WO2020239724A1

WO2020239724A1 - Compositions comprising 2 -fucosyllactose and gos

Info

Publication number: WO2020239724A1
Application number: PCT/EP2020/064502
Authority: WO
Inventors: Arjen Nauta
Original assignee: Frieslandcampina Nederland B.V.
Priority date: 2019-05-29
Filing date: 2020-05-26
Publication date: 2020-12-03
Also published as: CN113874024A; EP3976051A1

Abstract

A composition comprising 2'-fucosyllactose (2'-FL) and galacto-oligosaccharides (GOS) for use in the treatment of one or more of the indications selected from the 5 group consisting of overweight, a condition associated with overweight, the prevention of a condition associated with overweight, obesity, a condition associated with obesity, and the prevention of a condition associated with obesity; in a human subject with a healthy, overweight or obese body weight.

Description

COMPOSITIONS COMPRISING 2 -FUCOSYLLACTOSE AND GOS

FIELD OF THE INVENTION

The invention disclosed herein relates to a composition comprising 2’-fucosyl lactose for use in the treatment of one or more of the indications selected from the group consisting of overweight, a condition associated with overweight, the prevention of a condition associated with overweight, obesity, a condition associated with obesity, and the prevention of a condition associated with obesity; in a human subject with a healthy, overweight or obese body weight.

BACKGROUND

Obesity is a major health problem in developed countries, and is rapidly becoming a significant health problem in developing countries. Overweight and obesity are reaching epidemic proportions. According to the Behavioral Risk Factor Surveillance System (BRFSS), one out of three adults in the USA is obese.

Obesity is a complex, multifactorial disease. Obesity is typically defined as an excess of body weight for a height. However, this simple definition does not take into account complex genetic phenotypes primarily associated with excess adiposity, or body fatness, that can manifest metabolically and not just in terms of body size. Excessive adipose tissue is associated with a number of chronic diseases including hyperlipidemia, high blood pressure, glucose/carbohydrate intolerance, diabetes and cardiovascular diseases. The precise association between obesity and these chronic diseases is poorly understood, but solid epidemiological data support the existence of this association. Adipocytes (also referred to as lipocytes or fat cells) are the cells that primarily compose adipose tissue, specialized in storing energy as fat.

Adipose tissue morphology correlates with insulin measures and is linked to the total adipocyte number independently of sex and body fat level. Low generation rates of adipocytes associate with adipose tissue hypertrophy, whereas high generation rates associate with adipose hyperplasia. Adipose tissue expands by increasing the volume of preexisting adipocytes (adipose hypertrophy), by generating new small adipocytes (hyperplasia), or by both. Although the amount and distribution of adipose tissue associate independently with insulin resistance, type 2 diabetes, and other metabolic disorders, the size of adipocytes within the adipose tissue is also important (E. Arner et al. 2010 Diabetes vol 59 pp 105-109).

Adipose tissue, skeletal muscle and the liver are major organs involved in substrate metabolism. Research has revealed the presence of a strong inter-organ relationship in substrate metabolism. Functional disturbances in either of these organs can theoretically cause or contribute to the development of insulin resistance. The obese insulin resistant state is characterized by adipose tissue dysfunction, systemic lipid overflow and ectopic lipid accumulation in metabolically active organs such as the liver, pancreas and skeletal muscle. Additionally, skeletal muscle exhibits an impaired mitochondrial function and an impaired ability to adjust lipid oxidation to lipid supply. This leads to the accumulation, altered composition/localization of bioactive lipid metabolites interfering with insulin action.

The gut microbiota is increasingly being recognized as an important factor in fat distribution, insulin sensitivity and glucose and lipid metabolism. Accordingly, the intestinal microbiota could play an important role in the development of obesity and diabetes. One of the important functions of the human microbiota is the fermentation of indigestible carbohydrates, e.g. dietary fiber or GOS. The major products of this fermentation process are the short-chain fatty acids (SCFAs), such as acetate, propionate and butyrate.

Several in vivo rodent studies have shown that SCFA supplementation prevented diet-induced obesity and insulin resistance. Especially, increasing the acetate availability of the colonic and system ically most abundant SFCAs prevents diet-induced body weight gain, counteracts adiposity, and improves glucose homeostasis and insulin sensitivity.

For example sodium acetate (5% (w/w)) added to high-fat fed mice for 12 weeks reduced body weight gain and improved insulin sensitivity without changing food intake or physical activity (G. Den Besten et al. Diabetes. 2015, 64:2398-408).

