Pea protein peptides with anti-helicobacter pylori activity

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention deals with application of composition, which includes hydrolysate of pea protein and/or peptide for obtaining composition for treatment and/or prevention of infection Helicobacter pylori (versions), as well as such compositions (versions). Characterised compositions include lipid, protein and carbohydrate component, in which protein component includes protein source, which consists of pea protein hydrolysate, obtained by hydrolysis with protease, different from chymotrypsin, or from peptides, selected from group, which consists of Xaan-Asp-Phe-Leu-Glu-Asp-Ala-Phe-Asn-Val-Asn-Arg-Xaam and Xaan-Glu-Leu-Ala-Phe-Pro-Gly-Ser-Ala-Gln-Glu-Val-Asp-Arg-Xaam, where each Xaa independently can be any amino acid, and n and m are integer numbers, independently varying from 0-10, where peptide is contained in pea protein hydrolysate, or from both.

EFFECT: claimed inventions make it possible to treat or prevent diseases, caused by Helicobacter pylori infection, and/or diseases, associated with infection Helicobacter pylori in mammals.

17 cl, 2 tbl, 1 ex

 

Field of the invention

The present invention relates to the treatment and prevention of infections, gastrointestinal pathogens, in particular Helicobacter pylori.

Prior art inventions

Infections of the gastrointestinal tract are a major problem for many people and, in particular, in children and patients with immune system disorders or diseases of the gastrointestinal tract. Emerging diseases can be life-threatening. Infections of the gastrointestinal tract often cause Escherichia coli, Salmonella, Campylobacter, Porphyromonas gingivalis, Clostridium, Enterobacter and Helicobacter, such as Helicobacter pylori.

Helicobacter pylori (H. pylori) is a gram-negative, microaerophilic flagellate bacterium that infection colonizes the gastric mucosa of man. Infection with H. pylori is associated with severe diseases of the stomach, such as gastritis, peptic ulcer and stomach cancer. The world Health Organization classifies H. pylori as a group I carcinogen. The H. pylori infection is usually chronic and usually cannot be cured without specific therapy.

The H. pylori infection is mainly acquired in early childhood. The majority of children infected during the first 5 years of life. By the age of 10, the total frequency is more than 75% in developing countries, whereas in developed countries in�economony 10%, but the frequency may increase to 30-40% in children from lower socioeconomic groups.

In connection with the problems of antibiotic treatment and prevention by vaccination is necessary to prevent adhesion to the stomach mucosa and, therefore, infection with H. pylori, ideally by oral food intervention.

For eradication of H. pylori infection requires three or four drugs with antibiotics. The treatment is very expensive and there is also the risk of increasing antibiotic resistance of bacterial strains and reinfection after treatment failure. The treatment of children may be the most cost-effective method of reducing the frequency of infection and the morbidity and mortality associated with diseases associated with H. pylori. To date, there are no guidelines on the need to treat children. Hitherto inaccessible to the human vaccine. Prevention and therapeutic vaccination have been successful in animal models, but the transition to human vaccines remains difficult, partly due to the fact that immunology of the stomach is still poorly understood.

In connection with the problems of antibiotic treatment and prophylaxis by vaccination, it is necessary to prevent the adhesion of H. pylori to the gastric mucosa. Without the adhesion of bacteria to the risk associated inflammation, leading to gastritis� or maybe cancer, can be minimised. It is proved that dietary modification may be applicable in the maintenance treatment or prophylaxis of H. pylori infection in vivo and in vitro.

EP 1178104 relates to a food composition comprising a specific essential oil and/or specific pure compound isolated from the essential oils for the prevention or treatment of infection by the microorganism, Helicobacter similar. The food composition may also contain a source of carbohydrates, a source of fat and/or source of dietary protein, pea protein is one of them.

In JP 2005255679 described polypeptide obtained by processing the buttermilk protease, which has not only the effect of inhibiting the adhesion of Helicobacter pylori to the gastric mucosa, but also the effect of detachment of Helicobacter pylori from the gastric mucosa of carriers of Helicobacter pylori.

In JP 2001335504 described inhibitor of proliferation of Helicobacter pylori, which comprises as active ingredient the enzymatic hydrolysate of soy protein.

In WO 2008/043424 described composition for the treatment and/or prevention of infection by gastrointestinal pathogens, in particular Helicobacter pylori, and/or infections associated with infection with specified gastrointestinal pathogen in mammals. Composition comprising pea protein hydrolysate, intact pea protein and/or protein hydrolysate camel milk. Described hydrolysis�ZAT pea protein obtained by hydrolysis by chymotrypsin and trypsin.

In DE 10317935 described the use of casein for the production of the composition for the prevention or treatment of Helicobacter infection and for the prevention of diseases caused by Helicobacter.

Summary of the invention

The present invention relates to the use of compositions comprising specific pea protein peptides for the treatment and/or prevention of infection by gastrointestinal pathogens and/or diseases associated with infections specified gastrointestinal pathogens in a mammal, particularly infections of the gastro-intestinal pathogens, selected from the group consisting of Helicobacter, Escherichia coli, Salmonella, Porphyromonos gingivalis, Campylobacter, Clostridium and Enterobacter. The invention furthermore relates to the use of specific pea protein hydrolysate, obtained by hydrolysis with chymotrypsin to obtain compositions for the treatment and/or prevention of infection or disease specified above.

The authors present invention found that specific hydrolyzed pea protein is able to inhibit the adhesion of H. pylori to the cells of the gastric mucosa. Inhibition of adhesion makes these protein components particularly suitable for use in a method for the treatment and/or prevention of Helicobacter infection. Hydrolyzed pea protein get guide�the new sushi menu isolate pea protein by trypsin. Allocated and identified peptides that are responsible for inhibition of adhesion. Found that the peptides of the present invention Asp-Phe-Leu-Glu-Asp-Ala-Phe-Asn-Val-Asn-Arg (SEQ ID NO: 1) and Glu-Leu-Ala-Phe-Pro-Gly-Ser-Ala-Gln-Glu-Val-Asp-Arg (SEQ ID NO:2), forming part of the pea legumin A and vicilin, inhibit the adhesion of H. pylori to stomach cells. Since these peptides have a site of cleavage by chymotrypsin, pea protein hydrolysate can be obtained by hydrolysis with protease, preferably trypsin, with the condition that the protease is chymotrypsin, or using drugs proteases, preferably preparations of trypsin, which essentially do not possess the activity of chymotrypsin.

The present pea protein hydrolysate and/or pea protein peptides can be easily added to infant formulas, baby foods and foods for young people. Simple and safe use of such protein component makes the present invention especially important because drugs usually are problems side effects, and expensive treatment breaks down many drugs. The present pea protein hydrolysate and/or pea protein peptides also can be used by adults. The present pea protein hydrolysate and/or pea protein peptides do not need to enter the Department�but (for example, children), but you can enter together with food compositions.

