U.S. patent application number 10/609195 was filed with the patent office on 2004-03-25 for probiotic strains, a process for the selection of them, compositions thereof, and their use.
Invention is credited to Boza Puerta, Julio, Jimenez Lopez, Jesus, Martin Jimenez, Rocio, Rodriguez Gomez, Juan Miguel, Xaus Pey, Jordi.
Application Number | 20040057943 10/609195 |
Document ID | / |
Family ID | 29797105 |
Filed Date | 2004-03-25 |
United States Patent
Application |
20040057943 |
Kind Code |
A1 |
Xaus Pey, Jordi ; et
al. |
March 25, 2004 |
Probiotic strains, a process for the selection of them,
compositions thereof, and their use
Abstract
The present invention relates to a novel process for the
selection of new probiotic strains which comprises the following
steps: a) selecting for non-pathogenic strains which are capable of
surviving in breast milk and/or amniotic fluid, and b) selecting
for non-pathogenic strains which are able to be transferred to
breast milk and/or amniotic fluid after oral intake in healthy
individuals without colonizing other internal organs except
mucousas. The invention also provides new Lactobacillus strains,
which are: CECT5711 (Lactobacillus coryniformis), CECT5713
(Lactobacillus salivarius subsp. salivarius), CECT5714:
(Lactobacillus gasseri, formerly L. acidophilus), CETC5715:
(Lactobacillus gasseri), and CECT5716: (Lactobacillus fermentum);
and refers to their use for the prophylaxis or treatment against
digestive, infective, neuro-degenerative and immune related
diseases such as allergies or inflammatory diseases.
Inventors: |
Xaus Pey, Jordi; (Granada,
ES) ; Martin Jimenez, Rocio; (Granada, ES) ;
Rodriguez Gomez, Juan Miguel; (Madrid, ES) ; Boza
Puerta, Julio; (Granada, ES) ; Jimenez Lopez,
Jesus; (Granada, ES) |
Correspondence
Address: |
KATTEN MUCHIN ZAVIS ROSENMAN
575 MADISON AVENUE
NEW YORK
NY
10022-2585
US
|
Family ID: |
29797105 |
Appl. No.: |
10/609195 |
Filed: |
June 26, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10609195 |
Jun 26, 2003 |
|
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PCT/EP02/07169 |
Jun 28, 2002 |
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Current U.S.
Class: |
424/93.45 ;
435/252.3 |
Current CPC
Class: |
A23Y 2220/77 20130101;
A23L 33/135 20160801; A23C 9/1234 20130101; A23L 29/065 20160801;
A61P 25/00 20180101; A61P 31/12 20180101; A23C 9/1232 20130101;
A61P 11/06 20180101; A61P 9/10 20180101; A61P 37/04 20180101; A61P
25/28 20180101; C12R 2001/25 20210501; A61P 37/02 20180101; A61P
37/08 20180101; A23C 9/206 20130101; A61P 1/10 20180101; A61P 31/04
20180101; A61P 35/00 20180101; A61K 35/747 20130101; A61P 25/16
20180101; A61P 33/00 20180101; A61P 29/00 20180101; A61K 35/744
20130101; A61P 25/14 20180101; A61K 35/745 20130101; A61P 3/00
20180101; Y10S 435/853 20130101; A61P 17/06 20180101; A61P 19/02
20180101; A61P 31/10 20180101; A61P 1/04 20180101; C12N 1/205
20210501; C12R 2001/225 20210501 |
Class at
Publication: |
424/093.45 ;
435/252.3 |
International
Class: |
A61K 045/00; C12N
001/20 |
Claims
1. A method for the selection of probiotic microbial strains,
comprising the following steps: a. selecting for non-pathogenic
strains which are capable of surviving in breast milk and/or
amniotic fluid, and b. selecting for non-pathogenic strains which
are able to be transferred to breast milk and/or amniotic fluid
after oral intake in healthy individuals without colonizing other
internal organs except mucousas.
2. A method according to claim 1, wherein both breast milk and
amniotic fluid are from human sources.
3. A method as claimed in claim 2, wherein the probiotic tested
strains are any lactic acid bacteria selected from the genera:
Lactobacillus, Lactococcus, Leuconostoc, Enterococcus,
Streptococcus and Bifidobacterium.
4. A method according to claims 3, wherein the probiotic strains
tested had previously been obtained from: breast milk, feces of
breastfed babies or amniotic fluid.
5. A method according to claim 4, wherein the strains tested had
been obtained from human samples.
6. A bacterial strain selected by a method according to any of
claims 1 to 5
7. A strain of bacteria deposited in the CECT under Accession
N.sup.o 5711 (Lactobacillus coryniformis) or a variant thereof.
8. A strain of bacteria deposited in the CECT under Accession
N.sup.o 5713 (Lactobacillus salivarius subsp. salivarius) or a
variant thereof.
9. A strain of bacteria deposited in the CECT under Accession
N.sup.o 5714 (Lactobacillus gasseri, formerly L. acidophilus) or a
variant thereof.
10. A strain of bacteria deposited in the CECT under Accession
N.sup.o 5715 (Lactobacillus gasseri) or a variant thereof.
11. A strain of bacteria deposited in the CECT under Accession
Nu.sup.o 5716 (Lactobacillus fermentum) or a variant thereof.
12. A biologically pure culture of a strain according to any of
claims 6-11.
13. Use of mammal milk and of mammal amniotic fluid as a source to
obtain non-pathogenic probiotic bacteria, especially probiotic
bacteria selected from the genera Lactobacillus, Lactococcus,
Enterococcus, Streptococcus and Bifidobacterium
14. Use of mammal milk and of mammal amniotic fluid according to
claim 13 where the mammal milk and the mammal amniotic fluid are
human.
15. Use of mammal milk and of mammal amniotic fluid according to
claim 14 as a source to obtain the microbial strains defined in any
of the claims 6 to 11.
16. A composition comprising at least one of the bacterial strains
defined in claims 6 to 11, where the composition comprises
preferably from 2 to 6 strains, more preferably from 2 to 4
strains, most preferably from 2 to 3 strains, and where each of the
strains is present in the composition in a proportion from 0.1% to
99.9%, preferably from 1% to 99%, more preferably from 10% to
90%.
17. A composition comprising at least one of the bacterial strains
defined in claims 6 to 11 together with another strain or mixture
of strains where the mixture comprises preferably from 2 to 6
strains, more preferably from 2 to 4 strains, most preferably from
2 to 3 strains and where each of the strains is present in the
composition in a proportion from 0.1% to 99.9%, preferably from 1%
to 99%, more preferably from 10% to 90%.
18. A composition comprising a strain as claimed in any one of
claims 6 to 11 or a composition as defined in any of the claims 16
to 17 in a lyophilized form.
19. A composition comprising a strain as claimed in any one of
claims 6 to 11 or a composition as defined in any of the claims 16
to 17 in a frost form.
20. A composition comprising a strain as claimed in any one of
claims 6 to 11 or a composition as defined in any of the claims 16
to 17 in an inactivated form or dead.
21. A composition obtainable from the supernatant of a culture of a
bacterial strain according to any of the claims 6 to 11, or from a
composition according to any of the claims 16 to 17.
22. A composition obtainable by extraction of a culture of any of
the bacterial strains according to claims 6 to 11, or by extraction
of a composition according to claims 16 to 17.
23. A product obtainable from the metabolic activity of any of the
strains specified in claims 6 to 11, from a culture according to
claim 12 or from a composition according to claims 16 to 17,
wherein the product is preferably an enzyme.
24. Food product comprising support material and at least one
strain according to any of the claims 6 to 11, a culture, a
composition or a product according to claims 12 and 16 to 24.
25. A product according to claim 24 wherein the support material is
a food composition selected from milk, yoghourt, curd, cheese,
fermented milks, milk based fermented products, meat based
fermented products, fermented cereals based products, milk based
powders, cereal based powders, infant formulae, clinical nutrition
formula, ice-creams, juices, flours, bread, cakes, sugar, candies
or chewing-gums.
26. A product according to claim 24, wherein the microbial strain
according to claims 6 to 11 is contained in the support material in
an amount from about 10.sup.5 cfu/g to about 10.sup.12 cfu/g
support material, preferably from about 10.sup.6 cfu/g to about
10.sup.11 cfu/g support material, more preferably from about
10.sup.6 cfu/g to about 10.sup.10 cfu/g support material.
27. A pharmaceutical composition comprising at least one strain
according to any of the claims 6 to 11, a culture, a composition or
a product according to claims 12 and 16 to 26 and pharmaceutically
acceptable excipients.
28. A pharmaceutical composition according to claim 27 wherein the
microbial strains are contained in an amount from 10.sup.5 cfu/g to
10.sup.14 cfu/g support material, preferably from 10.sup.6 cfu/g to
10.sup.13 cfu/g support material, more preferably from 10.sup.7
cfu/g to 10.sup.12 cfu/g support material.
29. A strain of bacteria as claimed in anyone of claims 6 to 11, a
culture, a composition or product according to claims 12 and 16 to
26 for therapeutic or prophylactic treatment.
30. A composition according to claim 29 for topic, oral, ocular,
nasal, enteral, urogenital, vaginal or rectal administration
31. A composition according to claim 29 designed to be administered
to pregnant woman for the therapeutic or prophylactic treatment of
their foetus.
32. A composition according to claim 29 designed to be administered
to lactating woman for the therapeutic or prophylactic treatment of
their breastfed babies.
33. Use of a strain according to any of the claims 6 to 11, a
culture, a composition or a product according to claims 12 and 16
to 27 in the manufacture of a product for the therapeutic or
prophylactic treatment of human and animal diseases.
34. Use according to claim 33 for the treatment and/or prophylaxis
of chronic or acute infection, or of undesirable microbial
colonization, wherein the infection or colonization is caused by
parasites, bacteria, yeasts, fungi or viruses, of a mucosal surface
in a subject or animal in need thereof, wherein the mucosal surface
is selected from but not restricted to the group consisting of
oral, nasopharyngeal, respiratory, gastric, intestinal, urogenital
and glandular.
35. Use according to claim 33 for the treatment and/or prophylaxis
of temporally depressed immune levels in individuals subjected to
physiological stress.
36. Use according to claim 33 for the improvement of the immune gut
barrier in a subject or animal in need thereof; for the treatment
and/or prophylaxis of down-regulating hypersensitivity reactions to
food and metabolic intolerance such as: lactose intolerance; of
constipation and other gastro-intestinal disorders; of inflammatory
or auto-immune disorders such as: 113D, ulcerative colitis,
arthritis, atherosclerosis, multiple sclerosis, psoriasis or
sarcoidosis; and of tumor growth, metastasis and cancer in a
subject or animal in need thereof.
