RU2801538C2 - Fermented formula with indigestible oligosaccharides for use in treatment of rotaviral infection - Google Patents

Abstract

FIELD: nutrition.

SUBSTANCE: invention relates to the field of nutrition of infants and young children. The following is proposed: the use of a fermented composition and indigestible oligosaccharides for the manufacture of a food composition that is at least partially fermented by lactic acid producing bacteria and contains indigestible oligosaccharides for the treatment and/or prevention of rotavirus infection or diarrhea caused by rotavirus in an infant or young child. Indigestible oligosaccharides in the composition of the nutritional composition are selected from the group consisting of fructooligosaccharides, indigestible dextrins, galactooligosaccharides, xylooligosaccharides, arabinooligosaccharides, arabinogalactooligosaccharides, glucooligosaccharides, gentiooligosaccharides, glucomannooligosaccharides, galactomannooligosaccharides, mannanoligosaccharides, isomaltooligosaccharides, nigerooligosaccharides, glucomannooligosaccharides, chitooligosaccharides, soy oligosaccharides, sialyloligosaccharides, and fucooligosaccharides, and mixtures thereof. The food composition of the above composition is used to reduce the incidence, severity and duration of diarrhea caused by rotavirus or rotaviral infection, as well as to prevent rotavirus-induced slow growth and development or poor growth and development, increase blocking of rotavirus particles, reduce rotavirus load and/or increase in intestinal barrier function in rotavirus infection in an infant or a young child.

EFFECT: nutritional composition exhibits a synergistic effect when the fermented composition with indigestible oligosaccharides is used for the treatment and/or prevention of rotavirus infection or diarrhea caused by rotavirus in an infant or a young child.

10 cl, 3 tbl, 3 ex

Description

FIELD OF TECHNOLOGY TO WHICH THE INVENTION RELATES

The present invention relates to the field of infant and young child nutrition, infant formula for the treatment or prevention of rotavirus infection.

BACKGROUND OF THE INVENTION

Viral infections are common among people of all ages, but often seem to be concentrated in infants and children. Many viral infections result in fever and body aches or discomfort and are not as severe, and most children with viral infections get better without treatment. Particularly in infancy, it is difficult for parents to determine if their child has a potentially serious infection and needs immediate medical attention. Rotavirus (RV) is the world's leading cause of severe dehydrating diarrhea in children under 5 years of age. RV infects and replicates primarily in non-dividing mature enterocytes near the villus tips of the small intestine. As a result, babies develop diarrhea, vomiting, and fever for 3 days to 1 week, usually with reinfections that tend to be less severe than the first infection. The most common treatment consists of oral rehydration solutions (ORS). Since the pathogenesis of RV in humans is still unclear and vaccines against RV are not used on a global scale, modulation through the introduction of biologically active components into the diet is of interest.

Breast milk is the preferred food for babies. Breast milk contains several biologically active factors that benefit the relatively immature immune system of newborns at an early age and may prevent or reduce the severity of rotavirus infection, such as breast milk lactoferrin and oligosaccharides. However, breastfeeding an infant is not always possible or desirable. In such cases, Tier 1 formulas or Tier 2 formulas are good alternatives. These formulas should be optimally formulated to mimic the beneficial effects of breast milk as closely as possible.

In recent years, several introductions to the diet of biologically active compounds have been investigated. Among these compounds, probiotics have been the most extensively studied due to their known immunomodulatory effects and direct action, often on the small intestine, which is the primary site of RV infection. WO 00/53201 discloses strains of lactic acid bacteria capable of preventing diarrhea and preventing infection with rotavirus. WO 2004/2168 discloses bifidobacteria for the treatment of inflammatory diseases, including rotavirus-associated diarrhea. WO 02/39834 discloses probiotic bacteria for the prevention or inhibition of rotavirus diarrhea. EP 2407532 discloses that a probiotic strain and its B. breve supernatant prevents in vitro growth of rotavirus in vitro. Rigo-Adrovers et al., 2017, British Journal of Nutrition, 117, 209-217, explored two different concepts. The former is a heat-inactivated fermented blend and the latter is a specific blend of short chain galactooligosaccharides, long chain fructooligosaccharides and pectin derived acid oligosaccharides. Both nutritional concepts resulted in an improvement in the clinical symptoms of rotavirus infection. In Rigo-Adrover et al 2018, Frontiers in Immunology, doi:10.3389/fimmu.2018.01318, a study on the preventive role of a mixture of short-chain galactooligosaccharides, and long-chain fructooligosaccharides, and the probiotic Bifidobacterium breve M-16V, and a combination of a mixture of prebiotics and a probiotic was described in a double P model. B infections in rats. Dietary modification improved clinical symptoms after the first infection.

SUMMARY OF THE INVENTION

The inventors investigated the combination of a fermented mixture and a mixture of indigestible oligosaccharides in relation to the effects of rotavirus infection. Surprisingly, the combination of the fermented mixture with indigestible oligosaccharides resulted in a much higher binding of rotavirus particles compared to binding with the individual components of the fermented mixture or indigestible oligosaccharides. Rotavirus infection and its induced symptoms were investigated using a model with young rats. Rotavirus infection caused weight loss, which was mainly observed after a period of diarrhea, and the rotavirus-induced effect on organ weight improved in the group that received the combination of the fermented mixture and the mixture of indigestible oligosaccharides. Administration of the fermented mixture with indigestible oligosaccharides resulted in a reduced incidence, reduced duration, and reduced severity of rotavirus diarrhea. In addition, a decrease in viral shedding was observed. In addition, intestinal barrier permeability in rotavirus-infected animals was most reduced in the group treated with the combination of fermented mixture and indigestible oligosaccharides, as determined by alpha-1 antitrypsin.

DETAILED DESCRIPTION OF THE INVENTION

Thus, the present invention relates to a method for the treatment and/or prevention of rotavirus infection or diarrhea caused by rotavirus in an infant or young child, comprising the introduction of a food composition that is at least partially fermented by lactic acid producing bacteria and contains indigestible oligosaccharides for an infant or young child.

The invention can also be formulated as the use of a fermented composition and non-digestible oligosaccharides for the manufacture of a food composition that is at least partially fermented by lactic acid producing bacteria and contains non-digestible oligosaccharides, for use in the treatment and/or prevention of rotavirus infection or diarrhea caused by rotavirus in an infant or young child.

The invention may also be formulated as a food composition that is at least partially fermented by lactic acid producing bacteria and contains indigestible oligosaccharides, for use in the treatment and/or prevention of rotavirus infection or diarrhea caused by rotavirus in an infant or young child. .

In one embodiment, the treatment and/or prevention of rotavirus infection or diarrhea caused by rotavirus is

A. reducing the incidence of diarrhea caused by rotavirus or rotavirus infection,

b. reducing the duration of diarrhea caused by rotavirus or rotavirus infection,

c. reducing the severity of diarrhea caused by rotavirus or rotavirus infection,

d. prevention of slow growth or poor growth caused by rotavirus and / or slow development or poor development,

e. strengthening the blocking of rotavirus particles,

f. reduction in rotavirus load, and/or

g. strengthening the barrier function of the intestine in rotavirus infection.

The present invention also relates to a method

A. reduce the incidence of diarrhea caused by rotavirus or rotavirus infection,

b. reducing the duration of diarrhea caused by rotavirus or rotavirus infection,

c. reduce the severity of diarrhea caused by rotavirus or rotavirus infection,

d. prevention of slow growth or poor growth caused by rotavirus and / or slow development or poor development,

e. increasing the blocking of rotavirus particles,

f. reduce rotavirus load, and/or

g. increase in intestinal barrier function in rotavirus infection

in an infant or young child, comprising administering to the infant or young child a nutritional composition that is at least partially fermented by lactic acid producing bacteria and contains indigestible oligosaccharides.

The invention can also be formulated as the use of a fermented composition and non-digestible oligosaccharides to produce a food composition that is at least partially fermented by lactic acid producing bacteria and contains non-digestible oligosaccharides, when used for

A. reduce the incidence of diarrhea caused by rotavirus or rotavirus infection,

b. reducing the duration of diarrhea caused by rotavirus or rotavirus infection,

c. reduce the severity of diarrhea caused by rotavirus or rotavirus infection,

d. prevention of slow growth or poor growth caused by rotavirus and / or slow development or poor development,

e. increasing the blocking of rotavirus particles,

f. reduce rotavirus load, and/or

g. increase in intestinal barrier function in rotavirus infection

in an infant or young child.

The invention can also be formulated as a food composition which is at least partially fermented by lactic acid producing bacteria and contains indigestible oligosaccharides, when used for

A. reduce the incidence of diarrhea caused by rotavirus or rotavirus infection,

b. reducing the duration of diarrhea caused by rotavirus or rotavirus infection,

c. reduce the severity of diarrhea caused by rotavirus or rotavirus infection,

d. prevention of slow growth or poor growth caused by rotavirus and / or slow development or poor development,

e. increasing the blocking of rotavirus particles,

f. reduce rotavirus load, and/or

g. increase in intestinal barrier function in rotavirus infection

in an infant or young child.

