Saccharomyces cerevisiae yeast used as probiotic and composition based thereon

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

 

The present invention relates to the field of nutrition and health of human and/or animal.

The invention more preferably relates to new strains of yeast and new yeast obtained on the basis of new strains. Such yeasts are particularly useful for a good condition of the digestive tract and/or for preventing and/or treating disorders of the digestive tract of a human or animal.

The literature describes many microorganisms for useful application of the person in the digestive tract and for power, see, for example, WO 2006/021965.

Such microorganisms are traditionally denoted by the term "probiotic"related to the active microorganisms that can bring the body's master of the benefits in relation to health in the case when their introduction is sufficient (Joint FAO/WHO Expert Consultation Probiotics in food, FAO Food and nutrition paper Nr 85, ISBN 92-5-105513-0).

A useful result in oral introduction of microorganisms to a large extent depends not only on the strain of microorganism used, but also on the form of the introduction. Even among the same species, depending on the strains, the effects observed in practice vary greatly and are sometimes positive, sometimes negative or neutral, as, for example, for the species Escherichia coli, in which you can find as patoh is installed strains (for example, type enterotoxinogenic or Enterohaemorrhagic)and useful strains, such as strain Nissle 1917 (M. de Vrese; P.R. Marteau. Probiotics and Prebiotics: Effects on Diarrhea. 2007, J. Nutr., 137 (3 Suppl. 2), 803S-811S). Thus, at the present time in relation to a given strain is impossible to predict that the introduction of such a strain, you can count on useful effect on human health, and even to predict the nature of its possible beneficial effect or its intensity.

Some strains of microorganisms, in particular among yeast and dairy bacteria, have been identified in relation to some useful effect on the gastrointestinal tract. However, the total useful effect on the gastrointestinal tract often requires concomitant introduction of several strains of different nature (I. Goktepe; V.K. Juneja; M. Ahmedna (eds). Probiotics in Food Safety and Human Health. 2006, CRC Taylor & Francis, ISBN 1-57444-514-6).

In addition, it was observed that quite a few of the microorganisms, in particular lactic bacteria, possess anti-inflammatory action. This Pro-inflammatory effect may be particularly harmful and undesirable, for example, in the case of autoimmune diseases or immunodeficita.

Describes some fraction of yeast and/or derivative works of yeast in respect of their beneficial actions in the digestive tract.

So, n is the sample, mannoprotein derived products of yeast described in relation to their inhibitory effect on the adhesion of pathogens. It also describes the products of yeast cell walls in relation to their actions like fibers. However, there are many strains of the yeast Saccharomyces cerevisiae, which do not have a useful effect or similar effects.

In addition, depending on the strains and forms the input of yeast effects also can vary greatly.

Thus, there is a need for opportunities to place new strains of microorganisms, which may have beneficial effects in health prophylactically and/or therapeutically on known or anticipated pathology or impairment, or the General state of both physical and mental health.

Thus, an object of the present invention is a new Saccharomyces cerevisiae strain deposited at the National collection of cultures of microorganisms under the number CNCM I-3856, and a new strain of Saccharomyces cerevisiae var. boulardii, deposited in the National collection of cultures of microorganisms under the number CNCM I-3799.

The object of the present invention are also yeast Saccharomyces cerevisiae, obtained from the strain deposited at the National collection of cultures of microorganisms under the number CNCM I-3856, and the yeast Saccharomyces var. boulardii obtained is stepping down from the strain, deposited in the National collection of cultures of microorganisms under the number CNCM I-3799.

Another object of the present invention is a composition comprising the yeast Saccharomyces cerevisiae, obtained from the strain deposited at the National collection of cultures of microorganisms under the number CNCM I-3856, and/or yeast Saccharomyces var. boulardii, derived from the strain deposited at the National collection of cultures of microorganisms under the number CNCM I-3799, and/or at least one derivative product of the yeast Saccharomyces cerevisiae selected from extracts of yeast derived products cell wall, parietal glucans, parietal mannoproteins, lipid fractions of yeast fractions of nucleic acids yeast (RNA, DNA).

The composition according to the present invention has the following advantages:

- the ability, in particular, in dry form to resist and survive during the transition of gastric barrier, which allows to optimize its effect on the gastrointestinal tract;

- anti-inflammatory;

- the absence of proinflammatory action or very weak such action;

- the ability to reduce pain in the intestines;

- ability to impede and reduce the adhesion and colonization by bacteria, pathogenic and/or having invasive nature, the gastrointestinal tract, particularly the small intestine and then the stand intestine.

This new composition having such a combination of characteristics, up to the present time has not yet been described or identified.

Thus, this song is one of exceptional interest.

Another object of the present invention is the use of these compositions for the production of dietary supplements and/or probiotic, and/or product for preventive nutrition, and/or biologically active additives, and/or functional ingredients, and/or treatment, the cosmetic and/or pharmaceutically active substances intended for human and/or animal.

At the same time, the present invention relates to the use of a composition such as previously defined, to obtain food compositions intended to improve the comfort of the gastrointestinal tract and/or improving the intestinal microflora.

The object of the present invention is also the use of a composition such as previously defined, to obtain a medicinal product intended for the treatment and/or prevention of intestinal disorders, functional bowel disorders or diseases of the gastrointestinal tract.

The object of the present invention is also the use of a composition such as previously defined, to obtain a medicinal product intended for the treatment and/or prevention of diseases or disorders of the intestine, characterized by hyperalgesia.

The last object of the present invention is a set containing at least one kind of yeast and/or at least one derivative product of yeast, such as defined previously, in a format suitable for oral administration.

Strain, deposited by the applicant in accordance with the Budapest Treaty at the National collection of cultures of microorganisms (Institute Pasteur, Paris) under the number CNCM I-3856, in the further description, for brevity referred to as the "ScPro1".

Strain, also deposited by the applicant in accordance with the Budapest Treaty at the National collection of cultures of microorganisms (Institute Pasteur, Paris) under the number CNCM I-3799, in the further description, for brevity referred to as the "SCB1".

Also for brevity, a derivative of the yeast Saccharomyces cerevisiae selected from extracts of yeast derived products cell wall, parietal glucans, parietal mannoproteins, lipid fractions of yeast fractions of nucleic acids yeast (RNA, DNA) and their mixtures, further description is referred to as "derived".

Under the probiotic understand active microorganisms, which, if their introduction in sufficient quantities, have a positive effect on health, a comfortable condition and good health in addition to traditional nutrient efficiency is tov.

Under the product for preventive nutrition or dietary Supplement or functional food product or cosmetic means understand food product that contains ingredients that provide beneficial effects on health or able to improve physiological functions.

Under the food additive understand food product, the purpose is to Supplement the normal food regime. A food additive is a concentrated source of nutrients or other substances with a nutritional or physiological effect, in the case when they are used individually or in combination in small quantities.

Under food products intended for special nutrition (DDAP), understand food, the purpose of which is special food that is intended for a defined group of the population, such as infants, young children, athletes.

Food composition, such as referred to in the present invention may be a food additive or DDAP.

The strains of the present invention were identified by the applicant due to their numerous advantages and, in particular, due to their ability to induce beneficial effects on the human digestive tract and, in particular, the tone is exactly the intestines and the colon, and on the whole organism.

In practice, it was observed that yeast ScPro1 and/or SCB1 and/or derivatives thereof in an unexpected way is able to induce an anti-inflammatory effect in contrast to quite a large number of yeast strains and without Pro-inflammatory actions.

In practice, the yeast ScPro1 and/or SCB1 and/or their derivatives cause increased secretion of interleukin IL-10, involved in anti-inflammatory signals. In addition, in contrast to the action of the probiotic bacteria of the type Lactobacillus yeast ScPro1 and/or SCB1 and/or their derivatives do not induce the synthesis of Pro-inflammatory cytokine IL-12. The production of Pro-inflammatory cytokines TNFα and IFNγ also significantly less in comparison with bacterial probiotics. At the same time, the study allowed to prove an anti-inflammatory effect in vivo yeast ScPro1 and, in particular, halving the extent of the inflammation of the colon and reduce by one third of intestinal necrosis.

In addition, yeast ScPro1 and/or SCB1 and/or their derivatives in their dry form are able to overcome the gastric barrier without any negative effects on their survival or their integrity, data from yeast are not embedded in the internal environment of the colon.

The applicant for the first time and unexpectedly proved that the yeast ScPro1 and/or SCB1 and/or their derivatives can increase with recepsionist to pain, in particular, the model with the rat in vivo.

In addition to this beneficial effect yeast ScPro1 and/or SCB1 and/or their derivatives are able to inhibit the colonization and/or invasion at the level of gut microorganisms which are pathogenic and/or having invasive nature. The introduction of yeast causes a reduction in the number of enterobacteria on the level of the colon and the intestinal microflora, antibiotic-resistant.

In particular, the applicant was shown prophylactic and therapeutic activity against colonization of the intestine by the yeast Candida albicans and inflammation provoked and supported by the pathogen. At the same time, these yeasts have shown inhibitory effect on the ability to adhesion and invasion of strains of pathogenic and/or having invasive nature of pathotypes Escherichia coli isolated from biopsy material of the small intestine of patients with Crohn's disease.

According to the present invention, the yeast ScPro1 and/or SCB1 and/or their derivatives can be introduced into the active or reaktiviram form preferably by mouth.

The terms "active" or "active" according to the present invention realize the fact that the yeast have active or reaktiviram metabolism or capable of reproduction. We are talking in particular about the yeast in a dry or fresh form.

the AK rule, the yeast in fresh form are in the form of extruded or graded yeast. They can also be in the form of yeast in suspension, water-based, also called liquid yeast. In this case, the yeast are preferably encapsulated. Methods of encapsulation and different types of capsules are well known to specialists in this field of technology.

Among the dry forms of yeast can be mentioned yeast, which can be in a dry, instant or active dry form. Under dry yeast understand any yeast having a dry matter content greater than 90% and preferably in the range from about 92 to 96%.

Among dry yeast can also mention frozen or unfrozen yeast with average humidity.

Instant dry yeast is intended mainly for large and small bakeries. Other uses and sales potential based on food purposes (technology of medicinal forms, alcoholic fermentation). The feature data of dry yeast is that they do not require moisture before adding to the flour.

These yeast is produced by dehydration of yeast under the action of the gradient hot air, allowing you to turn pasty product (pressed or liquid yeast) in the product in the form of a thin su is th vermicelli, which remains active. Then the product with the aim of ensuring stability must be packaged in the absence of oxygen.

Active dry yeast are active yeast, dried at low temperatures to preserve their fermentation capacity and ensure sufficient long-term storage. Yeast is in the form of spherical particles.

These yeast is produced by dehydration of yeast under combined action of heat and mechanical impact, which allow to transform the yeast in pasty form dry product, while maintaining their viability.

Selected active dry yeast produced by extrusion and drying in the fluidized bed biomass (active yeast cells). Such active dry yeast, i.e. dry yeast having a high content of active yeast cells are in the form of granules in the General case with a diameter of from 0.1 μm to 2.5 mm and the content of H2O from 4 to 8% of the mass.

Such dry form have the advantage that they provide more good gastrorresistente compared with the form of fresh yeast and optimize the beneficial effects of yeast according to the present invention. The yeast of the present invention preferably are in the form of active dry yeast.

In General it is known that proinflammatory cytok the us stimulate inflammatory mechanisms who can be responsible for many clinical problems, particularly in cases of autoimmune diseases or immunodeficita.

For example, the yeast of the present invention can be used for the prevention and/or treatment of chronic and acute diseases or inflammatory disorders of the intestine, not necessarily associated with diarrhea or constipation.

In the first embodiment of the present invention illness or disease is not necessarily associated with diarrhea.

