Classifications

• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
  • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
  • A23L33/20—Reducing nutritive value; Dietetic products with reduced nutritive value
  • A23L33/21—Addition of substantially indigestible substances, e.g. dietary fibres
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
  • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
  • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
  • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
  • A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
  • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
  • A23L33/40—Complete food formulations for specific consumer groups or specific purposes, e.g. infant formula
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
  • A61K31/702—Oligosaccharides, i.e. having three to five saccharide radicals attached to each other by glycosidic linkages
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
  • A61K35/66—Microorganisms or materials therefrom
  • A61K35/74—Bacteria
  • A61K35/741—Probiotics
  • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
  • A61K35/745—Bifidobacteria
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
  • A61P37/02—Immunomodulators
  • A61P37/04—Immunostimulants

Definitions

• the present invention relates to the field of nutrition for infants and young children for improving the immune system.
• 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.
• slgA functions in the mucosal immune defense system and intestinal homeostasis, without provoking, but even ameliorating, an inflammatory response.
• standard infant formula based on cow's milk ingredients have a much lower concentration of slgA and infants fed standard infant formula have a reduced level of slgA compared to breast fed infants.
• EP 2 318 046 discloses a combination of probiotics with secretory IgA to treat or prevent inflammation.
• EP 2 315 595 discloses the use of live probiotic bacteria to increase immunoglobulin secretion in C section infants. This document does, however, not disclose a fermented ingredient nor lactic acid.
• WO 2014/201037 discloses the use of probiotics, prebiotics, synbiotics and antibiotics to treat mammalian microbiota changes associated with antibiotic treatments and various immunological conditions. Fukushima et al.
• probiotics are live lactic acid producing bacteria such as live lactobacilli or live bifidobacteria.
• infant formula with probiotics are marketed as powders with dried probiotic cells, in which fermentation is not possible due to the circumstances being unfavourable for fermentation, e.g. a too low water activity, and no lactic acid is produced.
• 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
• Bakker-Zierikzee et al. 2006, Pediatr Allergy Immunol 17: 134-140
• Mullie et al. (2004, Ped Res 56:791-795) disclosed that consumption of fermented formula in healthy new-borns was associated with an enhanced production of intestinal slgA antibodies specific to poliovirus after vaccine administration, but no effect on the total IgA titers.
• WO 2009/151330 discloses a composition that is obtained by fermentation by lactic acid producing bacteria and non-digestible oligosaccharides for use in caesarean section infants. Aim is induce tolerance for aiding the subsequent colonization by live bacteria. IgA secretion is not mentioned.
• WO 2009/151331 discloses an immune stimulating composition comprising non-digestible oligosaccharides and a product obtained after incubating with bifidobacteria. By stimulating the Th 1 response and decreasing the Th2 response of the systemic immune system, the vaccination response is increased and allergy is decreased.
• WO 2009/151329 discloses a composit ion comprising non- digestible oligosaccharides and a product obtained by incubating with bifidobacteria, to reduce bacterial translocation and improving the intestinal barrier function. It is the aim to prevent systemic infections in this way. The document is silent on any effect on mucosal immune defense and IgA secretion. SUMMARY OF THE INVENTION
• slgA intestinal secretory IgA
• the secretion of slgA was synergisticaiiy increased and more similar to the slgA in the breast fed reference group. This is furthermore indicative for a synergistic increase in the mucosal immune defense system.
• the synergistic effect cannot be explained by a direct interaction between the non-digestible oligosaccharides and the lactic acid producing bacteria in the fermented composition, as the bacteria were inactivated, nor by an increased intestinal microbial activity as there was no clear correlation with the intestinal pH.
• the invention thus concerns a method for increasing IgA secretion in a human subject with an age of 0 to 36 months comprising administering to the human subject a nutritional composition comprising a fermented ingredient and non-digestible oligosaccharide, said nutritional composition comprising 0.02 to 1.5 wt% of the sum of lactic acid and lactate based on dry weight of the nutritional composition and comprising 2.5 to 15 wt% non- digestible oligosaccharide based on dry weight of the nutritional composition.
• the present method for increasing IgA secretion in a human subject with an age of 0 to 36 months is a non-therapeutic method.
• the invention can also be worded as the use of a fermented ingredient and non-digestible oligosaccharide in the manufacture of a nutritional composition for use in increasing IgA secretion in a human subject with an age of 0 to 36 months, wherein the nutritional composition comprises 0.02 to 1.5 wt% of the sum of lactic acid and lactate based on dry weight of the nutritional composition and comprises 2.5 to 15 wt% non-digestible oligosaccharide based on dry weight of the nutritional composition.
