Compositions comprising hmo, their preparation and use for preventing and / or treating viral and / or bacterial infections

Abstract

The human milk oligosaccharide mixture comprises or consists essentially of: a) LNTri II, LNT, pLNH II and optionally lactose, or b) LNTri II, LNnT, pLNnH and optionally lactose, which can be used to prevent or treat viral or bacterial intestinal and respiratory tract infections and to modulate the human gastrointestinal microbiota to increase Bifidobacterium abundance. Methods of making the mixture and methods of crystallizing LNnT and LNT are also described.

Description

[0001] This application is a divisional application of the invention patent application with the invention name “Compositions comprising HMO, their preparation and use for preventing and / or treating viral and / or bacterial infections” and application number “201780039296.X” filed on June 23, 2017. Field of the Invention

[0002] The present invention relates to ternary mixtures of human milk oligosaccharides (HMOs), in particular to a) GlcNAcβ1-3Galβ1-4Glc (i.e., lactose-N-triose II or LNTri II); b) component A, which is Galβ1-4GlcNAcβ1-3Galβ1-4Glc (i.e., lactose-N-neotetraose or LNnT) or Galβ1-3GlcNAcβ1-3Galβ1-4Glc (i.e., lactose-N-tetraose or LNT); and c) component B, when component A is LNnT, component B is Galβ1-4GlcNAcβ1-3Galβ1-4GlcNAcβ1-3Galβ1-4Glc (i.e., p-lactose-N-neohexose or pLNnH), or component B is Galβ1-3GlcNAcβ1-3Galβ1-3GlcNAcβ1-3Galβ1-4Glc (i.e., p-lactose-N-hexaose II or pLNH II); and optionally a mixture of Galβ1-4Glc (lactose). The invention also relates to a method for preparing the ternary mixture and the application of the ternary mixture in human health. Background Art

[0003] HMOs have become the subject of great interest in recent years due to their role in many biological processes occurring in the human organism. Mammalian milk contains at least 130 of these complex oligosaccharides (Urashima et al.: Milk Oligosaccharides, Nova Biomedical Books, New York, 2011, ISBN: 978-1-61122-831-1).

[0004] Previously, the only source of HMO was mammalian milk, which contained primarily water, together with 55-70 g / l lactose, 24-59 g / l lipids, approximately 13 g / l protein, 5-15 g / l HMO and approximately 1.5 g / l minerals.

[0005] However, in the last decade, efforts to develop methods for synthesizing HMOs have increased significantly due to the role of HMOs in many human biological processes. In this regard, methods for producing HMOs by biofermentation, enzymatic methods, chemical synthesis, or a combination of these techniques have been developed. For example, by chemical methods, LNnT can be produced, as described in WO 2011 / 100980 and WO 2013 / 044928, LNT can be synthesized, as described in WO 2012 / 155916 and WO 2013 / 044928, a mixture of LNT and LNnT can be produced, as described in WO 2013 / 091660, 2'-FL can be produced, as described in WO 2010 / 115934 and WO 2010 / 115935, 3-FL can be produced, as described in WO 2013 / 139344, and 6'-SL and its salts can be produced, as described in WO 2010 / 100979. As examples of biotechnological methods, WO 01 / 04341 and WO 2007 / 101862 describe how to use genetically modified E. coli to produce core human milk oligosaccharides optionally substituted with fucose or sialic acid. As an example of an enzymatic method, sialylated oligosaccharides can be produced as described in EP-A-577580.

[0006] Efforts have also been made to develop methods for the enzymatic synthesis of HMO mixtures without the need to synthesize all of the constituent HMOs of the mixture, as described in WO 2012 / 156897 and WO 2012 / 156898. These methods provide a reaction mixture containing a plurality of different HMOs.

[0007] Non-enzymatic methods have also been sought for the synthesis of HMO mixtures. In this regard, WO 01 / 04341 describes a microbial process for the production of HMO mixtures. The process involves: i) transforming E. coli with exogenous genes encoding β1,3-N-acetylglucosaminyltransferase and β1,4-galactosyltransferase; ii) culturing or fermenting the E. coli cells in an aqueous medium or a fermentation medium containing glycerol and lactose; and then iii) permeabilizing the cells by heat treatment to release their HMO content. The cell contents apparently contain the following two HMOs: LNnT and p-LNnH, as well as p-lactose-N-neooctaose (p-LNnO) and p-lactose-N-neodecasaccharide (p-LNnD), which are separated from each other by biogel chromatography.

[0008] WO 2015 / 049331 describes the application of continuous mode simulated moving bed chromatography with ion exchange resins to separate neutral HMO LNT produced by bacterial fermentation from other oligosaccharide components.

[0009] However, an alternative microbial process for producing a mixture of HMOs including LNT or LNnT has been sought, in which the E. coli will secrete the mixture of HMOs out of the cells into the culture medium without permeabilizing the cells. Thus, the mixture of HMOs can subsequently be easily separated from the culture medium with minimal contamination of the mixture of HMOs, in particular from cellular proteins and cellular DNA.

[0010] Furthermore, alternative microbial processes have been sought for the production of mixtures of different HMOs comprising LNT or LNnT and preferably also LNTri II, in particular mixtures containing a major amount of LNT or LNnT relative to the amount of the other HMOs in the mixture. Summary of the invention

[0011] A first aspect of the invention relates to a synthetic mixture of HMOs comprising, or preferably consisting essentially of: a) LNT, pLNH II, LNTri II and optionally lactose; or b) LNnT, pLNnH, LNTri II and optionally lactose. Advantageously, in either case, the HMO mixture contains a major amount of LNT or LNnT relative to the other HMOs and lactose in the mixture. More advantageously, the first HMO mixture has the following weight ratios:

[0012] a) Lactose, LNTri II and pLNH II relative to LNT are:

[0013] Lactose: LNT is not more than 0.6, LNTri II: LNT is not more than 0.2, pLNH II: LNT is not more than 0.05; or

[0014] b) Lactose, LNTri II and pLNnH relative to LNnT are:

[0015] Lactose:LNnT is not more than 0.8, LNTri II:LNnT is not more than 0.1, and pLNnH:LNnT is not more than 0.4.

[0016] A second aspect of the present invention relates to a method for producing the synthetic HMO mixture of the first aspect of the present invention, comprising the steps of:

[0017] a) providing a genetically modified cell, preferably a bacterial cell, more advantageously an E. coli cell, comprising:

[0018] - a first recombinant gene encoding a β1,3-N-acetyl-glucosaminyltransferase capable of transferring GlcNAc from UDP-GlcNAc to lactose in a cell, thereby forming LNTri II in the cell, and

[0019] - a second recombinant gene encoding a β1,3- or β1,4-galactosyltransferase capable of transferring a galactosyl residue from UDP-Gal to LNTri II to form LNT or LNnT, respectively, in the cell,

[0020] b) culturing the cells in a medium containing lactose, thereby inducing:

[0021] - internalization of lactose into the cell, preferably via an active transport mechanism, and

[0022] - Formation of LNT, pLNH II and LNTri II, or LNnT, pLNnH and LNTri II in cells, then

[0023] c) optionally, continuing the culturing according to step b) until no lactose remains, and

[0024] d) separating and isolating the mixture of LNT, pLNH II, LNTri II and optionally lactose, or the mixture of LNnT, pLNnH, LNTri II and optionally lactose from the culture medium.

[0025] Advantageously, the genetically modified cells have LacY + Genotype, preferably LacZ - , LacY + Genotype, especially LacZ - , LacY + 、LacI - Genotype. Advantageously, the cells are intact during step c). In one embodiment of step c), in either case, complete consumption of lactose is achieved by extending the fermentation until the cells provided in step a) convert all lactose added in step b) into LNT, pLNH II and LNTri II, or LNnT, pLNnH and LNTri II. In other embodiments of step c), excess lactose is enzymatically hydrolyzed by exogenously added β-galactosidase, or by β-galactosidase expressed by the cells provided in step a).

[0026] The third aspect of the present invention provides a crystallization method, which method:

[0027] a) crystallizing LNT using one or more monohydroxy C1-C4 alcohols as antisolvent, wherein the crystallization is carried out from a mixture comprising LNT, LNTri II, pLNH II and optionally lactose, wherein the weight ratio of lactose, LNTri II and pLNH II relative to LNT is:

[0028] Lactose: LNT = 0-0.2, LNTri II: LNT no greater than 0.15, and pLNH II: LNT no greater than 0.05; or

[0029] b) crystallizing LNnT using one or more monohydroxy C1-C4 alcohols and / or acetone as antisolvent, wherein the crystallization is carried out from a mixture comprising LNnT, LNTri II, pLNnH and optionally lactose, wherein the weight ratio of lactose, LNTri II and pLNnH relative to LNnT is:

[0030] Lactose: LNnT = 0-0.2, LNTri II: LNnT not greater than 0.1, and pLNnH: LNnT not greater than 0.03.

[0031] A fourth aspect of the invention relates to an anti-infective composition for preventing and / or treating viral and / or bacterial infections in humans, comprising LNT, pLNH II and LNTri II, or LNnT, pLNnH and LNTri II, advantageously a synthetic HMO mixture according to the first aspect of the invention. The composition comprises a plurality of different HMOs having novel properties and combinations of biological activities. The composition can be used in particular to target viral and bacterial intestinal infections by modulating the gastrointestinal microbiota, modulating the intestinal association of bacteria and viruses, and improving intestinal barrier function and immune function. In this aspect, the composition increases the abundance of Bifidobacterium in the gastrointestinal microbiota in humans. The composition can also be particularly useful against viral and bacterial respiratory infections by inhibiting pathogen colonization, to protect the host from infection and / or to modulate the immune system.

