FIELD

The present invention relates to the field of infant and/or toddler nutrition. In particular the present invention relates to a non-digestible oligosaccharide composition suitable for use as an infant and/or toddler nutrition.

BACKGROUND

The microflora of the human large intestine (typically divided into caecum, colon and rectum) plays a crucial role in both human nutrition and health.

The bacterial composition is influenced and can be modulated by dietary intake.

Carbohydrates which have passed through the stomach and small intestine are metabolised by the bacteria and as a major end-product of metabolism short-chain fatty acids (SCFA), such as acetate, propionate, butyrate and valerate, are formed, which are subsequently released into the blood. Other end products of bacterial fermentation include for example lactate and succinate. The total amounts and compositions (relative amounts) of these end products in turn can have an effect on bacterial growth, pH, exclusion of pathogenic species, etc.

A method to beneficially influence the microbial flora and human health is the administration of prebiotics. "Prebiotics" were defined as "non-digestible food ingredients that beneficially affect the host by selectively stimulating the growth and/or activity of one or a limited number of bacteria in the colon, and thus improve the hosts health (Gibson and Roberfroid 1995, J. Nutr. 125, 1401-1412).

The criteria which a compound must fulfil in order to be classified as a prebiotic are: 1) it must not be hydrolysed or absorbed in the upper part of the gastrointestinal tract

(stomach, small intestine), 2) it must be selectively fermented by one or more potentially beneficial bacteria in the colon, 3) it must alter the colonic microbiota towards a healthier composition and 4) it must preferably induce effects which are beneficial to the health of the subject.

Commonly used prebiotics are so-called non-digestible carbohydrates (or "soluble dietary fibres"), which pass undigested through the upper part of the gastrointestinal tract into the large intestine. These include for example fructooligosaccharides (FOS), oligofructose, inulin and transgalacto-oligosaccharides (TOS).

WO2005/1 10121 describes a composition comprising galacto-oligsaccharides and polyfructose. Administration of this composition results in a decrease in the sum of intestinal and or faecal butyrate, isobutyrate, valerate and/or isovalerate relative to total short chain fatty acids.

WO 2004/026316 discloses the use of slowly fermented complex oligomeric or polymeric carbohydrates in a composition for preventing disorders caused by "putrefactive fermentation" in the colon, such fermentation resulting in production of toxic compounds (including isovalerate). The composition disclosed in WO 2004/026316 is said to cause a reduction in putrefaction fermentation and a reduction of pH throughout the colon.

It was therefore considered desirable in the prior art to reduce valeric acid levels in the individual, such as by use of non-digestible oligosaccharide compositions. The prior art indicates that administration of such non-digestible oligosaccharide compositions to a subject results in the desired decrease in valeric acid levels.

SUMMARY

We have now surprisingly demonstrated that administration of non-digestible oligosaccharides to individuals in fact results in an increase of valeric acid levels in the gut (rather than a decrease). We further disclose that preventing the decrease of, increasing or maintaining valeric acid levels in an individual may have beneficial medicinal effects, such as preventing or alleviating stress, tension or anxiety.

According to a 1 ^st aspect of the present invention, we provide the use of a composition comprising non-digestible oligosaccharides for increasing, maintaining or preventing a decrease in valeric acid in an individual.

The non-digestible oligosaccharide composition may comprise a combination of a transgalacto-oligosaccharide and a fructo-oligosaccharide. The non-digestible oligosaccharide composition may comprise a combination of a short chain galacto-oligosaccharides (scGOS) and long chain fructo-oligosaccharides (IcFOS). Such a combination is referred to in this document as "scGOS/lcFOS".

The scGOS/lcFOS may be together present in the composition at between 0.01 g/100 ml to 10 g/100 ml (0.1 to 100 g/1). It may be present at between 0.10 g/100 ml to 1.0 g/100 ml.

The non-digestible oligosaccharide composition may comprise between about 19:1 to 1 : 19 scGOS:lcFOS by weight. It may comprise about 9:1 scGOS:lcFOS by weight.

The non-digestible oligosaccharide composition may comprise infant formula. It may comprise a children's nutritional product. It may comprise a pediatric nutritional formula. It may comprise a toddler nutritional formula. It may comprise a growing up milk. It may comprise a human milk supplement. It may comprise a medicinal food.

The non-digestible oligosaccharide composition may comprise a cereal. It may comprise a growing up milk.

The individual may be a human baby. He or she may be an infant. He or she may be a toddler. The individual may be a toddler between 12 to 36 months old.

The non-digestible oligosaccharide composition may further comprise long chain polyunsaturated fatty acids (LCPUFA). The composition may further comprise

eicosapentaenoic acid (EPA). It may comprise docosahexaenoic acid (DHA). It may comprise arachidonic acid (ARA). It may comprise docosapentanenoic acid (DP A). It may comprise any combination of one or more of the above.

The LCPUFA is present in the composition at between 0.1 mg/100 ml to 100 mg/100 ml. It may be present at between 2 mg/100 ml to 20 mg/100 ml.

The valeric acid may be increased in the gut, such as intestine, of the individual.

The non-digestible oligosaccharide composition may comprise a powder suitable for making a liquid composition after reconstitution with an aqueous solution, such as with water.

The non-digestible oligosaccharide composition may be capable of decreasing or alleviating stress or anxiety in the individual. It may be capable of treating, alleviating or preventing insomnia, sleeping disorders, restlessness and anxiety. It may be capable of treating, alleviating or preventing epilepsy. The non-digestible oligosaccharide composition may be used as a sedative such as for nervous tension, hysteria, excitabilityand stress. It may be used as an anxiolytic, sleeping aid, anticonvulsantor migraine treatment.

There is provided, according to a 2 ^nd aspect of the present invention, a method of increasing, maintaining or preventing a decrease in valeric acid in the gut of an individual, the method comprising administering a composition comprising non-digestible oligosaccharides to the individual.

We provide, according to a 3 ^rd aspect of the present invention, a method of decreasing or alleviating stress or anxiety in the individual, by administering a composition comprising non-digestible oligosaccharides to the individual. The method may be used for the treatment, alleviation or prevention of insomnia, sleeping disorders, restlessness and anxiety, epilepsy, nervous tension, hysteria, excitability stress, convulsions or migraine in an individual.

As a 4 ^th aspect of the present invention, there is provided a non-digestible

oligosaccharide composition for use in a method of decreasing or alleviating stress or anxiety in an individual, or treatment, alleviation or prevention of insomnia, sleeping disorders, restlessness and anxiety, epilepsy, nervous tension, hysteria, excitability, stress, convulsionsor migraine in an individual.

We provide, according to a 5 ^th aspect of the present invention, use of a non-digestible oligosaccharide composition in a method of preparation of a medicament for decreasing or alleviating stress or anxiety in an individual, or for the treatment, alleviation or prevention of insomnia, sleeping disorders, restlessness and anxiety, epilepsy, nervous tension, hysteria, excitability, stress , convulsions or migraine in an individual.

The non-digestible oligosaccharide composition for such uses may comprise a feature as described above. The practice of the present invention will employ, unless otherwise indicated, conventional techniques of chemistry, molecular biology, microbiology, recombinant DNA and immunology, which are within the capabilities of a person of ordinary skill in the art. Such techniques are explained in the literature. See, for example, J. Sambrook, E. F. Fritsch, and T. Maniatis, 1989, Molecular Cloning: A Laboratory Manual, Second Edition, Books 1 -3, Cold Spring Harbor Laboratory Press; Ausubel, F. M. et al. (1995 and periodic supplements; Current Protocols in Molecular Biology, ch. 9, 13, and 16, John Wiley & Sons, New York, N.Y.); B. Roe, J. Crabtree, and A. Kahn, 1996, DNA Isolation and Sequencing: Essential Techniques, John Wiley & Sons; J. M. Polak and James O'D. McGee, 1990, In Situ

Hybridization: Principles and Practice; Oxford University Press; M. J. Gait (Editor), 1984, Oligonucleotide Synthesis: A Practical Approach, Irl Press; D. M. J. Lilley and J. E. Dahlberg, 1992, Methods of Enzymology: DNA Structure Part A: Synthesis and Physical Analysis of DNA Methods in Enzymology, Academic Press; Using Antibodies : A Laboratory Manual : Portable Protocol NO. I by Edward Harlow, David Lane, Ed Harlow (1999, Cold Spring Harbor Laboratory Press, ISBN 0-87969-544-7); Antibodies : A Laboratory Manual by Ed Harlow (Editor), David Lane (Editor) ( 1988, Cold Spring Harbor Laboratory Press, ISBN 0- 87969-314-2), 1855. Handbook of Drug Screening, edited by Ramakrishna Seethala,

Prabhavathi B. Fernandes (2001, New York, NY, Marcel Dekker, ISBN 0-8247-0562-9); and Lab Ref: A Handbook of Recipes, Reagents, and Other Reference Tools for Use at the Bench, Edited Jane Roskams and Linda Rodgers, 2002, Cold Spring Harbor Laboratory, ISBN 0- 87969-630-3. Each of these general texts is herein incorporated by reference.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 is a diagram showing the overall methodology of the conduct of the study. Baseline characteristics were that there should be no significant differences between both active groups against the control group The daily intake recommended was 500 ml to 630 ml. Figure 2 is a diagram showing the overall impact on the microbial system.

Figure 3: Percentage of Bifidobacteria at baseline and week 12 of the 3 study groups. Middle line in box plots are medians; box are interquartile ranges (IQR); and the error bars show 1.5 times the IQR.

Figure 4. Structure of valeric acid. DETAILED DESCRIPTION

NON-DIGESTIBLE OLIGOSACCHARIDE COMPOSITIONS

We have therefore surprisingly demonstrated, in contrast to the prior art indications, that it is desirable to increase, maintain or prevent a decrease in valeric acid levels. We demonstrate that this may be achieved through administration of a composition comprising non-digestible oligosaccharides. Such a non-digestible oligosaccharide composition may be administered to reduce or alleviate stress, tension or anxiety in an individual, such as a toddler.

We demonstrate in the Examples that a mixture of scGOS/lcFOS is capable of maintaining valeric acid levels in young children. Example 8 below shows that there was no significant difference in valeric acid concentrations in the stool of young children fed with scGOS/lcFOS between weeks 0 and 12 (p =0.805, p = 0.449). On the other hand, there was a significant decrease in valeric acid concentration in the stool of subjects fed with control mixture (p= 0.040).

Accordingly, a mixture of scGOS and IcFOS is capable of maintaining the levels of valeric acid when fed to young children. We therefore provide for the use of a mixture of scGOS and IcFOS in maintaining the levels of valeric acid in the gut of young children.

