U.S. patent application number 13/841604 was filed with the patent office on 2013-10-24 for isolation, identification and characterisation of strains with probiotic activity, from faeces of infants fed exclusively with breast milk.
This patent application is currently assigned to HERO ESPANA, S.A.. The applicant listed for this patent is Miriam BERMUDEZ BRITO, Angel GIL HERNANDEZ, Carolina GOMEZ LLORENTE, Inmaculada LLAMAS COMPANY, Jose MALDONADO LOZANO, Sergio MUNOZ QUEZADA, Fernando ROMERO BRAQUEHAIS, Antonio Francisco SUAREZ GARCIA, Jose Maria VIEITES FERNANDEZ. Invention is credited to Miriam BERMUDEZ BRITO, Angel GIL HERNANDEZ, Carolina GOMEZ LLORENTE, Inmaculada LLAMAS COMPANY, Jose MALDONADO LOZANO, Sergio MUNOZ QUEZADA, Fernando ROMERO BRAQUEHAIS, Antonio Francisco SUAREZ GARCIA, Jose Maria VIEITES FERNANDEZ.
Application Number | 20130280790 13/841604 |
Document ID | / |
Family ID | 42727821 |
Filed Date | 2013-10-24 |
United States Patent
Application |
20130280790 |
Kind Code |
A1 |
VIEITES FERNANDEZ; Jose Maria ;
et al. |
October 24, 2013 |
ISOLATION, IDENTIFICATION AND CHARACTERISATION OF STRAINS WITH
PROBIOTIC ACTIVITY, FROM FAECES OF INFANTS FED EXCLUSIVELY WITH
BREAST MILK
Abstract
The invention relates to probiotic micro-organisms isolated from
faeces of children exclusively fed with breast milk. Said
microorganisms are used in the food or pharmaceutical industry,
especially for use in infant formula milk, due to their probiotic
properties which have beneficial effects on the health of those
ingesting them. Said microorganisms consist of Lactobacillus
rhamnosus HERO 22A (CNCM I-4036), Lactobacillus paracasei HERO 7
(CNCM I-4034) and Bifidobacterium breve HERO 15B (CNCM I-4035).
Inventors: |
VIEITES FERNANDEZ; Jose Maria;
(Granada, ES) ; MUNOZ QUEZADA; Sergio; (Granada,
ES) ; LLAMAS COMPANY; Inmaculada; (Granada, ES)
; MALDONADO LOZANO; Jose; (Granada, ES) ; ROMERO
BRAQUEHAIS; Fernando; (Alcantarilla (Murcia), ES) ;
SUAREZ GARCIA; Antonio Francisco; (Granada, ES) ; GIL
HERNANDEZ; Angel; (Granada, ES) ; GOMEZ LLORENTE;
Carolina; (Alcantarilla (Murcia), ES) ; BERMUDEZ
BRITO; Miriam; (Alcantarilla (Murcia), ES) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
VIEITES FERNANDEZ; Jose Maria
MUNOZ QUEZADA; Sergio
LLAMAS COMPANY; Inmaculada
MALDONADO LOZANO; Jose
ROMERO BRAQUEHAIS; Fernando
SUAREZ GARCIA; Antonio Francisco
GIL HERNANDEZ; Angel
GOMEZ LLORENTE; Carolina
BERMUDEZ BRITO; Miriam |
Granada
Granada
Granada
Granada
Alcantarilla (Murcia)
Granada
Granada
Alcantarilla (Murcia)
Alcantarilla (Murcia) |
|
ES
ES
ES
ES
ES
ES
ES
ES
ES |
|
|
Assignee: |
HERO ESPANA, S.A.
Alcantarilla (Murcia)
ES
|
Family ID: |
42727821 |
Appl. No.: |
13/841604 |
Filed: |
March 15, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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13255842 |
Nov 22, 2011 |
|
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PCT/ES2010/000097 |
Mar 9, 2010 |
|
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13841604 |
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Current U.S.
Class: |
435/252.9 ;
435/252.1 |
Current CPC
Class: |
A61P 3/10 20180101; A61P
3/02 20180101; C12N 1/20 20130101; C12R 1/01 20130101; A61P 1/00
20180101; A61P 31/00 20180101; A61K 35/745 20130101; A61P 37/04
20180101; A23L 33/135 20160801; A23V 2002/00 20130101; A61K 35/747
20130101; A23Y 2220/73 20130101; A23L 33/40 20160801; A23V
2200/3204 20130101; A23Y 2300/29 20130101; C12R 1/225 20130101;
A23V 2002/00 20130101; A61P 11/06 20180101; A61P 3/04 20180101 |
Class at
Publication: |
435/252.9 ;
435/252.1 |
International
Class: |
A61K 35/74 20060101
A61K035/74 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 10, 2009 |
ES |
PCT/ES09/000130 |
Claims
1. A strain of probiotic microorganism isolated from faeces of
children exclusively fed with breast milk characterized by
consisting of Bifidobacterium breve HERO 15B (CNCM I-4035).
2-4. (canceled)
5. The strain of microorganism according to claim 1 characterized
by being presented in the form of pure biological culture.
6. The strain of microorganism according to claim 1 characterized
by being isolated.
7. The strain of probiotic microorganism according to claim 1
characterized by being presented in the form of viable cells.
8. The strain of probiotic microorganism according to claim 1,
characterized by being presented in the form of non-viable
cells.
9-16. (canceled)
17. The strain of probiotic microorganism according to claim 1 for
use in diets.
18. The strain according to claim 17 characterized in that the use
thereof in infant and/or adult and/or special diets.
19. The strain according to claim 18 for use in the preparation of
infant formula milk.
20. The strain according to claim 19, characterized in that the
formula milk consist of ready-to-eat infant milk and/or infant
cereals, and/or infant foods.
21. The strain according to claim 17 for use in the preparation of
food supplements.
22. The strain according to claim 17 for use in the preparation of
special formulas for oral and/or enteral nutrition.
23. The strain of probiotic microorganism according to claim 1 for
use in the preparation of a pharmaceutical product.
24. The strain of probiotic microorganism according to claim 1
applicable in stimulating the immune system and/or in
preventing/treating asthma and/or in preventing/treating
gastrointestinal disorders, and/or in eliminating/modulating the
main digestive pathogens, and/or in preventing/treating obesity and
its co-morbidities including metabolic syndrome and diabetes and/or
in the typical aging-associated diseases.
25. The strain according to claim 24, characterized in that said
gastrointestinal disorders comprise alterations of intestinal
transit such as constipation and alterations of the bioavailability
of minerals, infections and malabsorption syndromes.
26. The strain according to claim 25, characterized in that said
infections comprise gastric infections and gastrointestinal
infections with acute or chronic diarrhea.
27. The strain according to claim 25, characterized in that said
malabsorption syndromes comprise disorders affecting the anatomy of
the intestine such as the short bowel syndrome, and disorders
affecting the physiology of the intestine such as cystic fibrosis
of the pancreas, malabsoprtion of sugars especially lactose,
alterations of lipid absorption, food allergies, and inflammatory
bowel diseases such as Crohn's disease and ulcerative colitis.
Description
TECHNICAL SECTOR OF THE INVENTION
[0001] The general objective of this study is to isolate probiotic
microorganisms for a subsequent use in the food and pharmaceutical
industry, especially for using them in infant formula milk. Said
microorganisms have high values of resistance to pH, bile salts and
intestinal cell adhesion, they are therefore especially suitable
for use in the aforementioned industries.
BACKGROUND OF THE INVENTION
[0002] Nutrition has had a very significant development which has
changed its concept in the recent decades. Diet was previously
considered as having the role of providing nutrients necessary to
maintain the health condition, whereas today this concept has
evolved into the idea that diet can contain foods which in addition
to providing nutrition, promote health. This is the reason why the
food industry has started to develop a large amount of products
promoting health and wellbeing. In this area the line of functional
foods has had a very significant development, where the consumption
of probiotics by the population increases daily. The real challenge
is to broaden the knowledge of these foods, among them those
containing probiotics have a special interest.
[0003] There are very old records relating to the beneficial
effects derived from the consumption of foods with high bacterial
content, such as in the version of the old testament wherein it is
said that Abraham attributes his long life to the consumption of
milk, or the Roman historian Plinius who in the year 76 B.C.
recommended the use of fermented milk products for treating
gastroenteritis (Senmier and De Vrese 2001).
[0004] At the beginning of the last century, the Russian
microbiologist Elie Metchnikoff (1845-1916) suggested that the
consumption of fermented milk modulated the intestinal microbiota
producing a positive effect in human health (Metchnikoff 1908). He
fixed his attention to the fact that there was an incredible number
of centenarians in Bulgaria, despite of it being one of the poorest
European countries. He observed that the Bulgarians consumed large
amounts of yoghurt. Metchnikoff successfully isolated the bacteria
responsible for producing the yoghurt and used it in his
researches. It was the start of the probiotic study. Metchnikoff
became a strong defender of the concept that diet can protect the
body from pathogen invasion and therefore improves and prolongs the
quality of life. He was also the first person to develop a
preparation using lactobacillus in the form of capsule to be
ingested orally, called Lactobacillin.
[0005] At the same time, the French microbiologist Tissier observed
that the fecal microbiota of breast-feeding newborns have more
bacteria from the genus Bifidobacterium than the fecal microbiota
of children who have received artificial milk and acknowledged the
beneficial role of this microorganism.
[0006] Later in 1940, Bifidus Milk appeared to alleviate children
nutritional deficiencies during the 1.sup.st World War. In 1950,
the Degusta factory prepares Biogur and Bio-garde. In 1989, the
consumption and production of fermented milk increases in
Switzerland. In 1993, two researchers, Modler and Vila-Garcia,
developed the first low acidity bio yoghurt.
[0007] In 1965, Lilly and Stillwell used the term "probiotic" for
the first time to name the products of gastric fermentation. But
the more valid and widely used definition of probiotic will be that
enunciated much later on by Fuller (Fuller 1992, Fuller 1989).
Probiotics are defined as: "supplements of live microorganisms
which upon being added to as they condition an improvement in
his/her intestinal microbial balance". For the adult human being,
this includes both products derived from fermented milk and
preparations lyophilized with these bacteria.
[0008] In 1998, the International Life Science Institute (ILSI) in
Brussels defined probiotics as live microorganisms, which when
ingested in sufficient amounts, have beneficial effects on health,
which effect is way above the conventional nutritional effects.
They beneficially affect one or several functions of the organism.
They provide a better health condition and wellbeing and/or reduce
the risk of disease. They can be functional for the general
population or for particular groups thereof.
[0009] Today there are criteria for the definition of probiotic
microorganisms:
1. They are of human origin. 2. They are of non-pathogenic nature.
3. They are resistant to destruction by technical processes. 4.
They are resistant to destruction by gastric acid and bile. 5. They
adhere to the intestinal epithelium. 6. They are capable of
colonizing the gastrointestinal tract. 7. They produce
antimicrobial substances. 8. They modulate the immune response. 9.
They influence the human metabolic activities (cholesterol
assimilation, vitamin production, etc).
[0010] The probiotic bacteria can influence all the intestinal
cells and the mechanisms of action of these cells including the
effects on the microbiota (Backhed and Ley 2005), the modulation of
the immune function (Picard et al. 2004; Kalliomaki 2004) and the
increase of the epithelial barrier function (Madsen et al. 2001;
Isolauri & Salminen 2005).
[0011] Among the bacteria with probiotic activity, those from the
genus Bifidobacterium are the most abundant in the intestine with
25% of bacteria in the adult colon and 95% in the breast-feeding
newborn. There are many food products (yoghurt and milk) today
which are supplemented with this type of bacteria. Other strains
which also have probiotic activity are those from the genus
Lactobacillus, which according to "in vitro" studies inhibit the
adhesion of other anaerobic bacteria such as clostridium,
bacteroides, bifidobacterium, pseudomonas, staphylococcus,
streptococcus and enterobacteriaceas (Silva et al. 1987).
[0012] The use of probiotics as medical tool in some pathologies is
very well-received and the proof of the effectiveness thereof is
strong, mainly as the result of the clinical studies and
meta-analysis, for malabsoprtion of lactose, (Adolfsson et al.
2004; Piaia et al. 2003) gastrointestinal infections (Brownlee et
al. 2003) and diarrhea associated to the use of antibiotics
(D'Souza et al. 2002). Furthermore, the use of probiotic bacteria
either bifidobacteria, lactobacillus and/or a mixture thereof have
shown beneficial effects on some digestive diseases. There is much
proof in the literature on the beneficial effects.
[0013] The understanding of the relationship existing between the
components of the intestinal microbiota as well as the interaction
with the host is very complex. The genome facilitates analyzing the
isolated bacteria response to the intestinal conditions, partly
revealing the metabolic capacity of the strains, however, the
conditions in which these capacities can be expressed, as well as
the conditions to enable isolating most of the strains forming the
intestinal microbiota is still largely unknown, it being able to be
identified only by means of molecular tools identifying their
genome partly or completely. Due to this reason the development in
the area of the probiotics and functional foods is in full
development.
[0014] Thus, taking into account that there are different effects
between the probiotic strains and that varieties of bacteria
belonging to the same species may present different physiological
characteristics rendering them different or improved probiotic
properties against other bacteria, the identification and
characterization of the effects of new prebiotic strains is very
important in view of their health and industrial interest.
[0015] The general objective of this study is to isolate probiotic
microorganisms with improve probiotic properties resistance to an
acidic pH, resistance to bile salts and intestinal cell adhesion
for a subsequent use in the food and pharmaceutical industry
especially for using them in infant formula milk.
[0016] The present invention provides and characterizes probiotic
microorganisms isolated from faeces of children fed exclusively
with breast milk.
[0017] The greater resistance of the strains object of the
invention to pH and bile salts confers the probiotic microorganisms
a greater survivability as they pass through the stomach and
intestine and thus increases their colonizing effect and therefore
their antagonic effects against other potentially pathogenic
bacteria. On the other hand, the greater adherence of the
probiotics strains making up the object of the invention to the
human intestinal cells enables a greater action on all immune
system modulation.
OBJECT OF THE INVENTION
[0018] The present invention provides probiotic microorganisms
isolated from faeces of children fed exclusively with breast milk.
Said microorganisms, are used in the food or pharmaceutical
industry, especially for use in infant formula milk, due to their
probiotic properties which have beneficial effects on the health of
those ingesting them.
[0019] For the isolation of said probiotic microorganisms, the
present invention proposes the following specific objectives: a)
isolating lactic acid bacteria stains obtained from faeces of
exclusively breast-fed children; b) evaluating the resistance to pH
and bile salts; and c) evaluating the adherence to intestinal
epithelial cells.
