U.S. patent application number 13/390210 was filed with the patent office on 2012-10-18 for composition having strains of lactobacillus fermentum.
Invention is credited to Knut Heller, Jurgen Schrezenmeir.
Application Number | 20120263695 13/390210 |
Document ID | / |
Family ID | 43500052 |
Filed Date | 2012-10-18 |
United States Patent
Application |
20120263695 |
Kind Code |
A1 |
Schrezenmeir; Jurgen ; et
al. |
October 18, 2012 |
COMPOSITION HAVING STRAINS OF LACTOBACILLUS FERMENTUM
Abstract
The invention relates to a pharmaceutical and/or dietetic
composition for increasing the impact of the immune defense of
higher living beings, wherein bacteria of the species Lactobacillus
fermentum from at least one of the strains K1-Lb1 or K1-Lb6 or
K2-Lb4 or K6-Lb4 or K7-Lb1 or K8-Lb1 or K9-Lb6 are contained in
order to control the adaptive and natural immune defense by means
of T helper 1 and T helper 2 cells and/or bacteria of the species
Lactobacillus fermentum from at least one of the strains K2-Lb6 or
K11-Lb3 are contained in order to strengthen the native immune
defense.
Inventors: |
Schrezenmeir; Jurgen;
(Karlsruhe, DE) ; Heller; Knut; (Kiel,
DE) |
Family ID: |
43500052 |
Appl. No.: |
13/390210 |
Filed: |
August 11, 2010 |
PCT Filed: |
August 11, 2010 |
PCT NO: |
PCT/DE2010/000953 |
371 Date: |
April 23, 2012 |
Current U.S.
Class: |
424/93.45 ;
435/252.9; 435/6.11 |
Current CPC
Class: |
A23L 33/135 20160801;
A61P 3/00 20180101; A61P 29/00 20180101; A61K 35/747 20130101; A61P
17/06 20180101; A61P 9/12 20180101; A61P 1/04 20180101; A61P 31/06
20180101; C12Q 1/689 20130101; A61P 5/48 20180101; A61P 1/00
20180101; A61P 3/10 20180101; A61P 3/06 20180101; A23Y 2220/67
20130101; A61P 21/04 20180101; A61P 31/00 20180101; A61P 37/00
20180101; A23Y 2220/35 20130101; A61P 5/16 20180101; A61P 9/10
20180101; A61P 17/00 20180101; A61P 1/12 20180101; A61P 31/18
20180101; A61P 27/02 20180101; A61P 11/00 20180101; A61P 31/10
20180101; A61P 25/00 20180101; A61P 19/04 20180101; A61P 37/08
20180101; A23V 2002/00 20130101; A61P 11/06 20180101; A61P 19/02
20180101 |
Class at
Publication: |
424/93.45 ;
435/252.9; 435/6.11 |
International
Class: |
A61K 35/74 20060101
A61K035/74; A61P 17/00 20060101 A61P017/00; A61P 11/06 20060101
A61P011/06; A61P 37/08 20060101 A61P037/08; A61P 31/06 20060101
A61P031/06; A61P 1/04 20060101 A61P001/04; A61P 1/00 20060101
A61P001/00; A61P 1/12 20060101 A61P001/12; A61P 31/18 20060101
A61P031/18; A61P 31/10 20060101 A61P031/10; A61P 29/00 20060101
A61P029/00; A61P 5/16 20060101 A61P005/16; A61P 27/02 20060101
A61P027/02; A61P 17/06 20060101 A61P017/06; A61P 3/10 20060101
A61P003/10; A61P 19/04 20060101 A61P019/04; A61P 25/00 20060101
A61P025/00; A61P 21/04 20060101 A61P021/04; A61P 19/02 20060101
A61P019/02; A61P 9/10 20060101 A61P009/10; A61P 3/00 20060101
A61P003/00; A61P 9/12 20060101 A61P009/12; A61P 5/48 20060101
A61P005/48; A61P 3/06 20060101 A61P003/06; C12N 1/20 20060101
C12N001/20; G01N 27/26 20060101 G01N027/26; C12Q 1/68 20060101
C12Q001/68; A61P 37/00 20060101 A61P037/00 |
Foreign Application Data
Date |
Code |
Application Number |
Aug 11, 2009 |
DE |
10 2009 037 089.7 |
Claims
1. Pharmaceutical and/or dietetic composition for strengthening the
effect of the human immune defense, characterized in that, to
strengthen the adaptive immune defence by means of T helper 1 and T
helper 2 cells, bacteria of the genus Lactobacillus fermentum of at
least one of the strains K1-Lb1 or K1-Lb6 or K2-Lb4 or K6-Lb4 or
K7-Lb1 or K8-Lb1 or K9-Lb3 and/or of the genus Lactobacillus
plantarum of the strain K4-Lb6 and/or, to strengthen the native
immune defence, bacteria of the genus Lactobacillus fermentum of at
least one of the strains K2-Lb6 or K11-Lb3 are contained.
2. Composition according to claim 1, characterized in that each
strain is contained in Kimere.
3. Composition according to one of the preceding claims,
characterized in that the strengthening of the adaptive immune
defense is a shift from the T helper 2 to the T helper 1
reactions.
4. Composition according to claim 3, characterized in that at least
one of the strains K1-Lb1 or K1-Lb6 or K2-Lb4 or K4-Lb6 or K6-Lb4
or K9-Lb3 is contained.
5. Composition according to one of the preceding claims,
characterized in that the strengthening of the adaptive immune
defense is a shift from the T helper 1 to the T helper 2
responses.
6. Composition according to one of the preceding claims,
characterized in that the strengthening of the adaptive immune
defense is an inhibition of inflammations.
7. Composition according to claim 5 or 6, characterized in that at
least one of the strains K6-Lb4 or K7-Lb1 or K8-Lb1 is
contained.
8. Composition according to one of the preceding claims,
characterized in that the strengthening of the adaptive immune
defense is an increased release of defensin (hBD) from intestinal
cells.
9. Composition according to claim 8, characterized in that at least
one of the strains K2-Lb6 or K11-Lb3 is contained.
10. Composition according to one of the preceding claims,
characterized in that the strains tolerate a 0.3% bile salt
solution.
11. Composition according to one of the preceding claims,
characterized in that the strain K4-Lb6 tolerates a 3.0% bile salt
solution.
12. Composition according to one of the preceding claims,
characterized in that at least 8% of the strains survive a pH of
3.0 for at least 3 h.
13. Composition according to claim 12, characterized in that at
least one of the strains K1-Lb1 or K1-Lb6 or K2-Lb4 or K2-Lb6 or
K4-Lb6 or K6-Lb4 or K7-Lb1 or K8-Lb1 or K9-Lb3 is contained.
14. Composition according to one of the preceding claims,
characterized in that, to influence the concentration and
composition of glycosidic compounds at the membrane or at the wall
of a bacterium, and therefore to modulate the immune response of a
host, at least one of the strains expresses a glycosyl transferase
or a sugar transferase or an acyl transferase or a lipoteichonic
acid-exporting protein.
15. Composition according to claim 14, charazterized in that, for
at least one of the strains for glycosyl transferase, the sugar
residue docking can be catalyzed by diacylglycerol synthesis
(DAG).
16. Composition according to claim 14, characterized in that, for
at least one of the strains, the acyl transferase can be catalyzed
by diacylglycerol synthesis (DAG).
17. Composition according to one of the preceding claims,
characterized in that for at least one of the strains, enzymes
catalyse further sugar transfer for the synthesis of microbial
peptidoglycans.
18. Composition according to one of the preceding claims,
characterized in that, in the case of at least one of the strains,
the expulsion from the cytoplasm is mediated by a lipoteichonic
acid synthesis and/or by a peptidoglycan synthesis as
transporter.
19. Method for examining the identity of the strains according to
one of the preceding claims, characterized in that the
effect-mediating genes have a strain-specific pattern of the
suppressive subtractive hybridization (SSH).
20. Method according to claim 19, characterzied in that the part of
SSH can be determined by pulse field gel electrophoresis.
21. Use of the composition according to claim, for preparing a
medication or a food supplement or a pharmaceutical preparation for
prophylactic treatment or reduction of the risk of manifestation or
for therapy of eczema and/or atopic dermatitis and/or asthma and/or
rhinitis allergica or other allergies and/or tuberculosis and/or
colitis ulcerosa and/or eosinophilic pneumonia and/or other
Th2-driven illnesses or complaints.
22. Use of the composition according to claim, for preparing a
medication or a food supplement or a pharmaceutical preparation for
prophylactic treatment or reduction of the risk of manifestation or
for therapy of intestinal infections and/or travel diarrhoea and/or
colds and/or urogenital infections and/or HIV-associated
complications and complains and/or candidiasis or other complaints,
infections or other illnesses the defence against which is
Th1-mediated.
