Pre-fermented juices (PFJs) are natural biological inoculants widely utilized to optimize silage fermentation processes. In this study, the active bacterial populations within PFJ samples produced from alfalfa, meadow grass, and corn silage were evaluated using RNA-based 16S rRNA Real-Time PCR (RT-qPCR) methodology, which directly reflects cellular metabolic activity. To stimulate fermentation, molasses and glucose were added to the prepared plant extracts, and the mixtures were subjected to anaerobic incubation at 30 °C for 5 days. Following total RNA isolation using TRIzol reagent with bead-beating homogenization, cDNA was synthesized and SYBR Green-based RT-qPCR analyses were conducted. The molecular findings demonstrated the generation of sigmoid amplification curves and singular melting curve peaks confirming target gene specificity across all examined PFJ variants. Upon analyzing the cycle threshold (Ct) data, which varied according to substrate compositions, the most intense microbial activity was detected in PFJ samples produced from corn silage due to its highly fermentable carbohydrate profile. Conversely, the relatively higher Ct values observed in alfalfa-derived samples were attributed to the inherent cellular buffering capacity of the material. Consequently, it was demonstrated that this RNA-targeted approach, which eliminates analytical biases caused by inactive or dead cells, serves as a highly specific, sensitive, and reliable method for the functional characterization of the microbiota in fermentative systems.

Pre-fermented juice, RNA, 16S rRNA, active microbiota, silage fermentation, RT-qPCR