Fermented composition

The food composition in the methods or uses of the present invention, hereinafter also referred to as the present food composition, or the food composition of the invention, or the final food composition, is at least partially fermented. A partially fermented food composition includes at least a portion of the composition that has been fermented with lactic acid producing bacteria. Therefore, in the context of the present invention, a food composition that is at least partially fermentable refers to a food composition that contains a milk substrate that is fermented by lactic acid producing bacteria. It has been shown that the presence of the fermented composition in the final food composition leads, after administration, to action against rotavirus diarrhea and its symptoms.

The fermentation preferably takes place during the preparation of the food composition. Preferably, the food composition does not contain significant amounts of viable bacteria in the final product, and this can be achieved by heat inactivation after fermentation or inactivation by other means. Preferably, the fermented composition is a product derived from milk, which is a milk substrate that is fermented by lactic acid producing bacteria, wherein the milk substrate contains at least one selected from the group consisting of milk, whey, whey protein, whey protein hydrolyzate, casein, casein hydrolyzate, or mixtures thereof. Accordingly, food compositions containing fermented compositions and an indigestible oligosaccharide and their method of preparation are described in WO 2009/151330, WO 2009/151331 and WO 2013/187764.

The fermented composition preferably contains bacterial cell fragments such as glycoproteins, glycolipids, peptidoglycan, lipoteichoic acid (LTA), lipoproteins, nucleotides and/or capsular polysaccharides. It is preferable to use a fermented composition containing inactivated bacteria and/or cell fragments directly as part of the final food product, as this will result in a higher concentration of bacterial cell fragments. When commercial preparations of lactic acid producing bacteria are used, they are usually washed and the material is separated from the aqueous culture medium containing bacterial cell fragments, thereby reducing or eliminating the presence of bacterial cell fragments. In addition, fermentation and/or other interactions of lactic acid producing bacteria with a milk substrate may produce additional biologically active compounds such as short chain fatty acids, biologically active peptides and/or oligosaccharides and other metabolites, which may also lead to gut microbiota function more similar to that of breastfed infants. Such bioactive compounds that are produced during fermentation by lactic acid producing bacteria may also be referred to as postbiotics. It is believed that a composition containing such postbiotics is advantageously closer to breast milk, since breast milk is not a pure synthetic mixture, but contains metabolites, bacterial cells, cell fragments, and the like. Therefore, a fermented composition, in particular a product derived from fermented milk, is believed to have an improved effect compared to a product derived from unfermented milk with no or only lactic acid producing bacteria in terms of anti-rotavirus activity.

Preferably, the final food composition contains from 5 to 97.5 wt.% of the fermented composition in terms of dry weight, more preferably from 10 to 90 wt.%, more preferably from 20 to 80 wt.%, even more preferably from 25 to 60 wt.%. As a way to indicate that the final food composition contains at least partially fermented composition, and the level of the sum of lactic acid and lactate in the final food composition can be taken to determine the degree of fermentation, since this is the metabolic end product produced by lactic acid producing bacteria. acid during fermentation. The present final food composition preferably contains from 0.02 to 1.5 wt.% of the sum of lactic acid and lactate, based on the dry weight of the composition, more preferably from 0.05 to 1.0 wt.%, even more preferably from 0.2 up to 0.5 wt.%. Preferably, at least 50 wt.%, even more preferably at least 90 wt.% of the sum of lactic acid and lactate is in the form of L(+)-isomer. Thus, in one embodiment, the sum of L(+)-lactic acid and L(+)-lactate is more than 50% by weight, more preferably more than 90% by weight, based on the sum of total lactic acid and lactate. In the present specification, L(+)-lactate and L(+)-lactic acid are also referred to as L-lactate and L-lactic acid.

Lactic acid producing bacteria used to produce a fermented composition

The lactic acid producing bacteria used for the preparation of the fermentation composition, in particular for the fermentation of the lactic substrate, are preferably presented as a mono- or mixed culture. Lactic acid producing bacteria consist of the genera Bifidobacterium, Lactobacillus, Carnobacterium, Enterococcus, Lactococcus, Leuconostoc, Oenococcus, Pediococcus, Streptococcus, Tetragenococcus, Vagococcus, and Weissella . Preferably, the lactic acid producing bacteria used for fermentation include bacteria of the genus Bifidobacterium and/or Streptococcus .

Preferably Streptococcus is a strain of S. thermophilus . The selection of a suitable strain of S. thermophilus is described in example 2 of EP 778885 and in example 1 of FR 2723960. In a further preferred embodiment according to the present invention, the food composition contains 10 ² -10 ⁵ cfu of live S. thermophilus bacteria per g dry weight of the final food composition , preferably the final food composition contains 10 ³ -10 ⁴ live bacteria S. thermophilus per g dry weight.

Preferred strains of S. thermophilus for preparing a fermented composition for the purposes of the present invention were deposited by Gervais Danon with the National Collection of Cultures of Microorganisms (CNCM) held by the Pasteur Institute, 25 rue Dr. Roux, Paris, France on August 23, 1995 under accession number I -1620 and August 25, 1994 as accession I-1470. Other strains of S. thermophilus are commercially available.

Bifidobacteria are gram-positive anaerobic rod-shaped bacteria. Preferred SpeciesBifidobacterium to obtain a fermented composition for the purposes of the present invention, preferably have at least 95% sequence identity of 16 S rRNA compared to the type strain of the respective speciesBifidobacterium, more preferably at least 97% identity, as defined in manuals on the subject, e.g., Sambrook, J., Fritsch, E.F., and Maniatis, T. (1989), Molecular Cloning, A Laboratory Manual, 2nd ed ., Cold Spring Harbor (N.Y.) Laboratory Press. Preferably used Bifidobacteria are also described by Scardovi, V. Genus Bifidobacterium. p.1418 - p.1434. In: Bergey's manual of systematic Bacteriology. Vol. 2. Sneath, P.H.A., N.S. Maire, M.E. Sharpe and J.G. Holt (ed.). Baltimore: Williams & Wilkins. 1986. 635 p. Preferably, the lactic acid producing bacteria used for fermentation comprise or are at least oneBifidobacteriumselected from the group consisting ofB. breve, B. infantis, B. bifidum, B. catenulatum, B. adolescentis, B. thermophilum, B. gallicum, B. animalis or lactis, B. angulatum, B. pseudocatenulatum, B. thermacidophilumAndB. longum,more preferablyB. breve, B. infantis, B. bifidum, B. catenulatum, B. longum,more preferablyB. longum and B. breve,more more preferablyB.breve,more preferablyB.breve, selected from the group consisting ofB.breve Bb-03 (Rhodia/Danisco),B.breve M-16V (Morinaga),B.breve R0070 (Institute Rosell, Lallemand),B.breve BR03 (Probiotic),B.breve BR92 (Cell Biotech) DSM 20091, LMG 11613 andB.breve I-2219 deposited with CNCM, Paris, France. Most preferredB.breve representsB.breve M-16V (Morinaga) orB.breve I-2219, even more preferablyB.breve I-2219.

Most preferably, the food composition of the invention comprises a fermented composition that is fermented by lactic acid producing bacteria containing both B. breve and S. thermophilus . In one embodiment, the fermentation with lactic acid producing bacteria is the fermentation of Streptococcus thermophilus and Bifidobacterium breve . In one embodiment, the final food composition comprises a fermented composition in which lactic acid producing bacteria are inactivated after fermentation.

Preferably the fermented composition is not fermented by Lactobacillus bulgaricus . It is believed that L. bulgaricus fermented foods are not suitable for infants because in young children the specific dehydrogenase that converts D-lactate to pyruvate is much less active than the dehydrogenase that converts L-lactate.

Preferably, the food composition of the invention contains inactivated lactic acid producing bacteria and/or bacterial fragments derived from lactic acid producing bacteria, which is equivalent to more than 1×10 ⁴ cfu of lactic acid producing bacteria per g based on the dry weight of the final composition , more preferably 1×10 ⁵ CFU, even more preferably 1×10 ⁶ CFU. Preferably, the inactivated bacteria or bacterial fragments are equivalent to less than 1 x 10 ¹³ cfu of lactic acid producing bacteria per g based on the dry weight of the final composition, more preferably 1 x 10 ¹¹ cfu, even more preferably 1 x 10 ¹⁰ cfu. Correlation of inactivated lactic acid bacteria and CFU equivalence can be determined using molecular methods known in the art, or by checking the manufacturing process.

fermentation process

Preferably, the fermented composition is a product derived from milk, which is a milk substrate that is fermented by lactic acid producing bacteria, said milk substrate comprising at least one selected from the group consisting of milk, whey, whey protein, whey protein hydrolyzate, casein, casein hydrolyzate, or mixtures thereof. The milk-derived product or milk substrate to be fermented is appropriately present in the aquatic environment. The fermented milk substrate includes at least one selected from the group consisting of milk, whey, whey protein, whey protein hydrolyzate, casein, casein hydrolyzate, or mixtures thereof. The milk may be whole, semi-skimmed and/or skimmed milk. Preferably the fermented milk substrate contains skimmed milk. Whey can be sweet and/or sour whey. Preferably whey is present at a concentration of 3 to 80 g dry weight per liter of the aqueous medium containing the milk substrate, more preferably 40 to 60 g per liter. Preferably, the whey protein hydrolyzate is present in an amount of 2 to 80 g dry weight per liter of the aqueous medium containing the milk substrate, 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 substrate, more preferably 1 to 30 g/l. Preferably, the aqueous medium containing the milk substrate includes buffer salts to maintain the pH in the desired range. Preferably sodium or potassium dihydrogen phosphate is used as buffer salt, preferably 0.5 to 5 g/l, more preferably 1.5 to 3 g/l. Preferably, the aqueous medium containing the milky substrate contains cysteine in an amount of 0.1 to 0.5 g per liter of aqueous substrate, more preferably 0.2 to 0.4 g/L. The presence of cysteine results in a low redox potential of the substrate, which is favorable for the activity of lactic acid producing bacteria, in particular bifidobacteria . Preferably, the aqueous medium containing the milk substrate comprises yeast extract in an amount of 0.5 to 5 g/l of the aqueous medium containing the milk substrate, more preferably 1.5 to 3 g/l. Yeast extract is a rich source of enzyme cofactors and growth factors for lactic acid producing bacteria. The presence of yeast extract will enhance fermentation by lactic acid producing bacteria.

