Synbiotic composition for children for prevention and treatment of diaper skin rash

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to using a preparation containing the strain Bifidobacterium breve and a mixture of two soluble ingredients A and B for preparing a composition for prevention and treatment of diaper skin rash in the children. The content of the carbon ingredient A in the preparation makes 5-95 wt % of total content of the ingredients A and B. The ingredients A and B differ by an average number of monosaccharide carbohydrate links with an average chain length of the ingredient A is at least 5 monosaccharide links less than the average chain length of the ingredient B, and a structure of the monosaccharide carbohydrate links.

EFFECT: invention provides reducing allergic reactions in skin rash in the children.

9 cl, 2 dwg, 7 tbl, 29 ex

The technical field

The present invention relates to preparations containing probiotic and prebiotic for babies, especially those not receiving breast-feeding.

Prior art

Babies do not have the intestinal flora at birth. As a result of contact with the mother at birth and subsequent breast feeding is the rapid development and proliferation of intestinal flora. During the development of the intestinal flora remains immature, are in delicate balance with the environment, prone to rapid changes and, consequently, to diseases and lesions in the presence of pathogens. It is known that infants are breastfed, less likely to get infections or diseases than children receiving artificial nutrition. Therefore, infants are breastfed, are less susceptible to gastrointestinal infections in regard to both the incidence and duration of illness, they are less frequent these atopic diseases, such as allergies, eczema, allergic asthma, and they are less prone to constipation than infants receiving non-breast feeding. Normally, the intestinal flora of infants receiving breast feeding mainly consists of bifidobacteria and lactic acid bacteria. Breast milk contains milk oligosaccharides (HMO), which is a growth factor for the of epidemicity in the intestines of infants. Flora of infants fed milk mixture, more varied and usually contains more species ofBacteroides, ClostridiumandEnterobacteriaceae. In the body of infants receiving infant formula, contains from one-tenth to two thirds of the number of bifidobacteria in the body of infants receiving breast-feeding. It is believed that bifidobacteria are important microorganisms that help maintain a balanced intestinal microflora, and postulated that bifidobacteria have a revitalizing effect, including prevention and/or treatment of diarrhoea and intestinal infections. In addition, it is shown that bifidobacteria play a role in the immune system of the host.

The intestinal flora of children can be modified by changing the composition of food by consumption of probiotics or prebiotics. As an example, the probiotic approach in EP-A-0904784 described the application of a mixture of strains of microorganisms, including strains ofBifidobacterium. However, the problem with this is that the mixture of microbes, providing some beneficial effects on health can also have an adverse effect on the still immature intestinal flora of infants receiving artificial nutrition, which is associated with a broad spectrum of activity of this mixture. In addition, many probiotic supplements have a short with the OK of date and contain too little of living organisms, as a result they are not able to provide the expected probiotic action.

Prebiotics are defined as newsweaver.ie food ingredients that selectively stimulate the growth and/or activity of one or more bacteria in the colon and as a result have a beneficial effect on the host (Gibson and Roberfroid, J. Nutr. 125:1401-1412, 1995). The preferred way to improve intestinal flora artificially fed children is to stimulate bifidobacteria already present in the intestines of children receiving artificial nutrition, specific nevereverever the oligosaccharides, i.e. prebiotics. Also as prebiotics was offered a mixture of oligosaccharides and polysaccharides, for example, according to WO 00/08948. An example of such a mixture can serve as a combination of galactooligosaccharides with fructooligosaccharide. It was shown that the levels of bifidobacteria in the body of infants fed formula containing prebiotics, was higher than in the case of supply standard formula (see Moro et al., J. Pediatr. Gastroenterol. Nutr. 34:291-295, 2002).

Existing up to the present time, the approach consisted in General stimulation of bifidobacteria, i.e. at the level of genus. The genusBifidobacteriumconsists of many different kinds, different metabolism, enzyme activity, utilization of oligo - and polisher the Dov, the composition of cell membranes and interaction with the immune system of the host. In this regard, it can be expected that not every kind ofBifidobacteriumhas the same functional effect on the child. Examples of various species ofBifidobacteriumcan serve asB. longum, B. breve, B. infantis, B. adolescentis, B. bifidum, B. animalisandB. dentium. B.adolescentisprevails in the flora of adults and is rarely found in the feces of healthy infants and children.B. animalis andB. lactisnot found in humans, andB. dentiumis a pathogenic bacterium. In healthy infants bifidobacterial flora mainly consists ofBifidobacteriuminfantis, B. breveandB. longum. Kalliomaki et al. (Cur Opin Allergy Clin Immunol. 2003 Feb; 3(1):15-20 and cited there links) reported that suffer from allergic infants haveBifidobacteriumflora similar to adults, while children typicallyBifidobacteriumflora was detected in healthy children, indicating a correlation between the presence of certain species ofBifidobacteriumand the likelihood of developing allergies. These results indicate that stimulation of the genusBifidobacteriumin the colon of a child may be inadequate. The aim of the invention is the provision of infants receiving artificial nutrition, flora, reminiscent of the flora of breastfed babies, at the species level.

In the present invention, the term "child breast-fed is", applies to infants receiving exclusively breast milk. The term "child, not fed or partially breastfed" refers to infants who are not receiving or not receiving exclusively breast milk. This definition refers to infants who receive at least the contents of the bottle of artificial feeding in a day, i.e., at least 80 ml of formula per day, the rest of the food, if any, provided solid food or liquid food, such as milk, i.e., the definition refers to infants, partly feeding on mother's milk.

The invention

Found that the increase in the content ofBifidobacteriumwhen using mixtures neoslavery carbohydrates also regulates the population ofBifidobacteriumtowards populations are more similar to the child, i.e., depletedB. catenulatum,B.pseudocatenulatumandB. adolescentiswhereas infants fed standard formula, have flora, more like an adult, which is dominated byB. catenulatum,B. pseudocatenulatumandB. adolescentis. Also found that the population ofBifidobacteriumin infants, the power of which includes prebiotics contains insufficient amounts of one specific microorganism, namelyBifidobacterium breve.

In accordance with one aspect of the invention OEM home button Flex cable is hearing preparation containingBifidobacteriumbreve and the mixture neoslavery carbohydrate prebiotics. Discovered that this drug is very useful and suitable for the regulation of populations ofBifidobacteriumat the species level in the gastrointestinal tract of children. In addition, it has been unexpectedly discovered that the addition of other species ofBifidobacteriumbesidesB. breveis not necessary because already held sufficient regulation by the drug.

According to another aspect of the invention provides a product containingBifidobacteriumbreve and the mixture neoslavery carbohydrate prebiotics, which contains at least two different, fairly soluble carbohydrate components a and B.

Another aspect of the invention provides for the use of the drug for children who are not receiving fully or partially breast-feeding.

According to another aspect of the invention provides for the use of the drug for the manufacture of a composition intended for the regulation of populations of speciesBifidobacteriumin the gastrointestinal tract of children who do not receive fully or partially breast-feeding.

According to another aspect of the invention provides for the use of the drug for the manufacture of a composition intended for the prevention or treatment of immune system.

According to another aspect of the invention is provided the use of a mixture of carbohydrates to regulate populations of BifidobacteriumcatenulatumB. pseudocatenulatumand/orBifidobacterium adolescentisin the gastrointestinal tract of children who do not receive fully or partially breast-feeding.

According to another aspect of the invention provides a method of species-specific detection and quantification of species of the genusBifidobacteriumfound in humans, especially children, as well as diagnostic kit for detection and quantification of speciesBifidobacterium.

Detailed description of the invention

Drug

1)Bifidobacterium breve

Bifidobacteriumbreve is a necessary ingredient of the invention. The method proposed by the authors, it was found that this bacterium is present in limited quantities in infants receiving artificial nutrition. Accordingly, the introduction of this bacterium together with a mixture of carbohydrates helps to normalize the population of the speciesBifidobacteriumto a level equivalent to that present in the gastrointestinal tract of breastfed infant.

