Sustainable extracts from oliviculture by-products: phenolic content and its antimicrobial activity against foodborne pathogens

Sustainable extracts from oliviculture by-products: phenolic content and its antimicrobial activity against foodborne pathogens | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Sustainable extracts from oliviculture by-products: phenolic content and its antimicrobial activity against foodborne pathogens Luara Simões, Natália Fernandes, Daniel Sousa, Bruna da Silva, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8619319/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Olive leaves and olive oil residues are abundant by-products that can serve as alternative sources of antimicrobials. This study characterized extracts from Ascolano and Grappolo olive leaves and olive oil residues, focusing on phenolic content and antimicrobial activity. Aqueous and ethanolic extracts were tested against Listeria monocytogenes , Staphylococcus aureus , Bacillus cereus , Escherichia coli , and Salmonella Enteritidis , with HPLC (UV-vis) used for chemical analysis. Ethanolic extracts exhibited greater inhibition and higher phenolic compound concentrations. The Grappolo leaves ethanolic extract had a minimum bactericidal concentration of 50% for B. cereus , E. coli , and S. aureus and contained 9.24 ± 0.58 g.ml⁻¹ hydroxytyrosol. Olive oil residue ethanolic extracts showed high oleuropein content (16.47 ± 0.76 g.ml⁻¹) and inhibited B. cereus by 75%. The results highlight the potential use of olive leaves and residues of olive oil production extracts as natural and sustainable antimicrobials against some microorganisms that cause foodborne illness. Food Science & Technology Applied & Industrial Microbiology Antimicrobial activity Food additives Olive leaves Residue Sustainability Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Olive trees, Olea europaea L. , belong to the botanical family Oleaceae and are native to Mediterranean countries, therefore, this region is the largest producer of olives and olive oil, accounting for 98% of the total area of olive tree production in the world. Olive tree products are rich in important substances from a nutritional perspective [ 14 ] since they have antioxidant and antimicrobial properties attributed to their phenolic compounds, particularly hydroxytyrosol, and oleuropein [ 6 ]. Olive by-products often referred to as residues or waste products, are derived from the cultivation and processing of olive trees and olive oil, and are produced annually in large quantities [ 37 ]. Unfortunately, most of these by-products do not have practical applications and go to waste, therefore the use of both can be the focus of a sustainable valuation of new products [ 42 ]. One of the most abundant by-products of olive growing is olive leaves, which have antioxidant, anti-inflammatory, antimicrobial, and antiviral activity, therefore they can be considered a cheap and natural raw material of antioxidants [ 1 ]. In addition, there are residues from the processing of olives for the extraction of olive oil, which represent a huge environmental problem, as they are produced in large quantities and have a high content of organic matter, such as polyphenols, which may be responsible for the high toxicity [ 5 ]. Therefore, the use of this residue requires a significant economic effort to avoid severe environmental pollution, and it can be used as an economic source with antimicrobial and antioxidant activity, in order to prevent environmental damage arising from its misdirection [ 32 ]. Usually, for the production of olive oil, the centrifugation method is used, which can be a continuous three-phase process, where the waste produced comprises the liquid wastewater from vegetation olive mill waste-water and bagasse, which corresponds to solid waste [ 17 ]. The process can also be carried out in a two-phase system for the production of olive oil, originating a solid residue called two-phase olive pomace, which consists of a thick mass made up of the pulp and stone of the olive, as well as the water from the fruit and residual oil [ 8 ]. Due to the increased resistance of pathogenic microorganisms to numerous drugs and the susceptibility of acting on various types of substrates, there is a concern for the search for new alternatives, which encourages the search for natural antibiotics, according to the World Health Organization (WHO), foodborne infections remain one of the leading causes of disease worldwide [ 34 ]. Different approaches have been used for contamination by pathogenic microorganisms, such as the use of preservatives. In this context, due to concerns about the toxicity and dangers of synthetic preservatives, the demand for natural food preservatives has increased, and plant extracts emerge as a better option, rich in bioactive compounds, acting as natural preservatives [ 43 ]. Olive-growing by-products, being natural compounds, can emerge as an alternative to obtain antimicrobials, resulting in the replacement of chemical preservatives, which may contain toxic and carcinogenic properties. Given the above, the present study aimed to characterize extracts from olive leaves ( Olea europaea ) and residue of the olive oil production in a two-phase system, concerning phenolic content, as well as antimicrobial activity on food-contaminating bacteria. Materials and Methods Collection and sampling location The residues of the olive oil production and the olive leaves used to manufacture the extracts were both kindly provided by the Experimental Farm of EPAMIG (Empresa de Pesquisa Agropecuária de Minas Gerais) in Maria da Fé, Brazil (22◦18’ south latitude, 45◦23’ west longitude, an average elevation of 1.276 m). Olive leaves were randomly picked from olive branches of the Ascolano and Grappolo 546 cultivars. Approximately 1 kg of the residue of the olive oil production in a two-phase system (solid fraction) from unripe olives (green olives) containing olive pulp, skin, stone, water, and some concentration of olive oil, was collected. All materials were collected in sterile plastic bags and taken under refrigeration to the Laboratory of Microbiology of Fermentations, in the Microbiology sector, Department of Biology, Federal University of Lavras (UFLA), Lavras, Brazil, for the manufacture of extracts. Preparation of extracts Two extraction methods were used, resulting in aqueous extracts (using deionized water) and another ethanolic extract (using 70% ethanol). Olive leaves were washed with deionized water and dried at 37°C for 3 days, and the residue of the olive oil production was directly placed in an oven for drying in the same conditions. Then the dried leaves and residue were pulverized in a conventional blender in order to decrease the particle size to 90–150 µm. For each extraction method, 3 different extracts were prepared: olive leaves Ascolano extract; olive leaves Grappolo extract, and the residues of the olive oil production extract. Aqueous extract The methodology described by Al-Attar AM, et al. [ 3 ] was used with some modifications. 50 g of dried olive leaves and residues of the olive oil production were added to 2 L of hot water. After 3 h, the mixture was slowly boiled for 30 min. After boiling, the mixture was cooled to room temperature and subjected to an electric mixer for 20 min. Subsequently, the solutions were centrifuged (5 min at 5000 rpm) and filtered through a sterile syringe filter (0.2 µm, PES). The filtrate was evaporated in a rotary evaporator at 40°C to produce dry residue (active ingredients) and stored at 4°C to be used throughout the experiment. Ethanol extract The extracts were obtained using the methodology proposed by Ling LT, et al. [ 29 ] with some modifications. Dried olive leaves and residues of the olive oil production (50 g) were added with 750 ml of ethanol (70% v/v). The extraction was performed at room temperature, where the samples were left to rest for 24 h, so the extracts were centrifuged (5 min at 5000 rpm) and filtered through a sterile syringe filter (0.2 µm, PES). Then, the solvent was removed using a rotary evaporator at 38°C with a rotation of 120 rpm under a vacuum. The extracts were kept at 4°C temperature to be used throughout the experiment. Tested microorganisms The following bacteria were used: Gram-positive ( Listeria monocytogenes ATCC 19117, Staphylococcus aureus ATCC 8702, and Bacillus cereus ATCC 14579) and Gram-negative bacteria ( Escherichia coli EPEC055 and Salmonella Enteritidis ATCC 564). The inoculums were standardized with the aid of a growth curve, following the absorbance (O.D. 600nm) and the plate count using Brain Heart Infusion (BHI) agar (Sigma Aldrich, Darmstadt, Germany). The plates were incubated at 37°C for 24 h and the inoculum was standardized at 10 8 CFU.mL -1 . The inoculums were stored in a freezer at -70ºC and thawed at room temperature during the experiment. Antimicrobial activity To determine the percentage of inhibition, the protocol established by CLSI [ 9 ] was used, with some adaptations, using the broth microdilution technique (sterile 96-well microplates). The culture medium used was BHI broth (Sigma Aldrich, Darmstadt, Germany). To each well was added 100 µl of BHI broth. In the first well of each line of microplates, a volume of 100µL of the respective extract (resuspended in sterile water) to be analyzed was also added, obtaining the initial concentration of each extract (50%). From the initial wells, serial dilutions were performed obtaining decreasing concentrations of 50%, 25%, 12.5%; 6.25% (v/v). Aliquots of 10 µL of each bacterial suspension were inoculated into wells containing BHI medium and the respective concentration of extracts. Positive controls (PC) consisted of 10 µL of each bacterial suspension inoculated in BHI medium without the addition of extracts, and negative controls (NC) consisted of BHI medium supplemented with 10 µL of the different extract concentrations without the addition of bacterial suspension. The microplates were sealed and incubated in a BOD incubator at 37ºC for 24 h. All experiments were conducted in triplicate three repetitions. After incubation, after the incubation, absorbance at 600 nm was measured using a UV spectrophotometer (Thermo Scientific, Waltham, MA, USA), and the value of the percentage of inhibition was expressed as: Inhibition (%) = [1 - (Ac - NC/PC)] x 100 Where Ac represents the absorbance of the well with different concentrations of extracts, NC is the negative control, and PC is the absorbance of positive control [ 45 ]. Minimum Bactericidal Concentration (MBC) After the incubation period was determinate of the minimum bactericidal concentration (MBC), The micro drop technique was used with plating on BHI agar, 10 µL aliquots of cultures from the wells were plated where there was no turbidity were put in the plates and incubated at 37°C/24h. After incubation, the MBC of the extracts was determined, as its lowest concentration capable of promoting the absence of growth of the bacteria tested in plate [ 7 ]. The experiment was carried out in triplicate and with three repetitions. Identification of phenolic compounds contained in each extract by HPLC (UV-vis) High-pressure liquid chromatography (HPLC) equipped with UV-visible absorption (UV-vis) was used for the analytical qualification and quantification of phenolic compounds in each olive leaf extract. The extracts (10mg) were dissolved in 1 mL of 100 mM perchloric acid (Sigma Aldrich, Darmstadt, Germany) solution and filtered with 0.45µm filters into vials before analysis by HPLC (UV-vis). The separation of phenolic compounds was carried out in Shimpack SCR-101H column (7.9mm × 30 cm) Shimadzu, the mobile phase was 100mM perchloric acid (Sigma Aldrich, Darmstadt, Germany) solution, with a flow rate of 0.6mL/min over 20 min run, the oven temperature was 50 ◦C, detected with a 210nm UV detector (30 ◦C). All phenolic compounds were identified by comparing their retention times with the respective standards (chlorogenic acid, caffeic acid, coumaric acid, hydroxytyrosol, oleuropein, and rutin) (Merck, Darmstadt, Germany), as shown in Fig. 1 . Before running the experiments, calibration curves were established for each analyte so that relative response factors for each analyte could be considered in reporting analyte concentrations. Analyses were performed in triplicate. Calibration curves The calibration of the phenolic compounds method was conducted with 7 levels for chlorogenic acid, caffeic acid, coumaric acid, hydroxytyrosol, oleuropein, and rutin. The fit of the applied linear regression model was described as R 2 (> 0.99). Each set was made by dissolving the given mass in nanopure water to obtain L7. Levels 6 − 1 were then created by serial dilution into the same size volumetric flask used to create L7. Each level was syringe filtered through a 0.45 µm nylon filter. Calibration curves were obtained from the area of each standard peak in each level. Each compound was identified by retention time (min). Statistical analyses The results were submitted to statistical analyses using GraphPad Prism version 8.01 for Windows (La Jolla, CA, USA). The two-way ANOVA and Tukey’s multiple comparisons test were performed and the significant differences were reported as: **** for p < 0.0001; *** for p < 0.001; ** for p < 0.01; and * for p < 0.05. Results were expressed as mean values ± standard deviation (n = 3). Results and discussion Identification of phenolic compounds To our knowledge, this is the first time that Ascolano and Grappolo olive leaves have been studied. Table 1 showed the phenolic composition of the extracts used in this study, the compounds identified were chlorogenic acid, caffeic acid, coumaric acid, hydroxytyrosol, oleuropein, and rutin. In the aqueous extracts, the compounds caffeic acid, coumaric acid, and oleuropein were not present in olive leaf extracts, for the olive oil production residues aqueous extracts, caffeic acid was the only compound that was not present. The use of 70% ethanol (v/v) provided a higher concentration and variety of compounds in the extracts; therefore, ethanol was the solvent with the highest extraction capacity, which contributes to better antimicrobial capacity. This fact is in line with the work of Ortega-García F, et al. [ 36 ], which showed that the use of water combined with alcohols contributes to increased swelling of plant materials, resulting in the increased contact area between plant matrix and solvent, improving extraction yield and higher concentration of compounds. The compounds identified in the present study were also found in olive leaf extracts