Using lactobacillus paracasei cncm i-2116 to treat irritable bowel syndrome

FIELD: chemistry.

SUBSTANCE: invention relates to use of the Lactobacillus paracasei CNCM I-2116 strain to treat irritable bowel syndrome. A probiotic includes dead Lactobacillus paracasei CNCM I-2116 bacteria, a fermentation substrate and/or material made from Lactobacillus paracasei CNCM I-2116.

EFFECT: invention provides the capacity to normalise post-infection hyper-contractible state of intestinal muscles.

2 cl, 4 dwg, 2 ex

 

The technical field

This invention relates to the use of selected probiotic for the preparation of the nutritional composition or the medicinal product for the prevention or treatment of pain bowel or intestinal discomfort, motor dysfunction in the gut, neuromuscular disorders of the intestine and complications after infection of the intestine.

This invention relates also to a method of prevention or treatment of pain bowel or intestinal discomfort and persistent motor dysfunction in the gut.

Prior art

Irritable bowel syndrome (IBS, mucous colitis) is a complex disease of the lower section of the intestinal tract, which includes the combination of abdominal pain with alternating constipation and diarrhea, and these symptoms are often complicated by emotional stress. Other symptoms include bloating and distension of the abdomen. This is the most common functional bowel disease, which establish the diagnosis the doctors and gastroenterologists, and affects up to one in five Americans. The diverse nature of the symptoms together with psychological disorders, or without interfering with the progress in the understanding of the mechanisms, but as if there is a neuromuscular problem is in the lower part of the intestinal tract or decrease the resistance to stretching and irritative symptoms such the ICA. A small but significant subgroup of patients with IBS is marked by the sudden appearance of IBS symptoms after a bout of gastroenteritis (Spiller, "Post-infectious irritable bowel syndrome, Gastroenterology 2003, 124: 1662-1671).

Infant colic is a behavioral syndrome that is characterized by paroxysmal, excessive and inconsolable crying without a cause in healthy infants in the first months of life. Usually starts crying at the same time each day, increasing after lunch, evening and night and lasts 2 or 3 hours. This is a very common problem that affects between 15 and 30% of children in the first 3 months of life. Despite its frequency, tangible suffering and more than 40 years of research, the etiology of infantile colic is still not revealed until the end, however, it is assumed that when infant colic, as with IBS, plays the role of a violation of gastrointestinal function (Lindberg, "Infantile colic and small intestinal function: a nutritional problem?", Acta Paediatr. 1999, Suppl. 430: 58-60).

In order to study neuromuscular disorders in the gastrointestinal tract and to explore the effectiveness of a potential treatment, researchers have developed a model in mice, have neuromuscular disorders caused by infection of mice by the parasitic nematode Trichinella spiralis. As noted, for example, Barbara et al. (Barbara et al, "Persistent intestinal neuromuscular dysfunctions after acute nematode infection in mice", Gastroenterology 1997, 113: 224-1332). it is known that such contamination leads to a transient inflammation of the intestine, but inflammation-induced changes in the functioning of smooth muscle and enteric nerves persist after termination of the inflammatory response in the mucosa and after the restoration of its structure, and in the absence of parasites in the intestinal lumen. Barbara et al. of the opinion that this model resembles observations on the people who have maintained a neuromotor dysfunction rectum and sigmoid colon after recovery from acute gastritis caused by Salmonella, or disorders of gastric emptying and peristalsis in patients in remission from ulcerative colitis. In a later article in the same group offers this model as a model of post-infectious bowel dysfunction, resembling the clinical category of post-infectious irritable bowel syndrome (Barbara et al., "Role of immunologic factors and cyclooxygenase 2 in persistent postinfective enteric muscle dysfunction in mice", Gastroenterology 2001, 120: 1729-1736).

Probiotics are usually defined as a food additive with live microorganisms, which is a beneficial effect on a man or an animal host by improving its intestinal microbial balance. Up to the present time reported or anticipated several different beneficial action of probiotics, such as suppression of infection Heliobacter (EP 05903), increased resistance to colonization, in particular, in respect of Clostridium species, reduction of serum cholesterol, the effect on the host immune system, for example, on the level of humoral and cellular immune system.

EP 0768375 describes Bifidobacteria, which can be implanted in the flora of the intestine to attach to intestinal cells and competitive elimination of pathogenic bacteria to intestinal cells.

In WO 98/00035 described enteral compositions containing several lactic acid bacteria, which have been shown to stimulate the immune system, as measured by the number of T CD4+ peripheral blood lymphocytes.

When people and animals are suffering from intestinal discomfort or pain of the intestines, these symptoms often are the symptoms of intestinal disorders or, in other words, neuro-mepacrine disorders of the intestine.

Individuals of any age and in many circumstances suffer from neuromuscular disorders of the intestine. Examples are infants suffering from spastic abdominal pain (colic) and recurring abdominal pain, women suffering from pain in the intestines, caused by hormonal cycle, and many others.

In the context of irritable bowel syndrome (IBS) prior art has not considered the effect of probiotics on this specific the initial syndrome. In a recent study (Niedzielin K et al, A controlled, double-blind, randomized study on the efficacy of Lactobacillus plantarum 299V in patients with irritable bowel syndrome, European Journal of Gastroenterology & Hepatology 2001, 13:1143-1147) found that probiotics may play a role in the regulation of peristalsis of the digestive tract.

On the other hand, the article O'sullivan MA and O Morain (Bacterial supplementation in the irritable bowel syndrome. A randomized double-blind placebo-controlled crossover study, Dig Liv Dis 2000 May; 32(4):302-4) were not found significant differences between the mean values of the strain Lactobacillus casei GG and placebo. Other messages of the previous level confirmed by the recent discovery.

Thompson WG (Probiotics for irritable bowel syndrome: a light in the darkness Eur. J gastroenterol Hepatol 13:1135-1136, 2001) discusses the potential treatment of IBS with probiotics.

The purpose of this invention is to alleviate any pain or any discomfort associated with altered neuromuscular regulation and altered motor function in the intestine.

Another purpose of this invention is the reduction and/or weakening of neuromuscular disorders of the intestine associated with any possible circumstance in the life of the individual.

Brief description of the invention

Notable is the fact that the probiotic microorganisms and their metabolites and/or their substrates growth affect neuromuscular regulation in the intestine. In particular, it was shown that specific probiotics applicable for humanistinen musculoskeletal disorders in the gastrointestinal tract, in particular, after the infection.

Thus, in the first aspect, the invention provides the use of selected probiotic or a mixture of selected probiotics in the preparation of a nutritional composition or the medicinal product for the prevention or treatment of pain bowel or intestinal discomfort.

In the second aspect, the invention provides the use of selected probiotic or a mixture of selected probiotics in the preparation of a nutritional composition or the medicinal product for the prevention or treatment of motor dysfunction in the gut.

