CN118102891A

CN118102891A - Method for providing amorphous HMO product by drying

Info

Publication number: CN118102891A
Application number: CN202280069621.8A
Authority: CN
Inventors: C·Y·拉森; P·沙萨捏; M·诺沃提尼; J·T·赫尔曼斯佩格; A·M·C·詹斯; P·坦纳
Original assignee: DSM IP Assets BV
Current assignee: DSM IP Assets BV
Priority date: 2021-10-18
Filing date: 2022-10-18
Publication date: 2024-05-28

Abstract

A process for providing a solid amorphous HMO product, the process comprising drying an aqueous composition of the HMO. The solid amorphous HMO product is obtained by the process, as well as the solid amorphous HMO product itself.

Description

Method for providing amorphous HMO product by drying

Technical Field

A process for providing a solid amorphous HMO product is provided.

Background

Human Milk Oligosaccharides (HMOs) are heterogeneous mixtures of soluble glycans found in human milk. They are the third most abundant solid component after human milk has relayed lactose and lipids, at a concentration of 5-25g/l (Bode: human milk oligosaccharides and their beneficial effects, in Handbook of DIETARY AND nutritional aspects of human breast milk (Zibadi et al), pp.515-31,Wageningen Academic Publishers (2013).

Human Milk Oligosaccharides (HMOs) have attracted considerable interest in the last few years due to their important function in human development. To date, the structure of about 130 HMOs has been established (see Urashima et al :Milk Oligosaccharides,Nova Biomedical Books,New York,2011,ISBN:978-1-61122-831-1;Chen Adv.Carbohydr.Chem.Biochem.72,113(2015)), and considerably more substances may be present in human milk.

There is a constant search for a low cost way to industrialize as much HMO as possible so that researchers around the world can find, develop and utilize its use in nutritional and therapeutic formulations for infants and possibly children and adults. Some HMOs have recently been chemically synthesized in high yields, while other methods employ fermentation techniques using cultured microorganisms.

Regardless of the manner in which HMO is produced, the final step is to obtain purified HMO in solid form prior to packaging or formulation (e.g., using HMO to make infant formula). Some HMOs may crystallize, while others may only be provided as amorphous solids. However, HMOs are typically isolated as amorphous solids, typically separated from their aqueous solutions by removal of water in a drying step.

One of the drying methods is direct (or convection) drying, in which a hot process gas (air, inert gas or a mixture thereof) is applied to carry off the steam as moisture. Typical examples of direct drying commonly used to obtain amorphous HMO powder are spray drying (see e.g. WO2015/106943, WO 2019/160922), wherein a liquid solution of HMO to be dried is atomized into tiny droplets and the droplets are mixed in direct contact with hot gas of a drying medium, the moisture is evaporated, and the dried product is then collected by gas-solid separation. Although frequently used, some disadvantages are faced: the heat efficiency is not high, the energy consumption is high, two-stage dust removal is generally needed, and the requirement on drying gas/air is high. Another direct drying method is belt drying, which can provide a dry crystalline material instead of a dry amorphous powder (WO 2021/186258).

In indirect (or contact) drying, heat is applied through the solid surface/wall to dry a concentrated aqueous solution of HMO (see e.g. WO2021/094459, WO 2021/155157). Also, the process can easily provide a crystalline end product.

It is an object of the present invention to provide a process for providing a solid amorphous HMO product, preferably by indirect drying, which is versatile, gentle, economical, easy to scale up, easy to handle and suitable for industrial use.

Disclosure of Invention

In a first aspect, the present invention relates to a process for drying an aqueous composition of one or more Human Milk Oligosaccharides (HMOs) and thereby providing an amorphous powder product comprising solids of said one or more HMOs, the process comprising:

a) Providing the dried amorphous powder comprising the one or more HMOs in a dryer suitable for mechanically agitating the dried amorphous powder, to obtain a dry agitated amorphous powder, preferably the dryer is designed to produce the largest possible dryer wall contact of the powder by mechanical mixing,

B) Feeding one or more aqueous compositions of HMOs into, onto or onto the dry stirred amorphous powder to obtain a wet stirred mixture of amorphous powders,

C) Water is removed from the wet stirred mixture of amorphous powder at elevated temperature and/or vacuum while an aqueous solution of one or more HMOs is added continuously or sequentially to the wet stirred mixture of amorphous powder.

In a second aspect, the present invention relates to a dried solid amorphous HMO product comprising one or more HMOs obtained or obtainable by the process according to the first aspect of the invention.

Also provided are food products, particularly nutritional formulations, such as infant nutritional formulations, comprising the dry solid amorphous HMO product described herein.

Further features of the invention will become apparent from the following description, numbered aspects, embodiments and claims.

Detailed Description

The inventors elaborated on a particularly advantageous drying method to dry an aqueous composition, such as an aqueous solution or suspension, comprising one or more HMOs, thereby providing a dry solid amorphous HMO powder. The aqueous composition of one or more HMOs is dried in an indirect manner, wherein heat is emitted from the dryer wall and is conducted mainly through the mass of dried HMO powder pre-filled in the dryer and is conducted during the drying process together with the mass of dried HMO powder. Thus, the aqueous composition is dried on or within the previously dried powder. The efficiency of the drying process may be enhanced by the application of vacuum.