In the second embodiment of the present invention illness or disease not associated with diarrhea. In particular, yeast ScPro1 and/or SCB1 and/or their derivatives may be useful for prevention or treatment of colitis, which is characterized primarily by inflammation of the colon.

In particular, the yeast is in a good degree acceptable for the prevention and/or treatment of chronic inflammatory bowel disease (MICI), in particular, ulcerative colitis, hemorrhagic rectorite, celiac disease or Crohn's disease.

These diseases are characterized, in particular, a heightened immune response, involving many inflammatory cascades. For example, in the prevention or treatment of these diseases probiotic and/or product for the treatment of prophylact the economic power, and/or functional food product, and/or biologically active additive, and/or cosmetic agent is important that Pro-inflammatory effects are weaker.

Thus, yeast ScPro1 and/or SCB1 and/or their derivatives of the present invention are most preferably acceptable for this application. These yeasts have several additional advantages.

The first advantage is that they have the ability to increase resistance to pain. The second advantage, in particular in the case of Crohn's disease is that the yeast is able, in particular, to inhibit the ability of adhesion and invasion of strains of pathogenic and/or having invasive nature of E. coli originating from patients suffering from this disease.

The inflammatory response can be, in particular, due to the invasion of any pathogenic microorganisms.

So, for example, yeast ScPro1 and/or SCB1 and/or their derivatives according to the present invention show good efficacy in the prevention or treatment of gastrointestinal disorders or diseases caused by colonization of the intestine by microorganisms that are pathogenic and/or having invasive nature of prokaryotes, such as bacteria or eukaryotes, such as fungi.

Infection is a rule-intestinal disorders or diseases may represent a chronic inflammatory intestinal diseases, such as ulcerative colitis, celiac disease, Crohn's disease, hemorrhagic rectocele.

Besides the fact that yeast ScPro1 and/or SCB1 and/or their derivatives allow to increase the resistance to pain, they are also of interest for the prevention or treatment of diseases or disorders of the gut, followed by a state of hyperalgesia. Such pathologies or disorders may be a preferable functional bowel disorders, chronic inflammatory bowel disease (MICI) or food intolerance (Allergy, elaboration of conditioned reflexes and so on), characterized by chronic visceral pain.

These yeast preferably acceptable for the prevention or treatment of hyperalgesia, in particular irritable bowel syndrome (SII) of any form (constipation, diarrhea or a combination of them), and chronic visceral pain, not related to the SII, such as functional abdominal pain without difficulty urinating (FAPS: Functional Abdominal Pain) and pain associated with food intolerance and celiac disease.

Yeast ScPro1 and/or SCB1 and/or their derivatives, or any compositions containing them, can also be acceptable as prophylaxis in patients with a predisposition or higher susceptibility to disorders or diseases of this type, or as a treatment, e.g. the, in the case of crises or for longer periods. Compositions and methods of the present invention can reduce the suffering of patients, alleviating the symptoms or the cause of such disorders.

Combining these yeast and/or derivatives thereof of the present invention to pain, inflammation and micro-organisms pathogenic and/or having invasive nature, causes some way to improve well-being, health and/or comfort of the gastrointestinal tract of a human or animal.

The composition of the present invention may contain yeast ScPro1 and/or SCB1 yeast, and/or at least one derivative of the yeast Saccharomyces cerevisiae selected from extracts of yeast derived products cell wall, parietal glucans, parietal mannoproteins, lipid fractions of yeast fractions of nucleic acids yeast (RNA, DNA), in amounts in the range from about 107up to 6·1010SOME and preferably in the range from 108up to 2·1010SOME or in the range of from 1 mg to 10 g and preferably in the range of from 1 mg to 1, This number can be a daily quantity taken in one or more doses during the day.

Preferably the yeast ScPro1 and/or SCB1 and/or their derivatives used in therapeutic or non-therapeutic nae is the increase in the daily dose in the range from 10 7up to 6·1010CFU (colony forming units) and preferably in the range from 108up to 2·1010SOME.

When the yeast and/or their derivatives are active and not in reaktiviram form, daily dose, appropriate for therapeutic or non-therapeutic purpose, is preferably in the range of from 1 mg to 10 g and more preferably in the range of from 1 mg to 1, the Effective daily dose can be entered in one, two, three or four installments.

Yeast and/or their derivatives of the present invention or compositions containing them, preferably administered orally by. Their number may be therapeutically effective dose, which means that at least one of the symptoms is reduced or suppressed.

Yeast ScPro1 and/or SCB1 and/or their derivatives may be included in the food composition intended for human or animal, and/or with excipients or fillers, acceptable for oral administration.

Composition intended for human consumption, may be a liquid, paste or solid. In particular, the composition may be a dairy product such as cheese, butter, yogurt, or cream, the product on the basis of fruit, such as fruit juice, compote or fruit paste, drink or solid food product is t, for example, snacks, crackers, or other product. Thus, the composition contains yeast ScPro1 and/or SCB1 and/or their derivatives and the components of the food product or beverage.

Yeast ScPro1 and/or SCB1 and/or their derivatives may also be included in the pharmaceutical composition. The pharmaceutical composition is acceptable for oral administration. Thus, it contains yeast ScPro1 and/or SCB1 and/or their derivatives, as well as the corresponding traditional basis, selected from excipients, permitted for use in pharmaceutical compositions. The composition may be made in the form of a liquid, such as syrup or capsules for drinking, or in the form of tablets, gelatin capsules, sachets, capsules or powders and other acceptable herbal preparations.

In addition, yeast ScPro1 and/or SCB1 and/or their derivatives can be introduced with other probiotics and/or other functional ingredients, in particular, with probiotic bacteria, in particular for the most comprehensive preventive actions.

As an example we can mention dairy bacteria of the genera Lactobacillus, Bifidobacterium, Pediococcus, Propionibacterium, or Leuconostoc.

Yeast ScPro1 and/or SCB1 and/or their derivatives can also be entered with other active substances such as antibiotics, analgesic tools, anti-diarrhoeal remedies, laxatives is the means and mixtures thereof.

Further, the present invention is illustrated by examples and figures are presented in the order notes and permissive follows:

- figure 1 shows the variation in the survival of yeast ScPro1 in the digestive system, simulating the colon of a person, according to the example 2;

- figure 2 shows the action of yeast ScPro1 on the microflora of the colon, respectively, in example 2;

- figure 3 and 4 shows the change in the number of cells of Candida albicans in the faeces of mice in experiments 1 and 2 of example 5, the corresponding prevention model (figure 3) and treatment (figure 4);

- figure 5 shows the percentage of residual adhesion of cells of Escherichia coli AIEC LF82 on the epithelial cells of the human intestine, depending on the quantity of yeast ScPro1 when pre-incubation of the yeast cells were incubated with the epithelial cells of the intestine in an hour. The infection of cells by AIEC strain LF82 was carried out in the presence of yeast ScPro1 example 6, respectively;

- figure 6 shows the percentage of residual adhesion of cells of Escherichia coli AIEC LF82 on the epithelial cells of the human intestine, depending on the quantity of yeast ScPro1 co-incubation of yeast cells and cells of Escherichia coli were incubated simultaneously with the epithelial cells of the intestine within hours, respectively, example 6;

- 7 shows the measurement of the intensity of inflammation to the of Chechnya mice according to the macroscopic scale Wallace after the introduction of yeast ScPro1 and SCB1 accordingly, example 4;

on Fig shows the estimated rate of putting inflammation of the intestinal epithelium of mice according to the histological scale Ameho after the introduction of yeast ScPro1 and SCB1 accordingly, example 4;

- figure 9 shows the measurement of the intensity of inflammation of the intestines of the mice according to the macroscopic scale Wallace after the introduction of yeast ScPro1 and SCB1 entered separately or in combination according to example 4;

- figure 10 shows the measurement of the intensity of inflammation putting epithelium of mice according to the histological scale Ameho after the introduction of yeast ScPro1 and SCB1 entered separately or in combination according to example 4;

- figure 11 shows the expression level of mRNA of the gene encoding the protein of IL-10, after one and three hours after bringing into contact of yeast, or derivatives thereof of the present invention with the epithelial cells of the intestine of man according to example 7;

on Fig shows the level of expression of mRNA of the gene encoding the nuclear receptor PPARα, one and three hours after the bringing into contact of yeast, or derivatives thereof of the present invention with the epithelial cells of the intestine of man according to example 7;

on Fig shows the modulation of the expression of mRNA of the gene encoding the protein of IL-10, after one and three hours after bringing into contact the derivatives of yeast under this image is ateneu with epithelial cells of the intestine of man according to example 7;

on Fig shows the expression of the gene encoding the protein of IL-10, in the epithelial cells of the intestine of mice after injection of yeast and/or derivatives thereof of the present invention (example 4);

on Fig shows the expression of the gene encoding the nuclear receptor PPARα, in the epithelial cells of the intestine of mice after injection of yeast and/or derivatives thereof of the present invention (example 4);

on Fig shows the number of cytokine IL-10 PG/ml secreted putting cells in biopsy material of patients, not necessarily suffering from Crohn's disease, after bringing them in contact with yeast and/or their derivatives according to the present invention (example 8);

on Fig shows the number of cytokine TNF-α in PG/ml secreted putting cells in biopsy material of patients, not necessarily suffering from Crohn's disease, after bringing them in contact with yeast and/or their derivatives according to the present invention (example 8);

on Fig shows the results of tests to determine the ability of the binding of type 1 pili from mannoproteins factions (and EL05 EL06) yeast ScPro1;

on figa and 19B shows respectively the average percentage of residual adhesion and residual invasion of AIEC strain LF82 compared to T84 cells in co-incubation with increasing concentrations of yeast (example 6) - *p < 0,05 **p < 0,01;

on figa and 20B shown, respectively, in average percentage of adhesion and invasion of AIEC strain LF82 compared to T84 cells in co-incubation with increasing concentrations of mannoproteins yeast EL05 (example 6);

on figa and 21B shown, respectively, in the average percentage of residual adhesion and residual invasion of AIEC strain LF82 compared to T84 cells in the case of pre-incubation with increasing concentrations of yeast (example 6) - *p < 0,05, **p < 0,01;

on figa and 22B shown, respectively, in average percentage of adhesion and invasion of AIEC strain LF82 compared to T84 cells in the case of pre-incubation with increasing concentrations of mannoproteins yeast EL05 (example 6) - *p < 0,05, **p < 0,01;

on Fig shows the percentage residual adhesion of AIEC strain LF82 in relation to cells CHO-K1 and CHO-K1/CEACAM6 in the case of pre-incubation with increasing concentrations of yeast ScPro1 in dry soluble form (example 6) - *p < 0,05, **p < 0,01;

on Fig shows the adhesion of AIEC strain LF82 or not having drank mutant LF82-δfimH on the brush border of enterocytes 3 sample cells of patients with Crohn's disease (example 6);

on Fig shows the results of measurements of the threshold of pain perception (in mm RT. Art.) healthy rats in case of different yeast (example 9);

on Fig shows the result of the measurement of the threshold of pain perception (in mm RT. Art.) rats with visceral hypersensitivity in case of different yeast (example 9).

EXAMPLES

EXAMPLE 1. The survival rate of yeast ScPro1 and/or SCB1 in artificial digestive environment that simulates the human intestine

Study of the change of yeast ScPro1 and/or SCB1 during the passage of the gastrointestinal tract

Yeast ScPro1 and SCB1 were tested and investigated in vivo in artificial digestive environment that simulates human digestion, and, in particular, by examining the survival of the subjects of viable yeast in the process of passing through the gastrointestinal tract.

Were tested two samples of active dry yeast ScPro1 and two samples of active dry yeast SCB1.

