In this study, the fermentation characteristics of pine needles were investigated for the first time and the possibility of using them as plant-derived feed additives was confirmed. Four types of fermented pine needle were prepared: (1) natural fermentation (CON); (2) Lactobacillus plantarum SK4315 fermentation (LPF); (3) Saccharomyces cerevisiae SK3587 fermentation (SCF); and (4) co-culture fermentation (CCF). Fermentation lasted 48 h, and samples were taken at 4-h intervals until 12 h, then at 24 and 48 h. As analysis items, fermentation characteristics, antibacterial, antioxidant, and enzymatic activities were investigated. As a result, all pine needle cultures were fermented with changes in the number of viable cells and pH. LPF inhibited the growth of the most pathogens, and the activity became stronger with fermentation. Total polyphenol content (TPC) was the highest in the 48 h SCF and LPF samples, ABTS radical scavenging ability and intracellular antioxidant activity were higher in SCF. Enzymatic activities were different depending on each pine needle culture medium and the fermentation time. In summary, all pine needle cultures were normally fermented, and as fermentation progressed, LPF strengthened antibacterial activity and SCF strengthened antioxidant activity. This study confirmed the potential of fermented pine needles as a feed additive, showing an enhancement of biological activity.
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