WO2007108764A2 - Thermostable lactobacillus strains - Google Patents

Thermostable lactobacillus strains Download PDF

Info

Publication number
WO2007108764A2
WO2007108764A2 PCT/SE2007/050156 SE2007050156W WO2007108764A2 WO 2007108764 A2 WO2007108764 A2 WO 2007108764A2 SE 2007050156 W SE2007050156 W SE 2007050156W WO 2007108764 A2 WO2007108764 A2 WO 2007108764A2
Authority
WO
WIPO (PCT)
Prior art keywords
lactobacillus
food product
xlo
bread
products
Prior art date
Application number
PCT/SE2007/050156
Other languages
French (fr)
Other versions
WO2007108764A3 (en
Inventor
Eva GRAHN HÅKANSSON
Jennie Ekbeck
Original Assignee
Probac Ab
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Probac Ab filed Critical Probac Ab
Priority to EP07716125.5A priority Critical patent/EP1996696B1/en
Priority to US12/225,221 priority patent/US8137952B2/en
Priority to LTEP07716125.5T priority patent/LT1996696T/en
Priority to ES07716125.5T priority patent/ES2619185T3/en
Priority to DK07716125.5T priority patent/DK1996696T3/en
Publication of WO2007108764A2 publication Critical patent/WO2007108764A2/en
Publication of WO2007108764A3 publication Critical patent/WO2007108764A3/en
Priority to NO20084335A priority patent/NO343526B1/en
Priority to CY20171100355T priority patent/CY1118795T1/en

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • A—HUMAN NECESSITIES
    • A21—BAKING; EDIBLE DOUGHS
    • A21D—TREATMENT, e.g. PRESERVATION, OF FLOUR OR DOUGH, e.g. BY ADDITION OF MATERIALS; BAKING; BAKERY PRODUCTS; PRESERVATION THEREOF
    • A21D8/00—Methods for preparing or baking dough
    • A21D8/02—Methods for preparing dough; Treating dough prior to baking
    • A21D8/04—Methods for preparing dough; Treating dough prior to baking treating dough with microorganisms or enzymes
    • A—HUMAN NECESSITIES
    • A21—BAKING; EDIBLE DOUGHS
    • A21D—TREATMENT, e.g. PRESERVATION, OF FLOUR OR DOUGH, e.g. BY ADDITION OF MATERIALS; BAKING; BAKERY PRODUCTS; PRESERVATION THEREOF
    • A21D8/00—Methods for preparing or baking dough
    • A21D8/02—Methods for preparing dough; Treating dough prior to baking
    • A21D8/04—Methods for preparing dough; Treating dough prior to baking treating dough with microorganisms or enzymes
    • A21D8/045—Methods for preparing dough; Treating dough prior to baking treating dough with microorganisms or enzymes with a leaven or a composition containing acidifying bacteria
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L7/00—Cereal-derived products; Malt products; Preparation or treatment thereof
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L7/00—Cereal-derived products; Malt products; Preparation or treatment thereof
    • A23L7/10—Cereal-derived products
    • A23L7/104—Fermentation of farinaceous cereal or cereal material; Addition of enzymes or microorganisms
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L7/00—Cereal-derived products; Malt products; Preparation or treatment thereof
    • A23L7/10—Cereal-derived products
    • A23L7/109—Types of pasta, e.g. macaroni or noodles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
    • A61K35/747—Lactobacilli, e.g. L. acidophilus or L. brevis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2400/00—Lactic or propionic acid bacteria
    • A23V2400/11—Lactobacillus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/225—Lactobacillus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/225—Lactobacillus
    • C12R2001/25—Lactobacillus plantarum

