PL233261B1 - Lactic bacterial strain of Lactobacillus pentosus - Google Patents

Description

Description of the invention

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

Probiotic preparations administered to farm animals in the feed or applied orally, have a positive effect on their growth by stimulating the effect on the digestive tract and the processes taking place in it. Properly selected probiotic bacteria determine feed absorption, while also acting as a regulator of the balance of microorganisms in the animal's digestive tract. The effects of probiotic preparations are similar to those obtained as a result of the use of fodder antibiotics, because both reduce the number of pathogenic bacteria, but the way they work is different. Probiotics do not cause any side effects and do not imply the accumulation of harmful foreign substances, hence they have no grace period and there is no risk of overdosing. From the point of view of the consumer and producer of food, it is also important that the use of probiotics, thanks to the possibility of reducing or even completely eliminating the need for antibiotics, is a way of obtaining safe food.

So far, the bacterial strains of the Lactobacillus pentosus species are known, such as Lactobacillus pentosus LMG 10755T, Lactobacillus pentosus LMG 18401, Lactobacillus pentosus AJ292254, Lactobacillus pentosus ITA23, Lactobacillus pentosus ITA23, Lactobacillus pentosus LPSCC3, Lactobacillus pentosus LPSCC3, Lactobacillus pentosus LPSCC3 ™ patent application 407121, Lactobacillus pentosus strain LOCK 0976.

The subject of the invention is a new strain of lactic acid bacteria of the species Lactobacillus pentosus, marked with the symbol ŁOCK 1094 and deposited in the Polish Collection of Microorganisms (PCM) at the Institute of Immunology and Experimental Therapy of the Polish Academy of Sciences. Ludwik Hirszfeld in Wrocław under the number B / 00124.

LOCK 1094 strain was isolated from broiler chicken poultry.

The ŁOCK 1094 strain is a gram-positive rod, immobile, non-sporulating. It is a strain with probiotic properties, which is a homologue of bacteria of the Lactobacillus pentosus species with the nucleotide sequence of the DNA region encoding the 16S rRNA gene presented at the end of the description.

To determine the species affiliation of the new LOCK 1094 strain, the DNA region encoding the 1415 bp 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 Baser DNA v4.36.0 program. Based on the comparison of nucleotide sequences of the 16S rRNA genes of bacteria from the Lactobacillus pentosus JCM 1558 species, the similarity of the new bacterial strain to the species Lactobacillus pentosus was found with a probability of 99.87%.

The new LOCK 1094 strain is grown on the Rogosa medium intended for the detection of transparent yellow-brown 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 sulfate - 0.575, iron II sulfate - 0.034, manganese sulfate - 0, 12, agar 15.0, pH 5-6, preferably 5.5, at a temperature of 15-45 ° C, preferably 37 ° C during 48 hours. On Rogosa medium, the strain grows as white-cream, round colonies with a smooth surface and regular edges, about 1-3 mm in diameter.

The ŁOCK 1094 strain is also grown in the MRS liquid medium, which is a modification of the Rogosa medium, 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, with a pH of 5-6, preferably 5.5, 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 a 5% (v / v) CO2 enriched environment. However, it grows both under aerobic and relatively anaerobic conditions.

Morphological features of strain ŁOCK 1094

On a microscopic image, the bacteria appear as straight sticks, 4-5 µm long and up to 1 µm wide. The sticks do not produce spores.

PL 233 261 B1

Physiological and biochemical features of the new ŁOCK 1094 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 LOCK 1094 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 1094 strain is capable of fermenting the following saccharides and their derivatives: glycerol, arabinose, ribose, xylose, galactose, glucose, fructose, mannose, mannitol, sorbitol, N-acetylglucosamine, amygdalin, arbutin, esculin , salicin, cellobiose, maltose, lactose, melibiosis, sucrose, trehalose, inulin, melecytose, raffmose, gentianose, D-turanose, gluconate.

The ŁOCK 1094 strain is characterized by relatively heterofermentative metabolism. Fermented glucose to form lactic acid at 10.43 μmol / ml, acetic acid (3.77 μmol / ml), butyric acid (0.49 μmol / ml), acetaldehyde (0.09 μmol / ml) and ethanol ( 0.14 μmol / ml).

The API ZYM test showed that the LOCK 1094 strain exhibits the activity of the following enzymes: alkaline phosphatase, esterase, α-galactosidase, β-galactosidase, α-glucosidase, β-glucuronidase, acid phosphatase, phosphoamidase, leucine arylamidase, arylamidylglucine arylamidase, a-mannosidase, a-fucosidase. Moreover, it was found that the strain ŁOCK 1094 does not produce hydrogen sulphide, nor ammonia from arginine, does not show the ability to decompose H2O2.

