Fermented formula with non-digestible oligosaccharides for rotavirus induced infections

Abstract

Disclosed is a method of treating and / or preventing rotavirus infection and its associated symptoms in an infant or young child comprising administering a nutritional composition at least partially fermented by a lactic acid-producing bacterium and comprising a non-digestible oligosaccharide.

Description

[0001] This application is a divisional application of the invention patent application No. 202080005729.1 entitled "Fermented formula food with non-digestible oligosaccharides for infection caused by rotavirus". The original application corresponds to international application PCT / EP2020 / 051034, with a filing date of January 16, 2020 and a priority date of January 16, 2019. Technical Field

[0002] The invention relates to the field of infant nutrition and infant formula food, and is used for treating or preventing rotavirus infection. Background Art

[0003] Viral infections are common in people of all ages but appear to be typically concentrated in infants and children. Many viral infections cause fever, body aches, or malaise and are not serious, and most children with viral infections will get better without treatment. Especially during infancy, it can be difficult for parents to tell whether their infant has a potentially serious infection that requires immediate medical attention. Rotavirus (RV) is a global pathogen that causes severe, dehydrating diarrhea in children younger than 5 years of age. RV infects and replicates primarily in mature, nondividing enterocytes near the tips of the villi of the small intestine. As a result, infants experience diarrhea, vomiting, and fever for 3 days to 1 week, often with reinfection, which is often less severe than the initial infection. The most common treatment involves oral rehydration solutions (ORS). Because the pathogenesis of RV in humans remains unclear and RV vaccines have not yet been implemented worldwide, there is interest in the modulation of nutritional interventions with bioactive ingredients.

[0004] Human milk is a preferred food for infants. Human milk provides a variety of biologically active factors that are beneficial to the relatively immature immune system of newborns in the early stages of life and can prevent or reduce the severity of rotavirus infection, such as lactoferrin and human milk oligosaccharides. However, it is not always possible or desirable to breastfeed infants. In this case, infant formula or follow-on formula foods are good choices. These formula foods should have the best ingredients to simulate the beneficial effects of human milk as closely as possible.

[0005] In recent years, several nutritional interventions using bioactive compounds have been studied. Among these compounds, probiotics are widely studied due to their known immunomodulatory effects and direct effects on the small intestine (which is the focus of RV infection). WO 00 / 53201 discloses lactic acid bacteria strains that can prevent diarrhea and prevent rotavirus infection. WO2004 / 2168 discloses bifidobacteria for the treatment of inflammatory diseases (including diarrhea associated with rotavirus). WO 02 / 39834 discloses probiotics for preventing or inhibiting diarrhea rotavirus. EP 2407532 discloses that the probiotic strains of Bifidobacterium breve and their supernatants prevented the in vitro growth of rotavirus in in vitro experiments. In Rigo-Adrovers et al., 2017, British Journal of Nutrition, 117, 209-217, two different concepts were tested. The first is a heat-inactivated fermented formula, and the second is a specific mixture of short-chain galacto-oligosaccharides, long-chain fructo-oligosaccharides and pectin-derived acidic oligosaccharides. Both nutritional concepts improve the clinical symptoms of rotavirus infection. In Rigo-Adrover et al., 2018, Frontiers in Immunology, doi: 10.3389 / fimmu.2018.01318, a study of the preventive effects of a mixture of short-chain galacto-oligosaccharides and long-chain fructo-oligosaccharides and a combination of a prebiotic Bifidobacterium breve M-16V and a prebiotic mixture and a combination of probiotics in a rat double-RV infection model is described. After the first infection, dietary intervention can improve clinical symptoms. Summary of the invention

[0006] The inventors have tested the effect of fermented formula food combined with non-digestible oligosaccharide mixture against rotavirus infection. Surprisingly, the binding of rotavirus particles produced by the combination of fermented formula food and non-digestible oligosaccharides is much higher than the binding of the individual components of fermented formula food or non-digestible oligosaccharides. Rotavirus infection and the symptoms caused by it were studied using a young mouse model. Rotavirus infection-induced weight loss (mainly observed after the diarrheal period) and rotavirus-induced effects on organ weights were improved in the group that received the combination of fermented formula food and non-digestible oligosaccharide mixture. Giving fermented formula food together with non-digestible oligosaccharides can reduce the incidence, duration and severity of rotavirus-induced diarrhea. In addition, reduced viral shedding was observed. In addition, the intestinal barrier permeability of animals infected with rotavirus decreased the most in the group given a combination of fermented formula food and non-digestible oligosaccharides, as determined by α1 antitrypsin. DETAILED DESCRIPTION

[0007] Thus, the present invention relates to a method for treating and / or preventing rotavirus infection or rotavirus-induced diarrhea in an infant or young child, comprising administering to the infant or young child a nutritional composition at least partially fermented by lactic acid producing bacteria and comprising non-digestible oligosaccharides.

[0008] The present invention can also be formulated as the use of the fermented composition and the non-digestible oligosaccharides for the preparation of a nutritional composition at least partially fermented by lactic acid producing bacteria and comprising non-digestible oligosaccharides for the treatment and / or prevention of rotavirus infection or rotavirus-induced diarrhea in infants and young children.

[0009] The invention may also be formulated as a nutritional composition at least partially fermented by lactic acid producing bacteria and comprising non-digestible oligosaccharides for use in the treatment and / or prevention of rotavirus infection or rotavirus-induced diarrhea in infants or young children.

[0010] In one embodiment, the treatment and / or prevention of rotavirus infection or rotavirus-induced diarrhea is

[0011] a. Reduce the incidence of rotavirus-induced diarrhea or rotavirus infection,

[0012] b. Reduce the duration of rotavirus-induced diarrhea or rotavirus infection,

[0013] c. Reduce the severity of rotavirus-induced diarrhea or rotavirus infection,

[0014] d. Prevention of rotavirus-induced growth retardation or poor growth and / or developmental retardation or poor development,

[0015] e. Improve the blocking of rotavirus particles,

[0016] f. Reduction of rotavirus load, and / or

[0017] g. Improve intestinal barrier function during rotavirus infection.

[0018] The present invention also relates to a method for:

[0019] a. Reduce the incidence of rotavirus-induced diarrhea or rotavirus infection,

[0020] b. Reduce the duration of rotavirus-induced diarrhea or rotavirus infection,

[0021] c. Reduce the severity of rotavirus-induced diarrhea or rotavirus infection,

[0022] d. Prevention of rotavirus-induced growth retardation or poor growth and / or developmental delay or poor development,

[0023] e. Improve the blocking of rotavirus particles,

[0024] f. Reduction of rotavirus load, and / or

[0025] g. Improving intestinal barrier function during rotavirus infection, the method comprising administering to an infant or young child a nutritional composition that is at least partially fermented by lactic acid producing bacteria and includes non-digestible oligosaccharides.

[0026] The present invention can also be expressed as the use of the fermented composition and the non-digestible oligosaccharides for the preparation of a nutritional composition at least partially fermented by lactic acid producing bacteria and comprising non-digestible oligosaccharides for use in infants or young children.

[0027] a. Reduce the incidence of rotavirus-induced diarrhea or rotavirus infection,

[0028] b. Reduce the duration of rotavirus-induced diarrhea or rotavirus infection,

[0029] c. Reduce the severity of rotavirus-induced diarrhea or rotavirus infection,

[0030] d. Prevention of rotavirus-induced growth retardation or poor growth and / or developmental retardation or poor development,

[0031] e. Improve the blocking of rotavirus particles,

[0032] f. Reduction of rotavirus load, and / or

[0033] g. Improve intestinal barrier function during rotavirus infection.

[0034] The invention may also be described as a nutritional composition at least partially fermented by lactic acid producing bacteria and comprising non-digestible oligosaccharides for use in infants or young children.

[0035] a. Reduce the incidence of rotavirus-induced diarrhea or rotavirus infection,

[0036] b. Reduce the duration of rotavirus-induced diarrhea or rotavirus infection,

[0037] c. Reduce the severity of rotavirus-induced diarrhea or rotavirus infection,

[0038] d. Prevention of rotavirus-induced growth retardation or poor growth and / or developmental retardation or poor development,

[0039] e. Improve the blocking of rotavirus particles,

[0040] f. Reduction of rotavirus load, and / or

[0041] g. Improve intestinal barrier function during rotavirus infection.

[0042] Fermented composition

[0043] The nutritional composition according to the method or use of the present invention (hereinafter also referred to as the present nutritional composition, or the nutritional composition of the present invention or the final nutritional composition) is at least partially fermented. A partially fermented nutritional composition comprises at least a portion of a composition fermented by lactic acid producing bacteria. Therefore, in the context of the present invention, an at least partially fermented nutritional composition refers to a nutritional composition comprising a milk base fermented by lactic acid producing bacteria. The presence of a fermented composition in the final nutritional composition has been shown to be effective against rotavirus diarrhea and its symptoms when administered.

