PL229020B1 - New strain of Bifidobacterium breve - Google Patents

Description

Description of the invention

The subject of the invention is a new strain of Bifidobacterium breve marked with the symbol PB 04. Strains of the genus Bifidobacterium are present in large amounts in the human intestinal bacterial flora. They are believed to have a very beneficial effect on health. The conducted research has shown that bacteria of the genus Bifidobacterium increase the resistance of the human body to infections.

The Bifidobacterium breve strain deposited at the CNCM in Paris under the number 1-2219 is known from the Polish patent description PL 207831. This strain has the following characteristics: morphology: short sticks of rare Y and V shapes, metabolism: anaerobiosis; production of L - (+) - lactic and acetic acids, fermentation of sugars.

The dairy product according to the invention inoculated with the strain Bifidobacterium breve CNCM 1-2219 was administered to mice and then the development of the faecal flora and the regulation of the translocation phenomenon were examined. An increase in the number of Bifidobacterium and a very large decrease in the number of Clostridium in the lymphatic organs was observed.

The aim of the invention is to test bacteria of the genus Bifidobacterium breve having a beneficial effect on organisms and to select a strain with unique properties manifested in the ability to regulate the intestinal microflora.

The essence of the invention is a new strain of Bifidobacterium breve marked with the symbol PB 04 deposited in the Polish Collection of Microorganisms in Wrocław under the number B / 00028, showing the ability to survive in the gastrointestinal tract and its significant colonization.

The Bifidobacterium breve PB 04 strain has been deposited in accordance with the Budapest Treaty on the international recognition of the deposit of microorganisms for the purposes of patent proceedings at the Polish Collection of Microorganisms (PCM) at the Institute of Immunology and Experimental Therapy of the Polish Academy of Sciences in Wrocław (53-114 Wrocław, ul. Rudolfa Weigla 12) . The new strain Bifidobacterium breve PB04 was deposited on 04.06.2009 and received the number B / 00028. On March 1, 2013, the depository authority, the Polish Collection of Microorganisms, issued an attestation document regarding the announcement or amendment of the scientific description and / or the proposed taxonomic designation. The attestation carried out confirms the data contained in the deposit document of the Bifidobacterium breve PB 04 strain. I. Strain origin.

The Bifidobacterium breve PB 04 strain was isolated from the intestinal microflora of a healthy, natural-born infant, breast-fed, with no symptoms of gastrointestinal inflammation. II. Genetic identification of the strain Bifidobacterium breve PB 04. 1. Confirmation of the species identity of the strain, using an internationally recognized molecular method, ie sequencing of DNA encoding 16S rRNA.

The species affiliation of the Bifidobacterium breve PB 04 strain was determined by the method of gene sequencing encoding 16S rRNA. The DNA sequencing reaction, performed according to the EMQN standards, resulted in a sequence of 1383 bp in length which was compared with the actual deposits listed in the GenBank database. The obtained result confirmed that the Bifidobacterium breve PB 04 strain belongs to the species Bifidobacterium breve.

The following sequence pattern was obtained:

