CN108697141A - Nutritional formula containing non-digestible oligosaccharides and non-replicating lactic acid producing bacteria - Google Patents

Abstract

A nutritional composition comprising non-digestible oligosaccharides and non-replicating Bifidobacterium breve and/or Streptococcus thermophilus synergistically stimulates secretory IgA in infants.

Description

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

technical field

The present invention relates to the field of infant nutrition for improving the mucosal immune system.

Background technique

It is generally accepted that the best nutrition for newborn babies is human milk. Infant formula (IF) based on mature human milk is considered the best alternative when mothers are unable to breastfeed their infants or choose not to breastfeed. Research to improve the quality of infant formula is not necessarily to mimic the precise composition of human milk, but to achieve functional effects beyond the nutritional aspects only observed in breastfed infants.

Human milk is rich in secretory IgA (sIgA), which serves as the first line of defense to protect the intestinal barrier of infants from intestinal toxins and pathogenic microorganisms. sIgA is resistant to digestive enzymes and facilitates clearance of antigens and pathogenic microorganisms from the gut by blocking access to epithelial cell receptors, trapping them in mucus, and facilitating their removal through peristalsis and mucociliary activity. In addition, sIgA plays a role in the mucosal immune defense system and intestinal homeostasis without provoking and even ameliorating inflammatory responses. In contrast, standard infant formula based on cow's milk components has much lower sIgA concentrations, and infants fed standard infant formula have reduced sIgA levels compared to breastfed infants.

EP 2 318 046 discloses the combination of probiotics and secretory IgA for the treatment or prevention of inflammation. EP 2 315595 discloses the use of live probiotics for increasing immunoglobulin secretion in infants born by Caesarean section (C section). At birth, infants delivered by caesarean section are sterile and, in the absence of a microbiota, have severely reduced IgA secretion, requiring the intake of live probiotics. Fukushima et al. (1998, Int J Food Micribiol 42:39-44) studied the effect of the probiotic Bifidobacterium lactis strain on IgA secretion in a cohort of older children and found that total IgA and anti-poliovirus The level of IgA increases. Probiotics are live lactic acid producing bacteria such as live Lactobacillus or live Bifidobacterium.

Nondigestible oligosaccharides also affect intestinal sIgA responses through their effects on microbiota composition. Scholtens et al. (2008, J Nutr 138:1141-1147) and Bakker-Zierikzee et al. (2006, PediatrAllergy Immunol 17:134-140) found that fecal sIgA levels were higher in healthy infants fed formula supplemented with scGOS/lcFOS Fecal sIgA levels in infants fed standard formula support this hypothesis.

Mullié et al. (2004, Ped Res 56:791-795) disclosed that in healthy neonates, consumption of fermented formula was associated with increased production of intestinal sIgA antibodies specific to poliovirus following administration of the vaccine , but had no effect on total IgA titers.

WO 2009/151330 discloses a composition obtained by fermentation of lactic acid producing bacteria and non-digestible oligosaccharides for caesarean section infants. The aim is to induce tolerance, thereby facilitating subsequent colonization with viable bacteria. No effect on IgA secretion is disclosed. WO 2009/151331 discloses an immunostimulatory composition comprising non-digestible oligosaccharides and the products obtained after incubation with bifidobacteria. By stimulating Th1 responses of the systemic immune system and reducing Th2 responses of the systemic immune system, vaccination responses are increased and allergies are reduced. WO 2009/151329 discloses a composition comprising non-digestible oligosaccharides and products obtained by incubation with bifidobacteria for reducing bacterial translocation and improving intestinal barrier function. The aim is to prevent systemic infection in this way. WO 2008/153377 discloses an infant formula comprising inactive Bifidobacterium breve and non-digestible oligosaccharides. The document does not mention effects on mucosal immune defense and IgA secretion. US 2014/193542 relates to the provision of allergen-free infant nutritional compositions based on free amino acids and containing prebiotics and probiotics. Although IgE and IgG levels are monitored, the document does not mention secretory IgA. WO2014/201037 discloses the use of probiotics, prebiotics, synbiotics and antibiotics for the treatment of changes in the microbiota of mammals associated with antibiotic therapy and various immune disorders. Where combinations of prebiotics and probiotics are disclosed, the prebiotics are said to stimulate the growth or metabolic activity of the probiotics.

Contents of the invention

In a clinical trial of healthy full-term infants, it was found that when administered with a control formula containing no non-digestible oligosaccharides and no non-replicating lactic acid bacteria, containing non-replicating bacteria selected from Bifidobacterium breve and Streptococcus thermophilus When comparing infant formula containing lactic acid bacteria without non-digestible oligosaccharides or formula containing non-digestible oligosaccharides without non-replicating lactic acid bacteria, it was observed that administration of at least one species selected from the group consisting of Bifidobacterium breve and Infant formula containing a non-replicating lactic acid producing bacterial strain of Streptococcus thermophilus and containing non-digestible oligosaccharides produces a large increase in intestinal secretory IgA (sIgA). The secretion of IgA increased synergistically and was more similar to sIgA in the breastfed reference group. This further suggests a synergistic enhancement of the mucosal immune defense system.

Surprisingly, the synergistic effect could not be explained by a direct interaction between non-digestible oligosaccharides and lactic acid-producing bacteria, due to the inactivation of the bacteria, nor by increased intestinal microbial activity, due to the lack of a significant relationship with intestinal pH. Correlation.

Detailed ways

Accordingly, the present invention relates to a method of increasing IgA secretion in a human subject from 0 to 36 months of age, which comprises administering to the human subject a non-replicating agent comprising at least one non-replicating strain selected from Bifidobacterium breve and Streptococcus thermophilus. A nutritional composition that produces a strain of lactic acid bacteria and includes non-digestible oligosaccharides, the composition having 2.5 to 15% by weight of non-digestible oligosaccharides based on the dry weight of the nutritional composition.

In one embodiment, the method of the invention for increasing IgA secretion in a human subject between 0 and 36 months of age is a non-therapeutic method.

The invention may also be expressed as at least one non-replicating lactic acid producing bacterial strain selected from Bifidobacterium breve and Streptococcus thermophilus and non-digestible oligosaccharides in the preparation for increasing IgA in human subjects aged 0 to 36 months Use in a secreted nutritional composition comprising 2.5 to 15% by weight of non-digestible oligosaccharides, based on the dry weight of the nutritional composition.

The present invention can also be expressed as a nutritional composition comprising at least one non-replicating lactic acid producing bacterial strain selected from Bifidobacterium breve and Streptococcus thermophilus and non-digestible oligosaccharides, wherein the nutritional composition comprises 2.5 to 15% by weight of non-digestible oligosaccharides, based on the dry weight of the composition, for increasing IgA secretion in human subjects from 0 to 36 months of age.

In another aspect, the present invention relates to a method for improving the mucosal immune defense of a human subject aged 0 to 36 months, which comprises administering to the human subject comprising at least one species selected from Bifidobacterium breve and Streptococcus thermophilus. A nutritional composition comprising a non-replicating lactic acid producing bacterium strain and comprising non-digestible oligosaccharides having 2.5 to 15% by weight of non-digestible oligosaccharides based on the dry weight of the nutritional composition.

In one embodiment, the method of the invention for improving mucosal immune defenses in a human subject between 0 and 36 months of age is a non-therapeutic method.

