Olive leaves and two-phase olive pomace (alperujo) are abundant agro-industrial by-products with potential as sustainable sources of bioactive compounds. In this study, aqueous and 70% ethanolic extracts obtained from leaves of the Ascolano and Grappolo cultivars and from alperujo were characterized for their phenolic composition and evaluated for antimicrobial activity against foodborne pathogens, including Listeria monocytogenes, Staphylococcus aureus, Bacillus cereus, Escherichia coli, and Salmonella Enteritidis. Phenolic compounds were analyzed by HPLC-UV/Vis, and antimicrobial activity was assessed by broth microdilution and minimum bactericidal concentration assays. In general, ethanolic extracts showed broader phenolic profiles and stronger antimicrobial effects than aqueous extracts, indicating more efficient recovery of active compounds by hydroalcoholic extraction. The ethanolic extract of Grappolo leaves showed the highest antibacterial activity, achieving > 90% inhibition against B. cereus, E. coli, and S. aureus, and was the only extract to exhibit bactericidal activity at 50% against these microorganisms. This extract was also characterized by high hydroxytyrosol content, whereas the ethanolic alperujo extract showed the highest oleuropein concentration. The stronger activity of ethanolic extracts is likely related to their greater ability to solubilize phenolic compounds with antimicrobial relevance, particularly hydroxytyrosol and oleuropein, which may act through membrane disruption, interference with permeability, and impairment of essential cellular functions. Overall, the results support olive leaves and alperujo as promising low-value raw materials for recovering phenolic-rich extracts with potential applications as natural antimicrobial agents in food systems. To improve reproducibility and practical applicability, standardization of extract yield and concentration should be considered in future studies.
Citation: Luara Simões, Natalia Fernandes, Daniel Sousa, Bruna da Silva, Angélica Souza, Disney Ribeiro Dias. Sustainable extracts from oliviculture by-products: Phenolic content and its antimicrobial activity against foodborne pathogens[J]. AIMS Agriculture and Food, 2026, 11(3): 447-464. doi: 10.3934/agrfood.2026023
Olive leaves and two-phase olive pomace (alperujo) are abundant agro-industrial by-products with potential as sustainable sources of bioactive compounds. In this study, aqueous and 70% ethanolic extracts obtained from leaves of the Ascolano and Grappolo cultivars and from alperujo were characterized for their phenolic composition and evaluated for antimicrobial activity against foodborne pathogens, including Listeria monocytogenes, Staphylococcus aureus, Bacillus cereus, Escherichia coli, and Salmonella Enteritidis. Phenolic compounds were analyzed by HPLC-UV/Vis, and antimicrobial activity was assessed by broth microdilution and minimum bactericidal concentration assays. In general, ethanolic extracts showed broader phenolic profiles and stronger antimicrobial effects than aqueous extracts, indicating more efficient recovery of active compounds by hydroalcoholic extraction. The ethanolic extract of Grappolo leaves showed the highest antibacterial activity, achieving > 90% inhibition against B. cereus, E. coli, and S. aureus, and was the only extract to exhibit bactericidal activity at 50% against these microorganisms. This extract was also characterized by high hydroxytyrosol content, whereas the ethanolic alperujo extract showed the highest oleuropein concentration. The stronger activity of ethanolic extracts is likely related to their greater ability to solubilize phenolic compounds with antimicrobial relevance, particularly hydroxytyrosol and oleuropein, which may act through membrane disruption, interference with permeability, and impairment of essential cellular functions. Overall, the results support olive leaves and alperujo as promising low-value raw materials for recovering phenolic-rich extracts with potential applications as natural antimicrobial agents in food systems. To improve reproducibility and practical applicability, standardization of extract yield and concentration should be considered in future studies.
