HUE033028T2 - Termostabil lactobacillus törzsek - Google Patents

Description

(12) EUROPEAN PATENT SPECIFICATION (45) Date of publication and mention (51) Int Cl.: of the grant of the patent: C12N 1120 <200601> C12R 1125 <200601> 21.12.2016 Bulletin 2016/51 A61K 35I64<201501> (21) Application number: 07716125.5 (86) International application number: PCT/SE2007/050156 (22) Date of filing: 14.03.2007 (87) International publication number: WO 2007/108764 (27.09.2007 Gazette 2007/39)

(54) THERMOSTABLE LACTOBACILLUS STRAINS

TEMPERATURSTABILE LACTOBACILLUS-STAMME SOUCHES DE LACTOBACILLUS THERMOSTABLES (84) Designated Contracting States: (72) Inventors: AT BE BG CH CY CZ DE DK EE ES FI FR GB GR · GRAHN HAKANSSON, Eva HU IE IS IT LI LT LU LV MC MT NL PL PT RO SE S-90331 Umea (SE) SISKTR ♦ EKBECK, Jennie S-90440 Umea (SE) (30) Priority: 17.03.2006 SE 0600606

(74) Representative: Potter Clarkson LLP (43) Date of publication of application: The Belgrave Centre 03.12.2008 Bulletin 2008/49 Talbot Street

Nottingham NG1 5GG (GB) (73) Proprietor: Probi Aktiebolag 223 70 Lund (SE) (56) References cited: WO-A1-98/55131 DE-A1- 3 335 351 US-A- 4 110 477 US-B1- 6 761 885

Description

TECHNICAL FIELD

[0001] The present invention relates to the Lactcobacillus genus that has the ability to survive in high temperature environments. These thermostable microorganisms have the ability to produce viable antibacterial agents in different food products after heat treatment. The thermostable microorganisms of the Lactobacillus genus may be included in food products that demand high temperature. A further object of the present invention is the ability of a food product comprising the thermostable microorganisms of the Lactobacillus genus to promote immuntolerance and homeostasis. Furthermore, two novel strains of Lactobacillus genus are disclosed.

BACKGROUND OF THE INVENTION

[0002] Probiotics are defined as "live microorganisms administered in adequate amounts which confer a beneficial health effect on the host". Most probiotics are bacteria, which are small, single-celled organisms of the genera Lactobacillus, Lactococcus or Bifidobacterium.

[0003] At a minimum, probiotic products should be safe, effective, and should maintain their effectiveness and potency until they are consumed. This requires a responsible approach both by the producer and the consumer.

[0004] At birth Lactobacillus strains together with Bifidobacteria and Lactococcus are the first to colonise the sterile intestines. In adults Lactobacillus strains are also the dominating normal flora in the intestine. It has been known that it is beneficial to include lactic acid producing bacteria such as Lactobacillus in the diet. Today a lot of the products that have included lactic acid bacteria are administered in dairy products or are administered in the form of concentrates of the microorganisms, in the form of suitably formulated preparations including powders, granulates tablets or capsules containing a high number of one or more species of the beneficial microorganisms. A lot of these applications include protein or sugar from milk, against which many people are intolerant or allergic. It is advantageous to administer the beneficial microorganisms as a part of the normal diet. Therefore, it is desirable to incorporate the beneficial microorganisms in types of food products which are consumed universally and regularly in considerable quantities by a majority of consumers such as bread or other cereal products. However, several food products are subjected to temperatures which often kill the microorganisms in the food product before they reach the consumer.

[0005] People suffering from coeliac disease (CD) are not able to eat a diet containing the protein gluten which is often represented in bread and cereal products. Further, commercially available gluten-free food products such as pasta are of a low sensory and cooking quality and usually much more expensive than normal pasta. Thus, there is a need to provide new and less expensive food products such as pasta, bread and cereal products that can be consumed by people suffering from coeliac disease.

[0006] WO 94/00019 relates to baked products containing desirable viable microorganisms. It is concluded that microorganisms such as lactic acid bacteria are killed during the baking step as a result of heat inactivation. Accordingly, a fresh baked product does not contain any viable microorganisms at all. Therefore, a method is disclosed wherein a suspension of the viable microorganisms is injected into the baked product. It is an essential feature that the bakery product is cooled down to a temperature below +70 °C before the viable microorganisms are injected. There are several problems when injecting a suspension of viable microorganisms with a high concentration into a baked product. The injected microorganisms do not show any significant growth in the bread. It is difficult to achieve an equal distribution in the baked bread and the taste could be affected. Further, expensive equipment is needed to inject the viable microorganism suspension and it is not likely that such a method could be used outside a large-scale manufacturing process. The expensive equipment and the high concentration of the suspension with microorganisms will often result in an expensive product.

[0007] Therefore, it is an object of the present invention to solve these problems.

SUMMARY OF THE INVENTION

[0008] The inventors of the present invention have surprisingly found that thermostable microorganisms of the Lactobacillus genus that are able to survive temperatures from +85 °C for more than 25 minutes have the ability to produce viable antibacterial agents in different food products after such heat treatment.

[0009] Particularly, Lactobacillus plantarum and Lactobacillus rhamnosus have the ability to survive in high temperature and in microwave radiated environment and still produce an antibacterial agent that also inhibit growth of mould in food products. Further, two specific examples of known Lactobacillus strains are disclosed, Lactobacillus plantarum LB931 and Lactobacillus rhamnosus, LB21. Furthermore, two novel strains of Lactobacillus are disclosed, referred to as Lactobacillus plantarum LB7c and Lactobacillus plantarum LB3e.

[0010] The inventors have also shown that food products comprising the thermostable viable Lactobacillus strains chosen from the species Lactobacillusplantarum and Lactobacillus rhamnosus and particularly Lactobacillusplantarum LB931, Lactobacillus rhamnosus, LB21, Lactobacillus plantarum LB7c and Lactobacillus plantarum LB3e can reduce the immunological response in the intestine due to gluten. A food product containing such thermostable viable Lactobacillus strains promoting immuntolerance in autoimmune diseases such as coeliac disease.

DEFINITIONS

[0011] As disclosed herein, the term "LB" relates to bacteria of the genus Lactobacillus.

[0012] As disclosed herein, the term "thermostable" relates to bacteria that survive in temperatures higher than +80°C for more than 25 minutes.

[0013] As disclosed herein, the term "lactic acid bacteria" relates to bacteria producing lactic acid, such as bacteria belonging to the genera Lactobacillus and Lactococcus.

[0014] As disclosed herein, the term "CFU" relates to colony-forming units.

[0015] As disclosed herein, the term "food products" relates to food products such as bread, dough, cereal products such as porridge, gruel, muesli, granola, powdered cereal based product, pasta, semi products such as soups, bakeoff products, plain bread and stewed fruit.

[0016] As disclosed herein, the term "heat source" relates to heat sources that heat the food product such as a stove plate, oven, microwave radiated environment, microwave oven or a water bath.

[0017] A dough used herein usually contains a fluid such as milk or water, flour and yeast.

[0018] As disclosed herein, the term "a slice of bread" relates to a typical slice of bread which weighs about 15-40g.

[0019] As disclosed herein, the term "bake-off products" relates to semi-finished products for which the baking will be completed after supply to the consumer..

[0020] As disclosed herein, the term "soluble substances" relates to organic acids, inorganic acids, such as lactic acid, succinic acid, acetic acid, and propionic acid.

BRIEF DESCRIPTION OF THE DRAWINGS

[0021]

Figure 1 discloses the survival capability of freeze dried LB21 on grains stored in room temperature.

Figure 2 discloses the survival capability of freeze dried LB21 on grains stored in +4°C.

Figure 3 discloses the survival capability of freeze dried LB931 and LB21 in snack bars stored in +4°C.

Figure 4 discloses the survival capability of LB931 and LB21 in microwave radiated environment.

Figure 5 discloses the survival capability of LB931, LB7c, LB3e and L1A in +80°C.

Figure 6 discloses the survival capability of LB931 in +90°C.

Figure 7 discloses the Lactobacillus ability to survive the passage through the gastrointestinal tract in sourdough bread.

Figure 8 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 9 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 10 discloses the production of cytokines by polyclonal activated T cells in the presence of the probiotic bacteria strains LB931, LB3, LB7 and LB21.