[0044] Fermentation is preferably carried out during the production of the nutritional composition. Preferably, the nutritional composition does not contain a large amount of live bacteria in the final product, which can be achieved by heat inactivation after fermentation or inactivation by other means. Preferably, the fermented composition is a product of milk origin, which is a milk matrix fermented by lactic acid bacteria, wherein the milk matrix comprises at least one selected from the following: milk, whey, whey protein, whey protein hydrolysate, casein, casein hydrolysate or a mixture thereof. Suitably, nutritional compositions comprising fermented compositions and non-digestible oligosaccharides and methods for producing the same are described in WO 2009 / 151330, WO 2009 / 151331 and WO 2013 / 187764.

[0045] The fermented composition preferably includes bacterial cell fragments, such as glycoproteins, glycolipids, peptidoglycans, lipoprotein acid (LTA), lipoproteins, nucleotides and / or capsular polysaccharides. It is advantageous to directly use a fermented composition including inactivated bacteria and / or cell fragments as a part of the final nutritional product, because this will produce a higher concentration of bacterial cell fragments. When using a commercial preparation of lactic acid bacteria, it is usually washed, and the material is separated from the aqueous growth medium including bacterial cell fragments, thereby reducing or eliminating the presence of bacterial cell fragments. In addition, during fermentation and / or other interactions of lactic acid bacteria with milk matrix, other bioactive compounds can be formed, such as short-chain fatty acids, bioactive peptides and / or oligosaccharides, and other metabolites, which can also produce intestinal flora functions more similar to those of breast-fed infants. This bioactive compound produced by lactic acid bacteria during fermentation can also be referred to as post-biotics. Compositions including this post-biotic are considered to be advantageously closer to breast milk, because breast milk is not a clean synthetic formula food, but contains metabolites, bacterial cells, cell fragments, etc. Therefore, fermented compositions, in particular fermented milk-derived products, are believed to have an improved effect on anti-rotavirus action compared to non-fermented milk-derived products without or with only lactic acid-producing bacteria.

[0046] Preferably, based on dry weight, the final nutritional composition includes 5 to 97.5% by weight of the fermented composition, more preferably 10 to 90% by weight, more preferably 20 to 80% by weight, and even more preferably 25 to 60% by weight. As a way to indicate that the final nutritional composition includes at least part of the fermented composition and indicates the degree of fermentation, the level of the sum of the lactic acid and lactate content in the final nutritional composition can be used, because this is the metabolic end product produced by lactic acid bacteria during fermentation. Based on the dry weight of the composition, the final nutritional composition preferably includes 0.02 to 1.5% by weight of lactic acid and lactate, more preferably 0.05 to 1.0% by weight, and even more preferably 0.2 to 0.5% by weight. Preferably at least 50% by weight, even more preferably at least 90% by weight of the sum of lactic acid and lactate is in the form of L(+)-isomers. Therefore, in one embodiment, based on the sum of lactic acid and lactic acid, the sum of L(+)-lactic acid and L(+)-lactate is greater than 50% by weight, more preferably greater than 90% by weight. Herein, L(+)-lactate and L(+)-lactic acid are also referred to as L-lactate and L-lactic acid.

[0047] Lactic acid producing bacteria for preparing fermented compositions

[0048] Preferably, lactic acid producing bacteria for preparing a fermented composition, in particular for fermenting a milk matrix, are provided as a single or mixed culture medium. The lactic acid producing bacteria are composed of the genera Bifidobacterium, Lactobacillus, Carnobacterium, Enterococcus, Lactococcus, Leuconostoc, Oenococcus, Pediococcus, Streptococcus, Tetragenococcus, Vagococcus and Weissella. Preferably, the lactic acid producing bacteria for fermentation include bacteria of the genera Bifidobacterium and / or Streptococcus.

[0049] Preferably, the Streptococcus is a thermophilic Streptococcus (S. thermophilus) strain. The selection of suitable thermophilic Streptococcus strains is described in Example 2 of EP 778885 and Example 1 of FR2723960. In another preferred embodiment according to the present invention, the nutritional composition comprises 10 2 -10 5 cfu of live bacteria of Streptococcus thermophilus / g dry weight of the final nutritional composition, preferably the final nutritional composition comprises 10 3 -104 Live bacteria of Streptococcus thermophilus / g dry weight.

[0050] Preferred strains of thermophilic Streptococcus for preparing the fermented compositions for the purposes of the present invention have been deposited by Compagnie Gervais Danone with the accession number 1-1620 on August 23, 1995 and with the accession number 1-1470 on August 25, 1994 at the National Collection of Microorganisms (CNCM), managed by the Institut Pasteur, 25 rue du Docteur Roux, Paris, France. Other thermophilic Streptococcus strains are commercially available.

[0051] Bifidobacteria are Gram-positive anaerobic rod-shaped bacteria. Preferred bifidobacterium species for preparing the fermentation composition for the purpose of the present invention preferably have at least 95% identity with the 16S rRNA sequence when compared to the model strain of the respective bifidobacterium species, more preferably, as described in manuals on the subject (e.g. Sambrook, J., Fritsch, EF and Maniatis, T. (1989), Molecular Cloning, A Laboratory Manual, 2 ndScardovi, V. Genus Bifidobacterium, p. 1418-1434. In: Bergey's manual of systematic Bacteriology. Vol. 2. Sneath, PHA, NS Maier, ME Sharpe and JG Holt (ed.). Baltimore: Williams & Wilkins. 1986. 635 p. also describes preferably used bifidobacteria. Preferably, the lactic acid-producing bacteria used for fermentation include or are at least one bifidobacterium selected from the following: Bifidobacterium breve (B.breve), Bifidobacterium infantis (B.infantis), Bifidobacterium bifidum (B.bifidum), Bifidobacterium catechins (B.catenulatum), Bifidobacterium adolescentis (B.adolescentis), Bifidobacterium thermophilum (B.thermophilum), Bifidobacterium gallicum (B.gallicum), Bifidobacterium animalis (B.animalis) or Bifidobacterium lactis (B.lactis), Bifidobacterium angulata (B.angulat breve, Bifidobacterium breve Bb-03 (Rhodia / Danisco), Bifidobacterium breve M-16V (Morinaga), 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 in CNCM, Paris, France. Most preferably, the Bifidobacterium breve is Bifidobacterium breve M-16V (Morinaga) or Bifidobacterium breve 1-2219, even more preferably Bifidobacterium breve 1-2219.

[0052] Most preferably, the nutritional composition of the present invention comprises a composition fermented by lactic acid producing bacteria, the lactic acid producing bacteria comprising both Bifidobacterium breve and Streptococcus thermophilus. In one embodiment, the fermentation performed by lactic acid producing bacteria is a fermentation performed by Streptococcus thermophilus and Bifidobacterium breve. In one embodiment, the final nutritional composition comprises a fermented composition, wherein the lactic acid producing bacteria are inactivated after fermentation.

[0053] Preferably, the fermented composition is not fermented by Lactobacillus bulgaricus. Products fermented by Lactobacillus bulgaricus are considered unsuitable for infants because in young infants the activity of a specific dehydrogenase that converts D-lactate to pyruvate is much lower than the activity of a dehydrogenase that converts L-lactate.

[0054] Preferably, the nutritional composition of the present invention comprises: based on the dry weight of the final composition, an amount equivalent to greater than 1×10 4 cfu, preferably 1x10 5 cfu, even more preferably 1x10 6 cfu of lactic acid-producing bacteria and / or bacterial fragments derived from lactic acid-producing bacteria. Preferably, based on the dry weight of the final composition, the inactivated bacteria or bacterial fragments are equivalent to less than 1 x 10 per gram. 13 cfu, preferably 1x10 11 cfu, even more preferably 1x10 10 cfu of lactic acid producing bacteria. The correlation of inactivated lactic acid bacteria to cfu equivalents can be determined by molecular techniques known in the art or by examining the production process.

[0055] Fermentation method

[0056] Preferably, the fermented composition is a product of milk origin, which is a milk matrix fermented by lactic acid bacteria, the milk matrix comprising at least one selected from milk, whey, whey protein, whey protein hydrolysate, casein, casein hydrolysate or a mixture thereof. The milk-derived product or milk matrix to be fermented is appropriately present in an aqueous medium. The milk matrix to be fermented comprises at least one selected from milk, whey, whey protein, whey protein hydrolysate, casein, casein hydrolysate or a mixture thereof. The milk can be whole milk, semi-skimmed milk and / or skimmed milk. Preferably, the milk matrix to be fermented comprises skimmed milk. The whey can be sweet whey and / or acidic whey. Preferably, the whey is present at a concentration of 3 to 80 g dry weight per liter of aqueous medium containing the milk matrix, more preferably 40 to 60 g per liter. Preferably, the whey protein hydrolysate is present at 2 to 80 g dry weight per liter of aqueous medium containing the milk matrix, more preferably 5 to 15 g / l. Preferably, lactose is present in an amount of 5 to 50 g dry weight per liter of aqueous matrix, more preferably 1 to 30 g / l. Preferably, the aqueous medium containing the milk matrix includes a buffer salt in order to keep the pH within the desired range. Preferably, sodium dihydrogen phosphate or potassium dihydrogen phosphate is used as a buffer salt, preferably 0.5 to 5 g / l, more preferably 1.5 to 3 g / l. Preferably, the aqueous medium containing the milk matrix includes cysteine ​​in an amount of 0.1 to 0.5 g per liter of aqueous matrix, more preferably 0.2 to 0.4 g / l. The presence of cysteine ​​results in a low redox potential of the matrix, which is beneficial for the activity of lactic acid producing bacteria, in particular bifidobacteria. Preferably, the aqueous medium containing the milk matrix includes yeast extract in an amount of 0.5 to 5 g / l, 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 improve the fermentation performed by lactic acid producing bacteria.