23. Use of the composition according to claim, for preparing a
medication or a food supplement or a pharmaceutical preparation for
prophylactic treatment or reduction of the risk of manifestation or
for therapy of rheumatoid arthritis and/or hashimoto thyreoiditis
and/or uveitis and/or psoriasis and/or type 1 diabetes and/or
sjogren disease and/or. coeliac disease and/or systemic lupus
erythematosus and/or ankylosing spondylitis and/or Crohn's disease
and/or inflammatory intestinal diseases and/or sclerodera and/or
sarcoidosis and/or multiple sclerosis and/or vitiligo and/or
Grave's autoimmune thyroiditis and/or endocrinous opthalmopathy
and/or myasthenia gravis and/or osteoarthritis and/or
arteriosclerosis and therefore also cardiac infarction and/or
peripheral arterial occlusive diseases and/or cerebral infarction
(stroke) and/or metabolic syndrome and therefore also adipositas
and/or hypertonia and/or insulin resistance and/or type 2 diabetes
and/or dyslipoproteinaemia, as well as amyotrophic lateral
sclerosis and/or interstitial cystitis and/or irritable bowel
syndrome (IBS) other T helper 1-driven complains and illnesses.
24. Use of the composition according to claim 6, for preparing a
medication or a food supplement or a pharmaceutical preparation for
the reduction or elimination of the effects of autoimmune diseases
such as arthritis or dermatitis or of allergies or of illnesses
with inflammatory components such as a metabolic syndrome or
arteriosclerosis or those complaints and illnesses with
inflammatory component mentioned in claim 23.
25. Use of the composition according to claims 11, for preparing a
medication or a food supplement or a pharmaceutical preparation for
the prophylaxis and therapy or osteoporosis by acidification of the
milieu and displacement of other microorganisms with the
consequence of better mineral and trace element resorption.
26. Use of the composition according to claims 11, for preparing a
medication or a food supplement or a pharmaceutical preparation for
supportive therapy in the case of liver failure and hepatic
encephalopathy due to lower resorption of toxins.
27. Use of the composition according to claims 11, for preparing a
medication or a food supplement or a pharmaceutical preparation for
the reduction of complaints in the case of obstipation and/or
irritable bowel syndrome (IBS) and/or halitosis.
28. Use of the composition according to claims 11, for Preparing a
medication or a food supplement or a pharmaceutical preparation for
reduction of lactose intolerance by improving the lactose
exploitation and lactose digestion.
29. Use of the composition according to claims 11, for preparing a
medication or a food supplement or a pharmaceutical preparation
and/or for therapy of vaginosis or vaginitis.
30. Use of the composition according to claims 11, for preparing a
medication or a food supplement or a pharmaceutical preparation
and/or for intensifying and accelerating the bile acid and
cholesterol metabolism.
Description
[0001] The invention relates to a pharmaceutical and/or dietetic
composition for strengthening the effect of the human immune
defence.
[0002] The immune defence is an ever present and active biological
defence system of higher living organisms, which prevents tissue
damage by pathogens. It consists of various organs as well as cells
and molecules of various types that form a complex network. It
eliminates microorganisms and other foreign substances that have
penetrated into the body and is also capable of destroying the
body's own cells that have become defective. The immune defence has
great importance for the physical intactness of living organisms,
since virtually all organisms are continually subject to the
influences of the living environment, of which some even pose an
existential threat.
[0003] If harmful microorganisms penetrate into the body, this can
lead to functional disturbances and illness. Typical pathogens are
bacteria, viruses and fungi as well as single-cell parasites, for
example protozoa or other plasmodia, or multicellular parasites,
such as tapeworms.
[0004] Currently new genera of harmful bacteria and other unwelcome
microorganism are increasingly growing up, which are resistant not
only to one but often even to a plurality of medicines, which are
still very effective against the genera known hitherto. A
threatening example in July 2009 is the pathogen N1H1, also known
as swine flu, the mutants of which could lead to a pandemic.
[0005] There is therefore currently increased interest in a
strengthening and increasing the effectiveness of the body's own
defence.
[0006] Active substances with an immune modulating effect are
appropriate for this. The immune modulating effect can be
distinguished into a so-called natural or innate immunity, which a
newborn baby already has, and the adaptive or acquired immunity,
which only forms in the course of life due to contact with various
invaders. Adaptive immunity makes use of the T helper cells, which
are an essential part of the learning capacity of the body's
defence system. Of the group of T helper cells, the T helper 1 and
T helper 2 cells are particularly important.
[0007] In the case of illnesses, a known and efficient treatment is
to influence the so-called Th1/Th2 response such that the
respective illness is combatted much faster and more extensively.
To this end, their effect is increased by the administration of
stimulating substances.
[0008] Thus, the patent application KR 1007 42900 discloses the
Lactobacillus rhamnosus DCC 3201 as a bacterial strain, which is
added to foods as a probiotic supplement in order to counteract and
prevent atopical dermatitis. Laboratory tests suggest that this
bacterial strain acts to alleviate and prevent allergic reactions
by maintaining the balance of T helper 1 and T helper 2 cells.
[0009] The place where this bacterial strain was found in the
faeces of Korean children should actually not be a disadvantage
with appropriate preparation according to the prior art, however,
from the start, it does not arouse exclusively positive
expectations.
[0010] In any case, this bacterial strain is not expected to
provide an effect if the aim is a different, targeted shifting of
the T helper 1 to T helper 2 ratio, which acts on other
illnesses.
[0011] Against this background, it is the object of the invention
to find active substances from a group which have an
immune-modulating effect in that they strengthen the immune
responses of the body and in the process either reinforce the
natural immunity or support the adaptive immunity by stimulating T
helper cells as directly as possible to increased activity and in
the process selectively and primarily direct the effect either to T
helper cells 1 or to T helper cells 2.
[0012] As a solution, the invention provides a pharmaceutical
and/or dietetic composition in which, for increasing the impact of
the immune defence by means of T helper 1 and T helper 2 cells,
bacteria of the genus Lactobacillus fermentum from at least one of
the strains K1-Lb1 or K1-Lb6 or K2-Lb4 or K6-Lb4 or K7-Lb1 or
K8-Lb1 or K9-Lb3 and/or from the genus Lactobacillus plantarum of
the strain K4-Lb6 and/or, for strengthening the native immune
response, bacteria from the genus Lactobacillus fermentum of at
least one of the strains K2-Lb6 or K11-Lb3 are contained.
[0013] The designations KxLby used here are taken from the system
used in the original investigation of Kimere. K stands for Kimere,
the source of the hitherto unknown strains presented here, Lb
stands for Lactobacillus. From the large number of investigated
species, some were chosen so that the first parameter of the
designation--Kx--and the second parameter of the
designation--Lby--do not contain sequential numbers.
[0014] The designations Kx-Lby of the strains of the genus
Lactobacillus fermentum lead from two alternative kinds to an
unambiguous identification of the respective bacterial strain.
[0015] The first alternative is the known method of depositing a
derivative of the respective genus at the DSMZ, Deutsche Sammlung
von Mikroorganismen and Zellkulturen GmbH in Inhoffenstra.beta.e
7B, D-38124 Braunschweig. According to the procedure prescribed for
a patent application, intact and living examples of the bacterial
strains mentioned here were deposited at the DSMZ, specifically on
Aug. 6, 2009, under the following file numbers: [0016]
Lactobacillus fermentum K1-Lb1 [0017] Lactobacillus fermentum
K2-Lb6 [0018] Lactobacillus fermentum K7-Lb1 [0019] Lactobacillus
fermentum K8-Lb1 [0020] Lactobacillus fermentum K11-Lb3 [0021] and
Lactobacillus plantarum K4-Lb6
[0022] The other strains have not yet been deposited at the DSMZ.
For their disclosure, there serves the second alternative method of
making known the PFGE band pattern.
[0023] An alternative for depositing living bacterial is clear
identification by means of pulse field gel electrophoresis pattern
(PFGE). In the accompanying FIG. 3, for each of the bacterial
strains mentioned here, the so-called band patterns in each case,
which are analysed by the PFGE method and therein show an identical
band pattern, are thereby unambiguously identified as this strain
of the respective genus.
[0024] The method of typifying microorganisms by PFGE has been
known since 1984 and is part of the recognized prior art. The
researchers Schwartz and Cantor have observed that, on application
of electrical pulses, which periodically changed their orientation
at a particular angle in ratio to the agarose gel, large intact DNA
molecules were isolated as band pattern. This method is based on
U.S. Pat. No. 4,473,452 and comprises four key steps, namely [0025]
1. Preparing the samples by culturing the microorganisms in a
nutrient broth, embedding the cells in gels and obtaining
immobilized, deproteinized, intact DNA molecules [0026] 2.
Specifying the progress of electrophoresis for an optimum
separation between the molecules [0027] 3. Loading the samples into
the gels and performing pulse-field gel electrophoresis with the
result of a typical band pattern [0028] 4. Analysing the band
pattern by comparison with the band patterns of similar genera of
microorganisms. It is generally accepted in the prior art that the
band pattern thereby obtained permits a clear identification of the
respective genus, somewhat comparable with a fingerprint of a
person.
