CZ33363U1 - A food product or nutritional supplement containing the Lactobacillus plantarum strain - Google Patents

Description

Food product or nutritional supplement containing a strain of the micro-organism Lactobacillus plantarum

Field of technology

The technical solution relates to a preparation containing an isolated strain of microorganisms Lactobacillus plantarum LS / 07, collection number CCM 7766, which is also a means for the preparation of preparations and food additives for the purpose of preventive strengthening of the intestinal barrier. The Lactobacillus plantarum strain LS / 07 CCM 7766 was cultured from the faeces of a healthy human.

Prior art

The health of the human population is one of the main attributes of the functioning of human society. The high social demands placed on a person bring many negative effects on his overall health. Stress, nutritionally unbalanced food and unhealthy habits have an adverse effect on the intestinal microflora of humans, which can later have a negative effect on the overall health of the human population. Diseases of civilization arise as a result of the long-term effects of these factors. The treatment of diseases of civilization is long-term and financially highly expensive, which is why prevention in the fight against diseases of civilization is currently gaining ground. The use of selected probiotic strains can greatly ensure the stability of a healthy intestinal microflora with a consequent positive effect on the health of the individual.

Currently, isolated strains of lactobacilli are widely used as part of functional foods or nutritional supplements. Some probiotic strains used as part of nutritional supplements have a beneficial effect on the microflora of the human digestive tract, such strains are Lactobacillus plantarum 299 and Lactobacillus plantarum 299 in DSM 9843, these closely related strains have been isolated from the intestinal mucosa of healthy humans (Molin et al. 1993 and Johanson et al. col. 1993).

European patent specification EP 1904074 discloses the use of lactobacilli to increase the absorption of iron, zinc and calcium ions, relates to the use of at least one specified strain of Lactobacillus plantarum to increase the absorption of metal ions in mammals and a pharmaceutical composition comprising at least one specified strain of Lactobacillus plantarum for use in treatment of anemia. The patent discloses the use of already known patented strains of Lactobacillus plantarum DSM 695, L. plantarum 299v, DSM 9843, L. plantarum Heal 9, DSM 153121, L. plantarum, Heal 19, DSM 15 313, to increase the absorption of iron ions.

WO 2015/067947 discloses an invention relating to a strain of Lactobacillus plantarum and a composition for use in modifying cholesterol absorption in an individual, or in the treatment of heart disease, diabetes or obesity.

Published document RU 2491079 C1 describes a complex probiotic preparation and a method of its use. The preparation consists of a mixture of lactobacilli (Lacobacillus plantarum, L. fermentum, L. acidophilus) and bifidobacteria (Bifidobacterium bifidum, B. longum). These strains were obtained from plants.

EP 2309870 discloses an isolated strain of Lactobacillus plantarum Tensia DSM 21 380 as an antimicrobial and antihypertensive probiotic, a food product. It further describes a composition comprising said microorganism for the preparation of an antihypertensive drug and a method for controlling pathogens and non-starter pathogens in food products. This strain was isolated from the faeces of a healthy child in a comparative study performed in Estonian and Swedish children.

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The essence of the technical solution

The technical solution concerns a preparation containing a strain of microorganisms Lactobacillus Plantarum LS / 07 deposited in the collection of microorganisms under serial number CCM 7766 in the Czech Collection of Microorganisms Brno on 2.7.2009 under the terms of the Budapest Treaty, which metabolizes healthy substances that distinguish it from other strains of microorganisms of the genus Lactobacillus isolated from humans, animals and plants. The microorganism strain Lactobacillus plantarum LS / 07 was deposited under the number CCM 7766 in the Czech Collection of Microorganisms, Masaryk University, Faculty of Science, Tvrdého 14 (currently Kamenice 5, building A25), 602 00 (currently 625 00) Brno, Czech Republic.

