阅读说明：本技术 用于改善肠屏障完整性的营养组合物、组合物的制备以及治疗方法 (Nutritional compositions for improving intestinal barrier integrity, methods of making compositions, and methods of treatment ) 是由 F·J·普利兹卡诺 C·T·克尼平 B·斯塔尔 于 2018-07-12 设计创作，主要内容包括：本发明涉及包含2’-岩藻糖基乳糖的组合物,其用于治疗病毒引起的肠屏障破坏。还描述了2’-岩藻糖基乳糖在用于制备组合物中的用途以及通过给予组合物的治疗方法。(The present invention relates to a composition comprising 2' -fucosyllactose for use in the treatment of virus-induced breakdown of the intestinal barrier. Also described is the use of 2' -fucosyllactose in the preparation of the composition and methods of treatment by administering the composition.)

1. A nutritional composition comprising fucosylated human lacto-oligosaccharides, preferably 2' -fucosyllactose, for use in the treatment of virus-induced breakdown of the intestinal barrier in a human subject.

2. Nutritional composition for use according to claim 1, wherein the virus-induced breakdown of the intestinal barrier is a rotavirus-induced breakdown of the intestinal barrier.

3. Nutritional composition for use according to claim 1 or 2, wherein the virus-induced breakdown of the intestinal barrier is a virus-induced breakdown of the intestinal epithelial cells.

4. A nutritional composition comprising fucosylated human milk oligosaccharides, preferably 2' -fucosyllactose, for use in improving the integrity of the intestinal barrier in a human subject.

5. Nutritional composition for use according to claim 4, wherein the improvement of the integrity of the intestinal barrier is a reduction of the permeability of the intestinal barrier.

6. Nutritional composition for use according to any one of the preceding claims, wherein the nutritional composition further comprises at least one, more preferably at least two non-digestible oligosaccharides having a DP between 2 and 250, wherein non-digestible oligosaccharides are selected from the group consisting of: fructo-oligosaccharide, galacto-oligosaccharide, xylo-oligosaccharide, arabino-galacto-oligosaccharide, gluco-oligosaccharide, chito-oligosaccharide, glucomannan-oligosaccharide, galacto-mannan-oligosaccharide and oligomannose.

7. Nutritional composition for use according to claim 6, wherein the non-digestible oligosaccharides are selected from the group consisting of fructooligosaccharides and galactooligosaccharides.

8. Nutritional composition for use according to any one of the preceding claims, wherein the nutritional composition further comprises at least one long chain polyunsaturated fatty acid (LC-PUFA) selected from the group consisting of: eicosapentaenoic acid (EPA, 20:5n3), docosahexaenoic acid (DHA, 22:6n3), arachidonic acid (ARA, 20:4n6) and docosapentaenoic acid (DPA, 22:5n3), preferably DHA, EPA and/or ARA.

9. Nutritional composition for use according to any one of the preceding claims, wherein the human subject is an infant or a young child, preferably an infant.

10. Nutritional composition for use according to any one of the preceding claims, wherein the nutritional composition is an infant formula or a follow-on formula, preferably an infant formula.

11. Use of fucosylated human lactooligosaccharide (HMO), preferably 2' -fucosyllactose, for the preparation of a nutritional composition for the treatment of virus-induced breakdown of the intestinal barrier in a human subject.

12. A method for treating virus-induced intestinal barrier disruption in a human subject by administering a nutritional composition comprising fucosylated human lactooligosaccharide (HMO), preferably 2' -fucosyllactose.

13. Use of fucosylated Human Milk Oligosaccharides (HMOs), preferably 2' -fucosyllactose, for the preparation of a nutritional composition for improving the integrity of the intestinal barrier in a human subject.

14. A method for improving the integrity of the intestinal barrier in a human subject by administering a nutritional composition comprising fucosylated human lactooligosaccharides (HMOs), preferably 2' -fucosyllactose.

Technical Field

The present invention relates to infant nutrition containing non-digestible oligosaccharides, in particular to their use for improving the integrity of the intestinal barrier.

