Mixtures of fucosylated HMO

Abstract

The present invention relates to a mixture of human milk oligosaccharides ("HMO") comprising five fucosylated HMO wherein the synthesis comprises an alpha1-3 / 4-to-fucosidase enzymatic reaction between an HMO donor and an acceptor, as well as to an enzymatic process for preparing said mixture.

Description

Technical Field

[0001] The present invention relates to a mixture of human milk oligosaccharides ("HMO") comprising five fucosylated HMOs, and an enzymatic method for preparing the mixture, wherein the synthesis comprises an α1-3 / 4-transfucosidase enzymatic reaction between an HMO donor and an acceptor. Background Art

[0002] In recent years, HMOs have become the focus of attention due to their role in many biological processes in human organisms. Mammalian milk contains at least 130 of these complex oligosaccharides (see Urashima et al.: Milk Oligosaccharides. Nova Science Press (2011); or Chen, Adv. Carbohydr. Chem. Biochem. 72, 113 (2015)).

[0003] There is growing evidence that the community of microorganisms in the human digestive tract, or microbiome, plays an important role in health and disease. When the normal composition of the microbiome is out of balance, the human host can suffer adverse consequences. Recent studies have linked an imbalanced microbiome to a variety of diseases, such as cancer, obesity, inflammatory bowel disease, psoriasis, asthma, and possibly even autism. HMOs are thought to actively modulate the microbiome, and they have received increasing attention for this purpose. However, the remarkable diversity of HMOs, coupled with their lack of availability, particularly of the more complex HMOs, has hampered research into the specific functions of individual HMOs, thereby limiting exploration of their full potential to promote human health.

[0004] The natural source of HMO is mammalian milk, which contains mainly water, as well as 55-70 g / l lactose, 24-59 g / l lipids, about 13 g / l protein, 5-15 g / l HMO and about 1.5 g / l minerals.

[0005] In the past few years, efforts to develop industrial-scale production processes for HMOs have increased significantly. Processes have been designed to produce HMOs by microbial fermentation, enzymatic methods, chemical synthesis, or a combination of these techniques.

[0006] In particular, E. coli fermentation has been successfully used for large-scale industrial production of HMOs. WO 01 / 04341 describes how to use transgenic E. coli to produce core human milk oligosaccharides optionally substituted with fucose.

[0007] WO 2012 / 158517 and WO 2013 / 154725 describe prebiotic compositions containing 3-FL, 2'-FL and DFL, and WO 2012 / 112777 describes a mixture of 3-FL, 2'-FL, DFL and lactose produced in transgenic E. coli.

[0008] In order to be able to synthetically produce mixtures of HMOs without having to synthesize all of the component oligosaccharides of the mixture, enzymatic methods for the enzymatic synthesis of mixtures of HMO oligosaccharides have been developed and described in WO 2012 / 156897 and WO 2012 / 15698. As a further development, WO 2016 / 063261 and WO 2016 / 063262 describe optimized enzymes and enzymatic methods for producing fucosylated HMOs. These methods provide a reaction mixture containing a plurality of different HMO oligosaccharides.

[0009] Nonetheless, these approaches involve the use of highly specialized enzymes, which in turn require sensitive stoichiometry and reaction conditions, as well as termination of the reaction at the desired equilibrium point in the reaction and removal of secondary unwanted metabolites.

[0010] Therefore, there remains a clear need for further specific HMOs or combinations of HMOs that modulate the microbiome in a desired manner to address specific human health problems, as well as simplified and more precise methods for their production. Summary of the invention

[0011] A first aspect of the invention relates to a mixture consisting of or consisting essentially of component A which is 3-FL, component B which is LNFP-I, component C which is 2'-FL, component D which is LNDFH-I, component E which is DFL and optionally component F which is lactose.

[0012] A second aspect of the present invention relates to a composition comprising a mixture consisting of or consisting essentially of 2'-FL, 3-FL, LNFP-I, DFL, LNDFH-I and optionally lactose. The composition may be a nutritional composition, a pharmaceutical composition or a cosmetic composition.

[0013] A third aspect of the present invention relates to a method for enzymatically synthesizing a mixture of the above-mentioned HMOs, preferably under substantially buffer-free conditions. The enzymatic synthesis comprises an α1-3 / 4-transfucosidase enzymatic reaction between a fucosylated donor HMO and two fucosylated acceptor HMOs. The enzymatic synthesis for preparing a mixture of HMOs according to the present invention comprises the steps of reacting component A, component B and component C in the presence of an α1-3 / 4-transfucosidase to produce a reaction mixture, and removing the α1-3 / 4-transfucosidase and optionally lactose from the reaction mixture. Therefore, the present invention also relates to a mixture consisting of or essentially consisting of 2'-FL, 3-FL, LNFP-I, DFL, LNDFH-I and optionally lactose obtained or obtainable by the enzymatic synthesis disclosed herein.

[0014] A special feature of the enzymatic synthesis according to the invention is that it can be carried out under substantially unbuffered conditions. Typically, the enzymatic synthesis according to the invention is carried out in a pH range of pH 4.5 to pH 7, such as preferably in a pH range of pH 5.0 to pH 6.0.

[0015] The α1-3 / 4-transfucosidase used in the enzymatic synthesis according to the invention preferably lacks hydrolytic activity, or at least has a significantly reduced hydrolytic activity.

[0016] In one aspect of the invention, the enzymatic synthesis according to the invention further comprises a removal step, which comprises a step of inactivating the α1-3 / 4-transfucosidase. The inactivation step may comprise heating the reaction solution to a temperature of 60°C to 100°C for a period of 5 minutes to 180 minutes.

[0017] In the enzymatic synthesis according to the present invention, the α1-3 / 4-transfucosidase is removed by, for example, microfiltration, nanofiltration, centrifugation or ultracentrifugation. The enzymatic synthesis may include more than one step to remove the α1-3 / 4-transfucosidase. One step may include batch removal of the α1-3 / 4-transfucosidase with powdered activated carbon. Alternatively, the step may include removal of the α1-3 / 4-transfucosidase with powdered activated carbon in column mode.

[0018] Another aspect of the present invention relates to the use of a mixture consisting of or essentially consisting of 2'-FL, 3-FL, LNFP-I, DFL, LNDFH-I and optionally lactose for treating, preventing and / or ameliorating diseases and / or conditions associated with an imbalance in the microbiome, or a composition comprising a mixture consisting of or essentially consisting of 2'-FL, 3-FL, LNFP-I, DFL, LNDFH-I and optionally lactose for treating, preventing and / or ameliorating diseases and / or conditions associated with an imbalance in the microbiome.

[0019] Another aspect of the present invention relates to the use of a mixture consisting of or consisting essentially of 2'-FL, 3-FL, LNFP-I, DFL, LNDFH-I and optionally lactose for improving the viability of probiotics in aqueous solution.

[0020] Another aspect of the present invention relates to the use of a mixture consisting of or essentially consisting of 2'-FL, 3-FL, LNFP-I, DFL, LNDFH-I and optionally lactose, or a composition comprising a mixture consisting of or essentially consisting of 2'-FL, 3-FL, LNFP-I, DFL, LNDFH-I and optionally lactose, in human dietary management or cosmetic applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The following will refer to the attached Figure 1 Describing the present invention in more detail, Figure 1 Shown are colony counts of Lactobacillus rhamnosus after 3 h incubation at 37°C, pH 3.0, with or without HMO. DETAILED DESCRIPTION

[0022] Generally, unless explicitly defined or illustrated otherwise, all terms used herein should be interpreted according to their ordinary meaning in the technical field and apply to all aspects and embodiments of the present invention.

[0023] Unless expressly stated otherwise, all references to "a / an / the" [a sequence, an enzyme, a step, etc.] should be openly interpreted as referring to at least one instance of the stated sequence, enzyme, step, etc.

[0024] The steps of any method disclosed herein do not have to be performed in the exact order disclosed, unless explicitly stated.

[0025] The word “comprise” means “include” rather than “consisting of”.

[0026] In the context of the present invention, the term "oligosaccharide" refers to a sugar polymer comprising a plurality of monosaccharide units linked together by interglycosidic bonds. A preferred oligosaccharide of the present invention is human milk oligosaccharide (HMO).

[0027] In the present context, the term "human milk oligosaccharide" or "HMO" refers to a complex carbohydrate found in human breast milk (see Urashima et al.: Milk Oligosaccharides. Nova Science Press (2011); or Chen, Adv. Carbohydr. Chem. Biochem. 72, 113 (2015)).

[0028] Specifically, 2'-FL is 2'-fucosyllactose (Fucα1-2Galβ1-4Glc), 3-FL is 3-fucosyllactose (Galβ1-4[Fucα1-3]Glc), DFL is difucosyllactose (Fucα1-2Galβ1-4[Fucα1-3]Glc), LNFP-I is lacto-N-fucopentaose I (Fucα1-2Galβ1-3GlcNAcβ1-3Galβ1-4Glc), and LNDFH-I is lacto-N-difucohexaose (Fucα1-2Galβ1-3[Fucα1-4]GlcNAcβ1-3Galβ1-4Glc). In the above structure, Fuc represents L-fucopyranosyl, Gal represents D-galactopyranosyl, Glc represents D-glucopyranosyl, and GlcNAc represents D-(2-acetylamino)-2-deoxy-glucopyranosyl unit. In the context of the present invention, lactose is not considered as an HMO species.

[0029] In the context of the present invention, the term "donor" refers to a fucosylated sugar in which an α1-3-fucosyl group is transferred to another sugar by an enzyme-catalyzed reaction, after which the resulting product is a sugar containing an α1-3-fucosyl group or an α1-4-fucosyl group. The term "acceptor" refers to a sugar to which the fucosyl group from the donor is transferred.

[0030] “Dietary management” means the complete or partial feeding of a patient who, as a result of a disease, disorder or medical condition, is:

[0031] - limited, impaired or disturbed ability to ingest, digest, absorb, metabolize or excrete normal food or certain nutrients or metabolites contained therein, or

[0032] - Have other medically determined nutritional needs

[0033] (See: Commission Notice on the classification of Food for Special Medical Purposes of the European Commission, Official Journal of the European Union C 401, 25.11.2017, p.10-11).

[0034] "Microbiota," "microbial community," and "microbiome" refer to the community of living microorganisms that typically inhabit an organ or region of the body, particularly the gastrointestinal organs in humans. The most important members of the gastrointestinal microbiota include Firmicutes, Bacteroidetes, Actinobacteria, Proteobacteria, Synergistetes, Verrucomicrobia, Fusobacteria, and Euryarchaeota. At the genus level, Bacteroides, Faecalibacterium, Bifidobacterium, Roseburia, Alistipes, Collinsella, Blautia, Coprococcus, Ruminococcus, Eubacterium, and Dorea are also present. At the species level, Bacteroides uniformis, Alistipes putredinis, Parabacteroides merdae, Ruminococcusbromii, Dorea longicatena, Bacteroides caccae, Bacteroides thetaiotaomicron, Eubacterium hallii, Ruminococcus torques, Faecalibacterium prausnitzii, Ruminococcus lactaris, Collinsella aerofaciens, Dorea formicigenerans, Bacteroides vulgatus, and Roseburia intestinalis. The gastrointestinal microbiota includes mucosa-associated microbiota located in or attached to the mucosal layer covering the gastrointestinal epithelium, and lumen-associated microbiota found in the lumen of the gastrointestinal tract.

