WO2017017662A1 - Microorganism strain lactobacillus plantarum ls/07 ccm 7766, method of its use and food product - Google Patents
Microorganism strain lactobacillus plantarum ls/07 ccm 7766, method of its use and food product Download PDFInfo
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- WO2017017662A1 WO2017017662A1 PCT/IB2016/054600 IB2016054600W WO2017017662A1 WO 2017017662 A1 WO2017017662 A1 WO 2017017662A1 IB 2016054600 W IB2016054600 W IB 2016054600W WO 2017017662 A1 WO2017017662 A1 WO 2017017662A1
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- ccm
- lactobacillus plantarum
- strain lactobacillus
- microorganism strain
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- 240000006024 Lactobacillus plantarum Species 0.000 title claims abstract description 51
- 235000013965 Lactobacillus plantarum Nutrition 0.000 title claims abstract description 48
- 229940072205 lactobacillus plantarum Drugs 0.000 title claims abstract description 48
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- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 claims description 18
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- ZMMJGEGLRURXTF-UHFFFAOYSA-N ethidium bromide Chemical compound [Br-].C12=CC(N)=CC=C2C2=CC=C(N)C=C2[N+](CC)=C1C1=CC=CC=C1 ZMMJGEGLRURXTF-UHFFFAOYSA-N 0.000 description 1
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Classifications
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- 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
- 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
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
-
- 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
- A23V2400/00—Lactic or propionic acid bacteria
- A23V2400/11—Lactobacillus
- A23V2400/169—Plantarum
-
- 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
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Definitions
- Plantarum LS/07, strain number CCM 7766 which is also a mean for preparation of preparations and food additives for the purposes of preventive strengthening of the intestinal barrier.
- Strain Lactobacillus Plantarum LS/07 CCM 7766 has been cultivated from the stool of the healthy human.
- the invention also describes food products containing Lactobacillus Plantarum LS/07, CCM 7766.
- the health of the human population is one of the main attributes of the functioning of the human society. High social demands on the man bring many negative impacts on his overall health. Stress, nutritionally imbalanced diet and unhealthy habits have negative impacts on the human intestinal microflora, which can furthermore have negative impact on the overall health of the human population.
- the civilization diseases arise as a result of long-term operation of the stated factors. The treatment of the civilization diseases is long-term and financially highly costly, therefore the prevention is more promoted these days in a fight against civilization diseases. Usage of the selected probiotic strains can ensure the stability of the healthy intestinal microflora with the subsequent positive impact on the health of an individual.
- the isolated strains of lactobacilli are used on large scale as a part of the functional food or food supplements.
- Some probiotic strains used as a part of the food supplements have positive impact on the microflora of the digestive tract of a man; such strains are Lactobacillus plantarum 299 and Lactobacillus plantarum 299v DSM 9843 - both closely related strains were isolated from the intestinal mucosa of a healthy man (Molin et al. 1993 and Johanson et al. 1993).
- European patent file EP 1 904 074 discloses a use of the lactobacilli for the increase of the absorption of the ions of iron, zinc and calcium; it concerns the use of at least one specified strain of the Lactibacillus plantarum for the increase of the absorption of the ions in mammals and the pharmaceutical composition, which contains at least one specified strain of the Lactibacillus plantarum for the use during the treatment of the anemia.
- the patent discloses the use of the known, patented strains Lactobacillus plantarum DSM 695, L. plantarum 299v, DSM 9843, L. Plantarum Heal 9, DSM 153121 L. plantarum, Heal 19, DSM 15 313 for the increase of the absorption of the ions of the iron.
- Patent file WO2015067947 discloses an invention concerning the strain Lactobacillus plantarum and composition for the use during the modification of the absorption of the cholesterol in an individual, or for treating heart diseases, diabetes or obesity.
- Published file RU 2491079 C1 is Complex probiotic preparation and a method of its use.
- the preparation consists of the mixture of the lactobacilli (Lacobacillus plantarum, L. fermentum, L. acidophilus) and bifidobacteria (Bifidobacterium bifidum, B. longum). These strains were gathered from the plants.
- Published file EP 2 309 870 is called Isolated strain of Lactobacillus plantarum
- Tensia DSM 21 380 as an anti-microbial and anti-hypertensive probiotic, food product and composition containing the stated microorganism for the preparation of the antihypertensive medicament and a method for suppression of the pathogens and non- starter pathogens in the food products.
