Mixture of fucosylated HMOs

FIELD OF THE INVENTION

The present invention relates to a mixture of human milk oligosaccharides ("HMOs") comprising five fucosylated HMOs, as well as to an enzymatic process for making said mixture, wherein said synthesis comprises an a1-3/4-transfucosidase enzymatic reaction between HMO donors and acceptors.

BACKGROUND

HMOs have become the subject of much interest in recent years due to their roles in numerous biological processes occurring in the human organism. Mammalian milk contains at least 130 of these complex oligosaccharides (see Urashima et al: Milk Oligosaccharides. Nova Science Publisher (2011); or Chen, Adv. Carbohydr. Chem. Biochem. 72, 113 (2015)).

Evidence is accumulating that the resident community of microbes, called the microbiome, in the human digestive tract plays a major role in health and disease. When the normal composition of the microbiome is thrown off balance, the human host can suffer consequences. Recent research has implicated microbiome imbalances in disorders as diverse as cancer, obesity, inflammatory bowel disease, psoriasis, asthma, and possibly even autism. HMOs are believed to positively modulate the microbiome, and they are of increasing interest for this purpose. However, the remarkable diversity of HMOs, coupled with their lack of availability, especially of the more complex HMOs, has hampered studies of the specific functions of individual HMOs and thus limited the exploration of their full potential for use in promoting human health.

The natural source of HMOs is mammalian milk, which contains mostly water, together with 55- 70 g/l lactose, 24-59 g/l lipids, ca. 13 g/l proteins, 5-15 g/l HMOs and ca. 1.5 g/l minerals.

Efforts to develop processes for industrial scale production of HMOs have increased significantly in the last years. Processes have been designed for producing HMOs by microbial fermentation, enzymatic processes, chemical syntheses, or combinations of these technologies.

In particular, fermentation in E. coli has been successfully employed for large scale industrial production of HMOs. WO 01/04341 describes how to make core human milk oligosaccharides optionally substituted by fucose using genetically modified E. coli.

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 a genetically modified E. coli. In an effort to be able to synthetically produce mixtures of HMOs, without having to synthesize all of the component oligosaccharides of the mixture, enzymatic processes for enzymatic synthesis of HMO oligosaccharide mixtures have been developed and are described in WO 2012/156897 and WO 2012/156898. As a further development, WO 2016/063261 and WO 2016/063262 describe optimized enzymes and enzymatic processes for the generation of fucosylated HMOs. Such processes have provided reaction mixtures containing a plurality of different HMO oligosaccharides.

Still, these processes include the use of highly specialized enzymes which in turn brings on the need for sensitive stoichiometry and reaction conditions, as well as the need for terminating reactions at the point of desired equilibrium in the reactions and the removal of secondary unwanted metabolites.

There is thus still a clear need for further specific HMOs or combinations of HMOs to modulate a microbiome in a desired manner, so as to address specific human health issues, as well as for simplified and more precise methods for producing them.

SUMMARY OF THE INVENTION

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

The second aspect of the invention relates to a composition comprising the mixture consisting of or consisting essentially of 2’-FL, 3-FL, LNFP-I, DFL, LNDFH-I and optionally lactose. The composition can be a nutritional, a pharmaceutical or a cosmetic composition.

The third aspect of the invention relates to a process for the enzymatic synthesis of the above mixture of HMOs, preferably under an essentially buffer-free condition. Said enzymatic synthesis comprises an a1-3/4-transfucosidase enzymatic reaction between one fucosylated donor HMO and two fucosylated acceptor HMOs. The enzymatic synthesis for preparing a mixture of HMOs according of the present invention comprises the steps of reacting a component A, a component B and a component C in the presence of an a1-3/4-transfucosidase to produce a reaction mixture and of removing the a1-3/4-transfucosidase and optionally lactose from the reaction mixture. The present invention in consequence also relates to a mixture consisting of or consisting essentially of 2’-FL, 3-FL, LNFP-I, DFL, LNDFH-I and optionally lactose obtained or obtainable by the presently disclosed enzymatic synthesis.

A special feature of said enzymatic synthesis according to the present invention is that it may be performed under essentially buffer-free conditions. Typically, the enzymatic synthesis according to the present invention is performed 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.

The a1-3/4-transfucosidase used in the enzymatic synthesis according to the present invention preferably lacks hydrolytic activity, or at least has a significantly reduced hydrolytic activity.

In one aspect of the present invention, the enzymatic synthesis according to the present invention further comprises a removal step which contains a step for deactivating the a1-3/4- transfucosidase. Said inactivation step can comprise heating the reaction solution to temperatures in the range of 60 °C to 100 °C, for a period of time in the range of 5 minutes to 180 minutes.

In the enzymatic synthesis according to the present invention, the a1-3/4-transfucosidase is removed by e.g. microfiltration, nanofiltration, centrifugation or ultracentrifugation. Said enzymatic synthesis can comprise more than one step for the removal of the a1-3/4- transfucosidase. One step can consist of removing the a1-3/4-transfucosidase by powdered activated charcoal in batch mode. Alternatively, the step can consist of removing the a1-3/4- transfucosidase by powdered activated charcoal in column mode.

A further aspect of the invention relates to a mixture consisting of or consisting essentially of 2’- FL, 3-FL, LNFP-I, DFL, LNDFH-I and optionally lactose for the use in, or a composition comprising the mixture consisting of or consisting essentially of 2’-FL, 3-FL, LNFP-I, DFL, LNDFH-I and optionally lactose for the use in treating, preventing and/or ameliorating a disease and/or condition related to microbiome imbalance.

A further aspect of the 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 enhancing the viability of probiotics in aqueous solution.

A further aspect of the invention relates to a use of a mixture consisting of or consisting essentially of 2’-FL, 3-FL, LNFP-I, DFL, LNDFH-I and optionally lactose, or a composition comprising the mixture consisting of or consisting essentially of 2’-FL, 3-FL, LNFP-I, DFL, LNDFH-I and optionally lactose, in the dietary management of a human or in a cosmetic application.

BRIEF DESCRIPTION OF THE FIGURE

The invention will be described in further detail hereinafter with reference to the accompanying Figure 1 which shows colony counting of L. rhamnosus after 3 h incubation with or without HMOs at 37 °C at pH 3.0. DETAILED DESCRIPTION

Generally, all terms used herein are to be interpreted according to their ordinary meaning in the technical field, and applicable to all aspects and embodiments of the invention, unless explicitly defined or stated otherwise.

All references to "a/an/the [sequence, enzyme, step, etc.]" are to be interpreted openly as referring to at least one instance of said sequence, enzyme, step, etc., unless explicitly stated otherwise.

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

The word “comprising” is used in the sense of “including” rather than in to mean “consisting of”.

In the context of the invention, the term “oligosaccharide” means a saccharide polymer containing a number of monosaccharide units attached together via interglycosidic linkages. Preferable oligosaccharides of the invention are human milk oligosaccharides (HMOs).

The term “human milk oligosaccharide" or "HMO" in the present context means a complex carbohydrate found in human breast milk (for reference, see Urashima et al.: Milk Oligosaccharides. Nova Science Publisher (2011); or Chen, Adv. Carbohydr. Chem. Biochem. 72, 113 (2015)).

Specifically, 2’-FL is 2’-fucosyllactose (Fuca1-2Gaipi-4Glc), 3-FL is 3-fucosyllactose (Gaipi- 4[Fuca1-3]Glc), DFL is difucosyllactose (Fuca1-2Gaipi-4[Fuca1-3]Glc), LNFP-I is lacto-N- fucopentaose I (Fuca1-2Gaipi-3GlcNAcpi-3Gaipi-4Glc) and LNDFH-I is lacto-N- difucohexaose (Fuca1-2Gaipi-3[Fuca1-4]GlcNAcpi-3Gaipi-4Glc). In the above structures, Fuc means L-fucopyranosyl, Gal means D-galactopyranosyl, Glc means D-glucopyranose and GIcNAc means D-(2-acetylamino)-2-deoxy-glucopyranosyl unit. In the context of the present invention, lactose is not regarded as an HMO species.

In the context of the invention, the term “donor” relates to a fucosylated saccharide, wherein an a1-3-fucosyl is transferred, through an enzyme catalysed reaction, onto another saccharide, whereafter the produced product is an a1-3-fucosyl or a 1-4-fucosyl containing saccharide. The term “acceptor” relates to the saccharide onto which the fucosyl group from the donor is transferred.

“Dietary management" means exclusive or partial feeding of patients who, because of a disease, disorder or medical condition are suffering from: either have a limited, impaired or disturbed capacity to take, digest, absorb, metabolise or excrete ordinary food or certain nutrients contained therein, or metabolites, or have other medically-determined nutrient requirements

(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).

