Amorphous mixture comprising neutral monosaccharide or oligosaccharide and acidic non-carbohydrate component

Abstract

Provided are i) amorphous carbohydrates having improved chemical stability and / or physical properties, ii) methods for making amorphous carbohydrates having improved chemical stability and / or physical properties, and iii) methods for improving the chemical stability and / or physical properties of amorphous carbohydrates.

Description

[0001] This application is a divisional application of the invention patent application with the invention name “Amorphous mixture comprising neutral monosaccharides or oligosaccharides and acidic non-carbohydrate components” and application number “201880046571.5” filed on July 12, 2018. Technical Field

[0002] The present invention relates to an amorphous, preferably spray-dried or freeze-dried composition comprising neutral oligosaccharides, advantageously human milk oligosaccharides, and a method for improving the stability of amorphous neutral oligosaccharides. Background Art

[0003] In recent years, due to the role of complex carbohydrates including naturally secreted oligosaccharides in many biological processes occurring in living organisms, their manufacture and commercialization have increased significantly. Secreted oligosaccharides such as human milk oligosaccharides (HMOs) are carbohydrates that have received widespread attention in recent years and are becoming an important commercial target for the nutrition and treatment industry. In particular, due to the role of HMOs in many biological processes occurring in humans, the synthesis of these HMOs has increased significantly. The high importance of HMOs is directly related to their unique biological activities such as antibacterial, antiviral, immune system and cognitive development enhancement activities. It was found that human milk oligosaccharides act as prebiotics in the human intestinal system, helping to develop and maintain intestinal flora. In addition, they have also been shown to have anti-inflammatory effects, so these compounds are attractive components in the nutrition industry, whether synthetic compounds or naturally occurring compounds and their salts can be used to manufacture, for example, infant formula, infant cereals, clinical infant nutrition products, toddler formulas or as dietary supplements or health functional foods for children, adults, the elderly or lactating women. Similarly, due to the prognostic use of these compounds as immunomodulators, they are also paid attention to in the pharmaceutical industry for the manufacture of therapeutic agents. To date, the structures of more than 140 HMOs have been determined (see Urashima et al.: Milk Oligosaccharides, Nova Biomedical Books, New York, 2011; Chen Adv. Carbohydr. Chem. Biochem. 72, 113 (2015)), and many more HMOs are likely to exist in human milk.

[0004] The search for low-cost methods for manufacturing as many HMOs as possible in industrial quality is ongoing so that their use in nutritional and therapeutic formulations for infants and potentially children and adults can be discovered, developed and utilized by researchers worldwide. Biotechnological methods have proven to be promising and cost-effective in synthesizing a wide range of HMOs. Specifically, HMOs can be produced by fermentation of genetically modified bacteria, yeast or other microorganisms in aqueous media. See for example WO 01 / 04341, WO 2007 / 101862, WO 2010 / 070104, WO 2010 / 142305, WO 2012 / 112777, WO 2014 / 153253, WO 2015 / 032412, WO 2015 / 032413, WO 2015 / 036138, WO 2015 / 150328, WO 2015 / 197082, WO 2016 / 008602, WO 2017 / 101958, EP-A-2722394, Priem et al., Glycobiology 12, 235 (2002), Drouillard et al., Angew. Chem. Int. Ed. 45, 1778 (2006), Han et al., Biotechnol. Adv. 30, 1268 (2012), Lee et al., Microb. Cell Fact. 11: 48 (2012) and et al., Microb. Cell Fact. 12:40 (2013). Chemical and ex vivo enzymatic synthesis of HMOs are also known.

[0005] The final step of the above techniques is usually the crystallization or recrystallization of the individual HMOs from an aqueous organic solvent such as an alcohol, typically methanol (see, for example, WO 2011 / 100980, WO 2011 / 150939, WO 2014 / 009921, WO 2014 / 075680, WO 2014 / 086373, WO 2014 / 069625, WO 2015 / 188834, WO 2016 / 086947, WO 2016 / 095924, EP-A-1405856, Kuhn et al. Chem. Ber. 95, 513 and 518 (1962), Takamura et al. Chem. Pharm. Bull. 28, 1804 (1980)). On the other hand, HMOs can be provided in solid forms other than crystals, for example, as spray-dried or freeze-dried amorphous powders (see, for example, WO 2013 / 185780, WO 2015 / 049331, WO 2015 / 106943, CN102154163 A). The properties of crystalline and amorphous substances are different. Although amorphous forms of substances are generally expected to show better solubility and bioavailability, their crystalline counterparts are more stable chemically and physically and have low hygroscopicity (Bauer J. Valid. Technol. 63 (Summer 2009)).

[0006] Therefore, there is a need to improve the chemical stability and / or physical properties of amorphous carbohydrates, preferably neutral HMOs, while maintaining their beneficial properties. Summary of the invention

[0007] The object of the present invention is to provide i) an amorphous carbohydrate having improved chemical stability and / or physical properties, ii) a method for preparing an amorphous carbohydrate having improved chemical stability and / or physical properties, and iii) a method for improving the chemical stability and / or physical properties of an amorphous carbohydrate.

[0008] Thus, a first aspect of the present invention relates to an amorphous mixture comprising a neutral monosaccharide or oligosaccharide and an acidic non-carbohydrate component, wherein the pH of the mixture in a 5 w / w% aqueous solution thereof is about 3.5-5.8. Preferably, the amorphous mixture is a spray-dried or freeze-dried mixture, more preferably spray-dried.

