| Summary: | 碩士 === 國立中興大學 === 食品暨應用生物科技學系所 === 98 === Lactic acid bacteria ( LAB ) strains with probiotics functions have been used for the processing of fermented food, milk products as well as food and feed supplements. Initial people used plate counts method to enumerate bacteria, but it often causes underestimation. Real-time quantitative PCR is a newly molecular technique that can be used to enumerate bacteria. Many studies indicated that real-time quantitative PCR with good operating conditions provided high sensitivity and accuracy. For quantitative analysis, we used PCR and real-time quantitative PCR to indentified and quantitative bifidobacteria in simulated fermented milk. At the same time, we also used plate count method to compare with real-time quantitative PCR. This study highlighted the advantage of real-time quantitative PCR and investigated the detection of simulated fermented milk products contain bifidobacteria.
The simulated fermented milk are composed of whole milk and bifidobacteria reference strains. For extracting complete DNA, we tried three methods and determine to the value of A260 /A280. For Qualitative analysis , PCR products were 231 bp by genus-specific primers F_allbif_IS and R_allbif_IS ; PCR products were 67 to 118 bp by species-specific primers, respectively. For quantitative, real-time quantitative PCR performed with species-specific primers to analyze 7 reference strains in simulated fermented milk products. By using the Ct (cycle threshold) and the concentration of bacteria cells could generate the standard curve of bifidobacteria reference strains. Substitution Ct value of simulated fermented milk into standard curve could evaluate the concentration of bacteria. The Student’s t-test was used to compare each quantitative analysis between plating enumeration and real-time quantitative PCR. The result (p > 0.05) indicated that there was no significant difference between the two methods at a confidence level of 95 %. Besides, plate count method spent more time than real-time quantitative PCR.
This study describes a detection method for bifidobacteria in simulated fermented milk. The PCR analysis combined species -specific PCR is showing a great detection and identification potential using for seven species of bifidobacteria. The species-specific real-time quantitative PCR for evaluating the concentration of bacteria is no significant difference with the plate count method for enumeration. The result indicated that it has potential for developing a culture-independent bacteria enumeration procedure and set a foundation for future studies.
|
|---|
