Strain lactobacillus paracasei cncm i-2116 (ncc 2461) eliciting ability to prevent infection of epithelial cells intestine with diarrhea-inducing rotaviruses and agent for treatment and/or prophylaxis of diarrhea-associated disorders caused by rotaviruses

FIELD: biotechnology, microbiology.

SUBSTANCE: the strain Lactobacillus paracasei CNCM I-2116 (Ncc 2461) is able to attach with mammal intestine mucosa and to grow in the presence of up to 0.4% of bile acid salts and to prevent the infection of intestine epithelial cells with rotaviruses. Agent shows the content from 1 x 105 to 1 x 1012 CFU of the strain L. paracasei I-2116 (NCC 2461) and can be used for preparing an agent and foodstuff taken among milk, yogurt, cheese, dry milk, children's nutrition products or a pharmaceutical preparation taken among a liquid bacterial suspension, dry oral supplement, liquid oral supplement, dry product for feeding through a tube or liquid product for feeding through a tube. Invention provides the enhanced viability of the strain in its using and effectiveness in prevention of adhesion to intestine cells and invasion of pathogenic microorganism cells in intestine causing diarrhea. Invention can be used in food industry and medicine in prophylaxis and/or treatment of diarrhea-associated diseases.

EFFECT: valuable medicinal properties of strain and agent.

3 cl, 5 dwg, 7 ex

 

The technical field to which the invention relates.

This invention relates to the field of Microbiology, and in particular to new microorganisms of the family Lactobacillaceae, in particular microorganisms of the genus Lactobacillus, which can be used to prevent or treat diarrhea. This invention also relates to the use of these microorganisms for the preparation of accepted means to prevent or treat diarrhea.

The level of technology

Organisms that produce as a primary metabolic component lactic acid, are well known. Such bacteria can be found in milk or milk-processing 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”, represent a rather heterogeneous group and include, for example, the genera Lactococcus, Lactobacillus, Streptococcus, Bifidobacterium, Pediococcus, etc.

Lactic acid bacteria used as fermenting agents for canning food at low pH under the influence of fermentation products formed during the enzymatic activity, to inhibit the growth of spoiling food bacteria. For this purpose lactic acid bacteria used for the preparation of moisturising food from milk, such as cheese, yogurt and other formatiruem dairy products.

Recently, lactic acid bacteria have attracted much attention because they found certain strains that are useful 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 bifidobacteria promote immunomodulation, competitive eliminating the adhesion of pathogenic bacteria to intestinal cells and thereby contributes to the maintenance of human health.

During the last few years, the study also focused on the potential use of lactic acid bacteria as symbiotic agents. Under symbiotic agents understand viable microbial drugs that stimulate human health by preserving the natural intestinal microflora. Microbial drug recognizes the symbiotic agent if known effective microbes and their mode of action. It is believed that the symbiotic agents join mucous oblock the intestines, inhabit the intestinal tract and, apparently, prevents the attachment of harmful 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 of the stomach.

In this respect, WO 97/00078 as symbiotic agent described specific strain called 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 is characterized by adhesive and colonizing properties and has octopoteuthis enzymatic activity, so that the protein material contained in the insertion of the food product, optionally hydrolyzed by proteases secreted by 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 considered for application of lactic acid BA is clear, the ability to replace Heliobacter pylori, infectious agent, contributing to the development of ulcers, for making tools for therapeutic or prophylactic treatment of ulcers associated with the action of Heliobacter pylori.

The invention

Useful properties of lactic acid bacteria necessitates the search for and discovery of new strains of lactic acid bacteria beneficial to human health and/or animals.

Thus, the objective of the present invention consists in the search of new bacterial strains possessing properties obtained for humans and/or animals.

The above problem was solved by the discovery of new microorganisms, namely lactic acid bacteria of the genus Lactobacillus having the ability to attach to the intestinal mucosa, to populate it and prevent infection of epithelial cells of the intestinal rotavirus.

According to a preferred variant, detected Lactobacillus strains are able to grow in the presence of up to 0.4% bile salts so that they can easily pass through the gastrointestinal tract and stay active.

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

It has been shown that sm is the organisms of the present invention exhibit the following properties: are gram-positive, catalase-negative, negative for NH3-form of arginine and negative for the production of CO2. They produce L(+)-lactic acid, able to grow in the presence of bile salts at concentrations of up to about 0.4% and can prevent infection of epithelial cells with rotavirus.

New microorganisms can be used to prepare various received inside funds, such as milk, yoghurt, cheese, fermented milk products based on fermented milk, fermented products based on cereals, milk powder, baby food, in the amount of from about 105CFU/g to about 1011CFU/g of product. The reduction in CFU means colony forming unit”, which is defined as the number of colonies of bacterial cells detected on plates with agar medium.