Preferred strains ofBifidobacteriumbreve can be selected from isolates faeces of healthy infants, breast-fed. Typically, these strains are commercially available from manufacturers of lactic acid bacteria, but they can be directly isolated from feces, identified, characterized by small, high the us and produced. Examples of commercially available B. breve can serve asB. breveBb-03 from Rhodia,B. breveMV-16 from Morinaga andB. brevefrom Institut Rossel, Lallemad, butB. brevecan also be obtained from these collections of cultures as DSM 20091 and LMG 11613.

The number ofB. brevein the preparation of the invention is based on the total amount of soluble neoslavery carbohydrates and preferably is 10⁷-10¹¹, more preferably 10⁸-10¹⁰cfu (colony forming units) of bacteria on grams of total carbohydrates. In the case when the drug is used as an additive, it is preferable thatBifidobacterium breveattended number C⁶-1,C¹¹cfu/g, preferably C⁷-C¹⁰cfu/g, more preferably C⁸-C¹⁰cfu/g In the case of the drug as a (full) baby food, it is most preferable that the number of B. breve food was C⁴-C¹⁰cfu/g, preferably C⁶-C⁹cfu/g, more preferably C⁷-C⁸cfu/g of baby food. This concentration is chosen so that the daily dose was C⁶-1,C¹¹cfu/g, preferably C⁷-5x10¹⁰cfu/g, more preferably C⁸-C¹⁰cfu/g

2)The mixture neoslavery carbohydrate prebiotics

The mixture neoslavery carbohydrate prebiotics that the same is an essential element of the invention. The term "neoslavery" means that the carbohydrates in the gastrointestinal tract remain undigested and pass into the colon in preserverance form.

According to the invention the mixture is not digested carbohydrates contains at least two different, fairly soluble carbohydrate component a and b, which are not digested in the gastrointestinal tract and reach the colon in preserverance. The mixture of carbohydrates according to the invention may also consist solely of these two carbohydrate components a and B.

In a mixture of at least two neoslavery soluble carbohydrate components a and b, the carbohydrate component a is present in amounts of 5-95 wt% of the amount of the carbohydrate components a and B. moreover, at least 50%, preferably at least 75% of the total number neoslavery soluble carbohydrate components a and b are selected from disaccharides until eicosanoides (polysaccharides containing 20 monosaccharide units); the remaining amount can refer to neoslavery monosaccharides and neoslavery polysaccharides containing more than 20 links. Also preferably, more than 95%, preferably more than 98% of the total soluble neoslavery carbohydrates had a chain length of not more than 100 links. When specified in the description percentage and average LVEF who each refer to weight percents and averages, besides the obvious use of a different basis, or where otherwise indicated.

The carbohydrate components can vary in three ways:

(i) to (average) number of monosaccharide units in the carbohydrate with an average chain length of the component And at least 5 monosaccharide units shorter than the average chain length of the component; this means that if carbohydrates a and b contain the same structural units, i.e. form a mixture of homologues that differ only in chain length, the distribution of homologues should have two maxima, one of which corresponds to a value of less than 7, and the other more than 7, and two such maximum is located at a distance of at least 5 links from each other; in this case, carbohydrates, containing up to 6 links (hexasaccharide) are part of the component, and carbohydrates of 7 links (Gateshead) are part of the component;

(ii) the structure of the monosaccharide units of the carbohydrate component a And constructed of various structural units of the component; in the case when a and/or built from repeating combinations of different monosaccharide units, for example, in the case of galactomannans and arabinogalactans at least 50% of the monosaccharide units of these two components must be different from each other (in the example above, or one or both components must content shall be less than 50% anhydrogalactose links);

(iii) both a and b differ from each other (average) the chain length and structure; such an embodiment is preferred.

Preferably, component a is chosen from neoslavery monosaccharides up to hexasaccharides the same carbohydrate structure, and component To choose from Gateshead and higher polysaccharides with the same carbohydrate structure. Thus, the carbohydrate component a is at least one newsvideos of monosaccharide or at least one newsvideos oligosaccharide. The term "oligosaccharide" refers to carbohydrates containing from 2 to 6 monosaccharide units, inclusive. Preferably, the carbohydrate component a can also be formed by a mixture of two or more of these sugars. Therefore, it may consist of any number of monosaccharides and/or oligosaccharides of this type, i.e. the same structure.

According to this preferred variant implementation of the carbohydrate component comprises at least one polysaccharide comprising 7 or more monosaccharide units. The term "polysaccharides" refers to carbohydrates, ranging from Gateshead (for example, Gateshead, octasaccharide, nonshared, decesare and so on). The upper limit of the chain length polysaccharides is not discussed specifically, and they can have a length of at few with the Yong and even several thousand monosaccharide units. However, according to the invention, the chain length of more than 100 (about 16 kDa), especially over 700 (about 100 kDa) are less preferred. Preferably, component b contains no more than 5% or even not more than 2% of the homologues consisting of more than 100 monosaccharide units. The carbohydrate component must consist only of a single polysaccharide of this type or, preferably, of two or more polysaccharides of the specified type with different chain length, i.e., having the same structure.

Carbohydrate component And makes up 95% of the mass of the total amount of carbohydrate component and a carbohydrate component In (a+b=100% mass). The carbohydrate component comprises 5-95 wt% of the total amount of carbohydrate component and a carbohydrate component Century. According to a preferred variant implementation of the component quantity And is 95-60 wt%, preferably 95-80% of the mass and especially preferably 95-90% of the mass, and the quantity of the component In amounts of 5-40 wt%, preferably 5-20%, and particularly preferably 5-10% of the mass of the total amount of carbohydrates, provided that a+b=100% of the mass.

The term "soluble carbohydrate in the context of the invention refers to carbohydrates with a solubility of at least 50% according to the method described by L. Prosky et al, J. Assoc. Anal. Chem 71:1017-1023, 1988.

At least 80% carbohydrates or saccharides from the amount of carbohydrate components and To have a prebiotic effect. Preferably, at least 80% of the carbohydrates included in the carbohydrate component a and at least 80% carbohydrates, related to carbohydrate In, have a prebiotic effect. In other words, it is preferable that at least 80% of the mass of each carbohydrate or saccharide of the carbohydrate components a and b have reached the colon in newsvideo (hence, not capable of absorption in the small intestines). Thus, the carbohydrates or saccharides of the carbohydrate components a and b, located in the gastrointestinal tract and not absorbed and is not digested in the stomach or the small intestines and into the colon unchanged.

According to the invention, the term "prebiotically active carbohydrate" refers to a carbohydrate that enters the large intestine in an undigested form (and, therefore, not absorbed in the small intestines), and selectively stimulates the growth and/or activity of one or a limited number of bacterial species present in the gut, and thus contributes to maintaining good health. Consider prebiotic effect of such carbohydrates and specific mechanisms of their action are described in detail in “G. F. Gibson & M. B. Roberfroid, J. Nutr. 1995; 125: 1401-1412, which is specifically referenced and the contents of which are included in this description.

The maximum number is abioticheskie inactive carbohydrates or sugars in the carbohydrate components a and b is 20%. Such carbohydrates or sugars are water-soluble substances which can be allocated in undigested form. Such carbohydrates can produce a physical effect, consisting, for example, to increase the volume of feces or accelerated water absorption.

To estimate the ratio of the carbohydrate components a and b In the diet or pharmaceutical product was carried out by the following stages. In the first stage, soluble carbohydrates were extracted from the product water. Fats and proteins were removed from the extract. In the second stage, soluble carbohydrates or extract, respectively, were worked by human enzymes, for example, human amylase, pancreatic juice or drugs ciliated edges of the cells of the small intestines. The result has been newsweaver.ie carbohydrates (with the exception of in vivo absorbed monosaccharides obtained in the experiment in vitro), representing two of the carbohydrate component a and B. it is Assumed that 80% of their number has prebiotic activity.

Thus, a mixture of carbohydrates designed for use in the preparation of the invention are mixtures in which carbohydrates, soluble and newsweaver.ie in the above sense, satisfy the above criteria and constitute the components a and B.