[0009] Also preferably, the amorphous mixture consists essentially of one or more neutral monosaccharides or oligosaccharides and one or more acidic non-carbohydrate components.

[0010] A second aspect of the present invention relates to a method for preparing an amorphous mixture comprising a neutral monosaccharide or oligosaccharide and an acidic non-carbohydrate component, wherein the pH of the mixture in a 5 w / w% aqueous solution thereof is about 3.5-5.8, the method comprising the steps of:

[0011] a) preparing an aqueous solution of the neutral monosaccharide or oligosaccharide,

[0012] b) adding an acidic non-carbohydrate component to the aqueous solution so that the pH is not lower than –log(x / 5·10 -3.5 ) and not higher than –log(x / 5·10 -5.8 ), wherein x is the concentration of neutral monosaccharides or oligosaccharides in the aqueous solution in w / w %, then

[0013] c) spray drying or freeze drying the pH adjusted solution obtained in step b), preferably spray drying.

[0014] The third aspect of the present invention relates to the use of an acidic component for improving the chemical stability and / or physical properties of amorphous neutral monosaccharides or oligosaccharides, comprising mixing the acidic component and the neutral monosaccharides or oligosaccharides in an aqueous solution and spray drying or freeze drying, preferably spray drying.

[0015] Specifically, the present application provides the following implementation modes.

[0016] 1. An amorphous mixture comprising a neutral monosaccharide or oligosaccharide and an acidic non-carbohydrate component, wherein the pH of the mixture in a 5 w / w% aqueous solution thereof is about 3.5-5.8.

[0017] 2. The amorphous mixture according to item 1, which consists essentially of one or more neutral monosaccharides or oligosaccharides and one or more acidic non-carbohydrate components.

[0018] 3. The amorphous mixture according to item 1 or 2, which is spray-dried or freeze-dried.

[0019] 4. The amorphous mixture according to any one of items 1 to 3, wherein the oligosaccharide is a trisaccharide or higher oligosaccharide, preferably a reducing trisaccharide or higher reducing oligosaccharide, and more preferably the monosaccharide unit at the reducing end of the oligosaccharide is an aldose.

[0020] 5. The amorphous mixture according to item 4, wherein the monosaccharide unit at the reducing end is glucose, and more preferably a galactose unit is linked to the glucose via a β1-4 interglycosidic bond to form a lactose moiety.

[0021] 6. The amorphous mixture according to item 4 or 5, wherein the oligosaccharide is human milk oligosaccharide (HMO).

[0022] 7. An amorphous mixture according to any of the preceding items, wherein the acidic non-carbohydrate component is an inorganic acid, preferably sulfuric acid and its monosalts, HCl, HBr, nitric acid or phosphoric acid and its monosalts and disalts, or an organic acid, preferably formic acid, acetic acid, oxalic acid, tartaric acid, lactic acid, malic acid or citric acid.

[0023] 8. The amorphous mixture according to any one of the preceding items, wherein the pH is 4.5-5.8, more preferably 5.0-5.8, even more preferably 5.0-5.5.

[0024] 9. A method for preparing an amorphous mixture according to any one of items 1 to 8, comprising the following steps:

[0025] a) preparing an aqueous solution of neutral monosaccharides or oligosaccharides,

[0026] b) adding an acidic non-carbohydrate component to the aqueous solution so that the pH is not lower than –log(x / 5·10 -3.5 ) and not higher than –log(x / 5·10 -5.8 ), wherein x is the concentration of the neutral oligosaccharide in the aqueous solution after step a) in w / w %, then

[0027] c) spray-drying or freeze-drying the pH-adjusted solution obtained in step b).

[0028] 10. Use of an acidic non-carbohydrate component for improving the chemical stability and / or physical properties of amorphous neutral monosaccharides or oligosaccharides, comprising mixing the acidic component and the neutral monosaccharides or oligosaccharides in an aqueous solution, followed by spray drying or freeze drying, preferably spray drying.

[0029] 11. The use according to item 10, wherein the oligosaccharide is human milk oligosaccharide (HMO).

[0030] 12. A method for improving the chemical stability and / or physical properties of amorphous neutral monosaccharides or oligosaccharides, the method comprising:

[0031] a) preparing an aqueous solution of the neutral monosaccharide or oligosaccharide,

[0032] b) adding an acidic non-carbohydrate component to the aqueous solution so that the pH is not lower than –log(x / 5·10 -3.5 ) and not higher than –log(x / 5·10 -5.8 ), wherein x is the concentration of the neutral monosaccharide or oligosaccharide in the aqueous solution after step a) in w / w %,

[0033] c) spray drying or freeze drying the pH adjusted solution obtained in step b), preferably spray drying, and

[0034] d) obtaining an amorphous mixture comprising, preferably consisting essentially of, said neutral mono- or oligosaccharides and said acidic non-carbohydrate components.

[0035] 13. The method according to item 12, wherein the oligosaccharide is human milk oligosaccharide (HMO). DETAILED DESCRIPTION

[0036] Terms and Definitions

[0037] The term "monosaccharide" refers to a sugar of 5-9 carbon atoms, which is an aldose (e.g., D-glucose, D-galactose, D-mannose, D-ribose, D-arabinose, L-arabinose, D-xylose, etc.), a ketose (e.g., D-fructose, D-sorbose, D-tagatose, etc.), a deoxy sugar (e.g., L-rhamnose, L-fucose, etc.), or a deoxy amino sugar (e.g., N-acetylglucosamine, N-acetylmannosamine, N-acetylgalactosamine, etc.). "Neutral monosaccharide" refers to a monosaccharide lacking an acidic or basic functional group in free or salt form. Thus, for example, uronic acid, keturonic acid (e.g., sialic acid), or a deoxy amino sugar (e.g., glucosamine) with a free amino group is not considered a neutral monosaccharide. When these functional groups are modified so that they can no longer form acid addition salts (due to, for example, esterification of the carboxyl group or N-acylation of the amino group), the monosaccharide with this group is considered neutral.

