Synbiotic composition for children

FIELD: food industry.

SUBSTANCE: preparation for usage as feeding contains Bifidobacterium breve in quantity 1×104 -1×1010 cfu/g and a mixture of indigestible carbonhydrates. Carbohydrates are chosen from indigestible monosaccharides up to hexasaccharides of the same carbohydrate structure and from indigestible heptasaccharides and top polysaccharides of the same structure including inuline. Addition to children feeding and infant food containing mentioned preparation are proposed as its usage for healing or prevention of children immune disturbances as for usage for children obtaining artificial of partly breast-feeding.

EFFECT: usage of the preparation for producing of composition for prevention and healing of insufficient energy consumption, also for preparation of composition for inhibiting of eosinophils infiltration when having allergy.

16 cl, 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, disease, 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 nagradne food. 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 the growth factor d is I bifidobacteria in the intestine 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 neperevershenymy 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. animalisandB. 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 the 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 the population that are more similar to children, i.e. depleted inB. 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 include trivet drug 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 brevepresent in quantities of 1·106of 1.5·1011cfu/g, preferably 3·107-5·1010cfu/g, more preferably 5 x 108-1·1010cfu/g In the case of the drug as a (full) baby food, it is most preferable that the number of B. breve in the diet was 1·104-1·1010cfu/g, preferably 5·106-3·109cfu/g, more preferably 1·107-5·108cfu/g of baby food. This concentration is chosen so that the daily dose was 1·106of 1.5·1011cfu/g, preferably 3·107-5·1010cfu/g, more preferably 5 x 108-1·1010cfu/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 carbohydrate component a is present in amounts of 5-95 wt.%. from the amount of 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 of the interest and the average value is tions 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 units 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 etc). 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 up to 95% wt. from the total amount of carbohydrate component and a carbohydrate component In (a+b=100% wt.). The carbohydrate component comprises 5-95 wt.%. from 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% by weight. and particularly preferably 95-90% wt., and the quantity of the component In amounts of 5-40 wt.%, preferably 5-20 wt.%. and particularly preferably 5-10 wt.%. from the total amount of carbohydrates, provided that a+b=100% wt.

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 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 Is 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% wt. 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 intestine and enter the colon unchanged.

According to the invention, the term "prebiotically active carbohydrate" refers to a carbohydrate that enters the colon in 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.

Maximum if estvo prebiotics 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, such as 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

arabinoxylanes, 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,galactose, 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% wt. 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 and the like), 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, the carbohydrate mixture is harakteryzuyutsya 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 the-glucan (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, is 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 wt.%. carbohydrate components As belonging to the group of galactooligosaccharides. The preferred mixture may also contain at least 60 wt.%, preferably 80-100 wt.%. 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 (galacteros the sugars, 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 their 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 amount, in each case depending on the desired final product. Preferably, the insoluble carbohydrates was 0-10% wt. the number of carbohydrate mixtures.

Typical 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, triglycerides of 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% by weight. by weight of a source of lipids.

Preferably, the IP is the source of lipids 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 of, preferably, the lipid is present in the amount of 20-40% by 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% wt. 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 and the like, These components are particularly useful for liquid mixtures.

Baby food may optionally contain other substances that have a beneficial effect, such as (neugeborne) 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 inhibition of 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 ofBifidobacterim 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 people, ieBifidobacterium 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), real-time

When functioning as a probe of 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 obtained from the enzymatic cleavage of genes relevant species Bifidobacterium.

According to the invention the creation of primers and probes specific to the species ofBifidobacterium,for use in 5'-nuclease the analysis is performed 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 the 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]aB. 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 homola the Gia with other species, 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 1,5a (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 temperature 10°C 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 VICTMin position 5' (Applied Biosystem, NL) and the quencher NFQ-MGBTMin position 3 (Applied Biosystem, NL), as well as probes for various species ofBifidobacteriumoligonucleotides with a 5' reporter dye, 6-carboxyfluorescein (FAMTM) and a 3' quencher - NFQ-MGBTM(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 (FAMTM), and a 3' quencher dye, 6-carboxytetramethyl (TAMRATM). Designed probes are presented in table 1.