GTGGTGAGAGTGGCGAACGGGTGAGTAATGCGTGACCGACCTGCCCCATGCAC

CGGAATAGCTCCTGGAAACGGGTGGTAATGCCGGATGCTCCATCACACCGCAT

GGTGTGTTGGGAAAGCCTTTGCGGCATGGGATGGGGTCGCGTCCTATCAGCTT

GATGGCGGGGTAACGGCCCACCATGGCTTCGACGGGTAGCCGGCCTGAGAGG

GCGACCGGCCACATTGGGACTGAGATACGGCCCAGACTCCTACGGGAGGCAG

CAGTGGGGAATATTGCACAATGGGCGCAAGCCTGATGCAGCGACGCCGCGTGA

GGGATGGAGGCCTTCGGGTTGTAAACCTCTTTTGTTAGGGAGCAAGGCACTTTG

T GTT G AGT GT ACCTTTCG AATAAGCACCGGCT AACT ACGTGCCAGCAGCCGCG

GTAATACGTAGGGTGCAAGCGTTATCCGGAATTATTGGGCGTAAAGGGCTCGTA

GGCGGTTCGTCGCGTCCGGTGTGAAAGTCCATCGCTTAACGGTGGATCCGCGC

CGGGTACGGGCGGGCTTGAGTGCGGTAGGGGAGACTGGAATTCCCGGTGTAA

CGGTGGAATGTGTAGATATCGGGAAGAACACCAATGGCGAAGGCAGGTCTCTG

GGCCGTTACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATA

CCCTGGTAGTCCACGCCGTAAACGGTGGATGCTGGATGTGGGGCCCGTTCCAC

GGGTTCCGTGTCGGAGCTAACGCGTTAAGCATCCCGCCTGGGGAGTACGGCC

GCAAGGCTAAAACTCAAAGAAATTGACGGGGGCCCGCACAAGCGGCGGAGCAT

GCGGATTAATTCGATGCAACGCGAAGAACCTTACCTGGGCTTGACATGTTCCCG

ACGACCCCAGAGATGGGGTTTCCCTTCGGGGCGGGTTCACAGGTGGTGCATGG

TCGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAA

CCCTCGCCCCGTGTTGCCAGCGGATTATGCCGGGAACTCACGGGGGACCGCC

GGGGTTAACTCGGAGGAAGGTGGGGATGACGTCAGATCATCATGCCCCTTACG

TCCAGGGCTTCACGCATGCTACAATGGCCGGTACAACGGGATGCGACAGTGCG

AGCTGGAGCGGATCCCTGAAAACCGGTCTCAGTTCGGATCGCAGTCTGCAACT

CGACTGCGTGAAGGCGGAGTCGCTAGTAATCGCGAATCAGCAACGTCGCGGTG

AATGCGTTCCCGGGCCTTGTACACACCGCCCGTCAAGTCATGAAAGTGGGCAG

CACCCGAAGCCGGTGGCCTAACCCCTTGCGGGAGGGAGCCGTCTAAGGTGAG

GCTCGTGA 2. Genetic identification of the Bifidobacterium breve PB 04 strain by bacterial genome sequencing.

The genetic identification of the Bifidobacterium breve PB 04 strain was performed using the whole genome sequencing method. Bifidobacterium breve PB 04 genome sequencing was performed by mass pyrosequencing in 454 Titanium technology using Roce's GS Junior apparatus in conjunction with standard chain germination sequencing for selected genome fragments. This resulted in 93,227,556 bases in 290,855 sequence readings, giving 41.5 fold average coverage. This allowed the consensus sequence to be established with an accuracy equivalent to the presence of at most one nucleotide error per million bases.

The genetic identification of the Bifidobacterium breve PB 04 strain by the above methods showed that it belongs to the species Bifidobacterium breve. III. The invention is illustrated by the following examples, in which the experiments carried out documenting the unique properties of the Bifidobacterium breve PB 04 strain were described. Research on the resistance of Bifidobacterium breve PB 04 and the control strain against selected antibiotics and chemotherapeutic agents using the E-test method.

The aim of the study was to determine the resistance patterns of the probiotic Bifidobacterium breve PB 04 strain to selected antibiotics and chemotherapeutic agents by means of a quantitative method based on determination of the minimum growth inhibitory concentration (MIC) values using E-tests. The study was carried out for Bifidobacterium breve PB 04, as well as for the reference strain Bifidobacterium breve DSM 20213.

Table 1

Susceptibility of strains classified as Bifidobacterium breve to selected antimicrobial drugs (MIC) determined by E-tests.

The MIC values for the tested strain Bifidobacterium breve PB 04 are comparable to the MIC values read for the control strain Bifidobacterium breve DSM 20213. The tested strain Bifidobacterium breve PB 04 showed no abnormal increase in resistance in terms of the tested antimicrobial drugs. 2. Studies on the determination of resistance of the Bifidobacterium breve PB 04 strain to artificial gastric juice and bile salts. 2.1. Determination of resistance of Bifidobacterium breve PB 04 strain to artificial gastric juice.