Figure 11 discloses that both live LB931 and live LB21 gave the same cytokine responses on activated T cells. Figure 12 discloses that the lactobacillus did not increase the IFN-γ levels or IL-4 levels of resting T cells.

DETAILED DESCRIPTION OF THE INVENTION

[0022] The present invention relates to thermostable microorganisms of the Lactobacillus genus that are able to survive temperatures from +80 °C for more than 25 minutes.

[0023] It is disclosed that selected species of microorganisms of the Lactobacillus genus chosen from the species Lactobacillus plantarum and Lactobacillus rhamnosus have the ability to produce viable antibacterial agents in different food products after heat treatment.

[0024] Furthermore, two novel strains of Lactobacillus are disclosed, Lactobacillus strain chosen from the species Lactobacillus plantarum LB7c and Lactobacillus plantarum LB3e.

[0025] An embodiment is directed to microorganisms of the Lactobacillus strain chosen from the species Lactobacillus plantarum LB3e and Lactobacillus plantarum LB7c which have been deposited at Deutsche Sammlung von Mikroor-ganismen und Zellkulturen, and have been assigned accession number 17852 and 17853 respectively that are able to survive temperatures from +80 °C for more than 25 minutes.

[0026] Any of the two above mentioned Lactobacillus strains could be used in food products such as bread, dough, cereal products, porridge, gruel, muesli, granola, powdered cereal based product, pasta, semi products, soups, bakeoff products, plain bread, stewed fruit.

[0027] Further, a food product such as bread, dough, cereal products, porridge, gruel, muesli, granola, powdered cereal based product, pasta, semi product, soups, bake-off products, plain bread, stewed fruit characterized in that said food product also contains a viable 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 and 17853 respectively.

[0028] A bread or dough product wherein said bread or dough product also contains a viable Lactobacillus strain chosen from the species Lactobacillus plantarum LB931 which has been deposited at Deutsche Sammlung von Mikroorganismen und Zellkulturen and has been assigned accession number DSM 11918, Lactobacillus rhamnosus LB21 which has been deposited at NCIMB Ltd, Ferguson Building, Craibstone Estate and has been assigned accession number NCIMB 40564, Lactobacillus plantarum LB3e (DSM 17852) and Lactobacillus plantarum LB7c (DSM 17853), wherein said deposited strains are able to survive temperatures from +85°C for more than 25 minutes and said lactobacilli of at least one of said strains are incorporated into said bread and dough product before a heat treatment.

[0029] A food product prepared by heat treatment of a food product starting material using a heat source exposing said food product to a temperature above +85 °C wherein that said food product also contains a viable Lactobacillus strain preferably chosen from the Lactobacillus plantarum LB931 (DSM11918), Lactobacillus rhamnosus LB21 (NCIMB 40564), Lactobacillus plantarum LB7c (DSM 17853) and Lactobacillus plantarum LB3e (DSM 17852) respectively.

[0030] In further embodiments the use of the known viable Lactobacillus strains chosen from the species Lactobacillus plantarum (DSM 11918), Lactobacillus rhamnosus LB21 (NCIMB 4056) for manufacturing a food product such bread, dough, cereal products, porridge, gruel, muesli, granola, powdered cereal based product, pasta, semi product, soups, bake-off products, plain bread, stewed fruit wherein the food product is prepared by heat treatment of a food product starting material using a heat source exposing said food product to a temperature above +85°C, and wherein lactobacilli of at least one of said strains are incorporated into said food product before a heat treatment.

[0031] A food product mentioned above wherein the viable Lactobacillus strain is present in an amount above 1,0x103 CFU/g, preferably above 1,0x104 CFU/g and most preferably above 1,0x105 CFU/g, living bacteria per gram of the food product after the heat treatment.

[0032] A further object of the present invention is the ability of a food product mentioned above wherein the thermostable viable Lactobacillus strain chosen from the species Lactobacillus plantarum and Lactobacillus rhamnosus reduces the immunological response in the intestine due to gluten and particularly, Lactobacillus plantarum LB931 (DSM11918), Lactobacillus rhamnosus, LB21 (NCIMB 40564), Lactobacillus plantarum LB7c(DSM 17853) and Lactobacillus plantarum LB3e (DSM 17852).

[0033] A food product prepared by heat treatment of a food product starting material using a heat source exposing said food product to a temperature above +85°C characterized in that said food product also contains a viable thermostable Lactobacillus strain chosen from the species of Lactobacillus plantarum LB931 (DSM 11918), Lactobacillus rhamnosus, LB21 (NCIMB 40564), Lactobacillus plantarum LB7c (DSM 17853) and Lactobacillus plantarum LB3e (DSM 17852), wherein said food product is for promoting immuntolerance in autoimmune diseases such as coeliac disease.

[0034] The identity and deposit numbers of the strains of the present invention are listed here.

[0035] The LB931 has been deposited on Jan. 9, 1998 at DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen, Mascheroder Weg 1b D-38124 Braunschweig. It has been assigned accession number DSM11918.

[0036] The LB21 has been deposited on June 11, 1993 at NCIMB Ltd, Ferguson Building, Craibstone Estate, Bucks-burn, Aberdeen, AB21 9YA, UK. It has been assigned accession number NCIMB 40564.

[0037] The LB3e has been deposited on Jan. 6, 2006 at DSMZ-Deutsche Sammlung von Mikroorganismn und Zellkulturen, Mascheroder Weg 1b D-38124 Braunschweig. It has been assigned accession number DSM 17852.

[0038] The LB7c has been deposited on Jan. 6, 2006 at DSMZ-Deutsche Sammlung von Mikroorganismen und Zel-Ikulturen, Mascheroder Weg 1b D-38124 Braunschweig. It has been assigned accession number DSM 17853.

[0039] The Lactobacillus strains mentioned above could be referred to as LB931 (DSM11918), LB21 (NCIMB 40564), LB3e (DSM 17852) and LB7c (DSM 17853).

[0040] Food products according to the present invention may comprise one or more strains selected from the above defined LB strains. Other mixtures or single thermostable LB strains could be used advantageously within the scope of the present invention.

[0041] The above mentioned four thermostable Lactobacillus strains have the ability to survive in a high temperature environment i.e. above +80 °C for more than 25 minutes and still 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 8 discloses expression of organic acids and Figure 9 discloses expression of lactic acids in an Auto-Scaled Chromatogram of supernatants of the Lactobacillus strains LB931, LB3e, LB7c and LB21.

[0042] These strains are endurable and readily survive long periods of storage in room temperature. Accordingly products containing LB931, LB21, LB3e and LB7c have a long shelf life. The strains can easily be included in different food products.

[0043] The ability of the Lactobacillus to survive in different applications was determined, see Example 2. No major reduction of the CFU/g was detected in the wholemeal gruel powder preparation with freeze-dried LB931 and LB21 that were stored in +37° for 31 days.

[0044] The ability for Lactobacillus to survive in snack bars when they are put on oat grains was determined, see Example 3. It is possible to put Lactobacillus strains on grains and they will survive for 8 months in room temperature and more than 8 months in +4°C.

[0045] Both LB931 and LB21 have the ability to survive in snack bars for 25 days. The snack bars containing LB931 had 7,6 x 106 CFU/ml at day 46.

[0046] Thus, the thermostable LB strains can endure storage for a long period of time, and it is possible to induce the strain in different kinds of preparations. It could be used in bake-off products.

[0047] The survival capacity in a microwave radiated environment was also evaluated, see Example 4. Milk cereal drinks were prepared together with the Lactobacillus strains at a start concentration of 107 CFU/ml. LB931 showed a higher CFU/ml than LB21 until 80 seconds. The CFU/ml of LB21 remained unchanged from 30 seconds to 90 seconds. The Lactobacillus has the ability to survive in cereal products in microwave radiated environment. The results are presented in Figure 4.

[0048] The killing of the four different strains of lactic acid bacteria with UV-lightwere investigated and an interference test was used to test the four lactic acid strains for their ability to inhibit growth of pathogens and thus also their capacity to function as probiotics, the results are shown in Example 5.

[0049] The high temperature resistance of the Lactobacillus strains have been investigated, see Example 6. LB931, LB3e and LB7c have the ability to survive in +80°C for more than 25 minutes and LB931 survives for more than 5 minutes in +90°C. The control strain, L1A, do not even survive for 5 minutes in +80°C. The results are presented in Figure 5 and 6.