Another study showed that dietary supplementation of acetate (5% (w/w)) for 12 weeks to high-fat fed mice inhibited body weight gain, reduced plasma glucose and insulin concentrations, as well as reduced pro-inflammatory cytokines and chemokines (Y. Lu et al. Scientific Reports. 2016, 6:37589).

A study wherein sodium acetate was administered to the proximal or the distal colon of overweight men showed that fat oxidation and circulating plasma

concentrations of the satiety hormone peptide YY (PYY) was significantly increased when acetate was administered in the distal part of the colon. In contrast, no effect on energy expenditure or substrate oxidation was seen when acetate was

administered in the proximal colon (van der Beek, C. M., et al. Clinical Science 2016, 130(22), 2073-2082). In another study, distal colonic infusions of mixtures of SCFAs, all highest in acetate, increased energy expenditure, fat oxidation and plasma PYY in men (Canfora EE et al., Sci Rep. 2017, 7:2360).

Thus, SCFAs that are administered or generated in sufficient concentrations in the distal colon are associated with a beneficial effect on controlling body weight in a subject.

Since dietary intervention is clearly the preferred (first-line) option for the treatment and/or prevention of obesity (and related disorders), treatment of overweight, and prevention of a condition associated with overweight, it is a challenge to provide a composition, preferably an edible composition, that has the capability of delivering/liberating sufficient quantities of SCFAs in the distal part of colon.

In addition, it is desired that such a composition 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 obesity or overweight or of related disorders of any of these. Additionally it is desired to provide a non-therapeutic method of losing body weight or of maintaining a healthy body weight and / or to provide a non-therapeutic method of increasing the concentration of SCFAs in the distal colon of a human subject.

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) and galacto-oligosaccharides (GOS) for use in the treatment of one or more of the indications selected from the group consisting of overweight, a condition associated with overweight, and the prevention of a condition associated with overweight,; in a human subject with healthy or overweight body weight.

In one aspect the invention provides a composition comprising 2’- fucosy I lactose (2’-FL) and galacto-oligosaccharides (GOS) for use in the treatment of one or more of the indications selected from the group consisting of obesity, a condition associated with obesity, and the prevention of a condition associated with obesity; in a human subject with obese body weight.

In another aspect, the invention provides a non-therapeutic method for losing body weight or maintaining a healthy body weight in human subject, the non- therapeutic method comprising the step of administering to the subject a composition comprising 2’-fucosyllactose (2’-FL) and galacto-oligosaccharides (GOS) wherein the human subject has a healthy, overweight or obese body weight; preferably wherein the human subject is overweight or obese, more preferably obese.

In another aspect, the invention provides a non-therapeutic method for losing body weight or maintaining a healthy body weight in human subject, the non- therapeutic method comprising the step of administering to the subject a composition comprising 2’-fucosyllactose (2’-FL) and galacto-oligosaccharides (GOS) wherein the human subject has a healthy or overweight body weight; preferably wherein the human subject is overweight.

In a further aspect, the invention relates to a non-therapeutic method for increasing the concentration of SCFA, in particular of acetate, in the distal colon of a subject, the non-therapeutic method comprising the step of administering to the human subject a composition comprising 2’-FL and galacto-oligosaccharides (GOS) wherein the human subject has a healthy, overweight or obese body weight, preferably wherein the human subject is overweight or obese, more preferably obese.

In a further aspect, the invention relates to a non-therapeutic method for increasing the concentration of SCFA, in particular of acetate, in the distal colon of a subject, the non-therapeutic method comprising the step of administering to the human subject a composition comprising 2’-FL and galacto-oligosaccharides (GOS) wherein the human subject has a healthy or overweight body weight, preferably wherein the human subject is overweight. 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 be predisposed to the disorder or disease but has not yet been diagnosed as having the disorder or disease, and/or preventing further disease/disorder development if already present.

The invention, is based on the finding that a specific certain dietary fibers i.e. 2’-fucosyllactose (2’-FL) together with galacto-oligosaccharides (GOS) 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 and galacto-oligosaccharides (GOS) substantially increases the level of acetate in the distal part of the colon in human subjects. Therefore, in a first aspect, the invention relates to a composition comprising 2’-fucosyllactose (2’-FL) and galacto- oligosaccharides (GOS) for use in the treatment of one or more of the indications selected from the group consisting of overweight, a condition associated with overweight, and the prevention of a condition associated with overweight, in a human subject with a healthy or overweight body weight. In another aspect, the invention relates to a composition comprising 2’-fucosyllactose (2’-FL) and galacto- oligosaccharides (GOS) for use in the treatment of one or more of the indications selected from the group consisting of obesity, a condition associated with obesity, and the prevention of a condition associated with obesity; wherein the human subject is obese wherein the composition is further comprising galacto-oligosaccharides (GOS).

