EP3397076A1

EP3397076A1 - Fermented formula with non-digestible oligosaccharides

Info

Publication number: EP3397076A1
Application number: EP16822212.3A
Authority: EP
Inventors: Jan Knol; Houkje Bouritius; Akhtar Raish OOZEER; Alma Jildou Nauta
Original assignee: Nutricia NV
Current assignee: Nutricia NV
Priority date: 2015-12-29
Filing date: 2016-12-29
Publication date: 2018-11-07
Also published as: BR112018013501B1; RU2018127650A3; RU2018127650A; BR112018013501A2; CN108882740A; WO2017114900A1; RU2724536C2

Abstract

A fermented formula comprising non digestible oligosaccharides synergistically stimulates secretory IgA in infants.

Description

FERMENTED FORMULA WITH NON DIGESTIBLE OLIGOSACCHARIDES

FIELD OF THE INVENTION

The present invention relates to the field of nutrition for infants and young children for improving the immune system.

BACKGROUND OF THE FNVENTION

It is universally accepted that the optimum nutrition for a new-born infant is human milk. When a mother is unable to breast-feed her infant, or chooses not to breast-feed, an infant formula (IF) based on the composition of mature human milk is recognised as the best alternative. Research to improve the quality of in ant formulas is aimed not necessarily at mimicking the exact composit ion of human milk but at achiev ing the functional effects beyond merely the nutritional aspects that are observed in breast-fed infants. Human milk is rich in secretory IgA ( slgA) that serves as the first line of defense in protecting the intestinal barrier of the infant from enteric toxins and pathogenic microorganisms. SlgA is resistant to digestive enzymes and promotes the clearance of antigens and pathogenic microorganisms from the intestine by blocking their access to epithelial receptors, entrapping them in mucus, and facilitating their removal by peristaltic and mucociliary activities. In addition, slgA functions in the mucosal immune defense system and intestinal homeostasis, without provoking, but even ameliorating, an inflammatory response. In contrast standard infant formula based on cow's milk ingredients have a much lower concentration of slgA and infants fed standard infant formula have a reduced level of slgA compared to breast fed infants.

EP 2 318 046 discloses a combination of probiotics with secretory IgA to treat or prevent inflammation. EP 2 315 595 discloses the use of live probiotic bacteria to increase immunoglobulin secretion in C section infants. This document does, however, not disclose a fermented ingredient nor lactic acid. WO 2014/201037 discloses the use of probiotics, prebiotics, synbiotics and antibiotics to treat mammalian microbiota changes associated with antibiotic treatments and various immunological conditions. Fukushima et al. (1998, Int J Food Micribiol 42:39-44) studied the effect of a probiotic Bifidobacterium lactis strain on IgA secretion in a cohort of older children and found increased levels of total IgA and anti-polio virus IgA. Probiotics are live lactic acid producing bacteria such as live lactobacilli or live bifidobacteria. Typically infant formula with probiotics are marketed as powders with dried probiotic cells, in which fermentation is not possible due to the circumstances being unfavourable for fermentation, e.g. a too low water activity, and no lactic acid is produced.

Also non-digestible oligosaccharides influence the intestinal slgA response through their effect on the composition of the microbiota. Scholtens et al. (2008, J Nutr 138: 1141- 1147), as well as Bakker-Zierikzee et al. (2006, Pediatr Allergy Immunol 17: 134-140), found higher faecal slgA levels in healthy infants fed a formula supplemented with scGOS/lcFOS than in infants fed a standard formula, thereby supporting this hypothesis.

Mullie et al. (2004, Ped Res 56:791-795) disclosed that consumption of fermented formula in healthy new-borns was associated with an enhanced production of intestinal slgA antibodies specific to poliovirus after vaccine administration, but no effect on the total IgA titers.

WO 2009/151330 discloses a composition that is obtained by fermentation by lactic acid producing bacteria and non-digestible oligosaccharides for use in caesarean section infants. Aim is induce tolerance for aiding the subsequent colonization by live bacteria. IgA secretion is not mentioned. WO 2009/151331 discloses an immune stimulating composition comprising non-digestible oligosaccharides and a product obtained after incubating with bifidobacteria. By stimulating the Th 1 response and decreasing the Th2 response of the systemic immune system, the vaccination response is increased and allergy is decreased. WO 2009/151329 discloses a composit ion comprising non- digestible oligosaccharides and a product obtained by incubating with bifidobacteria, to reduce bacterial translocation and improving the intestinal barrier function. It is the aim to prevent systemic infections in this way. The document is silent on any effect on mucosal immune defense and IgA secretion. SUMMARY OF THE INVENTION

In clinical trials with healthy, term infants it was found that administration of a fermented in ant formula comprising non-digestible oligosaccharides a large increase of intestinal secretory IgA (slgA) was observed, when compared to administration of non- ermented control formula without non-digestible oligosaccharides, a fermented formula without non-digestible oligosaccharides, or a non-fermented formula comprising non-digestible oligosaccharides. The secretion of slgA was synergisticaiiy increased and more similar to the slgA in the breast fed reference group. This is furthermore indicative for a synergistic increase in the mucosal immune defense system.

Surprisingly, the synergistic effect cannot be explained by a direct interaction between the non-digestible oligosaccharides and the lactic acid producing bacteria in the fermented composition, as the bacteria were inactivated, nor by an increased intestinal microbial activity as there was no clear correlation with the intestinal pH.

DETAILED DESCRIPTION OF THE INVENTION

The invention thus concerns a method for increasing IgA secretion in a human subject with an age of 0 to 36 months comprising administering to the human subject a nutritional composition comprising a fermented ingredient and non-digestible oligosaccharide, said nutritional composition comprising 0.02 to 1.5 wt% of the sum of lactic acid and lactate based on dry weight of the nutritional composition and comprising 2.5 to 15 wt% non- digestible oligosaccharide based on dry weight of the nutritional composition.

In one embodiment, the present method for increasing IgA secretion in a human subject with an age of 0 to 36 months is a non-therapeutic method.

The invention can also be worded as the use of a fermented ingredient and non-digestible oligosaccharide in the manufacture of a nutritional composition for use in increasing IgA secretion in a human subject with an age of 0 to 36 months, wherein the nutritional composition comprises 0.02 to 1.5 wt% of the sum of lactic acid and lactate based on dry weight of the nutritional composition and comprises 2.5 to 15 wt% non-digestible oligosaccharide based on dry weight of the nutritional composition.

The invention can also be worded as a nutritional composition comprising a fermented ingredient and non-digestible oligosaccharide, wherein the nutritional composition comprises 0.02 to 1.5 wt% of the sum of lactic acid and lactate based on dry weight of the nutritional composition and comprises 2.5 to 15 wt% non-digestible oligosaccharide based on dry weight of the nutritional composition, for use in increasing IgA secretion in a human subject with an age of 0 to 36 months. In another aspect the invention concerns a method for improving mucosal immune defense in a human subject with an age of 0 to 36 month comprising administering to the human subject a nutritional composition comprising a fermented ingredient and non- digestible oligosaccharide, said nutritional composition comprising 0.02 to 1.5 wt% of the sum of lactic acid and lactate based on dry weight of the nutritional composition and comprising 2.5 to 15 wt% non-digestible oligosaccharide based on dry weight of the nutritional composition. In one embodiment, the present method for improving mucosal immune defense in a human subject with an age of 0 to 36 months is a non-therapeutic method.