[0050] The growth capacities of the Lactobacillus in sourdough starters have been investigated, see Example 7. Two different types of flours, wheat and rye, were used to determine the best environment for the Lactobacillus. The type of flour did not affect the outcome of the experiment. The starters increased one log and ended on the same CFU/g independent of the type of flours. As a control, the Lactococcus Lactis L1A was used. The L1A did not grow in the starter. The CFU/g had decreased from day 0 to day 3.

[0051] In Example 8 the survival of the Lactobacillus in bread is shown. The temperatures in different breads were established, see table 13. The survival of the bacteria in the bread was determined at different times, see tables 14-16. Lactobacillus Lactis L1A was used as control. All the Lactobacillus strains that were tested survived in all the different temperatures and times. The control, L1 A, did not survive. There was no visual growth of the bacteria from day 0.

[0052] Depending on the food product and the purpose it is desirable that it comprises the viable Lactobacillus strain in a sufficient amount that is preferably as high as possible, to provide the desired effects. The experimental results show that amounts above 1,0x1010 CFU/g can be achieved. In a food product the viable Lactobacillus strain should be present in an amount above 1,0x103 CFU/g, preferably above 1,0x104 CFU/g and most preferably above 1,0x105 CFU/g, living bacteria per gram of the food product.

[0053] An example is a slice of bread that weighs at least 15 gram wherein the viable Lactobacillus strain is present in an amount above 1,0 x 103 CFU/g, preferably above 1,0 x 104 CFU/g and most preferably above 1,0 x 105 CFU/g, living bacteria per gram of a slice of bread.

[0054] For example, a viable Lactobacillus strain wherein the start concentration is about 1 x 106 has a CFU above 1x103 after being exposed to temperatures from +80 °C for more than 25 minutes or alternatively for more than 10 s in a microwave radiated environment.

[0055] The capability of Lactobacillus to promoting immuntolerance in autoimmune diseases such as coeliac disease has been investigated in in vivo studies. The Lactobacillus strains are able to reduce the inflammatory lesion in the upper intestinal mucosa and reduce the levels of antibodies associated with autoimmune diseases. Example 9a, 9b and 9c also indicate that the Lactobacillus have the capability to influence the immune system in a positive way. The children that were 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. The biopsy showed normal tissue in the group given Lactobacillus and the tissue showed indication of inflammation in the group given placebo. Products according to the invention may comprise one or more strains selected from the above defined group. Other mixtures or single LB strains could advantageously be used within the scope of the present invention.

[0056] The present invention will now be described in reference to thefollowing examples, which should not be regarded as limiting for the scope of the invention.

EXAMPLES

Example 1 [0057] Isolation and identification of the Lactobacillus strains.

[0058] LB931 was isolated from a healthy woman. The strain was classified as Lactobacillus plantarum according to the test kit API 50 CH (API systems, BioMerieux, FR), and was denominated LB931. The strain was further typed by DNA-analysis with SDS-page at BCCM/LMG (Belgium) to be Lactobacillus plantarum-pentosusparaplantarum.

[0059] LB21 was isolated from a healthy baby. The strain was classified as Lactobacillus rhamnosus according to the test kit API 50 (API systems, BioMerieux, FR), and was denominated LB21. The strain was further typed by DNA-analysis with SDS-page at NCIMB (UK) to be Lactobacillus rhamnosus.

[0060] LB3e was isolated from human. The strain was classified as Lactobacillus plantarum according the kit API 50 (API systems, BioMerieux, FR), and was denominated LB3e. The strain was further typed by 16S rDNAgene sequence at DSMZ (Germany) to be Lactobacillus plantarum.

[0061] LB7c was isolated from human. The strain was classified as Lactobacillus plantarum according the kit API 50 (API systems, BioMerieux, FR), and was denominated LB7c. The strain was further typed by 16S rDNAgene sequence at DSMZ (Germany) to be Lactobacillus plantarum.

Example 2 [0062] The Survival Capabilities of Lactobacillus in different preparations. a) Skim milk preparations of LB931 were freeze-dried according to standard methods. To determine the durability of the preparations the obtained powder was stored in wholemeal gruel powder at +37°C for 31 days to symbolise storage in room temperature for 6 month. The number of bacteria was determined after 31 days and the results are disclosed in table 1 below. TABLE 1

Days No. of bacteria CFU/g

DayO 7,0 x 1011

Day 31 1,0 x 1011 b) LB931 dissolved in a suspension of equal parts skim milk and 0.9% NaCI. The dissolved bacteria were then incubated at different temperatures. The amount of bacteria was continuously monitored by cell counting. The results are disclosed in table 2 below. TABLE 2

No. of bacteria CFU/ml

Temperature, °C DayO Day 2 Day 5 Day 32 4 7,8 x 1010 2,2 x 1010 20 1,8 x 1010 2,2 x 1010 27 1,8 x 1010 1,2 x 1010 3,3x 109

The results show that LB931 is stable in a mixture of skim milk and NaCI for a period of one month at +4°C. c) A skim milk preparation of LB931 was freeze-dried according to standard methods. The obtained powder was stored in Petri dishes at room temperature and at +6°C. The number of bacteria was determined after 7 days and 25 days, respectively. The results are disclosed in table 3 below. TABLE 3

No of bacteria CFU/g

Temperature °C Day 0 Day 7 Day 25 6 4,2x 108 2,2 x 108 1,2x108 22 4,2x108 1,9x108 1,4x108

The number of bacteria in the freeze-dried powder was also monitored every fourth week up to 68 weeks. After one year, at +6°C, more than 105 CFU/g LB931 could be found. d) A skim milk preparation of LB21 was freeze-dried according to standard methods. The obtained powder was stored in a glass bottle at +4°C. The number of bacteria was determined after 0, 10, 40 and 365 days, respectively. The results are disclosed in table 4 below.

Table 4

Day 0 Day 10 Day 40 Day 365 LB21 2,3 x 101° 2,3x101° 1,0x101° 1,2x101°

Example 3 [0063] The Survival Capabilities of Lactobacillus in different oats products. a) A freeze-dried preparation of LB21 was put on rolled oats. Six different methods were used to put the bacteria on the grains. The freeze-dried LB21 were mixed with maltodextrin/glucose so they would stick to the grains. The bacteria were put on the grains at different temperatures and were also dried in different temperature to find the best method. The results are shown in Table 5. TABLE 5

Preparation Dry temperature

Air Oil Air

LB21 25°C +25°C - +30°C

LB21 30°C +30°C - +38°C

LB21 33°C +33°C - +38°C

LB21 5 % +29°C +38°C +38°C

LB21 25 % hot +28°C +38°C +38°C

LB21 25 % cool +28°C +27°C +25°C

The grains with bacteria were stored in both +25°C and in +4°C for 244 days. The Survival capability was determined once a month. The results are disclosed in tables 6-7 below.

TABLE 6. Stored in +25°C

No. of bacteria CFU/g

Time (days) LB21 25°C LB21 30°C LB21 33°C LB21 5% LB21 25 % hot LB21 25 % cool 6 4 x 108 2 x 108 1 x 108 2x 108 3 x 108 3x 108 31 2 x 108 2 x 108 1 x 108 2x 108 2 x108 2x 108 59 3 x 108 5 x 108 2x 108 2x 108 3 x 108 4x 108 90 2 x 108 3 x 108 1 x 108 1 x 108 3 x 108 1 x 108 122 2 x 108 2 x 108 7x 107 7x 107 1 x 108 1 x 108 144 2 x 108 2 x 108 1 x 108 8x 107 8 x 107 8x 107 (continued)

No. of bacteria CFU/g

Time (days) LB21 25°C LB21 30°C LB21 33°C LB21 5% LB21 25 % hot LB21 25 % cool Ϊ80 2x1ο7 2x1ο7 3x 107 2x10® 3x10s 5x 10® 219 1 x 106 2 x 106 3x 105 8x 104 9 x105 9x 105 244 4 x 103 1 x 103 1 x 103 2x103 5x 103 6x 103

TABLE 7. Stored in +4°C

No. of bacteria CFU/g

Time (days) LB21 25°C LB21 30°C LB21 33°C LB21 5% LB21 25 % hot LB21 25% cool 6 4x10® 2 x 108 1 x 108 2x 108 3x 108 3x 108 31 2 x 108 2 x 108 2 x 108 2x 108 2x 108 2x 108 59 4 x 108 2 x 108 2x 108 3x 108 4x 108 3x 108 90 4 x 108 2 x 108 2 x 108 3x 108 4x 108 2x 108 122 2 x 108 2 x 108 6x 107 2 x 108 2x 108 1 x 108 144 2 x 108 2 x 108 8x 107 2 x 108 2x 108 1 x 108 180 2 x 108 2 x 108 6x 107 1 x 108 1 x 108 1 x 108 219 2 x 108 1 x 108 4x 107 1 x 108 1 x 108 1 x 108 244 4 x 108 2 x 108 2x 107 2x 108 4x 108 3x 107 b) Snack bars were made with LB931 and LB21. Each bar weighed 25g before they were split in 4 pieces. Each piece was put in a sterile bag in +4°C. The number of bacteria was determined after 0, 10, 21 and 46 days. The results are disclosed in table 8 below. TABLE 8

No. of bacteria CFU/g Time (days) LB931 LB21 0 8x10® 1,2x 107 10 2,7 x 108 1,3x 107 21 3,8 x 108 3,Ox 105 46 7,6 x 106 0

Thermostable LB strains can survive in high temperature in different cereal preparations.

