PL229020B1 - New strain of Bifidobacterium breve - Google Patents
New strain of Bifidobacterium breveInfo
- Publication number
- PL229020B1 PL229020B1 PL406050A PL40605013A PL229020B1 PL 229020 B1 PL229020 B1 PL 229020B1 PL 406050 A PL406050 A PL 406050A PL 40605013 A PL40605013 A PL 40605013A PL 229020 B1 PL229020 B1 PL 229020B1
- Authority
- PL
- Poland
- Prior art keywords
- bifidobacterium breve
- strain
- bifidobacterium
- breve
- resistance
- Prior art date
Links
- 241000186012 Bifidobacterium breve Species 0.000 title claims description 46
- 230000001580 bacterial effect Effects 0.000 claims description 8
- 241000186000 Bifidobacterium Species 0.000 description 7
- 239000003833 bile salt Substances 0.000 description 7
- 210000001035 gastrointestinal tract Anatomy 0.000 description 7
- 210000004051 gastric juice Anatomy 0.000 description 6
- 238000000034 method Methods 0.000 description 6
- 241000894006 Bacteria Species 0.000 description 5
- 229940093761 bile salts Drugs 0.000 description 4
- 230000002068 genetic effect Effects 0.000 description 4
- 244000005700 microbiome Species 0.000 description 4
- 241000741973 Bifidobacterium breve DSM 20213 = JCM 1192 Species 0.000 description 3
- 241000588724 Escherichia coli Species 0.000 description 3
- 230000003042 antagnostic effect Effects 0.000 description 3
- 230000015556 catabolic process Effects 0.000 description 3
- 238000006731 degradation reaction Methods 0.000 description 3
- 238000004445 quantitative analysis Methods 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 238000012163 sequencing technique Methods 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- 108020004465 16S ribosomal RNA Proteins 0.000 description 2
- 241000194032 Enterococcus faecalis Species 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- 241000191963 Staphylococcus epidermidis Species 0.000 description 2
- 241000193985 Streptococcus agalactiae Species 0.000 description 2
- 230000006978 adaptation Effects 0.000 description 2
- 239000003242 anti bacterial agent Substances 0.000 description 2
- 230000000845 anti-microbial effect Effects 0.000 description 2
- 229940088710 antibiotic agent Drugs 0.000 description 2
- 239000002246 antineoplastic agent Substances 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 229940127089 cytotoxic agent Drugs 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 238000012268 genome sequencing Methods 0.000 description 2
- 244000005709 gut microbiome Species 0.000 description 2
- LWGJTAZLEJHCPA-UHFFFAOYSA-N n-(2-chloroethyl)-n-nitrosomorpholine-4-carboxamide Chemical compound ClCCN(N=O)C(=O)N1CCOCC1 LWGJTAZLEJHCPA-UHFFFAOYSA-N 0.000 description 2
- 241000894007 species Species 0.000 description 2
- 102100022524 Alpha-1-antichymotrypsin Human genes 0.000 description 1
- 241000193163 Clostridioides difficile Species 0.000 description 1
- 241000273265 Clostridioides difficile ATCC 9689 = DSM 1296 Species 0.000 description 1
- 241000193403 Clostridium Species 0.000 description 1
- 108091035707 Consensus sequence Proteins 0.000 description 1
- 108020004414 DNA Proteins 0.000 description 1
- 238000001712 DNA sequencing Methods 0.000 description 1
- 206010064147 Gastrointestinal inflammation Diseases 0.000 description 1
- 101000678026 Homo sapiens Alpha-1-antichymotrypsin Proteins 0.000 description 1
- 241000699670 Mus sp. Species 0.000 description 1
- 102000057297 Pepsin A Human genes 0.000 description 1
- 108090000284 Pepsin A Proteins 0.000 description 1
- 241000191967 Staphylococcus aureus Species 0.000 description 1
- 241000194017 Streptococcus Species 0.000 description 1
- RTAQQCXQSZGOHL-UHFFFAOYSA-N Titanium Chemical compound [Ti] RTAQQCXQSZGOHL-UHFFFAOYSA-N 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 235000011054 acetic acid Nutrition 0.000 description 1
- 150000001243 acetic acids Chemical class 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 210000000481 breast Anatomy 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 238000012790 confirmation Methods 0.000 description 1
- 235000013365 dairy product Nutrition 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 229940032049 enterococcus faecalis Drugs 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000000855 fermentation Methods 0.000 description 1