- This strain has been isolated from the stool of the healthy child as a part of the comparative study conducted on Estonian and Swedish children.
- the invention concerns a microorganism strain Lactobacillus Plantarum LS/07 deposited in the collection of microorganisms under the number CCM 77 66 In Czech collection of microorganisms Brno on 2.7.2009 according to the conditions under Budapest Treaty; the microorganism metabolizes healthful substances which differentiate it from the other microorganism strains of the genus Lactobacillus isolated from a man, animal or a plant.
- microorganism strain Lactobacillus Plantarum LS/07 has been deposited under the number CCM 77 66 In Czech collection of microorganisms at Masaryk University, Faculty of Sciences, Tvrdeho 14 (currently Kamenice 5, building A25), 602 00 (currently 625 00) Brno, Czech Republic.
- Lactobacillus plantarum LS/07 CCM 7766 belongs to a group of the lactic acid fermentation and other organic acids fermentation bacteria. The conducted enzymatic tests has shown that the strain Lactobacillus plantarum LS/07 CCM 7766 does not produce the enzyme ⁇ -glucuronidase, which is considered an important marker of the development of the carcinogenesis in man.
- the mentioned microorganism strain is applied as an independent nutritional supplement, preferably in form of the capsules or as an additive to the nutritional supplements for the purposes of the preventive strengthening of the intestinal barrier and intestinal microflora against toxic mutagens and infectious pathogens.
- Lactobacillus Plantarum LS/07 CCM 77 66 is isolated from a man and not from an animal and it is applied into functional food such as energetic cereal bars, diary or meat products, preferably to pasteurized bryndza (Slovak sheep milk cheese, also called in other languages Brimsen, Liptauer Kase, vulgo Burenda).
- the advantage of the new microorganism strain Lactobacillus Plantarum LS/07 CCM 7766, which is the essence of this invention, is the modulation effect of the used strain, which manifests itself through the increase of the number of the lactic fermentation bacteria and diminishment of the coliforms in the intestinal contents of the colon.
- the application of the strain has effect not only on intestinal microflora, but also on metabolic activities with the positive effect on the lipid profile.
- Lactobacillus strain Lactobacillus plantarum LS/07 has been characterized by the inventors in the clinical studies on the rats, whereby subsequent advantages of the strain according to this invention have been determined: Strain Lactobacillus plantarum LS/07 CCM 7766 not only not produces the enzyme ⁇ -glucuronidase which can have carcinogenetic effects, but at the same time it diminishes its activity in the intestinal content during the experimentally chemically induced acute and chronic colitis in the laboratory rats. Histological results have shown that in the groups of animals where the strain
- Lactobacillus plantarum LS/07 has been applied, there have been significant diminishment and attenuation of the colitis in the fixed tissue of the rats' colon as compared to the control group where the strain has not been applied.
- Example 1 In this example of the realization of the invention the strain is in form of a biologically pure culture.
- the stated microorganism strain is applied as an independent nutritional supplement, preferably in form of capsules or as a an additive to the nutritional supplements for the purposes of the preventive strengthening of the intestinal barrier and intestinal microflora against toxic mutagens and infectious pathogens.
- the stated strain Lactobacillus Plantarum LS/07 CCM 77 66 is applied into food products such as energetic cereal bars, diary or meat products, preferably to pasteurized bryndza (Slovak sheep milk cheese).
- Bryndza also called in other languages Brimsen, Liptauer Kase, vulgo Burenda
- Bryndza includes many probiotics, it lowers the cholesterol and lowers the amount of sugar in the blood as well as inflammation parameters.
- pasteurization weakens many of these effects.
- the addition of the Lactobacillus Plantarum LS/07 CCM 7766 into bryndza after the end of the pasteurization achieves the activity of the lactic acid fermentation bacteria while bryndza's keeping quality is preserved.
- microorganism strain Lactobacillus Plantarum LS/07 CCM 6677 has been cultivated by the following procedure.
- a rectal swab from the man's rectum is realized by means of a sterile removal kit.
- the removal swob is dipped into the supplied tube with physiological saline in the volume of 5 ml.
- a sample with the volume of 1 ml is removed from the supplied tube by the means of the pipette and it is transferred by the pipette to the tube with the physiological saline in the volume of 9 ml.