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 (HMO) naturally present in human breast milk, making up about 30% of all of HMOs. 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.

FIMOs 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 preferred method of preparing 2’-FL is disclosed in WO 2012/112777. Alternatively, 2’-FL is commercially available e.g. from

FrieslandCampina, DuPont and others.

Whether a human subject has a healthy body weight, has overweight, or is obese can be assessed in different ways and is depending on the type of species. As used herein, a healthy body weight, overweight, or obese are defined for a human subject using the subject’s body mass index (BMI), which is defined as the body weight measured in kilograms divided by the height (in meters) squared. In a formula, BMI for a human subject, is defined as:

( Equation 1).

For humans aged 18 and above, a BMI in a range of from at least 18.5 kg/m² to less than 25 kg/m² is considered a healthy body weight. A human aged 18 and above having a BMI in a range of from at least 25 kg/m² to less than 30 kg/m² is considered overweight. A human aged 18 and above having a BMI of at least 30 kg/m² is considered obese. A human aged 18 and above having a BMI of less than 18.5 kg/m² is considered underweight. For humans aged 2 to 18 years old (children and teenagers), the BMI value as obtained using Equation 1 needs to be adjusted because girls and boys develop at different rates and have different amounts of body fat at different ages. So in one embodiment of the invention, the human subject is at least two years old (age > 2 years). BMI measurements during childhood and adolescence as used herein, take age and sex into consideration. For children and teenagers, aged 2 to 18, being male or female, a healthy body weight, overweight and obese is defined as a BMI value in accordance with the values listed in Table 1. Table 1. Definition of healthy body weight BMI values

Cole et al BMJ:

j.com first published as

10.1136/bmj.320.7244.1240

https://www.bmi.com/content/bmi/320/7244/1240.full.pdf The term“healthy body weight” as used herein is defined as a BMI value in the range starting from“lower BMI value for a healthy weight” up to the“lower BMI value for overweight”, exclusive, as defined in Table 1 for a human subject with that age and sex. As used herein, a numeric range starting from a certain number, means that this number is included in the range. A numeric range“up to another number, exclusive” is defined as a range ending with this“another number”, the“another number” not being part of the range.

The term“overweight” as used herein is defined as a BMI value in the range starting from“lower BMI value for overweight” up to the“lower BMI value for obesity”, exclusive, as defined in Table 1 for a human subject with that age and sex.

The term“obese” or“obesity” as used herein is defined as a BMI value equal to or higher than the“lower BMI value for obesity” as defined in Table 1 for a human subject with that age and sex. An obese subject generally has an excess of adipose tissue in the body. In this context, obesity is to be seen as the increased level of fatness, which leads to health risks. The health risks accompanying obesity are presumed to increase as the level of fatness increases.

BMI values for children younger than 2 years old are hard to define, hence in one preferred embodiment the human subject as referred to in the different aspects and embodiments of the invention is a human subject aged 2 or older.

Following the definition given above, overweight is a weight - length combination which has a BMI between a healthy and obese range. Conditions associated with overweight are the same conditions that are associated with obesity, as herein defined above. Normally the severity or risks of the condition are less when a person is overweight as compared to being obese.

In one embodiment, the human subject as referred to in the various aspects and embodiments of the invention are overweight, in another obese.

In another embodiment, the composition for use of the invention, is for use in the treatment of overweight, a condition associated with overweight, and/or the prevention of a condition associated with overweight; wherein the human subject is overweight, preferably wherein the human subject is > 18 years old and has a body mass index (BMI) between 25 and 30 kg/m². In another embodiment, the composition for use of the invention, is for use in the treatment of obesity, a condition associated with obesity, and/or the prevention of a condition associated with obesity; wherein the human subject is obese, preferably wherein the human subject has a body mass index (BMI) of at least 30 kg/m², even more preferably wherein the human subject is > 18 years old.

As stated above, 2’-FL is a dietary fiber. For most humans, there is a maximum amount of dietary fiber that can be consumed on a daily basis. 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. So, in one embodiment the amount of 2’-FL in the composition as used in the invention is more than 0.1 gram. In another embodiment, it is in the range of from 0.1 to 30 gram, preferably in a range of from 0.5 to 25 gram, more preferably in a range of from 1 to 20 gram, most preferably in a range of from 2 to 10 gram.

LNnT may be considered as a dietary fiber. In one embodiment the

composition for use according to the invention does not comprise lacto-N- neotetraose (LNnT).

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 thus, GOS prepared by B. circulans beta-galactosidase is sold worldwide. 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 dietary fibers in 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 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 SCFAs (short chain fatty acids) such as 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.