Two samples differed by time of storage at room temperature in a vacuum: the duration of storage up to 6 months to 2 years.

Experimental conditions

Digestion was carried out in the system, called TIM1 (stomach + small intestine), in accordance with the experimental conditions established on the basis of data obtained from literature sources, and reproducing the digestion liquid food product (water) adult healthy fasting removal of the products of digestion dialysis and absorption. Each cycle of digestion was carried out for 5 hours. All experiments with digestion which m was carried out in the same General experimental conditions, described below.

Temperature: the temperature was maintained equal to 37°C.

The parameters of gastric emptying: gastric Emptying was performed respectively according to defined Elashoff et al. (1982) and expressed according to the formula:

F=1-2-(t/T)b

where F denotes a portion of the removed food, t denotes time, T denotes the half-life of the food product, and b denotes the parameter that describes the shape of the curve. Accepted parameters are: T = 15 min; b = 1.

Parameters emptying of the small intestine: the emptying of the small intestine was carried out accordingly modified according Elashoff (with the introduction of the parameter d provides slow emptying at the end of digestion, Fm= F + d·t3). Accepted parameters are: T = 150 min; b = 2,4; d = -10-7(cf. figure 2).

The specified pH value:

stomach (min/unit pH): 0/6,0; 10/3,2; 20/2,4; 40/1,8; 60/1,6; 90/1,5; 300/1,5;

duodenum: 6,4;

jejunum: 6,9;

the ileum: 7,2.

Gastric secretion:

HCl;

pepsin;

lipase.

Intestinal secretion:

NaHCO3in the three sections of the small intestine;

extract galciv the duodenum;

the extract of the pancreas into the duodenum;

Dialysis/absorption

Remove small portions of the intestinal chyme was carried out on two levels TIM1 (jejunum and the ileum) through hemodialysate. Dialysis intestinal chyme carried out continuously with saline solution whose composition was close to the composition of blood plasma. The dialysates were collected in dialysis bags.

Sampling was carried out during digestion at different levels of the tract to control the survival of the tested yeast.

Estimates of the number of yeast was carried out, therefore, the traditional microbiological methods and the adopted sampling: in the stomach after 10, 20, 30, and 45 min, the output of the small intestine with the accumulation within hours and in the final residue.

The calculation method set forth herein.

For each sampled quickly implemented a serial tenfold dilution of sterile saline solution (NaCl, 8.5 g/l). Then 0.1 ml of each dilution was applied and then distributed across the surface of agar medium, poured into Petri dishes (two cups at one breeding). The cups were incubated for 48 h at 35°C before counting "colony forming units" (CFU).

The results were expressed in CFU/ml (raw data) and the percentage of cells active yeast compared guests for their arrival cells initially introduced yeast to determine the level of survival of yeast in the stomach and at the outlet of the small intestine.

The following table summarizes theoretical levels of survival (100%viability) and the actual values obtained for each strain at the level of the stomach, the accumulated sample output from the small intestine after 5 h of digestion and accumulated in the sample system after 5 h of digestion.

Results

The products of digestionPut the yeast, CFUAt the exit from the stomach through
T = 45 min
At the exit from the small intestine through
T = 5 h
Overall survival through
T = 5 h
ScPro1, sample 13,5·101089%100%106%
ScPro1, sample 22,0·101088%95%106%
SCB1 11,5·101083%76%81%
SCB1 21,5·101085%69% 76%

Conclusion

These results well demonstrate superior survival in the gastrointestinal tract yeast ScPro1 and SCB1.

EXAMPLE 2. The survival rate of yeast ScPro1 in artificial digestive environment that simulates the human intestine

The study of survival of yeast ScPro1 during fermentation in the colon and the influence of yeast on the intestinal microflora

Yeast ScPro1 in the dry active form were tested and investigated in vitro in artificial digestive environment that simulates human digestion, and, in particular, by examining the changes and impacts viable subjects of yeast to the environment during fermentation in the colon.

Fermentation in the colon is a continuous fermentation with a consistent flow environment for the preservation of flora. This environment contains mostly complex carbohydrate compound, is not digested in the upper digestive tract (starch, pectin, cellulose, etc), to a greater or lesser extent, hydrolyzed proteins and mucin.

Fermented compounds are extracted from the environment of the large intestine also consistently. Medium passes through the dialysis system for continuous removal of soluble fermentation products.

Dialysate collected for analysis of fatty acids with short chains is (AGCC). The environment is kept in anaerobic conditions created by its own fermentation gases, while the environment has a redox potential of less than -300 mV. In conclusion, the pH was adjusted to the preset value is 6.

Each cycle of digestion include: stabilization period microflora within 2-3 days after planting of the colon, treatment period (at least for 3 days) with at least one daily Supplement product and the period of the termination of the processing for 3 days. In each experiment was controlled and/or recorded with the following parameters:

the viability of yeast;

- change various aerobic and anaerobic bacterial populations;

- change the main fermentation products (AGCC and gases);

- detection of standard enzymatic activity;

temperature, pH and redox potential.

Fermentation was carried out in penicillin vial with a capacity of 60 ml, closed, resealable tube with a septum and filled with 30 ml of environment of the colon (the culture medium with fresh faecal microflora). A sample of yeast was added to 30 ml of medium.

The environment of the colon consisted, on the one hand, of the microbial suspensions obtained from fresh faeces, in phosphate buffer solution, and on the other hand, from a typical food product is, also used for the cultivation of the microflora of the colon in artificial colon.

After mixing environment of the colon with the test product, the bottle was corked and sealed.

All these manipulations produced in the exhaust anaerobic Cabinet (in an atmosphere of a mixture gas containing no oxygen). The vial was placed in a rotary incubator (37°C - 200 rpm) for 24 hours

For each product the test was carried out in two samples. At the same time in the same conditions also prepared 4 bottles with the control samples (without the tested product). Two bottles were processed immediately (initial time), and two vials were incubated in the same way as the subjects bottles.

The fermentation was stopped after 24 h, and then the vials were processed.

The production of fermentation gases. The volume of gas produced by fermentation, are determined through a system of MARRIOTT (the principle of measuring the volume of the water displaced by the pressure of the gases contained in penicillin vial). The analysis of the gases contained in the vial, was carried out by way of CPG (H2, CO2CH4O2).

The production of fatty acids with short chains. Made the selection of the first sample of the contents of the colon. Sample or froze or immediately processed for determining the concentration of AGCC (volatile fatty acids to the short circuits) in the supernatant culture fluid. This analysis was carried out by way of CPG. Investigated the metabolites represented acetic, propionic, butyric, isomaltol, Valerian, isovalerianic, nylon, isocaproate and heptane acid.

Microbiological analysis. Made the selection of the second sample of the contents of the colon and immediately processed (serial tenfold dilution dilution recovery environment) to count the number of total anaerobic, facultative aerobic-anaerobic and fungal microflora.

Survival rates of yeast ScPro1 presented in figure 1. In this figure, each vertical arrow indicates the introduction of yeast ScPro1.

It was noted that the yeast ScPro1 show good survival to 3 days after injection and a significant loss between the 4th and 7th days of the period of introduction. This fact shows that the yeast is not embedded in the internal environment of the colon.

The results of microbiological analysis are presented in figure 2. They show a decrease in the number of enterobacteria in the presence of yeast ScPro1 with increase after termination of the introduction of yeast. During the introduction of yeast ScPro1 it was also noted that decreased significantly microflora resistant to antibiotics (chloramphenicol, gentamicin).

Results on the effects of yeast ScPro1 on producing yuchih fatty acids with short chains (AGCC) are summarized in the following table (in mm) in the environment of the colon).

Before processingDuring processingAfter processing
Acetate71,4 ± 2,357,6 ± 4,260,6 ± 0,7
Propionate22,8 ± 0,626,5 ± 4,235,7 ± 1,1
Butyrate35,0 ± 1,636,5 ± 2,226,6 ± 4,2
Isobutyrate3,2 ± 0,33,3 ± 0,23,4 ± 0,1
Isovalerate5,6 ± 0,55,2 ± 0,25,3 ± 0,0
Valerate8,0 ± 0,67,8 ± 1,49,1 ± 0,9
The izokapronata0,1 ± 0,10,0 ± 0,00,0 ± 0,0
Kapronat9,0 ± 1,1 7,3 ± 0,35,7 ± 0,7
Heptanoate0,2 ± 0,20,0 ± 0,10,0 ± 0,0
Totalwas 155.3 ± 2,7144,1 ± 6,8146,4 ± 3,4

During treatment there was a marked reduction in the content of acetates, partially in favor of propionate, which implies a decrease in the activity of acetogenic microflora.

Among other monitored parameters was not observed a clear effect of treatment on:

- production of gases (based on the number and content);

- General and simple (stable over time) the concentration of sugars;

- enzymatic activity.

EXAMPLE 3. The study of the influence of yeast ScPro1 and SCB1 for inducing the production of cytokines

It was investigated the effect of active yeast ScPro1 and SCB1 for inducing the production of cytokines in peripheral mononuclear cells of human blood (PBMC).

Yeast ScPro1 and SCB1 were tested in the dry soluble form and dry active form in relation to their ability to induce the production of cytokines IL-10, IL-12, TNFα, TNFγ in human PBMC.

Obtaining peripheral mononuclear blood cells of a person

Fresh human blood, obtained from don the ditch with good health in the Center of blood transfusion, was diluted 2 times with PBS-Ca (GIBCO) and was purified by gradient ficoll (GIBCO). After centrifugation at 400 g for 30 minutes at 20°C peripheral mononuclear blood cells (PBMC) form in the serum layer in the form of a circle. PBMC were carefully collected with suction, suspended in 50 ml final volume using PBS-Ca and washed 3 times with the same buffer solution by centrifugation for 10 minutes at 20°C and 350 g. Then PBMC again suspended, using complete RPMI medium (GIBCO)enriched with 10% wt./about. fetal calf serum (inactivated at 56°C for 30 minutes), 1% wt./about. L-glutamine (GIBCO) and gentamicin (150 µg/ml) (GIBCO). The number of PBMC were counted under the microscope, adjusted to a concentration of 2·106cell/ml, and distributed (in 1 ml aliquots of solution) on 24-hole cups for cell culture (Corning, Inc.).

Microbiological preparations

After cultivation, carried out during the night, Lactobacillus, Lactococcus and Escherichia coli (reference strains) were washed 2 times with PBS buffer solution, pH = 7,2, before the subsequent suspendirovanie in PBS with a concentration of 2·109CFU/ml

The concentration of yeast used in the first series of experiments was 2·108CFU/ml For study comparing initial dose, it was possible to perform serial dilutions 10 on 10 for comparison of the action at the end is Tralach 2·10 7, 2·108and 2·109CFU/ml

Incubation of peripheral mononuclear blood cells of a person

10 µl of the suspension were placed in the wells of cups containing PBMC and incubated at 37°C in an atmosphere of a mixture of 5% CO2and 95% atmospheric air. After incubation for 24 hours the supernatant was aspirated, centrifuged at 2000 rpm (Eppendorf model), extracted sediment was stored at -20°C.

The control sample consisted of gram-positive bacteria (Lactobacillus and Lactococcus), gram-negative bacteria (Escherichia coli) and buffer solution in the absence of yeast.

Quantification of cytokines

The levels of expression of cytokines was determined by ELISA method. Tablets for ELISA covered antibody (during the night), and the antibody was saturated with 1%solution of PBS/BSA (bovine serum albumin). A calibration curve was obtained with known concentrations of cytokines with detection limits from 15.62 wide to 2000 PG/ml (incubation over night). Investigation and quantification of anticytokine made by measuring the activity of streptavidin with the substrate TMB (tetramethylbenzidine, Pharmingen2). Were used commercial kits Pharmingen, in accordance with the description of the manufacturer. Four were chosen cytokine: 3 Pro-inflammatory (TNFα, IFNγ, IL-12) and one anti-inflammatory (IL-10).