collected in the southeast of Sicily, Italy in the work of Palmeri R, et al. [ 38 ]. Chlorogenic acid, hydroxytyrosol, and rutin were identified in the work by Zhang C, et al. [ 48 ] in olive leaves from cultivars grown in China, these compounds have remarkable antimicrobial properties [ 27 ]. Rutin was one of the flavonoids found in olive leaves in the work of Pham D-C, et al. [ 40 ]. Caffeic acid, p -coumaric acid, and oleuropein were identified in ethyl alcohol extracts from olive leaves in the work of Korukluoglu M, et al. [ 25 ]. Olive leaves extract and its constituents, particularly oleuropein, and hydroxytyrosol, have been reported to have health benefits, including antioxidant and antimicrobial properties [ 49 ]. Normally, in olive leaf extracts, oleuropein is the compound present in most amounts [ 27 ]. On the other hand, in our study, the olive leaf extracts of the two studied cultivars (Ascolano and Grappolo), hydroxytyrosol was the compound present in higher (p < 0.05) quantity, mainly in the Grappolo cultivar (9.24 ± 0.58 g.ml-1). Hydroxytyrosol is present in almost all parts of the olive tree, mostly in the leaves, being this compound is the main product of oleuropein degradation [ 47 ]. Therefore, possibly part of the oleuropein present in large amounts in the extracts may have been converted into hydroxytyrosol, resulting in the high concentration of these compounds in the extracts. Several studies have revealed different classes of phenolic compounds in olive residues [ 13 , 26 ]. Normally, the phenolic compound oleuropein is the most abundant in olives from different cultivars [ 15 ]. Accordingly, our study showed a high concentration of oleuropein in olive oil production residues ethanolic extracts (16.47 ± 0.76 g.ml -1 ), which consists of a mass containing the pulp, pit, skin, and traces of olive oil. Ethyl acetate extracts of olive mill wastewater were studied in the work of Fki I, et al. [ 16 ], the hydroxytyrosol was present in higher concentrations, whereas, caffeic acid and coumaric acid were present at lower concentrations. In the work of Dermeche S, et al. [ 12 ], the solid residue was analyzed, p-coumaric acid, caffeic acid, hydroxytyrosol, and rutin were identified in the extracts, the same for our work, however, the oleuropein is not present. On the other hand, this compound was present in higher (p < 0.05) amounts in the extracts of residues of the olive oil production in our study. One explanation could be the difference in olive maturation, where in our work the olives used to manufacture olive oil were unripe olives (green olives), whereas in the work where the samples were collected in the final stage, using ripe olives (black olives), when oleuropein is degraded into elenolic acid and hydroxytyrosol [ 4 ]. The main phenolic compounds found in olive oil processing waste in the work of Medeiros RML, et al. [ 32 ] were hydroxytyrosol, oleuropein, tyrosol, caffeic acid, P-coumaric acid, vanillic acid, catechol, and rutin. Therefore, these compounds represent a natural source of antioxidants and antimicrobials and can be used in the food industry, thus replacing chemical preservatives that have been associated with undesirable effects on human health. Table 1 Phenolic compounds in extracts derived from olive leaves and residue of olive oil production (g.ml -1 ) Extracts Solvent Hydroxytyrosol Chlorogenic acid Rutin Caffeic acid Coumaric acid Oleuropein EA Aqueous 0.65 ± 0.08 d 0.076 ± 0.005 c 0.023 ± 0.003 b - - - EG 1.82 ± 0.13 c 0.076 ± 0.002 c 0.024 ± 0.001 b - - - ER 0.66 ± 0.11 d 0.072 ± 0.0006 c 0.019 ± 0.001 b - 1.59 ± 0.09 c 0.79 ± 0.04 c EA Ethanolic 3.28 ± 0.46 b 0.087 ± 0.004 b 0.025 ± 0.001 b 0.89 ± 0.04 c 1.79 ± 0.09 c 1.83 ± 0.02 b EG 9.24 ± 0.58 a 0.095 ± 0.003 b 0.033 ± 0.002 a 1.18 ± 0.04 b 2.75 ± 0.1 b 1.89 ± 0.04 b ER 0.37 ± 0.07 d 0.72 ± 0.02 a 0.037 ± 0.003 a 1.48 ± 0.07 a 3.21 ± 0.28 a 16.47 ± 0.76 a EA: Extract of olive leaves Ascolano; EG: Extract of olive leaves Grappolo; ER: Extract of residue of olive oil production. -: not identified. The results are expressed as means. ± SD (n = 3). Statistical significance was determined by Tukey’s multiple comparisons, different letters differ significantly (p < 0.05). Antibacterial activity There is a critical need to combat emerging pathogenic microorganisms through varied chemical structures with innovative mechanisms. Therefore, the use of plant-based antimicrobial agents is important, and researchers are dedicating efforts to using natural compounds against pathogenic microorganisms to be used as preservatives in food or even to develop new drugs, which can be a potential alternative to synthetic compounds, that cause harmful effects on human health [ 22 ]. The present study was conducted in order to evaluate the antimicrobial activity against Listeria monocytogenes ATCC 19117, Staphylococcus aureus ATCC 8702, Bacillus cereus ATCC 14579, Escherichia coli EPEC055 and Salmonella Enteritidis ATCC 564 to extracts of by-products generated in the production of olive oil and olive leaves. We can observe that the extract of the leaves of Grappolo was the one that presented a greater antimicrobial activity against all the microorganisms tested, this fact can be correlated to the higher concentration of hydroxytyrosol of these extracts, this compound has demonstrated the potent antimicrobial activity of hydroxytyrosol, against several pathogens bacteria [ 33 ] Figure 2 showed the antimicrobial activity against the gram-positive bacterium Listeria monocytogenes . The olive leaves ethanolic extracts showed higher antimicrobial activity (Fig. 2B), with a percentage of inhibition of 91.20 ± 1.10 and 74.84 ± 4.43% for Grappolo and Ascolano leaf extracts, respectively, for the higher tested concentration. The highest (p < 0.05) antimicrobial activity for the aqueous extracts (Fig. 2A) was observed for the Ascolano leaf extract (57.51 ± 6.62) at 50% of concentration. Regarding inhibition by residues of the olive oil production extract, inhibition ranged from 16.37 ± 2.83 to 36.45 ± 4.09% for the ethanolic extracts, and from 14.82 ± 2.66 to 41.93 ± 3.21% for the aqueous extracts. According to Gökmen M, et al. [ 19 ] there was a positive effect of olive leaf extracts on Listeria monocytogenes , the antimicrobial activity was evaluated using disk diffusion microdilution methods. On the other hand, in the work of Hussain A, et al. [ 22 ], the leaf extracts from Olea ferruginea , using the same solvents as in our work, have no effect on the growth of L isteria monocytogenes . In the work of Liu Y, et al. [ 31 ], when studying the effect of olive leaf extract against L. monocytogenes , the authors found that with the use of extracts, the motility and biofilm formation of the bacterium were decreased. It has been suggested that olive leaf extract has the potential to be used in the food industry as an antimicrobial, in order to inhibit the growth of foodborne pathogens in foods, on processing equipment, or in packaging materials. Figure 2 . Percentage of inhibition of extracts derived from olive leaves and residue of olive oil production on Listeria monocytogenes . EA: Extract of olive leaves Ascolano; EG: Extract of olive leaves Grappolo; ER: Extract of residue of olive oil production. A: Aqueous extract of olive by-products, B: Ethanolic extract of olive by-products. The results are expressed as means and bar indicates SD (n = 3). Statistical significance was determined by Tukey’s multiple comparisons test (**** for p < 0.0001; *** for p < 0.001; ** for p < 0.01; and * for p < 0.05). For the microorganism B. cereus , the results are presented in Fig. 3, the ethanolic extracts (Fig. 3B) showed high values of antimicrobial activity, with the highest (p < 0.05) percentage found for the Grappolo leaf extract (92.24 ± 2.02%), followed by the residues of the olive oil production (75.20 ± 3.62%), at the highest concentration tested. On the other hand, at a concentration of 50%, the aqueous extracts of Ascolano leaf exhibited higher (p < 0.0001) antibacterial activity (46.71 ± 4.61%), followed by Grappolo leaf extract (27.51 ± 1.24%), this last one was the extract that presented higher (p < 0.0001) inhibition at the lowest tested extract concentrations (25, 12.5 and 6.25%). The aqueous extract of c showed a low antimicrobial activity against B. cereus , with a maximum activity of approximately 8.85 ± 0.53% (Fig. 3A). Bacillus cereus is a food-borne bacterium that is highly resistant and causes food poisoning. This organism is found throughout the world, thus making it a major concern for food safety [ 41 ]. In the work of Pereira AP, et al. [ 39 ], olive leaf aqueous extracts were screened for their antimicrobial activity, and the B. cereus was the most sensitive microorganism. Oleuropein, the main phenolic compound present in the residues of olive oil production (Table 1 ), has the ability to inhibit Bacillus cereus sporulation [ 21 ]. Therefore, possibly the extracts can be used both to inhibit the growth of vegetative cells and to inhibit sporulation. Figure 3. Percentage of inhibition of extracts derived from olive leaves and residue of olive oil production on Bacillus cereus. EA: Extract of olive leaves Ascolano; EG: Extract of olive leaves Grappolo; ER: Extract of residue of olive oil production. A: Aqueous extract of olive by-products, B: Ethanolic extract of olive by-products. The results are expressed as means and bar indicates SD (n = 3). Statistical significance was determined by Tukey’s multiple comparisons test (**** for p < 0.0001; *** for p < 0.001; ** for p < 0.01; and * for p < 0.05). A: Aqueous extract of olive by-products, B: Ethanolic extract of olive by-products. The inhibition of the extracts against the S. Enteritidis bacterium is shown in Fig. 4. Of the aqueous extracts (Fig. 4A), those from Grappolo leaf showed the highest (p < 0.05) inhibition ranging from 12.61 ± 2.64% (6.25% concentration) to 41.14 ± 2.66% (50% concentration). The ethanolic extracts (Fig. 4B) showed excellent antibacterial activities, with emphasis on the Grappolo leaf extract, which showed the highest (p < 0.0001) inhibition at all concentrations tested, ranging from 41.47 ± 0.25% (for the lowest concentration of 6.25%) to 85.13 ± 4.61% (at the highest concentration of 50%). The antimicrobial activity of the other ethanolic extracts was similar (p˃ 0.05) when tested at concentrations of 50, 25 and 6.25%, with an inhibition of 55.22 ± 4.37 for the Ascolano leaf extract, and 51.20 ± 2.97 for the aqueous extract, at the highest concentration. The inhibition of bacteria Salmonella Enteritidis, Listeria monocytogenes , and Escherichia coli by olive leaf extract was confirmed in the work of Liu Y, et al. [ 30 ]. In most cases, gram-positive bacteria are more sensitive to natural plant compounds than gram-negative bacteria [ 44 ]. This is thought to be due to differences in the cell structure of these bacteria. In gram-positive bacteria, antibacterial substances can enter through the cell wall and attack the cytoplasmic membrane, resulting in cytoplasmic leakage [ 23 ], Gram-negative bacteria, on the other hand, have an outer lipopolysaccharide component membrane that protects them from various agents [ 46 ]. However, in our work, we observed that the extracts showed high antimicrobial activity against the gram-negative S. Enteritidis bacterium, with an emphasis on the antimicrobial activity at low extract concentrations, probably the antimicrobial compounds present in the extracts have hydrophobic characteristics, which may contribute to the activity against Gram-negative bacteria. The activity against S. Enteritidis of oleuropein and caffeic acid was confirmed in the work of Lee O-H, et al. [ 28 ]. Figure 4. Percentage of inhibition of extracts derived from olive leaves and residue of olive oil production on Salmonella Enteritidis. EA: Extract of olive leaves Ascolano; EG: Extract of olive leaves Grappolo; ER: Extract of residue of olive oil production. A: Aqueous extract of olive by-products, B: Ethanolic extract of olive by-products. The results are expressed as means and bar indicates SD (n = 3). Statistical significance was determined by Tukey’s multiple comparisons test (**** for p < 0.0001; *** for p < 0.001; ** for p < 0.01; and * for p < 0.05). A: Aqueous extract of olive by-products, B: Ethanolic extract of olive by-products. Figure 5 shows the percentage inhibition of the extracts against S. aureus . The aqueous extracts (Fig. 5A) of Grappolo leaf and residue of olive oil production extracts showed the highest (p < 0.001) inhibition with values of 39.83 ± 2.50% and 42.56 ± 4.48%, respectively, at a concentration of 50%. However, at the lowest concentration tested (6.25%), only the Ascolano leaf extract was able to inhibit the S. aureus bacterium (17.33 ± 2.16%), on the contrary, in the same concentration, the Ascolano leaves ethanolic extract showed no inhibition capacity (Fig. 5B). The highest antimicrobial activity was demonstrated by the ethanolic extracts at a concentration tested of 50%. Again, Grappolo leaf extracts showed the highest (p < 0.0001) inhibition capacity (91.95 ± 3.50%), followed by Ascolano leaf extracts (60.61 ± 2.98%). The ethanolic extracts of the residue of olive oil production showed an antimicrobial activity ranging from 13.25 ± 2.25 to 42.77 ± 1.40%, for the lowest and highest concentrations tested, respectively. Aqueous extract from "Chemlali" olive leaf showed the maximum antimicrobial activity against S. aureus in the work of Debib A, et al. [ 11 ]. The ethanolic extract in the concentration of 100% of “Dathier” olive leaves showed the greatest inhibition potential against S. aureus [ 20 ]. Hydroxytyrosol and oleuropein are phenolic compounds that have been proven to be effective against several human intestinal or respiratory tract pathogens by inhibiting or delaying their growth rate, and different targets have been detected for Staphylococcus aureus [ 49 ]. The effect of olive mill wastewater extract was tested on Staphylococcus aureus and Escherichia coli in the work of Abu-Lafi S, et al. [ 2 ], showed an inhibition zone equal to 23 mm and 25 mm, respectively, having a higher antibacterial activity compared to well-known antibiotics. Figure 5 . Percentage of inhibition of extracts derived from olive leaves and residue of olive oil production on Staphylococcus aureus . EA: Extract of olive leaves Ascolano; EG: Extract of olive leaves Grappolo; ER: Extract of residue of olive oil production. A: Aqueous extract of olive by-products, B: Ethanolic extract of olive by-products. The results are expressed as means and bar indicates SD (n = 