[0029] The PFGE process, however, is not the actual content of this
patent application but only one of two methods used for unambiguous
identification of the bacterial strains of the genus Lactobacillus
fermentum to be protected. The common inventive feature of all
genera is the stimulating effect on the T helper cells.
[0030] The T helper cells belong to the lymphocytes, a sub-group of
the "white blood corpuscles", which are the most important agents
of the human immune defence. An adult human has somewhat over a
thousand different lymphatic cells, which comprise about two
percent of his bodyweight. The lymphocytes, like all blood cells,
originate from the bone marrow and, before their maturity into
functioning cells of the immune defence, must pass through
additional development and differentiation stages.
[0031] Some of these precursor cells migrate from the blood-forming
tissue directly into the thymus gland and develop into so-called
T-lymphocytes. There, they learn, inter alia, to differentiate
between the body's own cells and "invaders". The remaining
lymphocytes mature in the bone marrow into so-called B cells.
[0032] The most important property of the lymphocytes is their
capability of reacting with a particular molecular shape according
to the key- and-lock principle. To this end, as "lock" they bear on
their surface receptors, which are specially adapted to the protein
structure of one of millions of different foreign bodies--the
"keys". Via the structure of the cell's own protein and protein
that is foreign to the cell on the surface of the macrophage, they
recognize a "new invader" and mount the specific immune
defence.
[0033] For the actual execution of the immune defence, T killer
cells are created, which selectively bind the invader and destroy
it. The strengthening of this human immune defence by active
substances according to the invention takes place indirectly,
namely via stimulating the increased formation of T helper cells,
the B lymphocytes, to transform themselves into plasma cells, the
body's antibody factories. In the course of their short lifetime of
only a few days, they can pour out thousands of specialized defence
molecules per second.
[0034] It is at this point that the effect of the bacterial strains
presented here starts, in that they act on one of the two types of
T helper cells, either increasing the number of T helper 1 or T
helper 2 cells, as a result of which the number of the respective
other kind is inevitably reduced, and additionally or alternatively
thereto, increasing the number of T helper cells that are grown
overall.
[0035] By means of an additionally growing proportion of a very
particular type within the group of T helper cells, their number
with respect to the normal state increases strongly, firstly due to
the absolute increase and secondly due to the increase of the
relative proportion of the total number of all T helper cells. As a
result, a very much larger number of plasma cells for defence
molecules is very rapidly formed. Thus, the number of these defence
molecules with a total of three factors is increased, namely the
absolute and the relative increase of the number of T helper cells
and their effect strengthening by multiplication in the plasma
cells.
[0036] A further, more advantageous strengthening factor for this
effect is that, due to the selection of one of the genera presented
here, those plasma cells that form precisely the required
antibodies are predominantly formed. Due to the larger number of
the bacterial strains presented here, it is possible to select that
bacterial strain that forms antibodies that are particular
effective for a particular type of invader. If, for example, a
patient suffers from an allergy, it is appropriate to select one of
the genera K1-Lb1, K2Lb4, K4-Lb6, K6-Lb4 or K9-Lb3.
[0037] In addition, the immune system is prepared for a further
contact with the invader in that, the characteristic features of
the invading foreign substance is stored in a portion of the B
cells, so that, with the next invasion, the production of the
suitable antibodies can start directly. This "secondary immune
response" is the basis of most protective inoculations.
[0038] A further advantage of the large number of the bacterial
kinds presented here is their multiple effect in multimorbid
patients, that is to say patients that suffer from more than one
single illness. If, for example, the above-mentioned allergy
patient additionally suffers from hyperactivity of the gall
bladder, the bacterial type K4-Lb6 is therefore of particular
advantage, since it has a particularly high bile salt resistance
and, despite this second problem, therefore still has the first
desirable antiallergenic effect.
[0039] Another advantageous property of the bacterial strains
presented here is their probiotic property. This property is proven
inter alia by the fact that all strains were isolated from a food
that has successfully been used for generations, namely from
Kimere, a dough produced from pearl millet (Pennisetum claucum) by
spontaneous fermentation, whose place of origin is the district of
Mbeere in Kenya. Since all types of bacterial are contained in a
greater or lesser concentration in this widespread and
preferentially used staple food, their compatibility is proven. It
is also proven thereby that, in comparison to a very large number
of allopathic medicines, it can be classified as almost free of
side effects.
[0040] The selection of bacilli of the genus Lactobacillus
fermentum was induced by the investigation of the typical
conditions for the traditional production of Kimere, namely the
hygienic conditions during the preparation of the starting material
pearl millet, its processing by water treatment in very few
vessels, which by our standards have only been inadequately
cleaned, its processing by grinding in a moist state usually with
one and the same mill, and its fermentation generally in one and
the same vessel reserved for this purpose. That would, by European
standards, raise fears of contamination by microorganisms
disadvantageous to humans.
[0041] However, the investigation has shown that this is very
clearly not the case, but at least 90% of the microorganisms found
belonged to the genus Lactobacillus fermentum. From this it can not
only be concluded that this genus is generally compatible with
humans, but that it supresses, or for the most part eliminates,
other microorganisms that are less compatible with humans.
[0042] In the studies, the population with microorganisms in the
case of Kimere has generally proved very stable, which is a
positive feature for the use of the kinds of bacteria found there.
A further advantage is the relatively acid milieu, that is to say a
relatively low pH. Since the microorganisms have proven long-term
stable in this acid milieu, they will also survive the acid
environment in the stomach and gut and in this manner will prove
effective on oral administration--for example as food
supplement.
[0043] Strains were even found that are characterised by such a
high degree of acid resistance that they pass through the stomach
and small intestine even into the large intestine in order to
develop their healing effect there.
[0044] Of the bacterial strains recognised as effective, almost all
act not only against a single type of complaint, but against a
plurality thereof, so that they are also suitable inter alia for
treatment of those with multiple illnesses. However, clear focuses
of the type of effect were found, so that the individual bacterial
types can be selectively assigned to a focal action.
[0045] The first of the discovered types of effect is a
strengthening of the immune defence by a clear shifting of the T
helper cells away from T helper 2 cells towards T helper 1 cells.
The strains K1-Lb1, K1-Lb6, K2-Lb4, K4-Lb6, K6-Lb4 and K9-Lb3 act
predominantly with this focus. FIG. 1 shows the result of the in
vitro simulated effect. The different intensities of this type of
effect can be read off in three stages.
[0046] These bacterial strains are thus suitable for medicines,
food supplements or pharmaceutical preparations for the
prophylactic treatment or reduction of the risk of the
manifestation or for therapy of T helper 2-driven illnesses, such
as, for example, eczema and/or atopical dermatitis and/or asthma
and/or rhinitis allergica or other allergies and/or tuberculosis
and/or colitis ulcers and/or eosinophilic pneumonia.
[0047] These bacterial strains are thus suitable for medicines,
food supplements or pharmaceutical preparations for the
prophylactic treatment or for therapy in complaints, infections or
other illnesses whose defence is Th1 mediated, for example gut
infections and/or travel diarrhoea and/or colds and/or urogenital
infections and/or HIV-associated complications and complaints
and/or candidiasis.
[0048] Of the bacterial strains found, some act with a contrary
accent of the T helper cells: they effect a displacement of the
response away from the T helper 1 cells towards the T helper 2
cells. Thereby, bacterial strains are presented which are also
usable for entirely different illnesses in the same advantageous
manner. This effect predominantly characterises the strains K6-Lb2,
K7-Lb1 and K8-Lb1.
[0049] These strains which predominantly effect a reinforcement of
the immune defence by shifting away from the T helper 1 and towards
the T helper 2 responses, are suitable for producing a medicine or
a food supplement or a pharmaceutical preparation for prophylactic
treatment or for reducing the risk of manifestation of autoimmune
illnesses such as Crohn's disease or other T Helper 1-driven
illnesses.
[0050] The aforementioned strains K6-Lb2, K7-Lb1 and K8-Lb1 also
have an inhibiting effect against inflammation. And the K6-Lb4
strain also proves anti-inflammatory. These bacterial strains are
therefore suitable for producing a medicine or a food supplement or
a pharmaceutical preparation for the reduction or elimination of
the effects of autoimmune illnesses such as arthritis or dermatitis
or allergies or illnesses with an inflammatory component, such as a
metabolic syndrome or arteriosclerosis.