Results

Answers 4 is Fokino on 5 different donors was evaluated by the ratio of yeast/PBMC", equal to 1/1.

The results of quantitative determination of 4 cytokines, secreted in the supernatant culture fluid are summarized in the following table A. Data are presented as mean value (Moy) quantitative determination of 5 donors. The table shows also the values of the standard errors of the mean (Sem).

Table A
IL-10 (PG/ml)IFNγ (PG/ml)TNFα (PG/ml)IL-12 (PG/ml)
MoySemMoySemMoySemMoySem
The negative control sample0050050000
E. coli2474 83957376295911118538751515
Lactococcus lactis11143136103627062536298181101543
Bifidobacterium longum107235533780271641451756012220
Lactobacilus acidophilus4352598554346838183696857529343
SCB1569291278071923164922698141
ScPro14422921521893043643184785

It was observed:

1) production in the case of yeast ScPro1 and SCB1 very small, in some cases, no detectable amounts of IL-12-induced PBMC, in contrast to comparative bacteria;

2) significant levels of IL-10 in both cases, active yeast, allowed to reveal that SCB1 has better results than ScPro1;

3) is clearly smaller compared to the various subjects of probiotic bacteria the number of IFNγ and TNFα, secreted by the action of yeast ScPro1 and SCB1.

Conclusions:

it is clear that the yeast ScPro1 and SCB1 in the presence of PBMC does not induce the expression of proinflammatory cytokine IL-12 in contrast to the fact that traditionally see in the case of probiotic lactobacilli;

- yeast ScPro1 and SCB1 in the presence of PBMC induce significant levels of IL-10 (anti-inflammatory);

- the number of IFN-γ and TNF-α, secreted PBMC in the presence of yeast ScPro1 or SCB1, clearly less than in the case of probiotic bacteria.

Example 4. Evaluation of the protective action of yeast ScPro1 and SCB1 in the case of chemically induced colitis (TNBS) is clothed with the mouse

Traditionally used the proposed model with the animal was adapted to measure the anti-inflammatory action of yeast.

In this experience were used mouse strain Balb/c mice aged 6 weeks. Mice were acclimatized in the laboratory during the week prior experience with free access to food and water. Each sample was tested on a group of 10 mice. Colitis was induced in a cycle with free access to drinking water containing 5% (wt./about.) TNBS, within 7 days. Yeast is administered orally once daily by stomach tube for 3 days before induction of colitis by TNBS and during the period of TNBS treatment (7 days).

In addition to the two groups of subjects were used as the control group (negative control), which was used only saline.

The test parameters obtained from the results of the processing that follows.

- Macroscopic assessment of intestinal inflammation (scale Wallace). The colon of each mouse was examined under preprofile magnifier (5x) for the evaluation of macroscopic damage on the scale of Wallace from 0 to 10 depending on the evaluation criteria, characterizing the degree of inflammation, such as redness, thickness of the wall of the rectum and the area of ulceration.

Histological assessment of inflammation (scale Ameho). For the implementation of the histo is oricheskogo assessment scale Ameho from 0 to 6 depending on the degree of infiltration of inflammation, the presence of erosion, ulceration or necrosis and depth, as well as the distribution of damage on the surface of the used part of the colon taken exactly 2 cm from the anal canal. Quantitative determination of degradation and damage of the intestine was performed by 2 independent expert.

- Quantification of the expression of genes encoding IL-10 and PPARα. With this purpose from the tissues of the colon was isolated total RNA by means of the RNeasy kit (Macherey Nagel, Herdt, France) according to the manufacturer's instructions. Quantification of the RNA was carried out using a spectrophotometer. After treatment at 37°C for 30 minutes with 20-50 units of the drug DNase has I RNase-free (Roche Diagnostics Corporation, Indianapolis, Indiana, USA) were used primers oligo-DT (Roche Diagnostics Corporation, Indianapolis, Indiana, USA) for the synthesis of simple single-stranded circular DNA. The RNA was quantitatively determined using the set of SYBR green Master Mix (Applera, Martabe, France) and oligonucleotides person intended for in vitro studies (see tab. B below), by means of the device GeneAmp Abiprism 700 (Applera, Martabe, France). In each experiment used a reference and petalonia control samples. For each sample was carried out by three dimensions. The intensity of the staining SYBR vert was determined using the Abiprism 7000 SDS (Applera Martabe, France). All results are normalized by comparison with the gene coding for β-actin.

Table B
GenesThe nucleotide sequence nucleating
β-actinF: 5'-AAgTCCCTCACCCTCCCAAAAg-3'
R: 5'-AAgCAATgCTgTCACCTTCCC-3'
PPARαF: 5'-ACgATgCTgTCCTCCTTgATg-3'
R: 5'-gTgTgATAAAgCCATTGCCgT-3'
IL-10F: 5'-CAgTCAgCCAgACCCACAT-3'
R: 5'-gCTCCACTgCCTTgCTTT-3'

Yeast ScPro1 and SCB1 were tested in the previously described standard model of prevention. The control of body weight of the animals before induction of colitis showed that the composition of yeast, administered to mice, are very well tolerated.

Inflammation of the intestine, evaluated on a scale of Wallace, in the case of yeast ScPro1 (dry active form, 1 mg/day) and SCB1 decreased by 60% compared with the positive control. The SCB1 yeast also caused the reduction of inflammation. Necrosis of the intestine, assessed according to the scale Ameho, in the case of yeast ScPro1 (instant dry form, from 1 mg to 100 mg/day) decreased by one third compared with the positive control.

Yeast ScPro1 and SCB1, entered individually or jointly, have enhanced the level of gene expression, codereuse the anti-inflammatory interleukin IL-10 and the nuclear receptor PPARα

7-10 visible excellent value macroscopic indicators on scales Wallace and Ameho for yeast ScPro1 and SCB1 with different daily doses.

7 and 8 show respectively the macroscopic rate on a scale of Wallace and histological indicator on a scale Ameho for yeast to ScPro1 and SCB1 in dry soluble form, administered daily in an amount of from 10 μg to 1 mg

The numbers in each column of graphs, shown in Fig.7 and 8, indicated the following options:

1 means TNBS, taken separately;

2 means TNBS + ScPro1 (1 mg);

3 means TNBS + ScPro1 (100 μg);

4 means TNBS + SCB1 (1 mg);

5 means TNBS + SCB1 (100 µg).

You may notice that the yeast ScPro1 in dry soluble form, injected with a dose of 100 µg/day, significantly reduce damage on macroscopic and histological levels.

Figure 9 and 10 shows respectively the macroscopic rate on a scale of Wallace and histological indicator on a scale Ameho for yeast ScPro1 and SCB1, taken individually or in combination in the dry soluble form or in dry active form and entered daily in the amount of from 100 μg to 1 mg

On Fig and 15 illustrate, respectively, the levels of expression of genes encoding anti-inflammatory interleukin, and the nuclear receptor PPARα, at the level of putting cells.

The numbers to the each column of graphs, shown in figures 9, 10, 14 and 15, indicated the following options:

1 means TNBS, taken separately;

2 means TNBS + ScPro1 in dry soluble form (100 μg);

3 means TNBS + ScPro1 in the dry active form (100 μg);

4 means TNBS + SCB1 in the dry active form (100 μg);

5 means TNBS + ScPro1 in the dry active form (1 mg);

6 means TNBS + ScPro1 in the dry active form (100 μg);

7 means TNBS + ScPro1 in the dry active form (100 μg) + SCB1 (100 µg).

Conclusions

It can be noted that the yeast ScPro1 in the dry active form largely cause damage at the macroscopic level and there is a synergistic anti-inflammatory effect of the combination ScPro1 and SCB1 as at the macroscopic level, and at the histological level.

Yeast ScPro1 and SCB1 increased respectively 2.9 and 3.1 times the level of expression of the gene encoding anti-inflammatory interleukin IL-10, at doses of 100 mcg. The combination ScPro1 + SCB1 (column No. 7) increased 2.7 times the level of expression (Fig).

Yeast ScPro1 and SCB1 increased, respectively, 1.5 and 1.6 times the level of expression of the gene encoding the nuclear receptor PPARα, at doses of 100 mcg. The combination ScPro1 + SCB1 (column No. 7) increased 1.7 times the level of expression (Fig).

EXAMPLE 5. The study of the influence of yeast ScPro1 and SCB1 settling of the yeast Candida albicans at the level of the intestine in the case of chemically induced in the spalania in the model mouse

The purpose of this study is to determine the effects of introducing yeast ScPro1 and SCB1 probiotic type on the colonization of the intestine by pathogenic yeast Candida albicans and its effect potentiation of inflammation in the case of chemically induced colitis in a mouse model.

Subjects yeast taken in dry soluble form.

Experimental conditions

Mouse-female line Balb/C mice were aged 4 to 6 weeks. Since J0 until the day J14 animals received DSS (Dextran Sodium Sulfate (sulfate of dextranase) with a concentration of 1.5% in drinking water for chemical induction of inflammation.

Was carried out three experiences.

In the first experiment since J5 mice through a cannula was introduced 5·107yeast cells ScPro1 in 200 μl of PBS (phosphate buffer solution). This procedure was repeated daily for 19 days. A day J0 mice through a cannula was introduced 5·107cells of yeast strain C. albicans SC5314 in 200 ál PBS.

In the second experiment in the day J0 mice through a cannula was introduced 5·107yeast cells of the strain C. albicans SC5314 in 200 ál PBS. After 4 days the part of the mice through the gastric tube was introduced on 5·107yeast cells ScPro1 in 200 ál PBS. This procedure was repeated daily for 14 days.

In the third experience in day J0 mice through a cannula was introduced 5·107cells of yeast strain C. albicans SC5314 in 200 ál PBS. An hour later, the part of the mice through the gastric tube was introduced on 5·107cell d is Ozga ScPro1 in 200 ál PBS. This procedure was repeated daily for 14 days.

Animals involved in experiments 1, 2 and 3) daily controlled according to the following criteria:

the stool consistency and rectal bleeding, weight (clinical indicator);

- retrocognition 1 mg of faeces, homogenised in 1 ml PBS, 10 μl of which were seeded on Wednesday Candi-select; after culturing for 24 h at 37°C was calculated CFU C. albicans (colored blue) and S. cerevisiae (colored green);

animals were scored at the end of the test. Once the blood was collected by puncture of the heart, decantation at room temperature, serum was separated by centrifugation and stored at -80°C. Selected from the colon and divide into 4 pieces, 3 of them froze, and 1 was placed in a fixative (PFA, 4%) for histological examination.

Results

As can be seen in figure 3, in the first experiment (test on preventive effect in this model of chemically induced colitis is observed that the introduction of DSS significantly increases the colonization of the mucous membranes of the intestinal yeast C. albicans, since the days J4 (DSS + Ca). Is of great interest that the introduction of the probiotic yeast ScPro1 for 19 days significantly reduces colonization by yeast C. albicans induced by DSS.

As can be seen from figure 4, the WTO is the first experience (test for therapeutic effect) is observed, introduction probiotic yeast ScPro1 or SCB1 reduces settling induced by DSS. In addition, the action of yeast ScPro1 noticeable even after termination of the processing by DSS in the day J14.

Conclusions

From the above it follows that the introduction of yeast ScPro1 or SCB1 yeast significantly reduces colonization by yeast C. albicans, both in terms of prevention and in therapy. It should be noted that this protective effect lasts even after cessation of therapy.

