Palmitoylethanolamide (PEA) is time-dependently released by engineered NAPE-LP probiotic under palmitate boost. (A) Released PEA levels were evaluated in bacterial supernatant at 1, 3, 6, and 12 h by HPLC–MS and the results are expressed as the mean ± SD of n = 4 experiments performed in triplicate. In comparison with pLP in absence of palmitate supply, exogenous palmitate (0.0003 μg/mL) time-dependently increased PEA release from pNAPE-LP probiotics, both *** p < 0.001 vs. pLP and pLP in presence of palmitate 0.0003 μg/mL. No detectable amount of PEA was revealed by pLP even in the presence of 0.0003 μg/mL supplementation of exogenous palmitate. (B) PEA tissue concentrations evaluated in tissue homogenates from stomach, duodenum, jejunum, ileum and colon in mice treated with pNAPE-LP + palmitate 0.0003 μg/kg or pLP + palmitate 0.0003 μg/kg by HPLC–MS. Results are expressed, for each two groups as the mean ± SD of n = 6 experiments performed in triplicate. A significantly increased tissue concentration of PEA was observed in the duodenum and ileum of pNAPE-LP + palmitate 0.0003 μg/kg-treated mice as compared to pLP + palmitate 0.0003 μg/kg (+200% and +148%, respectively, both * p < 0.05), while the highest tissue concentration was reached in the colon with a 123% increase vs. pLP + palmitate 0.0003 μg/kg (*** p < 0.0001).