The carbohydrate component a can consist, for example, from one or the escolca the following carbohydrates: β-galactooligosaccharides,

α-galactooligosaccharides, fructo-oligosaccharides,

isolariciresinol, focalisation, mannooligosaccharides,

xylooligosaccharides, sialyloligosaccharides,

N-glycoproteinoses, O-glycoproteinoses,

glycopeptidolipids, caloosahatchee,

fitosanitarios, heterosaccharides,

galactooligosaccharides, glukuronkongugatov,

β-glucan (for example, 1,3-) oligosaccharides

arabinosylcytosine, arabinogalactans,

xylopyranoside, galactopyranoside,

runoilijaneidon,

soybean oligosaccharides (stachyose, raffinose, verbascose) and

lacto-N-neoteris, and the carbohydrate component may, for example, include one or more of the following carbohydrates or sugars, such as fruit(oz)Ana, including inulin, galactanes, fucoidans, arabinan, Kilani, xanthane, β-glucan, newsviva Polydextrose, neoslavery maltodextrin, galacturonan, N-glikana, glikana, hyaluronic acid, chondroitin, xiloglucanes, arabinogalactan, gum Arabic, alginates, carragenan, galactomannan, glucomannan, arabinoxylane, glycosaminoglycan, glycoproteinoses, proteoglycans, soy polysaccharides. It should be noted that the digestible carbohydrates are not part of the components a and B. So, glucose, fructose, g is lactose, sucrose, lactose, maltose and maltodextrins are not part of these components, even if they are lower homologues of galactooligosaccharides, fructo-oligosaccharides (inulin), etc. Newsweaver.ie carbohydrates of the invention, generally do not contain significant quantities of glucose units linked by alpha 1,4 and/or alpha-1,6 provisions, as in derivatives of starch, such as carbohydrates are digested. However, some polysaccharides and starch maltodextrins type can be newsweaver.ie or "sustainable" by physical and enzymatic methods; such oligo - and polysaccharides are covered by the present invention as long as they have substantial solubility.

In the selective combination of oligosaccharides and polysaccharides, and the simultaneous presence of the carbohydrate components a and b in the colon can be stimulated positively affect the health of the microorganisms and/or can be suppressed pathogenic microorganisms, and these effects are much more effective than when only one of these carbohydrate components. With the introduction of carbohydrate combinations can provide very rapid normalization of the colonic flora to maintain or prevent changes of intestinal flora when R is slichnih stressful situations and thus, the impact on the colonies of bacteria in the colon in a way that is more efficient than when using previously known carbohydrates.

According to a preferred variant implementation, at least 80% of the mass of the carbohydrate component and a carbohydrate component, part of the carbohydrates, which are bifidogenic and/or stimulate lactic acid bacteria. As it has been unexpectedly discovered that the use of a combination of oligosaccharides and polysaccharides having the above properties can be stimulated a stronger effect on the growth of lactic acid bacteria than in the case of oligosaccharides or polysaccharides separately. The result is stimulated not only the lactic acid bacteria that are naturally present in the intestine, but is also stimulating, sometimes selective effect on the growth of the exogenous input of bacteria.

In addition to considering the indirect impacts through the bacteria and their metabolites such as organic acids (acetates, lactates, etc.,), the change of the pH value and the stimulation of colonization, also appears to be a direct positive physical effect on peristalsis, the water content, the amount of faeces and mechanical effect on the mucous membrane of the intestine.

Thus, carbohydrate specialinterests not only nourishing effect, but also a wide spectrum of activity. In addition to the above biological effects, using mixtures of the present invention can achieve the following effects: stabilization of natural microflora, preventing growth of pathogenic substances/organisms, such as toxins, viruses, bacteria, fungi, transformed cells and parasites, dissolution complexes toxins, viruses, bacteria, fungi and other pathogens that contain endogenous cells, and their removal from the body and accelerate the healing of wounds.

Therefore this mixture can be used for the prevention and/or treatment of symptoms or diseases associated with impaired intestinal flora, for example, which is a consequence of the Association or the adhesion of these substances and organisms with epithelium or other endogenous cells.

Discovered that carbohydrate mixture is particularly effective in the case when the carbohydrate component a has a structure different from the structure of the component C. This is a different structure may be connected, for example, with the composition of monosaccharides, when, on the one hand, use fructans, and on the other galactanes. In addition, structural differences can be related to the nature of the glycosidic bonds (for example, differences between the bonds in α-galactooligosaccharide and β-galactooligosaccharide, or α-glucan (starch) and β (cellulose)). The composition of the monomers, and the nature of glycosidic bonds can influence the chemical properties (e.g. solubility) or physiological characteristics (e.g., monthly).

It should be borne in mind that carbohydrates identical dwellings are homologues, which can vary the length of the chain, but consist of the same monosaccharide units or combinations thereof. As a rule, subsequent homologue differs from the previous one by adding a single monosaccharide link present in the previous homologue. However, one link, usually end may be different, as is the case in some fructans containing a chain of (anhydrous)fructose units with a terminal glucose unit.

Preferably, the length of the polysaccharide chain of the component or average chain length in the case of a mixture of polysaccharides was at least three, preferably at least five units greater than the length of the oligosaccharide chain of the component or the average chain length of the mixture of oligosaccharides. Preferably, the average chain length of oligosaccharides And was 2-6 units, and the average chain length polysaccharides was 7-30 units, more preferably 8-20 links. In the presence of both oligosaccharides and polysaccharides same structure, carbohydrates such structure rossmar is ment as a component And in that case, when the average chain length has a value of less than about 6.5, and the individual elements with the length of the chain 7 and above do not apply to component A; on the other hand, they are treated as a component In the case when the average chain length has a value of about 6.5, and in this case the individual components with a chain length of 6 and below do not apply to the component C. In the presence of oligosaccharides and polysaccharides same structure in the absence of sugars other structure shall be two high on each side of 7 units, or otherwise, as explained above, not satisfied the condition of having two different carbohydrate components.

In such carbohydrates inter alia can be a significant part of the mixtures. With the introduction of the mixture of carbohydrates of different sizes and/or different "classes" or "structures" may be a synergistic effect compared to the prebiotic effect of substances of groups a and b separately.

The carbohydrate component may belong to the same class of compounds, but can also consist of several classes (for example, And may be a mixture of galactooligosaccharides and focalisation), whereas the carbohydrate component In equally can be derived from compounds of one or more classes (for example, may be a mixture of inulin with xylanase)

Preferred carbohydrate blend consists of galactooligosaccharides and inulin.

Particularly effective mixture containing at least 60 wt%, preferably 80-100% of the mass of the carbohydrate components As belonging to the group of galactooligosaccharides. The preferred mixture may also contain at least 60 wt%, preferably 80-100% of the mass of the carbohydrate components belonging to the group of fructooligosacharides. To obtain carbohydrate mixtures can be used known carbohydrates and mixtures thereof, used for the production of food or food products. You can also use raw materials, pre-modified by special methods. Obtaining mixtures can be realized as a result of simple mixing appropriately selected carbohydrate or oligosaccharide, a polysaccharide or mixtures of carbohydrates. The original components have to be mixed with each other in such a way as to match the parameters of ready mixes. As raw materials can use a backup carbohydrates (fructans, galactooligosaccharides legumes, fucoidan, α-glucan, laminarin, carrageenan, Manana, galactomannan, agar), natural resin, the carbohydrates of glycoproteins with N-glycosidic bond, the carbohydrates of glycoproteins with O-glycosidic bond, glikana glycolipids, carbohydrates, obtained by fermentation (galactor hosahalli, glucooligosaccharide, fructo-oligosaccharides, xylooligosaccharides), bacterial carbohydrates (such as xanthane), and oligosaccharides (galactooligosaccharide, glucooligosaccharide (α 1-2 and α 1-3 glucose residues), xylooligosaccharide), and skeletal carbohydrates, such as cellulose, hemicellulose (arabinan, galactanes), pectins and chitina. Preferably, such substances were food purity (Complex Carbohydrates in Foods, British Nutrition Foundation; Chapman & Hall, London 1990).

