WO2007108763A1

WO2007108763A1 - Use of lactobacillus strains for promoting immunotolerance in autoimmune disease

Info

Publication number: WO2007108763A1
Application number: PCT/SE2007/050155
Authority: WO
Inventors: Eva GRAHN HÅKANSSON; Maria Isaksson
Original assignee: Probac Ab
Priority date: 2006-03-17
Filing date: 2007-03-14
Publication date: 2007-09-27
Also published as: WO2007108763B1

Abstract

The present invention relates to the use of Lactobacillus strains chosen from the species Lactobacillus plantarum LB3e, LB7c and LB931 and Lactobacillus rhamnosus LB21 for promoting immunotolerance in autoimmune diseases chosen from coeliac disease and atopic dermatitis.

Description

Title: Use of Lactobacillus strains for promoting immunotolerance in autoimmune disease.

TECHNICAL FIELD

The present invention relates to the use of Lactobacillus strains chosen from the Lactobacillus plantarum and Lactobacillus rhamnosus for preparing a medicament promoting immuntolerance chosen from coeliac disease and atopic dermatitis.

BACKGROUND OF THE INVENTION

The mucosal surfaces of the lungs and the GI-tract have several purposes including absorption, macromolecule transport, barrier and secretory functions. However, the large mucosal surfaces are continuously exposed to millions of more or less harmful antigens from the environment, food and microorganisms. To meet these challenges, the mucosa possesses an immune system that tightly controls the balance between responsiveness and tolerance. The immune system of the mucosa is part of the en- tire immune system and, consequently, immune responses in the mucosa are reflected in the entire body. It consists of an integrated network of tissues, lymphoid and non-lymphoid cells and effector molecules such as antibodies and cytokines (1).

Coeliac disease (CD) is a common genetically determined chronic inflammatory in- testinal disease that occurs in all ages (7). It is induced by an environmental precipitant, gluten and related proteins in rye, barley and, possibly, oats. The celiac symptoms in small children are different than in adults and older children. Small children often suffer from diarrhoea, fatigue and loss of weight. In adults and older children the symptoms are more diffuse which may delay the diagnosis. Both the innate and adaptive immune reactions seem to take place in the small intestinal mucosa in active CD. The hallmark for untreated CD in an inflammatory lesion in the upper small intestinal mucosa characterized by increased frequency of intraepithelial lymphocytes together with various degrees of small intestinal villous atrophy and crypt hyperplasia (2) Today CD is considered to be an autoimmune disease (3, 4). People suffering from CD are not able to eat a diet containing gluten, which is often represented in bread and cereal products. CD relapses if gluten in reintroduced into the diet after first having been withdrawn. The treatment is a lifelong diet free from gluten. To be considered gluten-free diet the food products must contain less then lOOmg gluten/day (5).

Components of the normal flora, Bifidobacteria, Lactococcus and Lactobacilli, have been associated with beneficial effects on the host. These beneficial effects include maintenance of intestinal homeostasis, competitive exclusion of pathogens, produc- tion of antimicrobial compounds, promotion of gut barrier function, and immune modulation. (6)

From US2004047849 it is known that Lactobacillus strains can be used for treating allergy. Bacteria are disclosed that express high levels of INF-γ, thereby providing an immune response of Th 1 -type.

Also, from US2005214271, the Lactobacillus paracasei strain GM-080 is disclosed for treating allergy related diseases. INF- γ secretion is obtained.

Di Cagno et al. (J. Agric. Food Chem. 2005, 53, 4393-4402) disclose pasta that has been made from durum wheat fermented with lactobacilli for decreasing gluten intolerance. Di Cagno et al. (Applied and Environmental Microbiology, Feb. 2004, p 1088-1096) disclose a sourdough bread that is tolerated by celiac sprue patients. In these two articles it is discussed that the lactobacilli has the effect of digesting prolamin, gluten.

Further, it is known from US20050191403 that a peptidase enzyme can be used in the manufacture of a medicament for gluten intolerance. Thus, the use of food products and medicaments that can decrease e.g. gluten intolerance. However, from these disclosures it is not known that lactobacilli can affect the immune system.

Therefore, there is a need to provide improved means to promote immuntolerance in disorders such as CD. Especially, it would be advantageous to provide immune effect-improving food products, such as bread and cereals that are easily accessible to people.