The present invention may also be expressed in the preparation of at least one non-replicating lactic acid producing bacterium strain selected from Bifidobacterium breve and Streptococcus thermophilus and non-digestible oligosaccharides for improving the mucous membranes of human subjects aged 0 to 36 months Use in a nutritional composition for immune defense, wherein the nutritional composition comprises 2.5 to 15% by weight of non-digestible oligosaccharides based on the dry weight of the nutritional composition.

The present invention can also be expressed as a nutritional composition comprising at least one non-replicating lactic acid producing bacterial strain selected from Bifidobacterium breve and Streptococcus thermophilus and non-digestible oligosaccharides, wherein the nutritional composition comprises 2.5 to 15% by weight of non-digestible oligosaccharides, based on the dry weight of the composition, for improving the mucosal immune defense of a human subject aged 0 to 36 months.

Lactobacillus

The nutritional composition in the method or use of the invention - hereinafter also referred to as nutritional composition of the invention - comprises a lactic acid producing bacterium selected from Bifidobacterium breve and Streptococcus thermophilus.

Preferably, the composition comprises a strain of S. thermophilus. Preferably, Streptococcus thermophilus exerts beta-galactosidase activity during fermentation of the substrate. Selection of suitable S. thermophilus strains is described in Example 2 of EP 778885 and Example 1 of FR 2723960. The nutritional composition comprises less than 10 ⁶ cfu Streptococcus thermophilus bacteria/g dry weight, preferably the nutritional composition comprises less than 10 ⁵ cfu Streptococcus thermophilus/g dry weight, even more preferably less than 10 ³ cfu Streptococcus thermophilus/g dry weight. In one embodiment, the nutritional composition comprises 0-10 ⁴ cfu Streptococcus thermophilus/g dry weight, preferably 0-10 ³ cfu/g dry weight of the composition.

The nutritional composition comprises non-replicating S. thermophilus in an amount of at least 10 ⁷ cfu S. thermophilus bacteria/g dry weight, preferably greater than 10 ⁸ cfu/g dry weight, even more preferably greater than 10 ⁹ cfu/g dry weight . Preferably, the nutritional composition comprises less than ¹⁰¹⁴ cfu non-replicating Streptococcus thermophilus/g dry weight, even more preferably less than ¹⁰¹² cfu/g dry weight of the nutritional composition.

Preferred strains of Streptococcus thermophilus for use in the present invention have been deposited by the 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 August 23, 1995 Deposited with accession number I-1620 and deposited on August 25, 1994 with accession number I-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, it is commercially available from lactic acid bacteria producers, but it can also be isolated, identified, characterized and produced directly from feces. According to a preferred embodiment, the composition of the invention contains at least one Bifidobacterium breve selected from the group consisting of Bifidobacterium breve Bb-03 (Rhodia/Danisco), Bifidobacterium breve M-16V (Morinaga), Bifidobacterium breve Bifidobacterium breve R0070 (Institute Rosell, Lallemand), Bifidobacterium breve BR03 (Probiotical), Bifidobacterium breve BR92 (Cell Biotech) DSM 20091, LMG 11613 and Bifidobacterium breve I-2219 deposited at CNCM, Paris, France. Most preferably, the inactive Bifidobacterium breve is inactive Bifidobacterium breve M-16V (Morinaga) or Bifidobacterium breve I-2219, even more preferably Bifidobacterium breve I-2219.

The nutritional composition comprises less than 106 ^cfu B. breve bacteria/g dry weight, preferably the nutritional composition comprises less than ¹⁰⁵ cfu B. breve/g dry weight, even more preferably less than ¹⁰³ cfu B. breve/g dry weight. In one embodiment, the nutritional composition comprises 0-10 ⁴ cfu Bifidobacterium breve/g dry weight, preferably 0-10 ³ cfu Bifidobacterium breve/g dry weight of the nutritional composition.

The nutritional composition comprises non-replicating Bifidobacterium breve in an amount of at least 10 ⁷ cfu/g dry weight, preferably greater than 10 ⁸ cfu/g dry weight, even more preferably greater than 10 ⁹ cfu/g dry weight. Preferably, the nutritional composition comprises less than ¹⁰¹³ cfu non-replicating Bifidobacterium breve/g dry weight, even more preferably less than ¹⁰¹¹ cfu/g dry weight of the nutritional composition.

Preferably, the nutritional composition of the invention comprises less than 10 ⁶ cfu total of S. thermophilus and B. breve/g dry weight, preferably the nutritional composition comprises less than 10 ⁵ cfu total of S. thermophilus and B. breve bacilli/g dry weight, even more preferably less than 10 ³ cfu/g dry weight of the nutritional composition. In one embodiment, the nutritional composition comprises a total of 0-10 ⁴ cfu of Streptococcus thermophilus and Bifidobacterium breve/g dry weight, preferably 0-10 ³ cfu/g dry weight of the nutritional composition.

The equivalent of cfu may suitably be determined by performing a 5' nuclease assay on a composition comprising inactive Bifidobacterium breve, such as infant formula, using a Bifidobacterium breve probe and primers as disclosed in WO 2005/039319 , and compared with a calibration curve obtained from a comparable product, such as a standard infant formula to which a known amount of dried active Bifidobacterium breve cfu has been added. Dried live bifidobacteria are commercially available as described above.

Most preferably, the nutritional composition comprises fermented ingredients fermented by lactic acid bacteria comprising both Bifidobacterium breve and Streptococcus thermophilus. In one embodiment, the lactic acid bacteria fermentation is by Streptococcus thermophilus and Bifidobacterium breve. In one embodiment, the nutritional composition comprises a fermented ingredient, wherein the lactic acid bacteria are inactivated after fermentation.

The nutritional composition of the present invention comprises non-replicating lactic acid producing bacteria and/or bacterial fragments from lactic acid producing bacteria, wherein the lactic acid producing bacteria are selected from Bifidobacterium breve and Streptococcus thermophilus, and consist of more than 10 ⁷ cfu/g, more Preferably 10 ⁸ cfu/g, even more preferably 10 ⁹ cfu/g or equivalents thereof are obtained, based on the dry weight of the final composition. Preferably, non-replicating lactic acid producing bacteria and/or bacterial fragments from lactic acid producing bacteria - wherein the lactic acid producing bacteria are selected from Bifidobacterium breve and Streptococcus thermophilus - consist of less than 1 x ¹⁰¹⁴ cfu/g, more preferably 1×10 ¹³ cfu/g, even more preferably 1×10 ¹² cfu/g or equivalents thereof are obtained, based on the dry weight of the final composition.

Preferably, the nutritional composition of the invention is not fermented by Lactobacillus bulgaricus. The products of L. bulgaricus fermentation are considered unsuitable for infants because in young infants the specific dehydrogenase that converts D-lactate to pyruvate is much less active than the dehydrogenase that converts L-lactate. Hydrogenase.

Inactivation method of Bifidobacterium breve and/or Streptococcus thermophilus

The living cells of the lactic acid producing bacteria Bifidobacterium breve and/or Streptococcus thermophilus in the nutritional composition preferably become substantially all non-replicating after fermentation. S. thermophilus and/or B. breve cells can be made inviable by methods known in the art, including heat treatment steps (including sterilization, pasteurization, UHT treatment), irradiation (UV), treatment with oxygen , treatment with bactericides such as ethanol, sonication, application of ultrahigh pressure, high pressure homogenization and use of a cell disruptor, preferably by heat treatment.