[0035] "Modulating the microbiome" refers to exerting a change or controlling influence on the microbial population, such as an influence that results in an increase in the local intestinal abundance of Bifidobacterium, Barnesiella, Faecalibacterium, and / or butyrate-producing bacteria. In another example, such an influence may result in a decrease in the intestinal abundance of Ruminococcus gnavus and / or Proteobacteria. "Proteobacteria" is a phylum of Gram-negative bacteria that includes a wide variety of pathogenic bacteria, such as Escherichia, Salmonella, Vibrio, Helicobacter, Yersinia, and many other well-known genera.

[0036] "Oral administration" refers to any conventional form of delivery of a composition to a human through the oral cavity.

[0037] "Preventative treatment" or "prevention" refers to treatment given or actions taken to reduce the risk of developing a disease or recurring it.

[0038] "Treatment" means to address a medical condition or disease with the goal of improving or stabilizing the outcome of the person being treated, or to meet an underlying nutritional need. Thus, treatment includes dietary or nutritional management of a medical condition or disease by addressing the nutritional needs of the person being treated. "Treating" and "treatment" have grammatically corresponding meanings.

[0039] All prior teachings acknowledged above are incorporated herein by reference. Acknowledgement of any previously published document herein is not to be taken as an acknowledgment or representation that its teachings were common general knowledge as of the date herein.

[0040] New HMO blends

[0041] According to the present invention, it has surprisingly been found that a novel mixture of five fucosylated HMOs can be prepared by enzymatic synthesis using one HMO initial donor and two HMO initial acceptors. The reaction is preferably carried out under substantially no buffer conditions. Also preferably, the enzyme has limited or no hydrolytic activity.

[0042] The novel mixture consisting of or consisting essentially of 5 fucosylated HMOs has a unique new combination of properties and biological activities. Specifically, the mixture can be used to modulate the human microbiome, such as increasing the abundance of Bifidobacterium and the abundance of Barnesiella in the human microbiome. In addition, the mixture can reduce the abundance of Firmicutes, especially Clostridia, in the human microbiome. The mixture can also be used to treat and / or reduce the risk of various human bacterial and viral infections, as well. The novel mixture of HMOs described herein has anti-infective properties. In addition, when combined with probiotics, the mixture significantly improves the viability of the probiotics in aqueous solution.

[0043] One aspect of the present invention relates to a mixture consisting of or consisting essentially of the following components:

[0044] a) component A, which is 3-FL,

[0045] b) component B, which is LNFP-I,

[0046] c) component C, which is 2'-FL,

[0047] d) component D, which is LNDFH-I,

[0048] e) component E, which is DFL, and optionally

[0049] f) Component F which is lactose.

[0050] Therefore, one embodiment of the present invention is a mixture consisting of or consisting essentially of the following components:

[0051] a) component A, which is 3-FL,

[0052] b) component B, which is LNFP-I,

[0053] c) component C, which is 2'-FL,

[0054] d) component D, which is LNDFH-I, and

[0055] e) Component E which is DFL.

[0056] Another embodiment of the present invention is a mixture consisting of or consisting essentially of the following components:

[0057] a) component A, which is 3-FL,

[0058] b) component B, which is LNFP-I,

[0059] c) component C, which is 2'-FL,

[0060] d) component D, which is LNDFH-I,

[0061] e) component E, which is DFL, and

[0062] f) Component F which is lactose.

[0063] Enzymatic synthesis of a mixture for producing HMOs according to the invention

[0064] In the following paragraphs, the word "portable" is equivalent to "optionally portable" and the word "replaceable" is equivalent to "optionally replaceable".

[0065] The novel mixture of fucosylated HMOs is prepared by an enzymatic synthesis comprising a fucosyl HMO donor, component A (3-FL), two fucosyl HMO acceptors, component B (LNFP-I) and component C (2'-FL), and a transfucosidase, preferably under substantially buffer-free conditions. Also preferably, in the reaction, the enzyme has limited or no hydrolytic (i.e., fucosidase) activity.

[0066] Therefore, the present invention also relates to a method for preparing a mixture of HMOs, the mixture of HMOs consisting of or essentially consisting of component A which is 3-FL, component B which is LNFP-I, component C which is 2'-FL, component D which is LNDFH-I, component E which is DFL and component F which is lactose, by reacting components A, B and C in the presence of α1-3 / 4-transfucosidase to produce a reaction mixture containing components A, B, C, D, E and lactose, and then removing the α1-3 / 4-transfucosidase from the reaction mixture. The α1-3 / 4-transfucosidase can be removed by conventional means, for example by denaturing the reaction mixture, followed by centrifugation or ultrafiltration.

[0067] Furthermore, one aspect of the present invention relates to a method for obtaining a mixture of HMOs, the mixture of HMOs consisting of or essentially consisting of component A being 3-FL, component B being LNFP-I, component C being 2'-FL, component D being LNDFH-I and component E being DFL, the method comprising the steps of reacting components A, B and C in the presence of α1-3 / 4-transfucosidase to produce a reaction mixture containing components A, B, C, D, E and lactose, and then removing lactose and α1-3 / 4-transfucosidase from the reaction mixture. α1-3 / 4-transfucosidase can be removed by, for example, denaturing it and then centrifuging or ultrafiltration. Lactose can be separated from components A, B, C, D and E, for example, by cascade ultrafiltration and / or nanofiltration, or lactose can be first treated with lactase to degrade it into glucose and galactose, and then separated from components A, B, C, D and E by ultrafiltration and / or nanofiltration.

[0068] Suitable enzymes (proteins) for carrying out the above enzymatic reaction are α1-3 / 4-fucosidase or α1-3 / 4-transfucosidase. These enzymes have the ability to transfer fucosyl residues from fucosyl donors to acceptors, wherein the fucosyl residues are linked with α1-3-bonds. Specifically, if the acceptor is an HMO, the fucosyl residue is transferred to the 4-position of GlcNAc in the HMO acceptor, or to the 3-position of Glc if this position is not free or there is no GlcNAc in the HMO acceptor structure.

[0069] α1-3 / 4-(trans)fucosidase is a natural α1-3 / 4-L-fucosidase classified according to EC 3.2.1.111, or an engineered mutant thereof. All-natural α1-3 / 4-L-fucosidase can act as a transfucosidase to a certain extent.

[0070] A preferred α1-3 / 4-(trans)fucosidase suitable for carrying out the enzymatic reaction according to the present invention is an α1-3 / 4-(trans)fucosidase having at least 60%, preferably at least 70%, more preferably at least 80%, in particular at least 90% identity to amino acid positions 56 to 345 of the α1-3 / 4 glycosidase from Bifidobacterium longum subsp. infantis ATCC 15697 listed in U.S. Pat. No. 8,361,756 as SEQ ID no. 18 (referred to as SEQ ID no. 1 in the present application).

[0071] In the enzymatic synthesis of the present invention, as the concentration of the components produced in the reaction increases, the hydrolysis of the components may become significant, wherein the α3 / 4-fucosylated components are then hydrolyzed into non-α3 / 4-fucosylated components, lactose, 2'-FL and LNFP-I, and free fucose. Therefore, the α1-3 / 4-transfucosidase of the present invention preferably has limited or no hydrolytic activity. The limited or lack of hydrolytic activity of the α1-3 / 4-transfucosidase allows the reaction to last longer than otherwise possible.

[0072] Therefore, even preferred α1-3 / 4-transfucosidases for producing the HMO mixtures of the present invention are α1-3 / 4-transfucosidases lacking hydrolytic activity or at least having a significantly reduced hydrolytic activity.

[0073] This α1-3 / 4-transfucosidase can be prepared by changing the amino acid sequence at one or more amino acid positions, so that the mutated amino acid sequence leads to an increase in transfucosidase activity and / or a decrease in hydrolysis activity.

[0074] a) has been mutated at least one or more of the amino acid positions in SEQ ID no. 1: 134, 135, 168, 170, 174, 216, 221, 236, 237, 244, 245, 282 and 413, preferably at one or more of the following amino acid positions: 134, 135, 174, 216, 221, 282 and 413;

[0075] b) providing an enzymatic reaction of the invention with a conversion rate of at least 20% to 70%, such as at least 35%, or at least 40%, or at least 50%.

[0076] Suitably, the mutant fucosidases used in the present invention are non-natural fucosidases, that is, they do not occur in nature or naturally, but are prepared as a result of chemical synthesis, genetic engineering or similar methods in the laboratory, thereby producing synthetic mutant fucosidases.

[0077] Preferably, the mutant α1-3 / 4-transfucosidase comprises the polypeptide of SEQ ID no. 1, and at least one or more amino acid mutations at the following amino acid positions: 134, 135, 174, 216, 221 and 282, or consists thereof.

[0078] More preferably, the α1-3 / 4-transfucosidase of the first aspect comprises the polypeptide of SEQ ID no. 1, and one or more amino acid mutations at at least amino acid position 174 and the following amino acid positions: 134, 135, 170, 216, 221, 236, 237, 241, 244, 245 and 282, preferably one or more amino acid mutations at the following amino acid positions: 134, 135, 216, 221 and 282, or consists of thereof.

[0079] Also more preferably, the α1-3 / 4-transfucosidase comprises the polypeptide of SEQ ID no. 1, and one or more amino acid mutations at least at amino acid position 135 and the following amino acid positions: 134, 170, 174, 216, 221, 236, 237, 241, 244, 245 and 282, preferably one or more amino acid mutations at the following amino acid positions: 134, 135, 216, 221 and 282, or consists thereof.

[0080] Even more preferably, the α1-3 / 4-transfucosidase comprises the polypeptide of SEQ ID no. 1, and at least at amino acid positions 135 and 174, and one or more amino acid mutations at the following amino acid positions: 134, 170, 216, 221, 236, 237, 241 and 282, preferably one or more amino acid mutations at the following amino acid positions: 134, 216, 221 and 282, or consists thereof.

[0081] Optionally, the α1-3 / 4-transfucosidase comprises the polypeptide of SEQ ID no. 1, and at least at amino acid position 135 and / or 174, and one or more amino acid mutations at the following amino acid positions: 134, 170, 216, 221, 236, 237, 241 and 282, preferably one or more amino acid mutations at the following amino acid positions: 134, 216, 211 and 282, and there is a further mutation at one or more of the following amino acid positions: 165, 168, 232, 237, 258, 260 or 274, or consists thereof.

