This research investigated the protecting properties of polyphenols and flavonoids in phytonutrient pellets formulated from lemongrass powder and mangosteen peel (LEMANGOS pellets) through the microencapsulation and named microencapsulated LEMANGOS (mLEMANGOS). For this purpose, the effects of mLEMANGOS supplementation at various R:C ratios of 60:40 and 20:80 were evaluated and compared with monensin (antibiotic) supplementation under an in vitro study technique. Treatments were randomly assigned in a 2 × 4 × 2 factorial arrangement in a completely randomised design consisting of factors A: R:C ratios (60:40 and 80:20), factor B: mLEMANGOS supplementation (0, 2, 4, and 6% DM), and factor C: monensin supplementation (0 and 20% DM). There was an interaction between the R:C ratio and both mLEMANGOS and monensin supplements on the in vitro gas production kinetics, ruminal by-product fermentation, methane production, and rumen microbial population (p < 0.001, 0.01, 0.05). Results indicated that each supplementation influenced the gas production kinetics, while there was decreased cumulative gas production in the mLEMANGOS supplemented. Consequently, the supplemented group buffered ruminal pH and increased the in vitro dry matter degradability (IVDMD) and ammonia nitrogen (NH3-N) concentrations. Moreover, the additional treatment of mLEMANGOS supplementation (6% DM at R:C ratios of 60:40 and 20:80) significantly reduced the number of Methanobacteriales to 53.5% and 50.4% after 24 h, respectively. Results from those supplements can reduce methane production to 99.2% and 97.9% (p < 0.001), respectively. This research suggests that phytonutrient-based antimicrobial in the mLEMANGOS supplement could potentially be used as ruminant feed additives and as antimicrobial substances. HIGHLIGHT Microencapsulated LEMANGOS was formulated by biopolymer using green technique to retain the phytonutrients and their long-term release. The mLEMANGOS supplementation (at 6% of total DM) can be used as a synthetic bio-antibiotic for inhibiting methanogens-archaea population. The mLEMANGOS supplementation (at 6% of total DM) can enhance rumen nutrients degradability, ruminal end-products, and mitigate methane production.
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