[0051] The aforementioned strains K6-Lb2, K7-Lb1, K8-Lb1 and K6-Lb4
can be suitably and efficiently used for the prophylaxis or therapy
of rheumatoid arthritis, hashimoto thyreoditis, uveitis, psoriasis,
type 1 diabetes, sjogren disease, coeliac disease, systemic lupus
erythematosus, ankylosing spondylitis, Crohn's disease,
inflammatory intestinal diseases, sclerodera, sarcoidosis, multiple
sclerosis, vitiligo, Grave's autoimmune thyroiditis, endocrinous
opthalmopathy, myasthenia gravis, osteoarthritis, arteriosclerosis
and therefore also cardiac infarction, other peripheral arterial
occlusive diseases, cerebral infarction (stroke), metabolic
syndrome and therefore also in the case of adipositas, hypertonia,
insulin resistance, type 2 diabetes, dyslipoproteinaemia, and
amyotrophic lateral sclerosis, interstitial cystitis and/or
irritable bowel syndrome (IBS).
[0052] Some of the bacterial strains presented here additionally
strengthen the immune defence in that they effect an increased
release of defensin from the intestinal cells. It is also known
that defensins carry many cationic and hydrophobic amino acid
residues. They are thus amphipathic peptides. These positive
charges interact with the negative charges of the exciter
membranes. The preference of the defensins is for membranes that
are characterised by a low proportion of cholesterol and thereby
differ from those of eukaryotic organisms. When they have
penetrated the membrane, they also interact with anionic molecules
within the pathogen cell, such as DNA and RNA. By this means the
action spectrum is broad and corresponds to that of a
broad-spectrum antibiotic, so that it is difficult for a pathogen
to counteract the mechanism of the defensins.
[0053] To this extent, the effect mechanism is similar to that of
the T helper cells, and they are therefore to be assigned to the
same inventive idea. This type of effect applies predominantly to
the strains K1-Lb7, K2-Lb6 and K11-Lb3.
[0054] A further outstanding property is tolerance to a 3% bile
salt solution. This feature applies particularly to the K4-Lb6
strain. It ensures its survival through the greatest portion of the
human digestive system as far as the large intestine. Because the
bacterium is still in the living state when it arrives there it can
still develop its healing effect, namely the strengthening of the T
helper 1 reaction and the weakening of the T helper 2
influence.
[0055] For a very much larger number of the bacterial strains
presented here, survival in the intestines is no longer possible,
but survival in the stomach is. At least 8% of them survive a pH of
3.0 for at least 3 hours. By this means they can still develop
their effectiveness in full even after penetrating the pylorus
within the stomach. This applies to the strains K1-Lb1, K1-Lb6,
K2-Lb4, K4-Lb6, K6-Lb2, K6-Lb4, K7-Lb1, K8-Lb1 and K9-Lb3.
[0056] These strains are therefore, inter aka, appropriate for
prophylaxis for the following complaints and illnesses, to avoid
passing to a chronic state and for system-related therapy of
osteoporosis by acidification of the milieu and displacement of
other microorganisms with the result of improved mineral and trace
element resorption. Likewise, they are appropriate for liver
failures and hepatic encephalopathy by reduction or inhibition of
the resorption of toxins.
[0057] By means of these strains, lesser complaints with
obstipation, irritable bowel syndrome and halitosis can be
reached.
[0058] By means of the strains, the effects of lactose intolerance
can be successfully suppressed by supporting lactose utilization
and lactose digestion.
[0059] Thanks to these bacterial strains, a positive influencing of
the bile acid and cholesterol metabolism can also be achieved.
[0060] In a medicine or another form of administration, they are
also helpful against vaginosis or vaginitis, specific with oral or
topical application.
[0061] Another type of effect is the influencing of the
concentration and composition at the membrane or wall of a
bacterium, and consequently for modulation of the immune response
of the recipient, at least one of the strains expresses a glycosyl
transferase or a sugar transferase or an acyl transferase or a
lipoteichoic acid-exporting protein.
[0062] It is also observed as an effect mechanism that in the case
of at least one of the strains for glycosyl transferase, the sugar
residue docking can be catalysed by the diacylglycerol synthesis
(DAG).
[0063] Another subvariant is that for at least one of the strains,
acyl transferases can be catalysed by the diacylglycerol synthesis
(DAG).
[0064] In another variant of the effect of the bacterial strains
presented here, for at least one of the strains, enzymes catalyse
further sugar transfer for the synthesis of microbial
peptidoglycans.
[0065] For at least one of the strains, the expulsion from the
cytoplasm can be mediated by means of a lipoteichonic acid
synthesis and/or by means of a peptidoglycan synthesis as
transporter.
[0066] At the beginning of the description, it was mentioned that
the bacterial strains presented here are identified by the
deposition of at least one living example with the DSMZ.
Alternatively the respective band pattern of the pulse field gel
electrophoresis serves as the feature to be clearly identified. For
this process, it is an appropriate intermediate step to examine
whether the effect- mediating genes have a strain-specific pattern
of the suppressive subtractive hybridization (SSH).
Methods and Special Features of the Underlying Study
[0067] 1. Derivation from Kimere
[0068] Kimere is a spontaneously fermented dough or porridge from
pearl millet, which is produced and consumed, inter alia in the
district of Mbeere in Kenya, East Africa, by a traditional method
by first dry, then wet, milling on a millstone and subsequent
fermentation.
[0069] A special feature is that Kimere is fermented 18-24 hours
before consumption and is assimilated in its actively fermented
state as food or is kept for up to three days. Otherwise it is
similar to the preparation of the normal East African porridge,
called Uji. Kimere only differs from Uji in its viscosity and
consistency, since Kimere is more viscous.
[0070] The differences result from the manufacturing method, which
influences the microbial population of the final product. The
production of Uji includes the mechanical milling of maize, sorgum,
millet or other cereal with a hammer mill, followed by mixing the
resulting meal with water and spontaneous fermentation or pouring
back some of the already ready prepared Uji portion. Finally, the
fermented product is boiled and sweetened before consumption.
[0071] In contrast thereto, Kimere is milled on a millstone in a
dry state, followed usually by three wet milling operations with
decanting and finally a fermentation. In a variant, a portion of
the product is converted to a thin slime, to which water is added
and then boiled. The boiled Kimere is then mixed with the unboiled
again.
[0072] The biggest difference from Uji is that Kimere is consumed
in its actively fermenting stage, and therefore contains living
microorganisms, by contrast Uji is consumed immediately after
boiling and does not therefore contain living fermenting
microorganisms. This method of manufacturing Kimere is typical of
the regions of Mbeere, Tharaka, Chuk and Embu east of Mount
Kenya.
[0073] After the first purification process and the dry grinding,
the broken grain easily absorbs water--about 30% by volume--and is
soaked for about 30 to 60 minutes at room temperature. As a result,
the broken grains are softened and prepared for the following wet
grinding processes.
[0074] This is followed by a total of three wet grinding processes
with a thick porridge as result. Between each wet grinding process,
further water is added and after each milling operation, decanting
is performed. The result is a milky suspension, of which two thirds
is boiled with stirring.
[0075] After boiling it is charged together with the further
unboiled third into a fermentation vessel and left to ferment for
18 to 24 hours. The unbolted portion contains the microbes that
were contained in the grains and which have been absorbed during
the milling operations.
[0076] Usually a portion of a previous preparation of Kimere is
added, which speeds up the fermentation and influences the ultimate
microbial population. During fermentation, as a result of the
lactobacilli, the proportion of phythates is reduced; pathogens are
reduced and the flavour is improved.
[0077] The nutritional value of Kimere was thus improved compared
to non-fermented products and is free of coli bacteria and other
enterobacteria, which is an indication of microbial safety. The
presence of living bacteria in Kimere is of particular interest,
since it is the basis of a probiotic application.
[0078] 2. Intake and Preparation of the Kimere Samples
[0079] The samples were taken in 11 yards in Kathera, a part of
Kiang'Ombe, the Evurore part of the district of Mbeere. The samples
were prepared and placed overnight in fermentation vessels. On the
following morning, they were collected and taken to the Max-Rubner
Institute in Kiel in a screw top glass jar, where they were stored
for a maximum of 24 hours at 4.degree. Celsius.
[0080] Transportation took less than eight hours, during which the
samples were conveyed at normal room temperature.
[0081] By this means, it was ensured that the isolated
microorganisms behaved in a similar way to the microbial population
during the otherwise conventional fermentation, since Kimere is
fermented for 18 to 24 hours before consumption and can be consumed
from the same fermentation vessel for another three days
thereafter.
[0082] The samples were very well mixed in a vortex mixer. 1 g of
the sample was added to 9 ml of sterile Ringer's solution to obtain
a dilution of 10.sup.-1. Then series of dilutions were made down to
10.sup.-8 to count the lactobacilli.
[0083] 3. Determining the pH of Kimere
[0084] To determine the pH of Kimere samples, the method of the
AOAC (Association of Analytical Communities) in the version of 1995
was applied. 10 g of the samples in each case was mixed with 40 ml
of double-distilled water; the mixture was measured after a waiting
time of 10 minutes using a Delta 320 pH measuring instrument. The
pH values were measured in triplicate and the average value was
calculated.