Thus, this invention provides the received inside funds, representing a food product or pharmaceutical preparation containing at least one of the strains of Lactobacillus, which is characterized by the above symptoms.

For the preparation of the received inside the means according to the invention at least one of the Lactobacillus strains of the present invention added to the received inside the vehicle in an amount of from about 105CFU/g to about 1011CFU/g, prepact the tion from about 10 6CFU/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 liquid product for insertion through the tube with a number of Lactobacillus up to about 1012CFU/g, preferably from about 107CFU/g to about 1011CFU/g, more preferably from about 107CFU/g to about 1010CFU/g

The new activity of microorganisms in the gut of an individual depends on the number of these microorganisms. That is, the greater the number of microorganisms contained in the received inside the vehicle (food material or pharmaceutical composica), the above protective or therapeutic activity of this tool. As new microorganisms are harmless to humans and animals, and were isolated from the faeces of a child, a significant number of them can be included in the tool so that essentially significant part of the intestine of the individual was settled (colonized) new microorganisms.

List of figures

Figure 1. Screening of cell cultures to determine the protective properties of the bacterial strains about the Yves rotavirus.

Figure 2. Acidification of different environments while growing strain of L. paracasei CNCM I-2116 (named ST11).

Figure 3. The curve of the survival of L. paracasei ST11 at 10°within 30 days.

Figure 4. The mRNA of IL-12 and IL-10 in mouse attached cells derived from bone marrow, after incubation of these cells with serial dilutions ST11.

Figure 5. Reduced production of IL-4 during Th2 differentiation.

Disclosure of inventions

While intensive research leading to this invention, the inventors examined the faeces of infants and isolated them from many different bacterial strains. The selected strains were then tested for ability to prevent infection of epithelial cells with rotavirus, which have been known to cause diarrhea.

Several bacterial genera, including Lactobacillus, Lactococcus, Streptococcus, skanirovali on inhibitory rotavirus activity. Tests for inhibitory activity was carried out on three serotypes of rotavirus, which includes major etiological agents of viral diarrhoea person (serotypes G1, G3 and G4).

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 stationary phase bacteria were collected by centrifugation and resuspendable the physiological NaCl solution. Among the various tests, the bacterial cells were stored frozen (-20°).

The original rods of various rotaviruses were prepared by infecting confluent monolayers of cells. Rotaviruses were incubated before infection. The cells were infected 20 infectious doses for tissue culture.

To assess antirotavirus activity used two different methods. According to one method, different bacterial strains were tested for their ability to interact directly with the rotavirus, while in the other method was determined by bacterial strains that interact with cellular receptors rotavirus.

The first method was based on contacting a bacterial suspension with different rotavirus strains in a suitable medium. Then the mixture is virus-bacterium was applied to the cell monolayer of undifferentiated cells HT-29 colon adenoma intestine of man and the incubation continued, and then evaluated the replication of the virus.

The second method was based on the incubation of bacterial suspension first with the cellular monolayer of undifferentiated cells HT-29 colon adenoma intestine of a person with the subsequent addition of the virus. After the second incubation was evaluated viral replication.

Replication of rotavirus can be easily estimated histo-immunological staining be the Cove rotavirus-infected cells.

Bacterial strain possesses inhibitory rotavirus activity, if inoculation with rotavirus together with the target bacterial strain, the number of infected cells was reduced by 90% compared with inoculation only by rotavirus.

The study found that, of 260 analyzed bacterial strains only 9 significantly inhibited the replication of rotaviruses. It was found that these bacteria belonged to the subspecies rhamnosus or paracasei of the genus Lactobacillus. It was also found that one strain called Lactobacillus paracasei ST11, deposited in accordance with the terms of the Budapest Treaty with the Deposit number NCC 2461 (1-2116), is extremely effective in preventing infection of human cells by rotavirus. In addition, this strain has excellent growth characteristics, as evidenced by acidification of the culture media when growing this strain. This strain also had a good curve of survival during storage at a temperature of about 10°With, making it an excellent candidate for inclusion introduction in the received inside money (food or pharmaceutical composition), to be stored in the refrigerator.

In addition to the above discoveries, the authors also unexpectedly found that the strains of this invention possess anti-allergic AK is ewnetu, because affect the synthesis of various immunological mediators.

It is recognized that the humoral immune response and allergic reactions mediated by CD4+T cells with a Th2 phenotype. Th2 cells are characterized by production of large amounts 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 cytokine, is produced by mutually exclusive IN a subpopulation of CD4+T-cells. These Th1 cells, in turn, 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 cells, and suggest 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 by influencing 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 vspomogatelnymi cells.

It is well known that some components of gram-negative bacteria, such as LPS (lipopolysaccharide), in uriroot the synthesis of large quantities 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 Thi-phenotype.

