Browse > Article
http://dx.doi.org/10.5713/ajas.2007.1887

Rapid Identification of Bifidobacteria in Dairy Products by Gene-targeted Species-specific PCR Technique and DGGE  

Hong, Wei-Shung (Department of Animal Science and Technology, National Taiwan University)
Chen, Ming-Ju (Department of Animal Science and Technology, National Taiwan University)
Publication Information
Asian-Australasian Journal of Animal Sciences / v.20, no.12, 2007 , pp. 1887-1894 More about this Journal
Abstract
In this paper, a rapid and reliable gene-targeted species-specific polymerase chain reaction (PCR) technique based on a two-step process was established to identify bifidobacteria in dairy products. The first step was the PCR assay for genus Bifidobacterium with genus specific primers followed by the second step, which identified the species level with species-specific primer mixtures. Ten specific primer pairs, designed from nucleotide sequences of the 16-23S rRNA region, were developed for the Bifidobacterium species including B. angulatum, B. animalis, B. bifidum, B. breve, B. catenulatum, B. infantis, B. longum, B. minimum, B. subtile, and B. thermophilum. This technique was applied to the identification of Bifidobacterium species isolated from 6 probiotic products, and four different Bifidobacterium spp. (B. bifidum, B. longum, B. infantis, and B. breve) were identified. The findings indicated that the 16S-23S rDNA gene-targeted species-specific PCR technique is a simple and reliable method for identification of bifidobacteria in probiotic products. PCR combined with Denaturing Gradient Gel Electrophoresis (DGGE) for identification of the bifidobacteria was also evaluated and compared with the gene-targeted species-specific technique. Results indicated that for fermented milk products consistency was found for both species-specific PCR and PCR-DGGE in detecting species. However, in some lyophilized products, the bands corresponding to these species were not visualized in the DGGE profile but the specific PCR gave a positive result.
Keywords
Bifidobacterium spp.; Species-specific PCR; DGGE;
Citations & Related Records
Times Cited By KSCI : 2  (Citation Analysis)
Times Cited By Web Of Science : 1  (Related Records In Web of Science)
Times Cited By SCOPUS : 1
연도 인용수 순위
1 Ventura, M. and R. Zink. 2002. Rapid identification, differentiation, and proposed new taxonomic classification of Bifidobacterium lactis. Appl. Environ. Microbiol. 68:6429-6434.   DOI   ScienceOn
2 Chen, M. J., K. N. Chen and C. W. Lin. 2006. Development and verification of an optimum composition model for a synbiotic fermented milk using sequential quadratic programming techniques. Asian-Aust. J. Anim. Sci. 19(10):1490-1495.   과학기술학회마을   DOI
3 Grand, M., M. Küffer and A. Baumgartner. 2003. Quantitative analysis and molecular identification of bifidobacteria strains in probiotic milk products. Eur Food Res. Technol. 217:90-92.   DOI   ScienceOn
4 Matsuki, T., I. K. Watanabe, R. Tanaka and H. Oyaizu. 1998. Rapid identification of human intestinal bifidobacteria by 16S rRNA-targeted species- and group-specific primers. FEMS Microbiol. Lett. 167:113-121.   DOI   ScienceOn
5 Muyzer, G. and K. Smalla. 1998. Application of denaturing gradient electrophoresis (DGGE) and temperature gradient gel electrophoresis (TGGE) in microbial ecology. Antonie van Leeuwenhoek 73:127-141.   DOI   ScienceOn
6 Muyzer, G., A. Teske, C. O. Wirwen and H. W. Jannasch. 1995. Phylogenetic relationships of Thiomicrospira species and their idnetification in deep-sea hydrothermal vent samples by denaturing gradient gel electrophoresis of 16S rDNA fragments. Arch. Microb. 164:165-171.   DOI   ScienceOn
7 Walter, J., G. W. Tannock, A. Tilsala-Timisjarvi, S. Rodtong, D. M. Loach, K. Munroand and T. Alassatova. 2000. Detection and identification of gastrointestinal Lactobacillus species by using denaturing gradient gel electrophoresis and species specific primers. Appl. Environ. Microbiol. 66:297-303.   DOI   ScienceOn
8 O'Sullivan, D. J. 1999. Method for analysis of the intestinal microflora. In: G. W. Tannock, probiotics: A critical review. Horizon Scientific Press, Whmondham, UK. 23-44.
9 Ventura, M., M. Elli, R. Reniero and R. Zink. 2001. Molecular microbial analysis of Bifidobacterium isolates from different environments by the species-specific amplified ribosomal DNA restriction analysis (ARDRA). FEMS Microbiol. Ecol. 36:113-121.   DOI
10 Venema, K. and A. H. Maathuis. 2003. A PCR-based method for identification of bifidobacteria from the human alimentary tract at the species level. FEMS Microbiology Letters 224:143-149.   DOI   ScienceOn
11 Ouwehand, A. C. and S. J. Salminen. 1998. The health effects of cultured milk products with viable and non-viable bacteria. Intl. Dairy J. 8:749-758.   DOI   ScienceOn
12 Prakitchaiwattana, C. J., G. H. Fleet and G. M. Heard. 2004. Application and evaluation of denaturing gradient gel electrophoresis to analyse the yeast ecology of wine grapes. FEMS Yeast Res. 4:865-877.   DOI   ScienceOn
13 Roy, D., J. L. Berger and G. Reuter. 1994. Characterization of dairy-related Bifidobacterium spp. based on their $\beta$- galactosidase electrophoretic patterns. Intl. J. Food Microbiol. 23:55-70.   DOI   ScienceOn
14 Masco, L., G. Huys, E. De Brandt, R. Temmerman and J. Swings. 2005. Culture-dependent and culture-independent qualitative analysis of probiotic produ ts claimed to contain bifidobacteria. Int. J. Food Microbiol. 102:221-230.   DOI   ScienceOn
15 Scardovi, V. and L. D. Trovatelli. 1965. The fructose-6-phosphate as peculiar patternof hexose degradation in the genus Bifidobacterium. Ann. Microbiol. Enzymol. 15:19-29.