[0038] The term "oligosaccharide" refers to a sugar polymer consisting of at least two, preferably three to eight, more preferably three to six monosaccharide units (see above). Trisaccharides or higher oligosaccharides may have a linear or branched structure comprising monosaccharide units connected to each other by interglycosidic bonds. "Neutral oligosaccharides" are oligosaccharides consisting of neutral monosaccharide units.

[0039] The term "human milk oligosaccharide" or "HMO" refers to complex carbohydrates found in human breast milk (Urashima et al.: Milk Oligosaccharides, Nova Medical Books, NY, 2011; Chen Adv. Carbohydr. Chem. Biochem. 72, 113 (2015)). Neutral HMOs have a core structure of a lactose unit at the reducing end, which is extended by one or more β-N-acetyl-lactosamine groups and / or one or more β-lacto-N-biose units, and the core structure may be substituted by an α-L-fucopyranosyl moiety. Therefore, this non-acidic (or neutral) HMO has no sialic acid residues. Non-acidic (or neutral) HMOs may be fucosylated or non-fucosylated. Examples of such neutral non-fucosylated HMOs include lacto-N-triose II (LNTri II, GlcNAc(β1-3)Gal(β1-4)Glc), lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT), lacto-N-neohexose (LNnH), p-lacto-N-neohexose (pLNnH), p-lacto-N-hexose (pLNH) and lacto-N-hexose (LNH). Examples of neutral fucosylated HMOs include 2'-fucosyllactose (2'-FL), lacto-N-fucopentose I (LNFP-I), lacto-N-difucohexose I (LNDFH-I), 3-fucosyllactose (3-FL), difucosyllactose (DFL), lacto-N-fucopentose II (LNFP-II), lacto-N-fucopentose III (LNFP-III), lacto-N-difucohexose I (LNDFH-I), lacto-N-fucopentose II (LNFP-II), lacto-N-fucopentose III (LNFP-III), lacto-N-difucohexose II ...difucohexose II (LNDFH-I), lacto-N-fucopentose II (LNFP-II), lacto-N-fucopentose III (LNFP-III), lacto-N-difucohexose II (LNDFH-I), lacto-N-difucohexose II (LNDFH-I), lacto-N-fucopentose II (LNFP-II), lacto-N-fucopentose III (LNFP-III), lacto-N-difucohexose II (LNDFH-I), lacto- Lactose is preferably not considered an HMO.

[0040] In one embodiment, the term "about" refers to a variance of ±10%, or in another embodiment, a variance of ±5% from the specified value.

[0041] Amorphous mono- or oligosaccharides with improved stability

[0042] Although amorphous compounds (especially hydrophilic compounds) generally show better water solubility, higher dissolution rate and better bioavailability, which are key features for product development in the pharmaceutical and food industries, their shelf life is expected to be shorter than that of the corresponding crystalline form. Amorphous compounds tend to undergo a higher degree of chemical degradation than their crystalline form, and their physical appearance may be adversely changed, for example, due to agglutination. The inventors have noticed that in spray-dried neutral oligosaccharide samples with aldose reducing ends, new carbohydrate-type by-products appear during long-term storage, and their amount in the sample increases over time. The results of careful analysis show that this contamination is proven to be a rearranged ketose derivative of the reducing oligosaccharide, and the rearrangement can be illustrated on the aldohexose as follows:

[0043]

[0044] The inventors surprisingly found that if the spray-dried or freeze-dried amorphous neutral oligosaccharide contains an acidic component, the above rearrangement does not occur in the solid state, or at least occurs to a significantly lower extent. Therefore, the chemical stability of the spray-dried or freeze-dried material can even reach the chemical stability of the corresponding crystalline form.

[0045] Thus, a first aspect of the present invention is to provide an amorphous mixture comprising a neutral mono- or oligosaccharide and an acidic non-carbohydrate component, wherein the pH of the mixture in a 5 w / w % aqueous solution thereof is about 3.5-5.8.

[0046] In one embodiment, the amorphous mixture is a spray dried mixture, and in another embodiment, is a freeze dried mixture.

[0047] Preferably, the neutral monosaccharide or oligosaccharide is a reducing monosaccharide or oligosaccharide.

[0048] In one embodiment, the monosaccharide or monosaccharide unit at the reducing end of the neutral oligosaccharide is an aldose. In another embodiment, the monosaccharide or monosaccharide unit at the reducing end of the neutral oligosaccharide is a ketose.

[0049] Also preferably, in another embodiment, the monosaccharide unit at the reducing end of the neutral oligosaccharide is glucose, and more preferably, a galactose unit is linked to said glucose via a β1-4 interglycosidic bond to form a lactose moiety.

[0050] In a preferred embodiment, the reducing neutral oligosaccharide is a trisaccharide or higher neutral oligosaccharide, advantageously having a lactose moiety at the reducing end.

