FIELD OF THE INVENTION

The invention disclosed herein relates to a composition comprising 2’-fucosyl lactose (2’-FL) for use in the prevention of asthma in a human subject.

BACKGROUND

Asthma is often described as an allergic disease in which allergens (such as pollens, or mites) or certain workplace exposures can trigger attacks of airway narrowing and, through continued exposure, lead to airway inflammation and enhanced airway responsiveness. However, this association came from observations predominantly in western high-income countries, and this association between allergy and asthma is much weaker in low and middle income countries. Some occupational causes of asthma do not appear to involve allergy. It is now widely recognized that allergic mechanisms are involved in half, or less, of the people with asthma. In many people, asthma probably involves non-allergic inflammation of the airways, although we do not understand well the mechanisms involved.

Asthma as used herein is defined as “a heterogeneous disease, usually characterized by chronic airway inflammation (in accordance with the Global Initiative for Asthma (GINA)). It is defined by the history of respiratory symptoms such as wheeze, shortness of breath, chest tightness and cough that vary over time and in intensity, together with variable expiratory airflow limitation”. Asthma symptoms most commonly develop for the first time in early childhood. Asthma is a common chronic disease that is estimated to affect as many as 339 million people worldwide. It is a cause of substantial burden of disease, including both premature death and reduced quality of life, in people of all ages in all parts of the world.

There are two key asthma treatments: (i) bronchodilators (most commonly b2 - agonists) that reverse airway narrowing by relaxing airway smooth muscle, and (ii) corticosteroids, which treat the underlying airway inflammation (globalasthmareport.org). As asthma is usually characterized by chronic airway inflammation, treatments of asthma also have a chronic character.

Thorburn et al have shown that feeding mice a high-fibre diet yields a distinctive gut microbiota, which increases the levels of short-chain fatty acids, including acetate. High-fibre or acetate-feeding of mice led to marked suppression of allergic airways disease (AAD, a model for human asthma). High-fibre/acetate feeding of pregnant mice imparted on their adult offspring an inability to develop robust AAD. Their results provide evidence for a role of a high-fibre diet and production of acetate in protecting against the development of airway disease in the offspring. The effect of maternal acetate levels equal to or above the median in mice were also observed in human. It was shown that human asthma associates with maternal diet and symptoms may be reduced by administering high dietary fiber levels to the mother during late pregnancy. Such a diet was associated with high acetate levels in serum and with a decrease in percentage of infants requiring two or more general practitioner (GP) visits for cough or wheeze during the first 12 months (Thorburn et al 2015 Nature Communications 6:7320 | DOI: 10.1038/ncomms8320).

So, there is a need for therapeutic and non-therapeutic ways to prevent or at least reduce the symptoms of asthma. There is also a desire to reduce or minimize the need to use asthma treatments, both in view of costs and in view of health comfort.

In addition, it is desired that such a treatment is compatible with a normal and/or healthy diet. Preferably without negative effects on taste (of food) and/or mouth feeling. Other desired properties or effects include, but are not limited to, maintaining other signs of general health in the subject (e.g. maintaining a normal blood pressure, maintaining a healthy bowel movement, normal defecation, etc.), ease of implementation, ease of preparation, increasing the diversity of microbiota, and commercial availability of the compounds, combinations, and/or compositions used therein.

It is further desired to provide a composition for use in the non-therapeutic treatment of asthma. Preferably the treatment or prevention of asthma does not require a the administration of medicaments by young children.

It is an objective of the present invention to provide a composition that better addresses at least one of the aforementioned desires.

SUMMARY OF THE INVENTION

In one aspect, the invention provides a composition comprising 2’- fucosy I lactose (2’-FL) for use in the prevention of asthma in a human subject by administering the composition to the mother while being pregnant with the subject. In another aspect, the invention provides a method of preventing asthma in a human subject comprising the step of administering a composition comprising 2’FL to the mother of the subject while being pregnant with the subject. The method may be therapeutic or non-therapeutic.

In a further aspect the invention relates to the use of a composition comprising 2’FL to prevent asthma in a human subject characterized in that the composition is administered to the mother of the subject while being pregnant with the subject.

The invention as described herein is based on the finding that a specific dietary fiber i.e. 2’-fucosyllactose (2’-FL) can be administered, preferably orally, to a human subject to achieve at least one of the above desires. In particular, it was found that a composition comprising 2’-FL substantially increases the level of acetate in the distal part of the colon in human subjects. Therefore, the invention relates to a composition comprising 2’-fucosyllactose (2’-FL) for use in the treatment or prevention of asthma in a human subject by administering the composition to the mother while being pregnant with the subject.

DETAILED DESCRIPTION OF THE INVENTION

The term "treatment", in relation a given disease or disorder, includes, but is not limited to, inhibiting the disease or disorder, for example, arresting the development of the disease or disorder; relieving the disease or disorder, for example, causing regression of the disease or disorder; or relieving a condition caused by, or resulting from, the disease or disorder, for example, relieving, preventing or treating symptoms of the disease or disorder.

The term "prevention" in relation to a given disease or disorder means preventing the onset of disease development if none had occurred, preventing the disease or disorder from occurring in a subject that may not yet have been diagnosed as having the disorder or disease, and/or preventing further disease/disorder development if already present.

