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The exploration of natural antimicrobial compounds is necessary due to the current bacterial resistance to synthetic antibiotics. For this reason, pineapple residues were evaluated as a natural source of phenolic compounds with antimicrobial capacity. Pineapple residues were fermented with A. niger GH1 and subjected to ultrasound for the extraction of phenolic compounds. In the solid-state fermentation (SSF), the peel and the core (70:30) were fermented for 32 h. Ultrasonic-assisted extraction (UAE) was optimized. The factors solid–liquid ratio (X1), ethanol concentration (X2), and extraction cycle (X3) were optimized through a Box–Behnken statistical design, using the total phenol content (TPC) as a response variable. The optimized and pre-purified extract with amberlite was characterized using HPLC-MS and we measured the antimicrobial activity against Listeria monocytogenes, Staphylococcus aureus, and Escherichia coli. The optimal values for X1, X2, and X3 were 82 mg/mL, 26%, and two cycles, respectively. The extract was effective as an antimicrobial agent against S. aureus and L. monocytogenes, with MICs of 50 mg/mL and 12.5 mg/mL, respectively; the bacterial growth curve and transmission electron microscopy (TEM) confirmed the antimicrobial activity. The main compounds identified in the antimicrobial extract were 3,4-DHPEA-EA, Phloretin 2′-O-xylosyl-glucoside, and Feruloyl tartaric acid. Overall, the combination SSF-UAE showed to be a promising strategy to recover phenolic compounds from pineapple residues, exhibiting great potential as a natural antimicrobial agent.

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