[0051] In particular, a trisaccharide or higher neutral oligosaccharide containing a lactose moiety at the reducing end is a neutral human milk oligosaccharide (HMO). The neutral HMO is preferably a trisaccharide to an octasaccharide HMO, more preferably a trisaccharide, a tetrasaccharide, a pentasaccharide or a hexasaccharide HMO.More preferably, the trisaccharide HMO may be selected from 2'-O-fucosyllactose (2'-FL), 3-O-fucosyllactose (3-FL) and lacto-N-triose II (LNTriII, GlcNAc(β1-3)Gal(β1-4)Glc); the tetrasaccharide HMO may be selected from lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT) and difucosyllactose (lacto-difucosyllactose, DFL); the pentasaccharide HMO may be selected from lacto- N-fucose I (LNFP-I, Fucα1-2Galβ1-3GlcNAcβ1-3Galβ1-4Glc), lacto-N-fucose VI (LNFP-VI, Galβ1-4GlcNAcβ1-3Galβ1-4[Fucα1-3]Glc), lacto-N-fucose II (LNFP-II, Galβ1-3[Fucα1-4]GlcNAcβ1-3Galβ1-4Glc) lc), lacto-N-fucose III (LNFP-III, Galβ1-4[Fucα1-3]GlcNAcβ1-3Galβ1-4Glc), lacto-N-fucose V (LNFP-V, Galβ1-3GlcNAcβ1-3Galβ1-4[Fucα1-3]Glc); the hexasaccharide HMO can be selected from lacto-N-hexose (LNH, Galβ1-3GlcNAcβ1-3[Galβ1 -4GlcNAcβ1-6]Galβ1-4Glc), lacto-N-neohexose (LNnH, Galβ1-4GlcNAcβ1-3[Galβ1-4GlcNAcβ1-6]Galβ1-4Glc), p-lacto-N-hexose (pLNH, Galβ1-3GlcNAcβ1-3Galβ1-4GlcNAcβ1-3Galβ1-4Glc), p-lacto-N-hexose II (pLNH II, Galβ1-3GlcNAcβ1-3Galβ1-3GlcNAcβ1-3Galβ1-4Glc), p-lacto-N-neohexose (pLNnH, Galβ1-4GlcNAcβ1-3Galβ1-4GlcNAcβ1-3Galβ1-4Glc), LNDFH II(Galβ1-3[Fucα1-4]GlcNAcβ1-3Galβ1-4[Fucα1-3]Glc), LNDFH III(Galβ1-4[Fucα1-3]GlcNAcβ1-3Galβ1-4[Fucα1-3]Glc), p-lacto-N-neohexose II (pLNnH II, Galβ1-3GlcNAcβ1-3Galβ1-4GlcNAcβ1-3Galβ1-4Glc)).

[0052] In other cases, the neutral HMO is a non-fucosylated HMO, a mono-fucosylated HMO or a poly-fucosylated HMO. The non-fucosylated HMO may preferably be lacto-N-triose II, LNT, LNnT, LNH, L:NnH, pLNH, pLNHII, pLNnH or pLNnH II. The mono-fucosylated HMO may preferably be 2'-FL, 3-FL, LNFP-I, LNFP-II, LNFP-III, LNFP-V or LNFP-VI. The poly-fucosylated HMO is particularly a di-fucosylated or tri-fucosylated HMO, examples of which are DFL, LNDFH II or LNDFH III.

[0053] The acidic non-carbohydrate component in the above-mentioned amorphous mixture, preferably the spray-dried or freeze-dried mixture, is an inorganic acid or an organic compound having an acidic characteristic different from that of a carbohydrate. Preferably, suitable inorganic and organic acids are those known to be safe when used in the pharmaceutical and food industries, such as those commonly used to prepare pharmaceutically acceptable acid addition salts of active pharmaceutical ingredients with alkaline characteristics. The inorganic acid can be selected from, for example, sulfuric acid and its monosalts (such as monosodium sulfate (monosodium sulphate)), HCl, HBr, nitric acid, phosphoric acid and its monosalts and disalts (such as monosodium phosphate or disodium phosphate) or perchloric acid. Also preferably, suitable organic acids are alkanoic acids, such as formic acid, acetic acid or propionic acid; alkanediol acids, such as oxalic acid, malonic acid or succinic acid; hydroxy acids, such as tartaric acid, lactic acid or malic acid; tricarboxylic acids, such as citric acid.

[0054] In one embodiment, the amorphous, preferably spray-dried or freeze-dried mixture may contain more than one neutral HMO, such as two, three, four, five, two to six, three to ten neutral HMOs. Likewise, the amorphous, preferably spray-dried or freeze-dried mixture may contain more than one acidic non-carbohydrate component.

[0055] In a preferred embodiment, the above-mentioned amorphous, preferably spray-dried or freeze-dried mixture consists essentially of one or more neutral mono- or oligosaccharides, advantageously a HMO as described above, and one or more acidic non-carbohydrate components.

[0056] Comparative forced stability tests at elevated temperatures showed that the presence of the acidic non-carbohydrate components in the spray-dried HMOs virtually prevented the formation of rearranged ulose. Thus, the spray-dried HMOs were chemically as stable as the corresponding crystalline forms of the HMOs.

[0057] The claimed amorphous, preferably spray-dried or freeze-dried mixture should have a pH of no more than about 5.8 in its 5 w / w% aqueous solution. A pH above this is very close to the neutral region, where aldose-ketose rearrangement occurs easily according to comparative tests. On the other hand, it is recommended that the pH as defined above is no less than 3.5. A pH below 3.5 may initiate acid-catalyzed degradation of oligosaccharides by hydrolyzing at least one of the interglycosidic bonds. Therefore, it is preferred when the pH of the solution is between 4.0-5.8, such as 4.2-5.8, 4.5-5.8, 4.0-5.5, 4.2-5.5, 4.0-5.5, 4.2-5.5, 4.5-5.5, 4.0-5.0, 4.2-5.0 or 4.5-5.0, and another preferred pH range is 5.0-5.8, such as 5.0-5.5 or 5.5-5.8.