Table 1
Designed primers and probes for use in 5'- nucleonic analysis
TargetPrimers and probesSequence
(51→3*)
TPL
(C)
% GCBLAST roomThe length of the ampliconSEQ ID No
B. adolescentisF_adol_ISATA GTGGAC GCG AGC AAG 59521015335678-6465-1890671 BP1
R_adol_ISAGA TTG AAG AGT59431015335740-7519-16242
P_adol_ISTTG GCG AAA TCG CTG AAA GAA CGT TTC TTT TTa69301015335863-95222-172073
B. angulatumF_angul_ISTGG TGG TTT GAG AAC TGG59461015336044-12581-14600117 BP4
R_angul_ISATA GTG TCG ACG AAC AAC59321015336147-14351-299325
P_angul_ISAAT AAA CAA AAC A AAG GCC GCC AA TC 70571015488648-557-21046
B. bifidumF_bif_ISGTT GAT TTC GCC GGA CTC60521015336612-215666-12828105 BP7
R-bif_ISTTC GCA AGC CTA60561015336668-22451-307318
P_bif_ISTCG CGC AAA AAC TCC GCT GGC AAC A70561015336773-24053-34169
B. breveF_breve_ISGTG GTG GCT TGA GAA CTG59521015243936-11550-20333118 BP10
R_breve_ISGAT AG CAA AAC GAT321015244110-13595-2951411
P_breve_ISCGA AAC AAA CAC TAA A TGA TTC CTC GTT CTT GCT GT69451015244238-15062-1685312
B. catanulatumF_cate_ISGTG GAC GCG AGC AAT GC58651015335268-99-2071867 BP13
R_cate_ISAAT AGA GCC TGG CCA AAT58501015335364-15714217514
P-CATE_ISCG AAG CAA ACG ATG ACA TCA68391015335455-2899-1785915
B. dentiumF_dent_ISCCG CCA CCC ACA GTC T 59711015399643-15856-19947150 BP16
R_dent_ISAGC AAA GGG AAA CAC CAT59411015399751-16991-1121017
P_dent_ISGTTT ACGCGTCCA ACGGA70641015399833-18158-519818
B. infantisF_inf_ISCGC GAG CAA AAC AAT GGT58471037961234-06371-1436476 BP19
R_inf_ISTa
AAC GAT CGA
58361037961263-06691-2546120
P_inf_ISAAC GAA CAA TAG AGT T GAA TTC ATC AAC AGC AAA
Aa
69321037961294-06967-1747721
B. longumF_long_ISTGG AAG ACG TCG TTG GCT59501015323391-27595-22257109 BP22
R_long_ISTT
ATC GCG CGA
58561015323469-28673-2314723
P_long_ISGGC AAA AACGC ACC CAC CGC A68771015488566-4529-1393424
AllBifidobacteriumF_allbif_ISGGG ATG CTG
CTG TGG AAG
60571015399960-19603-31240231 BPa25
R.alIbif_JSAGA
TGC TCG CGT
60571015400076-20827-1741826
P_allbif_ISCCA CTA TCC AGT
TCA AAC CAC CAC GCG CCA
70611015400166-21749-1842427
andIn these cases, the basic technique have made some adjustments (more than 3 consecutive nucleotides or amplicon length more than 150 BP), the purpose of which was to detect a corresponding set of primers and probes.

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, the use of antibodies labeled with enzyme, where the use permantely 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 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, 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 relevo the nucleic acid sequences, which is a characteristic species of the genus Bifidobacterium, where the specified probe chosen from:

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 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 is Yu;

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 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, erisoodustuste 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 withifidobacterium 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 Oli is nucleotidyl 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 fry the STV for the detection of amplified DNA.

Example 1. Certification is designed probes and primers for detection of bifidobacteria

Bacterial strains used for confirmation 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 4°C (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 4°C. 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% (wt./about.) 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 mixture was incubated for 30 minutes at 37°C. Then the solution was centrifuged for 5 minutes at 1000 rpm at 4°C (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 65°C for 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 37°C. After which the DNA was besieged by keeping at -20°C for at least 30 minutes after adding 2 volumes of ice ethanol (96%) and 0.1 volume of 0,3M sodium acetate solution (pH 5,2). Solutions in which the formed precipitate was centrifuged at 13000 rpm for 20 minutes at 4°C and supernatant washed with 500 µl 70% ethanol followed by centrifugation at 13000 rpm for 5 minutes at 4°C. 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 what heated at 50°C for 2 minutes, keeping the sample for 10 minutes at 95°C, followed by 45 cycles with duration of 15 seconds at 90°C and 60°C duration 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 the 5'-nukleazy 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 presented in figure 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 reproducibility and repeatability once the ranks 5'-nucleonic tests.

Table 3
The sensitivity of the 5'-nucleonic analyses in comparison with "conventional" PCR, as well as their reproducibility and repeatability
TargetSensitivityand
(x)
Reproducibilityb
[CV (%)]
Repeatabilityc
[CV (%)]
B. adolescentis100005,11of 5.68
B.angulatum100019,4820,92
B. bifidum10011,6511,20
B. breve100to 2.064,08
B. catenulatum10009,4214,83
B. dentium10012,6511,35
B. infantis10002,342,31
B. longum100009,108,18
amany times the sensitivity of the 5'-nuclease analysis is higher than the sensitivity of the "traditional" PCR
breproducibility was determined by testing monocultures (100%)taken in 10-fold excess, and the calculation of CV (%) on the basis of the obtained results
withrepeatability was determined by triple checked monocultures, taken in four-fold excess, and the calculation of CV (%) on the basis of the obtained results

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 opt the maximum concentration of primers and probe, used in the duplex 5'-nucleonic analyses.