The invention can also be worded as the use of a fermented ingredient and non-digestible oligosaccharide in the manufacture of a nutritional composition for use in improving mucosal immune defense in a human subject with an age of 0 to 36 month, wherein the nutritional composition comprises 0.02 to 1.5 wt% of the sum of lactic acid and lactate based on dry weight of the nutritional composition and comprises 2.5 to 15 wt% non- digestible oligosaccharide based on dry weight of the nutritional composition. The invention can also be worded as a nutritional composition comprising a fermented ingredient and non-digestible oligosaccharide, wherein the nutritional composition comprises 0.02 to 1.5 wt% of the sum of lactic acid and lactate based on dry weight of the nutritional composition and comprises 2.5 to 15 wt% non-digestible oligosaccharide based on dry weight of the nutritional composition, for use in improving mucosal immune defense in a human subject with an age of 0 to 36 month.

Fermented ingredient

The nutritional composition in the methods or uses according to the present invention, hereafter also referred to as the present nutritional composition, comprises a fermented ingredient. The fermented ingredient is a composition that is fermented by lactic acid producing bacteria. The fermentation preferably takes place during the production process of the nutritional composition. Preferably, the nutritional composition does not contain significant amounts of viable bacteria in the final product due to heat inactivation after fermentation or inactivation by other means. Preferably the fermented ingredient is a milk-derived product, which is a milk substrate that is fermented by lactic acid producing bacteria, and said milk substrate comprising at least one selected from the group consisting of mi lk, whey, whey protein, whey protein hydro iysate, casein, casein hydro lysate, and lactose or mixtures thereof. Suitably, nutrit ional composit ions comprising fermented ingredient and non-digest ible oligosaccharide and their way of producing them are described in WO 2009/151330, WO 2009/151331 and WO 2013/187764.

The fermented ingredient preferably comprises bacterial cell fragments like glycoproteins, giyco lipids, peptidoglycan. lipoteichoic acid (LTA), lipoproteins, nucleotides, and/or capsular polysaccharides. It is of advantage to use the fermented ingredient comprising inact ivated bacteria and or cell fragments direct ly as a part of the final nutrit ional product, since this will result in a higher concentration of bacterial cel l fragments. When commercial preparations are used, these are usually washed and material is separated from the aqueous growth medium, comprising the bacterial cell fragments, thereby reducing or eliminat ing the presence of bacterial cel l fragments. Furthermore, upon fermentation and/or other interactions of lact ic acid producing bacteria with the milk substrate, additional bio-active compounds are formed, such as bioactive peptides and/or oligosaccharides, which also stimulate the mucosal immune defense system. Therefore the fermented ingredient, in particular fermented milk-deriv ed product, is bel ieved to have an improved effect compared to non-fermented milk-derived product.

Preferably the nutrit ional composit ion comprises 5 to 97.5 wt% of the fermented ingredient based on dry weight, more preferably 10 to 95 wt% , more preferably 20 to 90 wt%, even more preferably 25 to 60 wt%. As a way to specify the extent of fermentation, the level of the sum of lactic acid and lactate in the nutrit ional composit ion can be taken, as this is the metabolic end product produced by the lact ic acid producing bacteria upon fermentation. The present nutritional composit ion comprises 0.02 to 1 .5 wt% of the sum of lact ic acid and lactate based on dry weight of the composition, more preferably 0.05 to 1 .0 wt%, ev en more preferably 0.05 to 0.5 wt%. Preferably at least 50 wt%, ev en more preferably at least 90 wt%, of the sum of lactic acid and lactate is in the form of the L(+)- isomer. Thus in one embodiment the sum of L(+)-lactic acid and L(+)- lactate is more than 50 wt%, more preferably more than 90 wt%, based on the sum of total lact ic acid and lactate. Herein [_.( + )- lactate and L( + )- lactic acid is also referred to as L- lactate and L- lact ic acid.

Lactic acid producing bacteria used for producing the fermented ingredient

Lact ic acid producing bacteria used for preparing the fermented ingredient, in particular for fermentation of the milk substrate are preferably provided as a mono- or mixed culture. Lactic acid producing bacteria consists of the genera Bifidobacterium, Lactobacillus, Carnobacterium, Enterococcus, Lactococcus, Leuconostoc, Oenococcus, Pediococcus, Streptococcus, Tetragenococcus, Vagococcus and Weissella. Preferably the lactic acid producing bacteria used for fermentation comprises bacteria of the genus Bifidobacterium and/or Streptococcus.

Preferably the Streptococcus is a strain of S. thermophilic. Preferably the Streptococcus develop beta-gaiactosidase act ivity in the course of fermentation of the substrate. Selection of a suitable strain of S. thermophilus is described in example 2 of EP 778885 and in example 1 of FR 2723960. In a further preferred embodiment according to the present invention, the nutritional composition comprises 10 - 10⁵ cfu living bacteria of S. thermophilus, per g dry weight of the nutritional composition, preferably the nutritional composition comprises 10 - 1 0 ' living bacteria of S. thermophilus per g dry weight.

Preferred strains of S. thermophilus to prepare the fermented ingredient for the purpose of the present invent ion have been deposited by Compagnie Gervais Danonc at the Col lection Nationale de Cultures de Microorganismes (CNCM) run by the !nst itut Pasteur, 25 rue du Docteur Roux, Paris, France on 23 August 1995 under the accession number 1-1620 and on 5 August 1994 under the accession number 1- 1470.

Bifidobacteria are Gram-positive, anaerobic, rod-shaped bacteria. Preferred Bifidobacterium species to prepare the fermented ingredient for the purpose of the present invent ion preferably hav e at least 95 % ident ity of the 16 S rRNA sequence when compared to the type strain of the respective Bifidobacterium species, more preferably at least 97% ident ity as defined in handbooks on this subject for instance Sambrook, J., Fritsch, E.F., and Maniatis, T. (1989), Molecular Cloning, A Laboratory Manual, 2nd ed., Cold Spring Harbor (N.Y.) Laboratory Press. The Bifidobacteria preferably used are also described by Scardov i. V. Genus Bifidobacterium, p.1418 - p. 1434. In : Bergey's manual of systematic Bacteriology. Vol. 2. Sneath, P.H.A., N.S. Mair, M.E. Sharpc and J.G. Holt (ed.). Baltimore: Williams & Wilkins. 1986. 635 p. Preferably the lactic acid producing bacteria used for fermentation comprises or is at least one Bifidobacterium selected from the group consisting of B. breve, B. infantis, B. hifidum, B. catenulatum, B. adolescentis, B. thermophilum, B. gallicum, B. animalis or lactis, B. angulation, B. pseudocatenuiatum, B. thermacidophilum and B. longum more preferably B. breve, B. infantis, B. bifidum, B. catenulatum, B. longum, more preferably B. longum and B. breve, even more preferably B. breve, more preferably B. breve selected from the group consisting of B. breve Bb-03 (Rhodia/Danisco), B. breve M-16V (Morinaga), B. breve R0070 (Institute Resell, Lallemand), B. breve BR03 (Probiotical), B. breve BR92 (Cell Biotech) DSM 20091, LMG 11613 and B. breve 1-2219 deposited at the CNCM, Paris France. Most preferably, the B. breve is B. breve M-16V (Morinaga) or B. breve 1-2219, even more preferably B. breve 1-2219. Most preferably the nutritional composition comprises fermented ingredient that is fermented by lactic acid bacteria comprising both B. breve and S. thermophilus. In one embodiment, the fermentation by lactic acid bacteria, is fermentation by Streptococcus thermophilus and Bifidobacterium breve. In one embodiment, the nutritional composition comprises fermented ingredient wherein the lactic acid bacteria are inactivated after fermentation.