We further demonstrate in the Examples that a mixture of scGOS/lcFOS is capable of preventing a decrease in valeric acid levels in young children. Thus, Example 9 shows that when comparing the difference in valeric levels between week 0 and week 12 in young children fed with scGOS/lcFOS and the difference in valeric levels between week 0 and week 12 in controls, there was a significant decrease in valeric acid concentrations in control subjects compared with those fed with scGOS/lcFOS (p = 0.040).

Accordingly, a mixture of scGOS and IcFOS is capable of preventing a decrease the levels of valeric acid when fed to young children. We therefore provide for the use of a mixture of scGOS and IcFOS in preventing a decrease in the levels of valeric acid in the gut of young children.

We further demonstrate in the Examples that a mixture of scGOS/lcFOS is capable of generally increasing valeric acid levels in young children. Thus, in Example 10, it is seen that in both GUMl and GUM2 subjects, actual valeric acid concentrations were generally found to have increased compared to those in control group.

Accordingly, a mixture of scGOS and IcFOS is capable of increasing the levels of valeric acid when fed to young children. We therefore provide for the use of a mixture of scGOS and IcFOS in increasing the levels of valeric acid in the gut of young children.

In general, the compositions and methods described here make use of a non-digestible oligosaccharide composition, which is described in detail below. Such a composition may comprise a mixture of oligosaccharides, such as a mixture of scGOS and lcFOS, optionally together with LCPUFA.

USES OF NON-DIGESTIBLE OLIGOSACCHARIDE COMPOSITIONS

As noted above, the Examples demonstrate that administration of such a non-digestible oligosaccharide composition to an individual raises, maintains, or at least prevents a decrease in valeric acid levels in his or her gut.

Valeric acid is described in further detail below ("Valeric Acid"). As noted in that section, valeric acid is found naturally in the perennial flowering plant valerian (Valeriana officinalis), from which it gets its name. The roots of this plant are processed and prepared into a herbal or dietary supplement, known as valerian (described in detail below).

Valerian has been used as a medicinal herb since at least ancient Greece and Rome, and is used as a treatment for a number of conditions, including insomnia, sleeping disorders, restlessness, stress, anxiety, tension, and epilepsy. Valerian is known to have sedative and anxiolytic effects and is used as a sleeping aid, sedative, anticonvulsant and migraine treatment. It is particularly used as a sedative for nervous tension, hysteria, excitabilityand stress.

As valeric acid is a component of valerian, which has medicinal properties, we propose that valeric acid may be increased or maintained or prevented from decreasing in an individual to provide any of the effects set out above for valerian. Accordingly, valeric acid levels may be increased, maintained or prevented from decreasing in an individual in order to treat or prevent or alleviate any of the above conditions or symptoms.

Likewise, as the non-digestible oligosaccharide composition described in this document is capable of increasing, maintaining or preventing the decrease of valeric acid in an individual, the non-digestible oligosaccharide composition may be administered to an individual to achieve any of the medicinal properties associated with valerian, e.g., to treat, prevent, alleviate etc any of the above symptoms or conditions.

Accordingly, we provide for the use of a non-digestible oligosaccharide composition such as an scGOS/lcFOS composition, for the treatment, prophylaxis, prevention or alleviation of insomnia, sleeping disorders, restlessness, stress, anxiety, tension, and epilepsy. We further provide for the use of a non-digestible oligosaccharide composition such as an scGOS/lcFOS composition, for use as a sedative (such as for nervous tension, hysteria, excitabilityor stress), an anxiolytic, a sleeping aid, sedative, anticonvulsant or migraine treatment or prophylactic. We also provide for the use of a non-digestible oligosaccharide composition such as an scGOS/lcFOS composition for decreasing or alleviating stress or anxiety in an individual.

For this purpose, a number of criteria may be designated, which reflect the progress of treatment or prophylaxis or the well-being of the individual. Useful criteria in the case of the conditions described above may include for example the level of tension or stress in the individual, whether measured quantitatively or qualitatively (e.g., by questionnaires) as known in the art. Other indications may reflect the amount of migraine, etc.

Thus, as an example, a treated individual may show a decrease in such a symptom as measured by an appropriate assay or test. A treated individual may for example show a 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more decrease in one or more symptoms, compared to an individual who has not been treated.

For example, a patient disease may be defined as being "treated" if a condition associated with the disease is significantly inhibited (i.e., by 50% or more) relative to controls. The inhibition may be by at least 75% relative to controls, such as by 90%, by 95% or 100% relative to controls. By the term "treatment" we mean to also include prophylaxis, prevention or alleviation of any of the conditions or symptoms specified.

The term "treating" encompasses not only treating a patient to relieve the patient of the signs and symptoms of the disease or condition but also prophylactically treating an asymptomatic patient to prevent the onset or progression of the disease or condition. The term "amount effective for treating" is intended to mean that amount of a substance that will elicit the biological or medical response of a tissue, a system, animal or human that is being sought by a researcher, veterinarian, medical doctor or other clinician. The term also encompasses the amount of a substance that will prevent or reduce the risk of occurrence of the biological or medical event that is sought to be prevented in a tissue, a system, animal or human by a researcher, veterinarian, medical doctor or other clinician. NON-DIGESTIBLE OLIGOSACCHARIDES

We describe the use of a composition of non-digestible oligosaccharides in the maintenance, increase or prevention of decrease of valeric acid in individuals. In particular, the composition may comprise a non-digestible oligosaccharide A and/or B, such as non- digestible oligosaccharide A and non-digestible oligosaccharide B.

The non-digestible oligosaccharide A may be such that it does not comprise an acidic oligosaccharide. The non-digestible oligosaccharide B may be such that it does not comprise an acidic oligosaccharide. The non-digestible oligosaccharide A and the non-digestible oligosaccharide B may be such that both do not comprise acidic oligosaccharides. The non-digestible oligosaccharide A may comprise a non-acidic oligosaccharide. The hon-digestible oligosaccharide B may comprise a non-acidic oligosaccharide. Both the non- digestible oligosaccharide A and the non-digestible oligosaccharide B may comprise non- acidic oligosaccharides.

In some embodiments, non-digestible oligosaccharide A comprises a neutral non- digestible oligosaccharide. In other embodiments, non-digestible oligosaccharide B comprises a neutral non-digestible oligosaccharide. In further embodiments, both non-digestible oligosaccharide A and non-digestible oligosaccharide B comprise neutral non-digestible oligosaccharides.

The non-digestible oligosaccharide A and/or B may be water-soluble, as determined according to the method disclosed in L. Prosky et al, J. Assoc. Anal. Chem 71 : 1017-1023, 1988. The non-digestible oligosaccharide A and/or B may comprise an oligosaccharide with a degree of polymerisation (DP) of 2 to 200. The average DP of the non-digestible

oligosaccharide may be below 200, such as below 100, such as below 60, such as below 40.

As used in this document, the term "degree of polymerisation" or "DP" is intended to refer to the total number of saccharide units in an oligo- or polysaccharide chain. The

"average DP" is intended to refer to the average DP of oligosaccharides or polysaccharide chains in a composition, without taking possible mono- or disaccharides into account (which may be removed if present). The average DP of a composition may be used to distinguish between compositions. In addition the % saccharide units, such as the % glucose and % fructose units, in a composition may be used to distinguish. The non-digestible oligosaccharide A and/or B may be one that is not digested in the intestine by the action of digestive enzymes present in the human upper digestive tract (small intestine and stomach). The non-digestible oligosaccharide A and/or B may be fermented by the human intestinal microbiota. For example, glucose, fructose, galactose, sucrose, lactose, maltose and the maltodextrins are considered digestible. The oligosaccharide raw materials may comprise monosaccharides such as glucose, fructose, fucose, galactose, rhamnose, xylose, glucuronic acid, GalNac etc., but these are not part of the non-digestible

oligosaccharides as used in the present description.

The non-digestible oligosaccharide A and/or B included in the compositions and methods described here may include a mixture of non-digestible oligosaccharides. The non- digestible oligosaccharide A and/or B may be selected from the group consisting of fructo- oligosaccharide, such as inulin, non-digestible dextrin, galacto-oligosaccharide, such as transgalacto-oligosaccharide, xylo-oligosaccharide, arabino-oligosaccharide, arabinogalacto- oligosaccharide, gluco-oligosaccharide, such as gentio-oligosaccharide and cyclodextrin, glucomanno-oligosaccharide, galactomanno-oligosaccharide, mannan-oligosaccharide, chito- oligosaccharide, uronic acid oligosaccharide, sialyloligosaccharide, such as 3-SL, 6-SL, LSTa.b.c, DSLNT, S-LNH, DS-LNH, and fuco-oligosaccharide, such as (un)sulphated fucoidan OS, 2-FL, 3-FL, LNFP I, II, III, V, LNnFPI, LNDH, and mixtures thereof, such as fructo-oligosaccharide, such as inulin, galacto-oligosaccharide, such as transgalacto- oligosaccharide, which is β linked, uronic acid oligosaccharide and fuco-oligosaccharide and mixtures thereof, such as transgalacto-oligosaccharide, inulin and/or uronic acid

oligosaccharides, such as transgalacto-oligosaccharide. When the non-digestible

oligosaccharide A and/or B is a mixture, the averages of the respective parameters may be employed. We provide for example a composition with two different non-digestible

oligosaccharides, i.e. non-digestible oligosaccharide A and non-digestible oligosaccharide B. Non-digestible oligosaccharide A and non-digestible oligosaccharide B may have a different type of glycosidic linkage, a different degree of polymerisation and/or a different

monosaccharide composition. The percentage of a particular monosaccharide in non-digestible oligosaccharide A may be at least 40% higher than the percentage of the same monosaccharide in non-digestible oligosaccharide B, such as at least 50%, such as at least 75%, such as at least 90%. The percentage of a monosaccharide in the non-digestible oligosaccharide may be calculated by dividing the number of the respective monosaccharide units, e.g. glucose, in the non-digestible oligosaccharide by the total number of the monosaccharide units in that non- digestible oligosaccharide and multiply it by 100. When the non-digestible oligosaccharide is a non-digestible oligosaccharide mixture, the contribution of each individual monosaccharide unit in the non-digestible oligosaccharide mixture must be taken into account. The percentage of a monosaccharide in a non-digestible oligosaccharide mixture can simply be determined by completely hydrolysing the mixture and determining the number percentage for each monosaccharide. Non-digestible oligo-saccharide A may contain at least 40% galactose, such as at least

67% galactose, such as at least 75% galactose. Non-digestible oligosaccharide B may contain at least 30 number% fructose, such as at least 67% fructose, such as at least 80% fructose.