[0032] A fifth aspect of the invention relates to a method for regulating the gastrointestinal microbiota of a human to increase the abundance of bifidobacteria. The method comprises administering to the human LNT, pLNH II and LNTri II, or LNnT, pLNnH and LNTri II, advantageously a synthetic HMO mixture according to the first aspect of the invention.

[0033] A sixth aspect of the present invention relates to a method for preventing and / or treating viral and / or bacterial infections, particularly intestinal and respiratory infections, in humans. The method comprises administering to humans LNT, pLNH II and LNTri II, or LNnT, pLNnH and LNTri II, advantageously a synthetic HMO mixture according to the first aspect of the present invention. DETAILED DESCRIPTION

[0034] According to the present invention, it has been unexpectedly found that a synthetic mixture of LNT, pLNH II and LNTri II or LNnT, pLNnH and LNTri II and optional lactose can provide an anti-infective composition for treating and / or preventing viral and / or bacterial infections of the human intestinal tract and respiratory tract. The composition works by specifically regulating the microbiota in the intestinal tract. In this regard, the mixture increases the abundance of bifidobacteria in the microbiota and can also reduce the abundance of Firmicutes, especially Clostridia. The mixture also inhibits the colonization of a series of respiratory pathogens by stimulating host defenses, including regulating the immune system.

[0035] Furthermore, it has been surprisingly found that mixtures of LNT, pLNH II and LNTri II or LNnT, pLNnH and LNTri II and optionally lactose can be advantageously used for the selective crystallization of LNT or LNnT from the respective mixtures. The process of isolating LNT or LNnT from the culture broth can be facilitated and simplified by eliminating expensive and cumbersome separation techniques such as biogel chromatography and simulated fluid bed chromatography proposed in the prior art for separating carbohydrate components from one another. On the contrary, it is sufficient to isolate LNT, pLNH II and LNTri II or LNnT, pLNnH and LNTri II and optionally lactose, from which LNT or LNnT can be easily obtained by selective crystallization.

[0036] The term "synthetic mixture" or "synthetic composition" refers to an artificially prepared mixture or composition, which preferably refers to a mixture or composition containing at least one compound produced ex vivo chemically and / or biologically, such as by chemical reaction, enzymatic reaction or recombinantly. In this regard, "synthetic" is the opposite of "natural", meaning that the synthetic mixture or composition of the present invention is not the same as a natural composition or mixture, such as human milk, or that at least one HMO of the mixture or composition is not derived from a natural source, such as human milk.

[0037] A first aspect of the present invention

[0038] The mixture of the present invention is an HMO mixture comprising, preferably consisting essentially of:

[0039] -GlcNAcβ1-3Galβ1-4Glc(LNTri II),

[0040] - component A, which is Galβ1-3GlcNAcβ1-3Galβ1-4Glc (LNT) or Galβ1-4GlcNAcβ1-3Galβ1-4Glc (LNnT),

[0041] - Component B, which is:

[0042] When component A is LNT, Galβ1-3GlcNAcβ1-3Galβ1-3GlcNAcβ1-3Galβ1-4Glc(pLNH II), or

[0043] When component A is LNnT, Galβ1-4GlcNAcβ1-3Galβ1-4GlcNAcβ1-3Galβ1-4Glc(pLNnH),

[0044] - and optionally lactose.

[0045] In the context of the present invention, the term "optionally" means that lactose is present or not present in the mixture. In this regard, in one embodiment, the mixture of the present invention may be a mixture comprising LNT, pLNH II, LNTri II and lactose, preferably consisting essentially of them, and in another embodiment, may be a mixture comprising LNnT, pLNnH, LNTri II and lactose, preferably consisting essentially of them. Moreover, the mixture of the present invention may be a mixture comprising LNT, pLNH II and LNTri II, preferably consisting essentially of them, or a mixture comprising LNnT, pLNnH and LNTri II, preferably consisting essentially of them. In either case, each mixture of the present invention preferably contains a major amount of LNT or LNnT, which is respectively relative to each of pLNH II and LNTri II or pLNnH and LNTri II and any lactose in the mixture, more preferably relative to the total amount of pLNH II, LNTri II and any lactose or pLNnH, LNTri II and any lactose in the mixture. In the case of a mixture comprising LNnT, it is further preferred that the mixture contains substantially no p-LNnO or p-LNnD. In either case, the term "mainly" means that the amount of LNT or LNnT by weight in the mixture of the present invention is greater than the amount of each of pLNH II and LNTri II or pLNnH and LNTri II and any lactose, respectively, such that the amount of LNT is at least about 2.5 times greater, or at least about 5 times greater, or at least about 10 times greater than the amount of each of pLNH II and LNTri II and any lactose in the mixture, or the combined amount of each of pLNH II and LNTri II and any lactose in the mixture, or the amount of LNnT is at least about 2.5 times greater, or at least about 5 times greater, or at least about 10 times greater than the amount of each of pLNnH and LNTri II and any lactose in the mixture, or the combined amount of each of pLNnH and LNTri II and any lactose in the mixture.

[0046] In the HMO mixture, the weight ratio of lactose, LNTri II and pLNH II relative to LNT is preferably:

[0047] Lactose:LNT is 0 to no greater than 0.6, LNTri II:LNT is no greater than 0.2, and pLNHII:LNT is no greater than 0.05.

[0048] In the HMO mixture, the weight ratio of lactose, LNTri II and pLNnH relative to LNnT is preferably:

[0049] Lactose:LNnT is 0 to not more than 0.8, LNTri II:LNnT is not more than 0.1, and pLNnH:LNnT is not more than 0.4.

[0050] According to the above definition of the HMO mixture of the first aspect, lactose is an optional ingredient, which means that the ratio lactose:LNT is 0-0.6 and the ratio lactose:LNnT is 0-0.8. When the ratio is 0, the HMO mixture of the present invention does not contain lactose, or at least substantially does not contain lactose (i.e., substantially lactose-free). When the HMO mixture of the present invention contains lactose, its weight ratio to LNT is greater than 0 but not greater than 0.6, or its weight ratio to LNnT is greater than 0 but not greater than 0.8. On the other hand, in a mixture containing LNT, both the ratios LNTri II:LNT and pLNH II:LNT are greater than 0, or in a mixture containing LNnT, both the ratios LNTri II:LNnT and pLNnH:LNnT are greater than 0 (in other words, cannot be 0), because in either case, both LNTri II and pLNH II, or both LNTri II and pLNnH are essential components of the HMO mixture claimed.

[0051] In certain embodiments of the first aspect, the weight ratio of lactose, LNTri II and pLNH II relative to LNT is:

[0052] lactose: LNT no greater than 0.4, LNTri II: LNT no greater than 0.2, and pLNH II: LNT no greater than 0.05, or

[0053] Lactose:LNT is 0, LNTri II:LNT is not greater than 0.2, and pLNH II:LNT is not greater than 0.05, or

[0054] lactose: LNT no greater than 0.4, LNTri II: LNT no greater than 0.15, and pLNH II: LNT no greater than 0.03, or

[0055] Lactose:LNT is 0, LNTri II:LNT is not greater than 0.15, and pLNH II:LNT is not greater than 0.03, or

[0056] lactose: LNT = 0.15-0.20, LNTri II: LNT = 0.05-0.12, and pLNH II: LNT = 0.005-0.03, or

[0057] lactose: LNT=0, LNTri II: LNT=0.05-0.12, and pLNH II: LNT=0.005-0.03, or

[0058] lactose: LNT = 0.10-0.15, LNTri II: LNT = 0.04-0.1, and pLNH II: LNT = 0.005-0.03, or

[0059] Lactose: LNT = 0, LNTri II: LNT = 0.04-0.1, and pLNH II: LNT = 0.005-0.03.

[0060] In certain other embodiments of the first aspect, the weight ratio of lactose, LNTri II and pLNnH relative to LNnT is:

[0061] lactose: LNnT not greater than 0.4, LNTri II: LNnT not greater than 0.1, and pLNnH: LNnT not greater than 0.4, or

[0062] Lactose:LNnT is 0, LNTri II:LNnT is not greater than 0.1, and pLNnH:LNnT is not greater than 0.4, or

[0063] Lactose: LNnT not greater than 0.4, LNTri II: LNnT not greater than 0.05, and pLNnH: LNnT not greater than 0.2, or

[0064] Lactose:LNnT is 0, LNTri II:LNnT is not greater than 0.05, and pLNnH:LNnT is not greater than 0.2, or

[0065] lactose: LNnT = 0.37-0.72, LNTri II: LNnT = 0.01-0.05, and pLNnH: LNnT = 0.15-0.25, or

[0066] Lactose: LNnT = 0, LNTri II: LNnT = 0.01-0.05, and pLNnH: LNnT = 0.15-0.25, or

[0067] lactose: LNnT = 0.43-0.72, LNTri II: LNnT = 0.02-0.04, and pLNnH: LNnT = 0.15-0.20, or

[0068] lactose: LNnT = 0, LNTri II: LNnT = 0.02-0.04, and pLNnH: LNnT = 0.15-0.20, or

[0069] lactose: LNnT = 0.43-0.72, LNTri II: LNnT = 0.03-0.05, and pLNnH: LNnT = 0.18-0.19, or

[0070] Lactose: LNnT = 0, LNTri II: LNnT = 0.03-0.05, and pLNnH: LNnT = 0.18-0.19.

[0071] In other embodiments, the synthetic HMO mixture is advantageously suitable for the selective crystallization of LNT, wherein the weight ratio of lactose, LNTri II and pLNH II relative to LNT is:

[0072] Lactose: LNT = 0-0.2, LNTri II: LNT not greater than 0.15, and pLNH II: LNT not greater than 0.05, e.g.

