Title: New product

TECHNICAL FIELD

The present invention relates to the Lactobacillus 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 pre- sent 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 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.

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.

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 mi- croorganisms. 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 consum- ers 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.

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.

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 ⁰ 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.

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

SUMMARY OF THE INVENTION

The inventors of the present invention have surprisingly found that thermostable microorganisms of the Lactobacillus genus that are able to survive temperatures from +80 ⁰ C for more than 25 minutes or alternatively for more than 10 s in a microwave radiated environment and have the ability to produce viable antibacterial agents in different food products after such heat treatment.

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, Lactoba- cillus plantarum LB931 and Lactobacillus rhamnosus, LB21. Furthermore, two novel strains of Lactobacillus are disclosed, referred to as Lactobacillus plantarum LB7c and Lactobacillus plantarum LB3e.

The inventors have also shown that food products comprising the thermostable vi- able Lactobacillus strains chosen from the species Lactobacillus plantarum and

Lactobacillus rhamnosus and particularly Lactobacillus plantarum 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

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

As disclosed herein, the term "thermostable" relates to bacteria that survive in temperatures higher than +80 ⁰ C for more than 25 minutes.

As disclosed herein, the term "lactic acid bacteria" relates to bacteria producing lac- tic acid, such as bacteria belonging to the genera Lactobacillus and Lactococcus.

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

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, bake-off products, plain bread and stewed fruit.

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.

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

As disclosed herein, the term "a slice of bread" relates to a typical slice of bread which weighs about 15-4Og.

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..

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

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 LlA in +80 ⁰ C.

Figure 6 discloses the survival capability of LB931 in +90 ⁰ 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

The present invention relates to thermostable microorganisms of the Lactobacillus genus that are able to survive temperatures from +80 ⁰ C for more than 25 minutes.

It is also disclosed that thermostable microorganisms of the Lactobacillus genus are able to survive for more than 10 s in a microwave radiated environment.

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.

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

An embodiment is directed to microorganisms of the Lactobacillus strain chosen from the species Lactobacillus plantarum LB3e and Lactobacillus plantarum LB 7c which have been deposited at Deutsche Sammlung von Mikroorganismen und Zeelkulturen, and have been assigned accession number 17852 and 17853 respectively that are able to survive temperatures from +80 ⁰ C for more than 25 minutes.

Any of the 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, bake-off products, plain bread, stewed fruit.

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 wherein said deposited strains are able to sur- vive temperatures from +80 ⁰ C for more than 25 minutes.

In further embodiments said deposited strains have been showed to survive temperatures from +80 ⁰ C for more than 30, 40, 50 and 60 minutes.

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 wherein said deposited strains are able to survive for more than 10 s in a microwave radiated environment.

A food product mentioned above wherein the viable Lactobacillus strain is 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 de- posited 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).

A food product mentioned above prepared by heat treatment of a food product start- ing material using a heat source exposing said food product to a temperature above +80 ⁰ C wherein that said food product also contains a viable Lactobacillus strain

preferably chosen from the Lactobacillus plantarum LB931 (DSMl 1918), Lactobacillus rhamnosus LB21 (NCIMB 40564), Lactobacillus plantarum LB7c (DSM 17853) and Lactobacillus plantarum LB3e (DSM 17852) respectively.

Examples of two known Lactobacillus strains are disclosed, Lactobacillus plantarum LB931 and Lactobacillus rhamnosus, LB21.

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 said deposited strains are able to survive temperatures from +80 ⁰ C for more than 25 minutes are disclosed.

A food product mentioned above wherein the viable Lactobacillus strain is present in an amount above 1,OxIO ³ CFU/g, preferably above 1,OxIO ⁴ CFU/g and most preferably above 1,OxIO ⁵ CFU/g, living bacteria per gram of the food product after the heat treatment.

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 (DSM 11918), Lactobacillus rhamnosus, LB21 (NCIMB 40564), Lactobacillus plantarum LB7c (DSM 17853) and Lactobacillus plantarum LB3e (DSM 17852).