The average DP of non-digestible oligosaccharide A may be at least 5 monosaccharide units lower than the average DP of non-digestible oligosaccharide B, such as at least 10, such as at least 15. Non-digestible oligosaccharide A may have an average DP of 2-10, such as 3-5. Non-digestible oligosaccharide B may have an average DP below 200, such as 1 1 -60, such as 20-30. Including a non-digestible oligosaccharide with an increased degree of polymerisation may be employed. The non-digestible oligosaccharide A and B with a different DP may have the same or different monosaccharide composition. Non-digestible oligosaccharide A and B may therefore have a different monosaccharide composition and a different DP.

For example, at least 80 wt.%, such as at least 95 wt.%, such as at least 98 wt.% of the cumulative weight of non-digestible oligosaccharide A and B may have a DP below 60, such as below 40, such as below 20. The lower DP may reduce viscosity and increase

fermentability of the non-digestible oligosaccharides. For example, at least 50 wt.%, such as at least 75 wt.% of the cumulative weight of non-digestible oligosaccharides A and B may comprise non-digestible oligosaccharides with a DP of 2-8.

The percentage of at least one glycosidic linkage of non-digestible oligosaccharide A based on total glycosidic linkages of non-digestible oligosaccharide A may be at least 40% higher or lower than the percentage of the same glycosidic linkage in oligosaccharide B, such as at least 50%, such as at least 75%. The term "glycosidic linkage" may be used in this document to refer to a C-O-C linkage formed between the rings of two cyclic monosaccharides by the elimination of water. An increased diversity in glycosidic linkages may be used. Glycosidic linkages differ in that they covalently bind carbon atoms in the monosaccharide units at differently numbered positions, and/or that they form or β bonds. Examples of different glycosidic linkages occurring in non-digestible saccharides are β (1 ,3), a (1 ,4), β (2,1 ), a (1 ,2), and β (1 ,4) linkages. The glycosidic linkages in non-digestible oligosaccharide A may comprise at least 40% β (1 ,4) and/or β (1 ,6) glycosidic linkages, such as at least 75%. The glycosidic linkages in non-digestible oligosaccharide B may comprise at least 40% β (2,1) glycosidic linkages, such as at least 75%. Non-digestible oligosaccharide A and B may differ in monosaccharide unit composition and in type of glycosidic linkage. For example, non-digestible oligosaccharide A and B may differ in type of glycosidic linkage and DP. For example, non-digestible oligosaccharide A and B may differ in type of glycosidic linkage, monosaccharide composition and DP.

For example, at least 60%, such as at least 75% such as 90%, such as 98% of the total monosaccharide units of the non-digestible oligosaccharide, such as non-digestible

oligosaccharide A and B, may comprise monosaccharides selected from the group consisting of galactose (gal), fructose (fru) and glucose (glu) monosaccharides.

Non-digestible oligosaccharide A may comprise an oligosaccharide selected from the group consisting of β -galacto-oligosaccharide, a-galacto-oligosaccharide, and galactan. For example, non-digestible oligosaccharide A may comprise β -galacto-oligosaccharide. β - galacto-oligosaccharide is also sometimes referred to as transgalacto-oligosaccharide. For example, non-digestible oligosaccharide A may comprise galacto-oligosaccharides with β (1 ,4), β (1 ,3) and/or β (1 ,6) glycosidic bonds and a terminal glucose. Transgalacto- oligosaccharide is for example available under the trade name Vivinal®GOS (Borculo Domo Ingredients, Zwolle, Netherlands), Bi2muno (Clasado), Cup-oligo (Nissin Sugar) and Oligo- mate55 (Yakult).

Non-digestible oligosaccharide B may comprise fructo-oligosaccharide. A fructo- oligosaccharide may in other context have names like fructopolysaccharides, oligofructose, polyfructose, polyfructan, inulin, levan and fructan and may refer to oligosaccharides comprising β -linked fructose units, which may be linked by β (2,1 ) and/or β (2,6) glycosidic linkages, and may comprise a DP of between 2 and 200. For example, the fructo- oligosaccharide may contain a terminal β (2, 1 ) glycosidic linked glucose. The fructo- oligosaccharide may contain at least 7 β -linked fructose units. For example, inulin may be used as non-digestible oligosaccharide B. Inulin is a type of fructo-oligosaccharide wherein at least 75% of the glycosidic linkages are β (2,1) linkages. Typically, inulin has an average chain length between 8 and 60 monosaccharide units. A suitable fructo-oligosaccharide for use in the compositions as described here may be commercially available under the trade name Raftiline®HP (Orafti). Other suitable sources are raftilose (Orafti), fibrulose and fibruline (Cosucra) and Frutafit and frutalose (Sensus).

For example, a transgalacto-oligosaccharide with an average DP below 10, such as 6 may be used as non-digestible oligosaccharide A. A fructo-oligosaccharide with an average DP above 7, such as above 1 1 , such as above 20, may be used as non-digestible

oligosaccharide B.

If the composition comprises non-digestible oligosaccharide A and B, the weight ratio of non-digestible oligosaccharide A to non-digestible oligosaccharide B may be from 1/99 to 99/1, such as from 1/19 to 19/1, such as from 1 to 19/1. Such a weight ratio may be used when non-digestible oligosaccharide A has a low DP and non-digestible oligosaccharide B has a relatively high DP.

For example, oligosaccharide A may comprise a transgalacto-oligosaccharide and oligosaccharide B may comprise a fructo-oligosaccharide.

Thus, we describe a composition which comprises the non-digestible oligosaccharides A and B, wherein the non-digestible oligosaccharides A and B may differ either: i) in the percentage of at least one monosaccharide of oligosaccharide A based on total monosaccharide units of oligosaccharide A, the monosaccharide being at least 40% higher that the percentage of the same monosaccharide in oligosaccharide B; and/or ii) in the percentage of at least one glycosidic linkage of oligosaccharide A based on total glycosidic linkages of oligosaccharide A, the glycosidic linkage being at least 40% higher than the percentage of the same glycosidic linkage in oligosaccharide B; and/or iii) in the degree of polymerisation of oligosaccharide A, degree of polymerisation of oligosaccharide A being at least 5 monosaccharide units lower than the degree of

polymerisation of oligosaccharide B.

GOS/FOS AND SCGOS/LCFOS COMPOSITION OR COMBINATION

As noted above, non-digestible oligosaccharide A may comprise β -galacto- oligosaccharide or transgalacto-oligosaccharide, such as a galacto-oligosaccharide (GOS). The non-digestible oligosaccharide B may comprise fructo-oligosaccharide (FOS).

In one embodiment, therefore, the non-digestible oligosaccharide composition comprises galacto-oligosaccharide (GOS) and fructo-oligosaccharide (FOS). Such a composition is referred to for convenience in this document as a "GOS/FOS" combination or composition.

The galacto-oligosaccharide (GOS) may comprise short chain galacto-oligosaccharide (scGOS). The fructo-oligosaccharide may comprise long chain fructo-oligosaccharide (IcFOS). Thus, in an embodiment, the non-digestible oligosaccharide composition may comprise a mixture of short chain galacto-oligosaccharides (scGOS). and long chain fructo- oligosaccharides (IcFOS). Such a composition is referred to for convenience in this document as "scGOS/lcFOS".

The ratio of scGOS to IcFOS in the scGOS/lcFOS composition may be as set out above, such as from 1/99 to 99/1 , such as from 1/19 to 19/1 , such as from 1 to 19/1. For example, the weight ratio scGOS to IcFOS may be about (20 to 2): 1, for example, 20: 1, 19: 1 , 18: 1 , 17: 1 , 16:1, 15:1 , 14:1, 13: 1, 12: 1, 1 1 : 1, 10: 1, 9: 1, 8: 1, 7: 1, 6: 1 , 5:1 , 4:1 , 3: 1 and 2: 1. For example, the weight ratio scGOS to IcFOS may be 9: 1.

The scGOS/lcFOS may be present in the composition at any suitable concentration. For example, where the composition comprises a liquid, such as made-up formula milk or a made-up growing up milk, the scGOS/lcFOS may be present at for example, between 0.01 g/100 ml to 10 g/100 ml (0.1 to 100 g/1), 0.05 g/100 ml to 5.0 g/100 ml, 0.10 g/100 ml to 2.0 g/100 ml, 0.20 g/100 ml to 1.0 g/100 ml or 0.10 g/100 ml to 1.0 g/100 ml, etc. The scGOS/lcFOS may be present at for example, about 0.15 g/100 ml, about 0.20 g/100 ml, about 0.25 g/100 ml, about 0.30 g/100 ml, about 0.35 g/100 ml, about 0.40 g/100 ml, about 0.45 g/100 ml, about 0.50 g/100 ml, about 0.55 g/100 ml, about 0.60 g/100 ml, about 0.65 g/100 ml, about 0.70 g/100 ml, about 0.75 g/100 ml, about 0.80 g/100 ml, about 0.85 g/100 ml, about 0.90 g/100 ml, about 0.95 g/100 ml, about 1.0 g/100 ml, about 1.05 g/100 ml, about 1.10 g/100 ml, about 1.15 g/100 ml, about 1.20 g/100 ml, about 1.25 g/100 ml, about 1.30 g/100 ml, about 1.35 g/100 ml, or more.

In one embodiment, the scGOS/lcFOS may be present at 0.95 g/100 ml. In another embodiment, the scGOS/lcFOS may be present at 0.4 g/100 ml. The scGOS/lcFOS combination may be provided in the form of an enteral

composition. We therefore provide for an enteral composition comprising a non-digestible oligosaccharide selected from fructo-oligosaccharide such as lcFOS and a galacto- oligosaccharide such as scGOS, or an enteral composition comprising both.

Compositions containing scGOS and lcFOS are described in detail in WO 2000/08948 (and English language equivalents such as AU 766924), WO 2005/039597 and WO

2005/110121 , each of which is herein incorporated by reference.

LONG CHAIN POLYUNSATURATED FATTY ACIDS (LCPUFA)

In some embodiments, the non-digestible oligosaccharide composition further comprises a lipid component. The lipid component may comprise polyunsaturated fatty acids. For example, the non-digestible oligosaccharide composition may further comprise long chain polyunsaturated fatty acids (LCPUFA).

We therefore disclose a composition comprising short chain galacto-oligosaccharides (scGOS), long chain fructo-oligosaccharides (lcFOS) and long chain polyunsaturated fatty acids (LCPUFA). Such a composition may be referred to for convenience as an

scGOS/lcFOS/LCPUFA composition.