3). Statistical significance was determined by Tukey’s multiple comparisons test (**** for p < 0.0001; *** for p < 0.001; ** for p < 0.01; and * for p < 0.05). A: Aqueous extract of olive by-products, B: Ethanolic extract of olive by-products. The results of the antimicrobial activity against E. coli are shown in Fig. 6. For the aqueous extracts (Fig. 6A), the residue of olive oil production extracts was the least efficient (p < 0.01) to inhibit the microorganism studied at a concentration of 50% (32.16 ± 3.59%). On the other hand, Ascolano and Grappolo leaf extracts showed similar (p˃ 0.05) inhibition values, showing 44.07 ± 1.71% and 38.94 ± 4.99% of inhibition, respectively. As observed for all studied microorganisms, Grappolo leaves ethanolic extracts were also more efficient (p < 0.0001) in inhibiting E. coli bacterium (91.43 ± 0.35%), followed by Ascolano leaves ethanolic extracts (80.16 ± 2.17%) (Fig. 6B). Extracts from olive mill residues were evaluated for phenolic compounds and their antimicrobial and antioxidant activity, resulting in broad-spectrum antibacterial activity against Staphylococcus aureus, Bacillus subtilis, Escherichia coli , and Pseudomonas aeruginosa [ 35 ]. The bacterium E. coli was sensitive to acetone, ethyl alcohol, and diethyl ether extracts from olive leaves in the work of Korukluoglu M, et al. [ 25 ]. However, the aqueous extract had no antimicrobial activity. In addition, the authors observed antimicrobial activity for p -coumaric acid, caffeic acid, and oleuropein against E. coli. Figure 6. Percentage of inhibition of extracts derived from olive leaves and residue of olive oil production on Escherichia coli. EA: Extract of olive leaves Ascolano; EG: Extract of olive leaves Grappolo; ER: Extract of residue of olive oil production. A: Aqueous extract of olive by-products, B: Ethanolic extract of olive by-products. The results are expressed as means and bar indicates SD (n = 3). Statistical significance was determined by Tukey’s multiple comparisons test (**** for p < 0.0001; *** for p < 0.001; ** for p < 0.01; and * for p < 0.05). A: Aqueous extract of olive by-products, B: Ethanolic extract of olive by-products. In Table 2 , it is observed that only Grappolo leaves ethanolic extracts presented an MBC of 50% for the microorganisms Bacillus cereus, Escherichia coli , and Staphylococcus aureus , the bactericidal activity was not observed for the other microorganisms. The other extracts only showed inhibitory activity, not being able to cause microbial death. Again, we can observe that ethanolic is a more effective extracting solvent when compared to water for the extracts of by-products generated in the production of olive oil and olive leaves. So ethanol showed great extraction efficiency, these results are in line with other authors [ 10 , 18 , 24 , 25 ]. The type of solvent affects the distribution and number of phenolic compounds present in the extracts, and consequently the antimicrobial activity [ 25 ]. Therefore, we can also confirm the strong antimicrobial effect of the phenolic compounds found in the Grappolo leaves ethanolic extracts, especially hydroxytyrosol. The development of a natural and sustainable preservative olive by-product extract for food applications is a potentially viable approach that would allow us to explore the bioactivities of Olea europaea , furthermore, the use of olive by-products appears to be a more reasonable approach to develop or exploit components for biotechnological applications than using more valuable materials such as olive fruit or oil. Table 2 Minimum bactericidal concentration (MBC) evaluated to extracts derived from olive leaves and residue of olive oil production. Extract Microorganisms L. monocytogenes B. cereus S. Enteritidis S. aureus E. coli EA Aqueous - - - - - EG - - - - - ER - - - - - EA Ethanolic - - - - - EG - 50% - 50% 50% ER - - - - - EA: Extract of olive leaves Ascolano; EG: Extract of olive leaves Grappolo; ER: Extract of residue of olive oil production. ( - ): the bactericidal activity was not observed Conclusion Overall, these results indicate that by-products generated in the production of olive oil and olive leave extracts (especially ethanolic) can be considered a potential source of antibacterial agents against some microorganisms that cause foodborne illness, thus being an economical, sustainable, viable, and accessible alternative for antimicrobial treatment, being able to replace chemical preservatives, which contain toxic and carcinogenic properties. Further research is needed to obtain more information on the antimicrobial activity of the extracts in practice under different application conditions. Declarations Conflict of Interest The authors report there are no competing interests to declare Funding This work was supported by the Brazilian agencies Conselho Nacional de Desenvolvimento Cientifico e Tecnologico do Brasil (CNPq) under Grant [312336/2014–4, 423095/2016–1]; Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG) under Grant [CAG—APQ-03478–16]; and Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior (CAPES) under Grant [PNPD20131289]. Author Contributions L.S. conceptualized the study and led all stages of the research, including methodology development, validation, investigation, data curation, formal analysis, and visualization. She drafted the original version of the manuscript and was responsible for reviewing and editing the final text. N.F., D. S., and B.S. contributed to data formal analysis and participated in the critical review and editing of the manuscript. Acknowledgments The authors thank EPAMIG Experimental Farm in Maria da Fé, Minas Gerais, Brazil, for providing the olive leaves and residues used in this work. Data Availability Data available within the article or its supplementary materials References Abaza L, Youssef NB, Manai H, Haddada FM, Methenni K, Zarrouk M (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 Abu-Lafi S, Al-Natsheh MS, Yaghmoor R, Al-Rimawi F (2017) Enrichment of phenolic compounds from olive mill wastewater and in vitro evaluation of their antimicrobial activities. Evidence-Based Complementary and Alternative medicine 2017 https://doi.org/10.1155/2017/3706915 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 research international 2013 https://doi.org/10.1155/2013/461415 Amiot M-J, Fleuriet A, Macheix J-J (1989) Accumulation of oleuropein derivatives during olive maturation. Phytochemistry 28:67–69. https://doi.org/10.1016/0031-9422(89)85009-5 Babić S, Malev O, Pflieger M, Lebedev AT, Mazur DM, Kužić A, Čož-Rakovac R, Trebše P (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 Bisignano G, Tomaino A, Cascio RL, Crisafi G, Uccella N, Saija A (1999) On the in-vitro antimicrobial activity of oleuropein and hydroxytyrosol. J Pharm Pharmacol 51:971–974. https://doi.org/10.1211/0022357991773258 Bona EAMD, Pinto FGS, Fruet TK, Jorge TCM, Moura AC (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 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 CLSI (2014) Methods for broth dilution susceptibility testing of bacteria isolated from aquatic animals. approved guideline De Bruno A, Romeo R, Fedele FL, Sicari A, Piscopo A, Poiana M (2018) Antioxidant activity shown by olive pomace extracts. J Environ Sci Health Part B 53:526–533. https://doi.org/10.1080/03601234.2018.1462928 Debib A, Boukhatem MN (2017) Phenolic content, antioxidant and antimicrobial activities of Chemlali olive leaf (Olea europaea L.) extracts. Int J Pharmacol Phytochemistry Ethnomed 6:38–46. https://doi.org/10. 18052/www.scipress.com/IJPPE.6.38 Dermeche S, Nadour M, Larroche C, Moulti-Mati F, Michaud P (2013) Olive mill wastes: Biochemical characterizations and valorization strategies. Process Biochem 48:1532–1552. https://doi.org/10.1016/j.procbio.2013.07.010 El-Abbassi A, Saadaoui N, Kiai H, Raiti J, Hafidi A (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 El SN, Karakaya S (2009) Olive tree (Olea europaea) leaves: potential beneficial effects on human health. Nutr Rev 67:632–638. https://doi.org/10.1111/j.1753-4887.2009.00248.x Ferro MD, Lopes E, Afonso M, Peixe A, Rodrigues FM, Duarte MF (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 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 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 Ghomari O, Sounni F, Massaoudi Y, Ghanam J, Kaitouni LBD, Merzouki M, Benlemlih M (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 Gökmen M, Kara R, Akkaya L, Torlak E, Önen A (2014) Evaluation of antimicrobial activity in olive (Olea europaea) leaf extract. Am J Microbiol 5:37–40 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 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 Hussain A, Qarshi IA, Liaqat R, Akhtar S, Aziz I, Ullah I, Shinwari ZK (2014) Antimicrobial potential of leaf and fruit extracts and oils of wild and cultivated edible olive. Pak J Bot 46:1463–1468 Kalemba D, Kunicka A (2003) Antibacterial and antifungal properties of essential oils. Curr Med Chem 10:813–829. https://doi.org/10.2174/0929867033457719 Khan H, Ahmad W, Hussain I, Imran M, Afridi MS, Ullah S (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 Korukluoglu M, Sahan Y, Yigit A, Ozer ET, GÜCER S (2010) Antibacterial activity and chemical constitutions of Olea europaea L. leaf extracts. J Food Process Preserv 34:383–396. https://doi.org/10.1111/j.1745-4549.2008.00318.x Ladhari A, Zarrelli A, Ghannem M, Ben Mimoun M (2021) Olive wastes as a high-potential by-product: Variability of their phenolic profiles, antioxidant and phytotoxic properties. Waste Biomass Valoriz 12:3657–3669. https://doi.org/10.1007/s12649-020-01256-2 Lama-Muñoz A, del Mar Contreras M, Espínola F, Moya M, Romero I, Castro E (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 Lee O-H, Lee B-Y (2010) Antioxidant and antimicrobial activities of individual and combined phenolics in Olea europaea leaf extract. Bioresour Technol 101:3751–3754. https://doi.org/10.1016/j.biortech.2009.12.052 Ling LT, Yap S-A, Radhakrishnan AK, Subramaniam T, Cheng HM, Palanisamy UD (2009) Standardised Mangifera indica extract is an ideal antioxidant. Food Chem 113:1154–1159. https://doi.org/10.1016/j.foodchem.2008.09.004 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 Liu Y, McKeever LC, Suo Y, Jin TZ, Malik NS (2018) Antimicrobial activities of olive leaf extract and its potential use in food industry. In Natural and Bio-Based Antimicrobials for Food Applications; ACS Publications: pp 119-132.10.1021/bk-2018-1287.ch006 Medeiros RML, Villa F, da Silva DF, Cardoso Filho LR (2016) Destinação e reaproveitamento de subprodutos da extração olivícola. Scientia Agrar Paranaensis 100–108. https://doi.org/10.18188/sap.v15i2.11905 Medina E, De Castro A, Romero C, Brenes M (2006) Comparison of the concentrations of phenolic compounds in olive oils and other plant oils: correlation with antimicrobial activity. J Agric Food Chem 54:4954–4961. https://doi.org/10.1021/jf0602267 Nnachi RC, Sui N, Ke B, Luo Z, Bhalla N, He D, Yang Z (2022) Recent progress on biosensors for rapid detection of bacterial pathogens in water, food and environment. Environ Int 107357. https://doi.org/10.1016/j.envint.2022.107357 Obied H, Bedgood D Jr, Prenzler PD, Robards K (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 Ortega-García F, Blanco S, Peinado MÁ, Peragón J (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 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 Palmeri R, Siracusa L, Carrubba M, Parafati L, Proetto I, Pesce F, Fallico B (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 Pereira AP, Ferreira IC, Marcelino F, Valentão P, Andrade PB, Seabra R, Estevinho L, Bento A, Pereira JA (2007) Phenolic compounds and antimicrobial activity of olive (Olea europaea L. Cv. Cobrançosa) leaves. Molecules 12:1153–1162. https://doi.org/10.3390/12051153 Pham D-C, Nguyen H-C, Nguyen T-HL, Ho H-L, Trinh T-K, Riyaphan J, Weng C-F (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 research international 2020 https://doi.org/10.1155/2020/3497107 Rahnama H, Azari R, Yousefi MH, Berizi E, Mazloomi SM, Hosseinzadeh S, Derakhshan Z, Ferrante M, Conti GO (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 Romero-García J, Niño L, Martínez-Patiño C, Álvarez C, Castro E, Negro M (2014) Biorefinery based on olive biomass. State of the art and future trends. Bioresour Technol 159:421–432. https://doi.org/10.1016/j.biortech.2014.03.062 Shah MA, Mir SA (2022) Plant extracts as food preservatives. In Plant Extracts: Applications in the Food Industry; Elsevier: pp 127–141. https://doi.org/10.1016/B978-0-12-822475-5.00010-7 Shan B, Cai Y-Z, Brooks JD, Corke H (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 Simões L, Fernandes N, Teixeira J, Abrunhosa L, Dias DR (2023) Brazilian table olives: a source of lactic acid bacteria with antimycotoxigenic and antifungal activity. Toxins 15:71. https://doi.org/10.3390/toxins15010071 Tortora GJ, Case CL, Funke BR (2016) Microbiologia-12ª Edição. Artmed Editora Yuan J-J, Wang C-Z, Ye J-Z, Tao R, Zhang Y-S (2015) Enzymatic hydrolysis of oleuropein from Olea europea (olive) leaf extract and antioxidant activities. Molecules 20:2903–2921. https://doi.org/10.3390/molecules20022903 Zhang C, Xin X, Zhang J, Zhu S, Niu E, Zhou Z, Liu D (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 Zorić N, Kosalec I (2022) The Antimicrobial Activities of Oleuropein and Hydroxytyrosol. In Promising Antimicrobials from Natural Products; Springer: pp 75–89. https://doi.org/10.1007/978-3-030-83504-0_5 Additional Declarations The authors declare no competing interests. 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08:21:59","extension":"html","order_by":16,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":142855,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8619319/v1/69202d4b31aeafe68d7a0c6c.html"},{"id":100533825,"identity":"22036c55-8e91-49bb-850e-608c397a9dbf","added_by":"auto","created_at":"2026-01-19 03:01:02","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":63313,"visible":true,"origin":"","legend":"\u003cp\u003eHPLC chromatograms of the standards. 1: Hydroxytyrosol; 2: Chlorogenic acid; 3: Rutin; 4: Caffeic acid; 5: Coumaric acid; 6: Oleuropein.