- the sample is diluted ten-fold. This procedure proceeds until the dilution of the sample reaches 10-2 to 10-5 degrees of dilution.
- Cultivation of the lactobacilli is realized at the temperature of 37 °C for 48 hours. After the 48 hour incubation the size and shape of the grown colonies is assessed, whereby the undergrowth is formed by small, round, low convex, white colonies up to size of 2 mm. Isolation of the lactobacillus strain is realized according to the following method:
- Inoculation soils, consumables and tools used for isolation of the strain are sterile. All actions concerning the work the microbial culture are conducted in the sterile inoculation box.
- Detection of the lactobacilli by the light microscope Light microscope Olympus with 1000 times magnification is used in order to assess the morphology of the lactobacilli. Lactobacilli are perceived as blue gram- positive (G+) coloring moderately strong and moderately long non-moving rod shaped microorganisms.
- the prepared broth culture of the strain Lactobacillus plantarurn LS/07 is incubated for approx. 18 hours and its turbidimetric value is measured to 0,5 McFarland degree, which corresponds to the value of 1 ,5x10 8 microorganisms.
- the broth culture in volume 0,050 ml is removed from the overall broth culture by micro- pipette and applied into the hole of 4 mm (cut into the middle of the plate) of the selective LS agar.
- Inoculated Petri dishes are placed into the incubation trays (GASPAK patent USA) and incubated in the biological thermostat at the temperature 37 °C for 48 hours.
- PCR polymerase chain reaction
- Concentrations of the organic acids are determined by means of a machine (CS ISOTACHOPHORETIC ANALYSER) by the technique of the capillary isotachophoresis.
- Isolated strain Lactobacillus plantarum LS/07 CCM 7766 produces mostly the lactic acid as compared to the acetic acid. The values are measured after the 24 hour (lactic acid - 78,66 mmol/l, acetic acid - 7,91 mmol/l) and 48 hour (lactic acid - 226,31 mmol/l, acetic acid - 95,44 mmol/l) incubation in the thermostat at the temperature of 37 °C.
- CCM 7766 a commercial kit API ZYM (BIOMERIEUX®SA, France) is used. It is obvious from the achieved results that the tested strain does not produce ⁇ - glucuronidase, which is considered an important marker in the process of creation of carcinogenesis of the colorectal carcinoma.
- Isolated strain Lactobacillus plantarum LS/07 CCM 7766 is used in in vivo experimental studies on the laboratory rats. Experimental work on the laboratory rats is greatly significant in the study of the effects of the isolated new strains of the lactobacilli in in vivo conditions.
- Strain Lactobacillus plantarum LS/07 CCM 7766 isolated from the stool of the healthy human, is used in the experiment with the laboratory rats. Rats used in the experiment (51 rats) were four months old Sprague Dawley rats. We have prepared the fresh strain daily in the growth broth for the lactobacilli (average concentration 2,88 x 109 CFU/ml) and supplied it to the rats daily five times a week.
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Abstract
Invention concerns a microorganism strain Lactobacillus Plantarum LS/07 CCM 7766 and a method of its use for the purposes of adjustment and stabilization of healthy intestinal microflora of a man. Microorganism strain Lactobacillus Plantarum LS/07 CCM 7766 is isolated from a man and it is applied in the food products such as energetic cereal bars, diary and meat products and, preferably, pasteurized bryndza. The advantage of a new microorganism strain Lactobacillus Plantarum LS/07 CCM 7766 is the modulation effect of the used strain, which is manifested by the increase of the lactic fermentation bacteria and decrease of coliforms in the intestinal content of the colon. Application of the strain has effect not only on the intestinal microflora, but also on the metabolic activities with the positive effect on lipid profile. Strain Lactobacillus plantarum LS/07 CCM 7766 does not produce β-glucurinodase enzyme which can have carcinogenic effects, but it also decreases this enzyme's activity in the intestinal content, mainly during the acute or chronic inflammation of the colon.
Description
Microorganism strain Lactobacillus Plantarum LS/07 CCM 7766, method of its use and food product
Field of technology The invention concerns an isolated microorganism strain Lactobacillus
Plantarum LS/07, strain number CCM 7766, which is also a mean for preparation of preparations and food additives for the purposes of preventive strengthening of the intestinal barrier. Strain Lactobacillus Plantarum LS/07 CCM 7766 has been cultivated from the stool of the healthy human. The invention also describes food products containing Lactobacillus Plantarum LS/07, CCM 7766.
