FR3136650A3 - Blend of fucosylated HMOs - Google Patents

Abstract

The present invention relates to a human milk oligosaccharide ("HMO") mixture comprising five fucosylated HMOs, and to an enzymatic process for preparing said mixture, wherein said synthesis comprises an enzymatic reaction of α1-3/4-transfucosidase between HMO donors and acceptors.

Description

Blend of fucosylated HMOs Field of the invention

The present invention relates to a human milk oligosaccharide ("HMO") mixture comprising five fucosylated HMOs, and to an enzymatic process for producing said mixture, wherein said synthesis comprises an enzymatic reaction by an α1-3/4-transfucosidase between HMO donors and acceptors.

Background

HMOs have been the subject of significant interest in recent years due to their roles in many biological processes occurring in the human body. Mammalian milk contains at least 130 of these complex oligosaccharides ( see Urashima et al: Milk Oligosaccharides . Nova
SciencePublisher (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 imbalanced, 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 thought to positively modulate the microbiome, and are of growing interest for this purpose. However, the remarkable diversity of HMOs, coupled with their lack of availability, particularly for more complex HMOs, has hampered research into the specific functions of individual HMOs and therefore limited exploration of their full potential for use in promoting good human health.

The natural source of HMOs is mammalian milk, which contains mainly water, together with 55 to 70 g/l lactose, 24 to 49 g/l lipids, approximately 13 g/l protein, 5 to 15 g/l of HMO and approximately 1.5 g/l of minerals.

Efforts to develop processes for the production of HMOs on an industrial scale have increased significantly in recent years. Processes have been designed to produce HMOs by microbial fermentation, by enzymatic processes, by chemical synthesis, or by combinations of these technologies.

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

Documents WO 2012/158517 and WO 2013/154725 describe prebiotic compositions containing 3-FL, 2'-FL and DFL, and document WO 2012/112777 describes a mixture of 3-FL, 2'-FL, DFL and lactose , produced in genetically modified E. coli .

In an effort to be able to synthetically produce HMO mixtures without the need to synthesize all of the constituent oligosaccharides of the mixture, enzymatic methods for the enzymatic synthesis of HMO oligosaccharide mixtures have been developed, and are described in the documents WO 2012/156897 and WO 2012/156898. As a further development, documents WO 2016/063261 and WO 2016/063262 describe optimized enzymes and enzymatic processes for the generation of fucosylated HMOs. These methods yielded reaction mixtures containing a plurality of different HMO oligosaccharides.

Nevertheless, these processes include the use of highly specialized enzymes which, in turn, lead to the need for sensitive stoichiometry and reaction conditions, as well as the need to terminate reactions at the desired equilibrium point in the reactions and the elimination of unwanted secondary metabolites.

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

The first object of the invention relates to a mixture consisting or essentially consisting 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 possibly a component F which is lactose.

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

The third object of the invention relates to a process for the enzymatic synthesis of the above mixture of HMOs, preferably under essentially buffer-free conditions. Said enzymatic synthesis comprises an enzymatic reaction by an α1-3/4-transfucosidase between a fucosylated HMO donor and two fucosylated HMO acceptors. The enzymatic synthesis for preparing an HMO mixture according to the present invention comprises the steps of reacting a component A, a component B and a component C in the presence of an α1-3/4-transfucosidase to produce an reaction mixture and elimination of α1-3/4-transfucosidase and optionally lactose from the reaction mixture. Consequently, the present invention also relates to a mixture consisting or essentially consisting of 2'-FL, 3-FL, LNFP-I, DFL, LNDFH-I and optionally lactose, obtained or capable of being obtained by the enzymatic synthesis disclosed here.

A special feature of said enzymatic synthesis according to the present invention is that it can be carried out under essentially buffer-free conditions. Typically, the enzymatic synthesis according to the present invention is carried out at a pH in the range of 4.5 to 7, such as preferably a pH in the range of 5.0 to 6.0.

The α1-3/4-transfucosidase used in the enzymatic synthesis according to the present invention is preferably devoid of hydrolytic activity, or at least has significantly reduced hydrolytic activity.

According to one aspect of the present invention, the enzymatic synthesis according to the present invention further comprises an elimination step which contains a step for deactivating the α1-3/4-transfucosidase. Said inactivation step may include heating the reaction solution to temperatures ranging from 60°C to 100°C, for a period of time ranging from 5 minutes to 180 minutes.

In the enzymatic synthesis according to the present invention, the α1-3/4-transfucosidase is eliminated for example by microfiltration, nanofiltration, centrifugation or ultracentrifugation. Said enzymatic synthesis may include more than one step for the elimination of the α1-3/4-transfucosidase. One step may consist of eliminating the α1-3/4-transfucosidase using powdered activated charcoal in batch mode. Alternatively, the step may consist of removing the α1-3/4-transfucosidase using powdered activated charcoal in column mode.

Another object of the invention relates to a mixture consisting or essentially consisting of 2'-FL, 3-FL, LNFP-I, DFL, LNDFH-I and optionally lactose, for use in, or a composition comprising the mixture consisting or essentially consisting of 2'-FL, 3-FL, LNFP-I, DFL, LNDFH-I and optionally lactose, for use in the treatment, prevention and/or improvement of a disease and/or 'a condition associated with an imbalance in the microbiome.

Another object of the invention relates to the use of a mixture consisting or essentially consisting of 2'-FL, 3-FL, LNFP-I, DFL, LNDFH-I and optionally lactose, to amplify the viability of probiotics in aqueous solution.

Another object of the invention relates to the use of a mixture consisting or essentially consisting of 2'-FL, 3-FL, LNFP-I, DFL, LNDFH-I and optionally lactose, or of a composition comprising the mixture consisting of or essentially consisting 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 below in more detail with reference to the attached which shows the colony count of L. rhamnosus after 3 hours of incubation with or without HMO at 37°C and pH 3.0.

detailed description

In general, all terms used herein shall be construed according to their usual meaning in the art, and this is applicable to all subject matter of embodiments of the invention, unless explicitly defined or otherwise stated.

All references to "a [sequence, enzyme, step, etc.]" shall be openly construed as referring to at least one instance of said sequence, enzyme, step, etc., unless otherwise noted explicit contrary.

The steps of any method disclosed herein need not be performed in the exact order disclosed unless explicitly stated.

The word "comprising" is used in the sense of "including", rather than in the sense of "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 in the invention are human milk oligosaccharides (HMO).

The term "human milk oligosaccharide" or "HMO", as used herein, 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'-fucosyl-lactose ((Fucα1-2Galβ1-4Glc), 3-FL is 3-fucosyl-lactose (Galβ1-4[Fucα1-3]Glc), DFL is difucosyl-
lactose (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 structures, Fuc means the L-fucopyranosyl motif, Gal means the D-galactopyranosyl motif, Glc means the D-glucopyranose motif, and GlcNAc means the D-(2-acetylamino)-2-deoxy-glucopyranosyl motif. In the context of the present invention, lactose is not considered a species of HMO.

In the context of the invention, the term "donor" relates to a fucosylated saccharide, in which an α1-3-fucosyl is transferred, via an enzyme-catalyzed reaction, to another saccharide, whereupon the The product thus produced is a saccharide containing an α1-3-fucosyl or α1-4-fucosyl. The term "acceptor" refers to the saccharide onto which the fucosyl group from the donor is transferred.

“Dietary management” means the exclusive or partial feeding of patients who, because of a disease, disorder or medical condition, suffer from:
- either have a limited, reduced or impaired ability to take, digest, absorb, metabolize or excrete ordinary foods or certain nutrients they contain, or metabolites,
- or have other nutritional requirements determined medically.
(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).

The terms "microbiota", "microflora" and "microbiome" mean a community of living microorganisms that typically inhabit an organ or part of the body, particularly the gastrointestinal organs of humans. The most dominant members of the gastrointestinal microbiote include microorganisms of firmicate, bacteroidetes , actinobacteria , proteobacteria , synergist , Verrucomicrobia , fusobacteria , and Euryarchaeota , in the gender Bacteroides , faecalibacterium , bifidobacterium , rosebaria , rosebaria Blautia , coprococcus , Ruminococcus , Eubacterium and Dorea , at the 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 aer ofaciens , Dorea formicigenerans , Bacteroides vulgatus and Roseburia intestinalis . The gastrointestinal microbiota includes the mucosa-associated microbiota, which is located in or attached to the mucosal layer covering the epithelium of the gastrointestinal tract, and the lumen-associated microbiota, which is found in the lumen of the gastrointestinal tract.

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

"Oral administration" means any conventional form of delivery of a composition to a human by mouth.

“Preventive treatment” or “prevention” means a treatment given or an action taken to reduce the risk of the onset or relapse of a disease.

By “treat” we mean remedying an illness or medical condition with the goal of improving or stabilizing the outcome in the person being treated or addressing an underlying nutritional need. Therefore, the term "treat" includes the dietary or nutritional management of the disease or medical condition by addressing the nutritional needs of the person being treated. The terms "treat" and "processing" have grammatically corresponding meanings

All prior teachings acknowledged above are incorporated herein by reference. No acknowledgment of any previously published material herein should be construed as an admission or representation that its teaching was common general knowledge at the date thereof.

The new HMO mix

In accordance with the present invention, it has surprisingly been discovered that a novel mixture of 5 fucosylated HMOs can be prepared by enzymatic synthesis using a single starting HMO donor and two starting HMO acceptors. The reaction can preferably be carried out under conditions essentially free of buffer. Also preferably, the enzyme has limited or absent hydrolytic activity.