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 galacto-oligosaccharides (GOS). In other words, the composition as used in the invention comprises 2’-FL and GOS.

The combination of 2’-FL and GOS in relation to the invention provides better results than other fibers known in the art 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 composition comprising 2’-FL and GOS preferably comprises a mixture of 2’-FL and GOS. In one embodiment 2’-FL and GOS are not mixed the composition comprising 2’-FL and GOS, i.e. 2’-FL and GOS 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 GOS. Such uses and methods of treatment are also within the scope of the present invention. Preferably, 2’-FL and GOS are administered simultaneously.

When 2’-FL and GOS 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 GOS are administered sequentially, it is typically arbitrary which one of the two components is administered first. Accordingly, in one suitable embodiment, GOS is administered first and 2’- FL is administered second, whilst in another suitable embodiment, 2’-FL is

administered first and GOS is administered second.

As already indicated herein before, the present invention resides in the finding that the (oral) administration of 2’-FL or in particular of 2’-FL and GOS, to a subject results in an increase in the SOFA levels in the distal part of the colon. Such an increase in SOFA levels is understood to be beneficial to a variety of diseases or conditions, including, in particular, obesity and overweight and related diseases and conditions.

In a preferred embodiment of the invention, the composition of the invention is for use in the treatment and/or prevention of obesity and/or a condition associated with obesity, in a subject.

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 5 wt.% of 2’-FL and 5 wt.% of GOS as compared to the total 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 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 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 ration of 1 : 1.

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 0.1 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 another embodiment, the composition for use of the invention comprises 2’- FL and GOS, wherein the amount of 2’-FL is between 2 and 8 gram and the amount of GOS is between 1 and 6 grams.

The composition for use in the invention, wherein the composition 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.

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

In one embodiment, the composition for use of the invention is for the treatment of obesity and/or a condition associated with obesity, in a subject.

Preferably, said subject has a body mass index of at least 30 kg/m².

In another embodiment, the composition for use of the invention is for the treatment of obesity, in a subject. Preferably, said subject has a body mass index of at least 30 kg/m².

In still another embodiment, the composition for use of the invention, is for the treatment of a condition associated with obesity, in a subject. Preferably, said subject has a body mass index of at least 30 kg/m².

In yet another embodiment, the composition for use of the invention is for use in the prevention of obesity and/or a condition associated with obesity, in a subject. Preferably, said subject has a body mass index in a range of from at least 25 kg/m² to less than 30 kg/m². In still another embodiment, the composition for use of the invention is for use in the prevention of obesity in a subject. Preferably, said subject has a body mass index in the range of from 25 kg/m² to 30 kg/m².

In a particular embodiment, the composition for use of the invention is for use in the prevention of a condition associated with obesity, in a subject. Preferably, said subject has a body mass index in the range of from 25 kg/m² to 30 kg/m².

In one embodiment, the composition for use of the invention is for use in the treatment of overweight and/or a condition associated with overweight, and/or the prevention of a condition associated with overweight, in a subject. Preferably, said subject has a body mass index in the range of from 25 kg/m² to 30 kg/m².

In another embodiment, the composition for use of the invention is for use in the treatment of overweight and/or a condition associated with overweight, in a subject. Preferably, said subject has a body mass index in the range of from 25 kg/m² to 30 kg/m².

In yet another embodiment, the composition for use of the invention is for use in the treatment of overweight, in a subject. Preferably, said subject has a body mass index in the range of from 25 kg/m² to 30 kg/m².

In yet another embodiment, the composition for use of the invention is for use in the treatment of a condition associated with overweight, in a subject. Preferably, said subject has a body mass index in the range of from 25 kg/m² to 30 kg/m².

In still another embodiment, the composition for use of the invention is for use in the prevention of a condition associated with overweight, in a subject. Preferably, said subject has a body mass index in the range of from 25 kg/m² to 30 kg/m².

In one embodiment, the subject has a body mass index (BMI) of at least 25 kg/m², e.g. at least 26 kg/m², at least 27 kg/m², at least 28 kg/m², at least 29 kg/m², at least 30 kg/m², at least 31 kg/m², at least 32 kg/m², at least 33 kg/m², at least 34 kg/m², at least 35 kg/m², at least 36 kg/m², at least 37 kg/m², at least 38 kg/m², at least 39 kg/m², or at least 40 kg/m².

In some embodiments, the obesity is prevalent obesity. In other embodiments, the obesity is prevalent abdominal obesity. In some other

embodiments, the obesity is new onset obesity.