| [1] |
Bisignano G, Tomaino A, Cascio RL, et al. (1999) On the in-vitro antimicrobial activity of oleuropein and hydroxytyrosol. J Pharm Pharmacolo 51: 971–974. https://doi.org/10.1211/0022357991773258 doi: 10.1211/0022357991773258
|
| [2] |
Özcan MM, Matthäus B (2017) A review: Benefit and bioactive properties of olive (Olea europaea L.) leaves. Eur Food Res Technol 243: 89–99. https://doi.org/10.1007/s00217-016-2726-9 doi: 10.1007/s00217-016-2726-9
|
| [3] |
Romero-García J, Niño L, Martínez-Patiño C, et al. (2014) Biorefinery based on olive biomass. State of the art and future trends. Bioresource Technol 159: 421–432. https://doi.org/10.1016/j.biortech.2014.03.062 doi: 10.1016/j.biortech.2014.03.062
|
| [4] |
Abaza L, Youssef NB, Manai H, et al. (2011) Chétoui olive leaf extracts: Influence of the solvent type on phenolics and antioxidant activities. Grasas Aceites 62: 96–104. https://doi.org/10.3989/gya.044710 doi: 10.3989/gya.044710
|
| [5] |
Sánchez-Gutiérrez M, Bascón-Villegas I, Rodríguez A, et al. (2021) Valorisation of Olea europaea L. olive leaves through the evaluation of their extracts: Antioxidant and antimicrobial activity. Foods 10: 966. https://doi.org/10.3390/foods10050966 doi: 10.3390/foods10050966
|
| [6] |
Borjan D, Leitgeb M, Knez Ž, et al. (2020) Microbiological and antioxidant activity of phenolic compounds in olive leaf extract. Molecules 25: 5946. https://doi.org/10.3390/molecules25245946 doi: 10.3390/molecules25245946
|
| [7] |
Greco M, Rabanal MF, Frey C, et al. (2024) Phenolic compounds-enriched extract recovered from two-phase olive pomace serves as plant immunostimulants and broad-spectrum antimicrobials against phytopathogens including Xylella fastidiosa. Plant Stress 14: 100655. https://doi.org/10.1016/j.stress.2024.100655 doi: 10.1016/j.stress.2024.100655
|
| [8] |
Greco M, Caminada G, Coculo D, et al. (2026) From waste to defense: Agro-industrial byproducts as sources of biopesticides and bioelicitors for crop protection. J Agr Food Chem 74: 10624–10644. https://doi.org/10.1021/acs.jafc.5c13266 doi: 10.1021/acs.jafc.5c13266
|
| [9] |
Babić S, Malev O, Pflieger M, et al. (2019) Toxicity evaluation of olive oil mill wastewater and its polar fraction using multiple whole-organism bioassays. Sci Total Environ 686: 903–914. https://doi.org/10.1016/j.scitotenv.2019.06.046 doi: 10.1016/j.scitotenv.2019.06.046
|
| [10] |
Medeiros RML, Villa F, da Silva DF, et al. (2016) Destinação e reaproveitamento de subprodutos da extração olivícola. Sci Agr Paranaensis 100–108. https://doi.org/10.18188/sap.v15i2.11905 doi: 10.18188/sap.v15i2.11905
|
| [11] |
Galanakis CM, Tornberg E, Gekas V (2010) A study of the recovery of the dietary fibres from olive mill wastewater and the gelling ability of the soluble fibre fraction. LWT-Food Sci Technol 43: 1009–1017. https://doi.org/10.1016/j.lwt.2010.01.005 doi: 10.1016/j.lwt.2010.01.005
|
| [12] |
Borja R, Raposo F, Rincón B (2006) Treatment technologies of liquid and solid wastes from two-phase olive oil mills. Grasas Aceites 57: 32–46.https://doi.org/10.3989/gya.2006.v57.i1.20 doi: 10.3989/gya.2006.v57.i1.20
|
