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Title:  Lactic acid bacteria strains capable of preventing diarrhoea United States Patent:  7,029,669
Issued: 
April 18, 2006

Inventors:
 Reniero; Roberto (Le Mont-Pelerin, CH); Bruessow; Harald (La Tour de Peilz, CH); Rochat; Florence (Montreux, CH); Von Der Weid; Thierry (Paudex, CH); Blum-Speriesen; Stephanie (Lausanne, CH)
Assignee:
 Nestec S. A. (Vevey, CH)
Appl. No.:
 936543
Filed:
 March 2, 2000
PCT Filed:
 March 2, 2000
PCT NO:
 PCT/EP00/01797
371 Date:
 January 7, 2002
102(e) Date:
 January 7, 2002
PCT PUB.NO.:
 WO00/53201
PCT PUB. Date:  September 14, 2000


 

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Abstract

Novel microorganisms of the family Lactobacillaceae, particularly microorganisms of the gnus Lactobacillus, that are useful in preventing or treating diarrhoea are provided. The microorganism can be utilized for the preparation of an ingestable support and to a composition containing same. The microorganism Lactobacillus paracasei CNCM I-2116 can be included.

SUMMARY OF INVENTION

Consequently, a problem of the present invention is to provide additional bacterial strains that exhibit new properties beneficial for man and/or animals.

The above problem has been solved by providing novel microorganisms, namely lactic acid bacteria, belonging to the genus Lactobacillus having the traits of being capable to adhere to and essentially colonize the intestinal mucosa and to prevent infection of intestinal epithelial cells by rotaviruses.

According to a preferred embodiment the Lactobacillus strains are capable to grow in the presence of up to 0.4% bile salts, so that they may easily pass the gastrointestinal tract and stay essentially active.

According to another preferred embodiment the lactic acid bacterium is selected from the group consisting of Lactobacillus rhamnosus or Lactobacillus paracasei, preferably Lactobacillus paracasei, and is more preferably Lactobacillus paracasei CNCM I-2116.

The microorganisms of the present invention have been shown to exhibit the following properties, they are gram positive, catalase negative, NH3 form arginine negative and CO2 production negative. They produce L(+) lactic acid, are capable to grow in the presence of bile salts in a concentration of up to about 0.4% and may essentially prevent infection of epithelial cells by rotaviruses.

The novel microorganisms may be used for the preparation of a variety of ingestable support materials, such as e.g. milk, yogurt, curd, fermented milks, milk based fermented products, fermented cereal based products, milk based powders, infant formulae and may be included in the support in an amount of from about 105 cfu/g to about 1011 cfu/g. For the purpose of the present invention the abbreviation cfu shall designate a "colony forming unit" that is defined as number of bacterial cells as revealed by microbiological counts 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 traits.

For preparing 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 of from about 105 cfu/g to about 1011 cfu/g, preferably from about 106 cfu/g to about 1010 cfu/g, more preferably from about 107 cfu/g to about 109 cfu/g.

In case of a pharmaceutical preparation the product may be prepared in forms of tablets, liquid bacterial suspensions, dried oral supplements, wet oral supplements, dry tube feeding or a wet tube feeding etc., with the amount of Lactobacillus strains to be incorporated therein being in the range of up to 1012 cfu/g, preferably from about 107 cfu/g to about 1011 cfu/g, more preferably from about 107 cfu 1 g to about 1010 cfu/g.

The activity of the novel microorganisms in the individual's intestine is naturally dose dependent. That is, the more the novel microorganisms are incorporated by means of ingesting the above food material or the pharmaceutical composition the higher the protective and/or curing activity of the microorganisms. Since the novel micro-organisms are not detrimental to mankind and animals and have eventually been isolated from baby feces a high amount thereof may be incorporated so that essentially a high proportion of the individuals intestine will be colonized by the novel microorganisms.

DETAILED DESCRIPTION OF THE INVENTION

During the extensive studies leading to the present invention the inventors have investigated baby feces and isolated a variety of different bacterial strains therefrom. These strains were subsequently examined for their capability to prevent infection of epithelial cells with rotaviruses that are known to cause diarrhoea.