16 Scardovi, V., L. D. Trovatelli, B. Biavati and G. Zani. 1979. Bifidobacterium cuniculi, Bifidobacterium choerinum, Bifidobacterium boum, and Bifidobacteriumpseudocatenulatum: four new species and their deoxyribonucleic acid homology relationships. Intl. J. Sys. Bacteriol. 29:291-311.   DOI
17 Kwon, H. S., E. H. Yang, S. W. Yeon, B. H. Kang and T. Y. Kim. 2005. Rapid identification of potentially probiotic Bifidobacterium species by multiplex PCR using speciesspecific primers based on the region extending from 16S rRNA through 23S rRNA. FEMS Microbiol. Lett. 250:55-62.   DOI   ScienceOn
18 Felske, A., A. D. L. Akkermans and W. M. de Vos. 1998. Quantification of 16S rRNAs in complex bacterial communities by multiple competitive reverse transcription- PCR in temperature gradient gel electrophoresis fingerprintings. Appl. Environ. Microbiol. 64:4581-4587.
19 Germond, J., O. Mamin and B. Mollet. 2002. Species specific identification of nine human Bifidobacteriim spp. in feces. System. Appl. Microbiol. 25:536-543.   DOI   ScienceOn
20 Cocolin, L., M. Manzano, C. Cantoni and G. Comi. 2001. Denaturing gradient gel electrophoresis analysis of the 16S rRMA gene V1 region to monitor the dynamic changes in the bacterial population during the fermentation of Italian sausages. Appl. Environ. Microbiol. 67:5113-5121.   DOI   ScienceOn
21 Tilsala-Timisjärvi, A. and T. Alatossava. 1997. Development of oligonucleotide primers from the 16S-23S rRNA intergenic sequences for identifying different dairy and probiotic lactic acid bacteria by PCR. Intl. J. Food Microbiol. 35:49-56.   DOI   ScienceOn
22 Di Cello, F. and R. Fani. 1996. A molecular strategy for the study of natural bacterial communities by PCR-based techniques. Minerva Biotecnol. 8:126-134.
23 Temmerman, R., B. Pot, G. Huys and J. Swings. 2003. Identification and antibiotic susceptibility of bacterial isolates from probiotic products. Intl. J. Food Microbiol. 81:1-10.   DOI   ScienceOn
24 Theunissen, J., T. J. Britz, S. Torriani and R. C. Witthuhn. 2005. Identification of probiotic microorganisms in South African Products using PCR-based DGGE analysis. Intl. J. Food Microbiol. 98:11-21.   DOI   ScienceOn
25 Vaughn, E. E. and B. Mollet. 1999. Probiotics in the new millennium. Nahrung 43:148-153.   DOI   ScienceOn
26 Charteris, W. P., P. M. Kelly, L. Morelli and J. K. Collins. 1997. Selective detection, enumeration and identification of potentially probiotic Lactobacillus and Bifidobacterium species in mixed bacterial populations. Intl. J. Food Microbiol. 35:1-27.   DOI   ScienceOn
27 Chen, M. J., K. N. Chen and C. W. Lin. 2004. Optimization of the viability of probiotics in a fermented milk drink by the response surface model. Asian-Aust. J. Anim. Sci. 17(5):705-711.   과학기술학회마을   DOI
28 Kobayashi, Y. and H. B. I. El-Sawy. 2007. Year-round monitoring of verotoxin-producing escherichia coli from feces of dairy cattle. Asian-Aust. J. Anim. Sci. 20(5):789-794.   과학기술학회마을   DOI
29 Chen, M. J., K. N. Chen and C. W. Lin. 2005. Optimization on Response Surface Models for the optimal manufacturing conditions of dairy tofu. J. Food Engineering 68(4):471-480.   DOI   ScienceOn
30 Kaufmann, P., A. Pfejerkon, M. Teuber and L. Meile. 1997. Identification and quantification of Bifidobacterium species isolated from food with genus-specific 16S rRNA-targeted probes by colony-hybridisation and PCR. Appl. Environ. Microbiol. 63:1268-1273.
31 Kok, R. G., A. de Waal, F. Schut, G. W. Welling, G. Weenk and K. J. Hellingwerf. 1996. Specific detection and analysis of a probiotic Bifidobacterium strain in infant feces. Appl. Environ. Microbiol. 62:3668-3672.
32 Fasoli, S., M. Marzotto, L. Rizzotti, F. Rossi, F. Dellaglio and S. Torriani. 2003. Bacterial composition of commercial probiotic products as evaluated by PCR-DGGE analysis. Intl. J. Food Microbiol. 82:59-70.   DOI   ScienceOn