Application of proline-specific endoproteses for peptide and protein hydrolysis

FIELD: medicine.

SUBSTANCE: invention concerns medicine and application of proline-specific endoproteases for peptide and protein hydrolysis. Invention involves application of proline-specific endoprotease with pH optimum under 5.5 for production of diet additive or medicine for in vivo treatment of celiac disease or diseases related to presence of proline-rich peptides in food, or as diet additive or medicine for celiac disease prevention.

EFFECT: enhanced hydrolysis of forage proteins with high proline content.

13 cl, 12 ex,10 tbl, 3 dwg

 

The technical field to which the invention relates

The present invention relates to proteolytic hydrolysis of peptides or polypeptides.

The level of technology

It is known that enriched in Proline proteins, which enter into the composition of food, such as casein cow's milk or gluten grains, difficult proteolytic cleavage in the gastrointestinal tract of man. The result can be an accumulation of peptides with a high content of Proline, which can lead to undesirable consequences for certain groups of people. Some of these effects have been attributed to the fact that peptides with a high content of Proline act as opioids that bind to receptors in peripheral tissues and the Central nervous system. For example, the syndromes observed in patients with autism and schizophrenia, have been associated with the consumption of dietary proteins with a high content of Proline. Other consequences are the result of intolerance of peptides with a high content of Proline. For example, specific sequences enriched in Proline, are responsible for the observed toxicity of gluten in celiac disease (coeliac disease). Celiac disease is a common autoimmune disease of the small intestine, which may be vile is prohibited only for life diet eliminating gluten. Besides celiac disease is sometimes accompanied by psychiatric and neurological symptoms, showing how far-reaching consequences of the violation of the metabolism of peptides with a high content of Proline.

Proteins of cow's milk is associated with growth and health and are an important ingredient in the human diet. Casein approximately 80% of the total protein in cow's milk as an important source of amino acids, calcium and phosphate. Casein consists of approximately 50% alpha-Caseins, 35% beta-Caseins, 13% of Kappa-casein and 3% gamma-Caseins. In human milk fraction of alpha-casein largely does not exist.

It is known that the metabolism of casein produced a number of new biologically active peptides. From the alpha and beta-casein fractions were isolated and identified opioid peptides, named, respectively, the alpha casomorphine and beta casomorphin. Pharmacological effects, in particular, of beta-casomorphins widely studied. Beta-casomorphin with the sequence Tyr-Pro-Phe-Pro-Gly-Pro-Ile is the main opioid peptide cow's milk, which is called BCM-7 (beta-casomorphin (1-7); Chang et al. (1985) Journal of Biological Chemistry, 260, 9706-9712). In the distance from the fragment BCM-7, 60-66 amino acid sequence of a molecule of beta-casein smaller pieces, podobn the e BCM-7, such as Tyr-Pro-Phe-Pro (beta-casomorphin (1-4)) and Tyr-Pro-Phe-Pro-Gly (beta-casomorphin (1-5), in the positions 60-63 and 60-64 respectively, as well as all larger peptides, related to BCM-7, up to chain length 11 amino acid residues (at positions 60-70), show at least some degree of opioid activity. The N-terminal Tripeptide of BCM-7, i.e. the sequence Tyr-Pro-Phe at positions 60-62 does not possess opioid activity. It was argued that a genetic variant of beta-casein, referred to as A1 (with his-tag, and not polynomy as the A2 beta-casein, the residue at position 67) leads to the formation of fewer molecules BCM-7.

The main reason for the formation of various beta-casomorphine lies in the fact that their amino acid sequence is relatively rich prolinamide remains. Because peptide bonds, including prolinnova remains, hardly undergo proteolytic cleavage of beta-casomorphine sequence tends to persist after exposure to gastrointestinal proteases in the stomach and intestinal canal. For the same reason we can assume that these beta-casomorphine sequence tend to persist after incubation with other proteases, such as proteases, which are typically used in industrial production of protein gidrolizovat assumption implies all are widely available protein hydrolysates or products containing these protein hydrolysates contain BCM-7 or closely related peptides. Because peptide fragment of BCM-7 and related molecules associated with specific diseases, the presence of such molecules in protein hydrolysates, which are quite often used in dietary risk groups such as young children, the elderly and the sick, is undesirable. The results of binding opioid receptors with the human and bovine beta-casomorphine show that fragments with opioid activity, bind to opiate receptors in the membrane of rat brain. It was shown that beta-casein more selective with respect to mu-ligands, with little affinity for Delta - and Kappa - receptor subtypes. In accordance with these and other studies, it is stated that beta-casomorphine have a variety of gastrointestinal, pain, respiratory, cardiovascular, endocrine, and immunomodulatory effects. The common structural feature of opioid peptides comprising the residue of Proline, is to fragment Tyr-Pro-Phe/Trp (Okada et al., Vitamins and Hormones 2002, 65, 257-279).

Although normal individuals peptidases in the intestinal epithelial layer and in the blood are able to cope with splitting of the ETA-casomorphins, I believe that this is not always the case in patients suffering from schizophrenia, autism, ADHD (attention deficit disorder and hyperactivity) or other affective disorders. For example, with the emergence of these disorders have been associated genetic changes in enzyme activity dipeptidylpeptidase plasma IV (DPP IV), leading to incomplete cleavage of peptides with a high content of Proline. In addition, the constantly found hypermature, i.e. increased concentration in the urine peptides derived from casein or gluten (Reichelt, W.H. et al; (1997) Dev. Brain Dysfunct; 10:44-55). Currently, the scientific literature provides overwhelming evidence that incomplete digestion of peptides with a high content of Proline may contribute to the development and severe course of these diseases. In addition to the casein fragment BCM-7 in this regard, reference was also made resistant to protease peptides derived from gluten. Already in 1979 Panksepp (Trends in Neuroscience 1979; 2:174-177) proposed a theory of excess opioids, in which he suggested that the violation of the metabolism of opioids is part of the pathogenesis in autism. Currently, we understand that many of the peptides with a high content of Proline is very resistant to splitting such peptidases of the stomach and pancreas, as pepsin, trypsin chymotrypsin and the like, and that only certain enzymes, such as, for example, present in, among others, on the border of the villi epithelial layer of the gastrointestinal tract, is able to hydrolyze peptide bonds formed by Proline.

Gluten is an insoluble protein fraction crops such as wheat, rye, oats, barley, corn and rice, which remains after washing to remove starch and water-soluble components. Gluten can be subdivided on the solubility of the 4 main fractions, i.e. albumin, globulin, prolamin and glutelin. Among these fractions prolamine and barley glutelin fractions of wheat, corn, barley and oats especially are relatively high content of the amino acids Proline and glutamine. The current data imply that enriched in Proline sequences are a major factor in the development of celiac disease. Celiac disease, also known as gluten enteropathy, is an autoimmune disease of the small intestine caused by the ingestion of gluten proteins. It usually manifests in early childhood with severe symptoms, such as chronic diarrhea and bloating; at a later period of life symptoms include fatigue, weight loss due to malabsorption in the digestive tract and neurological symptoms. Media is enriched in Proline fractions of different cereals most toxic, apparently, are alpha-gliadin in wheat, hordein barley, secalin in rye and Avenyn oats (Schuppan, D.; Gastroenterology 2000; 119:234-242). The only effective treatment for patients with celiac disease is a lifelong diet that excludes gluten. In patients with celiac disease are often observed in various autoimmune disorders, especially type 1 diabetes, dermatitis herpetiformis, autoimmune thyroiditis, disorders associated with collagen autoimmune hair loss and autoimmune hepatitis. This shows that due to unknown mechanisms, untreated celiac disease causes a predisposition to autoimmune diseases of other organs (Schuppan, D.; Gastroenterology 2000; 119:234-242). Moreover, there are indications that a mild form of celiac disease have a group of people suffering from irritable bowel syndrome (IBS). IBS is a disorder that interferes with normal operation of the colon and is characterized by spastic abdominal pain, constipation and diarrhea. IBS usually begins around the age of 20 years and causes a large amount of discomfort and suffering. Eating wheat, barley, rice or dairy products is associated with increased symptoms of IBS.

Recently Shan and employees (Science; vol.297, 27 September 2002:2275-2279) identified a peptide with a high content of Proline, consisting of 33 amino acid residues and which is of proizvodigoods, which is believed to be the source of the set of primary T-cell epitopes specific for coeliac patients. While the extract of enzymes derived from small border cells of the intestinal villi, unable to hydrolyze this 33-dimensional peptide, adding bacterial proletarisation fromFlavobacterium meningosepticumleads to rapid metabolism with a concomitant strong decrease stimulation of the relevant T-cell clones. In imitation of the earlier work on oral introduction of papain (Messer, M. and bell, P.E.; Lancet 1976; 2:1022) article shows the potential proletarisation as a food enzyme in detoxification of gluten enzyme therapy.

Proletarisation (EC 3.4.21.26) have the unique ability preferred cleavage of the peptides from the carboxyl side prolinnova residues. In proletarisation isolated from mammals, as well as in proletarisation isolated fromFlavobacterium meningosepticumwas identified specific peptidase domain, which does not allow large structured proteins to approach the active site of the enzyme. In fact, these enzymes are unable to break down proteins containing more than about 30 amino acid residues, therefore, these enzymes at the present time are called "shedoligopeptides" (Fulop et al Cell, Vol.94, 161-170, July 24, 1998). All known proletarisation are cytosolic enzymes, the optimum pH is near neutral, and they are characterized by the fact that they are unable to effectively break down a molecule containing more than about 30 amino acid residues. The fact that these enzymes optimum pH value, which corresponds to the pH prevailing in the most remote parts of the gastrointestinal tract, making them ideally suitable as food additives, to support the process of digestion of dietary gluten in the gut.

Another enzyme that may be useful for the inactivation of toxic peptides enriched in Proline, is the enzyme dipeptidyl peptidase IV (US2002/0041871A). The dipeptidyl peptidase IV, also called Xaa-Pro-dipeptidylpeptidase (EC 3.4.14.5), catalyzes the separation of the N-terminal dipeptide Xaa-Xbb from peptide with N-terminal sequence Xaa-Xbb-Xcc-if Xbb is preferably Proline, provided that Xcc is not a Proline. The dipeptidyl peptidase IV was selected from a large number of sources mammals, for example, a rich source of this enzyme are border membrane of the intestinal villi. In addition, the enzyme has been isolated from such microbial sources, ka is the food microorganisms Saccharomyces, Lactococcus and Aspergillus. As proletarisation, all known dipeptidylpeptidase IV are enzymes with almost neutral optimum pH and, thus, they are suitable to support digestion in the intestine.

Due to the possible applications of Proline-specific oligopeptides and dipeptidylpeptidase IV in the treatment of celiac disease or schizophrenia, autism, or other affective disorders, were filed several patent applications relating to various aspects of the topic. For example, the U.S. patents 6447772 and WO 01/24816 describe compositions containing dipeptidyl peptidase IV, WO 03/068170 describes compositions containing Proline-specific oligopeptides, optionally combined with dipeptidyl peptidase IV, WO 02/45523 describes low allergenic protein hydrolysates obtained with the use of a Proline-specific endoprotease, and WO 03/028745 describes compositions containing bacterial strains that are able to reduce the concentration toxic to intestinal peptides with a high content of Proline. WO 96/36239 describes the benefits of products derived from cattle, mostly free of alleles of the beta-casein A1.

The invention

The present invention relates to a method of proteolytic hydrolysis of peptides or polypeptides, and the above-mentioned peptides or polypep the IDA contain from 4 to 40, preferably from 5 to 35, amino acid residues, and the above-mentioned peptides or polypeptides do not undergo hydrolysis under the action of subtilisin according to which the above-mentioned peptides or polypeptides are subjected to hydrolysis under the action of Proline-specific endoprotease at a pH of 6.5 or lower, preferably of 5.5 or below, and more preferably 5.0 or lower, with the aim of hydrolysis of the above-mentioned peptides or polypeptides. Preferably, in this method, the hydrolysis is subjected to at least 70%, more preferably not less than 80% and, most preferably, at least 90% of the peptide or polypeptide.

In accordance with another embodiment, the method according to the present invention relates to methods of proteolytic hydrolysis of peptides or polypeptides, and these peptides or polypeptides contain from 4 to 40, preferably from 5 to 35 amino acid residues and include Tripeptide fragment Glu-Xxx-Pro, Gln-Xxx-Pro, Tyr-Pro-Phe or Tyr-Pro-Trp, according to which the above-mentioned peptides or polypeptides are subjected to hydrolysis under the action of Proline-specific endoprotease at a pH of 6.5 or lower, preferably of 5.5 or below, and more preferably 5.0 or lower, with the aim hydrolysis of the above-mentioned peptides or polypeptides. Preferably, in this method, the hydrolysis is subjected to at least 70%, more preferably at least 80% and most of predpochtitel is not less than 90% of the peptide or polypeptide.

In addition, in the present invention, a method of proteolytic hydrolysis of peptides or polypeptides, and these peptides or polypeptides contain from 4 to 40, preferably from 5 to 35 amino acid residues and amino acid residues of the peptides or polypeptides include at least 30%, preferably at least 40%, of the residues of Proline and/or glutamine, according to which the above-mentioned peptides or polypeptides are subjected to hydrolysis under the action of Proline-specific endoprotease at a pH of 6.5 or lower, preferably of 5.5 or below, and more preferably 5.0 or lower, with the aim of hydrolysis of the above-mentioned peptides or polypeptides, provided that peptides or polypeptides contain at least 10% Proline residues. Preferably, in this method, the hydrolysis is subjected to at least 70%, more preferably at least 80% and most preferably at least 90% of the peptide or polypeptide.

Peptides or polypeptides in the method according to the present invention preferably include a fragment of Gln-Xxx-Pro or Glu-Xxx-Pro and contain 9 or more amino acid residues. These peptides or polypeptides mainly hydrolyzed to peptides containing 8 or less amino acid residues. If the peptides or polypeptides include a fragment Tyr-Pro-Phe or Tyr-Pro-Trp, preferably undergoes hydrolysis link between Pro and Phe or Pro and Trp.

Before Occitania indopoly-specific endoprotease, used in the method according to the present invention, preferably is a Proline-specific endoprotease derived fromAspergillusor belonging to the family of semiprotect S28. This enzyme preferably has an optimum pH values below 6.5, preferably below 5.5, more preferably lower than 5.0, for hydrolysis of peptides or polypeptides comprising from 4 to 40, preferably from 5 to 35 amino acid residues, which do not undergo hydrolysis under the action of subtilisin.

Proline-specific endoprotease can be used at pH values below 5.5 for hydrolysis of peptides with a high content of Proline, which are relevant to psychiatric disorders, including autism, schizophrenia, ADHD, bipolar affective disorder and depression, as well as disorders associated with celiac disease, such as autoimmune disorders, especially type 1 diabetes, dermatitis herpetiformis, autoimmune thyroiditis, a disease associated with collagen autoimmune hair loss, as well as autoimmune hepatitis and IBS.

This enzyme is mainly used for production of food products such as beer or bread, which do not contain associated with celiac disease epitopes, preferably gluten epitopes, more preferably, the epitopes of wheat or barley.

The present invention also about what is worn to the use of a Proline-specific endoprotease, which, preferably, is an enzymeAspergillusmore preferablyAspergillus nigeras a medicament or for use in the production of pharmaceuticals.

In accordance with another embodiment of the present invention, the Proline-specific endoprotease used for the production of food additives or medicines for the treatment or prevention of psychiatric disorders, including autism, schizophrenia, ADHD, bipolar affective disorder and depression, as well as disorders associated with celiac disease, such as autoimmune disorders, especially type 1 diabetes, dermatitis herpetiformis, autoimmune thyroiditis, a disease associated with collagen autoimmune hair loss, as well as autoimmune hepatitis and IBS.

