Strain of lactobacilli lactobacillus paracasei cncm i-2116 (ncc 2461) eliciting ability to prevent intestine colonization with pathogenic microorganisms causing diarrhea, supernatant of its culture and oral agent for prophylaxis and/or treatment of diarrhea-associated disorders

FIELD: biotechnology, microbiology, medicine.

SUBSTANCE: invention relates to the strain Lactobacillus paracasei CNCM I-2116 used for diarrhea prophylaxis causing by pathogenic microorganisms. Supernatant of this strain culture elicits ability to prevent colonization of intestine with pathogenic microorganisms causing diarrhea also and this strain is designated for preparing agent used for prophylaxis and/or treatment of disorders associated with diarrhea. Agent for oral administration represents therapeutically effective dose of the strain L. paracasei CNCM I-2116 or supernatant of its culture and acceptable foodstuff. Invention provides the enhanced viability of the strain in its applying and effectiveness in prophylaxis of adhesion to intestine cells and invasion to intestine cells of pathogenic microorganisms causing diarrhea.

EFFECT: valuable medicinal properties of strain.

5 cl, 8 dwg, 10 ex

 

This invention relates to the field of Microbiology and, in particular, to new microorganisms of the genus Lactobacillus, used to prevent diarrhea caused by pathogenic bacteria. In particular, this invention relates to the use of these microorganisms for the preparation of the received inside funds and to compositions containing it.

The level of technology

Organisms that produce as a primary metabolic component lactic acid, known for a long time. These bacteria can be detected in milk or in obrabatyvaemykh milk devices, respectively, living or decaying plants, and also in the intestines of humans and animals. These microorganisms that are grouped under the term “lactic acid bacteria”, are a heterogeneous group and include, for example, the genera Lactococcus, Lactobacillus, Streptococcus, Bifidobacterium, Pediococcus, etc.

Lactic acid bacteria used as fermenting agents for canning food products in conditions of low pH and actions fermentation products formed during the enzymatic activity, to inhibit the growth of spoiling food bacteria. For this purpose, lactic acid bacteria are used for cooking various food products made from milk such as cheese, yogurt and other EN zymes the dummy dairy products.

Recently among lactic acid bacteria were detected strains with beneficial properties for human and animal ingestion. In particular, it was found that certain strains of the genera Lactobacillus or Bifidobacterium able to colonize the intestinal mucosa and to help maintain the health of man and animal.

In this respect, EP 0768375 described specific strains of the genus Bifidobacterium, which are able to settle down as intestinal flora and can attach to intestinal cells. It is reported that these bifidobacteria promote immunomodulation, competitive eliminating the adhesion of pathogenic bacteria to intestinal cells, contributing to the maintenance of human health.

In the last few years, the study also focused on the potential use of lactic acid bacteria as symbiotic agents. Symbiotic agents are considered viable microbial drugs that promote human health by preserving the natural microflora in the gut. Microbial drug can be considered symbiotic agent if known effective microbes and their mode of action. It is believed that the symbiotic agents attached to the intestinal mucosa, inhabit the intestinal tract and, apparently, prevent attaching it BP is the breaking of microorganisms. A critical prerequisite for their actions is that they should reach the mucous membrane of the intestine in the right and viable form and not break in the upper part of the gastrointestinal tract, in particular, under the action of low pH prevailing in the stomach.

In this respect, WO 97/00078 as symbiotic agent described a specific strain of Lactobacillus GG (ATSS 53103). This microorganism is used, in particular, in a method of prevention or treatment induced food allergic reactions, where it is administered to the recipient together with the food product, which was subjected to a hydrolysis treatment with pepsin and/or trypsin. The selected strain of Lactobacillus described as exhibiting adhesive and colonizing properties and detecting protease enzymatic system, so that the protein material contained in the food product, which must be entered, optionally hydrolysed by proteases, sekretiruemyi this strain of Lactobacillus. The method discussed in this document, will eventually lead to intestinal absorption of protein material, which does not detect a substantial number of allergenic material.

In addition, in EP 0577903 reference is made to the use of lactic acid bacteria, the ability to replace Heliobacter pylori, a recognized agent causing the development I have you, in the preparation of a carrier intended for therapeutic or prophylactic treatment of ulcers associated with the action of Heliobacter pylori.