The new mixture, consisting of or essentially consisting of 5 fucosylated HMOs, has a unique new combination of biological properties and activities. Specifically, the mixture may be used for modulation of a human's microbiome, including increasing the abundance of Bifidobacterium and Barnesiella in a human's microbiome. Additionally, the mixture may reduce the abundance of Firmicutes in the human microbiome, particularly Clostridia . The mixture may also be used to treat and/or reduce the risk of a wide range of human bacterial and viral infections, as well as. The new HMO blend described here is anti-infectious. Additionally, the mixture, when combined with a probiotic, significantly amplifies the viability of that probiotic in aqueous solution.

1. un composant A qui est le 3-FL,
2. un composant B qui est le LNFP-I,
3. un composant C qui est le 2'-FL,
4. un composant D qui est le LNDFH-I,
5. un composant E qui est le DFL, et éventuellement
6. un composant F qui est le lactose.

One aspect of the present invention relates to a mixture consisting or essentially consisting of:

1. a component A which is 3-FL,
2. a component B which is LNFP-I,
3. a component C which is 2'-FL,
4. a component D which is LNDFH-I,
5. a component E which is the DFL, and possibly
6. a component F which is lactose.

1. un composant A qui est le 3-FL,
2. un composant B qui est le LNFP-I,
3. un composant C qui est le 2'-FL,
4. un composant D qui est le LNDFH-I, et
5. un composant E qui est le DFL.

Consequently, one embodiment of the invention is a mixture consisting or essentially consisting of:

1. a component A which is 3-FL,
2. a component B which is LNFP-I,
3. a component C which is 2'-FL,
4. a component D which is LNDFH-I, and
5. a component E which is the DFL.

1. un composant A qui est le 3-FL,
2. un composant B qui est le LNFP-I,
3. un composant C qui est le 2'-FL,
4. un composant D qui est le LNDFH-I,
5. un composant E qui est le DFL, et
6. un composant F qui est le lactose.

Another embodiment of the invention is a mixture consisting or essentially consisting of:

1. a component A which is 3-FL,
2. a component B which is LNFP-I,
3. a component C which is 2'-FL,
4. a component D which is LNDFH-I,
5. a component E which is the DFL, and
6. a component F which is lactose.

Enzymatic synthesis to produce the HMO mixture according to the invention

In the following paragraphs, the expression "may carry" is equivalent to the expression "may possibly carry", and the expression "may be substituted" is equivalent to the expression "is optionally substituted".

The above-described new mixture of fucosylated HMOs is prepared by enzymatic synthesis comprising a fucosyl HMO donor which is component A (3-FL), two fucosyl HMO acceptors which are component B (LNFP-I) and the component C (2'-FL), and a transfucosidase, preferably under essentially buffer-free conditions. Also preferably, in said reaction, the enzyme has limited or absent hydrolytic (i.e. fucosidase) activity.

Therefore, this invention also relates to a process for preparing an HMO mixture consisting or essentially consisting 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 reaction of components A, B and C in the presence of an α1-3/4-transfucosidase to produce a reaction mixture containing components A, B, C, D, E and lactose, and then removing α1-3/4-transfucosidase from the reaction mixture. The α1-3/4-transfucosidase can be eliminated in a conventional manner, for example by denaturation of the reaction mixture followed by its centrifugation or its ultrafiltration.

Also, an object of this invention relates to a process for obtaining a mixture of HMO consisting or essentially consisting 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 α1-3/4-transfucosidase to produce a mixture reaction containing components A, B, C, D, E and lactose, and then removing lactose and α1-3/4-transfucosidase from the reaction mixture. The α1-3/4-transfucosidase can be eliminated for example by denaturation thereof followed by centrifugation or ultrafiltration. Lactose can be separated from components A, B, C, D and E for example by ultra- and/or nano-filtration in cascade, or the lactose can first be treated with a lactase so that it is degraded into glucose and galactose which can then be separated from components A, B, C, D and E by ultra- and/or nano-filtration.

The enzyme (protein) suitable for carrying out the above enzymatic reaction is an α1-3/4-fucosidase or an α1-3/4-transfucosidase. These enzymes are capable of transferring a fucosyl residue from a fucosyl donor, in which the fucosyl residue is attached by an α1-3 bond, to an acceptor. More precisely, if the acceptor is an HMO, the fucosyl residue is transferred to position 4 of a GlcNAc in the acceptor HMO, or if either this position is not free or there is no GlcNAc in the structure of the acceptor HMO, at position 3 of Glc.

α1-3/4-(trans)fucosidase is a naturally occurring α1-3/4-L-fucosidase as classified according to EC 3.2.1.111 or its genetically modified mutant. All α1-3/4-L-fucosidases can act as a transfucosidase to some extent.

A preferred α1-3/4-(trans)fucosidase suitable for carrying out the enzymatic reaction according to the invention is an α1-3/4-(trans)fucosidase which has an identity of at least 60%, preferably at least 70%, more preferably at least 80%, in particular at least 90%, with amino acid positions 56 to 345 of Bifidobacterium α1-3/4-fucosidase longum subsp . infantis ATCC 15697 as presented in US patent 8361756 as a protein having SEQ ID NO: 18 (referred to as SEQ ID NO: 1 in the present application).

In the enzymatic synthesis of the invention, hydrolysis of the components can become significant due to an increase in the concentrations of the product components of the reaction, in which the α3/4-fucosylated components are subsequently hydrolyzed to the non-α3/ components. 4-fucosylated, lactose, 2'-FL and LNFP-I and free fucose. Thus, the α1-3/4-transfucosidase of the present invention preferably has limited or absent hydrolytic activity. The limited or absent hydrolytic activity of α1-3/4-transfucosidase allows longer reaction times than would otherwise be possible.

Thus, a still preferred α1-3/4-transfucosidase for producing the HMO mixtures of this invention is an α1-3/4-transfucosidase lacking hydrolytic activity, or at least having significantly reduced hydrolytic activity.

Such an α1-3/4-transfucosidase can be produced by altering the amino acid sequence at one or more amino acid positions, such that the mutated amino acid sequence results in enhanced transfucosidase and/or reduced hydrolytic activity. According to this invention, α1-3/4-tranfucosidase
has. 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 one or more of the following amino acid positions: 134, 135, 174, 216 , 221, 282 and 413; thereby
b. provides a conversion rate of at least 20% and up to 70%, such as at least 35%, or such as at least 40%, or such as at 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 produced in nature or do not occur naturally, but are produced as a result of chemical synthesis, genetic manipulation or similar laboratory processes, resulting in synthetic mutant fucosidases.

Preferably, the mutated α1-3/4-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.

Preferably, the α1-3/4-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 positions d following amino acids: 134, 135, 216, 221 and 282.

Also preferably, the α1-3/4-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 of 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 amino acid positions following: 134, 135, 216, 221 and 282.

Even more preferably, the α1-3/4-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 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 α1-3/4-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 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 another mutation at one or more of the following amino acid positions: 165, 168, 232, 237, 258, 260 and 274.

Preferably, the α1-3/4-transfucosidase comprises, more preferably consists of, the sequence of SEQ ID NO: 1 having mutations:
- at amino acid positions 135 and/or 174, and
- at least at one amino acid position chosen from 168, 237 and 413.

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

Even more preferably, the α1-3/4-transfucosidase comprises, even more preferably consists of, the sequence of SEQ ID NO: 1, as described above, having mutations:
- at amino acid position 174,
- at 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 endows the mutated enzyme not only with further enhanced transfucosidase synthesis performance, but also with further enhanced stability, particularly temperature stability, while maintaining further enhanced transfucosidase synthesis performance. . The mutations at 135, 165, 174, 232, 258, 260 and 274 are preferably the following:
- at position 135, in the form W135F or W135Y,
- at position 165, in the form P165E,
- at position 174, in the form A174F, A174H or A174N,
- at position 232, in the form R232A,
- at position 258, in the form Q258R,
- at position 260, in the form D260P,
- at position 274, in the form N274A.

Also preferably, the mutated α1-3/4-transfucosidase which comprises or consists of the polypeptide of SEQ ID NO: 1, and a mutation at at least amino acid position 174 or 282, preferably of both amino acid positions, provides significantly or completely suppressed hydrolytic activity. From this point of view, Ala (A) at position 174 is preferably replaced by Phe (F), Asn (N) or His (H), and/or Val (V) at position 282 is preferably replaced by Arg (R), Glu (E), His (H) or Lys (K). The suppressed hydrolytic activity is advantageous because the mutated enzyme then does not significantly degrade the donor and/or product by hydrolysis. As a result, the transfucosidase reaction is no longer kinetically controlled, and a much better synthesis/hydrolysis ratio (meaning better synthesis performance) can be obtained. Mutation at one or both, preferably both, of the above amino acid positions can provide hydrolytic activity toward fucosylated products reduced by at least 100-fold, preferably d at least 1,000 times, more preferably at least 10,000 times.

1. le polypeptide de la SEQ ID NO : 1 et une mutation au moins au niveau de la position d'acide aminé 174 ou 282, et
2. une activité hydrolytique significativement ou complètement supprimée, en comparaison avec la protéine selon la SEQ ID NO : 1.