As used herein, “prevention of asthma” or “preventing asthma” is defined as ameliorating the risk of suffering from asthma after birth. Suffering from asthma in a young child may for example be measured by the number of GP visits for cough and/or wheeze during the first 12 or 24 months of a child’s life. 2'-Fucosyllactose (2'-FL) is an oligosaccharide, more precisely, a fucosylated, neutral trisaccharide composed of L-fucose, D-galactose, and D-glucose units, linked Fuc(a1-2)Gal(pi-4)Glc; CAS Nr 41263-94-9. It is the most prevalent human milk oligosaccharide (FIMO) naturally present in human breast milk, making up about 30% of all of HMOs, at least in so called secretor mothers (i.e. FUT2 positive). HMOs are non-digestible carbohydrates and are the third most abundant component in human milk after lactose and fat. More than 200 different oligosaccharides have currently been identified in human milk. It has been suggested in clinical trials that 2’-FL plays a key role in protecting and promoting the health of newborn infants, particularly in respect to the immune system. It has been shown that the addition of 2’-FL to infant formula is safe and well-tolerated. In addition, 2’-FL is safe and well-tolerated for all other age groups, especially for adults.

HMOs can be obtained using methods known to those of skill in the art. For example, HMOs can be purified from human milk. Individual HMOs can be further separated using methods known in the art such as capillary electrophoresis, HPLC (e.g., high-performance anion-exchange chromatography with pulsed amperometric detection; HPAEC-PAD), and thin layer chromatography. See, e.g., U.S. Patent Application No. 2009/0098240. Alternately, enzymatic methods can be used to synthesize HMOs. Another method to manufacture HMO’s is via biosynthesis in engineered bacteria. For example, a method of preparing 2’-FL is disclosed in WO 2012/112777. Alternatively, 2’-FL is commercially available e.g. from FrieslandCampina, or others.

An amount of a compound such as 2’FL, in an composition, expressed in “a number of grams (or milligrams) per daily serving” as used herein means that the amount of the compound in the composition is such that when administering the recommended daily serving (i.e. daily dosage) of the composition to a subject, the subject will be administered with the number of grams of the compound. So, if the recommended daily dosage is 100 gram of composition divided over 2 portions, then a single serving consists of 50 gram of composition; a daily serving consists of 2 of such single servings.

As used herein, dietary fibers refers to carbohydrates which are neither digested nor absorbed by humans but are utilized by gut microbes. They can be isolated from plants or synthesized from sugars and are comprising at least 3 monosaccharide units (i.e. degree of polymerization DP> 3). Their fermentation in the gastrointestinal tract impacts the composition of bacterial communities as well as microbial metabolic activities, including the production of fermentative end products in the gut of the host. Some dietary fibers can also be classified as prebiotic. A prebiotic is defined as a non-digestible food ingredient that promotes the growth of beneficial microorganisms in the intestines of the host. Consumption of prebiotics result in specific changes in the composition and/or activity of the gastrointestinal microbiota, thus conferring benefit(s) upon host health.

Sources of prebiotic dietary fiber can be selected from one or more of the group consisting of beta glucan, FOS (including inulin), GOS, isomaltooligosaccharides, guar gum, lactulose, resistant starches (RS), maltodextrin, xylooligosaccharides and arabinooligosaccharides. Preferably, the prebiotic dietary fiber is selected from one or more of the group consisting of FOS and GOS. Prebiotic dietary fibers are claimed to have several health effects such as effect on gut microbacteria, metabolite production such as short chain fatty acids (SCFAs) that can have many positive outcomes for the host, effects on mineral absorption, effects on protein fermentation, changes in bacterial pathogenic populations, effects on allergy risk, effects on gut barrier permeability, and effect on immune system defense. See for example Health Effects and Sources of Prebiotic Dietary Fiber by Calson et al 2018, Curr Dev Nutr 2018;2:nzy005 doi: https://doi.Org/10.1093/cdn/nzv005.

Fructooligosaccharides (FOS), also sometimes called oligofructose or oligofructan, are oligosaccharide fructans. They may be obtained using inulin degradation or transfructosylation processes, using methods known in the art. FOS can be produced by degradation of inulin, or polyfructose, a polymer of D-fructose residues linked by b(2 1) bonds with a terminal a(1 2) linked D-glucose. The degree of polymerization of inulin ranges from 10 to 60. Inulin can be degraded enzymatically or chemically to a mixture of oligosaccharides with the general structure Glu-Fru _n (abbrev. GFn), with n ranging from 1 to 7. The second class of FOS is prepared by the transfructosylation action of a b-fructosidase of an Aspergillus e.g. Aspergillus niger on sucrose. The resulting mixture has the general formula of GFm, with m ranging from 1 to 5.

It must also be noted that, as used in the specification and the appended claims, the singular form "a", "an," and "the" comprise plural referents unless the context clearly indicates otherwise. For example, reference to a component in the singular is intended to comprise a plurality of components. It will be understood that within this disclosure, any reference to a weight, weight ratio, and the like pertains to the dry matter, in particular the dry matter of the composition, unless specified otherwise.

Unless defined otherwise, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

In a first aspect the invention provides a composition comprising 2’- fucosy I lactose (2’-FL) for use in the prevention of asthma in a human subject by administering the composition to the mother while being pregnant with the subject.

So far, no transfer of health effects of using 2’FL from the mother to the unborn child were known.

In another aspect, the invention relates to a method of preventing asthma in a human subject comprising the step of administering a composition comprising 2’FL to the mother of the subject while being pregnant with the subject.

In still another aspect, the invention relates to the use of a composition comprising 2’FL to prevent asthma in a human subject characterized in that the composition is administered to the mother of the subject while being pregnant with the subject. The use of the composition in this aspect of the invention may be therapeutic and / or non-therapeutic. Preferably, the use in the various aspects and embodiments of the invention is non-therapeutic.