- 230000004151 fermentation Effects 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 230000002496 gastric effect Effects 0.000 description 1
- 230000035784 germination Effects 0.000 description 1
- 210000005260 human cell Anatomy 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 230000000968 intestinal effect Effects 0.000 description 1
- 235000014655 lactic acid Nutrition 0.000 description 1
- 230000001926 lymphatic effect Effects 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 229940111202 pepsin Drugs 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 239000006041 probiotic Substances 0.000 description 1
- 230000000529 probiotic effect Effects 0.000 description 1
- 235000018291 probiotics Nutrition 0.000 description 1
- 238000011321 prophylaxis Methods 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 238000012175 pyrosequencing Methods 0.000 description 1
- 235000000346 sugar Nutrition 0.000 description 1
- 150000008163 sugars Chemical class 0.000 description 1
- 239000013589 supplement Substances 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 229910052719 titanium Inorganic materials 0.000 description 1
- 239000010936 titanium Substances 0.000 description 1
- 230000005945 translocation Effects 0.000 description 1
- 238000011282 treatment Methods 0.000 description 1
- 238000012070 whole genome sequencing analysis Methods 0.000 description 1
Landscapes
- Coloring Foods And Improving Nutritive Qualities (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Description
Opis wynalazkuDescription of the invention
Przedmiotem wynalazku jest nowy szczep Bifidobacterium breve oznaczony symbolem PB 04. Szczepy z rodzaju Bifidobacterium są w znacznej ilości obecne w ludzkiej jelitowej florze bakteryjnej. Uważa się, że mają one bardzo korzystny wpływ na stan zdrowia. Przeprowadzone badania wykazały, że bakterie z rodzaju Bifidobacterium zwiększają odporność organizmu ludzkiego na infekcje.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.
Znany jest z polskiego opisu patentowego PL 207831 szczep Bifidobacterium breve zdeponowany w CNCM w Paryżu pod numerem 1-2219. Szczep ten ma następującą charakterystykę: morfologia: krótkie pałeczki o rzadko spotykanych kształtach Y i V, metabolizm: anaerobioza;wytwarzanie kwasów L-(+)-mlekowego i octowego, fermentacja cukrów.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.
Produkt mleczny według wynalazku inokulowany szczepem Bifidobacterium breve CNCM 1-2219 podawano myszom a następnie badano rozwój flory kałowej i regulację zjawiska translokacji. Zaobserwowano zwiększenie liczby Bifidobacterium i bardzo duże zmniejszenie liczby Clostridium w narządach limfatycznych.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.
Celem wynalazku jest przebadanie bakterii z rodzaju Bifidobacterium breve posiadających korzystny wpływ na organizmy i wyselekcjonowanie szczepu o unikalnych właściwościach przejawiających się w zdolności do regulacji mikroflory jelitowej.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.
Istotą wynalazku jest nowy szczep Bifidobacterium breve oznaczony symbolem PB 04 zdeponowany w Polskiej Kolekcji Mikroorganizmów we Wrocławiu pod numerem B/00028 wykazujący zdolność przeżycia w przewodzie pokarmowym i jego znaczne zasiedlenie.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.
Szczep Bifidobacterium breve PB 04 został zdeponowany zgodnie z traktatem budapeszteńskim o międzynarodowym uznawaniu depozytu drobnoustrojów dla celów postępowania patentowego w Polskiej Kolekcji Mikroorganizmów (PCM) w Instytucie Immunologii i Terapii Doświadczalnej Polskiej Akademii Nauk we Wrocławiu (53-114 Wrocław, ul. Rudolfa Weigla 12). Nowy szczep Bifidobacterium breve PB04 został zdeponowany w dniu 4.06.2009r i otrzymał numer B/00028. W dacie 1.03.2013 roku organ depozytowy Polska Kolekcja Mikroorganizmów wydał dokument z przeprowadzonej atestacji dotyczącej komunikatu o późniejszym wskazaniu lub zmianie opisu naukowego i/lub proponowanego oznaczenia taksonomicznego. Przeprowadzona atestacja potwierdza dane zawarte w dokumencie depozytowym szczepu Bifidobacterium breve PB 04. I. Pochodzenie szczepu.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.