The LCPUFA may be present in the scGOS/lcFOS composition at any suitable concentration. For example, where the composition comprises a liquid, such as made-up formula milk or a made-up growing up milk, the LCPUFA may be present at for example, between 0.1 mg/100 ml to 100 mg/100 ml, 0.5 mg/100 ml to 50 mg/100 ml, 1 mg/100 ml to 25 mg/100 ml or 2 mg/100 ml to 20 mg/100 ml, etc. The LCPUFA may be present in the scGOS/lcFOS composition at for example, about 0.5 mg/100 ml, 1.0 mg/100 ml, 1.5 mg/100 ml, 2.0 mg/100 ml, 2.5 mg/100 ml, 3.0 mg/100 ml, 3.5 mg/100 ml, 4.0 mg/100 ml, 4.5 mg/100 ml, 5.0 mg/100 ml, 5.5 mg/100 ml, 6.0 mg/100 ml, 6.5 mg/100 ml, 7.0 mg/100 ml, 7.5 mg/100 ml, 8.0 mg/100 ml, 8.5 mg/100 ml, 9.0 mg/100 ml, 9.5 mg/100 ml, 10.0 mg/100 ml, 10.5 mg/100 ml, 1 1.0 mg/100 ml, 1 1.5 mg/100 ml, 12.0 mg/100 ml, 12.5 mg/100 ml, 13.0 mg/100 ml, 13.5 mg/100 ml, 14.0 mg/100 ml, 14.5 mg/100 ml, 15.0 mg/100 ml, 15.5 mg/100 ml, 16.0 mg/100 ml, 16.5 mg/100 ml, 17.0 mg/100 ml, 17.5 mg/100 ml, 18.0 mg/100 ml, 18.5 mg/100 ml, 9.0 mg/100 ml, 19.5 mg/100 ml, 20.0 mg/100 ml, or more. In one embodiment, the LCPUFA may be present in the scGOS/lcFOS composition at

16.4/100 ml. In another embodiment, the scGOS/lcFOS may be present at 3.5 mg/100 ml.

The non-digestible oligosaccharide composition may therefore comprise at least one long chain polyunsaturated fatty acid such as with 20 or 22 carbon atoms (LCPUFA). The LCPUFA may for example be present in an amount exceeding 0.1 % weight based on total fatty acids. The LCPUFA may be selected from the group consisting of docosahexaenoic acid (DHA), docospentanenoic acid (DPA), arachidonic acid (AA) and eicosapentaenoic acid (EPA). The composition may for example contain DHA such as in an amount exceeding 0.1 wt. % based on total fatty acids; and optionally AA such as in an amount exceeding 0.1 wt. % based on total fatty acids. The composition may comprise at least one LCPUFA of this group included in an amount such as between 0.15 and 1 wt. % based on total fatty acid content of the composition. For example at least two of these LCPUFAs may be present in an amount of such as between 0.15 and 1 wt. % based on total fatty acid content of the composition. In some embodiments, the composition may contain AA and DHA, such as AA, DHA and EPA. Where present, the AA content may be such that it does not exceed 5 wt. %, such as not exceeding 1 wt. %, such as between 0.1 and 0.6 wt. % of the total fatty acids. In certain embodiments, EPA and/or DHA may be added to balance the action of AA, e.g. reduce the potential pro-inflammatory action of AA metabolites. Excess metabolites from AA may cause inflammation. We therefore disclose a non-digestible oligosaccharide composition comprising scGOS, lcFOS and AA, EPA and/or DHA, wherein the weight ratio AA/DHA may be above 0.25, such as above 0.5, such as above 1. The ratio AA/DHA may be below 25, such as below 10. The weight ratio AA/EPA may be between 1 and 100, such as between 5 and 20. The weight ratio EPA/DHA may be 1 or lower, such as below 0.5.

The content of LCPUFA may be such that does not exceed 3 wt. % of the total fatty acids. Such a composition may be used to mimic human milk as closely as possible. For the same reason, the composition may contain less than 1 gram omega-3 LCPUFA per 100 gram fatty acids, such as between 0.1 and 0.75 gram per 100 gram fatty acids. The omega-6

LCPUFA content may be such that it does not exceed 2 gram per 100 gram fatty acids and is for example between 0.1 and 0.75 gram per 100 gram fatty acids.

The LCPUFAs and the other fatty acids may be provided as free fatty acids, in triglyceride-form, in phospholipid form, or as a mixture of one of more of the above. The composition may therefore comprise at least one of AA and DHA in phospholipid form. Such a composition may reduce the incidence of inflammatory disorders of the intestine. The non- digestible oligosaccharide composition may therefore comprise between 0.1 and 5 mg AA from phospholipid per gram total fat and between 0.1 and 5 mg DHA from phospholipid per gram total fat. The AA and/or DHA may at least be partly present in the form of

phosphatidylcholine (PC) and/or phosphatidylethanolamine (PE), e.g. AA and/or DHA containing PE and/or PC.

The LCPUFA may be present in the composition at any suitable amount, as described in the paragraphs above and also below in the relevant section under "Composition". VALERIC ACID

Valeric acid, or pentanoic acid, is a straight-chain alkyl carboxylic acid with the chemical formula C ₅ Hio0 ₂ . Its IUPAC name is pentanoic acid, InChI:

InChI= 1 S/C5H10O2/c 1 -2-3-4-5(6)7/h2-4H2, 1 H3 ,(H,6,7), InChlKey : NQPDZGIKB AWPEJ- UHFFFAOYSA-N and Canonical SMILES : CCCCC(=0)0. Valeric acid has a molecular weight of 102.15. It is a colourless liquid with a odour described as unpleasant, similar to butyric acid and penetrating. Valeric acid has an unpleasant flavor, similar to butyric acid and a fruity and penetrating taste. The boiling point of valeric acid is 186-187 deg C and the melting point of valeric acid is -34.5 deg C. The

density/specific gravity of valeric acid is 0.939 at 20 deg C/4 deg C. Dissociation constant in water is pka = 4.842 at 25 deg C. It is freely soluble in alcohol, ether, soluble in oxygenated solvents, slightly soluble in carbon tetrachloride and soluble in water, 2.4x10E4 mg/1 at 25 deg C.

Valeric acid is found naturally in the perennial flowering plant valerian (Valeriana officinalis), from which it gets its name. Valeric acid is also found in the essential oil oiBoronia anemonifolia, pineapple fruits, and dalieb fruit pulp (Harper DB et al; J Sci Food Agric 37: 685-8 (1986)). Valeric acid and its esters are found in the essential oil of lemon petitgrain (Fenaroli 's Handbook of Flavor Ingredients. Volume 2. Edited, translated, and revised by T.E. Furia and N. Bellanca. 2nd ed. Cleveland: The Chemical Rubber Co., 1975., p. 558). Valeric acid has been identified as a volatile component of roasted filberts ( inlin TE et al; J Agric Food Chem 20: 1021-8 (1972)), Parma ham (Hinrichsen LL, Pedersen SB; J Agric Food Chem 43: 2932-40 (1995)), fried chicken flavour (Tang J et al; J Agric Food Chem 31 : 1287-92 (1983)), bacon flavour (Ho CT et al; J Agric Food Chem 31 : 336-42 (1983)), baked potato flavour (Coleman EC et al; J Agric Food Chem 29: 42-8 (1981)), and raw beef (King MF et al; J Agric Food Chem 41 : 1974-81 (1993)).

Valeric acid was identified as a volatile fatty acid occurring in dalieb fruit pulp at a concn of 5 mg/kg pulp (Harper DB et al; J Sci Food Agric 37: 685-8 (1986)). Pentanoic acid has been identified as a volatile flavor component of mutton, chicken, beef, and pork (Shahidi F et al; CRC Crit Rev Food Sci Nature 24: 141-243 (1986)). Valeric acid was identified as a volatile component of microwave popcorn at a concn of 130 ug/kg (Buttery RG et al; J Agric Food Chem 45: 837-843). Valeric acid was identified as a volatile component of two commercial rice cakes at concns of 210 ppb and 270 ppb (Buttery RG et al; J Agric Food Chem 47: 4353-6 (1999)).

Valeric acid is primarily used as an intermediate for flavors and perfumes, ester-type lubricants, plasticizers, pharmaceuticals and vinyl stabilizers (Lewis, R.J. Sr.; Hawley's

Condensed Chemical Dictionary 14th Edition. John Wiley & Sons, Inc. New York, NY 2001., p. 1 158). It is also used as a sugarcane ripening agent (US Patent Number 3,870,503).

Volatile esters of valeric acid tend to have pleasant odors and are used in perfumes and cosmetics. Ethyl valerate and pentyl valerate are used as food additives because of their fruity flavors. Valeric acid has a similar structure to both GHB and the neurotransmitter GABA. It differs from valproic acid simply by lacking a 3-carbon side-chain.

Valeric acid is also known as 0BDBDC9F0EA731 D23DA5D795DE73CA5 A, 1- Butanecarboxylate, 1 -Butanecarboxy lie acid, 1-Butanecarboxylic acid, 1-pentanoate, 1- pentanoic acid, 109-52-4, 1 10140_ALDRICH, 120630-EP2275401 Al, 120630-

EP2284146A2, 120630-EP2284147A2, 120630-EP2298763A1, 120630-EP2371805A1, 12124-87-7, 19455-21 -1 (potassium salt), 19DF97F61ED344F72017661A3C9B9EDA, lylv, 3D39D3179CCA193195A417BBD626D390, 4-02-00-00868 (Beilstein Handbook

Reference), 42739-38-8 (ammonium salt), 486C6AFD678521B2017573964296BF0F, 556- 38-7 (zinc salt), 5B3D0A7DDA1262F7CBC941995BB06861, 6106-41-8 (hydrochloride salt), 66993-EP2298772A1 , 66993-EP2305687A1 , 66993-EP2308839A1 , 66993-EP2374787A1 , 70268-41-6 (manganese(+2) salt), 7496CD00ADD24A63C14B73919CA262EE,

75054 FLUKA, 8DC8E744C9F23C837DF9EDD4E1DA42EE,

8F 169DDD3E6DC8D93104DBDC03D9C58D, A78A054A31 F868328C922A48D0386508, AC1L1Q0L, Acide valerique, acido valerico, AG-D-26480, AI3-08657,

AIOOZOLXBKJJTA-UHFFFAOYSA-N, AKOS000118960, bmse000345, BRN 0969454, Butane- 1-carboxylic acid, Butanecarboxylate, Butanecarboxylic acid, C00803, C5:0, CH3- [CH2]3-COOH, CHEBI: 17418, CHEMBL268736, DB02406, DB04344,