In one embodiment of any of the aspects of the invention, the human subject as referred to in the various aspects and embodiments of the invention may have any age, e.g. a baby (0-12 months), toddler (1-3 years), preschool child (3-5 years), grade-schooler (5-12 years), teen (12-18 years), young adult (18-21 years) or an adult (>21 years). Since it is difficult to administer asthma medicines to young children in one embodiment the subject preferably is a baby, toddler or preschool child, more preferably the subject is a baby or toddler, most preferably the subject is a baby. Alternatively, because of the chronic character of asthma, subjects suffering from asthma have to take medicines for many years, so in another embodiment the subject is a teen or young adult. In another embodiment the subject is an adult.

Preferably the mother as referred to in the various aspect and / or embodiments of the invention is not suffering from asthma as it is suggested that maternal acetate levels have more impact on the child’s asthma levels in the first year of life if the mother were not asthmatic (Thorburn et al 2015 Nature Communications 6:7320 | DOI: 10.1038/ncomms8320).

The composition comprising 2’FL may administered to the mother during the complete pregnancy. Alternatively, in one embodiment it may be administered to the mother during the first 3 months of the pregnancy, preferably during the first 6 months of the pregnancy. In yet another embodiment, the composition comprising 2’FL is administered to the mother during the last 2 months of the pregnancy, preferably during the last 3 months of the pregnancy, more preferably during the last 6 months of the pregnancy, even more preferably during the last 8 months of the pregnancy. Most preferably, the composition comprising 2’FL is administered to the mother during the 2 ^nd half of the pregnancy. The age of a pregnancy, also referred to as gestational age, is taken from the beginning of the woman's last menstrual period. The normal pregnancy duration for humans is assumed by medical professionals to be 40 weeks.

For most humans, there is a maximum amount of dietary fiber that can be consumed on a daily basis, an amount above which gastro-intestinal discomfort starts to develop. The maximum amount of dietary fiber that can be consumed per day may be determined by gradually increasing the daily amount of dietary fiber and monitoring if adverse effects occur, one may for example start with 1 gram of 2’FL per day and increase with 3 grams per week. For 2’FL a daily dosage of 20 g has been tested as safe. The amount of 2’-FL in the composition as used in the invention also depends on the body weight (i.e. mass) of the subject; it is believed that heavier persons tolerate more dietary fiber. So, in one embodiment the amount of 2’-FL per daily serving in the composition as used in the invention is more than 0.01 gram preferably more than 0.1 gram. In another embodiment, the amount of 2’FL per daily serving is in the range of from 0.01 to 30 gram per daily serving, preferably it is in the range of from 0.1 to 30 gram per daily serving, more preferably in a range of from 0.5 to 25 gram, even more preferably in a range of from 1 to 20 gram, most preferably in a range of from 1 to 10 gram per daily serving. In still another embodiment, the amount of 2’FL is between 0.01 and 1 .0 gram per daily serving.

In another embodiment the amount of 2’FL in the composition for use in the invention is at least 0.1 wt% compared to the total dry weight of the composition, such as at least 0.5 wt, or at least 1 wt%. In one embodiment it is at least 5wt% compared to the total dry weight of the composition, preferably at least 10 wt%, more preferably at least 15wt% even more preferably at least 20 wt%, particularly preferably at least 30wt%, most preferably at least 40 wt%.

The term “GOS” as used herein, stands for galacto-oligosaccharides (GOS), which generally comprise a chain of galactose units and a glucose unit at the reducing end. GOS arise through consecutive trans-galactosylation reactions catalyzed by a beta-galactosidase (enzyme class EC.3.2.1 .23). Beta-Galactosidase enzymes are produced in many microorganisms such as Bacillus circulans, Aspergillus oryzae, Kluyveromyces marxianus, Kluyveromyces fragilis, Sporobolomyces singularis, and Lactobacillus fermentum. Beta-galactosidases differ in their three-dimensional structures, resulting in stereo- and regioselectivity of the glycosidic bonds that are formed during the trans-galactosylation reactions. For example, typically a fungal beta-galactosidase derived from Aspergillus predominantly produces b1 -6 bonds (thus resulting in a GOS preparation that predominantly comprises b1 -6 bonds, which may be referred to as “6’-GOS”), while a bacterial beta-galactosidase derived from Bacillus predominantly produce b1 -4 bonds (resulting in a GOS preparation that predominantly comprises b1 -4 bonds, which may also be referred to as “4’-GOS”). Moreover, beta-galactosidase produced by B. circulans possesses particularly strong trans-galactosylation activity, and is often used to synthesize GOS. More recently, a beta-galactosidase derived from Cryptococcus terrestris (recently renamed Papiliotrema terrestris) was used in GOS synthesis, as described in EP3399032 and WO 2019/002304. As such, GOS is well- known in the art.

Some of the GOS components exist naturally in human breast milk and colostrum. Typical GOS preparations mainly comprise di- to hexa-saccharides, although larger oligosaccharides may also occur.

Various physiological functions of GOS have been reported, including the capacity to stimulate the growth of bifidogenic bacteria in the gut, to support normal gut transit, to contribute to natural defenses and to enhance mineral absorption. GOS has received particular attention for their prebiotic effects that promote the growth of Bifidobacterium, Lactobacillus, and other enteric bacteria. Therefore, GOS is commonly used in infant formula, beverages fermented by Lactobacillus, and yogurts. Some of these foods containing GOS are certified as Food for Specified Health Uses by the Consumer Affairs Agency in Japan, and GOS is certified as generally recognized as safe (GRAS) substances by the U.S. Food and Drug Administration (GRAS Notices: GRN 233, 236, 285, 286, 334, 484, 489, 495, 518, and 569).

Preferably, GOS has a polymerization degree (DP) in a range of from 2 to 10 more preferably in a range of from 3 to 8. Suitable GOS preparations are commercially available, for example Vivinal® GOS available from FrieslandCampina Nederland B.V.. In this respect, it is noted that a GOS preparation (e.g. Vivinal® GOS) may also contain lactose and/or monosaccharides in addition to galacto- oligosaccharides. In relation to the invention, the amounts of GOS mentioned in the present application relate to the actual GOS content in a preparation, i.e. the actual amount of galacto-oligosaccharides, excluding lactose and excluding monosaccharides, if present.