The new isolated strain, according to the technical solution, proved in laboratory tests a high ability of antibacterial activity against the pathogenic strain Escherichia coli (CNTC Eck 63/59 deposited in the Czechoslovak Collection of Microorganisms Prague). Lactobacillus plantarum LS / 07 CCM 7766 belongs to the group of lactic acid bacteria producing lactic acid and other organic acids. The enzymatic tests performed showed that the Lactobacillus plantarum strain LS / 07 CCM 7766 does not produce the enzyme β-glucuronidase, which is considered to be an important marker of carcinogenesis in humans.

Said strain of microorganisms is applied as a separate nutritional supplement, preferably in the form of capsules or as an addition to nutritional supplements for the purpose of preventively strengthening the intestinal barrier and intestinal microflora against toxic mutagens and infectious pathogens.

A variant of the technical solution is that Lactobacillus plantarum LS / 07 CCM 7766 isolated from humans and not from animals is applied to functional foods such as energy cereal bars, fortified beverages, dairy and meat products, preferably pasteurized bryndza. When Lactobacillus plantarum LS / 07 CCM 7766 is used in pasteurized bryndza, the desired probiotic effect of a functional food is restored, which is pasteurized free of living microflora of lactobacilli from animals.

The advantage of the new strain of microorganisms Lactobacillus plantarum LS / 07 CCM 7766, according to the essence of the technical solution, is the modulating effect of the used strain, which is manifested by increasing the number of lactic acid bacteria and reducing the number of coliform microorganisms in the intestinal contents. Application of the strain affects not only the intestinal microflora, but also metabolic activities with a beneficial effect on the lipid profile.

The lactobacillus strain Lactobacillus plantarum LS / 07 was characterized by the inventors in clinical studies in rats, and the following advantages of the strain according to the invention were found:

The Lactobacillus plantarum strain LS / 07 CCM 7766 not only does not produce the enzyme β - glucuronidase, which may have carcinogenic effects, but also reduces its activity in the intestinal contents in experimentally chemically induced acute and chronic inflammation of the colon in rats.

Histological results showed that in the groups of animals treated with Lactobacillus plantarum LS / 07 strains, there was a significant reduction and suppression of inflammation in the fixed tissue of the rat colon compared to the control group where the strain was not administered.

Application of Lactobacillus plantarum strain LS / 07 CCM 7766 resulted in a significant inhibition (P <0.05) of the proinflammatory cytokines IL-2, IL-17 and TNF-α, and a slight increase in the anti-inflammatory regulatory cytokines IL-10 and TGF-β compared to the positive control. group of animals.

In the groups of animals treated with Lactobacillus plantarum LS / 07 CCM 7766, a significantly reduced population of gram-negative coliform bacteria (4.96 ± 0.32 CFU / g) was found in

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Examples of technical solution

Example 1

In this exemplary embodiment, the strain is in the form of a biologically pure culture.

Example 2

Said strain of microorganisms is applied as a separate nutritional supplement, preferably in the form of capsules or as an addition to nutritional supplements for the purpose of preventively strengthening the intestinal barrier and intestinal microflora against toxic mutagens and infectious pathogens.

Example 3

Said strain of Lactobacillus plantarum LS / 07 CCM 7766 is applied to foods such as energy cereal bars, fortified beverages, dairy and meat products or pasteurized brynza. Brynza (also called Brimsen, Liptauer Kase, vulgo Burenda) is a soft, kneaded and salted sheep's cheese product made from lump sheep's cheese. Brynza contains many probiotics, lowers cholesterol, lowers blood sugar and inflammatory parameters. However, pasteurization significantly attenuates many of these effects. By adding the Lactobacillus plantarum strain LS / 07 CCM 7766 to the bryndza after pasteurization, the activity of lactic acid bacteria is achieved while maintaining its durability.

Example 4

In this example, a microorganism strain of the genus Lactobacillus plantarum LS / 07 CCM 6677 was cultured according to the following procedure.

