Quantification of Fungi and Bacteria in Moromi Fermentation

The quantifications of fungi and bacteria in moromi fermentation samples were determined by qPCR. Genomic DNA extracted from moromi samples were used as the templates. The V3–V4 region of the 16S rRNA bacterial gene was amplified by using the primers Eub338 (5′-ACTCCTACGGGA GGCAGCAG-3′) and Eub518 (5′-ATTACCGCGGCTGCTGG-3′) [28 (link)]. For fungi, the ITS2 region was amplified by using the primers of ITS1 f (5′-TCCGTAGGTGAACCTGCGG-3′) and 5.8 S (5′-CGCTGCGTTCTTCATCG-3′) [28 (link)]. The qPCR was performed by using the QuantiFast SYBR green PCR kit commercial kit (Vazyme Biotech Co. Ltd., Nanjing, China) according to our previous protocol [29 (link)]. Standard curves of bacterial and fungal biomasses in moromi were created through plotting the C_T values of different concentrations of Bacillus pumilus’s 16S rRNA gene and Candida zeylanoides’s ITS gene. The StepOnePlus instrument (Applied Biosystems, Foster, CA, USA) was used for the qPCR experiment [24 (link),30 (link)].