The invention

Valuable properties of lactic acid bacteria determine the need for new strains of lactic acid bacteria beneficial to human health and/or animals.

Thus, the present invention consists in the search of new bacterial strains with properties useful for man and/or animals.

The above problem was solved with the discovery of new microorganisms, namely lactic acid bacteria belonging to genus Lactobacillus, which are able to prevent colonization of the intestine by pathogenic bacteria that cause diarrhea.

According to a preferred variant, detected a strain of Lactobacillus is able to attach to the intestinal mucosa of a mammal and grow in the presence of up to 0.4% bile salts.

In accordance with another preferred lactic acid bacteria selected from the group consisting of Lactobacillus rhamnosus or Lactobacillus paracasei, preferably Lactobacillus paracasei, more preferably Lactobacillus parasei CNCM I-2116.

It was shown that the microorganisms of the present invention have the following properties: they are gram-positive, catalase-negative, negative, what about the NH 3-form of arginine and negative for the production of CO2they produce L(+)-lactic acid, able to grow in the presence of bile salts at concentrations of up to about 0.4%, and may effectively prevent the colonization of intestinal cells the bacteria that cause diarrhea, such as pathogenic E. coli, for example, enteropathogenic E. coli (EPEC), or Salmonella, for example, Salmonella typhimurium.

New microorganisms can be used to prepare various means, in the form received inside materials carriers, such as milk, yoghurt, cheese, fermented varieties of milk, fermented milk products, fermented products based on cereals, powders based on milk, baby formula, and can be included in such media in an amount of from about 105CFU/g to about 1011CFU/g In the context of this invention abbreviation CFU means colony forming an ennico”, which is defined as the number of colonies of bacterial cells grown on plates with agar medium.

This invention provides also accept inside funds in the form of a food or pharmaceutical composition containing at least one of the strains of Lactobacillus having the above characteristics and/or containing supernatant of the culture in which the microorganism has been grown, Riego fraction, respectively.

For the preparation of food compositions of this invention at least one of the Lactobacillus strains of the present invention include a suitable carrier in an amount of from about 105CFU/g to about 1011CFU/g, preferably from about 106CFU/g to about 1010CFU/g, more preferably from about 107CFU/g to about 109CFU/g

In the case of a pharmaceutical product this product can be prepared in the form of tablets, liquid bacterial suspensions, dried oral supplements, moist oral supplements, dry product for insertion through the tube or moist product for insertion through the tube with a number of strains of Lactobacillus that are included in them, in the range up to about 1012CFU/g, preferably from about 107CFU/g to about 1011CFU/g, more preferably from about 107CFU/g to about 1010CFU/g

Activity funds containing as active component new microorganisms in the intestine of the individual is, of course, dependent on dose. That is, the more microorganisms activated by oral administration of the above means food material or pharmaceutical composition, the above protective or healing activity of this tool. As new microorganisms are harmless on the I people and animals and were isolated from the faeces of a child, the developed tools can enable a large number of microbial cells so that a significant portion of the intestine of the individual was settled (colonized) new microorganisms.

In accordance with another preferred option, the culture supernatant of Lactobacillus of the present invention can be used to prepare one of the above taken inside funds. The supernatant can be used as such or may be dried under conditions which do not destroy the metabolic compounds secreted by these microorganisms in a liquid environment, such as lyophilization, and may be further introduced into the tool. To minimize the number of unknown compounds in the supernatant of Lactobacillus preferably grow in certain environments, the composition of which is known and has no negative effect on the cultured host. In addition, a specialist with expertise in this area on the basis of his General knowledge may not necessarily be free supernatant from unwanted products, for example, by chromatography.

List of figures

Figure 1 shows the results of the analysis in cell culture to inhibit the adhesion of pathogenic bacteria E. coli to epithelial cells of cultured cells ST11.

Figure 2 shows the results of analisa cell culture to inhibit the adhesion of pathogenic bacteria E. coli to epithelially cells with the supernatant of the culture ST11.

Figure 3 shows the results of the analysis in cell culture to inhibit the invasion of Salmonella typhimurium in epithelial cells of cultured cells ST11.

Figure 4 shows the results of the analysis in cell culture to inhibit the invasion of Salmonella typhimurium in epithelial cells from the supernatant of the culture ST11.