"Microbiota", "microflora” and "microbiome" mean a community of living microorganisms that typically inhabits a bodily organ or part, particularly the gastro-intestinal organs of humans. The most dominant members of the gastrointestinal microbiota include microorganisms of the phyla of Firmicutes, Bacteroidetes, Actino bacteria, Proteobacteria, Synergistetes, Verrucomicrobia, Fusobacteria, and Euryarchaeota, at genus level Bacteroides, Faecalibacterium, Bifidobacterium, Roseburia, Alistipes, Collinsella, Blautia, Coprococcus, Ruminococcus, Eubacterium and Dorea, at species level Bacteroides uniformis, Alistipes putredinis, Parabacteroides merdae, Ruminococcus bromii, 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 the mucosa- associated microbiota, which is located in or attached to the mucous layer covering the epithelium of the gastrointestinal tract, and luminal-associated microbiota, which is found in the lumen of the gastrointestinal tract.

“Modulating of microbiome” means exerting a modifying or controlling influence on microbiota, for example an influence leading to an increase in the indigenous intestinal abundance of Bifidobacterium, Barnesiella, Faecalibacterium and/or butyrate producing bacteria. In another example, the influence may lead to a reduction of the intestinal abundance of Ruminococcus gnavus and/or Proteobacteria. “Proteobacteria” are a phylum of Gram-negative bacteria and include a wide variety of pathogenic bacteria, such as Escherichia, Salmonella, Vibrio, Helicobacter, Yersinia and many other notable genera.

"Oral administration" means any conventional form for the delivery of a composition to a human through the mouth.

“Preventive treatment” or “prevention” means treatment given or action taken to diminish the risk of onset or recurrence of a disease.

“Treat” means to address a medical condition or disease with the objective of improving or stabilising an outcome in the person being treated or addressing an underlying nutritional need. Treat therefore includes the dietary or nutritional management of the medical condition or disease by addressing nutritional needs of the person being treated. “Treating” and “treatment” have grammatically corresponding meanings. All prior teachings acknowledged above are hereby incorporated by reference. No acknowledgement of any prior published document herein should be taken to be an admission or representation that the teaching thereof was common general knowledge at the date hereof.

The novel HMO mixture

In accordance with the present invention, it has surprisingly been discovered that a novel mixture of 5 fucosylated HMOs can be prepared by an enzymatic synthesis using one HMO starting donor and two HMO starting acceptors. The reaction can preferably be conducted under essentially buffer-free conditions. Also preferably, the enzyme is of a limited or no hydrolytic activity.

The novel mixture consisting or consisting essentially of 5 fucosylated HMOs has a unique novel combination of properties and biological activities. Specifically, the mixture can be used for modulation of the microbiome of a human, such as to increase Bifidobacterium abundance and Barnesiella abundance in the microbiome of a human. Furthermore, the mixture can reduce Firmicutes abundance in the human microbiome, especially Clostridia. The mixture can also be used to treat and/or reduce the risk of a broad range of human bacterial and viral infections as well as for. The novel mixture of HMOs described herein is anti-infective. Furthermore, the mixture, when combined with a probiotic, substantially enhance the viability of that probiotic in aqueous solution.

One aspect of the present invention relates to a mixture consisting or consisting essentially of: a) a component A which is 3-FL, b) a component B which is LNFP-I, c) a component C, which is 2’-FL, d) a component D, which is LNDFH-I, e) a component E, which is DFL, and optionally f) a component F, which is lactose.

Accordingly, one embodiment of the invention is a mixture consisting or consisting essentially of: a) a component A which is 3-FL, b) a component B which is LNFP-I, c) a component C, which is 2’-FL, d) a component D, which is LNDFH-I, and e) a component E, which is DFL.

Another embodiment of the invention is a mixture of consisting or consisting essentially of: a) a component A which is 3-FL, b) a component B which is LNFP-I, c) a component C, which is 2’-FL, d) a component D, which is LNDFH-I, e) a component E, which is DFL, and f) a component F, which is lactose.

An enzymatic synthesis for producing the mixture of HMOs according to the invention

In the following paragraphs, the expression “may carry” is equivalent with the expression “optionally carries”, and the expression “can be substituted” is equivalent with the expression “is optionally substituted”.

The above-described novel mixture of fucosylated HMOs is prepared by an enzymatic synthesis comprising a fucosyl HMO donor which is component A (3-FL), two fucosyl HMO acceptors which are component B (LNFP-I) and component C (2’-FL), and a transfucosidase, preferably under essentially buffer-free conditions. Also preferably, in said reaction, the enzyme has a limited or no hydrolytic (that is fucosidase) activity.

Accordingly, this invention also relates to a process for making a mixture of HMOs consisting or consisting essentially of a component A which is 3-FL, a component B which is LNFP-I, a component C, which is 2’-FL, a component D, which is LNDFH-I, a component E, which is DFL, and a component F, which is lactose, by reacting components A, B and C in the presence of an a1-3/4-transfucosidase to produce a reaction mixture containing the components A, B, C, D, E and lactose, and then removing the a1-3/4-transfucosidase from the reaction mixture. The a1- 3/4-transfucosidase can be removed in a conventional manner, e.g. by denaturing the reaction mixture followed by its centrifugation or ultrafiltration.

Also, an aspect of this invention relates to a process for obtaining a mixture of HMOs consisting or consisting essentially of a component A which is 3-FL, a component B which is LNFP-I, a component C, which is 2’-FL, a component D, which is LNDFH-I and a component E, which is DFL, comprising the steps of reacting components A, B and C in the presence of an a1-3/4- transfucosidase to produce a reaction mixture containing the components A, B, C, D, E and lactose, and then removing lactose and the a1-3/4 transfucosidase from the reaction mixture. The a1-3/4-transfucosidase can be removed e.g. by denaturing it followed by centrifugation or ultrafiltration. The lactose can be separated from the components A, B, C, D and E e.g. by cascade ultra- and/or nanofiltration, or the lactose can first be treated with lactase to degrade it to glucose and galactose which can then be separated from the components A, B, C, D and E by ultra- and/or nanofiltration.

The suitable enzyme (protein) to carry out the above enzymatic reaction is an a1-3/4-fucosidase or an a1-3/4-transfucosidase. These enzymes have the ability to transfer a fucosyl residue from a fucosyl donor, wherein the fucosyl residue is attached with an a1-3-linkage, to an acceptor. Specifically, if the acceptor is an HMO, the fucosyl residue is transferred to the 4-position of a GIcNAc in the HMO acceptor, or if either that position is not free or there is no GIcNAc in the HMO acceptor structure, to the 3-position of Glc.

The a1-3/4-(trans)fucosidase is a natural a1-3/4-L-fucosidase as classified according to EC 3.2.1.111 or its engineered mutant. All natural a1-3/4-L-fucosidase can act as a transfucosidase in a certain extent.

A preferred a1-3/4-(trans)fucosidase suitable to carry out the enzymatic reaction according to the invention is a a1-3/4-(trans)fucosidase which have at least 60 %, preferably at least 70 %, more preferably at least 80 %, particularly at least 90 %, identity with amino acid positions 56 to 345 of the a1-3/4-fucosidase from Bifidobacterium longum subsp. infantis ATCC 15697 as set forth in US patent 8361756 as protein of SEQ ID no. 18 (referred to as SEQ ID no. 1 in the present application).

In the enzymatic synthesis of the invention, the hydrolysis of the components can become significant due to increasing concentrations of the produced components of the reaction, wherein the a 3/4-fucosylated components are then hydrolysed into the non-a3/4-fucosylated components, lactose, 2’-FL and LNFP-I and free fucose. Thus, the a1-3/4-transfucosidase of the present invention preferably has a limited or no hydrolytic activity. The limited or lacking hydrolytic activity of the a1-3/4-transfucosidase enables longer reaction durations than what would elsewise be possible.

Thus, an even preferred a1-3/4-transfucosidase for producing the HMO mixtures of this invention is a a1-3/4-transfucosidase that lacks hydrolytic activity, or at least has significantly reduced hydrolytic activity.

Such an a1-3/4-transfucosidase can be made by altering the amino acid sequence at one or more amino acid positions, so that the mutated amino acid sequence results in improved transfucosidase activity and/or reduced hydrolytic activity. In accordance with this invention, the a1-3/4-transfucosidase a) has been mutated at least at one or more of the following amino acid positions of SEQ ID no.1: 134, 135, 168, 170, 174, 216, 221 , 236, 237, 244, 245, 282 and 413, preferably at least at one or more of the following amino acid positions: 134, 135, 174, 216, 221 , 282 and 413; and thereby b) provides a conversion rate of at least 20 % up to 70 %, such as at least 35 %, or such as at least 40 %, or such a least 50 %, for the enzymatic reaction of the present invention.

Suitably, the mutant fucosidases used in the invention are non-natural fucosidases, that is, they are not made in nature or naturally-occurring, but are made as a result of chemical synthesis, genetic engineering or similar methods in the laboratory, resulting in synthetic mutant fucosidases.