37. Use according to claim 33 for the treatment and/or prophylaxis
of allergic disorders and asthma in a subject or animal in need
thereof.
38. Use according to claim 33 for the treatment and/or prophylaxis
of neuro-degenerative diseases in an individual or animal in need
thereof, selected from, but not restricted to, the group consisting
of Parkinson, stroke, Alzheimer, Huntington and dementia.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to a novel process for the
selection of new probiotic strains, to new probiotic microorganisms
of the genus Lactobacillus selected according to this method and to
compositions comprising these microorganisms; to their use for the
prophylaxis or treatment against digestive, infective,
neuro-degenerative and immune related diseases such as allergies or
inflammatory diseases, and to a novel source to obtain said
microorganisms.
BACKGROUND OF THE INVENTION
[0002] For many years, lactic acid bacteria have been utilized as
fermenting agents for the preservation of food taking benefit of a
low pH and the action of fermentation products generated during the
fermentative activity thereof to inhibit the growth of spoilage
bacteria. With this aim, non-well characterized lactic acid
bacteria or "fermentum" have been used to prepare a variety of
different foodstuffs such as dry fermented meat products, cheese,
and other fermented dairy products from milk.
[0003] Recently, lactic acid bacteria have attracted a great deal
of attention because some strains have been found to exhibit
valuable properties to man and animals upon ingestion. In
particular, specific strains of the genus Lactobacillus or
Bifidobacterium have been found to be able to colonize the
intestinal mucosa and to assist in the maintenance of the
well-being of man and animal, and has been named such as
probiotics.
[0004] Probiotics are considered to be viable microbial
preparations which promote the individual's health by preserving a
healthier microflora in the intestine. A microbial preparation may
be commonly accepted as a probiotic in case the effectual thereof
and their mode of action are known. Probiotics are deemed to attach
to the intestine's mucosa, colonize the intestinal tract and
likewise prevent attachment of harmful microorganisms thereon. A
crucial prerequisite for their action resides in that they have to
reach the gut's mucosa in a proper and viable form and do not get
destroyed in the upper part of the gastrointestinal tract,
especially by the influence of the low pH prevailing in the
stomach.
[0005] During the extensive studies leading to new probiotic
strains, previous patent applications have described the isolation
of variety of different bacterial strains from baby feces
(JP04320642, JP05227946). Moreover, the probiotic strains obtained
up to now were mainly selected for their capability to adhere to
the intestinal mucosa, usually by in vitro experiments. Subsequent
selection has not always been performed, and if it has, it used to
be mainly based on individual properties of the strain. Finally,
and sometimes after the commercialization of the strain, the
beneficial effects of the selected strain have been proven in
vivo.
[0006] In this regard, several patent applications such as eg.
EP0768375, WO97/00078, EP0577903 and WO00/53200 disclose specific
strains of Bifidibacterium and Lactobacillus and their beneficial
effects on diarrhea, immunomodulation, hypersensitivity reactions
or infection by pathogen microorganisms.
[0007] Moreover, the beneficial effects of human breast milk on the
well-being of infants compared to those fed with milk-based formula
has also been extensively reported. In this regard, a reduction in
the risk of infection, allergy, asthma and related affections, and
an improvement of the intestinal maturation and gut functions has
been described. Also, it has been reported that the composition of
the gut flora is different to human-milk fed infants from those fed
with milk-based formula. The beneficial effects and the modulation
of the gut flora of breast human milk have been attributed to its
characteristic composition as compared to infant formulas. Thus,
the benefits of breast milk proteins such as lactoferrin or
maternal immunoglobulins, and the rich composition in
oligosaccharides that may act as prebiotic compounds, in the
regulation of the flora and gut functions have been reported.
[0008] However, to our knowledge there is no publication or work
that describes the presence of microbial strains in normal human
breast milk. Neither it has been reported that such microbial
strains could be beneficial for the breast-fed baby, and therefore
acting as probiotics modulating the gut flora of the breast feeding
infant. Our work suggests that the well-being effects of the human
breast-fed could be also mediated by microbial strains present
therein.
[0009] It has been suggested in several works that the initial
colonization of the neonate is due to cross-contamination with
vaginal microflora during labor. However, there are several studies
that show similar initial microbial colonization of the neonate
independently of the neonate delivery route (cesarean versus
natural labor). Moreover, the fact that it is not possible to
obtain germ-free animals from conventional pregnant mice
nevertheless they have been obtained by cesarean, and that these
animals could also be obtained after embryo delivery to sterile
recipient mice (Okamoto, M. and Matsumoto, T. 1999. Exp. Anim. 48:
59-62), suggest to us that it has to be other mechanisms than
vaginal contamination that also influences the initial colonization
of the neonate, and that this mechanism has to begin before
labor.
[0010] In this regard, to our knowledge, there is no publication or
work describing the presence of lactic acid bacterial strains in
normal human amniotic fluid. Neither it has been reported that such
non-pathogenic microbial strains could be beneficial for the
gestating baby, and therefore conditioning, just during the
gestation, the initial microbial populations able to colonize the
fetal gut.
[0011] In understanding the valuable properties that particular
strains of lactic acid bacteria may provide, there is a desire in
the art for additional lactic acid bacterial strains that are
beneficial to the well being of man and/or animal. Consequently, a
problem of the prior art was to provide rational methods for the
selection of additional new bacterial strains and novel sources for
the selection of them, that allow the obtention of bacterial
strains which exhibit individually a high number of beneficial
properties for man and/or animals. The above problem has been
solved by providing novel microorganisms, namely lactic acid
bacteria, belonging to the genus Lactobacillus.
[0012] These new strains have been obtained from different sources
apart from feces, such as goat cheese and from human breast milk
and amniotic fluid, and have been chosen by a method consisting in
the ability of these strains to survive in breast milk and/or
amniotic fluid, and by their ability to be transferred to breast
milk and/or amniotic fluid after oral intake.
[0013] This selection method ensures that the bacterial strains
obtained have implicitly most of the characteristics attributed to
a potential probiotic strain, namely good resistance to digestion
process and the ability of gut colonization, but also a more
natural human origin, safety aspects, and the ability to colonize
and regulate some human niches other than the gut. Finally, the
selected strains have also been tested not only for their adhesion
capabilities but for having a high degree of beneficial
characteristics.
BRIEF DESCRIPTION OF THE FIGURES
[0014] FIG. 1. Screening method: Survival of bacteria into breast
milk and amniotic fluid.
[0015] This figure illustrates the survival rate of the selected
probiotic strains of this invention in breast milk and amniotic
fluid. According to the selection method, the survival of the
potential probiotic strains was measured using human breast milk
(grey bars) and amniotic fluid (black bars). 10.sup.8 cfu of any
candidate bacterial strain were resuspended in 1 ml of MRS or
breast milk or amniotic fluid, and incubated for 1 hour at
37.degree. C. in anerobic conditions. Survival after culture was
assessed cultivating serial dilutions in MRS agar plates. The
results are represented as the mean.+-.SD of three independent
experiments. Those candidate bacteria that survive more than 75% in
at least one of human fluids were initially selected.
[0016] FIG. 2. Screening method: Transfer of bacteria to breast
milk.
[0017] This figure illustrates the transfer of selected bacteria to
breast milk after oral intake. A) Labelling of the strains. Those
strains that survive in breast milk and/or amniotic fluid were
genetically labeled using a PCR-detectable construction. Different
strains are labeled with DNA fragments of different sizes (159
bp:F159; 189 bp: F189; and 228 bp: F228). The PCR signal is only
detectable in the genetically labeled strains. The figure shows as
example the labeling of the bacterial strains CECT5711, CECT5713
and CECT5714. B) Transfer of selected bacteria to breast milk. The
capability of the labeled strains to be transferred to breast milk
and/or amniotic fluid was evaluated using pregnant mice as an
animal model. Pregnant mice were inoculated every two days with
10.sup.8 cfu/mice (in this figure we showed results of L.
salivarius CECT5713) for two weeks before labor. Presence of
bacteria in the milk was detected indirectly comparing the number
of bacteria PCR-detectable in the gut of neonates before and just
after lactating for the first time. Lane 1 in each panel
corresponds to the molecular weight marker.
[0018] FIG. 3. RAPD profile of the selected bacteria.
[0019] This figure illustrates the RAPD profiles of the 5 selected
probiotic strains of this invention using two different primers
(Argdei and OPL5). Lanes 1, 8 and 15 correspond to the molecular
weight marker, lanes 7 and 14 are the negative controls.
[0020] FIG. 4. Comparison of the RAPD profile of the selected
bacteria with other bacteria of the same species.
[0021] This figure illustrates the differences in RAPD profiles
observed between the selected probiotic strains of this invention
and those obtained with other bacterial strains of the same
species. Each specie is represented in one of the panels: L.
coryniformis (A), L. salivarius (B), L. gasseri (C) and L.
fermentum (D). The strains used are described in Table III.
[0022] FIG. 5. Adhesion of probiotic strains to intestinal
cells.
[0023] This figure illustrate the adhesin of probiotic strains to
intestinal cells. The adhesion of the probiotic strains of this
invention were assessed using Caco-2 (grey bars) or HT-29 (black
bars) intestinal cell lines and compared to commercial probiotic
strains. Twenty randomized fields were counted and the results
expressed as the mean of the number of bacteria attached to the
cells per field.+-.SD. The adhesion capability of a probiotic
strain to each intestinal cell line was considered high if the
number of attached bacteria was >250, moderate between 100 and
250, and slight >100.
[0024] FIG. 6. Survival of probiotics strains to digestion
condition.
[0025] This figure illustrates the survival of probiotic strains to
digestion conditions. The resistance of the probiotic strains of
this invention to acidic (grey bars) and high bile salt content
(black bars) was assessed in vitro by culture of bacteria in MRS pH
3.0 or 0.15% bile salts for 90 minutes. The results are represented
as the mean.+-.SD of three independent experiments. The resistance
of a probiotic strain was considered high if the survival was
>80%, moderate between 60% and 80%, and slight >60%.
[0026] FIG. 7. Generation time of probiotic strains.