Figure 5 shows the acidification of different environments while growing strain of L. casei CNCM 1-2116 (named ST11).

6 shows a graph of the survival of L. casei ST11 at 10°within 30 days.

Fig.7 shows the distribution of mRNA of IL-12 and IL-10 in mouse attached cells isolated from bone marrow, after incubation of these cells with serial dilutions ST11.

Fig shows the result of the differentiation of Th2 as leading to reduced production of IL-4.

Information confirming the possibility of carrying out the invention

While intensive research leading to this invention, the inventors examined the faeces of infants and isolated them from many different bacterial strains. These strains were then tested for their ability to prevent colonization of epithelial cells with bacteria known to cause diarrhea.

Several bacterial genera, including Lactobacillus, Lactococcus and Streptococcus were skanirovali on the ability Engibarov the ü diarrhea. Tests for the ability to inhibit diarrhea spent mostly with pathogenic E. coli and Salmonella typhimurium as a representative bacteria to pathogens causing diarrhea in the affected individual.

Various lactic acid bacteria were grown in a suitable medium, such as MRS, Hugo-Jago or M17, at a temperature of from about 30 to 40°appropriate to their optimal growth temperature. After reaching the stationary growth bacteria were collected by centrifugation and resuspendable in physiological NaCl solution. The obtained bacterial cells were stored frozen (-20°).

For evaluation of antibacterial properties the following methods were used.

According to one method, cultivated strains of Lactobacillus of the present invention were tested for their ability to prevent the adhesion of pathogenic bacteria causing diarrhoea, intestinal cells or their invasion in the intestinal cells, respectively. For this purpose, intestinal cells incubated with pathogenic bacteria and cultured Lactobacillus strains of the present invention and evaluated the degree of adhesion or invasion, respectively.

According to the second method, the cell culture supernatant of Lactobacillus strains of the present invention was added together with pathogenic microorganisms to the intestinal cells and evaluated the degree of adhesion or invasion matched with the public.

Thus, the authors were able to show that cultivated strains of Lactobacillus and the supernatant was extremely effective for preventing both adhesion to intestinal cells, and invasion of intestinal cells, which indicates that metabolic compounds that are excreted new microorganisms, apparently, responsible for the activity against diarrhoea.

In addition to the above discoveries, the authors were able to show that the strains of the present invention unexpectedly exhibit antiallergic properties by certain influence on the synthesis of various immunological mediators (intermediaries).

It is recognized that the humoral immune response and allergic reactions mediated by CD4+T-cells bearing the phenotype of type 2 (Th2). Th2 dysbalance cells are characterized by production of high levels of interleukin 4 (IL-4), a cytokine required for the secretion of IgE, which is the main class of antibodies involved in allergic reactions.

The differentiation of Th2 dysbalance-cells is impaired IFN-γ, specific cytokine, which is produced by mutually exclusive Th1-subpopulation of CD4+T-cells. These Th1 cells, in turn, is strongly induced by interleukin 12 (IL-12). It was shown that, in contrast, IL-10, another cytokine that has a strong suppressive effect on the proliferation of Th1-cleto is, and, therefore, it is assumed that it plays a role in the immunosuppressive mechanisms.

In General, as IL-12 and IL-10 have a strong modulating effect on the development of CD4+T cells via an effect on the development of Th1-subpopulations. IL-12 is a key regulatory cytokine for the induction of Th1 differentiation and, consequently, inhibits the generation of Th2 dysbalance responses. The main by inhibiting Th2 dysbalance-cells is, therefore, stimulation of the synthesis of IL-12 auxiliary cells.

It is well known that some components of gram-negative bacteria, such as LPS (lipopolysaccharide), induce high levels of IL-12 in attached cells, such as macrophages and dendritic cells. Accordingly, it was found that gram-negative bacteria can greatly shift CD4+T-cell differentiation towards a Th1-phenotype.

The microorganism ST11 as an example of the Lactobacillus strains of the present invention was tested for its potential role in the induction of cytokines involved in the regulation of differentiation of CD4+T-cells. In particular, investigated the effect ST11 on the phenotype of CD4+T cells exposed to Th2 dysbalance-differentiation.

