WO2011096808A1

WO2011096808A1 - Use of sialyl oligosaccharides in weight management

Info

Publication number: WO2011096808A1
Application number: PCT/NL2011/050078
Authority: WO
Inventors: Ruprecht Jules Joost Van Neerven; Hermiena Christina Schoterman; Arjen Nauta
Original assignee: Friesland Brands B.V.
Priority date: 2010-02-05
Filing date: 2011-02-04
Publication date: 2011-08-11
Also published as: NL2004200C2

Abstract

The invention relates to prebiotics and the use thereof for promoting human health. In particular, it relates to milk-, whey- or egg-derived oligosaccharides capable of enhancing the relative abundance of beneficial micro-organisms. Provided is the use of a sialyl - oligosaccharide for the manufacture of a dietetic, nutraceutical, pharmaceutical or nutritional composition, like an infant formula, for treating or preventing a condition associated with a reduced amount of Bacteroides ssp. in the gastrointestinal tract. Also provided is an infant formula for reducing the risk of development of overweight or obesity of an infant in later life. Said composition comprises living Bacteroides ssp. in an amount of at least 10+5cfu/g dry weight of the composition.

Description

Title: Use of sialyl oligosaccharides in weight management.

The invention relates to prebiotics and the use thereof for promoting human health. In particular, it relates to milk-derived oligosaccharides capable of enhancing the relative abundance of beneficial micro-organisms.

There is increasing awareness that the human gut microflora plays a critical role in maintaining host health, both within the gastrointestinal tract and, through the systemic absorption of metabolites. An "optimal" gut microflora establishes an efficient barrier to the invasion and colonisation of the gut by pathogenic bacteria, produces a range of metabolic substrates which in turn are utilized by the host (e.g. vitamins and short chain fatty acids) and stimulates the immune system in a non- inflammatory manner. Although little is known about the individual species of bacteria responsible for these beneficial activities, it is generally accepted that the bifidobacteria and lactobacilli constitute important components of the beneficial gut microflora. A number of diet-based microflora management tools have been developed and refined over recent decades including probiotic, prebiotic and synbiotic approaches. Each aims to stimulate numbers and/or activities of the bifidobacteria and lactobacilli within the gut microflora.

Of particular interest is the use of the gut microbiome as a therapeutic target for weight management. The obese population is growing worldwide as energy intake exceeds energy expenditure. Consequently, more people are at risk of developing obesity-related diseases such as diabetes mellitus type 2 and cardiovascular diseases. It is known in the art that there is a relationship between the diversity of the gut microbiota and obesity and that the energy balance of a subject may be modulated by altering the subject's gut microbiota population. See for example WO2007/043933 and EP1974734.

WO2008/076696 discloses that modulation of the relative abundance of bacteria within the Bacteroidetes or the Firmicutes division can be used to modulate the energy balance in a subject. Among others, WO2008/076696 teaches to decrease energy harvesting, decrease body fat, or promote weight loss by increasing the relative abundance of Bacteriodetes. It was thus proposed to administer a probiotic composition comprising Bacteriodetes to a subject.

Prebiotic supplements have also been implicated in weight management. Prebiotics have a number of advantages over probiotics; principally the fact that they are not alive means that they can be processed into a much wider range of foods than can the fragile probiotics. Furthermore, prebiotics do not share the problem of probiotic survival upon ingestion by the consumer. Since prebiotics stimulate growth of bacteria that are already present in the gut, they can be seen as more natural ingredients than probiotics, which necessitate administration of extraneous bacteria.

Parnell and Reimer (American Journal of Clinical Nutrition, June 2009,

Volume 89, Number 6, Pages 1751-1759), recruited 48 overweight and obese, but otherwise healthy, adults and randomly assigned them to receive daily supplements of oligofructose (21 grams) or placebo (maltodextrin) for 12 weeks. At the end of the study, people in the oligofructose group lost on average 1.03 kg of body weight, while people in the placebo group gained an average of 0.45 kg. While levels of ghrelin were suppressed by the prebiotic supplements, and PPY levels increased, no effects were observed on the satiety hormone glucagon-like peptide 1 (GLP-1). Furthermore, the oligofructose group reported a reduction in self-reported caloric intake, in addition to decreased levels of glucose and a mirroring effect on insulin concentrations.

EP2143341 discloses the use of an N-acetylated oligosaccharide, a galactose- oligosaccharide and a sialylated oligosaccharide in the manufacture of a nutritional composition such as an infant formula, for administration to an infant in the first six months of life to reduce the risk of obesity later in life.

Critical to the prebiotic concept is their selective fermentation within the gut microflora by what are seen as beneficial genera. Various preferred relationships between prebiotics and beneficial bacteria have been reported. For example, oligofructose can be utilized efficiently by Lactobacilli and Bifidobacteria.

WO2007/101675 relates to preparations comprising a probiotic and a prebiotic mixture which is specifically designed to enhance the efficiency and the efficacy of the probiotic and to food products comprising said preparation. Thus, prebiotics are suitably used to modulate the relative abundance of a certain micro-organism of interest. So far, the effects of prebiotic oligosaccharides on Bacteriodes spp. are limited to suppressive or reducing effects. For example, Tanaka et al. (Bif. Microflora 1983; 2: 17-24) reported that numbers of Bacteriodes decreased significantly upon ingestion of 10 g/day of trans-galacto-oligosaccharides (TOS). A reduction in Bacteriodes spp. was also observed after ingestion of lactulose (Ballongue et al. , Scand J Gastroenterol 1997; 32:41-4) or FOS (Gibson et al, Gastroenterology 1995; 108:975-82).

Recognizing the therapeutic potential of modulating the amount of

Bacteroidetes in the gut, e.g. in the treatment of obesity, the present inventors set out to identify a prebiotic capable of enriching the gut microbiota for bacteria within the Bacteroidetes division. In particular, they aimed at identifying prebiotics derivable from ruminant's milk which are useful for enriching the gut microflora for Bacteroides distasonis, Bacteroides fragilis, Bacteroides ovatus, Bacteroides prevotalla group, Bacteroides thetaiotaomicron and/or Bacteroides vulgatus.