[0073] Lactose: LNT = 0-0.2, LNTri II: LNT not greater than 0.12, and pLNH II: LNT not greater than 0.03, or

[0074] Lactose: LNT = 0-0.2, LNTri II: LNT not greater than 0.1, and pLNH II: LNT not greater than 0.02, or

[0075] Lactose: LNT = 0-0.2, LNTri II: LNT not greater than 0.075, and pLNH II: LNT not greater than 0.01.

[0076] In other embodiments, the synthetic HMO mixture is advantageously suitable for the selective crystallization of LNnT, wherein the weight ratio of lactose, LNTri II and pLNnH relative to LNnT is:

[0077] Lactose: LNnT = 0-0.2, LNTri II: LNnT not greater than 0.1, and pLNnH: LNnT not greater than 0.03, e.g.

[0078] lactose: LNnT = 0-0.2, LNTri II: LNnT = 0.005-0.1, and pLNnH: LNnT = 0.005-0.03, or

[0079] Lactose: LNnT = 0-0.2, LNTri II: LNnT = 0.005-0.05, and pLNnH: LNnT = 0.005-0.025, or

[0080] Lactose: LNnT = 0-0.2, LNTri II: LNnT = 0.01-0.03, and pLNnH: LNnT = 0.01-0.025.

[0081] Second aspect of the present invention

[0082] The HMO mixture of the present invention can be readily obtained by a process involving culturing or fermenting the genetically modified cells in an aqueous culture medium or fermentation medium containing lactose and one or more carbon substrates, and then separating them from the culture medium. The term "culture medium" refers to the aqueous environment of the fermentation process in the fermenter outside the genetically modified cells.

[0083] The term "genetically modified cell" refers to a cell in which at least one DNA sequence is added, deleted or changed in the cell genome, so that the cell has an altered phenotype. This phenotypic change changes the characteristics of the genetically modified cell compared to the wild-type cell. Therefore, when cultured or fermented, the genetically modified cell can perform at least one additional chemical transformation due to the expression of at least one enzyme not found in the wild-type cell encoded by the added or changed DNA, or the genetically modified cell cannot perform a chemical transformation due to the expression of an enzyme found in the wild-type cell encoded by the deleted, added or changed DNA. Genetically modified cells can be produced by conventional genetic modification techniques. The genetically modified cell can be a bacterium or a yeast, but is preferably a bacterium. Preferred bacteria include Escherichia coli, Bacillus spp. (e.g., B. subtilis), Campylobacter pylori, Helicobacter pylori, Agrobacterium tumefaciens, Staphylococcus aureus, Thermophilus aquaticus, Azorhizobium caulinodans, Rhizobium leguminosarum, Neisseria gonorrhoeae, N. meningitis, Lactobacillus spp., Lactococcus spp., Enterococcus spp., Bifidobacterium spp.), Sporolactobacillus spp., Micromomosporaspp., Micrococcus spp., Rhodococcus spp., Pseudomonas, and especially Escherichia coli.

[0084] Specifically, the genetically modified cells of the present invention contain:

[0085] a first recombinant gene encoding a β1,3-N-acetyl-glucosaminyltransferase capable of transferring GlcNAc from UDP-GlcNAc to lactose in cells, thereby forming LNTri II,

[0086] - A second recombinant gene encoding

[0087] β1,3-galactosyltransferase, which is able to transfer the galactosyl residue from UDP-Gal to LNTri II in cells, thereby forming LNT,

[0088] Alternatively, β1,4-galactosyltransferase is able to transfer the galactosyl residue from UDP-Gal to LNTri II in cells, thereby forming LNnT.

[0089] Preferably, the genetically modified cells have LacZ - Genotype, especially LacZ - , LacY + Genotype, more specifically LacZ - , LacY + 、LacI - genotype.

[0090] When the genetically modified cells of the present invention are cultured in an aqueous medium containing lactose, the lactose can be internalized, and then the GlcNAc residue of the activated sugar nucleotide in the cell is transferred to the internalized lactose to form LNTri II in the cell. In either case, the cell can also transfer the galactosyl residue of the activated sugar nucleotide in the cell to the LNTri II previously formed in the cell to form LNT or LNnT in the cell. Conventional expression vectors can be used to introduce recombinant genes or equivalent DNA sequences responsible for these transfers into the cell in a known manner. The source of these heterologous nucleic acid sequences can be, for example, any bacteria, for example, Neisseria meningitidis (N.meningitidis) or Helicobacter pylori (H.pylori).

[0091] In carrying out the method, the genetically modified cells are cultured in the presence of a carbon substrate such as glycerol, glucose, sucrose, glycogen, fructose, maltose, starch, cellulose, pectin, chitin, etc. Preferably, the cells are cultured with glycerol, glucose, sucrose and / or fructose.

[0092] The method also relates to initially transporting exogenous lactose from culture medium to genetically modified cells. Lactose can be added to the culture medium subsequently transported to the cell by it in a conventional manner exogenously. Of course, the internalization of lactose should not affect basic, important functions or destroy the integrity of the cell. Internalization can occur via a passive transport mechanism, in which lactose passively diffuses across the plasma membrane of the cell. Flow is directed by the concentration difference of the lactose to be internalized in the extracellular and intracellular space, so that lactose is passed from the place with higher concentration to the place with lower concentration. However, lactose is preferably internalized into the cell under the assistance of an active transport mechanism, and lactose diffuses across the plasma membrane of the cell under the influence of the transporter or lactose permease (LacY) of the cell by this mechanism.

[0093] In some embodiments, the genetically modified cells used in the method lack enzyme activities that significantly degrade the following substances: lactose, LNT, LNnT, LNTri II, pLNH II, pLNnH, and metabolic intermediates required for the production of LNT, LNnT, LNTri II, pLNH II, and pLNnH in the cell. In this regard, the native β-galactosidase (encoded by LacZ in E. coli) of the cultured cells that hydrolyzes lactose into galactose and glucose is preferably deleted or inactivated (LacZ - genotype). In one embodiment of the second aspect of the invention, the excess lactose added in step b) is not removed or degraded after fermentation, in either case, according to step d), the mixture of lactose, LNT, LNTri II and pLNH II or the mixture of lactose, LNnT, LNTri II and pLNnH is separated and isolated from the culture medium to produce a HMO mixture consisting essentially of lactose, LNT, LNTri II and pLNH II or a HMO mixture consisting essentially of lactose, LNnT, LNTri II and pLNnH, respectively. In other embodiments, a mixture of lactose, LNT, LNTri II and pLNH II or a mixture of lactose, LNnT, LNTri II and pLNnH is produced by fermentation as described above, and in either case, the mixture of LNT, LNTri II and pLNH II or the mixture of LNnT, LNTri II and pLNnH is separated and isolated from the culture medium and excess lactose to produce a HMO mixture consisting essentially of LNT, LNTri II and pLNH II or a HMO mixture consisting essentially of LNnT, LNTri II and pLNnH, respectively. In still other embodiments, a mixture of lactose, LNT, LNTri II and pLNH II or a mixture of lactose, LNnT, LNTri II and pLNnH is produced by fermentation as described above, and then, in step c),

[0094] i) exogenously adding a lactose degrading enzyme, such as β-galactosidase, which hydrolyzes lactose into monosaccharides, or

[0095] ii) allowing the fermentation to continue until all the lactose added in step b) is converted to the desired HMO,

[0096] Provide a broth that is substantially free of lactose. In this regard, in option ii), the LacZ disclosed above -The genotype genetically modified cells may also include a functional recombinant β-galactosidase. The functional β-galactosidase may be encoded by a heat-inducible exogenous LacZ gene (see, e.g., WO 2015 / 036138). At fermentation temperature, the cells do not express the functional β-galactosidase, and therefore, the lactose internalized during the production of HMOs is not degraded in the cells. When the desired HMO concentration or amount is reached in the broth, the culture is continued at an elevated temperature, and the excess lactose is degraded by its expression of a functional β-galactosidase. Alternatively, the LacZ disclosed above - The genotype-modified cells may include a recombinant β-galactosidase (see, e.g., WO 2012 / 112777) with low but detectable levels of activity to remove any residual lactose at the end of the fermentation. Both options thus provide a broth containing LNT, LNTri II and pLNH II that is substantially free of lactose, or a broth containing LNnT, LNTri II and pLNnH that is substantially free of lactose, from which a HMO mixture consisting essentially of LNT, LNTri II and pLNH II or a HMO mixture consisting essentially of LNnT, LNTri II and pLNnH, respectively, can be easily separated and isolated.

[0097] Other examples of strains producing LNT are disclosed in, for example, et al. ChemBioChem 15, 1896 (2014) and Enzyme Microb. Technol. 75-76, 37 (2015), WO 2014 / 153253, WO 2015 / 036138 or WO 2016 / 008602. Other examples of strains producing LNnT are disclosed in the following literature, for example, Priem et al. Glycobiology 12, 235 (2002); Gebus et al. Carbohydr. Res. 361, 83 (2012); WO 01 / 04341, WO 2014 / 153253, WO 2015 / 197082 or WO 2016 / 008602.

[0098] In order to achieve high yields, the method preferably involves providing the carbon substrate and at least 30, preferably at least 45, more preferably 50-70, up to about 100 grams of lactose per liter of the initial volume of the culture medium. Preferably, the method is also carried out at a temperature of 28-35°C, preferably under continuous agitation and continuous aeration for more than 2 and 1 / 2 days, more preferably at least 4 days. Further preferably, the final volume of the culture medium is no more than 3 times, more preferably no more than 2 times, more preferably less than 2 times the initial volume of the culture medium before lactose and the carbon substrate are provided to the culture medium.