Accordingly, the milk substrate, in particular the aqueous medium containing the milk substrate, is pasteurized before the fermentation step in order to exclude the presence of undesirable live bacteria. Accordingly, the product is pasteurized after fermentation in order to inactivate the enzymes. Accordingly, enzyme inactivation occurs at 75°C for 3 min. Accordingly, the aqueous medium containing the milk substrate is homogenized before and/or the milk-derived product is homogenized after fermentation. Homogenization results in a more stable substrate and/or fermented product, especially in the presence of fat.

The inoculation density is preferably 1×10 ² to 5×10 ¹⁰ , preferably 1×10 ⁴ to 5×10 ⁹ cfu of lactic acid producing bacteria/ml aqueous medium containing milk substrate, more preferably 1×10 ⁷ to 1 ×10 ⁹ CFU of bacteria producing lactic acid/ml of an aqueous medium containing a milky substrate. The final density of the bacteria after fermentation is preferably 1×10 ³ to 1×10 ¹⁰ , more preferably 1×10 ⁴ to 1×10 ⁹ CFU/ml of the aqueous medium containing the milk substrate.

The fermentation is preferably carried out at a temperature of from about 20°C to 50°C, more preferably from 30°C to 45°C, even more preferably from about 37°C to 42°C. The optimum temperature for the growth and/or activity of lactic acid producing bacteria, in particular lactobacilli and/or bifidobacteria, is between 37°C and 42°C.

The incubation is preferably carried out at a pH of 4 to 8, more preferably 6 to 7.5. This pH does not cause protein precipitation and/or off taste, while lactic acid producing bacteria such as lactobacilli and/or bifidobacteria are able to ferment the milk substrate.

The incubation time is preferably in the range of 10 minutes to 48 hours, preferably 2 hours to 24 hours, more preferably 4 hours to 12 hours. lipoteichoic acid (LTA), flagella, lipoproteins, DNA and/or capsular polysaccharides and metabolites (postbiotics) occur to a sufficient or greater extent, while the incubation time should not be unnecessarily long for economic reasons.

Preferably the milk-derived product or milk substrate, preferably skimmed milk, is pasteurized, cooled and fermented with one or more lactic acid producing strains, preferably S. thermophilus , to a certain degree of acidity at which the fermented product is cooled and stored. Preferably, the second milk-derived product is similarly prepared using one or more Bifidobacterium species for fermentation. Subsequently, the two fermented products are preferably blended together and blended with the other components constituting the infant formula, with the exception of the fat component. Preferably, the mixture is preheated and then the fat is added inline, homogenized, pasteurized, and dried. Alternatively, fermentation occurs with both Bifidobacterium , preferably B. breve , and S. thermophilus in the fermentation tank.

Methods for obtaining a fermented composition suitable for the purposes of the present invention are well known. EP 778885, which is included in the present description by reference, discloses in particular in example 7 a suitable method for obtaining a fermented composition. FR 2723960, which is included in the present description by reference, discloses in particular in example 6 a suitable method for obtaining a fermented composition. Briefly, a milk substrate, preferably pasteurized, containing lactose and optionally other macronutrients such as fats, preferably vegetable fats, casein, whey protein, vitamins and/or minerals, etc., is concentrated, for example, from 15 to 50% dry matter and then S. thermophilus is inoculated with, for example, a 5% culture containing 10 ⁶ to 10 ¹⁰ bacteria per ml. Preferably this milk substrate contains milk protein peptides. The temperature and duration of fermentation are as above. Accordingly, after fermentation, the fermented composition may be pasteurized or sterilized and, for example, spray dried or lyophilized to provide a form suitable for the preparation of the final product.

A preferred method for preparing a fermented composition for use in a food composition of the invention is disclosed in WO 01/01785, more specifically in Examples 1 and 2. A preferred method for obtaining a fermented composition for use in a food composition of the invention is described in WO 2004/093899, more specifically in Example 1.

Live cells of lactic acid producing bacteria in the fermented composition are preferably removed after fermentation, for example by inactivation and/or physical removal. The cells are preferably inactivated. The lactic acid producing bacteria are preferably killed by heating after fermentation of the milk substrate. Preferred methods of thermal killing are (flash) pasteurization, sterilization, ultra high temperature processing, high temperature/short heat treatment and/or spray drying at temperatures where bacteria do not survive. Cell fragments are preferably obtained by heat treatment. Such heat treatment preferably inactivates at least 90% of living microorganisms, more preferably at least 95%, even more preferably at least 99%. Preferably, the fermented food composition contains less than 1×10 ⁵ colony forming units (CFU) of live lactic acid bacteria per g dry weight. The heat treatment is preferably carried out at a temperature in the range from 70 to 180°C, preferably from 80 to 150°C, preferably for about 3 minutes to 2 hours, preferably in the range from 80 to 140°C for from 5 minutes to 40 minutes. Inactivation of lactic acid bacteria preferably results in less subsequent acidification and a safer product. This is particularly preferred when the nutritional composition is to be administered to infants or toddlers, and yet beneficial effects on rotavirus infections are observed with a fermented composition containing inactivated lactic acid bacteria. Accordingly, after fermentation, the fermented composition may be pasteurized or sterilized and, for example, spray dried or lyophilized to provide a form suitable for the preparation of the final product.

Indigestible oligosaccharides

The present food composition contains non-digestible oligosaccharides and preferably contains at least two different non-digestible oligosaccharides, in particular two different sources of non-digestible oligosaccharides. The presence of non-digestible oligosaccharides is necessary to improve the effects against rotavirus infection. The presence of both non-digestible oligosaccharides and an at least partially fermented composition, in particular a dairy product obtained by fermentation with lactic acid-producing bacteria, is necessary to improve the anti-rotavirus effect, in particular in relation to the binding of rotavirus particles or blocking particles from sites adhesion to intestinal epithelial cells.

As used herein, the term "oligosaccharides" refers to saccharides with a degree of polymerization (DP) of 2 to 250, preferably a DP of 2 to 100, more preferably 2 to 60, even more preferably 2 to 10. If an oligosaccharide with a DP of 2 up to 100 is included in the present food composition, this leads to compositions that can contain oligosaccharides with SP from 2 to 5, SP from 50 to 70 and SP from 7 to 60. As used in the present invention, the term "indigestible oligosaccharides" refers to oligosaccharides that are not digested in the intestine by the action of acids or digestive enzymes present in the human upper digestive tract, such as the small intestine and stomach, but which are preferentially fermented by the human gut microbiota. For example, sucrose, lactose, maltose, and maltodextrins are considered digestible.

Preferably, the present non-digestible oligosaccharides are soluble. Used in the present description, the term "soluble", when it refers to polysaccharides, fibers or oligosaccharides, means that the substance is at least soluble in accordance with the method described by L. Prosky et al., J. Assoc. off. Anal. Chem. 71, 1017-1023 (1988).

The non-digestible oligosaccharides included in the present food compositions in the methods or uses of the present invention preferably comprise a mixture of various non-digestible oligosaccharides. The indigestible oligosaccharides are preferably selected from the group consisting of fructooligosaccharides such as inulin, indigestible dextrins, galactooligosaccharides such as transgalactooligosaccharides, xylooligosaccharides, arabinooligosaccharides, arabinogalactooligosaccharides, glucooligosaccharides, gentiooligosaccharides, glyuk annooligosaccharides, galactomannooligosaccharides, mannanoligosaccharides, isomaltooligosaccharides, nigeroligosaccharides, glucomannooligosaccharides, chitooligosaccharides, soy oligosaccharides, fucooligosaccharides, sialyloligosaccharides and mixtures thereof. Such oligosaccharides have many biochemical properties and similar functional benefits, including improved gut microbiota function. However, it is clear that some non-digestible oligosaccharides and preferably some mixtures have an even more improved effect. Therefore, more preferably the non-digestible oligosaccharides are selected from the group consisting of fructooligosaccharides such as inulin and galactooligosaccharides such as betagalactooligosaccharides and mixtures thereof, even more preferably betagalactooligosaccharides and/or inulin, most preferably betagalactooligosaccharides. In one embodiment, in the food composition according to the present invention, the non-digestible oligosaccharides are selected from the group consisting of galactooligosaccharides, fructooligosaccharides and mixtures thereof, more preferably betagalactooligosaccharides, fructooligosaccharides and mixtures thereof.