Because obesity is associated with the onset or progression of other diseases, the combinations and methods of the invention are further useful in methods of reducing complications associated with obesity including vascular disease, such as coronary artery disease, stroke, peripheral vascular disease, ischemia reperfusion, etc.; hypertension; insulin resistance, pre-diabetes, onset and/or progression of diabetes type II; hyperlipidemia and musculoskeletal diseases. The present invention, in various embodiments, accordingly provides combinations for use in methods of treating or preventing these obesity-associated diseases or conditions.

In some embodiments, the disease or condition is hyperglycemic condition. In certain embodiments, the hyperglycemic medical condition is related to insulin resistance, pre-diabetes, diabetes, diabetes mellitus type I, diabetes mellitus type II, or gestational diabetes, either insulin-dependent or non-insulin-dependent.

Metabolic syndrome is a cluster of conditions that synergistically increase the risk of cardiovascular disease, type 2 diabetes, and premature mortality. The components are abdominal obesity, impaired glucose metabolism, dyslipidemia, and hypertension. Prediabetes, which is a combination of excess body fat, insulin resistance as well as an impaired glucose metabolism, is considered an important characteristic of the metabolic syndrome. Prediabetes manifests as impaired fasting glucose and/or impaired glucose tolerance. The invention provides a combinations for use in a method of preventing or treating Metabolic Syndrome, or reducing one, two, three or more risk factors thereof, in a subject.

In some embodiments of the invention the disease or condition is overweight or an unhealthy weight. Hence in one embodiment of the invention the treatment or prevention is a method of controlling weight, a method of losing weight, a method of reducing weight, a method of preventing weight gain, a method of limiting weight gain, a method of reducing overweight, a method of preventing overweight, a method of inducing weight loss, a method of increasing weight loss, a method of managing weight and/or a method of maintaining a healthy weight. In a preferred embodiment of the invention, the method is a method of losing weight within a predetermined interval, e.g. to lose weight within 12 months, within 6 months, within 4 months, within 3 months, within 2 months, within 1 month, within 4 weeks, within 3 weeks, within 2 weeks or within 1 week.

In some embodiments of the invention the disease or condition is excess body fat (mass), which may also be referred to as adiposity. Hence, in one

embodiment of the invention the treatment or prevention is a method of controlling body fat (mass), a method of losing body fat (mass) a method of reducing body fat (mass), a method of preventing a gain in body fat (mass), a method of limiting gain in body fat (mass), a method of reducing body fat (mass), a method of preventing excess body fat (mass), a method of inducing loss of body fat (mass), a method of increasing loss of body fat (mass), a method of managing body fat (mass) and/or a method of maintaining a healthy body fat (mass). In a further embodiment of the invention, the method is a method of losing body fat (mass) within a predetermined interval, e.g. to lose body fat (mass) within 12 months, within 6 months, within 4 months, within 3 months, within 2 months, within 1 month, within 4 weeks, within 3 weeks, within 2 weeks or within 1 week.

Other embodiments are envisaged wherein the disease or disorder is a digestive condition, such as a functional gastrointestinal disorders (FGID). FGIDs are diagnosed and classified using the Rome IV criteria (released in May 2016) and include functional bowel disorders such as functional diarrhea, functional

constipation, Irritable Bowel Syndrome (IBS), e.g. IBS with predominant diarrhea [IBS-D], IBS with predominant constipation [IBS-C], and IBS with mixed bowel habits.

Other embodiments are envisaged wherein the condition is low-grade inflammation, e.g. chronic low-grade inflammation. Clinically low-grade inflammation is defined as a two to four-fold elevation in circulating levels of proinflam matory and anti-inflammatory cytokines as well as numerous other markers of immune system activity. Chronic low-grade inflammation plays a role in the pathology of numerous age-related chronic conditions.

The term "subject" as used herein refers to a human, that is treatable by the method of the invention. The term "subject" refers to both the male and female sex unless one sex is specifically indicated. The human subject can be an infant (> 2 years old), a juvenile, an adolescent, an adult or an elderly subject.

In embodiments of the invention the human subject is at least 18 years of age, e.g. at least 25 years, at least 30 years, at least 35 years, at least 40 years, at least 45 years, at least 50 years, at least 55 years, at least 60 years or at least 65 years of age. There is no particular upper limit although in practice, human subjects treated in accordance with the invention will typically be at most 100 years of age, e.g. at most 95 or at most 90 years of age.

In one embodiment, the subject is obese and/or suffering from

complications associated with obesity. In another embodiment, the subject is suffering from or at risk of suffering from complications associated with obesity, such as a subject suffering from one or more complications selected from vascular disease, such as coronary artery disease, stroke, peripheral vascular disease, ischemia reperfusion, etc.; hypertension; onset and/or progression of diabetes type II; hyperlipidemia and musculoskeletal diseases.