[0058] In one embodiment, when the neutral HMO or at least one of the neutral HMOs in the amorphous, preferably spray-dried or freeze-dried mixture is 2'-FL, the preferred pH is not less than 4.5, i.e. the pH range may be 4.5-5.8, 4.5-5.5, 4.5-5.0, 5.0-5.8, 5.0-5.5 or 5.5-5.8, preferably 5.2-5.8, because due to the acid sensitivity of the α1,2-fucosyl moiety, a low degree of hydrolysis to fucose and lactose can be observed at a pH of about 3.5, and this hydrolysis, although to a lesser extent, is still detectable at a pH of about 4.0-4.5. This means that the amorphous 2'-FL according to the present invention having a pH of about 3.5-4.5 in a 5 w / w% aqueous solution has a slightly shorter shelf life than the amorphous 2'-FL having a pH of 4.5-5.8. However, DFL, which also has an α1,2-fucosyl moiety in its structure, was shown to be hydrolytically stable in the pH range of 3.5–4.5. Therefore, it can be concluded that fucosylated neutral HMOs other than 2′-FL are as hydrolytically stable as non-fucosylated neutral HMOs.

[0059] In other embodiments, the neutral HMO in the amorphous mixture according to the present invention is 3-FL or DFL, and the preferred pH of its 5 w / w% aqueous solution is 4.0-5.8, such as 4.2-5.8, 4.5-5.8, 4.0-5.5, 4.2-5.5, 4.0-5.5, 4.2-5.5, 4.5-5.5, 4.0-5.0, 4.2-5.0 or 4.5-5.0, another preferred pH range is 5.0-5.8, such as 5.0-5.5 or 5.5-5.8.

[0060] In other embodiments, the neutral oligosaccharide in the amorphous mixture according to the present invention is a mono- or di-fucosylated tetrasaccharide, such as a mono- or di-fucosylated LNT or LNnT, and the preferred pH of its 5 w / w% aqueous solution is 4.0-5.8, such as 4.2-5.8, 4.5-5.8, 4.0-5.5, 4.2-5.5, 4.0-5.5, 4.2-5.5, 4.5-5.5, 4.0-5.0, 4.2-5.0 or 4.5-5.0, another preferred pH range is 5.0-5.8, such as 5.0-5.5 or 5.5-5.8. Examples of mono-fucosylated LNT or LNnT are LNFP-I, LNFP-II, LNFP-III, LNFP-V or LNFP-VI. Examples of difucosylated LNT or LNnT are LNDFH II or LNDFH III.

[0061] In other embodiments, the neutral HMO in the amorphous mixture according to the present invention is LNT, and the preferred pH of its 5 w / w% aqueous solution is 4.0-5.8, such as 4.2-5.8, 4.5-5.8, 4.0-5.5, 4.2-5.5, 4.0-5.5, 4.2-5.5, 4.5-5.5, 4.0-5.0, 4.2-5.0 or 4.5-5.0, another preferred pH range is 5.0-5.8, such as 5.0-5.5 or 5.5-5.8.

[0062] In other embodiments, the neutral HMO in the amorphous mixture according to the present invention is LNnT, and the preferred pH of its 5 w / w% aqueous solution is 4.0-5.8, such as 4.2-5.8, 4.5-5.8, 4.0-5.5, 4.2-5.5, 4.0-5.5, 4.2-5.5, 4.5-5.5, 4.0-5.0, 4.2-5.0 or 4.5-5.0, another preferred pH range is 5.0-5.8, such as 5.0-5.5 or 5.5-5.8.

[0063] A second aspect of the present invention relates to a method for preparing an amorphous mixture comprising a neutral monosaccharide or oligosaccharide and an acidic non-carbohydrate component, wherein the pH of the mixture in a 5 w / w% aqueous solution thereof is about 3.5-5.8, the method comprising the following steps:

[0064] a) preparing an aqueous solution of the neutral monosaccharide or oligosaccharide,

[0065] b) adding an acidic non-carbohydrate component to the aqueous solution so that the pH value is not lower than –log(x / 5·10 -3.5 ) and not higher than –log(x / 5·10 -5.8), wherein x is the concentration of neutral monosaccharides or oligosaccharides in the aqueous solution after step a) in w / w %, then

[0066] c) spray-drying or freeze-drying the pH-adjusted solution obtained in step b).

[0067] In the step a) of the method, an aqueous solution of one or more neutral monosaccharides or oligosaccharides is prepared in a conventional manner. Preferably, the solution is clear and the neutral monosaccharides or oligosaccharides dissolve. Before adding water thereto or adding it to water, one or more neutral monosaccharides or oligosaccharides can be in any solid form (e.g., crystallization, amorphous [precipitation, freeze drying, spray drying] or its mixture), syrup form or even in aqueous solution. Before performing step a), one or more neutral monosaccharides or oligosaccharides can include a certain amount or residual amount of (volatile) organic solvents, because these solvents are substantially removed in the step c) of the method and will not be included in the amorphous final material. Preferably, before performing step a), one or more neutral monosaccharides or oligosaccharides do not include a large amount of acidic or alkaline pollutants. If included, less (in the case of acidic pollution) or more (in the case of alkaline pollution) acidic non-carbohydrate components can be added to reach the required pH range.