So how to increase the duration of exclusive breastfeeding in infancy may have a prolonged protective effect against chronic H. pylori infection and therefore the risk of gastric carcinoma, especially it is also desirable to protect against H. pylori infection babies who receive infant formulas.

Detailed description

Therefore, the present invention relates to a method for the treatment and/or prevention and/or reduction of risk of infection by gastrointestinal pathogens and/or diseases associated with infection by gastrointestinal pathogens in a mammal, said method includes introduction to the specified mammal a composition comprising pea protein hydrolysate, with the condition that the pea protein hydrolysate was obtained by hydrolysis by the protease chymotrypsin. Preferably, the pea protein hydrolysate is obtained by hydrolysis protease other than chymotrypsin.

The invention can also be worded as the use of a composition comprising pea protein hydrolysate for the production of the composition for treatment and/or prevention of infection by gastrointestinal pathogens and/or disease associated with infection by gastrointestinal pathogens in a mammal, provided that the criminal code�linked hydrolyzed pea protein is not obtained by hydrolysis by the protease chymotrypsin.

The invention may also be formulated as a composition comprising pea protein hydrolysate for use in the treatment and/or prevention of infection by gastrointestinal pathogens and/or disease associated with infection by gastrointestinal pathogens in a mammal, with the proviso that the pea protein hydrolysate is not obtained by hydrolysis by the protease chymotrypsin.

In one aspect, the invention relates to a method for the treatment and/or prevention and/or reduction of risk of infection by gastrointestinal pathogens and/or disease associated with infection by gastrointestinal pathogens in a mammal, said method includes introduction to the specified mammal a composition comprising at least one peptide selected from the group consisting of Xaan-Asp-Phe-Leu-Glu-Asp-Ala-Phe-Asn-Val-Asn-Arg-Xaamand Xaan-Glu-Leu-Ala-Phe-Pro-Gly-Ser-Ala-Gln-Glu-Val-Asp-Arg-Xaamwhere each Xaa independently can be any amino acid, and n and m are integers independently ranging from 0 to 10.

The invention can also be worded as the use of a composition comprising a peptide, to obtain a composition for the treatment and/or prevention of infection by gastrointestinal pathogens and/or disease associated with infection by gastrointestinal pathogens in a mammal, where �eptid selected from the group consisting of Xaan-Asp-Phe-Leu-Glu-Asp-Ala-Phe-Asn-Val-Asn-Arg-Xaamand Xaan-Glu-Leu-Ala-Phe-Pro-Gly-Ser-Ala-Gln-Glu-Val-Asp-Arg-Xaamwhere each Xaa independently can be any amino acid, and n and m are integers independently ranging from 0 to 10.

The invention may also be formulated as a composition comprising at least one peptide selected from the group consisting of Xaan-Asp-Phe-Leu-Glu-Asp-Ala-Phe-Asn-Val-Asn-Arg-Xaamand Xaan-Glu-Leu-Ala-Phe-Pro-Gly-Ser-Ala-Gln-Glu-Val-Asp-Arg-Xaamwhere each Xaa independently can be any amino acid, and n and m are integers independently ranging from 0 to 10, for use in the treatment and/or prevention of treatment of infection by gastrointestinal pathogens and/or diseases associated with gastrointestinal pathogens in a mammal.

In the following aspect the invention also relates to compositions containing lipid, protein and carbohydrate components, where the lipid component provides 5 to 50% of the total daily consumption of calories, the protein component provides 5 to 50% of total daily calorie intake and the carbohydrate component provides 15 to 90% of total daily calorie intake, where the protein component comprises (i) at least one source of protein, consisting of pea protein hydrolysate, with the condition that the hydrolysis�t pea protein obtained by hydrolysis by the protease chymotrypsin and (ii) at least one source of nitrogen selected from the group consisting of milk proteins, hydrolysates of milk protein, egg protein, hydrolyzed egg protein, soy protein, soy protein hydrolysate, wheat protein, hydrolyzed wheat protein, rice protein, rice protein hydrolysate, free amino acids and mixtures thereof.

In another aspect the invention relates to compositions containing lipid, protein and carbohydrate component, where the lipid component provides 5 to 50% of the total daily consumption of calories, the protein component provides 5 to 50% of total daily calorie intake and the carbohydrate component provides 15 to 90% of total daily calorie intake, where the protein component comprises (i) at least one peptide selected from the group consisting of Xaan-Asp-Phe-Leu-Glu-Asp-Ala-Phe-Asn-Val-Asn-Arg-Xaamand Xaan-Glu-Leu-Ala-Phe-Pro-Gly-Ser-Ala-Gln-Glu-Val-Asp-Arg-Xaamwhere each Xaa independently can be any amino acid, and n and m are integers independently ranging from 0 to 10; and (ii) at least one nitrogen source selected from the group consisting of milk proteins, hydrolysates of milk protein, egg protein, hydrolyzed egg protein, soy protein, soy protein hydrolysate, wheat protein, hydrolyzed wheat protein, rice protein, hydrolyzed rice protein�, free amino acids and mixtures thereof.

Protein

The present invention provides a composition and its use for this treatment that contains pea protein hydrolysate obtained by a protease other than chymotrypsin. Preferably, the pea protein hydrolysate is obtained by hydrolysis protease other than chymotrypsin, more preferably, the pea protein hydrolysate is obtained by hydrolysis by trypsin. More preferably, the present invention provides specific peptides degradation of pea protein obtained by hydrolysis by trypsin, namely Xaan-Asp-Phe-Leu-Glu-Asp-Ala-Phe-Asn-Val-Asn-Arg-Xaamand Xaan-Glu-Leu-Ala-Phe-Pro-Gly-Ser-Ala-Gln-Glu-Val-Asp-Arg-Xaamwhere each Xaa independently can be any amino acid, and n and m are integers independently ranging from 0 to 10. Therefore, n and m independently can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In a preferred embodiment, each of the peptides Xaan-Asp-Phe-Leu-Glu-Asp-Ala-Phe-Asn-Val-Asn-Arg-Xaamand Xaan-Glu-Leu-Ala-Phe-Pro-Gly-Ser-Ala-Gln-Glu-Val-Asp-Arg-Xaamhas a calculated molecular weight between 1 kDa and 2.5 kDa, preferably between 1 kDa and 2 kDa, preferably between 1 and 1.5 kDa. In one embodiment of the invention n and m are 0. As these peptides are the sites of cleavage by chymotrypsin, and since it was found that smaller fragments still� peptides are inactive, the pea protein hydrolysate can be obtained by hydrolysis with chymotrypsin or trypsin plus chymotrypsin. When consumption of these peptides, they will disintegrate in the intestinal tract of man. However, since chymotrypsin and other enzymes as carboxymethyl and aminopeptidase that are released only in the duodenum, the peptides are still present in their active form in the stomach, the place where most H. pylori is present. Since it was found that these peptides derived from pea protein legumin A or vicilin, pea protein hydrolysate is preferably a hydrolysate of pea legumin A and/or pea vicilin received by a protease other than chymotrypsin, preferably trypsin. Commercial preparations of protease other than chymotrypsin, may contain minimal contamination by chymotrypsin and therefore may show some chymotrypsinogen activity. It is acceptable to obtain these hydrolysates. Minimum chymotrypsinogen activity does not lead to significant hydrolysis in peptides SEQ ID NO: 1 and SEQ ID NO:2. Some residual chymotrypsinogen activity in preparations of proteases, in particular the preparations of trypsin, is regarded only as influencing the release of peptides in the hydrolysate comprising the sequence of SEQ ID NO: 1 or SEQ ID NO:2. Method� for the hydrolysis of pea protein known in the art. Therefore, preferably the peptides of SEQ ID NO: 1 and SEQ ID NO:2 are from pea protein, it can also be provided by different sources. Therefore, in one embodiment the peptides of SEQ ID NO: 1 and/or SEQ ID NO:2 is provided in the form of protein nutritional quality. In one embodiment the peptides of SEQ ID NO: 1 and/or SEQ ID NO:2 is provided in the form of pea protein hydrolysate.