In this regard, the ability ST11 to induce the synthesis of mRNA encoding these two regulatory cytokine in mouse attached cells derived from bone marrow, cranialis 4 other strains of Lactobacillus and control of gram-negative bacteria (E. coli K12). The amount of mRNA was determined by semi-quantitative RT-PCR after 6 hours of incubation of the cells with serial dilutions of bacteria from 107up to 109CFU/ml

Although all strains of Lactobacillus, to some extent, induced the transcription of mRNA of IL-12, it was shown that ST11 is the most potent inducer, because a strong PCR signal detected even at the highest breeding bacteria. In fact, ST11 induces the transcription of mRNA of IL-12 to the extent that, and E. coli. The induction of mRNA of IL-10 was usually lower than the induction of mRNA of IL-12, the signal could be detected only at lower dilutions. However, ST11 was the most potent inducer of mRNA of IL-10 compared with other strains of Lactobacillus and E. Coli (control).

Thus, it was shown that ST11 is an effective inducer of cytokines involved in the differentiation of CD4+T-cells. His significant IL-12 inducing activity makes ST11 likely candidate for inhibition of Th2 dysbalance reactions, and IL-10 inducing activity can be used to prevent inflammatory processes.

In addition to the above discoveries, the authors also determined, does ST11 inhibitory effect on CD4+T-stands exposed Th2 dysbalance-differentiation, and positive effect on the function of Th1. To resolve this ass is Chi used a well-known cultural system of differentiated cells, in which the precursor CD4+T-cell polyclonal activated and modulated to exposure to either Th1-or Th2-differentiation, depending on the type of inductor that is present in this culture medium. Th1/Th2 dysbalance-differentiation was induced in 7-day primary culture, after which cells re-stimulated for 2 days in the secondary culture containing only environment, and the acquisition of the specific phenotype (Th1 or Th2) was assessed by the number of typical cytokines produced in the supernatant (INFγ or IL-4).

It is well known that containing precursor CD4+T cells of mice BALB/ preferably are differentiated by the predominance of the Th2 phenotype (high levels of IL-4, low content of IFN-γ in the secondary culture supernatant) after activation in the neutral conditions (only environment in primary culture). This phenotype could be fully converted to a picture of Th1(high content of IFN-γlow levels of IL-4) when added to primary cultures of blocking monoclonal antibodies against IL-4.

To study the potential role of ST11 in the inhibition of Th2 purified precursor CD4+T cells in BALB/c mice were activated in the presence of the attached bone marrow cells as accessory cells in primary culture. These cells were cultured in the medium of the ez additives, in the presence of 1 mg/ml LPS, in the presence of 108CFU/ml ST11 or in the presence of 108CFU/ml other Lactobacillus. Then cells were washed, CD4+T-cells once cleaned and re-stimulated by secondary cultivation in medium without additives.

The amount of cytokines produced differentiated CD4+T-cells was determined 2 days later. As expected, cells that were cultured in the medium without additives, were differentiated in a dominant Th2 phenotype. Adding ST11 to primary cultures modulated Th2 dysbalance-phenotin that was identified by 8-fold decrease in IL-4. The same inhibition was observed when culturing cells in the presence of LPS. In contrast, culturing cells in the presence of another strain of Lactobacillus have an impact on the production of IL-4. Interestingly, the levels of IFN-γ did not increase when adding ST11 in primary culture.

In General, ST11 specifically violated the production of IL-4 in CD4+T-cells exposed to Th2 dysbalance-differentiation, but did not increase significantly the secretion of IFN-γ. The fact that ST11 did not increase the formation of ifn-γmay be caused by its ability to induce IL-10, leading to the fact that it can maintain a low inflammatory action, despite its activity against Th2.

The result was to show that ST11 is one of the shtam the Lactobacillus s, having a good anti-Th2 dysbalance profile, which makes them an excellent candidate for use as microorganisms having antiallergic and symbiotic activity.

Hereinafter the invention will be described using examples without limiting the invention to these examples.