Proline-specific endoprotease is mainly used for production of food additives or medicines for individuals under 25 years of age.

In accordance with the present invention Proline-specific endoprotease is also used to produce food additives or medicines intended for the treatment or prevention of psychiatric disorders, including autism, schizophrenia, ADHD, bipolar affective disorder and depression, as well as disorders associated with celiachia is, as, for example, autoimmune disorders, especially type 1 diabetes, dermatitis herpetiformis, autoimmune thyroiditis, a disease associated with collagen autoimmune hair loss, as well as autoimmune hepatitis and IBS.

In addition, the present invention relates to the use of a Proline-specific endoprotease for hydrolysis of proteins or peptides, including more than 30 amino acid residues. For the purposes of the present invention, preferably used Proline-specific endoprotease Aspergillus, more preferablyA. niger.

The present invention also relates to the use of a Proline-specific protease which is active at pH 5 or below 5 in the presence of pepsin.

The present invention mainly relates to a method of proteolytic hydrolysis of these peptides or proteins present in the number of dairy proteins, obtained from cattle carrying alleles of the beta-casein A1 or beta-casein A2.

In addition, in the method according to the present invention can be used proteins. First, the protein must be hydrolyzed to peptides comprising from 4 to 40 amino acid residues. The protein hydrolysis must be carried out prior to the hydrolysis process of the present invention, or simultaneously with it.

In addition, the present invention relates to skin is of a Proline-specific endoprotease, having the optimum pH value below 6.5, preferably below 5.5, more preferably lower than 5.0, as a food additive, as a medicine, as a food additive for the production of medicines or for the production of feed, including pet food intended for animals, preferably mammals, non-human.

Detailed description of the invention

The purpose of the previous prior art, in the main, was either in the removal of food toxic peptides with a high content of Proline to its use, either in oral introduction of corrective enzymes to compensate for the improper digestion in the intestine. It is understood that in the normal process of gastro-intestinal digestion, the first stage of the breakdown of dietary proteins is proteolysis in the stomach under the action of the enzyme pepsin. The second stage is carried out in the small intestine, i.e. the duodenum and jejunum, located directly behind the stomach. In the duodenum there is an increase in the acid pH of the stomach contents by adding juices of the pancreas, containing also a large number of endo - and carboxypeptidase. Catalyzed by these enzymes further splitting destroyed pepsin is m food proteins is carried out in the cavity of the duodenum and the jejunum at pH values of more than 5. Before transport across the intestinal wall is the third stage, which includes the further hydrolysis of the peptides in the boundary surface membrane of the villi of the intestinal epithelium. This stage is carried out by a number of proteases, including DPP IV, which are localized in the membranes of these epithelial cells. In the previous prior art, it was assumed that in the intestine of individuals suffering from certain diseases that are targeted by the present invention, some of these epithelial enzymes and, in particular, DPP IV are partially inactive or even absent. In patients with celiac disease the levels of these epithelial enzymes are probably normal, but this group of individuals even small amounts of certain peptides with a high content of Proline can cause severe inflammatory T-cell response. Available in the prior art solution was to minimize the levels of toxic peptides with a high content of Proline present in the cavity of the intestine, into the cavity of the human intestine was introduced corrective enzymes intended for oral administration. In the framework of this approach, in the prior art were selected enzymes, as it is possible more close to the natural enzymes of man, i.e. enzymes that act is wny in the intestine at a pH greater than 5. These enzymes known prior art is inactive in acidic medium, or dosage forms for oral administration containing these enzymes prior art, provided with the appropriate coating to prevent their activity in an acidic environment.

The authors of the present invention have found that the enzymes that develop the main activity in the acidic environment of the stomach, offer the best solution to the problem of inefficient cleavage of peptides with a high content of Proline. This approach is novel and opens the possibility of applying to resolve this issue, other enzymes, besides known in the literature.

As discussed above, several publications were focused on the possibility of applying for the prevention of disturbed metabolism of opioids such enzymes as dipeptidyl peptidase IV and proletarisation. For selected enzymes of dipeptidyl peptidase IV and proletarisation, which, as a rule, active at pH values close to neutral. The meaning of this is that these enzymes will effectively hydrolyze enriched in Proline proteins only at pH values above 5. Taking into account the pH-profile of the gastrointestinal tract of humans, these enzymes known prior art does not become active until reaching the distal part of dwenadzatiperstnuu the big intestine, i.e. far beyond the stomach. However, the distal part of the duodenum is the main site of absorption of proteins and, as is also known, it faces defeat in patients with celiac disease. Thus, in fact, enzymes prior art will begin to work where already caused some damage.

In addition, the enzymes of the prior art become active only after considerable preliminary destruction of proteins with a high content of Proline. The time required to achieve this preliminary destruction under the action of pancreatic enzymes, further limits the time period that has used oral enzymes for complete hydrolysis of sequences with a high content of Proline. In accordance with the present invention is applied, the enzyme is able to cleave the majority of protein sequences with a high content of Proline and/or glutamine before food enters the small intestine. Ordinary residence time of food in the stomach provides the a period of time necessary for sufficient hydrolysis of proteins with a high content of Proline or glutamine. Moreover, in accordance with the present invention preferably applies an enzyme able to cleave the peptides from the carboxyl side of raynovich residues and, in addition, is able to cleave the intact proteins with carboxyl side prolinnova residues, even in the presence of pepsin. Enzymes known prior art will be hydrolyzed at low pH of the stomach and under the action of the proteolytic enzyme pepsin, which is secreted in the stomach. However, up to now within the framework of this method is unknown, the application of such enzymes, resistant to acid/pepsin. The approach applied in the present invention, will lead to a significant degradation of peptides, polypeptides and proteins with a high content of Proline before reaching the intestine, instead of the approach of the prior art, which serves to mimic the natural process of hydrolysis of peptides with a high content of Proline in the gut. The preferred enzyme used in the method according to the present invention, is an enzyme that is active at pH values below 5 in the presence of pepsin, and which is able to break down food proteins or polypeptides, and peptides. Solutions of the known prior art, in all cases require the coenzyme that carries out a preliminary hydrolysis of food protein to peptides, and only after this additional Proline-specific enzymes can begin the manifestation of its activity.

The terms "peptide" Il is "Oligopeptide" in the framework of this application is defined as a chain, containing from two to thirty amino acid residues, connected by peptide bonds. The terms "peptide" and "Oligopeptide" are considered synonyms (which corresponds to a common interpretation), and each of these terms can be used instead of another, depending on the context. Under this proposal, a "polypeptide" is defined as a circuit that contains more than 30 amino acid residues.

Assume that the peptides or polypeptides comprising from four to forty amino acid residues, which do not undergo hydrolysis under the action of subtilisin (EC 3.4.21.62), preferably subtilisin Carlsberg, are peptides or polypeptides that remain intact after incubation for 2 hours at pH 8.0 and 60°C in suspension or solution containing 20 g/l protein, and the ratio of enzyme to substrate is 0.12 AU-A (Alkalizing units according to Anson) protease units per gram protein. AU-proteina unit determined in accordance with the specified in the Analytical Method LUNA #2003-32153-01 issued by Novozymes (Denmark). Intact means that after incubation of the original peptide or polypeptide forms more than 80%, preferably more than 90%, more preferably more than 95% of the final products of the enzyme reaction. Almost enzyme cleavage perform under specified conditions, using 40 microlitres alcalase per gram available Belko is on the substrate. An example of such is not subject to hydrolysis of the peptide is a peptide VYPFPGPIPN formed during the hydrolysis of beta-casein, which is described in example 4. Another example of such, is not subject to hydrolysis of the polypeptide, is a 33-dimensional product described in example 6.

Oligopeptides is an enzyme classified as EC 3.4.21.26 and belongs to the family of semiprotect clan SC, family S9.

All formulas or sequences (oligo)peptides and polypeptides recorded in this application from left to right in the direction from amino end to the carboxy-end, in accordance with conventional practice. Letter codes of the amino acids used in the application are well known in the art and can be found in Sambrook, et al. (Molecular Cloning: A Laborotary Manual, 2nded. Cold Spring Harbor Laborotary, Cold Spring Harbor Laborotary Press, Cold Spring Harbor, NY, 1989). It is understood that the amino acids Xxx, Xaa, Xbb or Xcc can be any amino acid. The term "fragment" refers to an amino acid sequence that is part of the peptide, polypeptide or protein. In this application we have in mind that glutamine (Q or Gln is glutamine or glutamate (E or Glu). For example, in chemical detalizirovannoi gluten significant portion of the available natural glutamic residues are transformed into a form of glutamate. Therefore, the fragment Gln-Xxx-Pro also includes frag the UNT Glu-Xxx-Pro. In addition, other peptides, polypeptides or proteins glutamine can be converted into glutamate, for example, due to the pH of the medium, temperature, or using an enzymatic reaction, for example, under the action of transglutaminase.

Under the peptide or polypeptide with a high content of Proline (enriched in Proline) refers to a peptide or polypeptide containing from 4 to 40 amino acid residues and amino acid residues of the peptide or polypeptide include at least 30%, preferably not less than 40%, prolinnova and/or glutamic residues, provided that the peptide or polypeptide contains at least 10% prolinnova residues. "Food additive", adapted according to the DSHEA definition, is a product (non-tobacco)that is intended to Supplement the diet that bears or contains one or more of the following ingredients in the diet: proteins, including enzymes, polypeptides, peptides, vitamins, minerals, herbs or other botanicals, amino acids, nutrients, intended for use by man to Supplement the diet by increasing the total intake, or a concentrate, metabolite, constituent part of the extract or combination of these ingredients.

In addition, dietary supplements:

- are designed to receive ADAP is R in pills, capsules, tablets or in liquid form;

- not intended for use as a conventional food or as the sole food intake or diet;

mostly are marked "food additive";

- include products such as an approved new drug, certified antibiotic, or licensed biologic products that are available in the market as food supplements or food before approval, certification, or license (unless the Secretary of health and human services has not denied such approval).

Toxic peptides or polypeptides with a high content of Proline are peptides or polypeptides involved in the binding with opioid receptors or in the development or increase the severity of psychiatric disorder or disease.

Psychiatric disorders include autism, schizophrenia, ADHD, bipolar disorder, and depression.

Disorders associated with celiac disease, are autoimmune disorders, especially type 1 diabetes, dermatitis herpetiformis, autoimmune thyroiditis, a disease associated with collagen autoimmune hair loss and autoimmune hepatitis.

Internationally recognized classification scheme and nomenclature of all enzymes, approved IUMB, includes FR the basics. Regularly updated text IUMB numbers EC proteases can be found on the Internet site. In this system the enzymes defined by the fact that they catalyze only one reaction. The system divides protease on endo - and ectoprocta. To endoproteases include those enzymes that carry out the hydrolysis of internal peptide bonds, ectoprocta hydrolyzing the peptide bond adjacent to the terminal α-amino group (amino peptidases) or peptide bond between the terminal carboxyl group and the penultimate amino acid ("carboxypeptidase"). Endoprotease subdivided into sub-subclasses on the basis of catalytic mechanism. There are sub-subclasses selinantis (EC 3.4.21), continentales (EC 3.4.22), aspartic of endoprotease (EC 3.4.23), metalloendopeptidases (EC 3.4.24) and trainingoperations (EC 3.4.25).

In WO 02/45524 described Proline-specific endoprotease derived fromAspergillus niger. The authors of the present invention unexpectedly found that these enzymesAspergillussuccessfully applied in the method according to the present invention under conditions of acidic environment in the stomach and can hydrolyze under these conditions, dietary intact proteins, polypeptides, as well as smaller peptide molecules. In addition, this enzyme is not destroyed in the presence of the enzyme pepsin in acidic environment and will likely continue the t its activities throughout the duodenum. The authors showed that the Proline-specific endoprotease isolated from A. niger, completely different from the known Proline-specific proteases, as well as from glutens described in WO 03/068170, from the point of view of activity, but also from an evolutionary point of view. The last feature is sufficiently shown by the fact that the homology of amino acid sequences between glutamate described in WO 03/068170, and the enzyme isolated from A. niger is generally less than 20% when using the General algorithm of matching sequences. This result is consistent with the modern view that proletarisation not found in these fungi, asAspergillus nigerof which in accordance with the present invention the selected Proline-specific endoprotease (Venäläinen, J.I. et al, Eur J Biochem 271, 2705-2715 (2004)).

Under the Proline-specific endoprotease of the present invention or used according to the present invention assumes, for example, the polypeptide referred to in claims 1 to 5, 11 and 13 of the claims of the application WO 02/45524, which is incorporated into the present application by reference. Hence, this Proline-specific endoprotease is a polypeptide that possesses a Proline-specific endoproteolytically activity selected from the group consisting of:

(a) a polypeptide having the amino acid is based sequence, which at least 40% identical to the amino acid sequence comprising amino acids 1 to 526 of SEQ ID NO:2, or its fragment;

(b) a polypeptide encoded by polynucleotides that hybridizes in conditions of low stringency (i) the sequence of the nucleic acid SEQ ID NO:1 or a fragment that is identical to at least 80% or 90% on a plot of over 60, preferably over 100 nucleotides, more preferably, identical, at least 90% on a plot of more than 200 nucleotides, or (ii) the sequence of nucleic acids complementary to the nucleic acid sequence of SEQ ID NO:1. Sequence SEQ ID NO:1 and SEQ ID NO:2 corresponds to those shown in WO 02/45524. The polypeptide preferably is present in an isolated form.

Preferred polypeptide has an amino acid sequence that is at least 50%, preferably not less than 60%, preferably at least 65%, preferably not less than 70%, more preferably not less than 80%, even more preferably at least 90%, most preferably at least 95% and even more preferably, at least about 97% identical to amino acids 1 to 526 of SEQ ID NO:2 or containing amino acid sequence of SEQ ID NO:2.

The polypeptide is preferably encoded polynucleate the Ohm, which hybridizes in conditions of low stringency, more preferably under conditions of medium hardness, and most preferably in stringent conditions with (i) the sequence of the nucleic acid SEQ ID NO:1 or fragment, or (ii) the sequence of nucleic acids that is complementary to the nucleic acid sequence of SEQ ID NO:1.

The term "capable of gibridizatsiya" means that the target polynucleotide according to the present invention can gibridizatsiya with nucleic acid used as a probe (for example, with the nucleotide sequence shown in SEQ ID NO:1 or a fragment or complement SEQ ID NO:1) at a level significantly above background. The invention also includes polynucleotides that encode a Proline-specific endoprotease of the present invention, as well as the nucleotide sequence, it is complementary. The nucleotide sequence may be a RNA or DNA, including genomic DNA, synthetic DNA or cDNA. Preferably, the nucleotide sequence is a DNA and, most preferably, the sequence of genomic DNA. As a rule, polynucleotide of the present invention contains a contiguous sequence of nucleotides which is capable of selectively gibridizatsiya with coding consistently is part of or complement coding sequence of SEQ ID NO:1. Such nucleotides can be synthesized well-known in the art methods.

Polynucleotide according to the present invention can gibridizatsiya with the coding sequence or complement coding sequence of SEQ ID NO:1 at a level significantly above background. Background hybridization may take place, for example, due to the fact that the other cDNA represented in the cDNA library. The signal level generated by the interaction between polynucleotides of the present invention and the coding sequence or complement coding sequence of SEQ ID NO:1, typically at least 10 times, preferably not less than 20 times, more preferably not less than 50 times, and still more preferably not less than 100 times the intensity level of interaction between other polynucleotides and the coding sequence of SEQ ID NO:1. The intensity of interaction may be measured, for example, by adding the probe radioactive label, such as32P. Selective hybridization, as a rule, can be achieved using conditions of low stringency (0.3 M sodium chloride and 0.03 M sodium citrate at a temperature of about 40°C), conditions of medium hardness (for example, 0.3 M sodium chloride and 0.03 M sodium citrate at a temperature of about 50°C) and harsh conditions (e.g., 0.3 M sodium chloride and 03 M sodium citrate at a temperature of about 60°C).