Preferably the fermented ingredient is not fermented by Lactobacillus bulgaricus. L. bulgaricus fermented products are considered not suitable for infants, since in young infants the specific dehydrogenase that converts D-lactate to pyruvate is far less active than the dehydrogenase which converts L-lactate.

Preferably the present nutritional composition comprises inactivated lactic acid producing bacteria and/or bacterial fragments derived from lactic acid producing bacteria obtained from more than 1 x 10¹ cfu lactic acid producing bacteria per g based on dry weight of the final composition, more preferably l lO⁵ cfu, even more preferably l lO⁶ cfu. Preferably the inactivated bacteria or bacterial fragments are obtained from less than lxlO¹⁴ cfu lactic acid producing bacteria per g based on dry weight of the final composition, more preferably lxl 0¹³ cfu, even more preferably lxl 0¹² cfu. Process of fermentation

The milk substrate to be fermented is suitably present in an aqueous medium. The milk substrate to be fermented is preferably selected from the group consisting of milk, whey, whey protein, whey protein hydro lysate, casein, casein hydro lysate, and lactose, and mixtures thereof. Milk can be whole milk, semi-skimmed milk and/or skimmed milk. Preferably the milk substrate to be fermented comprises skimmed milk. Whey can be sweet whey, and/or acid whey. Preferably the whey is present in a concentration of 3 to 80 g dry weight per 1 aqueous medium containing milk substrate, more preferably 40 to 60 g per 1. Preferably whey protein hydrolysatc is present in 2 to 80 g dry weight per 1 aqueous medium containing milk substrate, more preferably 5 to 1 5 g/1. Preferably lactose is present in 5 to 50 g dry weight per 1 aqueous substrate, more preferably 1 to 30 g/1. Preferably the aqueous medium containing milk substrate comprises buffer salts in order to keep the pH within a desired range. Preferably sodium or potassium dihydrogen phosphate is used as buffer salt, preferably in 0.5 to 5 g/1, more preferably 1 .5 to 3 g per 1. Preferably the aqueous medium containing milk substrate comprises cysteine in amount of 0. 1 to 0.5 g per 1 aqueous substrate, more preferably 0.2 to 0.4 g/1. The presence of cysteine results in low redox potential of the substrate which is advantageous for activ it y of lactic acid producing bacteria, particularly bifidobacteria. Preferably the aqueous medium containing milk substrate comprises yeast extract in an amount of 0.5 to 5 g/1 aqueous medium containing milk substrate, more preferably 1 .5 to 3 g/1. Yeast e tract is a rich source of enzyme co-factors and growth factors for lact ic acid producing bacteria. The presence of yeast extract will enhance the fermentation by lact ic acid producing bacteria. Suitably the milk substrate, in particular the aqueous medium containing milk substrate, is pasteurised before the fermentation step, in order to eliminate the presence of unwanted l iv ing bacteria. Suitably the product is pasteurised after fermentation, in order to inact ivate enzymes. Suitably the enzyme inact ivation takes place at 75 °C for 3 min. Suitably the aqueous medium containing milk substrate is homogenised before and. or the milk-derived product is homogenised after the fermentation. Homogenisat ion results in a more stable substrate and or fermented product, especially in the presence of fat.

The inoculation density is preferably between lxlO² to 5 10"', preferably between lxlO⁴ to 5x10⁹ cfu lact ic acid producing bacteria/ml aqueous medium containing milk substrate. more preferably between lxl 0⁷ to lxlO⁹ cfu lactic acid producing bacteria ml aqueous medium containing milk substrate. The final bacteria density after fermentation is preferably between lxlO³ to lxlO¹⁰, more preferably between I x 1 0¹ to lxlO⁹ cfu/ml aqueous medium containing milk substrate.

The fermentat ion is preferably performed at a temperature of approximately 20 °C to 50 °C, more preferably 30 °C to 45 °C, even more preferably approximately 37 °C to 42 °C. The optimum temperature for growth and/or activity for lact ic acid producing bacteria, more particularly lactobacill i and/or bifidobacteria is between 37 °C and 42 °C.

The incubat ion is preferably performed at a pi i of 4 to 8, more preferably 6 to 7.5. This pH does not induce protein precipitation and/or an adverse taste, hile at the same time lact ic acid producing bacteria such as lactobacilli and/or bifidobacteria are able to ferment the milk substrate.

The incubation t ime preferably ranges from 10 minutes to 48 h, preferably from 2 h to 24 h, more preferably from 4 h to 12 h. A sufficient long time enables the fermentation and the concomitant production of immunogenic cell fragments such as glycoproteins, glycol ipids, peptidoglycan, l ipoteichoic acid (LTA), fiagellae, lipoproteins. DNA and/or capsular polysaccharides to take place at a high extent, whereas the incubation time need not be unnecessarily long for economical reasons.

Preferably, a milk substrate, preferably skimmed milk, is pasteurized, cooled and fermented with one or more lact ic acid producing strains, preferably a strain of S. thermophilus, to a certain degree of acidity, upon which the fermented product is cooled and stored. Preferably a second milk-derived product is prepared in a similar way using one or more Bifidobacterium species for fermentation. Subsequently, the two fermented products are preferably mixed together and mixed with other components making up an infant formula, except the fat component. Preferably, the mixture is preheated, and subsequent ly fat is added in-line, homogenized, pasteurized and dried.

Procedures to prepare fermented ingredients suitable for the purpose of the present invention are known per se. EP 778885, which is incorporated herein by reference, discloses in particular in example 7 a suitable process for preparing a fermented ingredient. FR 2723960, which is incorporated herein by reference, discloses in particular in example 6 a suitable process for preparing a fermented ingredient. Briefly, a milk substrate, preferably pasteurised, containing lactose and optionally further macronutrients such as fats, preferably vegetable fats, casein, whey protein, vitamins and/or minerals etc. is concentrated, e.g. to between 15 to 50% dry matter and then inoculated with S. thermophilus, for example with 5% of a culture containing 10⁶ to 10¹⁰ bacteria per ml. Preferably this milk substrate comprises milk protein peptides. Temperature and duration of fermentation are as mentioned above. Suitably after fermentation the fermented ingredient may be pasteurised or sterilized and for example spray dried or lyophilised to provide a form suitable to be formulated in the end product.

A preferred method for preparing the fermented ingredient of the present invent ion is disclosed in WO 01/01785, more particular in examples 1 and 2. A preferred method for preparing the fermented product of the present invention is described in WO 2004/093899, more particularly in example I .