E 15BEE42E 15C02 A40D 102EB3EB62B922, E40A 16B0720CC885B7EDF81 1271 F233 E, EA 170FFE 1020E7097260FBDCAB262800, EINECS 203-677-2,

FB8A414136DCC9B19D1B7614C5E80AAB, FEMA No. 3101, FEMQKUVCIUPVCH- UHFFFAOYSA-N, FVSQJHGXUHZTDJ-UHFFFAOYSA-N, GEWWXUFUBAEWLC- UHFFFAOYSA-N, GWFAAYTXIVYRLJ-UHFFFAOYSA-N, GXMWVRMHXCXIAF- UHFFFAOYSA-N, HLAVYXFVQYWPRQ-UHFFFAOYSA-N, HMDB00892,

HMS2267A03, HSDB 5390, 104-1052, InChI=l/C5H10O2/cl-2-3-4-5(6)7/h2-

4H2,1H3,(H,6,7, Kyselina valerova, LEVULINIC ACID, LMFA01010005, LS-3150, MLSOO 1066335, n-BuCOOH, n-C4H9COOH, n-Pentanoate, n-Pentanoic acid, n-Valerate, N- VALERIANSAEURE, n- Valeric acid, NCGC00183281-01, NCGC00183281-02,

NQPDZGIKBAWPEJ-UHFFFAOYSA-N, NSC 406833, NSC 406833, NSC-406833, NSC406833, pentoate, pentoic acid, Propylacetate, Propylacetic acid, Propylacetic acid, PSEJBIODJNSJNT-UHFFFAOYSA-N, RPHKUCBTGDWOCL-UHFFFAOYSA-N,

SBB053585, SHF, SMR000471834, STL169350, UN 3265, UZBDGENPSPJRCO- UHFFFAOYSA-N, V0003, Valerate, Valerianate, Valerianic acid, VALERIANIC ACID, C5, Valeriansaeure, Valeriansaure, Valeriansaure, valeric acid, Valeric acid , Valeric acid normal, VALERIC ACID, N-, Valeric acid, normal, VPROFPRQXIOJQU-UHFFFAOYSA-N, W310107 ALDRICH and WLN: QV4.

ASSAYS FOR VALERIC ACID

Assays for determining the amount of concentration of valeric acid are known in the art. For example, gas chromatography may be used for such an assay. A gas chromatography assay for valeric acid is described in Hoshika Y., 1977. Simultaneous gas chromatographic analysis of lower fatty acids, phenols and indoles using a glass capillary column. J

Chromatogr. 1977 Dec 1 ; 144(2): 181 -9. Gas chromatography of fatty acids is reviewed in Shantha and Napolitano 1992, J

Chromatogr. 1992 Oct 30;624(l-2):37-51. Other methods such as differential photometry may also be used (see Belikov VG. 1979, Differential photometric method and the prospects for its use in pharmaceutical analysis, Farmatsiia. 1979 Sep-Oct;28(5):52-63).

Other methods such as paper chromatography may be used to detect and quantify valeric acid. Such a method is described as method AOAC 965.24 in Horwitz W, ed.; Official Methods of Analysis of AOAC International 17th ed. (2003). CD-ROM, AOAC International, Gaithersburg, MD. Chromatographic separation of C2 to C4 saturated fatty acids is also described as method AOAC 945.52 in the same reference.

For specific measurement of valeric acid in stool samples, the procedure described in Example 4 and Example 5 below may be employed.

VALERIAN

Valeric acid is found naturally in the perennial flowering plant valerian (Valeriana officinalis), from which it gets its name.

The following section is adapted from the Wikipedia entry for valerian (Valerian (herb). (2012, February 5). In Wikipedia, The Free Encyclopedia. Retrieved 04:20, February 21 , 2012, from

http://en.wikipedia.Org/w/index.php ?title=Valerian_(herb)&oldid=47519741 1). The roots of Valeriana officinalis may be prepared into a herbal or dietary supplement. The roots are subject to trituration and dehydration processes, and are packaged, usually into capsules.

Valerian has been used as a medicinal herb since at least ancient Greece and Rome. Hippocrates described its properties and Galen later prescribed it as a remedy for insomnia. Based on its pharmacological mode of action, valerian root has been demonstrated to possess sedative and anxiolytic effects.

Valerian has also historically been used as a sedative, anticonvulsant and migraine treatment. Valerian extracts appear to have some affinity for the GABAA receptor, a class of receptors on which benzodiazepines are known to act.

Valerian is used for insomnia and other disorders as an alternative to benzodiazepine drugs, and as a sedative for nervous tension, hysteria, excitability and stress.

In the United States, valerian is sold as a nutritional supplement. Therapeutic use has increased as dietary supplements have gained in popularity, especially after the Dietary Supplement Health and Education Act was passed in 1994. This law allowed the distribution of many agents as over-the-counter supplements, and therefore allowed them to bypass the regulatory requirements of the Food and Drug Administration (FDA).

Valerian is used for sleeping disorders, restlessness and anxiety. Certain data suggests that Valerian has an effect that is calming but doesn't cause sleepiness the following day. When used as a sleeping aid, valerian appears to be most effective on users who have difficulty falling asleep. Also noteworthy is that valerian has been shown to have positive results on users who wake up during the night. Valerian often seems only to work when taken over longer periods (several weeks), though many users find that it takes effect immediately.

Valerian is sometimes recommended as a first-line treatment when risk-benefit analysis dictates. Valerian is often indicated as transition medication when discontinuing benzodiazepines.

Valerian has uses in herbal medicine as a sedative. Results of investigations into its effectiveness have been mixed. It has been recommended for epilepsy, but that is not supported by research (although valproic acid— an analogue of one of valerian's constituents, valeric acid— is used as an anticonvulsant and mood-stabilizing drug). Valerian root generally does not lose effectiveness over time.

One study found valerian tends to sedate the agitated person and stimulate the fatigued person, bringing about a balancing effect on the system. [End adaptation of Wikipedia entry]

COMPOSITION

The composition described here comprising non-digestible oligosaccharide(s) may comprise an enteral composition, i.e., anything that is enterally administered, such as orally. In particular, the enteral composition may comprise a foodstuff, such as nutritional composition or nutritional food.

As used in this document, the term "enteral" is intended to refer to the delivery directly into the gastrointestinal tract of a subject (e.g. orally or via a tube, catheter or stoma).

The composition may comprise an infant and/or toddler nutrition, such as an infant and/or toddler formula. The composition may comprise a children's nutritional product. It may comprise a pediatric nutritional product or formula, a toddler nutritional formula, growing up milk, human milk supplement or medicinal food.

The composition may in one embodiment be used as an infant formula. The

composition may be applied as a complete nutrition for infants. Such food may comprise lipid, protein and carbohydrate and may be administered in liquid form. In a further embodiment, the composition may comprise a ready-to-use liquid food, e.g. is in a ready-to-feed liquid form. A packed ready-to-use liquid food may involve fewer steps for preparation than a powder to be reconstituted and hence may involve a reduced chance of contamination by harmful micro-organisms.

The composition described here may comprise an infant and/or toddler nutrition which for example comprises between 5 and 50 en% lipid, between 5 and 50 en% protein, between 15 and 90 en% carbohydrate and non-digestible oligosaccharide A and/or B. In some embodiments, the composition may comprise an infant and/or toddler nutrition comprising between 35 and 50 en% lipid, between 7.5 and 12.5 en% protein and between 35 and 80 en% carbohydrate (en% is short for energy percentage and represents the relative amount each constituent contributes to the total caloric value of the preparation).

The composition comprising non-digestible oligosaccharides may comprise lipids, such as vegetable lipids. The lipid component may comprise a combination of vegetable lipids and at least one oil selected from the group consisting of fish, animal, algae and bacterial oil. The lipid may comprise over 50 mg/100 kcal (such as over 1 wt.% based on total fatty acids) a -linolenic acid (ALA). The lipid composition may comprise a wt/wt ratio of linoleic acid (LA) and ALA between 4 and 15, such as between 5 and 8. The composition comprising non- digestible oligosaccharides may comprise long chain polyunsaturated fatty acids (LC-PUFA), such as eicosapentaenoic acid (EPA) and/or docosahexaenoic acid (DHA), and/or

docospentanenoic acid (DPA), and/or arachidonic acid (ARA).

The composition comprising non-digestible oligosaccharides may comprise, based on total fatty acids, 0.03 to 0.8 wt.%, such as 0.12 to 0.4 wt.% DHA. The composition may comprise, based on total fatty acids, 0.01 to 0.2 wt.%, such as 0.03 to 0.1 wt.% EPA. The composition may comprise 0.03 to 1.6 wt.%, such as 0.12 to 0.8 wt.% ARA based on total fatty acids.

The composition comprising non-digestible oligosaccharides may comprise proteins. The proteins used in the nutritional preparation may be selected from the group consisting of non-human animal proteins (such as milk proteins, including caseins and whey proteins, meat proteins and egg proteins), vegetable proteins (such as soy protein, wheat protein, rice protein, potato protein and pea protein), hydrolysates (partially and/or extensively), free amino acids and mixtures thereof. Cow milk derived nitrogen source, particularly cow milk protein proteins such as casein and whey proteins may be employed, as the amino acid composition of these proteins is well balanced. The protein of the infant nutrition may be selected from the group consisting of hydrolysed milk protein (e.g. hydrolysed casein and/or hydrolysed whey protein), hydrolysed vegetable protein and/or amino acids. The use of these proteins may reduce the allergic reactions of the infant and/or toddler and/or increase protein absorption. The protein source may be extensively and/or partially hydrolysed. The protein source may be extensively hydrolysed whey protein derived from cow's milk. The composition comprising non-digestible oligosaccharides may comprise digestible carbohydrates. The digestible carbohydrates used in the nutritional preparation may be selected from the group consisting of sucrose, lactose, maltose, galactose, glucose, fructose, corn syrup solids, starch and maltodextrins, and mixtures thereof, such as lactose. The composition comprising non-digestible oligosaccharides may comprise minerals, trace elements and vitamins, choline, taurine, carnitine, myo-inositol and/or mixtures thereof. The composition comprising non-digestible oligosaccharides may comprise nucleotides. The composition may comprise cytidine 5'-monophospate, uridine 5'-monophospate, adenosine 5'-monophospate, guanosine 5'-monophospate, and inosine 5'-monophospate. The composition may comprise 0.005 to 0.07, such as 0.01 to 0.035 wt.% nucleotides based on dry weight. The presence of nucleotides may affects the immune system, intestinal barrier and/or intestinal microbiota.

