MXPA01008927A - Lactic acid bacteria strains capable of preventing diarrhoea - Google Patents

Abstract

The present invention pertains to novel microorganisms of the family Lactobacillaceae, especially to microorganisms of the genus Lactobacillus, that are useful in preventing or treating diarrhoea. In particular, the present invention relates to the use of said micro-organisms for the preparation of an ingestable support and to a composition containing the same.

Description

ACIDOLACTIC BACTERIA CEPAS CAPABLE OF AVOIDING DIARRHEA DESCRIPTION OF THE INVENTION The present invention belongs to novel microorganisms of the family Lactobacillaceae, especially to microorganisms of the genus Lactobacillus which are useful for preventing or treating diarrhea. In particular, the present invention relates to the use of such microorganisms for the preparation of an ingestible support and with a composition containing them. For a long time organisms that produce lactic acid as the main metabolic component have been known. These bacteria can be found in milk or in milk processing factories, respectively, in living or decaying plants, but also in the intestines of man and animals. These microorganisms, grouped under the term "lactic acid bacteria", represent a rather heterogeneous group and comprise, for example, the genera Lactococcus, Lactobacillus, Stretpcoccus, Bifidobacterium, Pediococcus, etc. The lactic acid bacteria have been used with fermentation agents for food preservation taking advantage of the low pH and the action of the fermentation products generated during the fermentative activity of the same to inhibit the growth of harmful bacteria. For this purpose, lactic acid bacteria have been used to prepare various different food products such as cheese, yogurt and other fermented milk products, from milk. Very recently, lactic acid bacteria have attracted a lot of attention to the extent that some strains have been found to show valuable properties for humans and animals when they are ingested. In particular, it has been found that specific strains of Lactobacillus or Bifidobacterium are able to colonize the intestinal mucosa and help maintain the well-being of men and animals. In this regard, EP 0 768 375 describes specific strains of the genus Bifidobacterium that are capable of being implanted in the intestinal flora and can adhere to the intestinal cells. It has been reported that these Bifidobacteria help in immunomodulation, are able to competitively exclude the adhesion of pathogenic bacteria to intestinal cells and thus help in maintaining the health of the individual. In recent years, research has also focused on the potential use of lactic acid bacteria as probiotic agents. Probiotics are considered viable microbial preparations which promote the health of the individual by conserving the natural microflora in the intestine. A microbial preparation can be commonly accepted as a probiotic in case the microbes that produce the alterations thereof as well as their mode of action are known. It is considered that probiotics bind to the intestinal mucosa, colonize the intestinal tract and likewise prevent the union of microorganisms harmful to it. A crucial requirement for its action is based on the fact that it must reach the mucosa of the intestine in an appropriate and viable way and that it is not distributed in the upper part of the gastrointestinal tract, especially because of the influence of the low pH that prevails in the stomach. In this regard, WO 97/00078 describes a specific strain, called Lactobacillus GG (ATCC 53103), as a probiotic. The microorganism is particularly used in a method for preventing or treating food-induced hypersensitivity reactions insofar as it is administered to a recipient together with a food material which has been subjected to hydrolysis treatment with pepsin or trypsin or both. The selected Lactobacillus strain is described as showing adhesive and colonizing properties and shows a protease enzyme system so that the protein material contained in the food product to be administered is further hydrolyzed by proteases secreted by specific strains of Lactobacillus The method discussed in this document will eventually result in the uptake of protein material by the gut that no longer shows a substantial amount of allergenic material. further, in EP 0 577 903 reference is made to the use of such lactic acid bacteria with the ability to replace Heliobacter pylori, the recognized agent for the development of ulcer, in the preparation of a support designed for therapeutic or prophylactic treatment of an ulcer associated with the action of Heliobacter pylori. In the knowledge of the useful properties that particular strains of lactic acid bacteria can provide, there is a desire in the art for additional strains of lactic acid bacteria that are beneficial for the well-being of men or animals, or both. Accordingly, a problem of the present invention is to provide additional bacterial strains which show new beneficial properties for man or animals, or both. The above problem has been solved by providing novel microorganisms, specifically lactic acid bacteria, belonging to the genus Lactobacillus which have the traits of being able to adhere and colonize essentially the intestinal mucosa and to avoid the infection of intestinal epithelial cells by rotavirus. According to a preferred embodiment, the Lactobacillus strains are capable of growing in the presence of up to 0.4% of bile salts, so that they can easily pass the gastrointestinal tract and remain essentially active. According to another preferred embodiment, the lactic acid bacteria are selected from the group consisting of Lactobacillus rhamnosus or Lactobacillus paracasei, preferably Lactobacillus paracasei and more preferably Lactobacillus paracasei CNCM 1-2116. It has been shown that the microorganisms of the present invention show the following properties, are gram positive, catalase negative, NH3 form negative arginine and negative CO 2 production. They produce L (+) lactic acid, are able to grow in the presence of bile salts in a concentration of up to approximately 0.4% and essentially prevent the infection of epithelial cells by rotavirus. The novel microorganisms can be used for the preparation of various ingestible support materials such as, for example, milk, yogurt, cottage cheese, fermented milk, milk-based fermented products, fermented grain-based products, milk-based powders, infant formulas. and may be included in the support in an amount of about 105 cfu / g to about 10 11 cfu / g. For the purpose of the present invention, the abbreviation ufe will designate a "colony forming unit" which is defined as the number of bacterial cells as shown by a microbiological count on agar plates.