Therefore, the mutated α1-3/4-transfucosidase has

1. the polypeptide of SEQ ID NO: 1 and a mutation at least at amino acid position 174 or 282, and
2. a significantly or completely suppressed hydrolytic activity, in comparison with the protein according to SEQ ID NO: 1.

Furthermore, in accordance with a certain embodiment, the mutated α1-3/4-transfucosidase comprises or preferably consists of the polypeptide of SEQ ID NO: 1 and a mutation at the level of
- at least 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, confers not only significantly reduced, preferably virtually undetectable, hydrolysis of the fucosylated product, but also improved stability, particularly temperature stability.

1. le polypeptide de la SEQ ID NO : 1 avec une mutation au moins de l'acide aminé à la position 174 ou 282, et au moins au niveau de la position d'acide aminé 165, 168, 232, 237, 258, 260 ou 274, et
2. une activité hydrolytique significativement ou complètement supprimée, et/ou
3. une stabilité, en particulier une stabilité à la température, amplifiée,

en comparaison avec la protéine selon la SEQ ID NO : 1.Therefore, the mutated α1-3/4-transfucosidase has

1. the polypeptide of SEQ ID NO: 1 with a mutation at least in 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
2. significantly or completely suppressed hydrolytic activity, and/or
3. stability, in particular stability at temperature, amplified,

in comparison with the protein according to SEQ ID NO: 1.

Preferably, the mutated α1-3/4-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 Ala (A) at position 174 is preferably replaced by Phe (F), Asn (N) or His (H) and/or Val (V) at position 282 is preferably replaced by Arg (R ), Glu (E), His (H) or Lys (K).

A particularly preferred modified α1-3/4-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 in accordance with SEQ ID NO: 1.

In carrying out the method of this invention, the particular relative concentrations of the donor component A, the acceptor components B and C, the α1-3/4-transfucosidase, the aqueous solvent and the incubation buffer are not reviews. From this point of view, the process can be adequately carried out at room temperature (for example at 15-50, preferably 20-37°C) at a pH of 4 to 7, preferably 5 to 6, for 15 minutes to 24 hours. The reaction is typically carried out so that an equilibrium is reached.

In the enzymatic reaction, component A is provided as a fucosyl donor and components B and C are provided as fucosyl acceptors. During the α1-3/4-transfucosidase reaction, a fucosyl residue is transferred from the donor to both acceptors, consequently resulting in the generation of a D component, an E component, and lactose. However, the newly generated component E can also serve as a fucosyl donor, resulting in an enzymatic α1-3/4-transfucosidase 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 subsequent subequilibrium reactions.

Even more preferably, α1-3/4-transfucosidase, advantageously the mutant W135F-A174N-N274A-E413R (the numbering of the amino acids being in accordance with SEQ ID NO: 1) is used in an amount of approximately 0, 09 to 0.11% by mass relative to the initial overall weight of components A, B and C. The quantity of enzyme does not affect the final proportion of HMOs in the composition obtained, and only has an effect when the equilibrium is reached (a higher concentration of enzyme leads to a shorter time to reach equilibrium).

With regard to the preparation of the mixture consisting or essentially consisting of components A, B, C, D, E, and optionally lactose, preferably mixtures according to any one of the embodiments i) to iv) set out below. below, the enzymatic synthesis according to the invention is carried out in the presence of a suitable buffer, but preferably under conditions essentially free of buffer.

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 the concentration of free hydrogen ions as a result of internal and/or environmental factors. Additionally, a buffered solution essentially maintains the pH for a reaction system within a specific pH range, depending on the buffering agent used. Additionally, it is well known that buffered solutions for enzymatic reactions provide essential cofactors for enzymatic reactions, e.g. critical salts, and also resistant to changes in hydrogen ion concentration, and conditions that stabilize proteins in general . Consequently, when carrying out an enzymatic reaction, those skilled in the art expect that it will be necessary to introduce into the reaction medium components and/or buffering agents which are normally considered essential for the enzyme to function properly.

However, before further processing of the reaction products, these additional components and/or agents, and/or their residual products, must be removed from the reaction, which can be both time-consuming and tedious, adding unwanted costs to the process. production process.

It has surprisingly been found that the enzymatic synthesis according to the present invention can be carried out under essentially buffer-free conditions, thereby eliminating the need for providing and removing buffering components and/or agents.

According to one aspect of the present invention, the enzymatic synthesis described here is carried out in purified water, in which the purification step may comprise one or more purification treatments chosen from the group consisting of: reverse osmosis, filtration , ultrafiltration, sedimentation, chlorination, iodine addition, electrodialysis, boiling, desalination, and flocculation.

According to a currently preferred aspect of the invention, the enzymatic synthesis is carried out in water having a conductivity, before use in the enzymatic synthesis, less than 10 µS/cm, such as less than 5 µS/cm, such as less than 1 µS/cm, such as less than 0.1 µS/cm, and/or a silica content less than 0.05 mg/l, such as less than 0.04 mg/l, such as less than 0.02 mg/l or such as less than 0.01 mg/l.

In enzymatic synthesis according to the present invention, essentially buffer-free conditions are conditions in which no additional buffering agent is added to the purified water.

As used herein, the term "substantially buffer-free" also includes a solution to which inorganic salts are added to purify water having previously specified purity and conductivity ranges, to act as stabilizing agents for the enzyme, in which the salts do not contain buffering capacity. Examples of salts include common salts such as, but not limited to, sodium chloride (NaCl), calcium chloride (CaCl ₂ ), magnesium chloride (MgCl ₂ ), potassium chloride (KCl ), and sodium sulfate (Na ₂ SO ₄ ).

In one aspect of enzymatic synthesis in accordance with the present invention, the substantially buffer-free conditions may include an amount of salt in the range of 1 micromolar to 1 molar, with a preferred range such as 100 micromolar to 500 micromolar , or in a more preferred range of 1 millimolar to 300 millimolar. In another aspect, the essentially buffer-free condition refers to the presence of trace amounts of ions in water, at a concentration resulting in a conductivity less than 5 µS/cm.

According to another aspect of the invention, the expression "essentially buffer-free" also includes the use of tap water.

The presence of ions in a solution, with or without the addition of salts to the solution, can affect the pH of the solution, even at trace concentrations, resulting in a conductivity greater than 5 µS/cm, again without the addition of buffering capacities to essentially buffer-free conditions. Additionally, the addition of reagents to a solution in an essentially buffer-free condition can also change the pH of the solution as well as the conductivity. Therefore, in the present context, the conditions after the addition of ions and/or reagents to the reaction solution are always considered to be essentially buffer-free.

According to a preferred aspect of enzymatic synthesis, according to the present invention, the synthesis is carried out in a substantially buffer-free solution in which the pH is in the range of 4 to 7, preferably at a pH of 5 to 6 .

Before using an HMO mixture according to the invention as a treatment or otherwise, it is necessary to remove unwanted reaction components. Undesired products of the final product are, for example, enzymes, such as α1-3/4-transfucosidase, lactose, additional trace products and/or similar undesired products.

Additionally, an enzymatic synthesis according to the present invention may include one or more steps to deactivate and separate the α1-3/4-transfucosidase from the remainder of the reaction mixture.

Enzyme 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 processing vessel or a plastic container or any other container suitable for carrying out the reaction. synthesis, in a second container, with for example pressure regulation, temperature regulation and/or regulation of the liquid and/or gaseous environment. Thus, enzyme synthesis may be carried out in a manner in which the reaction vessel and the inactivation/deactivation vessel are the same, or may be carried out in a manner in which the reaction vessel and the inactivation/deactivation vessel are not the same.

The deactivation step may 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. According to a preferred aspect, the deactivation step involves heating the reaction mixture to a temperature in the range from 60°C to 100°C for a time of at least 1, 5, 10, 30 or 60 minutes.

After the step(s) for deactivating the α1-3/4-transfucosidase in the remaining reaction mixture, an enzymatic synthesis of the present invention further comprises one or more steps for removing the deactivated α1-3/4-transfucosidase from the mixture. of synthesized HMO, which thus gives a mixture which consists or essentially consists of components A, B, C, D, E and lactose, preferably the mixtures according to any one of the embodiments i) and ii) explained below below, including their favorite and more favorite embodiments.

In general, α1-3/4-transfucosidase can be removed from the synthesized HMO mixture by conventional separation methods, such as filtration (nano-, ultra- or micro-filtration), removal by centrifugation or ultracentrifugation. , treatment of the reaction mixture with powdered activated carbon in a batch mode and/or column mode and/or chromatographic methods, such as ion exchange, affinity chromatography, exclusion chromatography diffusion, forward or reversed phase chromatography, preferably after the enzyme used for synthesis has been denatured or deactivated. Separation processes can be combined in a multi-stage separation.

According to one aspect of the invention, the reaction mixture, containing an α1-3/4-transfucosidase, is treated with activated carbon, for example the reaction mixture passes into a column packed with activated carbon by means of a peristaltic flow, high pressure flow or gravity flow.

According to another aspect of the invention, when using ultrafiltration or microfiltration for the separation of an α1-3/4-transfucosidase from the reaction mixture, the separation may involve a prior step wherein the (inactivated) α1-3/4-transfucosidase has formed particles, such as aggregates, crystals and/or precipitates, allowing for more convenient separation by filtration. The pore size of the ultra- or micro-filtration membrane is chosen so that the carbohydrate components can pass through the membrane while ensuring the retention of the (inactivated) α1-3/4-transfucosidase.

