PL238589B1 - Strain of lactic bacteria Lactobacillus rhamnosus - Google Patents

Description

Description of the invention

The subject of the invention is the strain of lactic bacteria Lactobacillus rhamnosus ŁOCK 1146 deposited in the Polish Collection of Microorganisms, at the Institute of Immunology and Experimental Therapy of the Polish Academy of Sciences. Ludwik Hirszfeld PAS in Wrocław under number B / 00217, with probiotic properties. So far, the strains of bacteria from the Lactobacillus rhamnosus species are known, such as Lactobacillus rhamnosus ATCC 53103, Lactobacillus rhamnosus GG, Lactobacillus rhamnosus Lc705, Lactobacillus rhamnosus ATCC 8530, Lactobacillus rhamnosus ATCC 8530, Lactobacillus rhamnosus LOC887, and Lactobacillus rhamnosus Lobac6 from the description Lobac6 Lobac6 from the description Lobac6 patent application P.422602.

It is known that probiotics ensure the balance of the gut microbiota and have a beneficial effect on human and animal health. The term probiotic is reserved for preparations or products that meet the following criteria: contain live cells in an appropriate amount, improve the health of the host, have a beneficial effect in the digestive tract (administered as food or feed additives). The effectiveness of the beneficial effects of probiotic strains of microorganisms, mainly lactic acid bacteria (LAB) on the human and animal organism, depends on many factors, therefore it is important to properly select and select strains and systematically provide the body with a large number of live cells of these microorganisms. According to the proposals of the World Health Organization (WHO), the Food and Agriculture Organization of the United Nations (FAO) and the European Food Safety Authority (EFSA), probiotic strains in the selection process must meet both safety, functionality and technological suitability criteria. The safety of a strain is defined by its origin and its antibiotic resistance profile. Functional aspects determine their ability to survive in the gastrointestinal tract (including resistance to low pH and bile salts). Technological properties, on the other hand, include the ability of probiotics to survive and preserve their talents during storage and distribution. Probiotic products have been known for a long time, but nowadays, due to the increased nutritional awareness of the public, they are becoming very popular. Diseases of civilization associated with inadequate nutrition, stressful lifestyle and food improvers also contribute to the increase in demand. The production of probiotic food, feed additives and pharmaceutical products, however, requires the acquisition of new strains and the selection of strains with appropriate properties.

The subject of the invention is a new strain of lactic acid bacteria of the species Lactobacillus rhamnosus, marked with the symbol ŁOCK 1146 and deposited in the Polish Collection of PCM Microorganisms at the Institute of Immunology and Experimental Therapy of the Polish Academy of Sciences. Ludwik Hirszfeld in Wrocław under the number B / 00217.

The ŁOCK 1146 bacterial strain was isolated from adult human faeces.

The ŁOCK 1146 strain is a gram-positive rod, immobile, non-sporulating. It is a strain with probiotic properties, which is a homologue of the bacteria of the Lactobacillus rhamnosus species with the nucleotide sequence of the DNA region encoding the 16S rRNA gene presented at the end of the description.

In order to determine the species affiliation of the new LOCK 1146 strain, the DNA region encoding the 1432 long 16S rRNA gene was sequenced. The obtained nucleotide sequence of the 16S rRNA gene was compared using the BLASTN 2.6.0+ program with the sequences available in the National Center of Biotechnology Information (NCBI) database and the program CLC Main Workbench 8. Based on the comparison of the nucleotide sequences of the 16S rRNA genes of Lactobacillus rhamnosus KY348290.1, the new bacterial strain was similar to the Lactobacillus rhamnosus species with a probability of 99.16%.

The new ŁOCK 1146 strain is grown on a clear, yellow-brown Rogosa medium intended for the cultivation of bacteria of the genus Lactobacillus sp., With the composition in g / l: casein peptone - 10.0, yeast extract - 5.0, D (+ ) glucose - 20.0, potassium phosphate - 6.0, ammonium citrate - 2.0, Tween 80 - 1.0, sodium acetate - 15.0, magnesium sulphate - 0.575, iron II sulphate - 0.034, manganese sulphate - 0 , 12, agar - 15.0, pH 5-6, at a temperature of 15-45 ° C, preferably 37 ° C during 48 hours. On the Rogosa medium, the strain grows as white-cream, round colonies with a smooth surface and regular edges, about 2-3 mm in diameter.

The ŁOCK 1146 strain is also grown in the MRS liquid medium, which is a modification of the Rogosa medium, containing glucose as a source, with the composition in g / l: yeast extract - 4.0, meat extract

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- 10.0, casein peptone - 10.0, D (+) glucose - 20.0, Tween 80 - 1.0, ammonium hydrogen citrate - 2.0, dipotassium phosphate - 2.0, sodium acetate - 5.0 , 7-aqueous magnesium sulfate - 0.20, 4-aqueous magnesium sulfate - 0.05, pH 5-6, at a temperature of 15-45 ° C, preferably 37 ° C during 48 hours. In this medium, the strain grows, creating a homogeneous turbidity of the medium.