Accordingly, a first aspect of the present invention relates to a process for drying an aqueous composition comprising HMO and thereby providing a dried amorphous powder of HMO, the process comprising:

a) Providing the dried amorphous powder comprising said HMO in a dryer suitable for mechanically stirring the dried amorphous powder, to obtain a dry stirred amorphous powder, preferably designed to produce the largest possible dryer wall contact of the powder by mechanical mixing,

B) Feeding an aqueous composition of HMO into, onto or onto the dry stirred amorphous powder to obtain a wet stirred mixture of amorphous powder,

C) Water is removed from the wet stirred mixture of amorphous powder at elevated temperature and/or vacuum while the aqueous composition of HMO is added continuously or sequentially to the wet stirred mixture of amorphous powder.

The drying methods disclosed herein have a number of advantages over prior art direct drying methods (e.g., spray drying) or contact drying methods (e.g., drum drying or roller drying). Most importantly, the aqueous composition, preferably aqueous solution, of HMO to be dried is added, preferably sprayed, into, onto or onto the dried amorphous powder of HMO pre-filled in the dryer, rather than directly to the heated walls of the dryer. In this regard, the drying process is subtle, no product layer forms on the dryer walls, and significant degradation/browning of HMOs can be avoided. Furthermore, unlike other contact drying methods such as drum drying, the method consistently provides a dry amorphous powder. Furthermore, since the process is operated in a closed apparatus, the high hygiene standards required for food or food additive production, in particular for infant formula production, are met. Furthermore, no metal abrasion (relative to drum drying) occurs and no process gas/air treatment (relative to spray drying) is required, thereby reducing the overall carbon footprint of the process. The hybrid dryer may be compact due to efficient heat transfer. The drying process according to the invention effectively removes water in a gentle manner, which makes the process particularly suitable for drying heat-sensitive substances. Due to the delicate drying process, the dried material does not stick to the dryer walls and the dryer is easy to discharge and clean.

The methods described herein use an aqueous composition comprising one or more HMOs. The aqueous composition may be an aqueous solution or an aqueous suspension, preferably an aqueous solution, of one or more HMOs.

The aqueous composition suitably comprises one or more HMOs, for example two or more HMOs, or three or more HMOs, or four or more HMOs. The aqueous composition may even comprise 5,6 or more HMOs. Advantageously, the HMO or HMOs are the same as those contained in the pre-filled dry amorphous HMO powder.

The aqueous composition may comprise additional components, for example, salts, pH adjusting agents or solubilising agents. The pH of the aqueous composition is suitably between 3 and 7, preferably between 4 and 6.

The one or more HMOs in the pre-filled dry amorphous powder as well as in the aqueous composition may comprise additional non-HMO carbohydrates such as lactose, lactulose, monosaccharides (e.g. glucose, galactose, fucose, sialic acid). The total amount of non-HMO carbohydrates is advantageously not more than 10wt% (calculated on the basis of non-aqueous solids), preferably 8wt%. The total amount of non-HMO carbohydrates excluding lactose is advantageously not more than 2wt% (calculated on the non-aqueous solids). Thus, the HMO content of the powder obtainable by the present process will be the same as the HMO content of the pre-filled dry amorphous powder and the aqueous composition.

In the process according to the invention, the drying step is suitably carried out until the moisture content in the solid HMO product is below 10%, preferably below 8%, more preferably below 6%, for example below 4% as low as 1-3%.

The human milk oligosaccharides referred to in the art are synthetic, i.e. produced by chemical or biochemical processes in vitro or in vivo. The synthetic HMO used in the methods and products of the invention may be selected from one or more of LNT, LNnT, 2'-FL, 3-FL, DFL, LNFP I, LNFP II, LNFP III, LNFP V, LNFP VI, 3' -SL, 6'-SL, FSL, LST a, LST b, LST c, and DS-LNT, preferably from one or more of LNT, LNnT, 2' -FL, 3-FL, DFL, LNFP I, 3'-SL, and 6' -SL.

Synthetic HMOs may be neutral or acidic (sialylated).

The term "neutral human milk oligosaccharide" refers to the non-sialylated (and therefore neutral) complex carbohydrate found in human milk (Urashima et al: milk oligosaccharides, nova Biomedical Books,2011;Chen Adv.Carbohydr.Chem.Biochem.72,113 (2015)) which comprises a core structure with a reducing end of a lactose unit, which a) is substituted with one or two α -L-fucopyranosyl moieties, b) is substituted with a galactosyl residue, or c) is extended via its 3' -OH group by an N-acetylglucosamine, a lacto-N-disaccharide (galβ1-3 GlcNAc) or an N-acetyllactosamine (galβ1-4 GlcNAc) moiety. Derivatives containing N-acetyllactosamine may be further substituted with N-acetyllactosamine and/or lacto-N-disaccharides (lacto-N-disaccharides always have non-reducing ends). Derivatives containing N-acetyllactosamine and lacto-N-disaccharides may optionally be substituted with one or more alpha-L-fucopyranosyl moieties.