Table 4
Optimized final concentration of primers and probe used in the duplex 5'-nucleonic analysis
Target5'-nukleazy analysisDirect primer
(nm)
Reverse primer
(nm)
Probe
(nm)
B. adolescentisB. adolescentis
AllBifidobacterium
300
300
150
600
100
100
B. angulatumB.angulatum
All Bifidobacterium
900
300
900
300
200
50
B. bifidumB. bifidum
AllBifidobacterium
600
300
600
300
200
100
B. breveB. breve
AllBifidobacterium
30
450
300
450
100
150
B. catenulatumB. catenulatum
All Bifidobacterium
300
600
300
600
100
100
B. dentiumB. dentium
AllBifidobacterium
900
300
900
300
200
50
B. infantisB. infantis
AllBifidobacterium
300
900
300
900
100
100
B. longumB. longum
AllBifidobacterium
300
600
300
600
100
200
All
Bifidobacterium
AllBifidobacterium
All bacteria
450
900
450
900
100
200

Example 2. Clinical trials

The study was performed double-blind, multicenter trial with a placebo control when using the two treatment groups. Infants receiving full is itania, aged from 28 to 90 days was chosen from four hospitals in Germany. The children participated in the experiments if their birth weight was 2600-4500 g and they ate a full milk powder 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.

Table 5
The composition of the experimental mixtures (per 100 ml of ready mix)
The mixture was blended with carbohydrates (Aptamil 1 with GOS/FOS, Milupa)The standard mix
(Aptamil 1, Milupa)
Energy value (kcal)7272
Protein (g)1,51,5
Carbohydrates (g)8,58,5
- lactose (g)7,57,5
- starch (g)11
Newsweaver.ie oligosaccharides (g)0,80
- galactooligosaccharide (g)0,720
- fructooligosaccharide (g)0,080
Fat (g)3,63,6

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). Conducting research the Finance 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 thawing of the samples in ice-cold water, followed 10× (wt./about.) dilution in PBS (0,37 M NaCl, 2.7 mm KCl, 8,1 mm Na2HPO4[pH 7,4]) and a 10-minute homogenization using 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 the extraction mixture f is Nol/chloroform. After phenol-chloroform 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 3 M sodium acetate (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=(thresholdAnd/thresholdIn)-(t1A-Ct1B)-1. Using the calculated values of the efficiency was calculated initial DNA content (Rothe formula Ro=threshold/(1+E)Ct. Then the initial number of DNA Bifidobacterium species can be separated on the original amount of DNA in all species of Bifidobacterium. Next, the resulting ratio can be normalized to 100% with respect to the number of monocultures.

The total number ofBifidobacteriumcan also be defined using oksanalove FISH technique (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 of the genusBifidobacteriumin 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.

Table 6
Prevalence (%) of species of Bifidobacteria in the faeces of children after 6 weeks of feeding mother's milk (BF), baby milk formula, containing diabeticheskoi mixture (GFCF) or a standard formula (SF)
BFGFSFSF
B. catenulatum806775/td>
B. adolescentis201150
B. breve707863
B. longum505663
B. bifidum101113
B.angulatum301113
B. infantis100100100
B. dentium201113

Table 7
The percentage of individual species of Bifidobacteria relative total number of Bifidobacteria in the feces 6 weeks supply
Breastfeeding
% (sd)
GFSF-Pete is their
% (sd)
SF-nutrition
% (sd)
B. catenulatum1,9 (1,0)1,5 (3,0)9,8 (12,6)
B. adolescentis0,3 (0,9)0,1 (0,2)2,9 (6,0)
B. breve11,7 (9,6)5,4 (10,8)4,9 (10,7)
B. longum7,3 (13,9)5,4 (10,7)6,2 (9,4)
B. bifidum<0,1 (0,0)<0,1 (0,0)<0,1 (0,0)
B. angulatum<0,0 (0,0)<0,1 (0,2)<0,1 (0,0)
B. infantis32,0 (18,9)32,1 (20,0)37,8 (18,4)
B. dentium<0,1 (0,0)<0,1 (0,0)<0,1 (0,0)

In three different groups there are a large number of different species ofBifidobacterium. Much of the considerable reduction of the prevalence and quantity of B. adolescentisobserved in children receiving breast-feeding, and children receiving GFSF, unlike children, 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. atenulatum(+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 on 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 use the hosted mice at the age of 6-9 weeks. All experiments were approved by the ethics Committee for the treatment of animals 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. Pavlova. 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 plexiglass 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 of 1·109(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 plethysmography the whole organism (BUXCO, EMKA, Paris, France). The respiratory response was expressed as enhanced pause (PenH).

The 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” 2nded. McGrow Hill Book Company, New York, USA). All other analyses were performed using student's criterion (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.

The results of 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.

Figure 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 relates the global PenH values were obtained by subtraction of control values, obtained for 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):

8% (energy) protein1.4 g (casein whey blend)
45% (energy) to digest carbohydrates7.5 g
47% (energy) fat3.5 g
GOS (90% of galactooligosaccharides, Borculo
Domno NL)/polymaltose (10% inulin, Raftilin HP, Orafti BE)
0.4 g
B. breve1,3·108cfu

Example 5

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

<>
10% (energy) protein1.8 g (casein whey blend)
46% (energy) to digest carbohydrates8.0 g
44% (energy) fat3.4 g
GOS/polymaltose (see example 4)0.4 g
B. breve1,4·108cfu

Example 6

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

10% (energy) protein1.9 grams (casein whey blend)
51% (energy) to digest carbohydratesto 9.9 g
39% of energy) fat3.3 grams
FOS (Raftilin, Orafti/galactomannan 9/10.4 g
B. breve1.6 x 108cfu