[0068] In step b) of the method, an acidic non-carbohydrate component is added to the solution obtained in step a). Preferably, the concentration of monosaccharides or oligosaccharides in the solution is not actually diluted by the addition of the acidic component, which means that the concentration of the monosaccharide or oligosaccharide solution in step a) and the concentration of the monosaccharide or oligosaccharide solution in step b) after the addition of the acidic component are actually the same. This can be achieved when the acidic component is added in its solid form or in its concentrated aqueous solution if it is solid, or in its concentrated aqueous solution if it is liquid or gaseous. If the monosaccharide or oligosaccharide solution is significantly diluted during the addition of the acidic component, the value x in step b) corresponds to the concentration of the monosaccharide or oligosaccharide after dilution. In any case, it is preferred that the acid is slowly added to the aqueous solution of the monosaccharide or oligosaccharide under stirring, and the pH is continuously checked, for example, by a pH meter. If the desired pH is reached, the addition of the acid is terminated. It is also preferred that the concentration x is close to 5w / w%. However, for economic reasons, the concentration x may be significantly higher than 5 w / w%, such as 20-50 w / w%, about 25 w / w%, about 30 w / w%, about 35 w / w% or about 40 w / w%.

[0069] In step c), the aqueous solution obtained in step b) is spray dried in a conventional manner: the aqueous solution obtained may be spray dried with hot air or hot inert gas, preferably hot air, at about 130-210° C., preferably 140-180° C., to produce a substantially dry amorphous free-flowing powder of a mixture comprising or consisting essentially of neutral HMO and acidic components. Thus, any conventional spray drying apparatus may be used, such as a co-current or multiple effect spray dryer, preferably a two-stage spray dryer (with a fluidized bed attachment). Likewise, the choice of atomizer or nozzle in the spray dryer is not considered critical, and any common rotating disk or high pressure cyclone nozzle may be suitably used. Alternatively, the aqueous solution obtained in step b) is freeze dried in a conventional manner.

[0070] The third aspect of the present invention relates to the use of acidic non-carbohydrate components for improving the chemical stability and / or physical properties of amorphous neutral monosaccharides or oligosaccharides, comprising mixing the acidic component and the neutral monosaccharides or oligosaccharides in an aqueous solution, and spray-drying or freeze-drying the aqueous solution to obtain an amorphous mixture, which comprises one or more neutral monosaccharides or oligosaccharides and one or more acidic non-carbohydrate components, preferably essentially consisting of one or more neutral monosaccharides or oligosaccharides and one or more acidic non-carbohydrate components.

[0071] The term "improving the chemical stability of an amorphous neutral mono- or oligosaccharide" preferably means that under the same conditions, in the presence of an acidic non-carbohydrate component, the amorphous mono- or oligosaccharide is less susceptible to chemical degradation or rearrangement reactions than in the absence of an acidic non-carbohydrate component, and thus these properties are improved. An example of chemical degradation is hydrolytic decomposition by breaking at least one of the interglycosidic bonds; an example of chemical rearrangement is rearrangement of an aldose to a ketose or vice versa.

[0072] The term "improving the physical properties of amorphous neutral monosaccharides or oligosaccharides" preferably means that under the same conditions, in the presence of an acidic non-carbohydrate component, the amorphous monosaccharides or oligosaccharides exhibit better physical properties than in the absence of an acidic non-carbohydrate component, and thus these properties are improved. Examples of improved physical properties are a lower tendency to phase transition (e.g., vitrification, crystallization) or a lower degree of agglomeration.

[0073] The inventors surprisingly found that the chemical stability of amorphous spray-dried neutral monosaccharides or oligosaccharides can be improved if acidic components other than carbohydrates are added to the monosaccharides or oligosaccharides. This can be achieved when one or more neutral monosaccharides or oligosaccharides and one or more acidic non-carbohydrate components, preferably only one acidic component, are dissolved in water and the resulting solution is spray-dried or freeze-dried. In a first aspect of the present invention, preferred and more preferred embodiments of neutral monosaccharides or oligosaccharides, acidic components and obtainable spray-dried or freeze-dried mixtures are disclosed; in a second aspect of the present invention, preferred and more preferred embodiments of methods for preparing spray-dried or freeze-dried mixtures are disclosed.

[0074] A fourth aspect of the invention relates to an amorphous mixture obtainable by the process disclosed in the second aspect of the invention, the amorphous mixture comprising, preferably consisting essentially of, one or more neutral mono- or oligosaccharides and one or more acidic non-carbohydrate components.

[0075] A fifth aspect of the present invention relates to a method for improving the chemical stability and / or physical properties of amorphous neutral monosaccharides or oligosaccharides, the method comprising:

[0076] a) preparing an aqueous solution of the neutral monosaccharide or oligosaccharide,

[0077] b) adding an acidic non-carbohydrate component to the aqueous solution so that the pH value is not lower than –log(x / 5·10 -3.5 ) and not higher than –log(x / 5·10 -5.8 ), wherein x is the concentration of neutral monosaccharides or oligosaccharides in the aqueous solution after step a) in w / w %,

[0078] c) spray drying or freeze drying the pH adjusted solution obtained in step b), preferably spray drying, and

[0079] d) obtaining an amorphous mixture comprising, preferably consisting essentially of, said neutral mono- or oligosaccharides and said acidic non-carbohydrate components.