Several bacterial genera comprising Lactobacillus, Lactococcus, Streptococcus were screened for their rotavirus inhibitory properties. The tests for the inhibitory property were essentially performed with three rotavirus serotypes representing the major etiological agents of human viral diarrhoea (serotypes G1, G3 and G4).

The various lactic acid bacteria were grown in a suitable medium, such as MRS, Hugo-Jago or M17 medium at temperatures of from about 30 to 40 C. corresponding to their optimal growth temperature. After reaching stationary growth the bacteria were collected by centrifugation and resuspended in physiological NaCl solution. Between the different tests the bacterial cells were stored frozen (-20 C.).

The various rotavirus stocks were prepared by infection of confluent cell monolayers. The rotaviruses were incubated before infection. The cells were infected with 20 tissue culture infectious doses.

For assessing anti-rotaviral properties two different protocols were applied. According to one protocol the various bacterial strains were examined for their direct interaction with the rotavirus while in the second protocol the bacteria were screened for those strains that interact with cellular rotavirus receptors.

The first protocol involved contacting the respective bacterial suspension each with a different rotavirus strain and incubating in suitable media. Subsequently, the virus-bacterium mixture was applied to a monolayer of cells of the human undifferentiated colon adenoma cells HT-29 and incubation was continued. Virus replication was then assayed.

The second protocol involved incubating the respective bacterial suspension first together with a monolayer of cells of the human undifferentiated colon adenoma cells HT-29 and adding the virus subsequently. After continued incubation virus replication was assayed.

Rotavirus replication may easily be assessed by histo-immunological staining of rotavirus proteins in infected cells.

A rotavirus inhibitory effect was attributed to a given bacterium when the number of infected cells was reduced by 90% in the cell culture inoculated with rotavirus plus the indicated bacteria in comparison with cells inoculated only with rotavirus.

Out of a total of 260 different bacterial strains primarily isolated merely 9 could be shown to essentially inhibit rotaviral replication. The different bacteria were ascertained to belong to the genus Lactobacillus subspecies rhamnosus or paracasei. One strain, termed Lactobacillus casei ST 11, that has been deposited in accordance with the Budapest Treaty and has received the deposit numbers NCC 2461 (I-2116), has been shown to be extremely effective in preventing infection of human cells by rotavirus. Moreover, this particular strain shows excellent growing properties as may be shown by acidification in different media. The strain also shows good performance as regards the survival rate during storage at low temperatures of about 10 C., which makes it an excellent candidate for being included in food stuff or pharmaceutical compositions to be stored at refrigerator conditions.

In addition to the above finding it could also be shown that the strains surprisingly also exhibit anti-allergenic properties in that said strains have an impact on the synthesis of different immunological mediators.

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

The differentiation of Th2 cells is impaired by IFN-γ, a particular cytokine that arises from the mutually exclusive Th1 subset of CD4+ T cells. Said Th1 cells are in turn strongly induced by interleukin 12 (IL-12). In contrast thereto IL-10, another cytokine, has been shown to have a strong suppressing impact on the proliferation of Th1 cells and is therefore deemed to play a role in immunosupressive mechanisms.

In summary, both IL-12 and IL-10 have strong modulatory effects on CD4+ T cell development by influencing the development of the Th1 subset. IL-12 is a key regulatory cytokine for the induction of Th1 differentiation and thus inhibits the generation of Th2 responses. A major pathway for inhibition of Th2 cells is therefore seen 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 levels of IL-12 in adherent cells, such as macrophages and dendritic cells. Consistently, it has been found that gram negative bacteria can strongly bias CD4+ T cell differentiation towards the Th1 phenotype.

The microorganism ST11 as an example of the Lactobacillus strains of the present invention has been tested for a potential role in the induction of cytokines involved in the regulation of CD4+ T cell differentiation. In particular, the effect of ST11 on the phenotype of CD4+ T cells undergoing Th2 differentiation has been studied.