The indigestible oligosaccharide is preferably selected from the group consisting of beta-galactooligosaccharide, alpha-galactooligosaccharide and galactan. According to a more preferred embodiment, the indigestible oligosaccharide is beta-galactooligosaccharide. Preferably, the indigestible oligosaccharide comprises a galactooligosaccharide with beta(1,4), beta(1,3) and/or beta(1,6) glycosidic linkages and terminal glucose. Transgalactooligosaccharide, for example, is available under the trade names Vivinal®GOS (Domo FrieslandCampina Ingredients), Bi2muno (Clasado), Cup-oligo (Nissin Sugar) and Oligomate55 (Yakult).

Non-digestible oligosaccharides preferably include fructooligosaccharides. Fructooligosaccharides may in other contexts be referred to as fructopolysaccharides, oligofructose, polyfructose, polyfructan, inulin, levan and fructan and may refer to oligosaccharides containing beta-linked fructose units which are preferably linked to beta(2,1) and/or beta( 2,6)-glycosidic bonds, and preferably SP from 2 to 200. Preferably, fructooligosaccharides contain terminal beta(2,1)-glycosidic linked glucose. Preferably, the fructooligosaccharides contain at least 7 beta-linked fructose units. In a further preferred embodiment, inulin is used. Inulin is a type of fructooligosaccharide in which at least 75% of the glycosidic bonds are beta(2,1) bonds. Typically, inulin has an average chain length of 8 to 60 monosaccharide units. A suitable fructooligosaccharide for use in the compositions of the present invention is commercially available under the tradename Raftilin®HP (Orafti). Other suitable sources are Raftilose (Orafti), Fibrulose and Fibrulin (Cosucra) and Frutafit and Frutalose (Sensus).

Preferably, the present food composition comprises a mixture of galactooligosaccharides and fructooligosaccharides. Preferably the mixture of galactooligosaccharides and fructooligosaccharides 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 from 5/1 to 12/1, even more preferably from 8/1 to 10/1, even more preferably in a ratio of about 9/1. This weight ratio is most preferred when the galactooligosaccharides have a low average SP and the fructooligosaccharides have a relatively high SP. Most preferred is a mixture of galactooligosaccharides with an average SP below 10, preferably below 6, and fructooligosaccharides with an average SP above 7, preferably above 11, even more preferably above 20.

Preferably, the present food composition comprises a mixture of short chain fructooligosaccharides and long chain fructooligosaccharides. Preferably the mixture of short chain fructooligosaccharides and long chain fructooligosaccharides 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. A mixture of short chain fructooligosaccharides with an average SP below 10, preferably below 6, and fructooligosaccharides with an average SP above 7, preferably above 11, even more preferably above 20, is preferred.

Preferably, the present food composition comprises a mixture of short chain fructooligosaccharides and short chain galactooligosaccharides. Preferably the mixture of short chain fructooligosaccharides and short chain galactooligosaccharides 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. A mixture of short chain fructooligosaccharides and galactooligosaccharides with an average SP below 10, preferably below 6 is preferred.

Preferably the food composition does not contain acid oligosaccharides derived from pectin. Such oligosaccharides are rich in galacturonic acid oligosaccharides. In the context of the present invention, acid oligosaccharides derived from pectin are characterized as a mixture of galacturonic acid oligomers with a degree of polymerization of 2-30. Therefore, preferably the present composition does not contain galacturonic acid oligosaccharides. These oligosaccharides are well known for their ability to block the adhesion of pathogens to receptors in the gut. However, surprisingly, their presence was not required to obtain the maximum blocking effect of the rotavirus particles when the non-digestible oligosaccharides were combined with the fermented food composition.

The present food composition preferably contains from 2.5 to 20 wt.% of total indigestible oligosaccharides, more preferably from 2.5 to 15 wt.%, even more preferably from 3.0 to 10 wt.%, most preferably from 5.0 to 7.5 wt.% in terms of dry weight of the food composition. In terms of 100 ml, the present food composition preferably contains from 0.35 to 2.5 wt.% of total indigestible oligosaccharides, more preferably from 0.35 to 2.0 wt.%, even more preferably from 0.4 to 1.5 wt.% in terms of 100 ml of food composition. A smaller amount of non-digestible oligosaccharides will be less effective in improving anti-rotavirus effects, while too much will lead to the side effects of bloating and abdominal discomfort.

food composition

The food composition used in accordance with the present invention can also be considered 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.

The present nutritional composition is preferably a first level milk formula, a second level milk formula, a preschool milk or a third level milk formula, or a third level infant formula or an infant formula. More preferably, the food composition is a first level milk formula or a second level milk formula. The present nutritional composition may preferably be used as a complete nutrition for infants. Preferably the present food composition is a first level formula. First level formula is defined as formula for use in infants and may, for example, be initial formula intended for infants 0 to 6 or 0 to 4 months of age. Level 2 formula is for infants aged 4 or 6 months to 12 months. At this age, babies begin to wean and switch to other foods. Toddler milk or third level formula or infant formula is for children aged 12 to 36 months. The present composition preferably contains a lipid component, a protein component and a carbohydrate component and is preferably administered in liquid form. The present food composition may also be in dry form, preferably in the form of a powder, accompanied by instructions for mixing said dry form, preferably powder, with a suitable liquid, preferably water. The food composition used in accordance with the invention preferably contains other fractions such as vitamins, minerals, trace elements and other micronutrients to make it a complete food composition. Preferably first level milk formulas contain vitamins, minerals, trace elements and other micronutrients in accordance with international guidelines.

The present food composition preferably comprises a lipid, a protein and a digestible carbohydrate in which the lipid provides 5 to 50% of the total calories, the protein provides 5 to 50% of the total calories and the digestible carbohydrate provides 15 to 90% of the total calories. Preferably, in the present food composition, the lipid provides 35 to 50% of the total calories, the protein provides 7.0 to 12.5% of the total calories, and the digestible carbohydrate provides 40 to 55% of the total calories. To calculate the % of total calories for protein, the total energy provided by proteins, peptides, and amino acids must be taken into account. Preferably the lipid provides from 3 to 7 g of lipids per 100 kcal, preferably from 4 to 6 g per 100 kcal, the protein provides from 1.6 to 4 g per 100 kcal, preferably from 1.7 to 2.5 g per 100 kcal and digestible carbohydrate provides 5 to 20 g per 100 kcal, preferably 8 to 15 g per 100 kcal of the food composition. Preferably, the present food composition comprises a lipid providing 4 to 6 g per 100 kcal, a protein providing 1.6 to 2.0 g per 100 kcal, more preferably 1.7 to 1.9 g per 100 kcal, and digestible carbohydrate providing 8 to 15 g per 100 kcal of the food composition. In one embodiment, the lipid provides 3 to 7 g of lipid per 100 kcal, preferably 4 to 6 g per 100 kcal, the protein provides 1.6 to 2.1 g per 100 kcal, preferably 1.6 to 2.0 g per 100 kcal and the digestible carbohydrate provides 5 to 20 g per 100 kcal, preferably 8 to 15 g per 100 kcal of the food composition, and preferably the digestible carbohydrate component contains at least 60 wt.% lactose, calculated on the total the amount of digestible carbohydrates, more preferably at least 75 wt.%, even more preferably at least 90 wt.% lactose, based on the total amount of digestible carbohydrates. Total calories are determined by the sum of calories derived from proteins, lipids, digestible carbohydrates, and indigestible oligosaccharides.

The present food composition preferably contains 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 breast milk. The present food composition preferably contains lactose. Since the present food composition includes a fermented composition that is obtained by fermentation with lactic acid producing bacteria, the amount of lactose is reduced compared to its source due to fermentation, which converts lactose into lactate and/or lactic acid. Therefore, when preparing the present food composition, lactose is preferably added. Preferably, the present food composition does not contain large amounts of carbohydrates other than lactose. Compared to digestible carbohydrates such as maltodextrin, sucrose, glucose, maltose and other high glycemic index digestible carbohydrates, lactose has a lower glycemic index and is therefore preferred. The present food composition preferably contains a digestible carbohydrate, in which 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. On a dry weight basis, the present food composition preferably contains at least 25 wt. % lactose, preferably at least 40 wt. %, more preferably at least 50 wt. % lactose.