Examples of neutral trisaccharides HMO include 2 '-O-fucosyllactose (2' -FL, fucα1-2Galβ1-4 Glc), 3-O-fucosyllactose (3-FL, galβ1-4 (Fucα1-3) Glc) or milk-N-triose II (GlcNAcβ1-3Galβ1-4 Glc); examples of neutral tetrasaccharides HMO include 2', 3-di-O-fucosyllactose (DFL, fucα1-2Galβ1-4 (Fucα1-3) Glc), lacto-N-tetrasaccharide (LNT, galβ1-3GlcNAcβ1-3Galβ1-4 Glc) or lacto-N-neotetrasaccharide (LNnT, galβ1-4GlcNAcβ1-3Galβ1-4 Glc); examples of neutral pentasaccharide HMOs include milk-N-fucose pentasaccharide I (LNFP I, fucα1-2galβ1-3glcnacβ1-3galβ1-4 Glc), milk-N-fucose pentasaccharide II (LNFP II, galβ1-3 (fucα1-4) glcnacβ1-3galβ1-4 Glc), milk-N-fucose pentasaccharide III (LNFP III, galβ1-4 (fucα1-3) glcnacβ1-3galβ1-4 Glc), milk-N-fucose pentasaccharide V (LNFP V, galβ1-3GlcNAcβ1-3Galβ1-4 (Fucα1-3) Glc), lacto-N-fucopyranose VI (LNFP VI, galβ1-4GlcNAcβ1-3Galβ1-4 (Fucα1-3) Glc); examples of neutral hexasaccharide HMOs include lacto-N-difucose hexasaccharide I (LNDFH I, fucα1-2Galβ1-3 (Fucα1-4) GlcNAcβ1-3Galβ1-4 Glc), lacto-N-difucose hexasaccharide II (LNDFH II, galβ1-3 (Fucα1-4)) GlcNAcβ1-3Galβ1-4 (Fucα1-3) Glc), lacto-N-difucose hexasaccharide III (LNDFH III, galβ1-4 (Fucα1-3) GlcNAcβ1-3Galβ1-4 (Fucα1-3) Glc), milk-N-hexose (LNH, galβ1-3GlcNAcβ1-3 (Galβ1-4GlcNAcβ1-6) Galβ1-4 Glc), p-milk-N-hexose (pLNH, galβ1-3GlcNAcβ1-3Galβ1-4 Glc), milk-N-neohexose (LNnH, galβ1-4GlcNAcβ1-3 (Galβ1-4GlcNAcβ1-6) Galβ1-4 Glc) or p-milk-N-neohexose (pLNnH, galβ1-4GlcNAcβ1-3Galβ1-4GlcNAcβ1-4Glcβ1-4 Glc).

The term "sialylated human milk oligosaccharide" refers to sialylated complex carbohydrates found in human milk (Urashima et al: milk oligosaccharides, nova Biomedical Books,2011;Chen Adv.Carbohydr.Chem.Biochem.72,113 (2015)) that contain a core structure that is lactose units at the reducing end, which may be a) substituted with an α -N-acetyl-neuraminic acid (sialic acid) moiety or b) extended with one or more β -N-acetyl-lactosamine groups and/or one or more β -lacto-N-disaccharide units, and whose core structure is substituted with an α -N-acetyl-neuraminic acid (sialic acid) moiety and optionally may be substituted with an αl-pyran fucosyl moiety. In this regard, the acidic HMO has at least one sialic acid residue in its structure.

Examples of acidic HMOs include 3' -sialyllactose (3 ' -SL), 6' -sialyllactose (6 ' -SL), 3-fucosyl-3 ' -sialyllactose (FSL), LST a, fucosyl-LST a (FLST a), LST b, fucosyl-LST b (FLST b), LST c, fucosyl-LST c (FLST c), sialyl-LNH (SLNH), sialyl-lacto-N-hexasaccharide (SLNH), sialyl-lacto-N-neohexasaccharide I (SLNH-I), sialyl-lacto-N-neohexasaccharide II (SLNH-II) and disialyl-lacto-N-tetrasaccharide (DS-LNT).

Different mixtures of enzymatically produced HMOs are described in earlier patent applications WO2016/199071, WO2017/221208, WO2017/103850, WO2016/157108, WO2016/063262, which are incorporated by reference.

Any artificial mixture of HMOs synthesized by any available method is also included within the scope of the present invention.

In step a) of the present invention, the dried amorphous powder comprising one or more HMOs is provided in a dryer suitable for mechanically stirring the dried amorphous powder, for example by means of a stirrer. The dried amorphous powder suitably has a moisture content that is desirably achieved at the end of the present drying process. The dried amorphous powder may be the product obtained after the drying process according to the invention, a spray dried amorphous powder, a freeze dried amorphous powder or an amorphous powder dried by any other method providing a substance in amorphous form. The dryer is suitably a mixing dryer or a stirred dryer, which is equipped with means with which the dried amorphous powder and the wet mixture of amorphous powder in step b) of the method can be subjected to efficient mechanical stirring/mixing/stirring, such as shovels, propellers, turbines, paddles, helical stirring blades, etc. Preferably, the mixing dryer is designed such that it is capable of producing the largest possible dryer wall contact of the powder by mechanical mixing. Thus, the term "dry stirred amorphous powder" means that the dried amorphous powder comprising one or more HMOs placed in a dryer is thoroughly mixed/stirred.