[0085] The aerobic mesophiles were counted on a plate count agar
(PCA) and aerobically incubated at 30.degree. C. for 48 hours. The
number of lactobacilli was determined by the area counting method
on an MRS agar (Merck, Darmstadt, Germany) according to the process
of De Man et. al. of 1960, and on the M17 agar (Merck, Darmstadt,
Germany).
[0086] The MRS agar plates were incubated in an anaerobic chamber
(MACS MG500+TG Airlock, dw-scientific, Shipley, West Yorkshire,
England) at 37.degree. C. for 48 hours. The gas atmosphere
consisted of 10% hydrogen, 10% carbon dioxide and 80% nitrogen.
[0087] Then the M17 agar plates were aerobically incubated at
30.degree. C. for 48 hours. Only plates with colony-forming units
(cfu) between 10 and 300 per gram were analysed and the result was
plotted as a decadic logarithm of the cfu number per gram of the
wet weight of the sample.
[0088] 4. Isolation and Biochemical Characterization of the
Lactobacilli
[0089] 1 g of the sample of Kimere was weighed in 9 ml Ringer's
solution and a plurality of tenfold dilutions were prepared down to
10.sup.-8. Of each dilution of 10.sup.-5 to 10.sup.-8, 0.1 ml in
each case was spread two fold on an MRS agar plate. The plates were
incubated at 37.degree. C. for 48 hours in an aerobic chamber as
described above.
[0090] Individual, different colonies were chosen based on their
morphology and purified by spreading again on MRS agar, and
inspected with the microscope. The gram staining and the catalase
reaction were performed as described by Harrigan and McCance
1990.
[0091] All rod-shaped gram-positive and catalase-negative isolates
were stored at -80.degree. C. in the Cryo-Bank.RTM. vials from
Germany for further characterizations. The production of CO.sub.2
glucose in MRS solution was determined by means of Durham tubes.
The chemical characterisation of the chains was performed with API
50 CH kits with API 50 CHL medium in conformity with the guidelines
of the manufacturer Biomerieux, Nurtingen, Germany, and by the
fermentation of various carbohydrates in MRS solution. The isolates
were also tested for their capability for growth at 15.degree. C.
and 45.degree. C.
[0092] 5. Molecular characterisation of the lactobacilli by means
of the amplified ribosomal DNA restriction analysis (ARDRA)
[0093] The DNA material for the polymerase chain reaction (PCR) was
prepared according to the method of Ismail 2007 and Vaneechoutte
et. al. 1992: A colony from an MRS agar plate was dissolved in 500
.mu.l of PCR buffer solution to produce a turbid suspension
(McFarland 3) and incubated in a Thermomixer at 95.degree. C. for
ten minutes. This DNA material was stored at -20.degree. C. until
the PCA analysis was performed.
[0094] The PCR was performed in an Eppendorf Mastercycler 5330,
Hamburg, Germany, in volume units of 50 .mu.l, consisting of 20
.mu.l PCR master mixture (Fermentas, Sankt Leon-Roth, Germany), 26
.mu.l double-distilled water, 1 .mu.l of the respective primer and
2 .mu.l of the DNA sample. The PCR program was Initial
denaturisation at 93.degree. C. for five minutes, 35 cycles of
denaturisation at 92.degree. C. for one minute, hardening at
58.degree. C. for 1.5 minutes and elongation at 72.degree. C. for
2.5 minutes respectively, followed by a final elongation at
72.degree. C. for ten minutes and cooling and keeping at 4.degree.
C.
[0095] These sequences of oligonucleotide primers were:
forward-UP68 5'-TGG CTC AGA TTG AAC GCT GGC GGC-3' and
reverse--UP69 5'-CCT TTC CCT CAC GGT ACT GGT-3'. An amplification
of about 2.4 kb was produced, which for the most part consisted of
the 16S rDNA, 16S-23SrDNA spacer region and portions of the 23SrDNA
-stand (Ismail, 2007, Vaneechoutte et al 1992). The restriction was
performed overnight at suitable temperatures in 20 .mu.l volume
units, which contained 0.5 .mu.l restriction enzyme
(HaelII.,Hinfl.) (Fermentas) DdeI (New England Bio Labs Inc.), 2
.mu.l enzyme buffer solution (Buffers R for HaellI. and Hinfl and
Buffer 3 for DdeI), 2.5 .mu.l -15.5 Ml 1.times.TE buffer, and
2.5-15 .mu.l PCR product.
[0096] The used volume was based on the strength of the signal from
the PCR products, which were analysed on the pattern of the agarose
gel restriction fragment by electrophoresis in 1.5% agarose gel in
1.times. TAE buffer at 80 V for two hours. Staining was performed
with ethidium bromide for 30 minutes, followed by washing for 15
minutes in water. The photographs were taken under UV light.
[0097] 6. Strain-specific PCR for Lactobacillus fermentum
[0098] Strain-specific PCR was performed according to the method
described by Dickson et. al. 2005. Primary LF1 (nt196-215; 5'-AAT
ACC GCA TTA CAA CTT TG-3') and LF2 (nt529-510; 5'-GGT TAA ATA CCG
TCA ACG TA-3') were used specifically for Lactobacillus fermentum.
An amplification of 337 bp length was produced. The amplification
program was used without change. The PCR products were analysed as
described for ARDRA.
[0099] 7. 16S rDNA Sequences:
[0100] Based on the ARDRA profile data in each case (chart 2-2), 12
strains were chosen for sequencing. The strains were chosen based
on the profiles that had been chosen after application of the Hinfl
restriction enzyme. Five strains that represented each of the two
ARDRA profiles (A1 and A2) and one of the profiles B and C in each
case. The PCR products generated with the primers UP68 (forwards)
and UP 69 (backwards) (Vaneechoutte et. al. 1992), were purified
with a clean up kit (NucleoSpin.RTM. Extract II, Macherey-Nagel,
Duren, Germany) according to the manufacturer's operating
instructions. Purification was controlled by electrophoresis in 1%
agarose gel. Partial sequencing of 16S rDNA was performed using
UP68 as sequencing primer. Sequencing was performed at MWG Biotech
AG in Ebersberg, Germany.
[0101] 8. Pulse Field Gel Electrophoresis (PFGE)
[0102] PFGE analysis of lactobacillus fermentum strains was
performed according to the method of Hoppe-Seyler et al. 2003. The
restriction enzymes that were tested were NotI, AscI and SmaI. AscI
gave the best profiles and was used in this study. Electrophoresis
was performed in a Bio-Rad CHEF-DRII system which was adjusted to
the following conditions: Temperature 14.degree. C., pump rate 70
rpm, first switching time 2.0 seconds, final switching time 30
seconds, running time 24 hours and voltage 175 volt. After
electrophoresis, the gel was soaked in ethidium bromide (0.5 mg/l)
for 30 minutes. The gel was then washed in distilled water for 10
minutes and photographed under UV light.
[0103] 9. Data Analysis
[0104] The microbiological quality of the colony-forming units
(cfu) and the pH were analyzed using SAS version 9.1. The ARDRA
gels were visually analysed and the patterns compared with those of
typical strains and brought to agreement. The orientation of the
16S rDNA sequences and the generation of the dendogram were based
on Escherichia coli ATCC11775T (access number X80725) as a root of
the dendrogram (Tamura et. al. 2007) with the MEGA4
neighbour-joining method.
[0105] The sequences were also compared with the sequences in the
gene bank for the DNA data using the BLAST algorithm (Altschul et.
al. 1997). The PGGE profiles were analysed using the GelCompar II
program (Applied Maths, Kortrijk, Belgium). Similarities between
different chains were derived using Dice's coefficient. The
unweighted pair group method with arithmetic mean (UPGMA) was used
to cluster different profiles.
[0106] 10. Microbiological Quality of Kimere
[0107] Lactobacilli make up the biggest portion of the microbial
population of fermented Kimere. Coli bacteria and other
enterobacteria were detected neither in 0.1 gram of the sample in
testing in LST nor in 0.01 gram of the sample on VRB agar, nor in
0.01 gram of the sample on VRBD.
[0108] 11. Biochemical Characterization of the Lactobacilli
[0109] 48 strains of isolated lactobacilli were examined for growth
at growth at 15.degree. C. and at 45.degree. C. and for their gas
production from glucose and for the capability to metabolize
various sugars. In addition, they were characterized by API 50CHL.
All strains were capable of growing on fructose, galactose,
glucose, lactose, maltose, mannose, ribose and sucrose. All strains
with the exception of K1-Lb5 were capable of growing on melibiose
and raffinose. All strains with the exception of the isolated
K4-Lb6 were capable of growing on mannitol, melecitose, rhamnose,
sorbitol and trehalose. Only three isolates were capable of growing
on cellobiose. Most strains with the exception of K4-Lb6 were
capable of growing at 45.degree. C.
[0110] By the identification according to API 50CHL, 33 strains of
Lactobacillus fermentum could be isolated. The API identity points
extended from 38.9 to 99.9%.