In the third aspect, the invention provides the use of selected probiotic or a mixture of selected probiotics in the preparation of a nutritional composition or the medicinal product for the prevention or treatment of neuromuscular disorders of the intestine.

In the fourth aspect, the invention provides the use of selected probiotic or a mixture of selected probiotics in the preparation of a nutritional composition or the medicinal product for the prevention or treatment of complications after infection of the intestine.

In the following aspect, the invention provides a method for prevention or treatment of pain bowel or intestinal discomfort the introduction of the human or animal an effective amount of a selected probiotic sludge is a mixture of selected probiotics.

In another aspect, the invention provides a method for prevention or treatment of motor dysfunction or post-infection of hyperperistalsis in the gut which the stages of introducing an effective amount of a selected probiotic or a mixture of selected probiotics.

The advantage of this invention is that it provides the possibility of treating or preventing neuromuscular disorders of the intestine and associated symptoms, problems, or conditions of the bowel disease.

Another advantage of this invention is that it provides the possibility of treating or preventing neuromuscular disorders of intestine without the introduction of pharmaceutical medicines, but on the basis of probiotic microorganisms or their derivatives in the food category.

Description of figures

Figure 1 shows the area under the curve (AUC) of the intensity of the contraction of muscle tissue of the intestine, taken from the body of the host that was infected by the nematode parasite, and 10 days after infection, was received in the form of feed of different probiotics, and control. Stimulation was performed with karakalem in various concentrations (see details in example 2). AUC is a measure of the intensity and nepozadovali contraction of the muscles of the intestine and a measure of the degree of neuromuscular the breach is, developed during infection. The symbols have the following meanings: ♦ control, Lactobacillus acidophilus (johnsonii), × Bifidobacterium longum, * Bifidobacterium lactis, Δ Lactobacillus paracasei. You can see that probiotics usually reduce AUC, whereby different strains have more or less effect.

Figure 2 shows the tonic contraction (A) and phase decline (In) muscle tissue, as described for figure 1, but stimulated by an electric field, and compares organisms-owners receiving in the form of feed probiotic strain Lactobacillus paracasei (CNCM I-2116) in a live state (L.p.), dead state (dead L.p.) and only the supernatant of the medium (Sn). The terms "tonic increase" and "phase reduction" are explained in example 2 and figure 3. As you can see, all derived from probiotic feed (live or dead bacteria, the supernatant) are clearly lower tonic increase and the phase reduction than the control (MRS).

Figure 3 explains the principle method used to determine neuromuscular disorders after infection. This curve shows the recording of muscle stimulation in vitro. Before stimulation register base (source) tone (1) and baseline (reference) phase reduction (4). After stimulation of the muscles, which is registered as stimulated tone (2) and stimulated phase reduction (5) Can be also calculated the horse under the curve (AUC) (7). For statistics compare tonic contraction (3), the phase reduction (6) and AUC (7) control treatment options.

Detailed description of the invention

In the context of this description the word "includes" means "includes, among other things". It should not be regarded as "consists only of".

For purposes of this invention, the term "selected probiotic microorganisms" or simply "selected probiotic" refers to any microorganism that is able to show beneficial effects reported here, or combination or mixture of such probiotics. Thus, the probiotic may be selected from known probiotic strains. However, the microorganism, which is still unknown, it has probiotic properties, may be having a beneficial effect in accordance with this invention, and therefore will be included in the term probiotic.

In the context of this invention, the term "food composition" is intended to encompass any suitable food material. Thus, the food composition may be a product intended for human consumption, but this term also includes products to be used by animals, for example, domestic animals such as dogs, cats, rabbits, Guinea pigs, mice, rats, birds (e.g. parrots), reptiles of ireby. However, this term also includes food, eat other domesticated animals, for example, feed products for cattle, for example, cattle, horses, pigs, sheep, goats, buffaloes, camels, etc.

The food composition may be a food product intended for human consumption, for example, drink, tile, snack, ice cream, milk product, for example, chilled or storable dairy product, a confectionery product, a grain product, such as grain Breakfast, frozen product intended for consumption after heating in the microwave or oven-ready food product, frozen food product or nutritional mixture.

Nutrient mixture includes any nutritionally complete or additional mixture. It can be commonly used nutrient mixture, the mixture for children and infants mixture for elderly patients, for patients in intensive care or special adaptive mixture for patients suffering from, for example, from a particular disease. For example, nutrient mixture can be adapted for patients suffering from nutrition-related problems, such as Crohn's disease, hyperglycemia, obesity, weight loss, diarrhea, diarrhoea, phenylketonuria, hepatitis, acute or chronic the Skye renal failure, additional area. Such mixtures can be recovered, i.e. to be in a dried form, or ready to drink, for example, in the form of liquid mixtures.

In the context of this invention, the term "enteric neuromuscular disorders" includes all associated with pain or discomfort symptoms that are associated with abnormal or impaired intestinal muscle contractions, airway or peristalsis. For example, these violations associated with disturbing bloating or disturbed defecation, with spastic pains (colic) in infants and/or children with intussusception of the bowel in humans or domestic animals, with the broken time of passage through the intestine, after injection of intestinal parasites, such as nematodes and pathogenic bacteria.

Additional gastrointestinal neuromuscular disorders in the context of this invention include disorders associated with breast age, for example, as part of the problem spastic pain (colic) infants violations associated with exercise, including the manifestation of painful spasms induced by exercise, and neuromuscular problems associated with intensive exercise and athletics, disorders associated with pregnancy, and disorders associated with childbirth, violations of associerad is installed with clinical patients, with others, not related to this damage, injury or infection, but clinical treatment or situation cause the loss of intestinal neuromuscular regulation, including antibiotics, immobilization and parenteral or enteral feeding, disorders associated with aging and loss of neuromuscular regulation associated with reduced activity, diets low in fiber and changes in the microflora, disorders associated with unusual dietary habits or unusual lifestyle, including alcohol, drugs, giving neuromuscular side effects of altered gravity astronauts, intense heat or severe cold and problems rehydration.

"Motor dysfunction" is the equivalent of enteric neuromuscular disorders that usually accompany infection of the gastrointestinal tract of a human or animal pathogens, such as nematodes, cestodes and some bacteria, for example, Helicobacter pylori, or Salmonella. Motor dysfunction can also occur during or after inflammation due to other causes.

In many cases, intestinal neuromuscular disorders and motor dysfunction are persistent and last for a long time.

Preferably, the application is in accordance with this invention refers to pain or discomfort bowel, related to muscle disorders of the intestine or associated with muscle disorders of the intestine.

"Complications" are violations or deviations from the healthy state stored firmly after infection, even if parasites or other infectious agents have been eliminated from the host. It is assumed that they are usually irreversible damage that is induced in the host.

"Derived from probiotics material" in the context of this invention includes living or dead probiotics, environment, obtained by fermentation with probiotic metabolites detected in the medium after fermentation, and its derivatives, such as concentrates, such as fermentation substrate, supernatant and/or retentate environment after elimination of probiotic bacteria, for example, by filtration or centrifugation.

