Pre-processing cooling of harvested olives: effects on oil composition and quality parameters in two different genotypes

Pre-processing cooling of harvested olives: effects on oil composition and quality parameters in two different genotypes | 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 Pre-processing cooling of harvested olives: effects on oil composition and quality parameters in two different genotypes Mario Vendrell Calatayud, Gaia Meoni, Leonardo Tenori, Claudio Luchinat, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6835795/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 The quality of olive oil is influenced by various factors including the olive environmental growth conditions. High temperatures in the later stages of fruit development can hasten ripening, with a detrimental impact on the fatty acid and polyphenol profiles. Harvesting and processing olives at high internal temperatures further degrades the oil quality, and especially affects the aromatic traits. To mitigate this problem, trials were conducted with the pre-processing rapid cooling of harvested olives from Frantoio and Leccino cultivars using hydrocooling technology. The aim was to assess the effects of quick cooling treatments of olives on oil quality and composition. The results indicated that cooled olive samples consistently produced virgin olive oil, and exhibited improved chemical quality parameters, particularly in Frantoio cultivars. The cooling treatment showed genotype-dependent effects, improving specific aroma compounds, and reducing off-flavours, although different outcomes were reported based on the season and genotype. Overall, pre-processing cooling seems to be a promising method for improving positive aroma compounds and minimizing off-flavors. Olea europaea hydrocooler temperature olive oil VOCs off-flavors Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 1. Introduction The global olive oil industry faces two interrelated challenges that demand innovative solutions. First, there is a growing demand for olive oil of improved organoleptic quality and enhanced nutraceutical properties. Consumers worldwide seek the delightful taste and aroma of extra virgin olive oil (EVOO) and are increasingly more aware and informed of its health-promoting attributes, mainly related to the monounsaturated fatty acid, polyphenol and tocopherol content (Tsimidou, 2006 ; Kalua et al., 2007 ; Modesti et al., 2021 ). EVOO secoiridoids, such as oleocanthal, oleacein, hydroxytyrosol, and squalene, show therapeutic promise against cancer, arthropathy, cardiovascular diseases, as well as antitumor activity on highly invasive breast tumor cells when acting together (Lozano-Castellón et al., 2020 ; Sánchez-Quesada et al., 2022 ). Meeting this demand requires careful cultivation and harvesting practices (anticipated harvest, for example, to avoid collecting over-ripe olives) and innovative processing techniques that preserve and possibly enhance these valuable compounds. Second, the specter of climate change looms large, ushering in warmer temperatures, prolonged summer seasons, and, consequently, leading to accelerated ripening and biochemical processes that may negatively affect oil composition and quality (Fraga et al., 2020 ; Michalopoulos et al., 2020 ; Plasquy et al., 2021 ). The delicate balance of fatty acids and phenolic compounds that contribute to the taste and health benefits of olive oil may be jeopardized by this acceleration. Moreover, elevated temperatures can accelerate the degradation of desirable volatiles, or shift their biosynthesis, reducing fruity and floral notes while sometimes increasing less favorable rancid odors (Aparicio et al., 1997 ; Servili et al. 2007 ). This alteration is particularly important because the aroma profile plays a crucial role in the sensory quality of olives and olive oil, and such changes can diminish their market appeal and sensory acceptability. Similarly to the modifications observed in polyphenol content, the composition of volatile compounds in olives markedly changes during ripening (Beltrán et al., 2005 ; Boskou, 2006 ). Therefore, it is imperative to adapt olive processing methods to address these emerging challenges effectively (Fraga et al., 2020 ). A practice that can aid in addressing these two challenges is temperature control during olive processing. Traditionally, olive paste temperature management during malaxation is aimed to reduce oxidation and retain the oil sensory and nutritional properties. Managing the temperature of olive paste during malaxation is crucial for preserving the sensory and nutritional qualities of olive oil. Optimal temperature ranges from 20–27°C, where the preservation of volatile compounds and phenolics is maximized, resulting in oils with rich aromas and high antioxidant content. Moderate temperatures (28–30°C) offer a balance between yield and quality, though with slight reductions in these beneficial compounds. High temperatures (above 30°C) increase oil yield but degrade volatile compounds and phenolics, leading to lower-quality oil with diminished aroma and health benefits (Angerosa et al., 2001 ; Kalua et al., 2006 ; Veneziani et al., 2017 ). In contrast, little is known about the effects of temperature control applied directly on harvested olives in the immediate pre-processing phase. In the optimal oil production chain, olives should be processed soon after harvest, avoiding any fruit storage. In fact, storing harvested olives has been recognized to decrease oil quality, even if refrigeration is applied for a better management of the mill schedule with the main goal of avoiding keeping olives at high environmental temperatures for long periods (Masella et al., 2019 ). Furthermore, to produce high quality olive oil, nowadays early harvests of still green or partially pigmented olives are commonly performed in many olive-growing areas. This agronomical approach aims at obtaining oils with high levels of polyphenols, and with attractive aroma profiles (green, herbaceous), with hints of spicy and bitter, and, compared to oil obtained from ripe/overripe olives, reduced risks of off-flavors. However, harvesting early in the season could result in handling olives with high internal temperatures. In addition, since the harvested olives are, while waiting to be processed, often kept in bins in open air and under direct