Example 7

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

10% (energy) protein equivalent1.8 g (hydrolyzed whey protein with milk)
42% (energy) to digest carbohydrates7,1 g
48% (energy) fat3.6 g
Sealline oligosaccharides, newsweaver.ie maltodextrins 9/10.4 g
B. breveof 6.5·108cfu/g

Example 8

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

10% (energy) protein equivalent1.8 g (hydrolyzed whey protein with milk)
42% (energy) to digest carbohydrates8.6 g
44% (energy) fat3.6 g
Frukooligosaharidov (from algal fucoidan), galactomannan 8/20.4 g
B. breve7,5·108cfu

Example 9

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

10% (energy) protein equivalent1.8 g (hydrolyzed whey protein with milk)
42% (energy) to digest carbohydrates8.6 g
44% (energy) fat3.6 g
Mannooligosaccharide, arabinogalactan 9/10.4 g
B. breve1,5·108cfu

Example 10

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

10% (energy) protein1.7 g (hydrolyzed whey protein with milk)
48% (energy) to digest carbohydrates8,4 g
42% (energy) fat3.3 grams
GOS/Galaktionova oligosaccharides/polymaltose 7/2/11.0 g
B. breve7,5·108cfu

Example 11

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

11% (energy) protein1.9 grams (hydrolyzed whey protein with milk)
48% (energy) to digest carbohydrates8.7 g
41% (energy) fat3.3 grams
Xylooligosaccharide/galactan 9/10.8 g
B. breve8·108cfu

Example 12

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

10% (energy) protein1.7 g (casein whey blend)
48% (energy) to digest carbohydrates8,1 g
42% (energy) fat3.1 g
GOS/polymaltose 9/10,8G
Galactomannan0,42
B. breve1,5·108cfu

Example 13

Infant formula containing RA is couple on 100 ml of the final product (and 15.9 g of powder):

13% (energy) protein2.2 g (casein whey blend)
49% (energy) to digest carbohydrates8.6 g
37% of energy) fat3.0 g
GOS/polymaltose 9/10.8 g
Galactomannan0.4 g
B. breve1.6 x 108cfu

Example 14

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

9% (energy) protein equivalent1.5 g (hydrolyzed whey protein with milk)
42% (energy) to digest carbohydrates6,9 g
49% (energy) fat3.6 g
GOS/polymaltose/sialyllactose 7/2/10.8 g
B. breve1,4·108cfu

Example 15

Baby malonates, containing per 100 ml of the final product (and 13.7 g of powder):

9% (energy) protein equivalent1.4 g (free amino acids))
44% (energy) to digest carbohydrates7,1 g
47% (energy) fat3.4 g
GOS/polymaltose 6/40.8 g
B. breve1,4·108cfu

Example 16

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

11% (energy) protein1.8 g (soy protein)
40% (energy) to digest carbohydrates6.7 g
49% (energy) fat3.6 g
GOS/galactooligosaccharide/polymaltose 8/1/10.8 g
B. breve1,4·108cfu

Example 17

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

12% of energy) protein2.2 g (soy protein)
43% (energy) to digest carbohydrates7,7 g
45% energy) fat3.6 g
FOS/galactan 9/10.8 g
B. breve1,5·108cfu

Example 18

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

13% (energy) protein2.0 g (hydrolyzed whey protein with milk)
57% (energy) to digest carbohydrates8.6 g
30% (energy) fat2.0 g
GOS/polymaltose 9/11.0 g
Soy polysaccharides0.5 g
B. breve1,5·109cfu

Example 19

A product based on milk containing per 100 ml:

14% (energy) protein2.5 g (cow's milk protein)
43% (energy) to digest carbohydrates7.5 g
43% (energy) fat3.4 g
GOS/polymaltose 7/31.5 g
B. breve3·108cfu

Example 20

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

11% (energy) protein2.0 g (hydrolyzed collagen and soy protein)
46% (energy) to digest carbohydrates8.6 g
43% (energy) fat3.6 g
GOS/polymaltose 3/10.4 g
B. breve6·108cfu

Example 21

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

28% of energy) protein0.7 g (casein is whey mixture)
72% (energy) to digest carbohydrates2.0 g
GOS/polymaltose 65/350.3 g
B. breve3·109cfu

Example 22

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

Galactomannan0.26 g
Digested carbohydrates0,44 g
GOS/polymaltose 85/150.3 g
B. breve1,0·109cfu

Example 23

Supplement containing per 100 ml:

Example 24

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

100% (energy) to digest carbohydrates2.2 g
MineralsK, Na, Cl
Osmotic solution concentration261 mOsm/1
GOS/polymaltose 55/450.4 g
B. breve1·109cfu
4% (energy) protein4.7 g (cow's milk protein)
53% (energy) to digest carbohydrates68 g
43% (energy) fat24,6 g
GOS/polymaltose 9/10.8 g
B. breve1,2·109cfu

Example 25

Baby food in tubes per 100 ml

9% (energy) protein3.4 g (casein)
50% (energy) carbohydrates18,8 g
41% (energy) fat8 g
GOS/polymaltose 7/30.4 g
B. breve5·108cfu