The composition comprising non-digestible oligosaccharides may comprise a non fermented composition. Fermentation by micro-organisms results in a lowering of the pH. The composition may have a pH above 5.5, such as 6.0, such as 6.5 in order to reduce damage to teeth. The pH may be between 6 and 8.

The composition may be formulated to reduce stool irregularities (e.g. hard stools, insufficient stool volume, diarrhoea), which is a major problem in many babies. The composition may be administered in the form of in liquid food which has an osmolality between 50 and 500 m Θ sm/kg, such as between 100 and 400 m Θ sm/kg.

The composition may be formulated so that it does not have an excessive caloric density, however still provides sufficient calories to feed the subject. Hence, the liquid food may have a caloric density between 0.1 and 2.5 kcal/ml, such as a caloric density of between 0.4 and 1.2 kcal/ml, such as between 0.55 and 0.75 kcal/ml. The composition comprising non-digestible oligosaccharides may have a viscosity between 1 and 60 mPa.s, such as between 1 and 20 mPa.s, such as between 1 and 10 mPa.s, such as between 1 and 6 mPa.s. The low viscosity ensures a proper administration of the liquid, e.g. a proper passage through the hole of a nipple. Also this viscosity closely resembles the viscosity of human milk. Furthermore, a low viscosity results in a normal gastric emptying and a better energy intake, which is essential for infants and/or toddlers which need the energy for optimal growth and development. The composition may be prepared by admixing a powdered composition with water. Normally infant formula is prepared in such way.

The composition described here may be made up as a packaged power composition wherein said package is provided with instruction to admix the powder with a suitable amount of liquid, thereby resulting in a liquid composition with a viscosity between 1 and 60 mPa.s.

The viscosity of the liquid may be determined using a Physica Rheometer MCR 300 (Physica Messtechnik GmbH, Ostfilden, Germany) at shear rate of 95 s ^"1 at 20°C.

The composition comprising non-digestible oligosaccharides may have a long shelf life. For example, it may be shelf stable at ambient temperature for at least 6 months, such as least 12 months, where it is in a liquid, ready-to-feed form.

GROWING UP MILK AND CEREAL

The composition comprising non-digestible oligosaccharides may in particular comprise a cereal or a growing up milk. Cereals are described in more detail below.

The non-digestible oligosaccharide composition may therefore comprise a children's nutritional composition provided as a growing-up milk or cereal. Such a composition may have a standard serving size of 200 ml, providing from about 60 to 75 kcal/100 ml of energy, with a recommended intake of two to three servings per day.

In such an embodiment, the amounts and types of proteins, lipids and carbohydrates may vary. Protein may comprise from about 2.5 to 3.75 g/100 kcal, with carbohydrate providing from about 1 1 to about 16.5 g/100 kcal and lipids comprising from about 2.2 to about 4.4 g/100 kcal.

In an embodiment, sources of carbohydrate for use in the growing-up milk or cereal may include maltodextrin, fructose, lactose, prebiotics, resistant starch, starch, and any combinations thereof. In an embodiment, less than 10% of energy per serving of the growing- up milk or cereal may be contributed from sugars selected from the group consisting of: white sugar (glucose), brown sugar, corn syrup, corn syrup solids, high fructose corn syrup, malt syrup, maple syrup, liquid fructose, molasses, honey, anhydrous dextrose, and any

combinations thereof. When vitamin A is present in the composition such as growing-up milk or cereal, it may be present in a range of about 1 to about 150 meg/serving. In another embodiment, vitamin A may be present in amounts ranging from about 57 to about 65 meg/serving. Any source of vitamin A known in the art to have nutritional uses may be suitable for use in the composition. In an embodiment, sources of vitamin A for use in the growing-up milk or cereal may include preformed sources of vitamin A, such as retinyl acetate, retinyl palmitate, retinol and any combinations thereof.

When Vitamin C is present in the composition such as growing-up milk or cereal, it may be present in the range of about 0.1 to about 10 mg/serving. In another embodiment, vitamin C may be present at the level of 5 mg/serving. Any source of vitamin C known in the art to have nutritional uses may be suitable for use in the present composition. In an embodiment, sources of vitamin C for use in the growing-up milk or cereal include L-ascorbic acid, sodium L-ascorbate, calcium L-ascorbate, ascorbyl palmitate, and any combinations thereof. When thiamin is present in the composition such as growing-up milk or cereal, it may be present in the range of about 0.01 to about 0.5 mg/serving. In another embodiment, thiamin may be present in the range of 0.05 to about 0.15 mg/serving. In yet another embodiment, thiamin may be in the range of 0.08 to 0.10 mg/serving. Any source of thiamin known in the art to have nutritional uses may be suitable for use in the nutritional composition. In an embodiment, sources of thiamin for use in the growing-up milk or cereal include thiamin hydrochloride, thiamin mononitrate and any combinations thereof.

When riboflavin is present in the composition such as growing-up milk or cereal, it may be present in the range of about 0.01 to about 0.5 mg/serving. In another embodiment, riboflavin may be present in the range of 0.05 to about 0.15 mg/serving. In yet another embodiment, riboflavin may be in the range of 0.08 to 0.10 mg/serving. Any source of riboflavin known in the art to have nutritional uses may be suitable for use in the present composition. In an embodiment, sources of riboflavin for use in the growing-up milk or cereal include free riboflavin, sodium riboflavin, riboflavin-5 'phosphate, and any combinations thereof. When vitamin B ₆ is present in the composition such as growing-up milk or cereal, it may be present in the range of about 0.01 to about 0.5 mg/serving. In another embodiment, vitamin B may be present in the range of 0.05 to about 0.15 mg/serving. In yet another embodiment, the level of vitamin B may be in the range of 0.08 to 0.10 mg/serving. Any source of vitamin B known in the art to have nutritional uses may be suitable for use in the present composition. In an embodiment, sources of vitamin B for use in the growing-up milk or cereal include pyridoxine hydrochloride, pyridoxine-5' -phosphate and any combinations thereof.

When folate is present in the composition such as growing-up milk or cereal, it may be present in the range of 5 to 50 meg/serving. In another embodiment, the folate content may be 10 to 40 meg/serving. In yet another embodiment, the folate content may be within the range of 20 to 35 meg/serving. Any source of folate known in the art to have nutritional uses may be suitable for use in the present composition. In an embodiment, the source of folate for use in the growing-up milk or cereal is folic acid.

When vitamin D is present in the composition such as growing-up milk or cereal, it may be present in the range of 0.1 to about 2 meg/serving. In yet another embodiment, the vitamin D content of the growing-up milk or cereal may be 0.5 to 1 meg/serving. Any source of vitamin D known in the art to have nutritional uses may be suitable for use in the present composition. In an embodiment, sources of vitamin D for use in the growing-up milk or cereal include cholecalciferol, ergocalciferol and any combinations thereof.

When calcium is present in the composition such as growing-up milk or cereal, total calcium may be present in the range of about 165 to about 300 mg/serving. In another embodiment, the level of total calcium in the growing-up milk or cereal may be provided in the range of about 180 to 250 mg/serving. Any source of calcium known in the art to have nutritional uses may be suitable for use in the present composition.

When iron is provided in the composition such as growing-up milk or cereal, it may be present in the range of 0.1 to 2.2 mg/serving. In another embodiment, iron may be present in the range of 0.5 to 1.8 mg/serving. In yet another embodiment, the level of iron provided in the growing-up milk or cereal may be in the range of 1.0 to 1.4 mg/serving. Any source of iron known in the art to have nutritional uses may be suitable for use in the present

composition. In an embodiment, sources of iron for use in the growing-up milk or cereal include ferrous sulfate, ferrous fumarate, and any combinations thereof. When zinc is provided in the composition such as growing-up milk, it may be present in the range of 0.2 to 1.5 mg/serving. In another embodiment, zinc may be present in the range of 0.5 to 1.0 mg/serving. Any source of zinc known in the art to have nutritional uses may be suitable for use in the present composition. In an embodiment, zinc is provided as zinc sulfate. When iodine is present in the growing-up milk or cereal, it may be present in the range of 0.2 to 41 meg/serving. In another embodiment, iodine may be present in the range of 5 to 15 meg/serving. Any source of iodine known in the art to have nutritional uses may be suitable for use in the present composition. In an embodiment, sources of iodine for use with the growing-up milk or cereal include sodium iodide, potassium iodide and any combinations thereof.

Where the composition comprises a growing-up milk or cereal formulated for children between the ages of 1 to 6 years, vitamins and minerals may be added in varying amounts and ranges based on a per-serving basis. In an embodiment, one serving of the growing-up milk or cereal may contain from about 15% to about 50% of the Estimated Average Requirement (EAR) for children between the ages of 1 and 6 years for the following nutrients: vitamin E, vitamin K, niacin, pantothenic acid, vitamin Bi ₂ , biotin, choline, potassium, magnesium, phosphorus, chloride, copper, selenium, fluoride, and any combinations thereof. In an embodiment, one serving of the growing-up milk or cereal may contain from about 20% to about 30% of the EAR for children between the ages of 1 and 6 years for the following nutrients: vitamin E, vitamin , niacin, pantothenic acid, vitamin Bi ₂ , biotin, choline, potassium, magnesium, phosphorus, chloride, copper, selenium, fluoride, and any

combinations thereof. Any known sources of these nutrients having nutritional uses may be suitable for use in the composition.

The composition such as growing up milk or cereal may optionally contain other substances that may have a beneficial effect on the host such as lactoferrin, nucleotides, nucleosides, immunoglobulins, CMP equivalents (cytidine 5' -monophosphate, free acid), UMP equivalents (uridine 5' -monophosphate, disodium salt), AMP equivalents (adenosine 5'- monophosphate, free acid), GMP equivalents (guanosine 5 '-monophosphate, disodium salt), and combinations thereof. CEREALS

As noted above, the composition comprising non-digestible oligosaccharides may comprise a cereal.

Accordingly, the non-digestible oligosaccharide composition described here may comprise a cereal component. Cereal components are an important part of the infant's diet, and are usually one of the first non-breast milk and non-infant formulae components introduced into the diet of infants. Ultimately, the infant will consume a high cereal diet, including bread, rice and pasta.