In still another embodiment, the subject is suffering from or at risk of suffering from a hyperglycemic medical condition, such as diabetes, diabetes mellitus type I, diabetes 10 mellitus type II, or gestational diabetes, either insulin-dependent or non-insulin-dependent.

In yet another embodiment, the subject is suffering from or at risk of suffering from metabolic syndrome. In a preferred embodiment, the subject meets the criteria of either or both of the criteria set forth by the 2001 National Cholesterol Education Program Adult Treatment Panel or the WHO, that individual is considered as afflicted with Metabolic Syndrome, as defined here above.

In one embodiment, the subject is a prediabetic subject. The World Health Organization (WHO) has defined prediabetes as a state of intermediate

hyperglycemia using two specific parameters, impaired fasting glucose (IFG) defined as fasting plasma glucose (FPG) of 6.1 -6.9 mmol/L (110 to 125 mg/dL) and impaired glucose 25 tolerance (IGT) defined as 2 h plasma glucose of 7.8-11.0 mmol/L (140- 200 mg/dL) after ingestion of 75 g of oral glucose load or a combination of the two based on a 2 h oral glucose tolerance test (OGTT).

In another embodiment of the invention, the subject is suffering from impaired fasting glucose, meaning that the subject's blood sugar levels during fasting are consistently above the normal range, but below the diagnostic cut off for a formal diagnosis of diabetes mellitus. In a preferred embodiment, the subject has a blood sugar level during fasting within the range of 6.1 -6.9 mmol/L. In a preferred

embodiment, the subject has a two-hour glucose level of 7.8 to 11.0 mmol/L on the 75-g oral glucose tolerance test.

In a particular embodiment of the invention, the subject is a healthy subject, e.g. when the method is mainly carried out for prophylaxis.

In embodiments of the invention, the treatments as described herein comprise the step of identifying a subject that is in need of receiving treatment with the compositions of the invention and/or identifying a subject suffering from or at risk of suffering from any of the conditions as described here above and/or a subject meeting any of the above-described physiological characteristics as described here above.

The treatments as described herein, comprise the administration to the subject in need thereof of a composition or product as described herein in an amount effective to prevent or treat one or more of the conditions or pathologies as defined herein.

Typically, the treatment entails the administration of the 2’-FL and optionally GOS in unit dose form. In one embodiment the treatment entails

administering a composition according to the invention. In a preferred embodiment, the composition is provided in the form of a single serving, optionally each single serving is individually packaged each serving comprising the 2’-FL and optionally GOS in unit dose amount.

In one embodiment, the unit dose amount of the 2’-FL is at least 1 grams, e.g. 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 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 GOS 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 GOS 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 GOS 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 GOS 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.

Unit doses of the 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 2’-FL and optionally GOS.

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 1-32 g per day, preferably in an average amount of 2-24 g per day, more preferably in an average amount of 4-16 g per day, e.g. approximately 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.

Another aspect of the invention is directed to a non-therapeutic method for losing body weight or maintaining a healthy body weight, in a human subject, the non-therapeutic method comprising the step of administering to the human subject a composition comprising (i) 2’-FL and (ii) GOS as defined elsewhere herein, wherein the human subject has a healthy or overweight body weight; preferably wherein the human subject is overweight. In one embodiment of this aspect, the subject is aged 18 or older.

Yet another aspect of the invention is directed to a non-therapeutic method for increasing the concentration of Short Chain Fatty Acid (SCFA), in particular of acetate, in the distal colon of a human subject, the non-therapeutic method comprising the step of administering to the human subject a composition comprising (i) 2’-FL and (ii) GOS as defined elsewhere herein; wherein the human subject has a healthy or overweight body weight, preferably wherein the human subject is overweight. As will be understood by those skilled in the art, a subject can be overweight without such overweight causing any sufferings from indications associated with overweight, while said subject may still desire to lose weight, e.g. for purely cosmetic reasons or to reduce the risks for obtaining a disease associated with overweight.

In one embodiment the non-therapeutic method of the invention is carried out for non-medical reasons, e.g. for cosmetic purposes. 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 embodiment of the invention, the subject in the non-therapeutic method is a healthy subject. The subject may have a blood sugar level during fasting below 6.1 , below 6.0, below 5.9, below 5.8, below 5.7, below 5.6 or even below 5.5 mmol/L. In yet another embodiment, the subject has a 2-hour plasma glucose level below 7.8, below 7.6, below 7.5, below 7.4, below 7.3, below 7.2, below 7.1 or even below 7.0 mmol/L on the 75-g oral glucose tolerance test. It will be understood that the 2-hour plasma glucose level relates to the plasma glucose level two hours after glucose ingestion.