[0027] This figure illustrates the generation time of probiotic
strains. The time of generation of the probiotic strains of this
invention was assessed in vitro by cultivating bacteria in MRS 0.2%
glucose for 120 minutes. The results are represented in minutes and
as the mean.+-.SD of three independent experiments. The generation
time of a probiotic strain was considered rapid if the time was
<60, moderate between 60 and 120, and slow >120 minutes.
[0028] FIG. 8. Fermentation capabilities of probiotic strains
[0029] This figure illustrates the ability of probiotic strains to
fermentate complex carbohydrates. The fermentation capabilities of
the probiotic strains of this invention to use complex
carbohydrates as an unique source of carbohydrates was assessed in
vitro by cultivating bacteria in MRS without glucose and
supplemented with 2% of indicated carbohydrates for 24 and 48
hours. Reduction of the pH was assessed using bromcresol purple.
The results are represented as the fold-induction in absorbance
after 24 hours compared with a control culture without carbohydrate
source (A) and the sum of all independent fold-induction values
(B). The fermentation capability of a probiotic strain was
considered high if the total value was >30, moderate between 25
and 30, and slight <25.
[0030] FIG. 9. Resistance to antibiotic of probiotic strains.
[0031] This figure illustrates the resistance to antibiotic of
probiotic strains. The resistance of antibiotic treatment of the
probiotic strains of this invention was assessed in vitro by an
agar well diffusion assay in Mueller-Hinton plates for 24-48 hours.
The diameter of the hallo of inhibition determines the antibiotic
effect. The results are represented as R (resistant) if the hallo
has a diameter <12 mm, I (intermediate) from 12 to 15 mm, and S
(sensible) if>15 mm. After that, a numerical value was assigned
to each condition: R=3, I=2, and S=1. The resistance capability of
a probiotic strain was considered high if the total value was
>17, moderate between 15 and 17, and slight <15.
[0032] FIG. 10. Acid production by the probiotic strains.
[0033] This figure illustrates the acid production by the probiotic
strains. The production of acid (lactic, propionic, acetic and
butyric acid) by the probiotic strains of this invention was
assessed in vitro by the measurement of the pH in milk cultures for
24 (grey bars) and 48 (black bars) hours. The production of acid by
a probiotic strain was considered high if the milk pH value after
48 hours was <4, moderate between 4 and 4.5, and slight
>4.5.
[0034] FIG. 11. Production of bactericide metabolites by the
probiotic strains.
[0035] This figure illustrates the production of antimicrobial
metabolites by the probiotic strains. The production of
antimicrobial metabolites by the probiotic strains of this
invention was assessed in vitro by an agar well diffusion assay in
TSA plates cultured with S. typhimuriumi (black bars) or
Escherichia coli (grey bars). The diameter of the hallo (in
millimeters) of inhibition induced by the bacterial supernatants
determines the bactericide effect. The antimicrobial capability of
a probiotic strain was considered high if the hallo was >12,
moderate between 8 and 12, and slight <8 for both pathogenic
strains.
[0036] FIG. 12. Inhibition of the adhesion of pathogenic
bacteria.
[0037] This figure illustrates the inhibition of the adhesion of
pathogenic bacteria. The adhesion of the pathogenic strains E. coli
(grey bars) and S. typhimurium (black bars) to Caco-2 cells was
assessed in the presence of the probiotic strains of this invention
and compared to commercial probiotic strains. Ten randomized fields
were counted and the results expressed as the mean of the % of
adhered gram-negative bacteria attached to the cells compared to
the number of pathogenic bacteria adhered in absence of probiotics.
The capability of a probiotic strain to inhibit pathogenic bacteria
adherence was considered high if the % of both attached pathogenic
bacteria was <25, moderate between 25 and 75, and slight
>75.
[0038] FIG. 13. Gut colonization by L. Salivarius CECT5713.
[0039] This figure illustrates the gut colonization by L.
salivarius CECT5713. The number of fecal lactobacillus,
bifidobacteria and coliform bacteria in mice supplemented daily for
14 days with 10.sup.8 cfu of L. salivarius CECT5713 was analyzed by
bacterial platting. Fecal samples (200 mg aprox) were collected at
day 0, 7 and 14 of probiotic supplementation and also one and two
weeks (day 21 and 28) after supplementation was terminated.
(*p<0.05; **p<0.01).
[0040] FIG. 14. Effect of L. Fermentum CECT5716 on Salmonella
infection.
[0041] This figure illustrates the effect of L. fermentum CECT5716
on Salmonella infection. A) L. fermentum CECT5716 inhibits
Salmonella translocation to the spleen. The number of Salmonella
colonies was measured in the spleens of mice treated with L.
fermentum CECT5716 with or without vaccination with 108 inactivated
cfu of Salnonella after 24 hour of an oral challenge with 10.sup.10
cfu Salmonella. B) The same mice were used to measure the IgA
content in feces.
[0042] FIG. 15. Effect of probiotic strains on cytokine
expression.
[0043] This figure illustrates the effect of probiotic strains on
cytokine expression. The TNF-.alpha. (A) ot IL-10 (B) cytokine
production was analyzed in bone marrow derived macrophages
stimulated with LPS and the indicated probiotic strain for 12
hours. Cytokine production was detected by an ELISA technique.
[0044] FIG. 16. Effect of probiotic strains on Ig G expression.
[0045] This figure illustrates the effect of probiotic strains on
Ig G expression. The IgG production was analyzed in lymphocytes
obtained from the spleen of Balb/c mice (6-8 weeks old) stimulated
with LPS and the indicated probiotic strain for 6 days.
Immunoglobulin production was detected by an ELISA technique from
Bethyl.
SUMMARY OF THE INVENTION
[0046] The present invention provides, therefore, a method for the
selection of probiotic microbial strains, comprising the following
steps:
[0047] a. selecting for non-pathogenic strains which are capable of
surviving in breast milk and/or amniotic fluid, and
[0048] b. selecting for non-pathogenic strains which are able to be
transferred to breast milk and/or amniotic fluid after oral intake
in healthy individuals without colonizing other internal organs
except mucousas.
[0049] In a further aspect, the invention provides new
Lactobacillus strains, which are:
[0050] CECT5711 (Lactobacillus coryniformis),
[0051] CECT5713 (Lactobacillus salivarius subsp. salivarius),
[0052] CECT5714: (Lactobacillus gasseri, formerly L.
acidophilus),
[0053] CETC5715: (Lactobacillus gasseri), and
[0054] CECT5716: (Lactobacillus fermentum).
[0055] A further aspect of the invention relates to the use of
mammal milk and mammal amniotic fluid as a source to obtain
non-pathogenic probiotic bacteria.
[0056] Another aspect of the invention relates to compositions and
products contaning at least one of the strains mentioned above.
[0057] Finally, a last aspect of the invention relates to the use
of the strains mentioned above or of any culture, composition or
product containing them in the manufacture of a product for the
therapeutic or prophylactic treament of human and animal
diseases.
DETAILED DESCRIPTION OF THE INVENTION
[0058] As mentioned above, the present invention provides a method
of selection of new bacterial strains consisting in the ability of
these strains to survive in breast milk and/or amniotic fluid, and
by their ability to be transferred to breast milk and/or amniotic
fluid after oral intake, which ensures the special characteristics
of the selected strains obtained with it. Thus, the main aspect of
the present invention is defined as a method for the selection of
probiotic microbial strains, comprising the following steps:
[0059] a. selecting for non-pathogenic strains which are capable of
surviving in breast milk and/or amniotic fluid, and
[0060] b. selecting for non-pathogenic strains which are able to be
transferred to breast milk and/or amniotic fluid after oral intake
in healthy individuals without colonizing other internal organs
except mucousas.
[0061] According to a preferred embodiment of the invention, both
breast milk and amniotic fluid are from human sources. The
probiotic tested strains in the method of the invention can be any
probiotic bacteria selected from, but not restricted to, the genera
Lactobacillus, Lactococcus, Leuconostoc, Enterococcus,
Streptococcus and Bifidobacterium. These probiotic strains are
preferably obtained from breast milk, feces of breastfed babies or
amniotic fluid, most preferably from human samples. Further details
of the method of the invention are given below in "Method and
Examples".
[0062] In a further aspect, the present invention provides any
bacterial strain selected by the method of the invention. Some of
these new bacterial strains which exhibit a number of
characteristics which render them beneficial to human health, and
in particular in the prophylaxis or treatment against digestive,
infective, neuro-degenerative and other immune related diseases
such as allergies or inflammatory diseases, have been deposited
according to the Budapest Agreement at the CECT--Coleccin Espaola
de Cultivos Tipo-, Valencia (Spain) on Jun. 11, 2002. These
bacterial strains and their characteristics are:
[0063] CECT5711: (Lactobacillus coryniformis), said bacteria being
obtained from goat cheese, selected by the proposed process, and
characterized by the RAPD profile showed in FIG. 3 and the features
described in Table I, II and VIII.
[0064] CECT5713: (Lactobacillus salivarius subsp. salivarius), said
bacteria being obtained from human breast-fed baby feces, selected
by the proposed process, and characterized by the RAPD profile
showed in FIG. 3 and the features described in Table I, II and to
VIII.
[0065] CECT5714: (Lactobacillus gasseri, formerly L. acidophilus),
said bacteria being obtained from human breast milk, detected also
in human amniotic fluid, selected by the proposed process, and
characterized by the RAPD profile showed in FIG. 3 and the features
described in Table I, II and VIII.
[0066] CECT5715: (Lactobacillus gasseri), said bacteria being
obtained from human breast milk, detected also in human amniotic
fluid, selected by the proposed process, and characterized by the
RAPD profile showed in FIG. 3 and the features described in Table
I, II and VIII.
[0067] CECT5716: (Lactobacillus fermentum), said bacteria being
obtained from human breast milk, selected by the proposed process,
and characterized by the RAPD profile showed in FIG. 3 and the
features described in Table I, II and VIII.
[0068] Moreover we have also confirmed that by applying the method
of the invention, it is possible to select new specific strains as
the mentioned above. In this sense, we have compared genetic (RAPD
profiles) and biochemical aspects such as fermentation capabilities
(API profiles), enzymatic potential (APIZYM profiles) and
antibiotic resistance of the selected strains with other strains of
the same species deposited in several culture collections (namely
CECT, ATCC, LMG, NCFB, etc . . . ). Accordingly, we have stablished
that these new strains are different from those previously
reported.