By gel-filtration chromatography, chromatographic methods, based on a crosslinked matrix of agarose/dextran, defined molecular weight range of the majority of H. pylori adhesion peptides and/or glycoconjugates present in the protein hydrolysate. It was found that effective H. pylori adhesion peptides are in the range 300-10000 Yes. Preferably an effective H. pylori adhesion peptides are in the range of 500-5000 Da. Therefore, the present protein hydrolysate preferably contains at least 1 wt.% peptides and/or glycoconjugates, preferably peptides, based on the total weight of the hydrolysate of the present peptides, with a molecular weight of from 300 to 10,000 Da, preferably at least 5 mass%, more preferably at least 50 wt.%, most preferably at least 75 wt.%. More preferably, the present�schy protein hydrolysate comprises at least 1 wt.% peptides and/or glycoconjugates, preferably peptides, based on the total weight of this protein hydrolyzate with a molecular weight of from 500 to 5000 Da, preferably at least 5 mass%, more preferably at least 50 wt.%, most preferably at least 75 wt.%. Unless otherwise specified, molecular weights mentioned in this description, define gel-filtration chromatography.

The present pea protein hydrolysate is preferably administered in an amount of from 0.1 to 100 grams per day, preferably in an amount of from 0.5 to 10 grams per day. The present pea protein peptides can be administered in a lower dose, namely in an amount of from 0.005 to 50 grams per day, preferably in an amount of from 0.05 to 5 grams per day.

Most preferably pea peptides of the present invention is administered in the form of pea protein hydrolysate. Alternative, pea peptides of the present invention can be synthesized or expressed by genetically modified (micro)organisms.

Preferably the composition includes a hydrolyzed pea protein, more preferably the fraction of pea protein hydrolysate is less than 10 kDa, more preferably less than 7.5 kDa, when the introduction of the hydrolysate is the most active in the prevention of adhesion of H. pylori. The preferred fraction hydroly�ATA pea protein has an average molecular weight of about 2 kDa. The preferred fraction of pea protein hydrolysate has a high concentration in the range from 1 kDa to 2.5 kDa. Therefore, it is preferable to use pea peptides obtained by hydrolysis by trypsin, with a size between 1 and 2.5 kDa. Preferably, the pea protein hydrolysate includes more than 35 mass%, more preferably more than 40 wt.% peptides ranging in size from 1 kDa to 2.5 kDa in the calculation of the total mass of the pea protein hydrolysate.

Gastrointestinal pathogens

The present method relates to the treatment and/or prevention of infection by gastrointestinal pathogens and/or diseases associated with the specified infection by gastrointestinal pathogens in a mammal, particularly the treatment and/or prevention of infection by gastrointestinal pathogens, which is selected from the group consisting of Helicobacter, Escherichia coli, Salmonella, Porphyromonas gingivalis, Campylobacter, Clostridium and Enterobacter and/or disease associated with infection by the specified gastrointestinal pathogenic in mammals.

The present invention provides for the treatment and/or prophylaxis of Helicobacter infections and/or diseases associated with Helicobacter infection in mammals. Helicobacter preferably selected from the group consisting of Helicobacter pylori, Helicobacter bizzozeronii, H. salomonis, Helicobacter heilmannii and Helicobacter felis. Preferably the present invention provides treatment and/or �profilaktika infections Helicobacter pylori (H. pylori) and/or diseases associated with Helicobacter pylori in mammals.

Food composition

It was found that the present pea protein hydrolysate and/or pea protein peptides may advantageously be applied to food such as baby food and clinical nutrition, especially the nutritional status of infants. The present nutritional composition preferably comprises a lipid component, a protein component and a carbohydrate component.

Therefore, the present invention also relates to a nutritional composition comprising the present pea protein hydrolysate and/or pea protein peptides and its application in the present method, where the lipid component provides 5 to 50% of the total daily consumption of calories, the protein component provides 5 to 50% of total daily calorie intake, carbohydrate component provides 15 to 90% of total daily calorie intake. Real composition is preferably used in infant formula, where the lipid component provides 35 to 50% of the total daily consumption of calories, the protein component provides 7.5 to 12.5% of the total daily calorie intake and the carbohydrate component provides 40 to 55% of total daily calorie intake. To calculate % commenorating consumption of calories for the protein component, you must take the total number of proteins peptides and amino acids.

In addition, the present pea protein hydrolysate and/or pea protein peptides of the present compositions preferably contain an additional nitrogen source for nutritional purposes. An additional source of nitrogen is preferably selected from the group consisting of protein, peptide, amino acids and mixtures thereof. Therefore, in a preferred embodiment the protein component of the present invention includes: (i) at least one protein source selected from the pea protein hydrolysate, obtained by hydrolysis with chymotrypsin, and/or pea protein peptides; and (ii) at least a nitrogen source selected from the group consisting of milk proteins, egg proteins, soy protein, wheat protein, rice protein, free amino acids and mixtures thereof. Preferably the present composition comprises (i) a pea protein hydrolysate, obtained by hydrolysis with chymotrypsin, and/or pea protein peptides and (ii) at least one nitrogen source selected from the group of hydrolyzed cow's milk whey, not hydrolyzed cow's milk whey, hydrolyzed casein of cow's milk and not hydrolyzed soy protein. Preferably, the pea protein hydrolysate is obtained by hydrolysis by trypsin.

When the real BAA�component thereof and, in particular, the present pea protein hydrolysate and/or pea protein peptides are administered in combination with an additional nitrogen source, the present composition preferably comprises between 0.1 and 50 wt.% the present pea protein hydrolysate and/or pea protein peptides and, in particular, between 1 and 10 wt.% the present pea protein hydrolysate and/or pea protein peptides in the calculation of the total mass of the protein.