Szczep Bifidobacterium breve PB 04 został wyizolowany z mikroflory jelita zdrowego noworodka urodzonego siłami natury, karmionego mlekiem matki, bez objawów stanu zapalnego przewodu pokarmowego. II. Identyfikacja genetyczna szczepu Bifidobacterium breve PB 04. 1. Potwierdzenie przynależności gatunkowej szczepu, z zastosowaniem międzynarodowo uznanej metody molekularnej, tj. sekwencjonowania DNA kodującego 16S rRNA.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.
Przynależność gatunkową szczepu Bifidobacterium breve PB 04 określono za pomocą metody sekwencjonowania genu kodującego 16S rRNA. W wyniku reakcji sekwencjonowania DNA, przeprowadzonej zgodnie ze standardami EMQN, uzyskano sekwencję długości 1383 par zasad, którą porównano z aktualnymi depozytami zamieszczonymi w bazie danych GenBank. Otrzymany wynik potwierdził przynależność szczepu Bifidobacterium breve PB 04 do gatunku Bifidobacterium breve.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.
Uzyskano następujący wzór sekwencji:The following sequence pattern was obtained:
GTGGTGAGAGTGGCGAACGGGTGAGTAATGCGTGACCGACCTGCCCCATGCACGTGGTGAGAGTGGCGAACGGGTGAGTAATGCGTGACCGACCTGCCCCATGCAC
CGGAATAGCTCCTGGAAACGGGTGGTAATGCCGGATGCTCCATCACACCGCATCGGAATAGCTCCTGGAAACGGGTGGTAATGCCGGATGCTCCATCACACCGCAT
GGTGTGTTGGGAAAGCCTTTGCGGCATGGGATGGGGTCGCGTCCTATCAGCTTGGTGTGTTGGGAAAGCCTTTGCGGCATGGGATGGGGTCGCGTCCTATCAGCTT
GATGGCGGGGTAACGGCCCACCATGGCTTCGACGGGTAGCCGGCCTGAGAGGGATGGCGGGGTAACGGCCCACCATGGCTTCGACGGGTAGCCGGCCTGAGAGG
GCGACCGGCCACATTGGGACTGAGATACGGCCCAGACTCCTACGGGAGGCAGGCGACCGGCCACATTGGGACTGAGATACGGCCCAGACTCCTACGGGAGGCAG
CAGTGGGGAATATTGCACAATGGGCGCAAGCCTGATGCAGCGACGCCGCGTGACAGTGGGGAATATTGCACAATGGGCGCAAGCCTGATGCAGCGACGCCGCGTGA
GGGATGGAGGCCTTCGGGTTGTAAACCTCTTTTGTTAGGGAGCAAGGCACTTTGGGGATGGAGGCCTTCGGGTTGTAAACCTCTTTTGTTAGGGAGCAAGGCACTTTG
T GTT G AGT GT ACCTTTCG AATAAGCACCGGCT AACT ACGTGCCAGCAGCCGCGT GTT G AGT GT ACCTTTCG AATAAGCACCGGCT AACT ACGTGCCAGCAGCCGCG
GTAATACGTAGGGTGCAAGCGTTATCCGGAATTATTGGGCGTAAAGGGCTCGTAGTAATACGTAGGGTGCAAGCGTTATCCGGAATTATTGGGCGTAAAGGGCTCGTA