Preferably, the mutated a1-3/4-transfucosidase comprises or consists of the polypeptide of SEQ ID no.1, and an amino acid mutation at least at one or more of the following amino acid positions: 134, 135, 174, 216, 221 and 282.

More preferably, the a1-3/4-transfucosidase of this first aspect comprises or consists of the polypeptide of SEQ ID no.1, and an amino acid mutation at least at amino acid position 174, and at one or more of the following amino acid positions: 134, 135, 170, 216, 221, 236, 237, 241 , 244, 245 and 282, preferably at one or more of the following amino acid positions: 134, 135, 216, 221 and 282.

Also more preferably, the a1-3/4-transfucosidase comprises or consists of the polypeptide of SEQ ID no.1, and an amino acid mutation at least at amino acid position 135, and at one or more of the following amino acid positions: 134, 170, 174, 216, 221, 236, 237, 241, 244, 245 and 282, preferably at one or more of the following amino acid positions: 134, 135, 216, 221 and 282.

Even more preferably, the a1-3/4-transfucosidase comprises or consists of the polypeptide of SEQ ID no.1, and an amino acid mutation at least at amino acid positions 135 and 174, and at one or more of the following amino acid positions: 134, 170, 216, 221, 236, 237, 241 and 282, preferably at one or more of the following amino acid positions: 134, 216, 221 and 282.

Optionally, the a1-3/4-transfucosidase comprises or consists of the polypeptide of SEQ ID no.1, and an amino acid mutation at least at amino acid positions 135 and/or 174, and at one or more of the following amino acid positions: 134, 170, 216, 221, 236, 237, 241 and 282, preferably at one or more of the following amino acid positions: 134, 216, 221 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.

Preferably, the a1-3/4-transfucosidase comprises, more preferably consists of, the sequence of SEQ ID NO 1 having mutations: at amino acid position 135 and/or 174, and at least at an amino acid position selected from 168, 237 and 413.

In this aspect, at position 135 Trp (W) is preferably substituted by Ala, Asp, Asn, Glu, Gin, His, Phe, Leu, Lys, Vai or Tyr, more preferably Phe or Tyr; at position 168 Ser (S) is preferably substituted by Glu (E); at position 174, Ala (A) is preferably substituted by Arg, Asn, Cys, Glu, lie, His, Leu, Lys, Met, Phe, Trp, Tyr or Vai, more preferably Asn, His or Phe; at position 237, Glu (E) is preferably substituted by His (H); and at position 413, Glu (E) is substituted by Arg (R).

Even more preferably, the a1-3/4-transfucosidase comprises, more preferably consists of, the sequence of SEQ ID no.1 as described above having mutations:

- at amino acid position 174,

- at an amino acid position 135 or 168, and

- at amino acid position 413

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

The above combination of mutations imparts not only a further improved transfucosidase synthetic performance to the mutated enzyme but a further enhanced stability, particularly temperature stability while maintaining further improved transfucosidase synthetic performance. The mutations at 135, 165, 174, 232, 258, 260 and 274 are preferably the following:

- at position 135, as W135F or W135Y,

- at position 165, as P165E,

- at position 174, as A174F, A174H or A174N,

- at position 232, as R232A,

- at position 258, as Q258R,

- at position 260, as D260P,

- at position 274, as N274A.

Also preferably, the mutated a1-3/4-transfucosidase that comprises or consists of the polypeptide of SEQ ID no.1, and a mutation of at least at amino acid position 174 or 282, preferably at least at both amino acid positions, provides significantly or completely suppressed 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, Vai (V) is preferably replaced by Arg (R), Glu (E), His (H) or Lys (K). The suppressed hydrolytic activity is beneficial because the mutated enzyme then does not significantly degrade the donor and/or the product by hydrolysis. As a result, the transfucosidase reaction is no longer kinetically controlled, and a much better synthesis/hydrolysis ratio (meaning a better synthetic performance) can be achieved. Mutation at one or both, preferably both, of the above amino acid positions can provide at least a 100- fold, preferably at least a 1000-fold, more preferably at least a 10000-fold reduced hydrolytic activity towards the fucosylated products.

Accordingly, the mutated a1-3/4-transfucosidase has a) the polypeptide of SEQ I D no.1 , and a mutation at least at amino acid position 174 or 282, and b) a significantly or completely suppressed hydrolytic activity, when compared to the protein according to SEQ ID no. 1.

In addition, according to a certain embodiment, the mutated a1-3/4-transfucosidase comprises or preferably consists of the polypeptide of SEQ ID no.1 , and a mutation of

- at least the amino acid position 174 or 282, and

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

The combination of mutations as disclosed above imparts not only a significantly reduced, preferably practically undetectable, hydrolysis of the fucosylated product but an enhanced stability, particularly temperature stability.

Therefore the mutated a1-3/4-transfucosidase has a) the polypeptide of SEQ ID no.1 with mutation at least at the amino acid at position 174 or 282, and at least at the amino acid position 165, 168, 232, 237, 258, 260 or 274, and b) significantly or completely suppressed hydrolytic activity, and/or c) enhanced stability, particularly temperature stability, when compared to the protein according to SEQ ID no. 1.

Preferably, the mutated a1-3/4-transfucosidase comprises or preferably consists of the amino acid sequence of SEQ ID no.1, a mutation of at least the amino acid at position 174 or 282, more preferably at position 174, Ala (A) is preferably replaced by Phe (F), Asn (N) or His (H) and/or at position 282, Vai (V) is preferably replaced by Arg (R), Glu (E), His (H) or Lys (K). A particularly preferred modified a1-3/4-transfucosidase comprises or consists of the amino acid sequence of SEQ ID no.1 with the following mutations: W135F-A174N-N274A-E413R, said amino acid numbering being according to SEQ ID no. 1.

In carrying out the process of this invention, particular relative concentrations of the component A donor, component B and C acceptors, the a1-3/4-transfucosidase, the aqueous solvent and the incubation buffer are not critical. In this regard, the process 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 min to 24 hours. The reaction is typically conducted so that an equilibrium is reached.

In the enzymatic reaction, component A is provided as fucosyl donor and components B and C are provided as fucosyl acceptors. In the course of the reaction, the a1-3/4-transfucosidase a fucosyl residue is transferred from the donor to both acceptors, resulting in thereby the generation of component D, component E and lactose. However, the newly generated component E can also serve as a fucosyl donor, which results in an a1-3/4-transfucosidase enzymatic reaction between component E and component B to generate component D and component C, or between component E and lactose to generate component A and component C, in further sub-equilibrium reactions.

More preferably, the a1-3/4-transfucosidase, advantageously the W135F-A174N-N274A-E413R mutant (the amino acid numbering being according to SEQ ID no. 1), is utilized in around 0.09- 0.11 w/w% based on the initial overall weight of components A, B and C. The amount of enzyme does not affect the final proportion of the HMOs in the obtained composition, it has an effect only to when the equilibrium is reached (higher enzyme concentration leads to a shorter time to reach the equilibrium).

Concerning the preparation of the mixture that consists or consists essentially of components A, B, C, D, E and optionally lactose, preferably the mixtures according to any one of the embodiments i) to iv) disclosed below, the enzymatic synthesis according to the invention is conducted in the presence of a suitable buffer, but preferably under essentially buffer-free conditions.

In light of the invention, a buffered solution is a solution containing a buffering agent, characterized in that the pH of the solution resists changes in free hydrogen ion concentration as a result of internal and/or environmental factors. Further, a buffered solution essentially maintains pH for a reaction system within a specific pH range, according to the used buffering agent. In addition, buffered solutions for enzymatic reactions are well-known to provide essential cofactors for enzymatically driven reactions, e.g. critical salts, as well as resisting changes in hydrogen ion concentration and conditions that stabilize proteins in general. In consequence, when conducting an enzymatic reaction, the person skilled in the art expects the need for introducing buffering agents and/or components to the reaction milieu that are normally considered to be essential for the enzyme to work properly.

Before further processing of the reaction products, though, these additional agents and/or components and/or their residual products, need to be removed from the reaction, which can be both time consuming and troublesome, adding unwanted costs to the production process.

Surprisingly, it was found that the enzymatic synthesis according to the present invention can be conducted under essentially buffer-free conditions, thus eliminating the necessity for providing and removing the buffering agents and/or components.

In one aspect the present invention the enzymatic synthesis described herein is conducted in purified water, wherein the purification step may comprise one or more of the purification processes selected from the group consisting of: reverse osmosis, filtration, ultrafiltration, sedimentation, chlorination, iodine addition, electrodialysis, boiling, desalination and flocculation.

In a presently preferred aspect of the invention, the enzymatic synthesis is conducted in water with a conductivity prior to use in the enzymatic synthesis below 10 pS/cm, such as below 5 pS/cm, such as below 1 pS/cm, such as below 0.1 pS/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.

