PL209987B1 - New strain of lactic bacteria Lactobacillus casei - Google Patents

Description

The subject of the invention is a new strain of lactic acid bacteria Lactobacillus casei with probiotic properties.

Specific and controlled positive regulation of the intestinal microflora composition can be achieved by probiosis, i.e. consumption of preparations or food products containing live bacteria, mainly from the genus Lactobacillus and Bifidobacterium. Therefore, food producers, and in particular functional food, and the pharmaceutical industry are interested in acquiring new strains of these bacteria with proven probiotic properties.

The natural environment of the bacteria of the genus Lactobacillus is milk, cheese, fermented milk drinks, meat, and in particular fermented sausages and vegetable silage. These bacteria also colonize the mucous membranes of the digestive tract of humans and animals.

So far, strains of Lactobacillus bacteria with probiotic properties are known, such as L. acidophilus NCFM, L. acidophilus DDS-1, L. acidophilus SBT-2062, L. acidophilus R0011, L. rhamnosus R0052, L. acidophilus LA-1, L. paracasei CRL -431, L. casei Shirota, L. casei DN-114001, L. fermentum RC-14, L. rhamnosus GR-1, L. Johnson La1, L. plantarum 299V, L rhamnosus 271, L. reuteri SD2112, L. rhamnosus GG, L. rhamnosus LB21, L. salivarius UCC118, L. acidophilus LB, L. paracasein 19.

The subject of the invention is a new strain of Lactobacillus casei LOCK 0900 with probiotic properties, which is a homologue of Lactobacillus casei bacteria, with the following nucleotide sequence of the DNA region encoding the 16S rRNa gene, the sequence of which determines the species belonging to the bacteria

102 ctgagaatggctttaagagattagcttgacctcgcggtctcgcaactcgttgtaccatcc 161

162 attgtagcacgtgtgtagcccaggtcataaggggcatgatgatttgacgtcatccccacc 221

222 ttcctccggtttgtcaccggcagtcttactagagtgcccaactaaatgctggcaactagt 281

282 cataagggttgcgctcgttgcgggacttaacccaacatctcacgacacgagctgacgaca 341

342 accatgcaccacctgtcattttgcccccgaaggggaaacctgatctctcaggtgatcaaa 401

402 agatgtcaagacctggtaaggttcttcgcgttgcttcaaattaaaccacatgctccaccg 461

462 cttgtgcgggcccccgtcaattcctttgagtttcaaccttgcggtcgtactccccaggcg 521

522 gaatgcttaatgcgttagctgcggcactgaagggcggaaaccctccaacacctagcattc 581

582 atcgtttacggcatggactaccagggtatctaatcctgttcgctacccatgctttcaagc 641

642 ctaagcgtcagttacagaccaaacaagccgccttcgccactggtgttcttccatatatct 701

702 acgcatttaaccggtacacatggagttccactgtcctcttttgcactcaagtttcccagt 761

762 ttccgatgcacttcctcgggtaagccgagggctttcacatcaaacttaaaaaaccgcctg 821

822 ggctcgctttaccccaaataa-tccggataacgcttggcacctacctatta 871.

The new strain was isolated from an adult human stool sample. The new strain shows 97% homology to Lactobacillus casei bacteria. Bacteria are shaped like short gram-positive rods, often linked in pairs.

The new 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 is characterized by relatively heterofermentative metabolism. It is capable of fermenting the following saccharides and their derivatives: glycerol, L-arabinose, ribose, D-xylose, galactose, glucose, fructose, mannose, sorbose, rhamnose, duocytol, inositol, mannitol, sorbitol, α-methyl-D-glucoside, N-acetylglucosamine, amygdalin, arbutin, esculin, salicin, cellobiose, maltose, lactose, melibiosis, sucrose, trehalose, melecytose, raffinose, starch, gentiobiose, D-turanose, D-tagatose, gluconate using the API 50 CHL test. Lactose is fermented to form lactic acid in the amount of 3.7 g / l, the proportion of L (+) lactic acid is 96.8%, acetic acid (1.53 g / l), acetaldehyde (1.24 mg / l) ) and ethanol (6.37 mg / l), and the glucose is fermented to form lactic acid in the amount of 2.0 g / l, with the proportion of L (+) lactic acid being

83.5%, acetic acid (0.1 g / L), acetaldehyde (0.95 mg / L).