Prior state of the art
The health of the human population is one of the main attributes of the functioning of the human society. High social demands on the man bring many negative impacts on his overall health. Stress, nutritionally imbalanced diet and unhealthy habits have negative impacts on the human intestinal microflora, which can furthermore have negative impact on the overall health of the human population. The civilization diseases arise as a result of long-term operation of the stated factors. The treatment of the civilization diseases is long-term and financially highly costly, therefore the prevention is more promoted these days in a fight against civilization diseases. Usage of the selected probiotic strains can ensure the stability of the healthy intestinal microflora with the subsequent positive impact on the health of an individual.
In present, the isolated strains of lactobacilli are used on large scale as a part of the functional food or food supplements. Some probiotic strains used as a part of the food supplements have positive impact on the microflora of the digestive tract of a man; such strains are Lactobacillus plantarum 299 and Lactobacillus plantarum 299v DSM 9843 - both closely related strains were isolated from the intestinal mucosa of a healthy man (Molin et al. 1993 and Johanson et al. 1993). European patent file EP 1 904 074 discloses a use of the lactobacilli for the increase of the absorption of the ions of iron, zinc and calcium; it concerns the use of at least one specified strain of the Lactibacillus plantarum for the increase of the
absorption of the ions in mammals and the pharmaceutical composition, which contains at least one specified strain of the Lactibacillus plantarum for the use during the treatment of the anemia. The patent discloses the use of the known, patented strains Lactobacillus plantarum DSM 695, L. plantarum 299v, DSM 9843, L. Plantarum Heal 9, DSM 153121 L. plantarum, Heal 19, DSM 15 313 for the increase of the absorption of the ions of the iron.
Patent file WO2015067947 discloses an invention concerning the strain Lactobacillus plantarum and composition for the use during the modification of the absorption of the cholesterol in an individual, or for treating heart diseases, diabetes or obesity.
Published file RU 2491079 C1 is Complex probiotic preparation and a method of its use. The preparation consists of the mixture of the lactobacilli (Lacobacillus plantarum, L. fermentum, L. acidophilus) and bifidobacteria (Bifidobacterium bifidum, B. longum). These strains were gathered from the plants. Published file EP 2 309 870 is called Isolated strain of Lactobacillus plantarum
Tensia DSM 21 380 as an anti-microbial and anti-hypertensive probiotic, food product and composition containing the stated microorganism for the preparation of the antihypertensive medicament and a method for suppression of the pathogens and non- starter pathogens in the food products. This strain has been isolated from the stool of the healthy child as a part of the comparative study conducted on Estonian and Swedish children.
Essence of the invention
The invention concerns a microorganism strain Lactobacillus Plantarum LS/07 deposited in the collection of microorganisms under the number CCM 77 66 In Czech collection of microorganisms Brno on 2.7.2009 according to the conditions under Budapest Treaty; the microorganism metabolizes healthful substances which differentiate it from the other microorganism strains of the genus Lactobacillus isolated from a man, animal or a plant. The microorganism strain Lactobacillus Plantarum LS/07 has been deposited under the number CCM 77 66 In Czech collection of microorganisms at Masaryk University, Faculty of Sciences, Tvrdeho 14
(currently Kamenice 5, building A25), 602 00 (currently 625 00) Brno, Czech Republic.
In laboratory tests, the new isolated strain according to this invention has shown high ability of the antibacterial activity against the strain Escherichia coli (CNTC Eck 63/59 deposited in Czech collection of microorganisms Prague). Lactobacillus plantarum LS/07 CCM 7766 belongs to a group of the lactic acid fermentation and other organic acids fermentation bacteria. The conducted enzymatic tests has shown that the strain Lactobacillus plantarum LS/07 CCM 7766 does not produce the enzyme β-glucuronidase, which is considered an important marker of the development of the carcinogenesis in man.
