WO2017114901A1

WO2017114901A1 - Nutritional formula with non-digestible oligosaccharides and non-replicating lactic acid producing bacteria

Info

Publication number: WO2017114901A1
Application number: PCT/EP2016/082848
Authority: WO
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: 2017-07-06
Anticipated expiration: 2018-06-29
Also published as: CN108697141A; EP3397075A1

Abstract

A nutritional composition comprising non-digestible oligosaccharideandnon-replicating Bifidobacterium breveand/or Streptococcus thermophilussynergistically stimulates secretory IgA in infants.

Description

NUTRITIONAL FORMULA WITH NON-DIGESTIBLE OLIGOSACCHARIDES AND NON-REPLICATING LACTIC ACID PRODUCING BACTERIA

FIELD OF THE INVENTION

The present invention relates to the field of nutrition for infants and young children for improving the mucosal 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 o infant formulas is aimed not necessarily at mimicking the exact composit ion of human milk but at achieving 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. At birth, infants born via C section are sterile and in the absence of microbiota have a severely reduced secretion of IgA hence requiring that live probiotic bacteria are ingested. 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. 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 aid the subsequent colonization by live bacteria. Effect on IgA secretion is not disclosed. WO 2009/ 1 5 133 1 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 o the systemic immune system, the vaccination response is increased and allergy is decreased. WO 2009 15 1329 discloses a composit ion comprising non-digestible oligosaccharides and a product obtained by incubating with bifidobacteria, to reduce bacterial translocation and improv ing the intestinal barrier function. It is the aim to prevent systemic infections in this way. WO 2008/153377 discloses an infant formula comprising non-v iable Bifidobacterium breve and non-digestible oligosaccharides. The document is silent on the effect on the mucosal immune defense and IgA secretion. US 2014 193542 is concerned with providing allergen free infant nutrition compositions based on free amino acids and containing prebiotics and probiotics. Whereas IgE and IgG lev els are monitored, the document is silent on secretory IgA. 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. Where the combination of prebiotics and probiotics is disclosed, the prebiotics are supposed to stimulate the growth or metabolic activity of the probiotics. SUMMARY OF THE INVENTION

In clinical trials with healthy, term infants it was found that administration of an infant formula comprising at least one strain of non-replicating lactic acid producing bacteria selected from the group consisting of Bifidobacterium breve and Streptococcus thermophilus and comprising non-digestible oligosaccharides, a large increase of intestinal secretory IgA (slgA) was observed, when compared to administration of control formula without non-digestible oligosaccharides and without non-replicating lactic acid producing bacteria, an infant formula comprising non-replicating lactic acid producing bacteria selected from the group consisting of Bifidobacterium breve and Streptococcus thermophilus but without non-digestible oligosaccharides, or a formula comprising non-digestible oligosaccharides, but ithout non- replicating lact ic acid producing bacteria. The secretion of IgA was synergist ically 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, 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 at least one strain of non-replicating lactic acid producing bacteria selected from the group consisting of Bifidobacterium breve and Streptococcus thermophilus and comprising non-digestible oligosaccharide, said composition having 2.5 to 15 wt% of non-digestible oligosaccharides 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 at least one strain of non-replicating lactic acid producing bacteria selected from the group consisting of Bifidobacterium breve and Streptococcus thermophilus 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 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 at least one strain of non-replicating lactic acid producing bacteria selected from the group consisting of Bifidobacterium breve and Streptococcus thermophilus and non-digestible oligosaccharide wherein the nutritional composition comprises2.5 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 at least one strain of non-replicating lactic acid producing bacteria selected from the group consisting of Bifidobacterium breve and Streptococcus thermophilus and comprising non-digestible oligosaccharide, said composition having 2.5 to 15 wt% of non-digestible oligosaccharides 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 at least one strain of non-replicating lactic acid producing bacteria selected from the group consisting of Bifidobacterium breve and Streptococcus thermophilus 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 nutritional composition 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 at least one strain of non-replicating lactic acid producing bacteria selected from the group consisting of Bifidobacterium breve and Streptococcus thermophilus and non-digestible oligosaccharide wherein the nutritional composition comprises2.5 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 months. Lactic acid producing bacteria