Preferably, the Streptococcus thermophilus and/or the Bifidobacterium breve are heat-treated after the fermentation step. Preferred methods to render S. thermophilus and/or B. breve non-replicating are (flash) pasteurization, sterilization, ultra high temperature treatment, high temperature/short heat treatment and/or in non-viable lactic acid producing bacteria. Spray dry at low temperature. Cell debris is preferably obtained by heat treatment.

By this heat treatment, preferably at least 95%, more preferably at least 97.5%, even more preferably at least 99% of the Bifidobacterium breve and/or Streptococcus thermophilus become non-replicating. The heat treatment is preferably performed at a temperature of 70 to 180°C, preferably 80 to 150°C for about 3 minutes to 2 hours, preferably at a temperature of 80-140°C for 5 minutes to 40 minutes.

Inactivation of lactic acid producing bacteria advantageously results in less post-acidification and a safer product. This is particularly advantageous when administering the nutritional composition to infants or young children. Suitably after fermentation, the composition may be pasteurized or sterilized and eg spray dried or lyophilized to provide a form suitable for formulation in the final product.

Non-replicating lactic acid bacteria do not form colonies by conventional plating methods. The conventional plating method is summarized in Microbiology book: James Monroe Jay, Martin J. Loessner, David A. Golden. 2005. Modern food microbiology. 7th edition, Springer Science, New York, N.Y. p. 790. Typically, the lack of replicating cells can be manifested as follows: after inoculating different concentrations of bacterial preparations (“non-replicating” samples) and incubating them under appropriate conditions (aerobic and/or anaerobic atmosphere; appropriate temperature and growth culture medium, and an appropriate incubation time, at least 24 hours), there were no visible colonies on the agar plate or increased turbidity in the liquid growth medium.

Non-replicating bacteria are also sometimes referred to as non-viable bacteria, non-culturable bacteria, or non-growing bacteria. Non-replicating bacteria contain metabolically active cells, inactivated or dead cells or bacterial cell debris.

fermented ingredients

The nutritional composition of the present invention preferably comprises a fermented ingredient. Preferably, the fermented component is a component fermented by lactic acid producing bacteria, preferably Streptococcus thermophilus and/or Bifidobacterium breve. Fermentation is preferably carried out during the preparation of the nutritional composition. Preferably, the fermented ingredient is a milk-derived product which is a milk substrate fermented by lactic acid producing bacteria and which comprises at least one milk substrate selected from the group consisting of: milk, whey, whey protein, milk Albumin hydrolyzate, casein, casein hydrolyzate and lactose, or mixtures thereof. Suitably nutritional compositions comprising fermented ingredients and non-digestible oligosaccharides and methods for their preparation are described in WO 2009/151330, WO 2009/151331 and WO 2013/187764.

The fermentation components preferably comprise bacterial cell debris, such as glycoproteins, glycolipids, peptidoglycan, lipoteichoic acid (LTA), lipoproteins, nucleotides and/or capsular polysaccharides. It is advantageous to use fermented ingredients containing inactivated bacteria and/or cell debris directly as part of the final nutritional product, as this will result in a higher concentration of bacterial cell debris. When commercial preparations are used, these preparations are typically washed and the material is separated from the aqueous growth medium containing bacterial cell debris, thereby reducing or eliminating the presence of bacterial cell debris. Furthermore, upon fermentative and/or other interactions of the lactic acid producing bacteria with the milk substrate, additional bioactive compounds are formed, such as bioactive peptides and/or oligosaccharides, which likewise stimulate the mucosal immune defense system. Thus, fermented ingredients, especially fermented milk-derived products, are believed to have an improved effect compared to non-fermented milk-derived products.

Preferably, the nutritional composition comprises 5 to 97.5% by weight, more preferably 10 to 95% by weight, more preferably 20 to 90% by weight, even more preferably 25 to 60% by weight fermented ingredients on a dry weight basis. As a means of expressing the degree of fermentation, the total level of lactic acid and lactate in the nutritional composition can be used, since this is the metabolic end product produced by the lactic acid producing bacteria during fermentation. The nutritional composition of the invention comprises a total of 0.02 to 1.5% by weight, more preferably 0.05 to 1.0% by weight, even more preferably 0.05 to 0.5% by weight of lactic acid and lactate, based on the dry weight of the composition. Preferably, in total at least 50% by weight, even more preferably at least 90% by weight, of the 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 greater than 50% by weight, more preferably greater than 90% by weight, based on the sum of lactic acid and lactate. Herein, L(+)-lactate and L(+)-lactic acid are also referred to as L-lactate and L-lactic acid.

fermentation method

The milk substrate to be fermented is suitably present in an aqueous medium. The milk substrate to be fermented is preferably selected from milk, whey, whey protein, whey protein hydrolyzate, casein, casein hydrolyzate and lactose and mixtures thereof. The milk may be whole milk, semi-skimmed milk and/or skimmed milk. Preferably, the milk substrate to be fermented comprises skim milk. The whey may be sweet whey and/or acid whey. Preferably, the whey is present at a concentration of 3 to 80 g dry weight/L of aqueous medium containing the milk substrate, more preferably 40 to 60 g/L. Preferably, the whey protein hydrolyzate is present at 2 to 80 g dry weight/L of aqueous medium containing the milk substrate, more preferably 5 to 15 g/L. Preferably lactose is present at 5 to 50 g dry weight/L aqueous substrate, more preferably 1 to 30 g/L. Preferably, the aqueous medium containing the milk substrate contains buffer salts to maintain the pH in the desired range. Sodium dihydrogen phosphate or potassium dihydrogen phosphate is preferably used as buffer salt, preferably at 0.5 to 5 g/L, more preferably at 1.5 to 3 g/L. Preferably, the aqueous medium containing the milk substrate comprises cysteine in an amount of 0.1 to 0.5 g/L aqueous substrate, more preferably 0.2 to 0.4 g/L. The presence of cysteine results in a substrate with a low redox potential, which favors the activity of lactic acid producing bacteria, especially bifidobacteria. Preferably, the milk substrate-containing aqueous medium comprises yeast extract in an amount of 0.5 to 5 g/L of the milk substrate-containing aqueous medium, more preferably 1.5 to 3 g/L. Yeast extract is a rich source of enzyme cofactors and growth factors for lactic acid producing bacteria. The presence of yeast extract will enhance the fermentation of the lactic acid producing bacteria.

Suitably, the milk substrate, in particular the aqueous medium containing the milk substrate, is pasteurized prior to the fermentation step in order to eliminate the presence of unwanted live bacteria. Suitably, the product is pasteurized after fermentation to inactivate the enzymes. Suitably, enzyme inactivation is performed at 75°C for 3 minutes. Suitably, the aqueous medium containing the milk substrate is homogenized prior to fermentation and/or the milk-derived product is homogenized after fermentation. Homogenization results in a more stable substrate and/or fermentation product, especially in the presence of fat.

The inoculation density is preferably 1×10 ² to 5×10 ¹⁰ , preferably 1×10 ⁴ to 5×10 ⁹ cfu Streptococcus thermophilus and/or Bifidobacterium breve/ml aqueous medium containing the milk substrate, more preferably 1 x 10 ⁷ to 1 x 10 ⁹ cfu/ml aqueous medium containing milk substrate. The final bacterial density after fermentation is preferably 1×10 ³ to 1×10 ¹⁰ , more preferably 1×10 ⁴ to 1×10 ⁹ cfu/ml of the aqueous medium containing the milk substrate.