EXAMPLE 6. Study of the inhibitory effect of yeast ScPro1 or SCB1 or their derivatives on the ability to adhesion and invasion of pathogenic strains of E. coli isolated from biopsy material of the small intestine of patients with Crohn's disease

The influence of active yeast ScPro1, SCB1 and their derivatives were investigated for their inhibitory effect on the ability to adhesion and invasion of pathogenic strains of E. coli isolated from biopsy material of the small intestine of patients with Crohn's disease.

Strains of E. coli, commonly referred to as the AIEC from Adherent-Invasive E. coli and isolated from biopsy material of the small intestine of patients with Crohn's disease (MC), is able to bind and capture the epithelial cells of the intestine.

The E. coli strain LF82 isolated from chronically damaged portion of the small intestine from the pain of the CSOs, suffering from Crohn's disease, has all the characteristics of an invasive bacterial pathogen. Characterization of the phenotype of the "adhesion-invasion" strain LF82 and the lack of genetic determinants of invasion, already described by E. coli, Shigella and Salmonella, has led to determenirovana existence of a new pathogenic groups of E. coli, which may be associated with Crohn's disease and marked AIEC. After phagocytosis by macrophages of the mouse or human AIEC strain LF82 survives and multiplies in a wide vacuoles, while maintaining the integrity of the host cell. In response to infection macrophages secrete a significant amount of TNFα. The prevalence of AIEC strains on the level of damage of the small intestine of patients with MC equal to 36.4 per cent.

The process of adhesion of bacteria to eukaryotic cells is the result of a specific interaction between the ligand present on the surface of bacteria called adhesion, and the receptor protein, glycoprotein or glycolipid nature, expressed on the surface of epithelial host cells. Regarding bacteria has been proven that adhesin FimH type 1 pili are involved in the adhesion of AIEC bacteria on the epithelial cells of the intestine. Bacterial adhesin FimH recognizes InterAcademy the CEACAM6 receptor (also known as CD66c or NCA), which is a glycoprotein rich in mannose residues and the Academy of Sciences of the normal sverkhekspressiya at the level of the small intestine in 90% of patients suffering from MC.

Experimental conditions

As the strain of the prototype was used AIEC strain LF82, notable for its ability to adhesion and invasion in epithelial intestinal cells in culture.

This study was conducted with 10 AIEC strains isolated from patients suffering from MC, to confirm the results obtained with the AIEC strain LF82.

The strain of E. coli DAEC (Diffuse Adherent E. Coli) C1845, which is attached to the epithelial cells by a mechanism that is not dependent on mannose (adesina Afa/Dr), was used as negative control sample.

Tests agglutination

With active yeast ScPro1 and SCB1 tests were carried out on quantitative agglutination in the presence of AIEC bacteria and in the presence of extracts of purified pili of type 1, derived from the AIEC strain LF82 in accordance with the method described Boudeau et al. (2001, Mol. Environ. 39:1272-84). Index agglutination was determined at a fixed concentration of yeast and variable concentrations of bacteria or purified pili of type 1.

In the case of fractions of yeast mannoproteins type, which is not observed agglutination, determining the ability of the binding of type 1 pili were carried out by the ELISA method.

Such testing is traditionally performed in a microplate. Fractions of yeast is fixed on the microplate. Various breeding behaviour is the R type 1 pili is brought into contact with the fractions of yeast. After leaching drinking type 1 open through antibody anti-drinking type 1, obtained from rabbit (Boudeau et al., 2001). After washing, use a secondary antibody linked to peroxidase. Quantification is carried out using peroxidase substrate (H2O2) and Chromogen (tetramethylbenzidine) by measuring absorbance with a microplate photometer at 450 nm.

Test for inhibition of the interaction of AIEC bacteria with CEACAM6 receptor expressed on the surface of epithelial cells of the intestine, yeast ScPro1 or SCB1

Used cell

To test for inhibition in vitro (pre-and co-incubation) were taken undifferentiated epithelial cells of the intestinal T84, largely expressing CEACAM6 receptor. The T84 cells were cultured in an atmosphere with 5% CO2at 37°C in an alkaline medium DMEM (Wednesday Needle in the modification of Dulbecco), supplemented with 50% Ham-F12 (Life technologies) and 10% fetal calf serum, decompencirovannah when heated. To this medium was added 1% amino acids, non-essential (Life technologies), 1% glutamine (Life technologies), 200 units/l penicillin, 50 mg/l streptomycin, 0.25 mg/l amphotericin B and 1% vitamin mixture X-100 for MEM medium (Minimum Essential Medium) (Life Technology). Cells were seeded at 4·105cells per well in 1 ml and incubated for the tion 48 h at 37°C in an atmosphere with 5% CO 2. Then a layer of T84 cells were washed with PBS solution and then in each of the wells was added 1 ml of the infected medium (DMEM/F12 + 10% SVF). Bacterial suspension in PBS to an optical density DO6200.1, prepared on the basis of AIEC strain LF82, cultured overnight at 37°C in broth, Luria-Bertani (LB). The T84 cells infected multiplet source of infection (MOI) of 10 bacteria per 1 cell, adding 25 µl of the bacterial suspension with an optical density DO6200.1, infected environment. Tablet in 24 wells were incubated for 3 h at 37°C in an atmosphere enriched with CO2. Determination of the residual adhesion and residual invasion of bacteria was carried out respectively described below.

Carried out the experiment with cells CHO-K1, not expressing CEACAM6, and the same genetically modified cells, stably expressing CEACAM6 (CHO-K1/CEACAM6). Cells CHO-K1 were cultured in DMEM/F12 containing 5% fetal calf serum, 1% L-glutamine, 200 units/l penicillin, 50 mg/l streptomycin and 0.25 mg/l amphotericin B. Cells CHO-K1 were cultured in DMEM/F12 containing 5% fetal calf serum, 1% L-glutamine and 600 µg/l hygromycin. Cells were seeded in 24-well plate at the rate of 2·105cells per well. After incubation for 7-8 h at 37°C the medium was replaced with a new cultural environment, more is authorized 5 μm sodium butyrate, to induce the expression of CEACAM6. To control the expression of CEACAM6 protein transfitsirovannykh cells was performed by Western blotting.

After incubation for 20-24 h at 37°C cells were incubated with increasing concentrations of the strain of yeast ScPro1 in dry soluble form within 1 h (experience pre-incubation), then they were infected by MOI 20 (4·106bacteria per well) for compliance with the ratio of bacteria/yeast used when testing with T84 cells. After incubation for 3 h at 37°C attached bacteria was calculated in the absence or in the presence of yeast, respectively, described below.

In another experience used surgical material obtained from patients. The enterocytes obtained from biopsy material of the small intestine 3 patients suffering from Crohn's disease, washed with a solution of PBS and then pre-incubated in an Eppendorf tube with a capacity of 2 ml in 1 ml of DMEM medium containing 20% fetal calf serum, in the presence of 0, 1.25, 2.5, or 5 mg/ml of the strain of yeast ScPro1 in dry soluble form. The contents of the tubes were mixed by rotation for 15 min at 37°C, then the enterocytes infected in the presence of 50 ál of yeast culture in LB medium overnight AIEC strain LF82. Next was carried out by incubation for 3 h at p is remesiana. The enterocytes were washed 2 times with PBS solution, then put the between-subject plate and cover glass and observed with phase-contrast microscopy. Estimates of the number of bacteria attached to the brush border of enterocytes, carried out in the absence or in the presence of yeast. Experience has also carried out with not having drank a mutant LF82-delta fimH to determine the basal level of adhesion of AIEC bacteria that do not recognize drank type 1 receptors CEACAM6. Test for inhibition of adhesion were carried out in the presence of antibodies anti-CEACAM6.

The method of analysis to determine the residual adhesion and residual invasion of bacteria on the epithelial cells of the intestinal T84

The cell layer was washed with a solution of PBS 4 times in 1 ml, and then the cells were subjected to lysis by incubation for 5 min at room temperature with 500 μl of 1%solution of Triton X-100 in distilled water. Lysates were diluted and then applied to LB agar-Agar to determine the number of CFU, corresponding to the number of attached bacteria.

To calculate invasive bacteria cell layer was rinsed with PBS solution after 3 h after infection, and then for 1 h were incubated with 1 ml of infected medium containing 100 μg/ml of gentamicin for the destruction of extracellular bacteria. Invasive bacteria was counted after lysis of the cells, serial dilutions and application n the agar LB-agar.

The levels of adhesion and invasion of AIEC strain LF82 analyzed compared with cells infected with AIEC strain LF82 and not subjected to any processing by the yeast, or derived from yeast.

All results are expressed respectively value R:

R = number of attached or invasive bacteria in the presence of yeast ScPro1/number of attached or invasive bacteria in the absence of treatment.

Method 1. The model of co-incubation

The T84 cells and the suspension of bacteria was obtained according to the technique previously described in the test for adhesion and invasion. Yeast or derivatives of yeast suspended in PBS with a certain concentration and 25 μl of this suspension was added to the infected environment T84 cells (1 ml). Then the cells directly infected by the bacterial strain MOI 10. A mixture of "suspension of bacteria/yeast incubated in the presence of cells homogenized, and then 24-well plate were incubated for 3 h at 37°C. the Levels of adhesion and invasion of bacterial strain was determined, respectively, described previously and in the absence and in the presence of yeast or yeast extracts during infection. The correlation between the level of bacterial adhesion or invasion in the absence of yeast (100%) and the level of bacterial adhesion or invasion in the presence of yeast is a level of residual adhesion or the residual of an invasion of bacteria.

Method 2. Model preliminary incubation

The T84 cells and the suspension of bacteria was obtained according to the technique previously described in the test for adhesion and invasion. Suspension of yeast or derivatives of yeast was added to the infected medium (1 ml) of T84 cells in a volume of 25 µl. Suspension of yeast homogenized and then incubated for 1 h at 37°C in 24-hole tablet for culturing cells. After this incubation, the T84 cells infected bacterial strain MOI of 10 in the presence of yeast for 3 h at 37°C. Counting attached and invasive bacteria was carried out, respectively, previously described in the presence or in the absence of yeast to determine the percentage of residual adhesion or residual infestations, while 100% means the adhesion or invasion in the absence of yeast.

- Verification of expression of CEACAM6

Immunocytochemical labeling was carried out for each batch of cultured cells to test for the presence and estimating the number downregulation of CEACAM6. Cells were cultured on sterile glass plates. The cell layer was washed with a solution of PBS, then fixed 3%paraformaldehyde at pH = 7.4 for 10 minutes at room temperature. Cells were incubated with monoclonal antibody anti-CEACAM6 (clone 9A7, Genovac), diluted in a ratio of 1/100 mixture "PBS-5% HP savor the TCI", in a humid atmosphere for one hour. After washing with a solution of PBS, the cells were brought into contact with the secondary antibody associated with fluorochromes (anti-mouse FITC, Zymed) and diluted in the ratio of 1/500 mixture "PBS-5% horse serum for 1 hour in a humid atmosphere. Glass plates were fixed on a glass slide Moewiol and then visualized using a fluorescent microscope.

- Check the absence of cellular cytotoxicity

No cellular cytotoxicity induced by different doses of yeast was examined by quantitative analysis of lactate dehydrogenase (LDH) in the incubation media "yeast/cell" or "FDL/cells" (Glasser et al., 2001).

Results

Tests agglutination with LF82

The agglutination titers obtained with LF82 in the presence of yeast ScPro1 or SCB1 in culture (= fresh) or dry (drying to dry, instant or liofilizovannyh forms), are summarized in the following table, which presents the results from 3 to 5 independent experiments.