Definitions

  • 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.
  • two novel strains of Lactobacillus genus are disclosed.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • Lactobacillus strains Two specific examples of known Lactobacillus strains are disclosed, Lactoba- cillus plantarum LB931 and Lactobacillus rhamnosus, LB21.
  • two novel strains of Lactobacillus are disclosed, referred to as Lactobacillus plantarum LB7c and Lactobacillus plantarum LB3e.
  • 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.
  • the term “LB” relates to bacteria of the genus Lactobacillus.
  • the term “thermostable” relates to bacteria that survive in temperatures higher than +80 0 C for more than 25 minutes.
  • lactic acid bacteria relates to bacteria producing lac- tic acid, such as bacteria belonging to the genera Lactobacillus and Lactococcus.
  • CFU colony-forming units
  • 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.
  • 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.
  • a slice of bread relates to a typical slice of bread which weighs about 15-4Og.
  • the term "bake-off products” relates to semi-finished products for which the baking will be completed after supply to the consumer.
  • soluble substances relates to organic acids, inorganic acids, such as lactic acid, succinic acid, acetic acid, and propionic acid.
  • 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- ⁇ levels or IL-4 levels of resting T cells.
  • 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.
  • thermostable microorganisms of the Lactobacillus genus are able to survive for more than 10 s in a microwave radiated environment.
  • 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.
  • 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.
  • 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- make temperatures from +80 0 C for more than 25 minutes.
  • said deposited strains have been showed to survive temperatures from +80 0 C for more than 30, 40, 50 and 60 minutes.
  • 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.
  • 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.
  • Lactobacillus plantarum LB931 Examples of two known Lactobacillus strains are disclosed, Lactobacillus plantarum LB931 and Lactobacillus rhamnosus, LB21.
  • 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).
  • 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
  • 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.
  • 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.
  • 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
  • Figure 9 discloses expression of lactic acids in an Auto- Scaled Chromatogram of supernatants of the Lactobacillus strains LB931, LB3e, LB7c and LB21.
  • 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.
  • Example 2 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.
  • Lactobacillus 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.
  • 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.
  • Example 4 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.
  • Example 5 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 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • Lactobacillus 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.
  • 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.
  • 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.
  • 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.
  • 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
  • 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.
  • Thermostable LB strains can survive in high temperature in different cereal preparations.
  • 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.
  • 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.
  • 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 .
  • ITS interference test strains
  • a suspension of LB931 was prepared by suspending freeze-dried LB931 in
  • 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.
  • 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.
  • 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.
  • glu- ten 0.1% Ig gluten/day/kg bodyweight
  • PBMC peripheral blood mononuclerar cells
  • 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.
  • 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.

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Microbiology (AREA)
  • Food Science & Technology (AREA)
  • Biotechnology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Genetics & Genomics (AREA)
  • Polymers & Plastics (AREA)
  • Nutrition Science (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Mycology (AREA)
  • General Engineering & Computer Science (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Virology (AREA)
  • Biomedical Technology (AREA)
  • Biochemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Veterinary Medicine (AREA)
  • Immunology (AREA)
  • Molecular Biology (AREA)
  • Epidemiology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Bakery Products And Manufacturing Methods Therefor (AREA)
  • Food Preservation Except Freezing, Refrigeration, And Drying (AREA)
  • Coloring Foods And Improving Nutritive Qualities (AREA)

Abstract

The present invention relates to the Lactcobacillus genus that have 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 5 treatment. The thermostable microorganisms of the Lactobacillus genus may be in- cluded 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 microor- ganisms of the Lactobacillus genus for promoting immuntolerance in autoimmune diseases such as coeliac disease. Furthermore, two novel strains of Lactobacillus 10 genus are disclosed, Lactobacillus plantarum LB3e and Lactobacillus plantarum LB7c deposited at Deutsche Sammlung von Mikroorganismen und Zeelkulturen, which have been assigned accession number 17852 and 17853 respectively.