Fermentation is preferably carried out at a temperature of about 20°C to 50°C, more preferably 30°C to 45°C, even more preferably about 37°C to 42°C. Optimal temperature for growth and/or activity of Streptococcus thermophilus and/or Bifidobacterium breve.

Incubation is preferably performed at a pH of 4 to 8, more preferably 6 to 7.5. The pH does not cause protein precipitation and/or bad taste, while lactic acid producing bacteria such as Bifidobacterium breve and/or Streptococcus thermophilus are able to ferment the milk substrate.

The incubation time is preferably 10 minutes to 48 hours, preferably 2 hours to 24 hours, more preferably 4 hours to 12 hours. A time long enough to allow fermentation to a large extent with simultaneous production of immunogenic cellular debris such as glycoproteins, glycolipids, peptidoglycan, lipoteichoic acid (LTA), flagella, lipoproteins, DNA and/or pods Membrane polysaccharides, however for economical reasons the incubation time need not be too long.

Preferably, the milk substrate, preferably skim milk, is pasteurized, cooled and fermented with one or more strains of Streptococcus to a degree of acidity at which the fermented product is cooled and stored. Preferably, the second milk-derived product is prepared in a similar manner using one or more Bifidobacterium species used for fermentation instead. Subsequently, the two fermented products are preferably mixed together and mixed with the components other than the fat component that make up the infant formula. Preferably, the mixture is preheated and then the fat is added in-line, homogenized, pasteurized and dried.

Methods of preparing suitable fermented ingredients are known per se. EP 778885 - incorporated herein by reference - specifically discloses in Example 7 a suitable method for preparing a fermentation component. FR 2723960 - incorporated herein by reference - specifically discloses in Example 6 a suitable method for preparing a fermentation component. Briefly, milk-derived products, preferably pasteurized The milk-derived substrate is concentrated to eg 15 to 50% dry matter and then inoculated with Streptococcus thermophilus eg with a ⁵ % culture containing 106 to ¹⁰¹⁰ bacteria/ml. Preferably, the milk-derived product comprises milk protein peptides. The temperature and duration of fermentation are as described above. Suitably, following fermentation, the fermented protein-containing composition may be pasteurized or sterilized and eg spray-dried or lyophilized to provide a form suitable for formulation in the final product. .

A preferred method of preparing a fermentation product is disclosed in WO 01/01785, more particularly in Examples 1 and 2. A preferred method of preparing a fermentation product is described in WO 2004/093899, more particularly in Example 1.

indigestible oligosaccharides

The composition of the invention comprises non-digestible oligosaccharides, and preferably at least two non-digestible oligosaccharides, in particular two non-digestible oligosaccharides of different origin. The presence of non-digestible oligosaccharides stimulates the secretion of sIgA. The presence of non-digestible oligosaccharides stimulates the mucosal immune defense system. Thus, the simultaneous presence of non-digestible oligosaccharides and non-replicating lactic acid producing bacteria selected from Bifidobacterium breve and/or Streptococcus thermophilus act synergistically and advantageously lead to higher IgA secretion in the gut. Thus, the simultaneous presence of non-digestible oligosaccharides and non-replicating lactic acid producing bacteria selected from Bifidobacterium breve and/or Streptococcus thermophilus acts synergistically and advantageously leads to an increased mucosal immune defense.

The term "oligosaccharide" as used herein refers to saccharides having a degree of polymerization (DP) of 2 to 250, preferably a DP of 2 to 100, more preferably 2 to 60, even more preferably 2 to 10. If the nutritional composition of the invention comprises oligosaccharides with a DP of 2 to 100, this leads to compositions which may contain oligosaccharides with a DP of 2 to 5, 50 to 70 and 7 to 60. The term "non-digestible oligosaccharide" as used in the present invention means that it is not digested in the intestinal tract by the action of acids or digestive enzymes present in the human upper digestive tract (such as the small intestine and stomach), but preferably by the human gut microbes Group fermented oligosaccharides. For example, sucrose, lactose, maltose, and maltodextrin are considered digestible.

Preferably, the non-digestible oligosaccharides of the invention are soluble. The term "soluble" as used herein, when referring to polysaccharides, fibers or oligosaccharides, means according to L. Prosky et al., J. Assoc. Off. Anal. Chem. method, the substance is at least soluble.

In the method or use of the present invention, the non-digestible oligosaccharides contained in the nutritional composition of the present invention preferably comprise a mixture of non-digestible oligosaccharides. The non-digestible oligosaccharides are preferably selected from the group consisting of fructo-oligosaccharides, such as inulin; non-digestible dextrins; galacto-oligosaccharides, such as trans-galacto-oligosaccharides; xylo-oligosaccharides, arabino-oligosaccharides, arabino-oligosaccharides Galactose (arabinogalacto-oligosaccharide), gluco-oligosaccharide (gluco-oligosaccharide), gentio-oligosaccharide (gentio-oligosaccharide), glucomanno-oligosaccharide (glucomanno-oligosaccharide), galactomannooligosaccharide (galactomannooligosaccharide), mannan oligosaccharide , isomaltooligosaccharide, nigero-oligosaccharide, glucomanno-oligosaccharide, chito-oligosaccharide, soybean oligosaccharide, uronic acid oligosaccharide ( uronic acidoligosaccharide); sialyloligosaccharides, such as 3-sialyllactose (3-SL), 6-sialyllactose (6-SL), sialyllactosetetraose a, b, c (LST), di Sialyllactose N tetrasaccharide (DSLNT), sialyl-lactoNhexaose (S-LNH), DS-LNH; and oligofucose, such as (un)sulfated fucoidan oligosaccharides (fucoidan oligosaccharide), 2'-fucosyllactose (2'-FL), 3-fucosyllactose (3-FL), difucosyllactose, lactose-N-fucopentose (lacto -N-fucopenatose) (LNFP) I, II, III, V, lactose-N-neofucopenaose (Lacto-N-neofucopenaose) (LNnFP), lactose-N-difucosyl-hexaose (Lacto - N-difucosyl-hexaose) (LNDH) and mixtures thereof, even more preferably selected from fructo-oligosaccharides, such as inulin; galacto-oligosaccharides, such as trans-galacto-oligosaccharides; and fucooligosaccharides and mixtures thereof, even More preferred are trans-galacto-oligosaccharides and/or inulin, most preferred are trans-galacto-oligosaccharides. In one embodiment of the composition or method of the present invention, the non-digestible oligosaccharide is selected from trans-galacto-oligosaccharides, fructo-oligosaccharides and mixtures thereof.

The non-digestible oligosaccharides are preferably selected from β-galacto-oligosaccharides, α-galacto-oligosaccharides and galactan. According to a more preferred embodiment, the non-digestible oligosaccharides are β-galacto-oligosaccharides. Preferably, the non-digestible oligosaccharides comprise galacto-oligosaccharides having β(1,4), β(1,3) and/or β(1,6) glycosidic linkages and terminal glucose. Trans-galacto-oligosaccharides, for example, can be traded under the trade name GOS (Domo FrieslandCampina Ingredients), Bi2muno (Clasado), Cup-oligo (Nissin Sugar) and Oligomate55 (Yakult) were obtained. These oligosaccharides increased sIgA levels to a greater extent and enhanced mucosal immune defense activity.