sunlight, the fruit temperature and that of the bulk increase, with the risk of off-flavor development and fermentative processes induction (Monteleone et al., 1998 ; Kyriakidis & Dourou, 2002 ). The relationship between the inner temperature of the olives before crushing and its impact on the quality of the resulting olive oil has not been extensively investigated. Two studies previously reported the effect of reducing olive fruit temperature to 18°C and up to 6.5°C before the crushing step of the oil extraction workflow (Dourou et al., 2020 ; Guerrini et al., 2021 ). These studies, carried out at lab-scale, highlight the beneficial effects of lowering temperature management of the olives in enhancing the EVOO quality, in particular regarding the phenolic content and the volatile profile, with increases of compounds imparting green aromas. Additionally, a third study focuses on reducing the temperature during the mechanical extraction process, specifically using a cooling crusher (19°C) running experiments in an industrial mill (Nucciarelli et al., 2022 ). This approach led to improvements in the volatile profiles of EVOOs from Coratina, Peranzana, and Moresca cultivars by increasing desirable aldehydes like ( E )-2-hexenal and reducing alcohols such as 1-penten-3-ol and 1-hexanol. Additionally, phenolic compounds were preserved, particularly in Moresca and Peranzana oils, by 17.8% and 12.1%, respectively, due to a reduction in oxidative processes (Nucciarelli et al., 2022 ). Reducing inner temperatures of harvested perishable fruits (pre-cooling) is a widely practiced technique in postharvest management, particularly in refrigerated storage facilities. Among the various pre-cooling techniques, hydrocooling is commonly used for summer fruits like cherries, tomatoes, and berries. For example, hydrocooling has been shown to effectively reduce the temperature of cherries by 10–15°C within 5–10 minutes, depending on the initial temperature and the water temperature used in the process (Kader, 2002 ). Similar results have been observed in tomatoes and berries, where rapid cooling helps to extend shelf life by reducing respiration rates and slowing down the ripening process (Horvitz, 2017 ; Cherono et al., 2018 ). The technique consists of a rapid cooling down of fruit inner temperature by showering them under cold water (0°C) for few seconds/minutes (less than 1 minute for small fruits) before they enter the cold storage rooms, resulting in a more efficient and immediate effects of the refrigeration. To scale up the laboratory experiments previously performed by our research group (Dourou et al., 2020 ) into an industrial-scale level, the hydrocooling technique has been applied to olives in a commercial mill in substitution of the olive washing step. The trials were conducted during three harvesting years on two Italian olive cultivars, Frantoio and Leccino, and treatment effects on olive oil quality were investigated by monitoring chemical parameters (free fatty acids, peroxide value, K232, K268, total phenol and tocopherol content) and applying metabolomics to fresh olive oils and to oils stored for one year under optimal conditions. 2. Materials and Methods 2.1. Fruit Material Olive fruit belonging to Frantoio and Leccino cvs were harvested between the months of October and November during three harvesting seasons (2019 and 2020 for Frantoio; 2020 and 2021 for Leccino) in a commercial farm located in Scansano (Grosseto, South Tuscany, Italy; 42.64500511746627, 11.360858291024662). Table 1 reports the Jaen index (ripening index) of the harvested fruit used in the trials from both cultivars in the different harvesting years. Table 1 Ripening index of the harvested Frantoio and Leccino olives subjected to hydrocooler treatment in the trials. CULTIVAR YEAR RI (Jaen index) Frantoio 2019 2.55 2020 2.14 Leccino 2020 1.71 2021 2.70 2.2. Hydrocooler treatment The trials were conducted at a commercial mill facility (Scansano, Grosseto, Italy). After harvest, 150 kg of olives were kept in bins until hydrocooling treatment and/or regular oil extraction procedure. For each cultivar and for each year, two sets of samples were considered: control oil (C), extracted from olives washed with water at ambient temperature; and hydrocooler oil (Hy), produced from olives subjected to hydrocooler treatment. The olives were cooled using the hydrocooling technology Presto Fresco (Danese S.r.l., Verona, Italy) (Fig. 1 ). This hydrocooler boasts compact dimensions (2.5 x 0.8 x 1.5 m, length x width x height). The equipment, which has been conceptually and technically built for treating other fruit types in plastic boxes, has been equipped with a conveyor belt typically used in commercial olive mill plants. Applying this modification, the equipment was capable of processing about 50 kg/min of olives disposed in a single layer on the conveyor. The hydrocooler trial consisted in the application of a water shower at about 1 ± 0.5°C for 30 s. Time and temperature have been selected after a trial performed to optimize hydrocooler treatment protocol in order to achieve specific inner temperatures in the olive fruit. Olive inner temperature was measured with a probe thermometer (Qimei, NY) before entering the equipment and at the exit of hydrocooling to assess treatment efficiency. Additional measurements of temperatures were performed on olive paste after the crushing and malaxation steps, and on the oil after centrifugation process. Oil was extracted from cooled and control olives using a commercial mill produced by Mori-Tem, equipped with a hammer crusher, a malaxator with temperature control, and a centrifuge extractor. This system allows to process relatively small quantities of olives (10 q/h), with a required minimum volume of 150 kg per kneader. For each set of samples, two consecutive extractions were performed for C and Hy treatments by processing 150 kg of olives every time. The malaxation time was 20 minutes at 28°C. To ensure the collection of “clean” oil from each specific treatment, only the oils from the second extraction were collected and considered for further analysis. After extraction, C and Hy oil samples were stored in dark glass bottles and analyzed in correspondence of: A) 2–3 weeks after production (fresh oil) and, B) after storage for one year at 15°C in 0.75 L bottles, kept in the dark and with nitrogen on the headspace of the bottle. 