Description

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 0C 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 0C 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 +800C 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 +800C.
Figure 6 discloses the survival capability of LB931 in +900C.
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 0C 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 0C 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 0C for more than 25 minutes.
In further embodiments said deposited strains have been showed to survive temperatures from +800C 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 0C 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 0C for more than 25 minutes are disclosed.
A food product mentioned above wherein the viable Lactobacillus strain is present in an amount above 1,OxIO3 CFU/g, preferably above 1,OxIO4 CFU/g and most preferably above 1,OxIO5 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 0C 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 106 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 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.
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 +800C for more than 25 minutes and LB931 survives for more than 5 minutes in +900C. The control strain, Ll A, do not even survive for 5 minutes in +800C. 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,OxIO10 CFU/g can be achieved. In a food product the viable Lactobaci strain should be present in an amount above 1,OxIO3 CFU/g, preferably above 1 1,,OOxxIIOO44 CCFFUU//gg aanndd most preferably above 1,OxIO5 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 IO3 CFU/g, preferably above 1,0 x 104 CFU/g and most preferably above l,0 x 105 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 106has 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.
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
Figure imgf000015_0001
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, 0C Day 0 Day 2 Day 5 Day 32
4 7,8 x 10iυ 2,2 x 10iυ 20 1,8 x lO10 2,2 x 1010
27 1,8 x lO10 1,2 x lO10 3,3 x 109
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 0C Day 0 Day 7 Day 25
~6 4,2 x 10" 2,2 x 10* 1,2 x 10* 22 4,2 x 108 1,9 x lO8 1,4 x lO8
The number of bacteria in the freeze-dried powder was also monitored every fourth week up to 68 weeks. After one year, at +60C, 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 IQ10 2,3 x IQ10 1,O x IO10 1,2 x IQ10
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 0C - +30 0C
LB21 300C +30 0C - +38 0C
LB21 33°C +33 0C - +38 0C
LB21 5 % +29 0C +38 0C +38 0C
LB21 25 % hot +28 0C +38 0C +38 0C
LB21 25 % cool +28 0C +27 0C +25 0C 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 300C 33°C 5% 25 % hot 25% cool
6 4x10" 2x10" 1x10" 2x10" 3x10" 3x10"
31 2 xlO8 2 xlO8 IxIO8 2 xlO8 2 xlO8 2 xlO8
59 3 xlO8 5 xlO8 2 xlO8 2 xlO8 3 xlO8 4 xlO8
90 2 xlO8 3 xlO8 IxIO8 IxIO8 3 xlO8 IxIO8
122 2 xlO8 2 xlO8 7 xlO7 7 xlO7 IxIO8 IxIO8
144 2 xlO8 2 xlO8 IxIO8 8 xlO7 8 xlO7 8 xlO7
180 2 xlO7 2 xlO7 3 xlO7 2 xlO6 3 xlO6 5 xlO6
219 IxIO6 2 xlO6 3 xlO5 8 xlO4 9 xlO5 9 xlO5
244 4 xlO3 IxIO3 IxIO3 2 xlO3 5 xlO3 6 xlO3
TABLE 7. Stored in +4°C
No. of bacteria CFU/g
Time LB21 LB21 LB21 LB21 LB21 LB21
(days) 25°C 300C 33°C 5% 25 % hot 25% cool
6 4x10" 2x10" 1x10" 2x10" 3x10" 3x10"
31 2 xlO8 2 xlO8 2 xlO8 2 xlO8 2 xlO8 2 xlO8
59 4 xlO8 2 xlO8 2 xlO8 3 xlO8 4 xlO8 3 xlO8
90 4 xlO8 2 xlO8 2 xlO8 3 xlO8 4 xlO8 2 xlO8
122 2 xlO8 2 xlO8 6 xlO7 2 xlO8 2 xlO8 IxIO8
144 2 xlO8 2 xlO8 8 xlO7 2 xlO8 2 xlO8 IxIO8
180 2 xlO8 2 xlO8 6 xlO7 IxIO8 IxIO8 IxIO8
219 2 xlO8 IxIO8 4 xlO7 IxIO8 IxIO8 IxIO8
244 4 xlO8 2 xlO8 2 xlO7 2 xlO8 4 xlO8 3 xlO7
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 107
10 2,7 x 108 1,3 x 107
21 3,8 x 108 3,O x 105
46 7,6 x 106 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 dm3. 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 +600C. 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