[0099] According to one embodiment, during the process of the present invention, the genetically modified LacZ - Y + E. coli strains were cultured by:

[0100] (1) a first phase of exponential cell growth, which is ensured by the carbon substrate, preferably glucose, provided in the culture medium and preferably lasts until the glucose has been completely consumed, which is preferably at least 12 hours, more preferably at least 18 hours, even more preferably 20-25 hours, up to about 48 hours; and

[0101] (2) A second phase of cell growth, which is limited by the carbon substrate, preferably glycerol and lactose, which is preferably continuously provided in the culture medium after the first phase, and which lasts until the glucose and preferably most (e.g., at least 60%) of the lactose have been consumed, which is preferably at least 35 hours, more preferably at least 45 hours, and even more preferably 50-70 hours, up to about 130 hours.

[0102] The mixture of LNnT, LNTri II, pLNnH and optionally lactose produced extracellularly in an aqueous medium by the above method comprises at least 20 g, more preferably at least 30 g, even more preferably at least 40 g, even more particularly preferably at least 50 g of LNnT per liter of final volume of the medium. In the mixture of LNnT, LNTri II, pLNnH and lactose in the culture medium, the lactose is mainly unreacted lactose that is not transported from the culture medium into the genetically modified cells.

[0103] In the process of cultivating genetically modified cells, LNT, LNnT, LNTri II, pLNH II and pLNnH accumulate in the intracellular and extracellular matrix of the cell, preferably mainly in the extracellular matrix. The main part of the mixture is then transported out of the cell to the aqueous medium in a passive manner, that is, it diffuses across the cell membrane to the outside of the cell. This transport can be improved by providing one or more sugar efflux transporters in the cell, that is, a protein that promotes sugar derivatives to flow from the cell to the culture medium (see, for example, WO 2010 / 142305). Sugar efflux transporters can be overexpressed under fermentation conditions to increase the output of the mixture produced in the cell to the culture medium.

[0104] The LNT, LNTri II and pLNH II product mixture transported from the cells to the aqueous medium preferably contains a major amount of LNT. More preferably, in the culture medium, LNT, LNTri II, pLNH II and optionally lactose are present in the following ratios:

[0105] Lactose: LNT is not greater than 0.6, LNTri II: LNT is not greater than 0.2, and pLNH II: LNT is not greater than 0.05, e.g.

[0106] lactose: LNT no greater than 0.4, LNTri II: LNT no greater than 0.2, and pLNH II: LNT no greater than 0.05, or

[0107] lactose: LNT no greater than 0.2, LNTri II: LNT no greater than 0.2, and pLNH II: LNT no greater than 0.05, or

[0108] lactose: LNT no greater than 0.4, LNTri II: LNT no greater than 0.15, and pLNH II: LNT no greater than 0.03, or

[0109] lactose: LNT no greater than 0.2, LNTri II: LNT no greater than 0.15, and pLNH II: LNT no greater than 0.03, or

[0110] lactose: LNT = 0.15-0.20, LNTri II: LNT = 0.05-0.12, and pLNH II: LNT = 0.005-0.03, or

[0111] Lactose: LNT = 0.10-0.15, LNTri II: LNT = 0.04-0.1, and pLNH II: LNT = 0.005-0.03.

[0112] LNT, LNTri II, pLNH II and optionally lactose may then be separated from cells and impurities in the culture medium in a conventional manner, and optionally a mixture of LNT, LNTri II and pLNH II is separated from lactose in the culture medium.

[0113] The LNnT, LNTri II and pLNnH product mixture transported from the cells to the aqueous medium preferably contains a major amount of LNnT. More preferably, in the culture medium, LNnT, LNTri II, pLNnH and optionally lactose are present in the following ratios:

[0114] Lactose: LNnT is not greater than 0.8, LNTri II: LNnT is not greater than 0.05, and pLNnH: LNnT is not greater than 0.4, e.g.

[0115] Lactose: LNnT = 0.2-0.8, LNTri II: LNnT not greater than 0.05, and pLNnH: LNnT not greater than 0.4, or

[0116] lactose: LNnT = 0.37-0.72, LNTri II: LNnT = 0.03-0.05, and pLNnH: LNnT = 0.15-0.25, or

[0117] Lactose: LNnT = 0.43-0.72, LNTri II: LNnT = 0.03-0.05, and pLNnH: LNnT = 0.15-0.20, or

[0118] lactose: LNnT = 0.43-0.72, LNTri II: LNnT = 0.03-0.05, and pLNnH: LNnT = 0.18-0.19, or

[0119] Lactose: LNnT not greater than 0.2, LNTri II: LNnT = 0.03-0.05, and pLNnH: LNnT = 0.15-0.25, or

[0120] Lactose: LNnT not greater than 0.2, LNTri II: LNnT = 0.03-0.05, and pLNnH: LNnT = 0.15-0.20, or

[0121] Lactose: LNnT is no greater than 0.2, LNTri II: LNnT = 0.03-0.05, and pLNnH: LNnT = 0.18-0.19.

[0122] LNnT, LNTri II, pLNnH and optionally lactose may then be separated from cells and impurities in the culture medium in a conventional manner, and optionally a mixture of LNnT, LNTri II and pLNnH is separated from lactose in the culture medium.

[0123] Step d) of the second aspect of the present invention comprises at least one of the following separation steps:

[0124] -Ultrafiltration

[0125] -Nanofiltration,

[0126] - ion exchange treatment, or

[0127] - Activated carbon treatment,

[0128] But preferably 2 or 3, or all 4 of these steps are performed in any order. Advantageously, an ultrafiltration step is always included, preferably as a first separation step (which is applied to the fermentation broth), followed by nanofiltration, ion exchange treatment and / or activated carbon treatment.

[0129] The first separation step involves separating an aqueous culture medium containing LNT, LNTri II, pLNH II and optional lactose or LNnT, LNTri II, pLNnH and optional lactose from cells and suspended particulate matter and pollutants, insoluble materials and debris. In this step, the culture medium can be clarified in a conventional manner, for example, by membrane filtration or centrifugation of the broth. According to one embodiment, the membrane filtration is ultrafiltration (UF). In the ultrafiltration step, biomass and preferably other high molecular weight suspended solids are separated from the soluble components of the broth. An aqueous solution containing the produced LNT, LNTri II, pLNH II and optional lactose or LNnT, LNTri II, pLNnH and optional lactose is passed through an ultrafiltration membrane without substantially changing its relative weight ratio to each other, producing a UF permeate (UFP).

[0130] Any conventional ultrafiltration membrane with a molecular weight cutoff (MWCO) ranging from about 1 to about 500 kDa, such as 10-250, 50-100, 200-500, 100-250, 1-100, 1-50, 10-25, 1-5 kDa, any other suitable sub-range, can be used. The membrane material can be ceramic, or made of synthetic or natural polymers such as polysulfone, polypropylene, cellulose acetate or polylactic acid. The ultrafiltration step can be applied in a dead-end or cross-flow mode. The separation step can include more than one ultrafiltration step using membranes with different MWCOs, for example, using two ultrafiltration separations, wherein the MWCO of the first membrane is higher than that of the second membrane. This arrangement can provide better separation efficiency for higher molecular weight components in the broth. After this separation step, the permeate contains material with a molecular weight lower than the MWCO of the second membrane, including LNT, LNTri II, pLNH II and optionally lactose or a mixture of LNnT, LNTri II, pLNnH and optionally lactose, in essentially the same range as before applying the UF step.

[0131] The second separation step comprises nanofiltration (NF). This nanofiltration step can be advantageously used to concentrate and / or remove ions of the previously obtained UFP, mainly monovalent ions, and remove organic materials such as monosaccharides with molecular weights lower than lactose. The MWCO of the nanofiltration membrane ensures that LNT, LNnT, LNTri II, pLNH II and pLNnH are retained, that is, its MWCO is lower than the ultrafiltration membrane used in the previous step, approximately 25-50% of the molecular weight of LNTri II, LNT or LNnT. In this respect, LNT, LNTri II and pLNH II or LNnT, LNTri II and pLNnH accumulate in the NF retentate (NFR). Interestingly, most of lactose (if present) is also retained in the NFR, therefore, the range of the amount of LNT, LNTri II, pLNH II and optional lactose or LNnT, LNTri II, pLNnH and optional lactose is substantially the same as before the application NF step. Nanofiltration can be combined with diafiltration with water to more effectively remove permeable molecules, for example, until the conductivity of the permeate indicates the absence or minimal presence of salts.

[0132] The third separation step preferably involves removing any residual minerals, salts or other charged molecules and amino acids from the residual culture medium, preferably after the second separation step. This third separation step may be carried out in a conventional manner using ion exchange resins by passing the residual culture medium through H + The cation exchange resin is preferably a strong exchanger and the anion exchange resin is a weak exchanger. In addition to removing salts and charged molecules from the residual culture medium, the ion exchange resin can also physically adsorb proteins, DNA and coloring / caramel bodies remaining in the second HMO mixture in the residual culture medium.

[0133] According to one embodiment, the ion exchange resin is an anion exchange resin, preferably a weakly basic anion exchange resin. In this step, negatively charged substances can be removed from pretreated solution because they are bound to the resin. In any appropriate manner, an aqueous solution of LNT, LNTri II, pLNH II and optional lactose or LNnT, LNTri II, pLNnH and optional lactose is contacted with an anion exchange resin, which will make negatively charged substances adsorbed on the anion exchange resin, and neutral oligosaccharides are passed therethrough. The liquid produced after contacting the anion exchange resin mainly contains a mixture of water, cations and LNT, LNTri II, pLNH II and optional lactose or LNnT, LNTri II, pLNnH and optional lactose.