Background

Human milk is considered the gold standard for infant nutrition due to its unique and unequalized combination of nutritional and functional ingredients. Therefore, the World Health Organization (WHO) and the like recommend that only breast milk be provided to infants not older than 6 months. Unfortunately, this is not always possible, in which case parents need to rely on infant formula to provide an optimal human milk substitute or to provide additional nutrition to human milk.

Especially at the beginning of life, nutritional support is needed to mature and protect the gastrointestinal tract of infants. The gastrointestinal epithelium generally acts as a selective barrier to allow absorption of nutrients, electrolytes and water and to prevent exposure to dietary and microbial antigens (including food allergens). Human milk provides several bioactive factors that benefit the relatively immature immune system of early-life newborns. These components are divided into two distinct groups according to their protective effect or their ability to promote maturation. In this sense, Human Milk Oligosaccharides (HMOS) are thought to play a role in both protection and maturation. In order to mimic the beneficial effects of human milk as closely as possible, considerable attention has been given to human lactooligosaccharides in the search for infant formulas with optimal compositions.

The 162 HMOS structures found to date and described in Urshima et al (Trends in diabetes and diabetes technology, 2018, 30; 172, SE51-SE65) show a lactose, polylactosamine or lacto-N-disaccharide core, which can be further conjugated with fucose or sialic acid or both. This structure confers a high degree of chemical variability and protection from digestion. Thus, most HMOS is neither absorbed nor metabolized in the proximal intestine and reaches the distal intestine undigested, exerting a prebiotic effect on certain bacterial populations, strengthening the intestinal barrier and preventing infection by intestinal pathogens. HMOS is present in human breast milk at relatively high ratios (5-20 g/L). The most abundant HMOS is 2 '-fucosyllactose (2' -FL), which accounts for about 20% of the total oligosaccharides in human breast milk. In vitro and in vivo studies of 2' -FL demonstrated immunomodulatory effects including promotion of anti-inflammatory activity (He et al, Gut 2016; 65: 33-46) and inhibition of pathogen colonization (Ruiz-Palacios et al, J Biol Chem 2003; 278: 14112-20).

Holscher et al, J Nutr 2014; 144:586-591 investigated the effect of HMOS on the maturation of certain human intestinal cell lines. The effect as an enhancer of intestinal cell proliferation is attributed to 2' -fucosyllactose. The modest increase in transepithelial resistance, which can be seen as an indicator of barrier integrity, is attributed to lacto N-neotetraose (LNnT).

Documents such as WO 2012/092155, WO 2013/032674, WO 2016/066175 and WO 2012/092160 all describe human milk oligosaccharides (including 2 ' -fucosylgalactose) and mention barrier functions, but none of them establish a valid link between 2 ' -fucosylgalactose and barrier functions, in particular the effect of 2 ' -fucosylgalactose on barrier permeability and intestinal epithelial cell destruction is not disclosed in the art.

Summary of The Invention

Alpha-1 antitrypsin (A1AT) is a protease inhibitor and is resistant to degradation by digestive enzymes. It is an endogenous marker of the presence of blood proteins in the intestinal tract. The inventors found that in a neonatal rat model of rotavirus infection, as evidenced by high levels of alpha-1 antitrypsin (A1AT) in the intestinal wash (gut wash), the intestinal barrier function was disrupted and that a significant cancellation of this disrupted barrier function could be achieved by the presence of 2' -fucosyllactose. Also surprisingly, the inventors found that this improvement of intestinal barrier function was not only achieved in case of impaired intestinal barrier function, but actually improved intestinal barrier function compared to the control group as evidenced by a significant reduction of A1AT in the intestinal wash compared to the control group.

Furthermore, the inventors found that the presence of other non-digestible oligosaccharides results in an even further improvement of the intestinal barrier function in case of rotavirus induced breakdown of the intestinal barrier function and compared to the uninfected control group.

Detailed Description

Accordingly, the present invention relates to a method for the treatment of virus-induced breakdown of the intestinal barrier in a human subject by administering a nutritional composition comprising fucosylated human lactooligosaccharides (HMOs), preferably 2' -fucosyllactose.