The UWGCG package provides the BESTFIT program which can be used to calculate identity (for example, using its default settings).

Algorithms PILEUP and BLAST N can also be used to calculate the identity of the sequence or to compare sequences (as, for example, to identify equivalent or similar sequences, for example, if the default settings).

Software for performing BLAST analysis available to everyone through the national Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/). This algorithm includes, firstly, the identification of pairs of sequences with a high degree of conformity (HSPs) by identifying short sets of characters of length W in the requested sequence, which either match or satisfy some positive threshold value T when compared with the set of characters of the same length in a database sequence. T is called the threshold of the next set of characters. These initial matches ones of the sets of symbols act as a starting point for initiating a search to detect HSPs containing them. Matching character sets will be extended in both directions along each sequence up until can be increased if the total is estvo matches. The extension in each direction to match the character set shall be terminated, if: the total number of matches is reduced by the number of X compared to the maximum achieved value; the total score of the match goes to zero or below, due to the accumulation of one or more groups of residues that contribute to a negative account; or if at the end of any of the sequences. The BLAST algorithm parameters W,T, and X determine the sensitivity and speed of verification. In the program BLAST, the default length character set (W), is equal to 11, the matrix calculation matches BLOSUM62 (B)of 50, expectation (E)of 10, M=5, N=4, and the comparison of both chains.

The BLAST algorithm performs a statistical analysis of the similarity between the two sequences. One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), showing the probability of a match between two sequences of nucleotides or amino acids may appear randomly. For example, it is assumed that the sequence is similar to another sequence if the smallest sum probability in a comparison of the first sequence with the second sequence is less than about 1, preferably less than about 0.1 and more preferably less than about 0.01, and on the Sabbath. more preferably, less than approximately 0.001 in.

The enzyme of the present invention is preferably derived from fungi, preferablyAspergillusmore preferably ofAspergillus niger. Thus, it was found that the enzyme isolated fromAspergillusreally is endoproteases, stable to the action of acids, and preferably not exposed to pepsin at prevailing in the stomach pH values. Proline-specific oligopeptides, mainly involving the enzymes, which can be obtained fromFlavobacterium meningosepticumthat is hardly active at pH values equal to or less than 5, and are inactivated by a combination of low pH and the presence of the enzyme pepsin. In addition, the Proline-specific protease, as a rule, is not able to destroy the intact proteins, and found that they effectively hydrolyzing only peptides smaller, i.e. peptides with a length up to approximately 30 amino acid residues.

In the present invention developed an economical, suitable for use in food compositions intended to delay or minimize effects associated with toxic peptides with a high content of Proline or impaired metabolism of opioids. These compositions include oral enzyme compositions suitable for food, pharmaceutical and veterinary use, and t is the train enzyme formulations designed to obtain hydrolysates of proteins and food products with significantly reduced levels of opioids or toxic peptides with a high content of Proline.

In the present invention discloses methods of hydrolysis of peptides or polypeptides with a high content of Proline, which are associated with the development of psychiatric disorders or celiac disease, or impaired metabolism of opioids. The present invention also discloses methods of obtaining food, which can prevent or slow the development of such disorders in younger children or, in General, people under the age of 25 years. Such methods may also be obtained from foods, which are better tolerated by people suffering from celiac disease and abdominal symptoms associated with IBS. Variant implementation of the present invention is associated with the cleavage of these peptides or polypeptides with a high content of Proline before use, which prevents or minimizes the effects of toxic peptides with a high content of Proline in the body. In younger children this will allow to avoid the effects of opioids, and immature immune system will not gain increased sensitivity due to exposure to such toxic peptides with a high content of Proline. In addition, the diet containing gennoe the amount of toxic peptides with a high content of Proline, will be useful from a preventive point of view for teenagers and adults, for example, for people suffering from not detected celiac disease or IBS. The present invention also relates to the addition of the appropriate enzymes to break down these toxic peptides with a high content of Proline in the body (human or animal) or peptides in the stomach, i.e. at pH values corresponding to the acidic medium, preferably at pH values below 5. In accordance with the latter embodiment of the invention people suffering from celiac disease, diseases associated with celiac disease, or diseases caused by low levels of Proline-specific proteases in the body, and require the cleavage of these peptides or polypeptides, acquire the ability to split the corresponding peptides or polypeptides with a high content of Proline in the stomach and proximal part of the duodenum. In addition, the enzymes of the present invention do not require protective coatings.

Preferably, at least 80% of the peptides or polypeptides with a high content of Proline, which are formed during the incubation of the peptides, polypeptides or proteins with subtilisin (EC 3.4.21.62), preferably a subtilisinBacillus licheniformis(or subtilisin Carlsberg) in a medium with neutral pH value, hydrolyzed Proline-specific andapr what aazami of the present invention. The formation of such resistant subtilisin peptides is illustrated by example 4 of this application. Such resistant subtilisin peptides with a high content of Proline is often associated with the above diseases. Examples of such peptides are BCM-7 and related BCM-7 peptides, i.e. peptides containing the amino acid sequence of YPFP, which occupies the position with 60 63 in the molecule of beta-casein. In addition, examples of such peptides are peptides derived from gliadin, which contain the fragment Gln-Xxx-Pro (Q-X-P), for example, the epitope PYPQPQLPY, as well as other resistant subtilisin molecules that can be derived from gliadin, hordein, secalin or avenin, and containing the same fragment Q-X-P or E-X-P. More preferably, at least 90%, even more preferably at least 95% and most preferably at least 99% of peptides with a high content Proline, which are formed during the hydrolysis under the action of subtilisin, split or not formed when using the Proline-specific endoprotease of the present invention. More preferably, these peptides with a high content of Proline can be destroyed by the method of the present invention at pH values below 5.5.

One of the ways to obtain the required enzymes could be used in the form of funds for promotion of food is the jam, for example, in the form of a stabilized enzyme compositions which are taken together with food, to help gastrointestinal digestion of food peptides or polypeptides with a high content of Proline. Another way could be to prevent or limit the receipt problematic toxic sequences with a high content of Proline, for example, by use of protein food, "pre-digested" by the enzymeAspergillus. Such protein food would be consumed in the form of a hydrolysate, for example, hydrolyzed gluten or dairy protein, and include hydrolysates, proteins which largely destroyed by the action of endo - and ecoprocess for the release of significant quantities of free amino acids. A typical example of the latter application could be getting gluten hydrolysates with a high content of glutamate to produce food seasonings. The hydrolysate could be used by itself, or could serve as a food ingredient. In another aspect the invention relates to the use of a composition for improving the portability of food, which is characterised in that the composition contains resistant to acids Proline-specific endoprotease of the present invention. In another aspect the invention relates to a method of gaining the food, containing additives resistant to acids Proline-specific endoprotease, to improve portability food. In all such applications, the Proline-specific enzyme used as so-called technological additives.

Strains of the genusAspergillushave a status suitable for human consumption, and it is known that enzymes isolated from these microorganisms obtained from a source that is certainly suitable for human consumption. In accordance with another preferred embodiment of the invention, the enzyme is secreted producing his cell, and is not secretively, the so-called cytosolic or periplasmatic enzyme. Thus, enzymes can be re-obtained from the cell broth mostly in a pure state, without costly stages of purification. The enzyme preferably has a high affinity to its substrate at common values of pH and temperature conditions. Preferably, the enzyme does not undergo inactivation such gastro-intestinal proteolytic enzymes like pepsin or trypsin, elastase and chymotrypsin at pH values typical for the corresponding part of the gastrointestinal tract. More preferably, the enzyme remains active in the stomach and in the duodenum and does not require protective the surface.

Human digestive tract is a series of different departments. Food eaten and after swallowing reaches the stomach where it is mixed with acid and endoproteases pepsin. The usual time of solid food in the stomach is in the range from one to several hours. Taking place from time to time the opening of the pylorus is preserved allows food which is mixed with acid and partially hydrolysed, to enter into the small intestine. In the first part of the small intestine, i.e. in the duodenum to food is added to the bile and the pancreatic juice. The pancreatic juice contains bicarbonate, for partial neutralization of gastric contents. In addition, the pancreatic juice contains an additional set of proteases, i.e. endoprotease trypsin, chymotrypsin and elastase, and carboxypeptidase A and B for further cleavage of peptides and polypeptides formed in the stomach under the action of pepsin. After the duodenum to digest the food reaches the jejunum. Duodenum and jejunum are the primary places digestion of proteins in the gastrointestinal tract. The process of assimilation involves further proteolytic breakdown of food protein by different protease, attached to the edge cells of the villi of the intestinal epithelium. Poslednyaya of these stages of hydrolysis is accompanied by facilitating transport of small peptides, as well as free amino acids across the intestinal wall. The final part of the small intestine is formed ileum, after which the digested food enters the large intestine (the colon). In the colon is the site of intense fermentation, but there is no appreciable absorption of amino acids or peptides.

Eating gluten in patients with celiac disease, leads to lesions of the proximal region of the small intestine. Atrophy of the intestinal villi is one of the most characteristic features of such lesions. Atrophy of the villi is not limited to the jejunum, but can also occur in the distal part of the duodenum (Meijer, J.W.R. et al, Virchows Arch. 2003 Feb; 442:124 to 128). Observations show that patients with celiac disease the damaging effects of toxic peptides with a high content of Proline significantly already in the area, located directly behind the stomach in the direction of food. The inventors have found that enzymatic therapy, the goal of which is to prevent the symptoms of celiac disease, should be directed to the hydrolysis of the corresponding peptides with a high content of Proline in the stomach and not in the more remote parts of the gastrointestinal tract. The activity of the enzyme used in the enzyme treatment, mainly is that he begins to work in the stomach and continues in order to stavitsa active in the duodenum. The inventors have found that opioid peptides, which play a role in the development of psychiatric, respiratory and cardiovascular disorders, as it is better to destroy in the stomach. Because the duodenum and jejunum are the primary places assimilation of proteins, the content of toxic peptides in this part of the small intestine should be as low as possible to minimize symptoms associated with the presence of these toxic peptides.

When used as a means to promote digestion, corrective enzymes must have significant activity at 37°C and, preferably, should have a low optimum pH value not to be destroyed in the acidic conditions of the stomach. In accordance with published data acidity received into the stomach food is reduced from the original value of pH 5 to pH 3.5 within thirty minutes after a meal, with subsequent further decrease to pH 2 within 60 minutes after administration (abstracts Mans Minkus; Uiversity of Utrecht, the Netherlands; ISBN: 90-393-1666-X). Therefore, the ideal enzymes intended for enzyme therapy should be active at such a low pH value as 2. Studies on gastric emptying show that 45 minutes after admission, almost 90% of solid food is still the same is the RCU (R.Notivol et al., 1984. Scand J.Gastroenterol. 19:1107-1113). After the stomach pH to digest food slowly increasing, reaching pH 5 in the distal part of the duodenum, i.e. approximately 50 cm after the stomach (Handbook of discrimination, American Phisiologcal Society, Washington, D.C., 1968, Ed. Werner very reason; Section 6: Alimentary Canal, Vol.111, pp.1457-1490). In WO 03/068170 described various "glutenase"that can reduce the levels of toxic gluten oligopeptides in food either before or after ingestion by the patient. The term "glutenase" refers to a protease or peptidase, more specifically prolyl-specific protease that is able to decompose toxic gluten oligopeptides proteins on non-toxic fragments. Since all of the mentioned glutenase, usually designed for the activity in the intestine, they have optimal activity at almost neutral pH values. In WO 03/068170 proposed decomposition of toxic peptides with a high content of Proline in the duodenum or her, and not in the stomach or the proximal part of the duodenum. In addition, WO 03/068170 aimed at producing enzymes, protected by so-called enteric coating, which does not make itself enzyme activity at low pH values, so that the available enzymes will pass through the stomach and become active only in the intestine in a neutral environment.

When you use the attachment as processing AIDS in the production of protein hydrolysates, the enzyme should demonstrate significant activity under conditions which permit the incubation, safe from the point of view of the presence of microbes in non-sterile conditions of industrial production. Adequate enzymatic activity when the treatment temperature of 50°C and pH values significantly below 5.5 satisfies these requirements.

It was shown that the basidiomycetesAgaricus bisporus(Sattar et al, J.Biochem. 107, 256-261 (1990)), and not related records of AscomycetesAspergillus niger(WO 02/45524) produce extracellular polyangiitis. However, the enzyme isolated from basidiomycetes, not withstand pH values below 5 and, therefore, is less attractive. Preferably used polyangiitis A. niger, which has an optimum pH in the acid region.

In the present invention developed enzyme preparations that combine low cost and acceptability from the point of view of legislation with proven effectiveness against peptide sequences with a high content of Proline in the acidic conditions of the environment. Preferably, the same enzyme can be used for the destruction not only of beta-casomorphins types A1 and A2, but also a variety of gluten epitopes.

It was found that these Proline-specific endoprotease isolated fromAspergillus, have shown considerable activity in RA is splenii peptides polypeptides or even proteins with a high content of Proline. This enzyme is mainly secreted generating it a microorganism in a fermentation broth that has an optimum pH in an acidic region and can be obtained suitable for human consumption economical way. The corresponding peptides of beta-casomorphin contain up to four Proline residues in the molecule and, in addition, beta-casomorphine A1 and A2 have different amino acid sequences. Unexpectedly, the authors found that the enzymeAspergillusable to hydrolyze beta-casomorphine with C-terminal side prolinnova residue at position 61 and, thus, effectively inactivate BCM-7 and related BCM-7 peptides as for beta-casein A1 and A2. This fact is quite remarkable, since the authors found that such a widely used and highly aggressive endoprotease, having specificity to a large range of substrates, as subtilisin (EC 3.4.21.64), commercially available as, for example, Alcalase, not able to destroy BCM-7. Similarly, other industrial available protease is not able to cleave BCM-7. In fact, isolated fromAspergillusProline-specific endoprotease can hydrolyze beta-casomorphin, but that completely unexpectedly, only one of the four prolinnova residues, have already in BCM-7. However, due to the specificity of this particular enzymeAspergillusto this particular residue is Proline, BCM-7, as well as all related BCM-7 molecules are effectively destroyed by incubation with the enzyme as the enzyme cleaves the fragment Tyr-Pro-Phe. In addition, the molecules of BCM-7, derived from beta-casein A1 and A2, are inactivated upon incubation with the Proline-specific endoproteaseAspergillus.

The advantage of this Proline-specific endoprotease is that the Proline-specific endoprotease can start the hydrolysis of sequences with a high content of Proline immediately upon coming in contact of the enzyme with protein. Proletarisation can become active only after significant pre-splitting molecules of gluten or casein under the action of the other, for example, gastrointestinal endoprotease. Due to the need of deep decay and a limited amount of time before getting food into the small intestine, the application of this Proline-specific endoprotease has significant advantages over known Oligopeptide. Another advantage of this Proline-specific endoprotease is that it can be used in industry to reduce the levels of toxic peptides with a high content of Proline in the gluten without full p is zrusenie structure of gluten. For example, the extrusion gluten wheat pasta with allocated fromAspergillusthe enzyme will lead to a product with some residual structural properties, but with greatly reduced levels of toxic peptides with a high content of Proline. To achieve the same reduction in toxic enriched in Proline peptides using proletarisation would require almost complete preliminary hydrolysis of other proteases, in order to achieve the desired length of the peptides of less than 30 amino acid residues. Needless to say, this will result in a complete loss of all relevant physico-chemical properties of gluten, i.e. the loss of consistency of the test and won't be able to bake bread acceptable form and volume. Di Cango and co-authors (Appl. Environ. Environ., Vol.70(2)1088-1096, 2004) describe the making of bread from sourdough, which is well tolerated by patients with gluten enteropathy. In their method, the level of toxic epitopes with a high content of Proline minimized through the use of the test, made with a significant part of non-toxic flour and fermentation mixture with selected lactic acid bacteria within 24 hours. Although their results, of course, are of great scientific interest, it is obvious that such a long period of fermentation medium as 24 hours, is not feasible in PR the industry. In addition, they used a significant part of non-toxic flour implies that there will be a lack of "normal" gluten, making the resulting bread will be limited. Therefore, it would be advantageous to use a low cost method, which makes possible the manufacture of 100% wheat bread using existing industrial techniques, but with limited levels of toxic peptides with a high content of Proline. These kinds of bread can be an important component of diets aimed at reducing the daily arrivals of toxic peptides. The consumer group for which this diet may be particularly suitable include younger children, adolescents suffering from IBS, the elderly and individuals suffering from the above diseases and symptoms. Although for patients with celiac disease daily intake of gluten less than 50 milligrams considered safe, any diet that includes significantly less toxic peptides with a high content of Proline than 13 g of gluten per day on average takes place in Western countries is probably useful for the above-mentioned groups of consumers.