According to another aspect of the invention, when using ultracentrifugation or centrifugation for the separation of α1-3/4-transfucosidase, the separation may involve an earlier step in which the α1- 3/4-transfucosidase (inactivated) formed particles, such as aggregates, crystals and/or precipitates, allowing for more convenient separation by (ultra)centrifugation.

The purification steps set out above result in a purified aqueous solution from which a solid mixture which consists or consists essentially of components A, B, C, D, E and lactose, preferably the mixtures according to any of embodiments i) and ii) set forth below, including their preferred and more preferred embodiments, may be obtained after removal of water, for example by evaporation, spray drying, or freeze drying .

For obtaining the mixture which 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) explained below, including their preferred and more preferred embodiments, lactose should be separated from components A, B, C, D and E. A suitable method for this is for example gel filtration, or membrane filtration using a membrane which ensures retention components A, B, C, D and E and allows lactose to pass through. Another method is that before or after the elimination of the α1-3/4-transfucosidase from the reaction medium as explained above, but before the elimination of the water, a β-galactosidase is added to degrade the lactose into glucose and galactose, then these monosaccharides are separated by means of a membrane which ensures the retention of components A, B, C, D and E and allows glucose and galactose to pass. The elimination of β-galactosidase can be carried out before or after this membrane filtration, by using one of the steps described for the elimination of α1-3/4-transfucosidase.
Modes of carrying out the first aspect of the invention

Concerning the mixture which consists or consists essentially of components A, B, C, D, E and F, it is preferable that, with respect to components A, B, C, D, E and F taken together,
i) - component D represents 8 to 13 mol% or 14 to 22% by mass, and/or
- component E represents 9.5 to 17.5 mol% or 11.5 to 19% by mass, and/or
- the D+E components represent 17.5 to 30.5 mol% or 25.5 to 41% by mass,
Or
ii) - component D represents 2 to 22 mole% or 5 to 34% by mass, or
- component E represents 7 to 24 molar% or 6 to 29% by mass,
provided that the D+E components represent 23.5 to 33 mol% or 30.5 to 43 mass%.

The mixture according to embodiment i) is typically capable of being obtained when the molar ratio of the donor (component A) to the acceptor (components B and C) is located in the range from approximately 3/1 to 1 /3, and the acceptor components are approximately equimolar. The mixture according to embodiment ii) is typically capable of being obtained when the molar ratio of the donor (component A) to the acceptor (components B and C) is approximately equimolar, and, in the acceptor, the molar ratio of component B to component C is in the range of approximately 3.5:1 to 1:3.9.

Concerning embodiment i) explained above, it is preferable that the components A+B+C represent 41.5 to 67 mol% or 41 to 66% by mass relative to the components A, B, C, D, E and F taken together, particularly when there is more component B present than component C.

In a more preferred embodiment i),
- component A represents 52 to 58 mol% or 47 to 52% by mass,
- component B represents 4 to 6 mole% or 6.5 to 8.5% by mass,
- component C represents 1 to 2 mole% or 1 to 2% by mass,
- component D represents 8.5 to 10 mol% or 15.5 to 17.5% by mass,
- component E represents 10 to 11.5 mol% or 11.5 to 13% by mass, and
- component F represents 17.5 to 20.5 mol% or 12 to 13.5% by mass,
with respect to components A, B, C, D, E and F taken together.

This mixture is likely to be obtained when the molar ratio of donor (component A) to acceptor (components B and C) is approximately 3:1, and the acceptor components are approximately equimolar.

Also, in a more preferred embodiment i),
- component A represents 42 to 46 molar% or 36 to 40% by mass,
- component B represents 7 to 9 mole% or 11.5 to 13.5% by mass,
- component C represents 3 to 4 mole% or 2.5 to 3.5% by mass,
- component D represents 9 to 11 mol% or 16.5 to 19% by mass,
- component E represents 11 to 13.5 mol% or 13 to 16% by mass, and
- component F represents 20.5 to 23.5 mol% or 13 to 16% by mass,
with respect to components A, B, C, D, E and F taken together.

This mixture is likely to be obtained when the molar ratio of donor (component A) to acceptor (components B and C) is approximately 2/1, and the acceptor components are approximately equimolar.

Also, in a more preferred embodiment i),
- component A represents 7.5 to 9.5 mol% or 5 to 7.5% by mass,
- component B represents 21 to 24 mol% or 30 to 33% by mass,
- component C represents 16 to 19 mole% or 12.5 to 15.5% by mass,
- component D represents 10 to 12 mole% or 16.5 to 19.5% by mass,
- component E represents 14 to 16.5 mol% or 14.5 to 17% by mass, and
- component F represents 23 to 27 mole% or 13 to 16% by mass,
with respect to components A, B, C, D, E and F taken together.

This mixture is likely to be obtained when the molar ratio of donor (component A) to acceptor (components B and C) is approximately 1/2, and the acceptor components are approximately equimolar.

Also, in a more preferred embodiment i),
- component A represents 3 to 5.5 mole% or 2.5 to 4% by mass,
- component B represents 26 to 30 mol% or 36 to 40% by mass,
- component C represents 22.5 to 25.5 mol% or 17 to 20% by mass,
- component D represents 8.5 to 11 mol% or 14 to 16.5% by mass,
- component E represents 11 to 14 mole% or 11 to 14% by mass, and
- component F represents 20 to 23 mol% or 11 to 13.5% by mass,
with respect to components A, B, C, D, E and F taken together.

This mixture is likely to be obtained when the molar ratio of donor (component A) to acceptor (components B and C) is approximately 1/3, and the acceptor components are approximately equimolar.

In a particularly more preferred embodiment i),
- component A represents 19 to 24.5 mol% or 15 to 20% by mass,
- component B represents 12.5 to 15.5 mol% or 19 to 22% by mass,
- component C represents 7 to 10 molar% or 6 to 8% by mass,
- component D represents 11 to 14 molar% or 18 to 22% by mass,
- component E represents 14.5 to 17.5 mol% or 16 to 19% by mass, and
- component F represents 25 to 30 molar% or 15 to 19% by mass,
with respect to components A, B, C, D, E and F taken together.

This mixture is likely to be obtained when the molar ratio of donor (component A) to acceptor (components B and C) is approximately 1:1, and the acceptor components are approximately equimolar.

Concerning embodiment ii) explained above, it is preferable that the components A+B+C represent 40.5 to 54.5 mol% or 41 to 54% by weight relative to the components A, B, C, D , E and F taken together, in particular when component A represents 21 to 27 mol%.

In a more preferred embodiment ii),
- component A represents 21.5 to 25.5% in moles or 16 to 19.5% by mass,
- component B represents 18 to 22 mol% or 24.5 to 28.5% by mass,
- component C represents 2 to 4 mole% or 1.5 to 3.5% by mass,
- component D represents 18 to 22 mole% or 30 to 34% by mass,
- component E represents 6.5 to 9.5 mol% or 6 to 9% by mass, and
- component F represents 23 to 27 mol% or 13 to 15.5% by mass,
with respect to components A, B, C, D, E and F taken together.

This mixture is likely to be obtained when the molar ratio of donor (component A) to acceptor (components B and C) is approximately equimolar and, in the acceptor, the molar ratio of component B to component C is about 3.5/1.

Also, in a more preferred embodiment ii),
- component A represents 21 to 24.5 mol% or 16.5 to 20% by mass,
- component B represents 15 to 18 molar% or 21 to 25% by mass,
- component C represents 5.5 to 7.5 mol% or 4 to 6% by mass,
- component D represents 13 to 16.5 mol% or 22 to 26% by mass,
- component E represents 12 to 15 mole% or 12.5 to 15.5% by mass, and
- component F represents 24 to 28 molar% or 14 to 17% by mass,
with respect to components A, B, C, D, E and F taken together.

This mixture is likely to be obtained when the molar ratio of donor (component A) to acceptor (components B and C) is approximately equimolar and, in the acceptor, the molar ratio of component B to component C is about 1.4/1.

Also, in a more preferred embodiment ii),
- component A represents 21 to 24.5 mol% or 17 to 20.5% by mass,
- component B represents 10.5 to 13.5 mol% or 16.5 to 20% by mass,
- component C represents 9 to 12 molar% or 8 to 10% by mass,
- component D represents 8 to 10 molar% or 14 to 17% by mass,
- component E represents 17.5 to 20.5 mol% or 19.5 to 23% by mass, and
- component F represents 24.5 to 28.5 mol% or 14.5 to 17.5% by mass,
with respect to components A, B, C, D, E and F taken together.

This mixture is likely to be obtained when the molar ratio of donor (component A) to acceptor (components B and C) is approximately equimolar and, in the acceptor, the molar ratio of component B to component C is approximately 1/1.5.

Also, in a more preferred embodiment ii),
- component A represents 23 to 27 mole% or 21 to 25% by mass,
- component B represents 6 to 8 molar% or 10 to 12.5% by mass,
- component C represents 15 to 19.5 mol% or 14 to 16.5% by mass,
- component D represents 2.5 to 4.5 mol% or 5.5 to 7.5% by mass,
- component E represents 21 to 24.5 mol% or 25 to 28.5% by mass, and
- component F represents 23.5 to 27.5 mol% or 16 to 19% by mass,
with respect to components A, B, C, D, E and F taken together.