2.3. Chemical parameters Free fatty acids (FFA), peroxide value (PV), UV spectrophotometric indices (K232 and K268), total phenol (TPC) and tocopherol content (TTC) were analyzed at Analytical S.r.l. (Florence, Italy) in accordance with official methods described in Regulation EC 2568/91 of the Commission of the European Union 25 . 2.4. Volatile organic compounds analysis (HS-SPME/GC-MS) Olive oil volatile organic compounds (VOCs) were analyzed using the headspace solid-phase microextraction technique (HS-SPME) followed by gas chromatography coupled to mass spectrometry (GC-MS) analysis following a protocol which has been previously published by our research group (Dourou et al., 2020 ; Vendrell-Calatayud et al., 2022). Briefly, 2 g of olive oil were placed into a transparent 20 ml crimp vial. The samples were incubated at 60°C for 30 min. VOCs were extracted at the same temperature using a divinylbenzene/carboxen/polydimethylsiloxane fiber (50/30µm, 2 cm, Supelco Ltd., Bellefonte, PA) for 1 h. GC-MS analyses were performed using a Perkin Elmer Clarus 680 gas chromatography coupled to a Perkin Elmer Clarus 600S quadrupole mass selective spectrometer, equipped with a split-splitless PSSI injection port (kept at 250°C). Helium was used as a carrier gas at a constant flow of 1 ml/min. Compound separation was performed using a Supelcowax-10 capillary column (60 m 0.32 mm i.d., 0.5 µm film thickness, Supelco Ltd, Bellefonte, PA). Column temperature program was the following: 40°C for 6 min, increased to 120°C at a rate of 5°C/min, then increased to 160°C at a rate of 3°C/min and finally to 240°C at 15°C/min, holding this temperature for additional 5 min (46 min of total run time). The volatiles absorbed by the fiber were desorbed in the gas chromatograph injector for 5 min. Volatile compounds present in the headspace of the olive oil under study were identified by matching their mass spectra with the reference mass spectra from the NIST Mass Spectra library, also using the Retention index (Kovats index) and available published literature to improve identification quality. Three technical repetitions per each treatment and sample were performed. The quantification of the different identified VOCs was carried out by calculating the relative intensity, which was obtained through the ratio of the area of each specific compound on the sum of the areas of all the compounds identified in each specific chromatogram. This approach stands for a normalized assessment of the abundance of individual compounds within the chromatographic data, which allows for avoiding variation due to fiber decay. 2.5. Fatty acids determination by GC-FID Fatty acids were analyzed by Analytical s.r.l. (Firenze, Italy) using a methodology in accordance with the EU standard guidelines (Appendix es II and IX of European Community Regulation EEC/2568/91). The fatty acids methyl esters (FAME) were obtained by cold alkaline transesterification using methanolic potassium hydroxide solution and extracted with n-heptane. The analysis of the fatty acid profile was conducted using a Focus GC, Thermo Scientific (Milano, Italy) chromatograph equipped with a split/splitless injector, a FID detector, and a SP-2560 fused silica capillary column (100 m x 0.25 cm i. d. x 0.2 µm film thickness, Supelco, Bellefonte, PA). Helium was used as carrier gas at an internal pressure of 110 kPa. The detector and injector were set at 275°C and 260°C, respectively. The oven temperature was programmed to start at 70°C for the first 4 minutes, increasing to 110°C at a rate of 8°C/min, then increasing to 170°C at a rate of 5°C/min, with a 10-minute hold, and finally increasing to 250°C at a rate of 4°C/min with a 15-minute hold. The split ratio was 1:50, and the injected volume was 1 µL. The results are expressed as the relative percentage of each fatty acid. A control sample of fatty acid methyl ester standard mixture (Supelco 37 FAME Mix, Supelco, Bellefonte, PA) was used to calibrate and identify the FAME by their retention times. 2.6. Phenolic composition determination by RP-HPLC-DAD Phenolic composition was analyzed by Analytical s.r.l. (Firenze, Italy) based on the EC Regulation 432/2012. The polar fractions were extracted by dissolving 2.5 g of olive oil in 5 mL of hexane, followed by the addition of an equal volume of methanol/water (60:40 v/v). The resulting mixture was vigorously vortexed for 2 min and subsequently centrifuged for 10 min at 3500 rpm. The polar phenolic compounds were subjected to reverse-phase high-performance liquid chromatography diode array detector (RP-HPLC-DAD) analysis before and after acidic hydrolysis. An aliquot of 200 µL from the polar fraction was mixed with 200 µL of a 1 M sulfuric acid solution to prepare for RP-HPLC-DAD analysis (hydrolysis). This mixture was then maintained in a water bath at 80°C for 2 h. The hydrolysis was carried out in triplicate for each sample. Subsequently, the dry hydrolysate was resuspended in 200 µL of acetonitrile/water (50:50, v/v) have been added to each hydrolysate. The three replicates were combined to obtain a representative hydrolysate that was filtered through a 0.45 µm pore size regenerated cellulose membrane (Schleicher and Schell, MicroScience GmbH, Dassel, Germany) before injection into the chromatograph. Chromatographic separation of polar phenols was carried out using a Nucleosil C18 column (250 × 4.6 mm, 5 µm). The elution system consisted of a gradient of 1% aqueous acetic acid (solvent A) and acetonitrile (solvent B). The gradient program included various steps with specific percentages of solvent B over a 60 min period. The flow rate was 0.5 mL/min, and the injection volume was 20 µL. Detection was performed using both a diode array detector (UV 6000 LP model, cell volume = 10 µL, Thermo Separation Products, San Jose, CA, USA) and a fluorescence detector (SSI 502 model, cell volume = 8 µL, Scientific Systems Inc., State College, PA, USA) in line with the chromatograph. Different wavelengths have been monitored: 260 nm for vanillic and caffeic acids, 280 nm for pinoresinol, hydroxytyrosol, tyrosol, and secoiridoids derivatives; 310 nm for p -coumaric acid and o-coumaric acid, 325 nm for ferulic and cinnamic acid, 338 nm for apigenin and 350 nm for luteolin and methyl luteolin. The results of the quantification were expressed as mg/kg performing a calibration curve for each detected compound. 