[0020] It is common to look for probiotic bacteria in the faeces of
babies. Furthermore, there is certain tradition recommending that
the probiotics must be of human origin, supposedly because they
would be better implanted in human intestine. However, many of the
isolated strains do not meet the probiotic condition since they are
rarely or are not resistant to the digestive juices and many of
them do not adhere to the intestinal epithelium. In the present
invention babies fed exclusively with breast milk are selected to
assure that the bacteria isolated is not commercial bacteria.
Furthermore, it has been shown that the intestinal microbiota of
babies fed with breast milk is very rich in bifidobacteria and
lactobacillus.
[0021] Relating to the probiotics, an effective probiotic must be
characterized by:
1. Its capacity to exert a beneficial effect on the host eg.
resistance to diseases. 2. It does not cause pathogenicity or
toxicity. 3. Its capacity to survive upon passing through the
intestinal tract. Eg. resistance to gastric acid and bile acids. 4.
Its capacity to maintain the adherence to the cells of the
intestinal wall. 5. Its short, stable generation time and its
ability of being viable for long periods under storage conditions.
6. It is of human origin. 7. It produces antimicrobial substances
against pathogens, antineoplastic properties. 8. Its ability of
influencing the metabolic activity.
[0022] Among the health advantages associated to probiotic
ingestion are:
1. Relief of symptoms derived from the malabsorption of lactose. 2.
Increment of the natural resistance to infectious diseases of the
intestinal tract. 3. Reduction of the serum cholesterol
concentration. 4. Digestion improvement. 5. Stimulation of
gastrointestinal immunity. 6. Development of immunotolerance to
food antigens and reduction of the risk of allergies
[0023] Thus, the present invention relates to new probiotic
microorganisms isolated from faeces of babies. The invention
specifically relates to the Lactobacillus rhamnosus HERO 22A (CNCM
1-4036), Lactobacillus paracasei HERO 7 (CNCM 1-4034) and
Bifidobacterium breve HERO 15B (CNCM I-4035) microorganisms. Said
microorganisms have improved probiotic properties against
microorganisms of the same species.
[0024] Formulation relates to the compositions or group of one or
several ingredients, i.e., class and number of the elements present
in a complex substance (food product or dosage form, among others)
and proportion in which they are found.
[0025] "Carrier" is understood as any type of substance allowing
the growth, transportation and/or administration of the strains of
the present invention. Depending on the purpose and/or use to which
said strains are intended for, the "carriers" could be of different
nature. The present invention relates to pharmaceutically
acceptable "carriers" such as those commonly associated to
capsules, tablets or powder, as well as a "carriers" formed by
ingredients or food products.
[0026] The food products intended for special diets are food
products which due to their particular composition or the
particular process for manufacturing them, are clearly
distinguished from the staple food products, which are suitable for
the nutritive objective indicated and which are marketed indicating
that they fulfill said objective. (Council Directive 89/398/EEC of
3 May 1989 related to the approximation of the laws of the Member
States on the food products intended for special diets (DO series L
no. 186 of 30 June)).
[0027] Special diets is understood as that which must fulfill the
particular nutritional needs of:
i) determined classes of people who have assimilation process or
metabolism disorder, or ii) determined classes of people who are in
particular physiological conditions and who, therefore, obtain
special benefits from a controlled ingestion of determined food
substances, or iii) babies or young children with good health.
[0028] Food supplement relates to those food products the purpose
of which is to complement the normal diet and consisting of
concentrated sources or nutrients or other substances which have a
nutritional or physiological effect in a single or combined manner,
marketed in dosage form, i.e. capsules, pills, tablets, pastilles
and other similar forms, powder sachets, liquid ampoule, bottles
with droppers and other similar forms of liquids and powders which
must be taken in small unitary amounts; (Directive 2002/46/EC of
the European Parliament and Council of 10 Jun. 2002 related to the
approximation of the laws of the Member States in food supplement
matter).
[0029] Probiotic relates to those microbial cell preparations or
microbial cells or components, of microbial cells with a beneficial
effect on the health and the wellbeing of the host.
[0030] Prebiotic is understood as "a non-digestible ingredient from
the diet which benefits and stimulates the intestinal bacteria
growth improving the intestinal balance of the host". Among the
prebiotics used are: inulin, oligofructosaccharides,
galactooligosaccharides, oligosaccharides coming from the
hydrolysis of pectins and other rubbers and mucilages, and
resistant starches and maltodextrins as well as nucleotides.
[0031] An object of the present invention relates to the strain of
probiotic microorganism isolated from faeces of children fed
exclusively with breast milk characterized by consisting
Lactobacillus rhamnosus HERO 22A (CNCM 1-4036) or Lactobacillus
paracasei HERO 7 (CNCM 1-4034) or Bifidobacterium breve HERO 15B
(CNCM I-4035).
[0032] In a particular embodiment, said strain is Lactobacillus
rhamnosus HERO 22A (CNCM I-4036).
[0033] In another particular embodiment, said strain is
Lactobacillus paracasei HERO 7 (CNCM 1-4034).
[0034] In another particular embodiment, said strain is
Bifidobacterium breve HERO 15B (CNCM I-4035).
[0035] In an embodiment, the strain previously described is
presented in the form of pure biological culture. In another
embodiment the strain is isolated.
[0036] In an embodiment the strain of microorganism previously
described is presented in the form of viable cells; in another
embodiment the strain is presented in the form of non-viable
cells.
[0037] Another object of invention relates to the formulation
comprising a strain of microorganism as has been previously
described. In a particular embodiment, said formulation comprises
another probiotic material, in another embodiment, it additionally
comprises prebiotic material.
[0038] In another particular embodiment the formulation described
comprises a carrier suitable for ingestion. Said carrier is
pharmaceutically acceptable such as those commonly associated to
capsules, tablets or powder.
[0039] In another particular embodiment said carrier is a food
product. Said food product is selected from the group consisting of
milk and milk-derived products, especially fermented milk and
cheeses; cereals and derivatives including bread doughs; soups and
other similar products in dehydrated form; fermented meat products;
fruit derivatives, juices and soft drinks; foods for specific
nutritional uses.
[0040] Another object of invention relates to the strain of
probiotic microorganism or the formulation previously described for
use in diets. In an embodiment, said diets relate to infant and/or
adult and/or special diets.
[0041] In another embodiment, the strain of probiotic microorganism
or the formulation previously described are used for the
preparation of infant formula milk. In a particular embodiment,
said formulas consist of ready-to-eat infant milk and/or infant
cereals, and/or infant foods.
[0042] In another embodiment, the strain of probiotic microorganism
or the formulation previously described are used for the
preparation of food supplements.
[0043] In another embodiment, the strain of probiotic microorganism
or the formulation previously described are used for the
preparation of special formulas for oral and/or enteral
nutrition.
[0044] In another embodiment the strain of probiotic microorganism
or the formulation previously described are used for the
preparation for pharmaceutical application/applicable as medicinal
product/for use in the preparation of a pharmaceutical product.
[0045] In another embodiment, the strain of probiotic microorganism
or the formulation previously described are applicable in
stimulating the immune system and/or in preventing/treating asthma
and/or in preventing/treating gastrointestinal disorders, and/or in
eliminating/modulating the main digestive pathogens, and/or in
preventing/treating obesity and its co-morbidities included the
metabolic syndrome and diabetes and/or in the aging-associated
diseases.
[0046] Said gastrointestinal disorders comprise alterations of
intestinal transit such as constipation and alterations of the
bioavailability of minerals, infections and malabsorption
syndromes.
[0047] Said malabsorption syndromes comprise disorders affecting
the anatomy of the intestine such as short bowel syndrome and
disorders affecting the physiology of the intestine such as cystic
fibrosis of the pancreas, malabsoprtion of sugar especially
lactose, alterations of the lipid absorption, food allergies, and
inflammatory bowel diseases such as Crohn's disease and ulcerative
colitis.
DESCRIPTION OF THE DRAWINGS
[0048] FIG. 1 shows, through a table, the influence of pH on the
survival of the Lactobacillus rhamnosus CNCM I-4036 and
Lactobacillus paracasei CNCM I-4034 strains, object of the
invention, compared with two commercialized strains. Specifically,
said table shows the values of viability (in % of colony forming
units from the pH resistance studies of the isolated Lactobacillus
rhamnosus 22A (CNCM I-4036), Lactobacillus paracasei 7 (CNCM
I-4034) strains and their respective commercial controls. Said
values are shown as % of survival by comparing the number of
bacteria present in the control to the number of bacteria present
at the different pH tested. The results are expressed in units of
percentage in the % column. It can be observed that at pH 3 the
strains 7 and 22A have a similar or slightly higher resistance than
the tested commercial strains. Nevertheless, at pH 2 the strain 22A
has a very high viability compared with the rest of the strains
which do not survive at this pH.
[0049] FIG. 2 shows, through a table, the influence of the bile
salts (Oxgall) on the survival of the Lactobacillus rhamnosus CNCM
I-4036 and Lactobacillus paracasei CNCM 1-4034 strains, object of
the invention, compared with two commercialized strains. Thus, said
table shows the values of viability from the bile salt resistance
studies of the isolated strains Lactobacillus rhamnosus 22A (CNCM
1-4036), Lactobacillus paracasei 7 (CNCM 1-4034) and their
respective commercial controls. Said values are shown as % of
survival in comparison with the number of bacteria present in the
control to the number of bacteria present at the different
concentrations of bile salts tested. The results are expressed in
units of percentage in the % column. As inferred from the results,
both strains 7 and 22A show a much higher percentage of survival,
about two times higher, than the tested commercial strains both at
the concentrations of 0.3% and 0.7% of bile salts. Both strains
have a survival rate greater than 100% indicating that they can
even reproduce in the presence of these salts. This indicates,
together with their high resistance to pH, a high colonizing
potential of the strains.
[0050] FIG. 3 shows, through a table, the adhesion to human
intestinal HT-29 cells of the Lactobacillus rhamnosus CNCM I-4036
and Lactobacillus paracasei CNCM I-4034 strains, object of the
invention, compared with two commercialized strains. Said capacity
is shown through the values of viability from the intestinal
epithelial cell adhesion studies of the isolated Lactobacillus
rhamnosus 22A (CNCM 1-4036), Lactobacillus paracasei 7 (CNCM
1-4034) strains and their respective commercial controls. Said
values are shown as % of bacteria adhered in comparison with the
number of bacteria present in the control. Both strains present
percentages of adhesion to the human intestinal HT-29 cells much
higher than that of the tested commercial strains, which indicates
their potential action in modulating the intestinal cell activities
included immunomodulation.
[0051] FIG. 4 shows, through a table, the influence of pH on the
survival of the strain Bifidobacterium breve CNCM 1-4035, object of
the invention, compared with two commercialized strains. Said
influence is shown through the values of viability from the pH
resistance studies of said strain in comparison to its respective
commercial controls. Said values are shown as % of survival by
comparing the number of bacteria present in the control to the
number of bacteria present at the different tested pH. The results
are expressed in units of percentage in the % column. It can be
observed that at pH 3 the strain 15B shows a resistance
significantly much greater than that of the other two
bifidobacteria tested, its viability being 100% greater than 100%
indicating that the bacteria can even reproduce at that pH.
[0052] FIG. 5 shows, through a table, the influence of the bile
salts (Oxgall) on the survival of the strain Bifidobacterium breve
CNCM I-4035, object of the invention, compared with two
commercialized strains. This influence is shown by means of bile
salt resistance studies of the isolated strain Bifidobacterium
breve 15B (CNCM I-4035) and its respective commercial controls.
Said values are shown as % of survival by comparing the number of
bacteria present in the control to the number of bacteria present
at the different concentrations of bile salts tested. The results
are expressed in units of percentage in the % column. The values of
survival in the presence of bile salts at low concentrations are
similar to those of the other two bifidobacteria. However, the
strain 15B shows a greater survival at higher concentrations.
[0053] FIG. 6 show, through a table, the adhesion to human
intestinal cells of the Bifidobacterium breve CNCM I-4035 strain,
object of the invention, compared with two commercialized strains.
Said capacity is shown through the values of viability from the
intestinal epithelial cell adhesion studies of the isolated strain
Bifidobacterium breve 15B (CNCM I-4035) and its respective
commercial controls. Said values are shown as % of bacteria adhered
in comparison with the number of bacteria present in the control.
The strain object of the invention has a percentage of adhesion to
the human intestinal HT-29 cells much higher than that of the
commercial strains tested, which indicates its potential action in
modulating of intestinal cell activities including
immunomodulation.
[0054] FIG. 7 shows the enzymatic activities (in units of: Units/ml
of culture medium) of the strain Bifidobacterium breve CNCM I-4035
(Bifidobacterium breve 15B), object of the invention, compared with
two commercialized strains (its controls). The results are
expressed as described in Example 11. The results obtained allow
concluding that the fermentative activity of CNCM I-4035 coincides
with that of a species from the genus Bifidobacterium, allowing
classifying CNCM I-4035 inside said genus
[0055] FIG. 8 shows the enzymatic activities of the Lactobacillus
rhamnosus CNCM 1-4036 (Lactobacillus rhamnosus HERO 22A) and
Lactobacillus paracasei CNCM 1-4034 (Lactobacillus paracasei HERO
7) strains, object of the invention, compared with two
commercialized strains (controls). The results are expressed as
described in Example 11. The results obtained allow concluding that
the fermentative activity of HERO 7 and HERO 22A coincides with
that of a species from the genus Lactobacillus paracasei and
Lactobacillus rhamnosus, allowing to classify CNCM I-4036 and CNCM
I-4034 inside said respective genus and species.
[0056] FIG. 9-FIGS. 9A and 9B show the results of the fermentative
activities of carbohydrates and other substrates (API 50 CHL) of
the selected CNCM I-4034 (Lactobacillus paracasei HERO 7) and CNCM
I-4036 (Lactobacillus rhamnosus HERO 22A) strains and their
controls. The results are expressed as described in Example 11. The
results obtained allow concluding that the fermentative activity of
HERO 7 (CNCM I-4034) and HERO 22A (CNCM I-4036) coincide with that
of the species from the genus Lactobacillus paracasei and
Lactobacillus rhamnosus, allowing classifying CNCM I-4036 and CNCM
I-4034 inside said genus.
[0057] FIG. 10-FIGS. 10A, 10B and 10C show the results of the
effect exerted by the probiotic bacteria of the present invention
on L. monocytogenes CECT 4031 and S. sonnei CECT 457. (A) effect
exerted on L. monocytogenes CECT 4031 from supernatant concentrated
10.times. obtained after 17 h of L. paracasei CNCM I-4034 growth.