In one embodiment of the present invention the pain or discomfort of intestinal motor dysfunction and neuromuscular disorders of the intestine caused by infection of the intestine by pathogens.

Pathogens in the context of the present invention include microorganisms that can infect the intestines of individuals and call status of the disease. Thus, the term "pathogen" includes parasites, bacteria, viruses, multicellular organisms, such as nematodes and the other worms.

Selection of probiotic

As the probiotic may be selected from any suitable organism. Preferably, the probiotic in accordance with this invention are selected from the microorganisms with beneficial effects on health and well-being of humans or animals.

The literature mentions some of the microorganisms from which can be selected probiotics in accordance with this invention. For example, EP 0862863 A1, in particular, on page 3, in lines 25-37 contains a list from which can be selected probiotic in accordance with this invention.

Examples of suitable probiotic micro-organisms include yeast, such as Saccharomyces, Debaromyces, Candida, Pichia and Torulopsis, moulds such as Aspergillus, Rhizopus, Mucor and Penicillium, bacteria such as the genera Bifidobacterium, Bacteroides, Clostridium, Fusobacterium, Melissococcus, Propionibacterium, Streptococcus, Enterococcus, Lactococcus, Kocuria, Staphylococcus, Peptostreptococcus, Bacillus, Pediococcus, Micrococcus, Leuconostoc, Weissella, Aerococcus, Oenococcus and Lactobacillus.

Specific examples of suitable probiotic micro-organisms are: Specific examples of suitable probiotic micro-organisms are: Aspergillus niger, A. oryzae, Bacillus coagulans, B. lentus, B. licheniformis, B. mesentericus, B. pumilus, B. subtilis, B. natto, Bacteroides amylophilus, .capillosus, .ruminocola, .suis. Bifidobacterium adolescentis, B. animalis, B. breve, B. bifidum, B. infantis, B. lactis, B. longum, B. pseudolongum, B. thermophilum, Candida pintolepesti, Clostridium butyricum, Enterococcus cremoris, E. diacetylactis, E. faecium, E. intermedius, E. lactis, E. muntdi, E. thermophilus, E. coli, Kluyveromyes fragilis, Lactobacillus acidophilus, L. alimentarius, L. amylovorus, L. crispatus, L. brevis, L. casei, L. curvatus, L. cellobiosus, L. delbrueckii ss. bulgaricus, L. farciminis, L. fermentum, L. gasseri, L. helveticus, L. lactis, L. plantarum, L. johnsonii, L reuteri, L. rhamnosus, L. sakei, L. salivarius, Leuconostoc mesenteroides, P. cereviseae (damnosus), Pediococcus acidilactici, P. pentosaceus, Propionibacterium freudenreichii, Prop, shermanii, Saccharomyces cereviseae, Staphylococcus carnosus, Staph, xylosus, Streptococcus infantarius, Strep, salivarius ss. thermophilus, Strep, thermophilus, Strep. lactis.

For example, the probiotic strain or strains can be selected from the group consisting of Bacillus licheniformis (DSM 5749), B. subtilis (DSM 5750), Bifidobacterium lactis (DSM 20215), strains of Enterococcus faecium (e.g., NCIMB 10415; NCIMB 11181; NCIMB 30098; DSM 3520; DSM 4788; DSM 4789; DSM 5464; DSM 7134; CECT 4515), E. mundtii (CNCM MA 27/4E), strains of Saccharomyces cerevisiae (for example, BCCM/MUCL 39885; CBS 493 94; CNCM I-1077; CNCM I-1079; NCYC Sc47), Lactobacillus casei (NCIMB 30096), L. farciminis (CNCM MA 67/4 R), L. johnsonii (I-1225 CNCM), Lactobacillus paracasei (I-2116 CNCM), L. planterum (CNCM I-840), L. rhamnosus (DSM 7133), P. acidilactici (CNCM MA 18/5 M), Streptococcus infantarius (CNCM I-841), Streptococcus thermophilus (Chr. Hansen) and, for example, mixtures thereof.

Additional examples of probiotic species with sample deposited strains of these species in accordance with this invention can be selected from the group consisting of Lactobacillus reuteri (CNCM I-2452, CNCM I-2448, CNCM I-2450, CNCM I-2451), Lactobacillus rhamnosus (CNCM I-2449), Lactobacillus acidophilus (CNCM I-2453), and mixtures thereof. The strains mentioned in this paragraph can be, for example, suitable for Pets.

Effective probiotic in accordance with this invention can be selected using the screening of the Pref is given to the above list. Although it may potentially be used with any method of screening was relatively quick method developed in Barbara G. Vallance BA, Collins SM Persistent intestinal neuromuscular dysfunction after acute nematode infection in mice, Gastroenterology 1997; 113: 1224-1232. This reference describes a model for measuring the intensity of enteric neuromuscular disorders.

Thus, suitable probiotic may be selected using stages:

- select at least one organism separate species of animal or people who suffer from intestinal neuromuscular disorders

- enteral introduction of this organism obtained from the probiotic material

the first measurement of contractility using intestinal muscle tissue of the body,

- compare this first contractility of the second contractile ability of the negative control and

selection of probiotic strain that caused the first reduced contractility of the muscle tissue of the body that received the probiotic compared with the negative control.

The term "negative control" in the context of screening for selection of specific probiotic strains indicates intestinal muscle tissue from an organism suffering from intestinal neuromuscular disorders, which did not enter enterline obtained from probiotic Mat is Rial.

This screening stage provides the first measurement of contractility using intestinal muscle tissues and in addition stage of this first comparison contractility of the second contractile ability of the negative control.

Contractility can be measured in any suitable way. For example, using techniques of Barbara G. Vallance BA, Collins SM (see above). See, in particular, the Chapter "Tissue Preparation for Contractility Studies, "Measurement of Contraction, Parameters of Electrical Field Stimulation", "Drugs and Solutions" and "Data Expression and Statistical Analysis", pages 1225-1226, which is incorporated herein by reference.

Thus, contractility measured in vitro, i.e. excision of segments of the intestine, for example, proximal jejunum, the fixing of these segments in the bath tissue, inducing muscle contractions, for example, chemical or electrical stimulator, and the registration of the reduction using a suitable data processing unit.

Measurement of basal tone (1), enhanced tone (2) and the tonic contraction (tonic convulsions) (3)and basal phase (4), stimulated phase reduction (5), the phase reduction (6) and the area under the curve (7), as shown in figure 3, can serve as parameters for contractility.

Selected probiotic in with the accordance with this invention is a probiotic, which of the above method of screening reduces intestinal muscle contractility in the body, suffering from intestinal neuromuscular disorders, for example, in comparison with negative control.

In the embodiment of the present invention is selected probiotic is a probiotic that is capable of murine models to act on the pathogen-induced immune response in such a way that it significantly reduces the number of redundant Th2 cytokines. Preferably, released Th2 cytokines are IL-4 and/or IL-13.