Methods of inactivation and/or physically removal of living cells

Living cells of lactic acid producing bacteria in the fermented ingredient are after fermentation preferably eliminated, for example by inactivation and/or physical removal. The cells are preferably inactivated. Preferably the lactic acid producing bacteria are heat killed after fermentation of the milk substrate. Preferable ways of heat killing are (flash) pasteurization, sterilization, ultra high temperature treatment, high temperature/short time heat treatment, and/or spray drying at temperatures bacteria do not survive. Cell fragments are preferably obtained by heat treatment. With this heat treatment preferably at least 90 % of living microorganisms are inactiv ated, more preferably at least 95 %, even more preferably at least 99 %. Preferably the fermented nutritional composition comprises less than lxlO⁵ colony forming units (cfu) living lactic acid bacteria per g dry- weight. The heat treatment preferably is performed at a temperature ranging from 70 to 180 °C, preferably from 80 to 150 °C, preferably for about 3 minutes to 2 hours, preferably in the range of 80 to 140 °C fo 5 minutes to 40 minutes. Inactiv ation of the lactic acid bacteria advantageously results in less post acidification and a safer product. This is especially advantageous when the nutritional composition is to be administered to infants or young children. Suitably after fermentation the fermented ingredient may be pasteurised or sterilized and for example spray dried or lyophil ised to prov ide a form suitable to be formulated in the end product.

Non-digestible oligosaccharide

The present nutritional composition comprises non-digestible oligosaccharide and preferably comprises at least two different non-digestible oligosaccharides, in particular two different sources of non-digest ible oligosaccharide. The presence of non-digestible oligosaccharide stimulates the secretion of slgA. The presence of non-digestible oligosaccharide stimulates the mucosal immune defense system. Hence, the presence of both the non-digestible oligosaccharide and the fermented ingredient, in particular the milk-derived product obtained by fermentation with lactic acid producing bacteria, synergisticaily and advantageously results in a higher IgA secretion in the intest ine. Hence, the presence of both non-digestible oligosaccharide and the fermented ingredient, in particular the milk-deriv ed product obtained by fermentation with lactic acid producing bacteria, synergisticaily and advantageously results in an increased mucosal immune defense.

The term "oligosaccharide" as used herein refers to saccharides with a degree of polymerization (DP) of 2 to 250, preferably a DP 2 to 100. more preferably 2 to 60, even more preferably 2 to 10. I f oligosaccharide with a DP of 2 to 100 is included in the present nutritional composition, this results in compositions that may contain oligosaccharides with a DP of 2 to 5, a DP of 50 to 70 and a DP of 7 to 60. The term "non-digestible oligosaccharide^" as used in the present inv ention refers to oligosaccharides which are not digested in the intestine by the action of acids or digestive enzymes present in the human upper digest ive tract, e.g. small intest ine and stomach, but which are preferably fermented by the human intestinal microbiota. For example, sucrose, lactose, maltose and maitodextrins are considered digestible.

Preferably the present non-digestible oligosaccharide is soluble. The term "soluble" as used herein, when having reference to a polysaccharide, fibre or ol igosaccharide, means that the substance is at least soluble according to the method described by L. Prosky et a!., J. Assoc. Off. Anal. Chem. 71 , 1017-1023 (1988). The non-digestible ol igosaccharide included in the present nutritional compositions in the methods or uses according to the present invention preferably include a mixture of non- digestible oligosaccharides. The non-digestible oligosaccharide is preferably selected from the group consist ing of fructo-oligosaccharide, such as inuiin, non-digest iblc dextrins, ga!acto-ol igosaccharide, such as transgalacto-oligosaccharide, xylo- oligosaccharide, a ra b i n o - o I i go s a c c h a r i d e , arabinogaiacto-oligosaccharide, g lu cool igosaccharide, gent io-ol igosaceharide. glucomanno-oligosaccharide, galactomannool igosaccharide, mannan-o 1 igosaccharide, isomalto-ol igosaccharide. nigero -ol igosaccharide, glucomanno-oligosaccharide, chito-ol igosaccharide, soy ol igosaccharide, uronic acid oligosaccharide, sialylol igosaccharide, such as 3- sialyilactose (3-SL), 6-sialyl lactose (6-SL), lactosi a 1 y 1 tet rasaccha ri de a.b.c (LST), disialyilactoNtetraose ( DSLNT). sialyl-lactoNhexaose (S-LNH), DS-LNH, and fuco- oligosaccharide, such as (un )sulphatcd fucoidan. oligosaccharide. 2'-fucosyliactose (2'- FL), 3-fucosy I lactose (3-FL), difucosyllactose, lacto-N-fucopenatose, (LNFP) I, I I, I I I, V, Lacto-N-neofucopenaose (LNnFP), Lacto-N-difucosyl-hexaose (LNDH), and mixtures thereof, even more preferably selected from the group consist ing of fructo- ol igosaccharide, such as inuiin, galaeto-ol igosaccharide, such as transgalacto- oligosaccharide, and fuco-o 1 i gosacch a ri de and mixtures thereof, even more preferably transgalacto-oligosaccharide, and/or inui in, most preferably transgalacto- oligosaccharide. In one embodiment in the composition or methods according to the present invention, the non-digestible oligosaccharide is selected from the group consisting of transgalacto-oligosaccharide, fructo-oligosaccharide and mixtures of thereof. The non-digestible oligosaccharide is preferably selected from the group consist ing of β- galacto-ol igosaccharide, ot-ga 1 acto-o I i gosaccha ri de, and galactan. According to a more preferred embodiment non-digestible oligosaccharide is β-galacto-ol igosaccharide. Preferably the non-digestible oligosaccharide comprises galacto-ol igosaccharide 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 ( Do mo FriesIandCampina Ingredients). Bi2muno (Clasado), Cup-ol igo (Nissin Sugar) and Oligomatc55 (Yakult). These oligosaccharides increase slgA levels and strengthens the mucosal immune defense act ivity to a larger extent. The non-digestible ol igosaccharide preferably comprises fructo-ol igosaccharide. A fructo-oligosaccharide may in other context have names like fructopoiysaccharide, oligo fructose, polyfructose, polyfructan, inul in. levari, and true tan and may refer to oligosaccharides comprising β- 1 inked fructose units, which are preferably linked by β(2, 1) and/or β(2,6) glycosidic linkages, and a preferable DP between 2 and 200. Preferably, the fructo-oligosaccharide contains a terminal β(2, 1) glycosidic linked glucose. Preferably, the fructo-oligosaccharide contains at least 7 β-l inked fructose units. In a further preferred embodiment inulin is used. Inulin is a type of fructo-oligosaccharide wherein at least 75% of the glycosidic linkages are β(2, 1) linkages. Typical ly, inulin has an average chain length between 8 and 60 monosaccharide un its. A suitable fructo- oligosaccharide for use in the composit ions of the present invention is commercially available under the trade name Raftiline®HP (Orafti ). Other suitable sources are Raft ilose (Orafti), Fibrulose and Fibruline (Cosucra) and F rut a fit. and Frutaiose (Sensus). Preferably the present nutritional composition comprises a mixture of galacto- oligosaccharide and fructo-oligosaccharide. Preferably the mixture of galacto- ol igosaecharide and fructo-oligosaccharide is present in a weight ratio of from 1/99 to 99/1 , more preferably from 1/19 to 19/1 , more preferably from 1/1 to 19/1, more preferably from 2/1 to 15/1, more preferably from 5/1 to 12/1 , even more preferably from 8/1 to 10/1 , even more prefeably in a ratio of about 9/1. This weight ratio is particularly advantageous when galacto-oligosaccharide has a low average DP and fructo- oligosaccharide has a relativ ely high DP. Most preferred is a mixture of galacto- oligosaccharide w ith an average DP below 10, preferably below 6 and a fructo- ol igosaccharide with an average DP above 7. preferably above 1 1 , even more preferably above 20. Such a mixture synergist ically increases slgA levels and strengthens the mucosal immune defense act ivity.