The nutritional composition in the methods or uses according to the present invention, hereafter also referred to as the present nutritional composition comprises lactic acid producing bacteria selected from the group consisting of Bifidobacterium breve and Streptococcus thermophilus. Preferably the composition comprises a strain of S. thermophilus. Preferably the S. thermophilus develop beta-galactosidase activity in the course of fermentation of substrate. Selection of a suitable strain of S. thermophilus is described in example 2 of EP 778885 and in example 1 of FR 2723960. The nutritional composition comprises less than 10⁶ cfu bacteria of S. thermophilus, per g dry weight, preferably the nutritional composition comprises less than 10⁵ cfu S. thermophilus per g dry weight, even more preferably less than 10³ cfu per g dry weight. In one embodiment the nutritional composition comprises from 0 to 10⁴ cfu S. thermophilus per g dry weight, preferably from 0 to 10³ cfu per g fdry eweight of the composition.

The nutritional composition comprises non-replicating S. thermophilus in an amount of at least 10⁷ cfu bacteria of S. thermophilus, per g dry weight, preferably more than 10⁸ cfu per g dry weight, even more preferably more than 10⁹ cfu per g dry weight. Preferably the nutritional composition comprises less than 10¹⁴ cfu non-replicating S. thermophilus per g dry weight, even more preferably less than 10¹² cfu per g dry weight of the nutritional composition.

Preferred strains of S. thermophilus to apply in the present invention have been deposited by Compagnie Gervais Danone at the Collection Nationale de Cultures de Microorganismes (CNCM) run by the Institut Pasteur, 25 rue du Docteur Roux, Paris, France on 23 August 1995 under the accession number 1-1620 and on 25 August 1994 under the accession number 1-1470.

Preferably the composition comprises a strain of Bifidobacterium breve. Preferred B. breve strains are those isolated from the faeces of healthy human milk- fed infants. Typically, these are commercially available from producers of lactic acid bacteria, but they can also directly be isolated from faeces, identified, characterised and produced. According to a preferred embodiment, the present composition contains at least one B. breve selected from the group consisting of B. breve Bb-03 (Rhodia/Danisco), B. breve M-16V (Morinaga), B. breve R0070 (Institute Rosell, 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 non- viable B. breve is non- viable B. breve M-16V (Morinaga) or B. breve 1-2219, even more preferably B. breve 1-2219.

The nutritional composition comprises less than 10⁶ cfu bacteria of B. breve, per g dry weight, preferably the nutritional composition comprises less than 10⁵ cfu of B. breve per g dry weight, even more preferably less than 10³ cfu. In one embodiment the nutritional composition comprises from 0 to 10⁴ cfu B. breve per g dry weight, preferably from 0 to 10³ per g dry weight of the nutitional composition. The nutritional composition comprises non-replicating B. breve in an amount of at least 10⁷ cfu, per g dry weight, preferably more than 10⁸ cfu, even more preferably more than 10⁹ cfu per g dry weight. Preferably the nutritional composition comprises less than 10¹³ cfu non-replicating B. breve per g dry weight, even more preferably less than 10¹¹ cfu per g dry weight of the nutritional composition.

Preferably the present nutritional composition comprises less than 10⁶ cfu bacteria of the sum of S. thermophilus and B. breve, per g dry weight, preferably the nutritional composition comprises less than 10⁵ cfu of the sum of S. thermophilus and B. breve, per g dry weight, even more preferably less than 10³ cfu per g dry weight of the nutritional composition. In one embodiment the nutritional composition comprises from 0 to 10⁴ cfu of the sum of S. thermophilus and B. breve per g dry weight, preferably from 0 to 10³ cfu per g dry weight of the nutritional composition.

The equivalent of cfu can suitably be determined by performing the 5 'nuclease assay with the B. breve probes and primers as disclosed in WO 2005/039319 on a composition, e.g. an infant formula, comprising non-viable B. breve and compare this with a calibration curve obtained from a comparable product, for instance a standard infant formula, to which known amounts of dried, viable B. breve cfu have been added. The dried viable bifidobacteria can be commercially obtained as described above.

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.