[0057] Suitably, the milk base, in particular the aqueous medium containing the milk base, is pasteurized prior to the fermentation step to eliminate the presence of harmful live bacteria. Suitably, the product is pasteurized after fermentation to inactivate the enzymes. Suitably, the enzyme inactivation is carried out at 75°C for 3 minutes. Suitably, the aqueous medium containing the milk base is homogenized prior to fermentation and / or the source product is homogenized after fermentation. Homogenization may result in a more stable base and / or fermented product, particularly in the presence of fat.

[0058] The seeding density is preferably 1x10 2 Up to 5x10 10 , preferably 1x10 4 Up to 5x10 9 cfu lactic acid producing bacteria / ml aqueous medium containing milk matrix, more preferably 1x10 7 Up to 1x10 9 cfu lactic acid producing bacteria / ml aqueous medium containing milk matrix. After fermentation, the final bacterial density is preferably 1x103 Up to 1x10 10 , more preferably 1x10 4 Up to 1x10 9 cfu / ml of aqueous medium containing milk matrix.

[0059] Fermentation is preferably carried out at a temperature of about 20 to 50° C., more preferably 30 to 45° C., even more preferably about 37 to 42° C. The optimal temperature for growth and / or activation of lactic acid-producing bacteria (especially Lactobacillus and / or Bifidobacterium) is 37 to 42° C.

[0060] The incubation is preferably carried out at a pH of 4 to 8, more preferably 6 to 7.5. This pH value does not cause protein precipitation and / or bad taste, while at the same time the lactic acid producing bacteria such as Lactobacillus and / or Bifidobacterium are able to ferment the milk matrix.

[0061] The incubation time is preferably 10 minutes to 48 hours, preferably 2 hours to 24 hours, more preferably 4 hours to 12 hours. A sufficiently long time allows fermentation and the concomitant production of immunogenic cell debris (e.g., glycoproteins, glycolipids, peptidoglycans, lipoprotein acid (LTA), flagella, lipoproteins, DNA and / or capsular polysaccharides) and metabolites (postbiotics) to proceed to a sufficient or higher degree, while for economic reasons, the incubation time does not need to be too long.

[0062] Preferably, the product of milk origin or milk matrix (preferably skim milk) is pasteurized, cooled and fermented to a certain acidity with one or more lactic acid producing strains (preferably thermophilic Streptococcus strains), 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 for fermentation. Subsequently, the two fermented products are preferably mixed together and mixed with other components (except the fat component) constituting the infant formula. Preferably, the mixture is preheated, and then the fat is added online, homogenized, pasteurized and dried. Alternatively, bifidobacteria (preferably breve bifidobacteria) and thermophilic Streptococcus are fermented in a fermenter at the same time.

[0063] Methods for preparing fermented compositions suitable for the purposes of the present invention are known per se. EP 778885 (incorporated herein by reference) discloses a suitable method for preparing a fermented composition, in particular in Example 7. FR 2723960 (incorporated herein by reference) discloses a suitable method for preparing a fermented composition, in particular in Example 6. Briefly, a milk base (preferably pasteurized) containing lactose and optionally other macronutrients such as fat (preferably vegetable fat), casein, whey protein, vitamins and / or minerals, etc., is concentrated to, for example, 15% to 50% dry matter and then inoculated with thermophilic Streptococcus, for example containing 10 6 Up to 10 10Preferably, the milk matrix comprises milk protein peptides. The temperature and duration of fermentation are as described above. Suitably, after fermentation, the fermented composition may be pasteurized or sterilized and, for example, spray-dried or freeze-dried to provide a form suitable for formulation in a final product.

[0064] Preferred methods for preparing fermented compositions for use in the nutritional compositions of the present invention are disclosed in WO 01 / 01785, more particularly in Examples 1 and 2. Preferred methods for preparing fermented compositions for use in the nutritional compositions of the present invention are disclosed in WO 2004 / 093899, more particularly in Example 1.

[0065] After fermentation, the living cells of the lactic acid producing bacteria in the fermented composition are preferably eliminated, for example by inactivation and / or physical removal. The cells are preferably inactivated. Preferably, the lactic acid producing bacteria are heat-killed after fermentation of the milk matrix. Preferred heat-killing methods are (flash) pasteurization, sterilization, ultra-high temperature treatment, high temperature / short time heat treatment and / or spray drying at a temperature at which the bacteria cannot survive. The cell fragments are preferably obtained by heat treatment. By such heat treatment, preferably at least 90% of the live microorganisms are inactivated, more preferably at least 95%, even more preferably at least 99%. Preferably, the fermented nutritional composition comprises less than 1x10 5 The heat treatment is preferably carried out at 70 to 180°C, preferably 80 to 150°C for preferably about 3 minutes to 2 hours, and the heat treatment is preferably carried out at a temperature of 80 to 140°C for 5 minutes to 40 minutes. The inactivation of lactic acid bacteria advantageously makes post-acidification less and the product safer. This is particularly advantageous when the nutritional composition is to be given to an infant or a young child, and a beneficial effect on rotavirus infection is still observed using a fermented composition comprising inactivated lactic acid bacteria. Suitably, after fermentation, the fermented composition can be pasteurized or sterilized, and for example spray-dried or freeze-dried to provide a form suitable for formulation in the final product.

[0066] Non-digestible oligosaccharides

[0067] The present nutritional composition comprises non-digestible oligosaccharides, and preferably comprises at least two different non-digestible oligosaccharides, in particular non-digestible oligosaccharides of two different origins. Non-digestible oligosaccharides are required to improve the effect on rotavirus infection. The presence of both non-digestible oligosaccharides and at least part of the fermented composition, in particular a product of milk origin obtained by fermentation with lactic acid producing bacteria, is required to improve the anti-rotavirus effect, in particular the binding of rotavirus particles or the blocking of particles from the adhesion sites of intestinal epithelial cells.

[0068] As used herein, the term "oligosaccharide" refers to a sugar having a degree of polymerization (DP) of 2 to 250, preferably DP of 2 to 100, more preferably DP of 2 to 60, and even more preferably 2 to 10. The present nutritional composition includes oligosaccharides having a DP of 2 to 100, which may result in a composition comprising oligosaccharides having a DP of 2 to 5, DP of 50 to 70, and DP of 7 to 60. As used herein, "non-digestible oligosaccharides" refer to oligosaccharides that are not digested in the intestine under the action of acids or digestive enzymes present in the upper gastrointestinal tract of humans (e.g., small intestine and stomach), but the oligosaccharides are preferably fermented by human intestinal microflora. For example, sucrose, lactose, maltose, and maltodextrin are considered digestible.

[0069] Preferably, the non-digestible oligosaccharides of the present invention are soluble. The term "soluble" as used herein, when referring to polysaccharides, fibers or oligosaccharides, means that the material is at least soluble according to the method described by L. Prosky et al., J. Assoc. Off. Anal. Chem. 71, 1017-1023 (1988).

[0070] In the method or use of the present invention, the non-digestible oligosaccharide included in the present nutritional composition preferably includes a mixture of different non-digestible oligosaccharides. Non-digestible oligosaccharides are preferably selected from: fructooligosaccharides (such as inulin), non-digestible dextrins, galacto-oligosaccharides (such as trans-galacto-oligosaccharides), xylooligosaccharides, arabino-oligosaccharides, arabino-galacto-oligosaccharides, gluco-oligosaccharides, gentio-oligosaccharides, glucomannan-oligosaccharides, galacto-oligosaccharides, manno-oligosaccharides, isomaltooligosaccharides, nigero-oligosaccharides, glucomannan-oligosaccharides, chitosan-oligosaccharides, soybean oligosaccharides, fucoidan-oligosaccharides, sialic acid oligosaccharides and mixtures thereof. Such oligosaccharides have many common biochemical properties and have similar functional advantages, including improving intestinal microbial function. It should also be understood that some non-digestible oligosaccharides and preferably some mixtures have even further improved effects. Therefore, more preferably, the non-digestible oligosaccharide is selected from fructooligosaccharides (such as inulin) and galacto-oligosaccharides (such as β-galacto-oligosaccharides), and mixtures thereof, even more preferably β-galacto-oligosaccharides and / or inulin, most preferably β-galacto-oligosaccharides. In one embodiment, in the nutritional composition of the present invention, the non-digestible oligosaccharide is selected from galacto-oligosaccharides, fructooligosaccharides and mixtures thereof, more preferably β-galacto-oligosaccharides, fructooligosaccharides and mixtures thereof.