It was surprisingly observed that a milk-derived sialic acid-containing oligosaccharide is capable of selectively promoting the growth of various Bacteroides species in adult and infant human faeces. This finding opens up a new window of application for sialyl oligosaccharides, in particular as prebiotic substance to enhance growth of Bacteroides ssp. in the gastrointestinal tract.

In one aspect, the invention provides a dietetic, nutritional or pharmaceutical composition, preferably formulated for oral or enteral administration, comprising at least one sialyl oligosaccharide in an amount effective to increase the relative abundance of Bacteroides ssp. in the gastrointestinal tract, such as Bacteroides distasonis, Bacteroides fragilis, Bacteroides ovatus, Bacteroides prevotalla group, Bacteroides thetaiotaomicron and/or Bacteroides vulgatus. In a specific embodiment, it comprises at least one sialyl oligosaccharide in an amount effective to increase the relative abundance of Bacteroides fragilis, Bacteroides ovatus, Bacteroides

thetaiotaomicron and/or Bacteroides vulgatus in the gastrointestinal tract.

WO2008/076696 discloses a nutritional composition comprising oligofructose and siallyllactose and its use for increasing the amount of Bifidobacteria. Whereas WO2008/076696 generally states that no effect on Bacteroides was observed, no information is given about which species were investigated.

As used herein, the term "sialyl oligosaccharide" means an oligosaccharide having at least one sialic acid moiety with associated charge. Sialic acids comprise a family of about 40 derivates of the nine carbon sugar neuramic acid. Sialic acid is the generic term for the N- or O-substituted derivatives of neuraminic acid. Milk contains sialic acid moieties in different forms. It can be present as a constituent of water soluble free oligosaccharides (e.g. sialyllactose), or bound to either glycoproteins such as lactoferrin and κ-casein and/or glycolipids (GD3 and GM3). The present invention is preferably practiced using a sialyloligosaccharide selected from the group consisting of sialyloligosaccharides derived from milk (e.g. cow, goat, camel, human milk), whey or egg. The sialyloligosaccharides can for instance be directly separated from milk, whey or egg or produced from components derived here from. Suitable

sialyloligosaccharides for use in the present invention are disialyllacto-iV-tetraose, 3'- sialyllactose, 6'-sialyllactose, 3'-sialyllactosamine, 6'-sialyllactosamine, 3'-sialyl-3- fucosyllactose, sialyllacto-iV-tetraose a, sialyllacto-iV-tetraose b, sialyllacto-iV-tetraose c, disialyllactose, 3'-sialyl Lewis A, 3'-sialyl Lewis X, disialyllacto-N-hexaose I, disialyllacto-N-hexaose II, sialyl Lea tetra, sialyllacto-N-neotetraose c, disialyllacto-N- fucopentaose II, and any combination thereof. An exemplary commercial preparation comprising egg yolk sialyl oligosaccharides is Sunsial E from Tayio Kaguku Co, Japan. Another exemplary preparation is Vivinal SL, a product comprising sialyllactose (FrieslandCampina Domo, the Netherlands)

In the sialyl oligosaccharides of human milk, sialic acid is attached to a penultimate galactose residue or N-acetylglucosamine residue via a2-3 or a2-6 linkage formed by the action of sialyl transferase (Kunz et al., Annu Rev Nutr.

2000;20:699-722, Asakuma et al, Biosci Biotechnol Biochem. 2007; 71:1447-51).

Sialyllactose represents the main part of the sialyl-oligosaccharide fraction in human and cow's milk. In one embodiment, the invention provides a dietetic, nutritional or pharmaceutical composition comprising sialyllactose in an amount effective to increase the relative abundance of Bacteroides ssp. in the gastrointestinal tract. For example, (partially) purified 3'-sialyllactose, 6'-sialyllactose or a mixture thereof may be used. Sialyl oligosaccharide, such as sialyllactose, may be enzymatically produced. In a specific aspect, a product of FrieslandCampina Domo (The Netherlands) with the tradename Vivinal SL, comprising about 6 weight% sialyllactose, is used as source of the prebiotic sialyl-oligosaccharide.

The sialyloligosaccharides 3' sialyllactose and /or 6' sialyllactose may be isolated e.g. by chromatographic or filtration technology from a natural source such as animal milks, for example human milk, cow milk, goat milk, camel milk, or whey. Preferred sources include whey and cow's milk. The concentration of lactose in cow's milk is about 40 to 60 g/litre, whereas the concentration of sialylated oligosaccharides in cow's milk is between 30 and 60 mg/ litre. In this latter fraction, sialyllactose is the most abundant oligosaccharide. In a preferred embodiment, the sialyloligosaccharide is obtained by a method comprising ion exchange technology, as for example in WO 2009/113861. For example, whey permeate streams are used as source of sialyllactose, which is captured selectively from these streams via ion exchange technology.

Subsequent downstream processing may include removal of remaining minerals and water, and a drying step.

The structures of sialic acid, 3'-sialylactose and 6' -sialyllactose are presented in table 1.

Table 1

A composition according to the invention finds its use as a dietetic, nutritional, nutraceutical or pharmaceutical composition. In a particular aspect, it can be used in the treatment or prevention of overweight or obesity in a subject, preferably a human subject. In humans, waist circumference measurement and body mass index (BMI) are the recommended ways to estimate body fat. BMI values from 18.5 to 24.9 are normal. Overweight is defined as a body mass index of 25.0 to less than 30.0

(consistent with U.S. Dietary Guidelines for Americans). A BMI of about 25 kg/m2 corresponds to about 10 percent over ideal body weight. Obesity is defined as a BMI of 30.0 or greater (consistent with criteria of the World Health Organization), or about 30 pounds or more overweight. Extreme obesity is defined as a BMI of 40 or greater.