The present invention also provides a food or a pharmaceutical composition containing at least one of the Lactobacillus strains having the above features. To prepare a food composition according to the present invention, at least one of the Lactobacillus strains according to the present invention is incorporated in a suitable support, in an amount from about 10 5 cfu / g to about 10 11 cfu / g, preferably from about 10 cfu / g to about 10 10 cfu / g, more preferably from about 107 cfu / g to about 109 cfu / g. In the case of a pharmaceutical preparation, the product can be prepared in tablet forms, liquid bacterial suspensions, dry oral supplements, oral moist supplements, dry tube feeding or wet tube feeding, etc., with the amount of strains of Lactobacillus to be incorporated therein which is in the range of up to 1012 cfu / g, preferably from about 107 cfu / g to about 1011 cfu / g, and more preferably from about 107 cfu / g to about 1010 cfu / g. g. The activity of novel microorganisms in the intestine of the individual is naturally dose dependent. That is, the greater the amount of microorganisms that are incorporated through the ingestion of the above food material or the pharmaceutical composition, the greater the protective or curative life, or both, of the microorganisms. Since novel microorganisms are not harmful to mankind and animals, and have finally been isolated from baby feces, a large amount of them can be incorporated so that essentially a high portion of the individual's intestine will be colonized by microorganisms. new. In the figures: Figure 1 shows a diagram illustrating a cell culture screening to determine the retroviral protective properties of bacterial strains. Figure 2 shows the acidification of L. casei strain CNCM 1-2116 (named ST11) of different growth media. Figure 3 shows the survival rate of L. casei strain STll at 10 ° C, measured for 30 days. Figure 4 shows the mRNA pattern of IL-12 and IL-10 in mouse adherent cells derived from bone marrow after incubation of the cells with serial dilutions of STll. Figure 5 shows the result of Th2 differentiation as a result of a decreased production of IL-4. During the extensive studies that lead to the present invention, the inventors have investigated baby feces and isolated several different bacterial strains thereof. These strains were subsequently examined for their ability to prevent infection of epithelial cells with rotavirus that is known to cause diarrhea.

Several diverse bacterial genera comprising Lactobacillus, Lactococcus, Streptococcus were examined for their rotavirus inhibitory properties. The tests for the inhibitory property were carried out essentially with three rotavirus serotypes that represent the main etiological agents of human viral diarrhea (serotypes Gl, G3 and G4). The various lactic acid bacteria are grown in a suitable medium, such as MRS, Hugo-Jago or M17 medium at temperatures from about 30 to 40 ° C corresponding to their optimum growth temperature. After reaching the stationary growth, the bacteria are collected by centrifugation and resuspended in a physiological solution of NaCl. Among the different tests, the bacterial cells are stored frozen (-20 ° C). Various rotavirus concentrates are prepared by infecting monolayers of confluent cells. Rotaviruses are incubated before infection. The cells are infected with 20 infectious doses of tissue culture. To determine the anti-rotaviral properties, two different protocols are applied. According to a protocol, the various bacterial strains are examined for their direct interaction with the rotavirus, whereas in the second protocol, the bacteria are examined for those strains that interact with rotavirus cellular receptors.