The present nutritional composition preferably contains at least one lipid selected from the group consisting of lipids of animal origin (excluding human lipids) and lipids of plant origin. Preferably the present composition comprises a combination of vegetable lipids and at least one oil selected from the group consisting of fish oil, animal oil, algal oil, fungal oil and bacterial oil. The lipid of the present food composition preferably provides 3 to 7 g per 100 kcal of the food composition, preferably the lipid provides 4 to 6 g per 100 kcal. In liquid form, for example as a ready-to-use liquid, the nutritional composition preferably contains 2.1 to 6.5 g lipid per 100 ml, more preferably 3.0 to 4.0 g per 100 ml. On a dry weight basis, the present nutritional composition preferably contains 12.5 to 40% by weight lipids, more preferably 19 to 30% by weight. Preferably the lipid comprises the essential fatty acids alpha-linolenic acid (ALA), linoleic acid (LA) and/or long chain polyunsaturated fatty acids (LC-PUFA). LC-PUFA, LA and/or ALA can be provided as free fatty acids, triglycerides, diglycerides, monoglycerides, phospholipids, or a mixture of one or more of the above. Preferably the present food composition comprises at least one, preferably at least two lipid sources selected from the group consisting of rapeseed oil (such as rapeseed oil, low erucic acid rapeseed oil and canola oil), high oleic sunflower oils, high oleic safflower oil, olive oil, seafood oils, microbial oils, coconut oil, palm kernel oil. This food composition is not breast milk.

The present food composition preferably contains protein. The protein used in the food 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 protein and/or bovine casein. Thus, in one embodiment, the protein in the present food composition comprises a protein selected from the group consisting of whey protein and casein, preferably whey protein and casein, preferably whey protein and/or cow's milk casein. Preferably, the protein contains less than 5% by weight, based on the total amount of the protein, of free amino acids, dipeptides, tripeptides, or hydrolyzed protein. The present food composition preferably contains casein and whey proteins in a casein:whey protein weight ratio of 10:90 to 90:10, more preferably 20:80 to 80:20, even more preferably 35:65 to 55:45.

Wt % protein on a dry weight basis of the present food composition is calculated according to the Kjeldahl method by measuring total nitrogen and using a conversion factor of 6.38 for casein or a conversion factor of 6.25 for proteins other than casein. Used in the present invention, the term "protein" or "protein component" refers to the sum of proteins, peptides and free amino acids.

The present food composition preferably contains a protein providing 1.6 to 4.0 g of protein per 100 kcal of the food composition, preferably 1.6 to 3.5 g, even more preferably 1.75 to 2.5 g per 100 kcal food composition. In one embodiment, the present food composition comprises a protein providing 1.6 to 2.1 g of protein per 100 kcal of the food composition, preferably 1.6 to 2.0 g, more preferably 1.7 to 2.1 g, even more preferably 1.75 to 2.0 g per 100 kcal of the food composition. In one embodiment, the present food composition contains less than 2.0 g of protein per 100 kcal, preferably 1.6 to 1.9 g, even more preferably 1.75 to 1.85 g per 100 kcal of the food composition. Too little protein in terms of total calories will result in inadequate growth and development in infants and young children. Too much will cause a metabolic burden on, for example, the kidneys of infants and young children. In liquid form, for example as a ready-to-use liquid, the food composition preferably contains 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. On a dry weight basis, the present food composition preferably contains 5 to 20 wt% protein, preferably at least 8 wt% protein based on dry weight of the total food composition, more preferably 8 to 14 wt%, even more preferably from 8 to 9.5 wt.% protein, based on the dry weight of the total food composition.

To meet the caloric requirements of an infant or toddler, the food composition preferably contains 45 to 200 kcal/100 ml liquid. For infants, the food composition more preferably contains from 60 to 90 kcal/100 ml of liquid, even more preferably from 65 to 75 kcal/100 ml of liquid. This calorie content provides an optimal ratio between water consumption and calories. For preschool children, children aged 12 to 36 months, the food composition more preferably has a calorie content of 45 to 65, even more preferably 50 to 60 kcal/100 ml. The osmolarity of the present composition is preferably 150 to 420 mosmol/l, more preferably 260 to 320 mosmol/l. The low osmolarity aims to further reduce gastrointestinal stress, which can affect rotavirus infection.

When the nutritional composition is in ready-to-use liquid form, the preferred volume administered daily is in the range of from about 80 to 2500 ml, more preferably from about 200 to 1200 ml per day. Preferably, the number of meals per day is from 1 to 10, preferably from 3 to 8. In one embodiment, the food composition is administered daily for a period of at least 2 days, preferably for a period of at least 4 weeks, preferably in over a period of at least 8 weeks, more preferably over a period of at least 12 weeks in liquid form, in which the total volume administered daily is from 200 ml to 1200 ml and in which the number of feedings per day is from 1 to 10.

The present food composition, when in liquid form, preferably has a viscosity of 1 to 60 mPa.s, preferably 1 to 20 mPa.s, more preferably 1 to 10 mPa.s, most preferably 1 to 6 mPa.s . The low viscosity ensures proper fluid administration, such as proper passage through the entire nipple. Also, this viscosity is very similar to the viscosity of breast milk. In addition, low viscosity results in normal gastric emptying and better energy intake, which is very important for babies who need energy for optimal growth and development. Alternatively, the present food composition is in powder form suitable for dilution with water to a state ready for use. The present food composition is preferably prepared by mixing the powder composition with water. Normally, first-level formula is prepared in this way. Thus, the present invention also relates to a packaged energy composition, wherein said package is provided with instructions for mixing the powder with a suitable amount of liquid, resulting in a liquid composition with a viscosity of 1 to 60 mPa.s. The viscosity of the fluid is determined using a Physica Rheometer MCR 300 (Physica Messtechnik GmbH, Ostfildern, Germany) at a shear rate of 95 s ^-1 at 20°C.

Application

Methods or uses according to the present invention, including the administration of the present food composition, also relate to the administration of an effective amount of the food composition to an object in need of such treatment, preferably an infant or young child.

The inventors found that the combination of a fermented formula (providing postbiotic compounds) and a mixture of indigestible oligosaccharides showed the ability to attenuate rotavirus infection and diarrhea caused by rotavirus. The fermented formula enriched with indigestible oligosaccharides reduced the incidence, duration and severity of diarrhea. In addition, the combination could interact directly with the virus and boost the host's immune system. Surprisingly, an improved effect of a fermented mixture with non-digestible oligosaccharides was observed compared to a fermented mixture containing no non-digestible oligosaccharides or non-digestible oligosaccharides in the direct blocking assay of rotavirus particles. This indicates a decrease in the rotavirus load in the gastrointestinal tract. Viral load, also known as blood virus concentration or virus titer, is a numerical expression of the number of infectious viral particles per ml. A higher viral concentration, titer, or load often correlates with the severity of the infection.

In addition, the greatest anti-rotavirus effects in terms of duration, frequency of occurrence and severity were observed in the group that received both indigestible oligosaccharides and the fermented mixture. Especially the effect of reducing the duration of treatment was observed only in this group and cannot be explained by the effects of non-digestible oligosaccharides or the fermented mixture alone. Interestingly, the combination of the fermented mixture with non-digestible oligosaccharides increased the intestinal barrier function the most during rotavirus infection, as assessed by intestinal barrier permeability using alpha-1 antitrypsin assay. Without wishing to be bound by theory, the observed improvement in gut barrier function can be explained by an unexpected increase in butyric acid content in the gut. Butyrate is known as a fuel for intestinal epithelial cells and for improving the intestinal barrier. The elevated butyric acid level is greater than would be expected based on the levels found for the indigestible oligosaccharide production group or the fermented blend alone.

Without wishing to be bound by theory, the lower viral shedding after intervention suggests that the analyzed components may influence virus binding. This microbiota-independent anti-infective effect of the combination of indigestible oligosaccharides and the fermented mixture may involve direct interaction with pathogens and may be associated with receptor mimicry. Thus, some components of the administered mixture may act as soluble receptor analogs for certain host molecules to which the virus can attach. Therefore, this interaction between the pathogen and the combination of the fermented mixture and indigestible oligosaccharides can dislodge the adhering pathogen from small intestinal epithelial cells, thereby reducing the infectious burden or infectivity of rotavirus and thus reducing the incidence, duration and/or severity of diarrhea. In addition, decreased viral shedding and other systemic and intestinal immune responses were observed. Also important is the discovery that rotavirus infection caused delayed weight gain, mainly observed after a period of diarrhea, and that the effects caused by this rotavirus infection on weight gain improved in the group that received the combination of the fermented mixture and the mixture of indigestible oligosaccharides. Not only was there an effect on growth, in particular on body weight, but there was also an effect on relative organ weight in the group that was infected with rotavirus. The spleen, liver, small and large intestines were reduced in weight, even in relative weight (wt.% of total body weight) of the RV group compared to the control group. This indicates that not only growth but also development suffers after rotavirus infection or diarrhea caused by rotavirus. However, these effects were improved and more similar to the non-infected control group in the group that received the combination of the fermented mixture and the mixture of indigestible oligosaccharides. Prevention of weight loss, or prevention of growth retardation or poor growth (in particular weight gain), or prevention of stunted development of organs such as the liver, spleen, small intestine and/or large intestine is of paramount importance for the development and growth of the infant or young child . These observations indicate both improved growth (particularly weight gain) and improved development in an infant or young child following rotavirus infection or rotavirus diarrhea when the nutritional composition of the present invention is administered.