Example 26

Baby food containing per 100 ml of the product

11% (EN) - Rev. reticency) protein 2.8 g (casein)
49% (energy) carbohydrates12.3 g
40% (energy) fat4.4 g
GOS/polymaltose 85/150.8 g
B. breve5·108cfu

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)

Protein (plant)7,4 g
Carbohydrates83 g
Fat0.5 g
Fiber, comprising 1.5 g GOS/polymaltose 9/13 g
B. breve1·1010cfu

Example 28

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 tabloidesque formula contains milk for a beginner to go baby, or cow's milk)

Protein (plant)9.5 g
Carbohydrates74 g
Fat2.0 g
Fiber, comprising 1.5 g GOS/polyproline 8/23 g
B. breve2·1010cfu

Example 29

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

GOS/polymaltose 75/252.0 g
B. breve2·109cfu

1. Preparation for use as a food containing Bifidobacterium breve in quantities of 1·104-1·1010cfu/g and a mixture of at least two neoslavery soluble carbohydrate components a and b, in which
specified carbohydrate component And differs from the specified component In the structure of the monosaccharide units of the carbohydrate;
the contents of the specified carbohydrate component comprises 5-95 wt.% from the total content is of componentof a and b;
at least 50% of the total number neoslavery soluble carbohydrates are selected from carbohydrates from disaccharides to eicosanoides; and
the carbohydrate components a and b differ by the number of monosaccharide units, with an average chain length of the carbohydrate component And at least 5 monosaccharide units lower than average long chain component C.

2. The preparation according to claim 1, in which the carbohydrate component And choose from neoslavery monosaccharides up to hexasaccharides the same carbohydrate structure, carbohydrate component selected from neoslavery of Gateshead and higher polysaccharides same carbohydrate structure.

3. The preparation according to claim 1, in which the content of the carbohydrate component is 95-60 wt.%, and the content of carbohydrate In amounts of 5-40 wt.%, and a+b=100 wt.%.

4. The preparation according to claim 3, in which at least 60 wt.%, preferably 80-100 wt.%, carbohydrate component And belongs to the group of galactooligosaccharides preferably to a group of TRANS-galactooligosaccharides.

5. The preparation according to claim 1, in which at least 60 wt.%, preferably 80-100 wt.%, the carbohydrate component b belongs to the group of fructooligosacharides, including inulin.

6. The preparation according to claim 1, containing 105-1011cfu Bifidobacterium breve per gram total content neoslavery soluble carbohydrates.

<> 7. The preparation according to claim 1 for use as supplements containing probiotic Bifidobacterium breve in quantities of 1·102-1·1011cfu/g per Supplement.

8. The preparation according to claim 1 for use as baby food containing Bifidobacterium breve in quantities of 1·102-1·1012cfu/g of pediatric nutritional supplements.

9. Additive in children's diets containing the drug according to any one of claims 1 to 8 and additionally comprising assimilable carbohydrate, a source of lipids or protein source, or a mixture.

10. Baby food containing preparation according to any one of claims 1 to 8 and additionally comprising assimilable carbohydrate, a source of lipids and protein source.

11. The use of the drug according to any one of claims 1 to 8 for the manufacture of a composition for the normalization of Bifidobacterium species composition in the gastrointestinal tract of children receiving artificial or partially breast-feeding, compared with the same composition in children breast-fed.

12. The use of the drug according to any one of claims 1 to 8 for the manufacture of a composition for preventing or treating one or more disorders of the immune system.

13. The application indicated in paragraph 12, where the immune system disorders selected from allergies, atopy, allergic rhinitis, food hypersensitivity, atopic dermatitis, eczema and asthma.

14. The application of section 12, where an immune disorder selected from diarrhoea and viral is yarei.

15. The use of the drug according to any one of claims 1 to 8 for the manufacture of a composition for prevention and/or treatment of insufficient energy absorption.

16. The use of the drug according to any one of claims 1 to 8 for the manufacture of a composition for inhibiting infiltration of eosinophils, neutrophils and mononuclear cells in allergic diseases, inhibition of the immune response Th2 type and/or stimulation of a Th1-mediated immune response.



 

Same patents:

FIELD: chemistry; biochemistry.

SUBSTANCE: invention relates to biotechnology and design of agents with immunomodulating properties. A new fungal strain Penicillium verrucosum VKPM F-984 and a new immunomodulating agent based on the strain are proposed. The strain is extracted from microflora of ginseng roots and is kept in a medium which contains mineral salts, glucose and asparagine. Fungus mycelium is extracted with a water-alcohol solution (70% ethanol solution). The advantage of this agent is its natural occurrence and effecient stimulation of adaptive capabilities of the body.

EFFECT: obtaining an extract which has stimulating action on cell and humoral immunity, improves immune status of the body.

3 cl, 2 ex

FIELD: chemistry.

SUBSTANCE: invention relates to new derivatives of imidazo[1,2-c]pyrimidinyl acetic acid of formula (I) or to its salts: , where R1 is ,, in which n is an integer ranging from 0 to 6; Y is aryl, where the said aryl is optionally substituted at a substitutable position with one or more substitutes selected from a group which consists of halogen or C1-6alkyl, optionally substituted with mono-, di- or trihalogen; R2 is hydrogen; R3 is hydrogen or halogen; and R4 is hydrogen. The invention also relates to derivatives of imidazo[1,2-c]pyrimidinyl acetic acid of formula (I-i) or to its salts, to a drug, to use of compounds in paragraph 1, as well as to a drug in form of a standard single dosage.