• the invention can also be worded as a nutritional composition comprising a fermented ingredient and non-digestible oligosaccharide, wherein the nutritional composition comprises 0.02 to 1.5 wt% of the sum of lactic acid and lactate based on dry weight of the nutritional composition and comprises 2.5 to 15 wt% non-digestible oligosaccharide based on dry weight of the nutritional composition, for use in increasing IgA secretion in a human subject with an age of 0 to 36 months.
• the invention concerns a method for improving mucosal immune defense in a human subject with an age of 0 to 36 month comprising administering to the human subject a nutritional composition comprising a fermented ingredient and non- digestible oligosaccharide, said nutritional composition comprising 0.02 to 1.5 wt% of the sum of lactic acid and lactate based on dry weight of the nutritional composition and comprising 2.5 to 15 wt% non-digestible oligosaccharide based on dry weight of the nutritional composition.
• the present method for improving mucosal immune defense in a human subject with an age of 0 to 36 months is a non-therapeutic method.
• the invention can also be worded as the use of a fermented ingredient and non-digestible oligosaccharide in the manufacture of a nutritional composition for use in improving mucosal immune defense in a human subject with an age of 0 to 36 month, wherein the nutritional composition comprises 0.02 to 1.5 wt% of the sum of lactic acid and lactate based on dry weight of the nutritional composition and comprises 2.5 to 15 wt% non- digestible oligosaccharide based on dry weight of the nutritional composition.
• the invention can also be worded as a nutritional composition comprising a fermented ingredient and non-digestible oligosaccharide, wherein the nutritional composition comprises 0.02 to 1.5 wt% of the sum of lactic acid and lactate based on dry weight of the nutritional composition and comprises 2.5 to 15 wt% non-digestible oligosaccharide based on dry weight of the nutritional composition, for use in improving mucosal immune defense in a human subject with an age of 0 to 36 month.
• the nutritional composition in the methods or uses according to the present invention comprises a fermented ingredient.
• the fermented ingredient is a composition that is fermented by lactic acid producing bacteria.
• the fermentation preferably takes place during the production process of the nutritional composition.
• the nutritional composition does not contain significant amounts of viable bacteria in the final product due to heat inactivation after fermentation or inactivation by other means.
• the fermented ingredient is a milk-derived product, which is a milk substrate that is fermented by lactic acid producing bacteria, and said milk substrate comprising at least one selected from the group consisting of mi lk, whey, whey protein, whey protein hydro iysate, casein, casein hydro lysate, and lactose or mixtures thereof.
• nutrit ional composit ions comprising fermented ingredient and non-digest ible oligosaccharide and their way of producing them are described in WO 2009/151330, WO 2009/151331 and WO 2013/187764.
• the fermented ingredient preferably comprises bacterial cell fragments like glycoproteins, giyco lipids, peptidoglycan. lipoteichoic acid (LTA), lipoproteins, nucleotides, and/or capsular polysaccharides.
• LTA lipoteichoic acid
• the fermented ingredient in particular fermented milk-deriv ed product, is bel ieved to have an improved effect compared to non-fermented milk-derived product.
• the nutrit ional composit ion comprises 5 to 97.5 wt% of the fermented ingredient based on dry weight, more preferably 10 to 95 wt% , more preferably 20 to 90 wt%, even more preferably 25 to 60 wt%.
• the level of the sum of lactic acid and lactate in the nutrit ional composit ion can be taken, as this is the metabolic end product produced by the lact ic acid producing bacteria upon fermentation.
• the present nutritional composit ion comprises 0.02 to 1 .5 wt% of the sum of lact ic acid and lactate based on dry weight of the composition, more preferably 0.05 to 1 .0 wt%, ev en more preferably 0.05 to 0.5 wt%.
• at least 50 wt%, ev en more preferably at least 90 wt%, of the sum of lactic acid and lactate is in the form of the L(+)- isomer.
• the sum of L(+)-lactic acid and L(+)- lactate is more than 50 wt%, more preferably more than 90 wt%, based on the sum of total lact ic acid and lactate.
• [_.( + )- lactate and L( + )- lactic acid is also referred to as L- lactate and L- lact ic acid.
• Lactic acid producing bacteria used for producing the fermented ingredient
• Lact ic acid producing bacteria used for preparing the fermented ingredient, in particular for fermentation of the milk substrate are preferably provided as a mono- or mixed culture.
• Lactic acid producing bacteria consists of the genera Bifidobacterium, Lactobacillus, Carnobacterium, Enterococcus, Lactococcus, Leuconostoc, Oenococcus, Pediococcus, Streptococcus, Tetragenococcus, Vagococcus and Weissella.
• the lactic acid producing bacteria used for fermentation comprises bacteria of the genus Bifidobacterium and/or Streptococcus.
• the Streptococcus is a strain of S. thermophilic.
• the Streptococcus develop beta-gaiactosidase act ivity in the course of fermentation of the substrate. Selection of a suitable strain of S. thermophilus is described in example 2 of EP 778885 and in example 1 of FR 2723960.