Tested strains: Bifidobacterium breve PB 04 and control strain - Bifidobacterium breve DSM 20213. In order to determine the degree of resistance of bacteria of the genus Bifidobacterium to gastric pH, the modified Clark method was used. Artificial gastric juice with a pH of 2.5 was used instead of concentrated hydrochloric acid. The results are given in Table 2.

Table 2

Resistance of strains of the genus Bifidobacterium in artificial gastric juice.

The tested strain Bifidobacterium breve PB 04 showed high resistance to low pH and the effect of pepsin in artificial gastric juice. This strain after 20 minutes. reduced its population by only 1 log, which may indicate its perfect adaptation to the unfavorable conditions prevailing in the human digestive tract. 2. 2. Determination of resistance of the Bifidobacterium breve PB 04 strain to bile salts.

The bile salt resistance was determined based on the Dashkevicz and Feighner method. The growth intensity of the tested strains and the yellow color of the medium were assessed on a semi-quantitative scale from - to +++. The results are given in Table 3. T a b e l a 3.

Resistance of strains of the genus Bifidobacterium to bile salts in concentrations: 1 g / l, 2 g / l, 5 g / l, 10 g / l, 20 g / l.

The Bifidobacterium breve PB 04 strain showed bile salt degradation typical of the genus Bifidobacterium and the species Bifidobacterium breve, i.e. it showed complete bile salt degradation (+++) for a concentration equal to 1 g / l, and for the remaining concentrations, i.e. 2 g / l, 5 g / l, 10 g / l, 20 g / l there was a poor semi-quantitative degradation determined at (+).

The tested strain of Bifidobacterium breve PB 04 showed high resistance to both low pH, equal pH in gastric juice, and to bile salts, which proves its perfect adaptation to the conditions in the human digestive tract. 3. Studies of Bifidobacterium breve PB 04 adherence to HT-29 MTX and Caco-2 human tissue lines.

The prepared preparations were assessed under a light microscope under magnification of 1000x. The average number of bacterial cells in the field of view was assigned an appropriate degree of adherence according to the range given below.

Table 4

Adherence of Bifidobacterium breve PB 04 strain to CaCo-2 tissue

Table 5

Adherence of Bifidobacterium breve PB 04 strain to HT-29MTX tissue.

The tested strain Bifidobacterium breve PB 04 showed a high degree of adherence to the human cell line.

Study of the antagonistic properties of Bifidobacterium breve PB 04 against pathogens of the gastrointestinal tract using a quantitative method.

The strain Bifidobacterium breve PB 04 (abbreviated as B. breve PB04) was selected for the study. The following bacterial etiological factors (indicator strains) were used in the study: • Escherichia coli 25922B / 1 (in short: E.coli), • Streptococcus agalactiae B / 3 (in short: S.aga / actiae), • Staphylococcus epidermidis B / 2 (abbreviated: S. epidermidis), • Staphylococcus aureus B / 5 (abbreviated: S. aureus), • Enterococcus faecalis (abbreviated: E. faecalis), • Clostridium difficile ATCC 9689 (abbreviated: Cl.difficile).

The results of the study are presented in Table 6.

Table 6

Antagonistic activity of the Bifidobacterium breve PB04 strain against selected indicator bacteria using the quantitative method.

The tested strain shows very strong antagonistic properties against indicator bacteria belonging to the species Streptococcus agalactiae and Streptococcus epidermidis and strong against Escherichia coli and Clostridium difficile.

The tested Bifidobacterium breve PB 04 strain was selected from strains that naturally colonize the gastrointestinal tract. Individual, specific features of the strain Bifidobacterium breve PB04 prefer it to be used in the treatment and prophylaxis of conditions resulting from the lack of proper bacterial flora of the digestive tract. The conducted research has shown that the selected bacterial strain Bifidobacterium breve PB04 meets all safety criteria for bacterial strains administered as a supplement to the physiological flora of the gastrointestinal tract.

Claims (2)

Patent claim 1. New bacterial strain Bifidobacterium breve PB 04 PCM B / 00028