Environment and solutions:

MRS (Difco)

Hugo-Jago (tripton 30 g/l (Difco), yeast extract 10 g/l (Difco), lactose 5 g/l (Difco), K2HPO46 g/l, meat extract, 2 g/l (Difco)agar 2 g/l (Difco))

M17 (Difco)

DMEM (modified by way of Dulbecco Wednesday Needle)

CFA (according to Ghosh et al., Journal of Clinical Microbiology, 1993 31:2163-6)

Agar, Muller Hinton (Oxoid)

LB (Luria Bertami, Maniatis, A Laboratory Handbook, Cold Spring Harbor, 1992)

Antibiotics were obtained from Sigma

With14-acetate (53,4 CI/mmol, Amersham International PLC)

PBS (NaCl 8 g/l, KCl 0.2 g/l, Na2HPO41,15 g/l To2NRA40.2 g/l))

A solution of Trypsin-EDTA (Seromed)

Fetal calf serum FCS (Gibco)

E. coli DAEC 1845 received from Washington University, Seattle, a E. coli JPN15 received from the Center for vaccine development University of Maryland, USA).

Example 1

Selection of lactic acid bacteria from the feces of infants

Fresh faeces were collected from the cradle 16 healthy infants aged 15-27 days. 1 g fresh faeces were placed in anaerobic conditions for transportation to the laboratory within 2 hours from sampling the serial cultivation of fekali is in ringer's solution were sown on the selective medium. For isolation of lactic acid bacteria used MRS-agar with antibiotics (fosfomicin 80 µg/ml sulfamethoxazole 93 µg/ml, trimethoprim 5 mg/ml) by incubation for 48 hours at 37°C. Colonies were selected randomly and were purified. Next conducted physiological and genetic characterization of isolates.

Example 2

Cultivation of cells SASO-2

For assays for inhibition as a model of the intestine used a cell line SASO-2. This cell line has features which are characteristic for cells of the intestine, such as, for example, polarization, expressie intestinal enzymes, the production of certain structural polypeptides, etc.

Cells were grown on three different media, namely, in plastic cups (25 sq cm, Corning) for growth and reproduction, and low-fat sterilized 6-hole glass plates (22 × 22 mm, Corning) for tests on adhesion and 24-hole glass plates (Corning) for tests on inhibition.

After the second day of cultivation medium (DMEM) was replaced once a day. Before use the medium was added to a mixture of 100 u/ml penicillin/streptomycin, 1 μg/ml amphotercin and 20% FCS, inactivated at 56°C for 30 minutes. Cultivation was carried out at 37°C in an atmosphere of 10% CO2. The cells were passively every six days. For this purpose, the cells were separated from stanolone treatment PBS with 0.25% trypsin and 3 mm EDTA, pH of 7.2. To neutralize the trypsin to the obtained cell suspension was added an equal volume of FCS, the mixture was centrifuged for 10 minutes at 1000 rpm and the precipitated cells were placed in culture. Approximately 3.5×105cells was transferred into a new culture bottle and cultured to obtain a confluent monolayer.

Example 3

Cultivation of bacteria

ST11:

Bacterial strain ST11 kept at -20°C in MRS medium containing 15% glycerol. The strain was grown anaerobically in MRS and before use in assays for inhibition transferred twice into a new environment with intervals of 24 hours. For the analysis used a concentration of 2×109CFU/ml

The supernatant after centrifugation for 1 hour at 20,000 rpm were tested for the presence of bacteria.

E. coli:

Used two strains of E. Coli - E. coli DAEC 1845 (E. coli with diffuse adhesion) and E. coli JPN15 (EPEC; enteropathogenic strain of E. coli).

The first passage after thawing was carried out on CFA agar-Muller Hinton, which is suitable for the induction of expression of adhesion factors of these bacteria.

Before each experiment, the bacterial cells double-incubated for 24 h at 37°in the new environment. Because JPN15 contains the gene for resistance to ampicillin, the antibiotic used for selection during cultivation.

Salmonella:

For the experiments used a piece is mm Salmonella typhimurium SL1344, which previously were grown in LB-medium.

Example 4

Analysis of inhibition for E. coli

After the second passage in the new environment of pathogenic bacterial strains were labelled with radioisotopes using14-acetate in 10 µci/ml in LB-medium. The strains were incubated for 18 hours at 37°C.

Then the bacterial suspension was centrifuged (1041 g, 15 min) to remove the supernatant with necklacethis With14-acetate. Sediment suspended and washed in PBS and suspended cells at a concentration of approximately 108cells/ml in 1% sterile mannose. It is known that mannose inhibits nonspecific adhesion.