[0111] 12. PFGE Profiles of the Kimere Isolates
[0112] All isolates from Kimere, which was identified as
Lactobacillus fermentum, were subjected to PFGE analysis. Of the
restriction enzymes that were used for testing (AscI, NotI, SmaI),
only AscI produced results suitable for comparison. FIG. 3 shows
the profiles of the 10 strains disclosed here. Although they have
only been isolated from a single kind of fermented food, most kinds
show fairly different PFGE patterns, which illustrates the high
biodiversification within the strains of Lactobacillus fermentum
from Kimere.
[0113] The very significant differences of the band patterns
between the individual strains also make it plausible that a clear
identification is possible using this band pattern.
[0114] 12. Strain-Specific Immunomodulation of the Strains of
Lactobacillus fermentum Isolated from Kimere
[0115] It indicates that probiotic effects are strain-specific. So
far, very few studies have compared strains of the same genus to
reach this conclusion. With the study described here, the probiotic
potential of strains of the genus Lactobacillus fermentum are
investigated and demonstrated strain-specifically in vitro. Nine
strains of Lactobacillus fermentum and one strain of Lactobacillus
plantarum, which could grow in a 3% bile salt solution, were
selected from 48 strains by a bile tolerance test.
[0116] These strains were further investigated, namely for their
growth rate in an MRS medium (Man Rogosa and Sharpe), supplemented
with 0.3% and 3% bile salt solution and for their resistance to a
low-pH by counting the surviving colony-forming units (cfu) after
exposure to pH 2 and pH 3 for three hours.
[0117] The immune-modulating potential was investigated by
co-incubation of the strains with human peripheral blood
mononuclear cells (PBMCs) for 48 hours at 3TC and measurement of
the cytokines, specifically for Th 1 cytokines (interferon-.gamma.)
and Th2 cytokines (Interleukin-4) in the supernatants by
enzymatic-linked immunosorbent assay (ELISA).
[0118] All strains could tolerate 0.3 bile acid, with strain K4-Lb6
even withstanding 3.0% bile acid. The K7-Lb1 and K8-Lb1 strains
even showed a survival rate of about 53% and 27.2% at a pH of 2.0;
by contrast most strains only remained intact at a pH of 3.0.
[0119] As with LGG, the strains K7-Lb1 and K8-Lb1 reduced
interleukin-4. LGG and all other strains induced a changed from the
basal Th1/Th2 ratio towards Th1 with the exception of the strains
K7-Lb1 and K8-Lb1 induced a change towards Th2. The SEA-stimulated
Th1/Th2 ratio was reduced from the Lactobacillus fermentum strains,
while, by contrast, it was raised by LGG.
[0120] The study thus shows strain-specific peculiarities of the
strains of Lactobacillus fermentum and also shows the capability of
suppressing the production of interferon-.gamma. induced by
Staphylococcus entertoxin A (SEA), a property that has not been
reported by earlier studies of Lactobacillus fermentum and of most
other Lactobacilli. These various properties can be used to combat
illnesses that are driven by a Th1 response or a Th2 response.
[0121] 13. Fermented Probiotic Food Supplements and their Effect on
the Immune System
[0122] The application of lactic acid bacteria (LAB) for human
nutrition has been known for millennia--wherever fermented milk or
other fermented foods have been enjoyed. Their direct association
with human health was first observed in 1908 by the Russian Nobel
laureate Elie Metchnikoff, who ascribed the health and longevity of
Bulgarian herdsmen to the microorganisms in the yoghurt that they
frequently consumed.
[0123] The observations found new interest when the use of
Lactobacilli and Bifidobacteria gained importance as food
supplements. "Probiotic" is defined as "A live microbial feed
supplement which beneficially affects the host animal by improving
its intestinal microbial balance." (Fuller, R. 1989, Probiotics in
Man and Animals. Journal of Applied Bacteriology 66, 365-378).
[0124] The definition and application of "probiotics" continued to
develop as soon as scientific evidence had been obtained
(Schrezenmeir, J. and de Vrese, M., 2001, Probiotics, Prebiotics
and Synbiotics, approaching a definition. American Journal of
Clinical Nutrition 73, 361-364.) A more recent proposal by
Galdiano, C. M., de LeBlanc, A. D., Vinderola, G., Bonet, A. E. B,
and Perdigon, G., 2007, Proposed Model: Mechanisms of
immunomodulation induced by probiotic bacteria. Clinical and
Vaccine Immunology 14, 485-492) includes immunomodulation in the
definition of probiotics. They defined probiotic as "living
microorganisms that, when added to foods, influence the composition
and activity of the microbes in the digestive system, modulate the
inflammatory reaction, improve the non-specific barriers in the gut
and strengthen or improve the immune response of the mucosae and of
the system."
[0125] The selection of strains for use as probiotics concentrates
on two key properties: The adaptability of the strain itself and
the health-supporting or functional aspect. These selection schemes
include: Survival in a milieu with low pH, growth in the presence
of bile salts, adhesion to epithelial cells on the intestinal wall,
colonization within the digestion system, maintaining the microbial
equilibrium, non-pathogenicity towards the recipient, resistance to
technical stress during processing and distribution and, finally,
the capability to improve the health of the recipient. (FAO/WHO,
2002, Guidelines for the evaluation of Probiotics in food. Joint
FAO/WHO Working group report on Drafting Guidelines for the
evaluation of Probiotics in food
ftp://ftp.fao.org/es/esn/food/wgre-port2.pdf, 1-11).
[0126] It must be noted here that not every individual probiotic
strain must show all these properties. According to recent
scientific findings, bacteria must not be "living" to product
immunomodulating effects since both living and dead bacteria or
only bacterial DNA demonstrably shows some health benefits.
[0127] However, survival in the digestion system is essential to
ensure that the probiotics reach the target in the active state,
depending on the functional requirements such as colonization of
the digestion system and the exclusion of putrefactive bacteria or
pathenogens.
[0128] Immunomodulation by probiotics is achieved by interaction
with the immune cells of the recipient and the secretion of various
signal molecules, such as cytokines and immunoglobulins. The T
helper cells perform an outstanding role within the immunocompetent
cells in the immunomodulation, which produces cytokines by means of
probiotics.
[0129] In interaction with the antigens, available T helper cells
differ in various ways, depending on the type and number of the
antigens. The environment of the cytokines ultimately determines
the family relationship and the effect profile. The differentiation
into T helper 1 cells depends on the interleukin 12-effected
activation of transcription 4 (STAT4), which leads to
interferon-.gamma. production, which, via a series of cascades,
leads to a further growth of T-bet-guided transcription factors,
which leads to a further growth of interferon-.gamma.,
interleukin-12 receptors .beta.2, interleukin-4 supression and the
maintenance of family relationships to T helper 1 cells.
[0130] On the other hand, T helper 2 cells differ in the presence
of interleukin-4 by STATE, an increase of interleukin-4 production,
the suppression of interferon-.gamma. production and thus
maintenance of T helper 2 descent.
[0131] The other kind of differentiation of T helper cells is T
regulation (Treg), which produces interleukin 10, Treg cytokines
IL-10 regulate the immune system both due to the suppression of T
helper 1 cells as well as of T helper 2 cytokines by ensuring
peripheral tolerances. The fourth known kind of natural T helper
cells is differentiation via interleukin 23, which leads to the
production of interleukin 17.
[0132] It is assumed that a disequilibrium between T helper 1, T
helper 2, Treg and T17 cells is the precursor to a manifestation of
various pathological conditions. The probiotic function increases
cytokine production towards a reinforcement of the immune defence,
a reduced allergic or autoimmune reaction and/or a lower
inflammation reaction.
[0133] A modulation of cytokine production by probiotics in human
and animal models was found both in vitro (Pochard, E., Gosset, P.,
Grangette, C, Andre, C, Tonnel, A. B., Pestel, J., Mercenier, A.,
2002, Lactic acid bacteria inhibit T(H)2-cytokine-production by
mononuclear cells from allergic patients. Journal of Allergy and
Clinical Immunology 110, 617-623, and Ghadimi, D., Folster-Holst,
R., de Vrese, M., Winkler, P., Heller, K. J., Schrezenmeir, J.,
2008, Effects of probiotic bacteria and their genomic DNA on
TH1/TH2 cytokines production by peripheral blood mononuclear cells
(pbmcs) of healthy and allergic subjects, Immunobiology 213,
677-692) and in vivo (Kopp, M. V., Goldstein, M., Dietschek, A.,
Sofke, J., Heinzmann, A. and Urbanek, R., 2008, Lactobacillus GG
has in vitro effects on enhanced Interleukin-10 and
Interferon-.gamma.-release of mononuclear cells but no in vivo
effects in supplemented mothers and their neonates). Clinical and
Experimental Allergy 38, 602-610).