"Significant" in the context of this invention denotes a statistically significant difference in relation to P<0,1, preferably p<0,05, which is obtained when comparing infected mice that receive probiotics (option handling), with a negative control.

Usually cytokines can be measured in the product of the longitudinal muscles of the muscular layer of the intestine (LMMP) in a predetermined period after infection. Preferably, the cytokines measured in LMMP 14 days after infection. The concentration of cytokines can be measured using commercially produced sets and using the manufacturers instructions.

The preferred method of assessing whether the probiotic significantly reduce redundant Th2 cytokines, includes mouse model and presents n the same.

Female mice NIH swiss (aged 6-8 weeks) (each) administered daily feeding through a stomach tube 375 larvae of Trichinella spiralis (see how Barbara G. et al, above).

To determine the effect of probiotics infected mice administered via a stomach tube from day 10 to day 21 after infection, 100 μl of 1010 probiotic microorganisms (bacteria, yeast, etc.) using 100 ál of MRS as a negative control.

LMMP can be prepared by excision of the entire jejunum, washing in cold sterile SFR and cut into 4 sections. Brisgau removed and pieces of intestine is placed on a glass rod. Muscular layer soskrebajut using a clean cotton swab, quickly frozen and stored at -70°C until analysis. The successful allocation of muscles can be confirmed by histological evaluation. Muscle tissue was placed in 1 ml lisanova buffer containing 10% NP-40, 10 mg/ml PMSF in isopropanol, 1 mg/ml Aprotinin and 1 mg/ml leupeptin. After homogenization of muscle tissue to measure the total concentration of protein (protein analysis, Bio-Rad, Hercules, CA, USA), and the sample is divided into aliquots and stored at -70°C for further analysis.

The concentration of IL-4 and IL-13 (Th2 cytokines) and other inflammatory mediators of TGF-β1 and PGE2 can be measured in LMMP using commercial kits (Quantikine M Murine, Minneapolis, MN, USA) in accordance with the instructions Izgotovitel is.

In another embodiment, the present invention probiotic is a probiotic that is capable of murine models to influence pathogen-induced inflammation in such a way that it significantly reduces the expression or the concentration of MOR-2, TGF-β1 or PGE2 in the LMMP.

The concentration of TGF-β1 or PGE2 can be measured by analysis of the LMMP using commercially available kits listed above for IL-4 and IL-13.

The preferred way to assess significant differences, in particular, the expression of TGF-β1 and MOR-2 in LMMP infected mice below.

The expression of the messenger RNA (mRNA) of TGF-β1 and MOR-2 measured in LMMP 14 days after infection. Total RNA isolated from the LMMP using one-stage method (Chonczynski R, Sacci N "Single step method of RNA isolation by acid guanidium thiocyanate-phenol-chloroform extraction," Ann Biochem 162: 156-159; 1987).

Reverse transcription and PCR reactions performed as described Verdu EF et al (Modulatory effect of estrogen in two murine models of experimental colitis. Am J Physiol gastrointest Liver Physiol 2000; 283: G27-36).

Using the following primers. HPRT (gipoksantin-guanine-phosphoribosyltransferase used as a control for standardization): sense 5'-GTT GGA TAC AGG CCA GAC TTT GTT G-3', antisense 5'-GAT TC A ACT TGC GCT CAT CTT AGG C-3' (Svetic A, "Cytokine expression after immunization))' J unmun 147: 2391-7). TGFp: sense 5'-TCA CCC GCG TGC HUNDRED ATG GT-3' and antisense 5'-GGA GCT GAA GCA ATA GTT GG-3' (Derynck R, Rhee L, Nucleic Acids Res 1987; 15: 3187-97); MOR-2: sense 5'TGG TGC CGG GTC TGA TG TG-3' and antisense 5'-GCA ATG CGG TTC TGA TAC TG-3' (Gustafson-Svard et al., Cyclooxygenase-1 and cyclooxygenase-2 gene expression in human colorectal adenocarcinomas and azomethan induced colonic tumours in rats. Gut 1996; 38: 79-84).

To exclude amplification of genomic DNA contaminating samples, experiments were performed using RNA as a substrate for PCR. After amplification, 15 μl of PCR products were separated by electrophoresis in 2% agarose gel, visualized by staining with ethidium bromide and photographed using a Polaroid land film type 55 (Kodak, Rochester, NY). The negatives used for densitometric quantification of the intensity of the bands using software Kodak Digital Science ID 2.0 Image Analysis. These results were normalized relative to the HPRT gene "household" and was expressed as the ratio of mRNA expression of cytokines to HPRT mRNA.

In one embodiment, the present invention is selected probiotic is Bifidobacterium. Preferably, it is Bifidobacterium lactis or Bifidobacterium longum.

In the following embodiment, the present invention is selected probiotic is Lactobacillus paracasei.

In another embodiment, the present invention is selected probiotic selected from the group consisting of Bifidobacterium longum (CNCM I-2170), Bifidobacterium lactis (German Culture Collection: DSM20215), Lactobacillus paracasei (CNCM I-2116, CNCM I-1292), and mixtures thereof.

In another embodiment of the present invention the probiotic includes dead breaking the practical bacteria, fermentation substrate and/or obtained from the probiotic material.

Optional, probiotics include their fermentation substrate as a prebiotic. Specialist with expertise in this area is usually known fermentation substrates of probiotics. For example, Bifidobacteria can use inulin and/or oligofructose as a fermentation substrate.

Getting probiotics

Specialist qualification in this field know how to get the selected probiotic microorganism. Probiotics can be obtained from commercial suppliers or can be obtained usually fermentation process and, optionally, drying. Specific strains often have specific preferences regarding the environment or substrate, which is known to a specialist with expertise in this field.

These microorganisms may be in dried form or, for example, in the form of spores, microorganisms that form spores. Drying of microorganisms after fermentation is known to a specialist with expertise in this area. See, for example, EP 0818529 (SOCIETE DES PRODUITS NESTLE), which describes a method of spray drying, or WO 0144440 (INRA). Usually bacterial concentrate microorganisms from the environment and is dried by spray drying, drying in a fluidized bed, by lyophilization (freeze-drying) or the other adequate drying process. For example, microorganisms can be mixed with the material carrier, such as a carbohydrate, such as sucrose, lactose or maltodextrin, lipid or protein, e.g., milk, during drying or before drying.

However, these microorganisms must not necessarily be present in dried form. It may be convenient to mix them directly after fermentation with the food product for optional execution of the drying process after that. This approach is described in WO 02065840 (SOCIETE DES PRODUITS NESTLE). Similarly probiotics can, theoretically, be used for human consumption directly after fermentation. Further processing, for example, for the preparation of suitable food, is not a prerequisite for the beneficial properties of probiotics.

Many probiotics suitable for this invention are commercially available and can be obtained in powdered form from numerous suppliers, such as, Bifidobacterium lactis (DSM 20215) can be obtained from Ch.Hansen.

