<i>Bifidobacterium animalis </i>AMT30 strain and the composition containing the strain of <i>Bifidobacterium animalis </i>AMT30

Abstract

Disclosed herein is a new strain of Bifidobacterium animalis AMT30 bacteria deposited by the number PCM B/00109 as well as composition for production of creams and ointments, parapharmaceutical, pharmaceutical, food preparations/products and food and water additives for humans and animals, consisting of the new strain of bacteria, medium and bulking agent distinguished by that the bacterial strain contains Bifidobacterium animalis AMT30, PCM B/00107 in amount of 10.sup.1 to 10.sup.13 of colony forming units cfu/ml-g. The composition may contain one or more strains of Lactobacillus plantarum bacteria.

Claims

1. A composition comprising a bacteria strain, medium and bulking agent, wherein the bacterial strain comprises a lyophilized strain of Bifidobacterium animalis AMT30, (PCM Accession No. B/00109) in an amount of 10.sup.3 to 10.sup.13 of colony forming units cfu/ml-g.

2. The composition of claim 1, wherein the composition is a cream, an ointment, a parapharmaceutical, a pharmaceutical, a food preparation or product, or a food and/or water additive for humans or animals.

Description

DETAILED DESCRIPTION

(1) Determination of Species Affiliation of the Bifidobacterium animalis AMT30.

(2) The Identification of the Strain

(3) The Bifidobacterium animalis AMT30 strain was isolated from the stool of an adult man. The strain was deposited under the Budapest Treaty in Polish Collection of Microorganisms (PCM) in the Institute of Immunology and Experimental Therapy of Polish Academy of Sciences (ul. Weigla 12, 53-114, Wroclaw, Poland). The deposit was lodged on 31 May 2016 and the number B/00109 was assigned.

(4) Isolation of the Bifidobacterium animalis AMT30 Strain.

(5) The strain isolation was performed using multistage screening method. The procedure of Bifidobacterium strain isolating was carried out on the basis of the culturing of tenfold dilutions of the material sample on the Garche's medium. The plates were incubated at 37° C. for 72 hours under anaerobic conditions (sealed jar with GasPac AN0025A, Oxoid, cartridge). Colonies of bacteria potentially belonging to the Bifidobacterium genus were multiplied on the liquid Garche's medium and were incubated at 37° C. for 24 hours under anaerobic conditions (sealed jar with GasPac AN0025A, Oxoid, cartridge).

(6) Culturing and multiplying procedure was carried out until pure cultures of bacteria, classified by microscopic observation to Bifidobacterium genus, were obtained.

(7) Confirmation of Isolated Strain Properties on the Genetic Level (RAPD and/or rep-PCR).

(8) In order to isolate the genomic DNA reagent kit Bacterial & Yeast Genomic DNA Purification Kit from Eurx (Gdańsk) and purification procedure for Gram-positive bacteria were applied. The quality of the isolated DNA was checked by electrophoresis (1% agarose, 0.5×TBE, 100 V, 30 min.)

(9) Isolates typing was performed applying rep-PCR method with the primer (GTG).sub.5 based on amplification technique as well as RAPD method (random amplification of polymorphic sections) with the primer S1 (CGACGTCATC). The procedure applied was identical to the previously described. (Markiewicz L. H., Biedrzycka E., Wasilewska E., Bielecka M. Rapid molecular identification and characteristics of Lactobacillus strains. Folia Microbiol., 2010, 55 (5), 481-488).

(10) Identification of the Isolated Bifidobacterium animalis AMT30 Strain Using Sequencing of Encoding Gene 16S rRNA Fragment.

(11) Identification of the isolated strain by sequencing of the fragment of the encoding gene 16S rRNA of at least 500 pairs was performed in the following stages:

(12) 1) DNA isolation from the colonies on Petri dishes.

(13) Isolation of genetic material (DNA) from colonies of microorganisms supplied on Petri dishes was made. DNA was extracted applying CHELEX resin method (Biorad) and in the presence of the enzymes that break down the cell wall.

(14) 2) The PCR reaction with specific primers and PCR matrix sequencing.

(15) In order to confirm the presence of bacteria in the examined sample PCR amplification of 16S rDNA fragments applying specific primers on DNA matrix isolated from colonies was made.

(16) Positive result of amplification was obtained. PCR products were purified and then sequencing was performed using the following kit: BigDye Terminator Mix v3.1 and genetic analyzer ABI3730x1 as well as specific primers. Obtained readouts (readouts from specific starters for bacterial 16S rDNA: 27F and 1492R) were assembled into the corresponding contigs obtaining consensus sequence.

(17) 3) Comparison of the obtaining sequences with NCBI database

(18) The obtained consensus sequences were compared with NCBI database—GeneBank using BLAST program.

