 , 38 : 62, 37 : 63, 36 : 64, 35 : 65, 30 : 70 or 20 : 80. It is understood that the additive can be added to an aqueous solution containing L-fucose in solid form or in form of an aqueous solution that is blended with the aqueous solution containing the L-fucose.

[0033] The method for manufacturing a spray-dried powder that consists essentially of L- fucose and an additive enabling spray-drying of L-fucose further comprises the step of subjecting the aqueous solution containing the L-fucose and the additive enabling spray-drying of L-fucose to a spray dryer, and spray-drying the aqueous solution to obtain a powder consisting essentially of L-fucose and the additive.

[0034] The method for manufacturing a spray-dried powder that consisting essentially of L- fucose and an additive enabling spray-drying of L-fucose further comprises the step of collecting at least a portion of the spray-dried powder.

[0035] According to the third aspect, provided is the use of the spray-dried powder consisting essentially of L-fucose and an additive for manufacturing a nutritional composition and / or a pharmaceutical composition. Said additive is preferably selected from the group consisting of maltodextrins and skimmed milk powder.

[0036] The spray-dried powder is suitable for human consumption, because neither the L- fucose nor the additive is toxic. Hence, the spray-dried powder can be used as a or for the manufacturing of a nutritional composition or a pharmaceutical composition, wherein said composition is intended for oral administration. In some embodiments, the nutritional composition is an infant formula. In additional and / or alternative embodiments, the pharmaceutical composition is for use in treating fucosylation- related congenital diseases. Hence, according to the fourth aspect, provided is a nutritional composition and / or a pharmaceutical composition containing a spray-dried powder which consists essentially of L-fucose and an additive, preferably an additive selected from the group consisting of maltodextrins and skimmed milk powder.

[0037] The present invention will be described with respect to particular embodiments, but the invention is not limited thereto but only by the claims. Furthermore, the terms first, second and the like in the description and in the claims, are used for distinguishing between similar elements and not necessarily for describing a sequence, either temporally, spatially, in ranking or in any other manner. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments of the invention described herein are capable of operation in other sequences than described or illustrated herein.

[0038] It is to be noticed that the term “comprising”, used in the claims, should not be interpreted as being restricted to the means listed thereafter; it does not exclude other elements or steps. It is thus to be interpreted as specifying the presence of the stated features, integers, steps or components as referred to, but does not preclude the presence or addition of one or more other features, integers, steps or components, or groups thereof. Thus, the scope of the expression “a device comprising means A and B” should not be limited to devices consisting only of components A and B. It means that with respect to the present invention, the only relevant components of the device are A and B.

[0039] Reference throughout this specification to “one embodiment” or “an embodiment” means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, appearances of the phrases “in one embodiment” or “in an embodiment” in various places throughout this specification are not necessarily all referring to the same embodiment, but may. Furthermore, the particular features, structures or characteristics may be combined in any suitable manner, as would be apparent to one of ordinary skill in the art from this disclosure, in one or more embodiments. Similarly, it should be appreciated that in the description of exemplary embodiments of the invention, various features of the invention are sometimes grouped together in a single embodiment, figure, or description thereof for the purpose of streamlining the disclosure and aiding in the understanding of one or more of the various inventive aspects. This method of disclosure, however, is not to be interpreted as reflecting an intention that the claimed invention requires more features than are expressly recited in each claim. Rather, as the following claims reflect, inventive aspects lie in less than all features of a single foregoing disclosed embodiment. Thus, the claims following the detailed description are hereby expressly incorporated into this detailed description, with each claim standing on its own as a separate embodiment of this invention.

[0040] Furthermore, while some embodiments described herein include some but not other features included in other embodiments, combinations of features of different embodiments are meant to be within the scope of the invention, and form different embodiments, as would be understood by those in the art. For example, in the following claims, any of the claimed embodiments can be used in any combination.

[0041] Furthermore, some of the embodiments are described herein as a method or combination of elements of a method that can be implemented by a processor of a computer system or by other means of carrying out the function. Thus, a processor with the necessary instructions for carrying out such a method or element of a method forms a means for carrying out the method or element of a method. Furthermore, an element described herein of an apparatus embodiment is an example of a means for carrying out the function performed by the element for the purpose of carrying out the invention.

[0042] In the description provided herein, numerous specific details are set forth. However, it is understood that embodiments of the invention may be practiced without these specific details. In other instances, well-known methods, structures and techniques have not been shown in detail in order not to obscure an understanding of this description.

[0043] The invention will now be described by a detailed description of several embodiments of the invention. It is clear that other embodiments of the invention can be configured according to the knowledge of persons skilled in the art without departing from the true spirit or technical teaching of the invention, the invention being limited only by the terms of the appended claims.

[0044] Example 1: Evaluation of potential additives for spray-drying L-fucose

[0045] In a trial to assess the utility of different putative additives for enabling spray-drying of L-fucose, aqueous solutions containing certain putative additives but no L-fucose were subjected to spray-drying. For spray-drying, a Buchi spray dryer B-290 (Buchi Labortechnik GmbH, Essen, Germany) was used, and spray-drying was performed at an inlet temperature of 140 °C, an outlet temperature as indicated in Table 1 showing the results of this trial, a nozzle cleaner 3, and a pump speed set at 23 %.

[0046] Table 1 : Assessment of spray-drying putative carrier materials as additives for spray-drying L-fucose

[0047] Spray-drying of maltodextrin (Maltodextrin DE 19, Berco Arzneimittel Gottfried Herzberg GmbH, Kleve, Germany) and skimmed milk powder (Uelzena eG, Uelzen, Germany) did not cause any problems. Very fine powders were obtained which neither exhibited clumping nor electrostatic charges.