(B) effect exerted on L. monocytogenes CECT 4031 from supernatant
concentrated 10.times. obtained after 24 h of B. breve CNCM I-4035
growth. (C) effect exerted on S. sonnei CECT 457 from supernatant
concentrated 10.times. obtained after 24 hours of L. rhamnosus CNCM
I-4036 growth.
[0058] FIG. 11 shows the results of the inhibitory effects of the
1% and 4% neutralized and non-neutralized Lactobacillus paracasei
CNCM I-4034 supernatant at 17 and 24 h of culture time for the
bacteria Salmonella typhi CECT 725, Salmonella typhimurium CECT 443
and Salmonella typhimurium CECT 4594. (A) inhibitory effect of
non-neutralized Lactobacillus paracasei CNCM 1-4034 at 17 h of
culture time for the bacteria Salmonella typhimurium CECT 443. (B)
inhibitory effect of non-neutralized Lactobacillus paracasei CNCM
I-4034 at 24 h of culture time for the bacteria Salmonella
typhimurium CECT 4594. (C) inhibitory effect of non-neutralized
Lactobacillus paracasei CNCM 1-4034 at 24 h of culture time for the
bacteria Salmonella typhi CECT 725. (D) inhibitory effect of
neutralized Lactobacillus paracasei CNCM I-4034 at 24 h of culture
time for the bacteria Salmonella typhi CECT 725. (E) inhibitory
effect of non-neutralized Lactobacillus paracasei CNCM 1-4034 at 17
h of culture time for the bacteria Salmonella typhi CECT 725
[0059] FIG. 12 shows the inhibitory effects of the 1% and 4%
neutralized and non-neutralized Bifidobacterium breve CNCM I-4035
supernatant at 17 and 24 h of culture time for the bacteria
Salmonella typhi CECT 725. (A) inhibitory effects of the 1% and 4%
non-neutralized Bifidobacterium breve CNCM I-4035 supernatant at 17
h of culture time for the bacteria Salmonella typhi CECT 725. (B)
inhibitory effects of the 1% and 4% neutralized Bifidobacterium
breve CNCM I-4035 supernatant at 17 h of culture time for the
bacteria Salmonella typhi CECT 725. (C) inhibitory effects of the
1% and 4% non-neutralized Bifidobacterium breve CNCM I-4035
supernatant at 24 h of culture time for the bacteria Salmonella
typhi CECT 725. (D) inhibitory effects of the 1% and 4% neutralized
Bifidobacterium breve CNCM I-4035 supernatant at 24 h of culture
time for the bacteria Salmonella typhi CECT 725.
[0060] FIG. 13 shows the inhibitory effects of the 1% and 4%
neutralized and non-neutralized Lactobacillus rhamnosus CNCM I-4036
supernatant at 17 and 24 h of culture time for the bacteria
Salmonella typhi CECT 725, Salmonella typhimurium CECT 4594,
Escherichia coli ETEC CECT 501, Escherichia coli ETEC CECT 515,
Escherichia coli EPEC CECT 729 and Escherichia coli EPEC CECT 742.
(A) inhibitory effects of the 1% and 4% non-neutralized
Lactobacillus rhamnosus CNCM I-4036 supernatant at 17 h of culture
time for the bacteria Salmonella typhi CECT 725. (B) inhibitory
effects of the 1% and 4% non-neutralized Lactobacillus rhamnosus
CNCM I-4036 supernatant at 24 h of culture time for the bacteria
Salmonella typhi CECT 725. (C) inhibitory effects of the 1% and 4%
neutralized Lactobacillus rhamnosus CNCM I-4036 supernatant at 24 h
of culture time for the bacteria Salmonella typhimurium CECT 4594.
(D) inhibitory effects of the 1% and 4% non-neutralized
Lactobacillus rhamnosus CNCM I-4036 supernatant at 17 h of culture
time for the bacteria Escherichia coli ETEC CECT 501. (E)
inhibitory effects of the 1% and 4% non-neutralized Lactobacillus
rhamnosus CNCM I-4036 supernatant at 24 h of culture time for the
bacteria Escherichia coli ETEC CECT 501. (F) inhibitory effects of
the 1% and 4% non-neutralized Lactobacillus rhamnosus CNCM 1-4036
supernatant at 17 h of culture time for the bacteria Escherichia
coli ETEC CECT 515. (G) inhibitory effects of the 1% and 4%
non-neutralized Lactobacillus rhamnosus CNCM I-4036 supernatant at
17 h of culture time for the bacteria Escherichia coli EPEC CECT
729. (H) inhibitory effects of the 1% and 4% neutralized
Lactobacillus rhamnosus CNCM I-4036 supernatant at 24 h of culture
time for the bacteria Escherichia coli EPEC CECT 742.
[0061] FIG. 14 shows the reduction of focus of infection obtained
in the virus Ito, Wa and VA70 on the HT-29 line from supernatants
concentrated 1.times. of the strains of the present invention after
(A) 17 h of growth and (B) 24 h of growth.
DETAILED DESCRIPTION OF THE INVENTION
[0062] The present invention provides probiotic microorganisms with
improved probiotic properties of resistance to pH, bile salt and
adhesion. Specifically, the present invention isolates and
characterizes the bacteria Lactobacillus rhamnosus HERO 22A (CNCM
I-4036), Lactobacillus paracasei HERO (CNCM I-4034) and
Bifidobacterium breve HERO 15B (CNCM I-4035), isolated from faeces
of babies.
[0063] It is known that the mucosal surfaces are colonized by a
large amount and a great diversity of microorganisms. In adults,
there are numerically more prokaryotic cells than eukaryotic cells,
in fact it is estimated that 90% of human cells are microbial
cells, whereas only 10% correspond to eukaryotic cells (Savage
1977). The influence of this microbial community on human
physiology is probably more obvious in the intestine due to the
fact that this organ contains most of these organisms. The density
in the proximal and mid-small intestine is relatively low, but
there is a considerable increase in the distal small intestine
which can reach 10.sup.8 cfu/ml of luminal content and in the colon
up to 10.sup.11-10.sup.12/g.
[0064] During the first few days of life, there is a great change
in the composition of the intestinal microbiota. In birth, the
intestine is sterile and within the first few hours of life the
bacteria start to appear in the stools. The gastrointestinal tract
is first colonized by the maternal vaginal and fecal bacterial
flora. The first microorganisms colonizing the intestine are those
which have a high reducing power, including species such as
enterobacteria, streptococcus and staphylococcus. The consumption
of oxygen by these bacteria gradually changes the intestinal
environment, allowing the growth of anaerobic bacteria including
lactobacillus and bifidobacteria. These bacteria colonizing the
newborn are mainly from the mother and the environment, the type of
delivery being one of the main determinants of the intestinal
microbiota (Bezirtzoglou 1997).
[0065] The intestinal ecosystem is formed by the interaction
between the microbiota, the intestinal epithelium, mucosal immune
system, and enteric nervous system (Gordon et al. 1997). The
comparison of normal rats and rats with germ-free intestine has
revealed a series of anatomic, biochemical and physiological
differences. For example, the presence of the microbiota increase
the epithelial exchange, it also conjugates and removes the
hydroxyl group from the bile acids, metabolizes the bilirubin and
reduces the cholesterol to coprostanol.
[0066] Therefore, the relationship between intestine and microbiota
is very close and it can be seen as a symbiosis relationship
because, for example, the microbiota can degrade carbohydrates that
cannot be degraded by the intestine due to lack of enzymatic
machinery. The products generated by this degradation are mainly
used as nutrients for the intestinal epithelium as occurs with the
short chain fatty acids. Furthermore, the presence of this
microbiota has an immunomodulation effect since the main
physiological characteristic of the intestinal mucosa is the
capacity of starting an energy response against invasive pathogens
which can colonize the intestinal epithelium, and at the same time
having a zero response to bacteria contained in foods or against
the resident microbiota. This lack of response is an active process
of several mechanisms known as oral tolerance. This process is
essential so that the host does not develop an inflammatory
response to the presence of any microorganism and thus can have
different responses to different microorganisms, thus aiding the
stability of the intestinal flora. This is the reason why having a
microbiota of normal composition can aid the host in physiological,
immune and metabolic development. This ecosystem is maintained in
equilibrium and any cause disrupting this equilibrium can trigger a
pathology (diarrheas, inflammatory diseases).
[0067] It is of interest to understand the importance of this
ecosystem, the function of non-pathological strains or "good
bacteria". The concept of probiotics as human health mediators has
been developed on this idea. Within the different areas studied,
the influence of the probiotics on the modulation of the gene
expression in different situations is one of the most
interesting.
[0068] As indicated previously, the present invention isolates
lactic acid bacteria and bifidobacteria with improved probiotic
properties from faeces of exclusively breast-fed children. To that
end, the pH resistance, bile salt resistance and the intestinal
epithelial cell adhesion capacity of the isolated bacteria were
assessed. The fundamental results of the present invention indicate
that in the faeces of these babies there are bacteria which are
highly resistant to gastric pH, to bile salts and with intestinal
epithelial cell adhesion capacity, which can be used as
probiotics.
[0069] To check the probiotic activity of the bacteria, they must
first be subjected to a series of in vitro assays simulating the
conditions at which said bacteria will be subjected to in the
organism, they must remain viable in said conditions and therefore
preserve their beneficial health properties. In the present
invention, the control bacteria used (Example 12) which in
comparison to the bacteria of the invention show their improved
probiotic properties are, for Bifidobacteria: Bifidobacterium
bifidum and Bifidobacterium longum supplied by Hero Espana S. A.,
and for Lactobacillus, 2 commercial lactobacillus are used:
Lactobacillus casei (Danone.RTM.) and Lactobacillus rhamnosus GG
(LGG) (VALIO.RTM.).
[0070] By taking into account the interest of the bacteria making
up this invention in the stimulation of the immune system and in
its action on the main digestive pathogens, these control bacteria
have been selected because they are marketed today on an
international level in the form of fermented milk and in other
dosage forms and there are several publications on their probiotic
effects especially in preventing acute diarrhea in children and
modulating the immune system in both animals and humans
[0071] Thus, the in vitro assays indicated relate to:
Resistance to Gastric Acidity
[0072] Before reaching the intestinal tract, the probiotic bacteria
must survive their passage through the stomach (Henriksson et al.
1999). The gastric acid secretion in the stomach forms a first
defense mechanism against most of the microorganism load entering
through the oral route. Therefore, the survival of the bacterial
strains in gastric acid is the most accurate indication of their
ability to pass through the stomach. The University College of
Cork-based Probiotic Research Group successfully isolated and
identified lactic acid bacteria which showed ideal probiotic
features (Dunne et al. 1999). Preliminary experiments were carried
out to determine the degree of initial resistance which the
lactobacillus and bifidobacteria strains isolated from the human
ileum have. The human gastric juice was obtained from healthy
individuals by aspiration through a nasogastric tube. Since the pH
in the stomach fluctuates (it can reach 1.5), it was measured
before use. This acid was added to the RSM medium (Rogosa Sharpe
Medium). The initial survival of the strains was evaluated as 108%
and 106% for Lactobacillus and Bifidobacterium, respectively, in
RSM medium (De Man et al 1960) with HCl changing the pH value
between 2.0 and 3.4. The results showed a great Bifidobacterium
sensitivity to the acidity (Thornton 1996). Thus, the strains
making up the present invention have resistance values greater than
those of the strains studied in said experiments, i.e. they have
greater resistance to acidity (FIGS. 1 and 4).
Resistance to Bile Salts
[0073] As has been explained above, to characterize a probiotic
potential, this must also be capable of resisting bile salts (Lee
and Salminen 1995). Bile acids are synthesized in the liver from
cholesterol and are secreted from the gall bladder to the duodenum
in the form of conjugates (500-700 mL/day) these acids experience
more chemical modifications (deconjugation, dehydrolyzation,
dehydrogenation, and deglucuronidation) in the colon almost only as
a result of the microbial activity. Both conjugated and
deconjugated acids have antibacterial activity inhibiting the
growth of Escherichia coli, Klebsiella sp., and Enterococcus sp
strains in vitro (Lewis et al 1972; Stewart et al 1986). The
deconjugated forms are more inhibitory and the gram positive are
more sensitive than the gram negative (Floch et al. 1972;
Percy-Robb 1972). The Dunne group (Dunne et al. 1999) has chosen,
in a first assay, to use a solid growth medium supplemented with
bovine, porcine and human bile acid up to a final concentration of
0.3% and 7.5% to assess the bile salt resistance of the strains.
After allowing the lactobacillus and bifidobacteria to grow, the
result was that they showed resistance to the bovine bile acid and
that the result of porcine bile acid was much more inhibitory for
both bacterial groups (Thornton 1996). Thus, relating to the search
for the possible probiotic for human consumption, the most relevant
result is the capacity thereof to grow in human bile. By taking
into account that human bile is not standardized and that their
bile acid content greatly varies from one individual to another,
the use of bovine bile standardized in its bile acid content
(OXGALL) as the substitute of the human biles is a common practice
in the state of the art, allowing protocolizing reproducible
assays. This has been the method followed in the present invention
for studying the bile salt resistance of the bacteria making up the
present invention. The results obtained show that said bacteria
have greater resistance to bile salt than their commercial controls
(FIGS. 2 and 5).
Intestinal Epithelium Adherence of Probiotic Strains
[0074] The adhesion of the strains adhered to the intestinal
epithelia tissue and the ability to colonize the gastrointestinal
tract must also be evaluated in the selection. The importance of
this action resides in the fact further capable of colonizing their
target host. In fact, of the probiotics available today, it seems
that only L rhamnosus GG remains inside the gastrointestinal tract
for a significant time period (Berg et al. 1998; Goldin et al. 1992
L. rhamnosus GG adheres to Caco-2 cells, HT-29 cells and Caco-2
cells belonging to human intestinal cell lines, they express the
morphological and physiological characteristics of a normal human
colonocyte and are used to test the mechanisms mediating the
enteropathogen adhesion (Bernet 1994). In recent studies they have
been used for the selection performed, and thus evaluating the
possible lactic acid bacteria or bifidobacteria based on their
adhesion capacity (Coconnier et al 1992; Bernet et al. 1993; Greene
& Klaenhammer 1994; Crociani et al 1995; Sarem et al. 1996;
Tuomola & Salminen 1998).