Various pathogenic bacterial strains (E. coli) were incubated with the monolayer of cells SASO-2 (37°C, 10% CO2) for 3 hours. We also carried out experiments with the addition of supernatant (obtained by centrifugation at 20000 rpm for 40 minutes).

As a control pathogenic bacteria were incubated with a monolayer of SASO-2 without adding ST11 or culture supernatant, respectively.

After incubation for 3 hours the medium was removed and the monolayer was washed three times with PBS. To exclude nonspecific adhesion leaching was conducted by 20 shuffle PBS. Cells were literally by adding 1 ml of sodium carbonate and incubated for 40 minutes at 37°C. After homogenizer and an aliquot (250 μl) was added to 5 ml scintillation fluid (Hionicfluor Packcard) and calculated (Packcard). The percentage of adhesion of pathogenic cells to cells of SASO-2 was calculated by comparison with control taken as 100% (adhesion; or invasion for example 5).

Example 5

Analysis of inhibition for Salmonella

Salmonella is a bacterium which is embedded in the epithelial cells and multiplies them. To determine the inhibitory activity of ST11 strain of Salmonella typhimurium SL1344 were incubated as described above in a medium containing14-acetate, and analyzed as in example 4.

To remove reprecipitate cells after incubation cells SASO-2 were washed in PBS. Then the medium was added gentamicin (20 μg/ml) and incubated for 1 hour at 37°C. Gentamicin is an antibiotic, not penetrating into the intestinal cells, thus all neprecejusies cells of the microorganism were killed, while Salmonella, already established in the intestinal cells, survived. Cells were twice washed in PBS and literally by adding sterile distilled water, after which the radioactivity was measured as described in example 4.

The results of experiments 4 and 5 shown in figures 1-4. You can see that the cultivated cells ST11 and culture supernatant were extremely effective in preventing adhesion to intestinal cells and invasion into intestinal cells of the pathogens causing diarrhea.

Example 6

ST11 incubated in a model of gastric juice. For a model of gastric juice pepsin (3 g/l) suspended in sterile saline solution (0.5% wt./about.) and brought the pH with concentrated Hcl to 2.0 and 3.0, respectively. ST11 were grown in the above media and determined resistance.

The results are shown in the table.

Table
pHCFU/ml T 0 min CFU/ml T 1 minCFU/ml T 15 minCFU/ml T 30 minCFU/ml T 60 min
2,02,0×1091,8×1091,2×1093,7×1097,0×109
3,02,0×1091,9×1091,7×1091,7×1098,4×109

ST11 have the following properties defined in accordance with the methods described in the Genera of lactic acid bacteria, Ed. B.J.B.Wood and W.H.Holzapfel, Blackie A&p

- gram-positive,

- catalase-negative,

- negative for NH3-form of arginine

- negative for the production of CO2,

- p is obserwuj L(+)-lactic acid,

- growing in the presence of bile salts at concentrations of up to about 0.4%.

Example 7

Growth ST11 in various conditions

ST11 incubated at 37°in the medium tomato-based (4% powder tomatoes, registrationentry in distilled water)supplemented with sucrose (0, 0,5, 1 or 2%) or soy peptone (0.5%) or glucose (0.5%), and over different periods of time. The results are shown in figure 5.

ST11 was further added in the amount of 2.5% to a medium consisting of rice flour (3%), wheat flour (2%) and sucrose (3%), and incubated at 37°to achieve a pH of 4.4. After cooling, the product was Packed with or without added vitamin C and kept at 10°C.

Figure 5 shows the data on survival ST11 at 10°in wheat drink, Packed in different plastic materials (polyethylene high density(HDPE), polystyrene (PS).

Example 8

Induction of the synthesis of mRNA of IL-12 and IL-10 in mouse attached cells ST11

Bone marrow cells were isolated from femur and tibia 8 weeks, not containing specific pathogen of C57BL/6 mice, were incubated at a concentration of 2×106cells/ml in RPMI medium (Gibco)containing 10% fetal calf serum, 1 mm L-glutamine, 12 mm Hepes, 0.05 mm 2-mercaptoethanol, 100 u/ml penicillin and 100 µg/ml streptomycin (all reagents from Gibco)for 12 hours at 37°and in the atmosphere of 5% CO 2. Neprecejusies cells were removed 3 successive washes with warm culture medium, and the remaining adherent cells were collected and incubated at a concentration of 106cells/ml for 6 hours in the presence or in the absence of bacteria. Previously, it was determined that 6 hours are optimal vremenno point for the synthesis of mRNA of cytokines in mouse attached cells in response to LPS. Bacteria were added at concentrations ranging from 109up to 107CFU/ml Bacteria were grown and stored as described above.