[0134] According to another embodiment, the ion exchange resin is a cation exchange resin, preferably a strongly acidic cation exchange resin. In this step, positively charged substances can be removed from pretreated solution because they are bound to resin. In any appropriate manner, a solution of LNT, LNTri II, pLNH II and optional lactose or LNnT, LNTri II, pLNnH and optional lactose is contacted with the cation exchange resin, which will make the positively charged substances adsorbed on the cation exchange resin, and neutral oligosaccharides are passed through. Except for negatively charged ions, the liquid produced after contacting the cation exchange resin mainly also contains water and LNT, LNTri II, pLNH II and optional lactose or LNnT, LNTri II, pLNnH and optional lactose.

[0135] One of the ion exchange resin treatments disclosed above may be sufficient to obtain a mixture of LNT, LNTri II, pLNH II and optionally lactose or LNnT, LNTri II, pLNnH and optionally lactose of the desired purity. If necessary, both cation and anion exchange resin chromatography may be applied in any order. The ion exchange resin treatment does not substantially change the relative weight ratios of LNT, LNTri II, pLNH II and optionally lactose or LNnT, LNTri II, pLNnH and optionally lactose in the mixture.

[0136] When using ion exchange resins, their degree of crosslinking can be selected depending on the operating conditions of the ion exchange column. Highly crosslinked resins offer the advantages of durability and high mechanical integrity, but the disadvantage is reduced porosity and decreased mass transfer. Low crosslinked resins are more brittle and tend to swell by absorbing the mobile phase. The particle size of the ion exchange resin is selected to allow sufficient flow of the eluent while still effectively removing charged species. Appropriate flow rates can also be obtained by applying negative pressure to the column elution end, or applying positive pressure to the column loading end and collecting the eluent. Positive and negative pressures can also be used in combination. The ion exchange treatment can be carried out in a conventional manner, for example, batchwise or continuously.

[0137] Non-limiting examples of suitable acidic cation exchange resins can be, for example, Amberlite IR100, Amberlite IR120, Amberlite FPC22, Dowex 50WX, Finex CS16GC, Finex CS13GC, Finex CS12GC, Finex CS11GC, Lewatit S, Diaion SK, Diaion UBK, Amberjet 1000, Amberjet1200.

[0138] Non-limiting examples of suitable basic anion exchange resins may be, for example, Amberlite IRA67, Amberlite IRA 96, Amberlite IRA743, Amberlite FPA53, Diaion CRB03, Diaion WA10, Dowex 66, Dowex Marathon, Lewatit MP64.

[0139] The fourth separation step preferably involves removing any residual coloring / caramel and / or water-soluble contaminants such as salts from the remaining culture medium. This fourth step can be carried out in a conventional manner using activated carbon to decolorize the HMO mixture obtained in the previous step. The activated carbon treatment can be after any of the above-mentioned ultrafiltration, nanofiltration or ion exchange treatments, preferably after the ion exchange treatment.

[0140] Carbohydrate substances such as LNT, LNnT, LNTri II, pLNH II, pLNnH and lactose (if present) tend to be combined with the surface of carbon particles from their aqueous solutions such as UF, NF or ion exchange treatments. Similarly, colorants can also be adsorbed on the carbon. Although carbohydrates and coloring materials are adsorbed, they are not combined with carbon or can be eluted with water in combination with weaker water-soluble materials. By changing the eluent from water to aqueous ethanol, the adsorbed carbohydrates can be easily eluted and collected, and the relative weight ratio of the mixture about LNT, LNTri II, pLNH II and optional lactose or LNnT, LNTri II, pLNnH and optional lactose is basically the same. The adsorbed coloring material will remain adsorbed on the carbon, thereby decolorization and desalination can be achieved simultaneously in this step.

[0141] Under certain conditions, LNT, LNTri II, pLNH II and lactose (if present) or LNnT, LNTri II, pLNnH and lactose (if present) are not adsorbed or at least substantially not adsorbed on the carbon particles, and elution with water produces an aqueous solution of these oligosaccharides without significant loss of their amount, while the coloring matter remains adsorbed. It is routine practice in the art to determine the conditions under which oligosaccharides will be bound to carbon from their aqueous solution. For example, in one embodiment, a more diluted solution of LNT, LNTri II, pLNH II and lactose (if present) or LNnT, LNTri II, pLNnH and lactose (if present) is used, and in another embodiment, carbon with a lower amount relative to the amount of oligosaccharides is used. In this embodiment, the weight ratio of LNT, LNTri II, pLNH II and optional lactose or LNnT, LNTri II, pLNnH and optional lactose is substantially the same as the range before the application of this specific activated carbon treatment.

[0142] Moreover, in one embodiment of the activated carbon treatment, when the lactose:LNT ratio in the mixture of LNT, LNTri II, pLNH II and lactose obtained during the fermentation process and / or after any of the separation steps disclosed above is higher than 0.2, it can be reduced to 0.2 or lower by advantageously applying activated carbon treatment, for example by adopting the method proposed by Whistler et al., J. Am. Chem. Soc. 72, 677 (1950) or Morales et al. Chromatographia 64, 233 (2006). These methods are based on eluting / removing a considerable amount of disaccharide lactose from the higher oligosaccharides LNTri II, LNT and pLNH II, so that a mixture of LNTri II, LNT and pLNH II with very low or no lactose content (i.e., wherein the weight ratio of lactose:LNT is less than 0.2) can be obtained, while the weight ratio of LNT to LNTri II and pLNH II is substantially unchanged. Therefore, by applying the above-mentioned activated carbon treatment to an HMO mixture comprising or consisting of LNT, LNTri II, pLNH II and lactose, wherein the weight ratio of lactose, LNTri II and pLNH II relative to LNT in the mixture is: lactose: LNT is greater than 0.2 and less than 0.8, LNTri II: LNT is not greater than 0.15, and pLNH II: LNT is not greater than 0.05, an HMO mixture comprising or consisting of LNT, LNTri II, pLNH II and lactose can be obtained, wherein the weight ratio of lactose, LNTri II and pLNH II relative to LNT in the mixture is: lactose: LNT = 0-0.2, LNTri II: LNT is not greater than 0.15, and pLNH II: LNT is not greater than 0.05, wherein the mixture can be advantageously used for selectively crystallizing LNT. A mixture in which at least one ratio is higher than the above-mentioned provisions is a mixture not suitable for selective crystallization of LNT.

[0143] Moreover, in one embodiment of the activated carbon treatment, when the ratio of lactose:LNnT in the mixture of LNnT, LNTri II, pLNnH and lactose obtained during the fermentation process and / or after any of the separation steps disclosed above is higher than 0.2, it can be reduced to 0.2 or lower by advantageously applying activated carbon treatment, for example by adopting the method proposed by Whistler et al., J. Am. Chem. Soc. 72, 677 (1950) or Morales et al. Chromatographia 64, 233 (2006). These methods are based on eluting / removing a considerable amount of disaccharide lactose from the higher oligosaccharides LNTri II, LNnT and pLNnH, so that a mixture of LNTri II, LNnT and pLNnH with very low or no lactose content (i.e., wherein the weight ratio of lactose:LNnT is lower than 0.2), while the weight ratio of LNnT to LNTri II and pLNnH is substantially unchanged. Therefore, by applying the above-mentioned activated carbon treatment to an HMO mixture comprising or consisting of LNnT, LNTri II, pLNnH and lactose, wherein the weight ratio of lactose, LNTri II and pLNnH to LNnT in the mixture is: lactose:LNnT greater than 0.2 and less than 0.8, LNTriII:LNnT not greater than 0.1, and pLNnH:LNnT not greater than 0.03, an HMO mixture comprising or consisting of LNnT, LNTri II, pLNnH and lactose can be obtained, wherein the weight ratio of lactose, LNTri II and pLNnH to LNnT in the mixture is: lactose:LNnT=0-0.2, LNTri II:LNnT not greater than 0.1, and pLNnH:LNnT not greater than 0.03, wherein the mixture can be advantageously used to selectively crystallize LNnT. A mixture in which at least one ratio is higher than the above-specified mixture is not suitable for the selective crystallization of LNnT.

[0144] For example, by adding charcoal powder to the carbohydrate aqueous solution under stirring, filtering off the charcoal, resuspending in aqueous ethanol under stirring, and separating the charcoal by filtering, the charcoal treatment can be carried out. In larger-scale purification, the carbohydrate aqueous solution after UF, NF or ion exchange treatment is preferably loaded on a column filled with charcoal, which can be granular charcoal, or can be optionally mixed with diatomaceous earth, and the column is then washed with the desired eluent. The fraction containing LNT, LNTri II, pLNH II and lactose (if present) or LNnT, LNTri II, pLNnH and lactose (if present) is collected. If necessary, residual ethanol is removed from the fraction by, for example, evaporation to obtain an aqueous solution of LNT, LNTri II, pLNH II and lactose (if still present) or LNnT, LNTri II, pLNnH and lactose (if present).