Preferably the present nutritional composition comprises a mixture of short chain fructo- oligosaccharide and long chain fructo-oligosaccharide. Preferably the mixture of short chain fructo-oligosaccharide and long chain friicto-o 1 i gosacc ha ri dc is present in a weight ratio of from 1/99 to 99/1 , more preferably from 1/19 to 19/1 , even more preferably from 1/10 to 19/1, more preferably from 1/5 to 15/1, more prefrably from 1/1 to 10/1. Preferred is a mixture of short chain fructo-oligosaccharide with an average DP below 10, preferably below 6 and a fructo-oligosaccharide with an average DP above 7, preferably above 1 1 , ev en more preferably above 20.

The present nutritional composit ion comprises 2.5 to 20 wt% total non-digestible oligosaccharide, more preferably 2.5 to 15 wt%, even more preferably 3.0 to 1 0 wt%, most preferably 5.0 to 7.5 wt%, based on dry weight of the nutritional composit ion. Based on 100 ml the present nutritional composit ion preferably comprises 0.35 to 2.5 wt% total non-digest ible oligosaccharide, more preferably 0.35 to 2.0 wt%, even more preferably 0.4 to 1 .5 wt%, based on 100 ml of the nutrit ional composit ion. A lower amount of non- digestible oligosaccharide will be less effective in stimulating slgA formation or mucosal immune defense, whereas a too high amount will result in side-effects of bloat ing and abdominal discomfort.

Nutritional composition

The nutritional composit ion used according to the present inv ent ion may also be considered as being a pharmaceutical composition, is preferably suitable for administration to infants. The present nutrit ional composit ion is preferably enterally administered, more preferably orally. The present nutrit ional composit ion is preferably an infant formula, follow on formula or a growing up milk. The present nutritional com osition can be advantageously applied as a complete nutrit ion for infants. Preferably the present nutritional composit ion is an infant formula. An infant formula is defined as a formula for use in infants and can for example be a starter formula, intended for infants of 0 to 6 or 0 to 4 months of age. A follow on formula is intended for infants of 4 or 6 months to 12 months of age. A growing up milk is intended for children of 12 to 36 months of age. The present composition preferably comprises a lipid component, protein component and carbohydrate component and is preferably administered in liquid form. The present nutritional composition may also be in the form of a dry food, preferably in the form of a powder which is accompanied w ith instructions as to mi said dry food, preferbly powder, with a suitable liquid, preferably water. The nutrit ional composit ion used according to the inv ent ion preferably comprises other fractions, such as vitamins, minerals, trace elements and other micronutrients in order to make it a complete nutritional composition. Preferably infant formulas comprise vitamins, minerals, trace elements and other micronutrients according to international directives.

The present nutritional composition preferably comprises lipid, protein and digestible carbohydrate invention wherein the lipid provides 5 to 50% of the total calories, the protein provides 5 to 50% of the total calories, and the digestible carbohydrate provides 15 to 90% of the total calories. Preferably, in the present nutritional composition the lipid provides 35 to 50% of the total calories, the protein provides 7.5 to 12.5% of the total calories, and the digestible carbohydrate provides 40 to 55% of the total calories. For calculation of the % of total calories for the protein, the total of energy provided by proteins, peptides and amino acids needs to be taken into account. Preferably the lipid provides 3 to 7 g lipid per 100 kcal, preferably 4 to 6 g per 100 kcal, the protein provides 1.6 to 4 g per 100 kcal, preferably 1.75 to 2.5 g 15 per 100 kcal and the digestible carbohydrate provides 5 to 20 g per 100 kcal, preferably 8 to 15 g per 100 kcal of the nutritional composition. Preferably the present nutritional composition comprises lipid providing 4 to 6 g per 100 kcal, protein providing 1.6 to 1.9 g per 100 kcal, more preferably 1.75 to 1.85 g per 100 kcal and digestible carbohydrate providing 8 to 15 g per 100 kcal of the nutritional composition. In one embodiment, the lipid provides 3 to 7 g lipid per 100 kcal, preferably 4 to 6 g per 100 kcal, the protein provides 1.6 to 2.1 g per 100 kcal, preferably 1.6 to 2.0 g per 100 kcal and the digestible carbohydrate provides 5 to 20 g per 100 kcal, preferably 8 to 15 g per 100 kcal of the nutritional composition and wherein preferably the digestible carbohydrate component comprises at least 60 wt% lactose based on total digest ible carbohydrate, more preferably at least 75 wt%, even more preferably at least 90 wt% lactose based on total digestible carbohydrate. The amount of total calories is determined by the sum of calories derived from protein, lipids, digest ible carbohydrates and non-digestible oligosaccharide.

The present nutritional composition preferably comprises a digestible carbohydrate component. Preferred digestible carbohydrate components are lactose, glucose, sucrose, fructose, galactose, maltose, starch and maltodextrin. Lactose is the main digestible carbohydrate present in human milk. The present nutritional composition preferably comprises lactose. As the present nutritional composition comprises a fermented ingredient that is obtained by fermentation by lactic acid producing bacteria, the amount of lactose is reduced compared to its source due to the fermentation whereby lactose is converted into lactate and/or lactic acid. Therefore in the preparation of the present nutritional composition lactose is preferably added. Preferably the present nutritional composition does not comprise high amounts of carbohydrates other than lactose. Compared to digestible carbohydrates such as maltodextrin, sucrose, glucose, maltose and other digestible carbohydrates with a high glycemic index, lactose has a lower glycemic index and is therefore preferred. The present nutritional composition preferably comprises digestible carbohydrate, wherein at least 35 wt%, more preferably at least 50 wt%, more preferably at least 60 wt%, more preferably at least 75 wt%, even more preferably at least 90 wt% , most preferably at least 95 wt% of the digestible carbohydrate is lactose. Based on dry weight the present nutritional composition preferably comprises at least 25 wt% lactose, preferably at least 40 wt%, more preferably at least 50 wt% lactose.