At the end of the 6-hour period of cultivation, the cells were separated by centrifugation and literally using a set of reagents TRIzol (GibcoBRL, Cat. No. 15596-018) according to the manufacturer's instructions. Total RNA was isolated by precipitation with isopropanol and performed reverse transcription into cDNA for 90 minutes at 42°using 200 E. reverse transcriptase (Superscript II, BRL) in a reaction volume of 40 ál containing 200 mm Tris pH 8.3, 25 mm KCl, 1 mg/ml oligo-d(T)15 (Boehringer Mannheim), 1 mm DTT (Boehringer Mannheim), 4 mm of each dNTP (Boehringer Mannheim) and 40 u/ml Rnasin (Promega). Used PCR primers and conditions previously described in Kopf et al. (Journal of Experimental Medicine 1996 Sep. 1:184 (3):1127-36). The number of normalized cDNA samples using primers specific for the β-2-microglobulin (house-keeping gene). PCR products section which ranged in 2% agarose gel and bands were analyzed under UV.

As shown in figure 1, ST11 showed the strongest inhibition of mRNA of IL-12 and IL-10, which is comparable to the levels observed with the positive control (E. coli). The differences are best seen when the very low concentrations of bacteria (107CFU/ml).

Example 9

Suppression of the synthesis of IL-4 ST11

CD4+T cells were isolated from the spleen does not contain specific pathogen BALB/c mice using a set of Mini-MACS from Miltenui Biotec (Cat. No. 492-01). CD4+T cells were cultured at a concentration of 2×105cells/ml in RPMI medium containing 10% fatal calf serum, 1 mm L-glutamine, 12 mm Hepes, 0.05 mm 2-mercaptoethanol, 100 u/ml penicillin and 100 μg/ml streptomycin, and activated within one week the stitching associated with tablet monoclonal antibodies against CD3(clone C) and CD28 (clone 37,51, both antibodies from Pharmingen). While primary cultures of CD4+T cells were cultured with attached bone marrow cells (selected as described above) as the supporting cells and with 108CFU/ml La1 or 1 mg/ml LPS or with one environment. Then cells were washed and CD4+T cells were purified again using set MiniMACS and re-stimulated in the secondary culture containing only environment. The cytokines produced differentiated CD4+T-cells were measured in supernatant after 2 days use what Itanium sandwich ELISA (kits from Endogen and Pharmingen).

The results are shown in figure 5. Cells differentiated in the presence only of the environment, showed dominatly the Th2 phenotype, characterized by high levels of IL-4. Adding ST11 to primary cultures strongly modulated Th2 differentiation, as it resulted in 8-fold decrease in the production of IL-4. Similar inhibition was observed in cell cultures, differentsirovaniya in the presence of LPS. In contrast, another strain of Lactobacillus had no significant effect on the levels of IL-4. Interestingly, the levels of IFN-γ did not increase when adding ST11 in primary culture.

As you can see from the above results, the strains of the present invention can be used for cooking food and/or pharmaceuticals, uses the beneficial properties of these microorganisms.

Example 10

Strain ST11 tested in clinical trials for the residents of the suburbs of Guatemala City on its ability to influence the transmission and course of acute diarrhoea rainy season, which affects the majority of children in this area. 203 children aged from 35 to 70 months were registered for this study and received the target dose of 1010viable microorganisms (ST11) or placebo during the feeding period of 29 days. The children selected for sample introduction, and that call is to be placed placebo, respectively, had the typical characteristics of underweight and growth for the corresponding age, due to exhaustion.

Before testing in preschool children carried out a safety assessment on the basis of in vitro and in vivo. In vitro studies showed the profile of antibiotic resistance, similar to other Lactobacillus used in food applications, and have not found the ability for the education of biogenic amines in the degradation of mucin (mucous secretion) and in respect of deconjugation salts of bile acids. In clinical trials with placebo, which consisted of 42 adult volunteer, ST11 was well tolerated and did not induce harmful effects among potential manifestations, subjected to monitoring, such as bloating, stool frequency per day and stool consistency; the levels of acute phase proteins in serum did not cause any concern regarding the potential inflammatory response.