[0145] The fifth (optional) separation step may be chromatography on a neutral solid phase, advantageously a reverse phase chromatography. The aqueous solution comprising LNT, LNTri II, pLNH II and optionally lactose or LNnT, LNTri II, pLNnH and optionally lactose obtained in the fermentation step and / or after any of the separation steps disclosed above may contain a small amount of other soluble hydrophobic impurities that should be removed. Hydrophobic impurities are adsorbed and thus retained due to hydrophobic interactions with hydrophobic ligands such as alkyl or aryl side chains of the fixed neutral solid phase gel matrix (resin), while the more hydrophilic LNT, LNTri II, pLNH II and optionally lactose or a mixture of LNnT, LNTri II, pLNnH and optionally lactose will not bind to the solid medium and can therefore be used as an aqueous medium, preferably water, for elution as a mobile phase. In this fifth separation step, the weight ratios of LNT to LNTriII, to pLNHI II and to lactose (if present) or LNnT, LNTri II, pLNnH and lactose (if present) do not substantially change.

[0146] Reverse phase chromatography can be performed in a conventional manner. Preferably, a hydrophobic chromatographic medium selected from the group consisting of reverse phase silica and an organic polymer, in particular a copolymer of styrene or divinylbenzene and a methacrylate polymer is used. The silica is preferably derivatized with a linear alkyl hydrocarbon in the length range of C1-C18 (most commonly C1, C4, C5, C8 and C18) or other hydrophobic ligands (e.g. phenyl or cyano).

[0147] Organic solvents may be added to the aqueous medium used as mobile phase in reverse phase chromatography to alter its polarity, thereby improving the separation of oligosaccharides from more hydrophobic substances. Many organic solvents may be used for this purpose, preferably water-miscible solvents, for example lower alkanols such as methanol, ethanol and isopropanol, or acetonitrile, or tetrahydrofuran, or acetone.

[0148] Reverse phase chromatography may otherwise be performed in conventional manner, eg batchwise or continuously.Purification may be readily performed by using conventional laboratory or industrial grade chromatography columns or vessels, in which the hydrophobic chromatography medium may be packed or suspended (beads).

[0149] In one embodiment of chromatography on a neutral solid phase, when the pLNH II:LNT ratio in the mixture of LNT, LNTri II, pLNH II and lactose obtained in the fermentation step and / or after any of the separation steps disclosed above is higher than 0.05, gel filtration chromatography can be applied to reduce it to 0.05 or less. The method is based on the separation of the hexose pLNH II from the lower oligosaccharides lactose, LNTri II and LNT according to size. The weight ratio of LNTri II and lactose to LNT does not substantially change. Therefore, by applying the above-mentioned chromatography to an HMO mixture comprising LNT, LNTri II, pLNH II and lactose or consisting thereof, wherein the weight ratio of lactose, LNTri II and pLNH II relative to LNT in the mixture is: lactose:LNT is 0-0.2, LNTriII:LNT is not greater than 0.15, and pLNH II:LNT is greater than 0.05, an HMO mixture comprising LNT, LNTriII, pLNH II and lactose or consisting thereof can be obtained, wherein the weight ratio of lactose, LNTri II and pLNH II relative to LNT in the mixture is: lactose:LNT=0-0.2, LNTriII:LNT is not greater than 0.15, and pLNH II:LNT is not greater than 0.05, and the mixture can be advantageously used to selectively crystallize LNT.

[0150] In one embodiment of chromatography on a neutral solid phase, when the pLNnH:LNnT ratio in the mixture of LNnT, LNTri II, pLNnH and lactose obtained in the fermentation step and / or after any of the separation steps disclosed above is higher than 0.03, gel filtration chromatography can be applied to reduce it to 0.03 or less. The method is based on the separation of the hexose pLNnH from the lower oligosaccharides lactose, LNTri II and LNnT according to size. The weight ratio of LNTri II and lactose to LNnT does not substantially change. Therefore, by applying the above-mentioned chromatography to an HMO mixture comprising or consisting of LNnT, LNTri II, pLNnH and lactose, wherein the weight ratio of lactose, LNTri II and pLNnH in the mixture relative to LNnT is: lactose:LNnT is 0-0.2, LNTri II:LNnT is not greater than 0.1, and pLNnH:LNnT is greater than 0.03, an HMO mixture comprising or consisting of LNnT, LNTri II, pLNnH and lactose can be obtained, wherein the weight ratio of lactose, LNTri II and pLNnH in the mixture relative to LNnT is: lactose:LNnT=0-0.2, LNTri II:LNnT is not greater than 0.1, and pLNnH:LNnT is not greater than 0.03, and the mixture can be advantageously used to selectively crystallize LNnT.

[0151] Gel filtration chromatography can be carried out in a conventional manner.

[0152] If the mixture of LNT, LNTri II, pLNHII and lactose obtained in the fermentation step and / or after any of the separation steps disclosed above has a lactose:LNT ratio greater than 0.2 and less than 0.8, and a pLNH II:LNT ratio greater than 0.05, then both the activated carbon treatment and gel filtration described above should be applied to obtain an HMO mixture comprising or consisting of LNT, LNTri II, pLNH II and lactose suitable for LNT crystallization.

[0153] If the mixture of LNnT, LNTri II, pLNnH and lactose obtained in the fermentation step and / or after any of the separation steps disclosed above has a lactose:LNnT ratio greater than 0.2 and less than 0.8 and a pLNnH:LNnT ratio greater than 0.03, both the activated carbon treatment and gel filtration described above should be applied to obtain an HMO mixture comprising or consisting of LNnT, LNTri II, pLNnH and lactose suitable for LNnT crystallization.

[0154] The third aspect of the present invention

[0155] The third aspect of the present invention relates to:

[0156] A process for crystallizing: using one or more monohydric C1-C4 alcohols as antisolvents:

[0157] a) LNT, wherein the crystallization is carried out from a mixture comprising LNT, LNTri II, pLNH II and optionally lactose, wherein the weight ratio of lactose, LNTri II and pLNH II relative to LNT is:

[0158] Lactose: LNT = 0-0.2, LNTri II: LNT not greater than 0.15, and pLNH II: LNT not greater than 0.05;

[0159] or

[0160] A process for crystallizing: using one or more monohydroxy C1-C4 alcohols and / or acetone as antisolvents:

[0161] b) LNnT, wherein the crystallization is carried out from a mixture comprising LNnT, LNTri II, pLNnH and optionally lactose, wherein the weight ratio of lactose, LNTri II and pLNnH relative to LNnT is:

[0162] Lactose: LNnT = 0-0.2, LNTri II: LNnT not greater than 0.1, and pLNnH: LNnT not greater than 0.03.

[0163] The crystallization is typically solvent-antisolvent crystallization, wherein the above HMO mixture is used as an aqueous solution thereof. The antisolvent is preferably methanol.

[0164] If the crystallization of LNnT is carried out in the reverse order of addition (i.e., adding the aqueous oligosaccharide solution to the antisolvent), the antisolvent is preferably acetone, and the LNnT so crystallized is preferably its polymorph disclosed in EP-A-1405856. If the crystallization is carried out in the normal order of addition (i.e., adding the antisolvent to the aqueous oligosaccharide mixture), the antisolvent is preferably a monohydroxy C1-C4 alcohol, advantageously methanol, and the LNnT so crystallized is preferably its polymorph disclosed in WO 2011 / 100980.

[0165] Fourth aspect of the present invention

[0166] The HMO mixture of the present invention contains LNT, LNTri II, pLNH II and optionally lactose or LNnT, LNTri II, pLNnH and optionally lactose, preferably consisting essentially of them, and is an anti-infective composition that can be used to treat and / or prevent viral and / or bacterial intestinal infections by specifically modulating the gastrointestinal microbiota, modulating the intestinal binding of viruses and pathogens, and improving intestinal barrier function and immune function. In particular, the mixture increases the abundance of bifidobacteria in the intestinal microbiota, in particular members of the Bifidobacterium adolescentis phylogenetic group, in particular Bifidobacterium adolescentis and / or Bifidobacterium pseudocatenulatum, and after about 14 days, Bifidobacterium longum and / or Bifidobacterium bifidum. The mixture may also reduce the abundance of Firmicutes, in particular Clostridium and pathogens. Moreover, each mixture acts as a decoy receptor and binds to rotavirus to prevent rotavirus from adhering to intestinal cells. These characteristics are combined with the improvement of intestinal barrier function and immune function, making the mixture suitable for preventing and treating viral and / or bacterial infections. The mixture can also reduce the abundance of active rumen cocci (Ruminococcusgnavus), which is related to intestinal diseases such as inflammatory bowel disease and irritable bowel syndrome. This is combined with the improvement of intestinal barrier function and immune function, making the mixture suitable for preventing and treating diseases such as inflammatory bowel disease, irritable bowel syndrome and other diseases related to verification and impaired barrier function.

[0167] The HMO mixtures of the present invention contain LNT, LNTri II, pLNH II and optionally lactose or LNnT, LNTri II, pLNnH and optionally lactose, preferably consisting essentially of them, and can also be used to prevent and / or treat viral and / or bacterial infections of the respiratory tract. In this regard, the mixture affects host defenses, including modulation of the immune system, thereby causing inhibition of respiratory pathogen colonization.

[0168] Anti-infective composition of the present invention can be a pharmaceutical composition.Each pharmaceutical composition can contain a pharmaceutically acceptable carrier, for example, a phosphate buffered saline solution, a mixture of ethanol in water, water and emulsions such as oil / water or water / oil emulsions, and various wetting agents or excipients.The pharmaceutical composition can also contain materials that do not produce adverse, allergic or other adverse reactions when given to the patient.Carriers and other materials can include solvents, dispersants, coatings, absorption promoters, controlled release agents and one or more inert excipients, for example, starch, polyols, granulating agents, microcrystalline cellulose, diluents, lubricants, adhesives and disintegrants.If required, the tablet of the anti-infective composition can be coated by standard aqueous or non-aqueous technology.