In other words, the present invention relates to a nutritional composition comprising fucosylated human lactooligosaccharide (HMO), preferably 2' -fucosyllactose, for use in the treatment of virus-induced breakdown of the intestinal barrier in a human subject.

For some jurisdictions, the present invention may also be expressed as the use of fucosylated human lactooligosaccharides (HMOs), preferably 2' -fucosyllactose, in the manufacture of a nutritional composition for the treatment of virus-induced breakdown of the intestinal barrier in a human subject.

The present invention also relates to a method for improving the integrity of the intestinal barrier in a human subject by administering a nutritional composition comprising fucosylated Human Milk Oligosaccharides (HMOs), preferably 2' -fucosyllactose.

In other words, the present invention relates to a nutritional composition comprising fucosylated human lactooligosaccharide (HMO), preferably 2' -fucosyllactose, for use in improving the integrity of the intestinal barrier in a human subject.

For some jurisdictions, the present invention may also be expressed as the use of fucosylated human lactooligosaccharides (HMOs), preferably 2' -fucosyllactose, in the manufacture of a nutritional composition for improving the integrity of the intestinal barrier in a human subject.

The amount of fucosylated HMO (preferably 2' -fucosyllactose) is preferably an effective amount for treating virus-induced breakdown of the intestinal barrier and/or an effective amount for improving the integrity of the intestinal barrier in a human subject.

Fucosylated human milk oligosaccharides and 2' -fucosyllactose

Prebiotics are typically indigestible carbohydrate compounds, such as non-digestible oligosaccharides (NDO). These compounds pass through the first part of the gastrointestinal tract without being substantially digested. In the intestine, these compounds are fermented by the microorganisms to release short-chain fatty acids and the like which are absorbed by the human body.

NDO are derived from a wide variety of sources, including human breast milk. Typically, these oligosaccharides are referred to as Human Milk Oligosaccharides (HMOS). Typical NDO used in infant food are GOS and FOS.

Human milk is the first choice food for infants, also known as the gold standard. Human milk contains particularly high levels of oligosaccharides, about 10 to 15g/L, which are generally much higher than NDO levels in milk from livestock. Furthermore, HMOS is structurally different compared to NDO in the milk of livestock. Human NDO are very complex and consist of a heterogeneous set of many different compounds with different sugar compositions. Large scale synthesis is complicated by their complex and polymorphic structure. Therefore, it has not been technically and economically feasible to prepare a composition (e.g. infant formula) having the same NDO composition as human milk. In the method or use of the invention, fucosylated non-digestible human milk oligosaccharides are used.

Fucosyllactose (FL) is a fucosylated non-digestible oligosaccharide present in human milk. It is not present in cow milk. It consists of three monosaccharide units (fucose, galactose and glucose) linked together. Lactose is a galactose unit linked to a glucose unit by a β 1,4 linkage. The fucose unit is linked to the galactose unit of the lactose molecule by an α 1,2 linkage (2 '-fucosyllactose, 2' -FL) or to the glucose unit of lactose by an α 1,3 linkage (3-fucosyllactose, 3-FL). 2' FL is the most abundant NDO in human milk. The HMOS used in the present invention is 2' -FL. Specifically, administration of 2 '-FL resulted in an improvement in the integrity of the intestinal barrier, and administration of 2' -FL also resulted in a therapeutic effect on viral-induced breakdown of the intestinal barrier.

2' -FL, (β -L-Fuc- (1 → 2) - β -D-Gal- (1 → 4) -D-Glc) is commercially available from, for example, Sigma-Aldrich. Alternatively, it may be isolated from human milk, for example as described in Andersson & Donald,1981, J chromatography.211: 170. cndot.1744, or produced by a genetically modified microorganism, for example as described in Albermann et al, 2001, Carbohydrate Res.334: 97-103.