[0071] The non-digestible oligosaccharide is preferably selected from β-galacto-oligosaccharides, α-galacto-oligosaccharides and galactans. According to a more preferred embodiment, the non-digestible oligosaccharide is β-galacto-oligosaccharide. Preferably, the non-digestible oligosaccharide comprises galacto-oligosaccharides having β(1,4), β(1,3) and / or β(1,6) glycosidic bonds and terminal glucose. Trans-galacto-oligosaccharides are, for example, commercially available under the name GOS (Domo Friesland Campina Ingredients), Bi2muno (Clasado), Cup-oligo (Nissin Sugar) and Oligomate 55 (Yakult) were purchased.

[0072] The non-digestible oligosaccharides preferably include fructo-oligosaccharides. Fructo-oligosaccharides may have names such as fructo-oligosaccharides, oligofructose, polyfructose, polyfructan, inulin, levan and fructan in other cases, and may refer to oligosaccharides comprising β-linked fructose units, preferably via β(2,1) and / or β(2,6) glycosidic bonds, and preferably having a DP of 2 to 200. Preferably, the fructo-oligosaccharide comprises terminal β(2,1) glycosidically bonded glucose. Preferably, the fructo-oligosaccharide comprises at least 7 β-linked fructose units. In another preferred embodiment, inulin is used. Inulin is a class of fructo-oligosaccharides in which at least 75% of the glycosidic bonds are β(2,1) bonds. Typically, the average chain length of inulin is 8 to 60 monosaccharide units. Suitable fructo-oligosaccharides for use in the compositions of the present invention may be found under the trade name HP (Orafti) is commercially available. Other suitable sources are Raftilose (Orafti), Fibrulose and Fibruline (Cosucra) and Frutafit and Frutalose (Sensus).

[0073] Preferably, the present nutritional composition comprises a mixture of galacto-oligosaccharides and fructo-oligosaccharides. Preferably, the mixture of galacto-oligosaccharides and fructo-oligosaccharides is present in a weight ratio of 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, more preferably 5 / 1 to 12 / 1, even more preferably 8 / 1 to 10 / 1, even more preferably 9 / 1. This weight ratio is particularly advantageous when the average DP value of galacto-oligosaccharides is low and the DP value of fructo-oligosaccharides is high. Most preferably, a mixture of galacto-oligosaccharides having an average DP lower than 10, preferably lower than 6, and fructo-oligosaccharides having an average DP higher than 7, preferably higher than 11, even more preferably higher than 20.

[0074] Preferably, the present nutritional composition 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 is present in a weight 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 / 5 to 15 / 1, more preferably 1 / 1 to 10 / 1. Preferred is a mixture of short-chain fructo-oligosaccharides having an average DP lower than 10, preferably lower than 6, and fructo-oligosaccharides having an average DP higher than 7, preferably higher than 11, even more preferably higher than 20.

[0075] Preferably, the present nutritional composition comprises a mixture of short-chain fructo-oligosaccharides and short-chain galacto-oligosaccharides. Preferably, the mixture of short-chain fructo-oligosaccharides and short-chain galacto-oligosaccharides is present in a weight 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 / 5 to 15 / 1, more preferably 1 / 1 to 10 / 1. Preferred is a mixture of short-chain fructo-oligosaccharides and galacto-oligosaccharides having an average DP of less than 10, preferably less than 6.

[0076] Preferably, the nutritional composition does not include pectin-derived acidic oligosaccharides. Such oligosaccharides are rich in galacturonic acid oligosaccharides. In the context of the present invention, pectin-derived acidic oligosaccharides are characterized in that they are a mixture of oligomers of galacturonic acid with a degree of polymerization of 2-30. Therefore, preferably, the present composition does not include galacturonic acid oligosaccharides. These oligosaccharides are known for their ability to block pathogens from adhering to receptors in the intestine. Surprisingly, however, when non-digestible oligosaccharides are combined with the fermented nutritional composition, their presence is not required to achieve maximum blocking effect on rotavirus particles.

[0077] The present nutritional composition preferably comprises, based on the dry weight of the nutritional composition, 2.5 to 20 wt% of total non-digestible oligosaccharides, more preferably 2.5 to 15 wt%, even more preferably 3.0 to 10 wt%, most preferably 5.0 to 7.5 wt%. Based on 100 ml of the nutritional composition, the present nutritional composition preferably comprises 0.35 to 2.5 wt% of total non-digestible oligosaccharides, more preferably 0.35 to 2.0 wt%, even more preferably 0.4 to 1.5 wt%. Smaller amounts of non-digestible oligosaccharides are less effective in improving the anti-rotavirus effect, while too much content may cause side effects of bloating and abdominal discomfort.

[0078] Nutritional composition

[0079] The nutritional composition used according to the invention may also be regarded as a pharmaceutical composition and is suitable for administration to infants.The present nutritional composition is preferably for enteral administration, more preferably for oral administration.

[0080] The present nutritional composition is preferably an infant formula, follow-up formula, infant milk or infant formula, or a growing up milk for infants or infant formula. More preferably, the nutritional composition is an infant formula or follow-up formula. The present nutritional composition can advantageously be used as complete nutrition for infants. Preferably, the present nutritional composition is an infant formula. Infant formula is defined as a formula for infants, and for example can be a starting formula intended for infants aged 0 to 6 or 0 to 4 months. Follow-up formula is suitable for infants aged 4 or 6 months to 12 months. At this age, infants begin to wean and eat other foods. Infant milk or growing up milk or infant formula is suitable for children aged 12 to 36 months. The present composition preferably includes a lipid component, a protein component and a carbohydrate component, and is preferably given in liquid form. The present nutritional composition can also be in the form of dry food, preferably in powder form, with instructions for mixing the dry food (preferably powder) with a suitable liquid (preferably water). The nutritional composition used according to the invention preferably comprises other parts such as vitamins, minerals, trace elements and other micronutrients to make it a complete nutritional composition.Preferably, the infant formula comprises vitamins, minerals, trace elements and other micronutrients according to international standards.

[0081] The present nutritional composition preferably includes lipid, protein and digestible carbohydrate, wherein lipid provides 5% to 50% of total calories, protein provides 5% to 50% of total calories, and digestible carbohydrate provides 15% to 90% of total calories. Preferably, in the present nutritional composition, lipid provides 35% to 50% of total calories, protein provides 7.0% to 12.5% ​​of total calories, and digestible carbohydrate provides 40% to 55% of total calories. In order to calculate the total calorie % of protein, the total energy provided by protein, peptides and amino acids needs to be considered. Preferably, lipid provides 3 to 7g lipid / 100kcal, preferably 4 to 6g / 100kcal, protein provides 1.6 to 4g / 100kcal, preferably 1.7 to 2.5g / 100kcal, and digestible carbohydrate provides 5 to 20g / 100kcal, preferably 8 to 15g / 100kcal of the nutritional composition. Preferably, the present nutritional composition includes lipids providing 4 to 6g / 100kcal, proteins providing 1.6 to 2.0g / 100kcal, more preferably 1.7 to 1.9g / 100kcal, and digestible carbohydrates providing 8 to 15g / 100kcal nutritional composition. In one embodiment, lipids provide 3 to 7g / 100kcal, preferably 4 to 6g / 100kcal, proteins provide 1.6 to 2.1g / 100kcal, preferably 1.6 to 2.0g / 100kcal, carbohydrates provide 5 to 20g / 100kcal, preferably 8 to 15g / 100kcal nutritional composition, and wherein, preferably, the digestible carbohydrate component includes, based on total digestible carbohydrates, at least 60% by weight of lactose, more preferably at least 75% by weight, even more preferably based on total digestible carbohydrates, at least 90% by weight of lactose. The amount of total calories is determined by the sum of calories produced by protein, lipids, digestible carbohydrates and non-digestible oligosaccharides.

[0082] The present nutritional composition preferably includes 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 includes lactose. Since the present nutritional composition includes a fermented composition obtained by fermentation of lactic acid bacteria, the amount of lactose is reduced compared to its source due to the conversion of lactose into lactate and / or lactic acid by fermentation. Therefore, in the preparation of the present nutritional composition, lactose is preferably added. Preferably, in addition to lactose, the present nutritional composition does not include a large amount of carbohydrates. Compared with digestible carbohydrates (such as maltodextrin, sucrose, glucose, maltose and other digestible carbohydrates with high glycemic index), lactose has a lower glycemic index and is therefore preferred. The present nutritional composition preferably includes 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, and most preferably at least 95% by weight of digestible carbohydrates are 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.

[0083] The present nutritional composition preferably includes at least one lipid selected from animal lipids (excluding human lipids) and plant lipids. Preferably, the present composition includes a combination of plant lipids and at least one oil selected from fish oil, animal oil, algae oil, fungal oil and bacterial oil. The lipid of the present nutritional composition preferably provides a nutritional composition of 3 to 7g / 100kcal, and preferably lipid provides 4 to 6g / 100kcal. When in liquid form, for example, as ready-to-feed liquid, the nutritional composition preferably includes 2.1 to 6.5g lipid / 100ml, more preferably 3.0 to 4.0g / 100ml. Based on dry weight, the present nutritional composition preferably includes 12.5 to 40% by weight of lipid, more preferably 19 to 30% by weight. Preferably, lipid includes 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 in the form of free fatty acids, in the form of triglycerides, in the form of diglycerides, in the form of monoglycerides, in the form of phospholipids, or as a mixture of one or more of the above. Preferably, the nutritional composition comprises at least one, preferably at least two lipid sources selected from rapeseed oil (such as colza oil, rapeseed oil and canola oil), high oleic sunflower oil, high oleic safflower oil, olive oil, marine oil, microbial oil, coconut oil, palm kernel oil. The nutritional composition is not human milk.