EFFECT: obtaining new biologically active compounds, which are active towards CRTH2.

23 cl, 2 ex

FIELD: medicine.

SUBSTANCE: invention refers to medical products and concerns an ellagic acid composition for immune system enhancement, differing that additionally is contains chitosan beta-1.3/1.6-glucans or oligosaccharides.

EFFECT: offered composition possesses enhanced immunostimulating effect.

3 ex

FIELD: medicine.

SUBSTANCE: invention refers to pharmacology and can be used in veterinary science and medicine for chemotherapy. There is disclosed immunostimulating and antioxidant lithium composition containing lithium ascorbate and lithium aspartate in percentage ratio 50:50.

EFFECT: expansion of the list of low-toxicity immunomodulators applicable for manufacture of drugs.

1 dwg, 2 tbl

FIELD: medicine.

SUBSTANCE: invention concerns medicine, particularly therapy, and can be applied in chronic disease treatment. Method involves causative agent extraction or determination of antibodies to causative agents or extraction of genetic components from blood, body fluid or smear. Treatment is performed by vaccine drugs specific to one or several identified causative agents, additionally immunomodulators are administered.

EFFECT: activated repair processes, arrested development of disease state of organs and systems due to elimination of identified pathogen from organism and to immune correction.

3 cl, 8 ex

FIELD: medicine.

SUBSTANCE: invention concerns veterinary medicine, bioactive stimulation agent and method of secondary immunodeficiency correction applying the agent. Method involves subcutaneous injection of non-specific immunoglobulin and additional oral application of alcohol infiltration of minced herbal mix including grass and inflorescence of Echinacea purpurea, coltsfoot, harmala shrub grass and common licorice roots at equal amount in the form of aqueous solution of 7-8% concentration in the dosage of 1.5-2.0 ml per kg of live weight for 15 days at 24 hour interval. Bioactive stimulation agent renders positive effect on all animal organs and systems, adjusts metabolic processes and enhances organisms resistance (activation of B- and T-systems, phagocytosis), allowing for correction of secondary immunodeficiency to physiological standard level.

EFFECT: natural origin, easy metabolic assimilation and excretion, high compatibility with other medicines, absent sensibilisation and induction of immunopathological reactions.

2 cl, 6 tbl, 3 ex

FIELD: medicine.

SUBSTANCE: invention concerns obtainment of cells of monocyte origin, inducing transplant acceptance, expressing antigens CD3 and CD14, and can be applied in transplantology. Monocytes are extracted from blood, reproduced in cultural medium with 1-20 mcg/l of cell growth factor M-CSF and cultivated for 24-72 hours in cultural medium with 0.1-20 ng/ml of interferon γ-IFN. Further the cells are extracted from cultural medium. For cell CD3 and CD14 expression antibodies produced by hybridoma DSM ACC2542 are used. If necessary, obtained cells are suspended and freezed.

EFFECT: obtainment of cells of monocyte origin, inducing transplant acceptance and suppressing transplant rejection reaction.

26 cl, 29 dwg, 1 tbl, 13 ex

FIELD: pharmacology.

SUBSTANCE: invention concerns compounds of formula (I) , where A is optionally substituted monocyclic aryl and heteroaryl group, B is optionally substituted monocyclic nitrogen-containing heterocyclic group, and either a) R1 is hydrogen atom and R2 is group selected out of -NH2 and optionally substituted alkinyl group, or b) R2, R1 and -NH- group with linked R1 form fragment selected out of fragments of formulae , , and , where Ra is selected out of hydrogen atom and group selected out of optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted aryl, -OR3, where R3 is independently selected out of group including hydrogen atom and lower alkyl or cycloalkyl groups, Rb is selected out of hydrogen atom and group selected out of optionally substituted alkyl or optionally substituted cycloalkyl group; application of claimed compounds in medicine obtainment for treatment of pathological states or diseases, the course of which is alleviated by antagonistic effect on adenosine receptor A2B, and pharmaceutical composition with antagonistic effect on adenosine receptor A2B.

EFFECT: compounds applicable in treatment of such diseases as asthma, bronchostenosis, allergic diseases, hypertension, atherosclerosis, reperfusion injury, myocardial ischemia, retinopathy, inflammation, gastrointestinal disorders related to cell proliferation, diabetes and/or autoimmune diseases.

26 cl, 49 ex, 2 tbl

FIELD: pharmacology.

SUBSTANCE: invention concerns novel compounds of formula (1a) or (1b) or their pharmaceutically acceptable salts with inhibition effect on matrix metalloproteinases (MMP). In formula

or T is absent; G1 and G2 is independently CH or N; A is C1-6alkyl; B is a link; D is a link; E is phenyl substituted by condensed heterocyclic ring in the form of nitrogen-containing ring which can be substituted by carbonyl groups; or substituted 6-membered heteroaryl or bicyclic heteroaryl where second ring in bicyclic system is benzene ring, and heteroaryl is 5-6-membered ring containing 1, 2 or 3 nitrogen heteroatoms, and heteroaryl is substituted by 1, 2 and 3 groups selected out of oxo and C1-6alkyl-; R16 is C1-6alkyl; R18 is halogeno, cyano, nitro, OR16 , OCF3, SR16 or COR16; m is 0 or integer 1 or 2; n is 0.