The microorganism ST11 as an example of the Lactobacillus strains of the present invention investigated a potential activity to induce 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 regulatory cytokines in mouse attached cells derived from bone marrow, compared with 4 other strains of Lactobacillus and control of gram-negative bacteria (E. coli To 12). The mRNA content was measured by semiquantitative RT-PCR (RT-PCR) after 6 hours of incubation of these cells with bacterial strains bred from 107up to 109CFU/ml

It was shown that although all strains of Lactobacillus could, to some extent, to induce the transcription of mRNA of IL-12, ST11 was the most potent inducer, because a strong PCR signal could be detected even at the highest dilutions of bacteria. In fact, the ability ST11 to induce the transcription of mRNA of IL-12 was just as strong as the ability of E. coli. The induction of mRNA of IL-10 was usually more than iscoe, than mRNA of IL-12, the signal could be detected at lower dilutions. Nevertheless, ST11 was the most potent inducer of mRNA of IL-10 compared with other strains of Lactobacillus and control of E. coli.

Thus, it was found that ST11 is an effective inducer of cytokines involved in the differentiation of CD4+T-cells. His strong activity induction of IL-12 makes it a candidate for inhibition of Th2 dysbalance reactions, and measured the induction of IL-10 can prevent inflammatory processes.

In addition to the above-described opening, studied, does ST11 inhibitory effect on CD4+T-stands exposed Th2 dysbalance-differentiation, and positive effect on the function of Th1. Used a well-known system for the analysis of cell differentiation, in which the precursor CD4+T-cell polyclonal activated and modulated the direction of either Th1 or Th2 dysbalance-differentiation, depending on the type of modulator is added to the culture medium. Th1/Th2 dysbalance-differentiation was induced for 7 days in primary culture, after which cells re-induced for 2 days in the secondary culture containing only the environment, and the acquisition of the specific phenotype (Th1 or Th2) was evaluated by determining the number of two types of cytokines produced in the supernatant (IFN-γ or IL-4).

It is known that the precursor CD4+T cells isolated from BALB/c mice, preferably differentiate in the predominant Th2 phenotype (high levels of IL-4, low content of IFN-γ in the secondary culture supernatant) when activated neutral conditions (medium without additives in primary culture). This phenotype could be fully converted to the Th1 phenotype (high content of IFN-γlow levels of IL-4) adding a blocking monoclonal antibodies to IL-4 in primary culture.

To study the potential role of ST11 in the inhibition of the Th2 phenotype predecessor CD4+T cells isolated from BALB/c mice, activated in the presence of the attached bone marrow cells as accessory cells in primary culture.

Cells were cultured either in medium without 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. After incubation, the cells were washed, CD4+T-cells once cleaned and re-stimulated in the secondary culture medium without additives.

The cytokines produced differentiated CD4+T-cells was determined after 2 days. As expected, cells that were differentiated in the presence of no additive environment, developed a dominant Th2 phenotype. Adding ST11 to primary cultures Modulare the Alo Th2 dysbalance-differentiation, since resulted in 8-fold decrease in IL-4. Similar magnitude of inhibition was observed in the cultures, differencirovany in the presence of LPS. In contrast, the other analyzed Lactobacillus had no significant effect on the levels of IL-4. Interestingly, the content of IFN-γ did not increase when adding ST11 in primary culture.

In General, ST11 specifically inhibited 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 would not increase proderecho IFN-γmay be associated with its ability to induce IL-10, leading to the fact that it can support low inflammatory action, despite its activity against Th2.

Thus the authors were able to show that ST11 is one of the strains of Lactobacillus with good anti-Th2 dysbalance profile, which makes them excellent candidates for use as bacterial strains with anti-allergic, symbiotic activity.

Information confirming the possibility of carrying out the invention

Hereinafter the invention will be described using examples.

Environment and solutions:

MRS (Difco)

Hugo-Jago (tripton Difco 30 g/l, yeast extract Difco 10 g/l, lactose Difco 5 g/l To2NRA46 g/l, meat extract Difco) 2 g/l Difco agar 2 g/l)

M17 (Difco)

M (eromed)

Ringer's solution (Oxoid)

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

Tryptose phosphate broth (Flow)

A solution of Trypsin-EDTA (Seromed)

Rotavirus Wa man (serotype G1) and simian rotavirus SA-11 (serotype G3) was obtained from R.A. Offit, Children's Hospital of Philadelphia, USA Reassertion DS-IxRRV virus was obtained from A.Kapikan, NIH Bethesda, USA Rotavirus Hochi human serotype 4 was obtained from R. Bachmann, University of Munich, Germany.

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 of sampling were prepared by serial dilution in ringer's solution and were sown on their selective environment. For isolation of lactic acid bacteria on MRS agar with antibiotics (fosfomicin 80 µg/ml sulfamethoxazole 93 µg/ml, trimethoprim 5 mg/ml) were incubated at 37°C for 48 hours. Colonies were selected randomly and were purified. Physiological and genetic characterization was performed on the isolates.