In an enzymatic synthesis according to the present invention, essentially buffer-free conditions are conditions wherein no additional buffering agent(s) are added to the purified water.

In the present context, the term “essentially buffer-free”, also includes a solution wherein inorganic salts are added to purify the water of previously specified conductivity and purity ranges, to act as stabilizing agents for the enzyme, wherein the salts do not contain buffering capabilities. Examples of salts include common salts such as, but not limited to, sodium chloride (NaCI), calcium chloride (CaCh), magnesium chloride (MgCh), potassium chloride (KCI) and sodium sulphate (Na2SO4).

In an aspect of an enzymatic synthesis, according to the present invention, essentially buffer- free conditions may comprise an amount of salt in a range of 1 micromolar to 1 molar, in a preferred range such as 100 micromolar to 500 millimolar, or in an even more preferred range of 1 millimolar to 300 millimolar. In another aspect, the essentially buffer-free condition refers to the presence of trace ions in water, in a concertation resulting in a conductivity below 5 pS/cm.

In another aspect of the invention, the term “essentially buffer-free” also includes the use of tap water. Presence of ions in a solution, with or without addition of salts to the solution, can affect the pH of the solution, even at trace concentrations, resulting in a conductivity above 5 pS/cm, still without adding buffering capabilities to the essentially buffer-free conditions. Furthermore, addition of reactants to a solution under essentially buffer-free condition may also change the pH of the solution as well as the conductivity. Consequently, in the present context, the conditions after the addition of ions and/or reactants to the reaction solution are still considered essentially buffer-free.

In a preferred aspect of an enzymatic synthesis, according to the present invention, the synthesis is conducted in an essentially buffer-free solution wherein the pH is in the range of pH 4 to pH 7, preferably at a pH 5 to pH 6.

Before utilizing a mixture of HMOs according to the invention as a treatment or otherwise, removal of unwanted reaction components is necessary. Unwanted products of the final product are e.g. enzymes, such as the a1-3/4-transfucosidase, lactose, additional trace products and/or similar unwanted products.

Furthermore, an enzymatic synthesis according to the present invention can comprise one or more steps for deactivating and separating the a1-3/4-transfucosidase from the remaining reaction mixture.

The enzymatic synthesis are conducted in a reaction vessel, and in some cases, it can be advantageous to transfer the solution from the reaction vessel, which can be a process-tank or a plastic container or any other suitable vessel for conducting the synthesis in, into a second container, with e.g. pressure control, temperature control and/or control of a liquid and/or a gaseous environment. Thus, an enzymatic synthesis can be performed in a way in which the reaction vessel and the inactivation/deactivation vessel are the same, or it can be performed in a way in which the reaction vessel and the inactivation/deactivation vessel are not the same.

The deactivation step can involve heating to a temperature in the range of 60 °C to 100 °C for a time interval 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 deactivation step involves heating of the reaction mixture to a temperature in the range of 60 °C to 100 °C for a time of at least 1, 5, 10, 30 or 60 minutes.

After the one or more steps for deactivating the a1-3/4-transfucosidase from the remaining reaction mixture, an enzymatic synthesis of the present invention further comprises one or more steps for removing the deactivated a1-3/4-transfucosidase from the synthesized HMO mixture, thereby providing the mixture that consists or consists essentially of components A, B, C, D, E and lactose, preferably the mixtures according to any one of the embodiments i) and ii) disclosed below, including their preferred and more preferred embodiments.

In general, the a1-3/4-transfucosidase can be removed from the synthesized mixture of HMOs by conventional separation methods, such as filtration (nano-, ultra- or microfiltration), removal by centrifugation or ultracentrifugation, treating the reaction mixture with powdered activated charcoal in batch mode and/or in column mode and/or chromatographic methods, such as ion exchange, affinity chromatography, size exclusion chromatography, forward phase or reverse phase chromatography, preferably after the enzyme used for the synthesis has been denatured or deactivated. Methods for separation may be combined into a multi-step separation.

In one aspect of the invention, the reaction mixture, containing an a1-3/4-transfucosidase, is treated with activated charcoal, for example the reaction mixture is passed over a column packed with activated charcoal using a peristaltic flow, high pressure flow or gravity flow.

In other aspect of the invention, when utilizing ultrafiltration or microfiltration for the separation of an a1-3/4-transfucosidase from the reaction mixture, the separation may involve a previous step wherein the (inactivated) a1-3/4-transfucosidase has formed particles, such as aggregates, crystals and/or precipitates, which enables a more convenient separation by filtration. The pore size of the ultra- or microfiltration membrane is selected so that the carbohydrate components are allowed to pass through the membrane while ensuring the retention of the (inactivated) a1- 3/4-transfucosidase.

In other aspect of the invention, when utilizing ultracentrifugation or centrifugation for the separation of the a1-3/4-transfucosidase, the separation may involve a previous step wherein the (inactivated) a1-3/4-transfucosidase has formed particles, such as aggregates, crystals and/or precipitates, which enables a more convenient separation by (ultra)centrifugation.

The purification steps disclosed above result in a purified aqueous solution from which a solid mixture that consists or consists essentially of components A, B, C, D, E and lactose, preferably the mixtures according to any one of the embodiments i) and ii) disclosed below, including their preferred and more preferred embodiments, can be obtained after water removal, e.g. evaporation, spray-drying or freeze-drying.

To obtain the mixture that consists or consists essentially of components A, B, C, D and E, preferably the mixtures according to any one of the embodiments iii) and iv) disclosed below, including their preferred and more preferred embodiments, lactose should be separated from the components A, B, C, D and E. A suitable method for this is for example gel filtration, or membrane filtration using a membrane that ensures the retention of components A, B, C, D and E and allows lactose to pass. Another method is that before or after removing the a1 -3/4- transfucosidase from the reaction milieu as disclosed above but before water removal a p- galactosidase is added to degrade lactose into glucose and galactose, 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 the p-galactosidase can be performed before or after this membrane filtration using one of the steps disclosed for removal of the a1-3/4-transfucosidase.

Embodiments of the first aspect of the invention

Concerning the mixture that consists or consists essentially of components A, B, C, D, E and F, it is preferred that, relative to components A, B, C, D, E and F taken together, i) - 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

- components D+E are 17.5-30.5 mol% or 25.5-41 w/w%, or ii) - 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 components D+E are 23.5-33 mol% or 30.5-43 w/w%.

The mixture according to embodiment i) is typically obtainable when the donor (component A) to acceptor (components B and C) molar ratio ranges from around 3:1 to 1 :3, and wherein the acceptor components are around equimolar. The mixture according to embodiment ii) is typically obtainable when the donor (component A) to acceptor (components B and C) molar ratio is around equimolar, and wherein in the acceptor, the component B to component C molar ratio ranges from around 3.5:1 to 1:3.9.

Concerning embodiment i) disclosed above, it is preferred that the components A+B+C are 41.5-67 mol% or 41-66 w/w% relative to components A, B, C, D, E and F taken together, especially wherein more component B is present than component C.

In a more preferred embodiment i),

- component A is 52-58 mol% or 47-52 w/w%,

- component B is 4-6 mol% or 6.5-8.5 w/w%,

- component C is 1-2 mol% or 1-2 w/w%,

- component D is 8.5-10 mol% or 15.5-17.5 w/w%,

- component E is 10-11.5 mol% or 11.5-13 w/w%, and - component F is 17.5-20.5 mol% or 12-13.5 w/w% relative to components A, B, C, D, E and F taken together.

This mixture is obtainable when the donor (component A) to acceptor (components B and C) molar ratio is around 3:1, and wherein the acceptor components are around equimolar.

Also in a more preferred embodiment i),

- component A is 42-46 mol% or 36-40 w/w%,

- component B is 7-9 mol% or 11.5-13.5 w/w%,

- component C is 3-4 mol% or 2.5-3.5 w/w%,

- component D is 9-11 mol% or 16.5-19 w/w%,

- component E is 11-13.5 mol% or 13-16 w/w%, and

- component F is 20.5-23.5 mol% or 13-16 w/w% relative to components A, B, C, D, E and F taken together.

This mixture is obtainable when the donor (component A) to acceptor (components B and C) molar ratio is around 2:1, and wherein the acceptor components are around equimolar.

Also in a more preferred embodiment i),

- component A is 7.5-9.5 mol% or 5-7.5 w/w%,

- component B is 21-24 mol% or 30-33 w/w%,

- component C is 16-19 mol% or 12.5-15.5 w/w%,

- component D is 10-12 mol% or 16.5-19.5 w/w%,

- component E is 14-16.5 mol% or 14.5-17 w/w%, and

- component F is 23-27 mol% or 13-16 w/w% relative to components A, B, C, D, E and F taken together.

This mixture is obtainable when the donor (component A) to acceptor (components B and C) molar ratio is around 1:2, and wherein the acceptor components are around equimolar.