Example 4 [0064] The Survival Capabilities of Lactobacillus in microwave radiated environment.

[0065] A freeze-dried preparation of LB931 was included in the conventional milk cereal drink powder and in porridge oats. The milk cereal drink and the porridge oats were prepared according to the instruction on the package resulting in a volume of about 200 ml. A microwave oven (850W) with the internal measurement 30x29x20 cm was used as the source of heat. Thus, the internal volume of the microwave oven was approximately 17 dm3. The number of bacteria was determined after 30 seconds, 60 seconds, 70 seconds, 80 seconds and 90 seconds, respectively. The temperature of the milk cereal drink after 90 seconds in the microwave oven was about +60°C. The procedure was repeated with overnight culture of LB21. The thermostable LB strains are able to survive in microwave radiated environment. The results are disclosed in table 9 below. TABLE 9

No. of bacteria CFU/ml

Time (s) LB931 LB21 “0 3,5 x 107 1,1 x 107 (continued)

No. of bacteria CFU/ml

Time (s) LB931 LB21 “30 2,2 x 107 2,2 x 105 60 2,6 x 107 1,0 x 105 70 1,3 x 107 1,0 x 105 80 3,9 x 106 1,0 x 105 90 1,1 x 103 1,0 x 105

Example 5 [0066] The killing of the four different strains of lactic acid bacteria with UV-light were investigated. The four different strains of lactic acid bacteria Lactobacillus plantarum; LB931 (isolated from urinary tract of human), LB3e (isolated from saliva of child), LB7c (isolated from tooth surface of child) and LB21 (isolated from faeces of newborn children) used in this example were spread on MRS-agar plates and incubated for 20 h at 37°C, 5% C02. They were then inoculated in 3 ml MRS-broth at 37°C, 5 % C02 over night which generates about 109 CFU/ml. The overnight cultures of the strains were added to a final volume of 1 % in MRS broth and were incubated for 20 h at 37°C, 5% C02. The bacteria were centrifuged (3800 rpm, 20 min, +4°C) and then washed 2 times in 10 ml 0.9% NaCI. The bacteria were dissolved in 0.9% NaCI. The concentration of the bacteria was determined by colony-forming unit (CFU) counting. The bacteria were irradiated for different times at a distance of 5 cm from a UVC-lamp (Kendro laboratory products, 2x15W, 254 nm). Lactic acid bacteria LB7c and LB21 were irradiated for 9 h while lactic acid bacteria LB3e and LB931 were irradiated for 10 h. The death of the bacteria was controlled on MRS agar plates, incubated for 48 h at 37°C, 5% C02.

Agar overlay- interference test [0067] An interference test was used to test the four lactic acid strains (see table 10 for their ability to inhibit growth of pathogens. The four Lactobacillus strains in table 10 were inoculated in 3 ml MRS broth and then incubated in 37°C, 5 % C02 for 20 h.1 ml of the cultures were mixed with 23 ml MRS agar together with 2 ml 7,5M NaAc, the plates were thereafter incubated for 20 h in 37°C, 5% C02. Nine different interference test strains (ITS) (NUS, Umea, Sweden), isolated from people with urinary infections, were inoculated in M17 broth and incubated in 37°C over night. A second agar layer of 25 ml M17 with 750 μΙ 6,5M KH2P04 was found over the first one. The test strains, in stationary phase, were replicated with help of a Steers steel pin (Steers et al., 1959) on the M17 agar surface and also on a control plate and then incubated in 37°C, 5% C02 over night.

Inhibition of pathogens by lactic acid bacteria [0068] An interference test was done to test the four lactic acid bacteria strains for their ability to inhibit pathogens and thus also their capacity to function as probiotics. The result from this test is shown in table 10. LB931 totally inhibited all 8 pathogens tested. LB3, LB7 and LB21 showed total inhibition of all pathogens except for Candida albicans, which were partly inhibited. Lactobacillus acidophilus was used as control.

TABLE 10. Inhibition of pathogens by lactic acid bacteria Strain tested LB931 LB3e LB7c LB21 L. acidophilus 2060V

Candida albicans ---- +

Klebsiella species - ± ± +

Staphylococcus aureus - +

Coagulase-negative staphylococcus -..- ?

Enterococcus faecalis .... +

Enterobacteriaceae species .... ?

Proteus mirabilis .... ?

Escherichia coli .... + +, no inhibition; ±, partly inhibition; -, total inhibition; ?, not done

Example 6 [0069] The Survival Capabilities of Lactobacillus in different temperatures. a) Different suspensions of Lactobacillus were made of freeze-dried LB931 in NaCI0.9 % and overnight culture of LB931, LB7c, LB3e and L1A. The suspensions were at 109-1011 CFU/ml. 5ml of NaCI 0.9 % was heated up to +80°C in a water bath. As soon as the NaCI was +80°C 100 μΙ of bacteria suspension was added. The number of bacteria was determined after 5 minutes, 10 minutes, 15 minutes, 20 minutes and 25 minutes, respectively. The results are disclosed in table 11 below (NVG= no visible growth). TABLE 11

No. of bacteria CFU/ml

Time (min) LB931 LB7c LB3e L1A 0 2,30 x 108 1,1 x108 9,7x107 2,5x 107

5 4,33 x 106 1,0 x 104 1,4x104 NVG

10 1,77 x 106 3,3 x 104 1,4x104 NVG

15 2,00 x 106 2,5 x 104 1,8x102 NVG

20 8,20 x 104 1,2x 103 1,9x105 NVG

25 2,24 x 105 1,0 x 104 7,8x104 NVG b) +80°C in more than 20, 30, 40, 50, 60 minutes 5ml of MRS were heated to +85°C in a water bath. 500 μΙ of a overnight culture was added to the suspension. The ability to survive was determined after 20, 30, 40, 50 and 60 minutes respectively. The results are shown in table 12 below. TABLE 12

No. of bacteria CFU/ml

Time (min) LB3e LB7c LB931 LB21

20 VG VG VG VG

30 VG VG VG VG

40 VG VG VG VG

50 VG VG VG VG

60 VG VG VG VG VG= visible growth c) A suspension of LB931 was prepared by suspending freeze-dried LB931 in NaCI 0.9 % and also an overnight culture of LB931. The NaCI was heated up to +90°C in a water bath. As soon as the NaCI was +80°C 100μΙ of bacteria suspension was added. The number of bacteria was determined after 0.5 minutes, 1 minutes, 2 minutes, 3 minutes, 4 minutes and 5 minutes, respectively. The results are disclosed in table 13 below. TABLE 13

No. of bacteria CFU/ml Time (min) LB931 0 1,76 x 108 0,5 5,00 x 105 1 3,54 x 104 2 4,43 x 104 3 1,25 x 104 4 1,14 x 104 5 3,00 x 104 d) A freeze-dried preparation of LB931 and LB21 was included in the conventional milk cereal drink powder and in porridge oats. The milk cereal drink was prepared according to the instruction on the package. The stove was used as the source of heat. The number of bacteria was determined after the milk cereal drink was warmed up to +37°C. The results are disclosed in table 14 below. TABLE 14

No. of bacteria CFU/ml Temperature (°C) LB931 LB21 10 2,9 x 107 3,0 x 107 37 2,4 x 107 2,8 x 107