Example 2

Definition antirotavirus activity strains

Several bacterial strains, including Lactobacillus, Lactocjccus, Streptococcus, and tested on antirotavirus activity in the test of inhibition in cell culture. The genus Lactococcus was not only the n only (Lc. lactis), consisting of two subspecies (Lc. lactis supsp. lactis and cremoris). All analyzed 30 strains. The genus Streptococcus was represented by a single species (S. Thermophilus) with 45 strains. The genus Leuconostoc and Propionibacterium were represented by only one species (6 strains), whereas the genus Enterococcus and genus Staphylococcus were presented, each, two kinds in total of 17 strains.

Only inhibitory rotavirus activity were tested 260 bacterial strains.

Method 1

30 μl of bacterial suspension containing an average of 3×106bacteria were mixed with 70 μl of medium Ml 99 containing 10% tryptose phosphate broth (Flow) and 5% solution of trypsin-EDTA (Seromed) (diluted 1:4 for cells HT-29) and 100 μl of the virus in the environment 99 Ml with the above additives. The mixture of virus-bacteria were incubated for 1 hour at 4°and 1 hour at 37°C. Undifferentiated cells HT-29 colon adenoma intestine of man, growing in the form of a confluent monolayer in 96-well microtiter tablets, washed three times with PBS, pH of 7.2. The mixture of virus-bacteria were applied to these cells and the microtiter plates were incubated for 18 hours in CO2-thermostat (Heraeus). Viral replication was assessed as described below.

Method 2

30 µl of bacterial suspension (see above) was mixed with 70 μl environment M containing 10% tryptose phosphate broth (Flow) and 5% solution of trypsin-EDTA (Seromed) (diluted 1:4 for tile is to HT-29) and was applied directly to the cells in the microtiter plates. After one hour incubation at 37°With 100 μl of virus in medium M199 with the above additives were added to the cells in microtiter tablets and incubated for 18 hours in CO2-thermostat (Heraeus). Viral replication was assessed as described below.

Viral replication was assessed using histo-immunological staining of proteins of rotavirus-infected cells, as described below.

Later, one day after infection, culture medium was removed from the microtiter tablets and the cells were fixed with absolute ethanol for 10 minutes. The ethanol was removed and tablets thrice washed with PBS. Then to each well was added 50 μl of rabbit antirotavirus serum (mostly against protein VP6), (University of Lausanne ISREC), diluted 1:2000 in PBS, and incubated 1 hour at 37°With a cover glass to prevent. Anticigarette was removed and tablets thrice washed with PBS. Then to each well was added 50 μl of goat antisera against rabbit immunoglobulin G (IgG)conjugated with horseradish peroxidase (GAR-IgG-PO; Nordic), diluted 1:500 in PBS and the plates were incubated 1 hour at 37°C. the Serum was removed and tablets thrice washed with PBS. Then to each well was added 100 μl of the substrate mixture containing 10 ml of 0.05 M Tris-Hcl (pH 7,8), 1 ml of N2About2(30% extra pure, diluted 1:600 in N2About; Merck) and 200 μl of 3-amino-9-ethylcarbazole (0.1 g/10 m is ethanol, stored in the form of an aliquot of 200 μl at -80°C; Sigma). The plates were incubated for at least 30 minutes at room temperature. The substrate was removed and to each well was added 200 μl of N2About to stop reaction. Foci of infected cells were counted under invertations microscope (Diavert; Leitz).

Only a very small number of bacterial strains interacted with rotavirus. Only 9 of 260 initially selected bacterial strains inhibited the replication of rotavirus in at least one method. Lactobacillus paracasei NCC 2461 (ST11) showed very high activity against Rotavirus serotype 1 rotavirus SA-11 serotype 3 and rotavirus Hochi serotype 4.

Example 3

Properties ST11

ST11 incubated in a model of gastric juice. Model of gastric juice was prepared by suspendirovanie pepsin (3 g/l) in sterile saline solution (0.5% wt./about.) and bringing the pH to 2.0 and 3.0, respectively concentrated Hcl.

ST11 were grown in the above media and determined resistance.

The results are summarized in table I.

Table I
pHCFU/mlCFU/mlCFU/mlCFU/mlCFU/ml
 T 1 minT 15 the John T 30 minT 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 was characterized by the following properties 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,

- producing L(+)-lactic acid,

- growing in the presence of bile salts in

concentrations up to about 0.4%.

Example 4

Growth ST11 under various conditions

ST11 incubated at 37°in the medium tomato-based (4% powder tomatoes, resuspending in distilled water), 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 2.

ST11 was 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 a pH of 4.4. After cooling, the product was Packed with add and without added vitamin C and kept at 10° C.