It is known that the speed and place of fermentation of different prebiotic dietary fibers in the colon is dependent on the type of fibers. For example, it is known that galacto-oligosaccharides (GOS) are fermented quickly upon colonic arrival in the cecum and proximal part of the colon. Significant amounts of acetate being produced with the fermentation of GOS is converted to other metabolites. Fermentation of GOS does not result in an increase of acetate levels (i.e. acetate concentration) in the distal colon (Canfora et al. Gastroenterology, 2017, vol 153 No. 1 pp 87-97).

Without wishing to be bound by any particular theory, the inventors believe that when a mixture of dietary fibers, including GOS and 2’-FL is administered to a subject, the colonic microbiota first ferments GOS as an energy source. Thus, it is believed that (more of) the 2’-FL can reach the distal colon where it is fermented by other gut microbes which produce acetate. Acetate production in the distal colon may then lead to its metabolic effects. E.g. acetate may exert beneficial effects locally and/or may enter the systemic circulation. A similar effect may occur when 2’FL is combined with other dietary fibers, e.g. prebiotic dietary fibers such as fructo- oligosaccharides (FOS), inulin, xylooligosaccharides (XOS) or resistant starch.

As used herein, “distal colon” refers to the descending colon (the left side of the colon) and the sigmoid colon (the S-shaped section of the colon that connects to the rectum).

So, in another embodiment, the composition for use of the invention, further comprises one or more dietary fibers, preferably wherein the dietary fiber is a prebiotic dietary fiber. In yet another embodiment, it further comprises one or more prebiotic dietary fibers wherein the prebiotic dietary fiber is selected from one or more of the group consisting of GOS, FOS, inulin and resistant starch, preferably GOS, FOS and inulin, particularly it further comprises one or more of the group consisting of FOS, inulin and resistant starch. Particularly preferably the composition for use in the invention further comprises galacto-oligosaccharides (GOS). In other words, in some embodiments the composition as used in the invention comprises 2’-FL and GOS as a dietary fiber.

In one embodiment, in the combination of 2’-FL and prebiotic dietary fibers in relation to the invention, the weight ratio between 2’-FL and the dietary fiber is in the range of from 0.5:10 to 10:0.5. Preferably, the composition comprises at least 5 wt.% of 2’-FL and at least 5 wt% of dietary fiber as compared to the total dry weight of the composition, more preferably it comprises at least 10 wt% 2’FL and at least 10 wt% dietary fiber, even more preferably, it comprises at least 20 wt% 2’FL and at least 20 wt% dietary fiber.

The combination of 2’-FL and GOS in relation to the invention provides better results as it allows more 2’-FL to arrive in the distal part of the colon. In one embodiment, the weight ratio between 2’-FL and GOS in the composition is in the range of from 0.5:10 to 10:0.5.

The combination of 2’-FL and FOS in relation to the invention provides better results as it allows more 2’-FL to arrive in the distal part of the colon. In one embodiment, the weight ratio between 2’-FL and FOS in the composition is in the range of from 0.5:10 to 10:0.5. In another embodiment, the weight ratio between 2’- FL and inulin in the composition is in the range of from 0.5:10 to 10:0.5.

In the composition of the invention, GOS and FOS (including inulin) may be present in a ratio of from 1 :9 to 9: 1.

The composition comprising 2’-FL and dietary fiber may, in one embodiment, be a kit of parts wherein the 2’-FL and dietary fiber are not mixed in a single composition, i.e. 2’-FL and dietary fiber are comprised in two separate compositions, when taken together with optional other components, these separate compositions create the composition of the invention. As will be understood by those skilled in the art, the benefits of the present invention may be obtained by sequential or simultaneous administration of 2’-FL and dietary fiber. Such uses and methods of treatment are also within the scope of the present invention. Preferably, 2’-FL and dietary fiber are administered simultaneously. When 2’-FL and dietary fiber are administered sequentially, the time between administrating the first compound and the second compound is less than 1 hour, preferably less than 45 minutes, more preferably less than 30 minutes, more preferably still less than 20 minutes, more preferably still less than 15 minutes, more preferably still less than 10 minutes, more preferably still less than 5 minutes, most preferably less than 1 minute.

It will be understood that when 2’-FL and dietary fiber are administered sequentially, it is typically arbitrary which one of the two components is administered first. Accordingly, in one suitable embodiment, dietary fiber GOS is administered first and 2’-FL is administered second, whilst in another suitable embodiment, 2’-FL is administered first and dietary fiber is administered second.

As already indicated herein before, the present invention resides in the finding that the (oral) administration of 2’-FL or of 2’-FL and dietary fiber (preferably GOS), to a subject results in an increase in the acetate levels in the distal part of the colon. Such an increase in acetate levels in the mother being pregnant with a child is understood to be beneficial to preventing asthma in the child.

In one embodiment, when the composition of the invention is comprising 2’-FL and dietary fiber, these are preferably present in an amount of at least 0.1 wt.% of 2’-FL and 1 wt.% of dietary fiber as compared to the total dry weight of the composition. In one embodiment the composition comprises at least 0.01 wt.% of 2’- FL and at least 5 wt.% of dietary fiber as compared to the total dry weight of the composition. In one embodiment the composition comprises at least 0.1 wt.% of 2’- FL and at least 5 wt.% of dietary fiber as compared to the total dry weight of the composition. In another embodiment it comprises at least 5 wt.% of 2’-FL and 5 wt.% of dietary fiber as compared to the total dry weight of the composition. In yet another embodiment, 2’-FL is present in an amount of at least at least 0.01 wt%, at least 01. wt%, at least 1 wt%, 10 wt.%, at least 20 wt.%, at least 30 wt.%, at least 40 wt.%, at least 50 wt.%, at least 60 wt.%, at least 70 wt.%, at least 80 wt.%, at least 90 wt.%, e.g. up to 91 wt%, 92 wt%, 93 wt%, or 94 wt% as compared to the total dry weight of the composition.