Strain preparation procedure:

A swab is transferred from the human rectum using a sterile collection kit. The collection swab is immersed in a 5 ml saline storage tube. A 1 ml sample is taken from the stock tube using a pipette and transferred by pipette to a 9 ml saline tube. The dilution results in a 10-fold dilution of the sample. This is followed by continuing this procedure until the sample is diluted from 10 ^-2 to a dilution step of 10 ^-5 . 0.1 ml samples are taken from each dilution tube and applied to lactobacilli-selective Petri dishes for lactic acid bacteria growth. The inoculated plates are stored in special incubation vessels in which there is a developer providing an anaerobic culture medium. Airtight containers are stored in a biological thermostat.

The lactobacilli are cultured at 37 ° C for 48 hours. After 48 hours of incubation, the size and shape of the overgrown colonies are assessed, with the stand consisting of small, round, low-convex white colonies up to 2 mm in size.

Isolation of the lactobacillus strain is performed according to the procedure:

Carefully (under a magnifying glass) one colony of bacteria is removed from the petri dish with a sterile inoculum and inoculated into a test tube with lactobacillus-selective LS broth in an amount of 5 ml. This procedure is repeated several times, with each collected colony being inoculated into a separate broth tube. Inoculated colonies are incubated in a thermostat at 37 ° C for 18 hours.

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After incubation, a 0.1 ml growth broth culture is removed from each tube and inoculated onto Petri dishes containing selective LS agar. The plates are then cultured in a thermostat under anaerobic conditions at 37 ° C for 48 hours. After incubation, lactobacillus colonies are assessed. Labeled colonies are picked for their morphology by microscopic Gram staining. Staining is performed as follows according to the scheme:

Deployment of the culture on a slide

Drying and fixation over a flame

Application

Φ

Crystal violet - 30 sec

Φ

Rinse with distilled water

Application

Lugol's solution - 30 seconds

Φ

Rinse with distilled water

Application

Ethanol 96% - 20 seconds ¹

Φ

Rinse with distilled water

Application

Carbolfuchsin - 30 seconds

Φ

Rinse with distilled water Dry with filter paper

Vaccine media, consumables and tools used to isolate the strain are sterile. All operations related to work with microbial culture are performed in a sterile vaccination box.

Detection of lactobacilli by light microscope:

An Olympus light microscope with 1000x magnification is used to assess the morphology of lactobacilli. Lactobacilli are observed as blue gram-positive (G +) staining with medium-strength and medium-length immobile rod-shaped microorganisms.

Detection of inhibition zones of Lactobacillus plantarum strain LS / 07 CCM 7766 against pathogenic Escherichia coli CNTC Eck 63/59 agar by diffusion test:

The prepared broth culture of Lactobacillus plantarum LS / 07 is incubated for approximately 18 hours and its turbidimetric value is measured to the degree of 0.5 McFarland, which corresponds to a value of 1.5x10 ⁸ microorganisms. A 0.050 ml broth culture is removed from the broth culture with a micropipette and applied to a 4 mm well (cut into the center of the plate) of selective LS agar. Inoculated petri dishes are placed in incubation vessels (GASPAK

-4CZ 33363 UI US patent) and incubated in a biological thermostat at 37 ° C for 48 hours. After 48 hours, agar is layered on the surface of the agar plates in the composition: peptone 0.5 g, fermented autolysate 1 g, glucose 1 g, agar 1.8 g, 100 ml distilled water, pH 6.9, with a volume of 3 ml, in which dissolves a 0,3 ml broth containing an overnight culture of an overgrown strain of Escherichia coli serotype O6: K2: H1 ATC 1938 CNTC Eck 63/59. After application of the strain, the petri dishes are placed in a thermostat and incubated at 37 ° C for 18 hours. After incubation, the size of the inhibition zones is measured, and the average result is obtained by summing the measured values of the individual measurements divided by the number of plates tested. In the exemplary embodiment, the readings from the three plates are used.