The first protocol involves contacting the respective bacterial suspension, each with a different strain of rotavirus and incubating it in a suitable medium. Subsequently, the virus-bacteria mixture is applied to a monolayer of HT-2 cell cells from human undifferentiated colon adenoma and incubation is continued. Then a viral replication assay is performed. The second protocol involves incubating the respective bacterial suspension, first together with a monolayer of HT-29 cells from human undifferentiated colon adenoma, and the virus is subsequently added. After continuous incubation of the virus, replication is assayed. Rotavirus replication can be easily determined by histoimmunological staining of rotavirus proteins in infected cells. A rotavirus inhibitory effect is attributed to a given bacterium when the number of infected cells is reduced in 90% in the cell culture inoculated with rotavirus plus the indicated bacteria, compared to cells inoculated only with rotavirus. Of a total of 260 different bacterial strains isolated in a primary manner, only 9 can be shown to essentially inhibit rotaviral replication. It was determined that the different bacteria belong to the genus Lactobacillus, subspecies rhamnos us or to casei. A strain, called Lactobacillus casei STll, which has been deposited according to the Budapest treaty and has received deposit numbers NCC 2461 (1-2116), has been shown to be extremely effective in preventing infection of human cells by rotavirus. In addition, this particular strain shows excellent growth properties as can be demonstrated by acidification in different media. The strain also shows good performance with respect to the survival rate during storage at low temperatures of approximately 10 ° C, which makes it an excellent candidate to be included in food products or in pharmaceutical compositions that are to be stored under refrigeration conditions. In addition to the above finding, it can also be demonstrated that the strains also surprisingly show antiallergenic properties insofar as the strains have an impact on the synthesis of different immunological mediators. It is generally recognized that humoral immune responses and allergic reactions are mediated by CD4 + T cells presenting the type 2 (Th2) phenotype. Th2 cells are characterized by the production of high concentrations of interleukin 4 (IL-4), a cytokine necessary for the secretion of IgE, which is the class of major antibody involved in allergic reactions.

Differentiation of Th2 cells is damaged by IFN- ?, a particular cytokine arising from a mutually exclusive Thl subset of CD4 + T cells. Such Thl cells in turn are strongly induced by interleukin 12 (IL-12). In contrast to this, it has been shown that IL-10, another cytokine, has a strong suppressive impact on the proliferation of Thl cells and is therefore considered to play a role in immunosuppressive mechanisms. In summary, both IL-12 and IL-10 have strong modulatory effects on the development of CD4 + T cells by altering the development of the Thl subgroup. IL-12 is a key regulatory cytokine for the induction of Thl differentiation and thus inhibits the generation of Th2 responses. A main route for the inhibition of Th2 cells is therefore observed in the stimulation of IL-12 synthesis by accessory cells. It is well known that some components of gram-negative bacteria, such as LPS, induce high concentrations of IL-12 in adherent cells, such as macrophages and dendritic cells. Consistently, it has been found that gram-negative bacteria can strongly displace the differentiation of CD4 + T cells into the Thl phenotype. The STll microorganism as an example of strains of Lactobacillus of the present invention have been tested to determine their potential role in the induction of cytokines involved in the regulation of CD4 + T cell differentiation.