In still another embodiment, when the composition of the invention is comprising 2’-FL and GOS, these are preferably present in an amount of at least 0.1 wt.% of 2’-FL and 1 wt.% of GOS as compared to the total dry weight of the composition. In another embodiment it comprises at least 5 wt.% of 2’-FL and 5 wt.% of GOS as compared to the total dry weight of the composition. In yet another embodiment, 2’-FL is present in an amount of at least 10 wt.%, at least 20 wt.%, at least 30 wt.%, at least 40 wt.%, at least 50 wt.%, at least 60 wt.%, at least 70 wt.%, at least 80 wt.%, at least 90 wt.%, e.g. up to 91 wt%, 92 wt%, 93 wt%, or 94 wt% as compared to the total dry weight of the composition. In another embodiment, GOS is present in an amount of at least 10 wt.%, at least 20 wt.%, at least 30 wt.%, at least 40 wt.%, at least 50 wt.%, at least 60 wt.%, at least 70 wt.%, at least 80 wt.%, at least 90 wt.%, e.g. up to 91 wt%, 92 wt%, 93 wt%, or 94 wt% as compared to the total dry weight of the composition.

In one embodiment, 2’-FL and GOS are provided in a weight ratio in a range of from 0.5:10 to 10:0.5, preferably in a weight ratio in a range of from 1:8 to 8:1, more preferably in a range of from 2:6 to 6:2, most preferably in a range of from 3:5 to 5:3. In an alternative embodiment, in the composition for use according to the invention 2’-FL and GOS are provided in a weight ratio of about 1:1; or even in a ratio of 1:1.

In another embodiment, the composition for use of the invention comprises 2’- FL and dietary fiber, wherein i. the amount of 2’-FL is greater than 0.01 g and ii) the total amount of 2’-FL and dietary fiber is less than 30 gram, preferably less than 25 gram, more preferably less than 20 gram, most preferably less than 15 gram. In another embodiment, the composition for use of the invention comprises 2’- FL and dietary fiber, wherein i. the amount of 2’-FL is greater than 0.1 g and ii) the total amount of 2’-FL and dietary fiber is less than 30 gram, preferably less than 25 gram, more preferably less than 20 gram, most preferably less than 15 gram. In yet another embodiment, the composition for use of the invention comprises 2’- FL and dietary fiber, wherein the amount of 2’-FL is between 0.1 and 8 gram and the amount of dietary fiber is between 1 and 6 grams per daily serving.

In a particular embodiment, the composition for use of the invention comprises 2’- FL and GOS, wherein i. the amount of 2’-FL is greater than 0.01 g and ii) the total amount of 2’-FL and GOS is less than 30 gram, preferably less than 25 gram, more preferably less than 20 gram, most preferably less than 15 gram. In another embodiment, the composition for use of the invention comprises 2’- FL and GOS, wherein i. the amount of 2’-FL is greater than 0.1 g and ii) the total amount of 2’-FL and GOS is less than 30 gram, preferably less than 25 gram, more preferably less than 20 gram, most preferably less than 15 gram. In yet another embodiment, the composition for use of the invention comprises 2’- FL and GOS, wherein the amount of 2’-FL is between 0.1 and 8 gram and the amount of GOS is between 1 and 6 grams per daily serving.

The composition for use according to the invention is a food product, preferably selected from the group consisting of dairy product e.g. as milk-product, milkshake, chocolate milk, yoghurt, cream, cheese, pudding, ice cream etc ; bar, such as nutritional bar, energy bar, snack bar, cereal bar, bar for diabetics etc.; liquid product, such as nutritional drink, diet drink, liquid meal replacers, sports drink and other fortified beverages; savory snack, such as chips, tortillas, puffed and baked snacks, crackers, pretzels; savory biscuit, bakery products, such as muffins, cakes, biscuits; pasta, such as spaghetti; and food supplement e.g. pills, capsules, or dry powder. Food supplements may be ready for consumption or may need to be dissolved in a liquid like water. The product in dry powder form may be accompanied with a device, such as a spoon, to measure the desired amount of the powder (e.g. daily or unit dose). Food supplements may further comprise other ingredients commonly used in food supplements such as vitamins, minerals, salts, etc. The food product is preferably selected from the group consisting of dairy product, liquid product, and food supplement. Alternatively, the composition is a food supplement such as a pill or powder, preferably a pill. The food product preferably is accompanied with instructions specifying how much of the product should be consumed on a daily basis.

The composition as defined herein or the food product as defined herein may be provided in ajar, bottle, sachet, carton, wrapping, and the like.

Typically, the treatment entails the administration of the 2’-FL and optionally non-digestible oligosaccharides (in one embodiment being GOS) in unit dose form. In one embodiment the treatment entails administering a composition according to the invention in a unit dose form. In a preferred embodiment, the composition is provided in the form of a single serving, optionally each single serving is individually packaged, and each serving comprising the 2’-FL and optionally dietary fiber in unit dose amount. Alternatively, the composition for use according to the invention is present in a container comprising a multitude of single servings such as 5, 7, 10, 30, 50, 100, 200, 300, 365 single servings. Such a container preferably is accompanied with instructions on the amount of the composition represents a single serving or daily serving. In still another embodiment, the container comprising the composition of the invention comprises at least 7, for example at least 14 or at least 31 single servings. Preferably the dietary fiber is selected from one or more of the group consisting of GOS, FOS, inulin, and resistant starch, more preferably from one or more of the group consisting of GOS, FOS and inulin. Most preferably the dietary fiber is GOS.