[0080] In a fifth aspect of the present invention, preferred and more preferred embodiments of neutral monosaccharides or oligosaccharides, acidic components and obtainable spray-dried or freeze-dried mixtures are disclosed in the first aspect of the present invention; preferred and more preferred embodiments of methods for preparing spray-dried or freeze-dried mixtures are disclosed in the second aspect of the present invention.

[0081] Example

[0082] Example 1

[0083] 2'-FL was prepared by bacterial fermentation and isolated as disclosed in WO 2015 / 032412. The material contained DFL as an accompanying HMO. The concentration of (2'-FL+DFL) in the aqueous solution was 34.5 g / 100 g solution.

[0084] Sample A: The pH of a portion of the aqueous solution was adjusted to 3.2 by adding 25% aqueous sulfuric acid. The acidic solution thus obtained was spray dried on a Buchi micro spray dryer B290 (inlet temperature: 175°C, outlet temperature: 120°C) to obtain a white amorphous powder. The pH of its 5 w / w% solution was 4.2.

[0085] Sample B: A portion of the aqueous solution was spray dried on a Buchi micro spray dryer B290 (inlet temperature: 175°C, outlet temperature: 120°C) to obtain a white amorphous powder. The pH of a 5 w / w% solution thereof was 6.3.

[0086] Sample C: A portion of the aqueous solution was concentrated to about 55% of its weight. Methanol was then added to crystallize 2'-FL, which was obtained as a white crystalline material after washing and drying (see WO 2015 / 188834). The pH of its 5 w / w% solution was 6.4.

[0087] 1 gram of each sample was placed in a 4 ml glass vial and sealed with a screw cap lined with Teflon. The 8-week storage sample was placed in an oven at 60°C, and then the 4-week and 2-week storage samples were placed after 4 and 6 weeks, respectively, so that the incubation of all samples ended at the same time. The samples measured at 80°C (4-week, 2-week and 1-week samples) also followed a similar experimental design pattern. The samples were kept in a refrigerator until they were placed in an oven at a given time. The standards were kept in a refrigerator until analysis.

[0088] The samples showed no phase changes during the study.

[0089] Stability samples and standards were analyzed by HPAEC.

[0090] The table below shows the content (w / w %) of 2'-O-fucosyl-lactulose (a ketose rearranged derivative of 2'-FL) in the samples.

[0091]

[0092] The amorphous samples with acidic pH showed practically no 2'-O-fucosyl-lactulose isomerization during storage under forced conditions, whereas in the samples with near-neutral pH an increase in the amount of rearranged derivatives was observed. Thus, the behavior of the amorphous samples with acidic pH was identical to that of the crystalline samples.

[0093] Example 2

[0094] A mixture of 2'-FL and DFL was prepared by bacterial fermentation and separated as disclosed in WO 2015 / 032412. The DFL content was about 14 w / w%. The concentration of (2'-FL+DFL) in the aqueous solution was made to be 25 g / 100 g solution.

[0095] Sample A: The pH of a portion of the aqueous solution was adjusted to 3.7 by adding 25% aqueous sulfuric acid. The acidic solution thus obtained was spray dried on a Buchi micro spray dryer B290 (inlet temperature: 175°C, outlet temperature: 116°C) to obtain a white amorphous powder. The pH of its 5 w / w% solution was 4.5.

[0096] Sample B: A portion of the aqueous solution was spray dried on a Buchi micro spray dryer B290 (inlet temperature: 175°C, outlet temperature: 120°C) to obtain a white amorphous powder. The pH of a 5 w / w% solution thereof was 7.0.

[0097] 1 gram of each sample was placed in a 4 ml glass vial and sealed with a screw cap lined with Teflon. The 8-week storage sample was placed in an oven at 60°C, and then the 4-week and 2-week storage samples were placed after 4 and 6 weeks, respectively, so that the incubation of all samples ended at the same time. The samples measured at 80°C (4-week, 2-week and 1-week samples) also followed a similar experimental design pattern. The samples were kept in a refrigerator until they were placed in an oven at a given time. The standards were kept in a refrigerator until analysis.

[0098] The samples showed no phase changes during the study.

[0099] Stability samples and standards were analyzed by HPAEC.

[0100] The table below shows the respective contents of 2'-O-fucosyl-lactulose and 3,2'-di-O-fucosyl-lactulose (ketose rearranged derivatives of 2'-FL and DFL) in the samples (in w / w %).

[0101]

[0102] Amorphous samples with acidic pH showed practically no isomerization of 2'-O-fucosyl-lactulose and 3,2'-di-O-fucosyl-lactulose during storage under forced conditions, whereas in samples with neutral pH an increase in the amount of rearranged derivatives was observed.

[0103] Slight hydrolysis of 2'-FL to fucose and lactose was observed in acidic samples, while no such hydrolysis occurred in neutral samples. On the other hand, no hydrolysis of DFL occurred in either acidic or neutral samples. This data suggests that the preferred pH range for 2'-FL, where no significant hydrolysis and rearrangement occurs, can be estimated to be 5.2-5.8.