[0169] The anti-infective composition of the present invention can be administered orally, for example, as a tablet, capsule or pellet containing a predetermined amount of the first mixture, or as a powder or granule containing a predetermined concentration of the first mixture, or a gel, paste, solution, suspension, emulsion, syrup, pill (bolus), dry medicine confection or slurry in an aqueous or non-aqueous liquid containing a predetermined concentration of the first mixture. Orally administered compositions can include adhesives, lubricants, inert diluents, flavoring agents and wetting agents. Orally administered compositions such as tablets can be optionally coated and can be formulated to provide a sustained, delayed or controlled release of the first mixture therein.

[0170] The anti-infective composition of the present invention may also be administered via rectal suppository, aerosol tube, nasogastric tube or direct infusion into the GI tract or stomach.

[0171] The anti-infective pharmaceutical composition of the present invention can also include therapeutic agents, such as antiviral agents, antibiotics, probiotics, analgesics and anti-inflammatory agents. These compositions can be determined in a conventional manner for the patient's appropriate dosage, which is based on multiple factors such as the patient's immune status, body weight and age. In some cases, the dosage will be a concentration similar to the concentration found in the mixture component in human breast milk. The amount required usually ranges from about 200mg to about 20g every day, in some embodiments, from about 300mg to about 15g every day, from about 400mg to about 10g every day, in some embodiments, from about 500mg to about 10g every day, and in some embodiments, from about 1g to about 10g every day. Suitable dosage regimens can be determined by methods known to those skilled in the art.

[0172] The anti-infective composition of the present invention can also be added to the nutritional composition. For example, it can be added to a water replenishment solution or used for the diet maintenance or supplement of the elderly or immunocompromised individuals. These anti-infective compositions can also include macronutrients, such as edible oils, carbohydrates and proteins. Edible oils include coconut oil, soybean oil, monoglycerides and diglycerides. Carbohydrates include, for example, glucose, edible lactose and hydrolyzed corn starch. Proteins include, for example, soy protein, whey and skim milk. Vitamins and minerals (such as calcium, phosphorus, potassium, sodium, chlorine, magnesium, manganese, iron, copper, zinc, selenium, iodine and vitamins A, E, D, C and complex vitamin B) can also be included in these anti-infective compositions.

[0173] Fifth aspect of the present invention

[0174] A fifth aspect of the present invention relates to a method for regulating the human gastrointestinal microbiota to increase the abundance of bifidobacteria. The method comprises administering to the human a mixture of LNT, pLNH II and LNTri II or a mixture of LNnT, pLNnH and LNTri II, advantageously a synthetic HMO mixture of the first aspect of the present invention or a nutritional or pharmaceutical composition comprising the same.

[0175] Sixth aspect of the present invention

[0176] The sixth aspect of the present invention relates to a method for preventing or treating viral and / or bacterial infections, particularly intestinal and respiratory infections, in humans. The method comprises administering to humans a mixture of LNT, pLNH II and LNTri II or a mixture of LNnT, pLNnH and LNTri II, advantageously a synthetic HMO mixture according to the first aspect of the present invention or a nutritional or pharmaceutical composition comprising the same.

[0177] Although the invention has been described in conjunction with preferred embodiments, it will be appreciated that numerous modifications may be made within the scope of the invention.

[0178] In this specification, unless expressly stated otherwise, the word "or" is used in the sense of an operator that returns a true value when either or both of the conditions are met, in contrast to the operator "exclusive or" which requires only one condition to be met. The word "comprising" is used in the sense of "comprising" rather than "consisting of". The teachings of all prior art identified above are hereby incorporated by reference. The identification of any prior published document should not be taken as an admission or representation that its teachings were common general knowledge in Australia or elsewhere at the date of its use.

[0179] Example

[0180] Example 1 - Preparation of a polysaccharide consisting essentially of LNT, LNTriII, pLNHII and lactose or LNnT, LNTri II, pLNnH and Lactose mixture

[0181] Strains:

[0182] The strain was constructed from E. coli K12 strain DH1 obtained from the Deutsche Sammlung von Mikroorganismen (reference DSM 5346). The following genes were deleted by inserting the Plac promoter while maintaining the genes involved in the biosynthesis of UDP-GlcNAc and UDP-Gal: lacZ, nanKETA, lacA, melA, wcaJ and mdoH. Strains contained a plasmid including β-1,3-N-acetylglucosaminyltransferase (IgtA) and an antibiotic marker (plasmid 1), and a plasmid including β-1,3-galactosyltransferase and an antibiotic marker (plasmid 2) [for production of LNT], or the pBBR3-IgtA-tet plasmid carrying the Neisseria meningitidis IgtA gene for β-1,3-N-acetylglucosaminyltransferase and a tetracycline resistance gene, and the pBS-galT-amp plasmid carrying the Helicobacter pylori galT gene for β-1-4-galactosyltransferase and an ampicillin resistance gene [for production of LNnT].

[0183] Fermentation steps:

[0184] Glucose, glycerol and lactose were all sterilized at 120° C. Isopropylthio-β-D-galactopyranoside (IPTG) was filter sterilized.

[0185] Fermentation was carried out in a 3 l fermenter containing ≈0.9 l of aqueous mineral medium (Samain et al. J. Biotechnol. 72, 33 (1999)). The temperature was maintained at 33 ° C and the pH was adjusted to 6.8 with 28% NH4OH. The strain was inoculated in LB medium (20 ml) supplemented with ampicillin and tetracycline. The exponential growth phase started from the inoculation and stopped when the glucose carbon source initially added to the aqueous medium was exhausted. Then a lactose solution (70 g lactose / 500 ml water) was added to the aqueous medium, after which feeding was started with a 500 g / l solution and 4.5 g / h glycerol as a carbon source. In addition, 1-2 ml of a 50 mg / ml solution of isopropylthio-β-D-pyranogalactoside (IPTG) was added to the aqueous medium as an inducer. The glycerol-fed fermentation lasted for about 90 hours, producing a final aqueous medium containing:

[0186] a) [For LNT-producing strains] LNT, LNTri II, pLNH II and lactose in the following ratios:

[0187] Lactose: LNT = 0.15-0.20

[0188] LNTri II: LNT=0.05-0.12

[0189] pLNH II: LNT = 0.005-0.03; or

[0190] b) [For strains producing LNnT] LNnT, LNTri II, pLNnH and lactose in the following ratios:

[0191] Lactose: LNnT = 0.43-0.72

[0192] LNTri II: LNnT = 0.03-0.05

[0193] pLNnH:LNnT=0.19-0.20

[0194] Its LNnT titer was 45 g / l.

[0195] Purification method:

[0196] The final aqueous medium from each batch was purified by the following consecutive steps:

[0197] i) centrifuging the culture medium to separate the E. coli cells, suspended particles and contaminants, insoluble materials and debris from the residual culture medium;

[0198] ii) washing the separated cells with warm water and then adding the wash water to the residual culture medium;

[0199] iii) ultrafiltration of the residual culture medium using a ceramic membrane with a pore size of 50 kDA MWCO, and then ultrafiltration of the culture medium again using a ceramic membrane with a pore size of 1 kDA MWCO to remove cellular proteins, peptides, amino acids, RNA and DNA, endotoxins and glycolipids;

[0200] iv) treating the culture medium with a strongly acidic cation exchange resin DOWEX 50WX 12 and a weakly basic anion exchange resin DOWEX 66 to remove minerals, salts and other charged molecules as well as amino acids, residual proteins, DNA and coloring / caramel bodies; then

[0201] v) Decolorize the culture medium with activated charcoal.

[0202] The resulting culture medium is an aqueous second HMO mixture of the present invention consisting essentially of:

[0203] a) lactose, LNTri II, LNT and pLNH II, wherein the ratio of lactose, LNTri II, LNT and pLNH II is as follows:

[0204] Lactose: LNT = 0.15-0.20

[0205] LNTri II: LNT=0.05-0.12

[0206] pLNH II: LNT = 0.005-0.03; or

[0207] b) lactose, LNTri II, LNnT and pLNnH, wherein the ratio of lactose, LNTri II, LNnT and pLNnH is as follows:

[0208] Lactose: LNnT = 0.43-0.72

[0209] LNTri II: LNnT = 0.03-0.05

[0210] pLNnH:LNnT=0.18-0.20.

[0211] Example 2 - Preparation of a polysaccharide consisting essentially of LNT, LNTri II, pLNH II and lactose or LNnT, LNTri II, pLNnH Dry mixture consisting of lactose

[0212] The mixture of LNT, LNTri II, pLNH II and lactose or the aqueous mixture of LNnT, LNTri II, pLNnH and lactose from Example 1 was nanofiltered using a 200 Da membrane to remove most of the residual water. The resulting wet second HMO mixture of LNTri II, LNT, pLNH II and lactose or the wet second HMO mixture of LNTri II, LNnT, pLNnH and lactose of the present invention was then further dried by heating to remove substantially all of the residual water; the weight ratio of the resulting dry mixture was as follows:

[0213] Lactose: LNT = 0.15-0.20

[0214] LNTri II: LNT=0.05-0.12

[0215] pLNH II: LNT = 0.005-0.03; or

[0216] Lactose: LNnT = 0.43-0.72

[0217] LNTri II: LNnT = 0.03-0.05

[0218] pLNnH:LNnT=0.18-0.19.

[0219] Example 3

[0220] Thirty male and female patients were recruited for the study. After a screening visit and a run-in period of 1-2 weeks, patients were selected and randomly divided into 3 groups of 10 patients each. Two groups were given the treatment product and one group was given the placebo product for 8 weeks. The treatment product contained a) 5 grams of LNT, LNTri II and pLNH II in combination, or b) 5 grams of LNnT, LNTri II and pLNnH in combination, while the placebo product contained 2 grams of glucose. All products were in powder form in unit dose containers.