[0075] From the studies performed with these cell lines it is
inferred that the adherence of the lactobacillus strains of the
present invention which is of the order of 9% on Caco-2 cells and
of the order of 5% on HT-29 cells (Tuomola et al, 1998; Dunne et
al, 2001; Dotes et al, 2008) is very high (7.5% for L. rhamnosus
CNCM I4036 and 15.5% for L. paracasei CNCM I-4034 (FIG. 3) in
comparison with the well characterized Lactobacillus rhamnosus GG
strain. The state of the art contemplates that the adhesion of the
bifidobacteria is small in comparison with the lactobacillus,
regardless of their species (Dunne et al, 2001). However, the
Bifidobacterium bifidum and B. longum strains used by the company
HERO Espana, which have been considered as controls in the present
invention, adhere to HT-29 cells with values close to 9%. Likewise,
the bifidobacteria object of the present invention adhere to said
cells with a much greater value of 16.7% (FIG. 6).
Selection of the Bacteria
[0076] Thus, in the present invention, the bacteria was selected
using specific culture media (Example 4) both for bifidobacteria
and for lactobacillus. During the isolation three new culture media
described as being specific for bifidobacteria were used, these
are: BFM medium (Nebra and Blanch 1999), Modified Columbia medium
and Beerens medium (Beerens 1991. Examples 4.1, 4.2 and 4.3),
whereby better result in obtaining bifidobacteria colonies was
obtained. To select lactobacillus, the culture medium used was
Rogosa agar medium (Example 4.4).
[0077] In the present invention, the bacteria colonies from the
different children which were incubated and subjected to the
selection tests were 4680 colonies. After the first assay of
resistance to pH 3.0 and to bile salts, there were 758 colonies
with a viability of 90%, after the tests of adhesion to the
intestinal epithelial cells there are only 90 colonies (Examples 5,
6 and 7)
[0078] These colonies were separated into lactobacillus and
bifidobacteria according to the culture medium of origin. Their
molecular identification (Example 10) by means of amplifying the
16S rRNA gene of each colony for the subsequent sequencing and
homology search thereof in the NCBI (BLAST) database was directly
performed.
[0079] Finally, there were 29 bacterial strains isolated from the
Beerens medium, 13 from the Rogosa medium and 10 from the modified
Columbia medium, which successfully passed the selection. Given the
number of the selected colonies, as has been explained, their
molecular identification by means of amplifying the 16S rRNA gene
of each colony for the subsequent sequencing and homology search in
the NCBI (BLAST) database was directly performed.
[0080] The strains classified as lactobacillus were sequenced and
these sequences were aligned with each other to know whether
bacteria with the same 16s rDNA gene existed, and 41 bacteria which
can be separated into 2 groups were found:
[0081] A group which had a homology of 99% of a 1474 bp fragment of
the 16s rDNA gene with:
Lactobacillus rhamnosus strain R-11 Lactobacillus rhamnosus strain
La Lactobacillus rhamnosus, strain: MNFLM01 Lactobacillus rhamnosus
strain IDCC 3201 Lactobacillus rhamnosus strain: YIT 0105 (=ATCC
7469) Lactobacillus rhamnosus strain Lcr35 16S
[0082] With these results, a bacteria with the best values from the
resistance tests was selected from the group (Examples 7 and 9) and
was called Lactobacillus rhamnosus HERO 22A, (subsequently numbered
by the Pasteur Institute [CNCM National Collection of Microorganism
Culture PASTEUR INSTITUTE 25, Rue du Docteur Roux F-75724 Paris] as
Lactobacillus rhamnosus CNCM 1-4036 where it was deposited on 2
Jul. 2008).
[0083] The other group which had a homology of 1000 of a 1276 bp
fragment of the 16s rDNA gene with:
Lactobacillus paracasei, strain: T11-9 Lactobacillus paracasei,
strain: T7-10 Lactobacillus casei strain KLDS 1.0720 Lactobacillus
casei strain L5 Lactobacillus casei, strain: YIT 0209 (=NCDO 151)
Lactobacillus casei, strain: YIT 0180 (=ATCC 334) Lactobacillus
paracasei strain IMPC 2.1 Lactobacillus paracasei, strain: NRIC
1944 Lactobacillus paracasei, strain: NRIC 1942 Lactobacillus
paracasei, strain: NRIC 1938 Lactobacillus paracasei, strain: NRIC
1934 Lactobacillus paracasei, strain: NRIC 0638 Lactobacillus casei
ATCC 334 Lactobacillus paracasei strain DJ1 Lactobacillus casei
strain Ru2-2i Lactobacillus paracasei isolate 3C Lactobacillus
paracasei isolate 2C Lactobacillus paracasei Lactobacillus casei
strain MCRF-284 Lactobacillus sp. L02 Lactobacillus paracasei
Lactobacillus paracasei, strain SM20 Lactobacillus casei strain
BL23 Lactobacillus paracasei subsp. Paracasei Lactobacillus
paracasei subsp. Paracasei Lactobacillus casei Lactobacillus
paracasei subsp. Tolerans
[0084] With these results, a bacteria with the best values of the
resistance tests (Examples 7 and 9) was selected from this second
group and was initially called Lactobacillus paracasei HERO 7,
(subsequently numbered by the Pasteur Institute [CNCM National
Collection of Microorganism Culture PASTEUR INSTITUTE 25, Rue du
Docteur Roux F-75724 Paris] as Lactobacillus paracasei CNCM 1-4034
where it was deposited on 2 Jul. 2008).
[0085] Subsequently, the same was performed with the group of the
bifidobacteria, only a single group which had a homology of 100% of
a 1136 bp fragment of the 16s rDNA gene with the following being
found:
Uncultured bacterium clone rRNA235 Bifidobacterium breve, strain:
ATCC 15700
[0086] With these results, a bacteria of the group of
bifidobacteria with the best results of resistance (139.6% at pH
2.5) (Examples 7 and 9) was selected and was initially called
Bifidobacterium breve HERO 153, (subsequently renamed by the
Pasteur Institute [CNCM National Collection of Microorganism
Culture PASTEUR INSTITUTE 25, Rue du Docteur Roux F-75724 Paris] as
Bifidobacterium breve CNCM I-4035 where it was deposited the 2 Jul.
2008).
[0087] Thus, these bacteria classified as:
Lactobacillus rhamnosus HERO 22A Lactobacillus paracasei HERO 7
Bifidobacterium breve HERO 15B. and were sent to the Pasteur
Institute for their deposit where they were recognized as unique
and were assigned the following final denomination:
TABLE-US-00001 Initial Denomination: Final Denomination:
Lactobacillus paracasei HERO 7 CNCM I-4034 Bifidobacterium breve
HERO 15B CNCM I-4035 Lactobacillus rhamnosus HERO 22A CNCM
I-4036
Results of Resistance to pH, Bile Salts and Cell Adhesion
[0088] As indicated above, in order for the probiotic strains to
exert a beneficial effect on the intestine, they must survive
passage through the stomach, resisting its acidity (pH 2.5-3.5)
(Holzapfel et al. 1998) and on the other hand, they must be
resistant to bile salts present in the small intestine in order to
reach the colon (Otles et al. 2003).
[0089] In the present study, the bacteria were incubated at pH 3.0
for 3 hours, although it has been described that 90 minutes should
be enough to reproduce the time elapsing between the entrance to
and exit from the stomach (Jin at al. 1998). In this case, the
isolated strains and the controls presented a viability close to
100% (Example 12/FIGS. 1 and 4) but the exposure to pH 2.5 showed
it was very selective because no control presented viability and
only the Lactobacillus rhamnosus 22A (CNCM I-4036) strain did. In
other words, the strain of the invention Lactobacillus rhamnosus
22A is considerably more resistant to acid than the control
bacteria therefore its passage into the gastrointestinal tract and
subsequent colonization are aided. On the other hand, the strain L.
rhamnosus 7A of the invention presents greater viability at pH 3.0
than the strains used as controls (Example 12, FIGS. 1, 4), which
means greater passage into the small intestine.
[0090] The viability of probiotic cultures at pH 3.0 for 2 hours
and in media containing 500-1000 mg (0.05-0.1%) of bile acids per
liter, are considered standard tests of tolerance to acid and bile
salts (Snelling 2005), although concentrations of 0.3% of bile
salts would be suitable for selecting probiotics. The bile salt
tests conducted at different concentrations (0.3% and 0.7%)
presented values greater than 100% in all cases for the bacteria
object of the invention and greater than the commercial bacteria
used as controls (Example 12, FIGS. 2 and 5). In conclusion, the
lactobacillus strains of the invention are more resistant to pH and
bile salts than other bacteria used as probiotics today.
[0091] It has been described that the lactobacillus in general
present a greater resistance to the gastrointestinal conditions,
especially with respect to acidity and bile salts (Ross et al.
2005). The results found are consistent with this description since
the values of resistance to the gastrointestinal conditions are
slightly higher in lactobacillus than in bifidobacteria.
[0092] As also mentioned above, another very important aspect for
the entrance of probiotics into intestinal microbiota is the
adhesion capacity on intestinal epithelial cells, since they
prevent the probiotic strains from being eliminated due to
peristaltic movements and other bacteria forming the intestinal
microbiota. Furthermore, adhesion is the first step in colonization
and it is probably a prerequisite for the competitive exclusion of
enteropathogens (Forestier et al. 2001; Lee et al. 2003) and for
the immunomodulation of the host (Ouwehand et al. 1999; Plant and
Conway 2002).
[0093] In the present invention, the properties of adhesion of the
different strains were studied using HT-29 cells as an in vitro
model of intestinal epithelium (Example 12, FIGS. 3 and 6). The
adhesion pattern showed to be specific for each strain since they
presented very different values even though they are from the same
species. This can be understood by comparing the adhesion capacity
of different probiotics described, for example, Lactobacillus casei
(Fyos.RTM.) presents adhesion of 14.4%, whereas Lactobacillus casei
(Lactophilus.RTM.) presents adhesion of 2.6% (Morata De Ambrosini
et al., 1999). As indicated above, the adherence of the
lactobacillus strains of the present invention is much greater
(7.5% for L. rhamnosus CNCM I4036 and 15.5% for L. paracasei CNCM
I-4034) than the other bacteria such as Lactobacillus rhamnosus GG,
which is of the order of 5% on HT-29 cells (Dunne et al, 2001)
(Table 3). Likewise, the Bifidobacteria, which is one of the
objects of the present invention, B. breve CNCM I-4035, also
adheres to said cells with a value much greater than 16.7% (FIG. 5)
compared with 9% of the bacteria used as controls.
[0094] These data confirm the results of the present invention,
showing the variability existing in the different probiotic
strains. In this specific case, the lactobacillus controls present
adhesion (4%) corresponding to half that of the bifidobacteria
controls (8%), however, the isolated strains in the present
invention present greater adhesion than their controls because when
the 16s rRNA of the different groups were aligned, the ones with
the best values for epithelial adhesion were selected.
[0095] Thus, in the case of lactobacillus the strains which
presented adhesion of the order of 7.48 and 11.55% with respect to
4.80 and 4.09% for their controls (FIG. 3) were selected, and in
the case of bifidobacteria those which presented adhesion of 16.7%,
with respect to 8.8 and 9.1% of their controls (FIG. 6) were
selected. In other words, the bacteria making up the different
objects of the present invention has a greater capacity of
colonizing and remaining in the intestine and will therefore lead
to a better probiotic effect.
Results of the Characterization of the Isolated Bacteria
16s RNA Study
[0096] Different molecular techniques have emerged for the
identification, composition and enumeration of the entire bacterial
community of the intestine, most of which are based on the study of
16s ribosomal RNA gene (rRNA), because in the past decade the 16s
rRNA gene has revolutionized the way that taxonomists classify and
identify bacteria. The 16s rRNA gene comprises regions ranging from
highly variable to highly conserved, and the difference of
sequences are used for determining the phylogenetic relationships
and distinguishing bacteria from species to strains. There are
databases available with more than 200,000 16s rRNA genes, such as
for example NCBI/BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi),
ribosomal database project-RPD (http://rpd.cme.msu.edu/htlm) and
EMBL (http://www.embl-heidelberg.de/), these databases compare the
existing sequences of the 16s rRNA gene with new sequences
obtained. As shown in Example 2, the isolated strains in the
present invention presented high homology with Lactobacillus
rhamnosus, Lactobacillus paracasei and Bifidobacterium breve, which
is consistent with the background documents stating that in a child
fed with breast milk there are high levels of bifidobacteria in
faeces, between 40-60% of the total of the microbiota, where
Bifidobacterium breve is found in a considerable percentage
(Harmsen et al. 2000). There is also a high percentage of
lactobacillus, mainly L. casei, L. paracasei, L. acidophilus, among
others (Heiling et al. 2002, Satokari et al. 2002).
[0097] The percentage of homology of the 16s rRNA gene of the
isolated strains was very high (99-100%) according to the
NCBI/BLAST database (Example 2). The fragments of the 16s rRNA gene
are approximately 1.4 kb, and the secondary fragments range between
1474 bp for Lactobacillus rhamnosus 22A HERO (CNCM I-4036), 1274 bp
for (CNCM I-4034) 7 HERO and 1118 bp for Bifidobacterium breve 15B
HERO(CNCM I-4035). Precisely the latter presents homology of 100%
with Bifidobacterium breve ATCC 15700 and a non-cultivable clone of
Bifidobacterium, therefore amplifying the sequenced fragment of the
16s rRNA gene would be of interest, but in this case a larger
fragment could not be obtained because the oligonucleotides 27F
(SEQ. ID. NO 1) and 1492R (SEQ. ID. NO 2), which amplify a fragment
of approximately 1400 bp, were unable to amplify the 16s rRNA gene
of the Bifidobacterium breve 15B HERO strain (CNCM I-4035).
Therefore, other universal oligonucleotides which amplify smaller
fragments such as 39F (SEQ. ID. NO 3) and 1391R (SEQ. ID. NO 4)
were used.
[0098] The sequenced fragments of the 16s rRNA gene of the isolated
lactobacillus strains show a homology of 99% with a group of
Lactobacillus rhamnosus, and the other strain presents a homology
of 100% with a large variety of Lactobacillus paracasei, and with a
small number of Lactobacillus casei. The fragments of 16s rRNA of
the controls, of the Lactobacillus rhamnosus 22A HERO (CNCM I-4036)
and Lactobacillus paracasei 7 HERO (CNCM I-4034) strains, and a
fragment of L. paracasei which had a high homology with the
isolated strain were aligned. From this, it can be observed that
there is a difference of 4 bases between the control strains, L.
rhamnosus 22A HERO (CNCM I-4036) and L. paracasei 7 HERO (CNCM
I-4034). There is also a difference of 4 bases between the LGG and
L. casei controls. The Lactobacillus rhamnosus 22A HERO strain
(CNCM I-4036) presents a difference of 1 base with both controls, a
difference of 1 base with the LGG control and a difference of 3
bases with L. casei. In this case, there are few differences
between the aligned strains, which would indicate that in these
strains the 16s rRNA gene is fairly conserved.