Specialist with expertise in this area is known for numerous different suppliers of probiotics. Some suppliers deliver probiotics encapsulated in specific form to guarantee a high level of survival of microorganisms during passage through the gastro-intestinal t the act or during storage or high shelf life of this product.

An example of a product containing microorganisms having increased storage stability without excessive losses, is described in EP 0180743, as well as in WO 02065840 (SOCEETE DES PRODUITS NESTLE).

Probiotics in accordance with this invention may be used enterline in any form. They can be added to a food composition such as a food product. On the other hand, they may be used directly, for example, in dried form or directly after receipt of biomass through fermentation.

Probiotics can for example be used in the form of a fermented milk product, such as a chilled dairy product, yogurt or fresh cheese. In these latter cases, the probiotic can also be used directly for the preparation of the fermented product and therefore has at least a dual function: probiotic function in the context of the present invention and the function of the fermentation substrate, such as milk, to get yogurt.

If the probiotic is added to the food mixture to a specialist with expertise in this area will be known of the possibility of achieving this. Dried, for example, dried by spray drying, bacteria, such as obtained by the process described in EP 0818529, can be added directly to the food mixture in powder is obraznoi form or any other, optional dried food product. For example, powdered probiotic preparation may be added to the food mixture, grain breakfasts, salads, slice of bread before eating.

Nutrient mixture containing specific probiotics are currently commercially available. For example, mixtures for subsequent observations that contain probiotics are sold Nestle, for example, a product like "NAN2 or NIDINA2 - with Bifidus", which is specially adapted for babies, can be used for the purpose of this invention, yet provided an effective number.

Alternatively, the dried probiotics can be added to the liquid product, for example, soft drink or an alcoholic drink. If you use bacteria in a living state, a liquid product containing probiotics must be used relatively quickly after the addition of probiotics. However, if bacteria are added to the product, stable during storage, quick use of it is not needed until probiotics are stable in non-alcoholic or alcoholic beverage.

WO 9810666 describes the simultaneous method of drying food composition and culture of probiotic bacteria. Thus, probiotics can be dried simultaneously with juices, milk products and the milk and vegetable, for example, obtaining a dried product containing probiotics. This product can then be recovered using water liquid.

Probiotics

Although it is not mandatory, probiotic bacteria can be used in a live condition, in order of probiotic microorganisms in intact form achieved intestine and colon, the latter of which can be colonized by them. If this is the case, a sufficient dose of live bacteria is usually consumed per day to achieve successful colonization. Specialist with qualifications in this field are aware of these daily doses that depend on microorganisms, but are typically in the range 106-1014preferably 107-1013colony forming units (CFU) per day.

In the context of this invention an effective amount of a live probiotic, which must be entered man weighing about 65 kg, will preferably be in the range of 1010-1014, more preferably 1011-1013most preferably 4×1012SOME day.

The preferred number of live probiotic corresponds to approximately one Cup 2 tbsp yogurt a day, prepared with probiotic strain that is commercially available. One day then the Oia food product or, if preferred are several daily servings, all the portions together will usually enriched with an effective amount of probiotics mentioned above.

However, the effects of the present invention can be also achieved with the dead probiotics, fermented environments or simply with the substrate for probiotics, which usually is a prebiotic fiber.

Thus, fermented environment, even if they essentially do not contain probiotics, but contain metabolites of probiotics, can be used for this invention;

In other words, dead or alive, probiotics, their environment, the substrate or metabolites can be directly added to food in the same manner or a similar manner described above for live probiotics more specifically. Fermented medium, substrate or metabolites can be separated from the bacteria after fermentation, for example, by centrifugation or filtration. The supernatant or the filtrate can be concentrated, chilled, frozen, dried, e.g. by spray drying, or directly used for enteral administration individual. If the fermented medium is dried, it can be turned into powder and, as described above for live probiotics added to any food product.

If su is inatant or fermentation medium should be man, the effective amount is in the range of 0.5 to 3 DL, preferably 1-2 DL environment for growing, collected after 30-50 hours, preferably 45-50 hours of bacterial growth. When the density of bacteria evaluate when OD600 nm, routine get OD 2-7, what is the corresponding increase 2-7×10 bacteria ml of the Supernatant can be entered after the removal of bacteria, for example, by filtering.

An effective amount of supernatant corresponds to the Cup 1-2 tbsp yogurt a day, cooked with selected probiotic, commercially available.

In the case of animals, such as Pets, the corresponding effective number of live bacteria or supernatant is calculated as a function of body weight.

You can also homogenize fermented environment, including probiotics and further processed normally destroyed probiotics together with the environment.

As already mentioned, the substrate of probiotics, such as dietary fiber, which stimulates specific probiotics may be used for this invention. This method is an indirect way of achieving effects in accordance with this invention. By stimulating the growth of specific probiotic strains in the gastrointestinal tract can be achieved the same effects as the effects reported here.

The following PR the measures are given only as an illustration and in no way should be construed as limiting the subject matter of this application.

Examples 1 and 2 below have as its objective to test whether violations of the intestine, which develop after a transient infection of the intestinal mucosa, and intestinal neuromuscular disorders in General be prevented or treated probiotic Supplement.

To test these questions used a murine model, which is persistent neuromuscular disorders after acute cases of infection with Trichinella spiralis.

Thus, it was found that probiotic bacteria can turn persistent intestinal neuromuscular disorders after intestinal infections.

These first results suggest that probiotic bacteria can interfere with the load of parasites during intestinal infections, and also that some of the long-term gastrointestinal complications resulting from these infections can be converted probiotics, even when the introduction starts after the establishment of parasitic infection. These effects, as was shown for the first time, to a high degree depend on the specific probiotic.

Example 1: probiotics for the prevention of infection with T. spiralis in mice

MATERIALS AND METHODS

For this experiment took the following are deposited in the Collection Nationale de Cultures de Microorganismes" (CNCM) strains, as well as commercially available strain of probiotic:

Lactobacillus acidphilus (johnsonii) (CNCM I-1225);

Lactobacillus paracasei (CNCM I-2116);

Bifidobacterium longum (CNCM I-2170);

Bifidobacterium lactis (German Culture Collection: DSM20215), purchased from Christian Hansen BioSystems A/S (CHL), 10-12 Boge Alle, P.O. Box 407, DK-2970 Horsholm, Denmark.

The preparations of probiotics and two control environment MRS or PBS was administered via a stomach tube to female mice NIH swiss (n=5 per group) once daily for 10 days before infection with T. spiralis (375 larvae). The introduction of the probiotic continued throughout the experiment. Nine days after infection with T. spiralis, mice were killed to count worms and determining the activity of myeloperoxidase (MPO).

Enter once daily through a stomach tube quantities were 1×109bacteria/100 μl medium for growing/mouse/day of each of the bacteria and 100 µl of the filtered environment for growing/mouse/day in experiments only with the supernatant.