Also available for enzymatic modification of raw materials and products using hydrolases (for example, glycosides, transglucosidase and lipases), transferring enzyme, isomers (for example, aldolase and ketols), oxidoreductases (e.g., oxidase and reductase (for example, glucocerebrosidase), LiAZ (for example, polysaccharide lyase) and ligase. In addition, it is possible to implement a technological modification of raw materials and products by pressure (e.g., by extrusion), temperature (for example, when the caramelization), using organic syntheses, organic modification (for example, karboksimetilirovaniya and paramilitaries), acidic and/or basic hydrolysis and fractionation (e.g., size and/or physico-chemical parameters such as charge and hydrophobicity) or using a combination of modifications.

Thus, the carbohydrate mixture is composed, there is, of the following monosaccharides and composed of them, oligosaccharides and polysaccharides: D-glucose, D-fructose, D-galactose, D-mannose, L-fucose, D-N-acetylglucosamine, D-N-atsetilgalaktozamin, D-xylose, L-rhamnose, D-arabinose, D-allose, D-talose, L-idose, D-ribose, as well as monosaccharides containing carboxyl groups, such as D-Galaktionova acid, D-glucuronic acid, D-mannurone acid and/or methylated forms, such as N-acetylneuraminic acid, N-glycolylneuraminic acid and/or its O-acetylated derivative. In addition, the monomers and the higher echelons based on them can be modified groups-OSO₃H and/or OPO₃H.

Newsweaver.ie carbohydrates according to the invention are usually applied with a daily dose of 0.5-30 g, preferably 2 to 15 g, more preferably 3-9, Preferably the preparation is in the form of supplements. This Supplement is suitable for the reception of children receiving artificial nutrition or partially breastfeeding food, including premature and full-term infants receiving artificial or partially breast-feeding.

Consider the drug can also be used as baby food. In this case, baby food according to the invention additionally comprises one or more ingredients selected from the digestible carbohydrate, a source of lipids, istochnikov and mixtures thereof.

3)Other components

In addition to the carbohydrate components a and b may also be present other carbohydrates. These include 1) digestible carbohydrates, which are digested as described above, and (2) insoluble carbohydrates that can be absorbed/assimilated, and even those who are not able to resorbtive/digestion. Typical insoluble, nevereverever carbohydrates intended for use as additives to child nutrition, are soy polysaccharides and resistant starch, cellulose and hemicellulose; the most preferred materials are selected from polysaccharides and resistant starch.

Typical soluble and digestible carbohydrates designed for use as additives to child nutrition, selected from maltodextrins, starch, lactose, maltose, glucose, fructose and sucrose and other mono - and disaccharides, more preferably from maltodextrin, lactose, maltose, glucose, fructose, sucrose and mixtures thereof.

In addition to the carbohydrate components a and b, such carbohydrates, numbered as sub-items 1) and 2) may be present in the mixture as such in any arbitrary number, and in each case depending on the desired final product. Preferably, the insoluble carbohydrates was 0-10% of the mass from the number of carbohydrate mixtures.Type cnie ingredients used as a source of lipids in food supplements to children's nutrition, can be any lipid or fat, suitable for use in baby food. Preferred sources of lipids include milk fat, sunflower oil, egg yolk lipid, canola oil, olive oil, coconut oil, palm oil, stone fruit, palm oil, palm olein, soybean oil, sunflower oil, fish oil and the oil obtained from microbial fermentation, containing long-chain polyunsaturated fatty acids. Such oils may be in the form of higher aleinov, as, for example, high oleic sunflower oil and high oleic safflower oil. In addition, a source of lipids may represent a fraction oils such as palm olein, triglyeride with medium chain length (MCT), and esters of fatty acids such as arachidonic acid, linoleic acid, palmitic acid, stearic acid, docosahexaenoic acid, linolenic acid, oleic acid, lauric acid, capric acid, Caprylic acid, hexanoic acid, etc.,

The preferred source of lipids for dairy mixtures of long-term storage contains triglyeride with an average chain length is preferably in the amount of 15-35% of the mass of the mass of the source of lipids.

Preferably, the East is CNIC lipid has a molar ratio between n-6 and n-3 fatty acids in the range of 5:1 to 15:1, preferably 8:1 to 10:1.

In the presence preferably, the lipid is present in the amount of 20-40% weight by weight of the composition or quantity of 0.8-1.5 g/100 kcal in infant feeding.

Proteins that can be used in food products of the invention include any protein or nitrogen source, suitable for human consumption. Examples of suitable protein sources for baby food usually include casein, whey, condensed skim milk, nonfat milk, soy, pea, rice, corn, protein hydrolysate, free amino acids, protein sources that contain calcium in a colloidal suspension of a protein, or a mixture thereof. According to the invention, it is preferable to use protein in the form of a hydrolysate, which reduces the risk of developing allergies in children. Commercial protein sources are readily available and well known in this field.

Hydrolysates baby food based on milk usually contain 100% whey protein of cow's milk. In other dairy mixtures the ratio between casein and whey usually is 1.8: 0,3-3,0.

In the presence preferably, the protein source is present in the amount of 9-19% weight by weight of the composition. The preferred amount of the protein source used in baby food, is 0.45-1.0 g/100 kJ.

Preferred full pittel the blend contains dietologicheskie significant quantities of all vitamins and minerals, necessary for the daily diet. Some vitamins and minerals are the minimum requirements. Examples of optional minerals, vitamins and other nutrients present in baby food can serve as vitamin a, vitamin b, vitamin B2, vitamin B6, vitamin B12, vitamin E, vitamin K, vitamin C, vitamin D, folic acid, Inositol, Niacin, Biotin, Pantothenic acid, choline, calcium, phosphorous iodine, iron, magnesium, copper, zinc, manganese, chloride, potassium, sodium, selenium, chromium, molybdenum, taurine and L-carnitine. Minerals are usually added in the form of salts. The presence and amount of specific minerals and other vitamins largely depends on the type of the child population that is targeted for food.

If necessary, baby food may contain emulsifiers and stabilizers, such as soy lecithin, citric acid esters of mono and diglycerides, etc., These components are particularly useful for liquid mixtures.

Baby food may optionally contain other substances that have a beneficial effect, such as (non-sugar) fiber, lactoferrin, immunoglobulins, nucleotides, nucleosides, etc.,

Application

Found that the preparations according to the invention is particularly useful for the normalization of the populationBifidobacteriumin accordance with the species distribution of the children, receiving breastfeeding, which is considered "standard" in the gastrointestinal tract of children receiving artificial nutrition or partially fed mother's milk, especially in premature infants, full-term infants, and children during the period of adaptation to solids. The preparations of the invention are also suitable for children in the transition from breastfeeding to replacement feeding.

Accordingly, provides for the use of the drug or composition according to the invention for the manufacture of a composition intended for the normalization of the population of a species ofBifidobacteriumin the gastrointestinal tract of children receiving artificial nutrition or partially fed mother's milk. Also found that the preparations according to the invention is particularly useful for the prevention or treatment of immune system. It is assumed that the immune state is a consequence of differences in species compositionBifidobacteriumin the gastrointestinal tract of children receiving artificial nutrition or partially fed breast milk, compared with children fed breast milk. Typically, these immune condition include a condition selected from allergies, atopic dermatitis, eczema, asthma, atopy, allergic rhinitis, food allergies, skin irritation from Pele is OK diarrhea and combinations of these conditions.

Accordingly, the invention provides for the use of the drug for prevention or treatment of one or more States of the immune system, preferably selected from allergies, atopic dermatitis, eczema, asthma and skin irritation from the diaper. In addition, the drug of the invention can be used for treatment (bacterial) diarrhea and especially viral diarrhea. Also provides for the use of the preparation according to the invention for the prophylaxis and/or treatment of disorders of energy absorption. Discovered that the drug according to the invention can be successfully used for inhibition of infiltration of eosinophils, neutrophils, and mononuclear cells in allergic lesions, and/or inhibiting the immune response Th2 type and/or stimulation of a Th1-mediated immune response. Mainly provides for the use of the drug or composition according to the invention for the manufacture of a composition intended for inhibiting infiltration of eosinophils, neutrophils and mononuclear cells in allergic lesions, inhibition of the immune response Th2 type and/or stimulation of a Th1-mediated immune response.