30 2,2 x 107 2,2 x 105
60 2,6 x 107 l,0 x 105
70 1,3 x 107 l,0 x 105
80 3,9 x 106 l,0 x 105
90 1,1 x 103 l,0 x 105 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% CO2. They were then inoculated in 3 ml MRS-broth at 37°C, 5 % CO2 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 in- cubated for 20 h at 37°C, 5% CO2. 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% CO2.
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 % CO2 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% CO2. 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 KH2PO4 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% CO2 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 109-10π CFU/ml. 5ml of NaCl 0.9 % was heated up to +800C in a water bath. As soon as the NaCl was +800C 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 IO7 2,5 x 107
0 108
4,33 x 1,O x IO4 1,4 x lO4 NVG
5 106
1,77 x 3,3 x lO4 1,4 x lO4 NVG
10 106
2,0O x 2,5 x 104 1,8 x 102 NVG
15 106
8,2O x 1,2 x 103 1,9 x 105 NVG
20 104
2,24 x 1,O x IO4 7,8 x lO4 NVG
25 105
b) +800C 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 +900C in a water bath. As soon as the NaCl was +800C 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 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 (0C) LB931 LB21
10 2,9 x 107 3,0 x 107
37 2,4 x 107 2,8 x 107
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 107 6 x 107
3 3 xlO9 9 xlO8 2 xlO9
TABLE 16
No. of bacteria CFU/g
Time (days) LB931 +4°' C LB7c +4°C LB3e +4°C
0 3xlOy 6x10" 3xlOy
3 2 xlO9 3 xlO8 9 xlO8
6 2 xlO9 2 xlO7 8 xlO8
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 xlO8 2 xlO7 6 xlO7 Ix 107
3 7 xlO9 9 xlO8 2 xlO9 8x 105
TABLE 18
No. of bacteria CFU/g stored in +4° C
Time (days) LB931 LB7c LB3e
0 3 xlO9 6x 108 3 xlO9
3 2 xlO9 3x 108 9 xlO8
6 2 xlO9 2x 107 8 xlO8
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 - 2400C 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 2300C Baked in 2300C Baked in 2400C
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 78,00C
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 +2300C 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 +2300C 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 lO2 7 x 10"
3 I x IO5 5 x lO4 I x IO4 3 x lO4
7 9 x lO4 6 x lO4 9 x lO4 Not done
TABLE 22
Baked in +2300C for 35 minutes (Rye Sourdough starter) No. of bacteria CFU/g
Time (days) LB931 LB3e LB7c
0 9 x lO2 2 x lO2 I x IO2 3 8 x lO3 3 x lO3 7 x lO3
7 9 x lO3 7 x lO3 6 x lO3
TABLE 23
Baked in +2400C for 30 minutes growth No. of bacteria CFU/g
Wheat Sourdough starter Rye Sourdough starter
Time (days) LB931 LlA LB931
0 4 x lO3 No Visible Growth 3 x lO4
(NVG)
2 6 x lO4 NVG 7 x lO4
4 2 x lO5 NVG 9 x lO5
6 I x IO5 NVG 6 x lO5
8 7 x lO4 NVG 8 x lO4
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 0C 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 lO3
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 lO4 NVG Day 15
Sourdough bread 5 x lO4 NVG Day 15
LB21 Sourdough bread 7,6 x 104 NVG Day 15
Bread with 5ml 1,2 x 103 NVG Day 15 bacterial solution
LB3e Sourdough bread 4 x lO4 NVG Day 15
LB7c Sourdough bread 6 x lO4 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 103 6 x 103 Study person 2 0 2 x 102 4 x lO3 Study person 3 0 5 x IQ7 6 x IQ3
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 (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 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
1010 CFU/day and group 2 was given placebo. Both groups were challenged with gluten following a standardized scale. The children were given O.lg gluten/day/kg 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 1010 CFU/day. The children were challenged with gluten following a standardized scale. The children were given 0.025 g gluten/day/kg bodyweight for 2 months and 0.05 g gluten/day/kg bodyweight 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.