RESULTS

There were differences in the number of worms between mice pretreated MRS or PBS, and, therefore, all further experiments used the MRS as a single control group.

It was found that mice treated with Bifidobacterium lactis, tended to have a lower number of worms than mice, pre-treated and MRS PBS. On the other hand, Lactobacillus acidophilus, apparently, increased load of worms. Other strains within the tested time period and doses in view of the Momus not had a significant impact on the load of worms.

Thus, it was found that different strains of probiotics had differential effects on the load of worms in the preliminary introduction. Specific strains of probiotics, for example, Bifidobacterium lactis, is able to reduce the infectious load of intestinal parasites such as hookworms.

Example 2: probiotics for the treatment of complications of infections of T. spiralis in mice

MATERIALS AND METHODS

In the second experiment, mice were first infected Trichinella spiralis (375 larvae) and was administered via a stomach tube once daily for five probiotics mentioned above, or MRS from day 10 to day 21 after infection, and then mice were killed and took the cloth for the experiments on contractility in vitro. In the model T. spiralis despite the expulsion of parasites and inflammation of the intestinal mucosa after 21 days after infection remain neuromuscular disorders (hyperactivity).

Neuromuscular function was assessed by measurement of contractility in vitro after pharmacological (carbachol) or electrical stimulation (EFS) of intestinal tissue, placed in baths for muscle. Used method is the method according to Barbara G. Vallance BA, Collins SM Persistent intestinal neuromuscular dysfunction after acute nematode infection in mice, Gastroenterology 1997; 113: 1224-1232. See, in particular, the Chapter "Tissue Preparation for Contractility Studis" and "Measurement of Contraction".

Thus, mice were extracted part of the small intestine and placed in oxygenated (95% 02/5% CO2) Krebs solution at 37°C. the Opposite ends of this section of the intestine is pinched. One end of this fabric were connected to isometric force sensor (model FT03C; Grass, Quincy, MA), and the other to the lining of the bath. Reactions were recorded on the recorder Grass 7E. The stimulation produced by using EFS and carbachol (for details see link above). Stimulated contractions were analyzed using the computer, whereby measured baseline (reference) tone, phase contraction, tonic contraction and the maximum stress state (tension) immediately after reduction and calculated the area under the curve.

Figure 3 shows the concept of the baseline (reference) tone (1), enhanced tone (2) and the tonic contraction (3)and base (initial) phase of contraction (4), stimulated phase reduction (5), the phase reduction (6) and the area under the curve (7).

RESULTS

Figure 1 shows the area under the curve, which takes into account the reduction period after stimulation and tension reduction within this period. Found a clear difference (lower area under the curve) between mice receiving probiotic strains listed above, and control, which shows that in the first case the e reduction after stimulation are shorter and/or less intense. The symbols in figure 1 have the following meaning: ♦ control, Lactobacillus acidophilus (johnsonii), × Bifidobacterium longum, * Bifidobacterium lactis, Δ Lactobacillus paracasei.

Figure 2 shows the tonic contraction (A) and phase decline (In) muscle tissue, as described for figure 1, but stimulated by an electric field, and compares organisms-owners receiving the probiotic strain Lactobacillus paracasei (NCM I-2116) in a live state (L.p.), dead state (dead L.p.) and only the supernatant of the medium (Sn). The control environment is the MRS. You can see that after 21 days after infection tension reduction is clearly reduced in the muscles of the intestine from mice that had been obtained from the probiotic feeding (live, dead, Sn), in comparison with mice being fed only MRS. These values approach the values of uninfected mice.

CONCLUSIONS

These results led to the conclusion that probiotics are able to normalize post-infectious hypersecretion the condition of the muscles of the intestine. In other words, probiotics reduce complications, which steadfastly persist after infection of the gastrointestinal tract. These effects vary from strain to strain, and in this invention were most significant when using a strain of probiotic Lactobacillus paracasei (NSM I-2116), and are present with all Bifidobacterium strains that were selected for this experiment.

Common in what Bodom from this experiment is that, that specific probiotic strains such as Lactobacillus paracasei (NSM I-2116), capable of acting directly on the contractile ability of muscles. This is the total opening has a consequence, which is that, in General, gastrointestinal neuromuscular disorders (intestinal contractions), which are in many cases over the life of the individual, can be cured, treated and/or prevented by the introduction of suitable probiotics.

Abnormal contractions of the intestine occur in infants, adolescents and adults suffering from spastic abdominal pain (colic), pain in the intestines or intestinal discomfort and pain that is described in the case of IBS (irritable bowel syndrome). Abnormal reduction can lead to invagination of the intestine in humans and Pets, they can cause flatulence and wrong and unacceptable time of passage through the gut.

We can conclude that in these cases the introduction of probiotics is achieved General weakening of these symptoms.

1. The application of probiotic Lactobacillus paracasei CNCM I-2116 for the treatment of irritable bowel syndrome.

2. The use according to claim 1, in which Lactobacillus paracasei CNCM I-2116 includes dead bacteria Lactobacillus paracasei CNCM I-2116, fermentation substrate and/or obtained from Lactobacillus paracasei CNCM I-2116 material.



 

Same patents:

FIELD: chemistry.

SUBSTANCE: invention relates to Saccharomyces cerevisiae CNCM I-3856 and Saccharomyces cerevisiae var. boulardii CNCM I-3799 yeast strains that are used as probiotic which is suitable for preparing food or pharmaceutical compositions. Also disclosed is a composition which contains yeast strains Saccharomyces cerevisiae CNCM I-3856 and/or Saccharomyces cerevisiae var. boulardii CNCM I-3799 and/or at least one parietal mannoproteins EL 05 and EL 06 of the Saccharomyces cerevisiae CNCM I-3856 yeast strain.

EFFECT: invention enables to reduce relieve paint in the intestines, induction of anti-inflammatory action without pro-inflammatory action, difficult and reduced adhesion and population of the gastrointestinal tract with bacteria that are pathogenic and/or invasive in nature.

12 cl, 30 dwg, 6 tbl, 9 ex

FIELD: process engineering.

SUBSTANCE: invention relates to biochemistry. Effluents are cleaned of suspended substances, oil products, phenols and chlorides for water to be discharged into pool. Inner surface of filtration dam is processed by bacterial culture Pseudomonas fluorescens "ВКГ" RCAM 00538 with titre of 10-13-10-11 in amount of 30 mg/dm3 in dry weight to obtain biofilm. Said filtration dam is filled with water to be cleaned and kept therein for at least 3 days. Effluents are forced through filtration dam consisting of the following rocks: Crushed stone or sand-gravel mix, or mix of mudstone with siltstone.

EFFECT: efficient cleaning to MPC acceptable for water discharge into pool.

1 dwg, 14 tbl, 18 ex

FIELD: chemistry.