Preferably, the composition of the invention comprises 1mg to 3g 2 ' -FL/100ml, more preferably 10mg to 2g 2 ' -FL/100ml, even more preferably 20mg to 100mg 2 ' -FL/100 ml. The composition of the invention preferably comprises from 0.007 to 20% by weight, more preferably from 0.07 to 10% by weight, even more preferably from 0.15 to 1% by weight, based on dry weight, of 2' -FL. Lower amounts of fucosyllactose are less effective in ameliorating virus-induced diarrhea, while too high amounts result in unnecessarily high costs of the product.

In one embodiment, the nutritional composition for said use according to the invention does not comprise lacto-N-neotetraose (LNnT). In one embodiment, the nutritional composition for use according to the invention does not comprise a human milk oligosaccharide other than 2' -FL.

Non-digestible oligosaccharides other than human milk oligosaccharides

The nutritional composition preferably further comprises non-digestible oligosaccharides other than HMOS. According to the present invention, the presence of other non-digestible oligosaccharides than HMOS leads to a further improvement of the integrity of the intestinal barrier and the presence of other non-digestible oligosaccharides than HMOS leads to an improvement of the therapeutic effect on virus-induced breakdown of the intestinal barrier.

NDO is preferably not or only partially digested in the intestine by the action of acids or digestive enzymes present in the human upper digestive tract (particularly in the small intestine and stomach) and is fermented by the human intestinal microbiota. For example, sucrose, lactose, maltose and the common maltodextrins are considered digestible.

Preferably, the present composition comprises non-digestible oligosaccharides having a DP between 2 and 250, more preferably between 2 and 60. The non-digestible oligosaccharides are preferably selected from at least one, more preferably from at least two of the following: fructo-oligosaccharides (fructo-oligosaccharides), galacto-oligosaccharides (galacto-oligosaccharides), xylo-oligosaccharides (xylo-oligosaccharides), arabino-oligosaccharides (arabino-oligosaccharides), arabinogalacto-oligosaccharides (arabino-oligosaccharides), oligo-gluco-oligosaccharides (gluco-oligosaccharides), chito-oligosaccharides (chito-oligosaccharides), glucomannan oligosaccharides (glucomano-oligosaccharides), galacto-oligosaccharides (galactomano-oligosaccharides) and oligomannose (mannan-oligosaccharides), and non-digestible oligosaccharides selected from milk oligosaccharides from milk and non-milk mammalian species. The group of fructooligosaccharides comprises inulin and the group of galactooligosaccharides comprises transgalactooligosaccharides or beta-galactooligosaccharides.

More preferably, the composition of the invention comprises Fructooligosaccharides (FOS) and/or Galactooligosaccharides (GOS), preferably the galactooligosaccharides comprise beta-and/or alpha-galactooligosaccharides. More preferably, the fructooligosaccharide is a long chain fructooligosaccharide (lcFOS). More preferably, the galactooligosaccharide is a short chain galactooligosaccharide and the beta-galactooligosaccharide is a short chain beta-galactooligosaccharide. Most preferably, the composition comprises long chain fructooligosaccharides and short chain galactooligosaccharides. The weight ratio of short-chain galactooligosaccharides to long-chain fructooligosaccharides is preferably between 100:1 and 10:1, preferably between 20:1 and 1:1, preferably about 9: 1.

The galactooligosaccharide is preferably a beta-galactooligosaccharide. In a particularly preferred embodiment, the composition of the invention comprises beta-galactooligosaccharides ([ galactose ] n-glucose; wherein n is an integer from 2 to 60, i.e. 2, 3, 4, 5, 6,. multidot.. multidot.,. 59, 60; preferably n is selected from 2, 3, 4, 5, 6, 7, 8, 9 and 10), wherein the galactose units are connected together in majority by beta bonds. Beta-galactooligosaccharides are also known as Transgalactooligosaccharides (TOS). For example, β -galactooligosaccharides are sold under the trade name Vivinal (TM) (Borculo Domo Ingredients, Netherlands). Another suitable source is Bi2Munno (Classado). Preferably, the galactooligosaccharides comprise beta-1, 3, beta-1, 4 and/or beta-1, 6 linkages. In a preferred embodiment, the galactooligosaccharide comprises at least 80%, more preferably at least 90%, of beta-1, 4 and beta-1, 6 linkages, based on the total linkages. In another preferred embodiment, the galactooligosaccharides comprise at least 50% beta-1, 3 linkages, based on the total linkages; more preferably at least 60% of the beta-1, 3 bonds, based on total bonds.