The mentioned microorganism strain is applied as an independent nutritional supplement, preferably in form of the capsules or as an additive to the nutritional supplements for the purposes of the preventive strengthening of the intestinal barrier and intestinal microflora against toxic mutagens and infectious pathogens. It is a variation of the invention that Lactobacillus Plantarum LS/07 CCM 77 66 is isolated from a man and not from an animal and it is applied into functional food such as energetic cereal bars, diary or meat products, preferably to pasteurized bryndza (Slovak sheep milk cheese, also called in other languages Brimsen, Liptauer Kase, vulgo Burenda). In case of use of the Lactobacillus Plantarum LS/07 CCM 7766 in pasteurized bryndza, the desired probiotic effect of this functional food is reinstated, since pasteurized bryndza - through the process of pasteurization - looses the live microflora of the lactobacilli originating in animals.
The advantage of the new microorganism strain Lactobacillus Plantarum LS/07 CCM 7766, which is the essence of this invention, is the modulation effect of the used strain, which manifests itself through the increase of the number of the lactic fermentation bacteria and diminishment of the coliforms in the intestinal contents of the colon. The application of the strain has effect not only on intestinal microflora, but also on metabolic activities with the positive effect on the lipid profile.
Lactobacillus strain Lactobacillus plantarum LS/07 has been characterized by the inventors in the clinical studies on the rats, whereby subsequent advantages of the strain according to this invention have been determined:
Strain Lactobacillus plantarum LS/07 CCM 7766 not only not produces the enzyme β-glucuronidase which can have carcinogenetic effects, but at the same time it diminishes its activity in the intestinal content during the experimentally chemically induced acute and chronic colitis in the laboratory rats. Histological results have shown that in the groups of animals where the strain
Lactobacillus plantarum LS/07 has been applied, there have been significant diminishment and attenuation of the colitis in the fixed tissue of the rats' colon as compared to the control group where the strain has not been applied.
By application of the strain Lactobacillus plantarum LS/07 CCM 7766 a significant inhibition (P<0,05) of the pro-inflammatory cytokines IL-2, IL-17 and TNF-a has been achieved, as well as moderate increase of the anti-inflammatory regulatory cytokines IL-10 and TGF-β as compared to positive control group of the animals.
In the groups of animals where Lactobacillus plantarum LS/07 CCM 7766 has been applied, a significantly lower population of the gram-negative coliforms (4,96 ± 0,32 CFU/g) as compared to the control group (5,45 ± 0, 1 1 CFU/g) has been found.
Examples of realization
Example 1 In this example of the realization of the invention the strain is in form of a biologically pure culture.
Example 2
The stated microorganism strain is applied as an independent nutritional supplement, preferably in form of capsules or as a an additive to the nutritional supplements for the purposes of the preventive strengthening of the intestinal barrier and intestinal microflora against toxic mutagens and infectious pathogens.
Example 3
The stated strain Lactobacillus Plantarum LS/07 CCM 77 66 is applied into food products such as energetic cereal bars, diary or meat products, preferably to
pasteurized bryndza (Slovak sheep milk cheese). Bryndza (also called in other languages Brimsen, Liptauer Kase, vulgo Burenda) is soft, kneaded and salted sheep cheese product produced from the lump sheep cheese. Bryndza includes many probiotics, it lowers the cholesterol and lowers the amount of sugar in the blood as well as inflammation parameters. However, pasteurization weakens many of these effects. The addition of the Lactobacillus Plantarum LS/07 CCM 7766 into bryndza after the end of the pasteurization achieves the activity of the lactic acid fermentation bacteria while bryndza's keeping quality is preserved.
Example 4
In this example the microorganism strain Lactobacillus Plantarum LS/07 CCM 6677 has been cultivated by the following procedure.
Working procedure of the preparation of the strain: A rectal swab from the man's rectum is realized by means of a sterile removal kit. The removal swob is dipped into the supplied tube with physiological saline in the volume of 5 ml. A sample with the volume of 1 ml is removed from the supplied tube by the means of the pipette and it is transferred by the pipette to the tube with the physiological saline in the volume of 9 ml. As a result, the sample is diluted ten-fold. This procedure proceeds until the dilution of the sample reaches 10-2 to 10-5 degrees of dilution. 0, 1 ml of samples are removed from the tubes by individual dilutions and they are applied to the Petri dishes with the selective soil for the lactobacilli and for the growth of the lactic acid fermentation bacteria. The inoculated plates are placed in the special incubation trays where there is a producer ensuring the anaerobic cultivation environment. Hermetically sealed containers are stored in a biological thermostat.