The cereal component may comprise a component selected from the group consisting of whole cereal, cereal flour, milled cereal, ground cereals, cereal starch, and cereal fibre. The cereal component may for example comprise a component selected from the group consisting of cereal flour, ground cereal and milled cereal. The cereal flour may in some embodiments comprise cereal flour which is dextrinised by heat treatment and/or cereal flour which has been enzyme treated in order to degrade the cereal starch. The non-digestible oligosaccharide composition described here may comprise a precooked cereal component, such as precooked cereal flour. The term "precooked cereal flour" indicates flour obtained by the process whereby flour, in granular and crystalline structure is swelled and transformed, for example in a continuous amorphous phase, in the presence of heat and water, dried (e.g. using drum drying or extrusion cooking) and ground. The precooked flour may comprise between 5 and 15 wt.% protein based on the total dry weight of the precooked flour. The use of precooked flour may be such that the final product has a reduced content of thermo-resistant spores compared to the use of non-precooked flour. Furthermore, the use of precooked may be such that the viscosity of the composition is more stable after reconstitution of the product with a warm liquid. This is in contrast to the situation wherein solely non-precooked flour is used. In the latter case the viscosity gradually increases with time.

The precooked flour if present may have a degree of gelatinisation of at least 50%, such as at least 75%. This gives better water holding capacity (WHC), resulting in an improved product (e.g. stability and palatability). The WHC of the precooked material may be between 2 and 10 g water/g dry matter precooked material, such as between 2.5 and 5 g water/g dry matter precooked material. The WHC can be determined as described by

Pinnavaia and Pizzirani (Starch/Starke 50 (1998) nr. 2-3, S. 64-67).

The non-digestible oligosaccharide composition described here may comprise at least one cereal selected from the group consisting of rice, millet, sorghum, wheat, barley, buckwheat, maize (corn), fonio, oats, rye, triticale, teff, wild rice, spelt, amaranth, quinoa and starchy root crops. Starchy root crops may be selected from the group consisting of potato, sweet potato, cassava, yams, aroids, oca, ulluco and mashua.

The composition may be gluten free. The intake of gluten by infants below 6 month of age may result in gastro-intestinal damage. In some embodiments, therefore, the composition may comprise one or more cereal components selected from the group consisting of rice, maize and millet, sorghum, teff, oat and starchy root crops. For example, the composition may comprise one or more cereal components selected from the group consisting of rice, maize and millet, teff, and oat. The composition may consist of rice, maize and millet, sorghum, teff, oat, starchy root crops and mixtures thereof. The cereal may be selected from the group consisting of rice, maize, oat, teff and millet. The cereal part of the composition may comprise mixtures of cereal components. Typically the cereal is processed as defined in EU directive 96/5/EC.

The composition described here may comprise between 10 and 99 g cereal component per 100 g dry weight of the composition, such as between 20 and 90 g, such as between 25 and 80 g. VISCOSITY

The composition comprising non-digestible oligosaccharides may have a viscosity of between 150 and 100,000 mPas at 20 °C and at a shear rate of 10 s ^"1 , such as between 250 and 25,000 mPas, such as between 300 and 10,000 mPas, such as between 500 and 10,000 mPas such as between 1000 and 10,000 mPas. The composition may have a semi-liquid and/or semi-solid constitution. Solid food is still inappropriate for infants changing from breast milk or infant liquid, because of the infant's lack of teeth and its poor swallowing reflex. Semi- liquid may refer to food products that have a viscosity above 150 mPas, but are still pourable. Semi-solid may refer to products that are still formable or spreadable but not pourable, with a viscosity up to 100,000 mPas. Whenever the term viscosity is used in this document, this refers to the physical parameter which is determined according to the following method: Shear flow viscosities were determined in a Paar Physika MCR 300 Modular Compact Rheometer. The instrument was equipped with a concentric cylinder geometry with a diameter of 27 mm. A logarithmic shear rate ramp is used from 0.1 to 1000 s ^"1 in 20 minutes having 40 measurement points. Using the same geometry viscosities can also be measured in shear flow at a constant shear rate of 10 s ^"1 for 10 minutes. The rheometer's thermostat is set on the appropriate temperature (i.e. 20 °C).

To prevent intestinal discomfort, the osmolarity of the semi-liquid and/or semi-solid may be between 300 and 600 mOsm/1, such as between 400 and 500 mOsm/1.

The composition comprising non-digestible oligosaccharides may be in a ready-to-eat form, in which the liquid is already present. In such a form, the product needs only to be heated before consumption and has a stable viscosity during consumption.

The composition comprising non-digestible oligosaccharides may be in the form of granules, flakes, puffs and/or shreds, such as granules.

POWDER

The non-digestible oligosaccharide composition may be in the form of a powder composition.

This may comprise: 10 to 99 wt.% cereal based on dry weight of the powder composition; 1.0 to 30 wt.% fibre based on dry weight of the powder composition; and one or more non-digestible oligosaccharides as described in this document. Reconstitution of this powder with a liquid (such as water or milk) may yield a composition with a viscosity of between 150 and 100,000 mPas. For example, 10 to 100 g powder may be reconstituted with 140 ml liquid (such as water), such as 14 to 80 g powder, such as 30 to 65 g powder, such as 40 to 60 g is reconstituted with 140 ml liquid. For example, the liquid may have a temperature of 30 - 70 °C upon mixing with the powder. We describe a packaging containing powder composition, wherein the packaging indicates that the powder composition is to be mixed with a suitable amount of liquid.

The powder may be in an agglomerated and/or granulated form with an average particle size below 2 mm, such as below 1 mm. For example, the composition may comprise milk protein, calcium, lactose and fat. This has the advantage that the dried product can be reconstituted with water instead of milk. Water advantageously is more readily available and less prone to contamination than milk. For example, the fat is of vegetable origin. This has the advantage that a healthier product is obtained than when the dried product is reconstituted with cow's milk comprising more saturated fat.

ADMINISTRATION

The composition described here may be administered to a number of subjects.

For example, it may be administered to prematurely born babies, maturely born babies, infants which are in the adaptation period to solid food, infants and/or toddlers such as with an increased risk for or suffering from allergy, and/or infants and/or toddlers such as with an increased risk for infections, such as infants and/or toddlers attending day care centres, or suffering from infections. The subject may also be a child, teenager or adult.

We therefore provide a method for providing nutrition to a human infant and/or toddler, comprising administering to the infant and/or toddler the composition described here. The infant and/or toddler may have an age between 0 and 36 month, such as between 0 and 18 month, such as between 0 and 12 months. We therefore provide a method for providing nutrition to a human infant with the age of 0-12 months. We further provide a method for providing nutrition to a human toddler with the age of 12-36 months. We also provide for a method for stimulating the health of an infant and/or toddler, comprising administering a composition comprising a non-digestible oligosaccharide A and/or B to the infant and/or toddler.

We further provide for a method for stimulating the health in an infant and/or toddler comprising the steps a) admixing i) a nutritionally or pharmaceutically acceptable liquid; and ii) a dry composition, wherein the dry composition comprises a non-digestible oligosaccharide A and/or B, and step b) administering the composition obtained in step a) to an infant and/or toddler. EXAMPLES

Example 1. Introduction and Objectives

Infant milk formulas supplemented with a prebiotics mixture of specific short-chain galacto- and long-chain fructooligosaccharides (scGOS/lcFOS, ratio 9:1 , Immunofortis®) have been shown to benefit infants by reducing the incidence of allergic symptoms and infections (Moro et al. 2006, Arslanoglu 2007, Bruzzese 2009).

More recently, a reduced risk of developing at least one infection in a study in young children consuming growing up milk (GUM) supplemented with scGOS/lcFOS/long chain polyunsaturated fatty acids (LCPUFA) has been demonstrated (Lee et al. 201 1). These effects are thought to be mediated through the increase of health-promoting bacteria such as bifidobacteria. The aim of this study was to investigate the effect of a GUM supplemented with scGOS/lcFOS and LCPUFA on the composition of the intestinal microbiota of healthy young children.

Example 2. Study Population In this randomised, double-blind, placebo-controlled study (PEARL Study), 195 healthy children 1 to 3 years of age were randomised to receive one of the three study products (growing up milk formulas) for 12 weeks (Table El below and Figure 1 ).

Antibiotics intake and partial breastfeeding four weeks before the study were considered as exclusion criteria. This study was conducted in Hanoi, Vietnam.

Control Group 1 Group 2 scGOS/lcFOS NONE 0.95 g/100 ml (6 g) 0.4 g/100 ml (2.5 g)

LCPUFA 3.5 mg/100 ml (22 16.4 mg/100 ml (103 3.5 mg/100 ml (22 mg) mg) mg)

Number of Subjects n = 66 n = 64 n = 65

Age (weeks, median 123 (108-140) 130 (108-143) 131 (108-145) Q1-Q3)

Gender male/female) 38/28 31/33 29/36

Type of Birth 52/14 53/11 44/21

(natural/C-section)

Table El : Study products composition. Children were asked to consume 3 serving 210 mL of the Growing Up Milk formula resulting in a daily intake given in brackets.

Example 3. Materials and Methods - Microbiota Analysis

Faecal samples were collected at baseline and at week 12. Analysis of faecal microbiota was performed by fluorescent in situ hybridization (FISH) and image analysis using a set of 16S rRNA- targeted oligonucleotide probes to assess the major gut bacterial groups (Table E2 below).

Piobe Specificity Notes

Bdis656 Bacteroides distasonis group Applied together with Bfra602

Bfra602 Bacteroidesfragilis group Applied together with Bdis656

Bif164-mod Bifidobacterium spp. modified fro mBifl 64

Chis150 Clostridium histolyticum group Applied together with Clit135

Clitl35 Clostridium lituseburense group Applied together with Chis150

Ec1531 Subset enterobacterial (E. coli, Shigella, Salmonella, Klebsiella)

Erec482 Eubacterium rectale- Clostridium coccoides group

Lab158 Lactobacillus- Enterococcus group

Table E2: 16S-rRNA targeted oligonucleotide probes used to quantify targeted bacteria with FISH Faecal short chain fatty acids (SCFA) lactate and pH were also measured (see below).

The primary outcome was defined as the change of proportion of Bifidobacteria between week 12 and baseline in Group 1 compared to the change in Control.

Example 4. Materials and Methods - Sample Preparation for Analysis of Valeric Acid Concentration

Frozen faecal samples were defrosted on ice and stool pH was measured using a Handylab pH meter (Schott Glas, Mainz, Germany) equipped with an Inlab 423 pH electrode (Mettler-Toledo, Columbo).

The faecal samples were then processed as described by Thiel et. al.[l] with slight modifications: 0.5 g of faeces was diluted in 4.5 ml PBS buffer (150 mM NaCl, 10 mM Na2HP04, 20 mM NaH2P04, pH 7.4) and homogenised by the addition of 6 glass beads (3 mm in diameter) and vortexing for 3 min.