Claims

1. Microorganisms of the Lactobacillus strain chosen from the species Lactobacillus plantarum LB3e and Lactobacillus plantarum LB7c 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 0C for more than 25 minutes.
2. Lactobacillus strain according to claim 1 for use 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.
3. 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
4. Food product such as bread, dough, cereal products, plain bread characterized in that said food 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, wherein said deposited strains are able to survive tempera- tures from +80 0C for more than 25 minutes.
5. 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, Lacto- bacillus rhamnosus LB21 which has been deposited at NCIMB Ltd, Ferguson
Building, Craibstone Estate and has been assigned accession number 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 0C for more than 25 minutes.
6. Food product according to any of the preceding claims prepared by heat treatment of a food product starting material using a heat source exposing said food product to a temperature above +80 0C characterized in that said food product also contains a viable Lactobacillus strain preferably 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) respectively.
7. Food product according to any of the preceding claims wherein the viable Lacto- bacillus strain is present in an amount above l,0 x 103 CFU/g, preferably above
1,OxIO4 CFU/g and most preferably above 1,O x IO5 CFU/g, living bacteria per gram of the food product after the heat treatment.
8. Food product according to any of the preceding claims wherein the thermostable viable Lactobacillus strain chosen from the species Lactobacillus plantarum and
Lactobacillus rhamnosus for promoting immuntolerance in autoimmune diseases such as coeliac disease.
9. Food product according to claim 7 wherein the thermostable viable Lactobacillus strain is chosen from the species Lactobacillus plantarum LB931 (DSM 11918),
Lactobacillus rhamnosus, LB21 (NCIMB 40564), Lactobacillus plantarum LB7c (DSM 17853) and Lactobacillus plantarum LB3e (DSM 17852).
10. Microorganisms of the Lactobacillus strain chosen from the species Lactobacil- lus plantarum LB3e and Lactobacillus plantarum LB7c which have been deposited at Deutsche Sammlung von Mikroorganismen und Zeelkulturen, and have been assigned accession number 17852 and 17853 respectively.
PCT/SE2007/050156 2006-03-17 2007-03-14 Thermostable lactobacillus strains WO2007108764A2 (en)

Priority Applications (7)

Application Number Priority Date Filing Date Title
EP07716125.5A EP1996696B1 (en) 2006-03-17 2007-03-14 Thermostable lactobacillus strains
US12/225,221 US8137952B2 (en) 2006-03-17 2007-03-14 Thermostable lactobacillus strains
LTEP07716125.5T LT1996696T (en) 2006-03-17 2007-03-14 Thermostable lactobacillus strains
ES07716125.5T ES2619185T3 (en) 2006-03-17 2007-03-14 Thermostable Lactobacillus strains
DK07716125.5T DK1996696T3 (en) 2006-03-17 2007-03-14 Thermostable lactobacillus strains
NO20084335A NO343526B1 (en) 2006-03-17 2008-10-16 Microorganisms of the Lactobacillus strain, their use and products containing them.
CY20171100355T CY1118795T1 (en) 2006-03-17 2017-03-20 LACTOBACILLUS HEAT STRENGTH

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
SE0600606-8 2006-03-17
SE0600606 2006-03-17

Publications (2)

Publication Number Publication Date
WO2007108764A2 true WO2007108764A2 (en) 2007-09-27
WO2007108764A3 WO2007108764A3 (en) 2008-02-21

Family

ID=38522848

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/SE2007/050156 WO2007108764A2 (en) 2006-03-17 2007-03-14 Thermostable lactobacillus strains

Country Status (11)

Country Link
US (1) US8137952B2 (en)
EP (1) EP1996696B1 (en)
CY (1) CY1118795T1 (en)
DK (1) DK1996696T3 (en)
ES (1) ES2619185T3 (en)
HU (1) HUE033028T2 (en)
LT (1) LT1996696T (en)
NO (1) NO343526B1 (en)
PL (1) PL1996696T3 (en)
PT (1) PT1996696T (en)
WO (1) WO2007108764A2 (en)