[0099] In order to broaden the genomic information of the studied
bacterial strains, the intergenic space present between the 16s and
23s genes was amplified, this space being known for having a large
size variability (Barry et al. 1991 & Navarro et al. 1992), and
it was also used for differentiating species of prokaryotes (Barry
et al. 1991). In the isolated strains the fragments of the
intergenic space range in length between Lactobacillus rhamnosus
22A HERO (CNCM I-4036) 579 bp, Lactobacillus paracasei 7 HERO (CNCM
I-4034) 512 bp and Bifidobacterium breve 15B HERO (CNCM I-4035) 182
bp. The 16s-23s intergenic fragments were compared with those in
the NCBI/BLAST database, and the results show a homology of 100%
for the Lactobacillus rhamnosus 22A HERO strain (CNCM I-4036) with
isolated Lactobacillus rhamnosus TS1 and Lactobacillus rhamnosus
PS1 16S. When comparing the homology results shown for 16s rRNA,
the results are completely different, therefore it is possibly a
strain that is not in the database or that the NCBI/BLAST database
has more information about 16s rRNA and not about the 16s-23s
intergenic space. These results indicate that the isolated bacteria
object of the invention is unique, which has been confirmed by the
Pasteur Institute when specifically giving it the name
Lactobacillus rhamnosus CNCM I-4036.
[0100] In the case of the Lactobacillus paracasei 7 HERO strain
(CNCM I-4034), the results of the 16s-23s intergenic space show a
homology of 100% for Lactobacillus casei ATCC 334. The NCBI/BLAST
database contains the complete genome and it is on the list of L.
casei showing a homology of 100% for the 16s rRNA, therefore it is
very probable that the isolated strain is L. casei ATCC 334; in any
case when sequencing other fragments of the genome, such as the 23s
or 5s genes or others, they could ratify or discard the idea that
it corresponds exactly to this strain. In this sense, after the
pertinent analyses, the Pasteur Institute has acknowledged the
strain as unique.
[0101] For the case of the bifidobacteria strain, the fragment is
rather small, therefore the intergenic space of the controls was
amplified, giving 165 bp for Bifidobacterium longum and 298 bp for
Bifidobacterium bifidum, which ratifies the existence of a large
variation in the size of the fragment. The control strains, the
isolated strain, Bifidobacterium breve 15B HERO (CNCM I-4035), and
the strain which showed a homology of 99% (NCBI/BLAST) with the
strain of the invention were aligned, observing a large difference
between the controls and the Bifidobacterium breve strains;
however, there is a difference of only one base between the strain
of the invention and the strain presenting a homology of 99%
(Bifidobacterium breve 16S-23S internal transcribed spacer (ITS),
strain Y8). This strain is completely different from the strain
with a homology of 100% for the 16s rRNA, therefore it could be a
strain that is not entered in this database.
[0102] All these results demonstrate that the three isolated
strains in the present invention are new, as they have not
previously been described in the state of the art.
Phenotypic Identification
[0103] As shown in Example 11, the present used a carbohydrate
fermentation kit (API 50CH) (FIG. 8) and an enzymatic activity kit
(API Zym) (FIG. 9) to analyze the biochemical capacities of the
isolated species. They constitute a rapid and theoretically
reproducible method for the phenotypic identification of pure
bacterial cultures. These tests have been used for characterizing
and identifying lactobacillus in milk (Medina et al. 2001), yoghurt
and other fermented dairy products (Andrighetto et al. 1998), and
cheeses (Andrighetto et al. 1998, Bouton et al. 1998 and De Angelis
et al. 2001). However, the reliability of these tests has been
questioned, especially for API 50CH, because it was initially
developed to identify lactobacillus strains for clinical use, and
because the manufacturer's database has not been updated for some
lactobacillus species, showing ambiguous results for identification
(Andrighetto et al. 1998 and Collins et al. 1993), however, the
information offered is valuable for phenotypically characterizing
the isolated strains.
[0104] As observed in FIG. 8, the isolated strains as well as the
controls present low proteolytic activity functioning as trypsin
and .alpha.-chymotrypsin, although they presented activity that was
different from leucine, for valine they present minimal activity in
bifidobacteria and very high activity in lactobacillus.
[0105] Bifidobacteria also present high activity for .alpha. and
.beta.-galactosidase and for .alpha.-glycosidase. The
.alpha.-galactosidase and .alpha.-glycosidase activity could differ
from the bifidobacteria of other lactic acid bacteria, as described
by Desjardins et al. (1990).
[0106] It is observed that in bifidobacteria and in the isolated
Lactobacillus rhamnosus 22A HERO strain (CNCM I-4036) there is high
.alpha.-galactosidase activity. In other words important activity
because the hydrolysis of specific sugar such as
.alpha.-D-galactosyl-oligosaccharide allows selective proliferation
of bifidobacteria in the intestinal tract (Gopal et al. 2001;
Gulewicz et al. 2002) because bifidobacteria can use
galacto-oligosaccharides. The evaluation of the phenotypic
characteristics of bifidobacteria has allowed confirming their
prior classification by genetic means and determining that their
enzymatic actions allow using several hydrocarbon substrates,
preferably glucose polymers with a bonds for the production of
lactic acid.
[0107] It has been described that few strains of other origins and
genera, including lactobacillus, present .beta.-glucuronidase
capacity (Gopa et al., 2001; Hopkins et al. 1998). Thus, the
lactobacillus and bifidobacteria strains of the present invention
as well as their controls do not present levels of
.beta.-glucuronidase activity, which is interpreted as a favorable
characteristic. The lack of .beta.-glucuronidase activity is a
characteristic that all the probiotics considered to be good must
have because this enzyme is produced by faecal enzymes
(nitroreductase, azoreductase) of a microbial origin, which cause
pro-carcinogens to turn into carcinogens (Kurman 1988). Nanno et
al. (1986) demonstrated that extracts of positive
.beta.-glucuronidase bacteria increase the mutagenic activation of
biliary metabolites of benzopyrene, whereas extracts of negative
.beta.-glucuronidase do not present this activity. In summary, it
can be said that since these bacteria present low activity to other
carbon sources such as mannose, fucose and glucuronides, they
prefer lactose and glucose as carbon sources for metabolism,
although the isolated Lactobacillus rhamnosus 22A HERO strain (CNCM
I-4036) presents enzymatic activity for .alpha.-fucose, which
entails a favorable effect on the fermentation of fucosyl-lactose
derivatives like those present in breast milk, having a known
favoring effect on the growth of bifidobacteria. This property
means that the probiotics of the present invention have a very
particular application in infant diets, although they can also be
applied in adult diets and special diets.
[0108] Another aspect that should be mentioned is the result of API
50CH (FIG. 9B), where the Lactobacillus paracasei 7 HERO strain
(CNCM I-4034) ferments inulin, a capacity that its controls do not
have. The capacity of fermenting inulin is not a common property
among lactobacillus (Cebeci and Gurakan 2003; Makras et al. 2005).
This indicates that the strain of the invention has the capacity to
ferment fructo-oligo-saccharades (FOS), widely used as prebiotics,
in infant diets, promoting the development of intestinal microbiota
in which lactobacillus and bifidobacteria predominate. This
characteristic of the bacteria of the invention reinforces the fact
that the probiotics of the present invention have a very particular
application in infant diets, although they can also be applied in
adult diets and special diets. The capacity of inulin and FOS in
increasing the number of bifidobacteria in the colon has been
demonstrated (Roberfroid et al. 1998; Van Loo et al. 1999).
Probiotic Activity
[0109] Once the aforementioned requirements concerning resistance
to pH, resistance to bile salts, adherence to intestinal epithelial
cells and sequencing 16S have been met, the isolated and defined
probiotic microorganism is had. The next step consists of
characterizing its probiotic activities, specifically, it would be
necessary to characterize the probiotic properties of Lactobacillus
rhamnosus HERO 22A (CNCM I-4036) and/or Lactobacillus paracasei
HERO 7 (CNCM I-4034) and/or Bifidobacterium breve HERO 15B (CNCM
I-4035).
[0110] Taking into account that the bacteria of the invention meet
the requirements of a probiotic in relation to resistance to pH,
bile salts and adherence, said bacteria can be applied in different
areas in which the use of probiotics is known in the State of the
Art, among others, in the treatment and prevention of different
pathologies such as lactose malabsorption, reduction of cholesterol
plasma levels, different types of diarrheas, inflammatory bowel
diseases, cancer, disorders caused by pathogenic bacteria, etc.
Thus, said probiotics will be applicable in different fields, like
in the action on the main digestive pathogens and in the
stimulation of the immune system, among others.
Pathogenic Bacteria
[0111] Among the different types of studies conducted on the
probiotics, those involving pathogenic bacteria of the
gastrointestinal system are of special interest. When choosing
pathogenic bacteria, their mechanism of pathogenicity, which varies
from one to the other, must be taken into account. A bacteria of
interest is enterotoxigenic Escherichia coli because the production
of its enterotoxin is an important cause of diarrhea in humans.
Recent studies have described that certain lactic acid bacteria
have an antagonistic effect with respect to enterotoxigenic
Escherichia coli (Gopal et al. 2001; Todoriki et al. 2001; Chu et
al. 2005; Tsai et al. 2007) and with respect to other pathogenic
bacteria such as Salmonella typhimorium and Shigella flexneri (Tien
et al. 2006; Jankowska et al. 2008).
[0112] Another aspect of interest of the probiotic strains is the
possible production of substances called bacteriocins, which are
secreted by some bacteria in order to compete with other
microorganisms growing in the same niche. These substances can
inhibit the growth or adhesion of pathogenic bacteria on intestinal
epithelial cells, which can be produced by some lactic acid
bacteria (Klaenhamer 1993; Jack et al. 1995; Sablon et al.
2000).
[0113] An important aspect is to know the interaction of the
probiotic or pathogenic bacteria with the intestinal epithelial
cells because all the bacteria present in human microbiota directly
interact with them.
[0114] It has been described that the presence of pathogenic
bacteria in intestinal epithelial cells stimulates a
pro-inflammatory response profile (Th1), releasing cytokines such
as TNF-.alpha. and IL-8, activating NF-.kappa.B, (Tien et al. 2006;
O'Hara et al. 2006) and increasing their expression. This response
in most cases is partially reduced in the presence of a probiotic
bacteria, which would indicate a beneficial effect (Servin
2004).
[0115] Thus, some probiotics could modulate the properties of the
DC, including their capacity to activate a specific immune response
(Kelsall et al. 2002). A stimulation and tolerance equilibrium
after contact with probiotic bacteria in the intestine could be
important for maintaining their homeostasis and being able to carry
out their beneficial protection functions with respect to
pathogenic bacteria in the digestive system of the host.
[0116] The probiotic strains of the present invention shows to have
an inhibitory effect on the growth of intestinal pathogenic
microorganisms, such as pathogenic bacteria, among others,
Helicobacter pylori, Listeria monocytogenes, Shigella sonnei,
enterotoxigenic Escherichia coli, enteropathogenic Escherichia
coli, enteric Salmonella, as well as enteric viruses, such as
Rotavirus.
Lactose Malabsorption
[0117] Mammals are born with sufficient lactase activity for using
lactose from breast milk. After weaning, this activity is gradually
reduced with age, and the intake of food containing it leads to
signs and symptoms related to lactose intolerance (abnormal
increase of gas, abdominal pain, diarrhea, etc.). It is known that
the use of probiotics releasing lactase favors the digestion of
lactose in the lumen of the intestine, fighting the symptoms of
lactose intolerance.
Reduction of Cholesterol Levels
[0118] The high level of blood lipids, such as cholesterol and
triglycerides, implies a high risk to human health due to the
association thereof with heart disease. Since the consumption of
foods with a low fat content or with microorganisms participating
in lipid metabolism is very beneficial for preventing these
conditions, lactobacillus strains regulating serum lipids have been
characterized. This probiotic effect is closely related to the
hydrolysis of bile salts.
[0119] It has thus been observed in different studies in animals
(Akalin et al. 1997; Fukushina and Nakano 1996) and in humans (Lin
et al. 1989) that the administration of probiotics can reduce serum
cholesterol concentration.
Diarrhea
[0120] It is a fact that the best documented clinical application
of a probiotic is the treatment of acute diarrhea. Clinical trials
have shown efficacy of the use of probiotics in the treatment for
the prevention and/or treatment of several intestinal disorders
including antibiotic-induced diarrhea (McFarland et al. 1995),
diarrhea in adults (Hocter et al. 1990), children (Cetina-Sauri and
Sierra 1994) and traveler's diarrhea (Kollaritsch et al. 1993).
[0121] In these cases, the probiotic used as a biotherapeutic agent
affects the expression and the activity of a large number of
enzymes and proteins, regulating the intestinal epithelium and
possibly microbiota.
[0122] In relation to the antibiotic-associated diarrhea, taking
probiotics when an antibiotic is prescribed can reduce the onset
and/or shorten the duration of the diarrhea. The most widely used
microorganisms are: Enterococcus faecium SF6 (Wunderlich et al.
1989), Lactobacillus GG (Siitonen et al. 1990; Vanderhoof et al.
1999), Lactobacillus acidophilus, L. bulgaricus, and Saccharomyces
Boulardii. These agents contribute to reducing the alteration of
the microbiota in the intestine, the change in the consistency of
the stools and the frequency of the latter.
[0123] Diarrhea caused by Clostridium difficile, which is an
opportunistic pathogen that takes advantage of the alteration of
the intestinal microbiota due to taking antibiotics, presents a
broad spectrum of clinical symptoms, ranging from mild benign
diarrhea to intense colitis with the development of toxic
megacolon, intraabdominal and systemic complications that can lead
to patient death. Diarrheas usually occur a few weeks after
beginning antibiotic therapy. The pathogenic sequence begins with
an alteration of the intestinal bacterial microbiota induced by
antibiotics, which allows colonization by C. difficile if the
person is exposed to the intake of this agent. Subsequently, the
bacteria releases toxins causing tissue damage. The pathogenic
strains of C. difficile produce toxins called A and B. S. Boulardii
inhibits toxins A and B by releasing a 54 kDa protease which cuts
out these toxins and their membrane receptors (Castagliuolo et al.
1999).
[0124] It has been seen that the oral administration of
Lactobacillus rhamnosus GG and Saccharomyces boulardii has been
effective in restoring the normal microbiota in patients.