In step b) of the method according to the invention, an aqueous composition, preferably a solution, of HMO is added into, onto or to the dry stirred amorphous powder of HMO to obtain a wet stirred mixture of amorphous powder. Preferably, the HMO or HMOs contained in the aqueous composition are the same as those in the dry stirred amorphous powder.

The aqueous composition may be an aqueous solution or an aqueous suspension, preferably an aqueous solution, of one or more HMOs. The HMO content of the aqueous composition and whether a solution or slurry/suspension is formed depends on the solubility of the HMO in question. Preferably, the aqueous composition does not comprise a solvent other than water. In certain embodiments, the aqueous composition may comprise one or more solvents other than water; the solvent may be a water miscible solvent or a partially water miscible solvent, typically those that are useful for chemically producing and/or purifying HMO, or for use in HMO or one of its precursors in the manufacture of HMO, such as crystallization of C ₁-C₄ alcohol, acetone, acetic acid, and the like. In the aqueous composition of HMO, the combined concentration of solvents other than water is not more than 5w/w%, preferably not more than 4w/w%, more preferably not more than 3w/w%, even more preferably not more than 2w/w%, advantageously not more than 1w/w%. Although the concentration of HMO in the aqueous composition is not an important factor, in order to enhance the efficacy and feasibility of the process, it is preferred that the minimum concentration of HMO is at least 80%, preferably at least 90% with respect to the solubility of water (or optionally with respect to the water-solvent mixture). Preferably, the process is carried out at a saturated solution or at a concentration below saturation, but it is also conceivable to carry out on supersaturated metastable solutions or aqueous slurries.

The aqueous HMO composition is added to, onto, or onto the dry stirred amorphous HMO powder with continuous stirring/mixing, resulting in the formation of a wet stirred mixture of amorphous powder. Care should be taken to add the aqueous composition to the dry stirred amorphous powder so that no local deliquescence occurs and the aqueous composition is immediately absorbed or encapsulated by the dry stirred powder. Due to the continuous stirring, the water content of the aqueous composition is evenly distributed throughout the dry stirred amorphous powder, thereby forming a wet stirred powder, the resulting powder can still be sufficiently stirred, but its water content/humidity will be higher. Preferably, the wet stirred powder has a moisture/water content of not more than 15%, such as not more than 12%, preferably not more than 10%, such as about 6-8% or 4-6%. Advantageously, the aqueous composition is added in the form of tiny droplets, for example in the form of a spray or a spray. Thus, sticking, pilling or product sticking or skinning on the walls of the mixing dryer is largely avoided.

Preferably, in step b), the aqueous composition is an aqueous solution of HMO. In this case, the aqueous solution is sprayed into, onto or onto the prefilled stirred dry powder by means of compressed air through an atomizing nozzle. Or by adding the aqueous solution through a standard liquid addition lance, which does not require pressurized air to aid the addition, it is therefore more advantageous if at least steps b) and c) are operated under vacuum. Whichever type of nozzle is used, it is mounted in the mixer such that the liquid solution is mostly, preferably entirely sprayed or sprinkled into, onto or onto the pre-filled dry-stirred powder in step b) and the wet-stirred powder mixture in step c).

In step c), water is removed from the wet stirred powder, so that the walls of the mixer are heated while the wet powder is stirred. The skilled person is able to set or adjust the wall temperature to obtain the desired moisture content of the dried amorphous HMO product. Due to the efficient agitation, heat from the walls is then transferred uniformly to the dry powder mass and eventually to the wet powder. In order to effectively remove water at atmospheric pressure, the wet powder is maintained at a temperature of about 60-90 ℃, preferably 70-80 ℃, which requires a wall temperature set at about 80-140 ℃, more preferably about 90-120 ℃. The water removed from the wet stirred powder leaves the apparatus as a vapour through an outlet.

The method includes operating under vacuum as long as the mixing dryer is suitable for this. The method is performed under reduced pressure, advantageously avoiding problems associated with high temperatures. The vacuum mix dryer is then typically operated at a pressure of 20-100 mbar, for example 20-60 mbar, allowing water to evaporate at a much lower temperature, thus exposing the product to a lower temperature (40-80 ℃). Preferably, the process is carried out under such vacuum, maintaining the product temperature at 60-80 ℃. In addition to operating at lower temperatures, vacuum mix dryers are also more hygienic (as they operate in a closed environment) and are therefore more suitable for baby food production.

While water is removed from the wet stirred powder under the above conditions, an important aspect of step c) is to add the aqueous composition of HMO continuously or sequentially to the dry but still wet stirred mixture in the manner disclosed in step b) of the process as follows. Similarly, under continuous agitation, the water content of the aqueous composition is uniformly distributed throughout the dry but still wet-stirred amorphous powder. It is important to set the addition rate of the aqueous composition according to the drying capacity (water evaporation capacity) of the system, which depends on the rotational speed of the mixer, the temperature, the volume and optionally the vacuum, so as to maintain a dynamic balance between the water/moisture introduced with the aqueous composition and the water vapor left. In this regard, the amount of water introduced by the addition of the aqueous composition is about the same as the amount of water removed within a given time window. For example, the rate of addition of the aqueous composition is set based on continuously measured and monitored condensed water. Advantageously, the wet stirred powder has a moisture/water content of not more than 15%, for example not more than 12%, preferably not more than 10%, for example about 6-8% or 4-6%.