2.7. Sensory analysis Evaluation of the olive oil was performed through quantitative descriptive analysis (QDA) from the expert panel of the University of Pisa. QDA was conducted at room temperature, in a standard sensory laboratory (ISO 8589:2010) located in the Department of Agriculture, Food, and Environment (DAFE) following the method described in the EEC/2568/91 Regulation and later modifications (Cherono et al., 2018 ; Macaluso et al., 2021 ). The judges (10 assessors: 6 females and 4 males, aged between 23 and 63 years), primarily experts in oil tasting, were selected based on their availability from a larger pool of judges who regularly collaborate with the DAFE at the University of Pisa. All judges underwent standardized training to enhance their ability to recognize, describe, and quantify tastes, odors, and texture properties in accordance with ISO 8586:2023 standards and typically worked collaboratively. A final set of 18 descriptive parameters including both quantitative descriptors olfactory intensity, fruity (olfactory), aromatic richness, taste intensity, fruity, bitter, spicy, sweet, heating, mold, winey, sludge, metallic, rancid, evolutionary state) and hedonic descriptors (olfactory pleasantness, taste pleasantness, and overall pleasantness), were selected. The samples were presented in a different order at each tasting session, and 5 min intervals between each sample were set. Furthermore, a oil sample was randomly replicated to verify the performance of the panel at each tasting session. For evaluation, each assessor was provided with filtered water and asked to cleanse their palate between tastings. Each attribute was evaluated on a 0–9 scale. All ratings were digitally acquired by the Input Sensory Soft 2.0 (ISS, Centro Studi Assaggiatori, Brescia, Italy). Finally, the overall hedonic index (HI) of the oils, which represents the overall acceptability of the product, was calculated based on the mean of the hedonic parameters, which were converted to a scale from 0 to 10, as previously reported (Bianchi et al., 2024 ). The HI was calculated at time 0 and after 1 year of storage to assess whether the acceptability of the product changed over time. Personal data, including consent forms and identifying information, were securely collected and stored in compliance with national data protection regulations. The sensory evaluations were carried out in accordance with ethical standards regarding human subject involvement, following and health and safety protocols. The study was approved by the Ethics Committee of the University of Pisa (protocol no. 0088081/2019). 2.8. Statistical analysis Multivariate analyses were performed using R software (R core team, 2023, version 1.1.463-2009-2018 R-studio, Inc.). Differences between olives temperature in the different stages of the process were evaluated by t-test ( p ≤ 0.05). Metabolites and chemical parameter levels (TPC, TTC, FFA, PV, and UV spectrophotometric indices K232 and K268) were evaluated using ANOVA ( p ≤ 0.05) and Tukey HSD post hoc test ( p ≤ 0.05). Fatty acid data were visually represented using histograms to illustrate the relative percentage of different fatty acids in the oil samples. Heatmap representation of the fold change (Formula 1) values were used to better visualize differences between control and treated samples in terms of polyphenols and VOCs level, where the samples were evaluated by t-test ( p ≤ 0.05). Partial Least Square Discriminant Analysis (PLS-DA) has been used for processing metabolomic (polyphenols, fatty acids, and VOCs) data. Treatment has been used as response variables, whereas polyphenols, fatty acids, and VOCs have been used as predictor variables. Variable Importance in Projection (VIP) scores were employed to filter the most important variables for the exploratory data analysis. All the analyses were conducted in triplicate. $$\:{Log}_{2}\left(\frac{Treated\:sample}{Control\:sample}\right)\:$$ 1 The data processing of the sensory profile has been carried out by the software Big Sensory Soft 2.0 (Centro Studi Assaggiatori, Brescia, Italy) and the statistical analyses were performed by ANOVA interquartile two ways, choosing samples and panelists as main factors. 3. Results 3.1. Effects of the hydrocooler treatment on the temperature of olives, paste and oil During three consecutive harvesting seasons (2019, 2020, and 2021), olives from Frantoio and Leccino cultivars were treated using hydrocooler equipment immediately after harvest, just before starting the oil extraction, as described in section 2.2 . Despite the short treatment duration (approximately 30 s), the olive fruit mesocarp temperature decreased significantly with a Δ temperature ranging between 6.2 and 9.4°C (Table 2 ). No significant differences were found in the extraction yield between the control and treated olives (Supplementary Table 1). Table 2 Temperatures of the olives (mesocarp) from Frantoio and Leccino cultivars at harvest and following the hydrocooler treatment across the three experimental seasons. Year Cultivar Temperature at harvest (°C) Temperature post-hydrocooler (°C) Δ TEMPERATURE (°C) 2019 Frantoio 21.7 12.4 9.3 2020 Frantoio 24.8 17.8 7.0 Leccino 23.4 17.2 6.2 2021 Leccino 23.5 14.1 9.4 In addition to the internal temperature of the olives, the paste temperature was monitored immediately after crushing and during malaxation and centrifugation. Table 3 presents the paste and oil temperatures recorded throughout the various stages of olive processing, for both the paste and the resulting oil in the different trials and for the two cultivars tested. For both cultivars and considering all years, significantly lower paste and oil temperatures were recorded for the Hy samples, with paste temperature showing the greatest differences with C samples (of approximately 4°C). Although there were considerable differences in the temperatures recorded at harvest and immediately after the cooling treatment across the different years (Table 2 ), the variations in paste temperatures after the crushing step were much less pronounced within the same sample groups (C or Hy), and showed more consistent and uniform values. Table 3 Temperature of the paste after crushing and after malaxation, and oil temperature after centrifugation in different trials and for both tested cultivars. Different letters associated