[0084] The present nutritional composition preferably includes protein. The protein used in the nutritional composition is preferably selected from non-human animal proteins, preferably milk proteins, plant proteins, such as preferably soy proteins and / or rice proteins, and mixtures thereof. The present nutritional composition preferably comprises casein and / or whey protein, more preferably bovine whey protein and / or bovine casein. Therefore, in one embodiment, the protein in the present nutritional composition includes proteins selected from whey protein and casein, preferably whey protein and casein, preferably, whey protein and / or casein are from milk. Preferably, the total protein based on free amino acids, dipeptides, tripeptides or hydrolyzed proteins, the protein composition is less than 5% by weight. The present nutritional composition preferably includes casein and whey protein, and the weight ratio of casein: whey protein is 10:90 to 90:10, more preferably 20:80 to 80:20, even more preferably 35:65 to 55:45.

[0085] The protein weight % is calculated according to the Kjeldahl-method by measuring the total nitrogen and using a conversion factor of 6.38 in the case of casein or 6.25 in the case of proteins other than casein, based on the dry weight of the nutritional composition. The term "protein" or "protein component" as used in the present invention refers to the sum of proteins, peptides and free amino acids.

[0086] The present nutritional composition preferably includes a protein providing 1.6 to 4.0g protein / 100kcal nutritional composition, preferably providing 1.6 to 3.5g / 100kcal nutritional composition, and even more preferably 1.75 to 2.5g / 100kcal nutritional composition. In one embodiment, the present nutritional composition includes a protein providing 1.6 to 2.1g protein / 100kcal nutritional composition, preferably providing 1.6 to 2.0g, more preferably 1.7 to 2.1g, and even more preferably 1.75 to 2.0g / 100kcal nutritional composition. In one embodiment, the present nutritional composition includes a protein content of less than 2.0g / 100kcal protein, preferably providing 1.6 to 1.9g, and even more preferably 1.75 to 1.85g / 100kcal nutritional composition. Based on total calories, too low a protein content will result in insufficient growth and development of infants and young children. Too high a quantity can increase the metabolic burden of, for example, the kidneys of infants and young children. When in liquid form, e.g. as a ready-to-feed liquid, the nutritional composition preferably comprises 0.5 to 6.0 g, more preferably 1.0 to 3.0 g, even more preferably 1.0 to 1.5 g protein / 100 ml, most preferably 1.0 to 1.3 g protein / 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 the dry weight of the total nutritional composition, more preferably 8 to 14 wt%, even more preferably 8 to 9.5 wt% protein, based on the dry weight of the total nutritional composition.

[0087] In order to meet the caloric demand of infants or young children, the nutritional composition preferably includes 45 to 200kcal / 100ml liquid. For infants, the nutritional composition more preferably has 60 to 90kcal / 100ml liquid, even more preferably 65 to 75kcal / 100ml liquid. This caloric density ensures the optimal ratio between water and caloric consumption. For young children--human subjects aged between 12 to 36 months, the nutritional composition more preferably has a caloric density of 45 to 65, even more preferably 50 to 60kcal / 100ml. The osmotic pressure (osmolarity) of this composition is preferably 150 to 420mOsmol / l, more preferably 260 to 320mOsmol / l. The purpose of low osmotic pressure is to further reduce gastrointestinal pressure, which may affect rotavirus infection.

[0088] When the nutritional composition is in a ready-to-feed liquid form, the preferred volume administered per day is about 80 to 2500 ml, more preferably about 200 to 1200 ml per day. 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 volume administered per day is 200 ml to 1200 ml, and wherein the number of feedings per day is 1 to 10.

[0089] When in liquid form, the present nutritional composition preferably has a viscosity of 1 to 60 mPa.s, preferably 1 to 20 mPa.s, more preferably 1 to 10 mPa.s, and most preferably 1 to 60 mPa.s. Low viscosity ensures proper administration of the liquid, for example, proper passage through the entire nipple. Moreover, the viscosity is very similar to the viscosity of human milk. In addition, low viscosity leads to normal gastric emptying and better energy absorption, which is essential for infants who need energy for optimal growth and development. Alternatively, the present nutritional composition is in powder form, suitable for reconstitution with water as a ready-to-drink liquid. The present nutritional composition is preferably prepared by mixing the powdered composition with water. Typically, infant formula is prepared in this way. Therefore, the present invention also relates to a packaged powder composition, wherein the package also provides instructions - mix the powder with an appropriate amount of liquid to obtain a liquid composition with a viscosity of 1 to 60 mPa.s. The liquid composition was measured using a Physica Rheometer MCR 300 (Physica Messtechnik GmbH, Ostfilden, Germany) at 20°C for 95s -1 The shear rate is used to measure the viscosity of a liquid.

[0090] application

[0091] The method or use of the present invention comprising administering the present nutritional composition is also referred to as administering an effective amount of the nutritional composition to a subject, preferably an infant or a young child, in need of such treatment.

[0092] The inventors have found that the combination of fermented formula foods (providing postbiotic compounds) and non-digestible oligosaccharide mixtures shows the ability to improve rotavirus infection and rotavirus-induced diarrhea. Fermented formula foods rich in non-digestible oligosaccharides reduce the incidence, duration and severity of diarrheal processes. In addition, the combination can directly interact with the virus and enhance the host immune system. When the direct blockade of rotavirus particles was determined, an unexpected improvement effect of fermented formula foods with non-digestible oligosaccharides was observed compared to fermented formula foods or non-digestible oligosaccharides that do not include non-digestible oligosaccharides. This indicates that the rotavirus load in the gastrointestinal tract is reduced. Viral load, also known as viral load or viral titer, is a numerical expression of the number of infectious virus particles per milliliter. Higher viral load, titer or load is generally associated with severity or infection.

[0093] In addition, the greatest anti-rotavirus effect for duration, morbidity and severity was observed in the group given both non-digestible oligosaccharides and fermented formula. In particular, the effect of shortening duration was observed only in this group, which could not be explained by the effects of non-digestible oligosaccharides or fermented formula alone. Interestingly, the combination of fermented formula and non-digestible oligosaccharides maximized the intestinal barrier function during rotavirus infection, as assessed by intestinal barrier permeability using α1 antitrypsin test. Without wishing to be bound by theory, the observed improvement in intestinal barrier function can be explained by the unexpected increase in butyrate in the intestine. Butyrate is known to serve as a nutrient for intestinal epithelial cells and improve the intestinal barrier. The increase in butyrate levels exceeded the levels that could be expected based on the levels seen in groups fed non-digestible oligosaccharides or fermented formula alone.

[0094] Without wishing to be bound by theory, the lower viral shedding after the intervention indicates that the analyzed components may have an effect on the binding to the virus. This anti-infective effect of the combination of non-digestible oligosaccharides and fermented formula food, which is independent of microorganisms, may involve direct interaction with pathogens and may be related to receptor mimicry. Therefore, some components of the mixture given may serve as soluble receptor analogs of specific molecules in the host to which the virus can adhere. Therefore, this interaction between the pathogen and the combination of fermented formula food and non-digestible oligosaccharides may cause the adhered pathogen to be removed from the epithelial cells of the small intestine, thereby reducing the infection load or the infectivity of rotavirus, thereby reducing the incidence, duration and / or severity of diarrhea. In addition, reduced viral shedding and different systemic and intestinal immune responses were observed. It is also important to find that rotavirus infection causes a decrease in weight gain, which is mainly observed after the diarrhea period, and the effect on weight gain caused by this rotavirus infection is improved in the group receiving the combination of fermented formula food and non-digestible oligosaccharide mixture. In this group, not only the effect on growth, especially body weight, but also the effect on relative organ weight was observed. When compared to the control group, the weight of the spleen, liver, small intestine and large intestine in the RV group were all reduced, even in relative weight (% weight of total body weight). This suggests that after rotavirus infection or rotavirus-induced diarrhea, not only growth but also development is affected. However, in the group receiving the combination of fermented formula and non-digestible oligosaccharide mixture, these effects were improved and more similar to the uninfected control group. Prevention of weight loss or prevention of decreased growth rate or poor growth (especially weight gain), or prevention of stunted development of organs (such as liver, spleen, small intestine and / or large intestine) is crucial for growing and developing infants or young children. These observations indicate that when the nutritional composition of the present invention is administered, both growth (especially weight gain) and development of infants or young children are improved after rotavirus infection or rotavirus-induced diarrhea.

[0095] Therefore, in one embodiment, the present invention relates to a method or use for preventing growth retardation or poor growth and / or developmental delay or poor reduction (more preferably, reducing weight gain, or weight loss) caused by rotavirus infection in infants or young children. Preferably, the method or use for preventing growth retardation or poor growth and / or developmental delay or poor development caused by rotavirus infection in infants or young children is to prevent growth retardation or poor growth and / or developmental delay or poor development after rotavirus infection or rotavirus-induced diarrhea. The method or use can be achieved by administering a nutritional composition that is at least partially fermented by lactic acid-producing bacteria and includes non-digestible oligosaccharides.