Example 5

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

Bone marrow cells were isolated from femoral and tibial bones 8-week, 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), 12 hours at 37°C in an atmosphere of 5% CO2. Unattached cells were removed 3 successive washes with warm culture medium, and the remaining adherent cells were 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 is the optimal time interval to determine the synthesis of mRNA of cytokines mouse attached cells in response to LPS. Bacteria were added at various concentrations in the range of 109up to 107CFU/ml Bacteria were grown and stored as described above.

At the end of the 6-hour period of culture cells were isolated by centrifugation and literally using a set of reagents TRIzol (GibcoBRL, Cat. No. 15596-018) according to the manufacturer's instructions. Total RNA precipitated with isopropanol and back transcrib is listed in the cDNA for 90 minutes at 42° With 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 (hringer 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 (β-2-microglobulin) (genes household). PCR products were separated in 2% agarose gel and bands were analyzed under UV.

As shown in figure 4, ST11 had the strongest inhibitory effect on the synthesis of mRNA of IL-12 and IL-10, comparable to that observed for the positive control (E. coli). The differences are best seen when the very low concentrations of bacteria (107CFU/ml).

Example 6

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 MiniMACS of Miltenui Biotec (Cat. No. 492-01). CD4+T cells were cultured at a concentration of 2×105cells/ml in RPMI medium 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, and activated for one week the stitching associated with tablet monoclonal antibodies against CD3 (clone C) CD28 (clone 37, 51), both antibodies from Pharmingen. While primary cultures of CD4+T cells were acultural with attached bone marrow cells (selected as described above) as the supporting cells in a medium containing 108CFU/ml Lal or 1 mg/ml LPS or medium without additives. Then cells were washed and CD4+T cells were isolated using a set of MiniMACS and re-stimulated in the secondary culture medium without additives. The cytokines produced differentiated CD4+T-cells was determined in supernatant 2 days using a sandwich ELISA (kits from Endogen and Pharmingen).

The results are shown in figure 5. Cells differentsirovaniya in the presence of no additive environment, had dominates Th2 phenotype, characterized by high levels of IL-4. Adding ST11 to primary cultures modulated Th2 differentiation, because it resulted in 8-fold reduction of the production of IL-4. Similar inhibition was observed in the cultures, differentsirovaniya in the presence of LPS. In contrast, other Lactobacillus strains had no significant effect on the levels of IL-4. Interestingly, the content of IFN-γ did not increase when adding ST11 in primary culture.

As you can see from the above data, the strains of the present invention may be used to prepare NR accept try food and/or pharmaceutical agents with valuable properties of these microorganisms.

Example 7

Strain ST11 investigated in clinical trials for the residents of the suburbs of Guatemala City on the ability to affect the transmission and course of acute diarrhoea rainy season, which affects most children in this area. Just 203 children aged from 35 to 70 months were registered for the 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 the introduction of placebo, respectively, had the typical characteristics of underweight and growth of the appropriate age, due to exhaustion.

Before testing for feeding in children of preschool age conducted a safety assessment on the basis of in vitro and in vivo. In vitro studies showed a picture of antibiotic resistance, similar to that of other Lactobacillus used for food applications, and do not form biogenic amines in the degradation of mucin (mucous secretion) and were not deconjugate bile salts. In clinical trials with placebo, which consisted of 42 adult volunteer, ST11 was well tolerated and did not induce harmful effects, such as bloating, stool frequency per day and stool consistency; the levels of acute phase proteins in serum did not cause any concern in relation to the potential drop of the inflammatory response.

Samples and placebo were prepared in the form of pads (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 medium 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, it was necessary to dissolve in 100 ml of water, also put up by the company Nestle, does not contain bacterial contamination.

In accordance with the test conditions diarrhoea was defined as 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 priest who PA and then to estimate using the total duration. There was a case when a child 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 are characterized by the presence of blood, mucus or pus in the stools, along with symptoms of fever and vomiting. The intensity of 7 chairs for 24 hours or intervention of a professional in a clinic, health Center or hospital also characterize 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. histolytica, 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. It was shown that ST11 remained viable in pads during the whole study, so that at the end of the study, the pads were capable of transmitting 1010viable microorganisms when reconstituirea water.

Dan is th invention revealed, sample containing symbiotic microorganism, can reduce the occurrence of diarrhea, in contrast to the control group (placebo), approximately 30%. But in the control group also showed a decrease in the number of occurrences of diarrhea compared with the normal population who are not receiving any samples or placebo, respectively. This may be partly explained by 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 conclude that ST11 can reduce the occurrence of diarrhea in vivo.

1. The strain Lactobacillus paracasei CNCM I-2116 (NCC 2461), with the ability to prevent infection of epithelial cells of the intestinal rotavirus that cause diarrhea.