The new strain is obtained by culturing on a liquid modified medium according to Rogos under the trade name MRS, containing 2% glucose as a carbon source at temperatures of 35-37 ° C, preferably 37 ° C. The strain grows in the form of large (about 2-3 mm in diameter), slightly convex, light cream colonies. The edge and surface of the colony are smooth. It does not produce spores.

The new strain is resistant to low pH (gastric acidity) in the pH range from 2.5 to 3.5 and to bile salts at concentrations of 2%, 4%, 6% for up to 48 hours.

PL 209 987 B1

The new strain shows resistance to the following antibiotics and chemotherapeutic agents with antibacterial activity: vancomycin, kanamycin, cephalotin, ceftazidime, aztreonam, amikacin, streptomycin, clotrimazole.

The new strain is characterized by good adherence to the Caco-2 cell lines marked as (++), while the adherence was determined semi-quantitatively assuming that (-) means no adherence, (+) - single bacterial cells in the entire preparation, (++) - single bacterial cells in individual visual fields, numerous in the preparation, (+++) - numerous bacteria in individual visual fields.

The new strain shows antagonistic activity against food-borne pathogenic bacteria: Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Enterococcus faecalis, Salmonella enteritidis, Salmonella Typhimurium, Listeria monocytogenes, Listeria innocua.

The new strain has little fecal activity. Of the three enzymes β-glucosidase, β-glucuronidase and urease, only β-glucosidase is active.

The following examples illustrate the genetic identification of the strain and its morphological, biochemical and biotechnological characteristics with reference to the figure in which Fig. 1 shows the survival graph of the LOCK 0900 bacterial mixtures with calcium gluconate, milk powder and a fructo-oligosaccharide preparation, brand name Raftilose®, stored at refrigerated temperature, and Fig. 2 shows the survival of these mixtures when stored at room temperature.

P r z x l a d I.

In order to determine the species affiliation of the new LOCK 0900 strain, the nucleotide sequence of the DNA region encoding the 16S rRNA gene of this strain was compared, using an appropriate computer program, with the sequence of the same DNA region of the known Lactobacillus casei strain. Comparison of the nucleotide sequences of the new strain and the known strain of Lactobacillus casei:

New: 102 ctgagaatggctttaagagattagcttgacctcgcggtctcgcaactcgttgtaccatcc 161 I I I I I I I I I 1 I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I

Known: 1297 ctgagaatggctttaagagattagcttgacctcgcggtctcgcaactcgttgtaccatcc 1238

New: 162 attgtagcacgtgtgtagcccaggtcataaggggcatgatgatttgacgtcatccccacc 221 I I I I I I I I I I I I! I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I> I I I I