[0134] In most cases, a mixture of Th1, Treg and Th2 cytokines was
detected, although several authors include an exchange of Th1 or
Th2 in the action sequence. This immunomodulation depends on the
dose and time and also shows dependencies on the inoculation of the
strains.
[0135] 14. Bacterial Cultures
[0136] The bacterial strains used in this study are isolated from
Kimere, a fermented dough from Kenya and characterised according to
molecular methods and kept at -80.degree. Celsius in a MAST
Cryobank.RTM. (MAST Diagnostic, Reinfeld, Germany). As positive
comparison controls for T helper 1 cytokines and T helper 2
cytokines, the strains Lactobacillus rhamnosus GG (ATCC 5310, a
strain that is known to stimulate interferon-.gamma.) and
Escherichia coli TG1 (BU-00035, a strain that is known to stimulate
interleukin-4). They were procured from commercial sources.
[0137] The Lactobacilli were cultured anaerobically overnight in
MRS solution at 3TC in an MAC8-VA500 workstation (Don Whitley
Scientific Limited, UK). The Escherichia coil TG1 were cultured
aerobically overnight at 37.degree. in a Luria-Bertani (LB)
solution. The cells were centrifuged for two minutes at 14,000 g
(corresponding to 144,500 rpm on an Eppendorf Minispin Plus
centrifuge). The bacterial pellets were washed twice with a
phosphate-buffered salt solution (PBS) dissolved in 1 ml PBS,
containing 20% glycerine, then counted in an improved Neubauer type
hemocytometer and subsequently brought to a concentration of
10.sup.8 cells per millilitre in a PBS solution with 20% glycerine
and kept at -80.degree. Celsius until use.
[0138] 15. Resistance to Bile Salts
[0139] 48 isolates of Lactobacilli were tested for their resistance
to bile salts according to the above-described method with small
changes. 0.1 ml of an MRS growth culture was added to 9.9 ml of MRS
solution with 0.3 or 0.5 or 1.0 or 2.0 or 3% cattle bile (Siegmar).
They were incubated for 24 hours at 37.degree. Celsius. The growth
was recorded after visual checking of the turbidity of the
tubes.
[0140] Ten isolates were selected, which even showed growth with 3%
ox bile. Their growth was then monitored with 0.3% and 3% ox bile.
2 ml as aliquot (partial sample) of Lactobacilli from a fresh
culture grown overnight was suspended in PBS as spheres and 100
.mu.l thereof was inoculated into an MRS solution, to which 0.3%
(w/v) bile solution had been added. A 0.3% (w/v) bile salt
concentration has been classified as physiologically relevant and
in many studies serves for probiotic selection. The same applies to
a 3% ox bile (w/w).
[0141] The cultures were incubated in the water bath at 37.degree.
Celsius. The growth was observed hourly by measurement of the
absorption at 620 nm (A Index 620 nm). The tests were repeated 3
times. The absorption values were recorded over the incubation
times as described by Gilliand and Walker. (Gilliand, S. E. and
Walker, D K 1990, Factors to consider when selecting a culture of
Lactobacillus acidophilus as a dietary adjunct to produce a
hypocholesterolemic effect in humans, Journal of Dairy Science
73,905-911).
[0142] 16. Acid Resistance
[0143] 2 ml of a culture grown overnight was centrifuged for one
minute at 14,000 g (corresponding to 145,400 rpm on an Eppendorf
Minispin Plus centrifuge). The pellets were washed twice with 2 ml
Ringer's solution and suspended again in 1.5 ml Ringer's solution.
0.1 ml of the suspension was transferred to 5 ml of 0.35% NaCl
solution (supplemented with pepsin (0.1 gram per 50 ml) and
adjusted to the various pH values 2.0 and 3.0 and 6.5 with HCl),
followed by incubation at 37.degree. Celsius in a water bath.
[0144] After three hours, 5 ml of 0.1 M phosphate buffer solution
with a pH of 6.5 was added. The survival rates were detected by
counting on MRS agar. The colony-forming units (cfu) were expressed
as a percentage of the control values that had been obtained on
incubation with a pH of 6.5.
[0145] 17. Test of Immunomodulation with PBMCs (peripheral blood
mononuclear cells).
[0146] Complete blood was collected from healthy donors aged
between 21 and 52 years and mixed with EDTA
(ethylenediaminetetraacetic acid) for purposes of anticoagulation.
Individuals who had reported an allergy or had recently suffered
infections of the respiratory passages or had taken medication were
excluded. The enlisting of test subjects and taking of blood
samples was performed according to strict ethical points of view
which had been laid down by the ethics committee of the University
of Kiel for the use of human test subjects in research. A written
agreement was obtained from all test persons before their
registration for this study. The samples were kept at room
temperature (18-25.degree. Celsius) until isolation of the
PBMCs.
[0147] The PBMCs were isolated as described above. Blood was mixed
with sodium chloride (NaCl) and filled into 50 ml centrifuge tubes
at a mixing ratio of 1:1 and placed on a Ficoll Lymphoprep Plus
(Axis Shield PoC AS, Oslo, Norway) at a ratio of 1:2 (16 ml Ficoll
and 32 ml blood) after centrifuging at 179.5 g (1,300 rpm) on an
Eppendorf centrifuge 5810 R, r=9.5 cm) for 30 minutes at 18.degree.
C. The PBMC layers were filled into sterile centrifuge tubes with
50 ml content. Washing liquor (PBS plus 10% FCS) was added to fill
the tubes (about 25 ml) and then to centrifuge them at 179.5 g for
10 minutes at 4.degree. Celsius.
[0148] Washing was performed three times with PBS plus 10% FCS;
each time, half of the washing medium was discarded of and finally
the entire washing medium was removed. After the third washing, the
PBMCs were suspended in the complete medium (RPMI 1640 Medium+10%
FCS+1% (penicillin+streptomycin)) all supplied by Gibco, Karlsruhe,
Germany. The PBMCs were counted in a hemocytometer, 10 .mu.l :90
.mu.l, PBMCs:staining agent and expressed as PBMCs/ml of the
medium.
[0149] 18. Coincubators of the PBMCs and Bacteria:
[0150] The concentration of PBMCs that was used was
1.times.10.sup.8 lines/ml and the bacteria 2.times.10.sup.7
cells/ml (1:20 PBMC to bacteria ratio). Besides the underlying
condition (medium), with the use of Staphylococcus entertoxin A
(SEA), a stimulating state was measured at a concentration of 2
.mu.g/ml. The treatments were: PBMC's+medium as comparison
parameter; PBMC's+SEA+medium; PBMC's+bacteria+medium and
PBMCs+SEA+bacteria+medium, in each case in 1 ml volume units in a
plate with 24 wells and in duplicate. After incubation at
37.degree. C. for 48 hours, the supernatant was centrifuged at
179.5 g, 4.degree. Celsius for ten minutes and then filtered
through a sterile microfilter with 0.2 .mu.m mesh size, and filled
into Eppendorf tubes (1.5 ml) in volume units of 250 .mu.l and kept
at -20.degree.C. for the ELISA test.
[0151] 19. Cytokine Sample
[0152] Examination of the detection of interleukin and
interferon-.gamma. in the supernatants was performed with an ELISA
plate with 96 wells, corresponding to the manufacturer's
directions. All ELISA reagents were purchased from Mabtech AG,
Hamburg, Germany. The lower detection limits were 1 pg/ml for
interleukin and 4.2 pg/ml for interferon-.gamma., as recommended
for each kit.
[0153] The absorption was measured at 450 and 570 nm wavelength in
a microplate reader, Molecular Devices, Munich, Germany. For
correction of the wavelength, the reading of the optical density at
570 nm was subtracted from the value read for 450 nm. The mean
absorption values of the empty plate (background control) was
deducted from the standard values for the standard test specimens
and the values of the samples, and then a standard curve was
generated using the curve fitting program with four parameters and
the derivation of the cytokine concentration in the samples.
[0154] 20. Statistics
[0155] By means of ANOVA, the mean values of the bile resistance of
various strains were compared. For the data for immunomodulation,
the analysis was performed in two steps: First, all the strains and
the comparative test samples were compared with one another. In the
second step, the values of the control parameters LGG and TG1 were
excluded, which are classified as "blips" for interferon-.gamma.
and interleukin-4.
[0156] With the general linear model of the SPSS 9.0 simulation
software, the mean values of the SEMs were derived and the mean
values of different strains were compared using the "Mann-Whitney U
test". With Sigma Plot 11.0, the results were presented
graphically. Statistical significance was considered at P>0.05.
To define the direction of the displacement of the ratio of T
helper 1 to T helper 2 values in the immune response, the ratio of
interferon-.gamma. to interleukin-4 was used. The mean ratio was
defined at 100% and the values for the other test specimens were
expressed as a percentage of this comparison value. Values over
100% were classified as a T helper 1 shift and values smaller than
100% as a T helper 2 shift.