2. The strain according to claim 1 for the preparation applied to the inside of the tool.

3. For the treatment and/or prevention of disorders associated with diarrhea caused by rotavirus containing from 1×105up to 1×1012SOME of the strain according to claim 1/g of product and a food product selected from the milk, yogurt, curd, cheese, fermented milk products based on fermented milk, ice cream, fermented products based on cereals, powdered milk, baby food, or pharmaceutical product selected from liquid bacterial, susp is nsii, dry oral supplements, liquid oral Supplement, dry product for feeding through a tube or liquid product for feeding through a tube.



 

Same patents:

FIELD: biotechnology, microbiology.

SUBSTANCE: invention proposes a method for preparing exopolysaccharide by culturing microorganisms in nutrient medium containing one or more carbon source assimilated by microorganisms and caruba seeds fraction as nitrogen organic source. Applying the proposed method provides preparing exopolysaccharide eliciting improved organoleptic, sensor and visual properties. Invention can be used in building, paper, textile, cosmetic, food, oil output industry and agriculture.

EFFECT: improved preparing method.

15 cl, 4 ex

FIELD: biotechnology, microbiology.

SUBSTANCE: invention proposes a method for preparing exopolysaccharide by culturing microorganisms in nutrient medium containing one or more carbon source assimilated by microorganisms and caruba seeds fraction as nitrogen organic source. Applying the proposed method provides preparing exopolysaccharide eliciting improved organoleptic, sensor and visual properties. Invention can be used in building, paper, textile, cosmetic, food, oil output industry and agriculture.

EFFECT: improved preparing method.

15 cl, 4 ex

FIELD: biotechnology, microbiology.

SUBSTANCE: invention proposes a method for preparing exopolysaccharide by culturing microorganisms in nutrient medium containing one or more carbon source assimilated by microorganisms and caruba seeds fraction as nitrogen organic source. Applying the proposed method provides preparing exopolysaccharide eliciting improved organoleptic, sensor and visual properties. Invention can be used in building, paper, textile, cosmetic, food, oil output industry and agriculture.

EFFECT: improved preparing method.

15 cl, 4 ex

FIELD: biotechnology, microbiology.

SUBSTANCE: invention proposes a method for preparing exopolysaccharide by culturing microorganisms in nutrient medium containing one or more carbon source assimilated by microorganisms and caruba seeds fraction as nitrogen organic source. Applying the proposed method provides preparing exopolysaccharide eliciting improved organoleptic, sensor and visual properties. Invention can be used in building, paper, textile, cosmetic, food, oil output industry and agriculture.

EFFECT: improved preparing method.

15 cl, 4 ex

FIELD: biotechnology, microbiology.

SUBSTANCE: invention proposes a method for preparing exopolysaccharide by culturing microorganisms in nutrient medium containing one or more carbon source assimilated by microorganisms and caruba seeds fraction as nitrogen organic source. Applying the proposed method provides preparing exopolysaccharide eliciting improved organoleptic, sensor and visual properties. Invention can be used in building, paper, textile, cosmetic, food, oil output industry and agriculture.

EFFECT: improved preparing method.

15 cl, 4 ex

FIELD: biotechnology, microbiology.

SUBSTANCE: invention proposes a method for preparing exopolysaccharide by culturing microorganisms in nutrient medium containing one or more carbon source assimilated by microorganisms and caruba seeds fraction as nitrogen organic source. Applying the proposed method provides preparing exopolysaccharide eliciting improved organoleptic, sensor and visual properties. Invention can be used in building, paper, textile, cosmetic, food, oil output industry and agriculture.

EFFECT: improved preparing method.

15 cl, 4 ex

FIELD: biotechnology, microbiology.

SUBSTANCE: invention proposes a method for preparing exopolysaccharide by culturing microorganisms in nutrient medium containing one or more carbon source assimilated by microorganisms and caruba seeds fraction as nitrogen organic source. Applying the proposed method provides preparing exopolysaccharide eliciting improved organoleptic, sensor and visual properties. Invention can be used in building, paper, textile, cosmetic, food, oil output industry and agriculture.

EFFECT: improved preparing method.

15 cl, 4 ex

FIELD: biotechnology, microbiology.

SUBSTANCE: invention proposes a method for preparing exopolysaccharide by culturing microorganisms in nutrient medium containing one or more carbon source assimilated by microorganisms and caruba seeds fraction as nitrogen organic source. Applying the proposed method provides preparing exopolysaccharide eliciting improved organoleptic, sensor and visual properties. Invention can be used in building, paper, textile, cosmetic, food, oil output industry and agriculture.

EFFECT: improved preparing method.

15 cl, 4 ex

FIELD: biotechnology, microbiology.

SUBSTANCE: invention proposes a method for preparing exopolysaccharide by culturing microorganisms in nutrient medium containing one or more carbon source assimilated by microorganisms and caruba seeds fraction as nitrogen organic source. Applying the proposed method provides preparing exopolysaccharide eliciting improved organoleptic, sensor and visual properties. Invention can be used in building, paper, textile, cosmetic, food, oil output industry and agriculture.