The agglutination titer
YeastFormAverageThe minimum is the value of the titre The maximum value of the titre
ScPro1Fresh culture1/71/31/12
ScPro1Dry soluble form1/581/201/96
ScPro1Dry liofilizovannye form1/431/241/64
SCB1Fresh culture1/281/121/40
SCB1Dry soluble form1/161/121/20
SCB1Dry liofilizovannye form1/191/161/24

Were obtained reliably good results agglutination with LF82 in the case of dry yeast ScPro1 in dry soluble form and SCB1 in dry soluble form.

the La data of yeast has been proven the importance of the favorable effects of method and in particular, the method of drying on their potential agglutination.

Tests agglutination with purified drinking

The agglutination titers obtained with purified drinking in the presence of yeast ScPro1 in culture (= fresh) or in dry soluble form and SCB1 yeasts (in dry form) are summarized in the following table.

The agglutination titer
YeastFormExperience 1Experience 2Experience 3
ScPro1Fresh compressed yeast1/3001/3001/400
ScPro1Dry soluble form1/6001/6001/300
SCB1Dry liofilizovannye form1/3001/3001/200

This test confirms that for agglutination largely need of interaction the interaction "drinking-yeast". Given that drinking is necessary for recognition Manasseh structures that are recognized in yeast and participate in the observed phenomenon of agglutination. Best results are obtained in the case of yeast ScPro1 in dry soluble form.

The results of tests to determine the ability of the binding of type 1 pili from mannoproteins fractions of yeast ScPro1

On Fig clearly shown that the purified pili of type 1, obtained from AIEC strain LF82, specifically fixed on mannoprotein yeast. It should be noted that the method of obtaining (thermal or enzymatic) of such mannoproteins (and EL05 EL06) has little effect on the affinity constant to drink.

The results for inhibition of the interaction of AIEC bacteria with CEACAM6 receptor expressed on the surface of epithelial cells of intestine

1/ the Distribution of samples of yeast or derivatives of yeast on the ability of inhibition of adhesion and invasion of AIEC strain LF82 to the epithelial cells of the intestinal T84 in the model for co-incubation.

Were investigated yeast ScPro1 in dry soluble form (3,09·107cell/mg), ScPro1 in dry form (1,86·107cell/mg) and SCB1 in dry soluble form (of 5.83·107cell/mg), as well as mannoprotein yeast EL05 (in dry form).

As a comparative sample were added yeast Ultra-levuresup> ®(Biocodex, 2,054·107cell/mg).

Comparison of the inhibitory capacity of yeast with an equal amount of yeast in the model for co-incubation

After washing with a solution of PBS and centrifugation for 15 min at 7500 rpm samples of yeast again suspended in PBS at a concentration of 4·108cell/ml Dilution of yeast in PBS was carried out with a ratio of 1/2, 1/10, 1/20 and 1/100.

Was carried out three independent experience in accordance with the method 1. The results on figa and 19B (residual adhesion and residual infestation) are presented as averages of the levels of residual adhesion and residual invasion and spacing errors, the corresponding standard error of mean values.

On figa and 19B shows the following results:

adhesion:

- yeast ScPro1 in dry soluble form and ScPro1 in dry form strongly inhibit the adhesion of strain LF82 in T84 cells depending on the dose. Inhibition of yeast ScPro1 in dry soluble form in an amount of 5·105cell/ml is more significant compared to yeast ScPro1 in dry form in an amount of 5·106cell/ml;

- SCB1 yeast in dry soluble form inhibit the adhesion of less than 2 other sample yeast: residual adhesion at a dose of 1·107cell/ml is 45.7% compared to 18.7% and 8% residual adhesion to ScPro1 in dry soluble form and ScPro1 in dry form, respectively;

infestation:

- yeast ScPro1 in dry soluble form significantly inhibit the invasion of T84 cells AIEC strain LF82 at a dose of 1·105cell/ml At a dose of 1·107cell/ml level of residual infestation is 16,3%;

for yeast ScPro1 in dry form and SCB1 in dry soluble form inhibitory effect is slower at the dose of 1·106and 5·106cell/ml, respectively.

Test for inhibition of mannoproteins EL05 in the model for co-incubation

Mannoprotein yeast EL05 suspended in PBS with a concentration of 160 mg/ml was Performed by serial dilutions in PBS with a ratio of 1/2, 1/4, and 1/8 1/40 and was added 25 μl of each suspension mannoproteins to the infected environment in accordance with the method 1.

Was carried out three independent experience. The results shown figa and 20B (residual adhesion and residual infestation), presented as mean values of residual levels of adhesion and spacing errors, the corresponding standard error of mean values.

On these figures it is shown that mannoprotein yeast EL05 have the ability of inhibiting the adhesion and invasion of AIEC strain LF82 in T84 cells depending on the dose in the model for co-incubation.

2/ Distribution of samples of yeast or derivatives of yeast on the ability of inhibition of adhesion and invasion of AIEC strain LF82 is and epithelial cells of the intestinal T84 model pre-incubation.

In the model pre-incubation were used the same yeast samples and fractions, as in the model for co-incubation.

Comparison of the inhibitory capacity of yeast with an equal amount of yeast in the model pre-incubation

After washing with a solution of PBS and centrifugation for 15 min at 7500 rpm samples of yeast again suspended in PBS at a concentration of 4·108cell/ml Dilution of yeast in PBS was carried out with a ratio of 1/2, 1/10, 1/20 and 1/100. Was carried out three independent experience in accordance with the method 2.

The results shown figa and 21B (residual adhesion and residual infestation), presented as mean values of residual levels of adhesion and residual invasion and spacing errors, the corresponding standard error of mean values.

Pretreatment of T84 cells yeast provides a significant inhibition of adhesion of strain LF82 at a dose of 5·106cell/ml strain ScPro1 in dry soluble form and ScPro1 in dry form. However, in the case of SCB1 yeast in dry soluble form so dose any significant inhibition was not observed.

Pretreatment of T84 cells yeast provides the inhibition of invasion strain LF82 at a dose of 1·105cell/ml of the strain of yeast ScPro1 in dry form.

At the dose of 5·105CL is TKA/ml 3 sample of the yeast to induce a significant decrease in invasion strain LF82.

Test for inhibition of mannoproteins EL05 in the model pre-incubation

Mannoprotein yeast EL05 suspended in PBS with a concentration of 160 mg/ml was Performed by serial dilutions in PBS with a ratio of 1/2, 1/4, and 1/8 1/40 and was added 25 μl of each suspension mannoproteins to the infected medium according to method 2. Was carried out three independent experience. The results shown figa and 22B (residual adhesion and residual infestation), presented as mean values of residual levels of adhesion and spacing errors, the corresponding standard error of mean values.

Thus, mannoprotein EL05 allow to inhibit depending on the dose adhesion and invasion of strain LF82 at a concentration of 2 mg/ml

The test results on the inhibition of yeast adhesion of AIEC strain LF82 in the case of CHO-K1 expressing or not expressing CEACAM6 receptor, the model pre-incubation

Was performed five independent experiments according to the previously mentioned method 2.

On Fig shown that there was a significant inhibition of adhesion of AIEC strain LF82 on the cells of CHO/CEACAM6 when pre-incubation with 25 μg/ml of yeast. In the case of these cells, the observed inhibitory effect depending on the dose.

Adhesion of AIEC strain LF82 also observed in cells CHO-K1, which is undoubtedly due to judge what essay minnesotabased proteins, expressed on the surface of such cells. However, pre-incubation of strain LF82 with yeast ScPro1 in dry soluble form does not provide inhibition largely adhesion on nitrostilbene cells.

Thus, this circumstance shows that the yeast affect the adhesion of strain LF82 with CEACAM6 receptor expressing cells.

Results inhibition of adhesion of AIEC strain LF82-level brush borders of enterocytes of patients with Crohn's disease, in the model of pre-incubation

On Fig shows the average indexes of adhesion obtained in the course of the experiment and calculated in the presence or absence of increasing concentrations of yeast ScPro1 in dry soluble form (mg/ml) or in the presence of antibodies anti-CEACAM6. According to the results presented in this figure, it can be stated in a dose-dependent significant decrease in the adhesion of AIEC strain LF82 to the brush border of the enterocytes of patients in the presence of a strain of yeast ScPro1 in dry soluble form. When the dose of yeast 5 mg/ml residual adhesion of AIEC strain LF82 such adhesion observed in the presence of antibodies anti-CEACAM6, or adhesion observed in the case of the mutant, devoid of type 1 pili.

Conclusions

From this study it follows that:

- yeast ScPro1 and SCB1, in particular, in the Hoi an instant form demonstrate a greater ability agglutination strain LF82;

- yeast ScPro1 and SCB1 able to inhibit in vitro adhesion and invasion of epithelial cells (T84, the enterocytes of the biopsy materials of the small intestine) and CHO cells expressing the receptor for human CEACAM6, in the case of E. coli bacteria, depending on the dose;

- mannoprotein able to inhibit in vitro adhesion and invasion of epithelial cells (T84, the enterocytes of the biopsy materials of the small intestine) and CHO cells expressing the receptor for human CEACAM6, in the case of E. coli bacteria, depending on the dose;

- in vitro yeast ScPro1 capable at high concentrations, partly to protect approximately 80% of cells infected by bacteria.

EXAMPLE 7. Study the regulatory role of the yeast ScPro1, SCB1 and derivatives of yeast in the expression of genes encoding IL-10 and PPARα, in the epithelial cells of the human intestine in vitro

Was studied probiotic yeast character ScPro1 and SCB1, administered separately or in combination, and/or fractions of yeast and their ability to inhibit the occurrence of inflammation through interaction with some intestinal receptors.

Tests in vitro

In particular, it was investigated the action of yeast and derivatives of the yeast of the present invention on different receptors in the epithelial cells of the intestine by in vitro assays of two cell lines of colon cancer CaCo-2 (ATCC HTB-37 and HT-29 (ATCC HTB-38).

For this purpose we carried out transcriptional analysis by extraction of RNA by the following method.

Cells were subjected to lysis in trisole. Then, the soluble fraction was carried out stage by desoksiribonukleaza, adding 200 μl of a solution containing 10 units of ribonuclease inhibitor, and 10 units of deoxyribonuclease.

10 μg of RNA were retrotranscription in the presence of 200 units of inverse transcriptase, dithiothreitol, oligo-dT15 and deoxyribonucleotides.

Amplified cDNA by well-known methods of competitive polymerase chain reaction (English PCR, Polymerase Chain Reaction), using specific sense and antisense seed, in particular genes, such as IL-10 and PPARα.

After 40 cycles of amplification performed in the presence of 1.25 units of Ampli Taq Gold 5000, and separation of different samples on a 3%agarose gel, the intensity of the bands was determined using the dissector.

The results are expressed as the number of molecules of mRNA per 105molecules of β-actin as an internal standard.

The results of figure 11 represent the values of expression of mRNA in an hour (index A) and 3 hours (index B) after bringing into contact of yeast or derivatives thereof, with epithelial intestinal cells expressing the gene encoding anti-inflammatory protein IL-10.

Figure 11 numbers denote the tested yeast/derivatives, yeast, which showed the level of quick expression greater than 4 times the comparative signal:

3 means the yeast Saccharomyces cerevisiae;

5 means the yeast ScPro1 of the present invention;

6 means the extract of Saccharomyces cerevisiae;

12 indicates the fraction of RNA of the yeast Saccharomyces cerevisiae.

These results well demonstrate that the yeast and derivatives of the yeast Saccharomyces cerevisiae according to the present invention induce rapid expression, after an hour, the gene encoding anti-inflammatory cytokine IL-10.

Indeed, compared with the untreated control sample, the expression of mRNA in the case of yeast and their derivatives according to the present invention exceeds 4 times the value on the ordinate axis, which already indicates an excellent level of quick expression.

Other results are presented on Fig. This figure shows the change depending on the quantity of the applied derivatives of yeast expression of the endogenous gene encoding the protein of IL-10.