In one embodiment, the unit dose amount of the 2’-FL is at least 0.01 gram, preferably at least 0.1 gram, 1 gram, e.g. at least 1.5 gram, at least 2 gram, at least 2.5 gram, at least 3 gram, at least 3.5 gram, or at least 4 gram.

In another embodiment, the unit dose amount of the 2’-FL is at most 30 grams, e.g. at most 25 grams, such as at most 20 grams, at most 15 grams, at most 12.5 grams, at most 10 grams, at most 9 grams, at most 8 grams, at most 7 grams, at most 6 g or at most 5 grams.

In one embodiment, the unit dose amount of the dietary fiber is 0.5-10 grams and the unit dose amount of the 2’-FL is 3-8 grams, preferably 4-8 grams, more preferably 5-8 grams 2’-FL. In another embodiment, the unit dose amount of the dietary fiber is 2-8 grams and the unit dose amount of the 2’-FL is 0.5-10 grams, preferably 1- 7 grams, more preferably 2-4 grams of 2’-FL. In still another embodiment, the unit dose amount of the dietary fiber is at least 1 grams, at least 1.5 grams, at least 2 grams, at least 2.5 grams, at least 3 grams, at least 3.5 grams, or at least 4 grams. In another embodiment, the unit dose amount of the dietary fiber is at most 25 grams, at most 20 grams, at most 15 grams, at most 12.5 grams, at most 10 grams, at most 9 grams, at most 8 grams, at most 7 grams, at most 6 g or at most 5 grams. In one embodiment, the unit dose amount of the GOS is 0.5-10 grams, more particularly 2-8 grams. Preferably the dietary fiber is selected from one or more of the group consisting of GOS, FOS, inulin, and resistant starch, more preferably from one or more of the group consisting of GOS, FOS and inulin. Most preferably the dietary fiber is GOS.

Unit doses of the composition of the inventio e.g. 2’-FL and optionally GOS, are preferably administered at least once a week, preferably at least once every 3 days, more preferably at least once every other day, most preferably at least once daily. In one embodiment of the invention, the treatment comprises the daily administration of unit doses of the composition of the invention. It is believed that the daily administration of the composition of the invention results in a more continuous production of acetate in the distal colon which increases the health effect to the unborn child.

In accordance with the invention, the treatment as defined herein before, is preferably continued for a period of at least two weeks, e.g. at least 3 weeks, at least 4 weeks, at least 1 month, at least two months, at least three months, at least 4 months, at least 5 months, or even at least 6 months.

In one embodiment, the combination of the invention is administered to a subject in an amount in a range of from 0.1 to 20 grams per day, preferably in a range of from 1 to 15 grams per day.

In another embodiment of the invention, the treatment comprises the administration of the 2’-FL in an average amount of 0.01 -32 g per day, in another embodiment it comprises the administration of 2’FL in an average amount of 0.1 - 32 g per day, preferably in an average amount of 1-24 g per day, more preferably in an average amount of 2-16 g per day, e.g. approximately 5 or 12 g per day; and optionally the administration of the GOS in an average amount of 2-24 g per day, preferably in an average amount of 3-16 g per day, more preferably 4-12 g per day, e.g. approximately 7.5 g per day, over a period of at least 2 weeks, preferably at least 3 weeks, at least 4 weeks, at least 1 month, at least two months, at least three months, at least 4 months, at least 5 months, or at least 6 months. In still another embodiment the treatment comprises the daily administration of a composition comprising 2’FL in an amount of between 0.01 and 5 g and optionally dietary fiber in an amount of 2 to 24 g. Preferably the dietary fiber is selected from one or more of the group consisting of GOS, FOS, inulin, and resistant starch, more preferably from one or more of the group consisting of GOS, FOS and inulin. Most preferably the dietary fiber is GOS.

Human milk oligosaccharides (HMOs) are a key constituent of human milk. They are a structurally and biologically diverse group of complex indigestible carbohydrates. To date, more than 200 different oligosaccharides have been identified, varying in size from 3 to 22 monosaccharide units. The most common HMOs are the neutral fucosylated and non-fucosylated oligosaccharides. The quantity and structure of these HMOs differs significantly among women and is dependent upon Secretor and Lewis blood group status (L. Bode, J. Nutr. 136: 2127- 2130, 2006.). 2’FL is an HMO. In one embodiment, the composition as used in the aspects of the invention comprises one or more further HMOs other than 2’FL. The HMOs of human milk are composed of various monosaccharides, namely glucose, galactose, fucose, N-acetylglucosamine and sialic acids (N-acetylneuraminic acid). The sugar fucose is an unusual molecule in that it has the L-configuration, whereas the other sugar molecules in the body have the D-configuration. The structure of HMOs is a lactose unit which may be elongated with one or more galactose and / or N-acetylglucosamine residues (core structure). The HMO core structure may be decorated with one or more fucose residues (i.e. fucosylated HMO) and with one or more sialic acid units (i.e. sialylated HMO). A HMO may also be fucosylated and sialylated. In one embodiment, the further HMO in the composition of the invention is selected from one or more of the group consisting of core HMO, sialylated HMO, and fucosylated HMO. Nearly 200 HMOs have been identified from human milk. Fucosylated HMOs were found to be the most prominent component (~77%), while sialylated HMOs accounted for about 16% of the total abundance of HMOs. The fucosylated HMOs are neutral molecules, while the sialylated HMOs are acidic. Preferred HMOs are 3'-Sialyllactose (3'SL); 6'-Sialyllactose (6'SL); 3-Fucosyllactose (3-FL); lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT) and disialyllacto-N- tetraose (DSLNT). HMOs can be obtained using methods known to those of skill in the art. For example, HMOs can be purified from human milk e.g. using methods known in the art such as capillary electrophoresis, HPLC (e.g., high-performance anion-exchange chromatography with pulsed amperometric detection; HPAEC-PAD), and thin layer chromatography, alternatively, HMO’s may be commercially obtained.