Fructooligosaccharides are NDOs comprising beta-linked chains of fructose units with a DP or average DP between 2 and 250, more preferably between 2 and 100, even more preferably between 10 and 60. The fructooligosaccharides comprise inulin, levan (levan) and/or mixed polyfructose (polyfructan). A particularly preferred fructooligosaccharide is inulin. Fructooligosaccharides suitable for use in the composition are also commercially available, for example(Orafti). Preferably, the average DP of the fructooligosaccharides is higher than 20.

In a preferred embodiment, the composition comprises a mixture of inulin and short chain fructooligosaccharides. In a preferred embodiment, the composition comprises a mixture of galacto-oligosaccharides and fructo-oligosaccharides, wherein the fructo-oligosaccharides are selected from short chain fructo-oligosaccharides and inulin, more preferably from inulin. Preferably the weight ratio of the mixture of two different non-digestible oligosaccharides, preferably galacto-oligosaccharides and fructo-oligosaccharides, is between 25 and 0.05, more preferably between 20 and 1. Galactooligosaccharides, preferably beta-galactooligosaccharides, are more capable of stimulating bifidobacteria. Preferably, the composition of the invention comprises galacto-oligosaccharides, preferably beta-galacto-oligosaccharides, with a Degree of Polymerization (DP) of 2 to 10, and/or fructo-oligosaccharides with a DP of 2 to 60.

In a preferred embodiment, the composition comprises a combination of fucosylated human lactooligosaccharides (preferably 2' -fucosyllactose), short chain galactooligosaccharides and long chain fructooligosaccharides. In a preferred embodiment, the weight ratio of the sum of short chain galacto-oligosaccharides and long chain fructo-oligosaccharides to 2' -fucosyllactose contained in the nutritional composition is between 20:1 and 1:100, preferably between 10:1 and 1:50, more preferably between 4:1 and 1:20, most preferably between 1:1 and 1: 15.

LC-PUFA

The composition may also comprise long chain polyunsaturated fatty acids (LC-PUFA). LC-PUFA are such fatty acids: wherein the acyl chain length is from 20 to 24 carbon atoms (preferably 20 or 22 carbon atoms) and wherein the acyl chain comprises at least two unsaturated bonds between said carbon atoms in the acyl chain. More preferably, the present composition comprises at least one LC-PUFA selected from the group consisting of: eicosapentaenoic acid (EPA, 20:5n3), docosahexaenoic acid (DHA, 22:6n3), arachidonic acid (ARA, 20:4n6) and docosapentaenoic acid (DPA, 22:5n3), preferably DHA, EPA and/or ARA. Such LC-PUFAs have a further beneficial effect on improving the integrity of the intestinal barrier.

The preferred content of LC-PUFA in the composition of the invention is not more than 15 wt.%, preferably not more than 10 wt.%, even more preferably not more than 5 wt.% of the total fatty acids. Preferably, the present composition comprises at least 0.2 wt.% LC-PUFA (more preferably DHA), preferably at least 0.25 wt.%, more preferably at least 0.35 wt.%, even more preferably at least 0.5 wt.% of total fatty acids. The composition of the invention preferably comprises ARA and DHA, wherein the weight ratio ARA/DHA is preferably higher than 0.25, preferably higher than 0.5, more preferably from 0.75 to 2, even more preferably from 0.75 to 1.25. The weight ratio is preferably lower than 20, more preferably between 0.5 and 5. The amount of DHA is preferably higher than 0.2 wt.%, more preferably higher than 0.3 wt.%, more preferably at least 0.35 wt.%, even more preferably 0.35-0.6 wt.% of the total fatty acids.