[0082] Preferably, the α1-3 / 4-transfucosidase comprises, more preferably consists of, the sequence of SEQ ID NO 1 having the following mutations:

[0083] - at amino acid position 135 and / or 174, and

[0084] - at least at an amino acid position selected from the group consisting of 168, 237 and 413.

[0085] In this regard, at position 135, Trp (W) is preferably replaced by Ala, Asp, Asn, Glu, Gln, His, Phe, Leu, Lys, Val or Tyr, more preferably Phe or Tyr; at position 168, Ser (S) is preferably replaced by Glu (E); at position 174, Ala (A) is preferably replaced by Arg, Asn, Cys, Glu, Ile, His, Leu, Lys, Met, Phe, Trp, Tyr or Val, more preferably Asn, His or Phe; at position 237, Glu (E) is preferably replaced by His (H); and at position 413, Glu (E) is replaced by Arg (R).

[0086] Even more preferably, the α1-3 / 4-transfucosidase comprises, more preferably consists of, the sequence of SEQ ID no. 1 as described above with the following mutations:

[0087] - at amino acid position 174,

[0088] - at amino acid position 135 or 168, and

[0089] - at amino acid position 413

[0090] - and optionally at amino acid position 165, 232, 258, 260 or 274.

[0091] The combination of the above mutations not only further improves the synthetic performance of the mutant enzyme transfucosidase, but also further enhances the stability, especially the temperature stability, while maintaining the further improved synthetic performance of the transfucosidase. The mutations at 135, 165, 174, 232, 258, 260 and 274 are preferably as follows:

[0092] - W135F or W135Y at position 135,

[0093] - at position 165 is P165E,

[0094] - at position 174 is A174F, A174H or A174N,

[0095] - R232A at position 232,

[0096] - Q258R at position 258,

[0097] - at position 260 is D260P,

[0098] - At position 274 is N274A.

[0099] Also preferably, a polypeptide comprising SEQ ID no. 1, and a mutation at least at amino acid position 174 or 282, preferably at least at two amino acid positions, or a mutant α1-3 / 4-transfucosidase consisting thereof provides a significantly or completely inhibited hydrolytic activity. In this regard, at position 174, Ala (A) is preferably replaced by Phe (F), Asn (N) or His (H), and / or at position 282, Val (V) is preferably replaced by Arg (R), Glu (E), His (H) or Lys (K). The inhibited hydrolytic activity is beneficial because the mutated enzyme does not significantly degrade the donor and / or product by hydrolysis. Therefore, the transfucosidase reaction is no longer kinetically controlled, and a better synthesis / hydrolysis ratio (meaning better synthesis performance) can be achieved. Mutations in one or two, preferably two, of the above amino acid positions can reduce the hydrolytic activity of fucosylated products by at least 100 times, preferably at least 1000 times, and more preferably at least 10000 times.

[0100] Therefore, the mutant α1-3 / 4-transfucosidase has

[0101] a) a polypeptide of SEQ ID no. 1 and a mutation at least at amino acid position 174 or 282, and

[0102] b) The hydrolytic activity is significantly or completely inhibited when compared to the protein according to SEQ ID no. 1.

[0103] Furthermore, according to certain embodiments, the mutant α1-3 / 4-transfucosidase comprises the polypeptide of SEQ ID no. 1, and the following mutations, or preferably consists of:

[0104] - at least amino acid position 174 or 282, and

[0105] - at least amino acid position 165, 168, 232, 237, 258, 260 or 274.

[0106] The combination of mutations described above not only significantly reduces the hydrolysis of the fucosylated product, preferably to an almost undetectable level, but also increases the stability, in particular the temperature stability.

[0107] Thus, when compared to the protein of SEQ ID no. 1, the mutant α1-3 / 4-transfucosidase has

[0108] a) a polypeptide of SEQ ID no. 1, which is mutated at least at amino acid position 174 or 282 and at least at amino acid position 165, 168, 232, 237, 258, 260 or 274,

[0109] and

[0110] b) significantly or completely inhibited hydrolytic activity,

[0111] and / or

[0112] c) Enhanced stability, especially temperature stability.

[0113] Preferably, the mutant α1-3 / 4-transfucosidase comprises, preferably consists of, the amino acid sequence of SEQ ID no. 1, at least at amino acid position 174 or 282, more preferably at amino acid position 174, Ala (A) is preferably replaced by Phe (F), Asn (N) or His (H), and / or at position 282, Val (V) is preferably replaced by Arg (R), Glu (E), His (H) or Lys (K), or a mutation thereof.

[0114] Particularly preferred modified α1-3 / 4-transfucosidases comprise the amino acid sequence of SEQ ID no. 1 and have the following mutations: W135F-A174N-N274A-E413R, or consist thereof, the amino acids being numbered according to SEQ ID no. 1.

[0115] In carrying out the method of the invention, the specific relative concentrations of component A donor, component B and C acceptor, α1-3 / 4-transfucosidase, aqueous solvent and incubation buffer are not critical. In this regard, the method can be suitably carried out at room temperature (e.g. 15-50, preferably 20-37° C.) at a pH of 4-7, preferably 5-6, for 15 minutes to 24 hours. The reaction is usually carried out to reach equilibrium.

[0116] In the enzymatic reaction, component A is provided as a fucosyl donor, and components B and C are provided as fucosyl acceptors. During the reaction, α1-3 / 4-transfucosidase transfers the fucosyl residue from the donor to two acceptors, thereby producing components D, component E, and lactose. However, in further subequilibrium reactions, the newly generated component E can also serve as a fucosyl donor, which results in an α1-3 / 4-transfucosidase reaction between component E and component B to produce components D and component C, or an α1-3 / 4-transfucosidase reaction between component E and lactose to produce components A and component C.

[0117] More preferably, the α1-3 / 4-transfucosidase, advantageously the W135F-A174N-N274A-E413R mutant (amino acid numbering according to SEQ ID no. 1), is used at about 0.09-0.11 w / w %, based on the total initial weight of components A, B and C. The amount of enzyme does not affect the final proportion of HMO in the composition obtained, it only acts when equilibrium is reached (the higher the enzyme concentration, the shorter the time to reach equilibrium).

[0118] Concerning the preparation of a mixture consisting of or essentially consisting of components A, B, C, D, E and optionally lactose, preferably a mixture according to any of embodiments i) to iv) disclosed below, the enzymatic synthesis according to the invention is carried out in the presence of a suitable buffer, but preferably under essentially buffer-free conditions.

[0119] According to the present invention, buffer solution is a solution containing a buffer, characterized in that the pH value of the solution can resist the change of the free hydrogen ion concentration caused by internal and / or environmental factors. In addition, according to the buffer used, the buffer solution is basically maintained at the pH value of the reaction system within a specific pH range. In addition, it is well known that the buffer solution of the enzyme reaction provides necessary cofactors, such as key salts (critical salts), and resists the change of hydrogen ion concentration and the condition of stabilizing proteins usually for the enzyme-driven reaction. Therefore, when carrying out the enzymatic reaction, those skilled in the art estimate the need to introduce buffers and / or components in the reaction environment, and these buffers and components are generally considered to be necessary for the normal operation of the enzyme.

[0120] However, the need to remove these additional reagents and / or components and / or residual products thereof from the reaction prior to further processing of the reaction product is time consuming and cumbersome, adding unnecessary costs to the production process.

[0121] Surprisingly, it has been found that the enzymatic synthesis of the present invention can be performed under essentially buffer-free conditions, thereby eliminating the necessity to provide and remove buffers and / or components.

[0122] In one aspect of the invention, the enzymatic synthesis described herein is carried out in purified water, wherein the purification step may comprise one or more purification processes selected from the group consisting of: reverse osmosis, filtration, ultrafiltration, precipitation, chlorination, iodine addition, electrodialysis, boiling, desalting and flocculation.

[0123] In a currently preferred aspect of the invention, the enzymatic synthesis is carried out in water having a conductivity below 10 μS / cm, such as below 5 μS / cm, such as below 1 μS / cm, such as below 0.1 μS / cm, and / or a silica content below 0.05 mg / l, such as below 0.04 mg / l, such as below 0.02 mg / l or such as below 0.01 mg / l, prior to use in the enzymatic synthesis.

[0124] In the enzymatic synthesis according to the present invention, substantially buffer-free conditions are conditions in which no additional buffer is added to purified water.

[0125] In this context, the term "substantially buffer-free" also includes a solution in which an inorganic salt is added to purify water of the previously specified conductivity and purity range as a stabilizer for the enzyme, wherein the salt does not contain buffering capacity. Examples of salts include common salts such as, but not limited to, sodium chloride (NaCl), calcium chloride (CaCl2), magnesium chloride (MgCl2), potassium chloride (KCl) and sodium sulfate (Na2SO4).

[0126] In one aspect of the enzymatic synthesis, according to the present invention, substantially buffer-free conditions may include salt amounts in the range of 1 micromolar to 1 molar, preferably in the range of 100 micromolar to 500 millimolar, or even more preferably in the range of 1 millimolar to 300 millimolar. In another aspect, substantially buffer-free conditions refer to the presence of trace ions in water at concentrations resulting in a conductivity of less than 5 μS / cm.

[0127] In another aspect of the invention, the term "substantially buffer-free" also includes the use of tap water.

[0128] The presence of ions in solution, whether or not salts are added to the solution, will affect the pH of the solution, even at trace concentrations, and will result in conductivity above 5 μS / cm, but will still not add buffering capacity to the substantially unbuffered conditions. Furthermore, the addition of reactants to a solution under substantially unbuffered conditions may also change the pH and conductivity of the solution. Therefore, in this context, the conditions after the addition of ions and / or reactants to the reaction solution are still considered to be substantially unbuffered.

[0129] In a preferred aspect of the enzymatic synthesis, according to the present invention, the synthesis is carried out in a substantially buffer-free solution, wherein the pH is in the range of pH 4 to pH 7, preferably pH 5 to pH 6.

[0130] Before the mixture of HMOs according to the invention is used for therapeutic or other purposes, it is necessary to remove unwanted reaction components. Unwanted products of the final product are, for example, enzymes such as α1-3 / 4-transfucosidase, lactose, other trace products and / or similar unwanted products.

[0131] Furthermore, the enzymatic synthesis according to the invention may comprise one or more steps for inactivating and isolating the α1-3 / 4-transfucosidase from the rest of the reaction mixture.

[0132] The enzymatic synthesis is carried out in a reaction vessel, and in some cases it may be advantageous to transfer the solution from the reaction vessel (which may be a process tank or a plastic container or any other suitable container for carrying out the synthesis) to a second container, for example, which has pressure control, temperature control and / or control of the liquid and / or gaseous environment. Thus, the enzymatic synthesis may be carried out in a manner in which the reaction vessel and the deactivation / inactivation vessel are identical, or it may also be carried out in a manner in which the reaction vessel and the deactivation / inactivation vessel are different.