In particular, the effect of STll on the phenotype of CD4 + T cells undergoing Th2 differentiation has been studied. In this regard, the capacity of STll to induce the synthesis of mRNA coding for these two regulatory cytokines in mouse adherent cells derived from bone marrow, with 4 other strains of Lactobacillus and with a control of granmnegative bacteria (E. coli) was compared. K12). The mRNA is measured by semiquantitative RT-PCR after 6 hours of incubation of the cells with serial dilutions of bacteria ranging from 109 to 10 7 cfu / ml. Although all strains of Lactobacillus can induce transcription of mRNA for IL-12 to some degree, it can be shown that STll is the strongest inducer, since a strong PCR signal can be detected even at a lower bacterial dose. In fact, the capacity of STll to induce the transcription of mRNA for IL-12 is as strong as that of E. coli. The induction of mRNA for IL-10 is generally weaker than for mRNA for IL-12, since only at higher bacterial doses can a signal be detected. However, STll is the strongest inducer of mRNA for IL-10, compared to the other lactobacillus and the E. coli control. Therefore, it is considered that STII is efficient in inducing immunoregulatory cytokines involved in the differentiation of CD4 + T cells. Their great ability to induce IL-12 makes them a candidate to inhibit Th2 responses and their measurable IL-10 induction can prevent inflammatory responses. In addition to the above finding, it has also been determined whether STll has an inhibitory effect on CD4 + T cells that undergo Th2 differentiation and a positive effect on Th1 functions. A well-established cell differentiation culture system is used, wherein the precursor CD4 + T cells are activated polyclonally and modulated to undergo Thl or Th2 differentiation, depending on the type of co-stimuli that are provided in the culture medium. Thl / Th2 differentiation is induced during a primary culture of 7 days after which the cells are re-stimulated for 2 days in a secondary culture containing medium only and the acquisition of a specific phenotype (Th1 or Th2) is determined. measure the types of cytokines produced in the supernatant (IFN-? versus IL-4). It is known that precursor CD4 + T cells of BALB / c mice differ preferentially to the predominant Th2 phenotype (high concentration of IL-4, low concentration of IFN-α in secondary culture supernatants). After activation under neutral conditions (medium only in the primary culture). This phenotype can be completely reversed to a Thl pattern (high concentration of IFN- ?, low concentration of IL-4) by the addition of a blocking monoclonal antibody to IL-4 in the primary culture. To investigate a potential role for STll in the inhibition of Th2, purified precursor CD4 + T cells of BALB / c mice are activated in the presence of bone marrow adherent cells, as accessory cells during the primary culture. These cells are co-cultured with either medium alone or in the presence of 1 mg / ml of LPS, or 108 cfu / ml of STll, or 108 cfu / ml of other Lactobacillus. After this time, the cells are washed and the CD4 + T cells are purified once more and re-stimulated in the secondary culture in medium alone. The cytokines produced by differentiated CD4 + T cells are measured after 2 days. As expected, cells differentiated in the presence of medium only show a dominant Th2 phenotype. The addition of STll to the primary cultures strongly modulates the result of Th2 differentiation, which results in an 8-fold decrease in the production of IL-4. This inhibition is similar in magnitude to that observed in cultures derived from differentiated cells in the presence of LPS. By contrast, the other strain of Lactobacillus does not have a measurable impact on IL-4 concentrations. Interestingly, the IFN-α concentrations are not increased. before the addition of STll in primary crops. In summary, STll specifically damages the production of IL-4 by CD4 + T cells that undergo Th2 differentiation, but does not significantly increase the secretion of IFN-α. The fact that STll does not increase the production of IFN-? it may be due to its ability to induce IL-10, with the consequence that it can maintain a low inflammatory impact despite its activity against Th2. Consequently, it can be shown that STll is a strain of Lactojacillus that has a good profile against Th2, which makes it an excellent candidate for use as a bacterium with antiallergic and probiotic activity. The present invention will now be described by means of an example.

Means and solutions: MRS (Difco), Hugo-Jago (Triptona Difco 30 g / 1, Difco yeast extract 10 g / 1, Difco lactose 5 g / 1, KH2P04 5 g / 1, Difco beef extract 2 g / 1, Difco 2 agar g / 1) M17 (Difco) M199 (Seromed) Ringer solution (Oxoid) PBS (NaCl 8 g / 1, KCl 0.2 g / 1, Na2HP04 1.15 g / 1, KH2P04 0.2 g / 1)) Triptose phosphate broth (flow) Trypsin solution EDTA (Seromed) Human rotaviruses Wa (serotype Gl) and simian rotavirus SA-11 (serotype G3) are obtained from P.A. Offit, Children's Hospital of Philadelphia, United States. The reclassified DS-lxRRV virus is obtained from A. Kapikian, NIH Bethesda, U.S.A. Hochi human rotavirus serotype 4 is obtained from P. Bauchmann, University of Munich, Germany.