• the nutritional composition comprises 10 - 10 5 cfu living bacteria of S. thermophilus, per g dry weight of the nutritional composition, preferably the nutritional composition comprises 10 - 1 0 ' living bacteria of S. thermophilus per g dry weight.
• Bifidobacteria are Gram-positive, anaerobic, rod-shaped bacteria.
• Preferred Bifidobacterium species to prepare the fermented ingredient for the purpose of the present invent ion preferably hav e at least 95 % ident ity of the 16 S rRNA sequence when compared to the type strain of the respective Bifidobacterium species, more preferably at least 97% ident ity as defined in handbooks on this subject for instance Sambrook, J., Fritsch, E.F., and Maniatis, T. (1989), Molecular Cloning, A Laboratory Manual, 2nd ed., Cold Spring Harbor (N.Y.) Laboratory Press.
• the Bifidobacteria preferably used are also described by Scardov i. V.
• the lactic acid producing bacteria used for fermentation comprises or is at least one Bifidobacterium selected from the group consisting of B. breve, B. infantis, B. hifidum, B. catenulatum, B. adolescentis, B. thermophilum, B. gallicum, B. animalis or lactis, B.
• B. pseudocatenuiatum, B. thermacidophilum and B. longum more preferably B. breve, B. infantis, B. bifidum, B. catenulatum, B. longum, more preferably B. longum and B. breve, even more preferably B. breve, more preferably B. breve selected from the group consisting of B. breve Bb-03 (Rhodia/Danisco), B. breve M-16V (Morinaga), B. breve R0070 (Institute Resell, Lallemand), B. breve BR03 (Probiotical), B. breve BR92 (Cell Biotech) DSM 20091, LMG 11613 and B.
• the B. breve is B. breve M-16V (Morinaga) or B. breve 1-2219, even more preferably B. breve 1-2219.
• the nutritional composition comprises fermented ingredient that is fermented by lactic acid bacteria comprising both B. breve and S. thermophilus.
• the fermentation by lactic acid bacteria is fermentation by Streptococcus thermophilus and Bifidobacterium breve.
• the nutritional composition comprises fermented ingredient wherein the lactic acid bacteria are inactivated after fermentation.
• the fermented ingredient is not fermented by Lactobacillus bulgaricus.
• L. bulgaricus fermented products are considered not suitable for infants, since in young infants the specific dehydrogenase that converts D-lactate to pyruvate is far less active than the dehydrogenase which converts L-lactate.
• the present nutritional composition comprises inactivated lactic acid producing bacteria and/or bacterial fragments derived from lactic acid producing bacteria obtained from more than 1 x 10 1 cfu lactic acid producing bacteria per g based on dry weight of the final composition, more preferably l lO 5 cfu, even more preferably l lO 6 cfu.
• the inactivated bacteria or bacterial fragments are obtained from less than lxlO 14 cfu lactic acid producing bacteria per g based on dry weight of the final composition, more preferably lxl 0 13 cfu, even more preferably lxl 0 12 cfu.
• the milk substrate to be fermented is suitably present in an aqueous medium.
• the milk substrate to be fermented is preferably selected from the group consisting of milk, whey, whey protein, whey protein hydro lysate, casein, casein hydro lysate, and lactose, and mixtures thereof.
• Milk can be whole milk, semi-skimmed milk and/or skimmed milk.
• the milk substrate to be fermented comprises skimmed milk.
• Whey can be sweet whey, and/or acid whey.
• 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.
• whey protein hydrolysatc is present in 2 to 80 g dry weight per 1 aqueous medium containing milk substrate, more preferably 5 to 1 5 g/1.
• lactose is present in 5 to 50 g dry weight per 1 aqueous substrate, more preferably 1 to 30 g/1.
• the aqueous medium containing milk substrate comprises buffer salts in order to keep the pH within a desired range.
• 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.
• the aqueous medium containing milk substrate comprises cysteine in amount of 0.
• 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 lact ic acid producing bacteria. The presence of yeast extract will enhance the fermentation by lact ic acid producing bacteria.
• the milk substrate in particular the aqueous medium containing milk substrate
• the product is pasteurised after fermentation, in order to inact ivate enzymes.
• the enzyme inact ivation takes place at 75 °C for 3 min.
• the aqueous medium containing milk substrate is homogenised before and. or the milk-derived product is homogenised after the fermentation. Homogenisat ion results in a more stable substrate and or fermented product, especially in the presence of fat.
• the inoculation density is preferably between lxlO 2 to 5 10"', preferably between lxlO 4 to 5x10 9 cfu lact ic acid producing bacteria/ml aqueous medium containing milk substrate. more preferably between lxl 0 7 to lxlO 9 cfu lactic acid producing bacteria ml aqueous medium containing milk substrate.