This mixture is likely to be obtained when the molar ratio of donor (component A) to acceptor (components B and C) is approximately equimolar and, in the acceptor, the molar ratio of component B to component C is about 1/3.9.

In a particularly more preferred embodiment ii),
- component A represents 19 to 24.5 mol% or 15 to 20% by mass,
- component B represents 12.5 to 15.5 mol% or 19 to 22% by mass,
- component C represents 7 to 10 molar% or 6 to 8% by mass,
- component D represents 11 to 14 molar% or 18 to 22% by mass,
- component E represents 14.5 to 17.5 mol% or 16 to 19% by mass, and
- component F represents 25 to 30 molar% or 15 to 19% by mass,
with respect to components A, B, C, D, E and F taken together.

This mixture is likely to be obtained when both the molar ratio of the donor (component A) to the acceptor (components B and C) and the molar ratio of component B to component C are approximately equimolar.

Concerning the mixture which consists or consists essentially of components A, B, C, D and E, it is preferable that, with respect to components A, B, C, D and E taken together,
iii) - component D represents 10 to 17.5 mol% or 16 to 25.5% by mass, and/or
- component E represents 12 to 23.5 mol% or 13 to 22% by mass, and/or
- the D+E components represent 22 to 41 mol% or 29 to 47.5% by mass,
Or
iv) - component D represents 3.5 to 29.5 mole% or 6 to 39% by mass, or
- component E represents 9 to 32 mol% or 7.5 to 34.5% by mass,
provided that the D+E components represent 31.5 to 41 mole percent or 36.5 to 49 mass percent.

The mixture according to embodiment iii) is typically capable of being obtained when the molar ratio of the donor (component A) to the acceptor (components B and C) is located in the range from approximately 3/1 to 1 /3, the acceptor components are approximately equimolar, and lactose is practically eliminated from the composition thus obtained. The mixture according to embodiment iv) is typically capable of being obtained when the molar ratio of the donor (component A) to the acceptor (components B and C) is approximately equimolar, in the acceptor, the molar ratio from component B to component C is in the range from about 3.5:1 to 1:3.9, and lactose is substantially eliminated from the composition thus obtained.

Concerning embodiment iii) explained above, it is preferable that there be more component B present in the mixture than component C.

In a more preferred embodiment iii),
- component A represents 65 to 71 mol% or 53.5 to 59.5% by mass,
- component B represents 5 to 7 molar% or 7.5 to 9.5% by mass,
- component C represents 1.5 to 2.5 mole% or 1 to 2% by mass,
- component D represents 10 to 12.5 mol% or 18 to 20.5% by mass, and
- component E represents 12 to 14.5 mol% or 13 to 15.5% by mass,
with respect to components A, B, C, D and E taken together.

This mixture is likely to be obtained when the molar ratio of donor (component A) to acceptor (components B and C) is approximately 3/1, the acceptor components are approximately equimolar, and the lactose is practically eliminated from the composition thus obtained.

Also, in a more preferred embodiment iii),
- component A represents 54 to 58.5 mol% or 42 to 46.5% by mass,
- component B represents 9 to 11.5 mol% or 13 to 15.5% by mass,
- component C represents 4 to 5 molar% or 3 to 4% by mass,
- component D represents 11.5 to 14 mol% or 19 to 23% by mass,
- component E represents 15 to 18 mol% or 15 to 18.5% by mass,
with respect to components A, B, C, D and E taken together.

This mixture is likely to be obtained when the molar ratio of donor (component A) to acceptor (components B and C) is approximately 2/1, the acceptor components are approximately equimolar, and the lactose is practically eliminated from the composition thus obtained.

Also, in a more preferred embodiment iii),
- component A represents 10 to 12.5 mol% or 6.5 to 9% by mass,
- component B represents 28.5 to 32 mol% or 34.5 to 38.5% by mass,
- component C represents 22 to 25 mol% or 15 to 17.5% by mass,
- component D represents 13.5 to 16 mol% or 19.5 to 22.5% by mass,
- component E represents 19 to 21.5 mol% or 17 to 19.5% by mass,
with respect to components A, B, C, D and E taken together.

This mixture is likely to be obtained when the molar ratio of donor (component A) to acceptor (components B and C) is approximately 1/2, the acceptor components are approximately equimolar, and the lactose is practically eliminated from the composition thus obtained.

Also, in a more preferred embodiment iii),
- component A represents 4.5 to 6.5 mol% or 2.5 to 4.5% by mass,
- component B represents 34 to 38 mol% or 41.5 to 45.5% by mass,
- component C represents 29 to 32.5 mol% or 19 to 22.5% by mass,
- component D represents 11 to 13.5 mol% or 16 to 19% by mass,
- component E represents 14.5 to 17.5 mol% or 13 to 16% by mass,
with respect to components A, B, C, D and E taken together.

This mixture is likely to be obtained when the molar ratio of donor (component A) to acceptor (components B and C) is approximately 1/3, the acceptor components are approximately equimolar, and the lactose is practically eliminated from the composition thus obtained.

In a particularly more preferred embodiment iii),
- component A represents 29 to 33 mol% or 20 to 24.5% by mass,
- component B represents 18 to 21.5 mol% or 23 to 25.5% by mass,
- component C represents 10 to 12.5 mol% or 7.5 to 9.5% by mass,
- component D represents 15 to 17.5 mol% or 22 to 22.5% by mass,
- component E represents 20.5 to 23.5 mol% or 19.5 to 22% by mass,
with respect to components A, B, C, D and E taken together.

This mixture is likely to be obtained when the molar ratio of donor (component A) to acceptor (components B and C) is approximately 1/1, the acceptor components are approximately equimolar, and the lactose is practically eliminated from the composition thus obtained.

Concerning embodiment iv) explained above, it is preferable that components A+B+C represent 57 to 70 mol% or 51 to 64.5% by weight relative to components A, B, C, D and E taken together, in particular that component A represents 29 to 35.5 mol%.

In a more preferred embodiment iv),
- component A represents 29.5 to 33.5 mol% or 19 to 22.5% by mass,
- component B represents 25 to 28.5 mol% or 29 to 33% by mass,
- component C represents 3 to 5 molar% or 2 to 3.5% by mass,
- component D represents 26 to 29.5 mol% or 35 to 39% by mass,
- component E represents 9 to 11.5 mol% or 7.5 to 10% by mass,
with respect to components A, B, C, D and E taken together.

This mixture is likely to be obtained when the molar ratio of the donor (component A) to the acceptor (components B and C) is approximately equimolar, in the acceptor the molar ratio of component B to component C is approximately 3.5/1, and the lactose is practically eliminated from the composition thus obtained.

Also, in a more preferred embodiment iv),
- component A represents 29 to 32.5 mol% or 20.5 to 23% by mass,
- component B represents 21 to 24.5 mol% or 26 to 29.5% by mass,
- component C represents 7.5 to 10 molar% or 5 to 7% by mass,
- component D represents 18 to 21.5 mol% or 27 to 29.5% by mass,
- component E represents 17 to 19.5 mol% or 15.5 to 18% by mass,
with respect to components A, B, C, D and E taken together.

This mixture is likely to be obtained when the molar ratio of the donor (component A) to the acceptor (components B and C) is approximately equimolar, in the acceptor the molar ratio of component B to component C is approximately 1.4/1, and the lactose is practically eliminated from the composition thus obtained.

Also, in a more preferred embodiment iv),
- component A represents 29 to 33 mol% or 22 to 24.5% by mass,
- component B represents 15 to 18 mol% or 20.5 to 23% by mass,
- component C represents 13 to 15.5 mol% or 9.5 to 12% by mass,
- component D represents 11 to 13.5 mol% or 17 to 20% by mass,
- component E represents 24.5 to 27.5 mol% or 24 to 27% by mass,
with respect to components A, B, C, D and E taken together.

This mixture is likely to be obtained when the molar ratio of the donor (component A) to the acceptor (components B and C) is approximately equimolar, in the acceptor the molar ratio of component B to component C is approximately 1/1.5, and the lactose is practically eliminated from the composition thus obtained.

Also, in a more preferred embodiment iv),
- component A represents 31.5 to 35.5 mol% or 26 to 29.5% by mass,
- component B represents 8.5 to 10.5 mol% or 12.5 to 15% by mass,
- component C represents 21 to 24 molar% or 17 to 20% by mass,
- component D represents 3.5 to 5.5 mol% or 6 to 9.5% by mass,
- component E represents 28 to 32 mol% or 30.5 to 34.5% by mass,
with respect to components A, B, C, D and E taken together.

This mixture is likely to be obtained when the molar ratio of the donor (component A) to the acceptor (components B and C) is approximately equimolar, in the acceptor the molar ratio of component B to component C is approximately 1/3.9, and the lactose is practically eliminated from the composition thus obtained.

In a particularly more preferred embodiment iv),
- component A represents 29 to 33 mol% or 21 to 24.5% by mass,
- component B represents 18 to 21.5 mol% or 23 to 25.5% by mass,
- component C represents 10 to 12.5 mol% or 7.5 to 9.5% by mass,
- component D represents 15 to 17.5 mol% or 23 to 25.5% by mass,
- component E represents 20.5 to 23.5 mol% or 19.5 to 22% by mass,
with respect to components A, B, C, D and E taken together.