| [13] | World Health Organization W (2026) Food safety. Available from: https://www.who.int/news-room/fact-sheets/detail/food-safety. |
| [14] | Shah MA, Mir SA (2022) Plant extracts as food preservatives, Plant Extracts: Applications in the Food Industry, Elsevier, 127–141. https://doi.org/10.1016/B978-0-12-822475-5.00010-7 |
| [15] |
Liu Y, McKeever LC, Malik NS (2017) Assessment of the antimicrobial activity of olive leaf extract against foodborne bacterial pathogens. Front Microbiol 8: 113. https://doi.org/10.3389/fmicb.2017.00113 doi: 10.3389/fmicb.2017.00113
|
| [16] |
Madureira J, Margaça FM, Buelga CS, et al. (2022) Applications of bioactive compounds extracted from olive industry wastes: A review. Compr Rev Food Sci F 21: 453–476. https://doi.org/10.1111/1541-4337.12861 doi: 10.1111/1541-4337.12861
|
| [17] |
Al-Attar AM, Abu Zeid IM (2013) Effect of tea (Camellia sinensis) and olive (Olea europaea L.) leaves extracts on male mice exposed to diazinon. BioMed Res Int 2013. https://doi.org/10.1155/2013/461415 doi: 10.1155/2013/461415
|
| [18] |
Ling LT, Yap SA, Radhakrishnan AK, et al. (2009) Standardised Mangifera indica extract is an ideal antioxidant. Food Chem 113: 1154–1159. https://doi.org/10.1016/j.foodchem.2008.09.004 doi: 10.1016/j.foodchem.2008.09.004
|
| [19] | CLSI (2014) Methods for broth dilution susceptibility testing of bacteria isolated from aquatic animals, approved guideline, Clinical and Laboratory Standards Institute Wayne. |
| [20] |
Simões L, Fernandes N, Teixeira J, et al. (2023) Brazilian table olives: A source of lactic acid bacteria with antimycotoxigenic and antifungal activity. Toxins 15: 71. https://doi.org/10.3390/toxins15010071 doi: 10.3390/toxins15010071
|
| [21] |
Bona EAMD, Pinto FGdS, Fruet TK, et al. (2014) Comparação de métodos para avaliação da atividade antimicrobiana e determinação da concentração inibitória mínima (cim) de extratos vegetais aquosos e etanólicos. Arquivos do Instituto Biológico 81: 218–225. https://doi.org/10.1590/1808-1657001192012 doi: 10.1590/1808-1657001192012
|
| [22] |
Ortega-García F, Blanco S, Peinado MÁ, et al. (2008) Polyphenol oxidase and its relationship with oleuropein concentration in fruits and leaves of olive (Olea europaea) cv. 'Picual' trees during fruit ripening. Tree Physiol 28: 45–54. https://doi.org/10.1093/treephys/28.1.45 doi: 10.1093/treephys/28.1.45
|
| [23] |
Palmeri R, Siracusa L, Carrubba M, et al. (2022) Olive leaves, a promising byproduct of olive oil industry: Assessment of metabolic profiles and antioxidant capacity as a function of cultivar and seasonal change. Agronomy 12: 2007. https://doi.org/10.3390/agronomy12092007 doi: 10.3390/agronomy12092007
|
| [24] |
Zhang C, Xin X, Zhang J, et al. (2022) Comparative evaluation of the phytochemical profiles and antioxidant potentials of olive leaves from 32 cultivars grown in China. Molecules 27: 1292. https://doi.org/10.3390/molecules27041292 doi: 10.3390/molecules27041292
|
| [25] |
Lama-Muñoz A, del Mar Contreras M, Espínola F, et al. (2020) Content of phenolic compounds and mannitol in olive leaves extracts from six Spanish cultivars: Extraction with the Soxhlet method and pressurized liquids. Food Chem 320: 126626. https://doi.org/10.1016/j.foodchem.2020.126626 doi: 10.1016/j.foodchem.2020.126626