[0133] The inactivation step may involve heating to a temperature of 60°C to 100°C for a duration of 1 minute to 180 minutes, such as 5 minutes to 150 minutes, 10 minutes to 120 minutes, 15 minutes to 90 minutes, and 30 minutes to 60 minutes. In a preferred aspect, the inactivation step involves heating the reaction mixture to a temperature of 60°C to 100°C for at least 1, 5, 10, 30 or 60 minutes.

[0134] After one or more steps of inactivating the α1-3 / 4-transfucosidase from the remaining reaction mixture, the enzymatic synthesis of the present invention further comprises one or more steps of removing the inactivated α1-3 / 4-transfucosidase from the synthesized HMO mixture, thereby providing a mixture consisting of or essentially consisting of components A, B, C, D, E and lactose, preferably a mixture according to any one of embodiments i) and ii) disclosed below (including preferred and more preferred embodiments thereof).

[0135] In general, the α1-3 / 4-transfucosidase can be removed from the synthetic HMO mixture by conventional separation methods, such as filtration (nanofiltration, ultrafiltration or microfiltration), removal by centrifugation or ultracentrifugation, treatment of the reaction mixture with powdered activated carbon in batch mode and / or column mode and / or chromatographic methods, such as ion exchange, affinity chromatography, size exclusion chromatography, normal phase or reverse phase chromatography, preferably after denaturation or inactivation of the enzyme used for the synthesis. The separation methods can be combined into a multi-step separation.

[0136] In one aspect of the invention, the reaction mixture containing α1-3 / 4-transfucosidase is treated with activated carbon, for example by passing the reaction mixture through a column packed with activated carbon using peristaltic flow, high pressure flow or gravity flow.

[0137] In another aspect of the invention, when the α1-3 / 4-transfucosidase is separated from the reaction mixture by ultrafiltration or microfiltration, the separation may involve a previous step in which the (inactivated) α1-3 / 4-transfucosidase forms particles, such as aggregates, crystals and / or precipitates, which makes separation by filtration more convenient. The pore size of the ultrafiltration membrane or microfiltration membrane is selected to enable the carbohydrate components to pass through the membrane while ensuring the retention of the (inactivated) α1-3 / 4-transfucosidase.

[0138] In another aspect of the invention, when the α1-3 / 4-transfucosidase is separated by ultracentrifugation or centrifugation, the separation may involve a previous step in which the (inactivated) α1-3 / 4-transfucosidase forms particles such as aggregates, crystals and / or precipitates, which makes a more convenient separation by (ultra)centrifugation possible.

[0139] The purification step disclosed above produces a purified aqueous solution from which, after removal of water, e.g. evaporation, spray drying or freeze drying, a solid mixture consisting of or essentially consisting of components A, B, C, D, E and lactose can be obtained, preferably a mixture according to any one of the embodiments i) and ii) disclosed below (including preferred and more preferred embodiments thereof).

[0140] In order to obtain a mixture consisting of or essentially consisting of components A, B, C, D and E, preferably a mixture according to any one of the embodiments iii) and iv) disclosed below (including preferred and more preferred embodiments thereof), lactose should be separated from components A, B, C, D and E. One suitable method is, for example, gel filtration, or membrane filtration using a membrane to ensure the retention of components A, B, C, D and E and allow lactose to pass. Another method is to add β-galactosidase to degrade lactose into glucose and galactose before or after removing the α1-3 / 4-transfucosidase from the reaction environment as described above, but before removing water, and then these monosaccharides are separated using a membrane that ensures the retention of components A, B, C, D and E and allows glucose and galactose to pass. The removal of β-galactosidase can be performed before or after membrane filtration, using one of the steps disclosed for removing α1-3 / 4-transfucosidase.

[0141] Embodiments of the first aspect of the present invention

[0142] With respect to a mixture consisting of or consisting essentially of components A, B, C, D, E and F, relative to the sum of components A, B, C, D, E and F,

[0143] i) - component D is 8-13 mol% or 14-22 w / w%, and / or

[0144] - component E is 9.5-17.5 mol% or 11.5-19 w / w%, and / or

[0145] - Component D+E is 17.5-30.5 mol% or 25.5-41 w / w%,

[0146] or

[0147] ii) - component D is 2-22 mol% or 5-34 w / w%, or

[0148] - Component E is 7-24 mol% or 6-29 w / w%,

[0149] Provided that component D+E is 23.5-33 mol% or 30.5-43 w / w%.

[0150] The mixture according to embodiment i) can usually be obtained when the molar ratio of the donor (component A) to the acceptor (components B and C) is about 3:1 to 1:3, and wherein the acceptor components are approximately equimolar. The mixture according to embodiment ii) can usually be obtained when the molar ratio of the donor (component A) to the acceptor (components B and C) is about equimolar, and wherein in the acceptor, the molar ratio of component B to component C is about 3.5:1 to 1:3.9.

[0151] With regard to the above embodiment i), it is preferred that component A+B+C is 41.5-67 mol % or 41-66 w / w % relative to the sum of components A, B, C, D, E and F, especially wherein component B is present in excess of component C.

[0152] In a more preferred embodiment i), relative to the sum of components A, B, C, D, E and F

[0153] - Component A is 52-58 mol% or 47-52 w / w%,

[0154] - Component B is 4-6 mol% or 6.5-8.5 w / w%,

[0155] - Component C is 1-2 mol% or 1-2 w / w%,

[0156] - Component D is 8.5-10 mol% or 15.5-17.5 w / w%,

[0157] - Component E is 10-11.5 mol% or 11.5-13 w / w%, and

[0158] - Component F is 17.5-20.5 mol% or 12-13.5 w / w%.

[0159] Such a mixture can be obtained when the molar ratio of donor (component A) to acceptor (components B and C) is about 3:1, and wherein the acceptor components are approximately equimolar.

[0160] Likewise in a more preferred embodiment i), relative to the sum of components A, B, C, D, E and F

[0161] - Component A is 42-46 mol% or 36-40 w / w%,

[0162] - Component B is 7-9 mol% or 11.5-13.5 w / w%,

[0163] - Component C is 3-4 mol% or 2.5-3.5 w / w%,

[0164] - Component D is 9-11 mol% or 16.5-19 w / w%,

[0165] - Component E is 11-13.5 mol% or 13-16 w / w%, and

[0166] - Component F is 20.5-23.5 mol% or 13-16 w / w%.

[0167] Such a mixture can be obtained when the molar ratio of donor (component A) to acceptor (components B and C) is about 2:1, and wherein the acceptor components are about equimolar.

[0168] Likewise in a more preferred embodiment i), relative to the sum of components A, B, C, D, E and F

[0169] - Component A is 7.5-9.5 mol% or 5-7.5 w / w%,

[0170] - Component B is 21-24 mol% or 30-33 w / w%,

[0171] - Component C is 16-19 mol% or 12.5-15.5 w / w%,

[0172] - Component D is 10-12 mol% or 16.5-19.5 w / w%,

[0173] - Component E is 14-16.5 mol% or 14.5-17 w / w%, and

[0174] - Component F is 23-27 mol% or 13-16 w / w%.

[0175] Such a mixture can be obtained when the molar ratio of donor (component A) to acceptor (components B and C) is about 1:2, and wherein the acceptor components are approximately equimolar.

[0176] Likewise in a more preferred embodiment i), relative to the sum of components A, B, C, D, E and F

[0177] - Component A is 3-5.5 mol% or 2.5-4 w / w%,

[0178] - Component B is 26-30 mol% or 36-40 w / w%,

[0179] - Component C is 22.5-25.5 mol% or 17-20 w / w%,

[0180] - Component D is 8.5-11 mol% or 14-16.5 w / w%,

[0181] - Component E is 11-14 mol% or 11-14 w / w%, and

[0182] - Component F is 20-23 mol% or 11-13.5 w / w%.

[0183] Such a mixture can be obtained when the molar ratio of donor (component A) to acceptor (components B and C) is about 1:3, and wherein the acceptor components are approximately equimolar.

[0184] In a particularly preferred embodiment i), relative to the sum of components A, B, C, D, E and F

[0185] - Component A is 19-24.5 mol% or 15-20 w / w%,

[0186] - Component B is 12.5-15.5 mol% or 19-22 w / w%,

[0187] - Component C is 7-10 mol% or 6-8 w / w%,

[0188] - Component D is 11-14 mol% or 18-22 w / w%,

[0189] - Component E is 14.5-17.5 mol% or 16-19 w / w%, and

[0190] - Component F is 25-30 mol% or 15-19 w / w%.

[0191] Such a mixture can be obtained when the molar ratio of donor (component A) to acceptor (components B and C) is about 1:1, and wherein the acceptor components are about equimolar.

[0192] Regarding the above embodiment ii), it is preferred that component A+B+C is 40.5-54.5 mol% or 41-54 w / w% relative to the sum of components A, B, C, D, E and F, especially wherein component A is 21-27 mol%.

[0193] In a more preferred embodiment ii), relative to the sum of components A, B, C, D, E and F

[0194] - Component A is 21.5-25.5 mol% or 16-19.5 w / w%,

[0195] - Component B is 18-22 mol% or 24.5-28.5 w / w%,

[0196] - Component C is 2-4 mol% or 1.5-3.5 w / w%,

[0197] - Component D is 18-22 mol% or 30-34 w / w%,

[0198] - component E is 6.5-9.5 mol% or 6-9 w / w%, and

[0199] - Component F is 23-27 mol% or 13-15.5 w / w%.

[0200] Such a mixture can be obtained when the molar ratio of donor (component A) to acceptor (components B and C) is approximately equimolar and wherein the molar ratio of component B to component C in the acceptor is approximately 3.5:1.

[0201] Likewise in a more preferred embodiment ii), relative to the sum of components A, B, C, D, E and F

[0202] - Component A is 21-24.5 mol% or 16.5-20 w / w%,

[0203] - Component B is 15-18 mol% or 21-25 w / w%,

[0204] - Component C is 5.5-7.5 mol% or 4-6 w / w%,

[0205] - Component D is 13-16.5 mol% or 22-26 w / w%,

[0206] - Component E is 12-15 mol% or 12.5-15.5 w / w%, and

[0207] - Component F is 24-28 mol% or 14-17 w / w%.

[0208] Such a mixture can be obtained when the molar ratio of donor (component A) to acceptor (components B and C) is approximately equimolar, and wherein the molar ratio of component B to component C in the acceptor is approximately 1.4:1.

[0209] Likewise in a more preferred embodiment ii), relative to the sum of components A, B, C, D, E and F

[0210] - Component A is 21-24.5 mol% or 17-20.5 w / w%,

[0211] - Component B is 10.5-13.5 mol% or 16.5-20 w / w%,

[0212] - Component C is 9-12 mol% or 8-10 w / w%,

[0213] - Component D is 8-10 mol% or 14-17 w / w%,

[0214] - Component E is 17.5-20.5 mol% or 19.5-23 w / w%, and

[0215] - Component F is 24.5-28.5 mol% or 14.5-17.5 w / w%.