The present nutritional composition comprises non-replicating lactic acid producing bacteria and/or bacterial fragments derived from lactic acid producing bacteria wherein the lactic acid producing bacteria are selected from the group consisting of Bifidobacterium breve and Streptococcus thermophilus, and obtained from, or the equivalent of, more than 10⁷ cfu per g based on dry weight of the final composition, more preferably 10⁸ cfu, even more preferably 10⁹ cfu. Preferably the non-replicating lactic acid producing bacteria and/or bacterial fragments derived from lactic acid producing bacteria wherein the lactic acid producing bacteria are selected from the group consisting of Bifidobacterium breve and Streptococcus thermophilus, are obtained from, or the equivalent of, less than lxlO¹⁴ cfu per g based on dry weight of the final composition, more preferably lxl 0¹³ cfu, even more preferably lxl 0¹² cfu.

Preferably the present nutritional composition 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.

Methods of inactivation of B. breve and/or S. thermophilus

Living cells of B. breve and/or S. thermophilus lactic acid producing bacteria in the nutritional composition are after fermentation preferably essentially all rendered non-replicating. S. thermophilus and/or B. breve cells can be made non-viable by methods known in the art, including heat treatment steps (including sterilization, pasteurization, UHT treatment), radiation (UV), treatment with oxygen, treatment with bactericidals such as ethanol, sonication, ultra high pressure application, high pressure homogenization and use of a cell disruptor, preferably by a heat treatment.

Preferably the S. thermophilus and/or B. breve are subjected to a heat treatment after a fermentation step. Preferable ways of making the S. thermophilus and/or B. breve non- replicating are (flash) pasteurization, sterilization, ultra high temperature treatment, high temperature/short time heat treatment and/or spray drying at temperatures that the lactic acid producing bacteria do not survive. Cell fragments are preferably obtained by heat treatment. With this heat treatment preferably at least 95 % of B. breve and/or S thermophilus are rendered non-replicating, more preferably at least 97.5 %, even more preferably at least 99 %. The heat treatment preferably is performed at a temperature ranging from 70 to 180 °C, preferably 80 to 150 °C, preferably for about 3 minutes to 2 hours, preferably in the range of 80-140 °C for 5 minutes to 40 minutes. Inactivation of the lactic acid producing 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 composition may be pasteurised or sterilized and for example spray dried or lyophilised to provide a form suitable to be formulated in the end product.

Non-replicating lactic acid bacteria do not form colonies by classical plating methods. Such classical plating methods are summarized in the microbiology book: James Monroe Jay, Martin J. Loessner, David A. Golden. 2005. Modern food microbiology. 7th edition, Springer Science, New York, N. Y. 790 p. Typically, the absence of replicating cells can be shown as follows: no visible colony on agar plates or no increasing turbidity in liquid growth medium after inoculation with different concentrations of bacterial preparations ('non replicating' samples) and incubation under appropriate conditions (aerobic and/or anaerobic atmosphere; appropriate temperature and growth medium, and appropriate incubation time, at least 24 h).

Non-replicating bacteria can also sometimes be referred to as non-viable bacteria, non culturable bacteria or non-growing bacteria. Non replicating bacteria comprise metabolically active cells, inactivated or dead cells or bacterial cell fragments. Fermented ingredient

The present nutritional composition preferably comprises a fermented ingredient. Preferably the fermented ingredient is a composition that has been fermented by lactic acid producing bacteria, preferably by S. thermophilus and/or ?. breve. The fermentation preferably takes place during the production process of the nutritional composition. 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 milk, whey, whey protein, whey protein hydrolysate, casein, casein hydrolysate, and lactose or mixtures thereof. Suitably, nutritional compositions comprising fermented ingredient and non-digestible oligosaccharide and their way o 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, glyco!ipids, peptidog!ycan. lipoteiehoic acid (LTA), lipoproteins, nucleotides, and or capsular polysaccharides. It is o advantage to use the fermented ingredient comprising inactivated bacteria and/or cell fragments directly as a part of the final nutritional product, since this will result in a higher concentration of bacterial cell 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 eliminating the presence of bacterial cell fragments. Furthermore, upon fermentation and/or other interactions of lact ic acid producing bacteria with the milk substrate, addit ional bio-act ive 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-derived product, is bel iev ed to have an improved effect compared to non-fermented milk-derived product.