In this respect the capacity of ST11 to induce the synthesis of mRNA encoding these two regulatory cytokines in mouse adherent cells derived from bone marrow was compared with 4 other strains of Lactobacilli and with a control of gram negative bacteria (E. coli K12). The mRNA was measured by semi-quantitative RT-PCR after 6 hours of incubation of the cells with serial dilutions of bacteria ranging from 10′ to 107 cfu/ml.

Although all strains of Lactobacillus could induce transcription of IL-12 mRNA to a certain degree, ST11 could be shown to be the strongest inducer, since as a strong PCR signal could be detected even at the lowest bacterial dose. In fact, the capacity of ST11 to induce IL-12 mRNA transcription was as strong as that of E. coli. Induction of IL-10 mRNA was in general weaker than for IL-12 mRNA, as only at higher bacterial doses a signal could be detected. Nevertheless, ST11 was the strongest inducer of IL-10 mRNA, as compared to the other lactobactilli and the E. coli control.

Thus, ST11 is deemed to be efficient in inducing immunoregulatory cytokines involved in CD4+ T cell differentiation. Its strong capacity to induce IL-12 makes it a candidate to inhibit Th2 responses and its measurable IL-10 induction may prevent inflammatory responses.

In addition to the above finding it was also determined whether ST 11 had an inhibitory effect on CD4+ T cells undergoing Th2 differentiation and a positive effect on Th1 functions. A well established cell differentiation culture system was utilized, where precursor CD4+ T cells were polyclonally activated and modulated to undergo either Th1 or Th2 differentiation, depending on the type of co-stimuli provided in the culture medium. Th1/Th2 differentiation was induced during a 7-days primary culture, after which the cells were then restimulated for 2 days in a secondary culture containing medium alone and acquisition of a specific phenotype (Th1 or Th2) was assessed by measuring the types of cytokines produced in the supernatant (IFN-γ vs.

IL-4).

It is known that precursor CD4+ T cells from mice of the BALB/c background preferentially differentiate to predominant Th2 phenotype (high IL-4, low IFN-γ in the 2ry culture supernatants) after activation under neutral conditions (medium alone in the 1ry culture). This phenotype could be completely reverted to a Th1 pattern (high IFN-γ, low IL-4) upon addition of a blocking monoclonal antibody to IL-4 in the 1ry culture.

To investigate a potential role for ST11 on Th2 inhibition, purified precursor CD4+ T cells from BALB/c mice were activated in the presence of bone marrow adherent cells as accessory cells during the 1ry culture. These cells were co-cultured either in medium alone, or in the presence of 1 mg/ml LPS, or 108 cfu/ml ST11, or 108 cfu/ml of another Lactobacillus. After this time, the cells were washed and CD4+ T cells were purified once again and restimulated in the 2ry culture in medium alone. Cytokines produced by the differentiated CD4+ T cells were measured after 2 days. As expected, cells differentiated in the presence of medium alone displayed a dominant Th2 phenotype. Addition of ST11 to the 1ry cultures strongly modulated the outcome of Th2 differentiation, as it resulted in an 8-fold decrease in IL-4 production. This inhibition was of similar magnitude as that observed in cultures derived from cells differentiated in the presence of LPS. In contrast, the other Lactobacillus strain had no measurable impact on IL-4 levels. Interestingly, IFN-7 levels were not increased upon addition of ST11 in the 1ry cultures.

In summary, ST11 specifically impaired IL-4 production by CD4+ T cells undergoing Th2 differentiation, but did not significantly increase IFN-γ secretion. The fact that ST11 does not increase IFN-γ production may be due to its capacity to induce IL-10 with the consequence that it may keep a low inflammatory impact despite its anti-Th2 activity.

In consequence, it could be shown that ST11 is a Lactobacillus strain that has a good anti-Th2 profile which makes it an excellent candidate for its use as a bacterium with anti-allergic, probiotic activity.
 


Claim 1 of 10 Claims

1. A composition comprising a biologically pure culture of lactic acid bacterium strain belonging to a genus Lactobacillus capable of adhering to and essentially colonizing an intestinal mucosa and capable of preventing infection of intestinal epithelial cells by rotaviruses, wherein the lactic acid bacterium strain is Lactobacillus paracasei CNCM I-2116.

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