To achieve the desired health effect through enriching the gut microflora for Bacteroides, known to decrease energy harvesting, the composition comprises at least one sialyloligosaccharide in an amount sufficient or effective to increase the relative abundance of a Bacteroides ssp. in the gastrointestinal tract. A composition according to the present invention can contribute to enhance the balance of beneficial and deleterious bacteria in the gastrointestinal tract of an animal, preferably a human, having or being at risk for overweight or obesity, comprising administering to the animal a composition comprising at least one sialyloligosaccharide. The relative abundance of Bacteroides ssp. refers to the quantitative contribution of Bacteroides ssp. to the total number of bacteria. For weight loss, the abundance may be altered by an increase of from about 100% to about 5000%, from about 200% to about 5000%, from about 300% to about 5000%, from about 400% to about 5000%, from about 500% to about 5000%, from about 600% to about 5000%, from about 700% to about 5000%, or from about 800% to 5000%. A method for determining the relative abundance of gut Bacteroides is described in WO2008/076696. Stated in another way, for weight loss, the abundance may be altered by an increase of about a two fold difference to about a fifty fold difference, of about a three fold difference to about a fifty fold difference, of about a four fold difference to about a fifty fold difference, of about a five fold difference to about a fifty fold difference, or of about a six fold difference to about a fifty fold difference

In one embodiment, the composition is capable (i.e. upon ingestion) of increasing the relative abundance of a Bacteroides ssp., preferably Bacteroides distasonis, Bacteroides fragilis, Bacteroides ovatus, Bacteroides prevotalla group, Bacteroides thetaiotaomicron and/or Bacteroides vulgatus, by at least 2-fold, preferably at least 4-fold, or even at least 6-fold. This effect may be observed after several hours up to several days after ingestion. In one aspect, the composition comprises at least one sialyl oligosaccharide in an amount of 0.005 - 20 grams per dose unit, for example a daily dose unit. A dose unit may consist of 0,2-500 grams, depending among others on the type of composition (solid, liquid), intended use (complete food, pharmaceutical composition, food supplement) and/or the intended consumer (adult, infant). For example, sialyl oligosaccharide may be present in an amount of 0.01— 10 gram per daily dose unit or per 100 grams or ml of composition. In another aspect, the composition comprises at least one sialyl oligosaccharide in an amount of 0.005-20 grams per 100 ml liquid composition, and preferably 0.1-10 grams per 100 ml liquid composition.

In one embodiment, the composition comprises the one or more

sialyloligosaccharide(s) as the sole prebiotic compound(s). In another embodiment the composition may contain one or more further (i.e. other than sialyloligosaccharides) prebiotic ingredients known in the art. Examples of suitable prebiotics are fructo and/or galacto-oligosaccharides, with short or long chains, inulin, fucose-containing oligosaccharides, beta glycans, carob flour, gums, pectins, galactans with short or long chains, glucosamine galactans and other glucosamine-containing oligosaccharides and nucleotides. In one preferred embodiment, a composition comprises sialyllactose and GOS, optionally as the sole prebiotic oligosaccharides. The composition may also contain one or more milk or whey derived components. Examples are sialic acid, a- Lactalbumin, lactoferrin, glycoproteins, casein macropeptide, gangliosides, phospholipids, colostrum, immunoglobulins, milk calcium and nucleotides.

In addition to the sialyloligosaccharide having a prebiotic effect on Bacteroides resident in the Gl-tract, a composition according to the invention may also be supplemented with one or more living Bacteroides ssp. (i.e. not inactivated, non- fragmented and capable of colonizing the intestinal tract at least transiently), such that the relative abundance of Bacteroides in the intestinal tract is even further enhanced. Preferred species include Bacteroides distasonis, Bacteroides fragilis, Bacteroides ovatus, Bacteroides prevotalla group, Bacteroides thetaiotaomicron and/or Bacteroides vulgatus. Typically the probiotics are added in an amount of at least 10 exp5 cfu/g dry weight of formula, preferably 10 exp6, more preferably 10 exp 7 cfu/g although generally even larger amounts are preferred, for example up to 10expl2 cfu/g of formula. In contrast to the present invention, WO2010/002241 discloses a nutritional composition comprising inactivated (e.g. heat killed) Gram-negative bacteria and/or bacterial cell fragments in an amount of less than 10 exp3 cfu per gram dry weight of formula and its use in providing nutrition to an infant delivered via caesarean section. According to WO2010/002241, the non-living, inactivated bacteria are thought to induce tolerance of the intestinal tract against colonisation with Gram-negative bacteria. The inactivated bacterial material is preferably combined with one or more non- digestible oligosaccharides selected from fructo- oligosaccharides, galacto-oligosaccharides and sialic acid comprising oligosaccharides. Importantly, the combined use of a sialyl oligosaccharide and living Bacteroides ssp. is not taught or suggested. In contrast, WO2010/002241 teaches to avoid the inclusion of live bacteria.

Of course, other beneficial probiotic strains may also be included in a composition provided herein, including specific strains of the genera Lactobacilli and Bifidobacteria that have been found to be able to colonise at least transiently the intestinal mucosa, to reduce the capability of pathogenic bacteria to adhere to the intestinal epithelium, to have immunomodulatory effects and/or to assist in the maintenance of well-being. Thus, alternative or additional probiotic bacterial strains include a lactobacillus or a bifidobacterium. Preferably strains which produce only L (+) lactic acid are used. Exemplary Lactobacillus (L.) species are L. rhamnosus, L. paracasei, L. amylovorus, L. ultunensis, L. acidophilus, L. kalixensis, L. delbrueckii subsp. Lactis, L. oris, L. fermentum, L. gastricus, L. antri, L. fermentum, L.