Preferably the nutritional composition comprises 5 to 97.5 wt% of the fermented ingredient based on dry weight, more preferably 1 0 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 nutritional composition can be taken, as this is the metabolic end product produced by the lactic acid producing bacteria upon fermentation. The present nutritional composition comprises 0.02 to 1.5 wt% of the sum of lactic acid and lactate based on dry weight of the composit ion, more preferably 0.05 to 1.0 wt%, even more preferably 0.05 to 0.5 wt%. Preferably at least 50 wt%, even more preferably at least 90 wt%, of the sum of lact ic acid and lactate is in the form of the L(+)-isomer. Thus in one embodiment the sum of !.( + )- lact ic 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 L( + )- lactate and L(+)-lactic acid is also referred to as L-lactate and L-lact ic acid. Process of fermentation

The milk substrate to be fermented is suitably present in an aqeuous medium. The milk substrate to be fermented is preferably selected from the group consisting of milk, whey, whey protein, whey protein hydrolysate. casein, casein hydrolysate. and lactose, and mixtures thereof. M il k 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 hydrolysate 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 pi 1 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 activity of lact ic 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 extract is a rich source of enzyme co-factors and growth factors for lactic acid producing bacteria. The presence of yeast extract will enhance the fermentation by lactic 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 living bacteria. Suitably the product is pasteurised after fermentation, in order to inactivate 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-deriv ed product is homogenised after the fermentation. Homogenisation results in a more stable substrate and/or fermented product, especially in the presence of fat.

The inoculation density is preferably between lxlO² to 5xl0¹⁰, preferably between lxlO⁴ to 5x10⁹ cfu S. thermophilus and/or B. breve/ml aqueous medium containing milk substrate, more preferably between lxl0⁷to lxlO⁹ cfu /ml aqueous medium containing milk substrate. The final bacteria density after fermentation is preferably between lxlO³ to lxlO¹⁰, more preferably between lxl 0⁴ to lxl 0⁹ cfu/ml aqueous medium containing milk substrate.

The fermentation 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 of the S thermophilus and/or B. breve.

The incubation is preferably performed at a pH of 4 to 8, more preferably 6 to 7.5. This pH does not induce protein precipitation and/or an adverse taste, while at the same time lactic acid producing bacteria such as B. breve and/or S. thermophilus are able to ferment the milk substrate. The incubation time 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, glycolipids, peptidoglycan, lipoteichoic acid (LTA), flagellae, 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 Streptococcus strains 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 instead. 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 subsequently fat is added in-line, homogenized, pasteurized and dried.

Procedures to prepare suitable fermented ingredients 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 composition. FR 2723960, which is incorporated herein by reference, discloses in particular in example 6 a suitable process for preparing a fermented composition. Briefly, a milk derived product, 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 derived product comprises milk protein peptides. Temperature and duration of fermentation are as mentioned above. Suitably after fermentation the fermented protein comprising composition 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 product is disclosed in WO 01/01785, more particular in example 1 and 2. A preferred method for preparing the fermented product is described in WO 2004/093899, more particularly in example 1. Non-digestible oligosaccharides

The present composition comprises non digestible oligosaccharide and preferably comprises at least two non-digestible oligosaccharides, in particular two different sources of non-digestible 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 non-replicating lactic acid producing bacteria selected from the group consisting of Bifidobacterium breve and/or Streptococcus thermophilic acts synergisticaily and advantageously results in a higher IgA secretion in the intestine. Hence, the presence of both the non-digestible oligosaccharide and non-replicating lactic acid producing bacteria selected from the group consisting of Bifidobacterium breve and/or Streptococcus thermophilus acts 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. If 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 invention refers to oligosaccharides which are not digested in the intestine by the action of acids or digestive enzymes present in the human upper digestive tract, e.g. small intestine and stomach, but which are preferably fermented by the human intestinal microbiota. For example, sucrose, lactose, maltose and maltodextrins 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 oligosaccharide, 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 oligosaccharide 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 consisting of fructo-oiigosaccharide, such as inulin, non-digestible dextrins, gaiacto- oligosaccharide, such as transgaiacto-oligosaccharide, xylo-oligosaccharide, arabino- oligosaccharide, arabinogalacto-oligosaccharide, gluco-oiigosaccharide, gentio- ol igosaccharide, glucomanno-oligosaccharide, galactomannool igosaccharide, mannan- o!igosaccharide, i. so malto-oligo saccharide, nigero -ol igosaccharide, glucomanno- oligosaccharide, chito-oligosaccharide. soy oligosaccharide, uronic acid oligosaccharide, sialylol i go s a c c h a r i d e , such as 3-sialyliactose (3-SL), 6-sialyl lactose (6-SL), I actosi a ly it et rasaec ha ri de a,b,c (LST), d i si a 1 y 11 acto t et raosc ( DSLNT), sia!yl-!acto hexaose (S-LNH), DS-LNH, and fuco-oiigosaccharide, such as (un)suiphated fucoidan oligosaccharide, 2'-fucosyliactose (2'-FL), 3-fucosy I lactose (3-FL), difucosy I lactose, iacto-N-fucopenatose, (LNFP) I, II, I I I, V, Lacto-N-neofucopenaose (LNnFP), Lacto-N-difucosyl-hexaose (LNDH), and mixtures thereof, even more preferably selected from the group consisting of fructo- oligosaccharide, such as inulin, galacto-oiigosaccharide, such as transgalacto-oligosaccharide, and fuco-oligosaccharide and mixtures thereof, even more preferably transgalacto- oligosaccharide, and/or inulin, most preferably transgalacto-oligosaccharide. In one embodiment in the composition or methods according to the present invention, the non- digestible oiigosaccharide is selected from the group consisting of transgaiacto- oligosaccharide, fructo-oligosaccharide and mixtures of thereof.