The invention also provides for the use of the above-described carbohydrate mixture to control the population of some species ofBifidobacteium other thanB. brevein particular to reduce the relative quantities ofBifidobacterium catenulatum, B. Pseudocatenulatumand/orB. adolescentis.

Development of probe and diagnostic kit

The invention also provides a method for quantitative determination of speciesBifidobacteriumespecially found in humans, i.e.Bifidobacterium catenulatum, B. Pseudocatenulatum, B. Adolescentis, B. breve, B. longum, B. bifidum, B. angulatum, B. infantisandB. Dentium,using species-specific oligonucleotide primers and probes.

Such primers and probes can be used for the identification of bifidobacteria and bifidobacterial species using methods such as FISH, PCR, DGGE, TGGE, the hybridization method of the dot-blotting and real-time PCR. Common in all these methods is the stage of hybridization using nucleotides. The specific purpose is to determine the number of species of bifidobacteria using PCR (polymerase chain reaction) in real time.

When functioning as a probe, each of the following sequences may include additional grounds related to their 5'- or 3'-ends.

Such oligonucleotides can be obtained by conventional methods of chemical synthesis, for example using an automatic DNA synthesizer. DNA fragments containing the above sequence can be perceived by the s enzymatic cleavage of genes relevant species Bifidobacterium.

According to the invention the creation of primers and probes specific to the species ofBifidobacteriumfor use in 5' nuclease analysis carried out as follows:

Were developed duplex 5' nuclease analyses forBifidobacterium adolescentis, B. angulatum, B. bifidum, B. breve, B. catenulatum, B. dentium, B. longumandB. infantis,in relation to all bifidobacteria. The authors developed 5' nucleonic analyses on intergenic spacer 16S-23S gdnc instead of the 16S gene gdnc, which is usually used for phylogenetic analysis and specific definitions of bacteria. The choice of the intergenic spacer regions is largely determined by the fact that the results concerning pollution and sensitivity have been described for real-time PCR using 16S gdnc. It also describes the great similarity between sequences of 16S gdnc various species ofBifidobacterium(Leblond-Bourget et al. 1996), which makes it almost impossible to create a series of primers and probes specific to different kinds ofBifidobacterium. Unexpectedly, these problems were solved using the intergenic spacer elements pane.

To create primers and probes of different sequences of 16S-23S intergenic spacer elements of the various species ofBifidobacterium (B. adolescentis[U09511 U09512 (1), U09513 (1) and U09514 (1)]^a,B. angulatum[U09515 (1)]^a,B. animalis[AY225132 (2), L26967 (1) and UO9858 (1)]^a,B. asteroides[UO9516 (1)] ^a,B. breve[AJ245850 (3), UO9518 (1), UO9519 (1), UO9520 (1) and UO9521 (1)]^a,B. bifidum[U09517 (1), UO9831 (1)]^a,B. catenalatum[UO9522]^a,B. choerinum[L36968]^a,B. coryneforme[UO9523]^a,B. cuniculi[UO9790]^a,D. dentium[UO10434]^a,B. indicum[UO9791]^a,B. infantis[AJ245851 (3), UO9525 (1), UO9527 (1) and UO9792 (1)]^a,B. longum[AJ245849 (3), UO9832 (1)]^a,B. pseudolongum[UO9524 (1), UO9879 (1)]^a,B. magnum[UO9878] (1)]^a,B. thermophilum[UO9528 (1)]^a, were obtained from Genbank, based databases EMBL and DDBJ. All obtained sequences were aligned using DNASIS for Windows V2,5 (Hitachi Software Engineering Co., Ltd., Wembly, UK) (^a=access code, 1=Leblond-Bouget, N., H. Philippe, I. Mangin, B. Decaris. 1996. The analysis of 16S RNA and 16S-23S internal transcribed spacer elements of the sequences was established inter - and intraspecifically phylogeneti relative toBifidobacterium. Cm. Int. J. Syst. Bacteriol. 46:102-11, 2=Ventura, M. and R. Zink. 2002. “Rapid identification, differentiation, and proposed new taxonomic classification ofBifidobacterium lactis”. Appl. Environ. Environ, 68: 6429-6434., 3=Brigidi, P., B. Vitali, E. Swennen, L. Altomare, M. Rossi, D. Matteuzzi. 2000. “Specific detection ofBifidobacterium strainsin pharmaceutical probiotic product and in human faces by polymerase chain reaction.” Syst. Appl. Environ. 23:391-399). Full conservative region sequences were used to generate primers and probes for all kinds ofBifidobacterium. Conservative region sequences subspecies of various types, which showed low homology with other is and used for designing primers and probes to determineB. Adolescentis, B. Angulatum, B. breve, B. bifidum, B. catenulatum,(includingB. Pseudocatenulatum), B. dentium, B. infatisandB. longum(includingB. pseudolongumthat is due to the high sequence homology between these two species).

Primers and TagMan MGB probes were designed using Primer Express 1.5 a (Applied Biosystems, Nieuwerkerk a/d IJssel, NL). Used the following criteria: Primers and probes should have a GC content of 30-80% and should be excluded experiments using more than 3 identical nucleotides, especially for guanidine (G)). The melting temperature of the probes should be 68 -70 ° C, whereas the primers should have a melting point of at s below. In addition, at the 5'end of the probe must not be present G and select a circuit containing a larger amount of cytosine (C), the number of G. the last Five nucleotides at the 3'end of the primers should contain no more than two G and/or C bases. Finally, the length of the amplicon should be no more than 150 base pairs. Designed primers and TagMan MGB probes are presented in table 1 and tested for specificity using the Basic Local Alignment Search Tool (BLAST).

The probe is designed to detect all kinds ofBifidobacteriumconsists of the oligonucleotide with a reporter dye VIC^TMin position 5' (Applied Biosystem, NL) and the quencher NFQ-MGB^TMin position 3'(Applied Biosystem, NL), as well as probes for various species ofBifidobacteriumoligonucleotides with a 5' reporter dye, 6-carboxyfluorescein (FAM^TM), and 3' quencher - NFQ-MGB^TM(Applied Biosystem, NL). To determine the total bacterial load was used (universal) probe General-purpose and a set of primers, which are described Nadkarmi, M. A., F. E. Martin, N. A. Jacques, and N. Hunter in “Determination of bacterial load by real-time PCR using a broad-range (universal) probe and primer set. Microbiology 148: 257-266 (2002). Universal probe consists of an oligonucleotide with a 5' reporter dye, 6-carboxyfluorescein (FAM^TM), and a 3' quencher dye, 6-carboxytetramethyl (TAMRA^TM). Designed probes are presented in table 1.

Labeled drugs received traditional ways by labeling the oligonucleotide is capable of detecting the marker. For marks you can use markers, including radioisotopes, fluorescent substances, enzymes, Biotin and haptens.

Hybridization between the labeled drug and the sample can be performed with known methods, such as dot-blotting and Northern blotting. The formation of the hybrid can be confirmed by detection of labeled drug using known techniques such as autoradiography using radioisotopes, antibodies labeled with enzyme, which use an enzyme or Biotin, etc.

DNA fragments of these oligonucleotides presented as SEQ ID selected from SEQ ID No 1, SEQ ID No 2, SEQ ID No 4, SEQ ID No 5, SEQ ID No 7, SEQ ID No 8, SEQ ID No 10, SEQ ID No 11, SEQ ID No 13, SEQ ID No 14, SEQ ID No 16, SEQ ID No 7, SEQ ID No 19, SEQ ID No 20, SEQ ID No 22, SEQ ID No 23, SEQ ID No 25, SEQ ID No 26, respectively, can be used as primers in PCR method designed to identify species. More specifically, the microbial cells to be identified, are bacterials and any of the DNA fragments of SEQ ID selected from SEQ ID No 1, SEQ ID No 4, SEQ ID No 7, SEQ ID No 10, SEQ ID No 13, SEQ ID No 16, SEQ ID No 19, SEQ ID No 22, SEQ ID No 25, and SEQ ID selected from SEQ ID No 2, SEQ ID No 5, SEQ ID No 8, SEQ ID No 11, SEQ ID No 14, SEQ ID No 17, SEQ ID No 20, SEQ ID No 23, SEQ ID No 26 add as a primer, then spend processing DNA polymerase. If using electrophoresis, etc. were observed amplification of DNA, it is meant that the cells possess a part of the gene corresponding to the used DNA fragment, i.e., cells identified as belonging to the same species as the primer of the DNA fragment.