All these considerations taken together, show new and significant advantages of enzymes isolated fromA. nigerabove enzymes, known from ur is VNA technology. The compositions containing the enzymes of the present invention, have been successfully used to reduce or slow the increase of sensitivity to gluten, the phenomena of disturbed metabolism of opioids or phenomena IBS. Such compositions can be used as a means to facilitate the digestion in order to achieve in situ reduction of the levels of toxic peptides with a high content of Proline in the gastrointestinal tract. On the other hand, such compositions can be used as additives to obtain hydrolysates of proteins that do not contain the mentioned toxic peptides enriched in Proline. Among other protein hydrolysates special case is the manufacture of beer, in which the use is obtained fromA. nigerProline-specific endoprotease low optimum pH offers an unexpectedly large number of advantages. According to WO 02/046381 application of Proline-specific endoprotease or during the stage of preparation of wort, or before filtration of the beer, either before keeping beer, reduces the amount of turbidity in beer. Available from the authors present invention, the data show that conducted accordingly incubation with the enzyme having a low optimum pH also reduces the level of toxic peptides with a high content of Proline produced from crops. Suddenly m is found, the processing of beer or other drinks containing cereal proteins, Proline-specific endoprotease not only leads to a decrease in the number of Muti, but in addition allows you to get the products that will be safe for people suffering from celiac disease.

Example 1 of this application shows acidic optimum pH and the perfect temperature optimum Proline-specific protease derived fromAspergillus. In examples 2 and 3 the authors showed that the Proline-specific endoprotease produced byAspergillus nigerthat really is a Proline-specific endoprotease, which can split large intact proteins as effectively as smaller peptides or polypeptides. In fact, the data of the inventors showed that the enzymeAspergillusis a new member of the family S28, not family S9, to which belong the famous oligopeptides (N.D. Rawlings and A.J.Barret, Biochimica & Biophisica Acta 1298(1996)1-3). The authors found that in contrast to the well-known oligopeptides, PropylenediamineAspergillus nigeractive in the acidic conditions in the stomach and shows a high effectiveness against hydrolysis large fragments and polypeptides with a high content of Proline. This high efficiency is illustrated in examples 4, 5 and 6. In example 4, the authors showed that in the process of obtaining hydroly is the ATA milk protein with alcalase, i.e. aggressive protease of broad-spectrum, often used for obtaining protein hydrolysates, several peptides including sequences of BCM-7 has maintained its integrity as a result of hydrolysis. However, these peptides quickly disappeared when incubation in acidic medium with Propylenediamine isolated fromAspergillus. The data presented in example 5, revealed the unexpected fact that the enzymeAspergillusit is only one of the four prolinnova residues present in the molecule BCM-7, derived from beta-casein A2. This shows that incubation of the substrate with a high content of Proline with Proline-specific protease does not imply automatic splitting of all peptide bonds, including prolinnova remains, even in conditions of sharply increased the ratio of enzyme/substrate. Example 6 the authors demonstrated the effectiveness of Propylenediamine derived fromAspergillusin relation to the 33-dimensional derived gliadin, which, as stated, is a major epitope for patients with celiac disease. Although alcalase broad-spectrum again failed to break down the molecule, either in acid or in alkaline medium, the enzyme isolated fromAspergillusrapidly digested this molecule in the acidic environment, forming 99.5% of peptides with a maximum length of 6 amino acid residues. Thus, NASM is rubbing on the high effectiveness against peptides with a high content of Proline in an acidic environment, even the enzymeAspergillusleft untouched, at least 0.5% of heptamer with the amino acid sequence YPQPQLP. Since the sequence YPQPQLP is a known specific for patients with celiac disease T-cell epitope, the data obtained show that in case it is not the most optimal Proline-specific enzymes, such as, for example, known Proline-specific oligopeptides, including enzymesFlavobacterium meningosepticumpractical application ofin vivoto prevent the formation of toxic peptides from gluten molecules is likely to be impossible. This conclusion is confirmed by the experiments described in examples 7 and 8. In example 7, the authors compared the curves of the activity of the Proline-specific endoproteaseA. nigerand the Proline-specific oligopeptidesF. meningosepticumin the range of pH 2-12. While the enzymeA. nigerdemonstrates activity in the pH range of 2.5 to 7, the enzymeF. meningosepticumto become active, you must have pH values more than the 5.0. In example 8, the authors simulated the situation in the stomach, placing both the Proline-specific enzyme in an environment with a low pH in the absence and in the presence of gastric enzyme pepsin. The subsequent measurement of the activity at the optimum for each of the enzymes pH values of the environment showed that the enzymeF. meningosepticumunable to keep ustoichivosti pH 2 or pH 3. In contrast, the enzymeA. nigersuffered such a low pH, as pH 2, and even in the presence of pepsin, which underlines its value for hydrolysis of toxic peptides in the stomach. In example 9 the authors showed that the enzyme isolated fromA. nigerable to degrade a large number of known epitopes of gluten HLA-DQ2. Interestingly, the position of the observed sites splits allow you to predict the destruction of all known in the literature as T-cell epitopes. In example 10 authors separated gluten epitopes from 100% malt beer and 100% wheat bread and showed that there is a possibility of detection of these epitopes in the analysis using antibodies. In example 11 the authors showed that the beer obtained when the process of production of enzymesA. nigercontains a significantly lower amount of gluten epitopes. In example 12 the authors showed that a similar result can be obtained by the inclusion of the enzyme in the composition of wheat dough for baking Dutch tin bread with low content of gluten epitopes.

Gluten is a water-insoluble substance with a complex three-dimensional structure. These properties of gluten in combination with its amino acid composition, enriched in Proline, make the gluten molecules are resistant to proteolysis in the stomach and intestines. As it is in none of secreted into the gastrointestinal tract of natural substances, having proteolytic activity, is not capable of cleavage of peptide bonds formed by Proline, has the meaning of the synergistic use of exogenous Proline-specific enzyme. However, people with celiac disease can be very sensitive to many epitopes present in gluten. According to the present invention acts as a natural digestive proteases can be improved Propylenediamine isolated fromAspergillusmoreover , in the present application is disclosed further improve the hydrolytic ability of this proteolytic mixture.

It is well known that the peptide bond formed by negatively charged residues such as Glu (E) and Asp (D), are poor substrates for proteases. In addition, natural gastro-intestinal proteolytic enzymes are not able to cope with these residues, as evidenced by the selection of the gliadin peptide WQIPEQSR resistant to proteases of the stomach and pancreas (cf. Shanet al). The latter publication also makes obvious the fact that everywhere present glutamine residues (Q) in the gluten can be deliciousy to glutamate residue (E) under the action of transglutaminase. Unfortunately, this regiospecific deliciousa gialinovykh peptides increases their immunogenic potential. In response to atipamezole the authors have developed an effective combination of enzymes, consisting of Proline-specific endoprotease derived fromAspergillusand auxiliary endoprotease, preventing the formation of toxic peptides with a high content of Proline. In accordance with the present invention can be applied glutamate-specific endoprotease (EC3.4.21.19), oversecrete which is carried out by a number of microorganisms, nutritional condition, for example,BacillusandStreptomyces. These enzymes can be obtained in a cost efficient manner suitable for obtaining food. Enzymes that can pass through the stomach intact, taking into account their enzymatic activity, are preferred. Basically, these enzymes will be acidic or neutral optimum pH. In combination with PropylenediamineAspergillusthis category glutamate-specific endoprotease is applicable in obtaining protein hydrolysates with a low enriched in Proline peptides.

Completely unexpectedly, the present study demonstrates that in addition to the glutamate-specific endoprotease there are other endoprotease, which have a synergistic effect when the incubation with the Proline-specific endoprotease isolated fromAspergillus. The authors concluded that endoprotease (EC 3.4.21-99) capable of splitting the connection between amino acid residues Q (glutamine) is L (leucine) and combine well with the Proline-specific endoprotease from Aspergillus. Especially successfully applicable endoprotease, which are the optimal pH lower than 5.0, and prefer either glutamic or lacinova residues in positions P1 or P1' substrates, such as aspartic protease Aspergillus (EC 3.4.23.18 and 19) and macropeptide (EC 3.4.23.23).

One of the applications of enzymes of the present invention is to use them as tools to facilitate digestion. In this application of the composition according to the present invention preferably are introduced orally, but can be other direct ways. Composition, usually administered to people, but they can also enter and animals, preferably mammals, to facilitate symptoms of hypersensitivity to gluten, or disorders of the metabolism of casein or gluten, or IBS. When applied as a means to facilitate the digestion, the enzymes of the present invention can be part of the dry formulations in the form of, for example, pills, tablets, granules, sachets or capsules. On the other hand, the enzymes of the present invention can be included in the liquid formulation, for example, syrup or capsules, or they may be included in food products with water activity (Aw) below 0,85. Compositions used in various compositions containing enzymes of the present invention, m is may also include, at least one compound from the group consisting of a physiologically acceptable carrier, excipient, filler, stabilizer, buffer and diluent, and these terms are used in their ordinary sense, to refer to substances that contribute packing, delivery, absorption, stabilization or, in the case of auxiliary substances, improving physiological result of the action of enzymes. The corresponding data for the various compounds that can be used in combination with enzymes of the present invention in powder form, can be found in “Pharmaceutical Dosage Forms”, second edition, Volumes 1, 2 and 3, ISBN 0-8247-8044-2 Marcel Dekker, Inc. Although the enzymes of the present invention, is included in the dry powders may be stored for a relatively long period, you should avoid contact with moisture or moist air, due to the choice of suitable packaging, such as blister packaging. In the case of the inclusion in the composition of the fluids, an important role is played by the compounds used to stabilize enzyme activity and protection from germs. Stabilization of enzyme activity may require lowering the water activity, which can be achieved by the use of polyols, such as glycerin, or various sugars. In addition, his roll on estiem known divalent cations, such as Ca2+or Mg2+and reducing agents, such as sulfur-containing amino acids or phenolic compounds such as BHT or propylgallate. Protection from germs, appropriate nutritional condition, can be achieved through the use of well-known combination of environment with low pH and low water activity, with the help of solutes, benzoates or parabens. In addition, you may need food thickeners, such as a hydrocolloid. A relatively new form of oral administration is the use of gelatin capsules containing liquid. In this application liquid, as a rule, is an oil or polyethylene glycol, or lecithin, which can be suspended dried enzymes of the present invention. Examples of formulations of tablets with improved stability of the enzymes described in US 2002/0136800.

Description of the drawings

Figure 1: Graphical representation of the optimal pH value of the Proline-specific endoprotease isolated fromA. nigerobtained when using as the substrate a synthetic peptide Z-Gly-Pro-pNA (cf. example 1).

Figure 2: SDS-PAGE of intact ovalbumin and synthetic 27-dimensional peptide after incubation with purified chromatography Proline-specific endoprotease isolated fromA. niger(cf. example 3).

Figure 3: Graph of the static representation of the optimal pH value of the Proline-specific endoprotease, isolated fromA. nigerandF. meningosepticum(cf. example 7).

Materials and methods

Materials

The firm Sigma were obtained following enzymes: amyloglucosidase fromAspergillus niger, 300 U/ml, Sigma A-7095; pepsin of the gastric mucosa of pigs, 2331 U/mg, Sigma P-7012; transglutaminase Guinea pigs, Sigma T-5398. 2.5% solution of trypsin was obtained from Gibco (BRL 25090-028) and the cartridge Sep-Pak Plus tC18, Walters No. 036810 the firm Walters. Alcalase® AF 2,4L, possessing activity of 2.6 AU(A) (alkalizing Anson units) units/g of product was obtained from Novozymes A/S (Bagsvaerd, Denmark). According to Novozymes, the details of this measurement activity can be found in Novozymes Analitical Method (LUNA#2003-32153-01/SOP no. :EB-SM-0218.02/02).

Synthetic peptides were obtained from Pepscan Systems B.V. (Lelystad, The Netherlands). Chromogenic peptide substrates were obtained from Pepscan Systems, or from Bachem, Switserland.

Proline-specific endoprotease fromA. niger

Surgentes and chromatographic purification of the Proline-specific endoprotease fromAspergillus nigerwere performed as described in WO 02/45524. The activity of the enzyme (1 u/10 mg protein) was tested on the synthetic peptide Z-Glu-Pro-pNA at 37°C in buffer citrate/centripetal pH 4.6. For the product of the reaction was observed spectrophotometrically at 405 nm. The activity of commercially available enzyme proletarisation (purchased from ICN Biomedicals/MP Biomedicals, Aurora, Ohio, US) was 35 units per mg of product is rich it was tested on Z-Glu-Pro-pNA at 30°C in buffer with a pH of 7. For the product of the reaction was observed spectrophotometrically at 405 nm. For both enzyme unit is defined as the amount of enzyme that promotes the release of 1 µmol p-anilide per minute under the above conditions.

Analysis of LC/MS

To characterize the products of enzymatic hydrolysis of proteins, obtained by the action of a mixture of enzymes of the present invention, used HPLC, using a mass spectrometer ion trap (Thermo Electron®, Breda, the Netherlands)connected to a P4000 pump (Thermoquest®, Breda, the Netherlands). Peptides were separated using column PEPMAP C18 300A (MIC-15-03-C18-PM, LC Packing, Amsterdam, The Netherlands), in combination with a gradient of 0.1% formic acid in Milli Q water (Millipore, Bedford, MA, USA; solution A) and 0.1% formic acid in acetonitrile (solution B) for elution. The gradient started with a 95% solution A, within 140 minutes increased the content of solution B to 40% and kept the last relationship for another 5 minutes. Injected volume was 50 microliters, the flow rate was equal to 50 microliters per minute and the column temperature was maintained at 30°C. the protein Concentration in the injected sample was approximately 50 micrograms/milliliter.

Detailed information about individual peptides were obtained by applying the scan-dependent MS/MS algorithm, which is a standard algorithm for mass spectrometers with ion trap.

the and full scan analysis was followed by a more detailed scan analysis to determine the charge of the most intense ion in the full range of the scanned mass. The following then MS/MS analysis of this ion led to partial information about the peptide sequence, which could be used for database searches using application SEQUEST from Xcalibur Bioworks (Thermoquest®, Breda, the Netherlands). Used databases extracted from OWL.fasta databank, which is available in the NCBI (National Centre for Biotechnology informatics) and contains proteins of interest used for the application. In these experiments, when the measurement has been well characterized protein substrates, such as serum proteins or casein, the accuracy of the analytical methods has increased due to the exclusion from consideration of MS/MS spectra with line sequences less than 50%.

Angiotensin (M=1295,6) used for setting the optimal sensitivity in the MS mode and for optimal fragmentation mode MS/MS, continuing the introduction of 60 micrograms/ml, caused mainly by the particles with double and triple charge mode MS and optimal energy collisions of approximately 35% in mode MS/MS.