(19) Comparative analysis with DNA sequences deposited in the Gene Bank (NCBI) showed that the analyzed sequence is identical with the sequence of Bifidobacterium animalis.

(20) In Vitro Study of Antagonistic Effect of Bifidobacterium animalis AMT30 Strain Against Pathogens of the Gastrointestinal Tract and Urogenital System.

(21) The study of antagonistic properties of Bifodobacterium animalis AMT30 strain against the following pathogens: Escherichia coli 0157:H7 (enterohaemorrhagic strain), Candida albicans 637, Candida krusei 8 was performed on the shared cultures in hydrolyzed milk (10% milk regenerated from skimmed powdered milk, hydrolyzed using pancreatin). Pathogenic strains used in vitro studies came from the Institute of Animal Reproduction and Food Research of Polish Academy of Sciences in Olsztyn. While the strain of Bifidobacterium animalis AMT30 comes from the private collection of microorganism from PROBIOS ltd. Pathogenic strains were stored frozen at −80° C. and directly before the study were activated by twofold passage in hydrolyzed milk.

(22) The shared cultures (examined samples) were inoculated using lyophilized Bifidobacterium animalis AMT30 strain at a level of 10.sup.9 colony forming units/ml (described hereinafter by commonly accepted abbreviation cfu/ml) as well as using active monoculture of pathogenic strain of the following species: Escherichia coli O157:H7, Candida albicans 637, Candida krusei 8 in an amount of 10.sup.5 cfu/ml.

(23) The single strains of pathogenic microorganisms (Escherichia coli 0157:H7, Candida albicans 637, Candida krusei 8) and monoculture of Bifidobacterium animalis AMT30 using inoculum level and liquid medium as in the shared culture were the control samples in the experiment.

(24) The shared cultures as well as single ones were prepared in four parallel tubes in triplicate. Incubation was carried out under anaerobic conditions at 37° C. for 0 (inoculum determining blank test) 24, 48 and 72 hours. After the incubation the number of live cells of Bifidobacterim animalis AMT30 bacteria as well as the number of pathogens were determined in shared and control cultures using plate count test on appropriate agar media (Table 1).

(25) The analysed material was diluted with 1% peptone water applying the method of serial tenfold dilutions and cultured on the bottom of Petrie dish, then the liquid agar medium at a temperature of 45° C. was poured. Directly after medium solidifying the plates were inverted upside down and incubated at 37° C. for 24 or 48 hours under aerobic or anaerobic conditions. Conditions of the culture are reported in Table 1. After the incubation, bacteria colonies of the shared cultures were counted and compared to the number of bacteria in the control cultures (single ones).

(26) TABLE-US-00001 TABLE 1 Culture conditions of the analysed bacterial strains the strain type Medium applied incubation conditions Bifidobacterium Modified Garche's medium by 37° C./48 hours, animalis AMT30 Teroguchi et al. (1982). anaerobic conditions composition of multiplying medium: created in sealed jar Meat peptone 20 g/L using GasPac cat. No. Yeast extract 2 g/L AN0025A cartridge, L-cysteine hydrochloride 0.4 g/L Oxoid. Lactose 10 g/L Sodium acetate 6 g/L MgSO.sub.4 × 7H.sub.2O 0.12 g/L Na.sub.2 HPO.sub.4 × 12H.sub.2O 2.5 g/L KH.sub.2PO.sub.4 2 g/L distilled water 1000 mL Garche's medium with agar (10 g/L) for quantitative determination of Bifidobacterium animalis AMT30 Escherichia coli Macconkey Agar (Merck, cat. no. 37° C./24 hours, aerobic O157:H7 1054650500) conditions Candida albicans 637, Agar Sabouraud with chloramphenicol 37° C./24 hours, aerobic Candida krusei 8 (BTL, cat no. P-0176) conditions

(27) Overall reduction in the number of bacteria of Escherichia coli O157:H7 enterohaemorrhagic strain as well as Candida albicans 637 and Candida krusei 8 fungi during 72 hours of incubation of shared culture with Bifidobacterium animalis AMT30 was found in the in vitro studies. The results are presented in Table 2.