The present nutritional composition preferably comprises at least one lipid selected from the group consisting o animal lipid (excluding human lipids) and vegetable lipids. Preferably the present composit ion comprises a combinat ion o vegetable lipids and at least one oil selected from the group consist ing o fish oil, animal oil, algae oil, fungal oil. and bacterial oil. The lipid of the present nutritional composit ion preferably provides 3 to 7 g per 100 kcal of the nutritional composition, preferably the lipid provides 4 to 6 g per 100 kcal. When in liquid form, e.g. as a ready-to-feed liquid, the nutritional composition preferably comprises 2.1 to 6.5 g lipid per 100 ml, more preferably 3.0 to 4.0 g per 100 ml. Based on dry weight the present nutritional composition preferably comprises 12.5 to 40 wt% lipid, more preferably 1 to 30 wt%. Preferably the lipid comprises the essential fatty acids alpha- lino lenic acid (ALA), linolcic acid (LA) and/or long chain polyunsaturated fatty acids (LC-PUFA). The LC-PUFA, LA and or ALA may be provided as free fatty acids, in triglyceride form, in diglyceride form, in monog!yceridc form, in phospholipid form, or as a mixture of one of more of the above. Preferably the present nutritional composition comprises at least one, preferably at least two lipid sources selected from the group consisting of rape seed oil (such as colza oil, low erucic acid rape seed oil and canola oil), high oleic sunflower oil, high oleic safflower oil, olive oil, marine oils, microbial oils, coconut oil, palm kernel oil and milk fat. The present nutritional composit ion is not human milk. The present nutritional composition preferably comprises protein. The protein used in the nutritional composition is preferably selected from the group consisting of non-human animal proteins, preferably milk proteins, vegetable proteins, such as preferably soy protein and/or rice protein, and mixtures thereof. The present nutritional composition preferably contains casein, and/or whey protein, more preferably bovine whey proteins and/or bovine casein. Thus in one embodiment the protein in the present nutritional composition comprises protein selected from the group consisting of whey protein and casein, preferably whey protein and casein, preferably the whey protein and/or casein is from cow's milk. Preferably the protein comprises less than 5 wt% based on total protein of free amino acids, dipeptides, tripeptides or hydrolyzed protein. The present nutritional composition preferably comprises casein and whey proteins in a weight ratio casein : whey protein of 10 : 90 to 90 : 10, more preferably 20 : 80 to 80 : 20, even more preferably 35 : 65 to 55 : 45. The wt% protein based on dry weight of the present nutritional composition is calculated according to the Kjeldahl-method by measuring total nitrogen and using a conversion factor of 6.38 in case of casein, or a conversion factor of 6.25 for other proteins than casein. The term 'protein' or 'protein component' as used in the present invention refers to the sum of proteins, peptides and free amino acids.

The present nutritional composition preferably comprises protein providing 1.6 to 4.0 g protein per 100 kcal of the nutritional composition, preferably providing 1.6 to 3.5 g, even more preferably 1.75 to 2.5 g per 100 kcal of the nutritional composition. In one embodiment, the present nutritional composition comprises protein providing 1.6 to 2.1 g protein per 100 kcal of the nutritional composition, preferably providing 1.6 to 2.0 g, more preferably 1.75 to 2.1 g, even more preferably 1.75 to 2.0 g per 100 kcal of the nutritional composition. In one embodiment, the present nutritional composition comprises protein in an amount of less than 2.0 g per 100 kcal, preferably providing 1.6 to 1.9 g, even more preferably 1.75 to 1.85 g per 100 kcal of the nutritional composition. A too low protein content based on total calories will result is less adequate growth and development in infants and young children. When in liquid form, e.g. as a ready-to-feed liquid, the nutritional composition preferably comprises 0.5 to 6.0 g, more preferably 1.0 to 3.0 g, even more preferably 1.0 to 1.5 g protein per 100 ml, most preferably 1.0 to 1.3 g protein per 100 ml. Based on dry weight the present nutritional composition preferably comprises 5 to 20 wt% protein, preferably at least 8 wt% protein based on dry weight of the total nutritional composition, more preferably 8 to 14 wt%, even more preferably 8 to 9.5 wt% protein based on dry weight of the total nutritional composition. In order to meet the caloric requirements of an infant or young child, the nutritional composition preferably comprises 45 to 200 kcal/100 ml liquid. For infants the nutritional composition has more preferably 60 to 90 kcal/100 ml liquid, even more preferably 65 to 75 kcal/100 ml liquid. This caloric density ensures an optimal ratio between water and calorie consumption. For young children, human subjects with an age between 12 and 36 months, the nutritional composition more preferably has a caloric density between 45 and 65, even more preferably between 50 and 60 kcal/100 ml. The osmolarity of the present composition is preferably between 150 and 420 mOsmol/1, more preferably 260 to 320 mOsmol/1. The low osmolarity aims to further reduce the gastrointestinal stress. When the nutritional composition is in a liquid form, the preferred volume administered on a daily basis is in the range of about 80 to 2500 ml, more preferably about 200 to 1200 ml per day. Preferably, the number of feedings per day is between 1 and 10, preferably between 3 and 8. In one embodiment the nutritional composition is administered daily for a period of at least 2 days, preferably for a period of at least 4 weeks, preferably for a period of at least 8 weeks, more preferably for a period of at 25 least 12 weeks, in a liquid form wherein the total volume administered daily is between 200 ml and 1200 ml and wherein the number of feedings per day is between 1 and 10.

The present nutritional composition, when in liquid form, preferably has a viscosity between 1 and 60 mPa.s, preferably between 1 and 20 mPa.s, more preferably between 1 and 10 mPa.s, most preferably between 1 and 6 m a.s. The low v iscosity ensures a proper administration of the liquid, e.g. a proper passage through the whole 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 which need the energy for optimal growth and development. The present composition is preferably prepared by admixing a powdered composit ion with water. Normally infant formula is prepared in such a way. The present inv ention thus also relates to a packaged power composition wherein said package is prov ided with instructions to admi 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 is determined using a Physica Rheo meter MCR 300 (Physica Messtechnik GmbH, Ostfilden, Germany) at a shear rate of 95 s^"1 at 20 °C. Application

In the context of the present invention, 'prevention' of a disease or certain disorder also means 'reduction of the risk' of a disease or certain disorder and also means 'treatment of a person at risk' of said disease or said certain disorder. The inventors have found that upon consumption of the nutritional composition of the present invention the level of slgA is synergetically increased. This is indicative for an improved mucosal immune defense system.

The effects described herein, i.e. the increase in slgA and/or the improved mucosal immune system, are observed to the situation compared to the administering of a nutritional composition not comprising the combination of fermented product and the non-digestible oligosaccharide. It was found that these effects observed also come closer to the levels observed in human milk fed infants compared to standard infant formula fed infants.

Surprisingly, the synergistic effect cannot be e plained by a direct interaction between the non-digestible oligosaccharide and the lactic acid producing bacteria in the fermented composition, as the bacteria were inactivated, nor by an increased intestinal microbial activity as there was no clear correlation with the intestinal pH.

In one embodiment, the present nutritional composition is used for increasing IgA secretion in a human subject with an age of 0 to 36 months. In one embodiment the present nutritional composition is used for increasing IgA secretion in a human subject of 0 to 18 months, even more preferably an infant with an age of 12 months of age or below, even more preferably an infant with an age of 0 to 6 months. In one embodiment the present nutritional composition is used for increasing IgA secretion in a young child of 12 to 36 months, most preferably a young child with an age of 18 to 30, or 24 months. Preferably the present nutritional composition is further used for providing nutrition to said human subject. The present nutritional composition is preferably is enterally administered, more preferably orally.