When the present protein component and in particular, the present pea protein hydrolysate is administered in combination with an additional nitrogen source, the present composition preferably comprises between 1 and 50 wt.% the present pea protein hydrolysate and/or pea protein peptides and, in particular, between 1 and 10 wt.% the present pea protein hydrolysate in the calculation of the total mass of the protein.

When the present protein component and in particular, the present peptides from pea protein is administered in combination with an additional nitrogen source, the present composition preferably comprises between 0.1 and 50 wt.% the present pea protein hydrolysate and/or pea protein peptides and, in particular, between 0.1 and 10 wt.% the present pea protein hydrolysate and/or pea protein peptides in the calculation of the total mass of the protein.

To nutritional compositions may be added source of digestible small drop�Dov. The present composition preferably contains lactose.

In a preferred embodiment the anti-infective agent against gastrointestinal pathogen present protein component and in particular, the present pea protein hydrolysate and/or pea protein peptides improve through co-injection of soluble, indigestible, fermentable oligosaccharide. The introduction of such oligosaccharides stimulate the growth of lactic acid bacteria such as bifidobacteria and lactobacilli, preventing colonization and infection with gastrointestinal pathogens. Accordingly, the present pea protein hydrolysate and/or pea protein peptides and present oligosaccharide in this respect act synergistically.

Preferably the present composition comprises non-digestible oligosaccharides with degree of polymerization (SP) (DP) between 2 and 50, more preferably 3 and 60. Non-digestible oligosaccharide is preferably selected from the group consisting of fructo-oligosaccharides (such as inulin), galactooligosaccharides (such as transplantological or beta-galactooligosaccharides, glucooligosaccharides (such as gentio-, Niger - and cyclodextrin-oligosaccharides), arabino-oligosaccharides, mannan-oligosaccharides, Xylo-oligosaccharides, Foucault-oligosaccharides, arabinogalactan-oligosaccharides, �lucomagno-oligosaccharides, galactomannan-oligosaccharides, oligosaccharides, including sialic acid and oligosaccharides uronic acid. Preferably the composition includes gum Arabic in combination with the non-digestible oligosaccharide.

Preferably the present composition comprises fructo-oligosaccharides, galactooligosaccharides and/or the oligosaccharide galacturonic acid, more preferably galactooligosaccharides, most preferably transplantological. In a preferred embodiment the composition includes a mixture of transplantationfollow and fructooligosaccharides. Preferably the present composition comprises galactooligosaccharides with SP 2-10 and/or fructo-oligosaccharides with JV 2-60. Galactooligosaccharide preferably selected from the group consisting of transplantationfollow, lacto-N-tetrose (LNT), lacto-N-neoteris (neo-LNT), fucosyl-lactose, fokusirovannyi LNT and fokusirovannyi neo-LNT. In a particularly preferred embodiment of the present method comprises administering transplantationfollow ([galactose]n-glucose; where n is an integer between 1 and 60, i.e. 2, 3, 4, 5, 6,...., 59, 60; preferably n is selected from 2, 3, 4, 5, 6, 7, 8, 9, or 10). Transplantological (TOS), for example, sold under the trade name Vivinal™ (Borculo Domo Ingredients, Netherlands). Preferably the saccharides transg�of lactoridaceae are β-linked.

FOS is a non-digestible oligosaccharide, comprising a chain of β-linked fructose with JV JV or middle 2-250, more preferably 10-100. Fructo-oligosaccharides include inulin, Levan and/or a mixed type of polifruktan. Especially preferred is inulin FOS. FOS, suitable for use in the compositions, is also commercially available, e.g. Raftiline®HP (Orafti).

Oligosaccharides uronic acid, preferably derived from degradation products of pectin. Oligosaccharides uronic acid preferably represent the oligosaccharide galacturonic acid. Therefore, the present composition preferably comprises degradation products of pectin with SP between 2 and 100. Preferably, the degradation products of pectin is produced from Apple pectin, beet pectin and/or citrus pectin. Preferably the composition includes transplantological, fructooligosaccharides and degradation products of pectin. The weight ratio of transplantologiia:FOS:degradation product of pectin is preferably(20-2):1:(1-3), more preferably(12-7):1:(1-2).

Preferably, the oligosaccharide uronic acid has one, preferably two terminal units of uronic acid, which can be free elitedivisionen. Preferably a terminal unit of a uronic acid selected from the group consisting of galacturonic acid, glucuronic acid, guluronic acid, Euronaval acid, mannuronate acid, Rybarikova acid and ultranova acid. Such units can be free or esterified. In an even more preferred embodiment the terminal hexose unit (i.e. uronic acid) has a double bond, which is preferably situated between the C4 and C5 the provisions of the terminal hexose units. Preferably one of the terminal hexose units includes the double bond. The group of carboxylic acids such units of uronic acid may be free or (partly) esterified, and are preferably at least partially methylated.

Oligosaccharides uronic acid used in the present invention, preferably prepared from pectin, pectate, alginate, chondroitin, hyaluronic acid, heparin, heparan, bacterial carbohydrates, sialogogues, fucoidan, focalisation or carrageenan, more preferably from pectin and/or alginate. Oligosaccharides uronic acid preferably get enzymatic digestion of lisati, lyase and/or andprecautions. Preferably use the hydrolysed sludge� lysate of pectin. The present oligosaccharide uronic acid preferably get enzymatic digestion of pectin with pectin Lisesi, pectin lyase, andprecautions and/or pectinases.

Such oligosaccharides uronic acid prevents the adhesion of intestinal pathogens. Compositions comprising peptides/pea protein hydrolysate of the present invention and oligosaccharide uronic acid, will possess superior anti - H. pylori effect.

The preferred oligosaccharide is sialyllactose, more preferably 3'-sialyllactose, such as the oligosaccharide also affect the adhesion of H. pylori. The compositions of the present invention, further comprising sialyllactose, preferably 3' sialyllactose, therefore, will possess superior anti H. pylori effect.

Preferably the composition includes from 80 mg to 2 g non-digestible oligosaccharides per 100 ml, more preferably from 150 mg to 1.50 g, even more preferably 300 mg to 1 g per 100 ml based on the dry weight of the composition preferably comprises from 0.25 wt.% to 20 mass%, more preferably from about 0.5 wt.% to 10 mass%, even more preferably from 1.5 wt.% to 7.5 mass%.

Therefore, in another embodiment of the present invention relates to a composition comprising non-digestible oligosaccharide selected from the group�standing from transplantologiia, fructo-oligosaccharides, oligosaccharide uronic acid and sialyllactose and

a) hydrolyzed pea protein, with the proviso that the pea protein hydrolysate is not obtained by hydrolysis by the protease chymotrypsin, or preferably, the pea protein hydrolysate obtained by hydrolysis protease other than chymotrypsin; and/or

(b) at least one peptide selected from the group consisting of Xaan-Asp-Phe-Leu-Glu-Asp-Ala-Phe-Asn-Val-Asn-Arg-Xaamand Xaan-Glu-Leu-Ala-Phe-Pro-Gly-Ser-Ala-Gln-Glu-Val-Asp-Arg-Xaamwhere each Xaa independently can be any amino acid, and n and m are integers independently ranging from 0 to 10.