This mixture is likely to be obtained when both the molar ratio of the donor (component A) to the acceptor (components B and C) and the molar ratio of component B to component C are approximately equimolar, and the lactose is practically eliminated from the composition thus obtained.
Pharmaceutical, cosmetic and/or nutritional composition

A mixture according to the present invention consisting or essentially consisting 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 can contain a pharmaceutically, cosmetically and/or nutritionally acceptable carrier such as phosphate-buffered saline, unbuffered saline, ethanol-in-water mixtures, 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 may also contain other materials that do not produce an adverse, allergic or otherwise undesirable reaction when administered to a patient.

The carriers and other materials included in the pharmaceutical, cosmetic and/or nutritional composition may 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, the tablet dosage forms of the anti-infective mixtures may be coated by aqueous or non-aqueous techniques known to those skilled in the art.

A 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, a capsule, a suppository, an effervescent tablet, a pellet, a tablet, a lozenge, a gel, a paste, a solution, a suspension, an emulsion, a syrup, a bolus, an electuary , a slurry, in an aqueous or non-aqueous liquid, powder, or granules containing a predetermined concentration of the HMO mixture.

Also, a pharmaceutical, cosmetic and/or nutritional composition according to the present invention may contain binders, lubricants, inert diluents, flavoring agents, and humectants. A tablet, capsule, suppository or pellet containing the pharmaceutical, cosmetic and/or nutritional composition of the present invention may optionally be coated or formulated so as to allow prolonged, delayed or controlled release of the anti-infectious HMO mixture.

The HMO mixture according to the invention can be supplemented with additional pharmaceutical agents, active pharmaceutical ingredients or agents having an effect on undesirable conditions for the health of the patient to whom the composition is administered.

In one embodiment, the composition may be in the form of a nutritional composition. For example, the nutritional composition may be a food composition, a rehydration solution, a medical food or a food for a particular medical purpose, a nutritional supplement, and the like. The nutritional composition may contain sources of digestible proteins, lipids and/or carbohydrates, and may be in powder or liquid form. The composition may be designed to be the sole source of nutrition, or as a nutritional supplement.

Suitable protein sources include milk protein, 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 or casein proteins, or mixtures of both. Proteins can be whole proteins or hydrolyzed proteins, either partially hydrolyzed or extensively hydrolyzed. Hydrolyzed proteins offer the benefit of easier digestion, which may be important for humans with inflamed or compromised gastrointestinal tracts. Proteins can also be provided in the form of free amino acids. Protein can represent from about 5% to about 30% of the energy in the nutritional composition, normally 10% to 20%. Ideally, the protein source does not contain excessive amounts of lactose.

The protein source may be a source of glutamine, threonine, cysteine, serine, proline, or a combination of these amino acids. The source of glutamine may be a dipeptide glutamine and/or a glutamine-enriched protein. Glutamine can be incorporated due to the use of glutamine by enterocytes as an energy source. Threonine, serine and proline are important amino acids for mucin production. Mucin coats the gastrointestinal tract and may improve intestinal barrier function and mucosal healing. Cysteine is a major precursor to glutathione, which is key to the body's antioxidant defenses.

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, potato-derived carbohydrates, tapioca, sucrose, glucose, fructose, sucrose, honey, sugar alcohols (e.g. maltitol, erythritol, sorbitol), or their mixtures. Preferably, the composition has a reduced content of or is free of lactose or other FODMAP carbohydrates. In general, digestible carbohydrates provide about 35% to about 55% of the energy in 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 MCT and LCT. For example, MCTs can represent from about 30% to about 70% by weight of lipids, more specifically from about 50% to about 60% by weight. MCTs offer the benefit of easier digestion, which may be important for humans with inflamed or compromised gastrointestinal tracts. In general, lipids provide about 35% to about 50% of the energy in the nutritional composition. Lipids may contain essential fatty acids (omega-3 and omega-6 fatty acids). Preferably, these polyunsaturated fatty acids provide less than approximately 30% of the total energy of the lipid source.

Suitable sources of long-chain triglycerides are canola oil, sunflower oil, palm oil, soybean oil, milk fat, corn oil, higher olefin 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 ratio of omega-6 (n-6) to omega-3 (n-3) polyunsaturated fatty acids of about 4:1 to about 10:1. For example, the ratio of n-6 to n-3 fatty acids can be from about 6:1 to about 9:1.

The nutritional composition may also contain vitamins and minerals. If the nutritional composition is intended to be the sole source of nutrition, it preferably contains 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 acidic 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 may also contain a carotenoid such as lutein, lycopene, zeaxanthin, and beta-carotene. The total amount of carotenoid incorporated can vary from approximately 0.001 µg/ml to approximately 10 µg/ml. Lutein may be present in an amount from about 0.001 µg/ml to about 10 µg/ml, preferably from about 0.044 µg/ml to about 5 µg/ml lutein. Lycopene may be present in an amount of from 0.001 µg/ml to about 10 µg/ml, preferably from about 0.0185 µg/ml to about 5 µg/ml lycopene. Beta-carotene may be present at about 0.001 µg/ml to about 10 mg/ml, for example from about 0.034 µg/ml to about 5 µg/ml beta-carotene.

The nutritional composition also preferably contains reduced concentrations of sodium; for example, from around 300 mg/l to around 400 mg/l. The remaining electrolytes can be present at concentrations established to meet needs without providing undue renal solute load to renal function. For example, potassium is preferably present at a level of approximately 1180 to approximately 1300 mg/l; and the chloride is preferably present at about 680 to about 800 mg/l.

The nutritional composition may also contain various other conventional ingredients such as preservatives, emulsifying agents, thickening agents, buffers, fibers 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 HNOO1, 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), compounds antioxidants/anti-inflammatories, including tocopherols, carotenoids, ascorbate/vitamin C, ascorbyl palmitate, polyphenols, glutathione, and superoxide dismutase (melon), other bioactive factors (e.g. hormones growth, cytokines, TGF-β), colorants, flavors, and stabilizers, lubricants, and so on.

The nutritional composition can be formulated as a soluble powder, a liquid concentrate, or a ready-to-use formulation. The composition can be administered to a human in need via a nasogastric tube or orally. Various flavorings and other additives may also be present.

The nutritional compositions may be prepared by any manufacturing techniques commonly used to prepare 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 source of protein, with heating and stirring. A carbohydrate feed solution is then prepared by adding minerals, trace and ultra-trace elements, thickening or suspending agents, to water, with heating and stirring. The resulting solution is maintained for 10 minutes with continued heating and stirring before the addition of carbohydrates (e.g. HMOs and digestible carbohydrate sources). The resulting feed solutions are then combined together, with heating and stirring, and the pH is adjusted to 6.6-7.0, after which the composition is subjected to a short-term elevated temperature treatment, during which the composition is heat treated, emulsified and homogenized, then left to cool. Water-soluble vitamins and ascorbic acid are added, the pH is adjusted to the desired range, if necessary, flavorings are added, and water is added to achieve the desired total solids content.

For a liquid product, the resulting solution can then be packaged under aseptic conditions to form an aseptically packaged nutritional composition. In this form, the nutritional composition can be in a concentrated liquid form or ready for use. Alternatively, the composition may be spray dried and processed and packaged into a powder for reconstitution.

When the nutritional product is a ready-to-use nutritional liquid, it may be preferable that the total concentration of HMO in the liquid, by weight of the liquid, be from about 0.1% to about 1.5%, inclusive 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 preferable that the total concentration of HMO in the liquid, by weight of the liquid, be from about 0.2% to about 3.0%, including 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 unit dosage form. The unit dosage form may contain an acceptable food grade carrier, e.g., phosphate buffered saline, ethanol-in-water mixtures, water, and emulsions such as oil/water or water emulsion. /oil, as well as various wetting agents or excipients. The unit dosage form may also contain other materials that do not produce an adverse, allergic or otherwise undesirable reaction when administered to a human. Carriers and other materials may 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 disintegrating agents. Preferably, the unit dosage form comprises primarily HMOs with a minimal amount of binders and/or excipients. Unit dosage forms are particularly suitable when they are nutritionally incomplete or are not intended to serve as the sole source of nutrition.

A unit dosage form may be administered orally, for example as a tablet, capsule, or pellet containing a predetermined amount of the mixture, or as a powder or granules relating to a predetermined concentration of the mixture, or a gel, paste, a solution, suspension, emulsion, syrup, bolus, electuary, slurry, in an aqueous or non-aqueous liquid, containing a predetermined concentration of the mixture. An orally administered composition may contain one or more binders, lubricants, inert diluents, flavoring agents, and humectants. An orally administered composition such as a tablet may optionally be coated and may be formulated to allow prolonged, delayed or controlled release of HMOs.

A unit dosage form may also be administered by nasogastric tube or direct infusion into the gastrointestinal tract or stomach.

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

The appropriate dosage of a nutritional composition for a human may be determined in a conventional manner, based on factors such as the concentration of HMO, and the general condition, immune status, body weight and age of the human. the human. The required amount of HMO should generally be in the range of about 1 g to about 15 g per day, in some embodiments from about 2 g to about 10 g per day, e.g. from about 3 g to about 7 g per day. Suitable dosage regimens can be determined by methods known to those skilled in the art.

In another embodiment, the HMO mixture may be formulated as a pharmaceutical composition. The pharmaceutical composition may contain a pharmaceutically acceptable carrier, for example phosphate-buffered saline, 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 composition may also contain other materials that do not produce an adverse, allergic or otherwise undesirable reaction when administered to a human. Carriers and other materials may 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 disintegrating agents.