In one embodiment, the present nutritional composition is used for improving mucosal immune defense in a human subject with an age of 0 to 36 months. In one embodiment the present nutritional composition is used for improving mucosal immune defense in an human subject of 0 to 18 months, even more preferably an infant with an age of 12 months of age or below, even more preferably an infant of 0 to 6 months. In one embodiment the present nutritional composition is used for improving mucosal immune defense in a young child of 12 to 36 months, most preferably a young child with an age of 18 to 30, or 24 months. Preferably the present nutritional composition is further used for providing nutrition to said human subject. The present nutritional composition is preferably is enterally administered, more preferably orally. In a preferred embodiment, the methods or uses according to the present invention are for vaginally delivered infants.

In a preferred embodiment, the methods or uses according to the present invention are for term infants, preferably for healthy term infants. 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. Wt% means weight percentage.

EXAMPLES

Example 1: Synergistic effect of fermented infant formula and non- digestible oligosaccharides on secretory IgA levels in infants

In a randomised, double-blind, controlled, parallel-group, prospective, multi-centre, multi-country, intervention study subjects were equally randomized over the four treatment groups. In addition, infants who were exclusively breast-fed since birth (never received any infant formula) and their mothers had the intention to continue exclusive breast-feeding until the infant's age of at least 4 months were included in the breastfeeding reference group. In total, 350 subjects were enrolled, of which 280 subjects were randomised to either of the four test products and 70 subjects were included in the breastfeeding reference group.

Test group 1 : Infant formula 1 is a modified cow's milk-based infant formula for bottle- fed babies at the age of 0-6 months (Nutrilon 1, sold by Nutricia, the Netherlands). The formula contains non-digestible oligosaccharides (NDO), a mix of galacto- oligosaccharides, Vivinal® GOS from FrieslandCampina Domo, with an average degree of polymerisation below 6, and fructo-oligosaccharides, RaftilinHP from Orafti, with an average degree of polymerisation above 20 in a w/w ratio of about 9: 1 and in an amount of about 0.8 g per 100 ml. The formula does not contain a fermented composition.

Test group 2: Infant formula 2 is a modified cow's milk-based infant formula for bottle- fed babies at the age of 0-6 months and is a fermented infant formula Calisma® (sold by Gallia, France) which comprises the strains B. breve CNCM 1-2219 and S. thermophilus CNCM 1-1620, heat killed after a fermentation process, bacterial fermentation metabolites such as L-(+) lactate. The amount of L-lactate is above 0.05 wt% based on dry weight of the composition. No NDO is added. In the fermentation process an amount of about 2 wt% based on dry weight galacto-oligosaccharides is produced by the S. thermophilus.

Test group 3: Infant formula 3 is the experimental test formula and is a modified cow's milk-based infant formula for bottle-fed babies at the age of 0-6 months. The formula contains non digestible oligosaccharides (a mix of galacto-oligosaccharides with an average degree of polymerisation below 6, and fructo-oligosaccharides, RaftilinHP from Orafti, with an average degree of polymerisation above 20) in an amount of about 0.9 g per 100 ml and comprising fermented infant formula Calisma® (sold by Gallia, France), as in IF 2 of test group 2. The amount of added galacto-oligosaccharides takes into account the amount of galacto-oligosaccharides present due to the action of S. thermophilus.

Control group: Infant formula 4 is the control formula: a modified cow's milk-based infant formula for bottle-fed babies at the age of 0-6 months, not comprising non- digestible oligosaccharides and not being fermented. All four test formulas contained nucleotides and a fat blend that contained long chain fatty acids. The formula were similar in caloric content, protein content, fat blend and had a similar amount of digestible carbohydrates. The number of living bacteria in test product 2 and 3 was below 10³ cfu/g. The formulas further comprised vitamins, minerals, trace elements and other micronutrients according to international directive 2006/141/EC for infant formula.

At the end of the study, 198 of the randomised subjects completed the study, whereas 82 of the randomised subjects withdrew from the study prematurely. There were no statistically significant differences in number and nature of early withdrawals comparing the active test product groups with the control group. Subjects were well balanced over the study groups with respect to the demographic and baseline characteristics.

For each visit, a stool sample was collected by the parents into stool containers provided by Nutricia Research. The samples were frozen at -20 °C straight after collection by the parents and kept at this temperature until handing over the samples to the investigational staff. At the site, the samples were stored at -80 °C for later analysis. At each collection, 2 tubes had to be filled by half. If required due to practical issues at the study site, other means of transport and storage could be arranged. Faecal secretory IgA levels were determined with standard laboratory techniques. As the distribution of slgA measurements showed a potential skewness, for statistical analysis logarithmic transformation has been implemented before testing for differences across the study groups by using ANOVA. The slgA levels were statistically analyzed based on geometric means (effect size = group difference expressed as geometric mean ratio due to log- transformation).

The results are shown in Table 1. For the analysis of the primary data, the intention to treat (ITT) analysis was considered to be the principal analysis for the study. At 4 months, a time period where the infants are still fully fed with infant formula, there is a statistically significant difference (p<0.05) between the faecal slgA levels in the group of IF 3 (the experimental formula comprising fermented formula and non-digestible oligosaccharides) and the control group of IF 4 or the group of IF 2 (Calisma). When compared with the group of IF 1 (Nutrilon) there was a trend (p=0.07). There were no statistically significant differences when comparing IF 1 with IF 2, IF 1 with IF 4, or when comparing IF 2 with IF 4.

Table 1 : Concentration of faecal slgA ^g/g per visit - ITT Population

* Compared to test IF 3 p< 0.05

# Compared to test IF 3: p=0.07

No statistics are given when compared with the breast fed reference group, as the reference group is not randomised and should therefore not be used in a direct comparison with the randomised study groups. No statistic analysis for the median is performed, but a similar pattern is observed.

The difference in slgA production between test group 3 and 4 can be expressed as effect sizes, based on geometric means, which is equal to 1.65. This can be interpreted that use of the experimental formula, IF 3, at age of 4 months results in about 65% increment in slgA concentration when comparing to use of control group with IF 4. Similar the effect size of test group 3 versus test group 1 was 1.53 and of test group 3 versus test group 2 was 2.06. The slgA concentration in the group consuming IF 3 are synergistically higher than expected, based on the results of the group consuming IF 1 and 2 and control IF 4 and beneficially more similar to the levels observed in the breast fed reference group. This is indicative for a synergistically increased effect on mucosal immune defense.

As the bacteria present in IF 3 (and 2) were heat inactivated, this effect cannot be explained by an increased amount of intestinal lactic acid producing bacteria deriving from the formula nor an increased intestinal fermentation of the added non digestible oligosaccharides by these lactic acid producing bacteria deriving from the formula.

