Abstract
The aim of this study is to optimize molecular detection and quantification methods of probiotic bacteria in complex microbial communities that have long been difficult for traditional culture-based methods. Traditional and real-time PCR were optimized to detect and quantify Lactobacillus spp. and Bifidobacterium spp. in complex microbial community. Fish and shrimp sauce were used as a model for complex microbial community. Directly form samples, 4 DNA extraction methods, primers specificity, PCR, and real-time PCR procedures were optimized, tested in comparison with samples, enriched bacteria and related standard bacterial strains, E. coli, Bacteroides, Enterococcus and Salmonella. Results showed that extracted genomic DNA using Wizard® Genomic DNA Purification Kit showed the highest yield, quality and performance. Moreover, the specificity of the primer set specific for Lactobacillus spp. and Bifidobacterium spp. was checked and found highly specific. The sensitivity of real-time PCR was higher than the conventional PCR and its quantifying potential is very precise for the detection and quantification of Lactobacillus spp. but not Bifidobacterium spp. which was absent in the tested samples. In conclusion, PCR and real-time PCR assays could be used very efficiently in quantifying and detecting Lactobacillus spp. that are present in very PCR-suppressive and complex microbial environment.
Highlights
Probiotic bacteria are essential for healthy gastrointestinal function
The sensitivity of real-time PCR was higher than the conventional PCR and its quantifying potential is very precise for the detection and quantification of Lactobacillus spp. but not Bifidobacterium spp. which was absent in the tested samples
For detecting target bacteria indirectly through an enrichment step for comparison with DNA extracted directly from samples, 1ml of the homogenized mixture was inoculated into 9 ml of MRS Broth (Oxoid, UK) for Lactobacillus spp. and TPY Broth (Oxoid, UK) for Bifidobacterium spp. before incubated anaerobically for 72 h at 37°C in an anaerobic jar, which contained Anaerocult ® A (Merck, Germany)
Summary
Probiotic bacteria are essential for healthy gastrointestinal function. The action of probiotics on intestinal flora results in vital benefits, including protection against pathogens, development of the immune system (Isolauri et al, 2002) and positive effects on colonic health and host nutrition (Umesaki and Setoyama, 2000). The use of Bifidobacterium as probiotics in food or pharmaceutical products is of high value. Lactobacilli bacteria are extensively used as probiotics and used technologically as food-associated micro-organism as they are generally recognized as safe (GRAS) (Salminen et al, 1998). Probiotic bacteria are found in some diet abundantly rendering such diet as a health supply for proper function of the intestine. The use of fermentation as a preservation method for fish has been of great value from earliest times. Among these products, fish sauce and shrimp sauce are the most popular products which are being used as a condiment in Southeast Asia (Yung et al, 2006). As fish fermentation involves minimal bacterial conversion of carbohydrates to lactic acid but entails extensive tissue degradation by
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
