Journal of Probiotics & Health
Open Access
ISSN: 2329-8901
ISSN: 2329-8901
Stefan R Herbel1, Markus Von Nickisch-Rosenegk2, Matthias Kuhn3, Jayaseelan Murugaiyan1, Lothar H Wieler1 and Sebastian Guenther1
1Freie Universit�?¤t Berlin, Germany
2Fraunhofer Institute, Germany
3CONGEN Biotechnologie GmbH, Germany
Posters-Accepted Abstracts: J Prob Health
Probiotics are believed to promote beneficial influences on the gastrointestinal tract and thereby on health in general. Therefore, they are often used in dairy and non-dairy products in human nutrition. Besides usage of probiotics to increase well-being of humans feeding of animals using probiotic bacteria has increased steadily, since European Union approved a prohibition using antibiotics as growth promoters. Classic diagnostic methods used for identification of species of the genera Lactobacillus and Bifidobacterium are including phenotypic comparison with reference strains and Polymerase Chain Reaction (PCR). In addition, present isolation methods of probiotic bacteria are usage of selective media and different growth conditions resulting in time-consuming, laborintensive diagnostic schemes by working culture-dependent. Furthermore, phenotypic characterization and species differentiation are error-prone. As probiotic action depends on quality, quantity and viability species detection by conventional PCR-detection method is not feasible, as it does not allows species-specific quantification within DNA sample directly extracted from probiotic source of choice. Thus, there is a need to establish a real time PCR (qPCR) method to comply with the requirements to identify and quantify probiotic strains in food without a prior cultivation step. Screening of different target-sequences for a specific identification of probiotic species ruled out classic targets such as 23s-5s rRNA (intergenic spacer) due to lacking in species-specificity. Other ones as heat shock proteins (hsp60) were chosen for identification and quantification of different members of the genus Lactobacillus. The ATPase subunit of the ATP-dependent clpC-gene was selected for the specific identification of members of the genus Bifidobacterium. In addition, we established an identification tool by MALDI-TOF MS analysis including the possibility to detect food pathogens if embedded in the database. This procedure allows a rapid inside view assuring species identity of probiotic organisms and an absence of organisms known as food spoiling ones. Within 45 minutes probiotic products are easily screened regarding contain of wanted and unwanted species by MALDI-TOF MS. Established qPCRs had been designed based on the same annealing conditions offering a fast and reliable detection, identification and quantification of different probiotic species in a single qPCR run within 7 hours (including DNA isolation, qPCR preparation and run + analyzing results). Thus, both techniques are easy to handle, less cost- and labor-intensive offering good validation performance for each product to screen. In addition, MALDI-TOF MS allows to be equipped by new protein data or specific primer pairs (qPCR) offering repeatable and standardized results of new species being added assuring product��?s quality and safety.
Email: stefan.herbel@fu-berlin.de