In one embodiment the non-therapeutic method of the invention is carried out for non-medical reasons. The non-therapeutic method of the invention will typically rely on the use of the same combinations, compositions and products as well as the same routes of administration and the same dosage regimens as defined herein above (e.g. for the composition for use).

In another aspect, the invention relates to a method of preventing asthma in a human subject comprising the step of administering a composition comprising 2’FL (i.e. a composition as herein defined above) to the mother of the subject while being pregnant with the subject. Preferably the method is non-therapeutic.

In still another aspect, the invention relates to the use of a composition comprising 2’FL and optionally GOS, (i.e. a composition as herein defined above) to prevent asthma in a human subject characterized in that the composition is administered to the mother of the subject while being pregnant with the subject. The use of the composition in this aspect of the invention may be therapeutic and / or non-therapeutic. Preferably, the use is non-therapeutic.

In another aspect, the invention relates to the use of 2’-FL and optionally GOS (i.e. a composition as herein defined above) for the manufacture of a medicament for preventing asthma in a human subject characterized in that the composition is administered to the mother of the subject while being pregnant with the subject.

In a particular embodiment of the invention, the composition for use is presented in the form of individually packaged single servings. The term “single serving” as used herein refers to a certain quantity and/or size of the product that is adequate for consumption as a single portion for a single person. Such products may be in a form that is ready-to-eat or ready-to-consume or it may be in a form that requires further processing, such as heating or addition of a quantity of hot or cold water. In one embodiment, the composition of the invention is presented in the form of individually packaged single servings, wherein each serving contains a unit dose as defined herein elsewhere, preferably each comprising 2’-FL in an amount of 0.1-8 gram, preferably 0.5-8 gram, more preferably 1-4 grams, and comprising 0.5-8 grams, preferably 1-4 grams, of GOS.

A preferred product form of the composition used in the invention is an edible bar, such as a nutritional bar, energy bar, diet bar or food supplement bar, snack bar, etc., examples of which are well known to those of skill in the art. The edible bar may in one embodiment be a cereal composition comprising a cereal mix and a binding syrup. The binding syrup can include e.g. glucose syrup, granulated sugar, glycerol, water, emulsifier, fat and flavors. The dietary fiber composition of the invention can suitably be incorporated in the binding syrup. Products of this type are known by those skilled in the art, e.g. from international patent publication no. WO 2017/078519.

In one embodiment of the invention, the composition as used in the invention does not comprise resistant starch.

Practice within the numerical limits stated, is generally preferred. Also, unless expressly stated to the contrary: percent, "parts of," and ratio values are by weight; the description of a group or class of materials as suitable or preferred for a given purpose in connection with the invention implies that mixtures of any two or more of the members of the group or class are equally suitable or preferred; description of constituents in chemical terms refers to the constituents at the time of addition to any combination specified in the description, and does not necessarily preclude chemical interactions among the constituents of a mixture once mixed; the first definition of an acronym or other abbreviation applies to all subsequent uses herein of the same abbreviation and applies, mutatis mutandis, to normal grammatical variations of the initially defined abbreviation; and, unless expressly stated to the contrary, measurement of a property is determined by the same technique as previously or later referenced for the same property.

It is also to be understood that this invention is not limited to the specific embodiments and methods described herein, as specific components and/or conditions may, of course, vary. Furthermore, the terminology used herein is used only for the purpose of describing particular embodiments of the present invention and is not intended to be limiting in any way.

Throughout this application, where publications are referenced, the disclosures of these publications in their entireties are hereby incorporated by reference into this application to more fully describe the state of the art to which this invention pertains.

The invention is hereinafter illustrated with reference to the following, non limiting, examples.

EXAMPLES

In vitro fermentation model, TIM-2

The in vitro fermentation studies were done using the TIM-2 model. This is a validated, dynamic, computer-controlled model that simulates the human colon, mimicking body temperature, lumen pH, absorption of water and microbial metabolites through a semipermeable membrane inside the model, mixing and transporting the intestinal contents with peristaltic movements, using an anaerobic microbiota from human origin, it corresponds basically to the model as described in Minekus, M., et al. Appl. Microbiol. Biotechnol. 1999 53, 108-114. doi: 10.1007/ S002530051622 and Kortman et al., Frontiers in Microbiology 2016, 6, 1481. Characteristics of the movements of the contents in the TIM-2 system was simulated using an increase of the pH and peristaltic movements of the contents in the system using peristaltic pumps as described in Minekus, M (1998. Development and validation of a dynamic model of the gastrointestinal tract. PhD thesis, Delft University of Technology, The Netherlands).

SC FA analysis

SCFA analysis was performed at Brightlabs B.V., Venlo, The Netherlands), according to (Sayago-Ayerdi SG, etal. Food Research International, E-pub date 13 December 2017; Sayago Ayerdi et al Food Research International 118 (2019) 89-95).

Inoculum

Pooled fecal microbiota samples from 20 healthy subjects with a BMI >18.5 kg/m ² (including both lean (18.5 kg/m ² < BMI < 25 kg/m ² ) and obese (BMI > 30 kg/m ² ) subjects) not suffering from any metabolic disease was used to inoculated the TIM-2 in vitro fermentation model.