Also in a more preferred embodiment i),

- component A is 3-5.5 mol% or 2.5-4 w/w%,

- component B is 26-30 mol% or 36-40 w/w%,

- component C is 22.5-25.5 mol% or 17-20 w/w%, - component D is 8.5-11 mol% or 14-16.5 w/w%,

- component E is 11-14 mol% or 11-14 w/w%, and

- component F is 20-23 mol% or 11-13.5 w/w% relative to components A, B, C, D, E and F taken together.

This mixture is obtainable when the donor (component A) to acceptor (components B and C) molar ratio is around 1 :3, and wherein the acceptor components are around equimolar.

In an especially more preferred embodiment i),

- 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% relative to components A, B, C, D, E and F taken together.

This mixture is obtainable when the donor (component A) to acceptor (components B and C) molar ratio is around 1:1, and wherein the acceptor components are around equimolar.

Concerning embodiment ii) disclosed above, it is preferred that the components A+B+C are 40.5-54.5 mol% or 41-54 w/w% relative to components A, B, C, D, E and F taken together, especially wherein component A is 21-27 mol%.

In a more preferred embodiment ii),

- component A is 21.5-25.5 mol% or 16-19.5 w/w%,

- component B is 18-22 mol% or 24.5-28.5 w/w%,

- component C is 2-4 mol% or 1.5-3.5 w/w%,

- component D is 18-22 mol% or 30-34 w/w%,

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

- component F is 23-27 mol% or 13-15.5 w/w% relative to components A, B, C, D, E and F taken together. This mixture is obtainable when the donor (component A) to acceptor (components B and C) molar ratio is around equimolar, and wherein in the acceptor, the component B to component C molar ratio is around 3.5:1.

Also in a more preferred embodiment ii),

- component A is 21-24.5 mol% or 16.5-20 w/w%,

- component B is 15-18 mol% or 21-25 w/w%,

- component C is 5.5-7.5 mol% or 4-6 w/w%,

- component D is 13-16.5 mol% or 22-26 w/w%,

- component E is 12-15 mol% or 12.5-15.5 w/w%, and

- component F is 24-28 mol% or 14-17 w/w% relative to components A, B, C, D, E and F taken together.

This mixture is obtainable when the donor (component A) to acceptor (components B and C) molar ratio is around equimolar, and wherein in the acceptor, the component B to component C molar ratio is around 1.4:1.

Also in a more preferred embodiment ii),

- component A is 21-24.5 mol% or 17-20.5 w/w%,

- component B is 10.5-13.5 mol% or 16.5-20 w/w%,

- component C is 9-12 mol% or 8-10 w/w%,

- component D is 8-10 mol% or 14-17 w/w%,

- component E is 17.5-20.5 mol% or 19.5-23 w/w%, and

- component F is 24.5-28.5 mol% or 14.5-17.5 w/w% relative to components A, B, C, D, E and F taken together.

This mixture is obtainable when the donor (component A) to acceptor (components B and C) molar ratio is around equimolar, and wherein in the acceptor, the component B to component C molar ratio is around 1 :1.5.

Also in a more preferred embodiment ii),

- component A is 23-27 mol% or 21-25 w/w%,

- component B is 6-8 mol% or 10-12.5 w/w%,

- component C is 15-19.5 mol% or 14-16.5 w/w%, - component D is 2.5-4.5 mol% or 5.5-7.5 w/w%,

- component E is 21-24.5 mol% or 25-28.5 w/w%, and

- component F is 23.5-27.5 mol% or 16-19 w/w% relative to components A, B, C, D, E and F taken together.

This mixture is obtainable when the donor (component A) to acceptor (components B and C) molar ratio is around equimolar, and wherein in the acceptor, the component B to component C molar ratio is around 1:3.9.

In an especially more preferred embodiment ii),

- 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% relative to components A, B, C, D, E and F taken together.

This mixture is obtainable when both the donor (component A) to acceptor (components B and C) molar ratio and the component B to component C molar ratio are around equimolar.

Concerning the mixture that consists or consists essentially of components A, B, C, D and E, it is preferred that, relative to components A, B, C, D and E taken together, iii) - 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

- components D+E are 22-41 mol% or 29-47.5 w/w%, or iv) - 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 components D+E are 31.5-41 mol% or 36.5-49 w/w%.

The mixture according to embodiment iii) is typically obtainable when the donor (component A) to acceptor (components B and C) molar ratio ranges from around 3:1 to 1:3, wherein the acceptor components are around equimolar, and wherein lactose is substantially removed from the so-obtained composition. The mixture according to embodiment iv) is typically obtainable when the donor (component A) to acceptor (components B and C) molar ratio is around equimolar, wherein in the acceptor, the component B to component C molar ratio ranges from around 3.5:1 to 1:3.9, and wherein lactose is substantially removed from the so-obtained composition.

Concerning embodiment iii) disclosed above, it is preferred that more component B is present in the mixture than component C.

In a more preferred embodiment iii),

- component A is 65-71 mol% or 53.5-59.5 w/w%,

- component B is 5-7 mol% or 7.5-9.5 w/w%,

- component C is 1.5-2.5 mol% or 1-2 w/w%,

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

- component E is 12-14.5 mol% or 13-15.5 w/w% relative to components A, B, C, D and E taken together.

This mixture is obtainable when the donor (component A) to acceptor (components B and C) molar ratio is around 3:1, wherein the acceptor components are around equimolar, and wherein lactose is substantially removed from the so-obtained composition.

Also in a more preferred embodiment iii),

- component A is 54-58.5 mol% or 42-46.5 w/w%,

- component B is 9-11.5 mol% or 13-15.5 w/w%,

- component C is 4-5 mol% or 3-4 w/w%,

- component D is 11.5-14 mol% or 19-23 w/w%,

- component E is 15-18 mol% or 15-18.5 w/w% relative to components A, B, C, D and E taken together.

This mixture is obtainable when the donor (component A) to acceptor (components B and C) molar ratio is around 2:1, wherein the acceptor components are around equimolar, and wherein lactose is substantially removed from the so-obtained composition.

Also in a more preferred embodiment iii),

- component A is 10-12.5 mol% or 6.5-9 w/w%,

- component B is 28.5-32 mol% or 34.5-38.5 w/w%, - component C is 22-25 mol% or 15-17.5 w/w%,

- component D is 13.5-16 mol% or 19.5-22.5 w/w%,

- component E is 19-21.5 mol% or 17-19.5 w/w% relative to components A, B, C, D and E taken together.

This mixture is obtainable when the donor (component A) to acceptor (components B and C) molar ratio is around 1 :2, wherein the acceptor components are around equimolar, and wherein lactose is substantially removed from the so-obtained composition.

Also in a more preferred embodiment iii),

- component A is 4.5-6.5 mol% or 2.5-4.5 w/w%,

- component B is 34-38 mol% or 41.5-45.5 w/w%,

- component C is 29-32.5 mol% or 19-22.5 w/w%,

- component D is 11-13.5 mol% or 16-19 w/w%,

- component E is 14.5-17.5 mol% or 13-16 w/w% relative to components A, B, C, D and E taken together.

This mixture is obtainable when the donor (component A) to acceptor (components B and C) molar ratio is around 1 :3, wherein the acceptor components are around equimolar, and wherein lactose is substantially removed from the so-obtained composition.

In an especially more preferred embodiment iii),

- 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% relative to components A, B, C, D and E taken together.

This mixture is obtainable when the donor (component A) to acceptor (components B and C) molar ratio is around 1 :1, wherein the acceptor components are around equimolar, and wherein lactose is substantially removed from the so-obtained composition. Concerning embodiment iv) disclosed above, it is preferred that the components A+B+C are 57- 70 mol% or 51-64.5 w/w% relative to components A, B, C, D and E taken together, especially wherein component A is 29-35.5 mol%.

In a more preferred embodiment iv),

- component A is 29.5-33.5 mol% or 19-22.5 w/w%,

- component B is 25-28.5 mol% or 29-33 w/w%,

- component C is 3-5 mol% or 2-3.5 w/w%,

- component D is 26-29.5 mol% or 35-39 w/w%,

- component E is 9-11.5 mol% or 7.5-10 w/w% relative to components A, B, C, D and E taken together.

This mixture is obtainable when the donor (component A) to acceptor (components B and C) molar ratio is around equimolar, wherein in the acceptor, the component B to component C molar ratio is around 3.5:1 , and wherein lactose is substantially removed from the so-obtained composition.

Also in a more preferred embodiment iv),

- component A is 29-32.5 mol% or 20.5-23 w/w%,

- component B is 21-24.5 mol% or 26-29.5 w/w%,

- component C is 7.5-10 mol% or 5-7 w/w%,

- component D is 18-21.5 mol% or 27-29.5 w/w%,

- component E is 17-19.5 mol% or 15.5-18 w/w% relative to components A, B, C, D and E taken together.