Composition comprising a metal oxide and a metal oxide

The present invention advantageously relates to a composition for a given use, wherein lipids provide 5 to 50% of the total calories, proteins provide 5 to 50% of the total calories, and carbohydrates provide 15 to 90% of the total calories. Preferably, in the composition of the invention, the lipid provides 35 to 50% of the total calories, the protein provides 7.5 to 12.5% of the total calories, and the carbohydrate provides 40 to 55% of the total calories. To calculate the total percent calories of the protein component, the total energy provided by the protein, peptide, and amino acids needs to be considered.

The composition of the present invention preferably comprises at least one lipid selected from animal lipids (excluding human lipids) and vegetable lipids. Preferably, the composition of the invention comprises a combination of vegetable lipids and at least one oil selected from the group consisting of fish oil, animal oil, algal oil, fungal oil and bacterial oil. The present composition comprising 2' -FL is not human milk.

The composition of the invention preferably comprises a protein. The protein component used in the nutritional formulation is preferably selected from the group consisting of non-human animal proteins, preferably milk proteins, preferably proteins from bovine milk, vegetable proteins, preferably soy proteins and/or rice proteins, free amino acids and mixtures thereof. The composition of the invention preferably contains casein, whey, hydrolysed casein and/or hydrolysed whey protein. Preferably, the protein comprises intact protein, more preferably intact bovine whey protein and/or intact bovine casein protein.

The present composition preferably comprises digestible carbohydrates. The present composition preferably comprises a digestible carbohydrate component wherein at least 35 wt.%, more preferably at least 50 wt.%, more preferably at least 75 wt.%, even more preferably at least 90 wt.%, most preferably at least 95 wt.% is lactose. The composition of the invention preferably comprises at least 25 grams lactose per 100 grams dry weight of the composition of the invention, preferably at least 40 grams lactose per 100 grams.

When the nutritional composition is a liquid, its caloric density is preferably from 0.1 to 2.5kcal/ml, even more preferably from 0.5 to 1.5kcal/ml, most preferably from 0.6 to 0.8 kcal/ml. The amount of nutritional composition administered per day is preferably from 50 to 2000ml, more preferably from 200 to 1500ml, most preferably from 400 to 1000 ml.

In one embodiment, the present invention relates to a supplement suitable for fortifying human milk to fortify human milk fortified with a standard human milk fortifier or to fortify a standard preterm formula. In the context of the present invention, the supplement does not contain all the macro and micronutrients required for preterm infants to achieve growth similar to that of a fetus and satisfactory functional development. The term "preterm birth" herein is synonymous with premature birth and means any human infant born before the 37 th week of gestation.

Thus, in one embodiment the nutritional composition according to the invention or for said use according to the invention comprises protein, fat and/or digestible carbohydrates and is selected from the group consisting of infant initial formula, infant follow-up formula (follow on formula), baby milk, preterm formula, post-hospital formula (post discharge formula) and human milk fortifier.

Applications of

According to the present invention, virus induced breakdown of the intestinal barrier is treated by administering a nutritional composition comprising fucosylated human lacto-oligosaccharides, preferably 2' -fucosyllactose. In a preferred embodiment, the intestinal barrier disruption treated is caused by rotavirus, also known as rotavirus-induced intestinal barrier disruption. Following an intestinal infection with a virus, mainly the intestinal epithelial cells are infected and destroyed. In one embodiment of the invention, the viral-induced breakdown of the intestinal barrier is a viral-induced breakdown of the intestinal epithelial cells.

Also according to the invention, the intestinal barrier integrity is improved by administering a nutritional composition comprising fucosylated human lacto-oligosaccharides, preferably 2' -fucosyllactose. The detection of A1AT in intestinal washes is a measure of intestinal barrier permeability. In a preferred embodiment, the improvement in intestinal barrier integrity is a decrease in intestinal barrier permeability. In yet another preferred embodiment, the improvement in intestinal barrier integrity refers to a decreased transport of blood components from the blood to the intestinal lumen. In a preferred embodiment, the blood component is a protein. In a preferred embodiment, the improvement in intestinal barrier integrity refers to a decreased transport of proteins from the blood to the intestinal lumen. In the context of the present invention, the transport of proteins from the blood to the intestinal lumen may also be referred to as protein transport or protein extravasation.