Non-digestible oligosaccharides preferably include fructo-oligosaccharides. In other cases, fructo-oligosaccharides may have, for example, fructopolysaccharides, oligofructose, polyfructose, polyfructan, inulin, levan and The name of fructan and may refer to an oligosaccharide comprising β-linked fructose units, preferably linked by β(2,1) and/or β(2,6) glycosidic bonds and with a preferred DP of 2 to 200. Preferably, the fructo-oligosaccharides contain glucose linked by terminal β(2,1) glycosidic linkages. Preferably, the fructo-oligosaccharide contains at least 7 β-linked fructose units. In another preferred embodiment inulin is used. Inulin is a fructo-oligosaccharide in which at least 75% of the glycosidic linkages are β(2,1) linkages. Typically, inulin has an average chain length of 8 to 60 monosaccharide units. The fructo-oligosaccharides suitable for use in the compositions of the present invention may be available under the trade name Commercially available from HP (Orafti). Other suitable sources are Raftilose (Orafti), Fibrulose and Fibruline (Cosucra) and Frutafit and Frutalose (Sensus).

Preferably, the nutritional composition of the invention comprises a mixture of galacto-oligosaccharides and fructo-oligosaccharides. Preferably, the mixture of galactooligosaccharide and fructooligosaccharide is 1/99 to 99/1, more preferably 1/19 to 19/1, more preferably 1/1 to 19/1, more preferably 2/1 to 15/1 1. More preferably present in a weight ratio of 5/1 to 12/1, even more preferably 8/1 to 10/1, even more preferably about 9/1. This weight ratio is particularly advantageous when the galacto-oligosaccharides have a low average DP and the fructo-oligosaccharides have a relatively high DP. Most preferred are mixtures of galacto-oligosaccharides having an average DP below 10, preferably below 6, and fructo-oligosaccharides having an average DP above 7, preferably above 11, even more preferably above 20. This mixture synergistically increases sIgA levels and enhances mucosal immune defense activity.

Preferably, the nutritional composition of the invention comprises a mixture of short-chain fructo-oligosaccharides and long-chain fructo-oligosaccharides. Preferably, the mixture of short-chain fructo-oligosaccharides and long-chain fructo-oligosaccharides has a ratio of 1/99 to 99/1, more preferably 1/19 to 19/1, even more preferably 1/10 to 19/1, more preferably 1/1 A weight ratio of 5 to 15/1, more preferably 1/1 to 10/1 is present. Preference is given to mixtures of short-chain fructo-oligosaccharides having an average DP below 10, preferably below 6, and fructo-oligosaccharides having an average DP above 7, preferably above 11, even more preferably above 20.

The nutritional composition of the present invention comprises a total of 2.5 to 20% by weight, more preferably 2.5 to 15% by weight, even more preferably 3.0 to 10% by weight, most preferably 5.0 to 7.5% by weight, based on the dry weight of the nutritional composition. Recalculate. Based on 100 ml, the nutritional composition of the invention preferably comprises a total of 0.35 to 2.5 wt.-%, more preferably 0.35 to 2.0 wt.-%, even more preferably 0.4 to 1.5 wt.-%, based on 100 ml of the nutritional composition, of non-digestible oligosaccharides. Lower amounts of non-digestible oligosaccharides are less effective in stimulating sIgA formation or mucosal immune defenses, while too high amounts cause side effects of bloating and abdominal discomfort.

nutritional composition

The nutritional composition used according to the invention may also be considered a pharmaceutical composition, preferably suitable for administration to infants. The nutritional composition of the present invention is preferably administered enterally, more preferably orally.

The nutritional composition of the invention is preferably an infant formula, a follow on formula or a growing up milk. The nutritional composition according to the invention can advantageously be used as complete nutrition for infants. Preferably, the nutritional composition of the invention is an infant formula. Infant formula is defined as a formula for infants and may for example be a starting formula for infants aged 0 to 6 or 0 to 4 months. The second phase formula is used for infants from 4 or 6 months of age to 12 months of age. Growing up milk is for children between 12 and 36 months of age. The compositions of the invention preferably comprise a lipid component, a protein component and a carbohydrate component, and are preferably administered in liquid form. The nutritional composition of the invention may also be in the form of a dry food, preferably a powder, with instructions for mixing said dry food, preferably powder, with a suitable liquid, preferably water. The nutritional composition used according to the invention preferably comprises further ingredients, such as vitamins, minerals, trace elements and other micronutrients, so that it is a complete nutritional composition. According to international directives, preferred infant formulas contain vitamins, minerals, trace elements and other micronutrients.

The nutritional composition of the present invention preferably comprises lipids, proteins and digestible carbohydrates, wherein the lipids provide 5 to 50% of the total calories, the proteins provide 5 to 50% of the total calories, and the digestible carbohydrates provide 15% of the total calories to 90%. Preferably, in the nutritional composition of the invention lipids provide 35 to 50% of the total calories, proteins provide 7.5 to 12.5% of the total calories and digestible carbohydrates provide 40 to 55% of the total calories. To calculate protein as a percentage of total calories, the total energy provided by proteins, peptides, and amino acids needs to be considered. Preferably, 3 to 7g lipid/100kcal nutritional composition is provided, preferably 4 to 6g/100kcal nutritional composition; 1.6 to 4g protein/100kcal nutritional composition is provided, preferably 1.75 to 2.5g/100kcal nutritional composition, and 5 Up to 20g digestible carbohydrates/100kcal nutritional composition, preferably 8 to 15g/100kcal nutritional composition. Preferably, the nutritional composition of the present invention comprises 4 to 6 g of lipids/100 kcal, 1.6 to 1.9 g of protein/100 kcal, more preferably 1.75 to 1.85 g of protein/100 kcal, and 8 to 15 g of digestible carbohydrates/100 kcal of the nutritional composition . In one embodiment, the nutritional composition provides 3 to 7 g of lipid per 100 kcal, preferably 4 to 6 g of lipid per 100 kcal of the nutritional composition; 1.6 to 2.1 g of protein per 100 kcal of the nutritional composition, preferably per 100 kcal of the nutritional composition 1.6 to 2.0 g protein; and provides 5 to 20 g digestible carbohydrates per 100 kcal nutritional composition, preferably 8 to 15 g digestible carbohydrates per 100 kcal nutritional composition, and wherein preferably the digestible carbohydrate component comprises at least 60 wt. % lactose, based on total digestible carbohydrates, more preferably at least 75% by weight, even more preferably at least 90% by weight lactose, based on total digestible carbohydrates. The total amount of calories is determined by the sum of calories from protein, lipids, digestible carbohydrates and indigestible oligosaccharides.