parabuchneri, L. brevis, L. plantarum, L. curvatus, L. sakei, L. gasseri, L. vaginalis, L. acidophilus, L. johnsonii, L. casei, L. salivarius and Lactobacillus reuteri. Particularly preferred strains are Lactobacillus rhamnosus ATCC 53103, Lactobacillus rhamnosus CGMCC 1.3724, Lactobacillus reuteri ATCC 55730, L. amylovorus DSM20552, L. ultunensis DSM16047, L. acidophilus ATCC4356, L. kalixensis DSM16043, L.

delbrueckii subsp. lactis DSM20073, L. oris DSM4864, L. reuteri DSM20016, L.

reuteri DSM20053, L. fermentum DSM20055, L. gastricus DSM16045, L. antri DSM16041, L. paracasei subsp. paracasei 43362, L. paracasei subsp. paracasei 43332, L. paracasei subsp. paracasei 43338, L. parabuchneri DSM5707, L. brevis DSM20054, L. plantarum NCD0326, L. plantarum NCIMB8826, L. curvatus NCD02739, L. sakei DSM20100, L. ruminis L5, L. gasseri SR21, L. vaginalis SR213, L. fermentum SR22, L. gasseri CR159, L. RC- 14, L. rhamnosis GR- 1, L. plantarum 299V and Lactobacillus paracasei CNCM 1-2116. Examples of preferred Bifidobacterium (B.) species are B. lactis, B. breve B. lactis Bb-1, B. lactis Bbl2, B. animalis, B. digestivus, B. infantis, B. adolescentis and B. longum. Particularly preferred strains are the strain of B. lactis sold by the Christian Hansen company of Denmark under the trade mark Bbl2, Bifidobacterium longum ATCC BAA-999 obtainable from Morinaga Milk Industry Co. Ltd. of Japan under the trade mark BB536, B. Adolescentis CIP 64.61, B. Adolescentis DSM 20083, B. Adolescentis NCFB 2229, B. Adolescentis NCFB 2204, B. Adolescentis LMG 10502, B. animalis JCM 20097, B. animalis DSM 20105, B. bifidum NCIMB 8810, B. bifidum LMG 11041, B. breve UCC2003, B. breve JCM 7017, B. breve JCM 7019, B. breve CCUG 43878, B. breve NCIMB 8815, B. breve NCFB 2258, B. breve NCFB 2257, B. breve NCFB 11815, B. dentium NCFB 2843, B. longum JCM 7050, B. longum JCM 7052, B. longum JCM 7056, B. longum CIP 64.63, B. longum CCUG 30698, B. longum NCIMB 8809, B. longum CCUG 15137, B. longum JCM 7053, B. longum / infantis CCUG 18157, B. infantis NCDO 2205, B. pseudocatenulatum LMG 10505, B. pseudocatenulatum NCIMB 8811, B. pseudolongum NCIMB 2244, B.

pseudolongum DSM 20095, B. globosum JCM 5820, B. globosum JCM 7092, B.

thermophilum JCM 7027. In a specific embodiment, the combination of B. lactis strain deposited under ATCC number 27536 and the L. casei strain deposited under ATCC number 55544 is used (see WO2008/056983 in the name of the applicant). In another specific embodiment, the combination of B. /acitsBB12 and L. paracasei CRL431is used.

The composition of the invention may be in any form suitable for human

administration, and in particular for administration in any part of the

gastrointestinal tract. Enteral administration of the compositions of the invention, and preferably oral administration, and administration through a tube or catheter, are all covered by the present invention. It can be a solid, semi-solid or liquid nutritional formulation, such as infant nutrition (infant formula, follow on formula or growing up milk), nutraceutical, nutritional or dietary supplement, functional food, beverage product, meal replacement, or food additive. Such nutritional compositions may be nutritionally complete, i.e. may include vitamins, minerals, trace elements as well as nitrogen, carbohydrate and fatty acid sources so that they may be used as the sole source of nutrition supplying essentially all the required daily amounts of vitamins, minerals, carbohydrates, fatty acids, proteins and the like. Accordingly, the compositions of the invention may be provided in the form of a nutritionally balanced composition, e. g. a complete formula diet or a complete meal, e. g. suited for oral or tube feeding. Alternatively, the composition of the invention may be provided as part of a meal, i.e. a nutritional or dietary supplement, e. g. in the form of a health drink. It may be desirable to provide the composition of the invention in the form of a low calorie composition, e. g. meal replacement. In this case the nutritional composition, e.g. meal replacement, is preferably low fat, i.e. less than about 10 en%, or substantially fat-free, i.e. less than about 2.5 en% contributed by fat, such as about 2 en% fat, based on the total caloric content of the composition. Suitably, a single serving of a low calorie nutritional composition, e. g. meal replacement, will have a caloric value of less than about 1000 kcal, and preferably between about 200 kcal and about 500 kcal. Suitable low calorie nutritional composition may include soft drink, such as juice, smoothie or soy-based drink, or dispersed in foods of any sort, such as, dairy bars, soups, cereals, e.g. breakfast cereals, muesli, candies, tabs, cookies, biscuits, crackers, such as a rice crackers, and dairy products, such as milk-shake, yoghurt drink, yoghurts and fruit drinks. In one embodiment, the composition is a non-fermented composition. Alternatively, the compositions of the invention may be provided as high calorie compositions, e. g. high calorie dietary supplement or meal replacement, for instance with a caloric value of more than about 400 kcal, preferably more than about 600 kcal, more preferably more than about 800 kcal.