Thus, it provides oligonucleotides containing SEQ ID selected from SEQ ID No 1, SEQ ID No 2, SEQ ID No 4, SEQ ID No 5, SEQ ID No 7, SEQ ID No 8, SEQ ID No 10, SEQ ID No 11, SEQ ID No 13, SEQ ID No 14, SEQ ID No 16, SEQ ID No 17, SEQ ID No 19, SEQ ID No 20, SEQ ID No 22, SEQ ID No 23, SEQ ID No 25, SEQ ID No 26, and the corresponding complementary sequences.

According to the invention also provides olignucleotides probe for detecting target nucleic acid sequences, which is a characteristic species of the genus Bifidobacterium, where the specified probe chosen from:

1) labeled oligonucleotide, which is specifically hybridized with Bifidobacterium adolescentisDNA represented by SEQ ID No 3, or the corresponding complementary sequence;

2) labeled oligonucleotide, which specifically hybridized withBifidobacterium angulatumDNA represented by SEQ ID No 6, or the corresponding complementary sequence;

3) labeled oligonucleotide, which specifically hybridized withBifidobacterium bifidumDNA represented by SEQ ID No 9 or the corresponding complementary sequence;

4) labeled oligonucleotide, which specifically hybridized withBifidobacterium breve DNA represented by SEQ ID No 12 or the corresponding complementary sequence;

5) labeled oligonucleotide, which specifically hybridized withBifidobacterium catenulatumDNA represented by SEQ ID No 15, or the corresponding complementary sequence;

6) labeled oligonucleotide, which specifically hybridized withBifidobacterium dentiumDNA represented by SEQ ID No 18, or the corresponding complementary sequence;

7) labeled oligonucleotide, which specifically hybridized withBifidobacterium infantisDNA represented by SEQ ID No 21, or the corresponding complementary sequence;

8) labeled oligonucleotide, which specifically hybridized withBifidobacterium longumDNA represented by SEQ ID No 24, or the corresponding complementary on what sledovatelnot;

9) labeled oligonucleotide, which specifically hybridized with allBifidobacteriumDNA represented by SEQ ID No 27, or the corresponding complementary sequence.

In addition, the invention provides a method of species-specific detection of species of the genus Bifidobacterium found in humans, particularly in children, including the following stages:

(A) contacting the sample with an oligonucleotide probe in the solution for hybridization, where the specified probe selected from the group consisting of:

1) labeled oligonucleotide, which specifically hybridized withBifidobacterium adolescentisDNA represented by SEQ ID No 3, or the corresponding complementary sequence;

2) labeled oligonucleotide, which specifically hybridized withBifidobacterium angulatumDNA represented by SEQ ID No 6, or the corresponding complementary sequence;

3) labeled oligonucleotide, which specifically hybridized withBifidobacterium bifidumDNA represented by SEQ ID No 9 or the corresponding complementary sequence;

4) labeled oligonucleotide, which specifically hybridized withBifidobacterium breveDNA represented by SEQ ID No 12 or the corresponding complementary sequence;

5) labeled oligonucleotide, which specifically hybridized withBifidobacterium catenulatumDNA represented by SEQ ID No 15, is whether the corresponding complementary sequence;

6) labeled oligonucleotide, which specifically hybridized withBifidobacterium dentiumDNA represented by SEQ ID No 18, or the corresponding complementary sequence;

7) labeled oligonucleotide, which specifically hybridized withBifidobacterium infantisDNA represented by SEQ ID No 21, or the corresponding complementary sequence;

8) labeled oligonucleotide, which specifically hybridized withBifidobacterium longumDNA represented by SEQ ID No 24, or the corresponding complementary sequence;

9) labeled oligonucleotide, which specifically hybridized with all BifidobacteriumDNA represented by SEQ ID No 27, or the corresponding complementary sequence, and

(C) determining whether hybridization of the probe with nucleic acids in a specified sample to detect the presence in the sample of specified species of the genus.

The invention also includes a method of species-specific detection of species of the genusBifidobacteriumfound in humans, particularly in children, including the following stages:

a) amplification of nucleic acid sequences using a set of primers comprising oligonucleotide primer SEQ ID No selected from SEQ ID No 1, SEQ ID No 4, SEQ ID No 7, SEQ ID No 10, SEQ ID No 13, SEQ ID No 16, SEQ ID No 19, SEQ ID No 22, SEQ ID No 23, SEQ ID No 25, and nucleotide primer SEQ ID selected from SEQ ID No 2, SEQ ID No 5 SEQ ID No 8, SEQ ID No 11, SEQ ID No 14, SEQ ID No 17, SEQ ID No 20, SEQ ID No 23, SEQ ID No 26; and

b) determining whether hybridization of the above oligonucleotide probe with the target sequence of nucleic acids.

The described method is convenient for the manufacture of a diagnostic kit. Accordingly, the invention provides a diagnostic kit for detecting in a sample species ofBifidobacteriumselected from theBifidobacterium adolescentis,B. angulatum, B. bifidum, B. breve, B. catenulatum, B. Dentium,B. infantisandB. longumusing hybridization analysis, which contains at least the above-mentioned DNA probe, and one or more additional funds required for hybridization analysis, such as denatured liquid environment hybridization, wash liquid, solid carrier, the vessel labeled hybridization and detection.

Also provides a diagnostic kit for detecting in a sample from the above speciesBifidobacteriumusing PCR analysis, including a set of the above DNA primers, and one or more additional funds required for the PCR analysis, such as polymerase, curing environment, the upper oil layer, the reaction vessel and means for detecting the amplified DNA.

Example 1: Certification of the developed probes and primers for detection of bifidobacteria

Bacterial the major strains, used to confirm the analysis, representing the relative quantification of the different species of Bifidobacterium, are listed in table 2.

All strains of bifidobacteria cultured in Mann Rogosa Sharp (MRS) broth (Oxoid Basingstoke, UK) medium at 37°C for 24 hours under anaerobic conditions. Night culture was stored at -20 ° C until further use.

DNA was extracted from bacterial cultures by thawing 5 ml frozen night crops in a mixture of water with ice. Then the culture was centrifuged for 20 minutes at 4000 rpm at s (Sorvlall RT7, Du Pont, Stevenage, UK) for the deposition of bacterial cells. The precipitate was washed in 1 ml of TES (50 mm Tris-HCl [pH 8.0], 5 mm EDTA; 50 mm NaCl), followed by centrifugation at 4000 rpm for 10 minutes at s. The supernatant was removed and the residue re-suspended in 1 ml of THMS (30 mm Tris-HCl [pH 8.0], 3 mm MgCl₂, 25% (mass/volume) sucrose). After transferring the suspension into 2 ml Eppendorf tube was added 200 μl of lysozyme (0.1 g/ml; Sigma Aldrich Chemie, Stainheim; DE) and 40 μl of mutanolysin (1 mg/ml; Sigma Aldrich Chemie,; DE) and the resulting mixture is incubated for 30 minutes at C. Then the solution was centrifuged for 5 minutes at 10,000 rpm at s (Sigma 1-15, Sigma Laborzentrifugen GmbH, Osterode am Harz, DE). The supernatant was removed and the residue re-suspended in 100 ál of THMS, then add 400 ál TES (including 0.5 SDS) and 7.5 ál of Proteinase K (20 mg/ml); Boehringer Mannhein GmbH, Mannhein, DE). The mixture was shaken and incubated at C within 30 minutes. Then there was the standard extraction with a mixture of phenol/chloroform followed by treatment with 2.5 ál of RNase A (1 mg/ml; Roche Diagnostics, Mannhein, DE) for 30 minutes at C. Then DNA was besieged by keeping at-a for at least 30 minutes after adding 2 volumes of ice ethanol (96%) and 0.1 volume of 0.3 M sodium acetate solution (pH 5,2). Solutions in which the formed precipitate was centrifuged at 13000 rpm for 20 minutes at C and supernatant washed with 500 µl 70% ethanol and subsequent centrifugation at 13,000 rpm for 5 minutes at s. Supernatant was discarded and the precipitate after centrifugation was dried at room temperature. DNA resuspendable in 100 ál of sterile Milli-Q and stored at -20 ° C.