Therefore, in one embodiment, the present invention relates to a method or use for the prevention of rotavirus-induced stunted growth or poor growth and/or retarded development or poor development, more preferably retarded weight gain or poor weight gain in infants or young children. Preferably, the method or use for the prevention of rotavirus-induced stunted growth or poor growth and/or stunted development or poor development in infants or young children is the prevention of stunted growth or poor growth and/or delayed development or poor development after rotavirus infection, or diarrhea caused by rotavirus. This method or use can be achieved by administering a food composition that is at least partially fermented by lactic acid producing bacteria and contains non-digestible oligosaccharides.

Therefore, in one embodiment, the present invention relates to a method or use for the prevention and/or treatment of rotavirus infection or diarrhea caused by rotavirus. More specifically, in various embodiments, the effect on rotavirus infection or diarrhea caused by rotavirus is a reduction in the incidence of diarrhea caused by rotavirus or rotavirus infection, or a reduction in the duration of diarrhea caused by rotavirus or rotavirus infection, or a decrease in the severity of diarrhea caused by rotavirus, or rotavirus infection. In additional embodiments, the effect on rotavirus infection or diarrhea caused by rotavirus is the prevention of rotavirus-induced stunted growth or poor growth and/or delayed development or poor development, or increased blocking of rotavirus particles, or reduced rotavirus load, or increased intestinal barrier function when rotavirus infection. The invention in one embodiment relates to a method or use for blocking rotavirus particles, reducing rotavirus load and/or reducing shedding of rotavirus. This is achieved by administering a food composition as described above that is at least partially fermented by lactic acid producing bacteria and contains non-digestible oligosaccharides, in particular scGOS and lcFOS, to an infant or young child.

In further embodiments, the present invention relates to a method for reducing the incidence of rotavirus diarrhea or rotavirus infection, or shortening the duration of rotavirus diarrhea or rotavirus infection, or reducing the severity of rotavirus diarrhea or rotavirus infection, or preventing rotavirus infection. slow growth or poor growth and/or delayed development or poor development, or increased blocking of rotavirus particles, or reduced rotavirus load, or increased intestinal barrier function in rotavirus infection, by administering a nutritional composition as described above, which is at least Partially fermented by lactic acid producing bacteria and contains non-digestible oligosaccharides such as scGOS and lcFOS for infant or young child.

For all methods and uses, the preferred effects claimed are in comparison to infants who are not administered the nutritional composition of the present invention; in other words, compared to infants who are administered a food composition that is at least partially non-fermentable and does not contain indigestible oligosaccharides.

The present nutritional composition is administered to an infant or young child between 0 and 36 months of age. Preferably the nutritional composition is administered to an infant, ie a child between 0 and 12 months of age, more preferably an infant between 0 and 6 months of age.

In this document and in its claims, the verb "comprise" and its conjugations are used in a non-limiting sense to indicate that elements following the word are included, but elements not specifically mentioned are not excluded. Also, referring to an element of the singular does not preclude the possibility of more than one element, unless the context clearly requires one and only one of the elements. Singular, therefore, usually means "at least one. Mass% means mass percent. Unless otherwise specified, a day is a 24-hour period (starting and ending at midnight).

EXAMPLES

Example 1 Effect of a fermented mixture with indigestible oligosaccharides on rotavirus infection in rats

Materials and methods

The rotavirus (RV) strain selected for infection (SA-11 monkeys) was prepared as described in Pérez-Cano FJ, et al., 2007, Pediatr. Res. 2007;62: 658-663. Briefly, virus reproduction was performed in African green monkey embryonic kidney cells (MA-104) and titrated as TCID ₅₀ /ml (TCID, tissue culture infectious dose). The viruses have been produced in accordance with current Good Laboratory Practice principles.

Pregnant Lewis G14 rats were obtained from Janvier (Les Genes-Saint-Iles, France) and kept separately in cages (2184L Eurostandard Type II L, Tecniplast, West Chester, PA, USA) with coarse fibrous bedding and fine paper as decoration. They were observed daily and allowed to give birth at term. Birthday was recorded as day 1 of life. The offspring were united by seven cubs/female with the same number of representatives of each sex in each nest. The pups had free access to teats and a rat diet. The females had access to water and commercial food (Teklad Global Diet 2014, Envigo, Indianapolis, IN, USA), an American Institute of Nutrition (AIN)-93G diet without restriction. Each female and her litter were housed individually under controlled temperature and humidity conditions on a 12 h:12 h light:dark cycle. In addition, the animals were placed in a special safe isolation room, specially designed and approved for work in biosafety level 2 conditions.

Experimental food or food components have been investigated:

1) First level thermally fermented milk formula. Fermented milk was obtained by fermenting a first level milk formula with Bifidobacterium breve C50 and Streptococcus thermophilus 065 during the manufacturing process. As a result of this process, active bacterial metabolites and cell fragments (postbiotic compounds) were added to the composition of the mixture. It was marketed in France under the names Calisma and Apaisia as a product from Bledina. Suitable processes for the preparation of this first level milk formula are described in example 1 of WO 2004/093899.

2) A mixture of indigestible oligosaccharides scGOS (source Vivinal® GOS, Borculo Domo FrieslandCampina) and lcFOS (source Raftilin® HP, Orafti) in a ratio of 9:1 wt./mass.

3) Filler: The filler of the food composition or ingredients in 1 and 2 was mineral water.

Lactating neonates in G14 Lewis rats were divided into several different experimental groups:

1) reference group (REF), which was a control group that was not infected with rotavirus and did not receive dietary modification.

2) the rotavirus (RV) group, which was a control group that was infected with rotavirus and did not receive dietary modification

3) study group (FM+G/F) who were infected with rotavirus and received fermented milk (FM) as described above and scGOS/lcFOS indigestible oligosaccharide mixture (G/F). FM and G/F were administered at 3 g/100 g body weight/day and 0.8 g/100 g body weight/day, respectively.

Each group consisted of three litters of seven pups each (n=21/group). The REF and PB group received the same amount of filler as the FM+G/F group. Diet or filler was administered from day 3 of life until the last day of the experiment. RV inoculation was performed on day 7 of life.

SA-11 was inoculated (2×10 ⁸ TCID ₅₀ /rat in 100 μl of phosphate-buffered saline (PBS) on day 7 of life to fed rats from the PB and FM+G/F groups. PB inoculations were performed 1 h after separation from their females to avoid interaction between RS and milk components REF group received the same volume of PBS under the same conditions.

SA-11 infection was assessed on days 8-21 by growth rate and clinical scores obtained from fecal samples. Fecal sampling was carried out once a day by light pressure and abdominal massage. Samples were immediately evaluated, weighed and frozen at -20°C for further analysis.

Animals were sacrificed at two different time points: three animals from each litter were sacrificed 1 week after RV inoculation on day 14 (n=9/group), while the other four animals from each litter continued to be fed until the end of the nursing period on day 14. day 21 (n=12/group). On the day of sacrifice, blood was collected by cardiac puncture to determine hematocrit (HCT) and obtain serum, which was stored at -20°C until further analysis. The liver, spleen, large and small intestines were weighed. In addition, the small intestine was cut into pieces of 5 mm and incubated with PBS for 10 min at 37°C in a shaker to obtain a solution of the intestine (GW). After centrifugation, the supernatants were stored at -20° C. until analysis.

The severity of diarrhea was assessed by both stool weight and stool scale. Stool samples were scored from 1 to 4 (diarrhea index, DI) depending on color, texture and amount according to the following scale: normal (diarrhea index=1), loose yellow-green (diarrhea index=2), completely loose yellow- green (diarrhea index=3), large amount of watery (diarrhea index=4) feces. A diarrhea score of ≥2 indicates diarrheal feces, while a diarrhea index score of ˂2 indicates no diarrhea. In addition, an analysis of the area under the curve (AUC) of severity during the period of diarrhea (days 7-13) was performed to determine the global significance of the severity of diarrhea. The maximum diarrhea index was defined as the highest score during the period of diarrhea.

The incidence of diarrhea was expressed as % of animals with diarrhea (% DA), considering the number of animals in each group, and in % diarrheal feces (% DF), taking into account the number of total samples collected each day in each group. AUC % of animals with diarrhea and % of diarrheal feces during days 7-13 were calculated as the global incidence rate. The maximum % of animals with diarrhea and diarrheal feces was defined as the highest values during the diarrhea period. The diarrhea period was calculated for each animal as the interval between the first and last day of diarrhea. The actual diarrhea days during the diarrhea period were also taken into account.

In all cases, mean and AUC for severity, % DA and % DF were calculated from baseline due to inherent aspects of each treatment; essentially subtracting baseline values the day before rotavirus inoculation due to inherent aspects of each treatment (normalized results). This effect is especially noticeable in the G/F groups due to the inherent stool thinning effect.

UV-inactivated SA-11 particles at 10 ⁵ /ml were used to coat 96-well plates (Nunc MaxiSorp, Wiesbaden, Germany). After PBS-1% blocking with bovine serum albumin (BSA, 1 h, room temperature [RT]), appropriate diluted serum samples or bowel lavage samples (3 h, RT) were added. Peroxidase-conjugated rabbit anti-rat Ig from Dako (Barcelona, Spain), or biotinylated mouse anti-rat IgA (A93-2), IgG1/2a (R19-15), or IgM (G53-238) monoclonal antibodies ( Mab) from BD Biosciences (Heidelberg, Germany) was added after washing. Thereafter, peroxidase-conjugated extravidin (Sigma-Aldrich, Madrid, Spain) and substrate were added. Pooled sera from RV-infected animals from previous studies were used as positive controls in each plate. A quadratic polynomial adjustment was used. The concentration of total anti-PB Ab and anti-PB IgG, IgA and IgM Abs from the PB group was given a value of 100 conventional units (AU)/ml, and the results for the FM+P group and from day 21 were expressed relative to this value.