A food product containing such viable thermostable Lactobacillus strains promoting immuntolerance in autoimmune diseases such as coeliac disease.

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

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 ac- cession number NCIMB 40564.

The LB3e has been deposited on Jan. 6, 2006 at DSMZ-Deutsche Sammlung von Mikroorganismn 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).

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.

The above mentioned five four thermostable Lactobacillus strains have the ability to survive in a high temperature environment i.e. above +80 ⁰ C for more than 25 minutes or alternatively for more than 10 seconds in a microwave radiated environment 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 microor-

ganisms, 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.

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.

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.

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.

Both LB931 and LB21 have the ability to survive in snack bars for 25 days. The snack bars containing LB931 had 7,6 x 10 ⁶ CFU/ml at day 46.

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.

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 10 ⁷ 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.

The killing of the four different strains of lactic acid bacteria with UV-light were 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.

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 ⁰ C for more than 25 minutes and LB931 survives for more than 5 minutes in +90 ⁰ C. The control strain, Ll A, do not even survive for 5 minutes in +80 ⁰ C. The results are presented in Figure 5 and 6.

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 LlA was used. The LlA did not grow in the starter. The CFU/g had decreased from day 0 to day 3.

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 LlA was used as control. All the Lactobacillus strains that were tested survived in all the different temperatures and times. The control, LlA, did not survive. There was no visual growth of the bacteria from day 0.

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 possi-

ble, to provide the desired effects. The experimental results show that amounts above 1,OxIO ¹⁰ CFU/g can be achieved. In a food product the viable Lactobaci strain should be present in an amount above 1,OxIO ³ CFU/g, preferably above 1 1,,OOxxIIOO ⁴⁴ CCFFUU//gg aanndd most preferably above 1,OxIO ⁵ CFU/g, living bacteria per gram of the food product.

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,O x IO ³ CFU/g, preferably above 1,0 x 10 ⁴ CFU/g and most preferably above l,0 x 10 ⁵ CFU/g, living bacteria per gram of a slice of bread.

For example, a viable Lactobacillus strain wherein the start concentration is about 1 x 10 ⁶ has a CFU above 1x10 ³ 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.

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 mu- cosa 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.

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

EXAMPLES

Example 1

Isolation and identification of the Lactobacillus strains.

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-pentosus- paraplantarum.

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.

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

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

Example 2 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

b) LB931 dissolved in a suspension of equal parts skim milk and 0.9% NaCl. 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, ⁰ C Day 0 Day 2 Day 5 Day 32

4 7,8 x 10 ^iυ 2,2 x 10 ^iυ 20 1,8 x lO ¹⁰ 2,2 x 10 ¹⁰

27 1,8 x lO ¹⁰ 1,2 x lO ¹⁰ 3,3 x 10 ⁹

The results show that LB931 is stable in a mixture of skim milk and NaCl 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 ⁰ C Day 0 Day 7 Day 25

^~ 6 4,2 x 10" 2,2 x 10* 1,2 x 10*

22 4,2 x 10 ⁸ 1,9 x lO ⁸ 1,4 x lO ⁸

The number of bacteria in the freeze-dried powder was also monitored every fourth week up to 68 weeks. After one year, at +6 ⁰ C, more than 10 ⁵ 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 IQ ¹⁰ 2,3 x IQ ¹⁰ 1,O x IO ¹⁰ 1,2 x IQ ¹⁰

Example 3

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 temperature Dry

Air Oil Air

LB21 25°C +25 ⁰ C - +30 ⁰ C

LB21 30 ⁰ C +30 ⁰ C - +38 ⁰ C

LB21 33°C +33 ⁰ C - +38 ⁰ C

LB21 5 % +29 ⁰ C +38 ⁰ C +38 ⁰ C

LB21 25 % hot +28 ⁰ C +38 ⁰ C +38 ⁰ C

LB21 25 % cool +28 ⁰ C +27 ⁰ C +25 ⁰ 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 LB21 LB21 LB21 LB21 LB21 LB21