Example 7 [0070] The survival capabilities of Lactobacillus in sourdough starter. a) Different sourdough starters were made of wheat flour containing different strains of Lactobacillus and Lactococcus. Flour and water was mixed to a volume of 3dl. 10ml of thawed bacteria suspension was included in the starters. The sourdough starters were kept in room temperature for 3 days. Everyday the sourdough starters were given more honey, flour and water. The number of bacteria was determined after 3 days. After the 3 days in room temperature the starters were put in +4°C for 6 days. The number of bacteria was determined after 3days, and 6 days, respectively. The results are disclosed in tables 15-16 below. TABLE 15

No. of bacteria CFU/g

Time (days) LB931 + 25°C LB7c+25°C LB3e+25°C 0 5 x 108 2 x 107 6 x 107 3 3 x 109 9 x 108 2 x 109 TABLE 16

No. of bacteria CFU/g

Time (days) LB931 +4°C LB7c +4°C LB3e +4°C 0 3 x 109 6 x 108 3 x 109 3 2 x 109 3 x 108 9 x 108 6 2 x 109 2 x 107 8 x 108 b) Different sourdough starters were made of rye flour containing different strains of lactobacillus. Flour and water was mixed to a volume of 3dl. 10ml of thawed bacteria suspension was included in the starters. The sourdough starters were kept in room temperature for 3 days. The sourdough starters were everyday given more honey, flour and water. The number of bacteria was determined after 3 days. Then the starters were kept in +4°C for 6 days. The number of bacteria was determined after 3days and 6 days, respectively. The results are disclosed in table 17-18 below. TABLE 17

No. of bacteria CFU/g stored in +25°C Time (days) LB931 LB7c LB3e L1A 0 2 x 108 2x 107 6 x 107 1 x107 3 7 x 109 9x 108 2 x 109 8x105 TABLE 18

No. of bacteria CFU/g stored in +4°C

Time (days) LB931 LB7c LB3e "6 3 x 10a 6x 108 3 x 10a 3 2 x 109 3 x 108 9 x 108 6 2 x 109 2 x 107 8 x 108

Example 8 [0071] The Survival Capabilities of Lactobacillus in bread, visual detection of mould in the bread and isolation of Lactobacillus in stool samples a) A sourdough was prepared with 1 dl wheat sourdough starter containing LB931, water, flour of wheat and rye to a total volume of 6dl. The dough was rising 2x45 minutes and baked at the temperature between 230 - 240°C for 25-30 minutes. The temperature in the bread was measured after the bread was taken out of the oven, 5 minutes, 10 minutes, 15 minutes and 20 minutes, respectively. The results are disclosed in table 19 below. TABLE 19

Time(minutes) Baked in 230°Cfor25 minutes Baked in 230°Cfor35 minutes Baked in240°Cfor30 minutes

0 97,6°C 99,4°C 97,6°C

5 Not done Not done 86,3°C

10 Not done Not done 78,0°C

15 Not done Not done 68,1°C

20 Not done Not done 62,7°C b) Sourdough bread was baked with thermostable Lactobacillus. The survival of the bacteria in the bread was determined after 2 days, 3 days, 4 days, 6 days and 7 days, respectively. The results are disclosed in tables 20-25 below. TABLE 20

Baked in +230°C for 20 minutes (Sourdough starter)

Time (days) No. of bacteria CFU/g LB3e (rye) LB7c (wheat) 0 1,1 x 105 1,2 x 104 5 1,1 x 104 2,1 x 105 TABLE 21

Baked in +230°C for 25 minutes (Rye Sourdough starter)

No. of bacteria CFU/g

Time (days) LB931 LB3e LB7c LB21 0 9 x 103 3 x 103 7 x 102 7x 103 3 1 x 105 5 x 104 1 x 104 3x 104 7 9 x 104 6 x 104 9 x 104 Not done TABLE 22

Baked in +230°C for 35 minutes (Rye Sourdough starter)

No. of bacteria CFU/g

Time (days) LB931 LB3e LB7c 0 9 x 102 2 x 102 1 x 102 3 8 x 103 3 x 103 7 x 103 (continued)

Baked in +230°C for 35 minutes (Rye Sourdough starter)

No. of bacteria CFU/g

Time (days) LB931 LB3e LB7c ~1 9 x 103 7x 103 6x 103 TABLE 23

Baked in +240°C for 30 minutes growth No. of bacteria CFU/g

Wheat Sourdough starter Rye Sourdough starter

Time (days) LB931 L1A LB931 0 4 x 103 No Visible Growth (NVG) 3 x1ο4 2 6 x 104 NVG 7 x 104 4 2 x 105 NVG 9 x 105 6 1 x 105 NVG 6 x 105 8 7 x 104 NVG 8 x 104 c) Bread was made of wheat flour containing the strain LB21. Warm water, honey, yeast, salt, wheat and 5 ml LB21 at CFU 1 x 109 were combined in a large mixer bowl to a total volume of 1 litre. The CFU/ml of the dough was 1 x 106. The dough was kneaded for 10 minutes until it pulled away from the sides of the bowl and felt soft, pliable and smooth. The dough was covered and left to rise for 1 hour. The large batch was divided into smaller parts. The dough was covered again and was left for 45 minutes to rise. The bread was baked in 220 °C for 20 minutes and cooled on a rack. The survival of the bacteria in the bread was determined after 0 days and 3 days respectively. The results are disclosed in table 24 below. TABLE 24

No. of bacteria CFU/g Time (days) LB21 0 2 X 103 3 6 x 103

Possible the CFU/g could be higher if the bread can be cooled faster after baking. d) Visual detection of mould in the bread, no visual growth (NVG).

Slices of bread containing Lactobacillus were kept in air tight plastic bags. The bags were check each day and the growths of mould were determined. The results are disclosed in table 25 below. TABLE 25

Bread No. of bacteria CFU/g day 15 Detection of mould LB931 Sourdough bread 5x104 NVG Day 15

Sourdough bread 5 x 104 NVG Day 15 LB21 Sourdough bread 7,6 x 104 NVG Day 15

Bread with 5ml bacterial solution 1,2x103 NVG Day 15 LB3e Sourdough bread 4x104 NVG Day 15 LB7c Sourdough bread 6x104 NVG Day 15 e) 3 healthy individuals consumed bread containing LB931, LB21, LB3e and LB7c for ten consecutive days, the strains could be recovered from stool samples in all 3 of the tested volunteers. The results are disclosed in table 26 below. TABLE 26

Day 0 Day 5 Day 10

Study person 1 0 7x 103 6x 103

Study person 2 0 2x 102 4x 103

Study person 3 0 5x 107 6 x 103

Example 9 [0072] a) 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 endomysium, transglutaminas 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 (0.1g gluten/day/kg bodyweight) but after 4 months she was eating a normal diet. The levels of endomysium and transglutaminas 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. b) 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 0.1g gluten/day/kg bodyweight for 4 weeks and 0.2g gluten/day/kg bodyweight thereafter. The antibody levels of endomysium, transglutaminas 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 mucous membrane of the small intestine. The children also had increased levels of endomysium, transglutaminas and total IgA. The two children in the active group did not experience any symptoms and the levels of endomysium, transglutaminas 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. c) A study with 10 children suffering from CD was performed. The children were given Lactobacillus (LB21) of 101° 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 thereafter. The antibody levels of endomysium, transglutaminas 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 and symptom-free, and had normal growth

Example 10

Method [0073] An in vitro test was done to evaluate the potential immunomodulatory characteristics of the four different lacto-bacillus 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 [0074] Figure 10 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 11 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. From Figure 10 and 3 it can be seen 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.

[0075] The lactobacillus did not increase the IFN-γ levels or IL-4 levels of resting T cells remarkable as seen in Figure 12. This shows that none of the four lactic acid bacteria strains have the ability to influence resting T cells.

[0076] 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.