[0216] Such a mixture can be obtained when the molar ratio of donor (component A) to acceptor (components B and C) is approximately equimolar and wherein in the acceptor the molar ratio of component B to component C is approximately 1:1.5.

[0217] Likewise in a more preferred embodiment ii), relative to the sum of components A, B, C, D, E and F

[0218] - Component A is 23-27 mol% or 21-25 w / w%,

[0219] - Component B is 6-8 mol% or 10-12.5 w / w%,

[0220] - Component C is 15-19.5 mol% or 14-16.5 w / w%,

[0221] - Component D is 2.5-4.5 mol% or 5.5-7.5 w / w%,

[0222] - Component E is 21-24.5 mol% or 25-28.5 w / w%, and

[0223] - Component F is 23.5-27.5 mol% or 16-19 w / w%.

[0224] Such a mixture can be obtained when the molar ratio of donor (component A) to acceptor (components B and C) is approximately equimolar, and wherein in the acceptor, the molar ratio of component B to component C is approximately 1:3.9.

[0225] In a particularly preferred embodiment ii), relative to the sum of components A, B, C, D, E and F

[0226] - Component A is 19-24.5 mol% or 15-20 w / w%,

[0227] - Component B is 12.5-15.5 mol% or 19-22 w / w%,

[0228] - Component C is 7-10 mol% or 6-8 w / w%,

[0229] - Component D is 11-14 mol% or 18-22 w / w%,

[0230] - Component E is 14.5-17.5 mol% or 16-19 w / w%, and

[0231] - Component F is 25-30 mol% or 15-19 w / w%.

[0232] Such a mixture is obtained when the molar ratio of donor (component A) to acceptor (components B and C) and the molar ratio of component B to component C are both approximately equimolar.

[0233] With respect to a mixture consisting of or consisting essentially of components A, B, C, D and E, relative to the sum of components A, B, C, D and E,

[0234] iii) - component D is 10-17.5 mol% or 16-25.5 w / w%, and / or

[0235] - component E is 12-23.5 mol% or 13-22 w / w%, and / or

[0236] - Component D+E is 22-41 mol% or 29-47.5 w / w%,

[0237] or

[0238] iv) - component D is 3.5-29.5 mol% or 6-39 w / w%, and / or

[0239] - Component E is 9-32 mol% or 7.5-34.5 w / w%,

[0240] Provided that component D+E is 31.5-41 mol% or 36.5-49 w / w%.

[0241] The mixture according to embodiment iii) can usually be obtained when the molar ratio of donor (component A) to acceptor (components B and C) is about 3:1 to 1:3, wherein the acceptor components are approximately equimolar, and wherein lactose is substantially removed from the composition thus obtained. The mixture according to embodiment iv) can usually be obtained when the molar ratio of donor (component A) to acceptor (components B and C) is about equimolar, wherein in the acceptor, the molar ratio of component B to component C is about 3.5:1 to 1:3.9, and wherein lactose is substantially removed from the composition thus obtained.

[0242] With regard to embodiment iii) above, it is preferred that more component B than component C is present in the mixture.

[0243] In a more preferred embodiment iii), relative to the sum of components A, B, C, D and E

[0244] - Component A is 65-71 mol% or 53.5-59.5 w / w%,

[0245] - Component B is 5-7 mol% or 7.5-9.5 w / w%,

[0246] - Component C is 1.5-2.5 mol% or 1-2 w / w%,

[0247] - component D is 10-12.5 mol% or 18-20.5 w / w%, and

[0248] - Component E is 12-14.5 mol% or 13-15.5 w / w%.

[0249] Such a mixture can be obtained when the molar ratio of donor (component A) to acceptor (components B and C) is about 3:1, wherein the acceptor components are about equimolar and wherein lactose is substantially removed from the composition thus obtained.

[0250] Likewise in a more preferred embodiment iii), relative to the sum of components A, B, C, D and E

[0251] - Component A is 54-58.5 mol% or 42-46.5 w / w%,

[0252] - Component B is 9-11.5 mol% or 13-15.5 w / w%,

[0253] - Component C is 4-5 mol% or 3-4 w / w%,

[0254] - Component D is 11.5-14 mol% or 19-23 w / w%,

[0255] - Component E is 15-18 mol% or 15-18.5 w / w%.

[0256] Such a mixture can be obtained when the molar ratio of donor (component A) to acceptor (components B and C) is about 2:1, wherein the acceptor components are about equimolar and wherein lactose is substantially removed from the composition thus obtained.

[0257] Likewise in a more preferred embodiment iii), relative to the sum of components A, B, C, D and E

[0258] - Component A is 10-12.5 mol% or 6.5-9 w / w%,

[0259] - Component B is 28.5-32 mol% or 34.5-38.5 w / w%,

[0260] - Component C is 22-25 mol% or 15-17.5 w / w%,

[0261] - Component D is 13.5-16 mol% or 19.5-22.5 w / w%,

[0262] - Component E is 19-21.5 mol% or 17-19.5 w / w%.

[0263] Such a mixture can be obtained when the molar ratio of donor (component A) to acceptor (components B and C) is about 1:2, wherein the acceptor components are about equimolar and wherein lactose is substantially removed from the composition thus obtained.

[0264] Likewise in a more preferred embodiment iii), relative to the sum of components A, B, C, D and E

[0265] - Component A is 4.5-6.5 mol% or 2.5-4.5 w / w%,

[0266] - Component B is 34-38 mol% or 41.5-45.5 w / w%,

[0267] - Component C is 29-32.5 mol% or 19-22.5 w / w%,

[0268] - Component D is 11-13.5 mol% or 16-19 w / w%,

[0269] - Component E is 14.5-17.5 mol% or 13-16 w / w%.

[0270] Such a mixture can be obtained when the molar ratio of donor (component A) to acceptor (components B and C) is about 1:3, wherein the acceptor components are about equimolar and wherein lactose is substantially removed from the composition thus obtained.

[0271] In a particularly preferred embodiment iii), relative to the sum of components A, B, C, D and E

[0272] - Component A is 29-33 mol% or 20-24.5 w / w%,

[0273] - Component B is 18-21.5 mol% or 23-25.5 w / w%,

[0274] - Component C is 10-12.5 mol% or 7.5-9.5 w / w%,

[0275] - Component D is 15-17.5 mol% or 22-25.5 w / w%,

[0276] - Component E is 20.5-23.5 mol% or 19.5-22 w / w%.

[0277] Such a mixture can be obtained when the molar ratio of donor (component A) to acceptor (components B and C) is about 1:1, wherein the acceptor components are about equimolar and wherein lactose is substantially removed from the composition thus obtained.

[0278] Regarding the above embodiment iv), preferably component A+B+C is 57-70 mol% or 51-64.5 w / w% relative to the sum of components A, B, C, D and E, especially component A is 29-35.5 mol%.

[0279] In a more preferred embodiment iv), relative to the sum of components A, B, C, D and E

[0280] - Component A is 29.5-33.5 mol% or 19-22.5 w / w%,

[0281] - Component B is 25-28.5 mol% or 29-33 w / w%,

[0282] - Component C is 3-5 mol% or 2-3.5 w / w%,

[0283] - Component D is 26-29.5 mol% or 35-39 w / w%,

[0284] - Component E is 9-11.5 mol% or 7.5-10 w / w%.

[0285] Such a mixture can be obtained when the molar ratio of donor (component A) to acceptor (components B and C) is approximately equimolar, wherein in the acceptor the molar ratio of component B to component C is approximately 3.5:1, and wherein lactose is substantially removed from the composition thus obtained.

[0286] Likewise in a more preferred embodiment iv), relative to the sum of components A, B, C, D and E

[0287] - Component A is 29-32.5 mol% or 20.5-23 w / w%,

[0288] - Component B is 21-24.5 mol% or 26-29.5 w / w%,

[0289] - Component C is 7.5-10 mol% or 5-7 w / w%,

[0290] - Component D is 18-21.5 mol% or 27-29.5 w / w%,

[0291] - Component E is 17-19.5 mol% or 15.5-18 w / w%.

[0292] Such a mixture can be obtained when the molar ratio of donor (component A) to acceptor (components B and C) is approximately equimolar, wherein in the acceptor the molar ratio of component B to component C is approximately 1.4:1, and wherein lactose is substantially removed from the composition thus obtained.

[0293] Likewise in a more preferred embodiment iv), relative to the sum of components A, B, C, D and E

[0294] - Component A is 29-33 mol% or 22-24.5 w / w%,

[0295] - Component B is 15-18 mol% or 20.5-23 w / w%,

[0296] - Component C is 13-15.5 mol% or 9.5-12 w / w%,

[0297] - Component D is 11-13.5 mol% or 17-20 w / w%,

[0298] - Component E is 24.5-27.5 mol% or 24-27 w / w%.

[0299] Such a mixture can be obtained when the molar ratio of donor (component A) to acceptor (components B and C) is approximately equimolar, wherein in the acceptor the molar ratio of component B to component C is approximately 1:1.5 and wherein lactose is substantially removed from the composition thus obtained.

[0300] Likewise in a more preferred embodiment iv), relative to the sum of components A, B, C, D and E

[0301] - Component A is 31.5-35.5 mol% or 26-29.5 w / w%,

[0302] - Component B is 8.5-10.5 mol% or 12.5-15 w / w%,

[0303] - Component C is 21-24 mol% or 17-20 w / w%,

[0304] - Component D is 3.5-5.5 mol% or 6-9.5 w / w%,

[0305] - Component E is 28-32 mol% or 30.5-34.5 w / w%.

[0306] Such a mixture can be obtained when the molar ratio of donor (component A) to acceptor (components B and C) is approximately equimolar, wherein in the acceptor the molar ratio of component B to component C is approximately 1:3.9, and wherein lactose is substantially removed from the composition thus obtained.

[0307] In a particularly preferred embodiment iv), relative to the sum of components A, B, C, D and E

[0308] - Component A is 29-33 mol% or 21-24.5 w / w%,

[0309] - Component B is 18-21.5 mol% or 23-25.5 w / w%,

[0310] - Component C is 10-12.5 mol% or 7.5-9.5 w / w%,

[0311] - Component D is 15-17.5 mol% or 23-25.5 w / w%,

[0312] - Component E is 20.5-23.5 mol% or 19.5-22 w / w%.

[0313] Such a mixture is obtained when the molar ratio of donor (component A) to acceptor (components B and C) and the molar ratio of component B to component C are both approximately equimolar and wherein lactose is substantially removed from the composition thus obtained.