[0104] Example 3

[0105] LNT was prepared by fermentation using E. coli K12 strain DH1 obtained from Deutsche Sammlung von Mikroorganismen (reference number DSM 5346) in which the following genes were deleted: nadC, lacZ, nanKETA, lacA, lacI, melA, wcaJ and mdoH, while the genes involved in the biosynthesis of UDP-GlcNAc and UDP-Gal were maintained. This strain also contained the Neisseria meningitidis (N. meningitidis) lgtA gene for β-1,3-N-acetylglucosaminyltransferase and a plasmid carrying the Helicobacter pylori ATCC 43504 galTK gene for β-1,3-galactosyltransferase integrated into the genome of the strain and the native regulatory and coding sequences of nadC. Fermentation parameters for a 2-liter fermenter: 0.6 liters of aqueous basal medium (Samain et al., J. Biotechnol. 72, 33 (1999)) were placed in a fermenter, the temperature was maintained at 28°C, and the pH was adjusted to 6.8 with 28% NH4OH. The strain inoculum was in a defined basal medium (20 ml) supplemented with trace minerals. The exponential growth phase began with inoculation and stopped when the glucose carbon source initially added to the aqueous medium was exhausted. A feed solution of glucose (750 g / l) was added to the medium at a feed rate of 7 ml / h. Lactose was added in two portions: 80 g of lactose in 160 ml of water was added after 1 hour, and then 40 g of lactose in 80 ml of water was added after 42 hours. The fermentation lasted for about 85-95 hours and produced a final aqueous medium containing LNT, accompanied by LNTri II, pLNH II and the remaining lactose (LNT titer: 40-45 g / l).

[0106] The fermentation broth is subjected to ultrafiltration to separate biomass and / or high molecular weight suspended solids from the soluble components of the fermentation broth to obtain a UF permeate containing LNT and associated carbohydrates, which is subjected to nanofiltration to obtain a NF retentate containing LNT and associated carbohydrates. The UF permeate is directly treated with H + The product was purified by treatment with a strong cation exchange resin of the type 100-400 μg / ml and then with a weak anion exchange resin in the free base form. The combined neutralized fractions containing LNT and the accompanying carbohydrates were treated with activated carbon to give a clear solution.

[0107] Sample A: The above aqueous solution (25.4 g LNT + 2 g LNTri II + 0.9 g pLNH II + 2 g lactose / 100 g solution) was adjusted to pH 3.2 by adding 25% aqueous sulfuric acid. The acidic solution thus obtained was spray dried on a Buchi micro spray dryer B290 (inlet temperature: 175° C., outlet temperature: 109° C.) to obtain a white amorphous powder. The pH of its 5 w / w% solution was 4.5.

[0108] Sample B: The above aqueous solution (25.4 g LNT + 2 g LNTri II + 0.9 g pLNH II + 2 g lactose / 100 g solution) was spray dried on a Buchi small spray dryer B290 (inlet temperature: 175° C., outlet temperature: 112° C.) to obtain a white amorphous powder. The pH of its 5 w / w% solution was 6.8.

[0109] Sample C: LNT polymorph C was prepared according to WO 2017 / 101953. The pH of its 5 w / w% solution was 6.3.

[0110] 1 gram of each sample was placed in a 4 ml glass vial and sealed with a screw cap lined with Teflon. The 8-week storage sample was placed in an oven at 60°C, and then the 4-week and 2-week storage samples were placed after 4 and 6 weeks, respectively, so that the incubation of all samples ended at the same time. The samples measured at 80°C (4-week, 2-week and 1-week samples) also followed a similar experimental design pattern. The samples were kept in a refrigerator until they were placed in an oven at a given time. The standards were kept in a refrigerator until analysis.

[0111] The samples showed no phase changes during the study.

[0112] Stability samples and standards were analyzed by HPAEC.

[0113] The following table shows the content (w / w %) of the keto-rearranged derivative of LNT (3Galβ1-3GlcNAcβ1-3Galβ1-4Fru) in the samples.

[0114]

[0115] The amorphous samples with acidic pH showed practically no rearrangement during storage under forced conditions, whereas in the amorphous samples with neutral pH as well as in the crystalline samples, an increase in the amount of rearranged derivatives was observed. Thus, the behavior of the amorphous samples with acidic pH is more favorable than that of either the neutral amorphous or the crystalline samples.

[0116] No obvious hydrolysis products were detected in all three samples.

Claims

1. An amorphous mixture comprising one or more human milk oligosaccharides (HMOs) and an acidic non-carbohydrate component, wherein the pH of the mixture in a 5 w / w% aqueous solution thereof is 3.5-5.8, and the HMO is lacto-N-neotetraose (LNnT), or lacto-N-neotetraose (LNnT) and one or more selected from lacto-N-tetraose (LNT), 2'-fucosyllactose (2'-FL), 3-fucosyllactose (3-FL) and difucosyllactose (DFL).

2. The amorphous mixture according to claim 1, which is spray-dried or freeze-dried.

3. The amorphous mixture according to claim 1 or 2, wherein the acidic non-carbohydrate component is an inorganic acid or an organic acid.

4. The amorphous mixture according to claim 1 or 2, wherein the acidic non-carbohydrate component is an inorganic acid selected from sulfuric acid and its monosalts, HCl, HBr, nitric acid or phosphoric acid and its monosalts and disalts.

5. The amorphous mixture according to claim 1 or 2, wherein the acidic non-carbohydrate component is an organic acid selected from the group consisting of formic acid, acetic acid, oxalic acid, tartaric acid, lactic acid, malic acid and citric acid.

6. The amorphous mixture according to claim 1 or 2, wherein the pH is 4.5-5.

8.

7. The amorphous mixture according to claim 1 or 2, wherein the pH is 5.0-5.

8.

8. The amorphous mixture according to claim 1 or 2, wherein the pH is 5.0-5.

5.

9. The amorphous mixture according to claim 1 or 2, wherein the pH is 4.0-5.

0.

10. The amorphous mixture according to claim 1 or 2, wherein the pH is 4.0-4.5, preferably 4.2-5.5, more preferably 4.5-5.5, more preferably 4.2-5.0.

Images