PL233263B1 - Lactic bacterial strain of Lactobacillus reuteri - Google Patents

Description

Description of the invention

The subject of the invention is a new strain of lactic acid bacteria Lactobacillus reuteri 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, there are known strains of bacteria from the Lactobacillus reuteri species, such as Lactobacillus reuteri INLA 4, Lactobacillus reuteri ATCC55730, Lactobacillus reuteri DSM 17938, Lactobacillus reuteri M KKP 2048.

The subject of the invention is a new strain of lactic acid bacteria of the species Lactobacillus reuteri, marked with the symbol ŁOCK 1092 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 / 00123.

LOCK 1092 strain was isolated from piglet intestinal contents.

The ŁOCK 1092 strain is a gram-positive rod, immobile, non-sporulating. It is a strain with probiotic properties, being a homologue of Lactobacillus reuteri bacteria 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 Lactobacillus reuteri LOCK 1092 strain, the DNA region encoding the 1379 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 reuteri JCM 1112 species, the similarity of the new bacterial strain to the species Lactobacillus reuteri was found with a probability of 99.71%.

The new ŁOCK 1092 strain is grown on Rogosa medium, which is a selective medium for the cultivation of bacteria of the genus Lactobacillus sp., Transparent, yellow-brown in color, 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. In this medium, the strain grows as white-cream, round colonies with a smooth surface and regular edges, about 1-2 mm in diameter.

The ŁOCK 1091 strain is also grown on 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 the strain ŁOCK 1092

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

Physiological and biochemical characteristics of the ŁOCK 1092 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 1092 strain is able to break down glucose both in the presence of oxygen and under anaerobic conditions, i.e. by fermentation.

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According to the API 50 CHL test, the LOCK 1092 strain is capable of fermenting saccharides and their derivatives such as ribose, xylose, galactose, glucose, maltose, lactose, melibiosis, sucrose, raffinose.

The ŁOCK 1092 strain is characterized by relatively heterofermentative metabolism. Fermented glucose to form lactic acid 10.79 µmol / ml, acetic acid (4.04 µmol / ml), butyric acid (0.47 µmol / ml), acetaldehyde (0.08 µmol / ml) and ethanol ( 0.15 μmol / ml). The API ZYM test showed that the LOCK 1092 strain showed the activity of the following enzymes: alkaline phosphatase, esterase, a-galactosidase, β-galactosidase, a-glucosidase, β-glucosidase, acid phosphatase, phosphoamidase, leucine arylamidase , valine arylamidase, cystine arylamidase.In addition, it was found that LOCK 1092 strain does not produce hydrogen sulphide, nor ammonia from arginine, does not show the ability to degrade H2O2.

Ability to use prebiotics of ŁOCK 1092 strain

The ŁOCK 1092 strain is able to grow in a medium containing prebiotics such as starch, β-glucan, maltodextrin, inulin, pectin as the sole carbon source, but the growth varies depending on the type and concentration of the saccharide. The best source of carbon is inulin at a concentration of 2%. On the other hand, when comparing the growth of LOCK 1092 in the presence of prebiotics with the growth in the presence of glucose (reference test), it was found that all prebiotics tested showed better growth, as shown in Figure 1 in the figure. LOCK 1092 also produces the highest amounts of acid as a result of inulin fermentation. lactic acid, with the highest proportion of L (+) lactic acid, 88%. 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 1092

The ŁOCK 1092 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 pH range from 2.0 to 3.0. ŁOCK 1092 strain also shows high resistance to bile salts in concentrations of 1% and 2% for up to 4 hours. Strain ŁOCK 1092 is resistant to antibiotics most commonly used in farm animals, such as penicillin, kanamycin, amoxicillin, doxycycline, erythromycin, tetracycline This strain also shows resistance to coccidiostats such as diclazuril, decoquinate, narasin-nicrabazine.

In addition, the new strain exhibits antagonistic activity against the intestinal pathogens of animals and human zoonoses, such as Salmonella enteritidis, Salmonella typhimurium, Salmonella choleraesuis, Campylobacter jejuni, Campylobacter coli, Listeria monocytogenes.

The ŁOCK 1092 strain has little fecal activity. Of the three enzymes β-glucosidase, β-glucuronidase and urease, only β-glucosidase is active.

The strain also shows the ability to detoxify mycotoxins, such as aflatoxin Bi, ochratoxin A, fumonisin, T-2 toxin, zearalenone, deoxynivalenol, which can most often contaminate grains and animal feed.

Biotechnological features of strain ŁOCK 1092

The biotechnological characteristics of the LOCK 1092 strain were determined in three fermentation media (feed mixes (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 The composition of the fermentation media was based on 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 106 cfu / ml. Growth dynamics in fermentation broths (cfu / g feed) was determined in 4, 8, 12, 16 , 24 and 30 hours of cultivation, and the reference was cultivation in MRS medium (cfu / ml of medium). Growth dynamics are presented in the figure 2 in the figure, and the biomass yield and the culture productivity in the table below.