• the final bacteria density after fermentation is preferably between lxlO 3 to lxlO 10 , more preferably between I x 1 0 1 to lxlO 9 cfu/ml aqueous medium containing milk substrate.
• the fermentat ion is preferably performed at a temperature of approximately 20 °C to 50 °C, more preferably 30 °C to 45 °C, even more preferably approximately 37 °C to 42 °C.
• the optimum temperature for growth and/or activity for lact ic acid producing bacteria, more particularly lactobacill i and/or bifidobacteria is between 37 °C and 42 °C.
• the incubat ion is preferably performed at a pi i of 4 to 8, more preferably 6 to 7.5. This pH does not induce protein precipitation and/or an adverse taste, hile at the same time lact ic acid producing bacteria such as lactobacilli and/or bifidobacteria are able to ferment the milk substrate.
• the incubation t ime preferably ranges from 10 minutes to 48 h, preferably from 2 h to 24 h, more preferably from 4 h to 12 h.
• a sufficient long time enables the fermentation and the concomitant production of immunogenic cell fragments such as glycoproteins, glycol ipids, peptidoglycan, l ipoteichoic acid (LTA), fiagellae, lipoproteins.
• DNA and/or capsular polysaccharides to take place at a high extent, whereas the incubation time need not be unnecessarily long for economical reasons.
• a milk substrate preferably skimmed milk
• one or more lact ic acid producing strains preferably a strain of S. thermophilus, to a certain degree of acidity, upon which the fermented product is cooled and stored.
• a second milk-derived product is prepared in a similar way using one or more Bifidobacterium species for fermentation.
• the two fermented products are preferably mixed together and mixed with other components making up an infant formula, except the fat component.
• the mixture is preheated, and subsequent ly fat is added in-line, homogenized, pasteurized and dried.
• thermophilus for example with 5% of a culture containing 10 6 to 10 10 bacteria per ml.
• this milk substrate comprises milk protein peptides.
• Temperature and duration of fermentation are as mentioned above.
• the fermented ingredient may be pasteurised or sterilized and for example spray dried or lyophilised to provide a form suitable to be formulated in the end product.
• a preferred method for preparing the fermented ingredient of the present invent ion is disclosed in WO 01/01785, more particular in examples 1 and 2.
• a preferred method for preparing the fermented product of the present invention is described in WO 2004/093899, more particularly in example I .
• Living cells of lactic acid producing bacteria in the fermented ingredient are after fermentation preferably eliminated, for example by inactivation and/or physical removal.
• the cells are preferably inactivated.
• the lactic acid producing bacteria are heat killed after fermentation of the milk substrate.
• Preferable ways of heat killing are (flash) pasteurization, sterilization, ultra high temperature treatment, high temperature/short time heat treatment, and/or spray drying at temperatures bacteria do not survive.
• Cell fragments are preferably obtained by heat treatment. With this heat treatment preferably at least 90 % of living microorganisms are inactiv ated, more preferably at least 95 %, even more preferably at least 99 %.
• the fermented nutritional composition comprises less than lxlO 5 colony forming units (cfu) living lactic acid bacteria per g dry- weight.
• the heat treatment preferably is performed at a temperature ranging from 70 to 180 °C, preferably from 80 to 150 °C, preferably for about 3 minutes to 2 hours, preferably in the range of 80 to 140 °C fo 5 minutes to 40 minutes.
• Inactiv ation of the lactic acid bacteria advantageously results in less post acidification and a safer product. This is especially advantageous when the nutritional composition is to be administered to infants or young children.
• the fermented ingredient may be pasteurised or sterilized and for example spray dried or lyophil ised to prov ide a form suitable to be formulated in the end product.
• the present nutritional composition comprises non-digestible oligosaccharide and preferably comprises at least two different non-digestible oligosaccharides, in particular two different sources of non-digest ible oligosaccharide.
• the presence of non-digestible oligosaccharide stimulates the secretion of slgA.
• the presence of non-digestible oligosaccharide stimulates the mucosal immune defense system.
• the presence of both the non-digestible oligosaccharide and the fermented ingredient, in particular the milk-derived product obtained by fermentation with lactic acid producing bacteria synergisticaily and advantageously results in a higher IgA secretion in the intest ine.
• the presence of both non-digestible oligosaccharide and the fermented ingredient, in particular the milk-deriv ed product obtained by fermentation with lactic acid producing bacteria synergisticaily and advantageously results in an increased mucosal immune defense.
• oligosaccharide refers to saccharides with a degree of polymerization (DP) of 2 to 250, preferably a DP 2 to 100. more preferably 2 to 60, even more preferably 2 to 10. I f oligosaccharide with a DP of 2 to 100 is included in the present nutritional composition, this results in compositions that may contain oligosaccharides with a DP of 2 to 5, a DP of 50 to 70 and a DP of 7 to 60.