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20100254956A1 (en) * 2007-12-06 2010-10-07 Aria Foods Amba Probiotic bacteria and regulation of fat storage
CN102304480A (en) * 2011-02-16 2012-01-04 广西科学院 Lactobacillus rhamnose strain for producing L-lactic acid efficiently and method for producing L-lactic acid by fermenting cassava and sugarcane molasses
WO2014108937A1 (en) * 2013-01-11 2014-07-17 株式会社タカギ Novel lactobacillus
US8891925B2 (en) 2011-08-19 2014-11-18 Corning Incorporated Low bend loss optical fiber
CN105087452A (en) * 2015-09-30 2015-11-25 厦门和美科盛生物技术有限公司 L. plantarum HM6159 and application thereof
CN105238713A (en) * 2015-09-30 2016-01-13 厦门和美科盛生物技术有限公司 L.plantarum HM6008 (lactobacillus plantarum HM6008) and usage thereof
EP3237600A4 (en) * 2014-10-01 2018-08-01 Triphase Pharmaceuticals Pvt. Ltd. Thermo-stable strains, products and methods thereof
JP2020513780A (en) * 2017-01-12 2020-05-21 プロビ アーベー Probiotic composition and uses thereof

Families Citing this family (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
PT2211626T (en) * 2007-08-29 2019-10-01 Ganeden Biotech Inc Baked goods
ES2595733T3 (en) 2008-10-16 2017-01-03 Ganeden Biotech, Inc. Probiotic cereal-based compositions
ES2691301T3 (en) * 2010-03-12 2018-11-26 Compagnie Gervais Danone Lactic acid bacteria for celiac disease
ES2575828B1 (en) * 2014-12-31 2017-07-07 Universidad De León Use of probiotics in increasing male fertility
CN114287552A (en) * 2021-12-23 2022-04-08 西藏安琪生物科技有限公司 Lactobacillus rhamnosus separated from nadu yak milk knot and application thereof

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994000019A1 (en) 1992-06-24 1994-01-06 FæLLESFORENINGEN FOR DANMARKS BRUGSFORENINGER (FDB) Baked product containing viable microorganisms and process for preparing same

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4110477A (en) * 1977-05-13 1978-08-29 Kabushiki Kaisha Naruse Fermentation Laboratory Method for producing natto containing lactic acid bacteria
DE3335351A1 (en) 1983-09-29 1985-04-11 Diamalt AG, 8000 München Process for producing a ready-to-use, dried baking concentrate and use thereof
SE511524C2 (en) 1997-06-02 1999-10-11 Essum Ab Lactobacillus casei rhamnosus strain and pharmaceutical preparation for the control of pathogenic intestinal bacteria
SE519648C2 (en) * 1998-03-06 2003-03-25 Essum Ab New strain of Lactobacillus plantarum

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994000019A1 (en) 1992-06-24 1994-01-06 FæLLESFORENINGEN FOR DANMARKS BRUGSFORENINGER (FDB) Baked product containing viable microorganisms and process for preparing same

Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20100254956A1 (en) * 2007-12-06 2010-10-07 Aria Foods Amba Probiotic bacteria and regulation of fat storage
US9113641B2 (en) * 2007-12-06 2015-08-25 Arla Foods Amba Probiotic bacteria and regulation of fat storage
CN102304480A (en) * 2011-02-16 2012-01-04 广西科学院 Lactobacillus rhamnose strain for producing L-lactic acid efficiently and method for producing L-lactic acid by fermenting cassava and sugarcane molasses
US8891925B2 (en) 2011-08-19 2014-11-18 Corning Incorporated Low bend loss optical fiber
WO2014108937A1 (en) * 2013-01-11 2014-07-17 株式会社タカギ Novel lactobacillus
JP5988455B2 (en) * 2013-01-11 2016-09-07 株式会社タカギ New lactic acid bacteria
EP3237600A4 (en) * 2014-10-01 2018-08-01 Triphase Pharmaceuticals Pvt. Ltd. Thermo-stable strains, products and methods thereof
CN105087452A (en) * 2015-09-30 2015-11-25 厦门和美科盛生物技术有限公司 L. plantarum HM6159 and application thereof
CN105238713A (en) * 2015-09-30 2016-01-13 厦门和美科盛生物技术有限公司 L.plantarum HM6008 (lactobacillus plantarum HM6008) and usage thereof
JP2020513780A (en) * 2017-01-12 2020-05-21 プロビ アーベー Probiotic composition and uses thereof
US11166993B2 (en) 2017-01-12 2021-11-09 Probi Ab Methods for celiac disease using lactobacillus strains