(days) 25°C 30 ⁰ C 33°C 5% 25 % hot 25% cool

6 4x10" 2x10" 1x10" 2x10" 3x10" 3x10"

31 2 xlO ⁸ 2 xlO ⁸ IxIO ⁸ 2 xlO ⁸ 2 xlO ⁸ 2 xlO ⁸

59 3 xlO ⁸ 5 xlO ⁸ 2 xlO ⁸ 2 xlO ⁸ 3 xlO ⁸ 4 xlO ⁸

90 2 xlO ⁸ 3 xlO ⁸ IxIO ⁸ IxIO ⁸ 3 xlO ⁸ IxIO ⁸

122 2 xlO ⁸ 2 xlO ⁸ 7 xlO ⁷ 7 xlO ⁷ IxIO ⁸ IxIO ⁸

144 2 xlO ⁸ 2 xlO ⁸ IxIO ⁸ 8 xlO ⁷ 8 xlO ⁷ 8 xlO ⁷

180 2 xlO ⁷ 2 xlO ⁷ 3 xlO ⁷ 2 xlO ⁶ 3 xlO ⁶ 5 xlO ⁶

219 IxIO ⁶ 2 xlO ⁶ 3 xlO ⁵ 8 xlO ⁴ 9 xlO ⁵ 9 xlO ⁵

244 4 xlO ³ IxIO ³ IxIO ³ 2 xlO ³ 5 xlO ³ 6 xlO ³

TABLE 7. Stored in +4°C

No. of bacteria CFU/g

Time LB21 LB21 LB21 LB21 LB21 LB21

(days) 25°C 30 ⁰ C 33°C 5% 25 % hot 25% cool

6 4x10" 2x10" 1x10" 2x10" 3x10" 3x10"

31 2 xlO ⁸ 2 xlO ⁸ 2 xlO ⁸ 2 xlO ⁸ 2 xlO ⁸ 2 xlO ⁸

59 4 xlO ⁸ 2 xlO ⁸ 2 xlO ⁸ 3 xlO ⁸ 4 xlO ⁸ 3 xlO ⁸

90 4 xlO ⁸ 2 xlO ⁸ 2 xlO ⁸ 3 xlO ⁸ 4 xlO ⁸ 2 xlO ⁸

122 2 xlO ⁸ 2 xlO ⁸ 6 xlO ⁷ 2 xlO ⁸ 2 xlO ⁸ IxIO ⁸

144 2 xlO ⁸ 2 xlO ⁸ 8 xlO ⁷ 2 xlO ⁸ 2 xlO ⁸ IxIO ⁸

180 2 xlO ⁸ 2 xlO ⁸ 6 xlO ⁷ IxIO ⁸ IxIO ⁸ IxIO ⁸

219 2 xlO ⁸ IxIO ⁸ 4 xlO ⁷ IxIO ⁸ IxIO ⁸ IxIO ⁸

244 4 xlO ⁸ 2 xlO ⁸ 2 xlO ⁷ 2 xlO ⁸ 4 xlO ⁸ 3 xlO ⁷

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 8 x 10" 1,2 x 10 ⁷

10 2,7 x 10 ⁸ 1,3 x 10 ⁷

21 3,8 x 10 ⁸ 3,O x 10 ⁵

46 7,6 x 10 ⁶ 0

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

Example 4

The Survival Capabilities of Lactobacillus in microwave radiated environment.