• DP degree of polymerization
• non-digestible oligosaccharide refers to oligosaccharides which are not digested in the intestine by the action of acids or digestive enzymes present in the human upper digest ive tract, e.g. small intest ine and stomach, but which are preferably fermented by the human intestinal microbiota.
• sucrose, lactose, maltose and maitodextrins are considered digestible.
• the present non-digestible oligosaccharide is soluble.
• soluble when having reference to a polysaccharide, fibre or ol igosaccharide, means that the substance is at least soluble according to the method described by L. Prosky et a!., J. Assoc. Off. Anal. Chem. 71 , 1017-1023 (1988).
• the non-digestible ol igosaccharide included in the present nutritional compositions in the methods or uses according to the present invention preferably include a mixture of non- digestible oligosaccharides.
• the non-digestible oligosaccharide is preferably selected from the group consist ing of fructo-oligosaccharide, such as inuiin, non-digest iblc dextrins, ga!acto-ol igosaccharide, such as transgalacto-oligosaccharide, xylo- oligosaccharide, a ra b i n o - o I i go s a c c h a r i d e , arabinogaiacto-oligosaccharide, g lu cool igosaccharide, gent io-ol igosaceharide.
• fructo-oligosaccharide such as inuiin, non-digest iblc dextrins, ga!acto-ol igosaccharide, such as transgalacto-oligosaccharide, xylo-
• glucomanno-oligosaccharide galactomannool igosaccharide, mannan-o 1 igosaccharide, isomalto-ol igosaccharide. nigero -ol igosaccharide, glucomanno-oligosaccharide, chito-ol igosaccharide, soy ol igosaccharide, uronic acid oligosaccharide, sialylol igosaccharide, such as 3- sialyilactose (3-SL), 6-sialyl lactose (6-SL), lactosi a 1 y 1 tet rasaccha ri de a.b.c (LST), disialyilactoNtetraose ( DSLNT).
• 3- sialyilactose 3- sialyilactose (3-SL), 6-sialyl lactose (6-SL), lac
• sialyl-lactoNhexaose S-LNH
• DS-LNH sialyl-lactoNhexaose
• fuco- oligosaccharide such as (un )sulphatcd fucoidan.
• the non-digestible oligosaccharide is selected from the group consisting of transgalacto-oligosaccharide, fructo-oligosaccharide and mixtures of thereof.
• the non-digestible oligosaccharide is preferably selected from the group consist ing of ⁇ - galacto-ol igosaccharide, ot-ga 1 acto-o I i gosaccha ri de, and galactan.
• non-digestible oligosaccharide is ⁇ -galacto-ol igosaccharide.
• the non-digestible oligosaccharide comprises galacto-ol igosaccharide with ⁇ (1 ,4), ⁇ (1,3) and/or ⁇ (1,6) glycosidic bonds and a terminal glucose.
• Transgalacto- oligosaccharide is for example available under the trade name Vivinal®GOS ( Do mo FriesIandCampina Ingredients). Bi2muno (Clasado), Cup-ol igo (Nissin Sugar) and Oligomatc55 (Yakult). These oligosaccharides increase slgA levels and strengthens the mucosal immune defense act ivity to a larger extent.
• the non-digestible ol igosaccharide preferably comprises fructo-ol igosaccharide.
• a fructo-oligosaccharide may in other context have names like fructopoiysaccharide, oligo fructose, polyfructose, polyfructan, inul in. levari, and true tan and may refer to oligosaccharides comprising ⁇ - 1 inked fructose units, which are preferably linked by ⁇ (2, 1) and/or ⁇ (2,6) glycosidic linkages, and a preferable DP between 2 and 200.
• the fructo-oligosaccharide contains a terminal ⁇ (2, 1) glycosidic linked glucose.
• the fructo-oligosaccharide contains at least 7 ⁇ -l inked fructose units.
• inulin is used.
• Inulin is a type of fructo-oligosaccharide wherein at least 75% of the glycosidic linkages are ⁇ (2, 1) linkages. Typical ly, inulin has an average chain length between 8 and 60 monosaccharide un its.
• a suitable fructo- oligosaccharide for use in the composit ions of the present invention is commercially available under the trade name Raftiline®HP (Orafti ).
• Other suitable sources are Raft ilose (Orafti), Fibrulose and Fibruline (Cosucra) and F rut a fit.
• the present nutritional composition comprises a mixture of galacto- oligosaccharide and fructo-oligosaccharide.
• the mixture of galacto- ol igosaecharide and fructo-oligosaccharide is present in a weight ratio of from 1/99 to 99/1 , more preferably from 1/19 to 19/1 , more preferably from 1/1 to 19/1, more preferably from 2/1 to 15/1, more preferably from 5/1 to 12/1 , even more preferably from 8/1 to 10/1 , even more prefeably in a ratio of about 9/1.