This mixture is obtainable when the donor (component A) to acceptor (components B and C) molar ratio is around equimolar, wherein in the acceptor, the component B to component C molar ratio is around 1.4:1 , and wherein lactose is substantially removed from the so-obtained composition.

Also in a more preferred embodiment iv),

- component A is 29-33 mol% or 22-24.5 w/w%,

- component B is 15-18 mol% or 20.5-23 w/w%,

- component C is 13-15.5 mol% or 9.5-12 w/w%, - component D is 11-13.5 mol% or 17-20 w/w%,

- component E is 24.5-27.5 mol% or 24-27 w/w% relative to components A, B, C, D and E taken together.

This mixture is obtainable when the donor (component A) to acceptor (components B and C) molar ratio is around equimolar, wherein in the acceptor, the component B to component C molar ratio is around 1:1.5, and wherein lactose is substantially removed from the so-obtained composition.

Also in a more preferred embodiment iv),

- component A is 31.5-35.5 mol% or 26-29.5 w/w%,

- component B is 8.5-10.5 mol% or 12.5-15 w/w%,

- component C is 21-24 mol% or 17-20 w/w%,

- component D is 3.5-5.5 mol% or 6-9.5 w/w%,

- component E is 28-32 mol% or 30.5-34.5 w/w% relative to components A, B, C, D and E taken together.

This mixture is obtainable when the donor (component A) to acceptor (components B and C) molar ratio is around equimolar, wherein in the acceptor, the component B to component C molar ratio is around 1:3.9, and wherein lactose is substantially removed from the so-obtained composition.

In an especially more preferred embodiment iv),

- component A is 29-33 mol% or 21-24.5 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 23-25.5 w/w%,

- component E is 20.5-23.5 mol% or 19.5-22 w/w% relative to components A, B, C, D and E taken together.

This mixture is obtainable when both the donor (component A) to acceptor (components B and C) molar ratio and the component B to component C molar ratio are around equimolar, and wherein lactose is substantially removed from the so-obtained composition. Pharmaceutical, cosmetic and/or nutritional composition

A mixture according to the present invention consisting of or consisting essentially of 3-FL, LNFP-I, 2’-FL, LNDFH-I, DFL and optionally lactose can be formulated as a pharmaceutical, cosmetic and/or nutritional composition which may contain a pharmaceutically, cosmetic and/or nutritional acceptable carrier such as phosphate buffered saline solution, unbuffered saline solution, mixtures of ethanol in water, water and emulsions such as an oil/water or water/oil emulsion, as well as various wetting agents and/or excipients. The pharmaceutical, cosmetic and/or nutritional composition can also contain other materials that do not produce an adverse, allergic or otherwise unwanted reaction when administered to a patient.

Carriers and other materials comprised in the pharmaceutical, cosmetical and/or nutritional composition can include one or more of the following: solvents, dispersants, coatings, absorption promoting agents, controlled release agents and one or more inert excipients, such as starches, polyols, granulating agents, microcrystalline celluloses, diluents, lubricants, binders, and disintegrating agents. If desired, tablet dosages of the anti-infective mixtures can be coated by aqueous or non-aqueous techniques known to the skilled person.

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

Also, a pharmaceutical, cosmetic and/or nutritional composition according to the present invention can include binders, lubricants, inert diluents, flavouring agents, and humectants. A tablet, capsule, suppository or pellet containing the pharmaceutical, cosmetic and/or nutritional composition of the present invention can optionally be coated or formulated, so as to provide sustained, delayed or controlled release of the anti-infective mixture of HMOs.

The mixture of HMOs according to the invention may be supplemented with additional pharmaceutical agents, active pharmaceutical ingredients or agents having an effect to adverse health conditions of the patient whom the composition is administered.

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 solution, a medical food or food for special medical purposes, a nutritional supplement and the like. The nutritional composition can contain sources of protein, lipids and/or digestible carbohydrates and can be in powdered or liquid forms. The composition can be designed to be the sole source of nutrition or as a nutritional supplement. Suitable protein sources include milk proteins, soy protein, rice protein, pea protein and oat protein, or mixtures thereof. Milk proteins can be in the form of milk protein concentrates, milk protein isolates, whey protein or casein, or mixtures of both. The protein can be whole protein or hydrolysed protein, either partially hydrolysed or extensively hydrolysed. Hydrolysed protein offers the advantage of easier digestion which can be important for humans with inflamed or compromised Gl tracts. The protein can also be provided in the form of free amino acids. The protein can comprise about 5 % to about 30 % of the energy of the nutritional composition, normally about 10 % to 20 %. Ideally, the source of protein does not include excessive amounts of lactose.

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 a glutamine dipeptide and/or a glutamine enriched protein. Glutamine can be included due to the use of glutamine by enterocytes as an energy source. Threonine, serine and proline are important amino acids for the production of mucin. Mucin coats the Gl tract and can improve intestinal barrier function and mucosal healing. Cysteine is a major precursor of glutathione, which is key for the antioxidant defences of the body.

Suitable digestible carbohydrates include maltodextrin, hydrolysed or modified starch or corn starch, glucose polymers, corn syrup, corn syrup solids, high fructose corn syrup, rice-derived carbohydrates, pea-derived carbohydrates, potato-derived carbohydrates, tapioca, sucrose, glucose, fructose, sucrose, honey, sugar alcohols (e.g. maltitol, erythritol, sorbitol), or mixtures thereof. Preferably the composition is reduced in or free from added lactose or other FODMAP carbohydrates. Generally digestible carbohydrates provide about 35 % to about 55 % of the energy of the nutritional composition. A suitable digestible carbohydrate is a low dextrose equivalent (DE) maltodextrin.

Suitable lipids include medium chain triglycerides (MCT) and long chain triglycerides (LCT). Preferably the lipid is a mixture of MCTs and LCTs. For example, MCTs can comprise about 30 % to about 70 % by weight of the lipids, more specifically about 50 % to about 60 % by weight. MCTs offer the advantage of easier digestion which can be important for humans with inflamed or compromised Gl tracts. Generally, the lipids provide about 35 % to about 50 % of the energy of the nutritional composition. The lipids can contain essential fatty acids (omega-3 and omega- 6 fatty acids). Preferably these polyunsaturated fatty acids provide less than about 30 % of total energy of the lipid source.

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

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

The nutritional composition can also include a carotenoid such as lutein, lycopene, zeaxanthin, and beta-carotene. The total amount of carotenoid included can vary from about 0.001 pg/ml to about 10 pg/ml. Lutein can be included in an amount of from about 0.001 pg/ml to about 10 pg/ml, preferably from about 0.044 pg/ml to about 5 pg/ml of lutein. Lycopene can be included in an amount from about 0.001 pg/ml to about 10 pg/ml, preferably about 0.0185 pg/ml to about 5 pg/ml of lycopene. Beta-carotene can comprise from about 0.001 pg/ml to about 10 mg/ml, for example about 0.034 pg/ml to about 5 pg/ml of beta-carotene.

The nutritional composition preferably also contains reduced concentrations of sodium; for example, from about 300 mg/l to about 400 mg/l. The remaining electrolytes can be present in concentrations set to meet needs without providing an undue renal solute burden on kidney 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.

The nutritional composition can also contain various other conventional ingredients such as preservatives, emulsifying agents, thickening agents, buffers, fibres and prebiotics (e.g. fructooligosaccharides, galactooligosaccharides), probiotics (e.g. B. animalis subsp. lactis BB- 12, B. lactis HN019, B. lactis Bi07, B. infantis ATCC 15697, L. rhamnosus GG, L. rhamnosus LGG DSM 33156, L. rhamnosus HNOOI, L. acidophilus LA-5, L. acidophilus NCFM, L. fermentum CECT5716, B. longum BB536, B. longum AH1205, B. longum AH1206, B. breve M- 16V, L. reuteri ATCC 55730, L. reuteri ATCC PTA-6485, L. reuteri DSM 17938), antioxidant/anti-inflammatory compounds including tocopherols, carotenoids, ascorbate/vitamin C, ascorbyl palmitate, polyphenols, glutathione, and superoxide dismutase (melon), other bioactive factors (e.g. growth hormones, cytokines, TFG-P), colorants, flavours, and stabilisers, lubricants, and so forth. The nutritional composition can be formulated as a soluble powder, a liquid concentrate, or a ready-to-use formulation. The composition can be fed to a human in need via a nasogastric tube or orally. Various flavours and other additives can also be present.

The nutritional compositions can be prepared by any commonly used manufacturing techniques for preparing nutritional compositions in solid or liquid form. For example, the composition can be prepared by combining various feed solutions. A protein-in-fat feed solution can be prepared by heating and mixing the lipid source and then adding an emulsifier (e.g. lecithin), fat soluble vitamins, and at least a portion of the protein source while heating and stirring. A carbohydrate feed solution is then prepared by adding minerals, trace and ultra-trace minerals, thickening or suspending agents to water while heating and stirring. The resulting solution is held for 10 minutes with continued heat and agitation before adding carbohydrates (e.g. the HMOs and digestible carbohydrate sources). The resulting feed solutions are then blended together while heating and agitating and the pH adjusted to 6.6-7.0, after which the composition is subjected to high-temperature short-time processing during which the composition is heat treated, emulsified and homogenized, and then allowed to cool. Water soluble vitamins and ascorbic acid are added, the pH is adjusted to the desired range, if necessary, flavours are added, and water is added to achieve the desired total solid level.