EFFECT: obtaining compounds and pharmaceutical composition based on them.

13 cl, 18 ex

FIELD: medicine.

SUBSTANCE: invention concerns medicine, particularly gynecology, and can be applied in treatment of autoimmune oophoritis of inflammatory genesis for patients of childbearing age suffering with long-term recurrent inflammatory diseases of pelvic organs. After infect eradiation, interleukin receptor antagonist is injected daily intramuscularly in 50 mg amount until circulating autoimmune antibody level in blood serum falls below 10 U/ml.

EFFECT: enhanced efficiency of treatment at different disease stages, prevented complications inherent to hormonal immunosuppressive therapy.

2 ex

FIELD: chemistry; biochemistry.

SUBSTANCE: Bifidobacterium pseudocatenulatum OV-2 strain is extracted from intestine contents of a healthy nursing infant, deposited and stored at the State collection of microorganisms normal microflora of the Federal State Research Institution Moscow G. N. Gabrichevskii Research Institute of Epidemiology and Microbiology Rospotrebnadzor under No. 211. The strain suits modern requirements, accumulates biomass on culture media in short periods with high concentration of bifidobacteria, forms organic acids, has antagonist properties towards pathogenic and opportunistic microflora, is resistant to a wide range of antibiotics and is not toxic. This allows for using B. pseudocatenulatum OV-2 strain in making bacterial preparations, biologically active additives to food, unfermented food products, hygienic and cosmetic agents.

EFFECT: provision for probiotic effect and normalisation of microbiocenoses of the human body, including gastrointestinal and urogenital tracts, skin and mucous coatings, as well as wider range of agents for correcting microflora of the human body.

4 tbl, 5 ex

FIELD: chemistry; biochemistry.

SUBSTANCE: Bifidobacterium angulatum OV-15 strain is extracted from intestine contents of a healthy nursing infant, deposited and stored at the State collection of microorganisms normal microflora of the Federal State Research Institution Moscow G. N. Gabrichevskii Research Institute of Epidemiology and Microbiology Rospotrebnadzor under No. 212. The strain suits modern requirements, accumulates biomass on culture media in short periods with high concentration of bifidobacteria, has acid-forming and antagonist properties towards pathogenic and opportunistic microflora, is resistant to a wide range of antibiotics, acidic medium and is not toxic. This allows for using B. angulatum OV-15 strain for making bacterial preparations, biologically active additive to food, unfermented food products, hygienic and cosmetic agents.

EFFECT: provision for probiotic effect and normalisation of microbiocenoses of the human body, including gastrointestinal and urogenital tracts, skin and mucous coatings, as well as wider range of agents for correcting microflora of the human body.

4 tbl, 5 ex

FIELD: chemistry; biochemistry.

SUBSTANCE: Bifidobacterium pseudocatenulatum OV-17 strain is extracted from intestine contents of a healthy nursing infant, deposited and stored at the State collection of microorganisms normal microflora of the Federal State Research Institution Moscow G. N. Gabrichevskii Research Institute of Epidemiology and Microbiology Rospotrebnadzor under No.213. The strain suits modern requirements, accumulates biomass on culture media in short periods with high concentration of bifidobacteria, has antagonist properties towards pathogenic and opportunistic microflora, is resistant to a wide range of antibiotics, acidic medium and is not toxic.

EFFECT: possibility of using B pseudocatenulatum OV-17 strain in making bacterial preparations, biologically active additives to food, fermented and unfermented food products, hygienic and cosmetic agents, which provides for a probiotic effect and normalisation of microbiocenoses of the human body, including gastrointestinal and urogenital tracts, skin and mucous coatings, and also widens the range of agents for correcting microflora of the human body.

4 tbl, 5 ex

FIELD: chemistry; biochemistry.

SUBSTANCE: Bifidobacterium breve OV-12 strain is extracted from intestine contents of a healthy nursing infant, deposited and stored at the State collection of microorganisms normal microflora of the Federal State Research Institution Moscow G. N. Gabrichevskii Research Institute of Epidemiology and Microbiology Rospotrebnadzor under No.217. The strain suits modern requirements, accumulates biomass on culture media in short periods with high concentration of bifidobacteria, has acid-forming and antagonist properties towards pathogenic and opportunistic microflora, is resistant to a wide range of antibiotics and is not toxic.

EFFECT: possibility of using B brever OV-12 strain in making bacterial preparations, biologically active additives to food, fermented and unfermented food products, hygienic and cosmetic agents, which provide for probiotic effect and normalisation of microbiocenoses of the human body, including gastrointestinal and urogenital tracts, skin and mucous coatings, and also widens the range of similar agents.

4 tbl, 6 ex

FIELD: medicine.