Example 1 Isolation of lactic acid bacteria from baby feces Fresh diaper feces are collected from 16 healthy babies 15 to 27 days old. 1 g of fresh faeces are placed under anaerobic conditions for transport to the laboratory and microbiological analysis is carried out in the following two hours from the time of sampling by serial dilutions in Ringer's solution and planted on selective media. The MRS agar plus antibiotics (fosfomycin 80 μg / ml, sulfamethoxazole 93 μg / ml, trimethoprim 5 μg / ml) is incubated at 37 ° C for 48 hours and used to isolate lactic acid bacteria. The colonies are taken randomly and purified. Physiological and genetic characterization is carried out in the isolates.

Example 2 Strain test in cell culture to determine antiretroviral activity Several bacterial genera comprising Lactobacillus, Lactococcus, Stretpcoccus, are selected and tested to determine the members which show anti-rotavirus activity in the cell culture inhibition test. The genus Lactococcus is presented by a single species. { Lc. lactis) consisting of two subspecies. { Lc. lactis subspecies lactis and cremoris). A total of 30 strains are tested. The genus Streptococcus is represented by a single species. { S. thermophi l us) with 45 strains. The genus Leu cones toc and Propionibacterium are represented only by a single species (6 strains), while the genera Enterococcus and Staphilococcus are represented by two species each and a total of 17 strains. In total, 260 bacterial strains are tested for rotavirus inhibitory activity.

First protocol μl of bacterial suspension (containing on average 3 x 106 bacteria) is mixed with 70 μl of M199 medium supplemented with 10% tryptose phosphate broth (Flow) and a solution of trypsin-5% EDTA (Seromed) (1: 4). diluted 1: 4 in the case of HT-29 cells) and 100 μl of virus in supplemented M199 medium. The virus-bacteria mixture is incubated for 1 hour at 4 ° C and for 1 hour at 37 ° C. Cells of cells HT-29 of human undifferentiated colon adenoma grows as a confluent monolayer in 96-well microtiter plates and washed three times with phosphate-buffered saline (PBS, pH 7.2). The virus-bacteria mixture is applied to the cells and the microtiter plates are incubated for 18 h in a C02 incubator (Heraeus). Virus replication is assayed as described in the following.

Second protocol μl of the bacterial suspension (supra) is mixed with 70 μl of M199 medium supplemented with 10% tryptose phosphate broth (Flow) and a solution of 5% EDTA trypsin (Seromed) (diluted 1: 4 in the case of cells HT-29) and is applied directly on the cells in the microtiter plates. After one hour of incubation at 37 ° C, 100 μl of virus in supplemented M199 medium is added to the cells in the microtiter plates. The incubation continues for 18 h in an incubator with C02 (Heraeus). Virus replication is assayed as described below.