Cultivation of the lactobacilli is realized at the temperature of 37 °C for 48 hours. After the 48 hour incubation the size and shape of the grown colonies is assessed, whereby the undergrowth is formed by small, round, low convex, white colonies up to size of 2 mm.
Isolation of the lactobacillus strain is realized according to the following method:
One colony of the bacteria is carefully (under the magnifying glass) removed from the Petri dish by the inoculation loop and it is inoculated to the tube with the lactobacillo-selective LS broth in the amount of 5 ml. The procedure is repeated multiple times, whereby each removed colony is inoculated to the independent tube with the broth. The inoculated colonies are incubated in the thermostat at the temperature 37 °C for 18 hours. After the incubation the grown broth culture in amount of 0, 1 ml is removed from each tube and inoculated on the Petri dish containing selective LS agar. Plates are then cultivated in the thermostat at anaerobic conditions at the temperature of 37 °C for 48 hours. After the incubation, the assessment of the colonies of the lactobacilli takes place. The marked colonies are removed for the purposes of assessment of their morphology by microscopically coloring technique Grama. The coloring is done according to the following scheme
Placement of the culture on the slide Drying and fixation above flame Application
I
30 seconds of crystallization
I
Rinse by distilled water Application 30 seconds of Lugol solution
I
Rinse by distilled water Application
20 seconds of 96% ethanol
I
Rinse by distilled water Application 30 seconds of carbolfuchsin
I
Rinse by distilled water Drying by the filter paper
Inoculation soils, consumables and tools used for isolation of the strain are sterile. All actions concerning the work the microbial culture are conducted in the sterile inoculation box.
Detection of the lactobacilli by the light microscope: Light microscope Olympus with 1000 times magnification is used in order to assess the morphology of the lactobacilli. Lactobacilli are perceived as blue gram- positive (G+) coloring moderately strong and moderately long non-moving rod shaped microorganisms.
Detection of the inhibition zones of the strain Lactobacillus plantarurn LS/07
CCM 7766 against pathogenic Escherichia coli CNTC Eck 63/59 by agar diffuse test:
The prepared broth culture of the strain Lactobacillus plantarurn LS/07 is incubated for approx. 18 hours and its turbidimetric value is measured to 0,5 McFarland degree, which corresponds to the value of 1 ,5x108 microorganisms. The broth culture in volume 0,050 ml is removed from the overall broth culture by micro- pipette and applied into the hole of 4 mm (cut into the middle of the plate) of the selective LS agar. Inoculated Petri dishes are placed into the incubation trays (GASPAK patent USA) and incubated in the biological thermostat at the temperature 37 °C for 48 hours. Agar composed of 0,5 g of peptone, 1 g of yeast autolysate, 1 g of glucose, 1 ,8 g of agar, and 100 ml of distilled water with pH 6,9 in volume of 3 ml
containing night culture of the grown strain Escherichia coli serotype 06:K2:H1 ATC 1938 CNTC Eck 63/59, is layered onto the surface of the agar plates after 48 hours. After the application of the strain, the Petri dishes are inserted to the thermostat and incubated at the temperature of 37 °C for next 18 hours. After the incubation the size of the inhibition zones is measured, whereby the average result gathered by the sum of the measured values in the individual measurements divided by the amount of the tested plates. In the example of realization the deducted values of the measurement form three plates are used.
Molecule-genetic detection of the strain Lactobacillus plantarum LS/07 CCM
7766:
For the molecule-genetic detection of the isolated strain a highly sensitive and specific method of the polymerase chain reaction (PCR) technique is used. PCR mixture, the specific primers used, the amplification of template and electrophoresis in agarose gel are realized according to sources in literature (Berthier and Ehrlich 1998). Amplification PCR products are colored by ethidium bromide and detected by ultraviolet light, whereby the specific PCR product at size of 265 base pairs is thereby gathered.
Determination of organic acids:
Concentrations of the organic acids (lactic acid and acetic acid) are determined by means of a machine (CS ISOTACHOPHORETIC ANALYSER) by the technique of the capillary isotachophoresis. Isolated strain Lactobacillus plantarum LS/07 CCM 7766 produces mostly the lactic acid as compared to the acetic acid. The values are measured after the 24 hour (lactic acid - 78,66 mmol/l, acetic acid - 7,91 mmol/l) and 48 hour (lactic acid - 226,31 mmol/l, acetic acid - 95,44 mmol/l) incubation in the thermostat at the temperature of 37 °C.