First tested the specificity of each pair 5' nuclease analysis by amplification of 25 µl of different strains (see table 2). PCR reaction volume of 25 μl was performed using a 2.5 ál of DNA templates, and 12.5 μl of TaqMan Universal Master Mix (Applied Biosystem), 900 nm of each primer and 200 nm of each probe and subsequent implementation of TaqMan Universal Temperature Profile, which was heated at 50ºc for 2 minutes, keeping the sample for 10 minutes at C followed by 45 cycles with duration of 15 seconds at s and 60C length is lnasty in 1 minute using the ABI Prism 7700 (Applied Biosystem, Nieuwerker a/d IJssel, NL). All 5' nucleonic analyses were relatively specific species ofBifidobacteriumfor which they were designed, and 5' nuclease analysis, designed to determine the total number of test speciesBifidobacteriumbut not for the other strains, such asPropioni bacteriumorLactobacillus. It should be noted that 5' nucleases analysis B. catenulatum also allows the detection of B. pseudocatenulatum. In addition, samples treated with Dnazol and RNase were tested to confirm that the analysis has not detected contaminated RNA. Preparing the mixture of monoculturesBifidobacterium adolescentis, B. angulatum, B. Breve,B. bifidum, B. catenulatum, B. dentium, B. infantisandB. longumto ensure that the mixture is about 100%. In this case, you can eliminate competition between the different species ofBifidobacteriumused as matrix. This corresponds to the case shown in Fig.1, which displays the set amount in the mixture of each of the speciesBifidobacteriumas well as the total number of speciesBifidobacteriumcontained in the mixture.

Table 3 shows the CV values for the reproducibility and repeatability of different 5' nucleonic tests.

Developed 5'-nuclease analyses were compared with traditional qualitative species-specific PCR (performed using the primers described by Matsuki, T., K. Watanabe, R. Tanaka, M. Fukuda and H. Oyazu. 1999. Distribution of bifidobacterial species in human intestinal microflora examined with rRNA-gene-tergeted species-specific primers. Appl. Environ. Environ. 65:4506-4512) to determine the sensitivity of different tests and checks for false positive or negative results. The results presented in table 3 indicate that the sensitivity of the 5'-nucleonic analysis differs from the conventional species-specific PCR. Table 4 shows the optimal concentration of primers and probe used in the duplex 5'-nucleonic analyses.

Example 2: Clinical trials

The study was performed double-blind, multicenter trial with a placebo control when using the two treatment groups. Infants, malnutrition, ranging in age from 28 to 90 days was chosen from four hospitals in Germany. The children participated in the experiments if their weight at the birth who was 2600-4500 g and they ate a full milk mixtures for at least four weeks before the beginning of therapeutic effects. From the experiment were excluded children with developmental disabilities or with proven or suspected Allergy to cow's milk, children, resulting mnogomodovykh birth, children who had received antibiotics in less than two weeks before the start of the study, as well as children exposed to mixtures containing Pro - or prebiotic, less than a month before the start of the study. After registering children in an arbitrary order determined in one of two experimental groups: a group receiving a diet supplemented 0.8 g/100 ml galactooligosaccharides and fructooligosaccharides (GFSE-group) and the group receiving standard baby food (SF group). The composition of the main nutrients in the formula are presented in table 5.

The group of infants of breast-feeding was used as the reference group (BF group). The stool samples were collected within three days after the start of the study, after 4 weeks and at the end of the probationary period (6 weeks). The study was approved by the Committee on medical ethics of the four hospitals. Prior to the study was obtained written consent from parents.

Nucleic acid was isolated from the faeces defrosting specimen is in the icy water, followed 10H (mass/vol.) dilution in PBS (0,37 M NaCl, 2.7 mm KCl, 8,1 mm Na₂HPO₄[pH 7,4]) and 10 minutes by homogenization using a device stomacher (IUL Instruments, Barcelona, Spain). Homogenized feces were stored at -20 ° C until DNA extraction. The extraction was started thawing a 1 ml sample of homogenised faeces in ice water, followed by centrifugation for 1 min at 1100 rpm to remove debris and large particles. Supernatant was transferred into a new tube and centrifuged for 5 minutes at 10000 rpm Precipitates after centrifugation resuspendable in 1 ml TN150 (10 mm Tris-HCl [pH 8.0], 10 mm RDTA) and transferred into sterile tubes containing 0.3 g of zirconium beads (diameter 0.1 mm, BioSpec Products, Bartlesville, US). In the resulting suspension were added to 150 µl of TE-buffered phenol (pH±7,5) and the samples for 3 minutes was placed in a beater with a mini-balls at 5000 rpm (BioSpec Products, Bartlesville, US). After grinding fraction samples were immediately cooled on ice before adding 150 ál of chloroform. The short samples were shaken and centrifuged for 5 minutes at 10,000 rpm, the upper phase was transferred into a clean Eppendorf tubes with a capacity of 2 ml and proceeded to extraction with a mixture of phenol/chloroform. After phenol-chloropropanol extraction was performed deposition DNA samples are cooled at -20 ° C for at least 30 minutes after adding 1 ml ice ethanol (96%) and 50 μl of 3M AC is Tata sodium (pH 5,2). Then the samples were centrifuged for 20 minutes at 13000 rpm and washed with 150 ál of 70% ethanol. After centrifugation for 5 minutes at 13,000 rpm supernatant was discarded and the sediments were air-dried at room temperature. DNA resuspendable in 100 ál of sterile Milli-Q and stored at -20 ° C.

The relative definition of the various species ofBifidobacteriumin stool samples was performed using duplex 5' nuclease analysis. The relative amount of each species was calculated according to Liu et al 2002. The efficiency of each amplification curve was calculated individually according to the formula E=(threshold_And/threshold_In)^-(t1A-Ct1B)-1. Using the calculated values of the efficiency was calculated initial DNA content (R_othe formula R_o=threshold/(1+E)^Ct. Then the initial number of DNA Bifidobacterium species can be divided into the initial amount of DNA in all species of Bifidobacterium. Then, the obtained value may be normalized to 100% with respect to the number of monocultures.

The total number ofBifidobacteriumcan also be determined using the above method FISH (Langendijk, F. Shut, G. J. Jansen, G. R. Raangs, G. R. Kamphuis, M. H. Wilkison and G. W. Welling “Quantitative fluorescence in situ hybridization of Bifidobacterium spp. with genus-specific 16S rRNA-targeted probes and its application in fecal samples,” Appl. Environ. Environ. 61 (8): 3069-75. (1995)).

The percentage R of the yea Bifidobacteriumin the calculation of the total content of bacteria was 75, 47, and 68% in BF, SF and GFCF group, respectively, which demonstrates that the GFCF group receiving the mixture neoslavery carbohydrates, has more bifidogenic flora, as in the case of BF group compared to the SF group.

Table 6 presents data showing the prevalence of each species in different groups at the beginning and at the end of the test. Table 7 summarizes the number of percentage of individual speciesBifidobacteriarelative to the total number ofBifidobacteria.