|
| [26] |
Pham DC, Nguyen HC, Nguyen THL, et al. (2020) Optimization of ultrasound-assisted extraction of flavonoids from Celastrus hindsii leaves using response surface methodology and evaluation of their antioxidant and antitumor activities. BioMed Res Int 2020. https://doi.org/10.1155/2020/3497107 doi: 10.1155/2020/3497107
|
| [27] |
Korukluoglu M, Sahan Y, Yigit A, et al. (2010) Antibacterial activity and chemical constitutions of Olea europaea L. leaf extracts. J Food Process Pres 34: 383–396. https://doi.org/10.1111/j.1745-4549.2008.00318.x doi: 10.1111/j.1745-4549.2008.00318.x
|
| [28] | Zorić N, Kosalec I (2022) The antimicrobial activities of oleuropein and hydroxytyrosol, Promising Antimicrobials from Natural Products, Springer, 75–89. https://doi.org/10.1007/978-3-030-83504-0_5 |
| [29] |
Yuan JJ, Wang CZ, Ye JZ, et al. (2015) Enzymatic hydrolysis of oleuropein from Olea europea (olive) leaf extract and antioxidant activities. Molecules 20: 2903–2921. https://doi.org/10.3390/molecules20022903 doi: 10.3390/molecules20022903
|
| [30] |
El-Abbassi A, Saadaoui N, Kiai H, et al. (2017) Potential applications of olive mill wastewater as biopesticide for crops protection. Sci Total Environ 576: 10–21. https://doi.org/10.1016/j.scitotenv.2016.10.032 doi: 10.1016/j.scitotenv.2016.10.032
|
| [31] |
Ladhari A, Zarrelli A, Ghannem M, et al. (2021) Olive wastes as a high-potential by-product: Variability of their phenolic profiles, antioxidant and phytotoxic properties. Waste Biomass Valori 12: 3657–3669. https://doi.org/10.1007/s12649-020-01256-2 doi: 10.1007/s12649-020-01256-2
|
| [32] |
Ferro MD, Lopes E, Afonso M, et al. (2020) Phenolic profile characterization of 'Galega vulgar' and 'Cobrançosa' portuguese olive cultivars along the ripening stages. Appl Sci 10: 3930. https://doi.org/10.3390/app10113930 doi: 10.3390/app10113930
|
| [33] |
Fki I, Allouche N, Sayadi S (2005) The use of polyphenolic extract, purified hydroxytyrosol and 3, 4-dihydroxyphenyl acetic acid from olive mill wastewater for the stabilization of refined oils: A potential alternative to synthetic antioxidants. Food Chem 93: 197–204. https://doi.org/10.1016/j.foodchem.2004.09.014 doi: 10.1016/j.foodchem.2004.09.014
|
| [34] |
Dermeche S, Nadour M, Larroche C, et al. (2013) Olive mill wastes: Biochemical characterizations and valorization strategies. Process Biochem 48: 1532–1552. https://doi.org/10.1016/j.procbio.2013.07.010 doi: 10.1016/j.procbio.2013.07.010
|
| [35] |
Amiot MJ, Fleuriet A, Macheix JJ (1989) Accumulation of oleuropein derivatives during olive maturation. Phytochemistry 28: 67–69. https://doi.org/10.1016/0031-9422(89)85009-5 doi: 10.1016/0031-9422(89)85009-5
|
| [36] | Hussain A, Qarshi IA, Liaqat R, et al. (2014) Antimicrobial potential of leaf and fruit extracts and oils of wild and cultivated edible olive. Pak J Bot 46: 1463–1468 |
| [37] | Gökmen M, Kara R, Akkaya L, et al. (2014) Evaluation of antimicrobial activity in olive (Olea europaea) leaf extract. Am J Microbiol 5: 37–40. |