Hence, in one embodiment of the invention the non-therapeutic method is for controlling weight, losing weight, reducing weight, preventing weight gain, limiting weight gain, inducing weight loss, increasing weight loss, managing weight and/or maintaining a healthy weight, in a subject, wherein said subject has overweight i.e. a BMI of from at least 25 kg/m² to less than 30 kg/m² for a subject aged 18 or older.

The non-therapeutic method can be a method for losing weight within a predetermined interval, e.g. to lose weight within 12 months, within 6 months, within 4 months, within 3 months, within 2 months, within 1 month, within 4 weeks, within 3 weeks, within 2 weeks or within 1 week.

Another aspect of the invention relates to a method of treating a human suffering from one or more of the indications selected from the group consisting of overweight, a condition associated with overweight, obesity, and a condition associated with obesity, by administering an effective amount of 2’-FL and GOS. Another aspect of the invention relates to a method of treatment a human desiring to loose body weight or maintaining a healthy body weight, by administering an effective amount of 2’-FL and GOS. The amount of 2’-FL and GOS is as defined elsewhere herein. In one embodiment of this aspect, the subject is aged 18 or older.

Yet another aspect of the invention is directed to a method of treatment a human subject suffering from low concentrations of Short Chain Fatty Acid (SCFA), in particular of acetate, in the distal colon, by administering to the human subject a composition comprising (i) 2’-FL and (ii) GOS as defined elsewhere herein; wherein the human subject has a healthy, overweight or obese body weight, preferably wherein the human subject is overweight or obese, more preferably obese. In one embodiment of this aspect, the subject is aged 18 or older. In another embodiment the subject is aged 18 or older and has a BMI of at least 30 kg/m².

In an embodiment of the invention, the subject in the method of treatment may have a blood sugar level during fasting below 6.1 , below 6.0, below 5.9, below 5.8, below 5.7, below 5.6 or even below 5.5 mmol/L. In yet another embodiment, the subject has a 2-hour plasma glucose level below 7.8, below 7.6, below 7.5, below 7.4, below 7.3, below 7.2, below 7.1 or even below 7.0 mmol/L on the 75-g oral glucose tolerance test. It will be understood that the 2-hour plasma glucose level relates to the plasma glucose level two hours after glucose ingestion.

In another aspect, the invention relates to the use of 2’-FL and GOS (i.e. a composition as herein defined above) for the manufacture of a medicament for losing weight. In yet another aspect, the invention relates to the use of 2’-FL and GOS (i.e. a composition as herein defined above) for the manufacture of a

medicament for treating overweight.

In one embodiment, composition as used in the different aspects and embodiments of the invention (i.e. the dietary fiber composition) comprises the combination of 2’-FL and GOS in an amount of at least 50 wt%, preferably at least 60 wt%, more preferably at least 70 wt%, more preferably still at least 80 wt%, more preferably still at least 85 wt%, more preferably still at least 90 wt%, as compared to the total weight of the dietary fiber composition.

The amount of the dietary fiber composition contained in a specific food product depends on the kind of food product, in particular its size and composition, as well as on the frequency and amount in which the product is or is supposed to be consumed.

In a particularly 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 comprising 0.5-8 grams, preferably 1 -4 grams, of 2’-FL 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 a further aspect, the invention pertains to a method of treating overweight or obesity or of a condition associated with overweight and/or obesity, or of preventing a condition associated with overweight, or of preventing obesity or of a condition associated with obesity, in a subject, wherein said combination comprises: (i) 2’-fucosyllactose (2’-FL); and (ii) galacto-oligosaccharides (GOS). Said method is as the treatment or prevention as defined above, including the amounts or ratio of 2’- FL and GOS in the combination, dosage regimens, subject, etc.

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

Except in the examples, or where otherwise expressly indicated, all numerical quantities in this description indicating amounts of material or conditions of reaction and/or use are to be understood as modified by the word "about" in describing the broadest scope of the invention. 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.

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 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.

As used herein, the term "comprising", which is synonymous with "including" or "containing", is open-ended, and does not exclude additional, unrecited element(s), ingredient(s) or method step(s), whereas the term "consisting of" is a closed term, which excludes any additional element, step, or ingredient which is not explicitly recited. As used herein, the term“essentially consisting of” is a partially open term, which does not exclude additional, unrecited element(s), step(s), or ingredient(s), as long as these additional element(s), step(s) or ingredient(s) do not materially affect the basic and novel properties of the invention.