In a specific aspect, the composition is an infant formula (including follow on formula or growing up milk). The bacterial composition in the infant digestive tract (also called the intestinal microflora) follows a pattern of change starting in the newborn, and varies depending on the infant diet. Development of the infant's intestinal microflora is initiated at birth. The aseptic, or sterile, digestive tract of the foetus is inoculated with bacteria during birth by the mother's intestinal and vaginal microflora. During the first week of life, enterobacteria and enterococci predominate in the gut of both breastfed and formula-fed infants. After this, the microflora changes rapidly. Some of the changes that occur depend on whether the infant is breastfed or formula-fed. An infant formula according to the present invention comprising a sialyl oligosaccharide, preferably a sialyllactose, can contribute to enhance the balance of beneficial and deleterious bacteria in the gastrointestinal tract of an infant having or at risk for overweight or obesity, e.g. at a later stage in life. Infant formulas are commonly used today to provide supplemental or sole source nutrition early in life. These formulas contain protein, carbohydrate, fat, vitamins, minerals, and other nutrients. They are commercially available as powders, ready-to-feed liquids, and liquid concentrates. There are currently a variety of commercially available infant formulas, each one designed to meet the specific nutritional needs of a particular infant group. Milk-based infant formulas, for example, represent the majority of commercially available infant formulas. Soy-based formulas also represent a large portion of the infant formula market by offering an alternative to milk-based formulas, especially in milk- intolerant infants. Lactose-free formulas are also available and can be useful in those infants with lactose sensitivity. Infant formulas with amino acids or partially hydrolyzed proteins are also available for certain infants.

The base infant formula comprises fat, protein, carbohydrate, vitamins and minerals, all of which are selected in kind and amount to provide a sole source of nutrition for the targeted infant or defined infant population. For example, the infant formula may be formulated for an infant of between 0 and 3 months of age, between 3 and 6 months of age, 6 and 9 months of age or 9 and 12 months of age (infant formula for infants between 6 and 12 months age are often called follow on formula). There are also infant formula for infants older than 12 months and even older than 24 months, and up to an age of 6 years (often called growing up milk). Infant formulas for use as base formulas include any known ready-to-feed infant formula, or any nutritional formula suitable for use in infants, provided that such a formula is a sole source nutritional having caloric density and osmolality values within the ranges defined herein. Many different sources and types of carbohydrates, fats, proteins, minerals and vitamins are known and can be used in the base formulas herein, provided that such nutrients are compatible with the added ingredients in the selected formulation and are otherwise suitable for use in an infant formula. Carbohydrates suitable for use in the base formulas herein may be simple or complex, lactose-containing or lactose-free, or combinations thereof, non-limiting examples of which include hydrolyzed, intact, naturally and/or chemically modified cornstarch, maltodextrin, glucose polymers, sucrose, corn syrup, corn syrup solids, rice or potato derived carbohydrate, glucose, fructose, lactose, high fructose corn syrup and indigestible oligosaccharides such as fructooligosaccharides (FOS), galactooligosaccharides (GOS), and combinations thereof. Particularly preferred is an infant formula comprising the combination of sialyllactose and GOS.

Proteins suitable for use in the base formulas herein include hydrolyzed, partially hydrolyzed, and non-hydrolyzed or intact proteins or protein sources, and can be derived from any known or otherwise suitable source such as milk (e.g., casein, whey, human milk protein), animal (e.g., meat, fish), cereal (e.g., rice, corn), vegetable (e.g., soy), or combinations thereof. In one embodiment, the composition of the invention comprises a whey fraction comprising the whey proteins a-lactalbumin (a- LA) and casein macropeptide (CMP), wherein the weight ratio between a-LA and CMP is > 2. In another embodiment, the composition of the invention comprises a whey fraction comprising the whey proteins a-lactalbumin (a-LA) and casein macropeptide (CMP), wherein the weight ratio between a-LA and CMP is < 2.

Proteins for use herein can also include, or be entirely or partially replaced by, free amino acids known for or otherwise suitable for use in infant formulas, non-limiting examples of which include alanine, arginine, asparagine, carnitine, aspartic acid, cystine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, taurine, threonine, tryptophan, taurine, tyrosine, valine, and combinations thereof. These amino acids are most typically used in their L-forms, although the corresponding D -isomers may also be used when nutritionally equivalent. Racemic or isomeric mixtures may also be used.

Fats suitable for use in the base formulas herein include coconut oil, soy oil, corn oil, olive oil, safflower oil, high oleic safflower oil, algal oil, MCT oil (medium chain triglycerides), sunflower oil, high oleic sunflower oil, palm and palm kernel oils, palm olein, canola oil, marine oils, cottonseed oils, and combinations thereof.

Vitamins and similar other ingredients suitable for use in the base formulas include vitamin A, vitamin D, vitamin E, vitamin K, thiamine, riboflavin, pyridoxine, vitamin B12, niacin, folic acid, pantothenic acid, biotin, vitamin C, choline, inositol, salts and derivatives thereof, and combinations thereof. Minerals suitable for use in the base formulas include calcium, phosphorus, magnesium, iron, zinc, manganese, copper, chromium, iodine, sodium, potassium, chloride, and combinations thereof.

Preferred probiotics for use in an infant formula include those capable of promoting the development of an early bifidogenic intestinal microbiota, e.g. the strains disclosed in EP 1974734.

A pharmaceutical composition as provided herein, e.g. clinical product, and nutritional compositions of the invention, e. g. dietary supplements, may be provided in the form of soft gel, sachets, powders, syrups, liquid suspensions, emulsions and solutions in convenient dosage forms. Oral pharmaceutical or dietary supplement forms may be made by conventional compounding procedures known in the pharmaceutical art, that is, by mixing the active substances together with edible pharmaceutical acceptable solid or liquid carriers and/or excipients, e. g. fillers such as cellulose, lactose, sucrose, mannitol, sorbitol, and calcium phosphates and binders, such as starch, gelatin, tragacanth, methylcellulose and/or polyvinylpyrrolidone (PVP). Optional additives include lubricants and flow conditioners, e. g. silicic acid, silicon dioxide, talc, stearic acid, magnesium/calcium stearates, polyethylene glycol (PEG) diluents, disintegrating agents, e. g. starch, carboxymethyl starch, cross-linked PVP, agar, alginic acid and alginates, colouring agents, flavouring agents, and melting agents. Dyes or pigments may be added to the tablets or dragee coatings, for example for identification purposes or to indicate different doses of active ingredient.