MALDI-TOF

Analysis spectroscopy MALDI-TOF was performed using the mass spectrometer Voyager De-Pro (Applied Biosystems). After mixing with a suitable matrix, samples of the peptides was measured in the linear regime. Weights determined using the software MassLynx, lined up consistently in the TB with the collapse after the ion source (PSD) to confirm the expected amino acid sequence of peptides.

Quantitative determination of gluten peptides in food samples.

Monoclonal antibodies specific for stimulating T-cell alpha-gliadin, γ-gliadin and LMW-putnikovic peptides, are available and used in competitive analysis for the definition of these peptides in food samples (E.H/A/ Spaenij-Dekking et al., GUT, 53:1267-1273 (2004))

Assays based on antibodies

For analysis on the basis of antibodies in mice Balb C was developed monoclonal antibodies against known for stimulating T-cell alpha-, gamma-gialinovykh and LMW-putnikovic peptides. After fusion of spleen cells of mice with a cell line of mouse myeloma, obtained hybridoma that produce antibodies. They were cloned by the limited dilution and monoclonal antibodies secreted by these cells was tested with the aim of applying them in the competitive analysis of monoclonal antibodies. For each specificdate chose one or two suitable monoclonal antibodies and determined the epitopes recognized by different monoclonal antibodies (see table below).

SpecificityT-cell epitopeEpitope antibodies
α-gliadin, Glia-alpha 2/9) QLQPFPQPQLPYQPFPQPQ
γ-gliadin (Glia-gamma 1)QPQQPQQSPFQQQRPFQQRPFI
LMW glutenin (Glt-156)QPPFSQQQQSPFSQQSPFS or PPFSQQ

EXAMPLES

Example 1

The optimum pH and temperature of the Proline-specific endoprotease derived fromA. niger

Retrieved fromA. nigerProline-specific endoprotease was sverkhekspressiya the owner of theA. niger, was isolated and subjected to chromatographic purification, using materials and methods described in WO 02/45524. To establish the optimal pH value of the thus obtained enzyme was used to buffer solutions with different pH values. Buffer solutions with pH 4,0-4,5-4,8-5,0-5,5 and 6,0 received using 0.05 mol/l Na acetate and 0,02M CaCl2; buffer solutions with pH 7.0 and 8.0 were obtained using 0.05 M buffers Tris/HCl containing 0.02 M CaCl2. The pH value was adjusted using acetic acid and HCl, respectively. As the substrate used chromogenic synthetic peptide Z-Glu-Pro-pNA. Buffer solution, substrate solution, and pre-diluted endoprotease (with an activity of 0.1 U/ml) was heated exactly to the temperature 37,0°C in a water bath. After stirring for PR what titanium reaction was observed spectrophotometrically at a wavelength of 405 nm and the temperature 37,0°C for 3.5 min, carrying out measurements every 0.5 minutes From the results shown in figure 1, it is clear that the Proline-specific endoprotease produced byA. niger, has an optimum pH of about 4.

In addition, the established temperature optimum of Propylenediamine. To this end, purified preparations of the enzyme were incubated in 0.1 mol/l Na acetate containing 0.02 mol/l CaCl2at pH 5.0 for 2 hours at different temperatures, using casein resorufin (Roche version 3) as the substrate and quantitatively determining the enzymatic activity measurement at 574 nm. According to the results of the Proline-specific endoprotease fromA. nigerhas a temperature optimum of about 50°C.

The optimum pH lying in the strongly acidic region, clearly suggests that the generatedA. nigerProline-specific endoprotease has the ideal properties for industrial applications, as well as for oral use, because it will show optimal activity under acidic environment, preferred for industrial applications, as well as prevailing in the stomach and the initial part of the small intestine. In addition, the temperature optimum of the enzyme makes this enzyme is highly suitable for both applications.

Example 2

The enzyme is derived fromA. nigeris representative of a new class of p is Olin-specific enzymes

Of the entire coding sequence of the Proline-specific endoprotease derived fromA. nigerthat is described in WO 02/45524 may be determined protein sequence of 526 amino acids. The novelty of the enzyme was confirmed by a search using the BLAST in such databases like SwissProt, PIR and trEMBL. To the surprise of the authors failed to detect obvious homology between enzymeA. nigerand famous proletarisation. A more thorough study of the amino acid sequence has revealed, however, a low but significant degree of homology with carboxypeptidases Pro-X (EC3.4.16.2), dipeptidylpeptidase I (EC3.4.14.2) and is specific to the thymus serinproteaza. All these enzymes are related to the family of serendipidous S28. Also, all of these enzymes and endoproteaseA. nigersaved configuration GxSYxG around the active site serine. In addition, members of the family S28 acidic optimum pH, have specificity against splitting with the carboxy-terminal side prolinnova residues and are synthesized with a signal sequence and propeptide, just like the Proline-specific endoprotease produced byA. niger. Finally, the enzymeA. nigerhas a size similar to the size of family members S28. Therefore, the Proline-specific endoproteaseA. nigerapparently is a member of the family semiprotect S28, and not the CE is ASTA S9, in which are grouped the majority of cytosolic proletarisation, including enzyme isolated fromFlavobacterium meningosepticum. Based on the above structural and physiological features we have come to the conclusion that the enzymeA. nigerbelongs to the family S28, and not to the family of semiprotect S9. Another feature that distinguishes the enzymeA. nigerfrom proletarisation, belonging to the family S9, is the fact that unlike cytosolic rollingupdates belonging to the latter family, recently identified enzymeA. nigersecreted in the growth medium. To date, it has been shown that only the basidiomycetesAgaricus bisporus(Sattar et al, J.Biochem. 107, 256-261 (1990)) and not related records of AscomycetesAspergillus niger(WO 02/45524) produce extracellular Propylenediamine. However, the enzyme obtained from basidiomycetes, cannot tolerate pH values below 5 and, therefore, is much less suitable for industrial applications, as well as for oral use.

This request is the first message on the allocation of family member S28 from lower eukaryotes and the description of its characteristics.

Example 3

Proline-specific endoprotease derived fromA. nigerable to hydrolyze large proteins and small peptides and, thus, is a real n what proteases

Due to the characteristic features of the structure of proletarisation belonging to the family S9, can't break down peptides larger than 30 amino acid residues. This limitation is an obvious disadvantage for the enzyme, which means, should hydrolyze all potentially toxic peptides with a high content of Proline as quickly and efficiently as possible. In order to find out whether these characteristic limitations related to the substrate molecule, for Proline-specific endoprotease derived fromA. nigerthe authors incubated chromatographically purified Propylenediamine fromA. nigerwith a small synthetic peptide, as well as with the large molecule of ovalbumin and subjected the resulting products of hydrolysis analyzed using the SDC-PAGE. Used synthetic peptide consisted of a 27-Mer, including the sequence of NH2-FRASDNDRVIDPGKVETLTIRRLHIPR-COOH, the company presented the Perscan (Lelystad, The Netherlands). As can be seen from its amino acid sequence, this peptide contains 2 prolinnova balance, one in the middle and one near the very end of the peptide. The intact molecule ovalbumin (Pierce Imject, vials containing 20 mg of frozen dried substance) consists of 385 amino acids and has a molecular mass of 42 750 Yes. This molecule contains 14 prolinnova residues, of the Institute of which is located in the C-terminal part of the molecule, and it can not be split Proline-specific endoprotease.

Ovalbumin and Oligopeptide separately incubated at 50°C with purified Proline-specific endoprotease derived fromA. niger. Through certain time periods were selected several samples, which were analyzed using SDS-PAGE.

Chromatographically purified Proline-specific endoprotease derived fromA. nigerwith the activity of 4.5 U/ml was diluted 100 times in 0.1m acetate buffer with pH 4, containing 20 mm CaCl2. Ovalbumin was dissolved in acetate buffer with a pH of 4 to a concentration of 1 mg/ml (22 μm). 27-dimensional peptide was dissolved in the same buffer to achieve a concentration of 0.48 mg/ml (152 microns). Polyarnosti solutions of ovalbumin and 27-measure was chosen so that both solutions had the same both molarity with respect to split prolinnova residues. Ovalbumin contains 13 potential customers cleavage of Proline, whereas the 27-dimensional peptide has only two. 0.5 ml of the solutions of both substrates were incubated with 10 μl (0,45 milied) solution of the enzyme in thermomixer Eppendorf at 50°C. Several times at certain intervals of time selected 10 ál samples inkubiruemykh mixtures and before SDS-PAGE was kept at 20°C. All materials used for SDS-PAGE and staining purchased from Invitrogen. The samples were obtained using a buffer LDS in accordance with the instructions the mi manufacturer, and separated on 12% bis-Tris gels using the buffer system MES-SDS according to the manufacturer's instructions. Staining was performed using the dye Simply Blue Safe Stain (Collodial Coomassie G250).

As you can see in figure 2, ovalbumin is cleaved by an enzyme derived fromAspergilluson a discrete line, approximately 35-36 KDa in the first 4.75 hours incubation (lane 3). The increase in the length of incubation leads to further disintegration into smaller products of different molecular weight (lane 7).

27-dimensional peptide also split, as can be judged by the weaker lines in the bands 4, 6 and 8 compared to lane 2. Shift corresponding to a very low molecular weight product (compare lanes 9 and 8), most likely caused by the removal of arginine residue from the carboxyl end of the peptide. The difference is approximately 200 Yes (measured using software Alphalmager 3.3d on the system Alphalmager 2000), and arginine has a molecular weight of 174. This shift corresponding to low molecular weight, probably, corresponds to the first stage in the cleavage of the peptide.

Further degradation of the product can be seen only by reducing the intensity of the lines on the SDS gel. The products of the further disintegration of the invisible, as in the gel cannot staining components with a molecular weight of about 1000 dye, Kumasi rilliantly blue.

From this experiment we can conclude that in contrast to known proletarisation, belonging to the family S9 isolated fromA. nigerProline-specific endoprotease has no specific preference for the cleavage of peptides small size compared to much larger proteins. Therefore, isolated fromA. nigerthe enzyme is a real endoprotease and is the preferred enzyme for cleavage of potentially toxic peptides with a high content of Proline.

Example 4

Beta casomorphin in the hydrolysates obtained after incubation with alcalase and combination of alcalase with the Proline-specific endoprotease fromA. niger

By analogy with the formation of resistant to protease beta-casomorphine during gastrointestinal proteolysis, the inventors were interested in, not observed whether such accumulation related BCM-7 peptide fragments in the process of industrial production of milk protein hydrolysates. To this end, the authors incubated beta-casein A2 from cow's milk is often used in industry subtilisin-alcalase, and alcalase in combination with Proline-specific endoprotease fromA. niger. The resulting peptides were subjected to analysis using LC/MS/MS.

Cow's milk which contains almost 10 g of beta-casein per kilogram of milk, that is 28% of the total available protein. To facilitate the analysis of the related BCM-7 amino acid sequence, the authors used in this experiment concentrated preparation (Sigma)containing at least 90% beta-casein (A2). This product was dissolved in water at a concentration of 20 g per liter, and then, using NaOH, brought the pH to 8 and added alcalase with 800 ál of concentrated enzyme per liter of solution of casein. Incubated for 2 hours at 60°C. Then lowered the pH to 4.5 using citric acid. Then the solution was divided into two parts: one part was heated for 5 minutes at 90°C for inactivation of alcalase and another part was added Proline-specific endoprotease fromA. nigerto achieve the concentration of the enzyme 1 u/g of casein present (the definition of a unit, see "Materials and methods"). Incubation with the Proline-specific endoprotease fromA. nigercontinued for 16 hours at 55°C and then was carried out by another heat treatment for inactivation of Proline-specific endoprotease. In conclusion, two samples with the measured concentration of beta-casein 20 mg/ml was sent to LC/MS/MS analysis. These samples were centrifuged for 10 minutes at 13000 rpm and before analysis LC/MS/MS was diluted 20 times in Milli Q. Analysis of LC/MS/MS was performed in accordance with the description of the time is eaten "Materials and methods".

On removal from the sequence of BCM-7 Tyr-Pro-Phe-Pro-Gly-Pro-Ile at amino acid positions 60-66 molecules of beta-casein, smaller pieces, such as Tyr-Pro-Phe-Pro (beta-casomorphin(1-4)) and Tyr-Pro-Phe-Pro-Gly (beta-casomorphin(1-5)amino acid positions 60-63 and 60-64 respectively, as well as peptides larger size up to a length of 11 amino acid residues (amino acid positions 60-70) show at least some the degree of opioid activity. It was reported that the Tripeptide Tyr-Pro-Phe at positions 60-62 does not possess opioid activity. To identify peptides used direct LC/MS/MS of the protonated molecules. Mass possibly suitable protonated peptides are shown in table 1. All spectra were obtained with the amplitude of the collision of 35% and the width of the peak 3 Yes. All the experimental data were compared with theoretical models of fragmentation based on the so-called B and Y ions. This process, usually carried out with the use of automatic data processing.

Amino acid sequence of beta-casomorphin begins with a tyrosine (Y; residue number 60 peptide chain beta-casein), and in the table it is highlighted in bold. The values of m/z presents for protonated molecules other possible candidate peptides. Detection prolinnova residue at position 67 shows that IP is alsoany substrate is a beta-casein A2.

The results obtained by LC/MS/MS analysis of the hydrolyzed beta-casein, which were formed after incubation with either alcalase, or with a combination of alcalase and Proline-specific endoprotease presented in table 2 and can be summarized as follows:

- by themselves casomorphine sequence, i.e. YPFPGPI, and derived no in beta-casein A2, processed or alcalase, or a combination of alcalase and Proline-specific endoprotease fromA. niger;

however, in the sample treated with alcalase, there are two peptide containing beta-casomorphine sequence, i.e. LVYPFPGPIPN and VYPFPGPIPN;

the intensity of the signal of these contain beta-casomorphin sequences is sharply reduced when processing the Proline-specific endoprotease fromA. niger.

Table 2
LC/MS/MS identification of peptides containing beta-casomorphin (1-7) amino acid sequence
Amino acid sequence of peptidem/zThe intensity after treatment with alcalaseThe intensity after treatment with alcalase + Proline-specific endoprotease
YPFPGPI790,4--
FPGPIPNS828,4--
YPFPGPIP887,5--
VYPFPGPI889,5--
VYPFPGPIP986,5--
LVYPFPGPI1002,5--
YPFPGPIPNS1088,5--
LVYPFPGPIP1099,6--
VYPFPGPIPN1100,6100·1070,05·107
VYPFPGPIPNS1187,6--
YPFPGPI 1201,7--
LVYPFPGPIPN1213,63,5·107-
VYPFPGPIPNSL1300,7--
LVYPFPGPIPNSL1413,8--

The results obtained clearly show that the combination of alcalase with the Proline-specific endoprotease fromA. nigerwith high efficiency destroys all potential beta casomorphine sequence, thereby providing the possibility of obtaining hydrolysates without potentially toxic sequences with a high content of Proline.

After a more thorough search of the resulting peptides, the authors were able to detect the presence of peptide VYP in the hydrolysate obtained when exposed to a combination of the two enzymes. Because this peptide could not be detected in the hydrolysate, which was formed using only alcalase, these findings suggest that cleavage at the C-end prolinnova residue at position 61 of the molecule of beta-casein by the enzyme derived fromAspergillus.

the example 5

Peptides obtained by incubation of the peptide VYPFPGPIPN formed under the action of alcalase, with the Proline-specific endoprotease fromA. niger

As shown in example 4 hydrolysis of beta-casein A2 with a combination of alcalase and Proline-specific endoprotease fromA. nigereffectively removes all potential casomorphine sequence. However, the complexity of the resulting peptides did not allow the authors to establish what position obtained fromAspergillusthe enzyme cleaves formed under the action of alcalase peptide VYPFPGPIPN. To this end, the peptide with this particular sequence was synthesized and incubated with the Proline-specific endoprotease at two concentrations of the latter. Subsequent analysis of the resulting peptides using LC/MS/MS revealed the exact site of cleavage by an enzyme.