[0157] 21. Resistance to Bile Salts and Low pH
[0158] To check their bile tolerance, all 48 isolates were
administered to an MRS medium overnight, which was adjusted to
0.3%, 0.5%, 1%, 2% and then to a proportion of 3% ox bile (w/v). By
checking the average turbidity, the growth of, first, 48, then 28,
26, 21 and 12 isolates was ascertained (see FIG. 4a).
[0159] The monitoring of their growth movement (change in
OD.sub.620 nm) in MRS solution, supplemented with 0.3% ox bile
(w/v) shows a good growth of all strains, which was indicated by a
short delay phase (FIG. 4b). Only the strains K1-Lb6 and K8-Lb1
required over three hours to reach 0.3 units of A.sub.620 nm. As
shown in FIG. 4c, the growth of the strain K4-Lb6 differed
significantly from the mean value of the others in a solution with
3% ox bile (w/v). The strain K4-Lb6 began to grow almost without a
delay phase. For all the other strains, the growth was only visible
after a prolonged incubation overnight.
[0160] If the strains were subject to a pH of 2 and a pH of 3 for
three hours, they showed the different stages of survival as shown
in FIG. 5. At a pH of 3, four strains, namely K1-Lb6, K7-Lb1,
K8-Lb1 and K9-Lb3, showed survival rates of over 80%. Two of these,
namely the K7-Lb1 and K8-Lb1 strains, showed the greatest stress
resistance towards most acids with survival rates of 52.9% at pH
2.0 and 27.2% at pH of 6.5, compared to the control strains.
[0161] 22. Cytokine Production Pattern
[0162] The Kimere strains supressed the SEA-induced
interferon-.gamma. production, a phenomenon that has so far not
been observed for strains of Lactobacillus fermentum. The Kimere
strains supressed interleukin-4 production. In the case of the
strains K7-Lb1 and K8-Lb1, the interleukin-4 was reduced to close
to the limit of detectability (FIG. 6b).
[0163] A strain-dependent shift of the interferon-.gamma. to
interleukin-4 ratio, and therefore of the accordingly proportional
ratio of T helper 1 to T helper 2, was observed (FIG. 6c).
[0164] The strains K7-Lb1 and K8-Lb1 displaced the T helper 1 to T
helper 2 balance towards T helper 2, while the others induced a
change towards T helper 1. All the tested strains were, in
comparison to TG1--which is known to suppress interleukin-4
production--capable of significantly suppressing the production of
interleukin-4. Compared to the control substances, the strains
K6-Lb4 and K8-Lb1 suppressed the basal secretion of interleukin-4
(FIG. 6b).
[0165] K7-Lb1 and K8-Lb1 suppressed the SEA-induced interleukin
secretion (FIG. 6b). Interleukin-4 extended from 1.96 pg/ml for
K8-Lb1 (almost not detectable) to 45 pg/ml for K1-Lb1 (FIG.
6b).
[0166] The basal interferon-.gamma. production was significantly
suppressed by the strains K7-Lb1 and K8-Lb1 (FIG. 6a).
[0167] A suppression of SEA-induced interferon-.gamma. production
by the tested bacterial strains could be observed. It was
particularly pronounced for the strains K7-Lb1 and K8-Lb1, but also
for all other Kimere strains (FIGS. 6a to 6c).
[0168] Since the overproduction of interferon-.gamma. plays a
leading role in the pathenogenesis of IBD, the reduction of
interferon-.gamma. by the strains of Lactobacillus fermentum from
Kimere presented here shows that these strains have an
immunomodulating property, which can be used in the battle against
illnesses that are dependent on T helper 1 cells. Various strains
of Lactobacillus fermentum from Kimere induced either a T helper 1-
or a T helper 2-driven response, depending on the respective
immunomodulation.
[0169] The various strains have different stimulation effects on
interferon-.gamma. and interleukin-4 (FIG. 6). The strains' ability
to suppress the basal interleukin-4 formation shows their possible
application for T helper 2-driven illnesses.
[0170] The ability of Lactobacillus fermentum isolates from Kimere
to suppress the formation of SEA-induced interferon-.gamma. and
simultaneously to block the production of interleukin-4 shows
unique possibility in the suppression of autoimmune illnesses and
atopic illnesses with inflammatory components.
[0171] Further details and features of the invention are explained
below in greater detail with reference to graphical descriptions.
However, they are not intended to limit the invention but only
explain it. In schematic, view:
[0172] FIG. 1 shows an overview of the properties of the individual
strains
[0173] FIG. 2 phylogenetic tree of the Lactobacilli presented here
in comparison with known bacteria
[0174] FIG. 3 band pattern of all 10 strains according to the PFGE
process
[0175] FIG. 4 tolerance of the strains to bile salt
[0176] FIG. 5 resistance of the strains to very acid milieu
[0177] FIG. 6 Th1 and Th2 cytokine production of human blood cells
(PBMCs) after coincubation with the strains
[0178] FIG. 1 shows, as a key property of the strains presented
here, characteristic effects and their intensities. It is clear
that almost all strains, with the exception of K11-Lb3, show three
characteristic properties that distinguish them from one another.
All of these strains have a more or less high bile salt tolerance
and a greater or lesser pH tolerance. All the strains have an
immunomodulating effect. This immunomodulating effect belongs
either to the natural or innate immunity, such as the elevated
defensin release due to the strains, in this case K2-Lb6 and
K11-Lb3, or is part of the adaptive or acquired immunity, such as
the influencing of the Th1/Th2 response.
[0179] FIG. 1 shows that, of the strains presented here, K1-Lb1,
K1-Lb6, K2-Lb4 and K4-Lb6, strengthen the Th1 helper reaction and
reduce the Th2 influence. The strains K2-Lb6, K6-Lb4, K7-Lb1 and
K8-Lb1, on the other hand, strengthen the Th2 response and reduce
the Th1 influence.
[0180] The intensity is shown in three stages. The highest of the
observed values in each case is designated +++. Correspondingly, ++
indicates approximately 2/3 and +corresponds to about 1/3.
[0181] In FIG. 2, some of the found strains are entered in a
phylogenetic tree, in which some other, adequately well know
bacterial strains have been entered. This phylogenetic tree is
based on partial sequences of the 16S rDNA of selected examples of
ARDRA-PCR grouping of isolates of the Lactobacilli from Kimere
compared with the BLAST database. The evolutionary distances were
derived using the UPMGA method, the bootstrap loader program being
based on 500 repetitions. To calculate the evolutionary distances,
the method of maximum combined probability was used. This
phylogenetic analysis was performed in MEGA4 according to Tamura
et. al. 2007.
[0182] FIG. 3 shows the band patterns of all 10 strains presented
here obtained by the PFGE process--pulse field gel electrophoresis.
From FIG. 3, it immediately becomes clear that the individual band
patterns differ from one another significantly. All the bacteria
that are analysed by the PFGE method, and which show precisely this
band pattern, are thereby clearly identified as this strain of the
respective genus.
[0183] The method of typifying microorganisms by PFGE has been
known since 1984 and is part of the recognized prior art. With the
extremely low effort of depositing a single image, it permits the
unambiguous identification of the respective bacterial strain.
[0184] FIGS. 4a to 4c show the tolerance of the strains to bile
salt.
[0185] FIG. 4a shows the number of the surviving strains on
exposure to cattle bile with increasing concentration of 0.3%, via
0.5%, 1% and 2%, up to 3% cattle bile concentration (w/v).
[0186] In FIG. 4b, by measurement of the absorption of the samples
at 620 nm, the specific growth of the strains in 0.3% cattle bile
solution is plotted against time. A somewhat similar behaviour is
shown for all strains.
[0187] FIG. 4c shows the results of an, in principle, identical
measurement, but with 3.0% concentration of cattle bile. A
particularly rapid growth of the strain K4-Lb6, which is very
obviously highly robust with respect to bile salt, is shown
here.
[0188] In FIG. 5, the resistance of the strains to very acid milieu
is shown, specifically for pH 2 and pH 3. The cells were incubated
in 0.35% NaCl (containing 3 g I.sup.-1 pepsin) and adjusted to pH
values of 2, 3 and 6.5 for 3 h at 37.degree. C. in each case. The
surviving cells were counted on an MRS agar plate at 37.degree. C.
for 48 to 72 h. The values at pH 6.6 defined as reference value
with 100% and the cells surviving at pH 2 and pH3 were compared
with this reference value.
[0189] FIGS. 6a to 6c show Th1 and Th2 cytokine production of human
blood cells (PBMCs) after co-incubation in vitro with Kimere
Lactobacilli strains, specifically with and without stimulation by
superantigenic Staphylococcus enterotoxin A (SEA). As an effect of
the bacterial strains on the immune system and on the shift of the
Th1/Th2 ratio, FIG. 6a shows the production of interferon-.gamma.
and FIG. 6b shows the production of interleukin-4 and FIG. 6c shows
the resulting ratio of interferon-.gamma. and interleukin-4.
* * * * *