| [38] | Liu Y, McKeever LC, Suo Y, et al. (2018) Antimicrobial activities of olive leaf extract and its potential use in food industry, Natural and Bio-Based Antimicrobials for Food Applications, ACS Publications, 119–132. https://doi.org/10.1021/bk-2018-1287.ch006 |
| [39] |
Rahnama H, Azari R, Yousefi MH, et al. (2022) A systematic review and meta-analysis of the prevalence of Bacillus cereus in foods. Food Control 109250. https://doi.org/10.1016/j.foodcont.2022.109250 doi: 10.1016/j.foodcont.2022.109250
|
| [40] |
Pereira AP, Ferreira IC, Marcelino F, et al. (2007) Phenolic compounds and antimicrobial activity of olive (Olea europaea L. Cv. Cobrançosa) leaves. Molecules 12: 1153–1162. https://doi.org/10.3390/12051153 doi: 10.3390/12051153
|
| [41] |
Holley RA, Patel D (2005) Improvement in shelf-life and safety of perishable foods by plant essential oils and smoke antimicrobials. Food Microbiol 22: 273–292. https://doi.org/10.1016/j.fm.2004.08.006 doi: 10.1016/j.fm.2004.08.006
|
| [42] |
Shan B, Cai YZ, Brooks JD, et al. (2007) The in vitro antibacterial activity of dietary spice and medicinal herb extracts. Int J Food Microbiol 117: 112–119. https://doi.org/10.1016/j.ijfoodmicro.2007.03.003 doi: 10.1016/j.ijfoodmicro.2007.03.003
|
| [43] |
Kalemba D, Kunicka A (2003) Antibacterial and antifungal properties of essential oils. Curr Med Chem 10: 813–829. https://doi.org/10.2174/0929867033457719 doi: 10.2174/0929867033457719
|
| [44] | Tortora GJ, Case CL, Funke BR (2016) Microbiologia-12a Edição: Artmed Editora. |
| [45] |
Lee OH, Lee BY (2010) Antioxidant and antimicrobial activities of individual and combined phenolics in Olea europaea leaf extract. Bioresource Technol 101: 3751–3754. https://doi.org/10.1016/j.biortech.2009.12.052 doi: 10.1016/j.biortech.2009.12.052
|
| [46] |
Debib A, Boukhatem MN (2017) Phenolic content, antioxidant and antimicrobial activities of "Chemlali" olive leaf (Olea europaea L.) extracts. Int J Pharmacology Phytochem Ethnomed 6: 38–46. https://doi.org/10.18052/www.scipress.com/IJPPE.6.38 doi: 10.18052/www.scipress.com/IJPPE.6.38
|
| [47] |
Himour S, Yahia A, Belattar H (2017) Oleuropein and antibacterial activities of Olea europaea L. leaf extract. Eur Sci J 13: 342–353. https://doi.org/10.19044/esj.2017.v13n6p342 doi: 10.19044/esj.2017.v13n6p342
|
| [48] |
Abu-Lafi S, Al-Natsheh MS, Yaghmoor R, et al. (2017) Enrichment of phenolic compounds from olive mill wastewater and in vitro evaluation of their antimicrobial activities. Evid-Based Compl Alt 2017. https://doi.org/10.1155/2017/3706915 doi: 10.1155/2017/3706915
|
| [49] |
Obied H, Bedgood Jr D, Prenzler PD, et al. (2007) Bioscreening of Australian olive mill waste extracts: biophenol content, antioxidant, antimicrobial and molluscicidal activities. Food Chem Toxicol 45: 1238–1248. https://doi.org/10.1016/j.fct.2007.01.004 doi: 10.1016/j.fct.2007.01.004
|
| [50] |
Ghomari O, Sounni F, Massaoudi Y, et al. (2019) Phenolic profile (HPLC-UV) of olive leaves according to extraction procedure and assessment of antibacterial activity. Biotechnol Rep 23: e00347. https://doi.org/10.1016/j.btre.2019.e00347 doi: 10.1016/j.btre.2019.e00347
|
| [51] |