(28) TABLE-US-00002 TABLE 2 Growth inhibition of pathogenic bacteria and fungi by Bifidobacterium animalis AMT30 The strain Inoculum 24 h of incubation 48 h of incubation 72 h of incubation Shared culture number of cfu/mL number of cfu/mL number of cfu/mL number of cfu/mL symbol AMT30 pathogen AMT30 pathogen AMT30 pathogen AMT30 pathogen Bifidobacterium 1.8 × 10.sup.9 2.4 × 10.sup.9 2.4 × 10.sup.9 2.1 × 10.sup.9 animalis AMT30 (monoculture) Escherichia coli 2.9 × 10.sup.5 1.1 × 10.sup.9 8.8 × 10.sup.8 5.2 × 10.sup.8 O157:H7 (monoculture) Candida albicans 676 6.1 × 10.sup.5 1.8 × 10.sup.7 1.5 × 10.sup.7 1.3 × 10.sup.7 (monoculture) Candida krusei 8 1.2 × 10.sup.5 4.1 × 10.sup.8 2.9 × 10.sup.8 2.4 × 10.sup.8 (monoculture) Bifidobacterium 1.8 × 10.sup.9 2.9 × 10.sup.5 2.7 × 10.sup.9 1.8 × 10.sup.6 2.5 × 10.sup.9 3.1 × 10.sup.2 2.1 × 10.sup.9 Abs* animalis AMT30 + Escherichia coli O157:H7 Bifidobacterium 1.8 × 10.sup.9 6.1 × 10.sup.5 2.7 × 10.sup.9 1.1 × 10.sup.6 3.1 × 10.sup.9 1.1 × 10.sup.3 2.8 × 10.sup.9 Abs* animalis AMT30 + Candida albicans 676 Bifidobacterium 1.8 × 10.sup.9 1.2 × 10.sup.5 2.5 × 10.sup.9 1.4 × 10.sup.6 3.6 × 10.sup.9 7.3 × 10.sup.2 3.8 × 10.sup.9 Abs* animalis AMT30 + Candida krusei 8 Abs*—absent in 1 ml of the culture shared with z Bifidobacterium animalis AMT30

(29) Data obtained on the basis of inhibiting the growth of pathogenic strains lead to the conclusion, that the selected strain of Bifidobacterium genus has antagonistic properties against selected strains of pathogenic bacteria and fungi.

Determination of Survivability of Bifidobacterium animalis AMT30 Strain at Low pH as Well as in the Presence of Bile Salts

(30) The survivability of Bifidobacterium animalis AMT30 strain at low pH was determined by acidity reduction of Bifidobacterium animalis AMT30 culture being in stationary phase of growth to a pH value of 3. However, in the case of survivability determination of Bifidobacterium animalis AMT30 strain in the presence of bile salts, at the beginning pH of Bifidobacterium animalis AMT30 culture was raised to a value of 6, then bile salts in an amount of 3% of the culture were added. Determination of survivability of Bifidobacterium animalis AMT30 strain at low pH was performed before lowering the pH of the culture (control sample), just after lowering the pH of the culture to a value of 3, so called minute 0, and after 40 and 180 minutes of incubation at 37° C. under anaerobic conditions. The survivability of Bifidobacterium animalis AMT30 in the presence of bile salts was determined prior to bile salts addition (control sample), just after addition of bile salts, so called minute 0 and after 1, 3 and 6 hours of incubation at 37° C. under anaerobic conditions. The live cells of Bifidobacterium animalis AMT30 was determined in colony forming units (cfu/ml) following the pour plate method. The survivability of Bifidobacterium animalis AMT30 strain was expressed as a percentage of the number of Bifidobacterium animalis AMT30 after 180 minutes in the case of survivability determining at pH value of 3, and after 6 hours in the case of the number determination of Bifidobacterium animalis AMT30 in the presence of bile salts compared with the number of Bifidobacterium animalis AMT30 strain in the control

(31) TABLE-US-00003 TABLE 3 Survivability of Bifidobacterium animalis AMT30 at pH = 3 number of bacteria (log10 cfu/ml) survivability before pH pH = 3 after180 lowering 40 180 minutes 0 0 minutes minutes % Bifidobacterium 9.17 9.16 9.18 9.28 100 animalis AMT30

(32) TABLE-US-00004 TABLE 4 Survivability of Bifidobacterium animalis AMT30 in the presence of 3% of bile salts number of bacteria (log10 cfu/ml) before bile survivability salts after bile salts addition after 6 h addition 0 h 1 h 3 h 6 h [%] Bifidobacterium 9.07 9.10 9.00 8.88 8.50 94 animalis AMT30
The examined strain of Bifidobacterium animalis AMT30 showed 100% of survivability at low pH=3 and 94% of survivability in the presence of bile salts in 3% concentration in the medium. The results showed high resistance of Bifidobacterium animalis AMT30 at low pH as well as in the presence of bile salts. This indicates that the Bifidobacterium animalis AMT30 strain adapts to the conditions of gastrointestinal tract.
Determination of Bifidobacterium animalis AMT30 Ability to Grow in the Presence of Selected Antibiotics Most Frequently Used in the Treatment of Animals