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 dm ³ . The number of bacteria was determined after 30 seconds, 60 seconds, 70 seconds, 80 seconds and 90 seconds, respectively. The tem- perature of the milk cereal drink after 90 seconds in the microwave oven was about +60 ⁰ 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 10 ⁷ 1,1 x 10 ⁷

30 2,2 x 10 ⁷ 2,2 x 10 ⁵

60 2,6 x 10 ⁷ l,0 x 10 ⁵

70 1,3 x 10 ⁷ l,0 x 10 ⁵

80 3,9 x 10 ⁶ l,0 x 10 ⁵

90 1,1 x 10 ³ l,0 x 10 ⁵

Example 5

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 planta- rum; 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% CO ₂ . They were then inoculated in 3 ml MRS-broth at 37°C, 5 % CO ₂ over night which generates about 10 ⁹ CFU/ml. The overnight cultures of the strains were added to a final volume of 1 % in MRS broth and were in- cubated for 20 h at 37°C, 5% CO ₂ . The bacteria were centrifuged (3800 rpm, 20 min, +4°C) and then washed 2 times in 10 ml 0.9% NaCl. The bacteria were dissolved in 0.9% NaCl. 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 1O h. The death of the bacteria was controlled on MRS agar plates, incubated for 48 h at 37°C, 5% CO ₂ .

Agar overlay- interference test 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 % CO ₂ for 20 h.l 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% CO ₂ . Nine different interference test strains (ITS) (NUS, Umea, Sweden), isolated from people with urinary infections, were inoculated in Ml 7 broth and incubated in 37°C over night. A second agar layer of 25 ml Ml 7 with 750 μl 6,5M KH ₂ PO ₄ 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 Ml 7 agar surface and also on a control plate and then incubated in 37°C, 5% CO ₂ over night.

Inhibition of pathogens by lactic acid bacteria

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 patho- gens 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

L. acidophilus

Strain tested LB931 LB3e LB7c LB21 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

The Survival Capabilities of Lactobacillus in different temperatures, a) Different suspensions of Lactobacillus were made of freeze-dried LB931 in NaCl 0.9 % and overnight culture of LB931, LB7c, LB3e and LlA. The suspensions were at 10 ⁹ -10 ^π CFU/ml. 5ml of NaCl 0.9 % was heated up to +80 ⁰ C in a water bath. As soon as the NaCl was +80 ⁰ C lOOμl 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 I l

No. of bacteria CFU/ml

Time (min) LB931 LB7c LB3e LlA

2,3O x 1,1 x 10" 9J x IO ⁷ 2,5 x 10 ⁷

0 10 ⁸

4,33 x 1,O x IO ⁴ 1,4 x lO ⁴ NVG

5 10 ⁶

1,77 x 3,3 x lO ⁴ 1,4 x lO ⁴ NVG

10 10 ⁶

2,0O x 2,5 x 10 ⁴ 1,8 x 10 ² NVG

15 10 ⁶

8,2O x 1,2 x 10 ³ 1,9 x 10 ⁵ NVG

20 10 ⁴

2,24 x 1,O x IO ⁴ 7,8 x lO ⁴ NVG

25 10 ⁵

b) +80 ⁰ C in more than 20, 30, 40, 50, 60 minutes

5ml of MRS were heated to +85°C in a water bath. 500μl 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

NaCl 0.9 % and also an overnight culture of LB931. The NaCl was heated up to +90 ⁰ C in a water bath. As soon as the NaCl was +80 ⁰ C lOOμl 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 re- suits are disclosed in table 13 below.

TABLE 13

No. of bacteria CFU/ml

Time (min) LB931

0 1,76 x 10"

0,5 5,00 x 10 ⁵

1 3,54 x 10 ⁴

2 4,43 x 10 ⁴

3 1,25 x 10 ⁴

4 1,14 x 10 ⁴

5 3,00 x 10 ⁴

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 ( ⁰ C) LB931 LB21

10 2,9 x 10 ⁷ 3,0 x 10 ⁷

37 2,4 x 10 ⁷ 2,8 x 10 ⁷

Example 7

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 5x10" 2 x 10 ⁷ 6 x 10 ⁷

3 3 xlO ⁹ 9 xlO ⁸ 2 xlO ⁹

TABLE 16

No. of bacteria CFU/g

Time (days) LB931 +4°' C LB7c +4°C LB3e +4°C

0 3xlO ^y 6x10" 3xlO ^y

3 2 xlO ⁹ 3 xlO ⁸ 9 xlO ⁸

6 2 xlO ⁹ 2 xlO ⁷ 8 xlO ⁸

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 3 days 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 LlA