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

The nutritional composition of the present invention preferably comprises at least one lipid selected from animal lipids (excluding human lipids) and vegetable lipids. Preferably, the composition of the invention comprises a combination of vegetable lipids and at least one oil selected from fish oils, animal oils, algal oils, fungal oils and bacterial oils. The nutritional composition of the present invention preferably provides 3 to 7 g lipid/100 kcal nutritional composition, preferably provides 4 to 6 g lipid/100 kcal nutritional composition. When in liquid form, eg as a ready-to-eat liquid, the nutritional composition preferably comprises 2.1 to 6.5 g lipid/100ml, more preferably 3.0 to 4.0 g/100ml. Based on dry weight, the nutritional composition of the invention preferably comprises 12.5 to 40% by weight, more preferably 19 to 30% by weight of lipids. Preferably, the lipid comprises the essential fatty acids alpha-linolenic acid (ALA), linoleic acid (LA) and/or long chain polyunsaturated fatty acids (LC-PUFA). LC-PUFA, LA and/or ALA may be provided as free fatty acids, in triglyceride form, diglyceride form, monoglyceride form, phospholipid form or as a mixture of one or more of the above. Preferably, the nutritional composition of the present invention comprises at least one, preferably at least two, lipid sources selected from the group consisting of rapeseed oil (such as colza oil, canola oil and canola oil), high Oleic sunflower oil, high oleic safflower oil, olive oil, marine oil, microbial oil, coconut oil, palm kernel oil, and milk fat. The nutritional composition of the present invention is not human milk.

The nutritional composition of the present invention preferably comprises protein. The protein used in the nutritional composition is preferably selected from non-human animal proteins, preferably milk proteins; vegetable proteins, such as preferably soy protein and/or rice protein; and mixtures thereof. The nutritional composition of the present invention preferably contains casein and/or whey protein, more preferably bovine whey protein and/or bovine casein. Thus, in one embodiment, the protein in the nutritional composition of the invention comprises a protein selected from whey protein and casein, preferably whey protein and casein, preferably whey protein and/or casein is from bovine milk. Preferably, the protein comprises less than 5% by weight of free amino acids, dipeptides, tripeptides or hydrolyzed proteins, based on total protein. The nutritional composition of the present invention preferably comprises casein and whey protein in a casein:whey protein weight ratio of 10:90 to 90:10, more preferably 20:80 to 80:20, even more preferably 35:65 to 55:45.

According to the Kjeldahl method, by measuring total nitrogen and using a conversion factor of 6.38 in the case of casein or 6.25 for other proteins than casein, based on the dry weight of the nutritional composition of the invention , to calculate the weight % of protein. The term "protein" or "protein component" as used in the present invention refers to the sum of proteins, peptides and free amino acids.

The nutritional composition of the invention preferably comprises 1.6 to 4.0 g protein per 100 kcal nutritional composition, preferably providing 1.6 to 3.5 g, even more preferably 1.75 to 2.5 g protein per 100 kcal nutritional composition. In one embodiment, the nutritional composition of the invention comprises 1.6 to 2.1 g protein per 100 kcal nutritional composition, preferably 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 nutritional composition of protein. In one embodiment, the nutritional composition of the invention comprises protein in an amount of less than 2.0 g/100 kcal, preferably 1.6 to 1.9 g, even more preferably 1.75 to 1.85 g/100 kcal nutritional composition. Protein levels that are too low on a total calorie basis will result in inadequate growth and development in infants and young children. When in liquid form, e.g. as a ready-to-eat liquid, the nutritional composition preferably comprises 0.5 to 6.0 g protein/100 ml, more preferably 1.0 to 3.0 g protein/100 ml, even more preferably 1.0 to 1.5 g protein/100 ml, most preferably 1.0 to 1.3 g Protein/100ml. Based on dry weight, the nutritional composition of the present invention preferably comprises 5 to 20% by weight of protein, preferably at least 8% by weight of protein, based on dry weight of the total nutritional composition, more preferably 8 to 14% by weight of protein, even More preferably 8 to 9.5% by weight 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/100ml of liquid. For infants, the nutritional composition more preferably has 60 to 90 kcal/100ml liquid, even more preferably 65 to 75 kcal/100ml liquid. This calorie density ensures an optimal ratio between water and calorie consumption. For young children - human subjects aged 12 to 36 months, the nutritional composition more preferably has a caloric density of 45 to 65, even more preferably 50 to 60 kcal/100ml. The osmolarity of the composition of the present invention is preferably 150 to 420 mOsmol/L, more preferably 260 to 320 mOsmol/L. The low osmolarity is designed to further reduce gastrointestinal stress.

When the nutritional composition is in liquid form, the preferred volume for daily administration is about 80 to 2500 ml per day, more preferably about 200 to 1200 ml. Preferably, the number of feedings per day is 1 to 10, preferably 3 to 8. In one embodiment, the nutritional composition is administered daily in liquid form for at least 2 days, preferably at least 4 weeks, preferably at least 8 weeks, more preferably at least 12 weeks, wherein the total daily volume administered is from 200ml to 1200ml, and Wherein the number of daily feedings ranged from 1 to 10.

When in liquid form, the nutritional composition of the invention preferably has a viscosity of 1 to 60 mPa.s, preferably 1 to 20 mPa.s, more preferably 1 to 10 mPa.s, most preferably 1 to 6 mPa.s. The low viscosity ensures proper fluid delivery, eg, fits through the entire nipple. The viscosity is also very similar to that of human milk. In addition, the low viscosity results in normal gastric emptying and better energy intake, which is necessary for infants who need energy for optimal growth and development. The compositions of the invention are preferably prepared by mixing a powdered composition with water. Typically, infant formula is prepared in this way. Therefore, the present invention also relates to a packaged powder composition, wherein said package provides instructions for mixing the powder with a suitable amount of liquid to obtain a liquid composition having a viscosity of 1 to 60 mPa.s. The viscosity of the liquids was determined using a Physica Rheometer MCR 300 (Physica Messtechnik GmbH, Ostfilden, Germany) at 20° C. with a shear rate of 95 s ⁻¹ .

application

In the context of the present invention, "prevention" of a disease or a condition also means "reducing the risk" of developing a disease or a condition, and also means being at risk of developing said disease or said condition human treatment".

The inventors of the present invention found that the level of sIgA increases synergistically after consumption of the nutritional composition of the present invention. This suggests an improvement in the mucosal immune defense system.

Compared to the administration of a nutritional composition not comprising a combination of non-replicating lactic acid producing bacteria selected from Bifidobacterium breve and/or Streptococcus thermophilus and non-digestible oligosaccharides, the effects described herein, namely an increase in sIgA and /or improved mucosal immune system. These effects observed were also found to be closer to the levels observed in human milk-fed infants than standard infant formula-fed infants.

Surprisingly, these synergistic effects cannot be explained by a direct interaction between the non-digestible oligosaccharides in the composition and the lactic acid-producing bacteria, due to the inactivation of the bacteria; nor by increased intestinal microbial activity, due to the interaction with the intestinal pH has no apparent correlation.

In one embodiment, the nutritional composition of the invention is used to increase IgA secretion in a human subject from 0 to 36 months of age. In one embodiment, the nutritional composition of the present invention is used to increase IgA secretion in human subjects from 0 to 18 months of age, even more preferably in infants under 12 months of age, even more preferably in infants from 0 to 6 months of age. In one embodiment, the nutritional composition of the present invention is used to increase IgA secretion in young children between 12 and 36 months of age, most preferably between 18 and 30 or up to 24 months of age. Preferably, the nutritional composition of the invention is also used to provide nutrition to said human subject. The nutritional composition of the present invention is preferably administered enterally, more preferably orally.