Liofilizovannye 10-dimensional peptide (Pepscan Systems; Lelystad, The Netherlands) was dissolved in citrate-phosphate buffer pH 4.5 with a concentration of 2 mg/ml To the solution was added Proline-specific endoprotease in concentrations of 1 and 10 u/g of peptide. Incubation was carried out for 4 hours at 55°C, and then used a heat treatment for 5 minutes at 90°C to inactivate the enzyme. After this, two samples were centrifuged for 10 minutes at 13000 rpm and before analysis of LC/MS and LC/MS/MS was diluted 20 times in Milli Q water. Initially, the mod is sci analyzed in the mode of LC/MS to monitor the decrease of signal intensity 10-measure using different amounts of the enzyme, as well as observing the signal peptides displayed on the LC/MS ion chromatogram after enzymatic cleavage.

Then was carried out by direct analysis LC/MS/MS of the protonated molecules of peptides found in girders LC/MS. All spectra were obtained with the amplitude of the collision of 35% and the width of the peak 3 Yes. All the experimental data were compared with theoretical model of fragmentation based on the so-called B and Y ions. This process is usually carried out with the use of automatic data processing for the identification of peptides, polypeptides and proteins.

Processing 10-measure VYPFPGPIPN (M=1099,5) 1 IU/g of the enzyme leads to a complete breakdown of the 10-measure for a few peptides. The intensity of the signal of the protonated molecule at m/z 1100,5 reduced by 3 orders of magnitude. Processing 10-measure 10 u/g does not lead to a further reduction in signal intensity of the protonated molecule and, in addition, not found signals of any additional peptides. When processing an enzyme concentration of 1 u/g was formed 4 peptide, and in the greatest quantity (almost 98%, see table 3) peptide VYP (M=377,2), characterized by the ratio of m/z 378,2. All four peptides were analyzed in the LC/MS/MS and found correct on the basis of the above criteria. Table 3 shows the weight protoni avannah peptides 10-measure VYPFPGPIPN M=1099,5, analyzed in modes of LC/MS and LC/MS/MS. In the second column presents the value of m/z of protonated molecules, in the third column of the intensity signals of the protonated molecules observed in mode, LC/MS, in the fourth column percentages on the basis of the peak areas of protonated molecules and in the fifth column position of the detected peptides in the total amino acid sequence of the 10-measure. It should be emphasized that the use of peak areas protonated molecules of peptides does not include the effect of differences in the efficiency of ionization.

Table 3
Peptides obtained by incubation of related HSR-7 10-measure VYPFPGPIPN c Proline-specific endoproteaseA. niger
Amino acid sequence of peptidem/zThe intensity of the mode LC/MSPercentage (%)The position in the General
amino acid sequence
VYP378,21·10897,71-3
VYPF 525,33·1050,31-4
VYPFP622,42·1062,01-5
VYPFPGP776,43·1040,031-7

However, we can conclude that the Proline-specific endoprotease fromA. nigerperforms splitting almost exclusively with C-terminal side of the Proline at position 61 (position 3 for this particular Decapeptide). The increase in the ratio of enzyme/substrate does not affect the implementation of cleavage.

Since all known molecules of beta-casomorphins with opioid activity have the same N-terminal sequence YPF, it is obvious that the effective splitting of this sequence between P and F (i.e. from the side of the carboxy-end prolinnova residue at position 61) under the action of Proline-specific endoprotease will effectively inactivate all BCM-7 and related BCM-7 peptides, regardless of whether they are formed from genetic variants of beta-casein A1 or A2.

A key role prolinnova residue at position 61 in cooperation with mu-receptor b is La also confirmed in modern Internet publication ("Synthesis and affinity for opioid receptors analogues of beta-casomorphins, contains beta-substituted amino acids" published Dipartamento di Scenza degli Alimenti Universita di Napoli "Frederico II" Facolta di Agraria).

Example 6

Peptides obtained by incubation of 33-dimensional derived gliadin LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF with the Proline-specific endoprotease fromA. niger

Reportedly Shan and employees (Science, Vol.297, 27 September 2002) treatment-resistant juices of the stomach and pancreas 33-dimensional derived gliadin LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF (M=3911) alcalase at pH 8 or pH 5 did not lead to any splitting of the molecule. However, as in the case of a 10-dimensional derived beta-casein, incubation with 1 unit of Proline-specific endoprotease fromA. nigerat pH 5 resulted in complete cleavage of the molecule into several peptides. The intensity of the signal three times protonated 33-measure at m/z 1304,4 decreased by 3 orders of magnitude in amplitude. When processing 33-measure 10 units of enzyme per gram protein was not observed further reduction of the signal intensity of the protonated molecules, as well as signals from other peptides.

After treatment, the enzyme was formed about 6 basic peptides and several minor peptides, and in the greatest number formed peptide, characterized by m/z 565,2. All 6 peptides were analyzed in the LC/MS/MS, it was found that they all contain Proline at the C-con is e, which confirms the specificity of the enzyme. The main resulting peptide is characterized by the ratio m/z 565,2 (sequence ...QPL table 4). Although the presence of the C-terminal sequence “LP” for this peptide could not be demonstrated unambiguously identified by mass theoretically can be formed by endoproteolytically the collapse of the 33-measure, so there remains some uncertainty about the exact N-terminal structure of the peptide. Most likely m/z 565,2 is an N-pyroglutamyl variant patterns QPQLP (M=581,3), although this was not investigated in more detail.

The emergence of QPQLP: LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF

The emergence of QPQLPYP: LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF.

Range of LC/MS/MS peptide with m/z 679 could be interpreted as PQPQLP. Despite the fact that the nature of the peptide with m/z 565,2 was not fully understood, the data obtained clearly demonstrated the preferred dissociation under the action of Proline-specific endoprotease fromAspergilluswith the C-terminal side prolinnova residues at positions 12, 19 and 26 (i.e. solely between prolinnova and tyrosinosis residues) of this particular 33-measure. On this model cleavage is not affected by the use of higher ratios of enzyme/substrate. All relevant information are summarized in table 4.

The first column lists the received peptide sequence, and the points used (as in column 5), in order to show that cannot be given a precise initial position of the peptide due to unexplained discrepancies masses. The second column shows the values of m/z of protonated molecules, in the third intensity signal peptides observed in mode, LC/MS, in the fourth percentages based on the peak areas of protonated molecules, and in the fifth column shows the position of the identified peptides in the total amino acid sequence.

Table 4
The peptides formed during the incubation of 33-dimensional derived gliadin LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF with the Proline-specific endoprotease fromA. niger
Amino acid sequence of peptidem/zThe intensity of the mode LC/MSPercentageThe position in the total amino acid
sequence
...YP523,21,9·1075,2..14, ..21, ..28
YPQPQLP 842,31,7·1060,513-19, 20-26
QPQP468,23,4·1079,329-32
PQPQLPof 679.23,4·1079,314-19, 21-26
..Quality learning project565,22.4 x 10865,9..12, ..19, ..26
...P599,23,5·1079,6

Splitting 33-measure, which, as mentioned, is a major epitope in patients with celiac disease may not be carried out by the juices of the stomach and the pancreas or by incubation with aggressive broad-spectrum protease action alcalase, neither alkaline nor acidic environment. However, these results showed efficient cleavage of Proline-specific endoprotease isolated fromA. nigerin the acidic conditions of the environment. This cleavage is carried out exclusively between prolinamide and tyrosinase residues of the molecule and leads to the formation of 99.5% of the peptides, length of not more than 6 amino acid residues. Thus, despite the high efficiency with respect to peptides with a high content of Proline in an acidic environment, even enzymeAspergillusleaves 0.5% of heptamer with the amino acid sequence YPQPQLP. Since the sequence PYPQPQLPY is a known T-cell epitope specific for coeliac patients, the findings again emphasize that, obviously, it is not possible practical application in vivo to prevent the formation of toxic peptides from gluten molecules is not quite optimal Proline-specific enzymes with a close to neutral optimum pH, such as known Proline-specific oligopeptides and the enzyme derived fromFlavobacterium meningosepticum.

Example 7

pH-spectrum of activity of the Proline-specific endoproteaseA. nigerand the Proline-specific oligopeptides fromF.meningosepticum

As shown in example 1, the optimum pH of the Proline-specific endoprotease isolated fromA. nigeris about 4.2. In this first test were not considered in pH in highly acidic region, so it is not clear behavior of the enzyme at extreme pH values that may exist in the stomach. In the application WO 03/068170 indicated that the Proline-specific proteases, such as proletarisation fromFlavobacterium meningosepticumor DPP IV fromAspergilusfumigatus or peptidyl dipeptidase species ofStreptomycesare the preferred candidates for destruction in situ toxic peptides with a high content of Proline. Typically, these proteases are characterized by an optimal pH value close to neutral, which apparently excludes any hydrolytic activity in the stomach. To show the differences in the optimal pH values that exist between the Proline-specific endoprotease of the present invention and enzymes prior art, the authors compared the pH-range of activities endoprotease derived fromAspergillusand oligopeptides isolated fromFlavobacterium. EndoproteaseAspergillusreceived as described in example 1. Oligopeptides fromFlavobacteriumwas purchased from ICN Biomedicals (35 U/mg; catalog No. 32082; Ohio, US).

To determine the pH-spectrum activity for the two considered enzymes, prepared buffer solutions with different pH values. The buffers in the pH range from 2.0 to 7.0 was obtained using 0.1 mol/l solution of citrate buffers in the pH range from 6.0 to 9.0 was obtained using 0.1 mol/l Tris, and buffers in the pH range from 8.0 to 12.0 received using 0.2 mol/l solution of glycine. The desired pH was obtained using HCl or NaOH. As a substrate for both enzymes was used chromogenic synthetic peptide Z-Gly-Pro-AMC (Bachem, Swizerland etc.). In each cell (tablets Cstar No. 3631) was administered to 85 ál of buffer, 10 μl of an enzyme solution and 5 μl of the substrate (4 mm Z-Gly-Pro-AMC in 60% methanol). The final concentration of the enzymeA. nigerwas 32 µg/ml (3.2 miles IU/ml), the resulting concentration of the enzymeF. meningosepticumamounted to 0.21 µg/ml (7,4 miles IU/ml). After stirring the reaction mixture was left for 30 minutes for reaction with 37,0°C, after which the measured fluorescence at multipleitem reader CytoFluor production PerSeptive Biosciences. The obtained comparative data shown in figure 3. The data obtained in these slightly different testing conditions, confirm the set in example 1 acid optimum pH Proline-specific endoproteaseA. niger. The data show that the enzymeA.nigerhas approximately 20% residual activity at pH of 2.2 and 7.5. In addition, the data shown in figure 3, confirm the previously published optimum pH of the enzymeF. meningosepticumin the area of pH 7.0. In the context of the present patent application is more important that at pH below 5.0 enzymeF. meningosepticumhas no activity.

The data obtained show that in contrast to the enzymeF. meningosepticumthe properties of the Proline-specific endoprotease fromA. nigerideal for full manifestation of enzyme activity in the acidic conditions prevailing in the stomach and part of the duodenum.

Example 8

The stability of the Rolin-specific endoprotease A. nigerand the Proline-specific oligopeptidesF. meningosepticumin the conditions present in the stomach

A necessary condition for successful enzyme therapy is an effective splitting of toxic peptides with a high content of Proline in the stomach and intestines before these peptides reach the distal part of the duodenum. This requires that the exogenous enzyme, which is planned to be used for enzyme therapy, was a Proline-specific protease that is active during their stay, food proteins in the stomach and, optionally, behind the stomach. To compare the activity of the Proline-specific proteases isolated fromA. nigerandF. meningosepticumin such conditions, reminiscent of conditions in the stomach, we analyzed the residual activity after incubation at 37°C for various periods of time at different pH values and in the presence and absence of gastric protease pepsin. Residual enzyme activity was measured at the optimum pH values and using chromogenic peptide optimal for the respective enzyme, i.e. pH 7.0 and substrate Z-Gly-Pro-pNA for enzymeF. meningosepticumand pH 4.0 and substrate Ala-Ala-Pro-pNA for enzymeA. niger. In such conditions, the enzymeA. nigerdemonstrates a high level of activity on the substrate Ala-Ala-Pro-pNA and specific activity (IU is it 10% of the activity on Ala-Ala-Pro-pNA) on the substrate Ala-Ala-Ala-pNA. In these conditions, the enzymeA. nigershows little activity on other substrates Ala-Ala-X-pNA.

To create the desired acidic environment used buffers citrate/HCl 0.2 mol/L. the Amount of enzymeA. nigerwas 1.5 U/ml, the amount of enzymeF. meningosepticum(MP Biochemicals, Ohio, US) 3,3 IU/ml (determination unit, see "Materials and methods"). Pepsin (Sigma) was added at a concentration of 180 μg/ml After sampling was added pepstatin (Sigma) at a concentration of 1,67 mg/ml to inactivate the pepsin. Under these conditions, pepstatin has no inhibitory effect on both the Proline-specific protease. Residual activity of two Proline-specific proteases was measured kinetically at 405 nm using synthetic substrates Ala-Ala-Pro-pNA, Z-Gly-Pro-pNA, respectively. For this purpose, the cells microtiter tablet was mixed with 200 µl of substrate solution (2 mmol/l Z-Gly-Pro-pNA in 0.05 mol/l phosphate buffer pH 7.0 or 1.5 mmol/l Ala-Ala-Pro-pNA in 0.05 mol/l acetate buffer pH 4.0) with 50 ál (pre-diluted from 10 to 100x) sample treated with acid/pepsin. To study the kinetics were measured absorption for 10 minutes at 405 nm and 30°C, using the reader tablets TECAN Genios (Salzburg, Vienna).

Displayed in table 5 the results show that pH 4 represents the lower limit at which it can preserve enzymeF. meningosepticu . It should be noted that according to that described in example 7, this enzyme has no activity at this pH environment. After 2 hours at pH 4.0 enzyme shows approximately 25% residual activity when tested under optimal conditions. However, in the presence of pepsin-this enzyme is completely inactivated at the specified pH for 15 minutes. By contrast with these results are the data obtained for the enzymeA. niger. This enzyme retains its activity at such low pH values, as 2, and even in the presence of pepsin. These results strongly suggest that only enzymes with optimal pH value below 5.5 are probably active in the stomach, so that well-known Proline-specific proteases that belong to classes of enzymes EC 3.4.21.26 (proletarisation), EC 3.4.14.5 (dipeptidyl peptidase IV) and EC 3.4.15.1 (peptidylglycine A), do not fall in the number of enzymes that destroy the peptides with a high content of Proline in the stomach.

Table 5
Residual enzyme activity oligopeptidesF. meningosepticumand endoproteaseA. nigerafter incubation for various periods of time in conditions similar to the conditions of the stomach
Conditions of incubationResidual enzyme activity oligopeptides
F. meningosepticumafter:
Residual enzyme activity endoproteaseA. nigerafter:
pHThe presence of pepsin15 min30 min60 min120 min15 min30 min60 min120 min
2No----++++
Yes----++++
3No- ---++++
Yes----++++
4No++++/-++++
Yes----++++
+ Indicates the presence of residual activity when tested under conditions optimum for this enzyme.
Indicates that there is no residual activity when tested under conditions optimum for this enzyme.