The pharmaceutical compositions can be administered orally, for example as a tablet, a capsule, or a pellet containing a predetermined quantity, or as a powder or granules relating to a predetermined concentration, or a gel, a paste, a solution, a suspension, emulsion, syrup, bolus, electuary, or slurry, in an aqueous or non-aqueous liquid, containing a predetermined concentration. Orally administered compositions may contain binders, lubricants, inert diluents, flavoring agents, and humectants. Compositions administered orally such as tablets may optionally be coated and may be formulated so as to allow prolonged, delayed or controlled release of the mixture they contain.

The pharmaceutical compositions may also be administered by means of a rectal suppository, an aerosol tube, a nasogastric tube or by direct infusion into the gastrointestinal tract or stomach.

The pharmaceutical compositions may also include therapeutic agents such as antibiotics, probiotics, analgesics, and anti-inflammatory agents. The appropriate dose of a pharmaceutical composition can be determined in a conventional manner, based on factors such as the concentration of the HMOs, and the general condition, immune status, body weight and age of the patient. The required amount of HMO should generally be in the range of about 1 g to about 15 g per day, in some embodiments from about 2 g to about 10 g per day, e.g. from about 3 g to about 7 g per day. Suitable dosage regimens can be determined by methods known to those skilled in the art.
Use of a mixture of HMOs according to the present invention

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

Individual HMOs are known to have health benefits and are known to have an effect on bacterial and viral infections. While isolated individual HMOs are beneficial on their own, combined mixtures of HMOs, as described herein, have a synergistic effect in the treatment of infections, compared to the isolated individual components.

An object of the invention relates to a mixture consisting or essentially consisting of 3-FL, LNFP-I, 2'-FL, LNDFH-I, DFL and optionally lactose, including the preferred and more preferred embodiments described above. , or a composition comprising the 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 described above, for use in the treatment, prevention and/or improvement of a disease and/or a condition linked to an imbalance of the microbiome. Alternatively, an object of the invention relates to a method of treatment, prevention and/or improvement of a disease and/or a condition linked to an imbalance of the microbiome in a human, comprising the administration to the human of 'a mixture consisting of or essentially consisting of 3-FL, LNFP-I, 2'-FL, LNDFH-I, DFL and optionally lactose, or a composition comprising the mixture consisting of or essentially consisting of 3-FL, LNFP- I, 2'-FL, LNDFH-I, DFL and possibly 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, for example to increase the abundance of Bifidobacterium and the abundance of Barnesiella, said modulation reducing the abundance of Firmicutes, in particular Clostridia.

Furthermore, another subject of the invention relates to a mixture consisting or essentially consisting of 3-FL, LNFP-I, 2'-FL, LNDFH-I, DFL and optionally lactose, including preferred embodiments and more described above, or a composition comprising the 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 described above, for use in treating and/or reducing the risk of a wide range of bacterial or viral infections in a human. Alternatively, an object of the invention relates to a method of treating and/or reducing the risk of a wide range of bacterial or viral infections in a human, comprising the administration to the human of a mixture consisting of or essentially consisting of 3-FL, LNFP-I, 2'-FL, LNDFH-I, DFL and optionally lactose, or of a composition comprising the mixture consisting or essentially consisting of 3-FL, LNFP-I, 2'- FL, LNDFH-I, DFL and possibly lactose. Infections can occur in several parts of the body, not limited to the gut microbiome, but also in other parts of the body exposed to the external environment, such as the 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 species of Salmonella and/or the pathogen responsible for Pseudomonas aeruginosa pneumonia, 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 Clostridium difficile toxins. One or more HMOs of the mixture according to the present invention can serve as a modulator of the immune system and affect intestinal health by stimulating bifidobacteria. One or more HMOs of the mixture according to the present invention can inhibit the growth of Group B Streptococcus both in infants and in breast milk. Group B Streptococcus is a major cause of neonatal septicemia, pneumonia and meningitis. One or more HMOs of the mixture according to the present invention can bind directly to the Shiga toxins Stx2 and Stx1B5 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 due to either bacterial or viral pathogens, most likely due to the binding of bacterial exotoxins.

Furthermore, another object of the invention relates to a non-medical use of a mixture consisting or essentially consisting of 3-FL, LNFP-I, 2'-FL, LNDFH-I, DFL and optionally lactose, including preferred and more preferred embodiments described above, or a composition comprising the mixture consisting of or essentially consisting of 3-FL, LNFP-I, 2'-FL, LNDFH-I, DFL and optionally lactose, including preferred and more preferred embodiments described above, for maintaining commensal homeostasis of the intestinal microbiota in a human.

Furthermore, another object of the invention relates to a non-medical use of a mixture consisting or essentially consisting of 3-FL, LNFP-I, 2'-FL, LNDFH-I, DFL and optionally lactose, including preferred and more preferred embodiments described above, or a composition comprising the mixture consisting of or essentially consisting of 3-FL, LNFP-I, 2'-FL, LNDFH-I, DFL and optionally lactose, including preferred and more preferred embodiments described above, in the dietary management of a human.

Additionally, 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 described above, enhances the viability useful probiotics in aqueous compositions. Probiotic strains, for example strains of Lactobacillus rhamnosus , when mixed with HMOs, have been found to have significantly increased regeneration and viability when coming into contact with a low pH liquid environment, such as lactobacillus rhamnosus. stomach acid or acidic drinks. Thus, the combination of the HMO mixture according to the present invention together with probiotic strains, preferably freeze-dried probiotics, can offer a reliable means of delivering a quantifiable quantity of probiotics to a host (human or animal), either in type form pharmaceutical, in nutritional compositions, or in a food-based form.

Although 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 objects of the invention are made available:
1. A mixture consisting or essentially consisting 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 possibly a component F which is lactose.
2. The mixture according to object 1, essentially consisting of 3-FL, LNFP-I, 2'-FL, LNDFH-I, DFL and lactose, preferably in which
- component D represents 8 to 13 molar% or 14 to 22% by mass, and/or
- component E represents 9.5 to 17.5 mol% or 11.5 to 19% by mass, and/or
- the D+E components represent 17.5 to 30.5 mol% or 25.5 to 41% by mass,
with respect to components A, B, C, D and E taken together,
Or
- component D represents 2 to 22 mol% or 5 to 34% by mass, or
- component E represents 7 to 24 molar% or 6 to 29% by mass,
with respect to components A, B, C, D, E and F taken together, provided that components D+E represent 23.5 to 33 mol% or 30.5 to 43% by mass.
3. The mixture according to object 1 or 2, in which
- the components A+B+C represent 41.5 to 67 molar% or 41 to 66% by weight, preferably in which there is more component B than component C present, or
- the components A+B+C represent 40.5 to 54.5 molar% or 41 to 54% by weight, preferably in which component A represents 21 to 27 mole%.
4. The mixture according to any of the preceding objects, in which
- component A represents 19 to 24.5 mol% or 15 to 20% by mass,
- component B represents 12.5 to 15.5 mol% or 19 to 22% by mass,
- component C represents 7 to 10 molar% or 6 to 8% by mass,
- component D represents 11 to 14 molar% or 18 to 22% by mass,
- component E represents 14.5 to 17.5 mol% or 16 to 19% by mass, and
- component F represents 25 to 30 mol% or 15 to 19% by mass.
5. The mixture according to object 1, essentially consisting of 3-FL, LNFP-I, 2'-FL, LNDFH-I and DFL, preferably in which
- component D represents 10 to 17.5 mol% or 16 to 25.5% by mass, and/or
- component E represents 12 to 23.5 mol% or 13 to 22% by mass, and/or
- the D+E components represent 22 to 41 mol% or 29 to 47.5% by mass,
with respect to components A, B, C, D and E taken together,

Or
- component D represents 3.5 to 29.5 mol% or 6 to 39% by mass, and/or
- component E represents 9 to 32 mol% or 7.5 to 34.5% by mass,
with respect to components A, B, C, D and E taken together,
provided that the D+E components represent 31.5 to 41 mole percent or 36.5 to 49 mass percent.
6. The mixture according to object 5, in which
- there is more component B than component C in the mixture, or
- the components A+B+C represent 57 to 70 mole% or 51 to 64.5% by weight, preferably in which component A represents 29 to 35.5 mole%.
7. The mixture according to object 1, 5 or 6, in which
- component A represents 29 to 33 mol% or 20 to 24.5% by mass,
- component B represents 18 to 21.5 mol% or 23 to 25.5% by mass,
- component C represents 10 to 12.5 mol% or 7.5 to 9.5% by mass,
- component D represents 15 to 17.5 mol% or 22 to 25.5% by mass,
- component E represents 20.5 to 23.5 mol% or 19.5 to 22% by mass.
8. A composition comprising the mixture according to any of the preceding objects, preferably said composition further comprising a probiotic, more preferably L. rhamnosus , in particular L. rhamnosus LGG DSM 33156.
9. A process for obtaining the mixture according to any one of objects 1 to 7, said process comprising the reaction of 3-FL, LNFP-I and 2'-FL in the presence of an α1-3/4-transfucosidase to producing 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 or consists of the sequence of acids amino acids of SEQ ID NO: 1 with the following mutations: W135F-A174N-N274A-E413R.
10. A mixture or composition according to any of objects 1 to 8, for use as a medicine, preferably
- in which a human's microbiome is modulated, or
- wherein the use is for the treatment and/or reduction of the risk of a wide range of bacterial or viral infections in a human.
11. A use of the mixture according to any one of objects 1 to 8 to amplify the viability of probiotics, preferably L. rhamnosus , more preferably L. rhamnosus LGG DSM 33156, in an aqueous composition.