For growth, the strain prefers an atmosphere enriched with CO2 introduced in the amount of 5% (v / v). However, it grows both under aerobic and relatively anaerobic conditions.

Morphological features of the strain ŁOCK 1146

In the microscopic image, the bacteria have the shape of short, straight rods, 4-5 μm long and up to 1 μm wide. The sticks do not produce spores.

Physiological and biochemical features of the ŁOCK 1146 strain

The strain does not produce catalase and cytochrome oxidase. It stains Gram (+).

The study of glucose metabolism under aerobic and anaerobic conditions in Hugh-Leifson's medium in tall columns showed that the ŁOCK 1146 strain is able to break down glucose both in the presence of oxygen and under anaerobic conditions, i.e. by fermentation.

The API 50 CHL test showed that the LOCK 1146 strain is capable of fermenting the following saccharides and their derivatives: ribose, galactose, glucose, fructose, mannose, sorbose, rhamnose, inositol, mannitol, sorbitol, N-acetylglucosamine, Methyl-a, D- glucopyranoside, amygdalin, arbutin, esculin, salicin, cellobiose, maltose, lactose, sucrose, trehalose, melecytose, gentianose, D-turanose, tagatose, gluconate.

The ŁOCK 1146 strain is characterized by relatively heterofermentative metabolism. The lactose is fermented to form lactic acid in the amount of 3.5 g / l, the proportion of L (+) lactic acid being 97%, acetic acid (1.5 g / l), acetaldehyde (1.0 mg / l) and ethanol (5.0 mg / L).

The API ZYM test showed that the LOCK 1146 strain exhibited the activity of the following enzymes: leucine arylamidase, valine arylamidase, cysteine arylamidase, naphthyl-AS-BI fusphohydrolase.

The ŁOCK 1146 strain does not produce hydrogen sulphide or ammonia from arginine, it does not have the ability to decompose H2O2.

Probiotic features of strain ŁOCK 1146

The ŁOCK 1146 strain meets the criteria for probiotic bacteria, which was found as a result of in vitro tests carried out according to the procedures recommended by FAO / WHO.

The new strain shows high resistance to low pH (gastric acidity) in the range from 2.0 to 3.0.

The ŁOCK 1146 strain also shows high resistance to bile salts in concentrations of 1% and 2%.

The ŁOCK 1146 strain is resistant to antibiotics and chemotherapeutic agents with antibacterial activity, such as: penicillin, kanamycin, vancomycin, amoxicillin, doxycycline, streptomycin, erythromycin, tetracycline, cephalotin, ceftazidime, aztimacreonam.

It shows antagonistic activity against food-borne pathogens, such as Salmonella Enteritidis, Salmonella Typhimurium, Salmonella Choleraesuis, Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Campylobacter jejuni, Listeria monocytogenes, Listeria innocua.

It has low fecal activity. Of the three enzymes β-glucosidase, β-glucuronidase and urease, only β-glucosidase is active.

The ŁOCK 1146 strain is characterized by good adherence to Caco-2 intestinal epithelial cells, and thus exhibits adhesive properties.

It demonstrates the ability to detoxify mycotoxins (aflatoxin Bi, ochratoxin A, fumonisin, toxin T-2, zearalenone, deoxynivalenol), which can most often contaminate food and feed.

Biotechnological features of strain ŁOCK 1146

The biotechnological properties of the ŁOCK 1146 strain, due to the possibility of using it in food and feed, were determined both in milk and in the compound feed.

In milk with 3.2% fat content, the LOCK 1146 strain reached the stationary phase after 19 hours of cultivation, and the cell yield was 2.10x109 cfu / ml. The duration of the adaptation phase of bacteria was 2 hours. The specific growth rate of the strain was 0.42 h ^-1 and the culture productivity was 0.45x10 ⁸ .

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In the feed mixture (ground grains: wheat 40%, barley 30%, corn 20% and rye 10%), the LOCK 1146 strain reached the stationary phase after 18 hours of cultivation, and the cell yield was 2.35x10 ⁹ CFU / ml. The duration of the adaptation phase of bacteria was 2 hours. The specific growth rate of the strain was 0.17 h ^-1 and the culture productivity was 0.50x108.

The ŁOCK 1146 strain is used as a food and feed additive.

The following examples illustrate the subject matter of the invention.

P r z k ł a d I

The ŁOCK 1146 strain was grown in the MRS liquid medium (Merck), with the composition in g / l: yeast extract - 4.0, meat extract - 10.0, casein peptone - 10.0, D (+) glucose - 20.0 , Tween 80 - 1.0, ammonium hydrogen citrate - 2.0, dipotassium phosphate - 2.0, sodium acetate - 5.0, magnesium sulphate 7-water - 0.20, magnesium sulphate 4-water - 0.05, o pH 5-6, in the presence of CO2 introduced in an amount of 5% (v / v), at a temperature of 37 ° C.

After 48 hours of culture, the cultured cells were separated from the medium on a shaker to obtain a density of 2.5x10 ⁹ cells / ml.