with each value (± standard deviation) indicate significant differences (within the same cv) between treatments and times (p ≤ 0.05, Tukey’s test). Values represent the mean of analyses conducted in triplicate for each year and cultivar. Year Cultivar Treatment Paste temperature after crushing (°C) Paste temperature after malaxation (°C) Oil temperature after centrifugation (°C) 2019 Frantoio C 26.4 ± 0.2a 25.3 ± 0.2a 27.8 ± 0.2a Hy 22.3 ± 0.4b 24.1 ± 0.2b 25.4 ± 0.3b 2020 Frantoio C 26.1 ± 0.4a 25.9 ± 0.2a 26.9 ± 0.2a Hy 23.5 ± 0.3b 25.1 ± 0.3b 25.7 ± 0.1b Leccino C 25.7 ± 0.2a 25.8 ± 0.2a 27.6 ± 0.1a Hy 21.4 ± 0.1b 23.7 ± 0.3b 26.3 ± 0.3b 2021 Leccino C 24.8 ± 0.2a 25.6 ± 0.1a 27.4 ± 0.3a Hy 20.8 ± 0.3b 23.5 ± 0.2b 26.1 ± 0.2b C: Control; Hy: Hydrocooler-treated 3.2. Evaluation of the quality and composition parameters of the oil samples 3.2.1. Effect of the hydrocooler treatment on chemical parameters and fatty acid profile of fresh oil Figures 2 and 3 show the free fatty acids (FFAs), peroxide values (PVs), spectrophotometric parameters K232 and K268, total phenol content (TPC) and total tocopherol content (TTC) analyzed 2–3 weeks after oil production in the Hy and C samples. Clear differences between Frantoio and Leccino cultivars in terms of responses to the hydrocooler treatment were present, as well as a strong effect of the harvesting year. This was potentially linked with the differences in the Jean index recorded at harvest (Table 1 ), and/or the differences registered in the different years in terms of Δ temperature after hydrocooling treatment (Table 2 ). Hy oil samples from the Frantoio cv consistently showed significant reductions in FFAs in both the 2019 and 2020 analyses. In contrast, the Hy Leccino oil samples showed no significant impact on FFA values in 2020 and a slight, but a significant increase in 2021. In terms of oxidative stability through PV, only the Frantoio Hy samples showed a significant decrease in both years. Additionally, K232 and K268 showed different behaviours in the two cultivars in the years studied, with a reduction in these indexes in the Hy samples only in 2019 for Frantoio, and for Leccino oils in 2021 (Fig. 2 ). The Hy Frantoio samples showed a significant and consistent reduction in TPC (Fig. 3 A) in both years. In contrast, the Hy Leccino samples presented a variable response, showing a slight but significant decrease in 2020, and a significant increase in 2021. Hy Frantoio samples showed a significant increase in TTC in 2019, but not in 2020. In comparison with the respective control oil (Fig. 3 B), a reduction in TTC was shown in the Hy Leccino oil samples in 2020, but an increase in 2021. Analysis of the fatty acid profile revealed a spectrum of 12 distinct molecules, categorized into monounsaturated, polyunsaturated, and saturated groups. Monounsaturated fatty acids included palmitoleic, margaroleic, oleic, and eicosanoic acids. The polyunsaturated category comprised linoleic and linolenic acids, while the saturated fatty acids comprised palmitic, stearic, arachic, beenic, and lignoceric acids. The isomer trans oleic + linoleic was also identified, showing a comprehensive profile of the different fatty acid composition in the samples examined. However, no significant effects of the hydrocooler treatments were detected in terms of the fatty acids analysed (data not shown). This was the case for both cultivars and in all years, with the only exception of a slight but significant decrease in lignoceric acid recorded for the Hy Leccino samples in 2021. 3.2.2. Effects of the hydrocooler treatment on the polyphenol profile The polyphenol analysis identified a total of 24 molecules, reported in Fig. 4 and Supplementary Table 2. This range of phenolic compounds comprised various classes, including acids, aldehydes, flavones, lignanes, and secoiridoids. The fold-change analysis reported in the heatmap (Fig. 4 ) varies depending on the treatment and time. Different trends were observed for the Frantoio and Leccino cultivars, also in relation to the different trial years. With the exception of luteolin, the Frantoio oil samples (Fig. 4 A) showed a general decrease in polyphenols in 2019 after the hydrocooler treatment, with significant decreases reported also in 2020, which however, involved different compounds. On the other hand, the Leccino samples (Fig. 4 B) showed a significant increase in specific polyphenols in 2021 such as cinnamic acid, apigenin, methyl luteolin, and oxidized dialdehyde decarboxy methyl lLigstroside aglycon (ODDMLA). As reported in Fig. 4 and supported by the measurements (mg/kg) in Supplementary Table 2, the Hy Frantoio oil samples from 2019 showed a reduction in both highly abundant polyphenols, such as pinoresinol, and less abundant compounds, such as apigenin, methyl luteolin, and cinnamic acid. The treatment reduced the abovementioned compounds by the same proportion. The highly abundant secoiridoid derivatives, such as AHOA (aldehydic hydroxylic oleuropein aglycon) and DDMLA (dialdehyde dicarboxymethyl ligstroside aglycon), and compounds such as AHLA (aldehydic hydroxylic ligstroside aglycon), followed the same trend and were greatly affected by the cooling treatment, as well as the less abundant phenolic compound, hydroxytyrosol which showed a decrease after the Hy treatment. In contrast, in the analysis of fresh Leccino oil produced in 2021 (Fig. 4 B), notable changes in phenolic compounds were identified. Polyphenols such as cinnamic acid and apigenin, as well as methyl luteolin and the secoiridoid derivate ODDMLA, showed a significant increase. Interestingly, very limited or no changes in the polyphenol profiles were detected in both Leccino and Frantoio oil samples in 2020 (Figs. 4 A and B). 