Example 2: Consumption of fermented infant formula with non-digestible oligosaccharides increases intestinal slgA levels

In a randomized, multi-centre, double-blinded, prospective clinical trial, infants were enrolled before 28 days of age and assigned to receive until 17 weeks of age one of three formulas:

Test group 1 : Infant formula 1 comprising per 100 ml 66 kcal, 1.35 g protein (bovine whey protein/casein in 1/1 weight ratio), 8.2 g digestible carbohydrate (of which 5.6 g lactose, and 2.1 g maltodextrin), 3.0 g fat (mainly vegetable fat), 0.8 g non-digestible oligosaccharides comprising scGOS (source Vivinal® GOS) and lcFOS (source RaftilinHP®) in a 9: 1 wt ratio. Of this infant formula about 50 % based on dry weight is derived from Lactofidus™, a commercially available infant formula marketed under brand name Gallia. Lactofidus™ is a fermented milk derived composition and is produced by fermenting with S. thermophilus and comprises B. breve. A mild heat treatment is employed to inactivate the lactic acid bacteria. The infant formula comprises about 0.55 wt% lactic acid + lactate based on dry weight, of which at least 95% is L(+)- lactic acid/lactate. The composition further comprises vitamins, minerals, trace elements and other micronutrients according to international directive 2006/141/EC for infant formula.

Test group 2: Infant formula 2, similar to infant formula 1, but without the non-digestible oligosaccharides scGOS and lcFOS.

Test group 3: Infant formula 3, a non fermented infant formula with 0.8 g non-digestible oligosaccharides comprising scGOS (source Vivinal® GOS) and lcFOS (source RaftilinHP®) in a 9: 1 wt ratio, and for the remainder similar in composition as infant formula 1. Stool samples were collected at baseline and after 17 weeks of intervention for physiological and microbiological analysis, in a way similar as described in example 1. Only samples of a subgroup of subjects were analysed that had a complete set of stool samples (both visits) with sufficient amount of stool for all analyses. In addition, samples from infants that used any systemic antibiotics any time after birth or that used thickeners added to formula during the study were excluded.

In the selected set of faecal samples the impact of the used infant formulas was assessed on secretory immunoglobulin A (slgA) and the results are shown in Table 2.

Table 2. Concentration of faecal slgA (μg/g) per visit

assumption and/or presence of outliers.

* Significant different values (p<0.05) between groups for the tested comparison of interest: IF 1 versus IF 3.

^# Significant different values (p<0.05) between groups for the tested comparison of interest: IF 1 versus IF 2. Again the slgA concentration in the group consuming the experimental infant formula 1 are higher than the concentrations of the group consuming infant formula 2 or 3, very similar to the outcomes in example 1. This is indicative for an increased effect on mucosal immune defense. Interestingly the effect of faecal slgA levels did not correspond with the effect on faecal pH. The pH was lowest in the group consuming infant formula 2 and intermediate in the group consuming the experimental infant formula 1. The pH reflects the activity of the intestinal microbiota. The lower faecal pH of test group 1 and 2 is due to the fermentation of the non-digestible oligosaccharides present in the test infant formula, and fermentation is highest in test group 2, whereas SIgA levels are highest in test group 1.

Claims

1. Use of a fermented ingredient and non-digestible oligosaccharide in the manufacture of a nutritional composition for use in increasing IgA secretion in a human subject with an age of 0 to 36 months, wherein the fermented ingredient is a composition that is fermented by lactic acid producing bacteria and wherein the nutritional composition comprises 0.02 to 1.5 wt% of the sum of lactic acid and lactate based on dry weight of the nutritional composition and comprises 2.5 to 15 wt% non-digestible oligosaccharide based on dry weight of the nutritional composition.

2. Use of a fermented ingredient and non-digestible oligosaccharide in the manufacture of a nutritional composition for use in improving mucosal immune defense in a human subject with an age of 0 to 36 months, wherein the fermented ingredient is a composition that is fermented by lactic acid producing bacteria and wherein the nutritional composition comprises 0.02 to 1.5 wt% of the sum of lactic acid and lactate based on dry weight of the nutritional composition and comprises 2.5 to 15 wt% non- digestible oligosaccharide based on dry weight of the nutritional composition.

3. The use according to any one of the preceding claims wherein the fermented ingredient is a ilk substrate that is fermented by lactic acid producing bacteria, and said milk substrate comprises at least one selected from the group consisting of milk, whey, whey protein, whey protein hydro lysate, casein, casein hydro lysate, and lactose or mixtures thereof.

4. The use according to any one of the preceding claims, wherein the nutritional composition is an infant formula, a follow on formula or a growing up milk.

5. The use according to any one of the preceding claims, wherein the non-digestible oligosaccharide is selected from the group consisting of fructo-oligosaccharide, non- digestible dextrin, galacto-oligosaccharide, xylo-o 1 igosaccharide. arabino- oiigosaccharide, arabinogalacto-oligosaccharide, gluco-oligosaccharide, gentio- o! igosaccharide. glucomanno-ol igosaccharide, galactomanno-ol igosaccharide, mannan-oligosaccharide, isomalto-ol igosaccharide, n igero-ol igosaccharide, glucomanno-oligosaccharide, chito-ol igosaccharide, soy oligosaccharide, uronic acid oligosaccharide, sialy!oligosaccharide, and fuco-o! igosaccharide, and mixtures thereof.

6. The use according to any one of the preceding claims, wherein the non-digestible oligosaccharide comprises galacto-oligosaccharide and/or fructo-oligosaccharide.

7. The use according to any one of the preceding claims, wherein at least 90 wt% of the sum of lactic acid and lactate is L(+)-lactic acid and/or L(+)-lactate.

8. The use according to any one of the preceding claims, wherein the fermented ingredient is fermented by Bifidobacterium and/or Streptococcus.

9. The use according to any one of the preceding claims wherein the nutritional composition comprises Bifidobacterium breve and/or Streptococcus thermophilus.

10. The use according to any one of the preceding claims wherein the lactic acid producing bacteria are inactivated to a level below 10⁵' more preferably below 10³ cfu/g dry weight of the nutritional composition.

11. The use according to any one of the preceding claims wherein the nutritional composition comprises protein, digestible carbohydrate and lipid and wherein the protein provides 1.6 to 4 g per 100 kcal, the digestible carbohydrates provides 5 to 20 g per 100 kcal and the lipid provides 3 to 7 g lipid per 100 kcal of the nutritional composition.

12. A method for increasing IgA secretion in a human subject with an age of 0 to 36 months comprising administering to the human subject a nutritional composition comprising a fermented ingredient and non-digestible oligosaccharide, said nutritional composition comprising 0.02 to 1.5 wt% of the sum of lactic acid and lactate based on dry weight of the nutritional composition and comprising 2.5 to 15 wt% non-digestible oligosaccharide based on dry weight of the nutritional composition and wherein wherein the fermented ingredient is a composition that is fermented by lactic acid producing bacteria.

13. A method for improving mucosal immune defense in a human subject with an age of 0 to 36 months comprising administering to the human subject a nutritional composition comprising a fermented ingredient and non-digestible oligosaccharide, said nutritional composition comprising 0.02 to 1.5 wt% of the sum of lactic acid and lactate based on dry weight of the nutritional composition and comprising 2.5 to 15 wt% non-digestible oligosaccharide based on dry weight of the nutritional composition and wherein wherein the fermented ingredient is a composition that is fermented by lactic acid producing bacteria.

14. The method according to claim 12 or 13, wherein the fermented ingredient is a milk substrate that is fermented by lactic acid producing bacteria, and said milk substrate comprises at least one selected from the group consisting of milk, whey, whey protein, whey protein hydro iysate, casein, casein hydro lysate, and lactose or mixtures thereof.