In one embodiment of the method, the dry stirred amorphous powder obtained in step a) is heated before adding the aqueous composition of HMO according to step b). For example, the dry powder is heated to about 60-90 ℃, preferably 70-80 ℃.

In a preferred embodiment, the aqueous composition comprising HMO to be added in step b) and/or step c) is heated to a temperature of 40-90 ℃, preferably 60-80 ℃, and fed into the mixer at that temperature. Advantageously, the dry stirred amorphous powder obtained in step a) and/or the wet stirred powder mixture obtained in step b) and/or c) are also maintained at the same temperature.

In one embodiment of the process, the dry powder fed into the mixer according to step a) is stirred at atmospheric pressure, and steps b) and c) are carried out under vacuum.

In one embodiment, steps a) and b) are performed at atmospheric pressure and step c) is performed under vacuum.

In one embodiment of the process, the dry powder fed into the mixer according to step a) is stirred under vacuum, for example 20-100 mbar or 20-60 mbar.

In other embodiments, the dry powder fed into the mixer according to step a) is stirred under vacuum, for example 20-100 mbar or 20-60 mbar, and the dry powder is warmed to about 60-90 ℃, preferably 70-80 ℃.

In other embodiments, step c) is operated under vacuum, preferably at 20-100 mbar or 20-60 mbar, while maintaining the wet stirred powder mixture obtained in step c) at about 60-90 ℃, preferably 70-80 ℃.

The above disclosed process, including preferred and more preferred embodiments thereof, produces a dried amorphous powder of one or more HMOs having a moisture content of less than 10%, preferably less than 8%, more preferably less than 6%, for example less than 4% down to 1-3%.

According to one embodiment, the above disclosed method is carried out in a vertical mixing dryer or a vertical vacuum mixing dryer. The vertical mixer dryer is equipped with a central mixing arm with mixing blades, such as helical mixing blades, to achieve efficient mixing and uniform heat distribution throughout the powder, thereby maximizing batch uniformity. The vertical mixer may be equipped with a rotary cutter which also helps homogenize the stirred powder, especially when agglomerates are formed locally.

According to step a), the vertical mix dryer is pre-filled with dry amorphous HMO powder. The amount of dry amorphous powder is selected so that the aqueous composition can be conveniently sprayed or sprinkled into, onto or onto the dry powder, and preferably the rotary cutter reaches the powder bed.

Once the dried amorphous powder of HMO is placed in the mixer and thoroughly stirred, the aqueous composition of HMO is sprayed or sprinkled as fine droplets into, onto or into the dry powder under constant stirring during step b), optionally at elevated temperature and/or vacuum as described above, and is continuously added, preferably continuously, throughout step c). Along with the continuous removal of the water vapor, the mass of the dry and wet powder mixture after stirring is also increased continuously. Advantageously step c) is continued until the volume of precipitated HMO powder reaches the maximum capacity of the mixing dryer. The addition of the aqueous solution is then terminated and water is continued to be removed until the desired water content of the dried powder is obtained. In this regard, operation of the process in a vertical hybrid dryer represents a batch drying mode.

A vertical mixer dryer equipped with a rotary cutter is suitable for obtaining the desired particle size range in addition to drying and homogenizing the dried powder. The speed of the rotary cutter may be varied to control the particle size distribution.

According to another embodiment, the above disclosed process is carried out in a horizontal contact dryer or a horizontal vacuum dryer. The horizontal dryer is equipped with a central rotating paddle system that can mix efficiently and distribute the heat evenly throughout the powder, thereby maximizing batch uniformity. The horizontal contact dryer is designed such that the feed inlet is at one end and the dried product exits the apparatus at the other end.

According to step a), the horizontal dryer is at least partially pre-filled with dry amorphous HMO powder. The dried amorphous HMO powder may be a product from a previous run. Once the dried amorphous powder of HMO is placed in a horizontal dryer and thoroughly stirred, the aqueous composition of HMO (preferably a solution) is sprayed or sprinkled as fine droplets into, onto or onto the dry powder under constant stirring during step b), optionally at elevated temperature and/or vacuum as described above, and is continuously added throughout step c). At the same time as the aqueous composition was added, the collection of dry powder was started. The agitated wet powder is transported through the dryer by paddles located on the rotating shaft while drying. The rotational speed of the shaft and/or the arrangement of the blades on the shaft affects the residence time. In this regard, the agitated wet powder mixture becomes increasingly dry as a function of distance from the inlet due to the movement of the agitated wet powder mixture from one end of the horizontal dryer to the other and the continuous water removal. Thus, the dried amorphous mixture exits the dryer through an outlet at the other end. The dried powder may be collected at the outlet as long as the aqueous composition is added through the inlet in step c). This mode of operation of the process in a horizontal dryer represents a continuous drying mode.

In a particular aspect, the one or more synthetic HMOs are produced by fermentation prior to the drying step described above.