[0096] Therefore, in one embodiment, the present invention relates to a method or use for preventing and / or treating rotavirus infection or rotavirus-induced diarrhea. More specifically, in different embodiments, the effect on rotavirus infection or rotavirus-induced diarrhea is to reduce the incidence of rotavirus-induced diarrhea or rotavirus infection, or to reduce the duration of rotavirus-induced diarrhea or rotavirus infection, or to reduce the severity of rotavirus-induced diarrhea or rotavirus infection. In a further embodiment, the effect on rotavirus infection or rotavirus-induced diarrhea is to prevent growth retardation or poor growth and / or developmental delay or developmental stunting caused by rotavirus, or to improve the blocking of rotavirus particles, or to reduce rotavirus load or improve intestinal barrier function during rotavirus infection. In one embodiment, the present invention relates to a method or use for blocking rotavirus particles, reducing rotavirus load and / or reducing rotavirus shedding. This is achieved by giving the above-mentioned nutritional composition to an infant or young child, which is at least partially fermented by lactic acid bacteria and includes non-digestible oligosaccharides, particularly scGOS and lcFOS.

[0097] In a further embodiment, the present invention relates to a method for reducing the incidence of rotavirus-induced diarrhea or rotavirus infection, or reducing the duration of rotavirus-induced diarrhea or rotavirus infection, or reducing the severity of rotavirus-induced diarrhea or rotavirus infection, or preventing rotavirus-induced growth retardation or poor growth and / or developmental retardation or poor development, or improving the blocking of rotavirus particles, or reducing rotavirus load, or improving intestinal barrier function during rotavirus infection by administering the above-mentioned nutritional composition to an infant or a young child, wherein the above-mentioned nutritional composition is at least partially fermented by lactic acid producing bacteria and comprises non-digestible oligosaccharides, in particular scGOS and lcFOS.

[0098] For all methods and uses, the claimed beneficial effects are compared to infants not given the nutritional composition of the invention, in other words compared to infants given a nutritional composition that is not at least partially fermented and does not comprise non-digestible oligosaccharides.

[0099] The present nutritional composition is administered to an infant or toddler aged 0 to 36 months. Preferably, the nutritional composition is administered to an infant, ie a human subject aged 0 to 12 months, more preferably an infant aged 0 to 6 months.

[0100] In this document and its claims, the verb "to comprise" and its variations are used in its non-restrictive sense, meaning that the items following the word are included, but items not specifically mentioned are not excluded. In addition, an element referred to 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 the presence of one and only one of the element. Thus, the indefinite article "a" or "an" usually means "at least one". Weight % (wt.%) means percentage by weight. Unless otherwise stated, a day means 24 hours (starting and ending at midnight).

[0101] Example

[0102] Example 1: Effects of fermented formula food and non-digestible oligosaccharides on rotavirus infection in rats

[0103] Materials and methods

[0104] Preparation of rotavirus (RV) strains (simian SA-11) for infection was described in Pérez-Cano FJ et al., 2007, Pediatr. Res. 2007; 62: 658-663. Briefly, virus propagation was performed in fetal African green monkey kidney cells (MA-104) and the TCID 50 The virus was prepared according to the current principles of Good Laboratory Practices.

[0105] G14 pregnant Lewis rats were obtained from Janvier (Le-Genest-Saint-Isle, France) and housed individually in cages (2184L Eurostandard Type II L, Tecniplast, West Chester, PA, USA) with large fiber particle bedding and tissue paper as enrichment. They were monitored daily and delivered at full term. The date of birth was registered as the first day of life. A litter of pups was divided into seven pups / mother rats, with similar numbers of males and females in each litter. The pups had free access to nipples and rat chow. The mother rats had access to water and commercial feed (Teklad Global Diet 2014, Envigo, Indianapolis, IN, USA) corresponding to the American Institute of Nutrition (AIN)-93G diet ad libitum. Each mother rat and its litter of pups were raised individually under controlled temperature and humidity conditions with a 12h:12h light:dark cycle. Additionally, the animals were housed in a dedicated safety isolation room that was specifically designed and approved to work under Biosafety Level 2 conditions.

[0106] The experimental nutrients or nutritional components tested were:

[0107] 1) Heat treated fermented infant formula. Fermented milk is obtained by fermenting infant formula with Bifidobacterium breve C50 and Streptococcus thermophilus 065 during the manufacturing process. This process adds active bacterial metabolites and cell debris (postbiotic compounds) to the composition of the formula. It is sold on the French market under the names Calisma and Apaisia, as a product of Bledina. A suitable method for obtaining this infant formula is described in Example 1 of WO 2004 / 093899.

[0108] 2) Non-digestible oligosaccharides scGOS (Source: GOS, Borculo Domo FrieslandCampina) and lcFOS (Source: HP, Orafti) in a weight / weight (wt. / wt.) ratio of 9:1.

[0109] 3) Excipient: The excipient of the nutritional composition or ingredients in 1 and 2 is mineral water.

[0110] Newborn lactating G14 Lewis rats were divided into several different experimental groups:

[0111] 1) Reference (REF) group, i.e., a control group that was not infected with rotavirus and did not receive dietary intervention

[0112] 2) Rotavirus (RV) group, i.e., control group infected with rotavirus and not receiving dietary intervention

[0113] 3) Study group (FM+G / F), i.e., infected with rotavirus and supplemented with the mixture of fermented milk (FM) and non-digestible oligosaccharide scGOS / lcFOS (G / F) described above. The dosage of FM and G / F was 3 g / 100 g body weight / day and 0.8 g / 100 g body weight / day, respectively.

[0114] Each group consisted of three litters with seven pups per litter (n=21 / group). REF and RV groups received the same amount of vehicle as the FM+G / F group until the last day of the experiment. RV vaccination was performed on day 7 of life.

[0115] The suckling mice in the RV and FM+G / F groups were inoculated with SA-11 (2x10 8 TCID 50 / rat, 100 μL phosphate buffered saline (PBS). To avoid interference between RV and milk components, RV inoculation was performed 1 hour after separation from its mother. The REF group received the same volume of PBS under the same conditions.

[0116] SA-11 infection was assessed on days 8-21 by growth rate and clinical indicators from stool samples. Fecal sampling was performed daily by gentle pressure and massage of the abdomen. The specimens were immediately scored, weighed, and frozen at -20°C for further analysis.

[0117] Animals were euthanized at two different time points: three animals per litter were euthanized one week after RV inoculation, on day 14 (n = 9 / group), while the other four animals per litter continued to receive feed until the end of lactation, on day 21 (n = 12 / group). On the day of sacrifice, blood was collected by cardiac puncture to determine the hematocrit (HCT) and serum was obtained and stored at -20°C until further analysis. The liver, spleen, large intestine and small intestine were weighed. In addition, the small intestine was cut into 5 mm segments and incubated in PBS at 37°C on a shaker for 10 minutes to obtain the intestinal wash fluid (GW). After centrifugation, the supernatant was stored at -20°C until analysis.

[0118] The severity of diarrhea was assessed by stool weight and stool scoring. According to color, texture and amount, stool samples were scored from 1 to 4 points (diarrhea index, DI) according to the following criteria: normal (diarrhea index = 1), loose yellow-green (diarrhea index = 2), completely loose yellow-green (diarrhea index = 3), and a lot of watery (diarrhea index = 4) stool. A diarrhea score ≥ 2 indicates diarrheal stool, while a diarrhea index < 2 indicates the absence of diarrhea. In addition, in order to find the overall value of diarrhea severity, the area under the severity curve (AUC) during diarrhea (days 7-13) was analyzed. The highest diarrhea index was defined as the highest score during diarrhea.

[0119] The incidence of diarrhea is expressed as % animals with diarrhea (%DA) (in terms of the number of animals in each group) and % feces with diarrhea (%DF) (taking into account the total sample size collected per day in each group). The AUCs for % animals with diarrhea and % feces with diarrhea were calculated for days 7-13 as overall values ​​for the incidence. The maximum percentage of animals with diarrhea and feces with diarrhea was defined as the highest value of the diarrhea period. The diarrhea period was calculated for each animal as the interval between the first and last day of diarrhea. The actual number of days with diarrhea within the diarrhea period was also counted.

[0120] In all cases, the mean values ​​and AUCs for severity, DA and DF were calculated taking into account the baseline values ​​of the intrinsic aspects of each treatment; in principle, the baseline values ​​of the day before rotavirus vaccination were subtracted due to the intrinsic aspects of each treatment (normalized results). This effect was particularly prominent in the group consuming G / F due to the intrinsic stool softening effect.