Determination of the enzyme activity For the study of the enzyme activity of the strain Lactobacillus plantarum LS/07
CCM 7766 a commercial kit API ZYM (BIOMERIEUX®SA, France) is used. It is
obvious from the achieved results that the tested strain does not produce β- glucuronidase, which is considered an important marker in the process of creation of carcinogenesis of the colorectal carcinoma.
Experiments on animal models
Isolated strain Lactobacillus plantarum LS/07 CCM 7766 is used in in vivo experimental studies on the laboratory rats. Experimental work on the laboratory rats is greatly significant in the study of the effects of the isolated new strains of the lactobacilli in in vivo conditions. Strain Lactobacillus plantarum LS/07 CCM 7766, isolated from the stool of the healthy human, is used in the experiment with the laboratory rats. Rats used in the experiment (51 rats) were four months old Sprague Dawley rats. We have prepared the fresh strain daily in the growth broth for the lactobacilli (average concentration 2,88 x 109 CFU/ml) and supplied it to the rats daily five times a week. Once a week (third to seventh week) we have applied chemical pro-carcinogen 1 ,2 dimethylhydrazine-dihydrochloride (1 ,2 DMH) to the rats by injection. During the whole course of the experiment (24 weeks) there has been no occurrence of clinical changes (diarrhea, death) in case of the tested group of the animals. This has confirmed that the strain is safe. The achieved results of the experiment confirm that the modulation effect of the used strain has been manifested by the increase in the number of lactic acid bacteria and decrease in the number of the coliforms in the intestine contents of the colon. The application of the strain had influence not only on the intestine microflora, but also on the metabolic activities with the positive effect on the lipid profile. The positive changes were measured in the immunity and histological laboratory examination of the tested animals.
Industrial applicability
Industrial applicability is obvious. According to this invention it is possible to industrially and repeatedly produce Lactobacillus Plantarum LS/07 CCM 7766. The abovementioned effects of the strain Lactobacillus Plantarum LS/07 CCM 7766 determine it for the use for the modulation of the intestine microflora as well as for the prevention of the inflammatory chronic diseases of humans.
Claims
1. A microorganism strain Lactobacillus Plantarum LS/07 CCM 7766, isolated from a human, which is deposited in the Czech collection of microorganisms at the Faculty of Science of the Masaryk University, Brno, Czech Republic.
2. A method of use of the microorganism strain Lactobacillus Plantarum LS/07 CCM 7766 according to the claim l i s c h a r a c t e r i z e d b y t h e f a c t , that the strain is used for a modulation of an intestinal microflora and for a prevention of inflammatory chronic diseases of humans.
3. The method of use of the microorganism strain Lactobacillus Plantarum LS/07 CCM 7766 according to the claim l i s c h a r a c t e r i z e d b y t h e f a c t , that it is used during a production of an independent nutritional supplement, preferably in form of capsules.
4. The method of use of the microorganism strain Lactobacillus Plantarum LS/07 CCM 7766 according to the claim l i s c h a r a c t e r i z e d b y t h e f a c t , that the strain is applied to food such as energetic cereal bars, diary and meat products or, preferably, to a pasteurized bryndza.
5. The lactic acid producing microorganism strain Lactobacillus Plantarum LS/07 CCM 7766 according to the claim l i s c h a r a c t e r i z e d b y t h e f a c t , that it is anibacterially active against a pathogenic strain Escherichia coli.
6. Microorganism strain Lactobacillus Plantarum LS/07 CCM 7766 according to the claim 5 i s c h a r a c t e r i z e d b y t h e f a c t , that it metabolizes healthy substances without production of β-glucuronidase.
7. Microorganism strain Lactobacillus Plantarum LS/07 CCM 7766 according to the claim 5 or 6 i s c h a r a c t e r i z e d b y t h e f a c t , that it lowers an activity of an enzyme of the β-glucuronidase in an intestinal content, mainly during acute and/or chronic inflammation of a colon.
8. Microorganism strain Lactobacillus Plantarum LS/07 CCM 7766 according to any of the claims 5to7 i s c h a r a c t e r i z e d b y t h e f a c t , that the number of lactic fermentation bacteria increases and at the same time the
number of coliforms in the intestinal content of the colon decreases with a positive effect on a lipid profile.