[0069] To our knowledge, this is the first time that an
experimental laboratory has observed that it is possible to obtain
non-pathogenic bacterial microorganisms present in normal mammal
breast milk or amniotic fluid, that exists a transfer of
non-pathogenic bacterial strains to breast milk and amniotic fluid
after oral intake, and that these microbial organisms are not
pathogenic strains that could act as probiotic bacterial strains
and thus, beneficially affect not only the subject who ingest them
but also to the fetus or breast feeding infant. For this reason, a
further aspect of the present invention refers to the use of the
mammal breast milk and amniotic fluid as new sources of bacterial
microorganisms having the ability to be used as probiotic strains,
the strains obtained from them, and the use thereof. The breast
milk and amniotic fluid are preferably human.
[0070] It is also an aspect of the invention comprising any of the
bacterial strains of the invention together with at least another
bacterial strain. In this regard, this invention refers to
biological pure cultures of each of the strains, or mixtures among
them or with other bacterial strains. Thus, said aspect of this
invention is the production of different compositions comprising at
least a strain or a mixture of the strains of the invention.
According to this aspect, the invention provides a composition
comprising at least one of the bacterial strains of the invention,
i.e., one of the strains mentioned above or any bacterial strain
selected by the method of the invention, where the composition
comprises preferably from 2 to 6 strains, more preferably from 2 to
4 strains, most preferably from 2 to 3 strains, and where each of
the strains is present in the composition in a proportion from 0.1%
to 99.9%, preferably from 1% to 99%, more preferably from 10% to
90%. In a preferred embodiment, the composition comprises at least
one of the bacterial strains of the invention together with another
strain or mixture of strains where the mixture comprises preferably
from 2 to 6 strains, more preferably from 2 to 4 strains, most
preferably from 2 to 3 strains and where each of the strains is
present in the composition in a proportion from 0.1% to 99.9%,
preferably from 1% to 99%, more preferably from 10% to 90%.
[0071] The compositions of the invention are preferably (no creo
que sea preferible que sean asi, simplemente lo pueden ser) in a
lyophilised form, in a frost form or even dead.
[0072] In a further aspect, the present invention provides a
composition obtainable from the supernatant of a culture of a
bacterial strain of the invention, or from any composition of the
invention. In a preferred embodiment the composition is obtainable
by extraction of a culture of any of the bacterial strains of the
invention or from a composition of the invention.
[0073] The supernatant of a culture of a Lactobacillus strain of
the present invention may be used for preparing an administrable
support. The supernatant may be used as such or may well be dried
under conditions that do not destroy the metabolic compounds
secreted or produced by the microorganisms into the liquid medium,
such as e.g. freeze drying. The present invention also refers to
the use of enzymes obtained from these probiotic strains and their
use in the production of protein hydrolysates or metabolites. The
present invention also refers to compositions of the strains of
this invention in a lyophilized form, freeze dried or inactivated
(dead bacteria) by conventional methods. Thus yet in another
aspect, the invention provides a product obtainable from the
metabolic activity of any of the strains of the invention, of a
culture of ay strain of the invention or of a composition according
to the present invention, wherein the product is preferably an
enzyme.
[0074] A further aspect of the invention consists in a food product
comprising support material and at least one strain according to
the invention, a culture, a composition or a product according to
the present invention. Preferably, the support material is a food
composition selected from milk, yoghourt, curd, cheese, fermented
milks, milk based fermented products, meat based fermented
products, fermented cereals based products, milk based powders,
cereal based powders, infant formulae, clinical nutrition formula,
ice-creams, juices, flours, bread, cakes, sugar, candies or
chewing-gums. In a preferred embodiment, the microbial strain
according to the instant invention is contained in the support
material in an amount from about 10.sup.5 cfu/g to about 10.sup.12
cfu/g support material, preferably from about 10.sup.6 cfu/g to
about 10.sup.11 cfu/g support material, more preferably from about
10.sup.6 cfu/g to about 10.sup.10 cfu/g support material.
[0075] For the purpose of the present invention the abbreviation
cfu shall designate a "colony forming unit" that is defined as the
number of bacterial cells as revealed by microbiological counts on
agar plates.
[0076] In another aspect the present invention provides
pharmaceutical compositions comprising at least one strain
according to the invention, a culture, a composition or a product
according to the invention and pharmaceutically acceptable
excipients. The required dosage amount in the food or
pharmaceutical composition described before will vary according to
the nature of the disorder or the proposed use of the composition,
whether used prophylactically or therapeutically and the type of
organism involved. For preparing a food composition according to
the present invention at least one of the Lactobacillus strains of
the present invention is incorporated in a suitable support, in an
amount of from 10.sup.5 cfu/g to about 10.sup.14 cfu/g support
material, preferably from about 10.sup.6 cfu/g to about 10.sup.13
cfu/g support material, more preferably from about 10.sup.7 cfu/g
to about 10.sup.12 cfu/g support material. The pharmaceutical
preparations can be prepared in forms of tablets, capsules, liquid
bacterial suspensions, dried oral supplements, wet oral
supplements, dry tube feeding or a wet tube feeding.
[0077] Nevertheless, the activity of the new microorganisms in the
individual is naturally dose dependent. That is, the higher the
number of the novel microorganisms that are incorporated by means
of ingestion or administration of the above food material or the
pharmaceutical composition, the higher protective and/or
therapeutic activity of the microorganisms. Since the
microorganisms of this invention are not detrimental to man and
animals and have eventually been isolated from baby feces, food or
human breast milk or amniotic fluid, a high amount thereof may be
incorporated so that essentially a high proportion of the
individual's mucosa will be colonized by the novel
microorganisms.
[0078] Preferably, the subject in need of treatment is selected
from the group consisting of individuals who suffer the disorder or
having risk to suffer the selected disorder, namely infection,
allergy, inflammation, etc. However, it will be recognized that the
present treatments are suitably employed in prophylaxis of those
disorders in any subject.
[0079] Moreover, due to the ability of the selected strains to be
transferred to and survive in breast milk and/or amniotic fluid,
the subjects in need of treatment could not only be those who
intake directly the selected strains but also the fetus or breast
feeding babies.
[0080] Preferably the probiotic, or the probiotic-containing
composition, is directed to the oral, gastric or to the intestinal
mucosal surface; however, it could also be directed to
naso-pharingeal, respiratory, genitourinary or glandular mucosa,
and it could be administered to human and animals by an oral,
rectal, topical, urethral or vaginal route.
[0081] Further, the probiotics of the present invention may be used
in conjunction with other treatments, to enhance or assist in their
efficacy.
[0082] Many people have a disturbed intestinal microflora, that is,
the balance between useful and harmful intestinal bacteria is
disturbed. A number of factors, among others stress, the presence
of bile salts, and specially diet, influence the bacterial flora.
In these situations the fermentation process could be disturbed and
the number of useful bacteria be reduced, the consequence would be
that the colon mucosa withers away and ceases to function at the
same time as the potentially malignant bacteria rapidly grow in
number. For this reason, one aspect of this invention is the use of
probiotics as prophylactic or therapeutic treatment of chronic or
acute infection, or of undesirable microbial colonization, of a
mucosal surface, comprising the administration of an effective
amount of a probiotic, or a probiotic-containing composition, to a
subject in need thereof.
[0083] The compositions of the present invention can also be used
effectively in the treatment of acute and chronic viral infections.
In particular, the treatment of chronic Epstein-Barr virus,
cytomegalovirus and other herpes-type virus infection, which are
ubiquitous in the population and are associated with a decrease on
the immune survillance.
[0084] Another embodiment of the invention is the use of the
probiotic bacteria of this invention for the prophylactic or
therapeutic treatment of diarrhea, independently whether this
disorder is due to the presence of a parasitic infestation and/or
bacterial or viral infection, the treatment with antibiotics or
quimio- or radio-therapy or to dietary or physical
complications.
[0085] The present invention also relates to the use of the stated
probiotics for the prevention and treatment of temporarily reduced
immune activity levels and normalizing immune activity levels that
are depressed in comparison with what may be considered normal,
such as that produced in aging or in healthy individuals who are
subject to intense exertion or in general to a great physiological
strain.
[0086] Moreover, through modulation of the immune response and the
balance between Th1 and Th2 cytokines, the probiotics of the
invention could also be used for the prophylactic or therapeutic
treatment of allergy and disorders related with the development of
tolerance against ingested proteins.
[0087] Another embodiment of the invention is the use of the
probiotics of this invention for the prophylactic or therapeutic
treatment of chronic inflammatory disorders such as, but not
restricted to, psoriasis, sarcoidosis, atherosclerosis,
inflammatory bowel disease, due to the ability of some of the
probiotic strains to reduce the production of pro-inflammatory
cytokines by activated macrophages.
[0088] The present invention also relates to the use of the strains
stated in this invention for the prophylactic or therapeutic
treatment of some cancer types. This use of the strains is based on
the described effects of some lactic acid bacteria counteracting
cancer due to their effects in the inhibition of carcinogenic
toxins in the intestines such as nitrosamines but also for the
effect of this probiotics in the modulation of the natural immune
defense.
[0089] Finally, the present invention also refers to the use of
these probiotic strains for the prophylactic or therapeutic
treatment of neuro-degenerative diseases due to the
hypocholesterolemic and the modulation of the oxidative stress
effect of some strains of probiotics. Both situations have been
related as risk factors for the development of neuro-degeneratives
diseases such as Parkinson or Alzheimer. Moreover, it has also been
described that commensal bacteria are able to deaminate
L-tryptophan producing indole-3-propionic acid which is a potent
neuroprotective agent.
[0090] We have also tested the potential of the strains selected by
the selection method of the present invention, analyzing the
probiotic properties of the selected strains using conventional
criteria. In this sense we have studied the following aspects: a)
Acid and bile stability, because the bacteria are mainly ingested
and must pass through the acidic environment of the stomach as well
as the bile-containing small intestine, and they must be able to
survive in these conditions; b) Adherence to intestinal mucosa,
because this property permits the bacteria to colonize and become
established in the gastrointestinal tract; c) Fermentative and high
proliferative capabilities, to enhance the establishment in the
mucosa; d) Resistance to antibiotics, because it could be necessary
for some indications; e) Reduction of pH (lactic acid production)
and production of antimicrobial metabolites by the strains of the
invention, since it can help them to form a protective barrier to
pathogens within the gastrointestinal tract; f) Immunomodulatory
capabilities.
[0091] The following methods and examples illustrate the
invention.