Violations of the chair (e.g. hard stools, insufficient stool volume, diarrhea) are a particular problem in many children and patients who suffer from or are at risk of H. pylori infection. Such patients often receive liquid foods. It was found that problems with defecation can be reduced by introducing a component of the present pea protein hydrolysate and/or pea protein peptides in the liquid food, which has an osmolality between 50 and 500 mOsm/kg, more preferably between 100 and 400 mOsm/kg. Prevention of diarrhea is particularly important when the present pea protein hydrolysate and/or pea protein peptides used together or after antibiotic treatment. In �Veta above, it is also important to liquid food had no excess calories, however, still provided enough calories for the patient. Hence, the liquid food preferably has a caloric content of between 0.1 and 2.5 kcal/ml, even more preferably a caloric content of between 0.5 and 1.5 kcal/ml, most preferably between 0.6 and 0.8 kcal/ml.

Application

The present invention provides a composition for and a method of treatment and/or prevention of infection by gastrointestinal pathogens (especially H. pylori infection and/or disease associated with infection by gastrointestinal pathogens (especially H. pylori), a mammal, preferably a human, said method includes the introduction of the present pea protein hydrolysate and/or pea protein peptides to a mammal or human. The diseases associated with infection by gastrointestinal pathogens in humans, include persistent chronic gastritis, diarrhea, abdominal pain, ulcers and/or stomach cancer. The diseases associated with H. pylori infection in humans include persistent chronic gastritis, ulcers and/or stomach cancer. The present invention also provides for the treatment and/or prophylaxis of such diseases in mammals, preferably humans, with the risk of developing, or needing their treatment.

The present invention relates to treatment and/or Pro�ractice of a mammal, preferably human or domestic animal, more preferably humans. Real composition is predominantly administered (a) children aged between 0 and 5 years, preferably children from 0 to 2 years, and/or (b) patients suffering from gastroduodenal diseases, especially patients suffering from peptic ulcer.

The present invention also is particularly suitable to prevent reinfection of the gastro-intestinal pathogens, especially H. pylori, after treatment of a mammal one or more antibiotics.

Example 1: Fraction of pea protein hydrolysate inhibit the adhesion of H. pylori to the gastric mucosa.

1.1: Study on anti-adhesion

Helicobacter pylori ATCC 700824 (J99) were cultivated for two or three passages to minimize the risk of an alternating phase shift OMP genes. H. pylori were incubated for 48 h in microaerophilic conditions at 37°C on tryptic soy agar (Becton Dickinson, Germany) supplemented with 5% defibrination sheep blood (Oxoid, UK). Human epithelial cells of the stomach (AGS cells) were grown in RPMI 1640 with L-glutamine (PAA, Germany) supplemented with 10% FCS, the culture dishes tissue (75 cm2, Sarstedt, USA) and 6-well plates (Sarstedt, USA) in 5% CO2.

Grown on agar H. pylori were collected and were resuspended in sterile carbonate buffer (pH 9,0) to a density of about 1.0×108bacter�th at ml. 10 μl of FITC solution (1% in DMSO) was added and incubated with the bacteria for 45 min with a Fluorescent label was terminali by the deposition of bacteria by centrifugation (3150×g, 5 min). Bacteria were washed twice in PBS to remove excess FITC was resuspended and carefully for future use. In vitro study of the anti-adhesive activity of the tested compounds relative to FITC-labeled H. pylori on AGS cells was performed by flow cytometry (Niehues & Hensel, 2009 J. Pharm. Pharamcol. 61: 1303-1307).

1.2 Fractionation sent biological samples of pea protein hydrolysate.

50 grams of isolate pea protein (Nutralys®F85F, 84% of the mass./mass. protein from Roquette Freres (Lestrem, France) was dissolved in 1.5 liters of distilled water at 50°C. Hydrolysis was started by adding 0.56 g of trypsin (PTN Novo 6. OS, Novozymes A/S, Bagsvaerd, Denmark). the pH was controlled as to 7.0 by adding NaOH. The reaction was allowed to continue for 2 h. the Process was stopped by heat inactivation of the enzyme at 85°C for 5 min. the Precipitated material was precipitated by centrifugation at 3,800×g for 20 min at 20°C and the supernatant was ultrafiltrated using the tablet device and the frame using a 700 cm2membrane 10 kDa NMWCO PES (UltranLab, Schleicher&Schuell, Dassel, Germany). The UF-retentate liofilizirovanny and used for the subsequent separation of pea peptides.

Activity �otnositelno H. pylori was determined as described in 1.1. Pea protein itself did not show any anti-adhesion activity against H. pylori. After ultrafiltration found that the antiadhesive activity of pea protein hydrolysate (56% adhesion of bacteria with 0.5 mg/ml) is within fractions of UF-retentate and in the permeate fraction.

The UF-retentate 10 kDa was fractionally by size exclusion chromatography (SEC) using 42×5.0 cm ID column Packed with Toyopearl®HW-50S (Tosoh Bioscience GmbH, Stuttgart, Germany). Ammonium bicarbonate 0.1 M, containing 2% (vol./about.) 2-propanol, was used as the mobile phase. The column was treated with a flow rate of 4 ml/min at 4°C. the Eluting compounds were investigated by UV 220 nm, and fractions were collected at intervals of 7 ml and were combined to obtain four major fractions, namely F1-F4. F1 is the elution volume of about 0-220 ml and exhibits adhesion 76% at 0.5 mg/ml, F2 is the elution volume of about 220-310 ml and active adhesion 94% at 0.5 mg/ml, F3 is the elution volume of about 310-500 ml and active adhesion 25% at 0.5 mg/ml and F4 is the elution volume 500-720 ml and active adhesion 118% at 0.5 mg/ml.

Subsequent purification of F3 was achieved by chromatography with reversed phase (RPC) using the column 11,8×1.0 cm ID, Packed with Amberchrom resin®CG-161S (Tosoh Bioscience GmbH, Stuttgart, Germany). Used mobile�and phases were 0.1% of (about./about.) TFA in distilled water (A) and 0.1% (vol./about.) TFA in 2-propanol (B). The elution was performed by linear gradient from 5% B to 60% B in 7 column volumes, the flow rate of 0.85 ml/min, UV 220 nm. Received fractions, each of 4 ml, and were pooled to give three main fractions: F3.1 to F3.3. Fraction liofilizirovanny before subsequent analysis.

