| Summary: | 博士 === 國立中興大學 === 食品暨應用生物科技學系所 === 97 === Lactic acid bacteria (LAB) strains with probiotic functions have been used for the processing of fermented food and milk products as well as food and feed supplements. In addition to viability, identity of the LAB species in products is important to consumers and regulatory agencies. 16S rRNA gene has been generally used as target for the identification of eubacteria. However, the identical 16S rDNA sequences of closely related species and the divergent 16S rDNA sequences of a single organism remain problems. The elongation factor Tu gene (tuf), which is present as a single copy in most gram-positive bacteria, has recently been an alternative for designing gene-based methods. In this study, we tried to develop and use of tuf gene-based molecular methods for the qualification and quantification of Lactobacillus and Bifidobacterium.
Based on tuf and recA gene sequences retrieved form the GenBank database, 12 species-specific and one genus-specific primer sets were designed for the PCR detection of L. acidophilus, L. brevis, L. casei group, L. delbrueckii, L. farciminis, L. fermentum, L. jensenii, L. reuteri, Lactobacillus spp. and L. amylovorus, L. pentosus, L. plantarum/L. brevis, L. rhamnosus. The specificities of these PCR primer sets were confirmed by assaying Lactobacillus spp. and other bacterial strains. All the targeted Lactobacillus generated PCR products with predicted sizes. Furthermore, the labels of commercial fermented milk products were examined with these PCR primer sets. The results indicated that label of most fermented milk products were correct with Lactobacillus species. Therefore, the specific PCR primers developed could be used to detect Lactobacillus in fermented milk products.
On the other hand, partial sequences of the tuf and 16S rRNA gene for 18 Bifidobacterium strains belonging to 14 species were determined. Phylogenetic tree was constructed using neighbor-joining method. The phylogenetic tree based on tuf gene has a profile similar to that determined on the basis of 16S rRNA gene. Sequence alignment for these sequences showed that the similarities among the 14 Bifidobacterium species were 82.24-99.72% for partial tuf genes and 92.33-99.05% for partial 16S rRNA genes, respectively. According to these partial tuf genes, 12 species-specific and one genus-specific primer sets were designed for the PCR detection of B. adolescentis, B. animalis, B. bifidum, B. breve, B. cuniculi, B. gallinarum, B. globosum, B. indicum, B. infantis, B. longum, B. minimum, B. subtile and Bifidobacterium spp.. While Bifidobacterium spp. and other bacterial strains were assayed, these PCR primer sets showed great specificities to targeted Bifidobacterium. Detection limits of species-specific PCR were N×103 CFU per ml (N=1-9) in artificially spiked milk targeted with B. longum and B. lactis, respectively. Bifidobacterial strains in commercial probiotic products could be identified with these tuf gene-based PCR primers and be enumeration by plating count method. Thus, the quality of these porbiotic products could be assured.
In addition, tuf gene-based PCR primers specific for the detection of L. acidophilus, L. casei group, L. delbrueckii, and B. longum were combined for the simultaneous detection of these LAB. The specificity of this multiplex PCR was confirmed and the PCR products generated from L. acidophilus, L. delbrueckii, L. casei group, and B. longum were 397, 230, 202, and 161 bp, respectively. The identification limit for each LAB strain with this multiplex PCR method was N× 103 CFU per ml in milk samples. When this multiplex PCR method were used for the simultaneous detection of the LAB in fermented milk products, the LAB species listed on the labels of these products could be identified without the preenrichment step. The results of the multiplex PCR method were further confirmed by PCR assay using primers based on the 16S rDNA or the 16S–23S intergenic spacer region and by biochemical tests using the API 50 CHL kit. In conclusion, this multiplex PCR method could be used for the identification and detection of LAB in commercial fermented milk products.
Denaturing gradienet gel electrophoresis (DGGE) is a technique able to differentiate DNA fragnments with sequences diversity. With polymerase chain reaction, DGGE was able to analyze the species in mixed cultures. PCR-DGGE targeting tuf gene was developed and used for species identification. When 16 Bifidobacterium species were assayed with the PCR-DGGE method, all these species could be differentiated to 13 separated patterns, except that the tuf gene sequences from B. adolescentis/B. thermophilum, B. longum/B. magnum and B. lactis/B. gallinarum which migrated the same distance with DGGE gel. Combing with species-specific PCR, all the 16 Bifidobacterium species could be identified. Detection limits of genus-specific PCR-DGGE were N×104 CFU per ml in artificially spiked milk targeted with B. longum and B. lactis, respectively. Furthermore, species-specific PCR and PCR-DGGE were used for the detection of bifidobacterial species in probiotic products. The Bifidobacterium in probiotic products could be faithfully detected by tuf gene-based PCR and PCR-DGGE.
Finally, SYBR Green I based Real-time PCR were developed for the detection of L. acidophilus, L. casei group, L. delbrueckii, B. lactis and B. longum using tuf gene-based specific primers. According to the melting temperature (Tm) of the PCR products generated with these primers, the specificity could be confirmed. Using Ct (cycle threshold) and the concentration of bacteria cells, the standard curves of LAB strains were generated and could be used in evaluating the concentration of bacteria. For the quantification of the LAB in fermented products, the enumeration results obtained from selective medium plating and Real-time PCR showed great agreement between two methods. The result suggested that it has potential for developing a culture-independent bacteria enumeration procedure by Real-time PCR.
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