3.2.3. Volatile Organic Compound (VOC) analysis VOCs play a crucial role in defining the olive oil aroma and flavour profile, which are key factors in its sensory quality and consumer acceptance. The analysis revealed a total of 31 VOCs, as reported in Supplementary Table 3 and Supplementary Figs. 1 and 2, with different chemical classes, including organic acids, alcohols, aldehydes, ketones, and other compounds. Figure 5 : Partial Least Square Discriminant Analysis (PLS-DA), Variable importance in projection (VIP), and heatmap representation of the VOCs composition. Frantoio (A and B) and Leccino (C and D). Treatment has been used as response variable, whereas all the quantified VOCs have been used as predictor variables. A and C: Score plots of the models created analysing fresh oil samples. B and D: Score of the variables that contributed the most to the grouping of the samples deriving from the different treatments with the heatmap representation (blue color: higher in hydrocooler; red color: lower in hydrocooler). Analysis was performed in triplicate per year and per cultivar; Frantoio (2019 and 2020) and Leccino (2020 and 2021). The asterisks indicate significant differences between the treated and control samples (t-test p ≤ 0.05). Considering factors1 and 2, for the Frantoio oil samples the model explained a total of 97% of the variability within the dataset (Fig. 5 A). Again, considering factors1 and 2, for Leccino a total of 95% of the total variability was explained by the model. Both models showed an important effect of the treatment, which induced a clear clustering of Hy (right side of the plot for both models) and C samples (left side of the plot for both models). In addition, another common feature in the two models was that the impact of the year led to a separation of the samples along factor 2. The Frantoio samples from 2019 showed positive values on factor 2, while samples from 2020 were characterized by negative values. Similarly, in Leccino, the influence of the year was evident, with the year 2020 reflecting positive values on factor 2 and the year 2021 being associated with negative values. This distinct positioning indicates a discernible difference between the two years. Figures 5 B and D report the ten variables that contributed the most to the cluster formation based on their different importance in the projection score (VIPs), together with the heatmap representation of the fold change (FC) between the C and Hy samples for these variables, and the results of the univariate statistical analysis. The values corresponding to the relative intensity recorded for each compound can be found in Supplementary Table 3. Regarding the Frantoio oil samples, among the variables that contributed the most to the separation of oil clusters, some VOCs also revealed statistically significant differences between the C and Hy samples. Specifically, 3-hexen-1-ol and 2-hexen-1-ol (which, except for 2-hexen-1-ol in 2019, increased in the Hy oils), and 1-penten-3-one and ethyl alcohol (which, except for 1-penten-3-one in 2020, decreased in the Hy oils) (Fig. 5 B). The Frantoio VIP list also shows other molecules, despite not being significant after the univariate analysis. Of these, 2-hexenal, which is one of the most important C6 VOC in olive oil, and 1-penten-3-ol, which is considered an off-flavour, appeared to increase and decrease, respectively, in Hy oils (Fig. 5 B). Regarding the Leccino oil samples, 3-hexen-1-ol and 2-hexen-1-ol were included in the VIP list and showed significant differences between the C and Hy samples, with Hy oils showing higher levels in both years. These C6 alcohols are important contributors to the olive oil aroma and derive from the lipoxygenase (LOX) pathway. In addition, a volatile ester derived from 3-hexen-1-ol, namely 3-hexen-1-ol acetate, was also found to be differentially accumulated in Hy oils, showing higher levels than the C samples in both years (Fig. 5 D). The ester 3-hexen-1-ol acetate accumulated in the Hy samples of the Frantoio cultivar, being present in the VIP list (Fig. 5 B), but this result was not supported by statistical significance running univariate analysis. In general, most of the modifications observed after the hydrocooler treatment and considering all the years of analysis (Fig. 5 B and D) regarded the class of C6 alcohols, which are associated with a herbal/green flavour. In the Hy Frantoio oil, the relative intensity of 3-hexen-ol and 2-hexen-1-ol, which are two highly abundant compounds, increased by approximately ten and two-fold, respectively. In the Frantoio oil samples, the treatment also induced a significant decrease (approximately ten-fold) in ethyl alcohol, which is characterized by an alcoholic note and considered to be an off-flavour. In addition, for the Frantoio cultivar, 1-penten-3-one, which is another highly abundant compound, decreased by about six-fold due to the treatment. Regarding the Leccino oil, 3-hexen-ol acetate, 3-hexen-ol, and 2-hexen-1-ol, which are highly abundant compounds, increased by about twenty, six, and two-fold, respectively. These three compounds contribute to grassy and green aromas. 3.2.4. Multivariate analysis of fatty acids, polyphenols, and VOC data To better understand the effects of the hydrocooler treatment, a multivariate analysis of the datasets composed of fatty acids, polyphenols, and VOCs was performed considering data from both cultivars and both seasons within the same analysis (Fig. 6 ). The aim of this analytical approach was to highlight the most influential variables involved in the olive response to hydrocooler treatment regardless of the cultivar and year of application. The PLS-DA model created (Fig. 6 A) explained approximately 50% of the total variability found in the dataset considering factors 1 and factor 2 together. In the score plot of the model, a clear separation of the oils from the different cultivars can be observed, with the Frantoio samples shown on the left of the graph and the Leccino oils positioned on the right. The effect of the year of analysis on the sample positioning, and consequently on the compound concentration, is evident, with important differences observed across different treatments. It is interesting to note that the Frantoio control and treated samples are located a long way from each other in the plot, while samples from Leccino were close together, with a less clear clustering of the different experimental theses. This thus suggests a stronger effect of the treatment on the Frantoio cultivar. Regarding the VIP score analysis (Fig. 6 B), DDMLA (dialdehyde decarboxy methyl ligstroside aglycon) and ODDMLA (oxidated dialdehyde decarboxy methyl ligstroside aglycon) emerged as the most influential polyphenols along with DDMOA (dialdehyde decarboxy methyl oleuropein aglycon) and OAHOA (oxidized aldehydic hydroxylic oleuropein aglycon). At the same time, 2-hexen-1-ol and 3-hexen-1-ol contributed greatly to the differentiation of the cultivars, which was also reflected in the specific VIP score in Fig. 6 B. In contrast, the fatty acids linolenic (C18:3) and oleic (C18:1) showed relatively lower scores, ranking among the least influential variables when considering the top 10 most significant factors, and showing very small changes in the heatmap analysis. 