Known: 1237 attgtagcacgtgtgtagcccaggtcataaggggcatgatgatttgacgtcatccccacc 1178

New: 222 Known 1177 new 282 well-known 1117 new 342 well-known 1057 new 402 well-known 997 new 462 well-known 937 new 522 well-known 877 new 582 well-known 817 ttcctccggtttgtcaccggcagtcttactagagtgcccaactaaatgctggcaactagt IIIIIIIIIIIIIIIIIIIII IIIIIIIIIIIIIIIIIIIII IIIIIIIIIIIIIIIIII ttcctccggtttgtcaccggcagtcttactagagtgcccaactaaatgctggcaactagt cataagggttgcgctcgttgcgggacttaacccaacatctcacgacacgagctgacgaca IIIIIIIIIIIIIIIIIIIII IIIIIIIIIIIIIIIIIIIII III! I I I I I I I I I I I I cataagggttgcgctcgttgcgggacttaacccaacatctcacgacacgagctgacgaca accatgcaccacctgtcattttgcccccgaaggggaaacctgatct I I I cataaggggaaa I I I I I I I I I Caggtgatcaaa I I I I I I I I I I I I I I 1 I I I I I I I I I I I I I I I I I I I accatgcaccacctgtcattttgcccccgaaggggaaacctgatctctcaggtgatcaaa agatgtcaagacctggtaaggttcttcgcgttgcttcaaattaaaccacatgctccaccg 1 I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I 1 I I I I I I I I agatgtcaagacctggtaaggttcttcgcgttgcttcgaattaaaccacatgctccaccg cttgtgcgggcccccgtcaattcctttgagtttcaaccttgcggtcgtactccccaggcg IIIIIIIIIIIIIIIIIIIIIIII I IIIII I and III I I I I I I I LII I 1 I I I I I I I I I I I I I cttgtgcgggcccccgtcaattcctttgagtttcaaccttgcggtcgtactccccaggcg gaatgcttaatgcgttagctgcggcactgaagggcggaaaccctccaacacctagcattc I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I gaatgcttaatgcgttagctgcggcactgaagggcggaaaccctccaacacctagcattc atcgtttacggcatggactaccagggtatctaatcctgttcgctacccatgctttcaagc IIII I I I I I I I I II IIII I I I I I I I I IIII II I I I I I I III I I I I I I I I IIII I I I III atcgtttacggcatggactaccagggtatctaa tcctgttcgctacccatgctttcgagc

281

1118

341

1058

401

998

461

938

521

878

581

818

641

758

PL 209 987 B1

New: 642 known 757 new 702 well-known 698 new 762 well-known 638 new 822 well-known 578 ctaagcgtcagttacagaccaaacaagccgccttcgccactggtgttcttccatatatct 701 II IIIIIIIIIIIIIIIIII III IIIIIIIIIIIIIIIII IIIIII IIIIIIIIIII ctcagcgtcagttacagaccagaca-gccgccttcgccactggtgttcttccatatatct 699 acgcatttaaccggtacacatggagttccactgtcctcttttgcactcaagtttcccagt 761 IIIIIII III II IIIII IIIIIIIIIIII IIIIIIIII II IIIII IIIIIIIIIII acgcatttcaccgctacacatggagttccactgtcctcttctgnactcaagtttcccagt 639 ttccgatgcacttcctcgggtaagccgagggctttcacatcaaacttaaaaaaccgcctg 821 IIIIIIIIIIIIIIIIIIIII IIIIIIIIIIIIIIIIIIIII IIIIIIIIIIIIIIII ttccgatgcacttcctcggttaagccgagggctttcacatcagacttaaaaaaccgcctg 579 ggctcgctttaccccaaataa-tccggataacgcttggcacctacctatta 871 IIIII III III II I IIIIIIIII lily Tl IIIIIIIIII lily cgctcgctttacgcccaataaatccggataacgcttgccacctacgtatta 528.

As a result of comparing the sequence of the new strain Ł OCK 0900 and the known strain, it was found for the new strain:

length = 1521, similarity = 754/771 (97%), unidentified = 2/771 (0%), thread = Plus / Minus

For the LOCK 0900 strain, 16 intracellular proteins with molecular weights of 69.36 were isolated; 62.57; 54.10; 48.66; 45.84; 42.97; 40.92; 38.87; 32.43; 30.85; 29.60; 27.41; 24.17; 23.64; 19.95; 16.87 kDa and 14 extracellular proteins with masses 174.59; 93.10; 78.05; 62.57; 56.25; 48.66; 47.69; 42.97; 40.92; 36.82; 34.49; 32.93; 32.43; 29.10 kDa.

P r z x l a d II.