GGCGGTTCGTCGCGTCCGGTGTGAAAGTCCATCGCTTAACGGTGGATCCGCGCGGCGGTTCGTCGCGTCCGGTGTGAAAGTCCATCGCTTAACGGTGGATCCGCGC
CGGGTACGGGCGGGCTTGAGTGCGGTAGGGGAGACTGGAATTCCCGGTGTAACGGGTACGGGCGGGCTTGAGTGCGGTAGGGGAGACTGGAATTCCCGGTGTAA
CGGTGGAATGTGTAGATATCGGGAAGAACACCAATGGCGAAGGCAGGTCTCTGCGGTGGAATGTGTAGATATCGGGAAGAACACCAATGGCGAAGGCAGGTCTCTG
GGCCGTTACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATAGGCCGTTACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATA
CCCTGGTAGTCCACGCCGTAAACGGTGGATGCTGGATGTGGGGCCCGTTCCACCCCTGGTAGTCCACGCCGTAAACGGTGGATGCTGGATGTGGGGCCCGTTCCAC
GGGTTCCGTGTCGGAGCTAACGCGTTAAGCATCCCGCCTGGGGAGTACGGCCGGGTTCCGTGTCGGAGCTAACGCGTTAAGCATCCCGCCTGGGGAGTACGGCC
GCAAGGCTAAAACTCAAAGAAATTGACGGGGGCCCGCACAAGCGGCGGAGCATGCAAGGCTAAAACTCAAAGAAATTGACGGGGGCCCGCACAAGCGGCGGAGCAT
GCGGATTAATTCGATGCAACGCGAAGAACCTTACCTGGGCTTGACATGTTCCCGGCGGATTAATTCGATGCAACGCGAAGAACCTTACCTGGGCTTGACATGTTCCCG
ACGACCCCAGAGATGGGGTTTCCCTTCGGGGCGGGTTCACAGGTGGTGCATGGACGACCCCAGAGATGGGGTTTCCCTTCGGGGCGGGTTCACAGGTGGTGCATGG
TCGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAATCGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAA
CCCTCGCCCCGTGTTGCCAGCGGATTATGCCGGGAACTCACGGGGGACCGCCCCCTCGCCCCGTGTTGCCAGCGGATTATGCCGGGAACTCACGGGGGACCGCC
GGGGTTAACTCGGAGGAAGGTGGGGATGACGTCAGATCATCATGCCCCTTACGGGGGTTAACTCGGAGGAAGGTGGGGATGACGTCAGATCATCATGCCCCTTACG
TCCAGGGCTTCACGCATGCTACAATGGCCGGTACAACGGGATGCGACAGTGCGTCCAGGGCTTCACGCATGCTACAATGGCCGGTACAACGGGATGCGACAGTGCG
AGCTGGAGCGGATCCCTGAAAACCGGTCTCAGTTCGGATCGCAGTCTGCAACTAGCTGGAGCGGATCCCTGAAAACCGGTCTCAGTTCGGATCGCAGTCTGCAACT
CGACTGCGTGAAGGCGGAGTCGCTAGTAATCGCGAATCAGCAACGTCGCGGTGCGACTGCGTGAAGGCGGAGTCGCTAGTAATCGCGAATCAGCAACGTCGCGGTG
AATGCGTTCCCGGGCCTTGTACACACCGCCCGTCAAGTCATGAAAGTGGGCAGAATGCGTTCCCGGGCCTTGTACACACCGCCCGTCAAGTCATGAAAGTGGGCAG
CACCCGAAGCCGGTGGCCTAACCCCTTGCGGGAGGGAGCCGTCTAAGGTGAGCACCCGAAGCCGGTGGCCTAACCCCTTGCGGGAGGGAGCCGTCTAAGGTGAG
GCTCGTGA 2. Identyfikacja genetyczna szczepu Bifidobacterium breve PB 04 metodą sekwencjonowania genomu bakterii.GCTCGTGA 2. Genetic identification of the Bifidobacterium breve PB 04 strain by bacterial genome sequencing.
