Study on identification of lactic acid bacteria by denaturing gradient gel electrophoresis and oligonucleotide microarray

• Main Authors: Hsi-Chia Chen, 陳希嘉
• Other Authors: 陳明汝
• Format: Others
• Language: zh-TW
• Published: 2009
• Online Access: http://ndltd.ncl.edu.tw/handle/69162826021385651302

博士 === 國立臺灣大學 === 動物科學技術學研究所 === 97 === Lactic acid bacteria (LAB) are widely employed as starter cultures in the dairy industry and supplemented in different commercial products. Detecting and identifying various species of lactic acid bacteria with rapid method is often important for quality control of dairy products and monitoring fermentation processes. However, identification of lactic acid bacteria in the complex bacterial communities is still difficult for both phenotypic methods and genotypic methods. The aim of this study focused on polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) and oligonucleotide microarray method to develop a rapid, reproducible and easy to handle molecular tool for identification of lactic acid bacteria in dairy products. For PCR-DGGE, selection of primers is the key to analyze highly conserved genetic profiles among LAB. Four primer sets, targeted for 20 reference LAB strains, were evaluated and primer set GC338f/518r was selected due to its highly discriminating power at species level. After selection of primer sets, LAB in kefir grains and commercial probiotic products were assessed using PCR-DGGE by a culture-independent and a culture-dependent way, and were further confirmed by DNA sequencing techniques. Kefir grains results indicated that a combined method of cultivation with PCR-DGGE and subsequent DNA sequencing could successfully identify four LAB strains, Lactobacillus kefiranofaciens, Lb. kefiri, Leuconostoc mesenteroides and Lactococcus lactis from three kefir grains from Taiwan (named Hsinchu, Mongolia and Ilan). It was interesting to find that all three kefir grains contain similar LAB species. Furthermore, the DGGE as a culture-independent method indicated that Lb. kefiranofaciens was found in all three kefir grains, whereas Lb. kefiri was only observed in Hsinchu kefir grain and Lc. lactis was found in both Mongolia and Ilan samples. Two additional strains, Pseudomonas spp. and E. coli, were also detected in kefir grains. The DGGE profiles for commercial probiotic products demonstrated that the samples contained most lactic acid bacteria that specified on the product label except of the lyophilized mixed starter powder. This may be due to a concentration below the detection limit or due to the fact that none of these bacteria were present in the probiotic product. For microarray method, oligonucleotide probes targeting variable regions of the 16S rRNA gene were designed and tested for the identification of LAB. The strategy involved designing possible oligomer probes, using two software (ARB and PRIMROSE), for each target at species and genus level. All candidate 25-mer probes were poly(T)-tailed to reach an overall length of 60 oligonucleotides. Microarrays were manufactured by in situ synthesis (Agilent Technologies). Eight samples, including 5 pure LAB (Lb. acidophilus, Lc. lactis, Str. thermophilus, Leu. mesenteroides and Bif. animalis), 1 non-targeted stain (B. cereus) and 2 mixed strain, were then assessed. Results indicated that, of the 21438 individual hybridization reaction (3573 validated probes × 6 references strains), 20082 probes (93.67%) yielded the expected result by showing significantly differential signal. Further inspection of each species probe, over 50 % of species probes designed for Lb. acidophilus, Str. thermophilus and Bif. animalis had true-positive signals with target bacteria. Whereas, true-positive signals were lower than 10% for probes designed for Lc. lactis and Leu. mesenteroides. For mixed strain samples, most of significantly positive signals would not be decreased when the amount of target DNA was down to 0.1