• This weight ratio is particularly advantageous when galacto-oligosaccharide has a low average DP and fructo- oligosaccharide has a relativ ely high DP.
• Most preferred is a mixture of galacto- oligosaccharide w ith an average DP below 10, preferably below 6 and a fructo- ol igosaccharide with an average DP above 7. preferably above 1 1 , even more preferably above 20.
• Such a mixture synergist ically increases slgA levels and strengthens the mucosal immune defense act ivity.
• the present nutritional composition comprises a mixture of short chain fructo- oligosaccharide and long chain fructo-oligosaccharide.
• the mixture of short chain fructo-oligosaccharide and long chain friicto-o 1 i gosacc ha ri dc is present in a weight ratio of from 1/99 to 99/1 , more preferably from 1/19 to 19/1 , even more preferably from 1/10 to 19/1, more preferably from 1/5 to 15/1, more prefrably from 1/1 to 10/1.
• Preferred is a mixture of short chain fructo-oligosaccharide with an average DP below 10, preferably below 6 and a fructo-oligosaccharide with an average DP above 7, preferably above 1 1 , ev en more preferably above 20.
• the present nutritional composit ion comprises 2.5 to 20 wt% total non-digestible oligosaccharide, more preferably 2.5 to 15 wt%, even more preferably 3.0 to 1 0 wt%, most preferably 5.0 to 7.5 wt%, based on dry weight of the nutritional composit ion.
• the present nutritional composit ion preferably comprises 0.35 to 2.5 wt% total non-digest ible oligosaccharide, more preferably 0.35 to 2.0 wt%, even more preferably 0.4 to 1 .5 wt%, based on 100 ml of the nutrit ional composit ion.
• a lower amount of non- digestible oligosaccharide will be less effective in stimulating slgA formation or mucosal immune defense, whereas a too high amount will result in side-effects of bloat ing and abdominal discomfort.
• the nutritional composit ion used according to the present inv ent ion may also be considered as being a pharmaceutical composition, is preferably suitable for administration to infants.
• the present nutrit ional composit ion is preferably enterally administered, more preferably orally.
• the present nutrit ional composit ion is preferably an infant formula, follow on formula or a growing up milk.
• the present nutritional com osition can be advantageously applied as a complete nutrit ion for infants.
• the present nutritional composit ion is an infant formula.
• An infant formula is defined as a formula for use in infants and can for example be a starter formula, intended for infants of 0 to 6 or 0 to 4 months of age.
• the present composition preferably comprises a lipid component, protein component and carbohydrate component and is preferably administered in liquid form.
• the present nutritional composition may also be in the form of a dry food, preferably in the form of a powder which is accompanied w ith instructions as to mi said dry food, preferbly powder, with a suitable liquid, preferably water.
• the nutrit ional composit ion used according to the inv ent ion preferably comprises other fractions, such as vitamins, minerals, trace elements and other micronutrients in order to make it a complete nutritional composition.
• infant formulas comprise vitamins, minerals, trace elements and other micronutrients according to international directives.
• the present nutritional composition preferably comprises lipid, protein and digestible carbohydrate invention wherein the lipid provides 5 to 50% of the total calories, the protein provides 5 to 50% of the total calories, and the digestible carbohydrate provides 15 to 90% of the total calories.
• the lipid provides 35 to 50% of the total calories
• the protein provides 7.5 to 12.5% of the total calories
• the digestible carbohydrate provides 40 to 55% of the total calories.
• the lipid provides 3 to 7 g lipid per 100 kcal, preferably 4 to 6 g per 100 kcal, the protein provides 1.6 to 4 g per 100 kcal, preferably 1.75 to 2.5 g 15 per 100 kcal and the digestible carbohydrate provides 5 to 20 g per 100 kcal, preferably 8 to 15 g per 100 kcal of the nutritional composition.
• the present nutritional composition comprises lipid providing 4 to 6 g per 100 kcal, protein providing 1.6 to 1.9 g per 100 kcal, more preferably 1.75 to 1.85 g per 100 kcal and digestible carbohydrate providing 8 to 15 g per 100 kcal of the nutritional composition.
• the lipid provides 3 to 7 g lipid per 100 kcal, preferably 4 to 6 g per 100 kcal
• the protein provides 1.6 to 2.1 g per 100 kcal, preferably 1.6 to 2.0 g per 100 kcal
• the digestible carbohydrate provides 5 to 20 g per 100 kcal, preferably 8 to 15 g per 100 kcal of the nutritional composition and wherein preferably the digestible carbohydrate component comprises at least 60 wt% lactose based on total digest ible carbohydrate, more preferably at least 75 wt%, even more preferably at least 90 wt% lactose based on total digestible carbohydrate.