The nutritional composition for use according to the invention is for use in a human subject. Preferably, the human subject is an infant or a young child. Infants are human subjects of 0-12 months of age. Young children are human subjects of 12-36 months of age. In a preferred embodiment, the human subject is a preterm or prematurely born infant. In particular, this subgroup of infants may benefit from the administration of fucosylated human milk oligosaccharides (preferably 2 '-fucosyllactose) or the administration of fucosylated human milk oligosaccharides (preferably 2' -fucosyllactose) in combination with long chain fructo-oligosaccharides and short chain galacto-oligosaccharides, since the organs constituting the intestine of preterm infants are immature compared to term born (term-born) infants, and there is a need to improve the integrity of the intestinal barrier, to reduce the permeability of the intestinal barrier and/or to reduce the disruption of the intestinal barrier.

Preferably, the fucosylated human lactooligosaccharide (preferably 2 '-fucosyllactose) or the nutritional composition comprising the fucosylated human lactooligosaccharide (preferably 2' -fucosyllactose) is administered to the human subject daily. Likewise, the administration of the nutritional composition comprising fucosylated human lactooligosaccharide, preferably 2' -fucosyllactose, or the use thereof, preferably starts before the occurrence of virus-induced diarrhea and lasts for a period of time during which the subject can be diagnosed with diarrhea.

In a preferred embodiment, the human subject is vaginally born, also known as naturally born.

The composition of the present invention is preferably enterally administered, more preferably orally administered. The composition of the invention is preferably a nutritional formula, preferably an infant formula. The composition of the invention can advantageously be applied as a complete nutrition for infants. The composition of the invention preferably comprises lipids, proteins and carbohydrates and is preferably administered in liquid form. The invention encompasses dry compositions (e.g., powders) with instructions for mixing the dry compositions (particularly nutritional formulas) with a suitable liquid (e.g., water).

Drawings

The figure shows the analysis of the concentration of alpha-1 antitrypsin (A1AT) in intestinal washes by ELISA as a measure of intestinal barrier disruption. Results are expressed as mean ± s.e.m. (n-4-8); p <0.05 compared to REF group; compared to the RV group, # p <0.05 (by MWU assay).

Detailed Description

Example 1

Newborn rats were divided into 5 groups (24 animals per group): reference (REF) group, rotavirus infection (RV) group and 3 rotavirus infection groups each supplemented with: a) mixture of scGOS and lcFOS (RV + GOS/FOS group); b)2 '-FL (RV + 2' -FL group); and c) scGOS/lcFOS and 2 '-FL (RV + GOS/FOS + 2' -FL group).

The same normalized volume/body weight (4.5 μ L/g/day) of all products was orally administered to lactating rats once daily from day two to day sixteenth of life (corresponding to a strict lactation period). The RV + GOS/FOS group was supplemented with 0.8g scGOS/lcFOS/100g body weight. The RV +2 '-FL group was supplemented with 0.2g of 2' -FL per 100g of body weight. The RV + GOS/FOS + 2' -FL group received both products at the same dose as given alone and maintained the administration volume (4.5. mu.L/g/day). The REF group and RV group were given the same volume of water.

RV (simian SA-11) was obtained as described previously (Perrez-Cano et al, Peditar Res 2007; 62:658-63) and inoculated (4X 10) on day 5 of life in all experimental groups except the REF group, which received the same volume of Phosphate Buffered Solution (PBS) under the same conditions⁸Tissue culture infectious dose 50[ TCID50]) Rat).

Body weights were recorded daily throughout the study to assess weight gain. Half of the animals of each group (n-12) were sacrificed on day 8 to analyze variables associated with peak diarrhea, and the other half (n-12 of each group) were sacrificed on day 16 to analyze the effect of the supplement after diarrhea was resolved. In addition, the length of the nose-anus and tail were measured to determine the body/tail ratio, in terms of body weight/length²(g/cm²) Calculated Body Mass Index (BMI) and^0.33length) × 1000 (g)^0.33/cm) calculated Lee index.