Ability to use prebiotics of the ŁOCK 1094 strain

The ŁOCK 1094 strain is able to grow in a medium containing prebiotics as the only carbon source, such as starch, β-glucan, maltodextrin, inulin, pectin, but the growth is variable and depends on the type and concentration of the saccharide. The best sources of carbon are inulin and pectin. On the other hand, when comparing the growth of LOCK 1094 in the presence of prebiotics and in the presence of glucose (reference test), it was found that for all prebiotics tested, the growth was better, as shown in Figure 1 in the figure.

Also as a result of fermentation of inulin and pectin, strain LOCK 1094 produces the highest amounts of lactic acid, with the highest proportion of L (+) lactic acid being 87%. Apart from lactic acid, the fermentation products of prebiotics (starch, β-glucan, maltodextrin, inulin and pectin) are acetic acid, butyric acid, acetic aldehyde and ethanol.

Probiotic features of strain ŁOCK 1094

The ŁOCK 1094 strain meets the criteria for probiotic bacteria, which was confirmed by in vitro tests performed according to the procedures recommended by FAO / WHO.

The ŁOCK 1094 strain shows high resistance to low pH (gastric acidity) in the pH range from 2.0 to 3.0.

The ŁOCK 1094 strain also shows high resistance to bile salts in concentrations of 1% and 2% for up to 4 hours.

The ŁOCK 1094 strain shows resistance to antibiotics most commonly used in farm animals, such as penicillin, kanamycin, amoxicillin, doxycycline, erythromycin, and tetracycline.

The ŁOCK 1094 strain also shows resistance to coccidiostats such as diclazuril, decoquinate, narasin-nicrabazine.

The new strain shows antagonistic activity against the intestinal pathogens of animals and human zoos, such as Salmonella enteritidis, Salmonella typhimurium, Salmonella choleraesuis, Campylobacter jejuni, Campylobacter coli, and Listeria monocytogenes.

The ŁOCK 1094 strain has a low faecal activity. Of the three enzymes β-glucosidase, β-glucuronidase and urease, only β-glucuronidase is active.

The ŁOCK 1094 strain shows the ability to detoxify mycotoxins, such as aflatoxin Bi, ochratoxin A, fumonisin, T-2 toxin, zearalenone, deoxynivalenol, which can most often be contaminating grains and animal feeds.

Biotechnological features of strain ŁOCK 1094

The biotechnological characteristics of the LOCK 1094 strain were determined in three fermentation media (feed mixtures (P1, P2 and P3) with the following composition of ground grains: feed P1 - 50% wheat, 30% barley and 20% maize, feed P2 - 40% wheat, 30% barley, 20% maize and 10% rye, feed P3 - 40% wheat, 30% barley, 20% maize and 10% soybean.

EN 233 261 Β1 fermentation was established on the basis of grains used in feeding monogastric animals. The feed mixtures were dissolved in water in the following proportions: 1: 1.0; 1: 1.5; 1: 2.0. The fermentation broths were inoculated with the bacterial suspension in the amount of 10 ⁶ cfu / ml. The growth dynamics in fermentation media (CFU / g feed) was determined at 4, 8, 12, 16, 24 and 30 hours of culture, and the reference test was the culture in MRS medium (CFU / ml of medium). The dynamics of the growth of the strain is presented in the form of graph 2 in the figure, and the yield of biomass and the productivity of cultivation in the table below.

T a be I a

Strain Cell yield N [cfu / g; CFU / ml] Productivity P [cfu / kgxh; jtk / fxh] Specific growth rate μ [h · ¹ ] Adaptation phase [h] Fermentation broth PI P2 P3 PI P2 P3 PI P2 P3 PI P2 P3 No. pentosus LOCK 1094 6.33 9.60 9.48 xl0® xl0 'x! 0 ^s 2.11 3.20 3.16 χΙΟ ¹⁰ χ1θ χ10 '° 0.16 0.23 0.18 7.27 4.55 2.81

MRS control medium

No. pentosus LOCK 4.75X10 ' 1.58χ10 ¹⁰ 0.22 1.57 1094

It turned out that the growth dynamics and the biomass yield were dependent on the composition of the fermentation medium and the highest values in relation to the MRS control medium were obtained for the cultivation in the fermentation medium P2. The cell yield in this medium is 9.60x10 ⁹ cfu / g, and the productivity of the strain increases to the level of 3.20x10 ¹¹ cfu / kgxh. Moreover, it was found that the optimal medium for the growth of LOCK 1094 is a medium with a ratio of ground grains (flour) to water of 1: 1.5. Under these conditions, the bacterium achieves the highest biomass growth, also the productivity is higher compared to the flour to water ratios of 1: 1.0 and 1: 2.0. The LOCK 1094 strain also exhibits storage stability in the form of a lyophilisate under refrigerated conditions (4-5 ° C) and at room temperature (25 ° C) for 4 months of storage.