EFFECT: improved preparing method.

15 cl, 4 ex

FIELD: biotechnology, microbiology.

SUBSTANCE: invention relates to producing ethyl alcohol or fodder for monogastral animals. The polyenzyme product with glucoamylase, proteolytic and xylanase activities is prepared by fermentation of wheat bran using microorganism Aspergillus niger. Indicated glucoamylase, proteolytic and xylanase activities have the following minimal values: glucoamylase activity - at least 100 U/g of dry matter; proteolytic activity - at least 100 U/g of dry matter; xylanase activity - at least 100 U/g of dry matter under condition that glucoamylase activity has to be at least 750 U/g of dry matter and/or xylanase activity has to be at least 300 U/g of dry matter. Invention provides enhancing the soluble nitrogen content in wort after saccharification, reducing viscosity and effectiveness in using.

EFFECT: improved preparing method, valuable properties of hydrolyzed bran.

14 cl, 10 tbl, 7 ex

FIELD: biotechnology, microbiology.

SUBSTANCE: invention proposes a method for preparing exopolysaccharide by culturing microorganisms in nutrient medium containing one or more carbon source assimilated by microorganisms and caruba seeds fraction as nitrogen organic source. Applying the proposed method provides preparing exopolysaccharide eliciting improved organoleptic, sensor and visual properties. Invention can be used in building, paper, textile, cosmetic, food, oil output industry and agriculture.

EFFECT: improved preparing method.

15 cl, 4 ex

FIELD: biotechnology, veterinary science, microbiology.

SUBSTANCE: the strain of bacterium Bacillus subtilis VKM B-2287 is isolated from soil. Bacterial cells are gram-positive with aerobic type of respiratory, don't form capsule but form round spores. The strain hydrolyzes glucose, mannitol and lactose but it doesn't ferment sucrose, inositol, sorbitol and maltose, it doesn't form gas in fermentation process and inhibits the growth of staphylococcus, Escherichia coli, enterobacteria, citrobacteria and aeromonas. The strain is used as industrial one for preparing a preparation named by authors as "Subtilis+". The preparation normalizes function of digestive tract in agricultural animals, poultries and fishes and can be used in treatment and prophylaxis of bacterial infections. Invention can be used for preparing the probiotic preparation.

EFFECT: valuable properties of strain and preparation.

1 tbl, 3 ex

FIELD: agricultural microbiology.

SUBSTANCE: the strain Azotobacter vinelandii IB 4 is obtained by analytical selection of natural isolates by method for selection of producers eliciting rather high antagonistic activity with respect to fungal phytopathogens. The strain is deposited in collection of microorganisms of Biology Institution UNTS RAN at number Azotobacter vinelandii IB 4. For preparing the preparation the strain is grown in nitrogen-free nutrient medium at 28-30oC for 60 h under aeration condition up to the titer value 1010 CFU/ml. The strain elicits high nitrogenase activity and ability for producing cytokinins that simulate the growth and development of plants.

EFFECT: valuable properties of microorganism strain.

7 tbl, 7 ex

FIELD: waste water treatment.

SUBSTANCE: cyanide effluents are treated with alkali or alkali-earth metal metabisulfite in presence of copper catalyst and residual cyanide and thiocyanate are subjected to bacterial destruction using strains Pseudomonas putida 21 and Pseudomonas stutzeri 18.

EFFECT: enabled detoxification of effluents within a wide cyanide and thiocyanate concentration range and therefore allowed use of the method for cleaning waste waters and slurries in gold mining, galvanic, pharmaceutical, and in a number of other industries.

FIELD: biotechnology, microbiology.

SUBSTANCE: invention relates, in particular, to preparing nutrient media used for culturing the plague microorganism vaccine strain and can be used in medicinal microbiology. The nutrient medium for culturing the plague microorganism vaccine strain comprises additionally as a stimulating additive sodium sulfite and as a nutrient base - soybean fruits enzymatic hydrolyzate in the following ratio of components, g/l: microbiological agar, 11.0-13.0; soybean fruits enzymatic hydrolyzate, 250.0-350.0; sodium chloride, 4.5-5.5; sodium hydrogen phosphate, 3.5-4.5; sodium sulfite, 0.0003-0.0005; distilled water, the balance. Invention provides enhancing the growth property of nutrient medium.

EFFECT: valuable properties of medium.

3 ex

FIELD: biotechnology, food and medicinal industry, microbiology.