Suitably, according to this aspect, the HMO fermentation broth, the enzymatic reaction environment or the synthesis reaction mixture is subjected to one or more purification steps prior to the drying step described above. The purification step performed on the fermentation broth, the enzymatic reaction environment or the synthesis reaction mixture may be selected from one or more of the following:

Removing solid material, for example from HMO synthesis environment, such as fermentation material (e.g. proteins and DNA) or enzymatically reacted material (proteins);

removal of salts and charged molecules (e.g. small DNA fragments, organic acids, peptides);

removing uncharged or uncharged materials (e.g. lipids, polysaccharides, e.g. endotoxins, colorants in fermentation);

-removing HMO precursors and/or byproducts;

-if a HMO mixture is produced, isolating HMO;

-removing excess water.

In some aspects, the one or more synthetic HMOs are produced by fermentation, and the fermentation broth is further processed to produce an aqueous composition comprising the one or more synthetic Human Milk Oligosaccharides (HMOs).

In one embodiment, the fermentation broth is treated prior to drying by the steps of:

A) Clarifying the fermentation broth to remove suspended particulates and contaminants, particularly cells, cellular components, insoluble metabolites and debris from the fermentation process; then

B) Removing substantially all proteins, as well as peptides, amino acids, RNA and DNA and any endotoxins and glycolipids that may interfere with subsequent purification steps from the aqueous solution obtained in step a).

In step A), the fermentation broth is clarified in a conventional manner, for example by centrifugation or filtration. Preferably, the aqueous medium is first flocculated and then centrifuged or filtered to remove any remaining insoluble particulates and contaminants, as well as cells and cell components and insoluble metabolites and debris.

In step B), proteins and related impurities are removed from the previously obtained aqueous medium in a conventional manner, e.g. by ultrafiltration, nanofiltration, tangential flow high efficiency filtration, tangential flow ultrafiltration, affinity chromatography, ion exchange chromatography, hydrophobic interaction chromatography, gel filtration, size exclusion chromatography and/or activated carbon treatment. If desired, the activated carbon treatment helps to remove or at least reduce the amount of colorants and/or water-soluble contaminants (e.g., salts). Ion exchange chromatography is effective in removing charged components such as salts, chromophores, proteins, amino acids, lipids and DNA.

Thus, the method may further comprise the following separation/purification steps of the fermentation broth in any order prior to drying:

i) Ultrafiltration (UF),

Ii) Nanofiltration (NF), and

Iii) Treating with ion exchange resin.

Advantageously, step i) is carried out before step ii). More advantageously, step i) is carried out before either of steps ii) and iii). Preferably, the process is carried out in the following order: step ii) follows step i) and step iii) follows step ii). The method may also include activated carbon treatment after UF, NF or ion exchange resin treatment.

The process provided herein may be supplemented by additional process steps performed on the solid amorphous HMO product obtained, i.e. after drying.

In one aspect, the methods described herein further comprise the step of milling the solid amorphous HMO product. Milling can provide solid amorphous HMO powder with various particle size distributions (D90), depending on the type of mill used, such as impact hammer mill, ball mill, forced screen (forced sieve), or jet mill. As another aspect, the method may further comprise the step of sieving the solid amorphous HMO powder and separating the HMO powder into at least a first HMO powder fraction and a second HMO powder fraction. Depending on the type of screen used, the screening may provide the HMO powder fraction with a particle size distribution (D90). Screening or classifying may be adapted to any customer's requirements. From a food safety point of view, a maximum of 1mm screening, preferably 0.5-0.7mm, will be required. However, if a mixing dryer, such as a vertical mixing dryer, is equipped with a rotary cutter or a rotary knife, a solid amorphous HMO powder having the desired particle size distribution (D90) can be provided without a separate milling step.

The solid HMO product can also be formulated in food products, in particular nutritional formulations, such as infant nutritional formulas.

The nutritional formulation comprising the solid amorphous HMO product may be a food, beverage or feed. The nutritional formulation may also contain edible micronutrients, vitamins and minerals. The amount of these ingredients may vary depending on whether the formulation is intended for normal, healthy infants, children, adults, or subjects with particular needs (e.g., suffering from metabolic disorders). Micronutrients include, for example, edible oils, fats or fatty acids (e.g., coconut oil, soybean oil, monoglycerides, diglycerides, palm oil, sunflower oil, fish oils, linoleic acid, linolenic acid, etc.), carbohydrates (e.g., glucose, fructose, sucrose, maltodextrin, starch, hydrolyzed corn starch, etc.), and proteins from casein, soybean, whey, or skim milk, or hydrolysates of these proteins, but proteins from other sources (intact or hydrolyzed) may also be used. Vitamins can be selected from vitamins A, B, B2, B5, B6, B12, C, D, E, H, K, folic acid, inositol, and niacin. The nutritional formulation may comprise the following minerals and trace elements: ca. P, K, na, cl, mg, mn, fe, cu, zn, se, cr or I.

In a preferred embodiment, the nutritional formulation is an infant nutritional formulation. Infant nutrition formulation refers to food products for infants to use with specific nutrition within 4-6 months after birth and to meet the needs of the infant itself. It may contain one or more probiotics bifidobacteria, prebiotics such as fructo-and galacto-oligosaccharides, proteins from casein, soy, whey or skim milk, carbohydrates such as lactose, sucrose, maltodextrin, starch or mixtures thereof, lipids (e.g. palm oil, sunflower oil, safflower oil), vitamins and minerals essential in the daily diet. The infant formula contains a total amount of solid HMO product of 0.1-3.0g/100g of formula.