F3.1 having an elution volume of about 3 to 33 ml showed activity adhesion 109% at 0.5 mg/ml, F3.2, having an elution volume of about 33 to 54 ml, showed the activity of adhesion of 112% at 0.5 mg/ml, and F3.3, having a volume of elution 54 to 85 ml, showed the adhesion activity of 40% at 0.5 mg/ml, 70% at 0.2 mg/ml and 78% at 0.1 mg/ml.

The size distribution of the molecules, as determined using the Calibration Superdex Peptide 10/300 GL (Amersham Biosciences 5Cat. No. 17-5176-01). A sample of 2% of the mass./about. (supernatant after centrifugation) were diluted in 0.1% TFA in 30% ACN/water. The peptides were determined by UV at 214 nm. Size distribution (percentage of total area) was as follows:

Range MMGPC F1 (A0622)GPC F2 (A0623)GPC F3 (A0624)GPC F4 (A0625)
> 10 kDa3,7A 16.40,00,0
10 kDa <>the 7.5 kDa3,9 15,20,20,1
The 7.5 kDa <>5 kDa11,616,91,40,4
5 kDa <> 2,5 kDa29,016,323,94,3
The 2.5 kDa <> 1 kDa29,8The 17.346,321,0
1 kDa <> 0,5 kDa11,39,8The 16.223,6
<a 0.5 kDa10,78,211,950,6

Consequently, the average molecular weight of fraction F3 was about 2 kDa with the highest number of peptides in the range of from 1 kDa to 2.5 kDa.

1.3 Identification of peptides

For unambiguous identification of the active peptides of fraction F3.1, F3.2 and F3.3 investigated by tandem MS analysis MALDI-TOF-TOF. The signal peptide present in the active fractions, but not detectable in the idle mixtures, specifically chose �La subsequent tandem sequencing of amino acids on the basis of MS. Six peptide sequences (S1-S6) were determined by this method (sequences are given in table. 1) and clearly identified with the software ProteinPilot™ with integrated algorithm Paragon™ (Applied Biosystems, Darmstadt, Germany). The following parameters were chosen to estimate the amino acids according to MALDI-tandem-MS-data through an algorithm Paragon™: sample type: identification; alkylation of cysteine: no; digestion: trypsin; instrument: 4800; special factors: none; types: no restrictions; ID focus: biological modifications; database: Uniprot/Swiss-Prot (version January 23, 2007, including a database of pollutants, both in FASTA format); search effort: thorough.

Peptides S1-S6 were identified as peptide fragments from pea legumin And or vicilin. Subsequently, the corresponding peptides were synthesized for further unambiguous study of anti-adhesive properties (Thermo Fisher Scientific (Ulm, Germany). It was found that undecapeptide S3 is the most active compound that reduces bacterial adhesion of H. pylori significantly to 81% and 83% respectively (75 ACC. 150 μm, corresponding to 0.1 resp. 0.2 mg/ml). Also S5, which inhibited the adhesion by 6-17% (75 resp. 150 μmol), was evaluated from two active peptides obtained by the digestion of pea protein by trypsin.

Table 1: Sequences of peptides S1-S6, from syntheti�die peptide S3A-S3H, with an average adhesion (±SEM) of FITC-labeled H. pylori to AGS cells after pre-treatment of bacteria relevant peptides. Data refer to the raw control H. pylori (=100%). Positive control: 3 sialyllactose (15 mm).

Test connectionThe amino acid sequenceRel. adhesion [%] (±SEM; n=3)
75 microns150 µm
S1Leu-Asp-Ala-Leu-Glu-Pro-Asp-Asn-Arg-Ile-Glu-Ser-Glu-Gly-Gly-Leu-Ile-Glu-Thr-Trp-Asn-Pro-Asn-Asn-Lys (SEQ ID NO:3)95±10104±7
S2Leu-Asn-Ile-Gly-Pro-Ser-Ser-Ser-Pro-Asp-Ile-Tyr-Asn-Pro-Glu-Ala-Gly-Arg (SEQ ID NO:4)93±894±6
S3Asp-Phe-Leu-Glu-Asp-Ala-Phe-Asn-Val-Asn-Arg (SEQ ID NO: 1)81±383±6
S4Trp-Glu-Arg-Glu-Glu-Asp-Glu-Glu-Gln-Val-Asp-Glu-Glu-Trp-Arg (SEQ ID NO:5)98±2107±2
S5Glu-Leu-Ala-Phe-Pro-Gly-Ser-Ala-Gln-Glu-Val-Asp-Arg (SEQ ID NO:2) 94±285±2
S6Gly-Asp-Phe-Glu-Leu-Val-Gly-Gln-Arg (SEQ ID NO:6)94±397±2
S3AAsp-Phe-Leu-Glu-Asp (SEQ ID NO:7)102±3108±6
S3BAla-Phe-Asn-Val-Asn-Arg (SEQ ID NO: 8)97±4104±1
S3CLeu-Glu-Asp-Ala-Phe-Asn-Val-Asn-Arg (SEQ ID NO:9)95±5100±4

S3DAsp-Ala-Phe-Asn-Val-Asn-Arg (SEQ ID NO: 10)89±489±3
S3EAsp-Phe-Leu-Glu-Asp-Ala-Phe-Asn-Val (SEQ ID NO: 11)91±491±4
S3FAsp-Phe-Leu-Glu-Asp-Ala-Phe (SEQ ID NO: 12)85±390±5
S3GLeu-Glu-Asp-Ala-Phe-Asn-Val (SEQ ID NO: 13)93±294±3
S3HAsp-Ala-Phe 104±6105±1
Untreated control-100±2100±2
Positive control-70±570±5

A similar experiment was performed as described in example WO 2008/043424. Briefly, the pea protein hydrolysate was microfractional through nanochromatography with reversed phase (C 18) and each fraction was applied online on MALDI target. Each of 832 stains were evaluated for the presence of the claimed peptides S3 (m/z 1339) and S5 (m/z 1418) by mass spectrometry MALDI-TOF. In conclusion, the peptide S5 absolutely are unable to identify. The S3 peptide was detected only in trace amounts.

1.4 Inhibition of adhesion of H. pylori in situ

To confirm the results obtained fraction F3 and S3 peptide was evaluated in relation to anti-adhesion activity in the study in situ on the fabric of the human stomach. Experiments in situ with histological sections of the gastric mucosa of man and FITC-labeled H. pylori was performed in accordance with Lengsfeld et al, 2004, J. Agricul. Food Chem: 52, 1495-1503 and evaluated by fluorescent microscopy. Adhesion of FITC-labeled H. pylori to the epithelium were evaluated by fluorescence microscopy and visualize�Oia. The number of bacteria adhered to the epithelial surface, was evaluated in double-blind conditions. Maximum adhesion was expressed as 100% adhesion. The intensity of the area of fluorescence was calculated by means of the software ImageJ®(Olympus, Germany), by standardizing the size of the fluorescence of the negative control as 100%.