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Blackboard

Strain Yield of N cells [CFU / g; CFU / ml] Productivity P [cfu / kgx | i; jtk / hh] Proper growth rate JLlh ± Adaptation phase [h] Fermentation broth PI P2 P3 PI P2 P3 PI P2 P3 PI P2 P3 No. reuteri LOCK 1092 1.10 3.13 4.96 χ! Θ 'x10 ¹⁰ x10 ^p 3.65 1.04 1.65 χΙΟ ¹⁰ χΙΟ ¹² x10 ¹⁰ 0.15 0.16 0.21 8.42 5.80 4.18

MRS control medium

No. reuteri LOCK 1092 1.95x10 ' _ 6,48xlO ¹⁰ _ 0.09 0.95

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 yield of cells in this medium was 3.13 × 10 ¹⁰ cfu / g, and the productivity of the strain increased to the level of 1.04 × 10 ¹² cfu / kgxh. Moreover, it was found that the optimal medium for the growth of LOCK 1092 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 1092 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 1092 strain is used as a feed additive for monogastric animals in the prevention of bacterial diseases and mycotoxin poisoning, and improving the safety of animal husbandry.

The following examples illustrate the subject matter of the invention.

Example I

LOCK 1092 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 grown cells were separated from the substrate on the shaker at a density of 1.9 x 10 ⁹ cells / ml.

Example II

LOCK 1092 strain was grown in MRS liquid medium (Merck), 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-water-0.20, magnesium sulfate 4-water-0.05, o 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 1.95 x 10 ⁹ cells / ml.

Example III

The bacteria obtained as in Example 1 were placed in a pH 2 and 3 environment. At pH 3.0, the cell viability after 60 minutes is 95% and after 240 minutes it is 90%. In contrast, at a pH of 2.0, 93% of cells survive after 60 minutes, and 88% 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 is 92%, and with a bile concentration of 1%, 98% of bacterial cells survive.

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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. Storage of ŁOCK 1092 for 4 months reduced the number of viable cells by 8% for the refrigerated temperature and by 15% for the room temperature.

List of the nucleotide sequence of the DNA region encoding the 16S rRNA gene of the Lactobacillus reuteri ŁOCK 1092 bacterial strain, which defines its species affiliation:

TGATTGTATGGTGCTTGCACCTGATTGACGATGGATCACCAGTGAGTGGCGGACGGGTGAGTAACACGTAGGTAACCTGCCCCGGAGCGGGGGATAACATTTGGAAACAGATGCTAATACCGCATAACAACAAAAGCCACATGGCTTTTGTTTGAAAGATGGCTTTGGCTATCACTCTGGGATGGACCTGCGGTGCATTAGCTAGTTGGTAAGGTAACGGCTTACCAAGGCGATGATGCATAGCCGAGTTGAGAGACTGATCGGCCACAATGGAACTGAGACACGGTCCATACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCACAATGGGCGCAAGCCTGATGGAGCAACACCGCGTGAGTGAAGAAGGGTTTCGGCTCGTAAAGCTCTGTTGTTGGAGAAGAACGTGCGTGAGAGTAACTGTTCACGCAGTGACGGTATCCAACCAGAAAGTCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTATCCGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGTTGCTTAGGTCTGATGTGAAAGCCTTCGGCTTAACCGAAGAAGTGCATCGGAAACCGGGCGACTTGAGTGCAGAAGAGGACAGTGGAACTCCATGTGTAGCGGTGGAATGCGTAGATATATGGAAGAACACCAGTGGCGAAGGCGGCTGTCTGGTCTGCAACTGACGCTGAGGCTCGAAAGCATGGGTAGCGAACAGGATTAGATACCCTGGTAGTCCATGCCGTAAACGATGAGTGCTAGGTGTTGGAGGGTTTCCGCCCTTCAGTGCCGGAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCTACGCGAAGAACCTTACCAGGTCTTGACATCTTGCGCTAACCTTAGAGATAAGGCGTTCCCTTCGGGGACGCAATGAC AGGTGGTGCATGGTCGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGTTACTAGTTGCCAGCATTAAGTTGGGCACTCTAGTGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGACGACGTCAGATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGACGGTACAACGAGTCGCAAGCTCGCGAGAGTAAGCTAATCTCTTAAAGCCGTTCTCAGTTCGGACTGTAGGCTGCAACTCGCCTACACGAAGTCGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTTTGTA in which A - represents adenine, G - guanine C - cytosine T - thymine.

Claims (1)

Patent claim 1. Lactobacillus reuteri LOCK 1092 strain of lactic bacteria deposited in the Polish Collection of Microorganisms at the Institute of Immunology and Experimental Therapy of the Polish Academy of Sciences. Ludwik Hirszfeld in Wrocław under the number B / 00123, with probiotic properties.