The LOCK 1094 strain is used as an additive to feed for monogastric animals in the prevention of bacterial diseases and poisoning caused by mycotoxins, and improving the safety of animal husbandry.

The following examples illustrate the subject matter of the invention.

Example I

LOCK 1094 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 5.5, in the presence of CO2 introduced in an amount of 5% (v / v), at a temperature of 37 ° C under aerobic conditions.

After 48 hours of culture, the cultured cells were separated from the medium on a shaker to obtain a density of 4.71 × 10 ⁹ cells / ml.

Example II

LOCK 1094 strain was grown in MRS liquid medium (Merck company), 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 sulfate 7, aqueous - 0.20, magnesium sulfate 4-water - 0.05, pH 5 , 5, in the presence of CO2 introduced at 5% (v / v), at a temperature of 37 ° C under relatively anaerobic conditions.

After 48 hours of culture, the grown cells were separated from substrate on a shaker to give a density of 4.75 x 10 ⁹ cells / ml.

PL 233 261 Β1

Example III

The bacteria obtained as in Example 1 were placed in a pH 2 and 3 environment. At pH 3.0, the cell survival was 97% after 60 minutes and 94% after 240 minutes. In contrast, at pH 2.0, 95% of cells survived after 60 minutes, and 90% after 240 minutes.

Example IV

The bacteria obtained as in Example 1 were exposed to bile concentrations of 1% and 2% for up to 4 hours. After 4 hours of incubation in the presence of 2% bile, cell survival was 91%, and at 1% bile, 93% of bacterial cells survived.

Example V

The lyophilisate of bacteria obtained as in Example 1 was stored at 4-5 ° C and at room temperature 25 ° C for 4 months. The cooling temperature provided greater stability and durability. The storage of LOCK 1094 for 4 months reduced the number of viable cells by 7% for the refrigerated temperature and by 14% for the room temperature.

List of the nucleotide sequence of the DNA region encoding the 16S rRNA gene of the Lactobacillus pentosus LOCK 1094 bacterial strain, which defines its species affiliation:

· TCGACGAACTCTGGTATTGATTGGTGCTTGCATCATGATTTACATTTGAGTGAGTGGCGAACTGGTGAGTAACACGTGGGAAACCTGCCCAGAAGCGGGGGATAACACCTGGAAACAGATGCTAATACCGCATAACAACTTGGACCGCATGGTCCGAGTTGGAAAGATGGCTTCGGCTATCACTTTTGGATGGTCCCGCGGCGTATTAGCTAGATGGTGGGGTAACGGCTCACCATGGCAATGATACGTAGCCGACCTGAGAGGGTAATCGGCCACATTGGGACTGAGACACGGCCCAAACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCACAATGGACGAAAGTCTGATGGAGCAACGCCGCGTGAGTGAAGAAGGGTTTCGGCTCGTAAAACTCTGTTGTTAAAGAAGAACATATCTGAGAGTAACTGTTCAGGTATTGACGGTATTTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGTTTTTTAAGTCTGATGTGAAAGCCTTCGGCTCAACCGAA GAAGTGCATCGGAAACTGGGAAACTTGAGTGCAGAAGAGGACAGTGGAACTCCATGTGTAGCGGTGAAATGCGTAGATATATGGAAGAACACCAGTGGCGAAGGCGGCTGTCTGGTCTGTAACTGACGCTGAGGCTCGAAAGTATGGGTAGCAAACAGGATTAGATACCCTGGTAGTCCATACCGTAAACGATGAATGCTAAGTGTTGGAGGGTTTCCGCCCTTCAGTGCTGCAGCTAACGCATTAAGCATTCCGCCTGGGGAGTACGGCCGCAAGGCTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCTACGCGAAGAACCTTACCAGGTCTTGACATACTATGCAAATCTAAGAGATTAGACGTTCCCTTCGGGGA CATGGATACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATTATCAGTTGCCAGCATTAAGTTGGGCACTCTGGTGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGATGGTACAACGAGTTGCGAACTCGCGAGAGTAAGCTAATCTCTTAAAGCCATTCTCAGTTCGGATTGTAGGCTGCAACTCGCCTACATGAAGTCGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTTGGAACCCCCGCCCCNCCACACCTGGAAAGTTTGGAAACCCCCAAAGGCGGGGGGGGAAACC in which A - represents adenine, G - guanine C - cytosine T - thymine.

Claims (1)

Patent claim 1. Lactobacillus pentosus LOCK 1094 strain of lactic bacteria deposited in the Polish Collection of Microorganisms at the Polish Institute of Immunology and Experimental Therapy. Academy of Sciences Ludwik Hirszfeld in Wrocław, under the number B / 00124, with probiotic properties.