Identyfikację genetyczną szczepu Bifidobacterium breve PB 04 przeprowadzono za pomocą metody sekwencjonowania całego genomu bakterii. Sekwencjonowanie genomu bakterii Bifidobacterium breve PB 04 przeprowadzono metodą masowego pirosekwencjonowania w technologii 454 Titanium przy użyciu aparatu GS Junior firmy Roce w połączeniu ze standardowym sekwencjonowaniem metodą germinacji łańcucha dla wybranych fragmentów genomu. Uzyskano w ten sposób 93 227 556 zasad w 290 855 odczytach sekwencji, co dało 41,5 krotnie średnie pokrycie. Umożliwiło to ustalenie sekwencji konsensusu z dokładnością odpowiadającą obecności najwyżej jednego błędnego nukleotydu na milion zasad.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.
Identyfikacja genetyczna szczepu Bifidobacterium breve PB 04 powyższymi metodami wykazała, że należy on do gatunku Bifidobacterium breve. III. Wynalazek ilustrują poniższe przykłady, w których opisano przeprowadzone doświadczenia dokumentujące unikalne właściwości szczepu Bifidobacterium breve PB 04 1 Badania oporności Bifidobacterium breve PB 04 i szczepu kontrolnego wobec wybranych antybiotyków i chemioterapeutyków przy zastosowaniu metody E-test.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.
Celem badania było określenie schematów oporności szczepu probiotycznego Bifidobacterium breve PB 04 na wybrane antybiotyki i chemioterapeutyki za pomocą metody ilościowej opartej na oznaczeniu wartości najmniejszych stężeń hamujących wzrost (MIC) przy użyciu E-testów. Badanie przeprowadzono dla Bifidobacterium breve PB 04, a także dla szczepu wzorcowego Bifidobacterium breve DSM 20213.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.
Tabela 1Table 1
Wrażliwość szczepów zaliczonych do gatunku Bifidobacterium breve na wybrane leki przeciwdrobnoustrojowe (MIC) określone metodą E-testów.Susceptibility of strains classified as Bifidobacterium breve to selected antimicrobial drugs (MIC) determined by E-tests.
Wartości MIC dla badanego szczepu Bifidobacterium breve PB 04 są porównywalne z wartościami MIC odczytanymi dla szczepu kontrolnego Bifidobacterium breve DSM 20213. Badany szczep Bifidobacterium breve PB 04 nie wykazywał nietypowego wzrostu oporności w zakresie badanych leków przeciwdrobnoustrojowych. 2. Badania oznaczania oporności szczepu Bifidobacterium breve PB 04 na sztuczny sok żołądkowy i sole żółci. 2.1. Oznaczenie oporności szczepu Bifidobacterium breve PB 04 na sztuczny sok żołądkowy.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.
Badane szczepy: Bifidobacterium breve PB 04 i szczep kontrolny - Bifidobacterium breve DSM 20213. W celu oznaczenia stopnia oporności bakterii z rodzaju Bifidobacterium na pH soku żołądkowego posłużono się zmodyfikowaną metodą Clarka. Zamiast stężonego kwasu solnego zastosowano sztuczny sok żołądkowy o pH równym 2,5. Wyniki podano w tabeli nr 2.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.
Tabela 2Table 2
Przeżvwalność szczepów z rodzaiu Bifidobacterium w sztucznym soku żołądkowym.Resistance of strains of the genus Bifidobacterium in artificial gastric juice.
Badany szczep Bifidobacterium breve PB 04 wykazał dużą oporność na niskie pH i działanie pepsyny sztucznego soku żołądkowego. Szczep ten po upływie 20 min. redukował swoją populację tylko o 1 log, co może świadczyć o jego doskonałym przystosowaniu do niesprzyjających warunków panującym w ludzkim przewodzie pokarmowym. 2. 2. Oznaczenie oporności szczepu Bifidobacterium breve PB 04 na sole żółci.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.
Oporność na sole żółci wyznaczono w oparciu o metodę Dashkevicz’a i Feighner’a. Intensywność wzrostu badanych szczepów i zabarwienia podłoża na kolor żółty oceniono w skali półilościowej od - do +++. Wyniki podano w tabeli Nr 3. T a b e l a 3.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.
Oporność szczepów z rodzaju Bifidobacterium na sole żółci w stężeniach: 1 g/l, 2 g/l, 5 g/l, 10 g/l, 20 g/l.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.