SUMMARY OF THE INVENTION

The inventors of the present invention have surprisingly found that microorganisms of the Lactobacillus genus chosen from the species Lactobacillus plantarum and Lactobacillus rhamnosus, particularly LB931 (DSMl 1918), LB21 (NCIMB 40564), LB3e (DSM 17852) and LB7c (DSM 17853) are suitable for promoting immuntol- erance in autoimmune diseases such as, coeliac, atopic dermatitis, disease diabetes, Ulcerative colitis and Crohn's disease, inflammatory bowel disease, psoriasis, rheumatoid arthritis and allergies. Good results have been disclosed among people suffering from coeliac disease (CD) and atopic dermatitis. The Lactobacillus strains are able to keep the inflammatory lesion normal in the upper intestinal mucosa and re- duce the levels of antibodies associated with autoimmune diseases.

DEFINITIONS

As disclosed herein, the term "LB" relates to bacteria of the genus Lactobacillus.

As disclosed herein, the term "lactic acid bacteria" relates to bacteria producing lactic acid, such as bacteria belonging to the genera Lactobacillus.

As disclosed herein, the term "CFU" relates to colony-forming units.

As disclosed herein, the term "CD" relates to coeliac disease. BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 discloses expression of organic acids in an Auto- Scaled Chromatogram of supernatants of the Lactobacillus strains LB931, LB3e, LB7c and LB21 (analysis completed by Steins Laboratorium).

Figure 2 discloses expression of lactic acids in an Auto- Scaled Chromatogram of supernatants of the Lactobacillus strains LB931, LB3e, LB7c and LB21 (analysis completed by Steins Laboratorium).

Figure 3 discloses the production of cytokines by polyclonal activated T cells in the presence of the probiotic bacteria strains LB931, LB3, LB7 and LB21.

Figure 4 discloses that both live LB931 and live LB21 gave the same cytokine re- sponses on activated T cells.

Figure 5 discloses that the lactobacillus did not increase the IFN-γ levels or IL-4 levels of resting T cells.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to pharmaceutical compositions comprising selected strains of microorganisms of the Lactobacillus genus chosen from the species Lactobacillus plantarum and Lactobacillus rhamnosus, particularly strains such as Lactobacillus plantarum LB931, Lactobacillus rhamnosus LB21, Lactobacillus planta- rum LB7c and Lactobacillus plantarum LB3e for promoting immuntolerance in autoimmune diseases chosen from coeliac disease and atopic dermatitis. The Lactobacillus strains are able to keep the inflammatory lesion normal in the upper intestinal mucosa and reduce the levels of antibodies associated with autoimmune diseases. According to the present invention the Lactobacillus plantarum and Lactobacillus rhamnosus strains can be included in a pharmaceutical composition, dietary supplement, food or the components thereof, which are normally administered to people. The food product can be chosen from bread, dough, cereal products, porridge, gruel, muesli, granola, powdered cereal based product, pasta, semi products, soups, bake-off products, plain bread, stewed fruit.

The food product mentioned above should comprise at least one of Lactobacillus plantarum LB931, Lactobacillus rhamnosus, LB21, Lactobacillus plantarum LB7c and Lactobacillus plantarum LB3e.

The food product mentioned above should contain a unit dosage CFU from 1x10⁵ to 1x10¹¹ CFU/dose of each single strain, preferably > lxl0⁷ CFU/dose of each single strain.

Compositions and a medicament according to the present invention may comprise one or more strains selected from the below said Lactobacillus strains. Other mixtures or single Lactobacillus strains could advantageously be used within the scope of the present invention. A pharmaceutical composition should contain at least one of Lactobacillus plantarum LB931 , Lactobacillus rhamnosus, LB21 , Lactobacillus plantarum LB7c and Lactobacillus plantarum LB3e together with at least one ex- cipient.

The identity and deposit numbers of the strains of the present invention are listed below.

The LB931 has been deposited on Jan. 9, 1998 at DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen, Mascheroder Weg Ib D-38124 Braunschweig. It has been assigned accession number DSMl 1918. The LB21 has been deposited on June 11, 1993 at NCIMB Ltd, Ferguson Building, Craibstone Estate, Bucksburn, Aberdeen, AB21 9YA, UK. It has been assigned accession number NCIMB 40564.

The LB3e has been deposited on Jan. 6, 2006 at DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen, Mascheroder Weg Ib D-38124 Braunschweig. It has been assigned accession number DSM 17852.