SUBSTANCE: invention relates to a method of altering immunomodulating properties of lipopolysaccharides of plague bacteria in vitro, which involves obtaining preparations of lipopolysaccharides (LPS) and mouse toxin (MT) Yersinia pestis with subsequent formation of a LPS-MT complex thereof. A modified form of LPS-MT is used as an inducer of synthesis of cytotoxins TNF-α and IFN-γ. To this end, a test sample is prepared, to which LPS is added in amount of 5 mcg (50 mcl from working dilution of 100 mcl/ml) and MT is added in amount of 50 ng (5 mcl from working dilution of 10 mcg/ml); the sample is then incubated for 30 min at 37°C. The volume of the sample in eppendorfs is then brought to 100 mcl with sterile buffered physiological solution of NaCl and the obtained mixture is added a tray dimple containing a culture of human monocyte cell line U-937 (1×106 cells in a dimple); the latter is cultured in a medium of PRMI 1640 with simultaneous double control. Further, 1, 4, 20 hours after the beginning of combined incubation of the preparations of LPS with monocytes, quantitative accounting of the synthesised cytotoxins is carried out, wherein change in the immunomodulating properties of LPS of plague bacteria in vitro is determined from the amount of cytotoxins produced and the dynamics of their synthesis.

EFFECT: invention enables to alter immunomodulating properties of lipopolysaccharides of plague bacteria in vitro, which enables to realise toxic potential of the endotoxin of plague bacteria.

2 cl, 8 dwg, 2 ex

FIELD: biotechnologies.

SUBSTANCE: strain Rhodococcus erythropolis 1-KP, extracted from podzol soil contaminated with oil from the territory of the Kolsky peninsula, having high speed of oil utilisation, is deposited in the Departmental Collection of Beneficial Microorganisms of Agricultural Purpose of the Russian Academy of Agricultural Sciences (RCAM) (GNU VNIISHM) under the number RCAM01142 and may be used for treatment of contaminated soils from oil.

EFFECT: improved quality of soil cleaning from oil.

3 tbl

FIELD: biotechnologies.

SUBSTANCE: strain pseudomonas citronellolis 48-U, having high speed of oil and diesel fuel recycling, is deposited in the Departmental Collection of Beneficial Microorganisms of Agricultural Purpose of the Russian Academy of Agricultural Sciences (RCAM) (GNU VIISHM) under the registration number RCAM 01441 and may be used for treatment of contaminated soils from oil and diesel fuel.

EFFECT: improved quality of soil cleaning from oil and diesel fuel.

3 tbl

FIELD: biotechnologies.

SUBSTANCE: strain Rhodococcus fascians 4-G, extracted from soil contaminated with black oil and sampled from the territory of a boiler plant located in the settlement Gorelovo, Leningrad region, is deposited in the Departmental Collection of Beneficial Microorganisms of Agricultural Purpose of the Russian Academy of Agricultural Sciences (RCAM) (GNU VNIISHM) under the number RCAM01140 and may be used for treatment of contaminated soils from oil.

EFFECT: invention makes it possible to increase quality of soil treatment from oil.

3 tbl

FIELD: biotechnologies.

SUBSTANCE: strain Pseudomonas aeruginosa RCAM01139 is proposed for decomposition of oil and diesel fuel.

EFFECT: strain is characterised by high efficiency in processes of oil and diesel fuel utilisation, which are the only sources of carbon and energy.

3 tbl

FIELD: biotechnologies.

SUBSTANCE: strain Micrococcus luteus PBT-1, having high catalase activity and aiding increasing productivity of the agroecosystem, participating in processes of transformation of organic remains of natural origin, is deposited in the Departmental Collection of Useful Microorganisms of Agricultural Purpose of the Russian Agricultural Academy (RCAM) (GNU VNIISHM) under the registration number RCAM 01016 and may be used in transformation of organic remains of natural origin.

EFFECT: invention makes it possible to increase productivity of an agroecosystem.

3 ex

FIELD: biotechnologies.

SUBSTANCE: strain Penicillium sp. PBT-2 extracted from samples of humic pod mixed with cereal straw and having polyfunctional properties and promoting increased productivity of agroecosystem, participating in processes of transformation of organic remains of natural origin, is deposited in the Departmental Collection of Beneficial Microorganisms of Agricultural Purpose of the Russian Academy of Agricultural Sciences (RCAM) (GNU VNIISHM) under the registration number RCAM 01017 and may be used in transformation of organic remains of natural origin.

EFFECT: improved productivity of agroecosystem.

3 ex

FIELD: biotechnologies.

SUBSTANCE: strain of nodule bacteria Bradyrhizobium japonicum 859 (VNIISHM No.24117) is proposed, used as a facility to produce a fertiliser for soya. When soya seeds are treated with the fertiliser produced on the basis of the specified strain Bradyrhizobium japonicum, the index of germination increases by more than 8%, and crop capacity increases by 17-30%.

EFFECT: higher germination of plants, improved crop capacity.

4 tbl

FIELD: biotechnologies.

SUBSTANCE: strain Pseudomonas aeruginosa RCAM01139 is proposed for decomposition of oil and diesel fuel.

EFFECT: strain is characterised by high efficiency in processes of oil and diesel fuel utilisation, which are the only sources of carbon and energy.

3 tbl

FIELD: biotechnologies.

SUBSTANCE: invention relates to the method to produce a brucellosis antigen for the rose bengal test (RBT). The method to produce the brucellosis antigen includes growing of the strain B. abortus 104M on the solid nutrient medium, washing of the culture with 0.5% phenolized solution, inactivation of biomass in a water bath at the temperature of 88±1°C for 30 minutes, performance of inspection for sterility, whirling of the bacterial suspension at 6-7 K rpm for 40 minutes, slurrying of the brucelli residue in 0.7% water solution of rose bengal at the ratio of 1:1 and dyeing of microbial cells at 4°C, re-slurrying of the microbial suspension in the buffer dissolvent and performance of standardisation.

EFFECT: method makes it possible to produce an antigen of higher activity and may be used in production of preparations for brucellosis diagnostics.

3 tbl

FIELD: biotechnologies.

SUBSTANCE: invention may be used to assess biological activity of lactobacilli and bifidus bacteria relative to Vibrio cholerae with the purpose to establish possibility to use probiotics for prevention and treatment of cholera. The method provides for testing of antagonistic and acid-forming activity of lactobacilli and bifidus bacteria strains. Antagonistic activity relative to choleraic vibrios is detected by the method of holes in dense nutrient media - CDM - agar and alkaline agar, and activity is accounted on the basis of quantitative data, namely: zone of growth inhibition (in mm) of a test strain V. cholerae from 6.0 to 15.0 - low antagonistic activity, from 15.1 to 29.0 - medium, ≥29.1 - high. Acid-forming activity of strains relative to choleraic vibrios expressed in Turner degrees (°T) is defined on the basis of quantitative data. So if acid formation ≤is 99.9 (°T), then activity of acid formation is assessed as low, if the values of this index are within (100-149.9) (°T) - as medium, and if the value is ≥150.0 (°T) - as high. At the same time the result of assessment of lactobacilli and bifidus bacteria biological activity relative to V. cholerae eltor, classica, O139, non O1/ non O139 is considered positive with 4 versions of combination of testing results according to the specified indices, namely: high antagonistic activity, high acid-forming activity, accordingly, high - medium, medium - high, medium - medium.