[0048] Based on these results, maltodextrin and skimmed milk powder were considered suitable as additives for spray-drying L-fucose to obtain amorphous, pulverulent L- fucose. Example 2: Assessing L-fucose : additive ratio for spray-drying

[0049] In this trial, maltodextrin was chosen as additive for spray-drying L-fucose to investigate suitable ratios of L-fucose and additive in spray-drying L-fucose.

[0050] Crystalline L-fucose (purity 97 %; Chr. Hansen HMO GmbH, Rheinbreitbach, Germany) and maltodextrin 19 (Berco Arzneimittel Gottfried Herzberg GmbH, Kleve, Germany) were dissolved in water in different ratios, and the resulting aqueous solutions were subjected to spray-drying using a Buchi spray dryer B-290 (Buchi Labortechnik GmbH, Essen, Germany). Spray-drying was performed at an inlet temperature of 140 °C, an outlet temperature as indicated in Table 2 showing the results of this trial, a nozzle cleaner 3, and a pump speed set at 23 %.

[0051] Table 2: Results of evaluating different ratios of L-fucose and maltodextrin in spray-drying of L-fucose.

[0052] The spray-dried powders obtained in trials 1 to 3 were fine, free flowing powders without any crystalline structures. The powders did not show any clumping nor were any electrostatic charges observed. The colors of all powders were white to slightly ivory.

[0053] T rial 4 provided a dry powder too. However, the yield of product was significantly lower than the yields in trials 1 to 3, because of a higher degree of powder being attached to the inner wall of the spray-dryer. The powder had a white to slight ivory color too, but was found to contain crystalline structures and exhibited notable stickiness and clumping. Example 3: Adapting spray-drying of L-fucose to manufacturing processes

[0054] For adapting spray-drying of L-fucose to an industrial scale production process, a concentrate of L-fucose (purity 97 %) made by fermentation as described in WO 2016 / 120448 A1 and WO 2019 / 101629 A1 was used. The different additives were dissolved in samples of the L-fucose concentrate (dry matter = 18.76 %-wt.), and the mixtures were subjected to spray-drying using a Buchi spray dryer B-290 (Buchi Labortechnik GmbH, Essen, Germany). Spray-drying was performed at an inlet temperature of 140 °C, an outlet temperature as indicated in Table 3 showing the results of this trial, a nozzle cleaner 3, and a pump speed set at 23 %.

[0055] Table 3: Results of trials assessing utility of different ratios of L-fucose and additive in spray-drying L-fucose (MDX = maltodextrin 19; SMP = skimmed milk powder)

[0056] No problems were encountered during spray-drying in trials 1 , 2, 4, and 5. The spray-dryings resulted in powders showing fine and fluffy structures. Neither clumping nor electrostatic charges were observed.

[0057] In trial 3, a hard and crystalline powder exhibiting low flowability but high stickiness was obtained. Due to the high degree of powder sticking to the inner wall of the spray-dryer, the yield of product recovered was low as compared to the recovery in trials 1 , 2, 4, and 5.

Claims

CLAIMS1 . A spray-dried powder consisting essentially of L-fucose and an additive for enabling spray-drying of L-fucose, wherein said additive is selected from the group consisting of maltodextrin and skimmed milk powder.

2. The spray-dried powder according to claim 1 , wherein the spray-dried powder contains at least 10 %-wt. of L-fucose, at least 20 %-wt. of L-fucose, at least 30 %-wt. of L-fucose, or at least 35 %-wt. L-fucose.

3. The spray-dried powder according to claim 1 or 2, wherein the spray-dried powder contains less than 40 %-wt. of L-fucose, less than 39 %-wt. of L- fucose, less than 38 %-wt. of L-fucose, less than 37 %-wt. of L-fucose, or less than 36 %-wt. of L-fucose.

4. The spray-dried powder according to any one of claims 1 to 3, wherein the ratio of L-fucose : additive by %-wt. is in the range of from 10 : 90 to less than 40 : 60.

5. The spray-dried powder according to any one of claims 1 to 4, wherein the L- fucose is present in amorphous form.

6. The spray-dried powder according to any one of claims 1 to 5, wherein the spray-dried powder comprises a water content of less than 4 %-wt.

7. A method for manufacturing a spray-dried powder that consisting essentially of L-fucose and an additive enabling spray-drying of L-fucose, the method comprising the steps of- providing an aqueous solution containing a mixture of L-fucose and the an additive for enabling spray-drying of L-fucose, wherein the additive is selected from the group consisting of maltodextrin and skimmed milk powder;- subjecting the aqueous solution to a spray dryer;- spray-drying the aqueous solution to obtain a powder consisting essentially of L-fucose and the additive; and- collecting at least a portion of the powder.

8. The method according to claim 7, wherein the aqueous solution contains the L-fucose in an amount of at least 4 %-wt.

9. The method according to claim 7 or 8, wherein the weight ratio of L-fucose and additive in the aqueous solution is from about 10 : 90 to less than 40 :

60.

10. The method according to any one of claims 7 to 9, wherein the L-fucose is obtained by microbial fermentation in that a population of microbial cells that were metabolically engineered to synthesize L-fucose are cultured in a medium and under conditions that are permissive for the microbial cell to synthesize L-fucose.

11. The method according to claim 10 wherein the L-fucose is recovered from the medium.

12. The method according to claim 11 , wherein the L-fucose being recovered from the culture medium has a purity of at least 80 %.

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