Example 9

The activity of the Proline-specific endo is noteasy in relation to various gluten epitopes

In several publications indicated peptides that are derived from wheat gluten that are recognized by T-cells of the small intestine of patients with coeliac disease. In this context, of particular interest is the binding of peptides with molecules HLA-DQ2, because people who carry only the haplotype HLA-DQ2, have a much higher chance of developing celiac disease (Maki et al.; N. Engl.J. Med. 2003 Jun 19; 348 (25): 2517-24). All studied adult HLA-DQ2 patients respond to a sequence of alpha-gliadin, i.e. Glia-alpha2 and Glia-alpha9 (Arentz-Hansen et al., J.Exp.Med. 2000; 6:337-342). Children who currently starts celiac disease react to peptides Glia-alpha 20, Glia-gamma 30, Glt-17, Glt-156 and Glu-21 (Vader et al, Gastroenterology 2002; 122:1729-1737). In example 6, the inventors have already demonstrated successful cleavage of the peptide Glia-alpha2 (PQPQLPYPQPQLPY) under the action of the enzyme derived fromA. niger. In this example, the authors conducted a test enzymeA. nigerthe ability to break down and also other relevant peptides derived from gluten, as well as homologues of these peptides derived from other cereals.

With this purpose, synthesized the corresponding peptides, after which their purity was confirmed by HPLC and mass spectroscopy (MALDI-TOF) in accordance with the methodology described in section "Materials and methods". Cleavage of the peptides was carried out by mixing 50 μl of the peptide solution (1 mg/ml) with 50 μl of rest the RA enzyme A. nigercontaining 0.43 mcg/ml (0,043 miles IU) of enzyme in 50 mmol/l buffer solution of ammonium acetate with pH 4.5. After incubation for various periods of time at room temperature was selected samples of 0.5 ml and added to a 9.5 µl of matrix solution (1:1 H2O and CH3CN containing 0.2% TFU). After desalting using granules of ion exchange resin Dowex, the liquid was applied on the plate LDI together with some suitable quantities of the reference peptides, after which all peptides were subjected to analysis. From the obtained data it was possible to establish the sites of cleavage, as shown in table 6. Peptides Glt 156 and Glia gamma-2 are 10-measures; Glt-17 is an 8-Merom, but for optimal binding requires C-terminal extensions residues of P and L. Clearly defined cleavage sites shown double vertical lines, cleavage sites, which are not so obvious, shows a single vertical lines. Known minimal T-cell epitopes on each peptide is shown underlined. The symbols “U”, used in Glu-5, display or Lazenby or solarenemy the rest.

From the obtained data it is clear that not only Glia-alpha2, but, in fact, all gluten epitopes that are important for the young and for adults HLA-DQ2 patients effectively hydrolyzed isolated fromA. nigerthe cast new is-specific protease. Since most of the cleavage sites can be accurately attributed to the known minimum binding sites of T-cell epitopes, it is likely that the influence of the enzyme on gluten epitopes will cancel the recognition and, therefore, will effectively prevent subsequent T-cell proliferation, which ultimately leads to the symptoms characteristic of patients with celiac disease.

td align="left"> Glu-21
Table 6
The sites of cleavage in various known the gluten epitopes of wheat under endoprotease isolated fromA. niger
Glia-α2PQ//PQL//YPQPQLPY
Glia-α9QLQP//FP//QPQLP//Y
Glia-α20PFRP//QQP//YP/QPQPQ
Glia-γ1QPQQP//QQSFP//QQQRP//F
Glia-γ2QQP//YPQQP//QQPFPQ
Glia-γ30VQGQGIIQP//QQPAQL
Glt-17QQPP//FSQQQQQP//LPQ
Glt-156QQPP//FSQQQQSP/FSQ
Glu-5QQUSQP//QUP//QQQQUP//QQPQQF
QPQP//FP//QQSEQSQQP//FQPQPF

Using this approach were established on the sites of cleavage by the enzyme, derived fromA. nigerat a number of homologues of gluten epitopes of wheat isolated from oats, barley and rye (Vader, W et al, Gastroenterology 2003; 125:1105-1113). The results obtained (see table 7) show that the enzymeA. nigerable to cleave not only responsible for celiac disease gluten epitopes of wheat, but also homologues of these peptides, which are found in oats, barley and rye. As in table 6, the sites of cleavage, not doubtful, shown by double lines.

Table 7
The sites of cleavage in various known not wheat gluten the epitopes under endoprotease isolated fromA. niger
Source of glutenNameAmino acid sequenceThe homologue of the number of gluten epitopes wheat
OatsAv-gamma2BQQP//FVQQQQP//FVQGlia-gamma2
OatsAv-gamma2AQQP//VEQQEQPFVQ Deliciously
Glia-gamma2
OatsAv-gamma2BQQP//FVQQQQP//FVQQGlia-gamma2
BarleyHor-alpha2QQFP//QPQQP//FPQQPGlia-alpha2
BarleyHor-alpha9PQQP//FP//QP//QQPFR//QGlia-alpha9
RyeSec-alpha2QP//FP//QP//QQPFPQSQGlia-alpha2

Example 10

The study of the allocation of gluten epitopes from 100% malt beer and 100% wheat bread.

As it was planned (see "Materials and methods"), the method of extraction was tested in combination with the analysis on the basis of antibodies in the sample treated PVPP 100% malt beer (see example 11) and on a sample of 100% wheat bread (see example 12). In accordance with the obtained results (see table 8), the retrieval technique in conjunction with the analysis on the basis of antibodies capable of detecting anti-alpha-gliadin, anti-gamma-gliadin and anti-glutinosae epitopes in beer and bread. The obtained data together suggest that the applied retrieval technique suitable for the OBN is pursued gluten samples, as beer and bread. Based on these results, in examples 11 and 12 were more samples of beer and bread, which in the course of manufacture were exposed to different concentrations of Proline-specific endoproteaseA. niger.

Table 8
The results of the analysis using antibodies. The measurement of the exposed samples at two dilutions: 1/16 and 1/64. The results are expressed as µg/ml of the original sample
Anti-alpha gliadinAnti-gamma gliadinAnti-glutenin
Dilution of the sample1/161/641/161/641/161/64
Beer>>24971063786166163
Bread>>10501738 1536404215

Example 11

Beer production with the use of a Proline-specific protease fromA. nigerleads to low content of gluten epitopes

Beer haze is formed by combining formed from gluten enriched in Proline proteins, polypeptides and peptides with polyphenols, which are extracted from the grain (mostly barley)used for the production of beer. As shown in WO 02/046381, education beer turbidity can be reduced or prevented, thanks to the introduction in the production process of beer resistant to acids Proline-specific endoprotease upon receipt of the wort, fermentation or under exposure. In the conventional brewing process education Muti prevent the processing of PVPP, i.e. a compound that binds a variety of available polyphenols, but had only a minor impact on the levels of peptides with a high content of Proline, which are involved in the formation of turbidity. Since the conventional method of brewing is not possible to eliminate toxic peptides with a high content of Proline, patients with celiac disease are not allowed to drink beer.

The purpose of this study is to establish whether the addition of Proline-specific endoprotease fromA. nigerin the process of manufacture of the population of beer to lower levels of toxic peptides with a high content of Proline in the final product. If this is true, patients with celiac disease could drink this beer.

The beer-making process was carried out for 20 sec pilot plant IFBM (Nancy, France). Used a sample of Proline-specific endoprotease derived fromA. nigerwas stable in 50% (by weight) glycerin and had a total activity of 5 u/g of fluid (the definition of a unit, see "Materials and methods").

During the execution of independent procedures beer was brewed five servings of 100% malt beer using either PVPP (standard)or different amounts of Proline-specific endoprotease to prevent the formation of turbidity. In all experiments, the method of preparing the wort was exactly the same. In different experiments, the Proline-specific protease was added either at the beginning of the process of education wort, or after the formation of the wort, just before fermentation beer. In the process of education's wort has been tested three different doses of the enzyme, namely a 2.5, 5.0 and 7.5 units of enzyme per kg of used malt. When fermentation has experienced only one dosage of the enzyme, namely, 0.75 units of enzyme per kg of added malt. The reference sample beer was stable 30 g/HL PVPP, which was added before filtration of beer.

Each cooking was carried out from 300 kg of barley malt and pellet hops. If coord is wort used the ratio of liquid/malt 3:1 (volume/weight) and pH of 5.6. Chart receiving wort included the first phase lasting for 20 minutes at 45°C, the second stage duration 15 min at 64°C, the third stage with a duration of 25 minutes at 76°C and then heated to 78°C. Between stages the heating rate was 1°C / minute. The wort is boiled for 90 minutes Good the precipitate was achieved using a hydrocyclone separator. Fermentation was performed using a strain of the bottom fermenting yeast, while the norm is adding 17×106cells/ml wort and survival with the addition of 97%. For the period of fermentation for 10 days at 12°C +/- 1°C was followed by cold maturing within 5 days at -1°C +/- 1°C. the Beer was saturated with carbon dioxide at a pressure of 5.2 g/inch and, after bottling, pasteurizable at 60°C for 20 minutes.

Table 9
Various samples of beer and content of gluten on the results of the analyses using antibodies
The method of stabilization of beerSample beer 1Sample beer 2Sample beer 3Sample beer 4Sample beer 5
fermentationobtaining wortobtaining wortobtaining wort
PVPP30 g/HL
Enzyme units/kg of malt0,752,55,07,5
Analyses using antibodies (μg/ml)
Anti-alpha gliadin
Dilution 1/16
71315553327199
Dilution 1/64225216378 71
Anti-gamma gliadin
Dilution 1/16
1148748742
Dilution 1/64453262022
Anti-glutenin
Dilution 1/16
144968
Dilution 1/6421313410

In accordance with the results of anti-alpha-gliadin and anti-gamma gliadin antibodies are formed in comparable amounts. Results related to anti-gljuteinovogo antibody, in this analysis are not decisive. It is obvious that the use of enzyme in persons is nasty after the stage of obtaining the wort, leads to very low levels in the beer toxic peptides enriched in Proline. The idea that the recognition sites of the antibody have a minimum length of 5 amino acid residues, but the site recognition by T cells requires at least 9 amino acid residues, makes it even less likely recognizing T-cells short peptides generated by enzymatic treatment. In fact, the data obtained confidently indicate that, using the above-mentioned enzymatic processing, you can cook a beer that is safe for celiacs.

Example 12

The production of bread when included in the composition of the test Proline-specific endoprotease fromA. nigerreducing the amount of gluten epitopes

The dough for baking bread made of 3500 g of wheat flour (80% KolibriTMand 20% IbisTM), 1990 ml of water (56%), 77 g of compressed yeast (2,2%), 70 g of salt (2%), 140 mg of ascorbic acid (40 MT) and varying amounts of enzyme, isolated fromA. nigeras shown in table 10. The number of added enzyme compensated by the decrease in the number added in the dough water.

To get the dough mixed the ingredients, using a spiral mixer Diosnar®for 2 minutes at speed 1 and then 6 minutes on speed 2. The pieces of dough weight of 875 g gave a spherical shape, kept within 35 minutes at 34°C and 85% relative humidity, was obmanuli, was formed and put into forms that are kept for 75 minutes at 38°C and 87% relative humidity and baked for 20 minutes at 220°C. the Evaluation of the test and the resulting bread was fulfilled by professional bakers. From the results shown in table 10, it is clear that the addition of Proline-specific endoprotease obviously does not affect the spatial structure of the gluten and the ability of the test to hold gases, because the values of the volume and elasticity of bread has not been influenced by the enzyme at a concentration of 225 IU/kg flour or below. In fact, only adding more enzyme had a negative effect on the dough and received bread, which would have seemed unacceptable to most consumers. Because of this observation have not been recorded for data analysis using antibodies for bread, obtained at high concentrations of enzyme.

The data shown in table 10, show a clear decrease in the number of toxic peptides with a high content of Proline, present in the bread obtained when the concentration of the enzyme is about 200 u/kg flour. It is very likely that this reduction would be insufficient to allow the use of such bread patients with celiac disease. However, such a decrease could be sufficient to obtain food from profilactic the treatment benefit for people, suffer not detected celiac disease or IBS, or even for young children with immature immune systems.

good
Table 10
Various samples of 100% wheat bread and the number of available gluten in them according to analysis using antibodies
The amount of the applied enzyme units/kg flour
Dosage045225450
The consistency testgoodgoodacceptableunacceptable
Compliance testgoodgoodhighvery high
The amount of breadgoodsignificantsignificantSmall
The structure of breadgoodgooda very rough structure of bread crumb
Analyses using antibodies (μg/ml)
Anti-alpha gliadin
Dilution 1/16
Dilution 1/64
4231
2347
4231
2212
237
124
Not defined
Anti-gamma gliadin
Dilution 1/16
Dilution 1/64
1926
3979
1509
2895
1183
888
Not defined
Anti-gluten
Dilution 1/16
Dilution 1/64
359
232
74
232
98
121
Not defined

1. Application polyspecific endoprotease having a pH optimum below 5.5, for the manufacture of dietary supplements to prevent celiac disease, diseases associated with celiac disease associated with the presence of Proline-rich peptides in the consumed food or due to reduced level polyspecific protease.

2. Application polyspecific endoprotease having a pH optimum below 5.5, the of drugs for in vivo treatment of celiac disease, diseases associated with celiac disease associated with the presence of Proline-rich peptides in the consumed food or due to reduced level polyspecific protease.

3. Application polyspecificity endoprotease having a pH optimum below 5.5, as dietary supplements to prevent celiac disease, disorders associated with celiac disease associated with the presence of Proline-rich peptides in the consumed food or due to reduced level of Proline-specific proteases.

4. Application polyspecificity endoprotease having a pH optimum below 5.5, as a drug for the treatment of celiac disease, disorders associated with celiac disease associated with the presence of Proline-rich peptides in the consumed food or due to reduced level polyspecific protease.

5. The use according to any one of claims 1 to 4, in which the pH is lower than 5.0.

6. The use according to any one of claims 1 to 4, wherein the disorder or the disease is localized in the stomach.

7. The use according to any one of claims 1 to 4, in which polyspecific endoprotease is the enzyme of Aspergillus, preferably Aspergillus niger.

8. The use according to claim 1 or 3, wherein polyspecific endoprotease used for the production of food additives intended for individuals under the age of 25 years.

9. Note the Addendum according to claim 1 or 3, when polyspecific endoprotease used for the production of medicinal products intended for individuals under the age of 25 years.

10. The use according to claim 1 or 3, wherein polyspecific endoprotease use as a food additive, a food additive, or for the production of feed, including feed for animals, not people, preferably mammals.

11. The use according to claim 2 or 4, in which polyspecific endoprotease used as medicines, for the production of medicines for animals, not people, preferably mammals.

12. The use according to any one of claims 1 to 4, in which polyspecificity endoprotease able to hydrolyze proteins or peptides having more than 30 amino acid residues.

13. The use according to any one of claims 1 to 4, which is rich in Proline polypeptide is a gluten.



 

Same patents:

FIELD: medicine.

SUBSTANCE: substance of polypeptide nature with molecular weight 14350 Da, with N-end amino acid sequence, homologous phospholipase A2 of snake venom, and possessing properties of direct thrombin inhibitor of mixed type as well as antiproliferative action is separated of cobra venom Naja haje by three-stage liquid chromatography.

EFFECT: invention enables to produce selective direct thrombin inhibitor, possessing antiproliferative action.

1 tbl, 3 dwg, 10 ex

FIELD: medicine.

SUBSTANCE: method of obtaining somatotrophic hormone (STH) with decreased content of aggregate of its isoforms involves separation of STH isoforms by means of anion exchange chromatography by using anion exchange resin for decreasing content of the above aggregate up to not more than 10% (wt), on the basis of total mass of the above isoforms and the above aggregate. At that, there performed is loading of the above STH including dephenylalanine and/or trisulphide impurity and/or its aggregate on anion exchange resin chosen from the group including diethylaminoethyl cellulose and Q - sepharose. Loading is performed at the value of loading conductivity of anion exchange resin less or equal to 10 m cm/cm, at pH of anion exchange resin loading of 5 to 10 and at loading of anion exchange STH resin, which includes the above impurity or the above aggregate comprising not more than 10 g of protein/l of the volume filled with anion exchange resin.