In one embodiment, the nutritional composition of the present invention is used to improve the mucosal immune defense of a human subject aged 0 to 36 months. In one embodiment, the nutritional composition of the invention is used to improve the mucosal immune defense of a human subject from 0 to 18 months of age, even more preferably an infant under 12 months of age, even more preferably an infant from 0 to 6 months of age . In one embodiment, the nutritional composition of the present invention is used to improve the mucosal immune defense of young children between 12 and 36 months of age, most preferably between 18 and 30 or up to 24 months of age. Preferably, the nutritional composition of the invention is also used to provide nutrition to said human subject. The nutritional composition of the present invention is preferably administered enterally, more preferably orally.

In a preferred embodiment, the method or use of the invention is for vaginally delivered infants.

In a preferred embodiment, the method or use of the invention is for term infants, preferably healthy term infants.

Herein and in the claims, the verb "comprise" and its conjugations are used in their non-limiting sense to mean that items that follow the word are included, but items not specifically mentioned are not excluded. Also, an element introduced by the indefinite article "a" or "an" does not exclude the presence of more than one element, unless the context clearly requires that one and only one element be present. Thus, the indefinite article "a" or "an" usually means "at least one". % by weight means percent by weight.

Example

Example 1: Synergistic effect of an infant formula comprising non-replicating lactic acid producing bacteria selected from Bifidobacterium breve and Streptococcus thermophilus and non-digestible oligosaccharides on secretory IgA levels in infants

In a randomized, double-blind, controlled, parallel-group, prospective, multicenter, multinational, interventional study, subjects were randomized equally into four treatment groups. In addition, infants who had been exclusively breastfed (never received any infant formula) since birth and whose mothers intended to continue exclusive breastfeeding until at least 4 months of age were included in the breastfeeding reference group. A total of 350 subjects were enrolled, of which 280 subjects were randomly given any 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 (Nutrilon 1 , marketed by Nutricia, The Netherlands) for bottle-fed infants aged 0-6 months. The formula contains non-digestible oligosaccharides (NDO) - galacto-oligosaccharides (available from FrieslandCampina Domo A mixture of GOS, average degree of polymerization lower than 6) and fructooligosaccharides (RaftilinHP from Orafti, average degree of polymerization higher than 20) in a w/w ratio of about 9:1 in an amount of about 0.8 g/100 ml. The formulation does not contain non-replicating lactic acid producing bacteria selected from Bifidobacterium breve and/or 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 infants aged 0-6 months, and is an infant formula containing strains Bifidobacterium breve CNCM 1-2219 and Streptococcus thermophilus CNCM 1- Partially fermented infant formula of 1620 (marketed by Gallia, France), said strain is heat-inactivated after the fermentation process, the bacterial fermentation metabolite being eg L-(+) lactic acid. The amount of L-lactate is higher than 0.05% by weight, based on the dry weight of the composition. NDO is not added. During the fermentation, galacto-oligosaccharides were produced by Streptococcus thermophilus in an amount of about 2% by weight based on dry weight. The equivalents of non-replicating Bifidobacterium breve and Streptococcus thermophilus were higher than 5.10 ⁷ cfu/g. The amount of replicating Bifidobacterium breve and Streptococcus thermophilus was below 1.10 ³ cfu/g.

Test Group 3: Infant Formula 3 is an experimental test formula and is a modified cow's milk-based infant formula for bottle-fed infants aged 0-6 months. The formula contained indigestible oligosaccharides (a mixture of galacto-oligosaccharides with an average degree of polymerization lower than 6 and fructo-oligosaccharides with an average degree of polymerization higher than 20 (Raftilin-HP from Orafti)) in an amount of about 0.9 g/100 ml , and containing fermented infant formula as in IF 2 of test group 2 (marketed by Gallia, France). The amount of galacto-oligosaccharides added takes into account the amount of galacto-oligosaccharides produced due to the action of Streptococcus thermophilus.

Control group: Infant formula 4 is the control formula: A modified cow's milk-based infant formula for bottle-fed infants aged 0-6 months without non-digestible oligosaccharides and free from Bifidobacterium breve and/or or Streptococcus thermophilus non-replicating lactic acid producing bacteria, and non-replicating lactic acid producing bacteria.

All four test formulations contained nucleotides and a fat mix containing long chain fatty acids. The formulas were similar in calorie content, protein content, fat mix, and had similar amounts of digestible carbohydrates. According to the International Directive 2006/141/EC for infant formula, the formula also contains vitamins, minerals, trace elements and other micronutrients.

At the end of the study, 198 randomized subjects completed the study, while 82 randomized subjects withdrew from the study prematurely. There was no statistically significant difference in the number and nature of early exits between the effective test product group and the control group. Subjects were well balanced in the study groups in terms of demographic and baseline characteristics.

At each visit, parents collected stool samples into stool containers provided by Nutricia Research. Samples were frozen at -20°C directly after parental collection and kept at this temperature until the samples were handed over to the investigator. On site, samples were stored at -80 °C for subsequent analysis. At each collection, 2 tubes must be half filled. Alternative means of transport and storage can be arranged if required due to practical issues at the study site. Fecal secretory IgA levels were measured using standard laboratory techniques. As the distribution of sIgA measurements showed potential skewness, for statistical analysis a log transformation had been performed before using ANOVA to test for differences between study groups. Statistical analysis of sIgA levels was performed based on geometric means (effect size = group difference expressed as geometric mean ratio due to logarithmic transformation).

The results are shown in Table 1. To analyze the raw data, ITT analysis was considered as the main analysis of this study. At 4 months, the period during which infants were still fully fed infant formula, the IF 3 group (experimental formula containing non-replicating lactic acid bacteria and non-digestible oligosaccharides) compared with the IF 4 control or IF 2 groups There was a statistically significant difference (p<0.05) between the fecal sIgA levels in (Calisma). There was a trend (p=0.07) when compared to the IF 1 group (Nutrilon). There were no statistically significant differences when IF 1 was compared to IF 2, IF 1 to IF 4, or when IF 2 was compared to IF 4.

When compared to a breastfed reference group, no statistics are given because the reference group was not randomized and should therefore not be used for direct comparison with a randomized study group. Statistical analysis of median values was not performed, but a similar pattern was observed.

The difference in sIgA production between test groups 3 and 4 can be expressed as an effect size based on the geometric mean, which is equal to 1.65. This could be explained by the fact that the use of the experimental formulation IF 3 at 4 months of age resulted in an increase in sIgA concentrations of about 65% when compared to the control group using IF 4 . Similarly, the effect size of Test Group 3 relative to Test Group 1 is 1.53, and the effect size of Test Group 3 relative to Test Group 2 is 2.06.

The sIgA concentrations in the group consuming IF 3 were synergistically higher than expected based on the results of the groups consuming IF 1 and IF 2 and the control IF 4, and were beneficially more similar to the levels observed in the breastfed reference group. This suggests a synergistic increasing effect on mucosal immune defenses.

Since the bacteria present in IF 3 (and IF 2) are heat-inactivated, this effect cannot be explained by an increase in the amount of intestinal lactic acid-producing bacteria derived from the formula, nor by the production of these formula-derived Increased intestinal fermentation of added non-digestible oligosaccharides by lactic acid bacteria.