[0314] Pharmaceutical, cosmetic and / or nutritional compositions

[0315] According to the present invention, a mixture consisting of or essentially consisting of 3-FL, LNFP-I, 2'-FL, LNDFH-I, DFL and optionally lactose can be formulated into a pharmaceutical composition, a cosmetic composition and / or a nutritional composition, which can contain a pharmaceutically, cosmetically and / or nutritionally acceptable carrier, such as a phosphate buffered saline solution, an unbuffered saline solution, an ethanol water mixture, water and an emulsion, such as an oil / water or water / oil emulsion, and various wetting agents and / or excipients. The pharmaceutical composition, the cosmetic composition and / or the nutritional composition can also contain other materials that do not produce adverse, allergic or other undesirable reactions when administered to a patient.

[0316] The carriers and other materials included in the pharmaceutical, cosmetic and / or nutritional compositions may include one or more of the following: solvents, dispersants, coatings, absorption enhancers, controlled release agents and one or more inert excipients, such as starch, polyols, granulating agents, microcrystalline cellulose, diluents, lubricants, binders and disintegrants. If desired, the tablet dosage of the anti-infective mixture may be coated by aqueous or non-aqueous techniques known to those skilled in the art.

[0317] The pharmaceutical, cosmetic and / or nutritional composition according to the present invention can be administered orally, buccally, sublingually, topically and / or rectally. It can be formulated as a tablet, capsule, suppository, effervescent tablet, pellet, lozenge, troche, gel, paste, solution, suspension, emulsion, syrup, pill, electuary, slurry, in an aqueous or non-aqueous liquid, as a powder or as granules containing a predetermined concentration of the HMO mixture.

[0318] In addition, the pharmaceutical composition, cosmetic composition and / or nutritional composition according to the present invention may include a binder, a lubricant, an inert diluent, a flavoring agent and a humectant. Tablets, capsules, suppositories or pellets containing the pharmaceutical composition, cosmetic composition and / or nutritional composition of the present invention may optionally be coated or formulated to provide sustained, delayed or controlled release of the HMO anti-infective mixture.

[0319] The HMO mixtures according to the present invention may be supplemented with additional drugs, active pharmaceutical ingredients or agents that have an effect on the adverse health condition of the patient to whom the composition is administered.

[0320] In one embodiment, the composition can be in the form of a nutritional composition. For example, the nutritional composition can be a food composition, a rehydration, a medical food or a food for special medical purposes, a nutritional supplement, etc. The nutritional composition can contain a source of protein, lipid and / or digestible carbohydrates and can be in powder or liquid form. The composition can be designed as a sole source of nutrition or as a nutritional supplement.

[0321] Suitable protein sources include milk protein, soy protein, rice protein, pea protein and oat protein, or mixtures thereof. The milk protein may be milk protein concentrate, milk protein isolate, whey protein or casein, or a mixture of the two. The protein may be whole protein or partially hydrolyzed or extensively hydrolyzed protein. Hydrolyzed protein has the advantage of being easier to digest, which is important for people with inflamed or damaged gastrointestinal tract. Protein may also be provided in the form of free amino acids. Protein may account for about 5% to about 30% of the energy of the nutritional composition, typically about 10% to 20%. Ideally, the source of protein does not include excess lactose.

[0322] The protein source can be a source of glutamine, threonine, cysteine, serine, proline, or a combination of these amino acids. The glutamine source can be glutamine dipeptide and / or a glutamine-rich protein. Glutamine can be included because intestinal epithelial cells use glutamine as an energy source. Threonine, serine, and proline are important amino acids for the production of mucins. Mucins coat the gastrointestinal tract and can improve intestinal barrier function and mucosal healing. Cysteine ​​is the main precursor of glutathione, which is key to the body's antioxidant defenses.

[0323] Suitable digestible carbohydrates include maltodextrin, hydrolyzed or modified starch or corn starch, glucose polymers, corn syrup, corn syrup solids, high fructose corn syrup, rice derived carbohydrates, pea derived carbohydrates and potato derived carbohydrates, tapioca starch, sucrose, glucose, fructose, sucrose, honey, sugar alcohols (e.g. maltitol, erythritol, sorbitol), or mixtures thereof. Preferably, the composition reduces or does not contain added lactose or other FODMAP carbohydrates. Generally digestible carbohydrates provide about 35% to about 55% of the energy of the nutritional composition. A carbohydrate suitable for digestion is low dextrose equivalent (DE) maltodextrin.

[0324] Suitable lipids include medium chain triglycerides (MCT) and long chain triglycerides (LCT). Preferably, lipid is a mixture of MCT and LCT. For example, MCT can contain about 30% to about 70% lipid by weight, more specifically, about 50% to about 60% by weight. MCT has the advantage of being easier to digest, which is important for people with inflamed or damaged gastrointestinal tract. Typically, lipid provides about 35% to about 50% of the energy of the nutritional composition. Lipid can contain essential fatty acids (ω-3 and ω-6 fatty acids). Preferably, these polyunsaturated fatty acids provide less than about 30% of the total energy of the lipid source.

[0325] Suitable sources of long-chain triglycerides are rapeseed oil, sunflower oil, palm oil, soybean oil, butterfat, corn oil, high oleic acid and soy lecithin. Fractionated coconut oil is a suitable source of medium-chain triglycerides. The lipid profile of the nutritional composition is preferably designed to have a ratio of polyunsaturated fatty acids ω-6 (n-6) to ω-3 (n-3) of about 4:1 to about 10:1. For example, the fatty acid ratio of n-6 to n-3 can be about 6:1 to about 9:1.

[0326] The nutritional composition can also include vitamins and minerals. If the nutritional composition is intended to be the only source of nutrition, it preferably includes a complete vitamin and mineral spectrum. Examples of vitamins include vitamin A, B complex (such as B1, B2, B6 and B12), C, D, E and K, niacin and acidic vitamins such as pantothenic acid, folic acid and biotin. The example of minerals includes calcium, iron, zinc, magnesium, iodine, copper, phosphorus, manganese, potassium, chromium, molybdenum, selenium, nickel, tin, silicon, vanadium and boron.

[0327] The nutritional composition can also include carotenoids such as lutein, lycopene, zeaxanthin and beta-carotene. The total amount of carotenoids contained can vary between about 0.001 μg / ml and about 10 μg / ml. The content of lutein can be about 0.001 μg / ml to about 10 μg / ml, preferably about 0.044 μg / ml to about 5 μg / ml of lutein. The content of lycopene can be about 0.001 μg / ml to about 10 μg / ml, preferably about 0.0185 μg / ml to 5 μg / ml of lycopene. Beta-carotene can include about 0.001 μg / ml to about 10 mg / ml, for example, about 0.034 μg / ml to about 5 μg / ml of beta-carotene.

[0328] The nutritional composition preferably also contains a reduced concentration of sodium, for example from about 300 mg / l to about 400 mg / l. The remaining electrolytes may be present in concentrations that meet the needs without causing excessive renal solute burden on renal function. For example, potassium is preferably present in a range of about 1180 to about 1300 mg / l, and chloride is preferably present in a range of about 680 to about 800 mg / l.

[0329] The nutritional composition may also contain various other conventional ingredients, such as preservatives, emulsifiers, thickeners, buffers, fibers and prebiotics (e.g., fructooligosaccharides, galacto-oligosaccharides), probiotics (e.g., B. animalis subsp. lactis BB-12, B. lactis HN019, B. lactis Bi07, B. infantis ATCC 15697, Lactobacillus rhamnosus GG, Lactobacillus rhamnosus LGG DSM 33156, Lactobacillus rhamnosus HNOO1, Lactobacillus acidophilus LA-5, Lactobacillus acidophilus NCFM, Lactobacillus fermentum CECT5716, Bifidobacterium longum BB536, Bifidobacterium longum AH1205, Bifidobacterium longum AH1206, Bifidobacterium breve M-16V, Lactobacillus reuteri ATCC 55730, Lactobacillus reuteri ATCC PTA-6485, Lactobacillus reuteri DSM17938), antioxidant / anti-inflammatory compounds including tocopherols, carotenoids, ascorbic acid / vitamin C, ascorbyl palmitate, polyphenols, glutathione and superoxide dismutase (melon), other bioactive factors (such as growth hormone, cytokine, TFG-β), colorants, flavors and stabilizers, lubricants, etc.

[0330] The nutritional composition can be formulated as a soluble powder, a liquid concentrate or a ready-to-use preparation. The composition can be administered to a person in need thereof via a nasogastric tube or orally. Various flavors and other additives may also be present.

[0331] Nutritional composition can be prepared by any conventional manufacturing technology for preparing solid or liquid nutritional composition. For example, the composition can be prepared by combining various raw material liquids. Fat-encapsulated protein raw material liquid can be prepared by heating and mixing lipid sources, and then adding emulsifiers (such as lecithin), fat-soluble vitamins and at least a portion of protein sources while heating and stirring. Then, a carbohydrate raw material liquid is prepared by adding minerals, trace and ultra-trace minerals, thickeners or suspending agents to water while heating and stirring. Before adding carbohydrates (such as HMO and digestible carbohydrate sources), the resulting solution is kept under continuous heating and stirring for 10 minutes. Then, the resulting raw material liquids are mixed together while heating and stirring, and the pH is adjusted to 6.6-7.0, and then the composition is subjected to high temperature short-time treatment, during which the composition is heat-treated, emulsified and homogenized, and then cooled. Water-soluble vitamins and ascorbic acid are added, and the pH is adjusted to the desired range if necessary, spices are added, and water is added to reach the desired total solid content.

[0332] For liquid products, the resulting solution can then be aseptically packaged to form an aseptically packaged nutritional composition. In this form, the nutritional composition can be in a ready-to-eat or concentrated liquid form. Alternatively, the composition can be spray dried, processed and packaged into a reconstitutable powder.

[0333] When the nutritional product is a ready-to-feed nutritional liquid, the total concentration of HMO in the liquid is preferably from about 0.1% to about 1.5%, including from about 0.2% to about 1.0%, such as from about 0.3% to about 0.7%, by weight of the liquid. When the nutritional product is a concentrated nutritional liquid, the total concentration of HMO in the liquid is preferably from about 0.2% to about 3.0%, including from about 0.4% to about 2.0%, such as from about 0.6% to about 1.5%, by weight of the liquid.

[0334] In another embodiment, the nutritional composition is a unit dosage form. The unit dosage form may include an acceptable food grade carrier, such as phosphate buffered saline, an ethanol water mixture, water and emulsions (such as oil / water or water / oil emulsions), and various wetting agents or excipients. The unit dosage form may also contain other materials that do not produce adverse, allergic or other unwanted reactions when applied to a person. The carrier and other materials may include solvents, dispersants, coatings, absorption promoters, controlled release agents and one or more inert excipients, such as starch, polyols, granulating agents, microcrystalline cellulose, diluents, lubricants, binders and disintegrants. Preferably, the unit dosage form mainly includes HMO and a minimum amount of binders and / or excipients. When nutrition is incomplete or not intended as the sole source of nutrition, the unit dosage form is particularly suitable.