METHODS AND EXAMPLES
Example 1
New Method of Selection of Probiotic Strains
[0092] We have developed a novel method of selection of new
bacterial strains consisting in the ability of these strains to
survive in breast milk and/or amniotic fluid, and by their ability
to be transferred to breast milk and/or amniotic fluid after oral
intake. The rationale of this novel method described in the present
invention is that ensures special characteristics of the selected
strains obtained with it, since the bacterial strains obtained have
implicitly most of the characteristics attributed to a potential
probiotic strain, namely good resistance to digestion process and
the ability of gut colonization, but also a more natural human
origin, safety aspects, and the ability to colonize and regulate
some human niches other than the gut. Moreover, these new strains
have been obtained from different sources apart from feces, such as
goat cheese and from human breast milk and amniotic fluid.
Example 1a: Resistance to Human Fluids
[0093] Colonies isolated from different sources were checked by
their ability to survive in human breast milk and also in human
amniotic fluid. To analyze the survival rate of the probiotic
strains of this invention, 10.sup.8 cfu of each bacteria were
cultured in 1 ml of human breast milk or human amniotic fluid for
60 minutes in anaerobic conditions at 37.degree. C. The survival
was calculated by MRS agar plating of serial dilutions and compared
to the number of colonies obtained in control conditions (MRS broth
pH 6.2). Plates were cultured 16-18 hours at 37.degree. C. in
anaerobic conditions. The experiment was repeated three times.
Strains were considered resistant when the survival at least in one
of the human fluids was higher than 75% compared with the control
conditions (FIG. 1).
Example 1b: Transfer to Human Fluids
[0094] The second criteria in the selection process described in
the present invention is that bacteria should be able to be
transferred to breast milk and/or amniotic fluid after oral intake.
In order to test this capability, the putative strains were
genetically labeled, as described latter, and orally administered
to pregnant mice as animal model. Transfer of bacteria was analyzed
by PCR screening of the colonies obtained from the amniotic fluid
and from the gut of breastfed mice.
[0095] Labeling of Bacteria:
[0096] Three primer couples were employed to obtain three different
PCR fragments (F159: 159 bp, F189: 189 pb, and F228: 228 bp,
respectively). The three fragments included the junction between
the 35S rRNA promoter of the Cauliflower Mosaic Virus (CaMV) and
the 5-enolpyruvylshikimate-3-p- hosphate synthase (EPSPS) gene from
Agrobacterium tumefaciens. The primers were designed from the
artificial sequence present in Roundup Ready soya (EMBL accession
number: AX033493). BamHI sites were added to the 5'-tails of all
the primers to facilitate the cloning of the PCR fragments. Once
obtained, the PCR products were purified using the QIAquick PCR
purification kit (Qiagen), digested with BamHI, and ligated into
pTG262, a plasmid that confers resistance to Chloramphenicol (Cm).
Subsequently, these plasmids were individually introduced into the
selected strains by electroporation following conventional
protocols. Identity of the transformants was confirmed by PCR (FIG.
2A).
[0097] Transfer of Bacteria:
[0098] Four pregnant Balb/c mice were orally inoculated with
10.sup.8 cfu of genetically-labelled strains vehiculated in 200
.mu.l of milk every two days from two weeks before labor. Just
before labor, amniotic fluid was aseptically collected from two of
the mice, and cultured on MRS agar plates. The other two pregnant
mice finalized gestation. The transfer of the genetically labeled
bacteria to breast milk was analyzed by comparison of the bacteria
isolated from the neonate's gut just before and after first
lactation. All the plates were incubated for 24 h at 37.degree. C.
under anaerobic conditions. For each sample obtained, 52 colonies
were randomly selected among those that grew on the MRS plates and
subcultured on Cm-MRS plates. Finally, to detect the
genetically-labeled colonies among the Cm-resistant colonies, PCR
analyses were performed using DNA from the Cm-resistant colonies as
template (FIG. 2B). Transfer was considered positive when at least
two PCR-positive colonies could be detected in at least one of the
samples.
[0099] The bacterial strains selected by the method described in
the present invention were further tested in order to establish
their singularity and their probiotic properties as described in
the examples 3 and 4.
Example 1c
Isolation of Lactic Acid Bacteria
[0100] The bacterial strains that have been submitted to the
selection method described in the present invention have been
obtained from different sources apart from feces, such as goat
cheese and from human breast milk and amniotic fluid. Tills
isolation process have been performed as described above:
[0101] Isolation from Human Breast Milk:
[0102] Two milliliter samples of human breast milk was collected
aseptically from a 35 year-old woman (15 days after delivery). In
order to isolate bacterial strains from this sample, serial
dilutions of 0.1 ml in peptone water were plated on MRS (pH 6.2),
MRS (pH 5.5), APT, RCM, LM17, GM17 and Elliker agar plates at
37.degree. C. in both aerobic and anaerobic conditions for 24-48
hours. From about 740 colonies in total, 74 colonies (10%) that
included at least two colonies of the appreciated different
morphologies were selected and further cultured in MRS agar at
37.degree. C. in anaerobic conditions and tested according to the
proposed method. Two of the colonies obtained from this sample were
able to fulfill the defined criteria.
[0103] The selected breast milk-derived Lactobacillus gasseri
CECT5714 and Lactobacillus fermentum CECT5716 were originally
isolated from the MRS (pH 6.2) agar plates cultured in aerobic
conditions, whereas the Lactobacillus gasseri CECT5715 was isolated
from the APT agar plates cultured in anaerobic conditions.
[0104] Isolation from Human Amniotic Fluid:
[0105] Isolation of bacterial strains from human amniotic fluid was
performed by dilution of 2 ml of human amniotic fluid collected
aseptically by the clinical staff during labor from two volunteers.
Serial dilutions of 0.1 ml in peptone water were plated on MRS (pH
6.2), MRS (pH 5.5), APT, RCM, LM17, GM17 and Elliker agar plates at
37.degree. C. in both aerobic and anaerobic conditions for 24-48
hours. From about 400 colonies in total, 40 colonies (10%) that
included at least two colonies of the appreciated different
morphologies were selected and further cultured in MRS agar at
37.degree. C. in anaerobic conditions and tested according to the
proposed method.
[0106] The two strains selected from this source were identical to
those previously selected from human breast milk, namely
Lactobacillus gasseri CECT5714 and CECT5715.
[0107] Isolation from Food Products (Goat Cheese):
[0108] Isolation of bacterial strains from food products was
carried out by homogeneization in peptone water of 20 g of a
central part of the food product collected aseptically. 0.1 ml of
serial dilutions were plated in MRS agar (Oxoid) plates and RCM
agar (Oxoid) plates and cultured in both aerobic and anaerobic
conditions at 32.degree. C. for 48 hours. From more than 500
colonies in total, 5 colonies of each condition were selected and
further cultured in MRS agar at 37.degree. C. in anaerobic
conditions. These colonies were tested according to the proposed
method. Only one of the colonies was able to fulfill the defined
criteria. The selected cheese-derived colony Lactobacillus
coryniformis CECT5711 was originally isolated from MRS agar plates
cultured in aerobic conditions.
[0109] Isolation from Human Breast-Fed Baby Feces:
[0110] Isolation of bacterial strains from human feces was
performed by homogenization of 2 g of feces collected aseptically
from three independent babies (15-45 day old) in peptone water. 0.1
ml of serial dilutions were plated on MRS (pH 6.2), MRS (pH 5.5),
APT, RCM, LM17, GM17 and Elliker agar plates at 37.degree. C. in
both aerobic and anaerobic conditions for 24-48 hours. From about
670 colonies in total, 67 colonies (10%) that included at least two
colonies of the appreciated different morphologies were selected
and further cultured in MRS agar at 37.degree. C. in anaerobic
conditions and tested according to the proposed method. Only one of
the colonies was able to fulfill the defined criteria.
[0111] The selected baby feces-derived Lactobacillus salivarius
subsp. salivarius CECT5713 was originally isolated from the MRS (pH
6.2) agar plates cultured in aerobic conditions.
Example 2
Physiological and Genetic Characterization
[0112] The phenotype of each selected bacterial strain grown on MRS
media (agar or broth) at 37.degree. C. in anaerobic conditions was
as described in Table I:
1TABLE I Phenotypic characteristics of the different probiotic
strains of the invention. The phenotypic characteristics of the
different probiotic strains of the invention were compared to that
observed in known commercial probiotic strains (Lactobacillus
rhamnosus LGG from Valio, Lactobacillus johnsonii La1 from Nestl
and Lactobacillus casei immunitas from Danone). TEST CECT5711
CECT5713 CECT5714 CECT5715 CECT5716 LGG LA1 LC Origen cheese feces
breast milk breast milk breast milk feces feces feces Gram + + + +
+ + + + catalase - - - - - - - oxidase - - - - - - - morphology rod
rod rod rod rod rod rod small rod size (.mu.m) 1 .times. 1.5 - 4
0.9 .times. 15 - 3 0.9 .times. 2 - 4 1 .times. 2 - 10 1 .times. 1.5
- 3 1 .times. 2 - 4 0.9 .times. 1.5 - 3.5 0.9 .times. 15 - 2
motility nonmotile nonmotile nonmotile nonmotile nonmotile
nonmolile nonmotile nonmotile agregation single/pairs single/pairs
single/pairs single/pairs single/pairs long chains single/pairs
single/pairs
[0113] For the identification of the selected probiotic strains a
fermentation API 50CH (BioMerieux) analysis at 37.degree. C. in
anaerobic conditions for 24 and 48 hours was carried out following
the specified instructions indicated by the manufacturer. The
results after 24 hours of culture are summarized in Table II. A
positive fermentable substrate is that with a value higher than
3.