The ŁOCK 0900 strain was grown on MRS liquid medium with the composition in g / l: yeast extract 4.0, meat extract - 8.0, peptone K -10.0, glucose - 20.0, ammonium hydrogen citrate - 2.0, phosphate dipotassium 2.0, sodium acetate - 5.0, magnesium sulphate 7-water - 0.20, magnesium sulphate 4-water - 0.05, agar - 15.0, at 37 ° C. After 24 hours of cultivation, the cultured cells were separated from the medium on a shaker at a density of 4x10 ⁹ cells / ml.

P r x l a d III.

The bacteria obtained as in Example 2 were placed in media with a pH of 2.5 and 13.5.

In an environment with a pH of 2.5, after 180 minutes, it survived at 3.7 x 10 ⁸ cfu (colony forming units) / ml with the starting amount of 4.0 x 10 ⁸ cfu / ml. In the environment with pH 3.5, after 180 minutes, 2.5 x 10 ⁸ cfu / ml survived with the initial amount of 4.6 x 10 ⁸ cfu / ml.

P r x l a d IV.

The bacteria obtained as in Example 2 were treated with bile salts at concentrations of 2%, 4% and 6% for up to 48 hours. As a result ACTIVITY salt of O LCI concentration of 2% after 48 hours by y L of 3.3 x 10 ⁶ cfu / ml at an initial quantity of 2.1 x 10 ⁸ cfu / ml. After 48 hours, 1.6 x 10 ⁶ cfu / ml survived under the influence of bile salts with a concentration of 4%, with the initial amount of 1.95 x 10 ⁸ cfu / ml. After 48 hours, 1.0 x 10 ⁶ cfu / ml survived under the influence of bile salts at a concentration of 6%, with the initial amount of 1.7 x 10 ⁸ cfu / ml.

P r z k ł a d V.

In order to determine the biotechnological properties of the new Ł OCK 0900 bacterial strain, the inoculum of this strain was inoculated with UHT milk with 2% fat content. The ŁOCK 0900 strain reached the stationary phase after 24 hours of cultivation in milk, and the cell yield was 7.7 x 10 ⁸ cfu / ml. The duration of the adaptation phase of bacteria was 2 hours.

The maximum value of the specific growth rate of the strain was 0.46 h ^-1 , and the productivity of the culture was 0.47 x 10 ⁸ .

The strain in the initial stage of the stationary phase reached an acidity of 24 ° SH, guaranteeing a purely acidic taste of the product. The specific acidic activity of the bacteria, expressed in ° SH per colony forming unit, in the stationary phase of the growth of the strain was 2.1 ° SH / 10 ⁸ cfu.

After 24 hours of incubation, the strain used 45.7% of milk lactose.

Moreover, it was found that:

- the strain is characterized by β-galactosidase activity at the level of 0.43 J, where the activity unit J is defined as the number of μmoles of o-nitrophenyl-β-galactopyranose (ONP) released in 1 minute by 1 mg of bacteria dry weight,

- is capable of the simultaneous use of glucose and galactose,

- does not synthesize bacteriocins and bacteriocin-like compounds,

- it is capable of synthesizing biosurfactants, compounds insensitive to the action of proteolytic enzymes - pepsin and trypsin, showing antagonistic activity against: Pseudomonas aeruginosa, Pseudomonas fluorescens, Lactobacillus acidophilus, Enterobacter aerogenes.

PL 209 987 B1

P r x l a d VI.

The storage stability of products and preparations containing the new strain ŁOCK 0900 was determined.

For this purpose, UHT milk with a fat content of 2% was inoculated with an inoculum of ŁOCK 0900 bacteria added in an amount of 10%, incubated until the milk was acidified, and then the acidified milk was stored at 4-5 ° C for 21 days. The non-acidified milk containing the inoculum of these bacteria was also stored under similar conditions.