The LB7c has been deposited on Jan. 6, 2006 at DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen, Mascheroder Weg Ib D-38124 Braunschweig. It has been assigned accession number DSM 17853.

The Lactobacillus strains mentioned above could be referred to as LB931

(DSMl 1918), LB21 (NCIMB 40564), LB3e (DSM 17852) and LB7c (DSM 17853).

Compositions according to the present invention may comprise one or more strains selected from the above defined LB strains. Other mixtures or single Lactobacillus strains could advantageously be used within the scope of the present invention. A preferred strain is Lactobacillus rhamnosus LB21.

In further embodiments the present invention relates to the use of a Lactobacillus strain chosen from the species Lactobacillus plantarum and Lactobacillus rhamnosus for preparing a medicament for promoting immunotolerance in autoimmune diseases chosen from diabetes, Ulcerative colitis and Crohn's disease, inflammatory bowel disease, psoriasis, rheumatoid arthritis and allergies. The Lactobacillus strains are able to keep the inflammatory lesion normal in the upper intestinal mucosa and reduce the levels of antibodies associated with autoimmune diseases in the patients.

For the practical use, the microorganisms of the present invention could be freeze- dried and formulated in a suitable means for administration such as capsules, tab- lets, powders, granulate, or similar. The bacterial cultures may be prepared according to conventional methods such as suspension, freeze-drying and filling of capsules.

Suspension

The lactobacilli are cultured to early stationary phase in optimised culture broth, washed in NaCl and then resuspended in physiologic saline solution (0.9%), approx. 10% milk solutions like skim milk, breast milk substitution (infant formula like Neocate), oat milk, soy bean milk, or similar. The suspension can be frozen at - 20⁰C or below, in tubes of 5-10 ml each, for thawing one at a time. The concentration can be up to 10¹⁰cfu/ml.

Freeze-drying

The bacteria are cultured to early stationary phase in optimised culture broth, washed in NaCl and then resuspended in cryoprotectant like a 10-20% carbohydrate solution, e.g. sucrose, trehalose, glucose, fructose, sackarose or inulin with or without addition of glutamate or potato or corn starch. The bacteria can also be resuspended in 10% skim milk. The suspension is freezed at between -55°C and -80⁰C, and then put in the freeze-drier, to evaporate all water. This leaves a dry crystallised material that can be ground into a fine powder. The yield generally is about

10¹⁰cfu/g. The powder can be stored at room temperature or for longer stability in fridge or freezer, preferably in moister-proof container to prevent the powder from absorbing water from the air. It can also be mixed in water-free substrates like vegetable oils such as olive oil, rapeseed oil, garlic oil, linseed oil or sunflower oil, for long-term storage.

Filling of capsules

The freeze-dried powder can be filled in gelatine capsules. This is done by using a manual capsule-filler, in which gelatine capsules are divided by hand and each half put in one of two matching plates. When all capsule spaces are occupied the powder is poured over one of the plates and put into the capsule halves. When they are full the empty halves are mounted onto the filled ones and the plates are pressed together so that the capsules are "locked". Each round makes 100 capsules. There are different sizes of capsules, but most suitable are size 2 and 3, that can hold about 250 and 120 mg respectively.

Other methods can be used to dry bacteria. One example is spray-drying, a technique where warm air is used to remove water contents from a finely sprayed suspension of bacteria.

A tablet comprising at least one Lactobacillus strains according to the present invention could further comprise at least one excipient such as cellulose, starch, lactose, diluent, disintegrating agent and lubricants and similar.

The pharmaceutical compositions of the invention may comprise from 1x10⁵ to 1x10¹¹ CFU/ dose of each single strain, preferably > 1x10⁷ CFU/dose of each single strain selected from the species Lactobacillus plantarum and Lactobacillus rhamno- sus, particularly strains such as Lactobacillus plantarum LB931, Lactobacillus rhamnosus, LB21, Lactobacillus plantarum LB7c and Lactobacillus plantarum LB3e. A preferred daily dosage is above 1x10⁷ CFU/dose of each single. A suitable excipient could be included in the composition.

A pharmaceutical composition could be in the form of capsules, suspension, freeze- dried powder, frozen suspension, granulate, tablet or similar.