Examples

Example 1

In a 20 liter fermenter, a solution of 3-FL (4.278 kg, corrected for dosage, 8.76 moles), LNFP-I (3.807 kg, corrected for dosage, 4.46 moles) and 2'-FL (2.072 kg, corrected for dosage, 4.24 moles) in water (17.5 kg). The pH of the solution is adjusted to 6.0 with a 7% HCl solution. A lyophilized powder (11.6 g) of the W135F-A174N-N274A-E413R mutant of Bifidobacterium longum subsp. is dissolved in water (100 g). infantis ATCC 15697 (the numbering of the amino acids being in accordance with SEQ ID NO: 1, see document WO 2016/063261) and everything is added to the solution above, then the reaction mixture is stirred at 25 ° C for 18 hours, after which it is heated to 55°C over 4 hours and stirring is continued at this temperature for 2 hours. Next, the pH of the mixture was established at 5.0 with a 7% HCl solution and the mixture was heated to 70°C over 1 hour and maintained at this temperature for 2 hours. The mixture is then allowed to cool to 15°C while the enzyme precipitates. The suspension obtained is then subjected to ultrafiltration (15 kDa ceramic membrane, 0.5 m ² , transmembrane pressure 5.8 to 5.9 bars, temperature 19 to 24°C) and 28.05 kg of permeate are collected. The permeate is subjected to microfiltration (0.2 μm PES membrane) and lyophilized, which gives 10.57 g of powder (saccharides by HPLC: 95% by weight among which 3-FL represents 16% by weight, LNFP-I represents 19% by weight, 2'-FL represents 7% by weight, LNDFH-I represents 18% by weight, DFL represents 17% by weight and lactose represents 18% by weight; protein content by the Bradford test : < 6 ppm).

Example 2

In accordance with the protocol set out in Example 1, the following compositions are obtained with different donor/acceptor ratios: Relative molar ratio Starting Product Product Giver Acceptor 3-FL LNFP-I 2'-FL 3-FL LNFP-I 2'-FL LNDFH-I DFL lactose 0.51 0.49 0.23 0.14 0.08 0.12 0.16 0.27 0.24 0.25 0.52 0.48 0.23 0.16 0.06 0.15 0.14 0.26 0.28 0.2 0.51 0.49 0.23 0.20 0.03 0.20 0.08 0.26 0.38 0.21 0.52 0.48 0.23 0.12 0.1 0.09 0.19 0.27 0.19 0.29 0.51 0.49 0.23 0.07 0.17 0.03 0.23 0.25 0.1 0.39 0.50 0.5 0.21 0.13 0.09 0.11 0.16 0.3 0.25 0.25 0.76 0.24 0.55 0.05 0.02 0.09 0.10 0.19 0.12 0.12 0.68 0.32 0.44 0.08 0.03 0.10 0.13 0.22 0.16 0.16 0.35 0.65 0.08 0.23 0.18 0.11 0.15 0.25 0.32 0.33 0.26 0.74 0.04 0.28 0.24 0.10 0.13 0.21 0.36 0.38

Example 3

Solutions of lyophilized Lactobacillus rhamnosus (Probio-Tec® LGG®, Chr. Hansen, deposited in the DSMZ culture collection with accession number DSM 33156, 0.4 mg/ml) and HMO are heated to 37°C. (2'-FL, 2'-FL/DFL in a weight ratio of 6.1/1 and the HMO mixture of Example 1, respectively, 5% w/v) in sterile buffered saline solution phosphate (PBS, pH = 3), and mix vigorously for about 30 seconds until no visible lumps remain. The control contains only the probiotic. The tubes are incubated at 37°C for 3 hours. The samples are further diluted and 100 µl is spread in duplicate on MRS agar plates incubated for 48 hours at 37°C in anaerobic chambers. Results are expressed as mean values with standard deviation of colony forming units (CFU) per milliliter, and calculated from LGG colonies on agar plates ( ).

The lyophilized LGG dissolved with a mixture of HMOs according to the invention presents a regeneration and a capacity of survival strongly and significantly amplified in comparison with the control or certain of the ingredients of the mixture of HMO after 3 hours of incubation at 37 ° C in Acidic conditions at pH 3.0, as determined by colony counts. The data suggest that the reduction in regeneration and viability of L. rhamnosus after exposure to low pH conditions, for example in the stomach or in an acidic beverage, can be prevented in the presence of HMO.

Claims (25)

Mixture consisting or essentially consisting 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 possibly a component F which is lactose. A mixture according to claim 1, comprising, essentially consisting of, or consisting of 3-FL, LNFP-I, 2'-FL, LNDFH-I, DFL and lactose. Mixture according to claim 1 or 2, in which
- component D represents 8 to 13 molar% or 14 to 22% by mass, and/or
component E represents 9.5 to 17.5 mol% or 11.5 to 19% by mass, and/or
- the D+E components represent 17.5 to 30.5 mol% or 25.5 to 41% by mass,
with respect to components A, B, C, D, E and F taken together Mixture according to claim 1 or 2, in which
- component D represents 2 to 22 mol% or 5 to 34% by mass, or
- component E represents 7 to 24 molar% or 6 to 29% by mass,
with respect to components A, B, C, D, E and F taken together, provided that components D+E represent 23.5 to 33 mol% or 30.5 to 43% by mass. Mixture according to claim 3, in which the components A+B+C represent 41.5 to 67 mol% or 41 to 66% by mass, preferably in which there is more component B than component C present. Mixture according to claim 4, in which the components A+B+C represent 40.5 to 54.5 mol% or 41 to 54% by mass, preferably in which the component A represents 21 to 27 mol%. Mixture according to any one of the preceding claims, in which
- component A represents 19 to 24.5 mol% or 15 to 20% by mass,
- component B represents 12.5 to 15.5 mol% or 19 to 22% by mass,
- component C represents 7 to 10 molar% or 6 to 8% by mass,
- component D represents 11 to 14 molar% or 18 to 22% by mass,
- component E represents 14.5 to 17.5 mol% or 16 to 19% by mass, and
- component F represents 25 to 30 mol% or 15 to 19% by mass. A mixture according to claim 1, comprising, essentially consisting of or consisting of 3-FL, LNFP-I, 2'-FL, LNDFH-I and DFL. Mixture according to claim 1 or 8, in which
- component D represents 10 to 17.5 mol% or 16 to 25.5% by mass, and/or
- component E represents 12 to 23.5 mol% or 13 to 22% by mass, and/or
- the D+E components represent 22 to 41 mol% or 29 to 47.5% by mass,
with respect to components A, B, C, D and E taken together. Mixture according to claim 1 or 8, in which
- component D represents 3.5 to 29.5 mol% or 6 to 39% by mass, or
- component E represents 9 to 32 mol% or 7.5 to 34.5% by mass,
relative to components A, B, C, D and E taken together, provided that components D+E represent 31.5 to 41 mol% or 36.5 to 49% by mass. A mixture according to claim 9, wherein there is more component B than component C present in the mixture. Mixture according to claim 10, in which the components A+B+C represent 57 to 70 mol% or 51 to 64.5% by mass, preferably in which the component A represents 29 to 35.5 mol%. Mixture according to any one of claims 1 and 8 to 12, in which
- component A represents 29 to 33 mol% or 20 to 24.5% by mass,
- component B represents 18 to 21.5 mol% or 23 to 25.5% by mass,
- component C represents 10 to 12.5 mol% or 7.5 to 9.5% by mass,
- component D represents 15 to 17.5 mol% or 22 to 25.5% by mass,
- component E represents 20.5 to 23.5 mol% or 19.5 to 22% by mass. Composition comprising the mixture according to any one of the preceding claims. Composition according to claim 14, further comprising a probiotic, preferably L. rhamnosus , more preferably L. rhamnosus LGG DSM 33156. Composition according to claim 14 or 15, which is a nutritional, pharmaceutical or cosmetic composition. A process for obtaining the mixture of any of claims 1 to 13, said process comprising reacting 3-FL, LNFP-I and 2'-FL in the presence of an α1-3/4-transfucosidase to produce a mixture reaction, and then eliminating the α1-3/4-transfucosidase and optionally lactose from the reaction mixture A method according to claim 17, wherein the α1-3/4-transfucosidase comprises or consists of the amino acid sequence of SEQ ID NO: 1 with the following mutations: W135F-A174N-N274A-E413R. A method according to claim 17 or 18 which is carried out under essentially buffer-free conditions. Mixture or composition according to any one of claims 1 to 16, for use as a medicament. A mixture for use or composition for use according to claim 20, wherein the microbiome of a human is modulated. A mixture for use or composition for use according to claim 20, wherein the use is the treatment and/or reduction of the risk of a wide range of bacterial or viral infections in a human. Non-medical use of the mixture according to any one of claims 1 to 13 or of the nutritional or cosmetic composition according to any one of claims 14 to 16 for modulating the microbiome of a human. Use of the mixture according to any one of claims 1 to 13 to amplify the viability of probiotics, preferably L. rhamnosus , more preferably L. rhamnosus LGG DSM 33156, in an aqueous composition. Use of the mixture according to any one of claims 1 to 13 for a cosmetic application.