In three different groups there are a large number of different species ofBifidobacterium. A significant decrease in the prevalence and number ofB. adolescentisobserved in children receiving breast-feeding, and children receiving GFSF unlike atdata, eating the standard mixture. After 6 weeks of feeding prevalence and the number ofB. Adolescentissignificantly higher in children receiving SF than in children receiving GFSF or breastfed. Analysis of samples of faeces of children receiving GFSF, found a great diversity in bifidobacterial flora, similar to the flora of children receiving breast feeding, this was not observed stimulation of only one or a few species. Apart from the effect on B. adolescentis profiles of children breastfed, and children receiving GFSF, also exhibit a lower content ofB. catenulatum(+B. pseudocatenulatum) than the profile of children eating the standard mixture.B. infantisandB. longumas it turned out , dominate children receiving breast-feeding, and children eating the standard milk formula (SF) or in the standard mixture, supplemented with prebiotics (GFSF).B. brevedominate in all three groups, however, in the group of breastfeeding percentage ofB. breveof the total number of bifidobacteria higher (11.7 per cent) than in SF (4.9%) and GFSF (5,4%) group.

Example 3: Experiments for allergies in animals

Male BALB/c mice, which do not contain specific pathogens, were obtained from Charles River (Maastrict, the Nederlands). Food and water were given without limitation and used mice at the age of 6-9 weeks. All experiments were approved by the ethics Committee for the treatment of animal and of the University of Utrecht, the Netherlands.

Egg albumin (type V) and acetyl-β-methylcholine (metafolin) were purchased from Sigma Chemical Co. (St. Lois, MO. USA). Aluminum hydroxide (AlumImject) was purchased from Pierce (Rockford, IL, USA).

Mice were senzibilizirani two i.p. injections containing 10 mg of egg albumin caused 2.25 mg aluminum hydroxide in 100 µl of saline solution, or only saline on day 0 and 7, from the beginning of the test. The mice were infected by 35, 38 and 41 day by inhalation of aerosols egg albumin when placed in a Plexiglas chamber for 20 minutes. Aerosols were obtained by spraying a solution of egg albumin (10 mg/ml) in physiological solution using device Pari LC Star (Pari respiratory Equipment, Richmond, VA, USA).

After 28 days and before the end of the experiment (i.e., day 42) mice were treated daily by oral administration C⁹(CFU)Bifidobacterium breveand 25 mg of a mixture of galactooligosaccharides and fructooligosaccharides (9:1) through a tube (0.2 ml physiological saline). As control over the probe was injected with 0.2 ml of physiological salt solution.

Respiratory susceptibility to rasparennouu methacholine was determined 24 hours after infection by the aerosol in consciousness and free mice using whole body plethysmography combined (BUXCO, EMKA, Paris, France). The respiratory response was expressed as enhanced pause(PenH).

Statistical analysis: Curves of the respiratory response to metacholine statistically analyzed using the General linear model or repeated measurements followed by post-hoc comparison between groups. Counting cells were statistically analyzed using the Mann-Whitney test (Siegel, S., Castellan Jr. N J, 1988 “Nonparametric statistics for the behavioral sciences” 2^nded. McGrow Hill Book Company, New York, USA). All other analyses were conducted using the criterion of Student (Abramowitz, M., Stegun, I. A., 1972, “Handbook of mathematical functions Dover publications, Inc. New York, USA). Statistically significant was considered a probability value p<0,05.

Results respiratory hypersensitivity: the results of the measurement of respiratory hypersensitivity showed that, compared with the control group, mice receivingB. breve+mix of galactooligosaccharides and fructooligosaccharides demonstrate a statistically significant decrease in respiratory hypersensitivity, indicating that the decrease asthmatic reactions.

In Fig.2 hypersensitivity displayed schedule relative value PenH (enhanced pause) on the concentration of methacholine for mice receiving a combination ofB. breve+mixture of GOS/FOS, and a control group of mice receiving saline. Shown in the graph relative PenH values were obtained by subtracting the control values obtained on the I mice, desensibilisation egg albumin, and normalization of the values obtained for the control group at the highest concentration of methacholine.

As is well known in this field and international directives, composition all of the following examples can optionally contain minerals, trace elements and vitamins, choline, taurine, carnitine, and/or monoset or mixtures thereof. In addition, the composition may optionally be present organic acids, flavorings and dyes.

Example 4

Infant formula containing per 100 ml of the final product (and 13.1 g of powder):

Example 5

Infant formula containing per 100 ml of the final product (and 14 g of powder):

Example 6

Infant formula containing per 100 ml of the final product (and 16.1 g of powder):

Example 7

Infant formula containing per 100 ml of the final product (13 g powder):

Example 8

Infant formula containing per 100 ml of the final product (15 g powder):

Example 9

Infant formula containing per 100 ml of the final product (and 15.1 g of powder):

Example 10

Infant formula containing per 100 ml of the final product (and 15.2 g of powder):

Example 11

Infant formula containing per 100 ml of the final product (and 15.8 g of powder is ka):

Example 12

Infant formula containing per 100 ml of the final product (15 g powder):

When is EP 13

Infant formula containing per 100 ml of the final product (and 15.9 g of powder):

Example 14

Infant formula containing per 100 ml of the final product (and 13.5 g of powder):

Example 15

Infant formula containing per 100 ml of the final product (and 13.7 g of powder):

Example 16

Infant formula containing per 100 ml of the final product (and 13.5 g of powder):

Example 17

Infant formula containing per 100 ml of the final product (and 15.1 g of powder):

Example 18

Infant formula containing per 100 ml of the final product (16.5 g of powder):

Example 19

A product based on milk containing per 100 ml:

Example 20

Infant formula containing per 100 ml (and 15.4 g of powder):

Example 21

Additive: 3 g of the product added to 100 ml of milk containing:

Example 22

Supplement containing: 0.4-0.8 g of material added to 100 ml of milk per gram:

Example 23

Supplement containing per 100 ml:

Example 24

Baby food containing per 100 g (85 g added to 240 ml of milk):

Example 25

Baby food in tubes per 100 ml:

Example 26

Baby food containing per 100 ml product:

Example 27

Baby food consisting of rice flour, containing per 100 g of dry product: (4-7 spoons add in 200 ml of warm infant formula containing milk for the kid starts to walk, or cow's milk):

Example 28/p>

Baby food, consisting of pre-cooked cereals (wheat, rye, rice, barley, oats, buckwheat), containing per 100 g of dry product (5-7 spoons add in 250 ml of warm infant formula containing milk for a beginner to go baby, or cow's milk):

Example 29

Baby food consisting of homogenised vegetables or fruits containing per 100 ml:

1. The application of the product containing Bifidobacterium breve and a mixture of at least two neoslavery soluble carbohydrate components a and b, and the content of the carbohydrate component comprises 5-95 wt.% from the total with the actual content of the components a and b; moreover, the components a and b differ
(i) by the average number of monosaccharide units of the carbohydrate with an average chain length of the carbohydrate component And at least 5 monosaccharide units lower than the mean chain length of the component, and
(ii) the structure of the monosaccharide units of carbohydrate,
for the manufacture of a composition for prevention or treatment of skin irritation from the diaper in children.

2. Application under item 1, where at least 50% of the total number neoslavery soluble carbohydrates are selected from carbohydrates from disaccharides to eicosanoides.

3. Application under item 1 or 2, where at least 60 wt.%, preferably 80-100 wt.%, carbohydrate component And belongs to the group of galactooligosaccharides, preferably, to the group of TRANS-galactooligosaccharides, and/or where at least 60 wt.%, preferably 80-100 wt.%, the carbohydrate component b belongs to the group of fructooligosacharides, including inulin.

4. Application under item 1 or 2, where the composition contains 10⁴-10¹¹cfu of strain Bifidobactenum breve per gram total content neoslavery soluble carbohydrates.

5. Application under item 1 or 2, where the composition is used as a probiotic Supplement containing probiotic strain Bifidobacterium breve in quantities of 1×10⁶of 1.5×10¹¹cfu/g per Supplement.

6. Application under item 1 or 2, where the components of ice further comprises a digestible carbohydrate, the source of the lipids and the protein source.

7. Application under item 6, where the protein is present in the hydrolysate.

8. Application under item 6, where the composition is intended for the treatment or prevention of skin irritation from the diaper in children with the likelihood of developing allergies.

9. Application under item 7, where the composition is intended for the treatment or prevention of skin irritation from the diaper in children with the likelihood of developing allergies.