[0125] Traveler's diarrhea is due to a bacterial, viral or
parasitic infection. There are many microorganisms causing it and
they are probably different from one country to another. By
frequency, they include: E. coli, Shigela, Salmonella,
Campylobacter, Rotavirus and Giardia lamblia. Traveler's diarrhea
affects half the travelers going to high-risk areas. The bacteria
used as probiotics in different studies are: Lactobacillus,
Bifidobacterium, Streptococci and Enterococci. Lactobacillus GG has
been the most effective probiotic with respect to traveler's
diarrhea.
Inflammatory Bowel Diseases
[0126] One of the main uses of probiotics involves an imbalance in
the microbiota and immune system. According to this interest, the
study of inflammatory bowel diseases is one of the most interesting
focal points for the possible use of probiotics as clinical
therapies. The studies conducted in this area have provided
important information on the clinical use of probiotics and the
gene expression of different intermediaries involved in these
diseases.
[0127] Inflammatory bowel diseases (IBD) are chronic inflammatory
disorders of the intestine with unknown origin (ulcerative colitis,
Crohn's disease), the pathogenesis is complex and involves at least
3 important elements: genetic susceptibility factors, enteric
microflora and immunity-mediated damaged. It has been hypothesized
that IBDs occur due to an abnormal response of the T cells with
respect to the microbiota, it has also been speculated that the
presence of pathogenic organisms could cause these diseases.
[0128] There is a reduced level of Lactobacillus and Bifidobacerium
in colon biopsies in patients with IBD (Fabia et al. 1993; Favier
et al., 1997). Conventional treatments for IBD focus on suppressing
or modulating the immunity of the host, and among these treatments,
the use of antibiotics is an effective treatment for Crohn's
disease. This would indicate that the use of probiotics for
modifying microflora may be important in the treatment of IBD.
[0129] There is a recent study in the background in which it has
been found that patients with Crohn's disease have reduced amounts
of .beta.-galactosidases in faeces during the active periods of the
disease. This reduction is correlated with the reduction of
Bifidobacteria, which are the source of .beta.-galactosidase
(Favier et al. 1997).
Cancer
[0130] Colorectal cancer is one of the most serious complications
of IBDs, including ulcerative colitis and Crohn's disease (Eaden et
al., 2001). The precise mechanism whereby IBD can generate a
carcinogenic process is not well understood. It is assumed that it
could be the cause of a chronic inflammatory process (Weitzman
& Gordon 1990), which in some experimental models may function
as a tumor promoter.
[0131] The intestinal microbiota and the immune system play an
important role in regulating carcinogenesis. Probiotics can have an
effect in both, hence great effort has been made in this field to
act against colon cancer. It has been found that probiotics can
reduce the concentrations of enzymes, mutagens, secondary bile
salts which are possibly involved in the carcinogenic process of
the colon (Wollowski et al. 2001). Epidemiological data supports
that the daily consumption of fermented products has a protective
effect against colon adenomas or cancer (Rafter and Glinghammar
1995).
[0132] A symbiotic combination has been used for the study of the
prevention of cancer, which is a mixture of a probiotic and a
prebiotic. This combination increased short-chain fatty acid
levels, which are the main products of bacterial fermentation,
their primary role being to act as a source of nutrients for the
intestinal epithelium. They are associated with an induction of
differentiation, suppression of proliferation and increase of in
vitro apoptosis (Heerdt et al. 1997; Medina et al. 1997), and they
may play a role in preventing some diseases such as
gastrointestinal disorders and cancer (Julia et al. 2006).
[0133] The present invention not only demonstrates the capacity of
the selected strains for inhibiting the growth of pathogenic
bacteria and intestinal enteric viruses, but it also demonstrates
superior characteristics which define the probiotic properties of
microorganisms, such as resistance to pH, bile salts and adherence
to the intestine, in comparison with control probiotic bacteria
known in the state of the art.
[0134] The results have shown in all cases that the probiotic
properties of the bacteria of the invention are better than said
control bacteria. It is thus known that the activity of the
bacteria used as controls is focused on the following
L. casei inmunitas of Danone
[0135] The beneficial effects associated with probiotic
compositions containing L. casei inmunitas (Actimel.RTM.) have the
best immune response to different infectious agents, increasing the
level of activating cytokines of the immune system, improving the
proliferative response of T cells and modulating the expression of
NK cells. It has been observed that drinking Actimel.RTM. improves
the prognosis of infant diarrheas associated with infections,
reducing the severity and duration thereof.
[0136] The compositions containing L. casei inmunitas in turn have
positive anti-inflammatory effects on the mucosa of the human colon
because they increase the immune response of the host, which is
beneficial for individuals with inflammatory bowel diseases and for
preventing colon cancer.
[0137] In view of the foregoing and of the different comparative
experiments conducted which have shown better probiotic properties
of Lactobacillus paracasei HERO 7 (CNCM I-4034) with respect to L.
casei inmunitas, Lactobacillus paracasei HERO 7 (CNCM I-4034) has
an excellent application, among others, in preventing different
pathologies such as lactose malabsorption, reduction of cholesterol
plasma levels, different types of diarrheas, inflammatory bowel
diseases, cancer, etc., in the improvement of the immune response
with respect to different infectious agents, in the improvement of
infant diarrheas, as an antiinflammatory of the mucosa of the human
colon (and accordingly in preventing colon cancer).
LGG
[0138] Lactobacillus GG adheres to the intestinal cells,
stimulating the immune response and preventing pathogenic diarrhea.
Different studies have demonstrated that the consumption of
compositions containing LGG, such as Bioactif of Kaiku.RTM.,
inhibits the competitive colonization of the intestine by
pathogenic microorganisms. These microorganisms in turn produce
antimicrobial compounds inhibiting the growth of pathogenic
strains, with the subsequent inhibition of the growth of pathogenic
strains. Thus, the consumption of compositions with LGG maintains
or restores intestinal microflora equilibrium, optimizing
absorption processes of the function of the intestinal mucosa.
[0139] In view of the foregoing and of the different comparative
experiments conducted which have shown better probiotic properties
of Lactobacillus rhamnosus HERO 22A (CNCM I-4036) with respect to
Lactobacillus GG, Lactobacillus rhamnosus HERO 22A (CNCM I-4036)
has an excellent application, among others, in stimulating the
immune response and preventing pathogenic diarrhea and maintaining
or restoring the intestinal microflora equilibrium.
Bifidobacterium Longum and Bifidobacterium bifidum
[0140] It is known from the state of the art that Bifidobacterium
Longum are resistant to antibiotics, therefore their consumption in
periods in which individuals are being treated with antibiotics
prevents the diarrhea that is occasionally caused in patients.
Other applications of these microorganisms are aimed at reducing
cholesterol, alleviating the symptoms of lactose intolerance,
stimulating the immune system and preventing cancer.
[0141] The consumption of compositions with B. bifidum alleviates
the symptoms associated with diarrhea. In turn, they are
microorganisms which increase the immunological response of the
individual by increasing phagocytic activity in peripheral
blood.
[0142] In view of the foregoing and of the different comparative
experiments conducted which have shown better probiotic properties
of Bifidobacterium breve HERO 15B (CNCM I-4035) with respect to
Bifidobacterium Longum and B. bifdum, Bifidobacterium breve HERO
15B (CNCM I-4035) has an excellent application, among others, in
stimulating the immune response and preventing diarrhea caused by
antibiotics, in reducing the cholesterol, improving the symptoms of
lactose intolerance, in preventing cancer, etc.
Probiotics in Foods, Drinks, Drugs, Etc.
[0143] Incorporating viable microorganisms in foods is a
long-standing practice. Yoghurt and other fermented milks are foods
which have traditionally included live microorganisms. The
development of functional foods in recent years has brought about
the development of new applications based on the use of
microorganisms capable of producing beneficial effects for the
organism.
[0144] The area of infant diets has not been an exception in the
field of the functional foods and ingredients of this type have
started to be included in different types of infant foods in recent
years. Probiotics have been one of the main lines of development,
being applied mostly in the field of milk formulas, primarily
continuation and growth formulas. The objective that is sought with
the incorporation of probiotics in foods is their implantation in
the colon of the host and the procurement of a series of beneficial
effects (reduction of pathogenic flora, production of vitamins and
other nutritional substances, reduction of the pH of the medium,
etc.).
[0145] The probiotic bacteria of the present invention are all
recognized as species comprised within the group of lactic acid
bacteria, and they have been known for some time on an
international level for their nil pathogenic power. Therefore, they
are susceptible to being used for the fermentation of dairy
products, among others, in an isolated manner or in conjunction
with other lactic acid bacteria, for example Streptococcus
thermophilus, Lactococcus lactis, Streptococcus lactis, etc.
Likewise, their use in infant milk, like any another lactic acid
bacteria, does not involve any potential food safety problem.
[0146] The incorporation of these probiotics in foods and beverages
must assure a particular number of live bacteria in the final
product after the maximum shelf-life period of the product by means
of using a suitable mixing (or fermentation, where appropriate)
process.
[0147] The probiotics of the present invention can be applied in
infant diets as well as in adult diets and special diets. Said
probiotics can be used in the form of powder alone or mixed with
other excipients known in the state of the art such as sugar,
proteins, milk powder, etc., or as active ingredients in the
fermentation of preferably dairy-based products. Thus, said
probiotics can be incorporated in powder or in liquid form in foods
used by the general population, particularly milk and milk-derived
products, especially fermented milk and cheeses; cereals and
derivatives, including bread doughs; soups and other similar
products in dehydrated form; fermented meat products; fruit
derivatives, juices and soft drinks; foods for specific nutritional
uses, including infant milk, infant cereals, ready-to-eat infant
foods, etc. They can also be found in food supplements and special
formulas for oral and enteral nutrition for clinical use.
[0148] If it is in a powder product (infant milk, cereals . . . ),
the probiotics will be incorporated by dry mixing them into the end
product. Thus, the probiotics of the present invention can be
incorporated in food powder intended for being reconstituted with
water or another liquid such as milk (infant milk powder, cereals .
. . ).
[0149] In their use for fermenting milk or dairy products and
preparing acidified milk, the probiotics are added to the liquid
milk base during an intermediate step of the process and producing
fermentation at controlled temperature and time to obtain acidified
milk.
[0150] The probiotics of the invention can also be applied in food
supplements and even in pharmaceutical products, which could be
presented in the form of powder preparations, tablets, sugar-coated
tablets, etc. These products have a gastric ulcers, acute diarrhea
and other diseases of the gastrointestinal tract.
EXAMPLES
Example 1
Amplifying 16S-23S Intergenic Fragments
[0151] The intergenic segments of the selected strains were
amplified, sequenced and the homology search was performed in the
NCBI (BLAST) database which delivered the following results:
[0152] Lactobacillus rhamnosus 22A (CNCM I-4036) has a homology of
100% of a 579 bp fragment with:
[0153] Lactobacillus rhamnosus isolate TS1
[0154] Lactobacillus rhamnosus isolate PS1 16S
[0155] Lactobacillus paracasei 7 (CNCM I-4034) has a homology of
100% of a 512 bp fragment with:
[0156] Lactobacillus casei ATCC 334
[0157] Bifidobacterium breve 15B (CNCM I-4035) has a homology of
99% of the 182 bp fragment 16s-23s intergenic space, with:
[0158] Bifidobacterium breve (ITS), strain Y8
[0159] Bifidobacterium longum (ITS), strain Y10
Example 2
Aligning Sequenced Sections
[0160] The Clustalw program on-line tool
(http://www.ebi.ac.uk/clustalw/) was used to align the sequenced
sections of the selected strains and controls.
[0161] The overall alignment of the sequences of rDNA 16S gene of
the strains with the controls showed the existence of differences
between the sequences of the controls and the samples L. rhamnosus
22A and L. paracasei 7, and a complete homology between the sample
7 and a selected sequence of L paracasei is observed.
[0162] Relating to the selected sample .beta.. breve 15B, a
difference between the sequences of the controls and the sample B.
breve 15B, and a homology of 100% between B. breve 15B and a
selected sequence of B. breve is observed.
[0163] The overall alignment of the sequences of the intergenic
space 16S-23S of the strain 15B and controls allowed observing a
large difference between the sequence of the controls and the
sample B. breve 15B.
[0164] In fact the sequencing of the 16S-23S intergenic space is
unique and does not coincide with anything described previously for
a bifidobacteria which indicates that the strain of the invention
is unique, an aspect recognized by the Pasteur Institute by giving
it a denomination which is also unique.
Example 3
Taking and Processing the Samples
Taking of Samples.
[0165] Faeces from exclusively breast-fed children between 2 and 4
months old were taken in anaerobic conditions at the clinic of the
pediatrician JM, the inventor of this patent. Parents were asked to
bring their children to the clinic first thing in the morning, the
children was expected to have a bowel movement after stimulation
and after the bowel movement the faeces were collected in a sterile
container by means of a plastic spoon which is adhered to its
cover. Once the collection ended, the container with the sample was
introduced in an anaerobic jar (Anaerojan.RTM., Oxoid, Hampshire,
United Kingdom) accompanied by a sachet generating anaerobic
atmosphere (Anaerogen.RTM., Oxoid, Hampshire, United Kingdom) and
the jar was sealed hermetically and transported to the laboratory
where the samples were processed in a time no longer than 2
hours.
Processing and Seeding the Samples
[0166] The samples to be analyzed can be handled once collected or
prior to cooling at -80.degree. C. in correctly identified
Eppendorf tubes.
[0167] Thus, a suspension of 10% faeces in PBS (phosphate saline
buffer (PBS, Sigma-Aldrich, Madrid, Spain)) and L-cysteine
hydrochloride (Scharlau CEIME, Barcelona, Spain) (0.05%) is
prepared. 7 dilutions from 10.sup.1 to 10.sup.7 are made from this
preparation, finally 50 .mu.l of each dilution are seeded in the
two selected culture media and are incubated in anaerobiosis for
culturing bifidobacteria (Anaerogen.RTM.) and CO.sub.2 rich medium
for lactobacillus (CO.sub.2Gen) for 72 hours at 37.degree. C.
Example 4
Preparing Culture Medium
[0168] 3 specific culture media for bifidobacteria and a culture
medium specific for lactobacillus are then indicated:
1. Beerens medium {Beerens 1990}: this medium is used to determine
bifidobacteria.
[0169] For its preparation, in a one liter Erlenmeyer flask 47 g of
Brain Heart Infusion Agar, 5 g of D-(+) Glucose, 0.5 g of iron
citrate III, 0.5 g of L-cysteine and one liter of distilled water
are mixed. This mixture is heated with constant stirring on a
heated stirring plate for a couple of minutes until it boils, it is
then left to cool at room temperature. Once 55.degree. C. is
reached, 5 ml of propionic acid and 2.2 ml of 2 Eq/L sodium
hydroxide is added therein, then the pH is adjusted to 5.0.