Claims 1. Microorganisms of the Lactobacillus strain chosen from the species Lactobacillus plantarum LB3e and Lactobacillus planatarum LB7c which have been deposited at Deutsche Sammlung von Mikroorganismen und Zellkulturen and have been assigned accession number 17852 and 17853 respectively. 2. Microorganisms of the Lactobacillus strain chosen from the species Lactobacillus plantarum LB3e and Lactobacillus plantarum LB7c according to claim 1 characterized in that they are able to survive temperatures from +85°C for more than 25 minutes. 3. Use of Lactobacillus strain according to claim 1 in food products such as bread, dough, cereal products, porridge, gruel, muesli, granola, powdered cereal based product, pasta, semi products, soups, bake-off products, plain bread, stewed fruit. 4. A method for preventing mould in food products wherein at least one Lactobacillus strain according to claim 1 is incorporated into said food products. 5. A method for preventing mould in food products according to claim 4, wherein the food product is selected from bread, dough, cereal products, porridge, gruel, muesli, granola, powdered cereal based product, pasta, semi products, soups, bake-off products, plain bread, and stewed fruit. 6. Food product such as bread, dough, cereal products, porridge, gruel, muesli, granola, powdered cereal based product, pasta, semi product, soups, bake-off products, plain bread, stewed fruit characterized in that said food product also contains a viable Lactobacillus strain according to claim 1. 7. Bread and dough products wherein said bread and dough products also contains a viable Lactobacillus strain chosen from the species Lactobacillus plantarum. LB931, which has been deposited at Deutsche Sammlung von Mikroorganismen und Zellkulturen and has been assigned accession number DSM 11918, Lactobacillus rhamnosus LB21, which has been deposited at (NCIMB Ltd, Ferguson Building, Craibstone Estate and has been assigned accession number (NCIMB 40564, 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 and 17853 respectively, wherein said deposited strains are able to survive temperatures from +85 °C for more than 25 minutes and said lactobacilli of at least one of said strains are incorporated into said bread and dough products before a heat treatment. 8. A method for preventing mould in bread and dough products wherein a Lactobacillus strain according to claim 7 is incorporated into said bread and dough products. 9. Use of a viable Lactobacillus strain chosen from the species Lactobacillus plantarum LB931, which has been deposited at Deutsche Sammlung von Mikroorganismen und Zellkulturen and has been assigned accession number DSM 11918, Lactobacillus rhamnosus LB21, which has been deposited at NCIMB Ltd, Ferguson Building, Craibstone Estate and has been assigned accession number (NCIMB 40564, 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 and 17853 respectively, for manufacturing a food product, wherein the food product is prepared by heat treatment of a food product starting material using a heat source exposing said food product to a temperature above +85 °C such as bread, dough, cereal products, porridge, gruel, muesli, granola, powdered cereal based product, pasta, semi product, soups, bake-ofF products, plain bread, or stewed fruit wherein lactobacilli of at least one of said strains are incorporated into said food product before a heat treatment. 10. A method for preventing mould in food products wherein at least one Lactobacillus strain according to claim 9 is incorporated into in said food products. 11. Food product prepared by heat treatment of a food product starting material using a heat source exposing said food product to a temperature above +85 °C characterized in that said food product also contains a viable Lactobacillus strain chosen from the species of Lactobacillus plantarum LB931 (DSM11918), Lactobacillus rhamnosus LB21 (NCIMB 40564), Lactobacillus plantarum LB3e (DSM 17852), and Lactobacillus plantarum LB7c (DSM 17853) respectively. 12. Food product according to claim 11 wherein the viable Lactobacillus strain is present in an amount above 1,0 x 103 CFU/g, preferably above 1,0x104 CFU/g and most preferably above 1,0 x 105 CFU/g, living bacteria per gram of the food product after heat treatment. 13. Afood product according to claims 7, 11 or 12 comprising the thermostable viable Lactobacillus strain chosen from the species Lactobacillus plantarum and Lactobacillus rhamnosus for promoting immuntolerance in autoimmune diseases such as coeliac disease.

Patentansprüche 1. Mikroorganismen des Lactobacillus-Stamms, ausgewählt aus den Spezies Lactobacillus plantarum LB3e und Lactobacillus plantarum LB7c, die in der Deutschen Sammlung von Mikroorganismen und Zellkulturen hinterlegt worden sind und denen die Zugangsnummern 17852 beziehungsweise 17853 zugeteilt worden sind. 2. Mikroorganismen des Lactobacillus-Stamms, ausgewählt aus den Spezies Lactobacillus plantarum LB3e und Lactobacillus plantarum LB7c nach Anspruch 1, dadurch gekennzeichnet, dass sie fähig sind, Temperaturen von +85 °C mehr als 25 Minuten lang zu überleben. 3. Gebrauch eines Lactobacillus-Stamms nach Anspruch 1 in Nahrungsmittelerzeugnissen, wie etwa Brot, Teig, Getreide Produkten, Haferbrei, Brei, Müsli, Müsliriegeln, Produkten auf Getreidepulverbasis, Teigwaren, Halbprodukten, Suppen, Backerzeugnissen, einfachem Brot, Kompott. 4. Verfahren zum Verhindern von Schimmel in Nahrungsmittelerzeugnissen, wobei wenigstens ein Lactobacillus-Stamm nach Anspruch 1 den Nahrungsmittelerzeugnissen zugegeben wird. 5. Verfahren zum Verhindern von Schimmel in Nahrungsmittelerzeugnissen nach Anspruch 4, wobei das Nahrungsmittelerzeugnis ausgewählt ist aus Brot, Teig, Getreideprodukten, Haferbrei, Brei, Müsli, Müsliriegeln, Produkten auf Getreidepulverbasis, Teigwaren, Halbprodukten, Suppen, Backerzeugnissen, einfachem Brot und Kompott. 6. Nahrungsmittelerzeugnis, wie etwa Brot, Teig, Getreideprodukte, Haferbrei, Brei, Müsli, Müsliriegel, Produkte auf Getreidepulverbasis, Teigwaren, Halbprodukte, Suppen, Backerzeugnisse, einfaches Brot, Kompott, dadurch gekennzeichnet, dass das Nahrungsmittelerzeugnis ferner einen lebensfähigen Lactobacillus-Stamm nach Anspruch 1 enthält. 7. Brot- und Teigprodukte, wobei die Brot- und Teigprodukte ferner einen lebensfähigen Lactobacillus-Stamm enthalten, ausgewählt aus den Spezies Lactobacillus plantarum LB931, der in der Deutschen Sammlung von Mikroorganismen und Zellkulturen hinterlegt worden ist und dem die Zugangsnummer DSM 11918 zugeteilt worden ist, Lactobacillus rhamnosus LB21, der bei NCIMB Ltd, Ferguson Building, Craibstone Estate hinterlegt worden ist und dem die Zugangsnummer NCIMB 40564 zugeteilt worden ist, Lactobacillus plantarum LB3e und Lactobacillus plantarum LB7c, die in der Deutschen Sammlung von Mikroorganismen und Zellkulturen hinterlegt worden sind und denen die Zugangsnummern 17852 beziehungsweise 17853 zugeteilt worden sind, wobei die hinterlegten Stämme fähig sind, Temperaturen von +85 °C mehr als 25 Minuten lang zu überleben, und die Lactobacilli von wenigstens einem der Stämme den Brot- und Teigprodukten vor einer Wärmebehandlung zugegeben werden. 8. Verfahren zum Verhindern von Schimmel in Brot- und Teigprodukten, wobei ein Lactobacillus-Stamm nach Anspruch 7 den Brot- und Teigprodukten zugegeben wird. 9. Gebrauch eines lebensfähigen Lactobacillus-Stammes, ausgewählt aus den Spezies Lactobacillus plantarum LB931, der in der Deutschen Sammlung von Mikroorganismen und Zellkulturen hinterlegt worden ist und dem die Zugangsnummer DSM 11918 zugeteilt worden ist, Lactobacillus rhamnosus LB21, der bei NCIMB Ltd, Ferguson Building, Craibstone Estate hinterlegt worden ist und dem die Zugangsnummer NCIMB 40564 zugeteilt worden ist, Lactobacillus plantarum LB3e und Lactobacillus plantarum LB7c, die bei der Deutschen Sammlung von Mikroorganismen und Zellkulturen hinterlegt worden sind und denen die Zugangsnummern 17852 beziehungsweise 17853 zugeteilt worden sind, zum Herstellen eines Nahrungsmittelerzeugnisses, wobei das Nahrungsmittelerzeugnis durch Wärmebehandlung eines Nahrungsmittelerzeugnis-Ausgangsmaterials unter Verwendung einer Wärmequelle, die das Nahrungsmittelerzeugnis, wie etwa Brot, Teig, Getreideprodukte, Haferbrei, Brei, Müsli, Müsliriegel, Produkte auf Getreidepulverbasis, Teigwaren, Halbprodukt, Suppen, Backerzeugnisse, einfaches Brot oder Kompott, einer Temperatur über +85 °C aussetzt, hergestellt wird, wobei Lactobacilli von wenigstens einem der Stämme dem Nahrungsmittelerzeugnis vor einer Wärmebehandlung zugegeben werden. 10. Verfahren zum Verhindern von Schimmel in Nahrungsmittelerzeugnissen, wobei wenigstens ein Lactobacillus-Stamm nach Anspruch 9 den Nahrungsmittelerzeugnissen zugegeben wird. 11. Nahrungsmittelerzeugnis, hergestellt durch eine Wärmebehandlung eines Nahrungsmittelerzeugnis-Ausgangsmaterials unter Verwendung einer Wärmequelle, die das Nahrungsmittelerzeugnis einer Temperatur über +85 °C aussetzt, dadurch gekennzeichnet, dass das Nahrungsmittelerzeugnis ferner einen lebensfähigen Lactobacillus-Stamm, ausgewählt aus den Spezies Lactobacillus plantarum LB931 (DSM11918), Lactobacillus rhamnosus LB21 (NCIMB 40564), Lactobacillus plantarum LB3e (DSM 17852) beziehungsweise Lactobacillus plantarum LB7c(DSM 17853), enthält. 12. Nahrungsmittelerzeugnis nach Anspruch 11, wobei der lebensfähige Lactobacillus-Stamm nach einer Wärmebehandlung in einer Menge über 1,0 x 103 KbE/g, vorzugsweise über 1,0 x 104 KbE/g und am stärksten bevorzugt über 1,0 x 105 KbE/g lebenden Bakterien pro Gramm des Nahrungsmittelerzeugnisses vorliegt. 13. Nahrungsmittelerzeugnis nach Anspruch 7, 11 oder 12, den thermostabilen lebensfähigen Lactobacillus-Stamm umfassend, ausgewählt aus den Spezies Lactobacillus plantarum und Lactobacillus rhamnosus zum Fördern der Immuntoleranz von Autoimmunkrankheiten, wie etwa Zölialkie.