In another preferred embodiment, the nutritional formulation may be a food supplement comprising a solid amorphous HMO product. The food supplement may comprise one or more probiotics in an amount sufficient to achieve the desired effect in the individual, preferably in children and adults. The food supplement may also contain vitamins, minerals, trace elements and other micronutrients. The food supplement may be in the form of, for example, a tablet, capsule, lozenge or liquid. The extender may contain conventional additives selected from, but not limited to, binders, coating agents, emulsifiers, solubilizers, encapsulating agents, film formers, adsorbents, carriers, fillers, dispersants, wetting agents, gelling agents, and the like. The daily dose of solid HMO product ranges from 0.1 to 5.0g.

The following numbered aspects of the invention are provided:

aspect 1. A method for drying an aqueous composition of one or more Human Milk Oligosaccharides (HMOs) and thereby providing a dried amorphous powder of said one or more HMOs, the method comprising:

a) Providing the dried amorphous powder comprising the one or more HMOs in a dryer suitable for mechanically agitating the dried amorphous powder to obtain a dry agitated amorphous powder,

B) Feeding one or more aqueous compositions of HMOs into, onto or onto a dry stirred amorphous powder to obtain a wet stirred mixture of amorphous powders, and

C) Water is removed from the wet stirred mixture of amorphous powder at elevated temperature and/or vacuum while the aqueous composition of one or more HMOs is added continuously or sequentially to the wet stirred mixture of amorphous powder.

The method of aspect 2, aspect 1, wherein the dryer is designed to produce the greatest possible dryer wall contact of the powder by mechanical mixing.

The method of aspect 3, aspect 1 or 2, wherein the dryer is a vertical hybrid dryer or a vertical vacuum hybrid dryer.

The method of aspect 4, aspect 3, wherein the vertical mixing dryer or the vertical vacuum mixing dryer is equipped with a central mixing arm with helical mixing blades.

The method of aspect 5, aspect 4, wherein the vertical hybrid dryer or the vertical vacuum hybrid dryer is equipped with a rotary cutter.

Aspect 6. The method of aspect 1 or 2, wherein the dryer is a horizontal contact dryer or a horizontal vacuum dryer.

The method of aspect 7, aspect 6, wherein the drying is performed continuously.

Aspect 8 the method of any one of the preceding aspects, wherein the aqueous composition of one or more HMOs is an aqueous solution.

The method of aspect 9.7, wherein the concentration of HMO in the aqueous solution is at least 80%, preferably at least 90% of its solubility with respect to water.

Aspect 10. The method of any one of the preceding aspects, wherein the wet stirred mixture of amorphous powder in step b) has a moisture content of no more than 15%.

The method of aspect 11, aspect 10, wherein the wet stirred mixture of amorphous powder in step b) has a moisture content of about 8-10%, 6-8% or 4-6%.

Aspect 12 the method of any one of the preceding aspects, wherein the temperature of the wet stirred mixture of amorphous powder is maintained at about 60-90 ℃.

The method of aspect 13, aspect 12, wherein the temperature of the wet stirred mixture of amorphous powder is maintained at 70-80 ℃.

Aspect 14 the method of any one of the preceding aspects, wherein step c) is operated under vacuum.

The method of aspect 15, aspect 14, wherein the vacuum is 20-100 mbar, preferably 20-60 mbar.

Aspect 16. The method of any one of the preceding aspects, wherein the wet stirred mixture of amorphous powder in step c) has a moisture content of no more than 15%.

The method of aspect 17, aspect 16, wherein the wet stirred mixture of amorphous powder in step c) has a moisture content of about 8-10%, 6-8%, or 4-6%.

The method of any of the preceding aspects, wherein in step c), the amount of water introduced by adding the aqueous composition is about the same as the amount of water removed.

Aspect 19. The method of any of the preceding aspects, wherein the dry stirred amorphous powder obtained in step a) is warmed to about 60-90 ℃, preferably 70-80 ℃.

Aspect 20. The method of any of the preceding aspects, wherein the aqueous composition, preferably the aqueous solution, to be added in step b) and/or step c) is preheated to a temperature of 40-90 ℃, preferably 60-80 ℃ before addition.

Aspect 21. The method of any one of the preceding aspects, wherein the dry powder fed to the mixer according to step a) is stirred at atmospheric pressure, and steps b) and c) are performed under vacuum.

Aspect 22. The method of any of the preceding aspects, wherein step c) is operated under vacuum, preferably at 20-100 mbar or 20-60 mbar, while the wet stirred powder mixture is maintained at about 60-90 ℃, preferably 70-80 ℃.

Aspect 23. The method of any of the preceding aspects, wherein the dried amorphous powder of the one or more HMOs obtained has a moisture content of less than 8%, preferably less than 6%, for example less than 4% down to 1-3%.

Aspect 24 the method of any one of the preceding aspects, wherein the HMO is a neutral HMO.

Aspect 25 the method of aspect 24, wherein the neutral HMO is a nonfucosylated HMO.