[0335] The unit dosage form can be administered orally, for example, in the form of tablets, capsules or pellets containing a predetermined amount of the mixture, or in the form of a powder or granules containing a predetermined concentration of the mixture, or in the form of a gel, paste, solution, suspension, emulsion, syrup, pill, electuary or suspension in an aqueous or non-aqueous liquid containing a predetermined concentration of the mixture. Orally administered compositions may include one or more binders, lubricants, inert diluents, flavoring agents and humectants. Orally administered compositions such as tablets may optionally be coated and may be formulated to provide sustained, delayed or controlled release of the HMO.

[0336] The unit dosage form may also be administered by nasogastric tube or direct infusion into the gastrointestinal tract or stomach.

[0337] The unit dosage form may also include therapeutic agents such as antibiotics, probiotics, analgesics, and anti-inflammatory agents.

[0338] The appropriate dosage of the nutritional composition for a human can be determined in a conventional manner based on factors such as the concentration of the HMO, the human condition, immune status, body weight and age. The desired amount of HMO is generally in the range of about 1 g to about 15 g per day, and in certain embodiments about 2 g to about 10 g per day, such as about 3 g to about 7 g per day. The appropriate dosage regimen can be determined by methods known to those skilled in the art.

[0339] In other embodiments, the mixture of HMOs can be formulated into a pharmaceutical composition. The pharmaceutical composition can include a pharmaceutically acceptable carrier, such as phosphate buffered saline, an ethanol water mixture, water, and an emulsion, such as an oil / water or water / oil emulsion, and various wetting agents or excipients. The pharmaceutical composition can also include other materials that do not produce adverse, allergic or other undesirable reactions when administered to a human. The carrier and other materials can include solvents, dispersants, coatings, absorption enhancers, controlled release agents, and one or more inert excipients, such as starches, polyols, granulating agents, microcrystalline cellulose, diluents, lubricants, binders, and disintegrants.

[0340] The pharmaceutical composition can be administered orally, for example in the form of tablets, capsules or pellets containing a predetermined amount of mixture, or in the form of powders or granules containing a predetermined concentration of mixture, or in the form of gels, pastes, solutions, suspensions, emulsions, syrups, pills, electuaries or slurries containing a predetermined concentration of mixture in an aqueous or non-aqueous liquid. Orally administered compositions can include adhesives, lubricants, inert diluents, flavoring agents and humectants. Orally administered compositions such as tablets can be optionally coated and can be formulated to provide a sustained, delayed or controlled release of the mixture herein.

[0341] The pharmaceutical compositions may also be administered by rectal suppository, aerosol tube, nasogastric tube, or direct infusion into the gastrointestinal tract or stomach.

[0342] The pharmaceutical composition may also include therapeutic agents, such as antibiotics, probiotics, analgesics, and anti-inflammatory agents. The appropriate dosage of the pharmaceutical composition can be determined in a conventional manner based on factors such as the concentration of the HMO, the patient's condition, immune status, body weight, and age. The required amount of the HMO is generally in the range of about 1 g to about 15 g per day, and in certain embodiments about 2 g to about 10 g per day, such as about 3 g to about 7 g per day. The appropriate dosage regimen can be determined by methods known to those skilled in the art.

[0343] Use of the mixture of HMOs according to the invention

[0344] The present invention also relates to the use of a mixture consisting of or essentially consisting of 3-FL, LNFP-I, 2'-FL, LNDFH-I, DFL and optionally lactose for medical purposes.

[0345] Individual HMOs are known to have beneficial health-related effects and are known to affect bacterial and viral infections. While the individual HMOs in isolation are beneficial on their own, the combined mixtures of HMOs, as described herein, have synergistic effects in treating infections compared to the individual components in isolation.

[0346] One aspect of the present invention relates to a mixture consisting of or substantially consisting of 3-FL, LNFP-I, 2'-FL, LNDFH-I, DFL and optionally lactose (including the preferred and more preferred embodiments disclosed above), or a composition comprising a mixture consisting of or substantially consisting of 3-FL, LNFP-I, 2'-FL, LNDFH-I, DFL and optionally lactose (including the preferred and more preferred embodiments disclosed above), for use in treating, preventing and / or improving diseases and / or conditions associated with an imbalance in the microbiome. Alternatively, one aspect of the present invention relates to a method for treating, preventing and / or improving diseases and / or conditions associated with an imbalance in the human microbiome, comprising administering to a human a mixture consisting of or substantially consisting of 3-FL, LNFP-I, 2'-FL, LNDFH-I, DFL and optionally lactose, or a composition comprising a mixture consisting of or substantially consisting of 3-FL, LNFP-I, 2'-FL, LNDFH-I, DFL and optionally lactose. Generally, the mixture of 3-FL, LNFP-I, 2'-FL, LNDFH-I and DFL according to the present invention can be used to modulate the human microbiome, for example, increasing the abundance of Bifidobacterium and Barnesella, which reduces the abundance of Firmicutes, especially Clostridium.

[0347] In addition, another aspect of the present invention relates to a mixture consisting of or substantially consisting of 3-FL, LNFP-I, 2'-FL, LNDFH-I, DFL and optionally lactose (including the preferred and more preferred embodiments disclosed above), or a composition comprising a mixture consisting of or substantially consisting of 3-FL, LNFP-I, 2'-FL, LNDFH-I, DFL and optionally lactose (including the preferred and more preferred embodiments disclosed above), for use in treating and / or reducing the risk of a wide range of bacterial or viral infections in humans. Alternatively, one aspect of the present invention relates to a method for treating and / or reducing the risk of a wide range of bacterial or viral infections in humans, comprising administering to a human a mixture consisting of or substantially consisting of 3-FL, LNFP-I, 2'-FL, LNDFH-I, DFL and optionally lactose, or a composition comprising a mixture consisting of or substantially consisting of 3-FL, LNFP-I, 2'-FL, LNDFH-I, DFL and optionally lactose. Infections can occur in several parts of the body, not only limited to the intestinal microbiome, but also in other parts of the body exposed to the external environment, such as the skin, hair, ears, eyes, nose and respiratory system. One or more HMOs in the mixture according to the invention can inhibit the adhesion of pathogenic bacteria, such as Pseudomonas aeruginosa or Campylobacter jejuni, uropathogenic and enteropathogenic E. coli, certain Salmonella species and / or pneumonia-causing pathogens Pseudomonas aeruginosa, and viruses such as norovirus. One or more HMOs in the mixture according to the invention can also directly bind to pathogenic toxins, such as toxins from Clostridium difficile. One or more HMOs in the mixture according to the invention can act as immune system modulators and affect intestinal health by stimulating bifidobacteria. One or more HMOs in the mixture according to the invention can inhibit the growth of Group B Streptococcus in infants and breast milk. Group B Streptococcus is the main cause of neonatal sepsis, pneumonia and meningitis. One or more HMOs of the mixture according to the invention can directly bind to Shigella dysenteriae Shiga toxins Stx2 and Stx1B5. One or more HMOs of the mixture according to the invention have the potential to reduce the risk of infectious diseases caused by bacterial or viral pathogens, most likely due to binding to bacterial exotoxins.

[0348] In addition, another aspect of the present invention relates to a mixture consisting of or essentially consisting of 3-FL, LNFP-I, 2'-FL, LNDFH-I, DFL and optionally lactose (including the preferred and more preferred embodiments disclosed previously), or a composition comprising a mixture consisting of or essentially consisting of 3-FL, LNFP-I, 2'-FL, LNDFH-I, DFL and optionally lactose (including the preferred and more preferred embodiments disclosed previously), for non-medical use in maintaining the symbiotic homeostasis of the human intestinal microbiota.

[0349] In addition, another aspect of the present invention relates to a mixture consisting of or essentially consisting of 3-FL, LNFP-I, 2'-FL, LNDFH-I, DFL and optionally lactose (including the preferred and more preferred embodiments disclosed above), or a composition comprising a mixture consisting of or essentially consisting of 3-FL, LNFP-I, 2'-FL, LNDFH-I, DFL and optionally lactose (including the preferred and more preferred embodiments disclosed above), for non-medical use in human dietary management.

[0350] Furthermore, a mixture consisting essentially of or consisting of 3-FL, LNFP-I, 2'-FL, LNDFH-I, DFL and optionally lactose (including the preferred and more preferred embodiments disclosed above) improves the viability of useful probiotics in aqueous compositions. It has been found that probiotic strains, such as Lactobacillus rhamnosus strains, when mixed with HMOs, have significantly increased regeneration and viability when in contact with low pH liquid environments (such as stomach acid or acidic beverages). Therefore, the combination of a mixture of HMOs according to the present invention with a probiotic strain (preferably freeze-dried probiotics) can provide a reliable way to deliver a quantifiable amount of probiotics to a host (human or animal) in a drug-like form, a nutritional composition form or a food-based form.

[0351] While the invention has been described with reference to embodiments, it will be appreciated that various modifications may be made within the scope of the invention.

[0352] The present invention provides the following numbered aspects:

[0353] 1. A mixture consisting essentially of 3-FL as component A, LNFP-I as component B, 2'-FL as component C, LNDFH-I as component D, DFL as component E and optionally lactose as component F.

[0354] 2. The mixture according to aspect 1, which consists essentially of 3-FL, LNFP-I, 2'-FL, LNDFH-I, DFL and lactose, preferably wherein

[0355] Relative to the sum of components A, B, C, D, E and F,

[0356] - component D is 8-13 mol% or 14-22 w / w%, and / or

[0357] - component E is 9.5-17.5 mol% or 11.5-19 w / w%, and / or

[0358] - Component D+E is 17.5-30.5 mol% or 25.5-41 w / w%,

[0359] or

[0360] Relative to the sum of components A, B, C, D, E and F,

[0361] - component D is 2-22 mol% or 5-34 w / w%, or

[0362] - Component E is 7-24 mol% or 6-29 w / w%,

[0363] Provided that component D+E is 23.5-33 mol% or 30.5-43 w / w%.

[0364] 3. A mixture according to aspect 1 or 2, wherein

[0365] - component A+B+C is 41.5-67 mol% or 41-66 w / w%, preferably wherein component B is present in excess of component C,

[0366] or

[0367] - Component A+B+C is 40.5-54.5 mol% or 41-54 w / w%, preferably component A is 21-27 mol%.

[0368] 4. A mixture according to any of the preceding aspects, wherein

[0369] - Component A is 19-24.5 mol% or 15-20 w / w%,

[0370] - Component B is 12.5-15.5 mol% or 19-22 w / w%,

[0371] - Component C is 7-10 mol% or 6-8 w / w%,

[0372] - Component D is 11-14 mol% or 18-22 w / w%,

[0373] - Component E is 14.5-17.5 mol% or 16-19 w / w%, and

[0374] - Component F is 25-30 mol% or 15-19 w / w%.