For a liquid product, the resulting solution can then be aseptically packed to form an aseptically packaged nutritional composition. In this form, the nutritional composition can be in ready-to- feed or concentrated liquid form. Alternatively, the composition can be spray-dried and processed and packaged as a reconstitutable powder.

When the nutritional product is a ready-to-feed nutritional liquid, it may be preferred that the total concentration of HMOs in the liquid, by weight of the liquid, is from about 0.1 % to about 1.5 %, including from about 0.2 % to about 1.0 %, for example from about 0.3 % to about 0.7 %. When the nutritional product is a concentrated nutritional liquid, it may be preferred that the total concentration of HMOs in the liquid, by weight of the liquid, is from about 0.2 % to about 3.0 %, including from about 0.4 % to about 2.0 %, for example from about 0.6 % to about 1.5 %.

In another embodiment, the nutritional composition is in a unit dosage form. The unit dosage form can contain an acceptable food-grade carrier, e.g. phosphate buffered saline solution, mixtures of ethanol in water, water and emulsions such as an oil/water or water/oil emulsion, as well as various wetting agents or excipients. The unit dosage form can also contain other materials that do not produce an adverse, allergic or otherwise unwanted reaction when administered to a human. The carriers and other materials can include solvents, dispersants, coatings, absorption promoting agents, controlled release agents, and one or more inert excipients, such as starches, polyols, granulating agents, microcrystalline cellulose, diluents, lubricants, binders, and disintegrating agents. Preferably the unit dosage form comprises primarily HMOs with a minimum amount of binders and/or excipients. Unit dosage forms are particularly suitable when nutritionally incomplete or not intended as a sole source of nutrition.

A unit dosage form can be administered orally, e.g. as a tablet, capsule, or pellet containing a predetermined amount of the mixture, or as a powder or granules containing a predetermined concentration of the mixture or a gel, paste, solution, suspension, emulsion, syrup, bolus, electuary, or slurry, in an aqueous or non-aqueous liquid, containing a predetermined concentration of the mixture. An orally administered composition can include one or more binders, lubricants, inert diluents, flavouring agents, and humectants. An orally administered composition such as a tablet can optionally be coated and can be formulated to provide sustained, delayed or controlled release of the HMOs.

A unit dosage form can also be administered by naso-gastric tube or direct infusion into the Gl tract or stomach.

A unit dosage form can also include therapeutic agents such as antibiotics, probiotics, analgesics, and anti-inflammatory agents.

The proper dosage of a nutritional composition for a human can be determined in a conventional manner, based upon factors such as the concentration of HMOs, the human’s condition, immune status, body weight and age. The required amount of HMOs would generally be in the range from about 1 g to about 15 g per day, in certain embodiments from about 2 g to about 10 g per day, for example about 3 g to about 7 g per day. Appropriate dose regimes can be determined by methods known to those skilled in the art.

In further embodiment, the mixture of HMOs can be formulated as a pharmaceutical composition. The pharmaceutical composition can contain a pharmaceutically acceptable carrier, e.g. phosphate buffered saline solution, mixtures of ethanol in water, water and emulsions such as an oil/water or water/oil emulsion, as well as various wetting agents or excipients. The pharmaceutical composition can also contain other materials that do not produce an adverse, allergic or otherwise unwanted reaction when administered to a human. The carriers and other materials can include solvents, dispersants, coatings, absorption promoting agents, controlled release agents, and one or more inert excipients, such as starches, polyols, granulating agents, microcrystalline cellulose, diluents, lubricants, binders, and disintegrating agents.

The pharmaceutical compositions can be administered orally, e.g. as a tablet, capsule, or pellet containing a predetermined amount, or as a powder or granules containing a predetermined concentration or a gel, paste, solution, suspension, emulsion, syrup, bolus, electuary, or slurry, in an aqueous or non-aqueous liquid, containing a predetermined concentration. Orally administered compositions can include binders, lubricants, inert diluents, flavouring agents, and humectants. Orally administered compositions such as tablets can optionally be coated and can be formulated to provide sustained, delayed or controlled release of the mixture therein.

The pharmaceutical compositions can also be administered by rectal suppository, aerosol tube, naso-gastric tube or direct infusion into the Gl tract or stomach.

The pharmaceutical compositions can also include therapeutic agents such as antibiotics, probiotics, analgesics, and anti-inflammatory agents. The proper dosage of a pharmaceutical composition can be determined in a conventional manner, based upon factors such the concentration of the HMOs, the patient’s condition, immune status, body weight and age. The required amount of HMOs would generally be in the range from about 1 g to about 15 g per day, in certain embodiments from about 2 g to about 10 g per day, for example about 3 g to about 7 g per day. Appropriate dose regimes can be determined by methods known to those skilled in the art.

The use of a mixture of HMOs according to the present invention

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

Individual HMOs are known to have beneficial health related effects and are known to affect bacterial and viral infections. While the isolated individual HMOs are beneficial in themselves, combinatorial mixtures of HMOs, as described herein, have a synergistic effect in the treatment of infections, compared to the isolated individual components.

One aspect of the invention relates to a mixture consisting of or consisting essentially of 3-FL, LNFP-I, 2’-FL, LNDFH-I, DFL and optionally lactose, including the preferred and more preferred embodiments disclosed earlier, or a composition comprising the mixture consisting of or consisting essentially of 3-FL, LNFP-I, 2’-FL, LNDFH-I, DFL and optionally lactose, including the preferred and more preferred embodiments disclosed earlier, for use in treating, preventing and/or ameliorating a disease and/or a condition related to microbiome imbalance. Alternatively, one aspect of the invention relates to a method for treating, preventing and/or ameliorating a disease and/or a condition related to microbiome imbalance in a human comprising administering the human a mixture consisting of or consisting essentially of 3-FL, LNFP-I, 2’-FL, LNDFH-I, DFL and optionally lactose or a composition comprising the mixture consisting of or consisting essentially of 3-FL, LNFP-I, 2’-FL, LNDFH-I, DFL and optionally lactose. Typically, a mixture of 3-FL, LNFP-I, 2’-FL, LNDFH-I and DFL according to the present invention, can be used for modulation of the microbiome of a human, e.g. to increase Bifidobacterium abundance and Barnesiella abundance, said modulation reduces Firmicutes abundance, especially Clostridia.

Furthermore, another aspect of the invention relates to a mixture consisting of or consisting essentially of 3-FL, LNFP-I, 2’-FL, LNDFH-I, DFL and optionally lactose, including the preferred and more preferred embodiments disclosed earlier, or a composition comprising the mixture consisting of or consisting essentially of 3-FL, LNFP-I, 2’-FL, LNDFH-I, DFL and optionally lactose, including the preferred and more preferred embodiments disclosed earlier, for use in treating and/or reducing the risk of a broad range of bacterial or viral infections in a human. Alternatively, one aspect of the invention relates to a method for treating and/or reducing the risk of a broad range of bacterial or viral infections in a human comprising administering the human a mixture consisting of or consisting essentially of 3-FL, LNFP-I, 2’-FL, LNDFH-I, DFL and optionally lactose or a composition comprising the mixture consisting of or consisting essentially of 3-FL, LNFP-I, 2’-FL, LNDFH-I, DFL and optionally lactose. Infections can arise in several body parts, not only limited to the gut microbiome, but also in other parts of the body exposed to the external environment, such as skin, hair, ears, eyes, nose and the respiratory system. One or more HMOs of the mixture according to the present invention can inhibit the adhesion of pathogenic bacteria, such as Pseudomonas aeruginosa or Campylobacter jejuni, uropathogenic and enteropathogenic Escherichia coli, certain Salmonella species and/or the pneumonia-causing pathogen Pseudomonas aeruginosa, as well as viruses such as norovirus. One or more HMOs of the mixture according to the present invention can also bind directly to pathogenic toxins, such as toxins from Clostridium difficile. One or more HMOs of the mixture according to the present invention may serve as an immune system modulator and affect intestinal health through stimulation of bifidobacteria. One or more HMOs of the mixture according to the present invention can inhibit the growth of Group B Streptococcus in both infants and breast milk. Group B Streptococcus are a leading cause of neonatal sepsis, pneumonia and meningitis. One or more HMOs of the mixture according to the present invention can bind directly to Shiga toxins Stx2 and Stx1 B5 of Shigella dysenteriae. One or more HMOs of the mixture according to the present invention have the potential to reduce the risk of infectious diseases caused by either bacterial or viral pathogens most likely due to the binding to bacterial exotoxins.