Rotavirus replication is assayed by histoimmunological staining of rotavirus proteins in infected cells, as described below. One day after infection, the cell culture medium is discarded from the microtitre plates and the cells are fixed with absolute ethanol for 10 min. The ethanol is discarded and the plates are washed three times in PBS buffer. Then 50 μl of an anti-rotavirus serum (directed mainly against the VP6 protein) produced in rabbits (obtained from ISREC University or Lausanne) is added to each well and diluted 1: 200 in PBS and incubated for 1 h at 37 ° C with a Sliding cover to prevent drying of the wells. The antiserum is subsequently discarded and the plates are washed three times with PBS. Subsequently, 50 μl of antiserum against rabbit immunoglobulin G (IgG), produced in goats, and coupled to peroxidase (GAR-IgG-PO) are added.; Nordic) at a dilution of 1: 500 in PBS to each well and the plates are incubated for 1 hour at 37 ° C. The serum is discarded and the plates are washed once more three times with PBS. Then add 100 μl of the following substrate mixture to each well: 10 ml of 0.05 M Tris-hydrochloride (pH 7.8), 1 ml of H202 (30% suprapur, diluted 1: 600 in H20, Merck) and 200 μl of 3-amino-9-ethylcarbazole (0.1 g / 10 ml of ethanol stored in aliquots of 200 μl a - 80 ° C; A-5754; Sigma). The plates are incubated for at least 30 min at room temperature. The substrate is discarded and the wells are filled with 200 μl of H20 to stop the reaction. The foci of infected cells are counted with an inverted microscope (Diavert, Leitz). Only very few bacterial strains interact with rotavirus. Only 9 of the 260 selected primary bacterial cells inhibit rotavirus replication in at least one protocol. Lactobacillus paracasei NCC 2461 (STll) shows an extremely high activity against rotavirus serotype 1, serotype 3, SA-11 and rotavirus Serotype 4 Hochi.

Example 3 STll properties STll was subjected to incubation in simulated gastric juice. The simulated gastric juice is prepared by suspending pepsin (3 g / 1) in sterile saline (0.5% w / v) and adjusting the pH to 2.0 and pH 3, respectively with concentrated HCl. STll has been grown in varying amounts in the above media and the resistance of the microorganisms has been determined. The results are summarized in table I below.

Table I STll has the following properties, as defined according to the methods described in the genera of lactic acid bacteria, Ed. B.J.B. Wood and W. H. Holzapfel, Blackie A &P. -grampositiva, -catalasa negative, -NH3 forms negative arginine -production of C02 negative -production of L (+) lactic acid -growth in the presence of bile salts, in a concentration of up to approximately 0.4%.

Example 4 Growth of STll under different conditions STll is incubated at 37 ° C in tomato-based medium (4% tomato powder rehydrated in distilled water) supplemented with sucrose (0, 0.5, 1 0 2%) or soy peptone (0.5%) or glucose (0.5%) for different periods of time. In Figure 2 the results are shown. STL is additionally added in an amount of 2.5% to a medium composed of rice flour (3%), wheat flour (2%) and sucrose (3%) and incubate at 37 ° C until a pH of 4.4 is reached. After cooling, the product is packed with or without the addition of vit. C and stored at 10 ° C.

Example 5 Induction of mRNA synthesis for IL-12 and IL10 in mouse adherent cells, by STII Bone marrow cells are isolated from the femur and tibia of 8 week-old specific pathogen-free C57BL / 6 mice and incubated at a concentration of 2 x 106 cells / ml in RPMI medium (Gibco) containing 10% fetal bovine serum. , 1 mM L-glutamine, 12 mM Hepes, 0.05 mM 2-mercaptoethanol, 100 U / ml penicillin and 100 μg / ml streptomycin (all Gibco reagents) for 12 hours at 37 ° C an atmosphere of C02 5% . The non-adherent cells are discarded by three consecutive washes with warm culture medium, and the remaining adherent cells are harvested and incubated at a concentration of 10 6 cells / ml for 6 hours, in the presence or absence of bacteria. It has previously been determined that 6 hours represent an optimal time point for the synthesis of mRNA for cytokine by mouse adherent cells, in response to LPS. Bacteria are added at different concentrations ranging from 109 to 107 cfu / ml. The bacteria are grown and stored as indicated above (page 6). At the end of the 6 hour culture period, cells are isolated by centrifugation and used using the TRIzol reagent kit (GibcoBRL, Cat. No. 15596-018) following the manufacturer's instructions. Total RNA is isolated by precipitation with isopropanol and subjected to reverse transcription in cDNA for 90 min at 42 ° C using 200 U reverse transcriptase (Superscript II, BRL) in a reaction volume of 40 μl containing 200 mM Tris, pH 8.3, 25 mM KCl, 1 μg / ml oligo d (T) 15 (Boehringer Mannheim), 1 mM DTT (Boehringer Mannheim), 4 mM each of dNTP (Boehringer Mannheim) and 40 U / ml Rnasin (Promega) The PCR primers and conditions are used as described in advance in Kopf et al. (Journal of Experimental Medecine 1996 Sep 1; 184 (3): 1127-36). The amounts of cDNA are normalized within the samples using primers specific for a constitutive gene (β-2-microglobulin). The PCR products are separated on a 2% agarose gel and the bands are analyzed under UV radiation. As shown in Figure 4, STll shows the strongest induction of mRNA for IL-12 and IL-10 which is comparable to the concentrations observed with the positive control. { E. col i). Differences are best seen at lower concentrations of bacteria (107 cfu / ml).