As used herein, the term“comprising” (or“comprise(s)”) hence includes the term“consisting of” (“consist(s) of”), as well as the term“essentially consisting of” (“essentially consist(s) of”). Accordingly, the term“comprising” (or“comprise(s)”) is, in the present application, meant as more particularly encompassing the term

“consisting of” (“consist(s) of”), and the term“essentially consisting of” (“essentially consist(s) of”).

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.

EXAMPLE

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, 108-1 14. 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, et al. Food Research International, E-pub date 13 December 2017; Sayago Ayerdi et al Food Research International 1 18 (2019) 89-95).

Inoculum

Pooled fecal microbiota samples from 1 1 healthy lean 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 K₂FIP0₄-3FI₂0, 4.5 g NaCI, 0.005 g FeS0₄-7H₂0, 0.5 g MgS0₄-7H₂0, 0.45 g CaCI₂-2H₂0, 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 1 M 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 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 NaFhPC (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 2.

Table 2. Results of the TIM-2 experiments, using microbiota samples from lean or obese subjects to which 2’-FL was administered.

In Table 2, the amounts of acetate and short chain fatty acids (SCFA) refer 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 in lean subjects (having a BMI in a range of from at least 18.5 kg/m2 to less than 25 kg/m2) SCFA levels and in particular acetate levels are increased in the distal colon when 2’-FL was added.

The results further show that the addition of 2’-FL to obese subjects results in a higher SCFA and acetate levels (at least 90% increase of acetate and SCFA) in the distal colon as compared to using microbiota from lean subjects.

Claims

1. A composition comprising 2’-fucosyllactose (2’-FL) and galacto- oligosaccharides (GOS) for use in the treatment of one or more of the indications selected from the group consisting of overweight, a condition associated with overweight, and the prevention of a condition associated with overweight;

in a human subject with a healthy or overweight body weight.

2. The composition for use of claim 1 , wherein the human subject is overweight.

3. The composition for use of claim 1 , for use in the treatment of overweight, a condition associated with overweight, and/or the prevention of a condition associated with overweight; wherein the human subject is > 18 years old and has a body mass index (BMI) between 25 and 30 kg/m².

4. A composition comprising 2’-fucosyllactose (2’-FL) and galacto- oligosaccharides (GOS) for use in the treatment of one or more of the indications selected from the group consisting of obesity, a condition associated with obesity, and the prevention of a condition associated with obesity; in a human subject with an obese body weight.

5. The composition for use of claim 4, wherein the human subject has a body mass index (BMI) of at least 30 kg/m².

6. The composition for use of any one of the preceding claims, wherein the

amount of 2’-FL is in the range of from 0.1 to 30 grams, preferably in a range of from 0.5 to 25 gram, more preferably in a range of from 1 to 20 gram, most preferably in a range of from 2 to 10 gram.

7. The composition for use of any of the preceding claims, wherein the

composition does not comprise lacto-N-neotetraose (LNnT).

8. The composition for use according to any of the preceding claims, wherein the weight ratio between 2’-FL and GOS is in the range of from 0.5:10 to 10:0.5.

9. The composition for use according to any of the preceding claims, wherein the composition comprises at least 5 wt.% of 2’-FL and at least 5 wt.% of GOS as compared to the total weight of the composition.

10. The composition for use according to any of the preceding claims, 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;

or the composition for use of anyone of claims 7, 8 or 9, wherein the amount of 2’-FL is between 2 and 8 gram and the amount of GOS is between 1 and 6 grams.

11. The composition for use of any one of the preceding claims, wherein the composition is a food product, preferably wherein the food product is selected from the group consisting of dairy product, bar, liquid product, savory snack, savory biscuit, bakery products, pasta and food supplement.

12. The composition for use of any one of the preceding claims, wherein the composition is in the form of in the form of single serving; optionally such single servings is individually packaged.

13. The composition for use of any one of the preceding claims, wherein the composition is orally administered.

14. A non-therapeutic method for losing body weight or maintaining a healthy body weight in a human subject, the non-therapeutic method comprising the step of administering to the subject a composition as defined in anyone of claims 1 to 13; wherein the human subject has a healthy or overweight body weight; preferably wherein the human subject is overweight .

15. A non-therapeutic method for increasing the concentration of Short Chain Fatty Acid (SCFA), preferably of acetate, in the distal colon of a human subject, the non-therapeutic method comprising the step of administering to the human subject a composition as defined in anyone of claims 1 to 13; wherein the human subject has a healthy or overweight body weight, preferably wherein the human subject is overweight.