[0375] 5. The mixture according to aspect 1, which consists essentially of 3-FL, LNFP-I, 2'-FL, LNDFH-I and DFL, preferably wherein

[0376] Relative to the sum of components A, B, C, D and E,

[0377] - component D is 10-17.5 mol% or 16-25.5 w / w%, and / or

[0378] - component E is 12-23.5 mol% or 13-22 w / w%, and / or

[0379] - Component D+E is 22-41 mol% or 29-47.5 w / w%,

[0380] or

[0381] Relative to the sum of components A, B, C, D and E,

[0382] - component D is 3.5-29.5 mol% or 6-39 w / w%, and / or

[0383] - Component E is 9-32 mol% or 7.5-34.5 w / w%,

[0384] Provided that component D+E is 31.5-41 mol% or 36.5-49 w / w%.

[0385] 6. The mixture according to aspect 5, wherein

[0386] - there is more component B than component C in the mixture

[0387] or

[0388] - Component A+B+C is 57-70 mol% or 51-64.5 w / w%, preferably component A is 29-35.5 mol%.

[0389] 7. The mixture according to aspect 1, 5 or 6, wherein

[0390] - Component A is 29-33 mol% or 20-24.5 w / w%,

[0391] - Component B is 18-21.5 mol% or 23-25.5 w / w%,

[0392] - Component C is 10-12.5 mol% or 7.5-9.5 w / w%,

[0393] - Component D is 15-17.5 mol% or 22-25.5 w / w%,

[0394] - Component E is 20.5-23.5 mol% or 19.5-22 w / w%.

[0395] 8. A composition comprising the mixture according to any one of the preceding aspects, preferably the composition further comprises a probiotic, more preferably Lactobacillus rhamnosus, in particular Lactobacillus rhamnosus LGG DSM 33156.

[0396] 9. A method for obtaining the mixture according to any one of aspects 1 to 7, wherein the method comprises reacting 3-FL, LNFP-I and 2'-FL in the presence of α1-3 / 4-transfucosidase to produce a reaction mixture, and then removing the α1-3 / 4-transfucosidase and optionally lactose from the reaction mixture, preferably, wherein the α1-3 / 4-transfucosidase comprises the amino acid sequence of SEQ ID no.1 having the following mutations: W135F-A174N-N274A-E413R, or consists of it.

[0397] 10. A mixture or composition according to any one of aspects 1 to 8, for use as a medicament, preferably

[0398] -wherein the human microbiome is modulated,

[0399] or

[0400] - wherein the use is to treat and / or reduce the risk of a wide range of bacterial or viral infections in humans.

[0401] 11. Use of a mixture according to any one of aspects 1 to 8 for enhancing the viability of a probiotic, preferably Lactobacillus rhamnosus, more preferably Lactobacillus rhamnosus LGG DSM 33156, in an aqueous composition.

[0402] Example

[0403] Example 1

[0404] A solution of 3-FL (4.278 kg, 8.76 mol, corrected by measurement), LNFP-I (3.807 kg, 4.46 mol, corrected by measurement) and 2'-FL (2.072 kg, 4.24 mol, corrected by measurement) in water (17.5 kg) was transferred to a 20-liter fermenter. The pH of the solution was adjusted to 6.0 with 7% HCl solution. A lyophilized powder (11.6 g) of the W135F-A174N-N274A-E413R mutant of Bifidobacterium longum subsp. infantis ATCC15697 (amino acid numbering according to SEQ ID no. 1, see WO 2016 / 063261) was dissolved in water (100 g) and added to the above solution, and the reaction mixture was then stirred at 25° C. for 18 hours, then heated to 55° C. over 4 hours, and stirred at this temperature for 2 hours. Subsequently, the pH value of the mixture was set to 5.0 with 7% HCl solution, the mixture was heated to 70°C over 1 hour and kept at this temperature for 2 hours. The mixture was then allowed to cool to 15°C while the enzyme precipitated. The obtained suspension was then subjected to ultrafiltration (15 kDa ceramic membrane, 0.5 m 2 , transmembrane pressure: 5.8-5.9 bar, temperature: 19-24° C.), collecting 28.05 kg of permeate. The permeate was microfiltered (0.2 μm PES membrane) and freeze-dried to obtain 10.57 g of powder (sugars determined by HPLC: 95 w / w%, of which 3-FL was 16 w / w%, LNFP-I was 19 w / w%, 2'-FL was 7 w / w%, LNDFH-I was 18 w / w%, DFL was 17 w / w% and lactose was 18 w / w%; protein content determined by Bradford test: <6 ppm).

[0405] Example 2

[0406] According to the protocol disclosed in Example 1, the following compositions were obtained at different donor-acceptor ratios:

[0407]

[0408] Example 3

[0409] Lyophilized Lactobacillus rhamnosus (Probio A solution of 1,000 μg / ml (2'-FL, 2'-FL / DFL in a weight ratio of 6.1:1 and a mixture of HMO from Example 1, 5 w / v%) in sterile phosphate buffered saline (PBS, pH = 3) was heated to 37°C and mixed vigorously for about 30 seconds until no lumps were visible. The control contained only the probiotics. The tubes were incubated at 37°C for 3 hours. The samples were further diluted and 100 μl were plated on MRS agar plates in duplicate and incubated at 37°C in an anaerobic chamber for 48 hours. The results are expressed as mean values ​​and the standard deviation of colony forming units (CFU) per ml was calculated from the LGG colonies on the agar plates ( Figure 1 ).

[0410] Freeze-dried LGG dissolved with the HMO mixture according to the invention showed significantly enhanced regeneration and viability after incubation for 3 hours at 37°C, pH 3.0, as assessed by colony counts, compared to the control or some of the HMO mixture components. The data suggest that the reduction in regeneration and viability of Lactobacillus rhamnosus after exposure to low pH conditions (e.g., in the stomach or in acidic beverages) can be prevented in the presence of HMOs.

Claims

1. A mixture comprising, consisting essentially of, or consisting of 3-FL as component A, LNFP-I as component B, 2'-FL as component C, LNDFH-I as component D, DFL as component E, and optionally lactose as component F.

2. The mixture of claim 1, comprising, consisting essentially of, or consisting of 3-FL, LNFP-I, 2'-FL, LNDFH-I, DFL, and lactose.

3. The mixture according to claim 1 or 2, wherein relative to the sum of components A, B, C, D, E and F - component D is 8-13 mol% or 14-22 w / w%, and / or - component E is 9.5-17.5 mol% or 11.5-19 w / w%, and / or - Component D+E is 17.5-30.5 mol% or 25.5-41 w / w%.

4. The mixture according to claim 1 or 2, wherein relative to the sum of components A, B, C, D, E and F, - component D is 2-22 mol% or 5-34 w / w%, or - Component E is 7-24 mol% or 6-29 w / w%, Provided that component D+E is 23.5-33 mol% or 30.5-43 w / w%.

5. A mixture according to claim 3, wherein component A+B+C is 41.5-67 mol% or 41-66 w / w%, preferably wherein component B is present in excess of component C.

6. The mixture according to claim 4, wherein the components A+B+C are 40.5-54.5 mol% or 41-54 w / w%, preferably wherein the component A is 21-27 mol%.

7. A mixture according to any one of the preceding claims, wherein - Component A is 19-24.5 mol% or 15-20 w / w%, - Component B is 12.5-15.5 mol% or 19-22 w / w%, - Component C is 7-10 mol% or 6-8 w / w%, - Component D is 11-14 mol% or 18-22 w / w%, - Component E is 14.5-17.5 mol% or 16-19 w / w%, and - Component F is 25-30 mol% or 15-19 w / w%.

8. The mixture of claim 1 comprising, consisting essentially of, or consisting of 3-FL, LNFP-I, 2'-FL, LNDFH-I, and DFL.

9. The mixture according to claim 1 or 8, wherein relative to the sum of components A, B, C, D and E - component D is 10-17.5 mol% or 16-25.5 w / w%, and / or - component E is 12-23.5 mol% or 13-22 w / w%, and / or - Component D+E is 22-41 mol% or 29-47.5 w / w%.

10. The mixture according to claim 1 or 8, wherein relative to the sum of components A, B, C, D and E, - component D is 3.5-29.5 mol% or 6-39 w / w%, and / or - Component E is 9-32 mol% or 7.5-34.5 w / w%, Provided that component D+E is 31.5-41 mol% or 36.5-49 w / w%.

11. The mixture of claim 9, wherein more component B is present than component C in the mixture.

12. The mixture according to claim 10, wherein the components A+B+C are 57-70 mol% or 51-64.5 w / w%, preferably wherein the component A is 29-35.5 mol%.

13. A mixture according to any one of claims 1 and 8 to 12, wherein - Component A is 29-33 mol% or 20-24.5 w / w%, - Component B is 18-21.5 mol% or 23-25.5 w / w%, - Component C is 10-12.5 mol% or 7.5-9.5 w / w%, - Component D is 15-17.5 mol% or 22-25.5 w / w%, - Component E is 20.5-23.5 mol% or 19.5-22 w / w%.

14. A composition comprising a mixture according to any one of the preceding claims.

15. The composition according to claim 14, further comprising a probiotic, preferably Lactobacillus rhamnosus (L. rhamnosus), more preferably Lactobacillus rhamnosus LGG DSM 33156.

16. The composition according to claim 14 or 15, which is a nutritional composition, a pharmaceutical composition or a cosmetic composition.

17. A method for obtaining a mixture according to any one of claims 1 to 13, wherein the method comprises reacting 3-FL, LNFP-I and 2'-FL in the presence of α1-3 / 4-transfucosidase to produce a reaction mixture, and then removing the α1-3 / 4-transfucosidase and optionally lactose from the reaction mixture.

18. The method according to claim 17, wherein the α1-3 / 4-transfucosidase comprises or consists of the amino acid sequence of SEQ ID no. 1 having the following mutations: W135F-A174N-N274A-E413R.

19. The method of claim 17 or 18, which is performed in the absence of substantially buffer.

20. Use of a mixture or composition according to any one of claims 1 to 16 as a medicament.

21. Use of the mixture or use of the composition according to claim 20, wherein the human microbiome is modulated.

22. Use of a mixture or use of a composition according to claim 20, wherein the use is to treat and / or reduce the risk of a wide range of bacterial or viral infections in humans.

23. Non-medical use of a mixture according to any one of claims 1 to 13 or a nutritional or cosmetic composition according to any one of claims 14 to 16 for modulating the microbiome of the human body.

24. Use of a mixture according to any one of claims 1 to 13 for enhancing the viability of a probiotic, preferably Lactobacillus rhamnosus, more preferably Lactobacillus rhamnosus LGG DSM 33156, in an aqueous composition.

25. Use of a mixture according to any one of claims 1 to 13 for cosmetic applications.

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