A further aspect of the invention relates to the use of a sialyl-oligosaccharide for the manufacture of a dietetic, nutritional of pharmaceutical composition for treating or preventing a condition associated with a reduced amount Bacteroides ssp. in the gastrointestinal tract. The sialyl oligosaccharide is preferably 3'-sialyllactose, 6'- sialyllactose, or a mixture thereof. The prebiotic sialyl oligosaccharide may be used in combination with one or more Bacteroides ssp. such that a synbiotic dietetic, nutritional of pharmaceutical composition is produced. In a specific embodiment, the use of a sialyl-oligosaccharide is provided for the manufacture of an infant formula for reducing the risk of development of overweight or obesity of an infant in later life. The formula may contain any of the components described herein above. The infant formula can be formulated to be administered to an infant of between 0 and 3 months of age, between 3 and 6 months of age, between 6 and 9 months of age, between 9 and 12 months of age or between 12 and 24 months of age.

Herewith, the invention also provides a method for enhancing the balance of beneficial and deleterious bacteria in the gastrointestinal tract of an animal having or at risk for overweight or obesity, comprising administering to the animal a composition comprising at least one sialyl oligosaccharide. Preferably, the

enhancement is associated with reduction of energy harvesting. Said enhancement for example comprises an increase in level of one or more of Bactericides spp.

Also within the scope of the present invention is the use of a sialyl

oligosaccharide to enhance the growth of a Bacteriodes spp. such as Bacteroides distasonis, Bacteroides fragilis, Bacteroides ovatus, Bacteroides prevotalla group, Bacteroides thetaiotaomicron and/or Bacteroides vulgatus. In a specific embodiment, it comprises the use of at least one sialyl oligosaccharide to increase the relative abundance of Bacteroides fragilis, Bacteroides ovatus, Bacteroides thetaiotaomicron and/or Bacteroides vulgatus. Growth can be in vitro or in vivo. A preferred sialyl oligosaccharide for use as prebiotic for a Bacteriodes spp. is a (partially) purified sialyllactose, such as 3'-sialyllactose, 6'-sialyllactose or a mixture thereof.

LEGEND TO THE FIGURES

Figure 1: Effects of sialyllactose and GOS on micoflora composition of batch cultures inoculated with a pool of adult faeces. The panels show the total amount of bacteria per vessel (A), bacteroides (B), and bifidobacteria (C) numbers quantified by QPCR. Values shown are averages of two separate runs, with error bars indicating the deviation of the mean of the two measurements.

Figure 2: Effects of sialyllactose and GOS on micoflora composition of batch cultures inoculated with a pool of adult faeces. The panels show the amount of bacteroides (A), and bifidobacteria (B) numbers quantified by QPCR. Values shown are values corrected for the growth in each vessel relative to t=0, and are averages of two separate runs, with error bars indicating the bacterial counts of the two separate runs. Figure 3: Effects of sialyllactose and GOS on micoflora composition of batch cultures inoculated with a pool of infant faeces as described in example 2. The panels show the total amount of bacteria per vessel (A), bacteroides (B), and bifidobacteria (C) numbers quantified by QPCR. Values shown are averages of two separate runs, with error bars indicating the deviation of the mean of the two measurements.

Figure 4 : Effects of sialyllactose and GOS on micoflora composition of batch cultures inoculated with a pool of infant faeces as described in example 2. The panels show the amount of bacteroides (A), and bifidobacteria (B) numbers quantified by QPCR.

Values shown are values corrected for the growth in each vessel relative to t=0, and are averages of two separate runs, with error bars indicating the bacterial counts of the two separate runs.

EXPERIMENTAL SECTION

Example 1: Effect of sialyl oligosaccharide on adult faeces microflora composition.

Improvement of the health of human can be accomplished by selective stimulation of the growth and/or activity of groups of bacteria in the colon. This stimulation can be induced by prebiotics like oligo- and polysaccharides. This example describes a comparison of the selective fermentation of GOS and sialyl-containing

oligosaccharides by adult human microflora. GOS (Vivinal GOS, FrieslandCampma Domo) and sialyllactose (Vivinal SL, FrieslandCampma Domo) were purified in order to make them suitable for use in the in vitro model and to exclude interference with other components like lactose and glucose. A pH-controlled batch fermentation model, which represents the distal part of the colon, was used to measure the effect of the oligosaccharides on the composition of the microflora, and the production of short chain fatty acids and lactate. Methods

Production of oligosaccharides

Both GOS (Vivinal GOS) and sialyllactose (Vivinal SL) were purified by means of chromatography into purified GOS and purified SL

Faecal Sample Preparation

Faecal samples were obtained from 16 adult volunteers (average BMI 23,4, range 18,0 - 27,8) free of known gastrointestinal disorders. None of the volunteers had taken prebiotic products within a three-month period prior to sampling. Individual faecal samples were diluted to a 1 in 10 weight/weight mixture using 0.1M, pH7, phosphate buffered saline (PBS) (Sigma laboratories, Gillingham, Dorset, UK) at pH7.4 and pooled. The PBS was reduced overnight in an anaerobic cabinet (10% H2, 10% CO2, 80% N2). This mixture was then homogenised for 120 seconds (Seward Stomacher 80 Biomaster), at normal speed, in the stomacher. The faecal slurry was aliquotted and frozen. The faecal slurry was thawed just prior to the start of the experiment and used to inoculate the batch cultures. pH Controlled, Stirred batch cultures

Chemostat nutrient medium was prepared containing the following ingredients: Peptone water 2g/l (Oxoid, Basingstoke, UK), yeast extract 2g/l (Oxoid), NaCl O.lg/1 (Fisher, Loughborough, UK), K2HPO4.3H20 0.05g/l (BDH, Poole, UK), KH2PO4 0.04g/l (BDH), MgS0₄.7H₂0 O.Olg/1 (Sigma), CaCl2.2H₂0 0.005g/l (BDH), NaHCOs 2g/l (Sigma), Tween 80 (BDH) 2ml, Hemin 0.02g/l, Vitamin Ki ΙΟμΙ (Sigma), Cystiene HC1 0.5g/l (Oxoid), Bile Salts 0.5g/l (Oxoid), Resazarin 4ml/l (Sigma), pH7.