[0114] The selected bacterial strains were taxonomically classified
according to their SDS-PAGE ID protein profiling and 16S rDNA
sequence by BCCMILMG (Belgium) and/or NIZO Food Research (The
Netherlands), respectively. The results obtained from these tests
lead to the taxonomical classification of the bacterial strains as
indicated above. With this classification the bacterial strains of
the invention were deposited according to the Budapest Agreement at
the CECT--Coleccin Espaola (le Clultivos Tipo-, Valencia (Spain) on
June 11.sup.Th 2002 and with the following accession numbers:
[0115] Lactobacillus coryniformis: CECT5711
[0116] Lactobacillus salivarius subsp. salivarius: CECT5713
[0117] Lactobacillus acidophilus. CECT5714
[0118] Lactobacillus gasseri: CECT5715
[0119] Lactobacillus fermentum: CECT5716
Example 3
Singularity of the Selected Strains
[0120] Although the analysis of the SDS-PAGE ID protein profiling
and 16S rDNA sequence performed in example 2 are suitable methods
to define bacterial species, they have not enough specifity to
discriminate between different strains of the same bacterial
species. For this reason, RAPD-PCR analysis of the strains was
performed using two different lactobacilli specific primers (ArgDei
and OPL5). For the Randomly Amplified Polymorphic DNA (RAPD)-PCR
analysis, genomic DNA was isolated from 10 ml of overnight MRS
cultures using the DNeasy tissue kit (Qiagen) and following the
protocol recommended by the supplier for isolation of genomic DNA
from Gram-positive bacteria. Total DNA was used in subsequent PCR
amplifications carried out in a Techne DNA Thermal Cycler. PCR
amplifications were performed using either primer OPL5
(5'-ACGCAGGCAC-3'), or ArgDei (5'-ACCYTRGAAGGYGGYGATGTB-3'). Five
.mu.l of the PCR mixtures were analyzed on a 1.2% (wt/vol) agarose
(Sigma) gel with ethidium bromide staining. A 100-bp ladder
(Invitrogen) was used as a molecular weight standard. Gels were run
for approximately 1 h at 100 V, and the DNA was visualized and
analyzed in a gel documentation system (Gel Doc 2000, Bio-Rad),
using the Diversity Database software package (Bio-Rad).The results
are showed in FIG. 3.
[0121] Moreover, in order to test the singularity of the bacterial
strains selected with this new process and compare them with
strains obtained by other selection criteria but that had been
previously assigned to the same species. These probiotic strains
were obtained from several culture collections such as CECT, ATCC,
LMG or DSM and described in Table III.
2TABLE III Probiotic strains used for testing the singularity of
the probiotics included in this invention. Lactobacillus
coryniformis DSM 20005: Lactobacillus coryniformis subsp. torquens
DSM 20007: Lactobacilus coryniformis subsp. coryniformis CECT 982:
Lactobadilus coryniformis subsp. coryniformis CECT 4129:
Lactobadilus coryniformis subsp. torquens Lactobacillus fermentum
LMG 8900: Lactobacillus fermentum = ATCC 11976 LMG 17551:
Lactobacilus fermentum = ATCC 23271 CECT 285: Lactobacilus
fermentum = ATCC 9338 CECT 4007: Lactobacilus fermentum = ATCC
14931 Lactobacillus gasseri LMG 11413: Lactobacillus gasseri LMG
13047: Lactobacillus gasseri = ATCC 19992 LMG 13134: Lactobacillus
gasseri = ATCC 9857 LMG 18176: Lactobacillus gasseri LMG 18194:
Lactobacilus gasseri CECT 4479: Lactobacilus gasseri Lactobacillus
salivarius DSM 20492: Lactobacillus salivarius CECT 4062:
Lactobacillus salivarius CECT 4063: Lactobacillus salivarius
[0122] All the selected strains included in this invention were
compared with the strains described in Table III regarding their
RAPD-PCR profiles using two different primers, results of this
analysis (FIG. 4) shown that the selected strains included in this
invention are different to those previously described. Moreover, we
extended our results and compared not only genetic characteristics
but also biochemical aspects of the selected strains with those
strains described in Table III. In this sense, we performed API
analyses (BioMerieux) (Table IV), APIZYM analyses (BioMerieux)
(Table V) and antibiotic resistance as described in example 5 g
(Table VI). The activities that differ to that observed with the
strains of this invention has been indicated in grey.
[0123] Probiotic strains used were described in Table III. The
activities that differ to that observed with the strains of this
invention has been indicated in grey.
3TABLE VI Comparison of antibiotic resistances of the selected
bacteria using the method described in this invention with other
bacteria of the same species. 1 2 3 4 Probiotic strains used were
described in Table III. The antibiotics used and the methodology is
described in example 4e. R = resistant, I = intermediate, S =
sensible. The activities that differ to that observed with the
strains of this invention has been indicated in grey.
[0124] Probiotic strains used were described in Table III. The
antibiotics used and the methodology is described in example 4e.
R=resistant, I=intermediate, S=sensible. The activities that differ
to that observed with the strains of this invention has been
indicated in grey.
Example 4
Probiotic Characteristics of the Strains
[0125] We also analyzed the suitability of the probiotic selection
process included in this invention regarding its ability to select
bacterial strains with desirable probiotic characteristics. In
order to evaluate this, the selected strains were analyzed for a
high number of different characteristics that could enhance their
capabilities to act as a probiotic strains. Moreover, we assigned
arbitrarialy (as indicated) a numerical value to the results
obtained in each test in order to compare the probiotic strain
included in this invention to those obtained by other selection
criteria. The results obtained are summarized in Table VIII at the
end of this example and described in the following sub-examples and
compared with some commercial strains.
Example 4a
Adhesion Analysis to Caco-2 and HT-29
[0126] Culture of Caco-2 and HT-29 Cells
[0127] For the adhesion and inhibition assays, the cell lines
Caco-2 (ATCC HTB-37) and HT-29 (ATCC HTB-38) were utilized as a
model of the intestine cells. Both cell lines presented features
characteristic for intestinal cells such as polarization,
expression of intestinal enzymes, and production of particular
structural polypeptides and mucins.
[0128] The cells were grown in plastic flasks (75 cm.sup.2, Nunc)
in DMEM (PAA laboratories) as culture medium supplemented with 10%
inactivated FCS (Fetal Calf Serum, PAA laboratories), non essential
aminoacids, 100 U/ml penicilline/streptomycine, 1 .mu.g/ml
amphoterine. Cell culture was performed at 37.degree. C. in an
atmosphere comprising 95% air and 5% CO.sub.2. Media was changed on
a two daily basis and the cells were splitted every week.
[0129] For the adhesion assays the cells were splitted to 35 mm
plastic dishes (Nunc) and cultured in similar conditions but
without antibiotics after confluence. Adhesion assays were
performed 10-14 days post-confluence.
[0130] Culture of Bacteria
[0131] Probiotic Strains:
[0132] The probiotic strains of this invention were cultured in MRS
broth (pH 6.2) in anaerobic conditions for 16-18 hours at
37.degree. C. after inoculation of a 0.1% (v/v) from the glycerol
stock. In this conditions, the concentration of the culture was
1-2.times.10.sup.9 cfu/ml, as observed by plating on MRS agar.
[0133] Gram-Negative Strains:
[0134] Escherichia coli 0157:H7 (non-pathogenic) (CECT4972), E.
coli 0157:H7 (entero-pathogenic) (CECT4783), E. coli 0157:H7
(entero-pathogenic) (CECT4782), Salinonella cholerasuis typhi
(CECT409) and S. cholerasuis typhimurium (CECT443) were all
obtained from the CECT-Coleccin Espaola de Cultivos Tipo-. All gram
negatives strains were cultured in TSB broth (AES Laboratoire) in
anaerobic conditions for 16-18 hours at 37.degree. C. after
inoculation of a 0.1% (v/v) from the glycerol stock. At this
conditions, the concentration of the culture was 1-2.times.10.sup.9
cfu/ml, as observed by plating on TSA agar (AES Laboratoire).
[0135] Adhesion Analysis
[0136] Caco-2 and HT-29 intestinal cell lines were cultured in 35
mm plastic dishes in 2 ml medium without antibiotics to confluence.
10-14 days post-confluence 1 ml of media was replaced with 1 ml of
a suspension of 10.sup.8 bacteria in DMEM. The cultures were
incubated 1 hour at 37.degree. C. After that, cells were washed
twice with PBS and fixed with ice-cold 70% methanol for 30 minutes.
Plates were air dried and Gram stained. The attached bacteria were
visualized using an optical Axiovert 200 (Zeiss) microscope at
1000.times. magnification in oil-immersion. Twenty randomized
fields were counted and the results expressed as the mean of the
number of bacteria attached to the cells per field.+-.SD. The
capability of a probiotic strain was considered high if the number
of attached bacteria was >250, moderate between 100 and 250, and
slight >100 (FIG. 5)
Example 4b
Resistance to Acid and Bile Salts
[0137] To analyze the resistance of the probiotic strains of this
invention to acidic and high bile salt content, conditions that
these bacteria will encounter during the digestive transit,
bacteria were cultured in MRS broth media either at pH 3.0 or in
MRS broth at pH 6.2 supplemented withO.15% bile salts (Sigma) for
90 minutes. The survival rate was calculated by MRS agar plating of
serial dilutions and compared to the number of colonies obtained in
control conditions (MRS broth pH 6.2). Plates were cultured 16-18
hours at 37.degree. C. in anaerobic conditions. The experiment was
repeated three times. Resistance was considered high when the
survival was >80%, moderate 80% to 60%, slight <60% compared
with the control conditions (FIG. 6).
Example 4c
Time of Generation
[0138] The time of generation, meaning the time that a bacterial
culture requires to duplicate the concentration of bacteria, is an
important characteristic for a probiotic bacteria. It is important
from an industrial point of view (production of a higher amount of
biomass in the same amount of time) and from a probiotic point of
view (higher colonization of the gut). In order to consider both
aspects we have analyzed the generation time of the probiotic
strains of the invention in a rich media (industrial point of view)
and in a poor media (probiotic point of view).
[0139] The probiotic strains of this invention were grown in MRS
broth (pH 6.2) with 2% (rich media) or 0.2% (poor media) glucose
for 0, 1, 2, 4 and 6 hours at 37.degree. C. in anaerobic conditions
and the concentration of bacteria was determined by plating serial
dilutions in MRS agar plates and incubation of the plates for 16-18
hours at 37.degree. C. in anaerobic conditions (FIG. 7). The
generation time was calculated as the time in minutes necessary in
order to duplicate the number of colonies at the initial time.
Example 4d
Fermentation Capabilities
[0140] The capacity of a bacterial strain to metabolize complex
carbohydrates (soluble and non-soluble fibers) ensures that these
probiotic strains could use them as a carbon source in the colon,
and thus enhance the efficiency of colonization. For this reason,
we have tested the capability of the probiotic strains of this
invention to use several non-digestible fibers as an unique source
of carbohydrates.
[0141] To assay the capability to the probiotic bacterial strains
to metabolize fiber we cultured, a liquid culture was carried out
in MRS broth media without glucose and supplemented with a 2% of
each fiber in 96 well flat-bottomed plastic dishes (Nunc) for 24
and 48 hours at 37.degree. C. in anaerobic conditions. The
fermentation process was controlled by pH decrease in the media and
determined by a colorimetric approach using 0.3% phenol red as
indicator and measuring the absorbance at 540 nm.