0 2 xlO ⁸ 2 xlO ⁷ 6 xlO ⁷ Ix 10 ⁷

3 7 xlO ⁹ 9 xlO ⁸ 2 xlO ⁹ 8x 10 ⁵

TABLE 18

No. of bacteria CFU/g stored in +4° C

Time (days) LB931 LB7c LB3e

0 3 xlO ⁹ 6x 10 ⁸ 3 xlO ⁹

3 2 xlO ⁹ 3x 10 ⁸ 9 xlO ⁸

6 2 xlO ⁹ 2x 10 ⁷ 8 xlO ⁸

Example 8

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 IdI 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 ⁰ 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

Baked in 230 ⁰ C Baked in 230 ⁰ C Baked in 240 ⁰ C

Time (minutes) for 25 minutes for 35 minutes for 30 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 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 ⁰ C for 20 minutes (Sourdough starter)

Time (days) No. of bacteria CFU/g

LB3e (rye) LB7c (wheat)

0 1,1 x 10 ⁵ 1,2 x 10 ⁴

5 1,1 x 10 ⁴ 2,1 x 10 ⁵

TABLE 21

Baked in +230 ⁰ C for 25 minutes (Rye Sourdough starter) No. of bacteria CFU/g

Time (days) LB931 LB3e LB7c LB21

0 9 x 10" 3 x 10" 7 x lO ² 7 x 10"

3 I x IO ⁵ 5 x lO ⁴ I x IO ⁴ 3 x lO ⁴

7 9 x lO ⁴ 6 x lO ⁴ 9 x lO ⁴ Not done

TABLE 22

Baked in +230 ⁰ C for 35 minutes (Rye Sourdough starter) No. of bacteria CFU/g

Time (days) LB931 LB3e LB7c

0 9 x lO ² 2 x lO ² I x IO ²

3 8 x lO ³ 3 x lO ³ 7 x lO ³

7 9 x lO ³ 7 x lO ³ 6 x lO ³

TABLE 23

Baked in +240 ⁰ C for 30 minutes growth No. of bacteria CFU/g

Wheat Sourdough starter Rye Sourdough starter

Time (days) LB931 LlA LB931

0 4 x lO ³ No Visible Growth 3 x lO ⁴

(NVG)

2 6 x lO ⁴ NVG 7 x lO ⁴

4 2 x lO ⁵ NVG 9 x lO ⁵

6 I x IO ⁵ NVG 6 x lO ⁵

8 7 x lO ⁴ NVG 8 x lO ⁴

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 10 ⁹ were combined in a large mixer bowl to a total volume of 1 litre. The CFU/ml of the dough was 1 x 10 ⁶ . 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 ⁰ 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 10 ³

3 6 x lO ³

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

No. of bacteria

Bread CFU/g day 15 Detection of mould

LB931 Sourdough bread 5 x lO ⁴ NVG Day 15

Sourdough bread 5 x lO ⁴ NVG Day 15

LB21 Sourdough bread 7,6 x 10 ⁴ NVG Day 15

Bread with 5ml 1,2 x 10 ³ NVG Day 15 bacterial solution

LB3e Sourdough bread 4 x lO ⁴ NVG Day 15

LB7c Sourdough bread 6 x lO ⁴ 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 O Day 5 Day 10

Study person 1 0 7 x 10 ³ 6 x 10 ³ Study person 2 0 2 x 10 ² 4 x lO ³ Study person 3 0 5 x IQ ⁷ 6 x IQ ³

Example 9 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 (10 ¹⁰ 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 glu- ten (0. Ig 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 in-

testine 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

10 ¹⁰ 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 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 10 ¹⁰ 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

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 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.

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.

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.