Fecal samples up to 10 days post virus inoculation (age 7-16 days) were diluted in PBS to 20 mg/mL and homogenized using a Polytron (Kinematica, Lucerne, Switzerland). Fecal homogenates were centrifuged (200 g , 5 min, 4°C) and supernatants were frozen at -20°C until use. Virus particles in the stool homogenates were quantified using the aforementioned ELISA method.

For statistical analysis, the PASW Statistics 20 software package (SPSS Inc, Chicago, Illinois, USA) was used. The Kolmogorov-Smirnov and Levene tests were used to assess the normal distribution and the equality of the variance, respectively. A standard one-way ANOVA analysis of variance was performed with the experimental group as an independent variable. When SA-11 inoculation/treatment had a significant effect on the dependent variable, Scheffe's retrospective analysis was used. The Kruskal-Wallis and Mann-Whitney U-tests were used when an abnormal distribution or different variance was found. Finally, a chi-square test was used to compare the incidence of diarrhea. Differences were considered significant at P˂0.05. All results are expressed as the mean and standard error of the mean number of animals.

results

Effect on growth :

Body weight was recorded in all animals, and neither diet nor viral infection affected their growth. No differences were observed until 1 week post-inoculation (day 14). This means that weight loss due to infection or the effect of diet can be ignored. However, some differences were observed at the end point of the experiment on day 21 (n=9/group). In the PB group, a significant decrease in body weight was observed compared to the REF and FM+G/F groups. If overall weight gain (% from day 2 to 21) is taken into account, the induction of weight loss in the PB group (p < 0.05) and protection by FM+G/F correction is still observed (see Table 1 ).

Table 1: Body weight (g) during the study, before and after virus inoculation on day 7. Results are expressed as means and standard error of the mean (n=21 animals/group).

Age MT (g) REF RV FM+G/F Day Average value SEM Average value SEM Average value 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 28.76⃰ 0.42 36.11 0.40

⃰p˂0.05 compared to REF

In the REF group, a relative increase in body weight of 411.11% was observed. In the RW group, this was significantly lower (p˂0.05) and 310.45%. In the FM+G/F group, this reduced body weight gain was not observed and the relative increase was 439.90%.

Effect on organs

Rotavirus infection also caused some changes in organ weight both at day 14 and 21. Specifically, 7 days after infection (day 14 of life), RV infection caused a lower relative mass of the spleen and a higher relative mass of the liver (p˂0, 05 compared to REF). The effect on liver weight was also observed in the FM+G/F group, but the effect on spleen improved in the FM+G/F group. Day 21 results showed a significant effect on the RV group with a lower relative mass of spleen, liver and small intestine (SI) compared to the REF group (p˂0.05). Also, the relative mass of the large intestine was lower. However, the addition of the FM+G/F mixture prevented the effect of RV infection on these variables (p˂0.05 compared to RV), and the relative weight was higher and did not differ statistically from the reference group.

Frequency of diarrhea

Two different approaches were used to estimate the incidence of diarrhea: % animals with diarrhea (% DA) and % diarrheal feces (% DF).

% DA showed that almost all animals inoculated with RV, 95-100%, exhibited diarrhea at some point. As expected, less than 5% of the animals in the REF group (no PB inoculation) had diarrhea. The % DA in the PB group was above 80% during the first 4 days immediately after SA-11 inoculation (from days 8 to 11 of life). Thereafter, this percentage decreased, and on day 13, none of the animals in the RV group had diarrhea-like feces. Animals from the FM+G/F group showed some improvement during the diarrhea period. In particular, this group showed a trend towards a decrease in % DA compared to the PB group starting two days after infection induction (day 8-11). An improvement in the incidence of diarrhea was observed, which was significantly lower than in the RV group at days 9 and 10 of life (p˂0.05).

Calculation of the incidence of diarrhea as % DF, which takes into account only the amount of diarrheal stool relative to the stool samples obtained, showed the same pattern as the above results for all groups, and an even more preferable effect was observed in the FM+G/F group; showing a lower % of diarrheal feces on days 8, 9, 10, 11 and 12.

The process of diarrhea in the RV group began 1-2 days after inoculation and ended approximately on day 11 of life. Both the period of diarrhea and the days with diarrhea were 3 to 4 days. In the FM+G/F group, these periods were slightly shorter, particularly when looking at % diarrheal stool (DF). In this case, the duration of diarrhea was shortened and no diarrheal feces were observed as early as day 11. In any case, the RV group had a maximum incidence (% DA and % DF) 3 days after RV induction (day 10), while the FM+G/F group showed the highest value on the first day after infection (day 8).

When both % DA-AUC (area under the curve) and % DF-AUC were calculated, the PB group had values of 328.57 and 362.06 conventional units, respectively, while the REF animals did not develop diarrhea and the AUC values were approximately 0. There was a clear overall decrease in the FM+G/F group. The % DA-AUC was reduced to 207.14 (63% compared to the PB group) and the % DF-AUC was reduced to 107.87 (30% of the PB group).

Severity of diarrhea

The diarrhea index (DI) in the PB group was above 2.5 from day 8 to day 10, after which the average dropped to approximately 2 on day 11 and even lower thereafter, indicating no diarrhea in most animals. In the FM+G/F group, a significant decrease was observed and the DI was below 2 on all days post-infection except for the first day.

The Severity Model AUC (sAUC) was 5.99 in the RV group. A significant decrease in sAUC to 3.27 (approximately 55%) was observed in the FM+G/F group (p˂0.05), demonstrating an overall decrease in the severity of the process.

Virus shedding

Virus shedding was assessed after inoculation (days 7-16). In all PB-inoculated animals, maximum virus clearance (p˂0.054 compared to REF) occurred on the first day after inoculation (Day 8), while thereafter no difference was observed between the REF and PB groups. On the first day after inoculation, the FM+G/F correction partly avoided this increase in recovery, showing a lower viral load than the PB group (approximately 35%; p˂0.05) and more resembling REF animals.

immune response

Serum specific anti-RV antibodies (total, IgG, IgM and IgA) and intestinal IgA and IgM were assessed at days 14 and 21 of life. The PB group had measurable levels of specific total anti-PB, IgA, IgG and IgM in serum and intestine even at day 14 of life compared to the REF group.

The FM+G/F nutritional supplement was able to modify the specific humoral immune response against RV. At day 14, while diet did not appear to affect total serum anti-RV antibodies, there was a trend towards increased IgM, a significant increase in IgG (p˂0.05 vs. RV), and a significant decrease in IgA ( p˂0.05 compared to RW).

With respect to the intestinal section, a trend was observed to induce higher levels of IgA on day 14, but lower levels on day 21 due to the FM+G/F diet. The FM+G/F mixture appears to enhance the early response (day 14) in terms of serum anti-PB IgG and intestinal anti-PB IgA.

Example 2: Rotavirus Blocking Experiments

An in vitro blocking assay was performed to investigate the ability of FM+G/F, FM or G/F to bind to SA11 rotavirus. A 96-well plate was co-incubated with 5×10 ⁴ particles/ml SA-11 in PBS-Tween 1 wt% with various dilutions of food or food components (mineral water dilutions) and incubated for 30 minutes. Unbound viral particles were quantified by ELISA (Pérez-Cano FJ, et al., 2007, Pediatr Res 2007;62: 658-663). SA-11 virus particles (10 ⁵ to 10 ³ /ml) were used as a standard in each plate. This assay quantifies the number of SA-11 particles detected after incubation with several product dilutions, as well as the percentage of inhibition. FM+G/F in undiluted form was 267 mg/ml, of which 211 mg/ml FM and 56 mg G/F. For FM and G/F, stock solutions were 211 mg/mL and 56 mg/mL, respectively. These quantities were chosen because they correspond to the daily intake in example 1.

The results are shown in Table 2. All dilutions of food or food components used in example 1 have inhibitory activity. In particular, FM+G/F showed high binding activity, indicating a high blocking potential of this mixture.

Table 2: Percent inhibition of detection of PB particles after incubation with G/F, FM and FM+G/F at various dilutions of food and/or food components. Results are expressed with their SEM duplicates from three independent experiments.

Product G/F FM FM+G/F Product dilution factor) Mean value (SEM) Mean value (SEM) Mean value (SEM) 1/60 10.87⃰ (2.86) 68.56⃰ (8.66) 91.67⃰ (1.94) 1/6 19.98⃰ (2.6) 41.16⃰ (8.7) 98.12⃰ (0.9) 1/3 28.44⃰ (2.35) 29.72⃰ (8.2) 98.9⃰ (0.37) 1/2 39.41⃰ (3.79) 29.68⃰ (10.36) 97.16⃰ (0.59)

⃰p˂0.05 compared to % inhibition without product addition. Nd: no data

#p˂0.05 compared to % inhibition of G/F or FM products alone.

