Title: Lactic acid bacteria
strains capable of preventing diarrhoea United States Patent:
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,
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,
PCT PUB.NO.: WO00/53201
PCT PUB. Date:
September 14, 2000
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
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
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
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
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.
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
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.
If you want to learn more
about this patent, please go directly to the U.S.
Patent and Trademark Office Web site to access the full