[0142] The fibers used were: .alpha.-celulose (raw cellulose, Campi
y Jove), Actilight (fructo-oligosaccharide, Beghin-Meiji), Ficao
(cocoa fiber, Natra), Fructafit (Inulin, Sensus), Lactose
(Bordulo), Pectine (YM100, Genu), Raftiline (Inulin oligofructose,
Orafti), Raftilose (Inulin oligofructose, Orafti), and Vitacel
(purified cellulose, Campi y Jove).
[0143] The fermentation capability (defined as fold-induction of
the pH reduction compared to the control without fiber) was
calculated. Results showed in FIG. 8 represent the individual
values for each fiber (panel A) and the sum of all these individual
values for each selected strain (Panel B). Fermentation capability
was considered high when the sum of individual values was >30,
moderate 30 to 25, slight <25.
Example 4e
Resistance to Antibiotics
[0144] The use of antibiotics leads to a reduction of the comensal
gut microflora which sometimes relates to diarrhea and other gut
disorders. Moreover, this reduction in the amount of gut bacteria
could be the consequence of opportunistic pathogenic bacteria and
viruses to infect the host. The use of antibiotics to block the
infection does not resolve this disorder but complicates it. In
other situations like intestinal inflammation where probiotics
could exert a beneficial role, this potential effect is sometimes
limited by the simultaneous therapy with antibiotics. For these
reasons, the selection of potential probiotic strains able to
resist common antibiotics would be an improvement in the art.
[0145] To analyze the resistance of the probiotic strains of this
invention an agar well diffusion assay was used. Mueller-Hinton
agar plates containing 10.sup.6 cfu/ml of each probiotic strain
were prepared. Then, antibiotic commercial discs corresponding to
the indicated concentrations were added to the wells and allowed to
diffuse into the agar during a preincubation period of 10 minutes
at room temperature, followed by anaerobic incubation of the plates
at 37.degree. C. for 16-18 hours. Diameter of inhibition halos was
measured and the resistance degree of the bacteria to each
antibiotic was graded as R (resistant), I (intermediate) or S
(sensible) according to the described sensibility of lactobacilli
to this antibiotics (Table VII). After that, a numerical value was
assigned to each condition: R=3, I=2, and S=1. Ten different
antibiotics were tested and the numerical values were added up to
get an overall value. The resistance capability of a probiotic
strain was considered high if the total value was >17, moderate
between 15 and 17, and slight <15.
[0146] The antibiotics and concentrations used were: Erythromycin
15 mg (E 15), Penicillin 10 .mu.g (P 10), Ciprofloxacin 5 .mu.g
(CiP 5), Chloramphenicol 30 .mu.g (C 30), Nalidixic 30 .mu.g Na
30), Amoxicilin 10 .mu.g (AM 10), Tetracycline 30 .mu.g (Te 10),
Vancomicin 30 .mu.g (Va 30), Cephoxithin 30 .mu.g (Fox 30), and
Cephalothin 30 .mu.g (CF 30) (FIG. 9).
4TABLE VII Resistance of the selected strains to antibiotics. TEST
CECT5711 CECT5713 CECT5714 CECT5715 CECT5716 LGG LA1 LC E 15 S S S
S S S R R P 10 S S S S S S S S CiP 5 R I R R R I R R C 30 S S S S S
S S S Na 30 R R R R R R R R AM 10 S S S S S S S S Te 30 I S S S S S
S S Va 30 R R I I R R S S Fox 30 R S R R R R R R CF 30 S S I I S I
I I Total 19 15 16 18 19 17 18 18
Example 4f
Production of Metabolic Acids
[0147] The production of metabolic acids by probiotic bacteria,
namely lactic, acetic, propionic and butyric acid, and the
subsequent reduction of the pH in feces has been extensively
associated with a beneficial effect of these bacteria due to a
reduction in the growth and infective capabilities of opportunistic
pathogenic microorganisms. Moreover, some of these acids, specially
butyric acid, are rapidly absorbed and used by the intestinal cells
as an energy source. In this sense, reduced pH in feces of the
breast feeding infants has been associated with the reduced risk of
gut disorders compared with formula feeding babies.
[0148] Acid producing capacity of the probiotic strains of the
invention was observed by measurement of the pH reduction during
milk fermentation. Five ml of skimmed milk were inoculated with
10.sup.8 cfu of each bacterial strain and fermented for 24 (grey
bars) and 48 (black bars) hours at 37.degree. C. in anaerobic
conditions and the pH was measured using a CyberScan 510 pHmeter
(VWR). The production of acid by a probiotic strain was considered
high if the milk pH value after 48 hours was <4.5, moderate
between 4.5 and 5.5, and slight >5.5 (FIG. 10).
Example 4g
Production of Antimicrobial Metabolites
[0149] It has been suggested that the main benefficial effect of
probiotics is the control of the balance between useful and harmful
intestinal bacteria is the gut. When the number of useful bacteria
is reduced, opportunistic bacteria could over-grow and disturb the
well-being of the host or even induce an infection. Most bacterial
organisms have adquired characteristics or mechanisms that reduce
the growth capabilities of other microorganisms that cohabitate
with them and thus, enabling their selective growth. As stated in
example 4f, the reduction of pH through acid production by lactic
acid bacteria is one of such mechanisms. Moreover, some lactic
bacteria also produce bioactive peptides components and other
metabolites that selective inhibit the growth of other bacteria,
yeast or fungi. This is the case of bacteriocins such as
pediocin.
[0150] The probiotic strains of this invention were assessed for
their capability to produce antimicrobial metabolites using an agar
well diffusion assay. MRS agar plates containing 10.sup.6 cfu/ml of
different pathogenic bacteria strain (Salmonella typhmurium and
Escherichia coli) were prepared. Wells, with a diameter of 5 mm,
where then cut in the agar using a sterile cork-borer. Then, 50
.mu.l of a 2 fold concentrate supernatant of each probiotic strain
culture were added to the wells and allowed to diffuse into the
agar during a 2 hours preincubation period at 4.degree. C.,
followed by aerobic incubation of the plates at 37.degree. C. for
16-18 hours. The antimicrobial activity of each supernatant was
considered high if the diameter of the inhibition hallo for both
pathogenic bacteria strains was >12, moderate between 8 and 12,
and slight <8 (FIG. 11)
[0151] Moreover, it was also tested if the antimicrobial activity
of the supernatants was due to a antimicrobial substance or to the
production of metabolic acids. In this sense, the inhibitory effect
of a dilution of each metabolic acid (acetic, lactic, propionic and
butyric) at pH 4.5 was assayed. None of these situations inhibited
the growth of Salmonella or E. coli in these conditions (data not
shown). It was also tested the antimicrobial capabilities of
supernatants obtained from bacterial cultures using glucose or
lactose as a carbohydrate source. In these circumstances, those
bacterial strains that do not ferment lactose (L. rhamnosus GG and
L. acidophilis CECT5714) did not showed antimicrobial activity in
the lactose-containing culture whereas they showed this activity it
in the glucose culture (data not shown).
Example 4h
Inhibition of Pathogen Adhesion to Caco-2
[0152] Caco-2 intestinal cell lines were cultured in 35 mm plastic
dishes in 2 ml complete medium without antibiotics to confluence.
10-14 days post-confluence 1 ml of media was replaced with 1 ml of
a suspension of 10.sup.8 probiotic bacteria in DMEM. The cultures
were incubated 1 hour at 37.degree. C. After that, 1 ml of a
suspension of 10.sup.8 pathogenic bacteria (E. coli or S.
typhimurium) in DMEM was added to the cultures and incubated 1 hour
more at 37.degree. C. The cells were washed twice with PBS and
fixed with ice-cold 70% methanol for 30 minutes. Plates were air
dried and Gram stained. The attached bacteria were visualized using
an optical Axiovert 200 (Zeiss) microscope at 1000.times.
magnification in oil-immersion. The number of gram-negative
bacteria in 10 randomized fields were counted and the results
expressed as the mean of % of pathogenic bacteria attached to the
cells compared to control cultures without probiotic strains. The
capability to inhibit the adhesion of pathogenic bacteria to
intestinal cells of a probiotic strain was considered high if the %
of both strains of Gram-negative attached bacteria as compared with
the control was <25%, moderate between 25% and 75%, and slight
>75% (FIG. 12).
[0153] All the results obtained in the example 4 are summarized in
Table VIII. Each test was performed as indicated in the
corresponding sub-example and described in this document. The
categories in each test were assigned as indicated. a) Example 4a;
number of bacteria attached per field; high >250, moderate: 100
to 250, slight <100. b) Example 4b; % of survival compared to
control conditions; high >80%, moderate: 80% to 60%, slight
<60%. c) Example 4c; minutes necessary to duplicate de initial
population; rapid <60, moderate: 60 to 120, slow >120. d)
Example 2; number of fermentable substrates; high >18, moderate,
12 to 18, slight <12. e) Example 4d; Accumulated fold-reduction
of the total fermentable substrates compared with the control; high
>30, moderate: 25 to 30, slight <25. f) Example 4e,
Accumulated resistance to each antibiotic (resistant=3,
intermediate=2, sensible=1); high >17, moderate: 15 to 17,
slight <15. g) Example 4f; pH value of milk after 48 hours
culture; high <4, moderate: 4 to 4.5, slight >4.5. h) Example
4g; mm of inhibition hallo high >12, moderate: 12 to 8, slight
<8, (*) only in presence of glucose but not lactose. i) Example
4h; % of adhesion; high <25, moderate: 25 to 75, slight <75.
The global probiotic capability was calculated by the sum of all
tests (high=3, moderate=2, slight=1).
5TABLE VII Potency to act as a probiotic of the different strains
of the invention. The different capabilities to act as a probiotic
of the different strains of the invention were compared to those
observed in some commercial probiotic strains (Lactobacillus
rhamnosus LGG from Valio, Lactobacillus johnsonii La1 from Nestk
and Lactobacillus casei immunitas from Danone) and quantificated
arbitrarially. TEST CECT5711 CECT5713 CECT5714 CECT5715 CECT5716
LGG LA1 LC Adhesion to high moderate high high high high high
slight Caco-2.sup.a Adhesion to HT-29.sup.a high high moderate
moderate high high moderate slight Resistance to acid.sup.b high
moderate high moderate high moderate slight slight Resistance to
bile.sup.b high high slight slight moderate slight high high Time
of generation.sup.c moderate rapid moderate stow moderate rapid
moderate moderate API CH50.sup.d slight high moderate high moderate
moderate moderate high Fermentation.sup.d moderate high slight
slight slight slight high high Antibiogram.sup.f high slight
moderate high high moderate high high Reduction of pH.sup.g high
moderate moderate moderate moderate slight moderate slight
Antimicrabiat prod..sup.h high high high (*) moderate moderate
moderate(*) slight slight Pathogen Inhibition.sup.i high high
moderate high high high moderate slight Probiotic capability 26 28
23 23 26 23 24 20
Example 5
Probiotic Colonization of Mice Gut
[0154] By definition, a probiotic must colonize the gut mucosa of
the host. Moreover, it has been described that the beneficial
actions exerted by probiotics require this colonization. Although
in vitro studies such as adhesion capabilities to intestinal cell
lines, or resistance to the digestion conditions are good
approaches to select probiotic strains, these tests do not ensure
the effectiveness of the selected strain to colonize in vivo the
gut mucosa. For this reason, we performed an analysis in vivo of
the colonization capacity of the probiotic strains of the invention
using mouse as an experimental animal model.