Vitamine mixture

A vitamin mixture was used containing (per liter): 1 mg menadione, 2 mg D-biotin, 0.5 mg vitamin B12, 10 mg pantothenate, 5 mg nicotinamide, 5 mg p-aminobenzoic acid and 4 mg thiamine.

Dialysate

The dialysate used in the TIM-2 system contained (per liter): 2.5 g K2HPO4 3H2O, 4.5 g NaCI, 0.005 g FeS0 ₄ -7H ₂ 0, 0.5 g MgS0 ₄ -7H ₂ 0, 0.45 g CaCI ₂ -2H ₂ 0, 0.05 g bile and 0.4 g cysteine-HCI, plus 1 ml_ of the vitamin mixture.

Example 1

The fecal microbiota was freshly sampled in and stored directly (within 2h) on ice and under anaerobic conditions. Next, in an anaerobic cabinet, samples were diluted 1:1 with dialysate, and pooled at approximately equal weight, after which glycerol was added (to a final concentration of 12-13 w/w) and aliquots (30 ml/tube) were frozen in liquid nitrogen and stored at -80 °C. Prior to inoculation, 4x 30-ml aliquots were taken from the -80 °C freezer and thawed in a water bath at 37°C for exactly 1 hour (still under anaerobic conditions). In an anaerobic cabinet, the microbiota from the 4 tubes was combined and the same volume of pre-reduced (i.e. oxygen free) dialysate was added, gently mixed and divided over 4 syringes each comprising ca. 60 ml of microbiota-containing liquid.

The syringes were sealed with a small flexible tube closed with a tubing clamp. Each TIM-2 unit was inoculated with 1 of the 4 syringes (i.e. 60 ml microbiota/dialysate mixture), using one single sample port to inoculate a TIM-2 unit. After the microbiota was introduced into the unit, another 60 ml of pre-reduced dialysate was added into the TIM-2 unit to get to a final volume of 120 ml per unit (i.e. system).

To simulate the conditions in the proximal region of the colon, the colon transversum and the distal part of colon, the pH of the microbiota/dialysate mixture was increased from pH 5.8 to pH 7.0 using 1M NaOH over a period of 24 hours. The increase in pH simulated the movement of fibers through the colon during the 24 hours’ experiment (wherein the last 16 hours simulated the more distal colonic site (i.e. transverse + distal)).

Samples (1 ml_) were taken for Short Chain Fatty Acid (SCFA) analysis after 1 , 2, 4, 6, 8 and 24 h after test product insertion; cumulative absolute amounts of SCFA were determined.

Samples were centrifuged at 14,000 rpm for 10 min, filtered through a 0.45 pm PFTE filter, and diluted in the mobile phase (1.5mM aqueous sulfuric acid). Ten microliters were loaded into the column with the help of an automatic sampler 730 (Metrohm, Herisa, Switzerland). The acids were eluted according to their pKa. The analysis was carried out by ion exclusion chromatography (IEC) using an 883 chromatograph (IC, Metrohm) equipped with a Transgenomic IC Sep ICE-ION-300 column (30 cmx7.8mmx7 pm) and a MetroSep RP2 Guard. A column flow of 0.4 mL/min with a column temperature of 65 °C was used. The acids were detected using suppressed conductivity detection. Analyses were performed by Brightlabs (Venlo, The Netherlands).

Addition of the test-product:

After an adaption period of 40h, 7.5 grams of 2’-fucosyllactose (2’-FL) was introduced into a TIM-2 unit through the sample port (on Wednesday) as a single shot. An experimental week contained the following steps:

Monday: Start up all 4 units of the TIM-2 system (pH 5.8).

Tuesday: Feeding of Simulated ileal efflux environment medium (SIEM) (Maathuis et al 2009 Journal of the American College of Nutrition 28(6):657-66 DOI: 10.1080/ 07315724.2009.10719798);

Simulated ileal efflux medium (SIEM) contained 5.7 g/liter BD Bacto tryptone (BD), 2.4 g/liter D-glucose (Sigma-Aldrich), 6.14 g/liter NaCI (Roth, Germany), 0.68 g/liter KH2PO4 (Merck, Germany), 0.3 g/liter NaHaPC (Merck, Germany), 1.01 g/liter NaHCC (Merck, Germany), 5.6 g/liter bile salts no. 3 (Difco), 0.2 g/liter lysozyme (Serva, Germany), 1,000 U a-amylase (Fluka, Germany), 110 U trypsin (Sigma- Aldrich), 380 U chymotrypsin (Calbiochem, Germany), and 960 U lipase (Sigma- Aldrich). D(+)-Glucose and enzymes were filter sterilized before addition.

Wednesday: 3 h starvation period followed by a single insertion of test product; 2’-FL (7.5 gram of 2’-FL) was added through the sample port]; following introduction of the test product, samples for SCFA analysis were taken after 1 , 2, 4, 6 and 8 h. Thursday: 24 h after insertion of the test product: last sample was taken for SCFA analysis;

Friday: cleaning (the experiment was executed in one week).

Results

The increase in cumulative absolute acetate amount between the last two sampling points (i.e. between 8 and 24 h after product insertion) was taken as an indication for the increase in SCFA and of acetate in the distal colon.

The results of the experiments are displayed below in Table 1.

Table 1. Results of the TIM-2 experiments, using microbiota samples subjects to which 2’-FL was administered. * the amount of acetate refers to the amounts produced in between 8 and 24 hours after insertion of the test product; representing the amounts of acetate and SCFA produced in the distal colon. The experiment shows that insertion of 2’FL (7.5 gram) in the model system results in an increased acetate production of 17 mmol acetate in the distal colon. An increase in the acetate production was observed both when fecal microbiota samples of lean and obese subjects was used in the TIM-2 model system.