3.2.5. Sensory analysis To better understand how the postharvest hydrocooling treatment affected the olive oil sensory profile, a qualitative descriptive analysis was conducted to compare the sensory attributes of the fresh olive oil of each sample type (Fig. 7 ). The results indicated that the hydrocooling treatment significantly enhanced the sensory attributes of olive oil from both cultivars. For the Frantoio 2019 oil samples (Fig. 7 A), hydrocooling (FHy) increased olfactory intensity, taste intensity, and spiciness compared to the control (FC), although a decrease in fruity notes was observed. This trend was confirmed in the Frantoio 2020 samples (Fig. 7 B), where the treated oil (FHy) showed significantly higher values for olfactory intensity, olfactory fruity, taste intensity, and bitterness. However, fruity (taste) and spiciness slightly decreased compared to the control (FC). In the Leccino cultivar, an increase in bitterness, olfactory intensity, and spiciness was also detected in both the 2020 and 2021 samples. Specifically, in 2020 (Fig. 7 C), the hydrocooling treatment (LHy) resulted in higher levels of spiciness and bitterness compared to the control (LC), but it negatively affected taste and olfactory intensity. In contrast, in 2021 (Fig. 7 D), the treated Leccino samples (LHy) showed the highest levels of taste and olfactory intensity, fruity notes, bitterness, and spiciness. Overall, regardless of cultivar and harvest year, hydrocooling generally proved effective in producing oils with enhanced sensory profiles, particularly in terms of fruitiness, bitterness, and spiciness, a trend that aligns with findings from chemical and volatile compound analyses. 3.3. Influence of temperature treatment on olive oil quality one year after production To assess the impact of the hydrocooler treatment on the oil quality and composition during storage, C and Hy oil samples from both cultivars were kept in 0.75 l dark-glass bottles at 15°C in the dark, also adding nitrogen gas in the headspace of the bottle for one year, as outlined in section 2.2 . 3.3.1. Chemical parameters Figure 8 presents the FFA (acidity), PV, and spectrophotometric parameters K232 and K268, while Fig. 9 shows the TPC and TTC analyzed in oil samples after one year of storage. To identify a general trend and possible significant differences induced by the treatment on oil quality, data were grouped by cultivar and separated per year of analysis (2019 and 2020 for Frantoio, and 2020 and 2021 for Leccino). The analysis of chemical quality parameters revealed clearly different responses of the Frantoio and Leccino oil samples. Notably, Hy Frantoio samples showed a lower FFA value than that of the control in 2019 (as observed in the fresh oil), while no difference was detected in 2020. In contrast, Leccino exhibited no change in FFA in 2020 but a reduction in 2021. The peroxide value decreased consistently in the Hy Frantoio samples for both years, while Leccino remained stable in 2020 and showed a decrease in PV only in 2021, when C samples exceeded the VOO limit. Considering the behaviour of the two spectrophotometric indexes in oils from Leccino, no clear trend induced by the cooling treatment was recorded, despite a significant decrease recorded for k232 and k268 in 2021 and 2020, respectively. Vice versa, In the Frantoio oil samples, a reduction in K232 and K268 after the cooling treatment was consistently detected in both years. The TPC in both Frantoio and Leccino samples was reduced by the hydrocooler treatment in the 2019/2020 season but showed an increase in 2020/2021 after the cooling, thus demonstrating a season-specific behaviour (Fig. 9 ). The TTC in Frantoio exhibited an increase in 2019 but a decrease in 2020 in response to the cooling treatment, while in Leccino the opposite trend was observed, with a decrease in 2020 and an increase in 2021. 3.3.2. Multivariate analysis of fatty acid, polyphenol, and VOC data PLS-DA analyses of the fatty acid, polyphenol, and VOC data of the oil one year after production were performed separately for the two cultivars (Fig. 10 ). The model in Fig. 10 A refers to the Frantoio cultivar and explained a total of 89% of the variability considering factors 1 and 2 together. On the other hand, the model in Fig. 10 C refers to the Leccino oil and explains a total of 92% of the variability considering the first two factors. The PLS-DA score plot shown in Fig. 10 A illustrates the substantial and persistent differences between C and Hy olive oil samples from the Frantoio cultivar. A clear distinction between Hy and C samples is shown even after one year of storage under optimal conditions, and also the differences between the harvest seasons is still visible. A similar result was also obtained for the Leccino cultivar. Considering the Frantoio oil, after one year of storage of the C samples, which were initially homogeneous in the fresh oil, now exhibited a notable difference between the two years, thus indicating a variable long-term modification depending on the specific season. VIP analysis of the Frantoio samples (Fig. 10 B) revealed beenic acid (C22:0), methyl luteolin, 2-hexenal, and ferulic acid as primary variables contributing to the distinction between C and Hy samples. Variations between years introduced inconsistency, with lower polyphenols such as methyl luteolin and hydroxytyrosol only in 2019, while ferulic acid was higher only in 2020 (phenolic concentration values and relative intensity of VOCs are shown in Supplementary Tables 4 and 5, respectively). On the other hand, for the Leccino samples, VIP analysis (Fig. 10 D) revealed important variables influencing cluster separation, with lignoceric acid (C24:0), DOA (dialdehyde oleuropein aglycon), ferulic acid, and hydroxytyrosol acetate emerging as primary contributors to the differences between the C and Hy oil samples. Interestingly, also after one year of storage some common variables in the top 10 VIP list were identified as being affected by the hydrocooler treatment in both cultivars, namely 2-hexenal, ferulic acid, and DOA. 3.3.4. Sensory