I believe that the binding of rotavirus particles is preferred. Blocking them by direct interaction will prevent or reduce rotavirus entry into enterocytes and replication in the small intestine. The binding of rotavirus particles was much higher with FM+G/F than would be expected based on data for G/F and FM separately. These results indicate an unexpected improved effect of consuming a composition containing both a combination of a partially fermented composition and indigestible oligosaccharides on the prevention and/or treatment of rotavirus infection or diarrhea caused by rotavirus. In particular, a prophylactic effect, a reduction in the viral load of rotavirus infection, can be expected, and this can lead to a reduced severity, reduced incidence and reduced duration of rotavirus diarrhea and/or reduced viral shedding compared to compositions that do not contain a fermented composition or indigestible oligosaccharides.

Example 3: Effect of a fermented mixture with indigestible oligosaccharides on rotavirus infection in rats

An experiment similar to Example 1 was carried out, except that the PB inoculation was performed on day 5. Additional intestinal permeability was measured and short chain fatty acids of the caecum were measured, as is known in the art.

Experimental food or food components have been investigated:

1) First level thermally fermented milk formula. Fermented milk was obtained by fermenting a first level milk formula with Bifidobacterium breve C50 and Streptococcus thermophilus 065 during the manufacturing process. As a result of this process, active bacterial metabolites and cell fragments (postbiotic compounds) were added to the composition of the mixture. It was marketed in France under the names Lactofidus or Lactofidia as a product from Bledina. An additional heating step was used to kill lactic acid producing bacteria after the fermentation step (10 min, 90°C). The difference with the formula in example 1 is that there is a slightly increased fermentation and production of lactic acid. The amount of L-lactic acid was approximately 1.1 g per 100 g.

2) A mixture of indigestible oligosaccharides scGOS (source Vivinal® GOS, Borculo Domo FrieslandCampina) and lcFOS (source Raftilin® HP, Orafti) in a ratio of 9:1 wt./mass.

3) Filler: The filler of the food composition or ingredients in 1 and 2 was mineral water.

Lactating neonates in G14 Lewis rats were divided into several different experimental groups:

1) reference group (REF), which was a control group that was not infected with rotavirus and did not receive dietary modification.

2) the rotavirus (RV) group, which was a control group that was infected with rotavirus and did not receive dietary modification.

3) study group (G/F) who were infected with rotavirus and received a mixture of indigestible oligosaccharides scGOS/lcFOS (G/F). G/F was administered at 0.8 g/100 g body weight/day, respectively.

4) study group (FM) who were infected with rotavirus and received fermented milk (FM). FM was administered at 3 g/100 g body weight/day.

5) study group (FM+G/F) who were infected with rotavirus and received fermented milk (FM) as described above and scGOS/lcFOS indigestible oligosaccharide mixture (G/F). FM and G/F were administered at 3 g/100 g body weight/day and 0.8 g/100 g body weight/day, respectively.

Also included as controls were 3 groups of rats that received the same supplements as groups 3, 4 and 5 but without rotavirus infection. The diarrhea values obtained in these groups were used as a baseline to normalize the diarrhea index DI and the incidence of diarrhea (% DA).

Results:

Based on the diarrhea index (DI), the episode of diarrhea lasted from day 6 (day 1 after RV inoculation) to day 9 in the 2 RV group. On day 10, diarrhea stopped in all groups. The duration of diarrhea, as determined by DI, was similar in groups 3 and 4. The maximum DI on diarrhea days (days 6-9) was observed in group 2. In group 5, diarrhea based on DI was already significantly lower on day 9 than in group 2 RV. On all days of diarrhea, DI was lowest in group 5. This indicates a shorter duration of diarrhea in experimental group 5.

The incidence of diarrhea (based on the number of animals with diarrhea, based on the total number of animals, % DA) was the lowest in group 5. On day 9, only in group 5, the incidence of diarrhea was already absent, while it was still present in groups 2 , 3 and 4 indicating shortened duration or rotavirus infection with the combination of FM and NDO.

Table 3: Rates of diarrhea with normalization

Index Group 2 (RV) Group 3 (G/F) Group 4 (FM) Group 5 (G/F+FM) MDA 58.33 8.33 34.79 4.17 DA-AUC 166.66 29.52 86.23 8.33

MDA, animals with maximum diarrhea (%), DA-AUC: area under the curve of the % DA model.

Maximum incidence of diarrhea (MDA), peak incidence was significantly lower (using a chi-square test) in group 5 compared to group 2. MDA was also reduced in groups 3 and 4 compared to group 2. Area under the index curve diarrhea during the experiment was lowest in group 5 (8.33) and highest in group 2 (166.66). This value was 29.52 in group 3 and 86.23 in group 4. In addition, the low value in group 5 is lower than expected based on the effects in groups 3 and 4 separately. This indicates a reduced severity in the group that consumes both FM and G/F.

Intestinal permeability was determined using alpha-1 antitrypsin levels during bowel lavage on day 8. Interestingly, the combination of FM with G/F increased gut barrier function the most during rotavirus infection.

The observed improvement in gut barrier function can be explained by the unexpected increase in gut butyric acid in group 5. Butyrate is known to fuel intestinal epithelial cells and to improve the gut barrier. The butyrate level of the cecum in group 2 was 239.21 µmol/g of the material of the cecum. The elevated butyric acid level in group 5 (484.39 µmol/g caecal material) is greater than what would be expected from the levels found for group 3 (164.75 µmol/g cecal material, i.e. a decrease butyrate) and 4 (309.72 µmol/g caecal material) separately.

Claims (19)

1. The use of an enzymatic composition and indigestible oligosaccharides for the manufacture of a food composition, which is at least partially fermented by bacteria producing lactic acid, and contains intolerant oligosaccharides, in which indigestible oligosaccharides are selected from a group consisting of fructoligosaccharides, non -digestible decorates, galamas. Oligosaccharids, xyloholigosaccharides , arabinooligosaccharides, arabinogalactooligosaccharides, glucooligosaccharides, gentiooligosaccharides, glucomannooligosaccharides, galactomannooligosaccharides, mannanoligosaccharides, isomaltooligosaccharides, nigerooligosaccharides, glucomannooligosaccharides, chitooligosaccharides, soy oligosaccharides saccharides, sialyloligosaccharides, and fucooligosaccharides, and mixtures thereof, for the treatment and/or prevention of rotavirus infection or diarrhea caused by rotavirus in an infant or young child. 2. The use of the fermented composition and indigestible oligosaccharides for the manufacture of a food composition, which is at least partially fermented by bacteria producing lactic acid, and contains intolerant oligosaccharides, in which indigestible oligosaccharides were selected from a group consisting of fructoligosaccharides, non -transcounted by deconstruction, galalctic Oligosaccharids, xyloholigosaccharides , arabinooligosaccharides, arabinogalactooligosaccharides, glucooligosaccharides, gentiooligosaccharides, glucomannooligosaccharides, galactomannooligosaccharides, mannanoligosaccharides, isomaltooligosaccharides, nigerooligosaccharides, glucomannooligosaccharides, chitooligosaccharides, soy oligosaccharides saccharides, sialyloligosaccharides, and fucooligosaccharides, and mixtures thereof, for A. reducing the incidence of diarrhea caused by rotavirus or rotavirus infection, b. reducing the duration of diarrhea caused by rotavirus or rotavirus infection, c. reducing the severity of diarrhea caused by rotavirus or rotavirus infection, d. prevention of rotavirus-induced stunted growth or poor growth and/or stunted development or poor development, e. increasing the blocking of rotavirus particles, f. reduce rotavirus load, and/or g. increase in intestinal barrier function in rotavirus infection in an infant or young child. 3. Use according to claim 2, wherein the reduction, prevention or increase is observed compared to an infant or young child who is administered a food composition that is at least partially non-fermentable and does not contain indigestible oligosaccharides. 4. The use according to any one of the preceding claims, wherein the food composition contains from 0.1 to 1.5% by weight of the sum of lactic acid and lactate, based on the dry weight of the composition, and preferably wherein the sum of L-lactic acid and L- lactate is more than 50 wt.% in terms of the sum of the total amount of lactic acid and lactate. 5. Use according to any one of the preceding claims, wherein the food composition contains lactic acid producing bacteria, preferably selected from the group consisting of Bifidobacterium and Streptococcus, preferably both. 6. Use according to any one of claims 1 to 4, wherein the food composition contains inactivated lactic acid producing bacteria. 7. The use according to any one of the preceding claims, wherein the food composition contains from 2.5 to 15 wt.% indigestible oligosaccharides, based on the dry weight of the food composition. 8. Use according to any one of the preceding claims, wherein the non-digestible oligosaccharides are selected from the group consisting of galactooligosaccharides and fructooligosaccharides, preferably both. 9. Use according to any one of the preceding claims, wherein the food composition does not contain acidic oligosaccharides derived from pectin, in particular galacturonic acid oligosaccharides. 10. Use according to any one of the preceding claims, wherein the food composition is a first level milk formula or a second level milk formula.