9. Microorganism strain Lactobacillus Plantarum LS/07 CCM 7766 according to any of the claims 5to8 i s c h a r a c t e r i z e d b y t h e f a c t , that it inhibits a pro-inflammatory cytokines IL-2, IL-17 and TNF-a.
10. Microorganism strain Lactobacillus Plantarum LS/07 CCM 7766 according to any of the claims 5to9 i s c h a r a c t e r i z e d b y t h e f a c t , that it supports a growth of an anti-inflammatory regulatory cytokines IL-10 and TGF-β.
11. KMicroorganism strain Lactobacillus Plantarum LS/07 CCM 7766 according to any of the claims 1 , 5 to 10 in a dried form.
12. Microorganism strain Lactobacillus Plantarum LS/07 CCM 7766 according claim 11 in a form of lyophylisate.
13. A food product containing the microorganism strain Lactobacillus Plantarum LS/07 CCM 7766 according to any of the claims 1 , 5 to 12.
14. The food product according to the claim 13 i s c h a r a c t e r i z e d b y t h e f a c t , that it is a fermented diary product.
15. The food product according to the claim 14 i s c h a r a c t e r i z e d b y t h e f a c t , that it is a cheese.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| SK50011-2018A SK500112018A3 (en) | 2015-07-30 | 2016-07-30 | Microorganism strains Lactobacillus Plantarum LS/07 CCM 7766, product containing this strains |
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| SKPP54-2015 | 2015-07-30 | ||
| SK54-2015A SK542015A3 (en) | 2015-07-30 | 2015-07-30 | Strains Lactobacillus Plantarum LS/07 CCM 7766 and their use |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| WO2017017662A1 true WO2017017662A1 (en) | 2017-02-02 |
| WO2017017662A4 WO2017017662A4 (en) | 2017-03-16 |
Family
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/IB2016/054600 WO2017017662A1 (en) | 2015-07-30 | 2016-07-30 | Microorganism strain lactobacillus plantarum ls/07 ccm 7766, method of its use and food product |
Country Status (3)
| Country | Link |
|---|---|
| CZ (2) | CZ2018101A3 (en) |
| SK (2) | SK542015A3 (en) |
| WO (1) | WO2017017662A1 (en) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1195095A2 (en) * | 2000-10-05 | 2002-04-10 | St. Ivel Limited | Food products with antimicrobial lactic acid bacteria |
| EP1997499A1 (en) * | 2007-05-31 | 2008-12-03 | Puleva Biotech, S.A. | Mammalian milk microorganisms, compositions containing them and their use for the treatment of mastitis |
| EP2455450A1 (en) * | 2009-07-14 | 2012-05-23 | CJ CheilJedang Corporation | Novel lactobacillus plantarum and composition containing same |
-
2015
- 2015-07-30 SK SK54-2015A patent/SK542015A3/en not_active Application Discontinuation
-
2016
- 2016-07-30 CZ CZ2018-101A patent/CZ2018101A3/en unknown
- 2016-07-30 CZ CZ2018-34733U patent/CZ33363U1/en not_active IP Right Cessation
- 2016-07-30 SK SK50011-2018A patent/SK500112018A3/en not_active Application Discontinuation
- 2016-07-30 WO PCT/IB2016/054600 patent/WO2017017662A1/en active Application Filing
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1195095A2 (en) * | 2000-10-05 | 2002-04-10 | St. Ivel Limited | Food products with antimicrobial lactic acid bacteria |
| EP1997499A1 (en) * | 2007-05-31 | 2008-12-03 | Puleva Biotech, S.A. | Mammalian milk microorganisms, compositions containing them and their use for the treatment of mastitis |
| EP2455450A1 (en) * | 2009-07-14 | 2012-05-23 | CJ CheilJedang Corporation | Novel lactobacillus plantarum and composition containing same |
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Also Published As
| Publication number | Publication date |
|---|---|
| SK500112018A3 (en) | 2018-12-03 |
| SK542015A3 (en) | 2017-02-02 |
| WO2017017662A4 (en) | 2017-03-16 |
| CZ2018101A3 (en) | 2018-10-31 |
| CZ33363U1 (en) | 2019-11-12 |
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