The chemostat nutrient medium autoclaved, then aseptically poured into the sterile batch culture vessels. This was left overnight with nitrogen pumping through the vessel to provide an anaerobic environment. 0.5ml of 10% cysteine-HCl was added to aid reduction of the medium. pH meters (Electrolab pH controller, Tewksbury, UK) were set to regulate the vessels between pH6.8 and pH7 by the addition of 0.5M HC1 or 0.5M NaOH. The vessels were maintained at 37°C, and were stirred using magnetic stirrers. The test material (purified GOS or purified SL were added to the vessel to a final concentration of 10 g/1 oligosaccharide just prior to the addition of 15 ml of faecal slurry of a pool of adult faecal samples (10% w/w). The vessels were left for 24h, with samples taken at 0, 3, 6, 9 and 24h. The samples were centrifuged for 5 min at 13 200 rpm and supernatants and pellet fractions were stored at—20 C until SCFA measurement (supernatants) or DNA preparation for QPCR analysis (pellet). The pH-controlled fermentation experiments were conducted in two independent experiments using samples from the same faeces pool as indicated.

QPCR analyses

DNA was isolated from frozen pellets of fermented faecal samples using the QIAamp DNA Stool Mini Kit (Qiagen). The number of total bacteria and bacteria of specific groups were quantified by quantitative PCR essentially as described in EP1997906 and EP1997907. The primers and probes for the detection of total bacteria,

Escherichia coli, Lactobacilli, Clostridia, Bifidobacteria, and Bacteroides were based on 16S rDNA gene sequences, retrieved by http ://gr eengene s . lfal, go v website. From these sequences forward primers, reverse primers and TaqMan probes were designed using the Primer Express software. To check specificity, the selected primers and probes were compared to all available 16S rDNA gene sequences using the BLAST database search program. Applied Biosystems manufactured primers and probes.

Results

Batch culture fermentation experiments were set up as described above. The results shown in figure 1 show that SL strongly enhances the growth of Bacteroides, and GOS strongly enhances the growth of Bifidobacteria.

During the batch fermentation only a small increase in total bacteria counts was seen, especially in the carbohydrate groups (Figure 1A). As the total number of bacteria increased in each of the vessels, the results are also shown as values corrected for the total bacteria number in each vessel relative to t=0 (Figure 2).

SL was the only compound tested that clearly promoted the growth of bacteroides (figure IB). This was also the case after correction for growth of bacteria in the vessel, but the effect was less prominent (Figures IB and 2A).

A clear stimulatory effect on bifidobacteria was seen in the vessels containing GOS (figure 1C). This was also clear after correction for the total growth of bacteria in the vessels compared to t=0 (Figure 2B). SL had only a minor effect on bifidobacteria (Figure 1C and 2B) compared to the control. The growth of Clostridia, Lactobacilli and E. coli was not significantly promoted by either SL or GOS.

These results demonstrate that SL can selectively promote the growth of bacteroides in faeces from human adults.

Example 2: Effect of sialyl oligosaccharide on baby faeces microflora composition.

This example demonstrates the effect of oligo- and polysaccharides on the microbial population of baby faeces by in vitro fermentation. The experiments were essentially carried out as described in Example 1, only now with a batch of in vitro prefermented infant faeces. The testgroups included a control vessel, a vessel containing purified GOS (10 g/L oligosaccharide), and a vessel containing purified SL (lOg/L

oligosaccharide). Results

After DNA isolation of the fermented samples the amount of bacteria were

determined and displayed in Figure 3, and as displayed as values corrected for total bacteria growth compared to t=0 in Figure 4.

Figure 3B shows the amounts of Bacteroides present during batch fermentation. Bacteroides were stimulated by SL and GOS when compared to the control fermentation. When corrected for the total bacteria numbers a stimulation of

Bacteroides was most consistent and prominent for SL (Figure 4A). Figure 3A shows that the total growth of bacteria was stimulated by GOS and SL up to a 2-5 fold increase during 24 h. The total bacteria count of control was stable. Figure 3C illustrates that the Bifidobacteria were stimulated by GOS and SL compared to the Bifidobacteria in the control vessel present. After correction for the total bacteria count (Figure 4B), there is an increase of the amount of Bifidobacteria during the first part of the fermentation (till 9 h), whereas in the second part of the fermentation the amount of Bifidobacteria decreased. These results demonstrate that SL can also increase the amount of Bacteroides in infant faeces. Example 3: Species analysis using DNA-Chip

The prebiotic effect of sialyllactose on Bacteroides outgrowth in batch culture experiments with infant faeces was further investigated using DNA micro array technology (TNO, Zeist, the Netherlands). DNA obtained from the in vitro batch cultures of Example 2 (T=24 h) were subjected to a DNA chip designed for the detection of more than 400 bacterial species. For each species represented on the microarray one or more unique short oligonucleotide sequences from within the 16S rDNA gene were selected. Criteria for sequence selection, apart from being unique, included length and melting temperature. Short oligonucleotide sequences (approx 20 nt) were used with a melting temperature of 60°C according to the Wallace rule for which a one nucleotide mismatch already resulted in an absence (or very strong decrease) of signal after hybridization.

Results are shown in Table 2. The numbers are based on duplicate analysis, and represent the fold increase over background level.

Table 2

Control SL GOS SL+GOS

Bacteroides 6 25 15 15 distasonis

Bacteroides 25 190 125 190 fragilis

Bacteroides 25 150 125 170 ovatus

Bacteroides 15 95 40 65 prevotalla group

Bacteroides 40 180 90 150 thetaiotao micron

Bacteroides 15 180 50 165 vulgatus

Bacteroides 45 165 90 175 species

Bacteroides 10 75 30 55 group The results shown in Table 2 demonstrate that SL strongly enhances the growth of Bacteroides, in particular B. fragilis, B. thetaiotaomicron and B. vulgatus. The prebiotic effect of SL on Bacteroides was stronger than that exerted by GOS and remained intact when SL was used in combination with the bifidogenic GOS.