Revendications 1. Microorganismes de la souche Lactobacillus choisie parmi les espèces Lactobacillus plantarum LB3e et Lactobacillus plantarum LB7c qui ont été déposées auprès de Deutsche Sammlung von Mikroorganismen und Zellkulturen, et auxquelles ont été attribués les numéros d’accès 17852 et 17853 respectivement. 2. Microorganismes de la souche Lactobacillus choisie parmi les espèces Lactobacillus plantarum LB3e et Lactobacillus plantarum LB7c selon la revendication 1, caractérisés en ce qu’ils sont capables de survivre à des températures à partir de + 85 °C pendant plus de 25 minutes. 3. Utilisation d’une souche Lactobacillus selon la revendication 1 dans des produits alimentaires tels que le pain, la pâte, les produits à base de céréales, le porridge, le gruau, le muesli, le granola, un produit à base de céréales en poudre, les pâtes, les demi-produits, les soupes, les produits prêts à cuire, le pain nature et la compote de fruits. 4. Procédé permettant d’empêcher la moisissure dans des produits alimentaires, dans lequel au moins une souche Lactobacillus selon la revendication 1 est incorporée dans lesdits produits alimentaires. 5. Procédé permettant d’empêcher la moisissure dans des produits alimentaires selon la revendication 4, dans lequel le produit alimentaire est choisi parmi le pain, la pâte, les produits à base de céréales, le porridge, le gruau, le muesli, le granola, un produit à base de céréales en poudre, les pâtes, les demi-produits, les soupes, les produits prêts à cuire, le pain nature et la compote de fruits. 6. Produit alimentaire tel que le pain, la pâte, les produits à base de céréales, le porridge, le gruau, le muesli, le granola, un produit à base de céréales en poudre, les pâtes, les demi-produits, les soupes, les produits prêts à cuire, le pain nature et la compote de fruits caractérisé en ce que ledit produit alimentaire contient également une souche Lactobacillus viable selon la revendication 1. 7. Produits de pain et de pâte dans lesquels lesdits produits de pain et de pâte contiennent également une souche Lactobacillus viable choisie parmi les espèces Lactobacillusplantarum LB931, qui a été déposée auprès de Deutsche Sammlung von Mikroorganismen und Zellkulturen et à laquelle a été attribué le numéro d’accès DSM 11918, Lactobacillus rhamnosus LB21, qui a été déposée auprès de NCIMB Ltd, Ferguson Building, Craibstone Estate et à laquelle a été attribué le numéro d’accès NCIMB 40564, Lactobacillus plantarum LB3e, et Lactobacillus plantarum LB7c, qui ont été déposées auprès de Deutsche Sammlung von Mikroorganismen und Zellkulturen, et auxquelles ont été attribués les numéros d’accès 17852 et 17853, respectivement, dans lesquels lesdites souches déposées sont capables de survive à des températures à partir de + 85 °C pendant plus de 25 minutes et lesdits lactobacilles d’au moins l’une desdites souches sont incorporés dans lesdits produits de pain et de pâte avant un traitement thermique. 8. Procédé permettant d’empêcher la moisissure dans des produits de pain et de pâte, dans lequel une souche Lactobacillus selon la revendication 7 est incorporée dans lesdits produits de pain et de pâte. 9. Utilisation d’une souche Lactobacillus viable choisie parmi les espèces Lactobacillus plantarum LB931, qui a été déposée auprès de Deutsche Sammlung von Mikroorganismen und Zellkulturen et à laquelle a été attribué le numéro d’accès DSM 11918, Lactobacillus rhamnosus LB21, qui a été déposée auprès de NCIMB Ltd, Ferguson Building, Craibstone Estate et à laquelle a été attribué le numéro d’accès NCIMB 40564, Lactobacillus plantarum LB3e et Lactobacillus plantarum LB7c, qui ont été déposées auprès de Deutsche Sammlung von Mikroorganismen und Zellkulturen, et auxquelles ont été attribués les numéros d’accès 17852 et 17853 respectivement, permettant de fabriquer un produit alimentaire, dans laquelle le produit alimentaire est préparé par traitement thermique d’un matériau de départ de produit alimentaire à l’aide d’une source de chaleur exposant ledit produit alimentaire à une température supérieure à + 85 °C tel que le pain, la pâte, les produits à base de céréales, le porridge, le gruau, le muesli, le granola, un produit à base de céréales en poudre, les pâtes, les demi-produits, les soupes, les produits prêts à cuire, le pain nature ou la compote de fruits, dans laquelle des lactobacilles d’au moins l’une des desdites souches sont incorporés dans ledit produit alimentaire avant un traitement thermique. 10. Procédé permettant d’empêcher la moisissure dans des produits alimentaires, dans lequel au moins une souche Lactobacillus selon la revendication 9 est incorporée dans lesdits produits alimentaires. 11. Produit alimentaire préparé par traitement thermique d’un matériau de départ de produit alimentaire à l’aide d’une source de chaleur exposant ledit produit alimentaire à une température supérieure à + 85 °C, caractérisé en ce que ledit produit alimentaire contient également une souche Lactobacillus viable choisie parmi les espèces de Lactobacillus plantarum LB931 (DSM11918), Lactobacillus rhamnosus LB21 (NCIMB 40564), Lactobacillus plantarum LB3e (DSM 17852) et Lactobacillus plantarum LB7c (DSM 17853) respectivement. 12. Produit alimentaire selon la revendication 11, dans lequel la souche Lactobacillus viable est présente dans une quantité supérieure à 1,0 x 103 CFU/g, de préférence supérieure à 1,0 x 104 CFU/g et idéalement supérieure à 1,0 X 105 CFU/g, de bactéries vivantes par gramme du produit alimentaire après traitement thermique. 13. Produit alimentaire selon les revendications 7, 11 ou 12 comprenant la souche Lactobacillus viable thermostable choisie parmi les espèces Lactobacillus plantarum et Lactobacillus rhamnosus permettant de favoriser une immunotolérance dans des maladies auto-immunes telles que la maladie coeliaque.

REFERENCES CITED IN THE DESCRIPTION

This list of references cited by the applicant is for the reader’s convenience only. It does not form part of the European patent document. Even though great care has been taken in compiling the references, errors or omissions cannot be excluded and the EPO disclaims all liability in this regard.