[0121] UV-inactivated SA-11 particles were 5 / mL was used to cover 96-well plates (Nunc MaxiSorp, Wiesbaden, Germany). After blocking with PBS-1% bovine serum albumin (BSA, 1 hour, room temperature [RT]), appropriately diluted serum or intestinal wash samples were added (3 hours, room temperature). After washing, peroxidase-conjugated rabbit anti-rat Ig purchased from Dako (Barcelona, ​​Spain) or mouse biotinylated anti-rat IgA (A93-2), IgG1 / 2a (R19-15) or IgM (G53-238) monoclonal antibodies (MAbs) purchased from BD Biosciences (Heidelberg, Germany) were added. Afterwards, peroxidase-conjugated extravidin (Sigma-Aldrich, Madrid, Spain) and substrate were added. Pooled sera from previously studied RV-infected animals were used as control positives in each plate. A quadratic polynomial adjustment was used. The concentrations of total anti-RV Ab and anti-RV IgG, IgA, and IgM Abs in the RV group were 100 arbitrary units (AU) / mL, and the results of the FM+P group and day 21 were expressed with respect to this given value.

[0122] Fecal samples up to 10 days after virus inoculation (7-16 days old) were diluted in PBS at a concentration of 20 mg / mL and homogenized using a Polytron (Kinematica, Luzern, Switzerland). Fecal homogenates were centrifuged (200 g, 5 min, 4°C) and the supernatant was frozen at -20°C until use. Viral particles in fecal homogenates were quantified by the above-mentioned ELISA technique.

[0123] For statistical analysis, the PASW Statistics 20 software package (SPSS Inc, Chicago, IL, USA) was used. The Komolgorov-Smirnov and Levene tests were used to assess normal distribution and equality of variance, respectively. Conventional one-way ANOVA was performed considering the experimental group as the independent variable. When SA-11 vaccination / treatment had a significant effect on the dependent variable, the Scheffé post hoc test was used. When nonnormal distribution or different variances were found, the Kruskal-Wallis and Mann-Whitney U tests were used. Finally, the chi-square test was used to compare the incidence of diarrhea. Differences were considered significant when P values ​​< 0·05. All results are expressed as the mean and SEM of the number of animals.

[0124] result

[0125] Effect on growth:

[0126] The body weight of all animals was recorded, and neither diet nor viral infection affected their growth. No differences were observed until day 1 after inoculation (day 14). This means that weight loss due to infection or dietary effects can be ignored. However, at the end of the experiment - day 21 (n=9 / group), some differences were observed. The body weight of the RV group decreased significantly compared to the REF and FM+G / F groups. If the overall body weight gain (% from day 2 to day 21) is taken into account, this weight loss induction in the RV group is still observed (p<0.05), and the protective effect of the FM+G / F intervention (see Table 1).

[0127] Table 1: Body weight (g) before and after virus inoculation on day 7 during the study period. Results are expressed as mean and SEM (n=21 animals / group).

[0128] age BW(g) REF RV FM+G / F day average SEM average SEM average SEM 2 6.45 0.10 6.92 0.24 6.22 0.10 7 11.66 0.24 11.83 0.22 11.10 0.20 14 23.32 0.37 23.17 0.41 22.96 0.48 21 34.6 0.66 28.76* 0.42 36.11 0.40

[0129] *p<0.05 vs. REF

[0130] In the REF group, a relative weight gain of 411.11% was observed. In the RV group, this value was significantly lower (p<0.05) and was 310.45%; in the FM+G / F group, no such decrease in weight gain was observed, and the relative increase was 439.90%.

[0131] Effects on organs

[0132] Rotavirus infection also caused some changes in organ weights on days 14 and 21. Specifically, RV infection caused a decrease in relative spleen weight and an increase in relative liver weight (p<0.05vs.REF) 7 days after infection (day 14 of life). Effects on liver weight were also observed in the FM+G / F group, but the effect on spleen was ameliorated in the FM+G / F group. Results on day 21 demonstrated a significant effect on the RV group compared to the REF group (p<0.05), with lower relative weights of spleen, liver and small intestine (SI). The relative weight of the large intestine was also lower. However, supplementation with the FM+G / F mixture prevented the effects of RV infection on these variables (p<0.05vs RV), and relative body weights were higher and not statistically different from the reference group.

[0133] The incidence of diarrhea

[0134] The incidence of diarrhea was assessed using two different methods: % of animals with diarrhea (%DA) and % of feces with diarrhea (%DF).

[0135] The %DA showed that almost all RV-vaccinated animals (95% to 100%) exhibited diarrhea at some time. As expected, less than 5% of animals in the REF group (no RV vaccination) developed diarrhea. During the first 4 days after vaccination with SA-11 (days 8 to 11 of life), the %DA in the RV group was above 80%. Thereafter, the percentage decreased, and on day 13, no animals in the RV group had diarrhea-like feces. Animals in the FM+G / F group showed some improvement during diarrhea. Specifically, this group showed a trend of decreased %DA relative to the RV group, which began two days after infection induction (days 8-11). Improvements in diarrhea incidence were observed on days 9 and 10 of life, which were significantly lower than those in the RV group (p<0.05).

[0136] Diarrhea incidence was calculated as %DF, which only takes into account the number of diarrheal stools regarding the stool samples obtained, and all groups showed the same pattern as the above results, with a higher beneficial effect observed in the FM+G / F group; showing a lower % diarrheal stools on days 8, 9, 10, 11 and 12.

[0137] The diarrheal process in the RV group started 1-2 days after inoculation and ended around the 11th day of life. Both the diarrheal period and the number of diarrheal days were between 3 and 4 days. In the FM+G / F group, the length of these periods was slightly shorter, especially when looking at the diarrheal feces (DF) %. In this case, the duration of diarrhea was shortened and no diarrheal feces were observed already on the 11th day. In any case, the RV group had the maximum morbidity (%DA and DF%) on the 3rd day after RV induction (day 10), while the FM+G / F group showed the highest values ​​on the first day after infection (day 8).

[0138] When %DA-AUC (area under the curve) and %DF-AUC were calculated, the values ​​for the RV group were 328.57 and 362.06 arbitrary units, respectively, while the REF animals did not develop diarrhea and had AUC values ​​of approximately 0. A significant overall decrease was observed in the FM+G / F group. The %DA-AUC decreased to 207.14 (63% compared to the RV group) and the %DF-AUC decreased to 107.87 (30% of the RV group).

[0139] Severity of diarrhea

[0140] The diarrhea index (DI) of the RV group was above 2.5 from day 8 to day 10, after which the average score decreased to approximately 2 on day 11 and even lower thereafter, indicating that most animals were free of diarrhea. In the FM+G / F group, a significant decrease was observed, and the DI was below 2 on all days post-infection except the first day.

[0141] The AUC of the severity mode (sAUC) for the RV group was 5.99. A significant decrease in the sAUC to 3.27 (approximately 55%) was observed for the FM+G / F group (p<0.05), indicating an overall decrease in the severity of the procedure.

[0142] Viral shedding

[0143] Viral shedding was assessed post-inoculation (days 7-16). In all RV-inoculated animals, maximal viral clearance (p<0.054 vs REF) occurred on the first day post-inoculation (day 8), whereas thereafter, no differences were observed between REF and RV groups. The FM+G / F intervention partially averted this increased shedding on the first day post-inoculation, indicating viral loads that were lower than those of the RV group (approximately 35%; p<0.05) and more similar to REF animals.

[0144] Immune response

[0145] Serum specific anti-RV antibodies (total, IgG, IgM, and IgA) and intestinal IgA and IgM were assessed on days 14 and 21 of life. Compared with the REF group, the RV group had measurable levels of anti-RV total, IgA, IgG, and IgM in serum and intestine even on day 14 of life.

[0146] Supplementation of the diet with FM+G / F was able to modify the specific humoral immune response against RV. On day 14, although total serum anti-RV antibodies did not seem to be affected by the diet, an increasing trend was observed for IgM, a significant increase in IgG (p<0.05 vs RV), and a significant decrease in IgA (p<0.05 vs RV).

[0147] For the intestinal lumen, there was a trend towards induction of higher levels of IgA at day 14 but lower levels at day 21 due to the FM+G / F diet. The FM+G / F mixture appeared to enhance the early response (day 14) in terms of serum anti-RV IgG and intestinal anti-RV IgA.

[0148] Example 2: Rotavirus blocking experiment

[0149] In vitro blocking assays were performed to test the ability of FM+G / F, FM, or G / F to bind SA11 rotavirus. 96-well plates were filled with 5x10 4 SA-11 particles / ml were incubated with nutrients or nutritional components (diluted with mineral water) at different dilutions in 1 wt% PBS-Tween and incubated for 30 minutes. Unbound viral particles were quantified by ELISA (Pérez-Cano F J et al., 2007, Pediatr Res 2007; 62: 658-663). SA-11 viral particles (10 5 Up to 103 / ml) as a standard in each plate. This assay allows quantification of the number of SA-11 particles detected after inoculation with the product of multiple dilutions as well as the percentage of inhibition. FM+G / F in undiluted form was 267 mg / ml, with 211 mg / ml FM and 56 mg G / F. For FM and G / F, the stock solutions were 211 mg / ml and 56 mg / ml, respectively. These amounts were chosen because they correspond to the daily intake in Example 1.

[0150] The results are shown in Table 2. All dilutions of the nutrients or nutritional components used in Example 1 had inhibitory activity. In particular, FM+G / F showed high binding activity, indicating that the mixture had a high blocking potential.

[0151] Table 2: Percent inhibition of RV particle detection after incubation with G / F, FM and FM+G / F at different dilutions of nutrients and / or nutrient components. Results are expressed with their SEM of replicates from three independent experiments.

