Identification and Enumeration Method of Both Eukaryotic and Prokaryotic Microorganisms in Food Sample

Abstract

The method to analyze both eukaryotic and prokaryotic microorganisms without preliminary microbial information of sample seemed to be useful not only for research and investigation of microorganisms but also for industry using microorganisms. In the present manuscript, preparation of a new DNA primers, new reference database for 18S rDNA for our newly developed method [1]- [3], and analyses of eukaryotic and prokaryotic microorganisms in fermentation products were presented. In komekouji, Aspergillus spp., was enumerated to be 46.5 × 106 MPN g -1 , and Penicillium spp., was enumerated to be 1.5 × 106 MPN g -1 . In dry yeast, Saccharomyces group, were enumerated to be 8600 × 106 MPN g -1 . In komekouji-miso, no eukaryotic microorganism was detected, while the other Bacillus spp., was numerically dominant (21.5 × 106 MPN g -1 ) as prokaryotic microorganisms, followed by B. subtilis group (4.65 × 106 MPN g -1 ), and the other Firmicutes (3.7 × 106 MPN g -1 ). The komekouji-miso included lower number of Actinobacteria (0.15 × 106 MPN g -1 ), Burkhokderia sp. (1.5 × 106 MPN g -1 ), and the other α,β,γ -proteobacteria (0.12 × 106 MPN g -1 ). In sake-kasu, both prokaryote and eukaryote were not detected by the method. Present results indicated that using both universal primers for eukaryotic and prokaryotic microorganisms, each groups of prokaryotic and eukaryotic microorganisms were enumerated without any preliminary information nor setting up standard curve, which were required for real time PCR.