\u003c/p\u003e","description":"","filename":"image1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8619319/v1/0b7d46ee0b20eb50a9a6468a.jpeg"},{"id":100533826,"identity":"c6ded423-d762-4fa1-92fd-a38bca141d4c","added_by":"auto","created_at":"2026-01-19 03:01:02","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":858790,"visible":true,"origin":"","legend":"\u003cp\u003ePercentage of inhibition of extracts derived from olive leaves and residue of olive oil production on \u003cem\u003eListeria monocytogenes\u003c/em\u003e. EA: Extract of olive leaves Ascolano; EG: Extract of olive leaves Grappolo; ER: Extract of residue of olive oil production. A: Aqueous extract of olive by-products, B: Ethanolic extract of olive by-products. The results are expressed as means and bar indicates SD (n = 3). Statistical significance was determined by Tukey’s multiple comparisons test (**** for p\u0026lt;0.0001; *** for p\u0026lt;0.001; ** for p\u0026lt;0.01; and * for p\u0026lt;0.05)\u003c/p\u003e","description":"","filename":"Fig.2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8619319/v1/dc63eba98456f3d32c565270.jpg"},{"id":100533831,"identity":"ea6db87b-2f36-4d0d-b9d2-aea0f3bf3113","added_by":"auto","created_at":"2026-01-19 03:01:02","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1072237,"visible":true,"origin":"","legend":"\u003cp\u003ePercentage of inhibition of extracts derived from olive leaves and residue of olive oil production on \u003cem\u003eBacillus cereus.\u003c/em\u003e EA: Extract of olive leaves Ascolano; EG: Extract of olive leaves Grappolo; ER: Extract of residue of olive oil production. A: Aqueous extract of olive by-products, B: Ethanolic extract of olive by-products. The results are expressed as means and bar indicates SD (n = 3). Statistical significance was determined by Tukey’s multiple comparisons test (**** for p\u0026lt;0.0001; *** for p\u0026lt;0.001; ** for p\u0026lt;0.01; and * for p\u0026lt;0.05). A: Aqueous extract of olive by-products, B: Ethanolic extract of olive by-products\u003c/p\u003e","description":"","filename":"Fig.3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8619319/v1/d6c8fbc5a2d9c2fe98e37657.jpg"},{"id":100533843,"identity":"22888e1e-a2de-470b-adad-cf1dd3594930","added_by":"auto","created_at":"2026-01-19 03:01:02","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1011631,"visible":true,"origin":"","legend":"\u003cp\u003ePercentage of inhibition of extracts derived from olive leaves and residue of olive oil production on \u003cem\u003eSalmonella\u003c/em\u003eEnteritidis. EA: Extract of olive leaves Ascolano; EG: Extract of olive leaves Grappolo; ER: Extract of residue of olive oil production. A: Aqueous extract of olive by-products, B: Ethanolic extract of olive by-products. The results are expressed as means and bar indicates SD (n = 3). Statistical significance was determined by Tukey’s multiple comparisons test (**** for p\u0026lt;0.0001; *** for p\u0026lt;0.001; ** for p\u0026lt;0.01; and * for p\u0026lt;0.05). A: Aqueous extract of olive by-products, B: Ethanolic extract of olive by-products\u003c/p\u003e","description":"","filename":"Fig.4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8619319/v1/b3773d1f13a62e43def5a9cd.jpg"},{"id":100533835,"identity":"75b958e0-cff9-4e66-89af-a2785f31d312","added_by":"auto","created_at":"2026-01-19 03:01:02","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1000994,"visible":true,"origin":"","legend":"\u003cp\u003ePercentage of inhibition of extracts derived from olive leaves and residue of olive oil production on \u003cem\u003eStaphylococcus aureus\u003c/em\u003e. EA: Extract of olive leaves Ascolano; EG: Extract of olive leaves Grappolo; ER: Extract of residue of olive oil production. A: Aqueous extract of olive by-products, B: Ethanolic extract of olive by-products. The results are expressed as means and bar indicates SD (n = 3). Statistical significance was determined by Tukey’s multiple comparisons test (**** for p\u0026lt;0.0001; *** for p\u0026lt;0.001; ** for p\u0026lt;0.01; and * for p\u0026lt;0.05). A: Aqueous extract of olive by-products, B: Ethanolic extract of olive by-products\u003c/p\u003e","description":"","filename":"Fig.5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8619319/v1/8823c575b297cb89e96ae829.jpg"},{"id":100549106,"identity":"344e426e-699f-435d-bb83-dbccad5e7f59","added_by":"auto","created_at":"2026-01-19 08:22:25","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":998092,"visible":true,"origin":"","legend":"\u003cp\u003ePercentage of inhibition of extracts derived from olive leaves and residue of olive oil production on \u003cem\u003eEscherichia coli.\u003c/em\u003eEA: Extract of olive leaves Ascolano; EG: Extract of olive leaves Grappolo; ER: Extract of residue of olive oil production. A: Aqueous extract of olive by-products, B: Ethanolic extract of olive by-products. The results are expressed as means and bar indicates SD (n = 3). Statistical significance was determined by Tukey’s multiple comparisons test (**** for p\u0026lt;0.0001; *** for p\u0026lt;0.001; ** for p\u0026lt;0.01; and * for p\u0026lt;0.05). A: Aqueous extract of olive by-products, B: Ethanolic extract of olive by-products\u003c/p\u003e","description":"","filename":"Fig.6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8619319/v1/824729e3b20fab794aba288b.jpg"},{"id":100554621,"identity":"449d923c-7e47-4c65-aa38-4f0004111475","added_by":"auto","created_at":"2026-01-19 08:38:52","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5824702,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8619319/v1/809cf766-d409-4151-b3a8-f270f202cf61.pdf"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003eSustainable extracts from oliviculture by-products: phenolic content and its antimicrobial activity against foodborne pathogens\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eOlive trees, \u003cem\u003eOlea europaea L.\u003c/em\u003e, belong to the botanical family Oleaceae and are native to Mediterranean countries, therefore, this region is the largest producer of olives and olive oil, accounting for 98% of the total area of olive tree production in the world. Olive tree products are rich in important substances from a nutritional perspective [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e] since they have antioxidant and antimicrobial properties attributed to their phenolic compounds, particularly hydroxytyrosol, and oleuropein [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eOlive by-products often referred to as residues or waste products, are derived from the cultivation and processing of olive trees and olive oil, and are produced annually in large quantities [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Unfortunately, most of these by-products do not have practical applications and go to waste, therefore the use of both can be the focus of a sustainable valuation of new products [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. One of the most abundant by-products of olive growing is olive leaves, which have antioxidant, anti-inflammatory, antimicrobial, and antiviral activity, therefore they can be considered a cheap and natural raw material of antioxidants [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn addition, there are residues from the processing of olives for the extraction of olive oil, which represent a huge environmental problem, as they are produced in large quantities and have a high content of organic matter, such as polyphenols, which may be responsible for the high toxicity [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Therefore, the use of this residue requires a significant economic effort to avoid severe environmental pollution, and it can be used as an economic source with antimicrobial and antioxidant activity, in order to prevent environmental damage arising from its misdirection [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eUsually, for the production of olive oil, the centrifugation method is used, which can be a continuous three-phase process, where the waste produced comprises the liquid wastewater from vegetation olive mill waste-water and bagasse, which corresponds to solid waste [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. The process can also be carried out in a two-phase system for the production of olive oil, originating a solid residue called two-phase olive pomace, which consists of a thick mass made up of the pulp and stone of the olive, as well as the water from the fruit and residual oil [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eDue to the increased resistance of pathogenic microorganisms to numerous drugs and the susceptibility of acting on various types of substrates, there is a concern for the search for new alternatives, which encourages the search for natural antibiotics, according to the World Health Organization (WHO), foodborne infections remain one of the leading causes of disease worldwide [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eDifferent approaches have been used for contamination by pathogenic microorganisms, such as the use of preservatives. In this context, due to concerns about the toxicity and dangers of synthetic preservatives, the demand for natural food preservatives has increased, and plant extracts emerge as a better option, rich in bioactive compounds, acting as natural preservatives [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Olive-growing by-products, being natural compounds, can emerge as an alternative to obtain antimicrobials, resulting in the replacement of chemical preservatives, which may contain toxic and carcinogenic properties.\u003c/p\u003e \u003cp\u003eGiven the above, the present study aimed to characterize extracts from olive leaves (\u003cem\u003eOlea europaea\u003c/em\u003e) and residue of the olive oil production in a two-phase system, concerning phenolic content, as well as antimicrobial activity on food-contaminating bacteria.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eCollection and sampling location\u003c/h2\u003e \u003cp\u003eThe residues of the olive oil production and the olive leaves used to manufacture the extracts were both kindly provided by the Experimental Farm of EPAMIG (Empresa de Pesquisa Agropecu\u0026aacute;ria de Minas Gerais) in Maria da F\u0026eacute;, Brazil (22◦18\u0026rsquo; south latitude,\u003c/p\u003e \u003cp\u003e45◦23\u0026rsquo; west longitude, an average elevation of 1.276 m). Olive leaves were randomly picked from olive branches of the Ascolano and Grappolo 546 cultivars. Approximately 1 kg of the residue of the olive oil production in a two-phase system (solid fraction) from unripe olives (green olives) containing olive pulp, skin, stone, water, and some concentration of olive oil, was collected.\u003c/p\u003e \u003cp\u003eAll materials were collected in sterile plastic bags and taken under refrigeration to the Laboratory of Microbiology of Fermentations, in the Microbiology sector, Department of Biology, Federal University of Lavras (UFLA), Lavras, Brazil, for the manufacture of extracts.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003ePreparation of extracts\u003c/h3\u003e\n\u003cp\u003eTwo extraction methods were used, resulting in aqueous extracts (using deionized water) and another ethanolic extract (using 70% ethanol). Olive leaves were washed with deionized water and dried at 37\u0026deg;C for 3 days, and the residue of the olive oil production was directly placed in an oven for drying in the same conditions. Then the dried leaves and residue were pulverized in a conventional blender in order to decrease the particle size to 90\u0026ndash;150 \u0026micro;m. For each extraction method, 3 different extracts were prepared: olive leaves Ascolano extract; olive leaves Grappolo extract, and the residues of the olive oil production extract.\u003c/p\u003e\n\u003ch3\u003eAqueous extract\u003c/h3\u003e\n\u003cp\u003eThe methodology described by Al-Attar AM, et al. [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e] was used with some modifications. 50 g of dried olive leaves and residues of the olive oil production were added to 2 L of hot water. After 3 h, the mixture was slowly boiled for 30 min. After boiling, the mixture was cooled to room temperature and subjected to an electric mixer for 20 min. Subsequently, the solutions were centrifuged (5 min at 5000 rpm) and filtered through a sterile syringe filter (0.2 \u0026micro;m, PES). The filtrate was evaporated in a rotary evaporator at 40\u0026deg;C to produce dry residue (active ingredients) and stored at 4\u0026deg;C to be used throughout the experiment.\u003c/p\u003e\n\u003ch3\u003eEthanol extract\u003c/h3\u003e\n\u003cp\u003eThe extracts were obtained using the methodology proposed by Ling LT, et al. [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e] with some modifications. Dried olive leaves and residues of the olive oil production (50 g) were added with 750 ml of ethanol (70% v/v). The extraction was performed at room temperature, where the samples were left to rest for 24 h, so the extracts were centrifuged (5 min at 5000 rpm) and filtered through a sterile syringe filter (0.2 \u0026micro;m, PES). Then, the solvent was removed using a rotary evaporator at 38\u0026deg;C with a rotation of 120 rpm under a vacuum. The extracts were kept at 4\u0026deg;C temperature to be used throughout the experiment.\u003c/p\u003e\n\u003ch3\u003eTested microorganisms\u003c/h3\u003e\n\u003cp\u003eThe following bacteria were used: Gram-positive (\u003cem\u003eListeria monocytogenes\u003c/em\u003e ATCC 19117, \u003cem\u003eStaphylococcus aureus\u003c/em\u003e ATCC 8702, and \u003cem\u003eBacillus cereus\u003c/em\u003e ATCC 14579) and Gram-negative bacteria (\u003cem\u003eEscherichia coli\u003c/em\u003e EPEC055 and \u003cem\u003eSalmonella\u003c/em\u003e Enteritidis ATCC 564). The inoculums were standardized with the aid of a growth curve, following the absorbance (O.D. 600nm) and the plate count using Brain Heart Infusion (BHI) agar (Sigma Aldrich, Darmstadt, Germany). The plates were incubated at 37\u0026deg;C for 24 h and the inoculum was standardized at 10\u003csup\u003e8\u003c/sup\u003e CFU.mL\u003csup\u003e-1\u003c/sup\u003e. The inoculums were stored in a freezer at -70\u0026ordm;C and thawed at room temperature during the experiment.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eAntimicrobial activity\u003c/h2\u003e \u003cp\u003eTo determine the percentage of inhibition, the protocol established by CLSI [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e] was used, with some adaptations, using the broth microdilution technique (sterile 96-well microplates). The culture medium used was BHI broth (Sigma Aldrich, Darmstadt, Germany).