[0221] Patients were eligible to participate if they were at least 18 years old. All recruited patients were able and willing to understand and comply with the study methods. Patients were excluded if they had: participated in a clinical study in the month before the screening visit; had abnormal results in screening tests clinically related to participation in the study; had serious medical conditions such as malignancy, diabetes, severe coronary heart disease, kidney disease, neurological disease, or severe psychiatric disease or any condition that could affect the results of the study; used high-dose probiotic supplements (yogurt was allowed) in the 2 months before the study; took antibiotic medications in the 3 months before the study; took any medications regularly in the 2 weeks before the study that could interfere with symptom evaluation; were pregnant or breastfeeding.

[0222] At the screening visit, medical history and concomitant medications were registered, and blood samples were collected for safety analysis. Stool specimen kits were distributed. Patients were instructed to store the collected stool specimens in the refrigerator until the second visit.

[0223] At the second visit, eligibility criteria were checked and eligible subjects were randomized into the three groups in the trial. Stool samples were collected and devices for new samples were distributed. Patients were familiarized with an interactive Internet support system, which recorded data daily, and provided treatment or placebo products. Subjects were reminded not to change their daily diet during the study. Blood samples were collected for biomarker studies. Stool samples were stored at -80°C until analysis. Stool samples were analyzed by 16S rRNA sequencing.

[0224] The study lasted for 8 weeks, with patients taking either a placebo or treatment product daily. Patients were instructed to take these products in the morning with breakfast. Compliance was monitored via an interactive internet support system. Patients also used the system to record the following information:

[0225] - Bristol Stool Form Scale (BSFS) information,

[0226] - symptom information, such as abdominal pain, discomfort, cramping, bloating, and feelings of fullness,

[0227] - Additional Gastrointestinal Symptom Rating Scale (GSRS) information.

[0228] The questionnaire consisted of 15 items covering five dimensions (abdominal pain, indigestion, reflux, diarrhea, and constipation) using a seven-point Likert scale.

[0229] At the end of the study, each patient had an exit visit by the medical team. Stool and blood samples were collected and analyzed as previously described.

[0230] Stool analysis showed an increased abundance of Bifidobacterium in patients in the treatment group.

[0231] Example 4

[0232] Thirty five-day-old Sprague-Dawley rats were housed individually to avoid contamination between rats and provided with irradiated food and water. The rats were divided into three groups, 10 rats each, two treatment groups and one control group.

[0233] The mixture of LNT, LNTri II, pLNH II and the mixture of LNnT, LNTri II, pLNnH were added to the drinking water of each treatment group at a total concentration of 40 mg / ml. The water of the control group (day 0) remained unchanged. Fresh water was given every day and all rats had free access to water. Rats were fed with rodent chow and given fresh food every day.

[0234] Two days after administration of the mixture (day 2), rats in all groups were infected with the encapsulated S. pneumoniae strains by oral gavage.

[0235] After 24 hours, the rats were nasally washed with saline solution. The solution was recovered and the number of viable bacteria was obtained by counting the colonies on the agar plate. The rats were monitored for 1 week and then euthanized (day 10). After euthanasia, the lungs were removed. The lung tissue was minced in 10 ml PBS and then homogenized with Stomacher (2 minutes / sample). The tissue homogenate was serially diluted and cultured on selective streptococcal agar for 48 hours to determine the colony forming units (cfu).

[0236] In treated rats, lung colonization with S. pneumoniae was reduced compared to control rats. Similarly, the number of viable S. pneumoniae found in nasal washes was reduced in treated rats compared to control rats.

Claims

1. A synthetic mixture comprising, preferably consisting essentially of: -GlcNAcβ1-3Galβ1-4Glc(LNTri II), - component A, which is Galβ1-3GlcNAcβ1-3Galβ1-4Glc (LNT) or Galβ1-4GlcNAcβ1-3Galβ1-4Glc (LNnT), -Component B: When component A is LNT, it is Galβ1-3GlcNAcβ1-3Galβ1-3GlcNAcβ1-3Galβ1-4Glc(pLNH II), or When component A is LNnT, it is Galβ1-4GlcNAcβ1-3Galβ1-4GlcNAcβ1-3Galβ1-4Glc(pLNnH), - and optionally lactose.

2. The mixture according to claim 1, comprising LNT or LNnT in a major amount relative to the other components in the mixture.

3. A mixture according to claim 1 or 2, consisting essentially of: -LNTri II, - component A which is LNT or LNnT, -Component B: When component A is LNT, it is pLNH II, or When component A is LNnT, it is pLNnH.

4. A mixture according to claim 1 or 2, consisting essentially of: -LNTri II, - component A which is LNT or LNnT, -Component B: When component A is LNT, it is pLNH II, or When component A is LNnT, it is pLNnH, and -lactose.

5. A mixture according to claims 1-3, comprising, or essentially consisting of: LNTri II, LNnT, pLNnH and optionally lactose, and wherein the weight ratio of LNTri II, pLNnH and optionally lactose relative to LNnT is: LNTri II: LNnT is not greater than 0.1; pLNnH:LNnT is not greater than 0.4, preferably not greater than 0.03; and Lactose: LNnT is 0 to not more than 0.8, preferably not more than 0.

2.

6. A mixture according to claims 1-3, comprising, or essentially consisting of: LNTri II, LNT, pLNH II and optionally lactose, and wherein the weight ratio of LNTri II, pLNH II and optionally lactose relative to LNT is: LNTri II: LNT is not greater than 0.2; pLNH II:LNT is not greater than 0.05; and Lactose: LNT is 0 to no more than 0.

6.

7. The mixture according to claim 4, which consists essentially of LNTri II, LNnT, pLNnH and lactose, and wherein the weight ratio of lactose, LNTri II and pLNnH relative to LNnT is: Lactose: LNnT = 0.2-0.8, LNTri II: LNnT = 0.01-0.05, and pLNnH: LNnT = 0.1-0.

2.

8. The mixture of claim 4, consisting essentially of LNTri II, LNT, pLNH II and lactose, and wherein the weight ratio of lactose, LNTri II and pLNH II relative to LNT is: Lactose: LNT = 0.15-0.20, LNTri II: LNT = 0.05-0.12, and pLNH II: LNT = 0.005-0.

03.

9. The mixture according to any one of claims 1 to 8, which is contained in a nutritional composition or a pharmaceutical composition.

10. A method for obtaining a mixture according to any one of claims 1 to 9, comprising the following steps: a) providing a genetically modified cell, preferably a bacterial cell, more preferably an Escherichia coli (E. coli) cell, comprising: a first recombinant gene encoding an N-acetyl-glucosaminyltransferase capable of transferring GlcNAc from UDP-GlcNAc to lactose to form LNTri II in said cell, - a second recombinant gene encoding a galactosyltransferase capable of transferring a galactosyl residue from UDP-Gal to LNTri II in said cell to form component A, said component A being LNT or LNnT, and - genes encoding the biosynthetic pathways leading to said UDP-GlcNAc and said UDP-Gal in said cell, b) culturing the cells in a medium containing lactose, thereby inducing: - said lactose is internalized into said cell, preferably via an active transport mechanism, and - forming LNTri II, component A and component B in said cell, component B being pLNH II when component A is LNT, or component B being pLNnH when component A is LNnT, and c) optionally, continuing the culturing according to step b) until no lactose remains, and d) separating and isolating the mixture of LNnT, component A, component B and optionally lactose from the culture medium.

11. The method according to claim 10, wherein in step d), the culture medium is treated by centrifugation, ultrafiltration, ion exchange and then decolorization.

12. The method according to claim 11, wherein after step d), the culture medium is treated by nanofiltration to remove water from the culture medium.

13. An anti-infection composition for preventing and / or treating bacterial and / or viral infections in the human body, especially intestinal and respiratory tract infections, the anti-infection composition comprising a mixture of LNTri II, component A and component B, preferably a mixture according to any one of claims 1 to 9, component A is LNT or LNnT, when component A is LNT, component B is pLNHII, or when component A is LNnT, component B is pLNnH.

14. A method for regulating the microbiota of a human to increase the abundance of Bifidobacterium, the method comprising administering to the human a mixture comprising LNTri II, component A and component B, preferably a mixture according to any one of claims 1 to 9, component A being LNT or LNnT, component B being pLNH II when component A is LNT, or component B being pLNnH when component A is LNnT.

15. A method for preventing and / or treating viral and / or bacterial infections, especially intestinal and respiratory tract infections, in humans, the method comprising administering to humans a mixture comprising LNTri II, component A and component B, preferably a mixture according to any one of claims 1 to 9, component A being LNT or LNnT, component B being pLNH II when component A is LNT, or component B being pLNnH when component A is LNnT.

16. A method for crystallizing LNnT, wherein crystallization is carried out from a mixture comprising LNnT, LNTri II, pLNnH and optionally lactose using one or more monohydroxy C1-C4 alcohols and / or acetone as antisolvent, wherein the weight ratio of lactose, LNTri II and pLNnH relative to LNnT is: Lactose: LNnT = 0-0.2, LNTri II: LNnT not greater than 0.1, and pLNnH: LNnT not greater than 0.

03.

17. A method for crystallizing LNT, wherein crystallization is carried out from a mixture comprising LNT, LNTri II, pLNH II and optionally lactose using one or more monohydroxy C1-C4 alcohols as antisolvents, wherein the weight ratio of lactose, LNTri II and pLNH II relative to LNT is: Lactose: LNT = 0-0.2, LNTri II: LNT not greater than 0.15, and pLNH II: LNT not greater than 0.05.