Investigation of microbial diversity in Korean sourdough and its monitoring by real-time quantitative PCR

Abstract

Sourdough is a bread product made by a long fermentation of dough using naturally occurring lactobacilli and yeasts. The yeasts generate carbon dioxide which leavens the dough, and the lactic acid bacteria (LAB) produce lactic acid which contributes to flavor of sourdough. In comparison with breads made with cultivated yeasts, it usually has a mildly sour taste because of the lactic acid produced by lactobacilli. Although sourdough has a good preservability and texture, it is not appealing to many Koreans because of a sour taste. Recently, a modified form of sourdough has been developed by inoculating Nuruk as a starter, hereafter called the Korean sourdough. In order to further improve the Korean sourdough, it is necessary to understand the dynamics of microbial populations during the sourdough fermentation. Among many microorganisms present in the Korean sourdough, LAB are of special interest because various properties of sourdough are related to the metabolites of LAB such as acetic acid and lactic acid. In order to obtain useful information to modulate the microbiota in the sourdough for desirable properties, it is important to develop a rapid and reliable quantification method to detect LAB in the sourdough. In this study, a quantitative assay using a real-time polymerase chain reaction (qRT-PCR) method was developed. First, the identification of the LAB in the Korean sourdough was performed by a randomly amplified polymorphic DNA polymerase chain reaction (RAPD-PCR) along with partial 16S rRNA sequencing. Lactobacillus sanfrnaciscensis was the most dominant species, representing 56% of the isolates followed by Lb. curvatus (27%) and Lb. brevis (9%). The LAB such as Lb. plantarum. Lb. sakei and Pediococcus pentosaceaus were also detected. Next, several primers were designed to detect individual species, based on the sequence information obtained from National Center for Biotechnology Information (NCBI). It was demonstrated that each primer set bound to its target microorganisms without cross reactivity. The newly developed method showed a strong correlation with the result obtained by a colony-forming counting method, which has been recognized as a reliable method but requires tedious procedures and a long time. In addition, the spike recovery values were higher than 95 percent, even in the presence of interfering substances such as high concentrations of sugars and proteins in the sourdough. Above all, the total time required for the assay was shorter than four hours, which allows a rapid and real time monitoring of the microbial populations. Finally, the qRT-PCR method was applied to examining the changes in microbial populations during the sourdough fermentation. It was found that Lb. curvatus decreased throughout the fermentation. It seemed that a poor tolerance against acids as well as the inefficient maltose utilization ability of Lb. curvatus might be attributed to the reduced population. To sum up, the dynamics of microbial populations during the Korean sourdough fermentation could be rapidly assayed by the newly developed qRT-PCR method. It would provide useful information to improve the properties of sourdough by monitoring and controlling the microbiota.