SUBSTANCE: the strain Bifidobacterium longum 379-IN is obtained by selection without using methods of genetic modification of the strain Bifidobacterium longum B379M and distinct by ability to utilize insulin. The strain is deposited in GKNM GU "MNIIEM named for G. N. Gabrichevskiy Russia Ministry of Public Health" at № 172. The strain shows high technological effectiveness, accumulates biomass with substrates of vegetable origin and artificial nutrient media for short periods with concentration of bifidobacteria, it elicits acid-forming and antagonistic properties with respect to pathogenic and putrid microflora. This allows its using in manufacturing bacterial preparations, biologically active supplements for food, fermented-dairy and nonfermented-dairy foodstuffs, ferments, hygienic and cosmetic agents providing probiotic effect and normalization of microbiocenosis in human body, among them in gastroenteric and urogenital tracts, cutaneous and mucosa integuments. Invention can be used in manufacturing bacterial preparations, biologically active supplements for food, fermented-dairy and nonfermented-dairy foodstuffs, hygienic and cosmetic agents.

EFFECT: valuable properties of strain, expanded assortment of similar agents.

6 ex

FIELD: biotechnology, microbiology.

SUBSTANCE: method for preparing liquid lactobacterin involves regeneration, culturing passages of lyophilized culture and culturing ferment of lactobacilli in liquid lyophilized nutrient medium containing dry defatted milk enzymatic hydrolyzate with the content of amine nitrogen 1 485 mg%, 30.0 ± 3.0 g/l; yeast concentrated autolyzate, 110.0 ± 10 g/l; food agar, 0.8 g/l, and distilled water, up to 1 l. Culturing ferment is carried out up to accumulation of biomass of lactobacilli 109-1010 CFU/ml. Then 10-30% of supernatant liquid is removed from the ready product and replaced it with equal volume of fresh nutrient medium. Invention provides simplifying technology in preparing liquid lactobacterin and to elevate the storage period of viable lactobacilli. Invention can be used in producing probiotic preparations.

EFFECT: improved preparing method.

1 tbl, 3 ex

FIELD: biotechnology, microbiology, veterinary science.

SUBSTANCE: for preparing a preparation cells of microorganism Escherichia coli VKM CR-322D is cultured in nutrient medium containing Hottinger's broth, glucose, yeast extract, manganese sulfate, potassium hydrophosphate, sodium chloride and tap water in the content of amine nitrogen 125-155 mg%. Glucose is added by batch portions in the process of culturing cells that is carried out at temperature 30-31oC at stirring and aeration for 10-12 h. Prepared cultural fluid containing 3 x 109 bacterial cells/ml is mixed with protective sucrose-gelatin medium and subjected for lyophilic drying. Dried mass is stored under nitrogen that enhances safety of viable cells in the preparation. Applying the preparation for prophylaxis and treatment of agricultural and domestic animals and poultries with gastroenteric diseases provides its effectiveness.

EFFECT: improved preparing method, valuable veterinary properties of preparation.

17 cl, 8 ex

FIELD: biotechnology, biochemistry, microbiology.

SUBSTANCE: the strain Bacillus circulans B-65 a producer of cyclodextrin glucanotransferase is isolated and selected form the soil sample by culturing in nutrient medium with amylolytic activity 12.17 U/ml and cyclodextrinogenic activity up to 0.530 U/ml. Cyclodextrin glucanotransferase isolated from B. circulans B-65 shows the high degree for conversion of starch to cyclodextrins and this enzyme is specific for formation of β-cyclodextrin. Invention can be used in food industry for preparing cyclodextrins and cyclodextrin glucanotransferase used in different branches of industry.

EFFECT: improved isolating method, valuable properties of strain and enzymes.

9 cl, 8 tbl, 2 ex

FIELD: biotechnology, microbiology, dairy industry.

SUBSTANCE: the strain of microorganism Lactobacillus lactis VKPM B-8354 is prepared without using mutagens and genetic methods and shows resistance against broad spectrum of lactophages. The strain ferments effectively milk from different trading sorts, with broad range of fatness and different methods of thermal treatment. Individual specific properties of the strain allows its applying as a monostrain ferment. Curd obtained with applying the strain L. lactis VKPM B-8354 shows good organoleptic qualities, nonacid taste and homogenous consistence. The strain is suitable especially for plants with small volume of manufacture but with varied assortment.

EFFECT: valuable properties of strain.

3 tbl, 2 dwg, 5 ex

FIELD: biotechnology, veterinary science, microbiology.

SUBSTANCE: the strain of bacterium Bacillus subtilis VKM B-2287 is isolated from soil. Bacterial cells are gram-positive with aerobic type of respiratory, don't form capsule but form round spores. The strain hydrolyzes glucose, mannitol and lactose but it doesn't ferment sucrose, inositol, sorbitol and maltose, it doesn't form gas in fermentation process and inhibits the growth of staphylococcus, Escherichia coli, enterobacteria, citrobacteria and aeromonas. The strain is used as industrial one for preparing a preparation named by authors as "Subtilis+". The preparation normalizes function of digestive tract in agricultural animals, poultries and fishes and can be used in treatment and prophylaxis of bacterial infections. Invention can be used for preparing the probiotic preparation.

EFFECT: valuable properties of strain and preparation.

1 tbl, 3 ex

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