H. pylori, pre-treated fraction F3 (1.0 mg/ml) showed significantly impaired adhesion in this system of study (70% inhibition). Also S3 (300 Μ significantly reduced the adhesion of bacteria (about 40% inhibition). The data obtained clearly confirm the antiadhesive activity of F3 and S3. On the other hand, the fractionation of the bioassay also showed the kind of nonlinear activity profiling: antiadhesive effects did not increase consistently during protein purification stages, but became less for individual selected and studied peptides. This is a clear indication of the presence of other active compounds in the mixture that affect adhesive properties synergistic way.

1.5 Relationship of the structure-activity

For the relationship of the structural activity of the synthesized various active fragments of undecapeptide S3 lengths (S3A-S3H, see tab. 1). Functional study demonstrated total loss of inhibitory power on with�avanyu with a native peptide S3.

Only peptides S3D, E, F and G are a bit blocked adhesion (10% reduction). When compared with the amino acid sequence of S3 can be identified homologous motif and integral with terminal Tripeptide sequence, namely Asp-Ala-Phe. Consequently, the synthesized Tripeptide (Asp-Ala-Phe) S3H researched and showed that he is inactive, that probably indicates that this sequence can only be functionalized, if embedded in a longer peptide chain. The authors assumed that the most likely complete sequence of the peptide of at least 11 amino acids, as detected in S3, necessary for its inhibitory activity against bacterial adhesion, because of the possible formation of three-dimensional folds of peptide required to interact with a target receptor.

The existence of a specific secondary structure and three-dimensional protein folds can change the functionality of the peptides. For these preliminary experiments, conformational modeling software molecular operating environment (MOE), various peptides showed a high degree of folding undecapeptide S3, which is clearly absent in the low-molecular peptide fragments S3A-S3H (data not shown).

1.6 Specificity of preventing adhesion

Specificity �active peptides against outer membrane proteins of H. pylori (OMP) was shown using dot-battenhouse analysis glycoconjugates in accordance with previously described methods [Walz, et al2005, Glycobiology, 15, 700-708; Valkonen et al., 1994, 62 (9), 3640-3648].

Membrane polyvinylidene fluoride (PVDF) with a mesh width of 0.2 μm were labeled with 2 μl of a solution containing 1 µg of glycoprotein and neoglycoproteins.

Typical range of typical ligands for H. pylori adhesins used for these experiments were Lewisband antigenic conjugates of H type I blood group that interacts with OMP BabA, 3'-sialyllactose interacting specifically with OMP HpaA, sialyl-Lewis and laminin, are known to interact with OMP SabA, and fibronectin with still uncertain affinity towards the bacterial adhesins. In addition, human serum albumin (HSA) and bovine serum albumin (BSA) was used as control to exclude nonspecific binding of H. pylori with applied on the membrane compounds. In addition, 6'-sialyllactose used to demonstrate the specificity of binding HpaA with 3'-sialyllactose. We measured the fluorescence of adherent pre-processed and unprocessed-labeled bacteria.

In cases of pre-treatment H. pylori using F3 observed a significant reduction in bacterial interaction with the conjugates Lewisb-, Η-type I-and 3'-sialyllactose-HAS, as well as with the fiber�actinon. F3 also to a lesser extent influenced the binding of the conjugate sialyl-Lewisabut not inflicted on laminin. These discoveries clearly show that F3 interacts specifically with H. pylori adhesins BabA, HpaA, fibronectine activity and also less strongly with SabA.

In contrast to peptides S3 and S5 showed only inhibition of adhesion, mediated Lewisb-HSA, suggesting inhibition of BabA adhesin, whereas on other adhesins were not significantly affected.

In conclusion, this inhibition involves expressed or mimicry, or additional binding sites of F3 peptides with known receptor structures BabA, HpaA, SabA and not yet identified by adhesion with affinity towards the ECM protein fibronectin. Purified peptides were significantly less active than the more complex fractions, due to monovalent inhibition only bacterial adhesin of the purified peptide. On the contrary, the use of complex heterogeneous mixture capable of reacting with H. pylori OMP in multiple strategies and, consequently, leads to the blockade of several proteins responsible for bacterial adhesion. This clearly demonstrates that in different scenarios OMP important for the adhesion process, a complex mixture can probably be used much more efficiently than purified individual� connection.

The present results of example 1 are illustrative of the benefits of pea protein hydrolysate and/or pea peptides for the treatment and/or prevention of H. pylori infections and/or diseases associated with H. pylori infection in mammals.

1. The use of a composition comprising pea protein hydrolysate for the production of the composition for treatment and/or prevention of infection by gastrointestinal pathogens and/or disease associated with infection by gastrointestinal pathogens in a mammal, where the pea protein hydrolysate is obtained by hydrolysis protease other than chymotrypsin, where the gastrointestinal pathogen Helicobacter pylori is a.

2. The use of claim 1, wherein the pea protein hydrolysate is obtained by hydrolysis of pea protein legumin And or vicilin.

3. The use according to claim 1 or 2, where the pea protein hydrolysate is obtained by hydrolysis by the protease trypsin.

4. The use according to claim 1 or 2, where the pea protein hydrolysate contains more than 35 mass. % of peptides with a size between 1 and 2.5 kDa in the calculation of the total mass of the pea protein hydrolysate.

5. The use of a composition comprising a peptide, to obtain a composition for the treatment and/or prevention of infection by gastrointestinal pathogens and/or disease associated with infection by gastrointestinal pathogens, melicope�ment, where the peptide is selected from the group consisting of Xaan-Asp-Phe-Leu-Glu-Asp-Ala-Phe-Asn-Val-Asn-Arg-Xaamand Xaan-Glu-Leu-Ala-Phe-Pro-Gly-Ser-Ala-Gln-Glu-Val-Asp-Arg-Xaamwhere each XAA independently can be any amino acid, and n and m are integers independently ranging from 0 to 10, where the peptide is contained in the pea protein hydrolysate and where gastrointestinal pathogen Helicobacter pylori is a.

6. The use according to claim 1 or 5, where the disease is associated with infection, selected from the group consisting of gastritis, peptic ulcers and stomach cancer.

7. The use according to claim 1 or 5, where the mammal is (i) a child aged between 0 and 5 years, or (ii) a patient suffering from gastroduodenal diseases.

8. The use according to claim 1 or 5 to prevent reinfection by a pathogen after treatment of a mammal with one or more antibiotics.

9. Composition for treating and/or preventing Helicobacter pylori infections that contains lipid, protein and carbohydrate component, where the lipid component provides 5 to 50% of the average daily consumption of calories, the protein component provides 5 to 50% of the average daily consumption of calories and the carbohydrate component provides 15 to 90% of the average daily consumption of calories, characterized in that the protein component includes: (i) �of at least one protein source consisting of pea protein hydrolysate, where the pea protein hydrolysate is obtained by hydrolysis protease other than chymotrypsin, and (ii) at least one nitrogen source selected from the group consisting of milk proteins, hydrolysates of milk protein, egg protein, hydrolyzed egg protein, soy protein, soy protein hydrolysate, wheat protein, hydrolyzed wheat protein, rice protein, rice protein hydrolysate, and mixtures thereof, and where the composition contains between 0.1 and 50 wt. % pea protein hydrolysate to total weight of the protein.