Example 4 : Infant formulas

Table 3 shows the composition of three exemplary nutritional formulas according to the invention, e.g. infant formulas for the age group between 0-6 months, for reducing the risk of development of overweight or obesity of an infant in later life.

Table 3: Composition of the formulas (per 100 ml)

Basic Formula A: SL Formula B: Formula C: SL+

SL+GOS probiotics

Energy, kcal 67 67 67 67

Protein (g) 1.4 1.4 1.4 1.4

Carbohydrates

(g) 7.6 7.4 6.7 6.4

Fat (g) 3.5 3.5 3.5 3.5

0.25 (range 0.005 -

Sialyllactose (g) 2.0 g/lOOml) 0.2 0.2

0.5 (range 0.1 -

Vivinal GOS (g) 1.0 g/100 ml) 0.5

Bacteroides (cfu) 10expl2

Claims

A dietetic, nutritional, nutraceutical or pharmaceutical composition, comprising:

- at least one sialyloligosaccharide in an amount effective to increase the relative abundance of Bacteroides ssp. in the gastrointestinal tract of an animal, preferably a human; and

- living Bacteroides ssp in an amount of at least 10 exp5 cfu/g dry weight composition.

Composition according to claim 1, wherein said at least one

sialyloligosaccharide is present in an amount effective to increase the relative abundance of Bacteroides distasonis, Bacteroides fragilis, Bacteroides ovatus, Bacteroides prevotalla group, Bacteroides thetaiotao micron, Bacteroides vulgatus or a combination thereof.

Composition according to claim 1 or 2, comprising one or more

sialyloligosaccharides selected from the group consisting of disialyllacto-iV- tetraose, 3'-sialyllactose, 6'-sialyllactose, 3'-sialyllactosamine, 6'- sialyllactosamine, 3'-sialyl-3-fucosyllactose, sialyllacto-iV-tetraose a, sialyllacto-iV-tetraose b, sialyllacto-iV-tetraose c, disialyllactose, 3'-sialyl Lewis A, 3'-sialyl Lewis X, disialyllacto-N-hexaose I, disialyllacto-N-hexaose II, sialyl Lea tetra, sialyllacto-N-neotetraose c, disialyllacto-N-fucopentaose II.

Composition according to claim 3, comprising 3'-sialyllactose and/or 6'- sialyllactose.

Composition according to any one of claims 1 to 4, comprising said at least one sialyloligosaccharide in an amount of 0.005 - 20 grams per dose unit, preferably 0.01— 10 grams per dose unit.

6. Composition according to any of the preceding claims, wherein said

composition is formulated as an infant formula. Composition according to any of the preceding claims, wherein said Bacteroides ssp. is Bacteroides distasonis, Bacteroides fragilis, Bacteroides ovatus,

Bacteroides prevotalla group, Bacteroides thetaiotao micron, Bacteroides vulgatus or a combination thereof.

Composition according to any one of the preceding claims, wherein the total amount of living Bacteroides ssp. is 10 exp6 cfu/g dry weight of formula, preferably 10 exp 7 cfu/g

Composition according to any one of the preceding claims, further comprising one or more prebiotics, preferably selected from the group consisting of fructo- oligosaccharides (FOS), galacto-oligosaccharides (GOS), inulin, fucose- containing oligosaccharides, beta glycans, carob flour, gums, pectins, galactans with short or long chains, glucosamine-containing oligosaccharides and nucleotides, more preferably GOS.

10. Composition according to any one of the preceding claims, comprising at least one further milk- or whey- derived compound, preferably a compound selected from the group consisting of sialic acid, a-Lactalbumin, lactoferrin,

glycoprotein, casein macropeptide, gangliosides, phospholipids, colostrum, immunoglobulins, milk calcium and nucleotides.

11. Composition according to any of the preceding claims, for use in the treatment or prevention of overweight or obesity in a human subject.

12. Use of at least one sialyloligosaccharide for the manufacture of a dietetic, nutraceutical, nutritional or pharmaceutical composition for treating or preventing a condition associated with a reduced amount of Bacteroides ssp. the gastrointestinal tract.

13. Use according to claim 11, wherein said condition is overweight or obesity.

14. Use according to claim 11 or 12, wherein said at least one sialyloligosaccharide is selected from the group consisting of disialyllacto-iV-tetraose, 3'-sialyllactose, 6'-sialyllactose, 3'-sialyllactosamine, 6'-sialyllactosamine, 3'-sialyl-3- fucosyllactose, sialyllacto-iV-tetraose a, sialyllacto-iV-tetraose b, sialyllacto-iV- tetraose c, disialyllactose, 3'-sialyl Lewis A, 3'-sialyl Lewis X, disialyllacto-N- hexaose I, disialyllacto-N-hexaose II, sialyl Lea tetra, sialyllacto-N-neotetraose c, disialyllacto-N-fucopentaose II.

15. Use according to claim 13, wherein said sialyloligosaccharide is 3'-sialyllactose, 6'-sialyllactose, or a mixture thereof.

16. Use according to any one of claims 11 to 14, wherein said composition further comprises Bacteroides ssp.

17. Use according to any one of claims 11- 15 for the manufacture of an infant

formula.

18. Use according to claim 16, wherein said infant formula is an infant formula, a follow on formula or a growing up milk, formulated to be administered to human being between 0 and 6 years, preferably an infant formula formulated for infants between 0 and 6 months of age.

19. Use according to claim 16 or 17, for the manufacture of an infant formula for reducing the risk of development of overweight or obesity of an infant in later life.

20. Use of a sialyloligosaccharide to enhance the growth of a Bacteroides spp.

21. Use of a sialyloligosaccharide according to claim 19, wherein said

sialyloligosaccharide is 3'-sialyllactose, 6'-sialyllactose or a mixture thereof.