Also Published As

Publication number Publication date
NO20084335L (en) 2008-10-16
CY1118795T1 (en) 2017-07-12
US8137952B2 (en) 2012-03-20
PT1996696T (en) 2017-03-17
NO343526B1 (en) 2019-04-01
US20090208606A1 (en) 2009-08-20
ES2619185T3 (en) 2017-06-23
PL1996696T3 (en) 2017-08-31
WO2007108764A3 (en) 2008-02-21
DK1996696T3 (en) 2017-03-20
EP1996696B1 (en) 2016-12-21
LT1996696T (en) 2017-03-10
EP1996696A2 (en) 2008-12-03
HUE033028T2 (en) 2017-11-28

Similar Documents

Publication Publication Date Title
EP1996696B1 (en) Thermostable lactobacillus strains
Sarkar Potentiality of probiotic yoghurt as a functional food–a review
Maragkoudakis et al. Probiotic potential of Lactobacillus strains isolated from dairy products
US6835376B1 (en) Lactobacillus paracasei strain for preventing diarrhea caused by pathogenic bacteria
Torres-Maravilla et al. Identification of novel anti-inflammatory probiotic strains isolated from pulque
JP7546829B2 (en) Probiotic Lactobacillus crispatus strains for the treatment of urogenital yeast and bacterial infections
KR20220047875A (en) Bifidobacterium brib 207-1 and uses thereof
CN110122877A (en) Lactobacillus rhamnosus and application thereof
MX2010010256A (en) Strain of lactobacillus rhamnosus.
EP1971231A1 (en) Probiotic bread and method of its production
EP1743042A1 (en) Lactic acid bacteria strains exhibiting probiotic properties and compositions comprising the same
CN105733983A (en) Isolated microorganism strains lactobacillus plantarum mcc1 dsm 23881 and lactobacillus gasseri mcc2 dsm 23882 and their use
Gregoret et al. A comprehensive approach to determine the probiotic potential of human-derived Lactobacillus for industrial use
Soares et al. Sporeforming probiotic bacteria: Characteristics, health benefits, and technological aspects for their applications in foods and beverages
JP7111699B2 (en) bacteria
Kuerman et al. Effects of prebiotic carbohydrates on the growth promotion and cholesterol-lowering abilities of compound probiotics in vitro
KR20130049554A (en) Composition for preventing or treating atopic dermatitis
Sherwani Probiotics in processed dairy products and their role in gut microbiota health
EP2742125B1 (en) New strain of l. bulgaricus capable of inhibiting the adhesion of h. pylori strains to epithelial cells
EP1332223B1 (en) Method for screening probiotic strains of the genus bifidobacterium
KR102495386B1 (en) Gluten reduced flour mixture by lactic acid bacteria, food additive utilizing it, and food comprising it, and preparation method thereof
JP2009082127A (en) New lactic acid bacterium, lactic acid fermentation product and food and drink comprising lactic acid fermentation product using the same
Saliba Safety aspects and beneficial features of lactobacilli isolated from Lebanese Baladi goat milk
GS Comparative Evaluation of the Efficacy of the Probiotic, Chlorhexidineand Fluoride Mouthwash Against Salivary Streptococcus Mutans-An in Vivo Study
MXPA01008976A (en) Lactobacillus strains preventing diarrhoea pathogenic bacteria

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 07716125

Country of ref document: EP

Kind code of ref document: A2

DPE2 Request for preliminary examination filed before expiration of 19th month from priority date (pct application filed from 20040101)
REEP Request for entry into the european phase

Ref document number: 2007716125

Country of ref document: EP

WWE Wipo information: entry into national phase

Ref document number: 2007716125

Country of ref document: EP

NENP Non-entry into the national phase

Ref country code: DE

WWE Wipo information: entry into national phase

Ref document number: 12225221

Country of ref document: US