10. A composition according to claim 9, where the pea protein hydrolysate is obtained by hydrolysis by the protease trypsin.

11. Composition for treating and/or preventing Helicobacter pylori infections that contains lipid, protein and carbohydrate components, where the lipid component provides 5 to 50% of the average daily consumption of calories, the protein component provides 5 to 50% of the average daily consumption of calories and the carbohydrate component provides 15 to 90% of the average daily consumption of calories, characterized in that the protein component includes: (i) at least one source of protein consisting of a peptide selected from the group consisting of Xaan-Asp-Phe-Leu-Glu-Asp-Ala-Phe-Asn-Val-Asn-Arg-Xaamand Xaan-Glu-Leu-Ala-Phe-Pro-Gly-Ser-Ala-Gln-Glu-Val-Ap-Arg-Xaa mwhere each XAA independently can be any amino acid and n and m are integers independently ranging from 0 to 10, and where the peptide is contained in the pea protein hydrolysate, and (ii) at least one nitrogen source selected from the group consisting of milk proteins, hydrolysates of milk protein, egg protein, hydrolyzed egg protein, soy protein, soy protein hydrolysate, wheat protein, hydrolyzed wheat protein, rice protein, rice protein hydrolysate, and mixtures thereof, and where the composition contains between 0.1 and 50 wt. % pea protein peptides relative to the total mass of the protein.

12. A composition according to any one of claims. 9 to 11, comprising at least one component selected from the group consisting of hydrolyzed cow's milk whey, not hydrolyzed cow's milk whey, hydrolyzed casein of cow's milk, not hydrolyzed casein of cow's milk, hydrolyzed soy protein and not hydrolyzed soy protein.

13. A composition according to any one of claims. 9-11, including soluble, nevereverever, fermented oligosaccharide.

14. Composition for treating and/or preventing infections by Helicobacter pylori, comprising indigestible oligosaccharide selected from the group consisting of transplantologiia, fructo-oligosaccharides uronic acid and�of allactaga and
a. the pea protein hydrolysate obtained by hydrolysis protease other than chymotrypsin; and/or
b. at least one peptide selected from the group consisting of Xaan-Asp-Phe-Leu-Glu-Asp-Ala-Phe-Asn-Val-Asn-Arg-Xaamand Xaan-Glu-Leu-Ala-Phe-Pro-Gly-Ser-Ala-Gln-Glu-Val-Asp-Arg-Xaamwhere each XAA independently can be any amino acid, and n and m are integers independently ranging from 0 to 10, and where the peptide is contained in the pea protein hydrolysate.

15. A composition according to any one of claims. 9 to 11 or 14 with an osmolality between 50 and 500 mOsm/kg.

16. A composition according to any one of claims. 9 to 11 or 14 in the form of a nutritional or pharmaceutical composition.

17. A composition according to any one of claims. 9 to 11 or 14 for use in the treatment and/or prophylaxis of H. pylori infection and/or diseases associated with H. pylori infection.



 

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6 ex

FIELD: biotechnology.

SUBSTANCE: method of obtaining SSI comprises the following steps. The strain Yersinis pestis KM 1279 is grown on 1.5% agar LB, the bacteria are washed three times with cold buffered normal saline. The bacteria are pelleted by centrifugation, suspended in a solution of 5 mM NaOH, kept at 37°C for two hours and the cells are pelleted by centrifugation. The supernatant is selected and the procedure as repeated three times, three supernatants are combined and filtered through the nitrocellulose membrane. The filtrate is extracted three times with the mixture of chloroform-methanol-water in a ratio of 5:2:1. The chloroform fractions are separated by centrifugation, combined and freed from water-soluble impurities. The aqueous fraction is separated by centrifugation and removed, and the chloroform fraction is dried in a vacuum rotary evaporator and the dry preparation SSI is obtained. The proposed SSI is characterised with brown colouring of dry crystals, hydrophobic properties, fluorescence in ultraviolet, lipopeptide nature, the presence of iron ions, the molecular weight of 380.6 Da.

EFFECT: inventions enable to obtain the natural regulator of virulence of plague agent.

2 cl, 6 dwg, 6 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to biotechnology. What is presented is a method for preparing a recombinant protein of type III interferon-like factor (ILF III) of the producing strain E. coli. The inclusion bodies E. coli are washed and dissolved with using 2% aqueous γ-cyclodextrin. That is followed by the sequential Ni-Sepharose, Q-Sepharose and SP-Sepharose chromatographic procedures. Refolding of a target protein is performed with using a mixture of cysteamine and cystamine at pH 10.5. The Amberchrome Profile XT20, Amberchrome Profile HPR10 and Kromasil 300-5C18 chromatographic procedures are sequentially performed.

EFFECT: invention enables optimising the ILF III purification environment at the stage of washing and dissolving the inclusion bodies Ecoli and provides 12% target protein yield.

3 dwg, 4 ex

FIELD: chemistry.

SUBSTANCE: invention relates to biotechnology, specifically to immunostimulating compounds and may be used in medicine. An immunostimulating peptide of an amino acid sequence XLYDKGYTSKEQKDCVGI, where N-terminal X is N-acetylalanine, and may be covalently linked to fatty acids, selected from C2-C25, to form PDAG (peptidyl-2,3-diacylglycerides). The resulted compound may be included in pharmaceutical formulations for stimulating an immune response.

EFFECT: invention provides efficient stimulation of an immune response in subjects and may enhance the immunogenicity of the antigenic peptide when administered with PDAG.

29 cl, 10 dwg, 9 ex

FIELD: biotechnology.

SUBSTANCE: method of production of peptides is proposed. Yeast autolysis is carried out. The cell membranes are separated by centrifugation. The autolysate is purified on the gel sulphocationite in the hydrogen form, containing 12-16% divinylbenzene. The resulting peptide aqueous solution is passed sequentially through the gel anion-exchange material to obtain the solution at pH 2.0-2.6, and then through the gel cation-exchange material with the divinylbenzene content of 1-2% or macroporous cation-exchange material.

EFFECT: obtaining highly purified peptides that have biological activity.

11 ex

FIELD: veterinary medicine.

SUBSTANCE: method comprises subcutaneous administration of antibiotic preparation enroxyl 5% at a dose of 0.1 ml/kg daily one time a day for 7 days and intramuscular administration of homeopathic preparation ovarinin at a dose of 1 ml/kg one time for 4 days, 4-fold.

EFFECT: use of the invention enables to increase the efficiency of treatment, to reduce treatment time and to restore reproductive function of dogs.

2 tbl, 1 ex

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