The experience that has been acquired so far shows as convenient and effective a treatment schedule comprising the administration of the composition of the present invention, to be administered directly to the subject or in the food product or before consuming the food product.

The capability of Lactobacillus to promote immuntolerance has been investigated in in vivo studies. Example 1, 2 and 3 also indicate that the Lactobacillus have the ca- pability to in vivo influence the immune system in a positive way. The children given the Lactobacillus did not experience any symptoms related to the coeliac disease. All the children that were given placebo became ill after 1-3.5 months of challenge with gluten, see Example 3. The biopsy showed normal tissue in the group given Lactobacillus and the tissue showed indication of inflammation in the group given placebo. Nothing has indicated that the same result would be achieved when using any of the other Lactobacillus strains disclosed in the present invention, LB931 (DSMl 1918), LB21 (NCIMB 40564), LB3e (DSM 17852) and LB7c (DSM 17853).

The Lactobacillus strains LB931, LB3e, LB 7c and LB21 have the ability to produce substances (which could be soluble, such as lactic acid, succinic acid, acetic acid, and propionic acid) that inhibit the growth of pathogenic microorganisms, such as enterobacteria, group B streptococci, staphylococci and yeast. Figure 1 discloses expression of organic acids and Figure 2 discloses expression of lactic acids in an Auto-Scaled Chromatogram of supernatants of the Lactobacillus strains LB931, LB3e, LB7c and LB21.

The present invention will now be described with reference to the following exam- pies, which are used for illustrating the invention and should not be construed as limiting for the scope of the invention.

EXAMPLES

To diagnose celiac disease a biopsy of the upper small intestine is performed to determine if the intestinal mucosa is inflamed due to the effect of gluten. Typically, there is a lesion in the mucosa characterized by an increased number of intraepithelial (IEL) and lamina propria lymphocytes, villous atrophy of various degree, and crypt hyperplasia (8,9). When gluten is excluded from the diet the intestine is re- stored and the symptoms will withdraw. A new biopsy is performed a year later to confirm the restored intestine. The last biopsy is performed after a controlled chal- lenge with gluten to reassure the preliminary diagnosis. During the evaluation of the patient blood samples are taken once a month for analysing Immunoglobulin A (IgA) antibodies against gliadin, the major prolamin in wheat gluten, and IgA anti- endomysial antibodies (EMA) and IgG anti-tissue transglutaminase antibody (anti- tTG).

Example 1

A case study was made of a nine year old girl who was diagnosed with coeliac disease (CD). She was given Lactobacillus (LB21) daily in a high concentration (10¹⁰ CFU/day) for 3 months before being challenged with gluten. Her antibody levels of EMA, anti-tTG and total IgA were measured before and each month for the first year and once a year for 6 years. Since she had been on a restricted diet for many years before the study no antibodies were detected before the study started. In the beginning she was given a low concentration of gluten (O.lg gluten/day/kg body- weight) but after 4 months she was eating a normal diet. The levels of EMA and anti-tTG were normal during this time and she did not experience any celiac symptoms. A biopsy of the small intestine was performed when the girl was 13 years old. The results showed no inflamed mucosa tissue and her antibody levels were normal.

Example 2

A pilot study with 4 children suffering from CD was performed. The children were divided into two groups. Group 1 was given Lactobacillus (LB21) with 10¹⁰ CFU/day and group 2 was given placebo. Both groups were challenged with gluten following a standardized scale. The children were given O.lg gluten/day/kg body- weight for 4 weeks and 0.2g gluten/day/kg bodyweight thereafter. The antibody levels of EMA, Anti-tTG and total IgA were measured during the study. If the antibody levels of the children increased or if the CD symptoms enhanced, a biopsy of the small intestine was performed. The two children in the placebo group became ill after 1-3.5 months of challenged gluten diet. The biopsy showed damage to the mu- cous membrane of the small intestine. The children also had increased levels of

EMA, Anti-tTG and total IgA. The two children that were given Lactobacillus did not experience any symptoms and the levels of EMA, Anti-tTG and total IgA were normal. After 7 months of active treatment a biopsy of the small intestine was performed. The biopsy showed normal intestinal tissue in the upper intestinal mucosa.