[0152]

[0153] *p<0.05 vs % inhibition without added product. Nd: not determined

[0154] #p<0.05 vs % inhibition of products with G / F or FM alone

[0155] The binding of rotavirus particles is considered to be beneficial. Their blocking caused by direct interaction will avoid or reduce the entry of rotavirus into enterocytes and replication in the small intestine. Based on the data of G / F and FM alone, the binding of FM+G / F to rotavirus particles is far beyond expectations. These results indicate that consuming a combination of partially fermented compositions and non-digestible oligosaccharides has an unexpectedly improved effect on the prevention and / or treatment of rotavirus infection or rotavirus-induced diarrhea. In particular, when compared to the situation without a fermented composition or without non-digestible oligosaccharides, it can be expected to have an effect on the prevention and reduction of the viral load of rotavirus infection, and this can result in a reduction in the severity, incidence and duration of rotavirus diarrhea, and / or a reduction in viral shedding.

[0156] Example 3: Effects of fermented formula food and non-digestible oligosaccharides on rotavirus infection in rats

[0157] An experiment similar to Example 1 was performed, except that RV inoculation was performed on day 5. Intestinal permeability was additionally measured and cecal short chain fatty acids were measured as known in the art.

[0158] The experimental nutrients or nutritional components tested were:

[0159] 1) Heat-treated fermented infant formula. Fermented milk is obtained by fermenting infant formula using Bifidobacterium breve C50 and Streptococcus thermophilus 065 during the manufacturing process. This process adds active bacterial metabolites and cell debris (postbiotic compounds) to the formula composition. It is sold on the French market under the name Lactofidus or Lactofidia as a product of Bledina. After the fermentation step (10 minutes, 90°C), an additional heating step is used to kill lactic acid-producing bacteria. The difference from the formula in Example 1 is that fermentation and lactic acid production are slightly increased. The amount of L-lactic acid is about 1.1g / 100g.

[0160] 2) Non-digestible oligosaccharides scGOS (Source: GOS, Borculo Domo FrieslandCampina) and lcFOS (Source: HP, Orafti) in a 9:1 weight / weight ratio.

[0161] 3) Excipient: The excipient in the nutritional composition or ingredients 1 and 2 is mineral water.

[0162] Newborn lactating G14 Lewis rats were divided into several different experimental groups:

[0163] 1) Reference (REF) group, i.e., a control group that was not infected with rotavirus and did not receive dietary intervention

[0164] 2) Rotavirus (RV) group, i.e., control group infected with rotavirus and not receiving dietary intervention

[0165] 3) Study group (G / F), i.e., this group was infected with rotavirus and supplemented with a mixture of non-digestible oligosaccharides scGOS / lcFOS (G / F). G / F were given at 0.8 g / 100 g body weight / day respectively.

[0166] 4) Study group (FM), i.e., infected with rotavirus and supplemented with fermented milk (FM). FM was administered at 3 g / 100 g body weight / day.

[0167] 5) Study group (FM+G / F), i.e., infected with rotavirus, supplemented with the above fermented milk (FM) and non-digestible oligosaccharide scGOS / lcFOS mixture (G / F). The dosage of FM and G / F was 3 g / 100 g body weight / day and 0.8 g / 100 g body weight / day, respectively.

[0168] As control groups, 3 groups of rats not infected with rotavirus but fed with the same supplements as groups 3, 4 and 5 were included. The values ​​for diarrhea obtained in these groups were used as baselines to normalize the diarrhea index DI and diarrhea incidence (%DA).

[0169] result:

[0170] According to the diarrhea index (DI), in RV group 2, the diarrhea episode lasted from day 6 (1 day after RV inoculation) to day 9. On day 10, diarrhea had stopped in all groups. The duration of diarrhea determined by DI was the same in groups 3 and 4. The maximum DI during the diarrhea period (days 6-9) was observed in group 2. In group 5, diarrhea based on DI was already significantly lower than in RV group 2 on day 9. Among all days of diarrhea, group 5 had the lowest DI. This indicates that the duration of diarrhea was shorter in experimental group 5.

[0171] The incidence of diarrhea (based on the number of animals with diarrhea out of the total animals, %DA) was the lowest in Group 5. On Day 9, only Group 5 had no incidence of diarrhea, while Groups 2, 3, and 4 still had it, indicating that the combination of FM and NDO shortened the duration or rotavirus infection.

[0172] Table 3: Normalized diarrhea incidence index]

[0173]

[0174] MDA, maximum diarrheal animals (%), DA-AUC: area under the %DA model curve.

[0175] The maximum diarrhea incidence (MDA) - the peak incidence - was significantly reduced in Group 5 compared to Group 2 (using a chi-square test). The MDA was also reduced in Groups 3 and 4 compared to Group 2. The area under the diarrhea index curve during the experiment was the smallest in Group 5 (8.33) and the largest in Group 2 (166.66). In Group 3, the value was 29.52 and in Group 4 it was 86.23. Again, the low value in Group 5 was lower than expected based on the effects of Groups 3 and 4 alone. This suggests that the group that consumed both FM and G / F had a reduced severity.

[0176] Intestinal permeability was determined by alpha-1 antitrypsin levels in intestinal washes on day 8. Interestingly, the combination of FM and G / F maximally increased intestinal barrier function during rotavirus infection.

[0177] The observed improvement in intestinal barrier function can be explained by the unexpected increase in intestinal butyrate in Group 5. Butyrate is a nutrient for intestinal epithelial cells and improves the intestinal barrier. The cecal butyrate level in Group 2 was 239.21 μmol / g cecal material. The increased level of butyrate in Group 5 (484.39 μmol / g cecal material) exceeded expectations based on the levels found in Group 3 (164.75; μmol / g cecal material, i.e., a decrease in butyrate) and Group 4 (309.72 μmol / g cecal material) alone.

Claims

1. Use of a fermented composition and non-digestible oligosaccharides for preparing a nutritional composition, the nutritional composition comprising a milk base fermented by lactic acid-producing bacteria and comprising non-digestible oligosaccharides for treating and / or preventing rotavirus infection or rotavirus-induced diarrhea in infants or young children.

2. Use of a fermented composition and non-digestible oligosaccharides for preparing a nutritional composition, the nutritional composition comprising a milk base fermented by lactic acid-producing bacteria and comprising non-digestible oligosaccharides for use in infants or young children a. Reduce the incidence of rotavirus-induced diarrhea or rotavirus infection, b. Reduce the duration of rotavirus-induced diarrhea or rotavirus infection, c. Reduce the severity of rotavirus-induced diarrhea or rotavirus infection, d. Prevention of rotavirus-induced growth retardation or poor growth and / or developmental retardation or poor development, e. Improve the blocking of rotavirus particles, f. Reduction of rotavirus load, and / or g. Improve intestinal barrier function during rotavirus infection.

3. Use according to claim 2, wherein the reduction, prevention or increase is observed when compared to an infant or toddler administered a nutritional composition which has not been at least partially fermented and does not include non-digestible oligosaccharides.

4. Use according to claim 1 or 2, wherein the nutritional composition comprises 0.1 to 1.5 wt. % lactic acid and lactate in total, based on the dry weight of the composition.

5. The use according to claim 4, wherein the sum of L-lactic acid and L-lactate is more than 50% by weight, based on the sum of all lactic acid and lactate.

6. Use according to claim 1 or 2, wherein the nutritional composition comprises lactic acid producing bacteria.

7. The use according to claim 6, wherein the lactic acid producing bacteria are selected from Bifidobacterium and Streptococcus.

8. The use according to claim 7, wherein the lactic acid producing bacteria include both Bifidobacterium and Streptococcus.

9. Use according to claim 1 or 2, wherein the nutritional composition comprises inactivated lactic acid producing bacteria.

10. Use according to claim 1 or 2, wherein the nutritional composition comprises 2.5 to 15 wt% non-digestible oligosaccharides, based on the dry weight of the nutritional composition.

11. The use according to claim 1 or 2, wherein the non-digestible oligosaccharide is selected from fructooligosaccharides, non-digestible dextrins, galacto-oligosaccharides, xylo-oligosaccharides, arabino-oligosaccharides, arabino-galacto-oligosaccharides, gluco-oligosaccharides, gentio-oligosaccharides, glucomanno-oligosaccharides, galacto-oligosaccharides, manno-oligosaccharides, isomaltooligosaccharides, niger oligosaccharides, glucomanno-oligosaccharides, chito-oligosaccharides, soybean oligosaccharides, sialic acid oligosaccharides and fucoidan oligosaccharides, and mixtures thereof.

12. The use according to claim 11, wherein the non-digestible oligosaccharides are selected from galacto-oligosaccharides and fructo-oligosaccharides.

13. The use according to claim 12, wherein the non-digestible oligosaccharides are galacto-oligosaccharides and fructo-oligosaccharides.

14. Use according to claim 1 or 2, wherein the nutritional composition does not comprise pectin-derived acid oligosaccharides, in particular galacturonic acid oligosaccharides.

15. Use according to claim 1 or 2, wherein the nutritional composition is an infant formula or a follow-on formula.

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