Patent documents cited in the description • WO 9400019 A [0006]

Claims (3)

1. I, Λ ;.*· ·νοοα<'·χ i / η.··: : ör asOös. tartósé miji:roorgaáia;:aüsok a tc?:niv;.::·i i .·va Sermestabil Ttmgteb&amp;oillua tömasek Bsabadaláji igérsypentok p ronta ra;s 7..77>3v és a iníotöbaoiiios plan&amp;tsnm LB7c farök kövüi válgsstv:a:, amelyek a :bsntsohe Sarssisng von Mi ktoorganlamen nnö ••Ák7iKo7 iarer.-ne:, vansa.k letétbe helyavvs és a 17832, illetve 1787:=3 letétbe belyssésí arájáén várinak: nyilvántsr'fcvá,
2. 3, Άκ 1. igénypont sserintl L&amp;ot&amp;b&amp;oílláÉ· béréphős tartósé •aikroergani atjusok a 3a dióba ¢:0:.12 isis plánt:;;íz:lm LB3s és a raesirkaei 11 íls plsntzriim LBTc fajok kösül válását:m, ars.ml begy a s§5 "C-tél k erébe; o nőmérsékleteket több, sünt 277 peroen át képesek tőiéini, 77:< Ás l . .igénypont sveriaki L&amp;?tohap£llus eervs alkaijáasása olyan é,kö iSiié sor terme kekken, pirt a kenyér, nyerstéavba, gabonatermékek, sakk áss, kasa, müzli, gr :wd a, porított gabona alap 77 temek, késs te, rélkéss termékek, . evesek, elősütött tér aékn k, sima kenyér, befért-.

   1, Áll árrés él e Imi se ért: érmé ke kb «η a peoésvesodée eegakadáj.yösására, sbol ás 1, igénypont aaerinti legalább egy iaeíetaoiii ve tömve·. kevernek s nevevett éielsiiasertermékekbe. 77, á 4, igénypont averinti, c 7.a7;:··.:.e aorta ·:síké k bor. r,7:ir:S:énp penésaeaeeás megakadásvonáséra svoigélő ed.jéeás, ahol as éle Imis sertermékaket a köve b ke s ők iközál vél észtjük.: kényét, nyerst észt a, gabom-atemékek, ssbkása, kása, müzli, gr eve !.a, porított gabona alapé térnék, tészta, fáikése termékek, Levesek, elosétött teröekek, sima kegyér, és be főtt.. 6, £lev,gisse:r:te:tgéksk, mint S: kenyér, nyelséésstá:, ggisryrsbériaákek, vabkása, kása, jjjősíí. granela, ροritort: gabona alapé termék, téssta, fél kési:?. terméket, levesek, elősütött termékek·, sión kenyér, befőtt, a vsa I jeXiemsKve, begy nevese:: - éleiísis ver termék tartalmas a s 1, igénypont sserinti életképed ieoto-baoi7Ina tíSrveet is, 7, kenyér és nysestéests. témáké;·;;, ahol n eve vett kenyér és gysretésvta termékek tartalmasnak életképes: iscte&amp;esiiles fcöívset is a követkevőfc keséi vásssrtV'é: .paeimbaoiiire trla:its-ram LBi> iL, amely a Deutsohé Semminng von Ml 1 morgás lemen uná Be 1 !ko11nrerm--né;i va;a letétbe helyezve és s SEM Ildié letétbe öe.;.yczé::.! számon var; nyilvántartva, L3úta&amp;s:áil.ius z'ósmnosba Ι-Γο·: i, amely az BC1MB Léd, Fergt;son Baiiding; üraibstone bstato"néi van letétbe halyszve és a a EÜiMB 40564 letétbe helyezési számon van nyilvántartva, iactobaéiiloá piahiérum bűze és Fsolohyoliiss ρ,ίοηΐρΓϋρ Ι,Βνο, ermhlyeh a Depisshe üammlnng tea Mikzootganismen and tgllkuiigtére'bál vannak letétbe helyette ás a 17852,· Illette 17850 Let étbe helyezési számokon tannak nyilvántartva* 'Sh.pl nevese;.·, letétbe helyezett törzsek képeseik a eSS 'h>'tdl kezdődd Ispmérsék 1 eteksn több- mint 11 peraen át: túlélni,· és a legalább egyik nevezeti törzsből való nevezett lect obao.:. Hozok·:,1 nevezett kenyér ás nyerstészta termékekbe keverjük a hőkezelés éléit,
3. 8- Eljárás kenyér és nysrstészta termékekben ·: értené penészesedés mega ládái pózává ·:.« - ahol a 7, Igénypont szerinti valamely haerabeciiias torsáét keverünk a nevezett kenyér és nyerstéssia termékekbe - t - á tévei koré fajok közül választott életképes iaoroizsoiilvs térés alkalmazása; i.nctcba;;i77 tv plantarum Lükéi* amely a beetsche Sammlzsng tón Hl k:rpo r gar:j.aman apd Beit knXturen-ruí! tan letétbe helyezte és a DSM 11SIS letétbe helyezési számon van nyilvántartva* Imrétóbaei7ias ;. tamnoanv LESI- amely az FG1ME Ltd, Fekgason Bsilding, Cizaibstone Estate~néi tan letétbe helyezte és az bClMÜ döüsi letétbe helyezési számon tar: nyilvántartva, La.otobs-odllü's plantarum LB3e és Laotobaeiiius p bn; b,;·· an :,B /e, amelyek a Dentscha Bammlnng tón Mikroorganismen und Kell knltnzen-ozéi varrnak .letétbe helyezve és a I78S-:, liléivé 17SS3 letétbe helyezési, számokon vannak nyiivántartva, é 1 e Isii·® z e r t érmék gyártására, ánoi az éieiíáiszsírterpéket egy élelmiszertermék kilndniáéi anyag hőkezelésével áIli tjük éld egy olyan hőforrás alkalma zásával, amely nevezett élelmiszertermékei -í-85 v C fői ét ti hőmérsékletnek teszi ki., mini amilyen a kenyér, nyernie szí·:;, gabona terméke k, zabkása, kása, müzli, granola, porított gabona a ingó termék, tészta, félkész te zmékek- levesek, elősütött termékek, sima kenyér·, vagy befőtt, ahol nevezett törzsök iegslább egyike ezerinti iactobaollinaokst hékeze les elét! nevezőit élelmiszertermékbe keverünk. 18, kijárás él.grlmiazé.kteté;ékehbsn történd: penészesedés me ga ka dé1yoaás ára - ahol a 8. igénypont szerint.;. legalább egy Lacsobseiiive törzset keverünk a nevezett: áleltriszez'termékekbó, 11 < Óleististartörssék, amelyet agy él<alsls£<srt:Hrméfe kiinöoliso -íoyag oőkosol.éeéve 1 iliitnnk elő olyan hőforrás áikalharisáva 1, annly neaefötf éielaíiooe?:· ov;nok?U: 081 <;C fölötti hömétáéklstnok kosai ki, ssssal 1 ollesassEhogy netésett ávlölíKlsSíi^rá-íiSSyák' tartíü.mas: életképes La ο;: οί.οο£ i .km: tömrset is a koostkesö fajok köaöi választás: lm--:1-.:1:,} cl jpla-rtaCim LB931 (DőMllhfk'í laotofami 1 fos rfamsosra: IBS! UkClMB 4058«·, iaotobaollloa pl&amp;-ni:armi fBoO Í ÖSK X785ák, ás Ls-PbábpóiZTüé salat iaruB LBlo (DSM 178:53} , 11* Λ 11. igénypont ooérinti o lola-Jao'or torssal, ahol az életképes: LeictQbác:-tilos toros l, OalsB CFü/g fölötti mennyiségben, előnyőse;·· 1, Dalik C-Fü/g fölötti Sierinyiségben, és ! sok onyösahbon 1,0x10"' CFU/g fölötti iáenöylségbóö van. jelen, élő faktor lökként ,·, v. eiátlsásastrtárrn'ék-gratíojaként a bökeselást kese:: ooe, Iá, á 7,, 11. vény 12:. igénypontok szerinti élolíaiaossrterxák, aisely tartalmazza a faotöáa öli is a pl mi. fcaam és Lcot&amp;basíllus rhamnosus fajok közül választott terisootasbil:, életképes ilaastofeaaaíl les; törtsék aütísiskkan lartegsógakhen, salat a eöliékia (iisistárz-ékertysésj} , az 1 hí:·nni to j, e karc i a á 1 öée g 11 éséx'é >