EFFECT: invention allows obtaining somatotrophic hormone with decreased content of aggregate of its isoforms, its antagonist with decreased content of aggregate of its isoforms and total content of trisulphide impurity and dephenylalanine impurity.

8 cl, 5 dwg, 8 tbl, 6 ex

FIELD: medicine.

SUBSTANCE: method of obtaining somatotrophic hormone (STH) with decreased content of aggregate of its isoforms involves separation of STH isoforms by means of anion exchange chromatography by using anion exchange resin for decreasing content of the above aggregate up to not more than 10% (wt), on the basis of total mass of the above isoforms and the above aggregate. At that, there performed is loading of the above STH including dephenylalanine and/or trisulphide impurity and/or its aggregate on anion exchange resin chosen from the group including diethylaminoethyl cellulose and Q - sepharose. Loading is performed at the value of loading conductivity of anion exchange resin less or equal to 10 m cm/cm, at pH of anion exchange resin loading of 5 to 10 and at loading of anion exchange STH resin, which includes the above impurity or the above aggregate comprising not more than 10 g of protein/l of the volume filled with anion exchange resin.

EFFECT: invention allows obtaining somatotrophic hormone with decreased content of aggregate of its isoforms, its antagonist with decreased content of aggregate of its isoforms and total content of trisulphide impurity and dephenylalanine impurity.

8 cl, 5 dwg, 8 tbl, 6 ex

FIELD: medicine.

SUBSTANCE: method of obtaining somatotrophic hormone (STH) with decreased content of aggregate of its isoforms involves separation of STH isoforms by means of anion exchange chromatography by using anion exchange resin for decreasing content of the above aggregate up to not more than 10% (wt), on the basis of total mass of the above isoforms and the above aggregate. At that, there performed is loading of the above STH including dephenylalanine and/or trisulphide impurity and/or its aggregate on anion exchange resin chosen from the group including diethylaminoethyl cellulose and Q - sepharose. Loading is performed at the value of loading conductivity of anion exchange resin less or equal to 10 m cm/cm, at pH of anion exchange resin loading of 5 to 10 and at loading of anion exchange STH resin, which includes the above impurity or the above aggregate comprising not more than 10 g of protein/l of the volume filled with anion exchange resin.

EFFECT: invention allows obtaining somatotrophic hormone with decreased content of aggregate of its isoforms, its antagonist with decreased content of aggregate of its isoforms and total content of trisulphide impurity and dephenylalanine impurity.

8 cl, 5 dwg, 8 tbl, 6 ex

FIELD: chemistry.

SUBSTANCE: invention refers to the selective method for preparation of "АХЭ" inhibitor - perindopril with usage as initial reagent of the sterospecific amino acid N-/1-(S)-ethoxycarbonylbutyl/-(S)-alanine which is activated by tetramethyl-uronium salts in the presence of tertiary organic base and following interreaction with (2S,3aS,7aS)-octahydroindolo-2-carbonic acid or its ester. After completing of the reaction the protective group is removed by the hydrogenation, interphase hydrogenation or extraction.

EFFECT: obtaining of perindopril with usage of tetramethyl-uronium salts as reagents of coupling reaction.

5 cl, 3 ex

Ra antigen peptides // 2359974

FIELD: biotechnologies.

SUBSTANCE: claimed invention relates to novel class II antigen peptides of main histocompatibility copmplex, which are bound with "МНС" class II molecule.

EFFECT: claimed invention allows expanding arsenal of technical means used in early diagnostics of rheumatoid arthritis.

2 cl, 5 dwg, 4 tbl, 3 ex

FIELD: chemistry.

SUBSTANCE: extraction is done using affinity chromatography with immobilised metal. The method can be realised in native conditions. Biologically active G-CSF is obtained with purity over 95%. Two more chromatography stages are done, cation-exchange and gel filtration, to remove trace amounts of impurities. The method allows for obtaining G-CSF with high output and over 99% purity.

EFFECT: described method is especially suitable for industrial production of G-CSF.

16 cl, 5 dwg, 5 ex

FIELD: medicine.

SUBSTANCE: claimed invention relates to field of molecular biology, virology and medicine. Claimed is application of adenovirus for manufacturing medication for tumor treatment. Said virus is replication-deficient in cells which do not contain YB-1 in nucleus and encodes oncogenic protein E1A, which transactivates at least one viral gene from group including E1B55kDa, E4orf6, E4orf6 and E3ADP.

EFFECT: invention can be used for treating tumors demonstrating resistance to many cytostatic medications.

19 cl, 19 dwg, 13 ex

FIELD: chemistry, pharmaceutics.

SUBSTANCE: claimed invention relates to method of acidifying of one or several amino groups of peptide which is selected from group including exendin-3, exendin-4, Arg34-GLP-1(7-37), Gly8-GLP-1(7-36)-amide, Gly8-GLP-1(7-37), Val8-GLP-1(7-36)-amide, Val8-GLP-1(7-37), Val8Asp22-GLP-1(7-36)-amide, Val8Asp22-GLP-1(7-37), Val8Glu22-GLP-1(7-36)-amide, Val8Glu22-GLP-1(7-37), Val8Lys22-GLP-1(7-36)-amide, Val8Lys22-GLP-1(7-37), Val8Arg22-GLP-1(7-36)-amide, Val8Arg22-GLP-1(7-37), Val8His22-GLP-1(7-36)-amide, Val8His22-GLP-1(7-37), des(B30)-human insulin and their analogues, in which reaction of acidifying is carried out in water mixture containing less than 10% wt/wt aprotonic polar solvent, and interaction of peptide with acidifying agent of general formula I is carried out, where n is 0-8; R1 represents COOR4; R2 represents lipophilic part of molecule; R3 together with carboxyl group, to which R3 is bound, represents reaction-able ester or reaction-able N-hydroxyimidoesther; and R4 is selected from group including hydrogen, C1-12-alkyl and benzyl; in base conditions in water solution, acidifying agent being added to reaction mixture in form of solution stabilised by adding acid.

EFFECT: obtaining efficient method of peptide acidifying.

17 cl, 2 tbl, 8 ex

FIELD: chemistry, biochemistry.

SUBSTANCE: invention refers to biochemistry and can be used in pharmaceutical industry for mixture purification of natural mixtures containing plasminogen and fibrinogen from plasminogen. Plasminogen is removed from mixture containing plasminogen and fibrinogen using insoluble chromatography matrix that is covalently cross-linked with tranexamic acid through amino group with linker of length exceeding three carbon atoms. Therefore mixture mentioned above is applied on chromatographic column containing specified insoluble matrix. Then column is washed with neutral solution containing salts, while unbound material is collected.

EFFECT: extended technical feasibilities of purification of natural mixtures containing plasminogen and fibrinogen from plasminogen thus keeping original amount of fibrinogen in mixture.

15 cl, 1 dwg, 13 tbl, 6 ex

FIELD: medicine.

SUBSTANCE: invention refers to medicine, particularly to experimental pharmacology, and concerns correction of normal microflora composition in experiment. It is ensured by introduction to laboratory animals of 6-piperidinomethyl-2,3-dihydroquercetin- C21H23NO7.

EFFECT: method provides extended range of chemical agents that provide normalisation of colon microflora composition.

1 tbl, 1 ex

FIELD: veterinary.

SUBSTANCE: medication includes the following components, wt %: Sulfadimezinum 11.5-15; Trimethoprim 2.0-3.6; Colistin 2.0-3.5; glucose up to 100. Before application the medication is dissolved in warm water or colostrums at 35-37°C or fed with forage. The medication is applied daily twice in the dosage of 100 mg/kg of body weight for 3-5 days, or for 5-7 days under severe course of disease.

EFFECT: enhanced efficiency of colibacteriosis treatment for form animals.

12 tbl, 4 ex

FIELD: chemistry, biochemistry.

SUBSTANCE: invention relates to area of microbiology and biotechnology and can be applied in agriculture. Novel strain Megasphaera elsdenii is characterised by ability to utilise lactate with degree from 40% to 90% even in presence of sugars. Strain with said ability is obtained by method, which includes culturing of sample of paunch liquid. Novel strain Megasphaera elsdenii is applied in method of treatment and prevention of lactic acidosis of ruminants, in composition of veterinary preparation for treatment and prevention of said disease, in method of milk productivity increasing and in method of increasing ruminants fattening efficiency, in method of increasing growth rate and reduction of time of ruminants fattening, in method of reducing morbidity, induced by digestion disorders, and death rate among ruminants from lactic acidosis, as well as in method of increasing efficiency of conversion of ruminants diet based on concentrated forages when transferring ruminant to said diet.

EFFECT: obtaining ability to carry out wide-scale treatment and prevention of lactic acidosis.

17 cl, 3 dwg, 18 tbl

FIELD: medicine.

SUBSTANCE: invention relates medicine area, in particular, gastroenterology, and concerns treatment of GI tract dysbacteriosis. A set containing agents, possessing antagonist action on a GI tract pathogenic microflora and agents for restoration of a normal GI tract microflora (eubiotics and bacteritic preparations), and also independent GI tract electrostimulators are used for this purpose. The method of GI tract dysbacteriosis treatment combines a diet, the specified agents, and also electrostimulator insertion before and-or in the course of treatment.

EFFECT: optimum conditions for effect of eubiotics and bacteritic preparations at the expense of preliminary normalisation of GE tract motor and evacuation functions.

2 cl

FIELD: chemistry.

SUBSTANCE: invention relates to novel compounds of general formula (I), their optically active stereoisomers, as well as to pharmaceutically acceptable salts possessing properties of ORL1 and µ-opiate receptors. In general formula , R1 represents H, alkyl(1-6C), []m represents-(CH2)m-, in which m equals 0 or 1, R2 represents halogen, CF3, alkyl(1-6C), phenyl, cyano, cyanoalkyl(1-3C), hydroxy, (1-3C)alkoxy, OCF3, acyl(2-7C), trifluoroacetyl, (1-3C)alkylsulfonyl or trifluoromethylsulfonyl, and n represents integer number 0-4 on condition that when n equals 2, 3 or 4, R2 substituents can be similar or different, A represents saturated ring, []0 and []p represents -(CH2)o and -(CH2)p, and o and p independently correspond to 0, 1 or 2, R3, R4, R5 and R6 independently represent hydrogen, alkyl(1-3C), or (R4 and R6) together can form alkylene bridge, containing 1-3 carbon atoms on condition that when o equals 2, R3 represents hydrogen, and when p equals 2, R5 represents hydrogen, R7 represents H, halogen, alkyl(1-6C). Invention also relates to pharmaceutical composition, intermediate compounds for obtaining formula (1) compounds.

EFFECT: compounds can be used for preparing medication for treating disorders and diseases such as alimentary behaviour disturbances, arterial hypertension.

8 cl, 3 dwg, 1 tbl, 45 ex

FIELD: medicine.

SUBSTANCE: invention concerns experimental medicine and can be used in veterinary science. The Ultrasorb preparation is fed to the animals that are on a vivarium standard diet, within 14 days, based on 3 g/day, admixed with forage. The way prevents development of intestinal dysbacteriosis at animals.

EFFECT: prevention of intestinal dysbacteriosis at animals.

3 ex, 2 tbl

FIELD: medicine.

SUBSTANCE: 24 hours prior to the examination, children aged 3 to 8 year old takes in 100 ml lactulose (Duphalac) with water of room temperature in amount 1-1.5 liters within 4-6 hours. The second dose lactulose (Duphalac) 100 ml follows with water of room temperature in amount 1-1.5 liters. To prevent overaerogenesis and to reduce discomfort, Espumisan (Simethicone) is prescribed in dosage 15 ml 3 times a day. For children aged 8 to 18 years old, there is prescribed 200 ml lactulose (Duphalac) with water of room temperature in amount 1.5-2.0 litres within 4-6 hours followed with the second dose of lactulose (Duphalac) 200 ml with water of room temperature in amount 1-1.5 litres. Additionally Espumisan (Simethicone) is introduced in dosage 15 ml 3 times a day or 3-6 capsules 3 times a day.

EFFECT: effective cleansing of gastrointestinal tract in children of different age, thus ensuring good visualisation of large intestine mucosa and decreasing drug load on organism of younger children, preserving normal microflora of large intestine and preventing overaerogenesis.

2 ex

FIELD: medicine.

SUBSTANCE: there is prescribed gluten-free diet with eliminating cereals and additional order of protein: meat 100.0 g, cottage cheese 100.0 g, or sour cream 100.0 g daily. Creon is dosed 10000 ME at mealtime within 2 weeks. It is combined with physiotherapy exercises (PTE) within 20-30 minutes. During afternoon, every second day circular douche is applied at pressure 1.5-2 atmospheres, water temperature 36-35°C for 3-4 minutes within therapeutic course 10-12 procedures. On another days, radon baths are taken at water temperature 36°C, dosed 0.75 kBq/l for 8-10 minutes within therapeutic course 8-10 procedures. Electrophoresis is applied daily with 50% Dimexide solution (±) for small intestine projection by transverse technique, current density 0.05 mA/cm2 within 12-15 minutes within therapeutic course 10-12. The BAPs VC-20, VC-22, E-36, Gi-4 are exposed to red and infrared light daily within 1.5-2 minutes per each point within therapeutic course 10-12 procedures.

EFFECT: reduced specific immunologic markers of autoimmune intestine inflammations and cytokine generation level, normalised immune response of an organism, normalised hormonal and exchange processes.

2 ex, 2 tbl

FIELD: medicine.

SUBSTANCE: invention relates to experimental medicine and concerns pyelonephritis prevention in experiment. During 10 days preparation "Ultrasorb" is introduced to experimental animals with food in terms of 3 g/day. Then pyelonephritis is modelled by rectal infection with uropathogenic strain of lactose-negative E.Coli at the background of cold stress.

EFFECT: absence or reliable reduction of kidney tissue inflammation in animals which obtained "Ultrasorb".

4 ex

FIELD: medicine.

SUBSTANCE: invention relates to medicine, namely to pediatrics and gastroenterology and can be used for treating intestine disbacteriosis in children less than 1 year old. For this purpose bifidumbacterin forte is introduced. First 2-3 days preparation is introduced by 25-30 doses per day in 4 takings. During next 3-4 weeks bifidumbacterin forte is introduced by 5 doses 2 times a day.

EFFECT: method allows increasing efficiency of treatment of disbacteriosis in children less than 1 year old due to application of definite scheme of preparation introduction first in optimally high dose mode, then in supporting doses.

FIELD: chemistry, biochemistry.

SUBSTANCE: invention relates to biotechnology, namely to obtaining composition of individual proteolytic ferments and can be applied in medicine, cosmetology. In contents of composition are included not less than five proteases with molecular weight from 23 to 36 kilodalton and with -N-end amino acid successions: I V G G T E V T P G E I P Y Q L S L Q D -; I V G G T E V T P G E I P Y Q L S F Q D -; I V G G Q E A S P G S W P X Q V G L F F -; I V G G S E A T S G Q F P Y Q X S F Q D -; I V G G Q E A T P H T W V H Q V A L F I -; I V G G Q E A T P H T X V H Q V A L F I -; A M D X T A Y X D Y D E I Q A X L K G L -; A F D X T N Y N T F E E I N S I L D G V -; A A I L G D E Y L X S G G V V P Y V F G - Obtained composition is applied for treatment of purulent-necrotic and cicatricial changes of tissues in contents of pharmacological composition or elimination of purulent-necrotic and cicatricial changes of tissues in contents of cosmetic composition.

EFFECT: obtaining possibility to hydrolyse protein substrates to individual amino acids, obtained substance is highly efficient and does not cause allergic reactions when applied for a long time

6 cl, 3 tbl, 4 ex

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