Aspect 26 the method of aspect 25, wherein the nonfucosylated HMO is LNT or LNnT.

Aspect 27 the method of aspect 24, wherein the neutral HMO is a fucosylated HMO.

The method of aspect 28, aspect 27, wherein the fucosylated HMO is 2' -FL and/or DFL.

Aspect 29 the method of any one of the preceding aspects, wherein the HMO is sialylated HMO.

The method of aspect 30, aspect 29, wherein the sialylated HMO is 3'-SL or 6' -SL.

Aspect 31 the method of any one of the preceding aspects, wherein the HMO is produced by fermentation.

Aspect 32. A dried amorphous powder of one or more HMOs obtained or obtainable by the process as defined in any one of aspects 1 to 31.

Aspect 33. The dried amorphous powder of one or more HMOs of aspect 32 has a moisture content of less than 8%, preferably less than 6%, for example less than 4% down to 1-3%.

Aspect 34. A nutritional formulation comprising a dried amorphous powder of one or more HMOs according to aspects 32 or 33.

Aspect 35 the nutritional formulation of aspect 34 which is a food, beverage, feed, infant formula, medical food or food supplement.

The present technology has been described with reference to various aspects and embodiments. These aspects and embodiments may be combined as desired by the skilled person while remaining within the scope of the invention as defined by the appended claims. In particular, features associated with the methods described herein are also applicable to solid HMO products, as the methods are characterized by producing identifiable differences in the solid HMO products themselves.

Examples

Example 1

VMT 200-1730 of vertical vacuum mixing drierEquipped with an S-blade and a horizontal rotary cutter, the dryer was filled with 70kg of amorphous 2' -FL. The apparatus was gradually heated (conduction oil in the jacket) to 130 ℃ (inlet temperature). The central mixer was set at 50rpm. When the temperature of the stirred powder reached 65 ℃ (inlet temperature 125 ℃) the rotary cutter was set to 1500rpm and vacuum was applied. The hose (/ >) is added by means of a heated liquid at a pressure of 220mbar (powder temperature 74 ℃)The addition of the preheated (80 ℃) 2' -FL (Brix 56) solution was started with a standard spray gun having a spray system nozzle size of 10, the air pressure was 2 bar and the flow rate of the solution per minute was 500g. The flow rate was increased to 800g/min and then to 1050g/min, while the internal pressure was maintained at 90-110 mbar and the powder temperature was 71-77 ℃. 50kg of 2' -FL solution was injected in one hour. A vacuum of 50-80 mbar is maintained for a further 10-15 minutes. The process was stopped and 93kg of dried 2' -FL powder was collected, which was amorphous as evidenced by X-ray powder diffraction analysis. The residual moisture of the final powder was 2.1% (moisture analyzer). No viscous residue was found in the mixing chamber. /(I)

Claims

1. A method for drying an aqueous composition of one or more Human Milk Oligosaccharides (HMOs) and thereby providing a dried amorphous powder of the one or more HMOs, the method comprising:

B) Feeding one or more aqueous compositions of HMOs into, onto or onto the dry stirred amorphous powder to obtain a wet stirred mixture of amorphous powder, and

2. The method of claim 1, wherein the dryer is a vertical hybrid dryer or a vertical vacuum hybrid dryer.

3. The method of claim 1, wherein the dryer is a horizontal contact dryer or a horizontal vacuum dryer.

4. The method according to any one of the preceding claims, wherein the aqueous composition of one or more HMOs is an aqueous solution.

5. The method according to claim 4, wherein the concentration of HMO in the aqueous solution is at least 80%, preferably at least 90% of its solubility with respect to water.

6. The method according to any one of the preceding claims, wherein the wet stirred mixture of amorphous powder in step b) and step c) has a moisture content of not more than 15%, preferably about 8-10%, 6-8% or 4-6%.

7. The method according to any one of the preceding claims, wherein the temperature of the wet stirred mixture of amorphous powder is maintained at about 60-90 ℃, preferably 70-80 ℃.

8. The method according to any of the preceding claims, wherein step c) is operated under vacuum, and the vacuum is preferably 20-100 mbar, more preferably 20-60 mbar.

9. The method of claim 8, wherein the wet stirred powder mixture is maintained at about 60-90 ℃, preferably 70-80 ℃.

10. The method according to any of the preceding claims, wherein the HMO is a neutral HMO.

11. The method according to any one of claims 1 to 9, wherein the HMO is sialylated HMO.

12. The method of any of the preceding claims, wherein the HMO is 2' -FL, 3-FL, DFL, LNT, LNnT, LNFP-I, 3' -SL, 6' -SL, or any mixture thereof.

13. The method according to any of the preceding claims, wherein the HMO is produced by fermentation.

14. A dried amorphous powder of one or more HMOs obtained or obtainable by the process as defined in any one of claims 1 to 13, in particular wherein the dried amorphous powder of one or more HMOs has a moisture content of less than 8%, preferably less than 6%, such as less than 4% down to 1-3%.

15. A nutritional formulation comprising the dried amorphous powder of one or more HMOs of claim 14, preferably wherein the nutritional formulation is a food, beverage, feed, infant formula, medical food, or food supplement.