EFFECT: invention makes it possible to establish availability or absence of anti-cholera activity in strains.

2 dwg, 2 tbl, 3 ex

FIELD: biotechnologies.

SUBSTANCE: strain of nodule bacteria Bradyrhizobium japonicum 859 (VNIISHM No.24117) is proposed, used as a facility to produce a fertiliser for soya. When soya seeds are treated with the fertiliser produced on the basis of the specified strain Bradyrhizobium japonicum, the index of germination increases by more than 8%, and crop capacity increases by 17-30%.

EFFECT: higher germination of plants, improved crop capacity.

4 tbl

FIELD: medicine.

SUBSTANCE: present invention refers to biotechnology and medicine. There are presented versions (aCt1 and aCt2) of one-domain antibodies specifically binding the Chlamydia trachomatis antigen. There are described versions of the method of inhibiting an infection caused by Chlamydia wherein the method involves the preparation of elementary bodies C.trachomatis by a therapeutically effective amount of the nanoantibody aCt1 or aCt2 before being attached to infected target cells.

EFFECT: use of the invention provides the antibodies to detect and block the infections Chlamydia trachomatis that can find application in medicine.

6 cl, 4 dwg, 5 ex

FIELD: biotechnology.

SUBSTANCE: basic fermentation medium is prepared on the basis of fermentative lysate of starch in an amount of 8-15% for glucose, and additional fermentation medium containing the components of basic fermentation medium, 2-3.5 times concentrated. They are sterilised. Biosynthesis of L-lysine is carried out using strain-producer Corynebacterium glutamicum of All-Russia classification of microorganisms Ac-2577D (BIGOR 55). The fermentor is loaded with the basic nutrient medium in amount of 1/3 of its volume. Then inoculation of the basic fermentation nutrient medium is carried out with 10-20% inoculum grown in two stages. Biosynthesis process is carried out with continuous feeding starting from 8-16 hours of cultivation. At that the inhibitory concentration of reducing substances in the culture fluid of 4-8% is maintained. After filling the device to the maximum volume after 60-66 hours of cultivation and then every 6-12 hours for the rest part of biosynthesis process a part of the culture fluid in amount of 10-15% of the maximum volume is pumped into the device for final fermentation. In this device, the biosynthesis process is continued for 6-12 hours under the same conditions as in the main device but without feeding the additional nutrient medium. Then L-lysine is isolated.

EFFECT: invention enables to carry out the biosynthesis process for a long time without reducing the rate of synthesis of L-lysine, and provides obtaining of the culture fluid with high concentration of L-lysine prior to the isolation.

4 ex

FIELD: biotechnology.

SUBSTANCE: isolated recombinant adenosine deaminase is described, which comprises polypeptide SEQ ID NO: 1 or a version polypeptide SEQ ID NO: 1 of isolated recombinant adenosine deaminase, where the version polypeptide SEQ ID NO: 1 comprises one or more amino acid substitutions selected from the group consisting of: Gin instead Lysl98; Ala instead Thr245; and Arg instead of Gly351, and DNA encoding it. The conjugate of polyalkylene oxide with the said adenosine deaminase for treatment of adenosine deaminase-mediated diseases is presented where adenosine deaminase comprises from 11 to 17 chains of polyalkylene oxide with a molecular weight of 5 kDa for ADA protein. The methods of purification of the recombinant adenosine deaminase are proposed, including protein purification using ion exchange chromatography, or protein purification using hydrophobic interaction chromatography. Also the preparations of recombinant adenosine deaminase produced by these methods are provided.

EFFECT: invention enables to obtain recombinant adenosine deaminase, having increased stability.

14 cl, 1 tbl, 10 ex

FIELD: biotechnology.

SUBSTANCE: culture fluid obtained after growth of the mixture of strains Lactobacillus acidophilus NKI, 100ash, K3"Ш"24, is frozen, the fat layer is removed, the defatted part is thawed and the cells are separated by centrifugation. The obtained supernatant is sterilised by membrane vacuum filtration, is concentrated with membrane ultrafiltration in the process of centrifugation, the concentrate of supernatant is treated with the components more than 30 KDa with fivefold excess of ice acetone. The resulting precipitate is diluted with 20 mM NaCl at a ratio of 2:1 by volume and is mixed with 10% Tween-80 to a final concentration of 1% Tween. Purification and isolation of the target product is carried by the method of horizontal isoelectrophoresis in a plate of 5% polyacrylamide gel with 7-8 M urea and 5% sucrose in gradient pH 4-8 at a temperature 8-12°C for 8 hours When isolation of the target product after electrophoresis the gel stripes are cut on isopoints, frozen at a temperature (-20°C) for 2 hours, thawed and dispersed, the solid parts are separated by centrifugation, concentrated by centrifugation at 5500 rev/min for 60 min at room temperature, low molecular weight impurities are removed, and the concentrate is mixed with phosphate buffer in 100-fold volume The gel stripes are cut on isopoints with pl (4.0-4.3) with the isolation of acid lectins and pl (7.6-8.0) with the isolation of alkaline pectins.

EFFECT: obtaining of lectins having anti-candidal activity is provided

5 cl, 1 tbl

FIELD: biotechnology.

SUBSTANCE: invention relates to a strain of the bacteria Lactobacillus plantarum Inducia DSM 21379 used as a probiotic. The strain enhances the natural protective potential of organism, produces polyamines from ornithine and glutamate, nitric monoxide (NO) from arginine, possesses antioxidative activity and enhances the intestinal barrier function, stimulating cellular immunity of the intestinal mucosa and blood, and stimulates adaptively the proinflammatory cytokines. The strain in an amount providing an effective daily dose of from 108 to 109 CFU/ml or CFU/g is used for preparing a composition for use as an antioxidant probiotic.

EFFECT: increased cellular immunity.

7 cl, 8 dwg, 14 tbl, 6 ex

FIELD: biotechnology.

SUBSTANCE: strain Bacillus licheniformis CL-13 which has high histidine decarboxylase activity is deposited in the Russian National Collection of Industrial Microorganisms (RNCIM) under the registration number of RNCIM B-11185 and can be used in histidine decarboxylase of enzymatic preparation.

EFFECT: invention enables to obtain a strain of histidine decarboxylase activity.

1 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to pharmaceutical industry, particularly to therapeutic preparations of mumiyo. The therapeutic preparation which contains purified mumiyo, grapefruit citrosept, edible glycerol, ethanol and water at specific proportions.

EFFECT: preparation has a prolonged shelf life.

8 tbl

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