Shown in Fig values of expression were measured after one hour (1 h) and three hours (3 hrs.) Ways of expression in the case of yeast and extracts of the yeast Saccharomyces cerevisiae according to the present invention are indicated by numbers:

5 means the yeast ScPro1 of the present invention;

6 means the extract of Saccharomyces cerevisiae;

8 means parietal β-glucan of Saccharomyces cerevisiae;

9 who appoints the parietal mannoprotein yeast Saccharomyces cerevisiae;

11 indicates the fraction of DNA of the yeast Saccharomyces cerevisiae;

12 indicates the fraction of RNA of the yeast Saccharomyces cerevisiae.

Expression was measured at various concentrations of yeast and/or their derivatives.

The darker the color column on Fig, the greater is the concentration of yeast/derived.

The results presented in Fig show that extracts of the yeast Saccharomyces cerevisiae according to the present invention induce rapid expression of the endogenous anti-inflammatory cytokine (IL-10).

The results shown Fig represent the values of expression of mRNA in an hour (index A) and 3 hours (index B) after bringing into contact of yeast or derivatives thereof, with epithelial intestinal cells expressing the gene encoding the nuclear receptor PPARα.

On pig numbers denote tested yeast/derivatives of yeast, which showed the level of a slow expression of more than 3-fold comparative signal:

1 means the yeast Saccharomyces cerevisiae ScPro1 of the present invention in the dry active form;

4 means the yeast Saccharomyces cerevisiae;

5 means the yeast Saccharomyces cerevisiae ScPro1 of the present invention in the dry active form;

8 indicates the fraction of the cell wall of the yeast Saccharomyces cerevisiae;

9 indicates the fraction of parietal β-glucans of yeast Saccharomyces cerevisiae;

10 indicates the fraction of parietal mannoproteins yeast Sacchromyces cerevisiae;

11 indicates the fraction of DNA of the yeast Saccharomyces cerevisiae;

12 indicates the fraction of RNA of the yeast Saccharomyces cerevisiae.

These results well demonstrate that the yeast and derivatives of the yeast Saccharomyces cerevisiae according to the present invention induce a slow expression after three hours, the gene encoding the nuclear receptor PPARα.

Indeed, the expression of mRNA in the case of yeast and their derivatives of the present invention is greater than 3-fold axis value y, which already indicates an excellent level slow expression.

EXAMPLE 8. The study ex vivo regulatory role of the yeast and derivatives of yeast in the expression of genes encoding IL-10 and TNF-α, in the epithelial cells of the human intestine isolated from biopsy material of patients suffering from Crohn's disease

Influence of yeast and/or derivative of the yeast on the secretion of cytokines IL-10 (anti-inflammatory) and TNF-α (Pro-inflammatory) was studied ex vivo in biopsy material of patients suffering or not suffering from Crohn's disease.

Biopsy materials bowel selected patients suffering or not suffering from Crohn's disease, then placed for 24 h in medium HBSS-CMF, supplemented with penicillin and streptomycin, at 37°C in atmosphere containing 5% CO2. After washing, biopsy materials were brought into contact with the yeast, or derived from yeast in the tech is of 4 hours in medium RPMI 1640. The supernatant was separated for analysis by ELISA method. Then the biopsy materials were subjected to lysis in order to extract as mRNA and total protein.

Confirmation of cytokine secretion was carried out by immunological analysis of the supernatant fluid of the culture cells and protein extracts by ELISA method. Proteins were denaturiruet for 5 minutes at 95°C in a precipitation buffer solution (about./vol.; 75 mm Tris, pH 6.8; 5% glycerol; 0.25% of bromophenol blue, 2% SDS, 5% β-mercaptoethanol), was applied (50 μg) was separated on 10%polyacrylamide gel. After separation, proteins were transferred to PVDF membrane (Hybond-P, Amersham Pharmacia Biotech, Orsay, France) by semi-dry method of elektroprenos (Hoefer TE77, Amersham Pharmacia Biotech, Orsay, France) for 1 hour at 16 Century PPARα and IL-10 were isolated using polyclonal antisera rabbit anti-human PPARα and anti-IL-10 man, diluted in the ratio of 1/500, and quantitatively determined by chemiluminescence (E.C.L. Amersham Pharmacia Biotech, Orsay, France) on film Biomax-MR (Kodak) via Gel Analyst (CLARA VISION, Paris, France).

On Fig and 17 show the results obtained for IL-10 and TNF-α respectively. On the y-axis of the pending number of cytokines measured in PG/ml Each point corresponds to a measurement result obtained from biopsy material of a patient suffering from Crohn's disease in the acute stage (circle cher the CSOs color), a patient suffering from Crohn's disease in remission (gray circle), and healthy people (white circle). The rectangle corresponds to the average value of the measurements.

On these figures:

- 1 means the yeast Saccharomyces cerevisiae ScPro1 of the present invention in the dry active form;

- 3 means the yeast Saccharomyces cerevisiae;

- 8 means the fraction of the cell wall of the yeast Saccharomyces cerevisiae;

- 11 indicates the fraction of DNA of the yeast Saccharomyces cerevisiae;

- 12 means the fraction of RNA of the yeast Saccharomyces cerevisiae.

On Fig shown that the yeast ScPro1 of the present invention increase 2 times the secretion of anti-inflammatory cytokine IL-10 epithelial cells of patients suffering from Crohn's disease or in remission, compared with healthy people and a negative control sample (-), corresponding to the measurements made in the presence of only saline solution.

On Fig shown that the yeast ScPro1 of the present invention does not cause an increase in the secretion of proinflammatory cytokine TNF-α in intestinal cells isolated from biopsy material of patients suffering from Crohn's disease or in remission. No yeast ScPro1, nor any other subjects yeast or derivatives of yeast does not induce any secretion of TNF-α.

EXAMPLE 9. Study of the analgesic properties of driven and derivatives of yeast in model rats with colorectal extension

Experience with healthy rats

1/ Materials and methods

In this research, we used rats-males line Sprague Dawley (Charles River, Arbresle, France) weighing in the range of from 175 to 200 g Rats were acclimatized to the conditions of the kennel during the week before the experiment. Animals were kept for five animals in a cage with free access to food and water. All tests were performed according to the recommendations of the Committee for Research and Ethical Issues included in the International Association for the Study of Pain [6]. To avoid or minimize discomfort of the animals were taken precautions.

2/ evaluation of the sensitivity of the colon

Pain reception of the animals was evaluated by measuring the pressure inside the colon that is required to evoke a behavioral response. Such pressure created colorectal expanded by inflating the balloon inserted into the large intestine. Behavioral response is characterized by raising the rear part of the body of the animal and clearly observable abdominal contraction, corresponding to strong contractions [7-9]. Rats were anestesiologi volatile anesthetic agent (2%isoflurane) and intrarectal entered the tank (the technique according to the procedure described Bourdu [8]) as it is less traumatic to a depth of 7 cm from the anus. The catheter was attached to the base of the tail with adhesive tape. After 5 minutes, the rat was placed and in the middle of the box out of plexiglass, and the catheter was attached to the e-barostat (Distender Series IIRTM, G & J Electronics). The pressure was increased continuously until disabled by pain reflex or before reaching the pressure limit value is set to 80 mm RT. Art.

3/ Input connections

The yeast was administered via a stomach tube once daily for 15 days.

For a positive control for 30 minutes before colorectal expansion intraperitoneally injected morphine with a dose of 1 mg/kg

The study used 8 groups of rats:

- 10 control rats treated with PBS;

- 10-treated rats ScPro1 in dry soluble form (100 µg/day) (group 1);

- 10-treated rats ScPro1 in dry form (100 µg/day) (group 2);

- 10-treated rats strain SCB1 (100 µg/day) (group 3);

- 10-treated rats strains ScPro1 in dry soluble form (50 µg/day) + SCB1 in dry soluble form (50 µg/day) (group 4);

- 10 rats receiving 1 injection of morphine (1 mg/kg, 30 min before the expansion) (group 5).

4/ Results

On Fig shown that the yeast ScPro1, on the one hand, put into dry soluble form separately (group 1) or in combination with strain SCB1 (group 4), and on the other hand, put into dry active form (group 2), increase the threshold of pain perception, greatly reducing, thus, the perception of visceral pain compared with rats that nothing is f was introduced.

The following results are given in mm RT. Art. compared with the control group:

to 74.5 ± 3,07 versus 53.6 ± 3,9, p = 0.07 for strain ScPro1 in dry soluble form (group 1);

- 66,5 ± 3,36 versus 53.6 ± 3,9, p = 0.04 for the combination of strains ScPro1 and SCB1 in dry soluble form (group 4);

- 72 ± 2,59 versus 53.6 ± 3,9, p < 0.01 for strain ScPro1 in dry soluble form (group 2).

On the other hand, this effect is comparable with the effect caused by group 5 morphine, used with a dose of 1 mg/kg, with pain threshold 72 ± 2,59 mm RT. Art.

Strain SCB1 in dry soluble form also induces an analgesic effect in healthy rats - 70 ± 3,16 mm RT. Art., p = 0,026.

Experience with rats with increased visceral sensitivity

1/ Materials and methods

Were used rat-female line Sprague Dawley (Charles River, Arbresle, France) weighing in the range of from 175 to 200 g Rats were acclimatized to laboratory conditions for a week before the experiment. Animals were kept for five animals in a cage with free access to food and water. All tests were performed according to the recommendations of the Committee for Research and Ethical Issues included in the International Association for the Study of Pain [6]. There have been substantial precautions in relation to living conditions to avoid or minimize discomfort to the animals.

2/ the Induction of enhanced sensitivity is lnasty colon by flushing with butyrate

For each lavage catheter (Fogarty catheter with a diameter of 2 mm) was injected into the colon at a depth of 7 cm from the anus and introduced animals 2 times daily for 3 days 1 ml 200 mm sodium butyrate with a neutral pH (pH = 6,9). Healthy animals were injected with saline solution.

3/ Treatment of animals yeast according to the present invention

It was used 10 groups of animals possessing increased visceral sensitivity (n = 10 animals per group). Experimental animals were injected via a stomach tube 100 mcg yeast once a day for 15 days. Control animals were injected with PBS according to the same method as before. Yeast suspended in PBS solution. Instillation of butyrate or saline was started 7 days after the first injection via a stomach tube for 3 days. Increased sensitivity of the colon was measured 14 days after the start of treatment by oral or 7 days after instillation into the large intestine.

4/ Group experimental animals

The study used seven groups of rats. Group numbers correspond Fig:

10 rats treated with PBS (control group);

10 rats treated with dry yeast ScPro1 in dry soluble form (100 µg/day) (group 1);

10 rats treated with dry yeast ScPro1 in the dry active form (100 µg/day) (group 2);

10 rats treated with SCB1 yeast in dry soluble form (100 µg/day) - (group 3);

10 rats receiving ScPro1 in dry soluble form (50 µg/day) and SCB1 in dry soluble form (50 µg/day) (group 4);

10 rats receiving morphine (group 5);

10 rats treated with fibrates - (group 6).

5/ Results

First of all, it can be noted that the control rats pain threshold in experiments with rats with high sensitivity less pain threshold in healthy rats.

Yeast ScPro1 in dry soluble form, administered separately or in combination with SCB1 yeast, have in this model promising analgesic effect.

The following results are obtained:

Groupmm RT. Art.
156,5 +/- 4,27; p = 0.03
459 +/- 4,33; p = 0.02

The yeast of the present invention allow you to restore the level of perceived pain, comparable to the levels observed in healthy rats. Analgesic effect induced by the yeast equivalent effect caused by morphine.