• the amount of total calories is determined by the sum of calories derived from protein, lipids, digest ible carbohydrates and non-digestible oligosaccharide.
• the present nutritional composition preferably comprises a digestible carbohydrate component.
• Preferred digestible carbohydrate components are lactose, glucose, sucrose, fructose, galactose, maltose, starch and maltodextrin. Lactose is the main digestible carbohydrate present in human milk.
• the present nutritional composition preferably comprises lactose.
• lactose is the main digestible carbohydrate present in human milk.
• the present nutritional composition preferably comprises lactose.
• 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.
• the present nutritional composition does not comprise high amounts of carbohydrates other than lactose.
• the present nutritional composition preferably comprises digestible carbohydrate, wherein at least 35 wt%, more preferably at least 50 wt%, more preferably at least 60 wt%, more preferably at least 75 wt%, even more preferably at least 90 wt% , most preferably at least 95 wt% of the digestible carbohydrate is lactose. Based on dry weight the present nutritional composition preferably comprises at least 25 wt% lactose, preferably at least 40 wt%, more preferably at least 50 wt% lactose.
• the present nutritional composition preferably comprises at least one lipid selected from the group consisting o animal lipid (excluding human lipids) and vegetable lipids.
• the present composit ion comprises a combinat ion o vegetable lipids and at least one oil selected from the group consist ing o fish oil, animal oil, algae oil, fungal oil. and bacterial oil.
• the lipid of the present nutritional composit ion preferably provides 3 to 7 g per 100 kcal of the nutritional composition, preferably the lipid provides 4 to 6 g per 100 kcal.
• the nutritional composition preferably comprises 2.1 to 6.5 g lipid per 100 ml, more preferably 3.0 to 4.0 g per 100 ml. Based on dry weight the present nutritional composition preferably comprises 12.5 to 40 wt% lipid, more preferably 1 to 30 wt%.
• the lipid comprises the essential fatty acids alpha- lino lenic acid (ALA), linolcic acid (LA) and/or long chain polyunsaturated fatty acids (LC-PUFA).
• the LC-PUFA, LA and or ALA may be provided as free fatty acids, in triglyceride form, in diglyceride form, in monog!yceridc form, in phospholipid form, or as a mixture of one of more of the above.
• the present nutritional composition comprises at least one, preferably at least two lipid sources selected from the group consisting of rape seed oil (such as colza oil, low erucic acid rape seed oil and canola oil), high oleic sunflower oil, high oleic safflower oil, olive oil, marine oils, microbial oils, coconut oil, palm kernel oil and milk fat.
• the present nutritional composit ion is not human milk.
• the present nutritional composition preferably comprises protein.
• the protein used in the nutritional composition is preferably selected from the group consisting of non-human animal proteins, preferably milk proteins, vegetable proteins, such as preferably soy protein and/or rice protein, and mixtures thereof.
• the present nutritional composition preferably contains casein, and/or whey protein, more preferably bovine whey proteins and/or bovine casein.
• 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.
• 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.
• 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.
• 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.
• the nutritional composition 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.
• the nutritional composition preferably comprises 45 to 200 kcal/100 ml liquid.
• 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.
• 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.
• 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.
• the number of feedings per day is between 1 and 10, preferably between 3 and 8.
• the nutritional composition is administered daily for a period of at least 2 days, preferably for a period of at least 4 weeks, preferably for a period of at least 8 weeks, more preferably for a period of at 25 least 12 weeks, in a liquid form wherein the total volume administered daily is between 200 ml and 1200 ml and wherein the number of feedings per day is between 1 and 10.
• the present nutritional composition when in liquid form, preferably has a viscosity between 1 and 60 mPa.s, preferably between 1 and 20 mPa.s, more preferably between 1 and 10 mPa.s, most preferably between 1 and 6 m a.s.
• the low v iscosity ensures a proper administration of the liquid, e.g. a proper passage through the whole of a nipple. Also this viscosity closely resembles the viscosity of human milk. Furthermore, a low viscosity results in a normal gastric emptying and a better energy intake, which is essential for infants which need the energy for optimal growth and development.
• the present composition is preferably prepared by admixing a powdered composit ion with water. Normally infant formula is prepared in such a way.
• the present inv ention thus also relates to a packaged power composition wherein said package is prov ided with instructions to admi the powder with a suitable amount of liquid, thereby resulting in a liquid composition with a viscosity between 1 and 60 mPa.s.
• the viscosity of the liquid is determined using a Physica Rheo meter MCR 300 (Physica Messtechnik GmbH, Ostfilden, Germany) at a shear rate of 95 s "1 at 20 °C.
• 'prevention' of a disease or certain disorder also means 'reduction of the risk' of a disease or certain disorder and also means 'treatment of a person at risk' of said disease or said certain disorder.