The non-digestible oligosaccharide is preferably selected from the group consist ing of β- galacto-oligosaccharide, a-ga I acto-o 1 i gosacch ari de, and galactan. According to a more preferred embodiment non-digestible oligosaccharide is β-galacto-oligosaccharide. Preferably the non-digest ible oligosaccharide comprises galacto-oiigosaccharide 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 ( Domo FrieslandCampina I ngredients), Bi2muno (Clasado), Cup-oligo (Nissin Sugar) and Oligomate55 (Yakult ). These oligosaccharides increase slgA levels and strengthens the mucosal immune defense act ivity to a larger e tent.

The non-digestible oligosaccharide preferably comprises fructo-oligosaccharide. A fructo- oligosaccharide may in other context have names like fructopolysaccharide. oligo fructose, polyfructose. polyfructan, inulin, lev an and fructan. and may refer to ol igosaccharides 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- ol igosaccharide contains a terminal β(2, 1) glycosidic l inked 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-ol igosaccharide wherein at least 75% of the glycosidic linkages are β(2, 1) linkages. Typically, inulin has an av erage chain length between 8 and 60 monosaccharide un its. A suitable fructo-oligosaccharide for use in the compositions of the present invent ion is commercially available under the trade name Raftiiine®HP (Orafti). Other suitable sources are Raft ilose (Orafti), Fibrulose and Fibrulinc (Cosucra) and Friitafit and

Frutaiose (Sensus).

Preferably the present nutritional composit ion comprises a mixture of gaiacto-oligosaccharide and fructo-oligosaccharide. Preferably the mixture of ga lac too I i gosacch a ri dc 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 , ev en more prefeably in a rat io of about 9/1. This weight ratio is particularly adv antageous when ga I ac to-o 1 i gosacc hari dc has a low average DP and fructo-oligosaccharide has a relat ively high DP. Most preferred is a mixture of gaiacto- oligosaccharide with an average DP below 1 0. preferably below 6 and a fructo-oligosaccharide with an average DP abov e 7, preferably above 1 1 , even more preferably above 20. Such a mixture synergist ically increases slgA lev els and strengthens the mucosal immune defense activity.

Preferably the present nutritional composition comprises a mixture of short chain fructo- oligosaccharide and long chain fructo-oligosaccharide. Preferably the mixture of short chain f rue to-o 1 i gosacch a ri de and long chain fructo-oligosaccharide is present in a weight ratio of from 1/99 to 99/1 , more preferably from 1/19 to 19/1 , ev en 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-o 1 i gosacch a ri dc with an average DP below 10, preferably below 6 and a fructo- oligosaccharide with an average DP abov e 7. preferably above 1 1 , ev en more preferably abov e 20.