Table 1: Fecal slgA Concentrations (μg/g) at Each Visit - ITT Population

*Compared to test IF 3p<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 Bifidobacterium breve and Streptococcus thermophilus and non-digestible oligosaccharides increases intestinal sIgA levels

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

Test group 1: Infant formula 1 contains 66kcal per 100ml, 1.35g protein (bovine whey protein/casein, weight ratio is 1/1), 8.2g digestible carbohydrates (of which 5.6g lactose and 2.1g maltodextrin) , 3.0g fat (mainly vegetable fat), 0.8g contains scGOS in a weight ratio of 9:1 (source GOS) and lcFOS (source ) non-digestible oligosaccharides. On a dry weight basis, approximately 50% in the infant formula is derived from Lactofidus ^™ , a commercially available infant formula sold under the trade name Gallia. Lactofidus ^™ is a fermented milk-derived composition prepared by fermentation with Streptococcus thermophilus and comprising Bifidobacterium breve. Mild heat treatment is used to inactivate the lactic acid producing bacteria. The amount of non-replicating lactic acid producing bacteria selected from Bifidobacterium breve and Streptococcus thermophilus is greater than 10 ⁷ cfu/g dry weight. The amount of Streptococcus thermophilus was about 10 ⁴ to 10 ⁵ cfu/g dry weight. The infant formula comprises about 0.55% by weight of lactic acid + lactate on a dry weight basis, of which at least 95% is L(+)-lactic acid/lactate. According to the International Directive 2006/141/EC on infant formula, the composition also contains vitamins, minerals, trace elements and other micronutrients.

Test group 2: Infant formula 2, similar to infant formula 1, but without 0.8 g of non-digestible oligosaccharides scGOS and lcFOS.

Test Group 3: Infant Formula 3, a standard infant formula containing 0.8 g of non-digestible oligosaccharides in a 9:1 weight ratio of scGOS (source GOS) and lcFOS (source ), and the remainder had a similar composition to Infant Formula 1. The formulation does not contain Bifidobacterium breve and/or Streptococcus thermophilus.

Fecal samples were collected for physiological and microbiological analysis in a manner similar to that described in Example 1 at baseline and after 17 weeks of intervention. Only samples from the subgroup of subjects with a complete set of stool samples (two visits) in which the amount of stool was sufficient for all analyses were analyzed. In addition, samples from infants receiving any systemic antibiotics at any time after birth or using thickeners added to the formula during the study were excluded.

The effect of the infant formula used was evaluated on secretory immunoglobulin A (sIgA) in a selected set of faecal samples and the results are shown in Table 2.

Also, the sIgA concentration in the group consuming the experimental infant formula 1 was higher than that in the group consuming the infant formula 2 or 3, very similar to the results in Example 1. This suggests an increased impact on mucosal immune defenses.

Interestingly, the effect on fecal sIgA levels was inconsistent with the effect on fecal pH. The pH was lowest in the group consuming infant formula 2 and intermediate in the group consuming experimental infant formula 1 . pH reflects the activity of gut microbiota. The lower fecal pH of test groups 1 and 2 was attributed to the fermentation of non-digestible oligosaccharides present in the test infant formula, with the highest fermentation in test group 2 and the highest sIgA levels in test group 1 .

Table 2: Fecal sIgA concentrations (μg/g) at each visit

^W For analysis, the Wilcoxon rank sum test (W) was used due to violation of the normality assumption and/or presence of outliers.

*For the comparisons of interest tested: IF 1 vs. IF 3, there was a significant difference between the groups (p<0.05).

^#For the tested comparison of interest: IF 1 vs. IF 2, there is a significant difference between the groups (p<0.05).

Claims (12)

1. at least one non-replicating lactic acid producing bacteria bacterial strain and indigestible widow selected from bifidobacterium breve and streptococcus thermophilus Purposes of the sugar in preparing alimentation composition, the alimentation composition are used to increase IgA points of the human experimenter at 0 to 36 monthly age It secretes, wherein the alimentation composition includes the indigestible oligosaccharides of 2.5 to the 15 weight % of dry weight meter based on alimentation composition.

2. at least one non-replicating lactic acid producing bacteria bacterial strain and indigestible widow selected from bifidobacterium breve and streptococcus thermophilus Purposes of the sugar in preparing alimentation composition, the alimentation composition are used to improve the mucous membrane of the human experimenter at 0 to 36 monthly age Immune defense, wherein the alimentation composition includes the indigestible of 2.5 to the 15 weight % of dry weight meter based on alimentation composition Oligosaccharides.

3. purposes according to any one of the preceding claims, wherein the alimentation composition includes the short bifid of non-replicating Bacillus.

4. purposes according to any one of the preceding claims, wherein the alimentation composition includes the short bifid of non-replicating Bacillus and/or streptococcus thermophilus, amount are equal at least about 10⁷The dry weight of cfu/g alimentation compositions, more preferably at least 10⁸Cfu/g is dry Weight.

5. purposes according to any one of the preceding claims, wherein bifidobacterium breve and/or streptococcus thermophilus are inactivated To less than 10⁶The dry weight of cfu/g alimentation compositions, more preferably less than 10⁵It is the dry weight of cfu/g alimentation compositions, even more preferably low In 10³The level of the dry weight of cfu/g alimentation compositions.

6. purposes according to any one of the preceding claims is infant formula, second stage formulation or growth Breast.

7. purposes according to any one of the preceding claims, wherein the indigestible oligosaccharides are selected from oligofructose, no It can peptic dextrin, galactooligosaccharide, xylo-oligosaccharide, arabinoxylan oligosaccharide, Arabic galactooligosacchari(es, glucose oligosaccharide, oligomeric rough gentian Sugar, mannan oligosaccharide, galactomannan-oligosaccharide, manna oligosacchride, oligoisomaltose, nigero-oligosaccharide, mannan oligosaccharide, shell are few Sugar, soyabean oligosaccharides, oligosaccharide, sialyloligosaccharide and oligomeric fucose and its mixture.

8. purposes according to any one of the preceding claims, wherein the indigestible oligosaccharides include galactooligosaccharide And/or oligofructose.

9. purposes according to any one of the preceding claims, wherein the alimentation composition includes by bifidobacterium breve And/or the fermented ingredient of streptococcus thermophilus fermentation, and the wherein described alimentation composition includes 0.05 to 1.5 weight %'s in total L (+)-lactic acid and L (+)-lactate.

10. purposes according to any one of the preceding claims, wherein the alimentation composition includes protein, can digest Carbohydrate and lipid, and 1.6 to 4g protein/100kcal alimentation compositions are wherein provided, providing 5 to 20g can digest Carbohydrate/100kcal alimentation compositions, and 3 to 7g lipids/100kcal alimentation compositions are provided.

11. a kind of method of the IgA secretions of human experimenter increasing by 0 to 36 monthly age comprising give human experimenter's one kind Including at least one non-replicating lactic acid producing bacteria bacterial strain selected from bifidobacterium breve and streptococcus thermophilus and include indigestible The alimentation composition of oligosaccharides, the composition are indigestible with 2.5 to the 15 weight % of dry weight meter based on alimentation composition Oligosaccharides.

12. a kind of method of the mucosa-immune defence of human experimenter improving for 0 to 36 monthly age comprising give human experimenter It is a kind of comprising at least one non-replicating lactic acid producing bacteria bacterial strain selected from bifidobacterium breve and streptococcus thermophilus and include can not Digest oligosaccharides alimentation composition, the composition with 2.5 to the 15 weight % of dry weight meter based on alimentation composition can not Digest oligosaccharides.