Identifikace vybraných genů v bakteriích mléčného kvašení se zaměřením na potravinové doplňky
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Vysoké učení technické v Brně. Fakulta chemická
Abstract
Baktérie mliečneho kvasenia sa prirodzene vyskytujú v gastrointektinálnom trakte človeka. Medzi najvýznamnejšie patria rody Lactobacillus a Bifidobacterium, ktoré obsahujú rad probiotických druhov. Probiotické druhy tvoria súčasť doplnkov stravy. Na presnú a rýchlu identifikáciu tohto rodu a jeho druhov sa využívajú DNA amplifikačné metódy. Cieľom tejto práce bola izolácia a purifikácia DNA z vybraných doplnkov stravy pomocou magnetického nosiča P(HEMA-co-GMA), pokrytého karboxylovými skupinami, v prítomnosti PEG 6000 (16%) a NaCl (2 M) (konečné koncentrácie). Na izoláciu boli použité hrubé lyzáty buniek. Zo 4 doplnkov stravy bol pripravený hrubý lyzát buniek pomocou lyzozýmu, SDS a proteinázy K. Bola uskutočnená optimalizácia množstva pridaného lyzozýmu: 6, 9, 10, 15, 20 a 25 mg/ml). Koncentrácia DNA vo vzorkách bola zmeraná spektrofotometricky. Izolovaná DNA bola použitá ako matrica pre PCR, a to pre PCR s univerzálnymi primermi (veľkosť PCR produktu: 466 bp), rodovo špecifickú PCR pre rod Lactobacillus, rod Bifidobacterium a druhovo špecifické PCR pre druh Lactobacillus rhamnosus, Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus fermentum, pre skupinu Lactobacillus casei/paracasei, pre druh Streptococcus thermophilus, Bifidobacterium longum, Bifidobacterium bifidum a Bifidobacterium infantis. Amplifikované PCR produkty boli preukázané pomocou gélovej agarózovej elektroforézy. Výsledky boli porovnané s údajmi uvedenými výrobcami. Vo všetkých výrobkoch boli preukázané baktérie deklarovaných rodov.
Lactic acid bacteria are natural habitants of human gastrointectinal tract. Among the most important are bacteria of genus Lactobacillus and genus Bifidobacterium that contain a lot of probiotic species. Probiotic species are used as food supplements. This work was focused on DNA separation from crude cell lysates of 4 food supplements using magnetic carrier P(HEMA-co-GMA) covered by carboxyl groups. DNA was reversible adsorbed to the carriers in the presence of PEG 6000 (16%) and NaCl (2 M) (final concentrations) and eluted into TE buffer. Lysis of cells from food supplements was performed by lysozyme, SDS and proteinase K. The amount of lysozyme was optimalized. Concentration of separated DNA was measured by spectrophotometric method. The amount of isolated DNA was suitable for PCR. Isolated DNA was used for PCR with universal primers, PCR specific for genus Lactobacillus and genus Bifidobacterium and for 9 different species-specific PCRs: Lactobacillus rhamnosus, Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus fermentum, Lactobacillus casei/paracasei, Streptococcus thermophilus, Bifidobacterium longum, Bifidobacterium bifidum and Bifidobacterium infantis. Amplicons were detected by agarose gel electrophoresis (1,8%). It was shown that DNA amplification methods are quick and precise for identification of studied species. The results of bacteria identification were compared with data provided by the manufacturer. In all food supplements, bacteria of genus Lactobacillus and Bifidobacterium were detected. However, only some species provided by manufacturer were identified by PCR in each tablet.
Lactic acid bacteria are natural habitants of human gastrointectinal tract. Among the most important are bacteria of genus Lactobacillus and genus Bifidobacterium that contain a lot of probiotic species. Probiotic species are used as food supplements. This work was focused on DNA separation from crude cell lysates of 4 food supplements using magnetic carrier P(HEMA-co-GMA) covered by carboxyl groups. DNA was reversible adsorbed to the carriers in the presence of PEG 6000 (16%) and NaCl (2 M) (final concentrations) and eluted into TE buffer. Lysis of cells from food supplements was performed by lysozyme, SDS and proteinase K. The amount of lysozyme was optimalized. Concentration of separated DNA was measured by spectrophotometric method. The amount of isolated DNA was suitable for PCR. Isolated DNA was used for PCR with universal primers, PCR specific for genus Lactobacillus and genus Bifidobacterium and for 9 different species-specific PCRs: Lactobacillus rhamnosus, Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus fermentum, Lactobacillus casei/paracasei, Streptococcus thermophilus, Bifidobacterium longum, Bifidobacterium bifidum and Bifidobacterium infantis. Amplicons were detected by agarose gel electrophoresis (1,8%). It was shown that DNA amplification methods are quick and precise for identification of studied species. The results of bacteria identification were compared with data provided by the manufacturer. In all food supplements, bacteria of genus Lactobacillus and Bifidobacterium were detected. However, only some species provided by manufacturer were identified by PCR in each tablet.
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Citation
KRISTOVÁ, M. Identifikace vybraných genů v bakteriích mléčného kvašení se zaměřením na potravinové doplňky [online]. Brno: Vysoké učení technické v Brně. Fakulta chemická. 2010.
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Document version
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Language of document
sk
Study field
Potravinářská chemie a biotechnologie
Comittee
prof. Ing. Peter Šimko, DrSc. (předseda)
doc. Ing. Jiřina Omelková, CSc. (místopředseda)
prof. RNDr. Ivana Márová, CSc. (člen)
doc. RNDr. Alena Španová, CSc. (člen)
doc. Ing. Bohuslav Rittich, CSc. (člen)
Date of acceptance
2010-06-09
Defence
1. Diplomantka seznámila členy komise s náplní a cílem diplomové práce.
2. Byly přečteny posudky na diplomovou práci.
3. Diplomantka akceptovala všechny připomínky oponenta a na všechny otázky odpověděla v plné šíři.
Result of defence
práce byla úspěšně obhájena
Document licence
Standardní licenční smlouva - přístup k plnému textu bez omezení