Identification and phylogenetic characterization of novel Lactobacillus plantarum species and their metabolite profiles in grass silage

Italian ryegrass (IRG), barley, and rye are types of forage that are difficult to ensile with an assurance of good quality. Therefore, the addition of lactic acid bacteria (LAB) is the best way to enhance the preservation of this silage. However, applications of LAB have been impeded due to its poor growth characteristics, the sudden decline in pH, and other non-beneficial microbial growth associated with its presence. To overcome these limitations, a new Lactobacillus sp. KCC-10 and KCC-19 strain was isolated from well-fermented IRG silage samples. Biochemical and physiological studies revealed that the strains were Gram-positive, catalase-negative, and produced gas from glucose, and produced more lactic acid in fermentation. The 16S rRNA gene-based phylogenetic affiliation was determined by using bioinformatic tools that identified Lactobacillus sp. KCC-10 and KCC-19 with 100 % sequence similarity to Lactobacillus plantarum. Novel L. plantarum strains were deposited in the Korean Collection for Type Cultures under the accession numbers KACC 91785P and KACC 91758P, respectively. The shake-flask cultivation of these new strains under aerobic, microaerobic, and anaerobic conditions showed a higher specific growth rate than that achieved using the well-studied L. plantarum KACC 91016 and KACC 91096 on MRS broth and grass juice. Lactic acid was detected as the dominant organic acid in IRG (78.45 mM), barley (51.28 mM), corn (16.28 mM), and rice paddy (11.05 mM), followed by acetic acid and succinic acid. The KCC-10 in the silage was observed to increase from 2.4 × 10⁵ CFU/g per sample at day 0 to 0.58, 0.60, and 0.59 × 10⁹ CFU/g at day 5 for IRG, barley, and rye, respectively. The growth of KCC-10 and KCC-19 in all the silages decreased, as the storage period increased from 5 to 50 days. Whereas, KCC-19 was noted to increase from 2.7 × 10⁵ CFU/g per sample at day 0 to 0.71, 0.72, and 0.711 × 10⁹ CFU/g at day 5 for IRG, barley, and rye. Among the total organic acids, lactic acid was detected as the dominant acid present in IRG, barley, and rye silages. From these results, we concluded that strains KCC-10 and KCC-19 can be used as appropriate inoculants to prolong the stability of silage and fermentation quality.