The present nutrit ional composit ion comprises 2.5 to 20 wt% total non-digest ible oligosaccharide, more preferably 2.5 to 1 5 wt%, even more preferably 3.0 to 1 0 wt%, most preferably 5.0 to 7.5 wt%, based on dry weight of the nutrit ional composit ion. Based on 1 00 ml the present nutrit ional composit ion preferably comprises 0.35 to 2.5 wt% total non-digestible oligosaccharide, more preferably 0.35 to 2.0 wt%, even more preferably 0.4 to 1 .5 wt%, based on 1 00 ml of the nutrit ional composit ion. A lower amount of non-digest ible oligosaccharide will be less effect iv e in stimulat ing slgA format ion or mucosal immune defense, whereas a too high amount will result in side-effects of bloat ing and abdominal discomfort. Nutritional composition

The nutritional composition used according to the present invention may also be considered as being a pharmaceutical composit ion, is preferably suitable for administration to infants. The present nutrit ional composit ion is preferably enteral ly administered, more preferably orally.

The present nutritional composition is preferably an infant formula, follow on formula or growing up milk. The present nutritional composit ion can be advantageously applied as a complete nutrition for infants. Preferably the present nutrit ional 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 1 2 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 l iquid form. The present nutritional composit ion may also be in the form of a dry food, preferably in the form of a powder which is accompanied with instructions as to mi said dry food, preferably powder, with a suitable liquid, preferably w ater. The nutrit ional composit ion used according to the invent 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 v itamins, minerals, trace elements and other micronutrients according to internat ional directives.

The present nutritional composition preferably comprises lipid, protein and digestible carbohydrate inv ent ion wherein the lipid prov ides 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 composit ion the lipid prov ides 35 to 50% of the total calories, the protein prov ides 7.5 to 12.5% of the total calories, and the digestible carbohydrate provides 40 to 55% of the total calories. For calculat ion of the % of total calories for the protein, the total of energy provided by proteins, pept ides and amino acids needs to be taken into account. Preferably the lipid provides 3 to 7 g l ipid per 100 kcal. preferably 4 to 6 g per 1 00 kcal, the protein provides 1 .6 to 4 g per 100 kcal. preferably 1 .75 to 2.5 g 1 5 per 100 kcal and the digestible carbohydrate provides 5 to 20 g per 1 00 kcal. preferably 8 to 1 5 g per 100 kcal of the nutrit ional composit ion. Preferably the present nutrit ional composit ion comprises lipid prov iding 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 prov ides 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 digestible 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, digestible 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 of animal lipid (excluding human lipids) and vegetable lipids. Preferably the present composit ion comprises a combinat ion of vegetable lipids and at least one oil selected from the group consisting of fish oil, animal oil, algae oil, fungal oil, and bacterial oil. The lipid of the present nutritional composition 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 19 to 30 wt%. Preferably the lipid comprises the essential fatty acids alpha- lino lenic acid (ALA), iinoleic 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 diglyceridc form, in monog!yceride 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 safflo ver oil, olive oil, marine oils, microbial oils, coconut oil. palm kernel oil and milk fat. The present nutritional composition 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 mPa.s. The low viscosity ensures a proper administration o the liquid, e.g. a proper passage through the whole of a nipple. Also this viscosity closely resembles the viscosity o 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 composition with water. Normally infant formula is prepared in such a way. The present invention thus also relates to a packaged power composition wherein said package is provided with instructions to admi the powder with a suitable amount of liquid, thereby result ing 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 Mcsstechnik GmbH, Ostfiiden, 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 synergistically 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 non replicating lactic acid producing bacteria selected from the group consisting of Bifidobacterium breve and/or Streptococcus thermophilus and the non- digestible oligosaccharides. 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, these synergistic effect cannot be explained by a direct interaction between the non-digestible oligosaccharides and the lactic acid producing bacteria in the composition, as the bacteria were inactivated, nor by an increased intestinal microbial activity as there was no clear correlation with the intestinal pl l.

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 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 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 infant formula comprising non-replicating lactic acid producing bacteria selected from the group consisting of Bifidobacterium breve and Streptococcus thermophilus 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 breast-feeding 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 breast-feeding 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 non-replicating lactic acid producing bacteria selected from the group consisting of Bifidobacterium breve and Streptococcus thermophilus. The formula also does not contain replicating lactic acid producing bacteria.

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 partially 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. The equivalent of non-replicating B. breve and S thermophilus is above 5.10⁷ cfu/g. The amount of replicating B. breve and S thermophilus is below 1.10³ cfu/g.