Example 3

A study with 10 children suffering from CD was performed. The children were given Lactobacillus (LB21) of 10¹⁰ CFU/day. The children were challenged with gluten following a standardized scale. The children were given 0.025 g gluten/day/kg bodyweight for 2 months and 0.05 g gluten/day/kg bodyweight thereaf- ter. The antibody levels of EMA, Anti-tTG and total IgA were measured during the study. If the CD symptoms enhanced, a biopsy of the small intestine was performed to confirm the typical CD lesions. The children did not experience any symptoms during the study and after 12 months of gluten challenge under intervention of LB21 the children still were healthy, symptom-free, had normal growth and the biopsy showed normal tissue in the upper intestinal mucosa.

Example 4

A study of 32 children under the age of 3 with moderate to severe atopic dermatitis was performed. The children was administrated either active treatment; drinking a suspension of LB21 ( 10¹ ^fu/day) in an oat based formula, or placebo; just oat based formula, every day for 8 weeks. The eczema was assessed 5 times, once before, three times during and once after the study, by SCORAD. The parents were instructed to continue treatment with topical corticosteroids during the study and the tubes were weighed at every visit. There were no obvious difference (p>0,05) be- tween the groups regarding SCORAD. All patients reduced the score during the study. However, surprisingly the children in the active group had used less corticosteroids than the placebo group, suggesting that LB21 could have a steroid-saving effect.

Example 5 Method An in vitro test was done to evaluate the potential immunomodulatory characteristics of the four different lactobacillus strains. Venous blood was collected from healthy women and the peripheral blood mononuclerar cells (PBMC) were isolated using Ficoll- Paque gradient. The T cells were polyclonally activated using mAb anti- CD 3 and mAb anti- CD 28. The PBMC and the probiotic bacteria were then mixed in ratio 1:1. Live, dead and isolated supernatants were used to investigate whether it is the bacteria itself or some substances it produces that influences the immune system. The sample set-up was done twice; one set-up was incubated for 6h and the other set-up was incubated for 18 h. After 6 h incubation, total RNA was isolated and used for the quantification of cytokines at mRNA levels using real-time quantitative reverse transcriptase-polymerase chain reaction. The supernatants from the samples incubated for 18 h were isolated and used for quantification on protein levels with cytometric bead array.

Results

Figure 3 shows the production of cytokines by polyclonal activated T cells in the presence of the probiotic bacteria strains LB931, LB3, LB7 and LB21. Data are from three blood donors and the vertical lines show activated T cells without any stimulation with probiotics and act as a negative control. All four strains show the same responses for the four cytokines measured at mRNA level. The same pattern is also seen at protein levels, where two of the four strains were measured. As seen in Figure 4 both live LB931 and live LB21 gave the same cytokine responses on activated T cells. Dead LB931 and dead LB21 also follow each other when it comes to increase in cytokine levels. Figure 3 and 4 shows that it is the live lactobacillus and dead lactobacillus that increase or decrease the cytokine responses of activated T cells. Supernatants isolated from the five different lactobacillus strains did not influence the cytokine responses from human T cells at all. The Lactobacillus did not increase the IFN-γ levels or IL-4 levels of resting T cells remarkable as seen in Figure 5. This shows that none of the four lactic acid bacteria strains have the ability to influence resting T cells.

No statistically significant differences between the four lactobacillus strains were found using Tukey's Multiple Comparison Test. This test together with the results from the cytokine responses indicating that LB931, LB3, LB7, LB21 strains are alike.

Reference list:

1. Lievin-Le MV at all. The front line of enteric host defense against unwelcome intrusion of harmful microorganisms: mucins, antimicrobial peptides and microbiota. Clin Microbiol Rev 2006 19:315-337 2. Forsberg G et al, Paradoxial coexpression of proinflammatory and down- regulatory cytokines in intestinal T cells in childhood celiac disease. Gastroenterology 2002;123:667-78

3. Marsh MN, Gluten, major histocompability complex, and the small intestine. A molecular and immunobiologic approach to the spectrum of gluten sensitivity. Gastroenterology 1992;102:330-354

4. Green PH at all. Coeliac disease. Lancet 362:383-391,

5. Branski D, Latest developments in the pathogenesis and treatment of celiac disease. J Pediatr 149:295-300

6. Hischenhuber CR et all. Review article: safe amounts of gluten for patients with wheat allergy or coeliac disease. Aliment Pharmacol Ther 2006 26:559-575

7. Blum S, Schiffrin EJ. Intestinal microflora and homeostasis of the mucosal immune response: implications for probiotic bacteria? Curr Issues Intest Microbiol. 2003 Sep;4 (2):53-60

