Novel strategy to understand the influence of pine needle essential oil supplementation on the consumption of nutritional components in paper mulberry silage by multi-omics analysis

Pine needle essential oils (PNEO) have undergone extensive safety evaluations and are recognized for their diverse bioactivities including antifungal, insecticidal, and antibacterial properties. Fermentation quality, 16S rRNA sequencing, and metabolome analyses of paper mulberry silage were performed to investigate the efficacy of PNEO in mitigating nutrient loss during ensiling. Paper mulberry was fermented, both spontaneously and aided by PNEO (at contents of 160, 320, and 480 mg/kg, respectively). The addition of PNEO (320 mg/kg) significantly mitigated the consumption of dry matter, crude protein, and water-soluble carbohydrates, reducing their respective losses by 1.45, 4.25, and 13.93%. Microbial community analysis revealed that supplementing mulberry silage with PNEO does not significantly inhibit lactic acid bacterial growth throughout silage fermentation. Conversely, it inhibited the proliferation of Priestia, Enterobacteriaceae, Klebsiella, Acinetobacter, and Bacillus throughout silage fermentation, facilitating the growth of Enterococcus, Weissella, and Pediococcus, accelerating pH reduction due to fermentation. Metabolic analysis revealed 134 differential metabolites between spontaneous fermentation and that supplemented with 320 mg/kg PNEO on day 60 of fermentation. Of these, 118 were upregulated, and 16 downregulated. Annotation of downregulated metabolites indicated that pathways including valine and leucine, as well as isoleucine and amino acid biosynthesis were most enriched. In contrast, upregulated metabolites were primarily associated with carbon metabolism, the citrate cycle, propanoate metabolism, and galactose metabolism. Results suggest that PNEO selectively inhibited Enterobacteriaceae, Priestia, Klebsiella, and Bacillus, shifting sugar utilization dominance to lactic acid bacteria (LAB) through enhanced metabolic pathway activity under limited glucose conditions.