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, Raftiline-HP 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 without the non-replicating lactic acid producing bacteria selected from the group consisting of Bifidobacterium breve and Streptococcus thermophilus and also without replicating lactic acid producing bacteria.

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 formulas further comprise 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 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 the IF 3 (the experimental formula comprising non-replicating lactic acid producing bacteria 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.

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.

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

Example 2: Consumption of infant formula comprising non-replicating lactic acid producing bacteria selected from the group consisting of Bifidobacterium breve and Streptococcus thermophilus and 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 producing bacteria. The amount of non-replicating lactic acid producing bacteria selected form the group consisting of Bifidobacterium breve and Streptococcus thermophiles was above 10⁷ cfu/g dry weight. The amount of S. thermophilus was about 10⁴ to 10⁵ cfu/g dry weight. 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 0.8 g non-digestible oligosaccharides scGOS and lcFOS.

Test group 3: Infant formula 3, a standard infant formula with 0.8 g non-digestible oligosaccharides of scGOS (source Vivinal® GOS) and lcFOS (source RaftilinHP®) in a 9: 1 wt ratio, and for the remainder with similar composition as infant formula 1. The formula does not contain Bifidobacterium breve and/or Streptococcus thermophilus.

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 infant 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.

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.

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

For the analysis a Wilcoxon rank sum tests (W) was used due to violation of normality 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.

Claims

1. Use of at least one strain of non-replicating lactic acid producing bacteria selected from the group consisting of Bifidobacterium breve and Streptococcus thermophilus 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 2.5 to 15 wt% non-digestible oligosaccharide based on dry weight of the nutritional composition.

2. Use of at least one strain of non-replicating lactic acid producing bacteria selected from the group consisting of Bifidobacterium breve and Streptococcus thermophilus 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 nutritional composition 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 nutritional composition comprises non-replicating Bifidobacterium breve.

4. The use according to any one of the preceding claims, wherein the nutritional composition comprises non-replicating Bifidobacterium breve and/or Streptococcus thermophilus in amount equivalent to at least 10⁷ cfu/g dry weight of the nutritional composition, more preferably at least 10⁸ cfu/g dry weight.

5. The use according to any one of the preceding claims wherein the Bifidobacterium breve and/or Streptococcus thermophilus are inactivated to a level below 10⁶ cfu/g dry weight, more preferably below 10⁵ cfu/g dry weight even more preferably below 10³ cfu/g dry weight of the nutritional composition.

6. The use according to any one of the preceding claims, which is an infant formula, a follow on formula or a growing up milk.

7. The use according to any one of the preceding claims wherein the non-digestible oligosaccharide is selected from the group consisting of fructo-oiigosaccharide, non- digestible dextrin, galacto-oiigosaccharide, xylo-o I igosaccharidc, a rab i no-o I i gosaccha ri de, arabinogalacto-oiigosaecharide. gluco-oligosaccharide, gent io-ol igosaccharidc, glucomanno-oligosaccharide, galactomanno-ol igosaccharidc. ma n n an-o 1 i gosacch a ri de. i so m a 11 o - o I i go s a e e h a ri d e . nigero -oligosaccharide, glucomanno-oligosaccharide, chito- oligosaccharide, soy oligosaccharide, uronic acid oligosaccharide, ia I y Io I i gosacc h arid e. and fiieo-ol igosaccharidc, and mixtures thereof.

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

9. The use according to any one of the preceding claims wherein the nutritional composition comprises fermented ingredient, fermented by Bifidobacterium breve and/or Streptococcus thermophilus, and wherein the nutritional composition comprises 0.05 to 1.5 wt% of the sum of L(+)-lactic acid and L(+)-lactate.

10. 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 li id per 100 kcal of the nutritional composition.

11. 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 at least one strain of non-replicating lactic acid producing bacteria selected from the group consisting of Bifidobacterium breve and Streptococcus thermophilus and comprising non- digestible oligosaccharide, said composition having 2.5 to 15 wt% of non-digestible oligosaccharides based on dry weight of the nutritional composition.

12. 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 at least one strain of non-replicating lactic acid producing bacteria selected from the group consisting of Bifidobacterium breve and Streptococcus thermophilus and comprising non-digestible oligosaccharide, said composition having 2.5 to 15 wt% of non- digestible oligosaccharides based on dry weight of the nutritional composition.