Claims

1. Use of a Lactobacillus strain chosen from the species Lactobacillus plantarum LB3e and Lactobacillus plantarum LB7c which have been deposited at Deutsche Sammlung von Mikroorganismen und Zellkulturen, and have been assigned accession number 17852, 17853, respectively, Lactobacillus plantarum LB931 which has been deposited at Deutsche Sammlung von Mikroorganismen und Zellkulturen and has been assigned accession number DSM 11918 and Lactobacillus rhamnosus LB21 which has been deposited at NCIMB Ltd, Ferguson Building, Craibstone Es- tate and has been assigned accession number NCIMB 40564, for preparing a medicament promoting immuntolerance in autoimmune diseases chosen from coeliac disease.

2. Use of a Lactobacillus strain chosen from the species Lactobacillus plantarum LB3e and Lactobacillus plantarum LB7c which have been deposited at Deutsche

Sammlung von Mikroorganismen und Zellkulturen, and have been assigned accession number 17852, 17853, respectively, Lactobacillus plantarum LB931 which has been deposited at Deutsche Sammlung von Mikroorganismen und Zellkulturen and has been assigned accession number DSM 11918 and Lactobacillus rhamnosus LB21 which has been deposited at NCIMB Ltd, Ferguson Building, Craibstone Estate and has been assigned accession number NCIMB 40564, for preparing a medicament promoting immuntolerance in autoimmune diseases chosen from atopic dermatitis.

3. Use of a Lactobacillus strain according to claim 1 wherein the Lactobacillus strain is used for reducing the immunological response in the intestine against gluten or changes induced by gluten.

4. Use of a Lactobacillus strain according to claim 1 or 2 wherein the Lactobacillus strain is used for preparing a medicament to promote immuntolerance related to coeliac disease or atopic dermatitis.

5. Pharmaceutical composition, containing at least one of Lactobacillus plantarum LB931, Lactobacillus rhamnosus, LB21, Lactobacillus plantarum LB7c and Lacto- bacillus plantarum LB3e together with at least one excipient.

6. Pharmaceutical composition according to claim 5, containing a unit dosage CFU from IxIO⁵ to IxIO¹¹ CFU/ dose of each single strain, preferably > IxIO⁷ CFU/dose of each single strain.

7. Food product, comprising at least one of Lactobacillus plantarum LB931, Lactobacillus rhamnosus, LB21, Lactobacillus plantarum LB7c and Lactobacillus plantarum LB3e.

8. Food product according to claim 7, containing a unit dosage CFU from 1x10⁵ to IxIO¹¹ CFU/dose of each single strain.

9. Food product according to claim 7 or 8, wherein the food product is chosen from bread, dough, cereal products, porridge, gruel, muesli, granola, powdered cereal based product, pasta, semi products, soups, bake-off products, plain bread, stewed fruit.

10. A method of treating or preventing autoimmune diseases chosen from coeliac disease or atropic dermatitis in a mammal for promoting immuntolerance compris- ing administering to the mammal an effective amount of a Lactobacillus strain chosen from the species Lactobacillus plantarum LB3e and Lactobacillus plantarum LB7c which have been deposited at Deutsche Sammlung von Mikroorganismen und Zellkulturen, and have been assigned accession number 17852, 17853, respectively, Lactobacillus plantarum LB931 which has been deposited at Deutsche Sammlung von Mikroorganismen und Zellkulturen and has been assigned accession number DSM 11918 and Lactobacillus rhamnosus LB21 which has been deposited at NCIMB Ltd, Ferguson Building, Craibstone Estate and has been assigned accession number NCIMB.

11. A Lactobacillus strain chosen from the species Lactobacillus plantarum LB3e and Lactobacillus plantarum LB7c which have been deposited at Deutsche

Sammlung von Mikroorganismen und Zellkulturen, and have been assigned accession number 17852, 17853, respectively, Lactobacillus plantarum LB931 which has been deposited at Deutsche Sammlung von Mikroorganismen und Zellkulturen and has been assigned accession number DSM 11918 and Lactobacillus rhamnosus LB21 which has been deposited at NCIMB Ltd, Ferguson Building, Craibstone Estate and has been assigned accession number NCIMB 40564, for promoting immun- tolerance in autoimmune diseases chosen from coeliac disease or atropic dermatitis.