SUBSTANCE: invention concerns medical and veterinary drugs and methods of their obtainment and can be applied in biotechnology, medicine and agriculture. Complex probiotic medicine includes carrier in the form of porous enterosorbent, and eubiotic bacteria cells with nutrient and protective media, immobilised at the enterosorbent. Eubiotic bacteria cells with nutrient medium comprise lyophilised concentrate of bifidobacteria or lactobacteria consortium, or their mix with the titre of 108-1010 colony forming units per gram. Carbon enterosorbent VNIITU-1 or 2 of All-Russian Scientific Research Institute of Carbon Black is used as porous enterosorbent, at the following dry medicine component ratio, wt %: cells of eubiotic bifido- or lactobacteria consortium, or their mix of 108-1010 colony forming units per gram titre, with nutrient medium - 1.0-15.0, protective medium - 0.1 - 10.0, carbon enterosorbent VNIITU-1 or 2 - the rest to 100%. Bifido- and lactobacterium consortiums are mixed at the ratio of 1:1 to 3:1.

EFFECT: higher bacterium cell sorption and desorption efficiency by enterosorbent, enhancing clinical effect, and higher colony forming activity of eubiotic bacteria.

5 cl, 2 tbl, 5 ex

FIELD: medicine.

SUBSTANCE: method involves preparation of nutrient medium including plant material, plant material fermentation with anaerobic gram-negative bacterium with simultaneous bacterium cultivation in the medium to obtain extracts of fermented plant material. Extraction is performed by water or salt buffer. Also the extract can be optionally dried to obtain extract powder. Edible plant material containing carbohydrates, mainly polysaccharides, is used as plant material. Gram-negative bacterium lives only in symbiosis with given plant material. Additionally invention claims application of extracts and powder of fermented plant material in obtainment of medicines, veterinary medicines, quasi medicines, cosmetics, food products, including functional food, forage and detergents.

EFFECT: obtaining products with immunostimulating effect.

17 cl, 1 dwg, 23 tbl, 23 ex

Vaginal composition // 2369389

FIELD: medicine.

SUBSTANCE: vaginal composition comprises viable microorganisms of lactobacillus and/or bifid bacteria, preferentially Lactobacillus bifidus, devitalised saccharomyces, preferentially Saccharomyces cerevisiae, saccharide(s), vitamin A retinal and zinc with specified ratio of composition components.

EFFECT: vaginal protection without negative effect or infliction of harm.

14 cl, 2 ex, 1 tbl

FIELD: medicine.

SUBSTANCE: invention is a skin moistening agent for oral delivery comprising fermented milk whey as active component and obtained by milk fermentation using the strain Lactobacillus, helveticus, CM-4 (deposited with International depositary institution for patents by National Institute of advanced science and technology under inventory number FERM BP-6060).

EFFECT: invention provides skin moistening.

6 cl, 2 ex, 5 tbl

FIELD: medicine.

SUBSTANCE: pharmacological preparations are applied for treatment of purulent wounds and burns, pyoinflammatory dermatopathies, and for acceleration of healing and improvement of wound healing conditions. The external and local pharmaceutical composition possesses antibacterial and necrolytic action; it contains an active complex of bacteriolytic and proteolytic enzymes - Lisoamidase, a base and at least one desired additive chosen from: a substance disarranging the membrane structure of gram-negative microorganisms, an antibiotic, an anesthetic, a reparative process stimulator. The base is hydrophilic substances and a perfluororganic compound emulsion or their physiologically acceptable mixture or their physiologically comprehensible mix in certain component ratio.

EFFECT: enhanced therapeutic activity and ease of use, lower consumption of the active substance, ensured anaesthetising effect, additional stimulation of reparative processes, improved microcirculation, reduced secondary tissue necrosis surrounding a wound caused by excess free radical oxidation.

7 cl, 12 ex

FIELD: medicine.

SUBSTANCE: antibacterial and necrolytic pharmacological composition contains an active complex of bacteriolytic and proteolytic enzymes - Lisoamidase and a base. The pharmacological composition is characterised that as a base, it contains hydrophilic substances or physiologically acceptable mixture thereof providing soft formulation of the composition. Its components are taken in certain mass ratio.

EFFECT: extended application, enhanced therapeutic activity and ease of use, lower consumption of the active substance, ensured anaesthetising effect, improved microcirculation in wound tissues, control of inflammatory processes, and additional stimulation of reparative processes.

6 cl, 14 ex

FIELD: chemistry; biochemistry.

SUBSTANCE: Bifidobacterium pseudocatenulatum OV-2 strain is extracted from intestine contents of a healthy nursing infant, deposited and stored at the State collection of microorganisms normal microflora of the Federal State Research Institution Moscow G. N. Gabrichevskii Research Institute of Epidemiology and Microbiology Rospotrebnadzor under No. 211. The strain suits modern requirements, accumulates biomass on culture media in short periods with high concentration of bifidobacteria, forms organic acids, has antagonist properties towards pathogenic and opportunistic microflora, is resistant to a wide range of antibiotics and is not toxic. This allows for using B. pseudocatenulatum OV-2 strain in making bacterial preparations, biologically active additives to food, unfermented food products, hygienic and cosmetic agents.

EFFECT: provision for probiotic effect and normalisation of microbiocenoses of the human body, including gastrointestinal and urogenital tracts, skin and mucous coatings, as well as wider range of agents for correcting microflora of the human body.

4 tbl, 5 ex

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