Samples and placebo were Packed in the form of pads in bags (sachet) in a manufacturing company Nestle Product Technology Center in Konolfingen, Switzerland and delivered in the refrigerator in Guatemala. Each ball weighing 10 g consisted of a taste of shokolada flavored carrier and either 0.2 g ST11 (1010SOME), or, in the case of placebo, 0.2 g of milk powder. With the taste of chocolate flavored but Italy consisted of cocoa powder, sugar, soy lecithin, vanilla and cinnamon. The pads are kept at 4-6°With up to two hours before use. Before using the pad had to be dissolved in 100 ml of water provided by Nestle, which did not contain bacterial contamination.

In accordance with the Protocol, diarrhea, which had to deal, was defined as having three or more loose or do not have a form of bowel movements over a period of 24 hours. Diarrhoeal attack was defined as an event that gave evidence of diarrhea (3 diarrheal bowel movements within 24 hours). Its total duration in hours counted from the moment the first three demonstration defecation before the first decorated a chair or a period of 24 hours without any bowel movement. For a child to have a “new” attack, must pass 48 hours from the end of the first attack. If it does not, then it is considered a continuation of the same attack and then to estimate using the total duration. There was a case when a child who has experienced one or more documented episodes of diarrhoea during the 29-day period of observation. The intensity of diarrhoeal attack is based on the total number of produced liquid chairs. The elements of the severity of the attack include the presence of blood, mucus or pus in the stools, along with symptoms of fever and R is the notes. The intensity of 7 chairs for 24 hours or intervention of a professional in a clinic, health center or hospital also classify the attack as heavy.

When diarrhoeal attack was diagnosed with a control system, a sample of diarrheal stools were collected for microscopic examination and culturing to identify potential causative pathogens for this attack. The sample was diagnosed on rotavirus antigen, Giardia and E. histo-lytica, in the case of dysentery samples, and bacterial pathogens, including Shigella, Salmonella, Aeromonas, Plesiomonas shigelloides, E. coli and possibly V. cholerae.

During the study period took samples for testing the viability of the included micro-organisms during the period of introduction. Were able to show that this organism remained viable in pads during the whole study, so also in the end of the study, the pads were capable of transmitting 1010viable microorganisms when reconstituirea water.

The invention revealed that the sample containing symbiotic microorganism, reduced diarrhoea, in contrast to the control group (placebo), at approximately 30%. But the control group showed a reduced number of occurrences of diarrhoea in comparison with the normal population who are not receiving any PR is b, neither placebo, respectively. This last finding may be partially explained by taking into account the fact that children received more healthy meals and do not contain contaminants water. However, since this study was performed in the field, clearly we can assume that ST11 can reduce the occurrence of diarrhea in vivo.

1. The strain of lactic acid bacteria Lactobacillus paracasei CNCM I-2116 (NCC 2461), which are able to prevent colonization of the intestine by pathogenic bacteria that cause diarrhea.

2. The strain of lactic acid bacteria according to claim 1, designed to obtain accepted inside a means for the prevention and/or treatment of disorders associated with diarrhea.

3. The culture supernatant of the strain of lactic acid bacteria according to claim 1, obtained by centrifugation of the cultures of the indicated strains capable of preventing the colonization of the intestine by pathogenic bacteria causing diarrhoea, designed to obtain accepted inside a means for the prevention and/or treatment of disorders associated with diarrhea.

4. Taken inside the remedy for the prevention and/or treatment of disorders associated with diarrhea, comprising a therapeutically effective amount of the strain of lactic acid bacteria according to claim 1 or supernatant according to claim 3 and a food product selected from milk, yoghurt, curd, cheese, fermented m is Loka, products based on fermented milk, ice cream, fermented products based on cereals, powdered milk, baby food, or pharmaceutically acceptable medium selected from tablets, liquid bacterial suspensions, dried oral supplements, liquid oral Supplement, dry product for feeding through a tube or liquid product for feeding through a tube.

5. Taken inside the tool according to claim 4, comprising a strain of lactic acid bacteria according to claim 1 in an amount of from 1·105up to 1·1012CFU/g of product.



 

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