\u003c/p\u003e \u003cp\u003eTo each well was added 100 \u0026micro;l of BHI broth. In the first well of each line of microplates, a volume of 100\u0026micro;L of the respective extract (resuspended in sterile water) to be analyzed was also added, obtaining the initial concentration of each extract (50%). From the initial wells, serial dilutions were performed obtaining decreasing concentrations of 50%, 25%, 12.5%; 6.25% (v/v). Aliquots of 10 \u0026micro;L of each bacterial suspension were inoculated into wells containing BHI medium and the respective concentration of extracts. Positive controls (PC) consisted of 10 \u0026micro;L of each bacterial suspension inoculated in BHI medium without the addition of extracts, and negative controls (NC) consisted of BHI medium supplemented with 10 \u0026micro;L of the different extract concentrations without the addition of bacterial suspension. The microplates were sealed and incubated in a BOD incubator at 37\u0026ordm;C for 24 h. All experiments were conducted in triplicate three repetitions.\u003c/p\u003e \u003cp\u003eAfter incubation, after the incubation, absorbance at 600 nm was measured using a UV spectrophotometer (Thermo Scientific, Waltham, MA, USA), and the value of the percentage of inhibition was expressed as:\u003c/p\u003e \u003cp\u003eInhibition (%) = [1 - (Ac - NC/PC)] x 100\u003c/p\u003e \u003cp\u003eWhere Ac represents the absorbance of the well with different concentrations of extracts, NC is the negative control, and PC is the absorbance of positive control [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eMinimum Bactericidal Concentration (MBC)\u003c/h3\u003e\n\u003cp\u003eAfter the incubation period was determinate of the minimum bactericidal concentration (MBC), The micro drop technique was used with plating on BHI agar, 10 \u0026micro;L aliquots of cultures from the wells were plated where there was no turbidity were put in the plates and incubated at 37\u0026deg;C/24h. After incubation, the MBC of the extracts was determined, as its lowest concentration capable of promoting the absence of growth of the bacteria tested in plate [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. The experiment was carried out in triplicate and with three repetitions.\u003c/p\u003e\n\u003ch3\u003eIdentification of phenolic compounds contained in each extract by HPLC (UV-vis)\u003c/h3\u003e\n\u003cp\u003eHigh-pressure liquid chromatography (HPLC) equipped with UV-visible absorption (UV-vis) was used for the analytical qualification and quantification of phenolic compounds in each olive leaf extract. The extracts (10mg) were dissolved in 1 mL of 100 mM perchloric acid (Sigma Aldrich, Darmstadt, Germany) solution and filtered with 0.45\u0026micro;m filters into vials before analysis by HPLC (UV-vis). The separation of phenolic compounds was carried out in Shimpack SCR-101H column (7.9mm \u0026times; 30 cm) Shimadzu, the mobile phase was 100mM perchloric acid (Sigma Aldrich, Darmstadt, Germany) solution, with a flow rate of 0.6mL/min over 20 min run, the oven temperature was 50 ◦C, detected with a 210nm UV detector (30 ◦C).\u003c/p\u003e \u003cp\u003eAll phenolic compounds were identified by comparing their retention times with the respective standards (chlorogenic acid, caffeic acid, coumaric acid, hydroxytyrosol, oleuropein, and rutin) (Merck, Darmstadt, Germany), as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Before running the experiments, calibration curves were established for each analyte so that relative response factors for each analyte could be considered in reporting analyte concentrations. Analyses were performed in triplicate.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eCalibration curves\u003c/h2\u003e \u003cp\u003eThe calibration of the phenolic compounds method was conducted with 7 levels for chlorogenic acid, caffeic acid, coumaric acid, hydroxytyrosol, oleuropein, and rutin. The fit of the applied linear regression model was described as R\u003csup\u003e2\u003c/sup\u003e (\u0026gt;\u0026thinsp;0.99). Each set was made by dissolving the given mass in nanopure water to obtain L7. Levels 6\u0026thinsp;\u0026minus;\u0026thinsp;1 were then created by serial dilution into the same size volumetric flask used to create L7. Each level was syringe filtered through a 0.45 \u0026micro;m nylon filter. Calibration curves were obtained from the area of each standard peak in each level. Each compound was identified by retention time (min).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analyses\u003c/h2\u003e \u003cp\u003eThe results were submitted to statistical analyses using GraphPad Prism version 8.01 for Windows (La Jolla, CA, USA). The two-way ANOVA and Tukey\u0026rsquo;s multiple comparisons test were performed and the significant differences were reported as: **** for p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001; *** for p\u0026thinsp;\u0026lt;\u0026thinsp;0.001; ** for p\u0026thinsp;\u0026lt;\u0026thinsp;0.01; and * for p\u0026thinsp;\u0026lt;\u0026thinsp;0.05. Results were expressed as mean values\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (n\u0026thinsp;=\u0026thinsp;3).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results and discussion","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eIdentification of phenolic compounds\u003c/h2\u003e \u003cp\u003eTo our knowledge, this is the first time that Ascolano and Grappolo olive leaves have been studied. Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e showed the phenolic composition of the extracts used in this study, the compounds identified were chlorogenic acid, caffeic acid, coumaric acid, hydroxytyrosol, oleuropein, and rutin. In the aqueous extracts, the compounds caffeic acid, coumaric acid, and oleuropein were not present in olive leaf extracts, for the olive oil production residues aqueous extracts, caffeic acid was the only compound that was not present. The use of 70% ethanol (v/v) provided a higher concentration and variety of compounds in the extracts; therefore, ethanol was the solvent with the highest extraction capacity, which contributes to better antimicrobial capacity. This fact is in line with the work of Ortega-Garc\u0026iacute;a F, et al. [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e], which showed that the use of water combined with alcohols contributes to increased swelling of plant materials, resulting in the increased contact area between plant matrix and solvent, improving extraction yield and higher concentration of compounds.\u003c/p\u003e \u003cp\u003eThe compounds identified in the present study were also found in olive leaf extracts collected in the southeast of Sicily, Italy in the work of Palmeri R, et al. [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Chlorogenic acid, hydroxytyrosol, and rutin were identified in the work by Zhang C, et al. [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e] in olive leaves from cultivars grown in China, these compounds have remarkable antimicrobial properties [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Rutin was one of the flavonoids found in olive leaves in the work of Pham D-C, et al. [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Caffeic acid, \u003cem\u003ep\u003c/em\u003e-coumaric acid, and oleuropein were identified in ethyl alcohol extracts from olive leaves in the work of Korukluoglu M, et al. [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eOlive leaves extract and its constituents, particularly oleuropein, and hydroxytyrosol, have been reported to have health benefits, including antioxidant and antimicrobial properties [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. Normally, in olive leaf extracts, oleuropein is the compound present in most amounts [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. On the other hand, in our study, the olive leaf extracts of the two studied cultivars (Ascolano and Grappolo), hydroxytyrosol was the compound present in higher (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) quantity, mainly in the Grappolo cultivar (9.24\u0026thinsp;\u0026plusmn;\u0026thinsp;0.58 g.ml-1). Hydroxytyrosol is present in almost all parts of the olive tree, mostly in the leaves, being this compound is the main product of oleuropein degradation [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. Therefore, possibly part of the oleuropein present in large amounts in the extracts may have been converted into hydroxytyrosol, resulting in the high concentration of these compounds in the extracts.\u003c/p\u003e \u003cp\u003eSeveral studies have revealed different classes of phenolic compounds in olive residues [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Normally, the phenolic compound oleuropein is the most abundant in olives from different cultivars [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Accordingly, our study showed a high concentration of oleuropein in olive oil production residues ethanolic extracts (16.47\u0026thinsp;\u0026plusmn;\u0026thinsp;0.76 g.ml\u003csup\u003e-1\u003c/sup\u003e), which consists of a mass containing the pulp, pit, skin, and traces of olive oil. Ethyl acetate extracts of olive mill wastewater were studied in the work of Fki I, et al. [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], the hydroxytyrosol was present in higher concentrations, whereas, caffeic acid and coumaric acid were present at lower concentrations.\u003c/p\u003e \u003cp\u003eIn the work of Dermeche S, et al. [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], the solid residue was analyzed, p-coumaric acid, caffeic acid, hydroxytyrosol, and rutin were identified in the extracts, the same for our work, however, the oleuropein is not present. On the other hand, this compound was present in higher (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) amounts in the extracts of residues of the olive oil production in our study. One explanation could be the difference in olive maturation, where in our work the olives used to manufacture olive oil were unripe olives (green olives), whereas in the work where the samples were collected in the final stage, using ripe olives (black olives), when oleuropein is degraded into elenolic acid and hydroxytyrosol [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe main phenolic compounds found in olive oil processing waste in the work of Medeiros RML, et al. [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e] were hydroxytyrosol, oleuropein, tyrosol, caffeic acid, P-coumaric acid, vanillic acid, catechol, and rutin. Therefore, these compounds represent a natural source of antioxidants and antimicrobials and can be used in the food industry, thus replacing chemical preservatives that have been associated with undesirable effects on human health.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePhenolic compounds in extracts derived from olive leaves and residue of olive oil production (g.ml\u003csup\u003e-1\u003c/sup\u003e)\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"9\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eExtracts\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSolvent\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eHydroxytyrosol\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eChlorogenic acid\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eRutin\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eCaffeic acid\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eCoumaric acid\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003eOleuropein\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAqueous\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.65\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08 \u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.076\u0026thinsp;\u0026plusmn;\u0026thinsp;0.005 \u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.023\u0026thinsp;\u0026plusmn;\u0026thinsp;0.003 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.82\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13 \u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.076\u0026thinsp;\u0026plusmn;\u0026thinsp;0.002 \u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.024\u0026thinsp;\u0026plusmn;\u0026thinsp;0.001 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eER\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.66\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11 \u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.072\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0006 \u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.019\u0026thinsp;\u0026plusmn;\u0026thinsp;0.001 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e1.59\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09 \u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0.79\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04 \u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEthanolic\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.28\u0026thinsp;\u0026plusmn;\u0026thinsp;0.46 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.087\u0026thinsp;\u0026plusmn;\u0026thinsp;0.004 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.025\u0026thinsp;\u0026plusmn;\u0026thinsp;0.001 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.89\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04 \u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e1.79\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09 \u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e1.83\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e9.24\u0026thinsp;\u0026plusmn;\u0026thinsp;0.58 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.095\u0026thinsp;\u0026plusmn;\u0026thinsp;0.003 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.033\u0026thinsp;\u0026plusmn;\u0026thinsp;0.002 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1.18\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e2.75\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e1.89\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eER\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.37\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07 \u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.72\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.037\u0026thinsp;\u0026plusmn;\u0026thinsp;0.003 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1.48\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e3.21\u0026thinsp;\u0026plusmn;\u0026thinsp;0.28 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e16.47\u0026thinsp;\u0026plusmn;\u0026thinsp;0.76 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eEA: Extract of olive leaves Ascolano; EG: Extract of olive leaves Grappolo; ER: Extract of residue of olive oil production. -: not identified. The results are expressed as means. \u0026plusmn; SD (n\u0026thinsp;=\u0026thinsp;3). Statistical significance was determined by Tukey\u0026rsquo;s multiple comparisons, different letters differ significantly (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eAntibacterial activity\u003c/h2\u003e \u003cp\u003eThere is a critical need to combat emerging pathogenic microorganisms through varied chemical structures with innovative mechanisms. Therefore, the use of plant-based antimicrobial agents is important, and researchers are dedicating efforts to using natural compounds against pathogenic microorganisms to be used as preservatives in food or even to develop new drugs, which can be a potential alternative to synthetic compounds, that cause harmful effects on human health [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. The present study was conducted in order to evaluate the antimicrobial activity against \u003cem\u003eListeria monocytogenes\u003c/em\u003e ATCC 19117, \u003cem\u003eStaphylococcus aureus\u003c/em\u003e ATCC 8702, \u003cem\u003eBacillus cereus\u003c/em\u003e ATCC 14579, \u003cem\u003eEscherichia coli\u003c/em\u003e EPEC055 and \u003cem\u003eSalmonella\u003c/em\u003e Enteritidis ATCC 564 to extracts of by-products generated in the production of olive oil and olive leaves.