On Fig also shown significant analgesic effect of fenofibrate, which increases 2 times the threshold of pain perception in rats with visceral hypersensitivity (70 +/- 4,48, p= 0.001).

This result confirms the role of the receptor PPARα in the modulation of visceral pain.

1. The Saccharomyces cerevisiae strain deposited at the National collection of cultures of microorganisms under the number CNCM I-3856 used as a probiotic, suitable for food or pharmaceutical compositions.

2. The strain Saccharomyces cerevisiae var. boulardii, deposited in the National collection of cultures of microorganisms under the number CNCM 1-3799 used as a probiotic, suitable for food or pharmaceutical compositions.

3. A composition comprising a yeast strain according to claim 1 and/or the yeast strain according to claim 2 and/or at least one of the parietal mannoproteins EL 05 and 06 EL yeast strain according to claim 1, for use in the production of food or pharmaceutical compositions.

4. The composition according to claim 3, characterized in that the yeast are in a dry or fresh form.

5. The composition according to claim 4, characterized in that the yeast are instant dry form or in dry active form.

6. Composition according to any one of p 5, characterized in that it contains from 107up to 6·1010SOME and preferably from 108up to 2·1010SOME yeast strain according to claim 1 and/or yeast strain according to claim 2.

7. Composition according to any one of p 5, characterized in that it contains from 1 mg to 10 g and preferably from 1 mg to 1 g yeast strain according to claim 1 and/or d is of ogga strain according to claim 2 and/or at least one of the parietal mannoproteins EL 05 and 06 EL yeast strain according to claim 1.

8. Composition according to any one of p-7 for use in food compositions intended to improve the comfort of the gastrointestinal tract and/or improving the intestinal microflora.

9. Composition according to any one of p-7 for use in the manufacture of pharmaceutical compositions intended for the treatment and/or prevention of intestinal disorders, functional bowel disorders or diseases of the gastrointestinal tract.

10. Composition according to any one of p-7, for use in the manufacture of pharmaceutical compositions intended for the treatment and/or prevention of diseases or disorders of the gut, followed by a state of hyperalgesia.

11. Composition according to any one of p-10 for the introduction of yeast in a daily dose of from 107up to 6·1010SOME and preferably from 108up to 2·1010SOME.

12. Composition according to any one of p-10 for the introduction of yeast and/or at least one of the parietal mannoproteins EL 05 and 06 EL yeast strain according to claim 1 in a daily dose of from 1 mg to 10 g



 

Same patents:

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: process engineering.

SUBSTANCE: set of invention relates to water treatment. Proposed method comprises immobilisation of biomass bearing oil-oxidisers taken in effective amount into organic hydrophobic peat-based sorbent and drying it. Note here that said organic hydrophobic sorbent is prepared by low-temperature pyrolysis in vacuum of peat minced to 0.2-0.5 mm size at 210-250°C for 60-90 minutes. Said immobilisation is performed by adding sorbent into biomass suspension during its fermentation at retardation of oil-oxidising microorganisms growth. Note here that mass of added sorbent is 6-10 times larger than that of microorganisms contained in biomass suspension at the moment of sorbent application. Said oil-oxidising microorganisms represent yeast culture Candida maltosa All-Russian collection of industrial microorganisms Y-3446 or culture of bacteria Dietzia maris All-Russian collection of industrial microorganisms Ac-1824. Besides, this invention covers biosorbant for water treatment thus produced.

EFFECT: faster preparation, higher absorption capacity and efficiency.

2 cl, 4 ex

FIELD: biotechnologies.

SUBSTANCE: method provides for degreasing of buckwheat straw with 100% acetone. The degreased straw is extracted in 5 l of 70% ethyl alcohol in boiling water bath for 1 hour. The extracted raw materials are separated by filtration to produce alcohol extract. The produced alcohol extract of the stimulant is concentrated under vacuum, cleaned from admixtures with ethyl acetate and dried to prepare target product.

EFFECT: invention makes it possible to increase yield of yeast biomass.

2 tbl, 2 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to biotechnology, more specifically to expression constructs, and may be used for immunoglobulin expression. An expression vector contains one open reading frame (sORF) insert which contains a first sequence of nucleic acid coding a first polypeptide; a first intermediate sequence of nucleic acid coding a first protein cleavage site containing an autoprocessing element with an intein segment providing proteolytic sORF polypeptide cleavage between the first polypeptide and the intein segment and the second polypeptide, but not ligation of said first polypeptide with said second polypeptide; and a second sequence of nucleic acid coding the second polypeptide. The expression vector is able to express a mammalian polypeptide coding sORF and cleaved in said first protein cleavage site in a host cell; consisting of the first polypeptide - an immunoglobulin heavy chain, and the second polypeptide - an immunoglobulin light chain able to be assembled into a multimer.

EFFECT: invention provides functional antibody production with 'correct' setup and assembly.

40 cl, 9 dwg, 57 tbl, 4 ex

FIELD: chemistry.

SUBSTANCE: group of inventions relates to feed production, particularly a method for microbiological production of feed yeast from grain wastes. The method involves preparation of grain material by grinding to 120-160 mcm particles. Further, the ground grain material is used to prepare an aqueous suspension containing 15-25% dry substances. The obtained suspension is saccharified with α-amylase and glucoamylase to 6-10% glucose content in the suspension. Nitrogen and phosphorus sources are added to the obtained suspension such that 90 mg of nitrogen and 45 mg of phosphorus are required for synthesis of 1 g of biomass. A culture is added to the obtained nutrient medium, said culture being a producer of the Saccharomyces cerevisiae yeast strain VKPM U-3585, having amylase activity, which is deposited in the Russian National Collection of Industrial Microorganisms (VKPM) and can be used in producing feed protein. The Saccharomyces cerevisiae yeast strain VKPM U-3585 is continuously grown in a multiple-section apparatus while feeding the nutrient medium simultaneously into several sections, followed by concentration of the suspension by vacuum evaporation and drying. Moisture freed at the vacuum evaporation and drying steps is used to prepare the aqueous suspension of grain material.

EFFECT: invention increases output of crude protein and true protein.

5 ex

FIELD: food industry.

SUBSTANCE: one proposes a composition for preparation of a food product using yeast, containing: instant active dry yeast particles, at least one biologically-compatible oil and/or at least one biologically-compatible paraffin where the weight ratio of the said oil and/or paraffin to instant active dry yeast is higher than 0.4:1; additionally, the composition optionally contains at least one biologically-compatible hydrophilous component. All the components are homogeneously dispersed; the dry substance content in the composition is higher than 90 wt %, and lower than 98 wt %. Additionally, one proposes a method for preparation of a food product using yeast, involving stages of mixing for homogeneous distribution of all the said components. The composition, according to the invention, is in a liquid, paste-like or powder form.

EFFECT: oil or paraffins presence in the composition protects yeast during storage and ensures good long-term stability of yeast enzyme capability under no special physical conditions of storage.

30 cl, 3 dwg, 33 tbl, 16 ex

FIELD: food industry.

SUBSTANCE: method for production of pressed bakery yeast enriched with selenium envisages cultivation of Saccharomyces cerevisiae yeast in a nutritional medium containing molasses, a nitrogen source and mineral salts. One performs introduction of a selenium source solution represented by a sodium selenate solution containing sodium selenate in an amount of 220-240 mg/kg of the yeast concentrate. The mixture is maintained during 25-30 minutes. One performs filtration and pressing of the yeast concentrate.

EFFECT: method allows to produce pressed bakery yeast with increased selenium content.

1 ex

FIELD: food industry.

SUBSTANCE: bakery yeast production method involves mill offals hydrolysis during heating in the presence of an acid, hydrolysate neutralisation, addition of nutrient salts and growing Saccharomyces cerevisiae kind bakery yeast. The raw material for hydrolysate production is represented by aspiration fines. Hydrolysis is performed using a 3-7% solution of acetic acid during 75-85 minutes. Yield of saleable yeast from reducing substances is equal to 54-75%.

EFFECT: method allows to increase the yeast yield.

1 tbl, 3 ex

Yeast extract // 2449010

FIELD: chemistry.

SUBSTANCE: invention relates to a yeast extract containing free proline in amount of 8.0% or more of a composition of free glycine in amount of 5.0% or more from a composition of free amino acids. The yeast extract is obtained by culturing Saccharomyces cerevisiae JT-YE-P-52 FERM BP-10725 yeast to obtain a cultured product by obtaining yeast cells therefrom and extracting yeast cells with boiling water.

EFFECT: obtained yeast extract can be used as seasoning in different food products.

5 cl, 5 tbl, 5 ex

FIELD: food industry.

SUBSTANCE: method for production of yeast for pizza involves stages whereat one performs primary mother yeast fermentation and drying at a temperature of 90-110°C. One adds an emulsifier to the yeast after mother yeast fermentation without additional fermentation and prior to drying. Prior to drying one performs stages of filtration or evaporation for obtaining yeast with dry substance content equal to 33%. Primary mother yeast is represented by sugar-free Saccharomyces cerevisiae yeast. The yeast activity is equal to 260-450 ml CO2/h.

EFFECT: method allows to obtain yeast for pizza with low activity and darker colour, a fermenter usage is excluded which simplifies the process technology.

8 cl, 3 tbl, 5 ex

FIELD: biochemistry.

SUBSTANCE: the yeast mixture used for high concentrated alcohol fermentation from sugar-containing material contains any kind of dry yeast and nutrients necessary for yeast growth. The nutrients includes 40-70 weight parts of dry yeast, 20-40 weight parts of nitrogen source, 5-10 weight parts of phosphorus source, 2.5-5 weight parts of inorganic salt different form the phosphorus-containing and nitrogen-containing inorganic salts, 1-2.5 weight parts of microvitamin and 0.5-1.2 weight parts of penicillin. The yeast is taken from the yeast group containing beer yeast Saccharomyces cerevisiae Hansen from the Saccharomyces cerevisiae species, vine yeast Saccharomyces uvarum Beijerinek. The yeast mixture is received through grinding the nutrients separately, excluding the dry yeast. The dry yeast is mixed with the grinded nutrients with a certain weight ratio, then the mixture is stirred mechanically or manually until it is homogeneous.

EFFECT: increased alcohol fermentation up to 14.5-15.7% in standard raw material, such as sucrose, and reduced sugar residue in fermented mesh down to 0 - 0.1%.

9 cl, 3 tbl, 6 ex

FIELD: food industry.

SUBSTANCE: invention relates to food industry. The pastry making method involves mixing eggs, sugar, salt, flour and an additive and dough kneading. Eggs are whipped with sugar and salt prior to mixture with flour. After kneading dough is handled into pieces, left for fermentation for 25-30 min, cut into balls or stripes, deep fried at a temperature of 180-190°C with subsequent syrup pouring and moulding as desired. The syrup is prepared of honey and sugar by way of heating till homogenous mass formation. The additive is represented by baker's yeast activated with potassium salt of 3,5-di-tert-butyl-4-hydroxiphenylpropionic acid with concentration 0.001% of yeast weight in an amount of 0.1% of flour weight.

EFFECT: invention enables pastry quality improvement due to protein content increase by 10% and reduction of energy value by 177 kcal and 27% deep fat expenditure reduction.

2 dwg, 2 tbl

FIELD: medicine.

SUBSTANCE: invention refers to pharmaceutical industry, more particularly to a method for preparing a phytopreparation of Hedysarum neglectum roots or Hedysarum theinum roots. The method for preparing the phytopreparation of Hedysarum neglectum roots or Hedysarum theinum roots by three-stage extraction of the ground roots by aqueous-alcoholic solutions at intermittent shaking at room temperature in a dark place under specific conditions.

EFFECT: invention enables optimising the method for recovering the low-molecular extractants with a maximum degree of extraction with improved pharmacological activity.

1 ex

Up!