Molecular genetic detection of Lactobacillus plantarum strain LS / 07 CCM 7766:

A highly sensitive and specific method of polymerase chain reaction (PCR) is used for the molecular genetic detection of an isolated strain. The PCR mixture, the specific primers used, the template amplification and the agarose gel electrophoresis are performed according to the literature (Berthier and Ehrlich 1998). Amplification PCR products are stained with ethidium bromide and detected by ultraviolet light to obtain a specific PCR product of 265 base pairs.

Determination of organic acids:

Concentrations of organic acids (lactic and acetic acid) are determined using a capillary isotachophoresis technique (CS ISOTACHOPHORETIC ANALYZER). The isolated strain of Lactobacillus plantarum LS / 07 CCM 7766 produced the most lactic acid compared to acetic acid. Values are measured after 24 hours (lactic acid - 78.66 mmol / l, acetic acid -7.91 mmol / l) and 48 hours (lactic acid - 226.31 mmol / l, acetic acid 95.44 mmol / l) incubation in a thermostat at 37 ° C.

Determination of enzyme activity

The commercial API ZYM kit (BIOMÉRIEUX®SA, France) is used to study the enzyme activity of the Lactobacillus plantarum strain LS / 07 CCM 7766. From the obtained results it is found that the tested strain does not produce the enzyme β-glucuronidase, which is considered an important marker in the process of development of carcinogenesis of colorectal cancer.

Experiments on animal models

The isolated strain of Lactobacillus plantarum LS / 07 CCM 7766 is used in in vivo experimental studies in laboratory rats. Experimental work on laboratory animals is of great importance in studying the effect of isolated new strains of lactobacilli in vivo. We used the Lactobacillus plantarum strain LS / 07 CCM 7766 isolated by us from the faeces of a healthy person in an experiment with laboratory rats. The rats used in the experiment (number 51) came from the Sprague Dawley strain and were four months old. The strain was prepared daily fresh in lactobacilli growth broth (mean concentration 2.88 x 10 ⁹ CFU / ml) and administered daily to rats 5 times a week. Once a week (third to seventh week), rats were injected with the chemical procarcinogen 1,2 dimethylhydrazine dihydrochloride (1.2 DMH). During the whole experiment (24 weeks) we did not notice any clinical changes (diarrhea, death) in the experimental groups of animals. This confirmed that the strain was safe. The results of the experiment confirm that the modulating effect of the strain used was manifested by an increase in the number of lactic acid bacteria and a decrease in the number of coliform microorganisms in the intestinal content of the large intestine. Application of the strain had an effect not only on the intestinal microflora, but also on metabolic activities with a beneficial effect on the lipid profile. Positive changes in immune and histological laboratory tests of experimental animals are noted.

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Industrial applicability

The industrial applicability of the technical solution is obvious. According to this technical solution, it is possible to industrially and repeatedly produce Lactobacillus plantarum LS / 07 CCM 7766. The above effects of the Lactobacillus plantarum LS / 07 CCM 7766 strain predispose it to its use in modulating the intestinal microflora as well as in preventing inflammatory chronic human diseases.

Claims (8)

CLAIMS FOR PROTECTION A food product or nutritional supplement for the modulation of intestinal microflora or for the prevention of inflammatory chronic diseases in humans, characterized in that it contains a strain of Lactobacillus plantarum LS / 07 CCM 7766 isolated from humans, which is deposited in the Czech Collection of Microorganisms of the Faculty of Science, Masaryk University. Brno, Czech Republic. Food product or nutritional supplement according to claim 1, characterized in that it is in dried form. Food product or nutritional supplement according to claim 1 or 2, characterized in that it is in the form of a lyophilisate. Food product or nutritional supplement according to claim 1, characterized in that it is a fermented dairy product. Food product or nutritional supplement according to claim 1 or 4, characterized in that it is cheese. Food product or nutritional supplement according to any one of claims 1 to 3, characterized in that it is a nutritional supplement in the form of capsules. Food product or nutritional supplement according to any one of claims 1 to 4, characterized in that it is in the form of an energy cereal bar, a dairy or meat product. Food product or nutritional supplement according to any one of claims 1 to 4, characterized in that it is pasteurized bryndza.