\u003c/p\u003e \u003cp\u003eWe can observe that the extract of the leaves of Grappolo was the one that presented a greater antimicrobial activity against all the microorganisms tested, this fact can be correlated to the higher concentration of hydroxytyrosol of these extracts, this compound has demonstrated the potent antimicrobial activity of hydroxytyrosol, against several pathogens bacteria [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]\u003c/p\u003e \u003cp\u003eFigure 2 showed the antimicrobial activity against the gram-positive bacterium \u003cem\u003eListeria monocytogenes\u003c/em\u003e. The olive leaves ethanolic extracts showed higher antimicrobial activity (Fig.\u0026nbsp;2B), with a percentage of inhibition of 91.20\u0026thinsp;\u0026plusmn;\u0026thinsp;1.10 and 74.84\u0026thinsp;\u0026plusmn;\u0026thinsp;4.43% for Grappolo and Ascolano leaf extracts, respectively, for the higher tested concentration. The highest (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) antimicrobial activity for the aqueous extracts (Fig.\u0026nbsp;2A) was observed for the Ascolano leaf extract (57.51\u0026thinsp;\u0026plusmn;\u0026thinsp;6.62) at 50% of concentration. Regarding inhibition by residues of the olive oil production extract, inhibition ranged from 16.37\u0026thinsp;\u0026plusmn;\u0026thinsp;2.83 to 36.45\u0026thinsp;\u0026plusmn;\u0026thinsp;4.09% for the ethanolic extracts, and from 14.82\u0026thinsp;\u0026plusmn;\u0026thinsp;2.66 to 41.93\u0026thinsp;\u0026plusmn;\u0026thinsp;3.21% for the aqueous extracts.\u003c/p\u003e \u003cp\u003eAccording to G\u0026ouml;kmen M, et al. [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e] there was a positive effect of olive leaf extracts on \u003cem\u003eListeria monocytogenes\u003c/em\u003e, the antimicrobial activity was evaluated using disk diffusion microdilution methods. On the other hand, in the work of Hussain A, et al. [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], the leaf extracts from \u003cem\u003eOlea ferruginea\u003c/em\u003e, using the same solvents as in our work, have no effect on the growth of L\u003cem\u003eisteria monocytogenes\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eIn the work of Liu Y, et al. [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e], when studying the effect of olive leaf extract against \u003cem\u003eL. monocytogenes\u003c/em\u003e, the authors found that with the use of extracts, the motility and biofilm formation of the bacterium were decreased. It has been suggested that olive leaf extract has the potential to be used in the food industry as an antimicrobial, in order to inhibit the growth of foodborne pathogens in foods, on processing equipment, or in packaging materials.\u003c/p\u003e \u003cp\u003e \u003cb\u003eFigure 2\u003c/b\u003e. Percentage of inhibition of extracts derived from olive leaves and residue of olive oil production on \u003cem\u003eListeria monocytogenes\u003c/em\u003e. EA: Extract of olive leaves Ascolano; EG: Extract of olive leaves Grappolo; ER: Extract of residue of olive oil production. A: Aqueous extract of olive by-products, B: Ethanolic extract of olive by-products. The results are expressed as means and bar indicates SD (n\u0026thinsp;=\u0026thinsp;3). Statistical significance was determined by Tukey\u0026rsquo;s multiple comparisons test (**** for p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001; *** for p\u0026thinsp;\u0026lt;\u0026thinsp;0.001; ** for p\u0026thinsp;\u0026lt;\u0026thinsp;0.01; and * for p\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003eFor the microorganism \u003cem\u003eB. cereus\u003c/em\u003e, the results are presented in Fig.\u0026nbsp;3, the ethanolic extracts (Fig.\u0026nbsp;3B) showed high values of antimicrobial activity, with the highest (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) percentage found for the Grappolo leaf extract (92.24\u0026thinsp;\u0026plusmn;\u0026thinsp;2.02%), followed by the residues of the olive oil production (75.20\u0026thinsp;\u0026plusmn;\u0026thinsp;3.62%), at the highest concentration tested. On the other hand, at a concentration of 50%, the aqueous extracts of Ascolano leaf exhibited higher (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) antibacterial activity (46.71\u0026thinsp;\u0026plusmn;\u0026thinsp;4.61%), followed by Grappolo leaf extract (27.51\u0026thinsp;\u0026plusmn;\u0026thinsp;1.24%), this last one was the extract that presented higher (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) inhibition at the lowest tested extract concentrations (25, 12.5 and 6.25%). The aqueous extract of c showed a low antimicrobial activity against \u003cem\u003eB. cereus\u003c/em\u003e, with a maximum activity of approximately 8.85\u0026thinsp;\u0026plusmn;\u0026thinsp;0.53% (Fig.\u0026nbsp;3A).\u003c/p\u003e \u003cp\u003e \u003cem\u003eBacillus cereus\u003c/em\u003e is a food-borne bacterium that is highly resistant and causes food poisoning. This organism is found throughout the world, thus making it a major concern for food safety [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. In the work of Pereira AP, et al. [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e], olive leaf aqueous extracts were screened for their antimicrobial activity, and the \u003cem\u003eB. cereus\u003c/em\u003e was the most sensitive microorganism. Oleuropein, the main phenolic compound present in the residues of olive oil production (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), has the ability to inhibit \u003cem\u003eBacillus cereus\u003c/em\u003e sporulation [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Therefore, possibly the extracts can be used both to inhibit the growth of vegetative cells and to inhibit sporulation.\u003c/p\u003e \u003cp\u003e \u003cb\u003eFigure 3.\u003c/b\u003e Percentage of inhibition of extracts derived from olive leaves and residue of olive oil production on \u003cem\u003eBacillus cereus.\u003c/em\u003e EA: Extract of olive leaves Ascolano; EG: Extract of olive leaves Grappolo; ER: Extract of residue of olive oil production. A: Aqueous extract of olive by-products, B: Ethanolic extract of olive by-products. The results are expressed as means and bar indicates SD (n\u0026thinsp;=\u0026thinsp;3). Statistical significance was determined by Tukey\u0026rsquo;s multiple comparisons test (**** for p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001; *** for p\u0026thinsp;\u0026lt;\u0026thinsp;0.001; ** for p\u0026thinsp;\u0026lt;\u0026thinsp;0.01; and * for p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). A: Aqueous extract of olive by-products, B: Ethanolic extract of olive by-products.\u003c/p\u003e \u003cp\u003eThe inhibition of the extracts against the \u003cem\u003eS.\u003c/em\u003e Enteritidis bacterium is shown in Fig.\u0026nbsp;4. Of the aqueous extracts (Fig.\u0026nbsp;4A), those from Grappolo leaf showed the highest (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) inhibition ranging from 12.61\u0026thinsp;\u0026plusmn;\u0026thinsp;2.64% (6.25% concentration) to 41.14\u0026thinsp;\u0026plusmn;\u0026thinsp;2.66% (50% concentration). The ethanolic extracts (Fig.\u0026nbsp;4B) showed excellent antibacterial activities, with emphasis on the Grappolo leaf extract, which showed the highest (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) inhibition at all concentrations tested, ranging from 41.47\u0026thinsp;\u0026plusmn;\u0026thinsp;0.25% (for the lowest concentration of 6.25%) to 85.13\u0026thinsp;\u0026plusmn;\u0026thinsp;4.61% (at the highest concentration of 50%). The antimicrobial activity of the other ethanolic extracts was similar (p˃ 0.05) when tested at concentrations of 50, 25 and 6.25%, with an inhibition of 55.22\u0026thinsp;\u0026plusmn;\u0026thinsp;4.37 for the Ascolano leaf extract, and 51.20\u0026thinsp;\u0026plusmn;\u0026thinsp;2.97 for the aqueous extract, at the highest concentration. The inhibition of bacteria \u003cem\u003eSalmonella\u003c/em\u003e Enteritidis, \u003cem\u003eListeria monocytogenes\u003c/em\u003e, and \u003cem\u003eEscherichia coli\u003c/em\u003e by olive leaf extract was confirmed in the work of Liu Y, et al. [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn most cases, gram-positive bacteria are more sensitive to natural plant compounds than gram-negative bacteria [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. This is thought to be due to differences in the cell structure of these bacteria. In gram-positive bacteria, antibacterial substances can enter through the cell wall and attack the cytoplasmic membrane, resulting in cytoplasmic leakage [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], Gram-negative bacteria, on the other hand, have an outer lipopolysaccharide component membrane that protects them from various agents [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. However, in our work, we observed that the extracts showed high antimicrobial activity against the gram-negative \u003cem\u003eS.\u003c/em\u003e Enteritidis bacterium, with an emphasis on the antimicrobial activity at low extract concentrations, probably the antimicrobial compounds present in the extracts have hydrophobic characteristics, which may contribute to the activity against Gram-negative bacteria. The activity against \u003cem\u003eS.\u003c/em\u003e Enteritidis of oleuropein and caffeic acid was confirmed in the work of Lee O-H, et al. [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cb\u003eFigure 4.\u003c/b\u003e Percentage of inhibition of extracts derived from olive leaves and residue of olive oil production on \u003cem\u003eSalmonella\u003c/em\u003e Enteritidis. EA: Extract of olive leaves Ascolano; EG: Extract of olive leaves Grappolo; ER: Extract of residue of olive oil production. A: Aqueous extract of olive by-products, B: Ethanolic extract of olive by-products. The results are expressed as means and bar indicates SD (n\u0026thinsp;=\u0026thinsp;3). Statistical significance was determined by Tukey\u0026rsquo;s multiple comparisons test (**** for p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001; *** for p\u0026thinsp;\u0026lt;\u0026thinsp;0.001; ** for p\u0026thinsp;\u0026lt;\u0026thinsp;0.01; and * for p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). A: Aqueous extract of olive by-products, B: Ethanolic extract of olive by-products.\u003c/p\u003e \u003cp\u003eFigure 5 shows the percentage inhibition of the extracts against \u003cem\u003eS. aureus\u003c/em\u003e. The aqueous extracts (Fig.\u0026nbsp;5A) of Grappolo leaf and residue of olive oil production extracts showed the highest (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) inhibition with values of 39.83\u0026thinsp;\u0026plusmn;\u0026thinsp;2.50% and 42.56\u0026thinsp;\u0026plusmn;\u0026thinsp;4.48%, respectively, at a concentration of 50%. However, at the lowest concentration tested (6.25%), only the Ascolano leaf extract was able to inhibit the \u003cem\u003eS. aureus\u003c/em\u003e bacterium (17.33\u0026thinsp;\u0026plusmn;\u0026thinsp;2.16%), on the contrary, in the same concentration, the Ascolano leaves ethanolic extract showed no inhibition capacity (Fig.\u0026nbsp;5B).\u003c/p\u003e \u003cp\u003eThe highest antimicrobial activity was demonstrated by the ethanolic extracts at a concentration tested of 50%. Again, Grappolo leaf extracts showed the highest (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) inhibition capacity (91.95\u0026thinsp;\u0026plusmn;\u0026thinsp;3.50%), followed by Ascolano leaf extracts (60.61\u0026thinsp;\u0026plusmn;\u0026thinsp;2.98%). The ethanolic extracts of the residue of olive oil production showed an antimicrobial activity ranging from 13.25\u0026thinsp;\u0026plusmn;\u0026thinsp;2.25 to 42.77\u0026thinsp;\u0026plusmn;\u0026thinsp;1.40%, for the lowest and highest concentrations tested, respectively.\u003c/p\u003e \u003cp\u003eAqueous extract from \"Chemlali\" olive leaf showed the maximum antimicrobial activity against \u003cem\u003eS. aureus\u003c/em\u003e in the work of Debib A, et al. [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. The ethanolic extract in the concentration of 100% of \u0026ldquo;Dathier\u0026rdquo; olive leaves showed the greatest inhibition potential against \u003cem\u003eS. aureus\u003c/em\u003e [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Hydroxytyrosol and oleuropein are phenolic compounds that have been proven to be effective against several human intestinal or respiratory tract pathogens by inhibiting or delaying their growth rate, and different targets have been detected for \u003cem\u003eStaphylococcus aureus\u003c/em\u003e [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e The effect of olive mill wastewater extract was tested on \u003cem\u003eStaphylococcus aureus\u003c/em\u003e and \u003cem\u003eEscherichia coli\u003c/em\u003e in the work of Abu-Lafi S, et al. [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e], showed an inhibition zone equal to 23 mm and 25 mm, respectively, having a higher antibacterial activity compared to well-known antibiotics.\u003c/p\u003e \u003cp\u003e \u003cb\u003eFigure 5\u003c/b\u003e. Percentage of inhibition of extracts derived from olive leaves and residue of olive oil production on \u003cem\u003eStaphylococcus aureus\u003c/em\u003e. EA: Extract of olive leaves Ascolano; EG: Extract of olive leaves Grappolo; ER: Extract of residue of olive oil production. A: Aqueous extract of olive by-products, B: Ethanolic extract of olive by-products. The results are expressed as means and bar indicates SD (n\u0026thinsp;=\u0026thinsp;3). Statistical significance was determined by Tukey\u0026rsquo;s multiple comparisons test (**** for p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001; *** for p\u0026thinsp;\u0026lt;\u0026thinsp;0.001; ** for p\u0026thinsp;\u0026lt;\u0026thinsp;0.01; and * for p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). A: Aqueous extract of olive by-products, B: Ethanolic extract of olive by-products.