2. BFM medium (Nebra & Blanch 1999): this medium is specific
for bifidobacteria. Said medium has the following components, in
the indicated proportions per liter of solution:
[0170] 2 g meat extract
[0171] 7 g yeast extract
[0172] 2 g Starch
[0173] 0.5 g L-cysteine hydrochloride
[0174] 5 g sodium chloride
[0175] 5 g peptone
[0176] 2 g tryptone
[0177] 5 g Lactulose
[0178] 1 mg riboflavin*
[0179] 1 mg thiamine*
[0180] 16 mg methylene blue
[0181] 2 g lithium chloride
[0182] 5 ml Propionic acid
[0183] 15 g agar
[0184] The corresponding amounts are used to prepare 500 ml of this
bifidobacteria selective medium. This mixture is heated with
constant stirring on a heated stirring plate for a couple of
minutes until it boils, the solution is then autoclaved. Finally,
the vitamins (*) are prepared in concentrated solutions (stock
solution 1 mg/ml), they are then filtered and added together with
the propionic acid to the culture medium when the solution reaches
approximately 55.degree. C.
3. Modified Columbia medium (pH 5.0): this medium is specific for
bifidobacteria. Said medium has the following components in the
indicated proportions per liter of solution:
[0185] Columbia Agar Medium (Oxoid, Hampshire, United Kingdom).
[0186] Glucose (5 g/L).
[0187] Cysteine (0.5 g/L).
[0188] Agar (up to 15 g/L).
[0189] The previously indicated amounts are used to prepare 1000 ml
of this bifidobacteria selective medium. The mixture is heated with
constant stirring on a heated stirring plate for a couple of
minutes until it melts, the solution is then autoclaved. Finally,
the propionic acid is added to the culture medium when the solution
reaches approximately 55.degree. C. and the pH is adjusted to 5.0
with a 1N NaOH solution.
4. Rogosa agar medium: this medium is used to determine
lactobacillus. For its preparation, the specifications supplied by
the commercial company are followed.
[0190] It is prepared following the specifications supplied by the
commercial company.
Example 5
Seeding the Samples
[0191] Once the dilutions are performed, each of them is seeded in
triplicate by means of an inoculation loop. All the plates with the
different culture media are then incubated in a incubator with
controlled temperature at 37.degree. C.
[0192] The plates with the bifidobacteria culture media are
previously introduced in anaerobiosis jars into which a sachet of
Anaerogen.RTM. (anaerobic atmosphere generating system) is
incorporated therein and in those containing the lactobacillus
culture plates a sachet of CO2Gen.RTM. (CO.sub.2 atmosphere
generating system), they are finally incubated for 72 hours at
37.degree. C.
Example 6
Determining the Number of Colony Forming Units
[0193] After the incubation, the dilutions having a growth greater
than 10 colony forming units (CFU) are selected and the CFU in each
of the media is counted through an electronic colony counter pen
(Colony counter model 608702, Bio Co, Kobe, Japan). Finally, the
total number of CFU is calculated by means of the following
formula:
CFU=No. of colonies.times.dilution factor.times.dilution
[0194] Once the cultures are performed, the remaining faeces
samples were stored at -80.degree. C. until performing the
molecular biology studies.
Example 7
Determining the Resistance to pH and Bile Salts
[0195] After 72 hours of incubation, 100 colonies are selected from
each of the culture media per child, taking into account the
morphology observed with the naked eye. Said colonies both
lactobacillus and bifidobacteria are incubated in liquid Man Rogosa
Sharpe (RSM) medium and in anaerobic conditions for 48 hours.
Subsequently, a stock of glycerol is immediately made from each of
them (RSM+ Glycerol 10%).
[0196] At the same time as making the stocks of glycerol from the
different colonies, their viability both at pH 3.0 and at a
concentration of 3% bile salts (Oxgall, Sigma-Aldrich, Spain) are
analyzed. To that end, the operation is performed in the following
manner:
1. The colonies are centrifuged at 5000 rpm for 5 minutes. 2. The
supernatant is removed and resuspended in sterile PBS. 3.
Centrifuging again in the conditions similar to the above. 4. Steps
1 to 3 are repeated three times. 5. Finally resuspending in 1 ml of
sterile PBS. 6. Inoculating 100 ml of the previous suspension in
900 ml of PBS at pH 7.0 and at pH 3.0 and 0.3. % Oxgall dissolved
in PBS. 7. Incubating in anaerobic conditions for 3 hours at
37.degree. C. 8. Different dilutions (10.sup.1 to 10.sup.5) are
performed for each of the conditions in which the incubations have
been performed. 9. Seeding 50 .mu.l of each dilution. 10.
Incubating 72 hours at 37.degree. C. in anaerobiosis. 11.
Determining by means of counting the number of colonies present
both in the control and at pH and Oxgall. 12. Determining the
viability of each colony by means of the quotient:
Viability = No . of colonies at pH / Oxgall No . of control
colonies .times. 100 ##EQU00001##
[0197] All those colonies showing a viability both at pH 3.0 and at
0.3% Oxgall greater than 90% are considered as positives, which
colonies are kept to perform the rest of the tests. The other
colonies are eliminated.
Example 8
Control Bacteria
[0198] The colonies used with positive controls were the
following:
[0199] Para Bifidobacteria: Bifidobacterium bifidum and
Bifidobacterium longum supplied by Hero Spain S.A.
[0200] Para Lactobacillus: 2 commercial lactobacillus:
Lactobacillus casei (Danone.RTM.) and Lactobacillus rhamnosus GG
(LGG) (KAI KU.RTM.) were used.
[0201] Once the initial screening with the selected colonies ended,
a second screening was performed. In this case, the viability at pH
2.5 and 2.0 and at 0.5% and 0.7% Oxgall is verified. The protocol
to be followed is similar to the one used in the first assay. Once
the viability is determined, viability ranges are determined. In
this second screening, the commercial bacteria controls presented
values less than zero, therefore the one which had percentages of
viability greater than 4% was determined as the initially optimal
range for the selection of positive colonies. Dividing the colonies
into 3 groups:
Group 1 Colonies with viability greater than 66%. Group 2 Colonies
with viability between 33 and 66%. Group 3 Colonies with viability
greater than the 4%.
[0202] FIGS. 1 and 4 show the results of resistance and survival to
pH of the strains object of the invention. In the case of the
strains Lactobacillus rhamnosus 22A (CNCM I-4036) as well as
Lactobacillus paracasei 7 (CNCM I-4034), the results are
illustrated in FIG. 1 and they demonstrate that at pH 3.0 the
strains have a resistance similar or slightly greater than the
commercially tested strains. Nevertheless, at pH 2.0, the strain
22A has a very high viability compared with the rest of the strains
which do not survive that pH. In the case of the strain
Bifidobacterium breve CNCM I-4035, the results are illustrated in
FIG. 4, where it can be observed that at pH 3.0 the strain 15B
shows a resistance significantly much greater than that of the
other two bifidobacteria tested, its viability being greater than
100% which indicates that the bacteria can reproduced at this
pH.
[0203] FIGS. 2 and 5 show the results of the influence of the bile
salts on the survival of the strains of the present invention. In
FIG. 2 the results related to the strains CNCM I-4036 and CNCM
I-4034 are presented. As inferred, both strains show a percentage
of survival much greater than the commercial strains tested, the
percentage of survival being greater than 100% which indicates that
they can even reproduce in the presence of these salts. In FIG. 5
the results related to the strain CNCM 1-4035 are presented. As
illustrated in this drawing, this strain shows a greater survival
at higher concentrations if compared with the control
bifidobacteria of the state of the art.
Example 9
Test of Adhesion to Intestinal Epithelial Cells
[0204] With the colonies already selected by the resistance to pH
and bile salts, the cell adhesion assay is performed. Said assay
has been performed with intestinal epithelial cells HT29. First, a
series of attempts to determine the cell adhesion by means of
different staining: Gram staining, methylene blue staining, giemsa
staining, etc, was performed. Observing that it was very difficult
to determine the percentage of adhesion to the HT29 cells through
this means.
[0205] The question of which could be the best method to determine
the percentage of adhesion was raised, the conclusion that the best
method would probably be that which will allow recovering all the
adhered bacteria being reached. Therefore the trypsinization method
was chosen, and to that end it was proceeded in the following
manner:
1. Incubating the HT29 cells at 37.degree. C. and 5% CO.sub.2 until
confluence in 24-well plates. 2. Incubating the different colonies
to be assayed in anaerobiosis. 3. Contacting the bacteria with the
cells following the steps described below: a. Centrifuging the
bacteria at 5000 rpm for 5 minutes. b. Removing the supernatant and
resuspending the bacteria in 1 ml of sterile PBS. c. Repeating
steps a and b two more times. d. Determining the O.D of each
bacteria at 600 nm. e. Diluting the bacterial culture to O.D of 0.8
in cell culture medium prepared previously without FBS (fetal
bovine serum) and antibiotics (1 to 5.times.10.sup.6 CFU/ml). f.
Removing the culture medium of the cells. g. Washing several times
with sterile PBS to remove the FBS and antibiotic residues. h.
Adding 250 ml of the bacterial suspension to each well. The
experiment was performed in triplicate. i. Incubating at 37.degree.
C. and 5% CO2 for 90 minutes. 4. Once the bacteria are incubated
with the cells, the following is performed: a. Removing the medium
by aspiration with a pasteur pipette. b. Washing 4 or 5 times with
1.times.PBS (pH 7.0). c. Adding 100 .mu.l of trypsin and incubating
10-15 minutes at 37.degree. C. d. Recovering the entire volume of
the well and passing to an Eppendorf. e. Washing the well with 150
.mu.l of PBS and incorporating into the same Eppendorf. f. Making
several dilutions of each sample (4 or 5). g. Seeding 50 .mu.l of
each dilution. h. Incubating in anaerobiosis at 37.degree. C. for
72 hours, i. counting the number of colonies. 5. Determining the %
of adhesion.
% Ahesion = No . of adherent colonies No . of inoculated colonies
.times. 100 ##EQU00002##
[0206] FIG. 3 shows the adhesion results of the strains CNCM I-4036
(strain 22A) and CNCM I-4034 (strain 7), object of the invention.
Both strains have percentages of adhesion much greater than the
percentages shown by the control strains, (in the case of the
strain 22A, it is greater by two times), which indicates its
potential action in modulating intestinal cell activities including
the immunomodulation.
[0207] FIG. 6 shows the adhesion results of the strain CNCM 1-4035
(strain 15B), also indicating a percentage of cell adhesion much
greater than that of the control strains.
Example 10
Identifying Lactic Acid Bacteria
[0208] Isolating DNA, Amplifying and Sequencing of the 16S rRNA
Fragment
[0209] Given the number of colonies selected in the adhesion test,
their identification by means of amplifying the 16S rRNA fragment
of each colony, sequencing and homology search in the National
Center of Biotechnology Information (NCBI) database is directly
performed.
[0210] First, the selected colonies are incubated in RSM medium for
48 hrs at 37.degree. C. in anaerobiosis. The are then washed with
PBS. To that end:
1. The colonies are centrifuged at 5000 rpm for 5 minutes. 2. The
supernatant are removed and the bacterial pellet is resuspended in
1 ml of sterile PBS. 3. Repeating steps 1 and 2 three times. 4.
Finally, resuspending the pellet in 1 ml of sterile PBS.
[0211] The Genomic DNA of the bacteria is then extracted, whereby
it is performed in the following manner:
1. Centrifuging the previous suspension at 5000 rpm for 5 minutes
and removing the supernatant. 2. The bacterial pellet is
resuspended in 567 ml of Tris-EDTA (TE) buffer. 3. Adding 30 ml of
10% sodium dodecyl sulfate (SDS) and 3 ml of proteinase K (20
mg/ml). 4. Incubating the mixture at 37.degree. C. for 1 hour. 5.
Adding 100 ml of 5M NaCl and 80 ml of cetyl trimethylammonium
bromide (CTAB)/NaCl. 6. Mixing and incubating at 65.degree. C. for
10 minutes. 7. Adding the same volume (780 ml) of the
chloroform/isoamyl alcohol (24:1) mixture. 8. Mixing and
centrifuging for 5 minutes at 10000 rpm. 9. Extracting the upper
aqueous phase and transferring to a new Eppendorf. 10. Adding the
same volume of the phenol/chloroform/isoamyl alcohol (25:24:1)
mixture. 11. Mixing and centrifuging for 5 minutes at 10000 rpm.
12. Extracting the upper aqueous phase and transferring to a new
Eppendorf. 13. Adding 0.6 volumes of isopropanol. 14. Centrifuging
for 13 minutes at 13000 rpm at 4.degree. C. 15. Removing the
supernatant and adding 1 ml of 70% ethanol to the precipitated DNA.
16. Centrifuging for 5 minutes at 13000 rpm at 4.degree. C. 17.
Removing the supernatant and the DNA precipitate, then leaving it
to dry at room temperature. 18. Resuspending between 20-50 ml of
water. 19. Measuring the concentration by means of
spectrophotometer at 260 nm and obtaining the ratio 260/280 to
verify its purity. Amplifying the 16s rDNA gene and 16s-23s
intergenic space by PCR.
[0212] Oligonucleotides used:
[0213] To amplify the 16s rDNA gene, the following sets of
universal oligonucleotides were used:
TABLE-US-00002 (SEQ. ID. NO. 1) 27F 5'-AGAGTTTGATCMTGGCTCAG-3' (M =
A + C) (SEQ. ID. No. 2) 1492R 5'-TACGGYTACCTTGTTACGACTT-3' (Y = C +
T)
[0214] A fragment of approximately 1450 bp is amplified at a
hybridization temperature of 55.degree. C., amplification time of
90 sec and 35 cycles.
TABLE-US-00003 (SEQ. ID. No. 3) 39F 5'-TGGCTCAGRWYGAACGCTRG-3' (R =
A + G, W = A + T, Y = C + T) (SEQ. ID. No. 4) 1391 R
5'-GACGGGCGGTGWGTRCA-3'
[0215] A fragment of approximately 1350 bp is amplified at a
hybridization temperature of 52.degree. C., amplification time of
90 sec and 35 cycles.
[0216] Furthermore bifidobacteria specific oligonucleotides were
designed, these are:
TABLE-US-00004 (SEQ. ID. No. 5) Bif 250 bp F
5'-CTCGTAGGCGGTTCGTCG-3' (SEQ. ID. No. 6) Bif 250 bp R
5--AACGGGCCCCACATCCAG-3'
[0217] A fragment of approximately 250 bp is amplified at a
hybridization temperature of 65.degree. C., amplification time of
20 sec and 30 cycles.