Sample collection and processing

On days 8 and 16, half of each litter was intramuscularly anesthetized with ketamine (90mg/kg) (meridian Laboratories SA, Barcelona, Spain) and xylazine (10mg/kg) (Bayer a.g., levikusen, Germany), bled and their intestines obtained. The intestine was opened longitudinally, cut into 5 mm pieces, and incubated with 2mL of PBS in a shaker (10 min, 37 ℃) to obtain intestinal washes (GW). After centrifugation, the supernatant was stored at-20 ℃ and-80 ℃ until analysis by alpha-1 antitrypsin (A1AT) was performed.

Quantification of A1AT in intestinal washes (which serves as a marker of intestinal permeability) WAs performed using the rat SERPINA1/α 1 antitrypsin ELISA kit (Life span Biosciences Inc., Seattle, WA, USA) according to the manufacturer's instructions. The standard concentration range is 100 to 1.563 ng/mL. The detection sensitivity is 1.56 ng/mL.

Statistical analysis

Statistical analysis was performed using statistical software package for social sciences (SPSS v22.0) (IBM, Chicago, IL, USA). Data were tested for homogeneity of variance and normal distribution by the Levene test and the Shapiro-Wilk test, respectively. When the data is homogeneous and behaving normally, a conventional one-way ANOVA test is performed, followed by post hoc Bonferroni. Otherwise, the nonparametric Kruskal-Wallis test is performed first, followed by the post-operative Mann-Whitney U (MWU) test. Finally, the frequency of diarrhea occurrence was compared using the chi-square test. When p <0.05, a significant difference was established.

Results

Growth of

As shown by the results for body weight, body/tail ratio, BMI and Lee index (table 1), RV infection did not produce any significant changes in growth, either during the peak diarrhea period (day 8) or at the end of the study (day 16). At the end of the study (day 16, p <0.05), the group supplemented with scGOS/lcFOS had slightly higher body weights, although none of these growth changes changed BMI, some differences were observed in body/tail length ratio and Lee index. At some time points, all supplements increased body/tail length ratio compared to REF or RV. Furthermore, the Lee index is only reduced during the peak diarrhea compared to REF.

TABLE 1 variables relating to growth

Results are expressed as mean ± s.e.m. (n-12); p <0.05 compared to REF (by MWU test); compared to RV, # p <0.05 (by MWU assay).

Intestinal barrier function

Alpha-1 antitrypsin (A1AT) belongs to the serine protease inhibitor (serpin) superfamily and is referred to as a protease inhibitor. It is resistant to degradation by digestive enzymes and is an endogenous marker for the presence of blood proteins in the intestinal tract.

The A1AT levels in intestinal washes were determined by ELISA (see figure) to assess changes in intestinal permeability. During the inflammatory response, serum A1AT levels were significantly elevated and could be transported through the intestinal epithelial layer into the intestinal lumen due to increased permeability of the intestinal epithelial barrier (Yang et al, Sci Rep 2015; 5: 15004). The RV group showed higher levels of A1AT in the intestinal wash but was not statistically different from the REF group. However, supplementation with 2 '-FL and GOS/FOS + 2' -FL reduced the concentration of A1AT in the intestinal washes (p <0.05) compared to RV and REF, suggesting that they are not only able to treat intestinal permeability disruption caused by RV, but they also promote an increase in intestinal barrier function, as evidenced by the reduction in extravasation of the A1AT protein and its transport to the intestinal lumen. Treatment of intestinal barrier disruption was studied on the third day of the diarrhea period, which corresponds to the peak period of diarrhea severity observed in the study animals.

Example 2: infant formula for the treatment of virus-induced intestinal barrier disruption

The infant formula according to the invention comprises per 100ml (13.9 dry weight):

1.4g protein (whey and casein)

7.3g digestible carbohydrates (including lactose)

3.6g fat (vegetable fat, fish oil)

0.6g non-digestible oligosaccharides, of which 60mg 2' -fucosyllactose and 480mg beta-galacto-oligosaccharide, and 60mg fructo-oligosaccharide

According to the guidelines, choline, inositol, taurine, minerals, trace elements and vitamins are also included.