\u003c/p\u003e \u003cp\u003eThe results of the antimicrobial activity against \u003cem\u003eE. coli\u003c/em\u003e are shown in Fig.\u0026nbsp;6. For the aqueous extracts (Fig.\u0026nbsp;6A), the residue of olive oil production extracts was the least efficient (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) to inhibit the microorganism studied at a concentration of 50% (32.16\u0026thinsp;\u0026plusmn;\u0026thinsp;3.59%). On the other hand, Ascolano and Grappolo leaf extracts showed similar (p˃ 0.05) inhibition values, showing 44.07\u0026thinsp;\u0026plusmn;\u0026thinsp;1.71% and 38.94\u0026thinsp;\u0026plusmn;\u0026thinsp;4.99% of inhibition, respectively. As observed for all studied microorganisms, Grappolo leaves ethanolic extracts were also more efficient (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) in inhibiting \u003cem\u003eE. coli\u003c/em\u003e bacterium (91.43\u0026thinsp;\u0026plusmn;\u0026thinsp;0.35%), followed by Ascolano leaves ethanolic extracts (80.16\u0026thinsp;\u0026plusmn;\u0026thinsp;2.17%) (Fig.\u0026nbsp;6B).\u003c/p\u003e \u003cp\u003eExtracts from olive mill residues were evaluated for phenolic compounds and their antimicrobial and antioxidant activity, resulting in broad-spectrum antibacterial activity against \u003cem\u003eStaphylococcus aureus, Bacillus subtilis, Escherichia coli\u003c/em\u003e, and \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e The bacterium \u003cem\u003eE. coli\u003c/em\u003e was sensitive to acetone, ethyl alcohol, and diethyl ether extracts from olive leaves in the work of Korukluoglu M, et al. [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. However, the aqueous extract had no antimicrobial activity. In addition, the authors observed antimicrobial activity for \u003cem\u003ep\u003c/em\u003e-coumaric acid, caffeic acid, and oleuropein against \u003cem\u003eE. coli.\u003c/em\u003e\u003c/p\u003e \u003cp\u003e \u003cb\u003eFigure 6.\u003c/b\u003e Percentage of inhibition of extracts derived from olive leaves and residue of olive oil production on \u003cem\u003eEscherichia coli.\u003c/em\u003e EA: Extract of olive leaves Ascolano; EG: Extract of olive leaves Grappolo; ER: Extract of residue of olive oil production. A: Aqueous extract of olive by-products, B: Ethanolic extract of olive by-products. The results are expressed as means and bar indicates SD (n\u0026thinsp;=\u0026thinsp;3). Statistical significance was determined by Tukey\u0026rsquo;s multiple comparisons test (**** for p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001; *** for p\u0026thinsp;\u0026lt;\u0026thinsp;0.001; ** for p\u0026thinsp;\u0026lt;\u0026thinsp;0.01; and * for p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). A: Aqueous extract of olive by-products, B: Ethanolic extract of olive by-products.\u003c/p\u003e \u003cp\u003eIn Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, it is observed that only Grappolo leaves ethanolic extracts presented an MBC of 50% for the microorganisms \u003cem\u003eBacillus cereus, Escherichia coli\u003c/em\u003e, and \u003cem\u003eStaphylococcus aureus\u003c/em\u003e, the bactericidal activity was not observed for the other microorganisms. The other extracts only showed inhibitory activity, not being able to cause microbial death.\u003c/p\u003e \u003cp\u003eAgain, we can observe that ethanolic is a more effective extracting solvent when compared to water for the extracts of by-products generated in the production of olive oil and olive leaves. So ethanol showed great extraction efficiency, these results are in line with other authors [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. The type of solvent affects the distribution and number of phenolic compounds present in the extracts, and consequently the antimicrobial activity [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Therefore, we can also confirm the strong antimicrobial effect of the phenolic compounds found in the Grappolo leaves ethanolic extracts, especially hydroxytyrosol.\u003c/p\u003e \u003cp\u003eThe development of a natural and sustainable preservative olive by-product extract for food applications is a potentially viable approach that would allow us to explore the bioactivities of \u003cem\u003eOlea europaea\u003c/em\u003e, furthermore, the use of olive by-products appears to be a more reasonable approach to develop or exploit components for biotechnological applications than using more valuable materials such as olive fruit or oil.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eMinimum bactericidal concentration (MBC) evaluated to extracts derived from olive leaves and residue of olive oil production.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"2\" morerows=\"1\" nameend=\"c2\" namest=\"c1\" rowspan=\"2\"\u003e \u003cp\u003eExtract\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"5\" nameend=\"c7\" namest=\"c3\"\u003e \u003cp\u003eMicroorganisms\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eL. monocytogenes\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eB.\u003c/em\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003ecereus\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eS.\u003c/em\u003e Enteritidis\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003eS.\u003c/em\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eaureus\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cem\u003eE.\u003c/em\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003ecoli\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAqueous\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eER\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEthanolic\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e50%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e50%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e50%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eER\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"7\"\u003eEA: Extract of olive leaves Ascolano; EG: Extract of olive leaves Grappolo; ER: Extract of residue of olive oil production. ( - ): the bactericidal activity was not observed\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eOverall, these results indicate that by-products generated in the production of olive oil and olive leave extracts (especially ethanolic) can be considered a potential source of antibacterial agents against some microorganisms that cause foodborne illness, thus being an economical, sustainable, viable, and accessible alternative for antimicrobial treatment, being able to replace chemical preservatives, which contain toxic and carcinogenic properties. Further research is needed to obtain more information on the antimicrobial activity of the extracts in practice under different application conditions.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eConflict of Interest\u003c/h2\u003e \u003cp\u003eThe authors report there are no competing interests to declare\u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis work was supported by the Brazilian agencies Conselho Nacional de Desenvolvimento Cientifico e Tecnologico do Brasil (CNPq) under Grant [312336/2014\u0026ndash;4, 423095/2016\u0026ndash;1]; Funda\u0026ccedil;\u0026atilde;o de Amparo \u0026agrave; Pesquisa do Estado de Minas Gerais (FAPEMIG) under Grant [CAG\u0026mdash;APQ-03478\u0026ndash;16]; and Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior (CAPES) under Grant [PNPD20131289].\u003c/p\u003e\u003ch2\u003eAuthor Contributions\u003c/h2\u003e \u003cp\u003eL.S. conceptualized the study and led all stages of the research, including methodology development, validation, investigation, data curation, formal analysis, and visualization. She drafted the original version of the manuscript and was responsible for reviewing and editing the final text. N.F., D. S., and B.S. contributed to data formal analysis and participated in the critical review and editing of the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgments\u003c/h2\u003e \u003cp\u003eThe authors thank EPAMIG Experimental Farm in Maria da F\u0026eacute;, Minas Gerais, Brazil, for providing the olive leaves and residues used in this work.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e \u003cp\u003eData available within the article or its supplementary materials\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAbaza L, Youssef NB, Manai H, Haddada FM, Methenni K, Zarrouk M (2011) Ch\u0026eacute;toui olive leaf extracts: influence of the solvent type on phenolics and antioxidant activities. Grasas Aceites 62:96\u0026ndash;104. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3989/gya.044710\u003c/span\u003e\u003cspan address=\"10.3989/gya.044710\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAbu-Lafi S, Al-Natsheh MS, Yaghmoor R, Al-Rimawi F (2017) Enrichment of phenolic compounds from olive mill wastewater and in vitro evaluation of their antimicrobial activities. Evidence-Based Complementary and Alternative medicine 2017\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1155/2017/3706915\u003c/span\u003e\u003cspan address=\"10.1155/2017/3706915\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAl-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 research international 2013\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1155/2013/461415\u003c/span\u003e\u003cspan address=\"10.1155/2013/461415\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAmiot M-J, Fleuriet A, Macheix J-J (1989) Accumulation of oleuropein derivatives during olive maturation. Phytochemistry 28:67\u0026ndash;69. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/0031-9422(89)85009-5\u003c/span\u003e\u003cspan address=\"10.1016/0031-9422(89)85009-5\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBabić S, Malev O, Pflieger M, Lebedev AT, Mazur DM, Kužić A, Čož-Rakovac R, Trebše P (2019) Toxicity evaluation of olive oil mill wastewater and its polar fraction using multiple whole-organism bioassays. Sci Total Environ 686:903\u0026ndash;914. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.scitotenv.2019.06.046\u003c/span\u003e\u003cspan address=\"10.1016/j.scitotenv.2019.06.046\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBisignano G, Tomaino A, Cascio RL, Crisafi G, Uccella N, Saija A (1999) On the in-vitro antimicrobial activity of oleuropein and hydroxytyrosol. J Pharm Pharmacol 51:971\u0026ndash;974. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1211/0022357991773258\u003c/span\u003e\u003cspan address=\"10.1211/0022357991773258\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBona EAMD, Pinto FGS, Fruet TK, Jorge TCM, Moura AC (2014) Compara\u0026ccedil;\u0026atilde;o de m\u0026eacute;todos para avalia\u0026ccedil;\u0026atilde;o da atividade antimicrobiana e determina\u0026ccedil;\u0026atilde;o da concentra\u0026ccedil;\u0026atilde;o inibit\u0026oacute;ria m\u0026iacute;nima (cim) de extratos vegetais aquosos e etan\u0026oacute;licos. Arquivos do Instituto Biol\u0026oacute;gico 81:218\u0026ndash;225. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1590/1808-1657001192012\u003c/span\u003e\u003cspan address=\"10.1590/1808-1657001192012\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBorja R, Raposo F, Rinc\u0026oacute;n B (2006) Treatment technologies of liquid and solid wastes from two-phase olive oil mills. Grasas Aceites 57:32\u0026ndash;46. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3989/gya.2006.v57.i1.20\u003c/span\u003e\u003cspan address=\"10.3989/gya.2006.v57.i1.20\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCLSI (2014) Methods for broth dilution susceptibility testing of bacteria isolated from aquatic animals. approved guideline\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDe Bruno A, Romeo R, Fedele FL, Sicari A, Piscopo A, Poiana M (2018) Antioxidant activity shown by olive pomace extracts. J Environ Sci Health Part B 53:526\u0026ndash;533. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1080/03601234.2018.1462928\u003c/span\u003e\u003cspan address=\"10.1080/03601234.2018.1462928\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDebib A, Boukhatem MN (2017) Phenolic content, antioxidant and antimicrobial activities of Chemlali olive leaf (Olea europaea L.) extracts. Int J Pharmacol Phytochemistry Ethnomed 6:38\u0026ndash;46. https://doi.org/10. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e18052/www.scipress.com/IJPPE.6.38\u003c/span\u003e\u003cspan address=\"http://18052/www.scipress.com/IJPPE.6.38\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDermeche S, Nadour M, Larroche C, Moulti-Mati F, Michaud P (2013) Olive mill wastes: Biochemical characterizations and valorization strategies. Process Biochem 48:1532\u0026ndash;1552. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.procbio.2013.07.010\u003c/span\u003e\u003cspan address=\"10.1016/j.procbio.2013.07.010\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEl-Abbassi A, Saadaoui N, Kiai H, Raiti J, Hafidi A (2017) Potential applications of olive mill wastewater as biopesticide for crops protection. Sci Total Environ 576:10\u0026ndash;21. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.scitotenv.2016.10.032\u003c/span\u003e\u003cspan address=\"10.1016/j.scitotenv.2016.10.032\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEl SN, Karakaya S (2009) Olive tree (Olea europaea) leaves: potential beneficial effects on human health. Nutr Rev 67:632\u0026ndash;638. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/j.1753-4887.2009.00248.x\u003c/span\u003e\u003cspan address=\"10.1111/j.1753-4887.2009.00248.x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFerro MD, Lopes E, Afonso M, Peixe A, Rodrigues FM, Duarte MF (2020) Phenolic profile characterization of \u0026lsquo;Galega vulgar\u0026rsquo;and \u0026lsquo;Cobran\u0026ccedil;osa\u0026rsquo;portuguese olive cultivars along the ripening stages. Appl Sci 10:3930. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/app10113930\u003c/span\u003e\u003cspan address=\"10.3390/app10113930\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFki 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\u0026ndash;204. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.foodchem.2004.09.014\u003c/span\u003e\u003cspan address=\"10.1016/j.foodchem.2004.09.014\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGalanakis 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\u0026ndash;1017. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.lwt.2010.01.005\u003c/span\u003e\u003cspan address=\"10.1016/j.lwt.2010.01.005\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGhomari O, Sounni F, Massaoudi Y, Ghanam J, Kaitouni LBD, Merzouki M, Benlemlih M (2019) Phenolic profile (HPLC-UV) of olive leaves according to extraction procedure and assessment of antibacterial activity. Biotechnol Rep 23:e00347. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.btre.2019.e00347\u003c/span\u003e\u003cspan address=\"10.1016/j.btre.2019.e00347\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eG\u0026ouml;kmen M, Kara R, Akkaya L, Torlak E, \u0026Ouml;nen A (2014) Evaluation of antimicrobial activity in olive (Olea europaea) leaf extract. Am J Microbiol 5:37\u0026ndash;40\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHimour S, Yahia A, Belattar H (2017) Oleuropein and antibacterial activities of Olea europaea L. leaf extract. Eur Sci J 13:342\u0026ndash;353. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.19044/esj.2017.v13n6p342\u003c/span\u003e\u003cspan address=\"10.19044/esj.2017.v13n6p342\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHolley RA, Patel D (2005) Improvement in shelf-life and safety of perishable foods by plant essential oils and smoke antimicrobials. Food Microbiol 22:273\u0026ndash;292. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.fm.2004.08.006\u003c/span\u003e\u003cspan address=\"10.1016/j.fm.2004.08.006\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHussain A, Qarshi IA, Liaqat R, Akhtar S, Aziz I, Ullah I, Shinwari ZK (2014) Antimicrobial potential of leaf and fruit extracts and oils of wild and cultivated edible olive. Pak J Bot 46:1463\u0026ndash;1468\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKalemba D, Kunicka A (2003) Antibacterial and antifungal properties of essential oils. Curr Med Chem 10:813\u0026ndash;829. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.2174/0929867033457719\u003c/span\u003e\u003cspan address=\"10.2174/0929867033457719\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKhan H, Ahmad W, Hussain I, Imran M, Afridi MS, Ullah S (2020) Phytochemical composition, antioxidant and antimicrobial activities of leaves of Olea europaea wild variety. J Food Meas Charact 14:640\u0026ndash;648. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s11694-019-00310-5\u003c/span\u003e\u003cspan address=\"10.1007/s11694-019-00310-5\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKorukluoglu M, Sahan Y, Yigit A, Ozer ET, G\u0026Uuml;CER S (2010) Antibacterial activity and chemical constitutions of Olea europaea L. leaf extracts. J Food Process Preserv 34:383\u0026ndash;396. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/j.1745-4549.2008.00318.x\u003c/span\u003e\u003cspan address=\"10.1111/j.1745-4549.2008.00318.x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLadhari A, Zarrelli A, Ghannem M, Ben Mimoun M (2021) Olive wastes as a high-potential by-product: Variability of their phenolic profiles, antioxidant and phytotoxic properties. Waste Biomass Valoriz 12:3657\u0026ndash;3669. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s12649-020-01256-2\u003c/span\u003e\u003cspan address=\"10.1007/s12649-020-01256-2\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLama-Mu\u0026ntilde;oz A, del Mar Contreras M, Esp\u0026iacute;nola F, Moya M, Romero I, Castro E (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. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.foodchem.2020.126626\u003c/span\u003e\u003cspan address=\"10.1016/j.foodchem.2020.126626\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLee O-H, Lee B-Y (2010) Antioxidant and antimicrobial activities of individual and combined phenolics in Olea europaea leaf extract. Bioresour Technol 101:3751\u0026ndash;3754. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.biortech.2009.12.052\u003c/span\u003e\u003cspan address=\"10.1016/j.biortech.2009.12.052\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLing LT, Yap S-A, Radhakrishnan AK, Subramaniam T, Cheng HM, Palanisamy UD (2009) Standardised Mangifera indica extract is an ideal antioxidant. Food Chem 113:1154\u0026ndash;1159. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.foodchem.2008.09.004\u003c/span\u003e\u003cspan address=\"10.1016/j.foodchem.2008.09.004\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu Y, McKeever LC, Malik NS (2017) Assessment of the antimicrobial activity of olive leaf extract against foodborne bacterial pathogens. Front Microbiol 8:113. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3389/fmicb.2017.00113\u003c/span\u003e\u003cspan address=\"10.3389/fmicb.2017.00113\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu Y, McKeever LC, Suo Y, Jin TZ, Malik NS (2018) Antimicrobial activities of olive leaf extract and its potential use in food industry. In Natural and Bio-Based Antimicrobials for Food Applications; ACS Publications: pp 119-132.10.1021/bk-2018-1287.ch006\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMedeiros RML, Villa F, da Silva DF, Cardoso Filho LR (2016) Destina\u0026ccedil;\u0026atilde;o e reaproveitamento de subprodutos da extra\u0026ccedil;\u0026atilde;o oliv\u0026iacute;cola. Scientia Agrar Paranaensis 100\u0026ndash;108. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.18188/sap.v15i2.11905\u003c/span\u003e\u003cspan address=\"10.18188/sap.v15i2.11905\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMedina E, De Castro A, Romero C, Brenes M (2006) Comparison of the concentrations of phenolic compounds in olive oils and other plant oils: correlation with antimicrobial activity. J Agric Food Chem 54:4954\u0026ndash;4961. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1021/jf0602267\u003c/span\u003e\u003cspan address=\"10.1021/jf0602267\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNnachi RC, Sui N, Ke B, Luo Z, Bhalla N, He D, Yang Z (2022) Recent progress on biosensors for rapid detection of bacterial pathogens in water, food and environment. Environ Int 107357. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.envint.2022.107357\u003c/span\u003e\u003cspan address=\"10.1016/j.envint.2022.107357\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eObied H, Bedgood D Jr, Prenzler PD, Robards K (2007) Bioscreening of Australian olive mill waste extracts: biophenol content, antioxidant, antimicrobial and molluscicidal activities. Food Chem Toxicol 45:1238\u0026ndash;1248. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.fct.2007.01.004\u003c/span\u003e\u003cspan address=\"10.1016/j.fct.2007.01.004\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOrtega-Garc\u0026iacute;a F, Blanco S, Peinado M\u0026Aacute;, Perag\u0026oacute;n J (2008) Polyphenol oxidase and its relationship with oleuropein concentration in fruits and leaves of olive (Olea europaea) cv.\u0026lsquo;Picual\u0026rsquo;trees during fruit ripening. Tree Physiol 28:45\u0026ndash;54. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1093/treephys/28.1.45\u003c/span\u003e\u003cspan address=\"10.1093/treephys/28.1.45\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e\u0026Ouml;zcan MM, Matth\u0026auml;us B (2017) A review: Benefit and bioactive properties of olive (Olea europaea L.) leaves. Eur Food Res Technol 243:89\u0026ndash;99. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s00217-016-2726-9\u003c/span\u003e\u003cspan address=\"10.1007/s00217-016-2726-9\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePalmeri R, Siracusa L, Carrubba M, Parafati L, Proetto I, Pesce F, Fallico B (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. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/agronomy12092007\u003c/span\u003e\u003cspan address=\"10.3390/agronomy12092007\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePereira AP, Ferreira IC, Marcelino F, Valent\u0026atilde;o P, Andrade PB, Seabra R, Estevinho L, Bento A, Pereira JA (2007) Phenolic compounds and antimicrobial activity of olive (Olea europaea L. Cv. Cobran\u0026ccedil;osa) leaves. Molecules 12:1153\u0026ndash;1162. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/12051153\u003c/span\u003e\u003cspan address=\"10.3390/12051153\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePham D-C, Nguyen H-C, Nguyen T-HL, Ho H-L, Trinh T-K, Riyaphan J, Weng C-F (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 research international 2020\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1155/2020/3497107\u003c/span\u003e\u003cspan address=\"10.1155/2020/3497107\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRahnama H, Azari R, Yousefi MH, Berizi E, Mazloomi SM, Hosseinzadeh S, Derakhshan Z, Ferrante M, Conti GO (2022) A systematic review and meta-analysis of the prevalence of Bacillus cereus in foods. Food Control 109250. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.foodcont.2022.109250\u003c/span\u003e\u003cspan address=\"10.1016/j.foodcont.2022.109250\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRomero-Garc\u0026iacute;a J, Ni\u0026ntilde;o L, Mart\u0026iacute;nez-Pati\u0026ntilde;o C, \u0026Aacute;lvarez C, Castro E, Negro M (2014) Biorefinery based on olive biomass. State of the art and future trends. Bioresour Technol 159:421\u0026ndash;432. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.biortech.2014.03.062\u003c/span\u003e\u003cspan address=\"10.1016/j.biortech.2014.03.062\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShah MA, Mir SA (2022) Plant extracts as food preservatives. In Plant Extracts: Applications in the Food Industry; Elsevier: pp 127\u0026ndash;141.\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/B978-0-12-822475-5.00010-7\u003c/span\u003e\u003cspan address=\"10.1016/B978-0-12-822475-5.00010-7\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShan B, Cai Y-Z, Brooks JD, Corke H (2007) The in vitro antibacterial activity of dietary spice and medicinal herb extracts. Int J Food Microbiol 117:112\u0026ndash;119. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.ijfoodmicro.2007.03.003\u003c/span\u003e\u003cspan address=\"10.1016/j.ijfoodmicro.2007.03.003\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSim\u0026otilde;es L, Fernandes N, Teixeira J, Abrunhosa L, Dias DR (2023) Brazilian table olives: a source of lactic acid bacteria with antimycotoxigenic and antifungal activity. Toxins 15:71. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/toxins15010071\u003c/span\u003e\u003cspan address=\"10.3390/toxins15010071\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTortora GJ, Case CL, Funke BR (2016) Microbiologia-12\u0026ordf; Edi\u0026ccedil;\u0026atilde;o. Artmed Editora\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYuan J-J, Wang C-Z, Ye J-Z, Tao R, Zhang Y-S (2015) Enzymatic hydrolysis of oleuropein from Olea europea (olive) leaf extract and antioxidant activities. Molecules 20:2903\u0026ndash;2921. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/molecules20022903\u003c/span\u003e\u003cspan address=\"10.3390/molecules20022903\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang C, Xin X, Zhang J, Zhu S, Niu E, Zhou Z, Liu D (2022) Comparative evaluation of the phytochemical profiles and antioxidant potentials of olive leaves from 32 cultivars grown in China. Molecules 27:1292. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/molecules27041292\u003c/span\u003e\u003cspan address=\"10.3390/molecules27041292\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZorić N, Kosalec I (2022) The Antimicrobial Activities of Oleuropein and Hydroxytyrosol. In Promising Antimicrobials from Natural Products; Springer: pp 75\u0026ndash;89.\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/978-3-030-83504-0_5\u003c/span\u003e\u003cspan address=\"10.1007/978-3-030-83504-0_5\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"Federal University of Lavras","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Antimicrobial activity, Food additives, Olive leaves, Residue, Sustainability","lastPublishedDoi":"10.21203/rs.3.rs-8619319/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8619319/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eOlive leaves and olive oil residues are abundant by-products that can serve as alternative sources of antimicrobials. This study characterized extracts from Ascolano and Grappolo olive leaves and olive oil residues, focusing on phenolic content and antimicrobial activity. Aqueous and ethanolic extracts were tested against \u003cem\u003eListeria monocytogenes\u003c/em\u003e, \u003cem\u003eStaphylococcus aureus\u003c/em\u003e, \u003cem\u003eBacillus cereus\u003c/em\u003e, \u003cem\u003eEscherichia coli\u003c/em\u003e, and \u003cem\u003eSalmonella Enteritidis\u003c/em\u003e, with HPLC (UV-vis) used for chemical analysis. Ethanolic extracts exhibited greater inhibition and higher phenolic compound concentrations. The Grappolo leaves ethanolic extract had a minimum bactericidal concentration of 50% for \u003cem\u003eB. cereus\u003c/em\u003e, \u003cem\u003eE. coli\u003c/em\u003e, and \u003cem\u003eS. aureus\u003c/em\u003e and contained 9.24\u0026thinsp;\u0026plusmn;\u0026thinsp;0.58 g.ml⁻\u0026sup1; hydroxytyrosol. Olive oil residue ethanolic extracts showed high oleuropein content (16.47\u0026thinsp;\u0026plusmn;\u0026thinsp;0.76 g.ml⁻\u0026sup1;) and inhibited \u003cem\u003eB. cereus\u003c/em\u003e by 75%. The results highlight the potential use of olive leaves and residues of olive oil production extracts as natural and sustainable antimicrobials against some microorganisms that cause foodborne illness.\u003c/p\u003e","manuscriptTitle":"Sustainable extracts from oliviculture by-products: phenolic content and its antimicrobial activity against foodborne pathogens","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-19 03:00:57","doi":"10.21203/rs.3.rs-8619319/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"7855779a-081f-4146-9ad1-f7199d363850","owner":[],"postedDate":"January 19th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":61252061,"name":"Food Science \u0026 Technology"},{"id":61252062,"name":"Applied \u0026 Industrial Microbiology"}],"tags":[],"updatedAt":"2026-01-19T03:00:57+00:00","versionOfRecord":[],"versionCreatedAt":"2026-01-19 03:00:57","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8619319","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8619319","identity":"rs-8619319","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}