Khan H, Ahmad W, Hussain I, et al. (2020) Phytochemical composition, antioxidant and antimicrobial activities of leaves of Olea europaea wild variety. J Food Meas Charact 14: 640–648. https://doi.org/10.1007/s11694-019-00310-5 doi: 10.1007/s11694-019-00310-5
|
| [52] |
De Bruno A, Romeo R, Fedele FL, et al. (2018) Antioxidant activity shown by olive pomace extracts. J Environ Sci Heal B 53: 526–533. https://doi.org/10.1080/03601234.2018.1462928 doi: 10.1080/03601234.2018.1462928
|
| [53] |
Medina E, De Castro A, Romero C, et al. (2006) Comparison of the concentrations of phenolic compounds in olive oils and other plant oils: Correlation with antimicrobial activity. J Agr Food Chem 54: 4954–4961. https://doi.org/10.1021/jf0602267 doi: 10.1021/jf0602267
|
| [54] |
Sudjana AN, D'Orazio C, Ryan V, et al. (2009) Antimicrobial activity of commercial Olea europaea (olive) leaf extract. Int J Antimicrob Ag 33: 461–463. https://doi.org/10.1016/j.ijantimicag.2008.10.026 doi: 10.1016/j.ijantimicag.2008.10.026
|
| [55] |
Yangchen J, Sarkar D, Rood L, et al. (2025) Listeria monocytogenes: A continuous global threat in ready-to-eat (RTE) foods. Foods 14: 3664. https://doi.org/10.3390/foods14213664 doi: 10.3390/foods14213664
|
| [56] | World Health Organization W (2018) Risk communication applied to food safety: Handbook, Food & Agriculture Org. |
| [57] |
Ribeiro TB, Bonifácio-Lopes T, Morais P, et al. (2021) Incorporation of olive pomace ingredients into yoghurts as a source of fibre and hydroxytyrosol: Antioxidant activity and stability throughout gastrointestinal digestion. J Food Eng 297: 110476. https://doi.org/10.3390/foods14213664 doi: 10.3390/foods14213664
|
| [58] |
Khalifa I, Barakat H, El-Mansy HA, et al. (2016) Enhancing the keeping quality of fresh strawberry using chitosan-incorporated olive processing wastes. Food Biosci 13: 69–75. https://doi.org/10.1016/j.fbio.2015.12.008 doi: 10.1016/j.fbio.2015.12.008
|
| [59] |
Khemakhem I, Fuentes A, Lerma-García MJ, et al. (2019) Olive leaf extracts for shelf life extension of salmon burgers. Food Sci Technol Int 25: 91–100. https://doi.org/10.1177/1082013218795816 doi: 10.1177/1082013218795816
|
| [60] |
Barukčić I, Filipan K, Jakopović KL, et al. (2022) The potential of olive leaf extract as a functional ingredient in yoghurt production: The effects on fermentation, rheology, sensory, and antioxidant properties of cow milk yoghurt. Foods 11: 701. https://doi.org/10.3390/foods11050701 doi: 10.3390/foods11050701
|
| [61] |
Albertos I, Avena-Bustillos RJ, Martín-Diana AB, et al. (2017) Antimicrobial olive leaf gelatin films for enhancing the quality of cold-smoked Salmon. Food Packaging Shelf 13: 49–55. https://doi.org/10.1016/j.fpsl.2017.07.004 doi: 10.1016/j.fpsl.2017.07.004
|
| [62] |
Khwaldia K, Attour N, Matthes J, et al. (2022) Olive byproducts and their bioactive compounds as a valuable source for food packaging applications. Compr Rev Food Sci F 21: 1218–1253. https://doi.org/10.1111/1541-4337.12882 doi: 10.1111/1541-4337.12882
|
| [63] |
Saleh E, Morshdy AE, El-Manakhly E, et al. (2020) Effects of olive leaf extracts as natural preservative on retailed poultry meat quality. Foods 9: 1017. https://doi.org/10.3390/foods9081017 doi: 10.3390/foods9081017
|