Szczep Bifidobacterium breve PB 04 wykazał rozkład soli żółci typowy dla rodzaju Bifidobacterium oraz gatunku Bifidobacterium breve tzn. wykazał on całkowity rozkład soli żółci (+++) dla stężenia równego 1 g/l, zaś dla pozostałych stężeń tj. 2 g/l, 5 g/l, 10 g/l, 20 g/l zaobserwowano słaby rozkład w metodzie półilościowej określony na poziomie (+).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 (+).
Badany szczep Bifidobacterium breve PB 04 wykazał dużą oporność zarówno na niskie pH, równe pH występujące w soku żołądkowym, jak i na sole żółci co świadczy o jego doskonałym przystosowaniu do warunków panujących w ludzkim przewodzie pokarmowym. 3. Badania adherencji Bifidobacterium breve PB 04 do ludzkich linii tkankowych HT-29 MTX i Caco-2.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.
Przygotowane preparaty oceniane były pod mikroskopem świetlnym pod powiększeniem 1000x. Średniej liczbie komórek bakteryjnych w polu widzenia przyporządkowano odpowiedni stopień adherencji zgodnie z podanym poniżej zakresem.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.
Tabela 4Table 4
Adherencja szczepu Bifidobacterium breve PB 04 do tkanki CaCo-2Adherence of Bifidobacterium breve PB 04 strain to CaCo-2 tissue
Tabela 5Table 5
Adherencja szczepu Bifidobacterium breve PB 04 do tkanki HT-29MTX.Adherence of Bifidobacterium breve PB 04 strain to HT-29MTX tissue.
Badany szczep Bifidobacterium breve PB 04 wykazał duży stopień adherencji do ludzkiej linii komórkowej.The tested strain Bifidobacterium breve PB 04 showed a high degree of adherence to the human cell line.
Badanie właściwości antagonistycznych szczepu Bifidobacterium breve PB 04 wobec patogenów przewodu pokarmowego za pomocą metody ilościowej.Study of the antagonistic properties of Bifidobacterium breve PB 04 against pathogens of the gastrointestinal tract using a quantitative method.
Do badań wybrano szczep Bifidobacterium breve PB 04 (w skrócie B. breve PB04). W badaniu zastosowano następujące bakteryjne czynniki etiologiczne (szczepy wskaźnikowe): • Escherichia coli 25922B/1 (w skrócie: E.coli), • Streptococcus agalactiae B/3 (w skrócie: S.aga/actiae), • Staphylococcus epidermidis B/2 (w skrócie: S. epidermidis), • Staphylococcus aureus B/5 (w skrócie: S. aureus), • Enterococcus faecalis (w skrócie: E. faecalis), • Clostridium difficile ATCC 9689 (w skrócie: Cl.difficile).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).
Wyniki badania przedstawiono w tabeli 6.The results of the study are presented in Table 6.
Tabela 6Table 6
Antagonistyczne działanie szczepu Bifidobacterium breve PB04 wobec wybranych bakterii wskaźnikowych, przy zastosowaniu metody ilościowej.Antagonistic activity of the Bifidobacterium breve PB04 strain against selected indicator bacteria using the quantitative method.
Badany szczep wykazuje bardzo silne antagonistyczne właściwości wobec bakterii wskaźnikowych należących do gatunku Streptococcus agalactiae i Streptococcus epidermidis oraz silne w stosunku do Escherichia coli i Clostridium difficile.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.
Badany szczep Bifidobacterium breve PB 04 został wybrany spośród szczepów, które w naturalny sposób kolonizują przewód pokarmowy. Indywidualne, specyficzne cechy szczepu Bifidobacterium breve PB04 preferują go do zastosowania w leczeniu i profilaktyce stanów będących wynikiem braku właściwej flory bakteryjnej przewodu pokarmowego. Przeprowadzone badania wykazały, że wybrany szczep bakterii Bifidobacterium breve PB04 spełnia wszystkie kryteria bezpieczeństwa stawiane szczepom bakterii podawanym jako uzupełnienie składu fizjologicznej flory przewodu pokarmowego.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.
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