[0218] To amplify the 16s-23s intergenic areas of lactobacillus
bacteria and bifidobacteria, the following sets of oligonucleotides
were used:
TABLE-US-00005 (SEQ. ID. No. 7) lactoF 5'-ACACCGCCCGTCACACCATG-3'
(SEQ. ID. No. 8) lactoR 5 -CCHSTTCGCTCGCCGCTACT-3' (H = A + T, S =
G + C)
[0219] Lactobacillus specific oligonucleotides. A fragment of
approximately 600 bp is amplified at a hybridization temperature of
65.degree. C., amplification time of 30 sec and 30 cycles.
TABLE-US-00006 (SEQ. ID. No. 9) ISBif F 5'-GGGATGCTGGTGTGGAAGAGA-3'
(SEQ. ID. NO. 10) ISBif R 5'-TGCTCGCGTCCACTATCCAGT-3'
[0220] Bifidobacteria specific oligonucleotides. A fragment of
approximately 240 bp is amplified at a hybridization temperature of
60.degree. C., amplification time of 30 sec and 30 cycles.
[0221] To amplify the 16s rDNA gene and the 16s-23s intergenic
space, between 50-100 ng of DNA were loaded for a PCR with final
volume of 50 .mu.l, a denaturation temperature of 94.degree. C. for
30 seconds was used in each cycle. It was then programmed according
to the conditions of each set of oligonucleotides specified
above.
[0222] The result of the amplification was verified in a 1.3%
agarose gel, the samples were stained with ethidium bromide and
they were viewed in an ultra violet transilluminator.
[0223] The amplifications which were negative were repeated, those
which were positive were amplified with the GE healthcare kit:
Ilustra.TM. GFX.TM. PCR DNA and gel Band Purification Kit following
the manufacturer instructions. Once purified, the samples were
resuspendended in 25 ml of water and the purification was confirmed
by viewing in a new 1.3% agarose gel.
[0224] The samples were then brought to the DNA Sequencing Service
of the Institute of Parasitology and Biomedicine "Lopez-Neyra"
(CSIC).
Example 11
Identifying by Means of Fermentation Tests
[0225] The API ZYM and API 50 CHL (bioMerieux's) systems are used.
The API ZYM system is a semi-quantitative method for measuring
enzymatic activities. This system has 20 wells, 19 of which contain
a dehydrated substrate for detecting the activity of 19 enzymes
(FIGS. 7 and 8), a colorimetric result is obtained which is
indicative of the degree of enzymatic activity and was measured
into a scale of 0-5 in comparison with the control. API 50 CH
strips and API CHL medium (bioMerieux's) which is a method for
obtaining a fermentation profile of 49 carbohydrates (FIGS. 9 and
10) was also used. A colorimetric result is obtained, but in this
case they are only classified as positive (+), negative (-), and
intermediate (V) in comparison with the control. The control
bacteria were used in all the tests.
Example 12
Evaluating the Antimicrobial Activity of the Strains L. paracasei
CNCM I-4034, B. breve CNCM I-4035 and L. rhamnosus CNCM I-4036
Study Strains and Culture and Storage Conditions
[0226] In the present study a total of 3 strains belonging to the
genus Lactobacillus and Bidifobaterium (Table 1) have been
analyzed.
TABLE-US-00007 TABLE 1 Strains used in the study and culture
conditions. Culture Species Strain medium Temperature Aeration L.
paracasei CNCM I-4034 RSM 37.degree. C. Anaerobiosis B. breve CNCM
I-4035 0.05% 37.degree. C. Anaerobiosis RSM- cysteine L. rhamnosus
CNCM I-4036 RMS 37.degree. C. Anaerobiosis
[0227] For these strains, their antimicrobial capacity with respect
to bacterial digestive pathogenic agents {Helicobacter pylori,
Listeria monocytogenes, Shigella sonnei, enterotoxigenic
Escherichia coli, enteropathogenic Escherichia coli and Salmonella
enterica) and virus (virus Ito, Wa and Va70) shown in Tables 2 and
3, has been evaluated.
TABLE-US-00008 TABLE 2 Strains of pathogenic microorganisms used
Species Helicobacter Listeria Shigella pylori monocytogenes sonnei
Rotavirus Strains LMG 8775 CECT 4031 CECT 457 Wa LMG 18041 CECT 935
CECT 4887 Va70 LMG 19449 CECT 911 CECT 413 Ito
TABLE-US-00009 TABLE 3 Strains of pathogenic microorganisms used
Species enterotoxigenic enteropathogenic Salmonella Escherichia
coli Escherichia coli enterica Strains CECT 434 CECT 443 CECT 443
CECT 501 CECT 729 CECT 725 CECT 515 CECT 742 CECT 4594
[0228] In the case of the bacteria, these were stored in a RSM
solution added with 20% (w/v) glycerol by means of freezing at
-80.degree. C. The virus were stored frozen in MEM medium at
-190.degree. C.
Obtaining the Cell-Free Supernatant for the Study
[0229] To obtain concentrated supernatant for the different assays,
the strains were cultured in liquid medium for 17 h and 24 h in RSM
medium (CNCM I-4034 and CNCM I-4036) or RSM added with 0.05%
cysteine (CNCM I-4035) at 37.degree. C. The supernatant of each of
the strains was collected by centrifugation and it was lyophilized.
The concentrate obtained was dissolved until obtaining a solution
concentrated 10.times., the pH was neutralized to a value of 6.0
and was sterilized by means of filtration through 0.22 .mu.m.
Aliquots from the neutralized and sterilized supernatant were
stored frozen at -20.degree. C. until their use.
Activity Assays in Liquid Medium with Respect to Bacterial
Digestive Pathogens
[0230] To carry out the assays of inhibition in liquid medium, a
modification of the Spinler et al. protocol (2008) was used.
Briefly, in multi-well plates of 250 .mu.l of volume the
supernatants obtained were separately added in increasing (0.2% to
4%) percentages (v/v) to culture medium inoculated at 5% with
overnight growth of each of the pathogens. The growth curves were
obtained in a manner monitored by means of measuring of OD at 595
nm using the Multiskan 5 Ascent plate reader. From the results
obtained in the different replicates, the inhibition exerted was
quantitatively assessed in the form of percentage of inhibition of
the growth with respect to the control without the addition of
supernatant of the inhibitory strain.
Activity Assays in Liquid Medium with Respect to Viral Digestive
Pathogens
[0231] The assays of the viral infection reduction from supernatant
of the study strains were carried out according to the protocol
published by Botic et al. (2007) with modifications for the
adaptation thereof to the work to be performed in this project. In
this case, to perform these assays the HT-29 human intestinal cell
line was used.
Results of the Activity Assays in Liquid Medium with Respect to the
Bacterial Digestive Pathogens: Listeria monocytogenes, Shigella
sonnei and Helicobacter pylori
[0232] To assess the effect of the growth supernatants of the
strains L paracasei, L rhamnosus and B. breve, neutralized and
concentrated 10.times. growth supernatants of 17 and 24 h,
respectively, were used.
[0233] The results varied greatly depending both on the probiotic
and on the pathogenic strain. In the case of L. monocytogenes, the
addition of the supernatant obtained after the growth for 17 hours
of L. paracasei had an inhibitory effect (FIG. 10A). In the case of
L. rhamnosus, the best results were obtained from the addition of
supernatant of 24 hours of culture. In the case of B. breve, the
inhibition in L. monocytogenes CECT 4031 T (FIG. 10B) stood out.
The results obtained for S. sonnei were similar to those of L.
monocytogenes, since for L. paracasei the best results were
obtained from the addition of the supernatant obtained after the
growth for 17 h, and in L. rhamnosus the best results were obtained
from the addition of supernatant of 24 hours of culture (FIG. 10C).
In the case of H. pylori, a significant reduction in the growth of
the pathogen from the supernatants of 17 h and 24 h of L. paracasei
and B. breve has been obtained, the greatest inhibition being in
the supernatants coming from 24 h of culture. The percentages of
inhibition obtained are described in the following table (Table
4).
TABLE-US-00010 TABLE 4 percentages of inhibition obtained for each
of the pathogens in added culture media of supernatants of the
study strains. L. paracasei L. rhamnosus B. breve 0.4% (v/v) 2%
(v/v) 4% (v/v) 0.4% (v/v) 2% (v/v) 4% (v/v) 0.4% (v/v) 2% (v/v) 4%
(v/v) 17 h 24 h 17 h 24 h 17 h 24 h 17 h 24 h 17 h 24 h 17 h 24 h
17 h 24 h 17 h 24 h 17 h 24 h L. monocytogenes 0.00 0.00 33.09 6.64
0.00 1.89 1.51 3.21 8.43 13.86 2.45 30.31 0.69 0.00 1.24 0.00 0.03
0.00 CECT 935 L. monocytogenes 0.00 0.00 0.00 0.00 9.48 0.00 0.00
0.00 1.24 10.67 8.21 15.29 3.32 16.63 1.06 9.88 4.54 28.05 CECT
4031 L. monocytogenes 2.60 5.73 7.31 9.92 25.64 16.81 8.46 5.18
11.99 18.36 15.53 28.13 4.22 0.00 0.00 0.00 0.00 0.00 CECT 911 S.
sonnei CECT 457 0.00 0.00 2.63 9.95 9.25 20.53 1.78 0.41 0.00 16.04
6.79 33.80 0.00 12.94 0.00 25.75 2.44 19.36 S. sonnei CECT 413
28.72 0.00 32.51 3.88 80.99 82.10 0.00 5.17 5.49 16.32 80.95 82.06
8.05 0.00 7.19 0.00 14.83 0.00 S. sonnei CECT 0.00 0.00 2.63 9.95
9.25 20.53 0.00 0.00 0.00 10.05 3.80 29.07 0.00 0.00 0.00 0.00 0.00
0.00 4887 H. pylori LMG 0.00 0.00 0.00 0.00 0.00 9.24 0.00 9.70
2.06 4.19 0.00 0.00 0.00 0.00 14.97 0.00 6.33 0.00 4081.sup.T H.
pylori LMG 21.15 13.24 51.70 41.05 75.80 70.89 17.36 36.67 54.84
61.13 83.48 85.28 12.56 37.80 37.79 71.92 57.33 77.56 19499 H.
pylori LMG 13.84 31.34 34.61 74.04 33.65 95.66 0.00 0.00 0.00 22.50
70.77 44.22 25.77 20.38 35.38 58.55 38.50 85.00 8775
Results of the Activity Assays in Liquid Medium with Respect to the
Bacterial Digestive Pathogens: Salmonella typhi, Salmonella
thyphimurium and Escherichia coli
[0234] For the supernatant of the bacteria L. paracasei, a
significant inhibition of the growth is observed for the Salmonella
group tested (FIG. 11). This effect is mainly due to the
non-neutralized supernatant, which suggests that it is due to the
production of acid coming from the fermentation, limiting the
pathogen growth. In the case of the Salmonella typhi CECT 725, the
inhibitory effect either at 1% and 4% of the supernatant is
observed regardless of whether this is neutralized or
non-neutralized which suggests that the inhibition is due to some
type of bacteriocin or other factor of unknown nature that would be
exerting this effect on the pathogen.
[0235] The supernatant of the bacteria B. breve exerts a growth
inhibition on the bacteria Salmonella typhi (CECT 725). This effect
is observed using the supernatant for all the conditions (17 and 24
h; neutralized and non-neutralized; 1% and 4%), the production of
any type of bacteriocin or other factor of different nature (FIG.
12) again not being ruled out.
[0236] For the supernatant of the bacteria L. rhamnosus a
significant growth inhibition mainly at 4% is seen for all the
groups (E coli ETEC, E. coli EPEC and Salmonella enterica) (FIG.
13). This effect is mainly observed with the non-neutralized
supernatant which again suggests that it is due to acid products
derived from the fermentation, limiting the growth of the
pathogen.
[0237] Although in the case of the E. coli EPEC (CECT 742) and
Salmonella typhimurium (CECT 4594) inhibition is observed with the
neutralized supernatant of 24 h, this effect can be attributed, as
in the previous cases, to the presence of any type of factor or
bacteriocin coming from the probiotic bacteria growth medium (FIG.
13).
Results of the Activity Assays in Liquid Medium with Respect to the
Viral Digestive Pathogens: Human Rotavirus Ito, Wa and Va70
[0238] The infection protocols, detection of focus of infection and
quantification of protective effect for the human rotavirus Ito, Wa
and Va70 have been optimized. To obtain results which are as
representative as possible, the infection and protection assays
have been carried out in the HT-29 human cell line.
[0239] Once the virus is amplified in MA-104 cells, they were
titrated in the HT-29 line. The titers obtained in focus of
infection forming units were 2.02.times.10.sup.6 ffu/mL for the
virus Ito, 6.80.times.10.sup.4 ffu/mL for the virus Wa and
2.33.times.10.sup.5 ffu/mL for the virus Va70. The virus were
carried to the concentrations suitable for infection from the
titration results obtained. To assure the correctness of the
assays, the assays of infection were performed using three
consecutive serial decimal dilutions and the assays were performed
in triplicate. FIG. 14 summarizes the results of reduction of focus
of infection obtained in supernatants without previously
concentrating neutralized supernantants, coming from cultures of 24
h. These results indicate that the strains of the present invention
reduce the focus of infection of all the virus tested (Wa, Ito and
Va70).
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Sequence CWU 1
1
10120DNAArtificial SequenceSynthetic oligonucleotide 1agagtttgat
cntggctcag 20222DNAArtificial SequenceSynthetic oilgonucleotide
2tacggntacc ttgttacgac tt 22320DNAArtificial SequenceSynthetic
oligonucleotide 3tggctcagnn ngaacgctng 20417DNAArtificial
SequenceSynthetic oligonucleotide 4gacgggcggt gngtnca
17518DNAArtificial SequenceSynthetic oligonucleotide 5ctcgtaggcg
gttcgtcg 18618DNAArtificial SequenceSynthetic oligonucleotide
6aacgggcccc acatccag 18720DNAArtificial SequenceSynthetic
oligonucleotide 7acaccgcccg tcacaccatg 20820DNAArtificial
SequenceSynthetic oligonucleotide 8ccnnttcgct cgccgctact
20921DNAArtificial SequenceSynthetic oligonucleotide 9gggatgctgg
tgtggaagag a 211021DNAArtificial SequenceSynthetic oligonucleotide
10tgctcgcgtc cactatccag t 21
* * * * *
References