Byline: Carmen Salinas-Salazar, Carmen Hernandez-Brenes, Dariana Graciela Rodriguez-Sanchez, Elena Cristina Castillo, Jesus Manuel Navarro-Silva, Adriana Pacheco Keywords: acetogenins; antimicrobial; avocado; centrifugal partition chromatography; Listeria monocytogenes Abstract High standards regarding Listeria monocytogenes control and consumer demands for food products without synthetic additives represent a challenge to food industry. We determined the antilisterial properties of an enriched acetogenin extract (EAE) from avocado seed, compared it to two commercial antimicrobials (one enriched in avocado acetogenins), and tested purified molecules. Acetogenin composition in pulp and seed of Hass avocado was quantified. EAE were obtained by two sequential centrifuge partition chromatography separations and molecules purified by preparative chromatography and quantified by HPLC-MS-TOF and HPLC-PDA. Avocado seed extracts which are the following two: 1) EAE and 2) the commercially available antimicrobial Avosafe[R], presented similar inhibition zones and chemical profiles. Minimum inhibitory concentration (MIC) values of extracts and two isolated acetogenins varied between 7.8 and 15.6 mg/L, were effective at 37 and 4 [degrees]C, and showed a bactericidal effect probably caused by increased membrane permeability and lytic effects, evidenced by flow cytometry at 10 and 100x MIC. Activity was comparable to Mirenat[R]. Most potent acetogenins were Persenone C (5) and A (6), and AcO-avocadenyne (1), the latter exclusively present in seed. Common features of bioactive molecules were the acetyl moiety and multiple unsaturations (2 to 3) in the aliphatic chain, some persenones also featured a trans-enone group. Seeds contained 1.6 times higher levels of acetogenins than pulp (5048.1 [+ or -] 575.5 and 3107.0 [+ or -] 207.2 mg/kg fresh weight, respectively), and total content in pulp was 199 to 398 times higher than MIC values. Therefore, acetogenin levels potentially consumed by humans are higher than inhibitory concentrations. Results document properties of avocado seed acetogenins as natural antilisterial food additives. CAPTION(S): Figure S1-Chromatographic profiles of acetogenins from Hass avocado pulp (A) and seed (B) obtained by HPLC-PDA at 220 nm. Figure S2-Cell concentration overtime of Listeria monocytogenes under the presence of different concentrations of Avosafe[R] (commercial avocado seed oil enriched in acetogenins) during the determination of minimum inhibitory concentration (MIC; 37 [degrees]C, 34 h). Table S1-Composition and partition coefficient (K) of solvent systems evaluated for centrifugal partition chromatography (CPC) fractionation of extract E03.