[0155] Six male Balb/c mice (6-8 weeks old) were daily supplemented
with 10.sup.8 cfu in 0.2 ml of skimmed milk of L. salivarius
CECT5713 for 14 days. After this period, the probiotic
supplementation was stopped but the animals were still kept in
observation for another 14 days. Feces samples were collected at 0,
7, 14, 21 and 28 days from the initiation of the experience. Aprox.
200 mg of feces were collected independently from each mice and
homogenized at 50 mg/ml in peptone water. Serial dilutions of the
collected supernatant were prepared, and 0,1 ml plated in selective
agar plates (MRS for Lactobacilli, Eugon agar+tomato juice for
Bifidobacteria and McConkey agar for coliform bacteria). Plates
were incubated at 37.degree. C. in anaerobic conditions for 24
hours. The number of cfu was determined by counting on selective
media plates and the verage was calculated.
[0156] FIG. 13 shows that supplementation with L. salivarius
CECT5713 caused a stadistically significant increase in the number
of total lactobacilli in feces which demonstrates that this strain
is able to survive its passage through the digestive tract and
reach the colon. Moreover, the fact that the increased lactobacilli
count was still observable one week after finalization of the oral
supplementation demonstrates that this probiotic strain is able to
temporally colonize the gut mucosa.
[0157] Concomitantly with the lactobacilli increase, a reduction in
the fecal count of coliform bacteria was also observed, and was
still statistically significant two weeks after finalization of the
probiotic treatment. These findings show that dietary
supplementation with CECT5713 cells causes not only stimulation of
the beneficial flora but also inhibition of the harmful
bacteria.
Example 6
Effect of Lactobacillus fermentum CECT5716 on translocation of
Salmonella typhimurium in Mice Following Immunization with
Inactivated Salmonella Vaccine
[0158] Translocation of Gram-negative bacteria across the gut
epithelium can occur especially in subjects following
gastrointestinal infection, disease or surgery. Left untreated it
can lead to endotoxemia. In this example, the effect of feeding L.
fermentum CECT5716 on the translocation of gut pathogen Salmonella
typhimurium was examined.
[0159] Male Balb/c mice (6-8 weeks old) were daily orally
inoculated with 1.times.10.sup.8 cfu in 0.2 ml of milk or milk
alone for two weeks. After that, mice were immunized orally or not
with an inactivated Salmonella vaccine (10.sup.8 cfu inactivated
with paraphormaldehyde in 0.2 ml milk). After immunization, mice
were orally inoculated two weeks more with the L. fermentum
CECT5716 preparation in alternate days for two weeks more. Two
weeks after oral immunization, all mice were orally challenged with
live S. typhimurium (10.sup.10 cfu in 0.2 ml milk). Then, after
24-48 hours, the level of colonization of S. typhimurium in the
spleen was determined by colony counting in SS agar (Oxoid). The
fecal concentration of IgA specific for Salmonella antigens were
also measured by ELISA techniques (Biosource).
[0160] The results obtained demonstrate that L. fermentum CECT5716
potentiates the beneficial effect of the vaccination of mice with
the inactivated Salmonella vaccine as shown in FIG. 15. The
inhibition on the translocation of S. typhimurium induced by the
inactivated vaccine and potentiated by L. fermentum CECT5716 was
due to the increase of the secretion of specific IgA and also, to
the inhibition or blocking effect of the probiotic strain on the
mucosal adhesion of Salmonella as described in Example 4h.
Example 7
Effect of Probiotic Bacteria on Inflammatory Cytokines
[0161] Besides the reduction of the risk of infection, many
clinical effects associated to probiotic treatments are due to
immuno-modulatory capabilities of selected probiotic strains. The
regulation of the immune response is usually mediated through a
change in the balance between pro-inflammatory cytokines (Th1) such
as TNF-.alpha., humoral cytokines (Th2) such as IL-4 or IL-13, and
regulatory cytokines (Th3) such as IL-10 and TGF-.beta.. For this
reason, the effect of some of the probiotic strains of this
invention in regulating the expression of some of these crucial
cytokines was also tested.
[0162] Bone marrow derived macrophages were stimulated with 100
ng/ml of LPS (Sigma) as a cellular model. 10.sup.5 macrophages/well
were cultured in 24-well plastic plates (Nunc) with 1 ml of DMEM.
Once attached, macrophages were stimulated or not with 100 ng/ml
LPS and with 10.sup.7 cfU/ml of the indicated probiotic strains for
12 hours at 37.degree. C. in a 5% CO.sub.2 atmosphere. Supernatants
were collected and the production of cytokines was analyzed using a
mouse TNF-.alpha. or mouse IL-10 ELISA (Biosource).The results
obtained (FIG. 15) show that the consumption of the probiotic
strains of this invention could have a beneficial effect in some
inflammatory situations since they induce a global
anti-inflammatory effect on immune cells such as macrophages,
inducing an increase in IL-10 expression without increasing the
levels of secreted TNF-.alpha..
Example 8
Effect of Probiotic Bacteria on Ig Production
[0163] The effect of the probiotic strains of this invention on the
immunoglobulin production was analyzed using lymphocyte cultures
obtained from the spleen of male Balb/c mice (6-8 weeks old).
2.times.10.sup.6 lymphocytes were cultured in 1 ml DMEM in 24 well
plastic plates and stimulated with inactivated probiotic cultures
(10.sup.8 cfu/ml) in presence or absence of 25 .mu.g/ml LPS for 6
days. The production of Ig G by lymphocytes was assessed using a
mouse Ig G ELISA from Bethyl.
[0164] The results obtained (FIG. 16) show that the effect on the
Ig G production induced in lymphocytes of the probiotic strains of
this invention is highly variable depending on the selected strain
used. In this regard, there is some strains (CECT5711 and CECT5714)
that have immune-stimulating activities since induce the expression
of Ig G, while others (CECT5713 and CECT5715) have
immune-suppressive effects.
Example 9
Preparation of a Fermented Liquid Milk Formula
[0165] A normal fermented liquid milk composition with probiotics
was prepared using the following formula:
6 Milk 1.5% Fat; 3.2% protein 997 g/kg Skim milk powder 3 g/kg
Probiotic strain (10.sup.12 cft/g) 0.1 g/kg
[0166] The fat and dry solids contents of the milk were
standardized according to the formulation described above. After
that, the milk was homogenized at 20-25 Mpa and 65-70.degree. C. to
obtain optimum physical properties in the product. The preparation
was heated at 90-95.degree. C. and a holding time of about 5
minutes. This period of time causes the denaturation of about
70-80% of whey proteins. Cooled milk (40-45.degree. C.) was
inoculated with the probiotic strain in absence of any starter
culture and fermented in the incubation tank at 40-45.degree. C.
for 10 hours without agitation until reaching a final pH (pH
4.5-5). After clot formation, is the mixture was homogenized by
mechanical methods. Once the homogenization was carried out, the
preparation was cooled down to a temperature below 10.degree. C. in
60 minutes. After that, the composition was packaged. Final
cooling, normally down to 5.degree. C., took place in a cold room,
where the products were held to caducity.
Example 10
Preparation of a Set Yogurts
[0167] A yogurt product with probiotics was prepared using the
following formula:
7 Milk 3.1% Fat; 3.2% protein 987 g/kg Skim milk powder 13 g/kg
Starter 0.1 g/kg Probiotic strain (10.sup.12 cfu/g) 0.1 g/kg
[0168] The fat and dry solids contents of the milk were
standardized according to the formulation described above. The milk
was homogenized at 20-25 Mpa and 65-70.degree. C. to obtain optimum
physical properties in the product and heat treatment was performed
at 90-95.degree. C. and a holding time of about 5 minutes which is
able to denature about 70-80% of whey proteins. After
pasteurization, milk was cooled to 40-45.degree. C. and the starter
and probiotic cultures were metered into the stream of milk as they
were pumped from an intermediate storage tank to the filling
machine. Following packaging in the filling machine, the packages
after crating and palletizing, were trucked into the system for
incubation and cooling. After that, filled pallets were fermented
in the incubation room at 40-45.degree. C. for 5-6 hours until a pH
of 4.5 was reached. Cooling of the packets were performed quickly
obtaining a temperature of 12-15.degree. C. in 55-70 minutes. Final
cooling, down to 5.degree. C., took place in the chill store.
Example 11
Preparation of an Infant Formula in Powder
[0169] An infant formula with probiotics was prepared using the
following formula:
8 Demineralised whey 512 g/kg Palm olein 135 g/kg Lactose 92 g/kg
Skimmed Milk 95 g/kg Rapeseed oil 52 g/kg Coconut oil 49 g/kg
Sunflower oil 28 g/kg Water 31 g/kg Vitamin premix 2 g/kg Mineral
premix 4 g/kg Probiotic strain (10.sup.12 cfu/g) 0.1 g/kg
[0170] To an appropriately sized blend tank with agitation and
heating all solid ingredients were mixed with the liquid milk and
water in the absence of any vitamins. Then, the vegetable oils were
admixed. The mixture was then heated at 60-70.degree. C. and
emulsified through a single stage homogenizer at 6 to 7 MPa in
absence of oxygen. After emulsification the mixture was
standardized by addition of vitamins and the pH was adjusted in the
range of about 6.7 to 7.2. Then, the mixture was reheated to
between about 65.degree. C. and 70.degree. C. The product was
finally was dried in a spray drier to obtain a final dry powder
product. Finally, the probiotic strain (10.sup.12 cfu/g)
[0171] (0.1 g/Kg) was dry mixed with the final dry powder product
and was packaged.
* * * * *