During storage in acidified and non-acidified milk, the survival of ŁOCK 0900 bacteria and the total acidity of the product were checked. The survival of the ŁOCK 0900 strain in non-acidified milk, stored for 21 days at 4-5 ° C, was 93%, and the number of bacterial cells was 10 ⁸ cfu / ml.

In the milk acidified with the ŁOCK 0900 strain, the number of viable bacteria was high and amounted to 10 ⁹ cfu / ml and it did not change significantly during the 21 days of storage. Moreover, it was found that the refrigerated storage of milk acidified with the ŁOCK 0900 strain stabilizes the total acidity at the level of 44.8 ° SH (acidity limits 30-46 ° SH), which guarantees a purely sour taste of the product and ensures very good sensory properties of the product.

Mixtures of ŁOCK 0900 strain containing 10% addition of calcium gluconate, Raftilose® preparation and powdered milk were prepared and subjected to the lyophilization process.

The stability of lyophilized preparations stored for 4 months under refrigerated conditions (4-5 ° C) and at room temperature (25 ° C) was assessed. The results of the evaluation of the survival of the LOCK 0900 bacteria in these mixtures at refrigerated temperature and room temperature are shown in the graphs of Fig. 1 and Fig. 2 of the drawing.

It turned out that the cooling temperature ensures greater stability and durability of the freeze-dried preparations regardless of the protective factor.

The storage of the freeze-dried bacterial preparation with milk as a protective medium for 4 months at cooling temperature resulted in a reduction of the number of viable cells by 17.9%, and at room temperature by 31.8%. During the 4 months of storage of the lyophilisate of a mixture of bacterial cells with Raftilose®, the reduction in the number of live cells LOCK 0900 increased by 25.8% at the refrigerated temperature and by 40.9% at room temperature. Mortality of ŁOCK 0900 bacterial cells, lyophilized with calcium gluconate, stored at refrigerated temperature was 20.7%, while stored at room temperature - 43.7%. In nucleotide sequences:

a - is a nucleotide containing adenine as the nitrogen base, g - is a nucleotide containing guanine as a nitrogen base, c - is a nucleotide containing cytosine as a nitrogen base, t - is a nucleotide containing thymine as a nitrogen base.

Claims (1)

A new strain of lactic acid bacteria Lactobacillus casei LOCK 0900 with probiotic properties, being a homologue of Lactobacillus casei bacteria, with the following nucleotide sequence of the DNA region encoding the 16S rRNa gene, the sequence of which determines the species belonging to the bacteria 102 ctgagaatggctttaagagattagcttgacctcgcggtctcgcaactcgttgtaccatcc 161 162 attgtagcacgtgtgtagcccaggtcataaggggcatgatgatttgacgtcatccccacc 221 222 ttcctccggtttgtcaccggcagtcttactagagtgcccaactaaatgctggcaactagt 281 282 cataagggttgcgctcgttgcgggacttaacccaacatctcacgacacgagctgacgaca 341 342 accatgcaccacctgtcattttgcccccgaaggggaaacctgatctctcaggtgatcaaa 401 402 agatgtcaagacctggtaaggttcttcgcgttgcttcaaattaaaccacatgctccaccg 461 462 cttgtgcgggcccccgtcaattcctttgagtttcaaccttgcggtcgtactccccaggcg 521 522 gaatgcttaatgcgttagctgcggcactgaagggcggaaaccctccaacacctagcattc 581 582 atcgtttacggcatggactaccagggtatctaatcctgttcgctacccatgctttcaagc 641 642 ctaagcgtcagttacagaccaaacaagccgccttcgccactggtgttcttccatatatct 701 702 acgcatttaaccggtacacatggagttccactgtcctcttttgcactcaagtttcccagt 761 762 ttccgatgcacttcctcgggtaagccgagggctttcacatcaaacttaaaaaaccgcctg 821 822 ggctcgctttaccccaaataa-tccggataacgcttggcacctacctatta 871.