Subsequently, the samples were centrifuged at 300 ^χ g for 1 min to remove glass beads and larger particles. Two 1 ml portions of the suspension were stored at -80 °C until use for analysis of n-valeric acid.

Example 5. Materials and Methods - n- Valeric Acid Analysis n-valeric acid levels were quantitatively determined by a Shimadzu GC2010 gas chromatograph (GC) (Shimadzu Benelux, 'sHertogenbosch, Netherlands) equipped with a flame ionization detector. 1 μΐ of the sample was injected (split ratio 1 : 10) at 90°C in the column (ZB-FFAB, 15 m x 0.53 mm, film thickness 1.00 mm, Restek Co.) using helium as carrier gas (69ml/min). New columns were conditioned overnight at 220°C.

After injection of the sample, a stepwise temperature gradient to 220°C follows in 12 minutes and finally maintained at a temperature of 220°C for 1.5 minutes. The temperature of the injector and the detector was 200°C resp. 250°C.

After every ten samples the column was cleaned by injection of 1 μΐ 1% (v/v) formic acid to avoid memory effects of the column. The levels of n-valeric acid were determined using 2-ethylbutyric acid as an internal standard. Reference: Thiel, R. and M. Blaut, An improved method for the automated

enumeration of fluorescently labelled bacteria in human faeces. J Microbiol Methods, 2005. 61(3): p. 369-79.

Example 6. Results - Microbiota Composition

The proportions of Bifidobacteria increased over time for both active groups

(p=0.004 ^& and p=0.002 ^& for Group 1 and for Group 2, respectively) (Figure 3).

These changes were significantly higher than the Control group after 12 weeks of intervention (p=0.018 and p= 0.01 1 for Group 1 and for Group 2, respectively) (Figure 3 and Table E3 below).

Control M=66] Groupl^ Grou s p-Jye ¹

Week 0 M il Wttkl Wert 12 Week! Week 12 1«C« 2¾$Cd

Ί _' ΐ: ^ » · - . ·- ' *· ' ] Γ " ^■ · ' ···'! J" 1 »" ^r · " 3:^3 ^" : 1: j J 3" 3.:ir

335 1 ft: 3 ^{" f}

Π I ' 1? ^*

I j j 0 D6 1j j.-i: 3.-£.U 3 33 3.'-3.11 33: 333-3. 3v 333-333 ^' 1 ^: i ^■ ;

£r*: t ·;,;: > ·. .:.5. : >: < ^■ . r :j *·.' J. . . i ·.-·; «i W 33:c

* · . '

' 31: 33 -31' 335 33^3; 013-332 ' MM 31: 035-3:: ,· "·,

^* :{3ί :·:« !33Π:3·Γ3 ΜΧ, Μ" 13 :3 ^33:* ^' 13:i 1331-' j'3 L :. j.ij- i. 31 ^' 3 3 ·:»

[NJ Number of subjects of the analysis population;

Median (Q1-Q3)

* Stands for significant difference between the two groups of the change in bacteria between weekO and week12(p<0.05)

^* Stands for significant change within the group in bacteria between week 0 and week 12 (p<0.05)

$ Mann-Whitney Test

⁸ Wilcoxon Signed-RankTest,

Table E3: Percentage of different bacterial groups and total count (loglO/g of wet feces) of bacteria measured by FISH

Besides the increase of bifidobacteria in Group 1 , the percentage of Lactobacillus- Enterococcus significantly increased while the percentage of Eubacterium rectale-Clostridium coccoides group significantly decreased at 12 weeks (p<0.05 ^& ). At 12 weeks, the change in % of Eubacterium rectale-Clostridium coccoides group was significantly lower in Group 1 than Control (p=0.014 ) (Table E3 above and Figure 2).

After 12 weeks, in the Control group, the percentage of Clostridium

histolyticumllituseburense significantly increased and Bacteroides distasonislfragilis significantly decreased (p<0.05 ^& ) (Table E3 above and Figure 2).

No significant difference was observed for the percentage of other bacterial groups tested (Table E3 above and Figure 2).

The intervention did not lead to any significant change in the percentage of SCFA, lactate or pH (data not shown). However, subgroup analysis of children below 2 years of age showed a significant reduction of pH in Group 1 from 6.69 to 6.24 (decrease in pH: -0.45 + 0.18 (Mean + SE), p=0.021 ^@ ), which was significantly different compared with Control (p=0.036 ^@ ).

-Based on Analysis of Variance (AN OVA)

In summary, both absolute count and relative proportions of bifidobacteria increased over time within the active group (p=0.002 and p=0.004, respectively).

These increases were significantly higher compared to the control group after 12 weeks of intervention (p=0.01 and p=0.018, respectively), while the percentage of

Eubacterium rectale-Clostridium coccoides was significantly lower (p=0.014 ).

There was also a trend for the Lactobacillus-Enterococcus group to be higher in the active group than in the control group (p=0.066 for counts and p=0.095 for %). The other bacterial groups investigated showed no significant differences between active and control groups.

Example 7. n- Valeric Acid Concentrations in Young Children Fed with

scGOS/lcFOS/LCPUFA The results are shown in Table E4 and Table E5 below, which show valeric acid concentrations in the stool of young children fed with:

• Control: LCPUFA: 3.5 mg/100 ml (22 mg) ^• GUMl : scGOS/lcFOS: 0.4 g/100 ml (2.5 g) + LCPUFA: 3.5 mg/100 ml (22 mg), corresponding to "Group 2" in Table El

• GUM2: scGOS/lcFOS: 0.95 g/100 ml (6 g) + LCPUFA: 16.4 mg/100 ml (103 mg), corresponding to "Group 1" in Table El

Table 327.1 Sommary statistics for Valeric add (mmolkg) jlTTj p_vik

WtekUvs GUM2vs GUMlvs

Product Statistics WeekO Week 12 Week eekD WeekO Control Control b

ControipW MedianfMir.— Max} 1.67(0.08-6.471 1.24(0.08-4.351 ■0,32 (-4,87 -2,86) 0.040 0.04 ³ ().163 ^a

:.(Q!-Q3] 63(0.08-2.53) 54(0.08- 1,85) 54(-l.28-0.62) b

GUMI[.\ ^' =65] MedianfMir.— Max) 1.26 (0,08 -7.16) 1.29(0.08-3.16) 0,00 (-5,78 -2.95) 0.80ο

"|Q:-Q3> 64(0.08 - 2.08) 54 (0,08 -2.06) 54 (-0.53 - 0.81) b

GUM¾N=64] MediarfMin— Max) 1.05(0,08-6.55) 1.16(0.08-5.02) 0.00 (-5.15— .94) 0.449

64(0,08 - 2,24) 55(0,08-2,20) 55 (-0.55 - 1.09)

[N] Numberof subjects of the anaiysis population;

[n| Numberof non-missing subjects:

a

Mann-W itney testthe comparison is based on Change of study product vs Change of Control;

b

Wilcoxon Signed-Rank Test.HO: there is no difference between baseline and week 12 for the given product,

C'eated 17:25, 15APR20H P ^r ogram: SASS Enterprise Guide4.3, File Name: Table3.27.1_VALAC1 N_ITT.pdf

Table E4. Summary statistics for valeric acid (mmol/kg) [ITT]

Table E5. Summary statistics for valeric acid (%) [ITT]

The metabolites investigated showed no significant differences between active and control groups, except for the concentration of valeric acid, which decreased in the stool of young children having consumed the control product (p=0.04).

Example 8. scGOS/lcFOS Maintains Valeric Acid Levels

Table E4 and Table E5 show that there was no significant difference in valeric acid concentrations in the stool of young children fed with 0.4 g/100 ml (2.5 g) scGOS/lcFOS + 3.5 mg/100 ml (22 mg) LCPUFA (Group 2/GUM1) between weeks 0 and 12 (p =0.805). Likewise, there was no significant difference in valeric acid concentration in subjects fed with 0.95 g/100 ml (6 g) scGOS/lcFOS + 16.4 mg/100 ml (103 mg) LCPUFA (Group 1 /GUM2) between weeks 0 and 12 (p = 0.449).

On the other hand, there was a significant decrease in valeric acid concentration in the stool of subjects fed with control mixture (Control, LCPUFA alone) between weeks 0 and 12 (p= 0.040). Example 9. scGOS/lcFOS Prevents Decrease in Valeric Acid Levels

Furthermore, when comparing the difference in valeric levels between week 0 and week 12 in young children fed with 0.95 g/100 ml (6 g) scGOS/lcFOS + 16.4 mg/100 ml (103 mg) LCPUFA (Group 1/GUM2) and the difference in valeric levels between week 0 and week 12 in young children fed with LCPUFA alone (Control), Table E4 shows that there was a significant decrease in valeric acid concentrations in control subjects compared with those in Group 1/GUM2 (p = 0.040),

Example 10. scGOS/lcFOS Increases Valeric Acid Levels

Finally, in both GUM1 and GUM2 subjects, actual valeric acid concentrations were generally found to have increased compared to those in control group, as shown in Tables E4 and E5.

REFERENCES

1. Moro G, et al. Arch Dis Child 2006. 91(10): 814-9.

2. Arslanoglu S, J Nutr 2007. 137(1 1): 2420-4. 3. Bruzzese E, 2009. Clin Nutr 2009. 28(2): 156-61.

4. W.S. Lee et al. Effect of Growing Up Milk (GUM) containing

scGOS/lcFOS/LCPUFA on the Occurrence of Infections in Young Children attending Day Care Centers. Abstract presented at the 7th World Congress of the World Society for Pediatric Infectious Diseases 201 1. A-352-0006-00466. Thiel, R. and M. Blaut, An improved method for the automated enumeration of fluorescently labelled bacteria in human faeces. J Microbiol Methods, 2005. 61(3): p. 369-79.

In this document and in its claims, the verb "to comprise" and its conjugations is used in its non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded. In addition, reference to an element by the indefinite article "a" or "an" does not exclude the possibility that more than one of the element is present, unless the context clearly requires that there be one and only one of the elements. The indefinite article "a" or "an" thus usually means "at least one". Each of the applications and patents mentioned in this document, and each document cited or referenced in each of the above applications and patents, including during the prosecution of each of the applications and patents ("application cited documents") and any manufacturer's instructions or catalogues for any products cited or mentioned in each of the applications and patents and in any of the application cited documents, are hereby incorporated herein by reference. Furthermore, all documents cited in this text, and all documents cited or referenced in documents cited in this text, and any manufacturer's instructions or catalogues for any products cited or mentioned in this text, are hereby incorporated herein by reference. Various modifications and variations of the described methods and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in molecular biology or related fields are intended to be within the scope of the claims.