• the inventors have found that upon consumption of the nutritional composition of the present invention the level of slgA is synergetically increased. This is indicative for an improved mucosal immune defense system.
• the synergistic effect cannot be e plained by a direct interaction between the non-digestible oligosaccharide and the lactic acid producing bacteria in the fermented composition, as the bacteria were inactivated, nor by an increased intestinal microbial activity as there was no clear correlation with the intestinal pH.
• 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.
• the present nutritional composition is used for improving mucosal immune defense in a human subject with an age of 0 to 36 months. In one embodiment the present nutritional composition is used for improving mucosal immune defense in an human subject of 0 to 18 months, even more preferably an infant with an age of 12 months of age or below, even more preferably an infant of 0 to 6 months. In one embodiment the present nutritional composition is used for improving mucosal immune defense in a young child of 12 to 36 months, most preferably a young child with an age of 18 to 30, or 24 months. Preferably the present nutritional composition is further used for providing nutrition to said human subject. The present nutritional composition is preferably is enterally administered, more preferably orally. In a preferred embodiment, the methods or uses according to the present invention are for vaginally delivered infants.
• the methods or uses according to the present invention are for term infants, preferably for healthy term infants.
• 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.
• 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.
• Example 1 Synergistic effect of fermented infant formula and non- digestible oligosaccharides on secretory IgA levels in infants
• 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.
• NDO non-digestible oligosaccharides
• Vivinal® GOS from FrieslandCampina Domo
• fructo-oligosaccharides RaftilinHP from Orafti
• the formula does not contain a fermented composition.
• Test group 2 Infant formula 2 is a modified cow's milk-based infant formula for bottle- fed babies at the age of 0-6 months and is a fermented infant formula Calisma® (sold by Gallia, France) which comprises the strains B. breve CNCM 1-2219 and S. thermophilus CNCM 1-1620, heat killed after a fermentation process, bacterial fermentation metabolites such as L-(+) lactate.
• the amount of L-lactate is above 0.05 wt% based on dry weight of the composition. No NDO is added. In the fermentation process an amount of about 2 wt% based on dry weight galacto-oligosaccharides is produced by the S. thermophilus.
• Test group 3 Infant formula 3 is the experimental test formula and is a modified cow's milk-based infant formula for bottle-fed babies at the age of 0-6 months.
• the formula contains non digestible oligosaccharides (a mix of galacto-oligosaccharides with an average degree of polymerisation below 6, and fructo-oligosaccharides, RaftilinHP from Orafti, with an average degree of polymerisation above 20) in an amount of about 0.9 g per 100 ml and comprising fermented infant formula Calisma® (sold by Gallia, France), as in IF 2 of test group 2.
• the amount of added galacto-oligosaccharides takes into account the amount of galacto-oligosaccharides present due to the action of S. thermophilus.
• Infant formula 4 is the control formula: a modified cow's milk-based infant formula for bottle-fed babies at the age of 0-6 months, not comprising non- digestible oligosaccharides and not being fermented. All four test formulas contained nucleotides and a fat blend that contained long chain fatty acids. The formula were similar in caloric content, protein content, fat blend and had a similar amount of digestible carbohydrates. The number of living bacteria in test product 2 and 3 was below 10 3 cfu/g. The formulas further comprised vitamins, minerals, trace elements and other micronutrients according to international directive 2006/141/EC for infant formula.
• 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.
• Example 2 Consumption of fermented infant formula with non-digestible oligosaccharides increases intestinal slgA levels
• 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.
• scGOS source Vivinal® GOS
• lcFOS source RaftilinHP®
• LactofidusTM is a fermented milk derived composition and is produced by fermenting with S. thermophilus and comprises B. breve. A mild heat treatment is employed to inactivate the lactic acid bacteria.
• the infant formula comprises about 0.55 wt% lactic acid + lactate based on dry weight, of which at least 95% is L(+)- lactic acid/lactate.
• the composition further comprises vitamins, minerals, trace elements and other micronutrients according to international directive 2006/141/EC for infant formula.
• Test group 2 Infant formula 2, similar to infant formula 1, but without the non-digestible oligosaccharides scGOS and lcFOS.
• Test group 3 Infant formula 3, a non fermented infant formula with 0.8 g non-digestible oligosaccharides comprising scGOS (source Vivinal® GOS) and lcFOS (source RaftilinHP®) in a 9: 1 wt ratio, and for the remainder similar in composition as infant formula 1.
• Stool samples were collected at baseline and after 17 weeks of intervention for physiological and microbiological analysis, in a way similar as described in example 1. Only samples of a subgroup of subjects were analysed that had a complete set of stool samples (both visits) with sufficient amount of stool for all analyses. In addition, samples from infants that used any systemic antibiotics any time after birth or that used thickeners added to formula during the study were excluded.

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