Further, another aspect of the invention relates to a non-medical use of a mixture consisting of or consisting essentially of 3-FL, LNFP-I, 2’-FL, LNDFH-I, DFL and optionally lactose, including the preferred and more preferred embodiments disclosed earlier, or a composition comprising the mixture consisting of or consisting essentially of 3-FL, LNFP-I, 2’-FL, LNDFH-I, DFL and optionally lactose, including the preferred and more preferred embodiments disclosed earlier, for maintaining the commensal homeostasis of the gut microbiota in a human.

Further, another aspect of the invention relates to a non-medical use of a mixture consisting of or consisting essentially of 3-FL, LNFP-I, 2’-FL, LNDFH-I, DFL and optionally lactose, including the preferred and more preferred embodiments disclosed earlier, or a composition comprising the mixture consisting of or consisting essentially of 3-FL, LNFP-I, 2’-FL, LNDFH-I, DFL and optionally lactose, including the preferred and more preferred embodiments disclosed earlier, in the dietary management of a human.

In addition, mixture consisting of or consisting essentially of 3-FL, LNFP-I, 2’-FL, LNDFH-I, DFL and optionally lactose, including the preferred and more preferred embodiments disclosed earlier, enhances the viability of useful probiotics in aqueous compositions. It has been found that probiotic strains, e.g. Lactobacillus rhamnosus strains, when admixed with HMOs, have a significantly increased regeneration and viability when coming into contact with a low pH liquid environment, such as stomach acid or acidic beverages. Thus, the combination of the mixture of HMOs according to the present invention with probiotic strains, preferably lyophilized probiotics, can offer both a reliable way of delivering a quantifiable amount of probiotics to a host (human or animal), as either in pharmaceutical-like forms, in nutritional compositions or in food-based form.

Whilst the invention has been described with reference to embodiments, it will be appreciated that various modifications are possible within the scope of the invention.

The following numbered aspects of the invention are provided:

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.

2. The mixture according to aspect 1 consisting essentially of 3-FL, LNFP-I, 2’-FL, LNDFH- I, DFL and lactose, preferably wherein

- component D is in 8-13 mol% or in 14-22 w/w%, and/or

- component E is in 9.5-17.5 mol% or in 11.5-19 w/w%, and/or

- components D+E are in 17.5-30.5 mol% or in 25.5-41 w/w%, relative to components A, B, C, D, E and F taken together, or

- component D is in 2-22 mol% or in 5-34 w/w%, or - component E is in 7-24 mol% or in 6-29 w/w%, relative to components A, B, C, D, E and F taken together, provided that components D+E are in 23.5-33 mol% or in 30.5-43 w/w%.

3. The mixture according to aspect 1 or 2, wherein

- components A+B+C are in 41.5-67 mol% or in 41-66 w/w%, preferably wherein more component B is present than component C, or

- components A+B+C are in 40.5-54.5 mol% or in 41-54 w/w%, preferably wherein component A is in 21-27 mol%.

4. The mixture according to any of the preceding aspects, wherein

- component A is in 19-24.5 mol% or in 15-20 w/w%,

- component B is in 12.5-15.5 mol% or in 19-22 w/w%,

- component C is in 7-10 mol% or in 6-8 w/w%,

- component D is in 11-14 mol% or in 18-22 w/w%,

- component E is in 14.5-17.5 mol% or in 16-19 w/w%, and

- component F is in 25-30 mol% or in 15-19 w/w%.

5. The mixture according to aspect 1 consisting essentially of 3-FL, LNFP-I, 2’-FL, LNDFH- I, and DFL, preferably wherein

- component D is in 10-17.5 mol% or in 16-25.5 w/w%, and/or

- component E is in 12-23.5 mol% or in 13-22 w/w%, and/or

- components D+E are in 22-41 mol% or in 29-47.5 w/w%, relative to components A, B, C, D and E taken together or

- component D is in 3.5-29.5 mol% or in 6-39 w/w%, and/or

- component E is in 9-32 mol% or in 7.5-34.5 w/w%, relative to components A, B, C, D and E taken together, provided that components D+E are in 31.5-41 mol% or in 36.5-49 w/w%.

6. The mixture according to aspect 5, wherein

- more component B is present in the mixture than component C or

- components A+B+C are in 57-70 mol% or in 51-64.5 w/w%, preferably wherein component A is in 29-35.5 mol%.

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

- component A is in 29-33 mol% or in 20-24.5 w/w%,

- component B is in 18-21.5 mol% or in 23-25.5 w/w%,

- component C is in 10-12.5 mol% or in 7.5-9.5 w/w%,

- component D is in 15-17.5 mol% or in 22-25.5 w/w%,

- component E is in 20.5-23.5 mol% or in 19.5-22 w/w%.

8. A composition comprising the mixture according to any of the precedent aspects, preferably said composition further comprises a probiotic, more preferably L. rhamnosus, especially L. rhamnosus LGG DSM 33156.

9. A process for obtaining the mixture according to any one of aspects 1 to 7, wherein said process comprises reacting 3-FL, LNFP-I and 2’-FL in the presence of an c -3/4- transfucosidase to produce a reaction mixture, and then removing the cd-3/4-transfucosidase and optionally lactose from the reaction mixture, preferably wherein the cd-3/4-transfucosidase comprises or consists of the amino acid sequence of SEQ ID no.1 with the following mutations: W135F-A174N-N274A-E413R.

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

- wherein the microbiome of a human is modulated, or

- wherein the use is treating and/or reducing the risk of a broad range of bacterial or viral infections in a human.

11. A use of the mixture according to any of the aspects 1 to 8 for enhancing the viability of probiotics, preferably L. rhamnosus, more preferably L. rhamnosus LGG DSM 33156, in an aqueous composition.

EXAMPLES

Example 1

A solution of 3-FL (4.278 kg, assay corrected, 8.76 mols), LNFP-I (3.807 kg, assay corrected, 4.46 mols) and 2’-FL (2.072 kg, assay corrected, 4.24 mols) in water (17.5 kg) was transferred to a 20-litre fermenter. The pH of the solution was adjusted to 6.0 with a 7 % HCI-solution. Freeze-dried powder (11.6 g) of the W135F-A174N-N274A-E413R mutant of Bifidobacterium longum subsp. infantis ATCC15697 (the amino acid numbering being according to SEQ ID no.

1 , see WO 2016/063261) was dissolved in water (100 g) and added to the above solution, then the reaction mixture was stirred at 25 °C for 18 hours, followed by heating it up to 55 °C through 4 hours and the stirring was continued at this temperature for 2 hours. Subsequently, the pH of the mixture was set to 5.0 with a 7 % HCI-solution and the mixture was heated up to 70 °C through 1 hours and kept at this temperature for 2 hours. The mixture then was allowed to cool to 15 °C while the enzyme precipitated. The obtained suspension was then ultrafiltered (15 kDa ceramic membrane, 0.5 m ² , trans-membrane pressure: 5.8-5.9 bars, temperature: 19-24 °C) and 28.05 kg of permeate was collected. The permeate was microfiltered (0.2 pm PES- membrane) and freeze-dried to give 10.57 g of powder (saccharides by HPLC: 95 w/w% from which 3-FL is 16 w/w%, LNFP-I is 19 w/w%, 2’-FL is 7 w/w%, LNDFH-I is 18 w/w%, DFL is 17 w/w% and lactose is 18 w/w%; protein content by Bradford test: < 6 ppm). Example 2

In accordance with the protocol disclosed in Example 1, the following compositions were obtained at different donor-acceptor ratios: Example 3

Solutions of lyophilized Lactobacillus rhamnosus (Probio-Tec® LGG®, Chr. Hansen, deposited at the DSMZ culture collection under accession number DSM 33156, 0.4 mg/ml) and HMOs (2’-FL, 2’-FL/DFL in 6.1:1 weight ratio and the HMO mixture from Example 1 , respectively, 5 w/v%) in sterile phosphate-buffered saline (PBS, pH = 3) were warmed to 37 °C and vigorously mixed for about 30 sec until no visible clumps remained. The control contained only the probiotic. The tubes were incubated at 37 °C for 3 h. The samples were further diluted and 100 l were spread in duplicates onto MRS agar plates which were incubated for 48 h at 37 °C in anaerobic chambers. Results are expressed as mean values with standard deviation of colony-forming units (CFU) per millilitre calculated from LGG colonies on agar plates (Figure 1).

Lyophilized LGG dissolved with HMO mixture according to the invention showed a strongly significant enhanced regeneration and survivability compared to control or some of the HMO mixture ingredients after 3 h incubation at 37 °C in pH 3.0 acidic condition as assessed by colony counting. The data suggest that the decreased regeneration and viability of L. rhamnosus after exposure to low pH conditions, e.g. in the stomach or in an acidic beverage, can be prevented in the presence of HMOs.