Example 6 Suppression of IL-4 synthesis by STII CD4 + T cells are purified from the spleen of specific pathogen-free BABL / c mice using the MiniMACS kit from Miltenyi Biotec (Cat. No. 492-01). The CD4 + T cells are cultured at a concentration of 2 x 10 5 cells / ml in RPMI medium containing 10% fetal bovine serum, 1 mM L-glutamine, 12 mM Hepes, 0.05 mM 2-mercaptoethanol, 100 U / ml penicillin and 100 μg / ml of streptomycin and activated for a week by crosslinking with monoclonal antibodies bound to plate, to CD3 (clone 2C11) and CD28 (clone 37.51, both Pharmingen antibodies). During this primary culture the CD4 + T cells are co-cultured with bone marrow adherent cells (isolated as described above) as accessor cells and with 108 cfu / ml of STll or 108 cfu / ml of Lal, or 1 mg / ml of LPS, or half alone. After this time, cells are washed and CD4 + T cells are purified once again using MiniMACS equipment technology and re-stimulated in a secondary culture containing only medium. The cytokines produced by the differentiated CD4 + T cells are measured in the supernatants after 2 days using the sandwich or sandwich ELISA test (Endogen and Pharmingen equipment). The results are shown in Figure 5. Cells differentiated in the presence of medium only show a dominant Th2 phenotype, characterized by high concentrations of IL-4. The addition of STll to the primary cultures strongly modulates the result of Th2 differentiation, as it results in an 8-fold decrease in the production of IL-4. This inhibition is similar in magnitude to that observed in cultures derived from differentiated cells in the presence of LPS. In contrast to this, the other strain of Lactobacillus have no measurable impact on IL-4 concentrations. Interestingly, the concentrations of IFN-? they are not increased by the addition of STll in the primary cultures. It can be seen from the foregoing that the strains of the present invention are well prepared for the production of a food or pharmaceutical carrier or both, taking advantage of the useful properties of the microorganisms.