P r x l a d II

ŁOCK 1146 strain was grown in Rogosa liquid medium (Difco company) with the composition in g / l: casein peptone - 10.0, yeast extract - 5.0, D (+) glucose - 20.0, potassium phosphate - 6.0, citrate ammonium - 2.0, Tween 80 - 1.0, sodium acetate - 15.0, magnesium sulfate - 0.575, iron II sulfate - 0.034, manganese sulfate - 0.12, agar - 15.0, pH 5-6, in the presence of CO2 introduced in an amount of 5% (v / v), at a temperature of 37 ° C.

After 48 hours of cultivation, the cultured cells were separated from the medium on a shaker to obtain a density of 1.5x10 ⁹ cells / ml.

P r z x l a d III

The bacteria obtained as in Example 1 were placed in an environment of pH 2.0 and 3.0. In the environment with pH 2.0, after 180 minutes, 3.5x10 ⁸ cfu / ml survived with the initial amount of 5.0x10 ⁸ cfu / ml. In the environment with pH 3.0, after 180 minutes, 2.5x10 ⁸ cfu / ml survived with the initial amount of 4.0x10 ⁸ cfu / ml.

P r x l a d IV

The bacteria obtained as in Example 1 were exposed to bile at concentrations of 1% and 2% for up to 4 hours. After 4 hours of incubation at a bile concentration of 2%, the cell viability was 92%, and at a bile concentration of 1%, 95% of the bacterial cells survived.

P r z k ł a d V

The lyophilisate of the bacteria obtained as in Example 1 was stored at 4-5 ° C and at room temperature 25 ° C for 4 months. After this time, there was a reduction in the number of viable cells by 5% for the cooling temperature and by 14% for the room temperature. The cooling temperature provided greater stability and durability.

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Sequence Listing

CTGATTTATTGAAAGGTGCTTGCATTCCTTGATTTAGATTTTGAACGAGTGGCGGA

CGGGTGAGTAACACGTGGGTAACCTGCCCTTAAGTGGGGGATAACATTTGGAAA

CAGATGCTAATACCGCATAAATCCAAGAACCGCATGGTTCTTGGCTGAAAGATGG

CGTAAGCTATCG CTTTTGG ATGGACCCG CGG CGTATTAGCTAGTTGGTG AG GTAA

CGGCTCACCAAGG CAATG ATACGTAG CCG AACTG AG AGGTTG ATCG GCCACATT

GGGACTGAGACACGGCCCAAACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCA

CAATGGACGCAAGTCTGATGGAGCAACGCCGCGTGAGTGAAGAAGGCTTTCGGG

TCGTAAAACTCTGTTGTTGGAGAAGAATGGTCGGCAGAGTAACTGTTGTCGGCGT

GACGGTATCCAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAAT

ACGTAGGTGGCAAGCGTTATCCGGATTTATTGGGCGTAAAGCGAGCGCAGGCGG

TTTTTTAAGTCTGATGTGAAAGCCCTCGGCTTAACCGAGGAAGTGCATCGGAAAC

TGGGAAACTTGAGTGCAGAAGAGGACAGTGGAACTCCATGTGTAGCGGTGAAAT

GCGTAGATATATGGAAGAACACCAGTGGCGAAGGCGGCTGTCTGGTCTGTAACT

GACGCTGAGGCTCGAAAGCATGGGTAGCGAACAGGATTAGATACCCTGGTAGTC

CATGCCGTAAACGATGAATGCTAGGTGTTTGGAAGGGTTTCCGCCCTTCAGTGCC

GCAGCTAACGCATTAAGCATTCCGCCTGGGGGAGTACGACCGCAAGGTTGAAAC

TCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAACATGTGGGTTTAATTCG

AAGCAACGCGAAGAACCTTACCAGGTCTTGACATCTTTTGATCACCTGAGAGATC

AGGTTTCCCCTTCGGGGGCAAAATGACAGGTGGTGCATGGTTGTCGTCAGCTCGT

GTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATGACTAGTTGC

CAGCATTTAGTTGGGCACTCTAGTAAGACTGCCGGTGACAAACCGGAGGAAGGT

GGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACA

ATGGATGGTACAACGAGTTGCGAGACCGCGAGGTCAAGCTAATCTCTTAAAGCC

ATTCTCAGTTCGGACTGTAGGCTGCAACTCGCCTACACGAAGTCGGAATCGCTAG

TAATCGCGGATCAGCACGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCG

CCCGTCACACCATGAGAGTTTGTAACACCCGGAAAGCCGGTGGCGTAAACCCTT

TAGGGAGCGAGCCGTC where: A - stands for adenine, G - guanine, C - cytosine, T - thymine.

Claims (1)

Patent claim 1. Lactobacillus rhamnosus LOCK 1146 strain of lactic bacteria deposited in the Polish Collection of PCM Microorganisms at the Institute of Immunology and Experimental Therapy of the Polish Academy of Sciences. Ludwik Hirszfeld in Wrocław under the number B / 00217, with probiotic properties.