(33) Studies on the ability to grow of Bifidobacterium animalis AMT30 in the presence on 20 selected antibiotics were performed using shared cultures method. For this purpose Garche's liquid medium was inoculated with both Bifidobacterium animalis AMT30 in an amount of 4.9×10.sup.6 colony forming units/ml and one of 20 antibiotics in concentration corresponding to an antibiotic dose used in animal treatment per kilogram of body weight. The culture was carried out in three parallel tubes under anaerobic conditions at 37° C. for 24 hours. In addition, parallel control culture of the studied strain without presence of selected antibiotics was carried out. The number of the live cells was determined directly after inoculation and after 24 hours. Incubation of bacteria on Petri dishes was carried out at 37° C. for 48 hours under anaerobic conditions (sealed jar with GasPack AN0025A cartridge, Oxoid). The results are presented in Table 5

(34) TABLE-US-00005 TABLE 5 Determination of Bifidobacterium animalis AMT30 ability to grow in the presence of selected antibiotics most frequently used in the treatment of animals The number of B. animalis AMT30 Inoculum after 24 hours AMT30 of incubation Active substance [cfu/ml] [cfu/ml] AMT30 control culture without 4.9 × 10.sup.6 2.4 × 10.sup.9 antibiotic Tiamulin 20.2 mg/kg b.w. 4.9 × 10.sup.6 7.6 × 10.sup.5 Enrofloxacin 10 mg/kg b.w. 4.9 × 10.sup.6 4.4 × 10.sup.4 Trimethoprim/sulfamethoxazole 4.9 × 10.sup.6 3.2 × 10.sup.6 100 mg + 50 mg/kg b.w. Colistin 0.37 ml/10 kg b.w. 4.9 × 10.sup.6 1.9 × 10.sup.9 Florfenicol 20 mg/kg b.w. 4.9 × 10.sup.6 5.8 × 10.sup.5 Tilmicosin 20 mg/kg b.w. 4.9 × 10.sup.6 1.0 × 10.sup.6 Toltrazuril 7 mg/kg b.w. 4.9 × 10.sup.6 1.1 × 10.sup.9 Amprolium 20 mg/kg b.w. 4.9 × 10.sup.6 1.5 × 10.sup.9 Levamisole 25 mg/kg b.w. 4.9 × 10.sup.6 1.8 × 10.sup.9 Flubendazole 1.43 mg/kg b.w. 4.9 × 10.sup.6 1.4 × 10.sup.9 Doxycycline 50 mg/1 kg b.w. 4.9 × 10.sup.6 3.6 × 10.sup.5 Amoxicillin 20 mg/kg b.w. 4.9 × 10.sup.6 4.4 × 10.sup.5 Neomycin 20 mg/kg b.w. 4.9 × 10.sup.6 3.3 × 10.sup.7 Sodium sulfachloropyrazine 50 4.9 × 10.sup.6 2.4 × 10.sup.9 mg/kg b.w. Amoxicillin + clavulanic acid 8 4.9 × 10.sup.6 1.8 × 10.sup.5 mg/kg b.w. Phenoxymethylpenicillin 20 mg/kg 4.9 × 10.sup.6 8.0 × 10.sup.5 b.w. Lincomycin 5 mg/kg b.w. 4.9 × 10.sup.6 1.4 × 10.sup.3 Lincomycin + spectinomycin 150 mg 4.9 × 10.sup.6 9.2 × 10.sup.4 of antibiotic activity/kg b.w. Tylvalosin 40 mg/kg b.w. 4.9 × 10.sup.6 3.2 × 10.sup.5 Tylosin 100 mg/kg b.w. 4.9 × 10.sup.6 1.4 × 10.sup.6

(35) Bifidobacterium animalis AMT30 strain showed unique, broad spectrum of antibiotic resistance against studied antibiotics. Outstanding activity to multiply was found for AMT30 strain in the presence of 7 (Colistin, Toltrazuril, Amprolium, Levamisole, Flubendazole, Neomycin, Sodium sulfachloropyrazine) of the 20 studied antibiotics in relation to the number which the strain has reached in the control culture. However in the presence of Trimethoprim/sulfamethoxazole, Tilmicosin and Tylosin the number of Bifidobacterium animalis AMT30 remained at the level of inoculum. In the case of the cultures shared with Tiamulin, Enrofloxacin, Florfenicol, Amoxicillin, Doxycycline, Amoxycillin+clavulanic acid, Phenoxymethylpenicillin, Licomycin and Tylvalosin decrease in the number of bacteria of AMT30 strain from 1 to 3 orders of magnitude in comparison to the control culture was recorded. Bifidobacterium animalis AMT30 showed also antibiotic resistance against the most frequently used antibiotics in poultry farming.