Example 7 The STll strain is tested in a clinical trial in a community in the suburbs of Guatemala City to determine its ability to influence the transmission and experience of a diarrheal disease in a rainy, acute season, experienced by most children in this area. A total of 203 children aged 35 to 70 months are included in the study and receive a target dose of 1010 viable organisms (STll) or none (placebo) over a 29-day feeding period. The children selected for the sample and for the placebo, respectively, have the typical deficiencies of weight for age and height for age, characteristics due to malnutrition. Before starting the feeding trial in preschool children, a safety assessment is carried out based on in vitro and in vivo studies. The in vitro studies show a pattern of resistance to antibiotics similar to that of other lactobacillus used in food applications and without the potential to form biogenic amines, to degrade mucin and to deconjugate bile salts. In a placebo-controlled clinical study involving 42 adult volunteers, STll is well tolerated and does not induce any adverse effects, among the potential monitored manifestations, such as flatulence, number of stools per day and stool consistency; the concentrations of the acute phase proteins in serum do not generate any concern regarding a potential inflammatory reaction. Samples and placebos have been packed in sacks at the Nestlé Product Technology Center manufacturing facility in Konolfingen, Switzerland, and transported refrigerated to Guatemala. Each bag of 10 g consists of a vehicle with chocolate flavor and 0.2 g of STll (1010 ufe) or, in the case of placebo, 0.2 g of milk powder. The chocolate-flavored vehicle consists of cocoa powder, sugar, soy lecithin, vanillin and cinnamon. The sacks are stored at 4 to 6 ° C for up to two hours before use. Before using the bag, it must be dissolved in 100 ml of water provided by Nestlé, which is free of any bacterial contamination. According to the protocol applied, diarrhea is defined as the presentation of three or more fluid or unformed fetal stools during a 24-h period. An episode of diarrhea is defined as an event that presents evidence of diarrhea (3 diarrheic bowel movements during 24 h). Its total duration in h is calculated from the moment of the first of the three index stools until the appearance of the first formed stool or a period of 24 hours without any defecation. In order for a child to present a "new" episode, 48 hours must have elapsed before the end of the previous episode. If not, it is considered a continuation of the same episode and the total duration is then used for evaluation. A case is a child who experiences one or more documented episodes of diarrhea through an observation period of 29 days. The intensity of the diarrheal episode is based on the total number of loose stools produced. The elements of severity of the episode include the presence of blood, mucus or pus in feces, together with symptoms of fever and vomiting. An intensity of 7 evacuations in 24 hours or the need for intervention by a health professional in a clinic, health center or hospital also classifies the episode as serious. When a diarrheal episode is diagnosed through the surveillance system, a sample of diarrheal stools is collected for microscopic examination and a culture to identify the potential etiologic pathogens for this episode. The sample is diagnosed for the determination of rotavirus antigen Giardia and E. histolytica, in case the sample is dysenteric, and for the determination of bacterial pathogens that include Shi gel la, Salmon el, Aeromonas, Pi es i omona s shigelloides, E. coli and possibly V. cholerae. During the investigation period, product samples are collected to examine the viability of the microorganisms included during the administration period. It can be demonstrated that the microorganism remains viable in the sachets throughout the study so that at the end of the study the sachets are capable of transporting 1010 viable microorganisms when they are reconstituted with water. The study makes it clear that the sample containing the probiotic microorganism can decrease the presentation of diarrhea, in contrast to the control group (to which it is administered placebo) in approximately 30%. Even so, the control group shows in advance a decreased number of diarrheic presentations with respect to the normal population that does not receive any of the samples or placebo, respectively. This latter finding can be explained partly on the basis of children receiving additional useful nutrition and water free of contamination. However, since the study was conducted in the field, it can be clearly deduced that STll can safely reduce the presentation of diarrhea in vivo.

Claims (10)

1. Acrolactic bacteria belonging to the genus Lactobacillus capable of adhering and essentially colonizing the intestinal mucosa, capable of preventing infections of the intestinal epithelial cells by rotavirus and able to grow in the presence of up to 0.4% of bile salts.

2. Lactobacillus, as described in claim 1, which is selected from the group consisting of Lactobacillus rhamnosus or Lactobacillus paracasei.

3. Lactobacillus, as described in claim 2, which is Lactobacillus paracasei.

4. The strain of Lactobacillus paracasei, as described in claim 3, which is Lactobacillus paracasei CNCM 1-2116.

5. The use of lactic acid bacteria, as described in any of the preceding claims, for the preparation of an ingestible support material.

6. The use as described in claim 5, wherein the Lactobacillus strain is contained in the support material in an amount of about 10S cfu / g to about 1012 cfu / g of support material.

7. Use as described in any of claims 5 or 6, wherein the support material is a food composition that is selected from milk, yogurt, cottage cheese, cheese, fermented milks, fermented milk-based products, ice cream, products based in fermented cereal, milk-based powders and formulas for infants.

8. Use as described in any of claims 5 or 6, wherein the support material is used for the treatment or prophylaxis, or both, of disorders associated with diarrhea.

9. A food or pharmaceutical composition, containing at least one lactic acid bacterium, as described in any of claims 1 to 3.

10. The composition as described in claim 9, which is selected from milk, yogurt, cottage cheese, cheese, fermented milks, milk-based fermented products, ice cream, fermented cereal-based products, milk-based powders, infant formulas, tablets , liquid bacterial suspensions, dry oral supplement, moist oral supplement, dry tube feeding or wet tube feeding.