analysis To verify the influence of hydrocooler treatment on sample shelf life, a descriptive analysis was performed after one year of storage only for the 2020 samples (Frantoio e Leccino (Fig. 11 ). After one year of storage, the control oils (FC and LC) developed sensory defects, regardless of the cultivar (Frantoio or Leccino). Specifically, the presence of rancid and winey notes negatively impacted the overall sensory evaluation, resulting in the oils losing their eligibility for the Extra Virgin Olive Oil (EVOO) classification. In contrast, the hydrocooling-treated samples (Hy) maintained higher sensory quality throughout the observation period, regardless of cultivar. More specifically, Frantoio oil treated with hydrocooling (FHy) showed significant improvements in olfactory and taste intensity, bitterness, fruitiness, and spiciness compared to the control oils (FC) (Fig. 11 A). A similar trend was observed in the Leccino 2020 oils (Fig. 11 B). As previously noted, the hedonic quality of a product is a key factor in determining consumer acceptance. Figure 12 presents the Hedonic Index (HI) calculated for all analyzed oils, with a threshold value of six set for acceptability. All oils -except LC-21, FC-20 (after 1 year), and LC-20 (after 1 year)-scored above this threshold. In general, oils produced with the hydrocooling treatment (Hy) were consistently more appreciated than those without treatment (C). This trend was maintained after one year of storage: both LHy-20 and FHy-20 samples scored above the acceptability threshold, while the control oils (LC-20 and FC-20) were rated below it, consistent with the presence of sensory defects such as rancid and winey notes. 4. Discussion The olive oil industry has increasingly recognized the significant impact on oil quality of temperature control during the malaxation process. Studies have shown it can enhance the oil's nutraceutical properties by preserving key volatile compounds and antioxidants (Angerosa et al., 2001 ; Kalua et al., 2006 ; Veneziani et al., 2017 ; Veneziani et al., 2018 ). Temperature reduction has been successfully applied in other sectors of the fruit industry, such as in viticulture where cooling harvested grapes ( Vitis vinifera ) for 24 h before vinification resulted in changes in the aroma of the resulting wines. This thus indicated that the management of fruit temperature before processing could have an impact on the composition and aroma of the final produce (Xu & Siegenthaler, 1997 ). The previously published research at the lab-scale has led to the application of these principles to olive oil production, and thus the mill trials discussed here (Dourou et al., 2020 ; Guerrini et al., 2021 ). These trials showed the strict relation between lowering the temperature of the olive and the increase in the green aromas of the olive oil, thus highlighting the potential interest in this treatment for the olive oil industry. This work demonstrates that hydrocooler technology can be directly used in the mill plant without delaying the olive oil processing, since the treatment could replace the washing step used in traditional mills. Based on the preliminary results obtained during the initial mill experiments (2019), the objective of the treatment, in terms of the optimal quality of the final oil, was to cool the olives until the fruit reached an internal temperature of approximately 17–18°C. The internal temperature of the mesocarp varies over the years, probably due to the climate and weather at harvest time, as well as cultivar-related differences. The analysis of temperature data demonstrated a consistent and significant reduction in temperature induced by the treatment of both cultivars in each year of analysis. This significance was observed at each step evaluated, highlighting the treatment efficiency in decreasing the temperatures of the olive, paste and oil throughout the extraction process (Tables 2 and 3 ). At the same time, the differences in terms of temperature at harvest and the different cooling treatments of the internal olive tissue appeared to change in the different years (Table 2 ). However, these differences were greatly reduced after crushing, when the temperature of the samples from different years and the cultivars appeared more homogeneous within each treatment type (C and Hy, Table 3 ). This helped in homogenizing the effects of the cooling in different seasons. Considering the different olive genotypes, the effects of the treatment appeared to be strongly influenced by the different cultivars used (Table 2 ). While Frantoio exhibited several improvements in chemical parameters, as indicated by decreased FFA levels in both years, Leccino showed some negative trends. Similarly, Frantoio demonstrated an improved oxidative stability with consistent decreases in PV across both years, whereas Leccino showed a worsening trend with increased values. Regarding K232, no consistent pattern was observed across cultivars or years. However, an improvement was noted in K268 levels for Frantoio in 2019 and Leccino in 2021, as both showed decreases (Fig. 2 ), thus suggesting positive changes in these parameters. Additionally, the use of the hydrocooler and olive cooling had an impact on the TPC and TTC. Two studies performed on several cultivars (Frantoio, Leccino, Gentile, Ogliarola Garganica, Moraiolo, San Felice, Coratina, Peranzana, and Ottobratica) by cooling the paste after crushing showed no impact on the peroxide, free fatty acid, and spectrophotometric values (Veneziani et al., 2017 ; Veneziani et al., 2018 ). However, another study regarding the cooled storage of Frantoio and Moraiolo olives using refrigerated cells before oil extraction showed higher spectrophotometric values, but no differences in FFA or PV were detected (Guerrini et al., 2021 ). This aligns with the findings of our study and suggests a strong effect of the genotype in response to the hydrocooler treatment. Another study on the effects of applying a cooling crusher on the quality of the final olive oil quality, showed no significant impact on chemical parameters. On the other hand, our results demonstrated several notable modifications, as outlined in the results section (Nucciarelli et al., 2022 ). This discrepancy may be attributed to the different responses of the olive genotypes used in each study and/or to the different cooling strategies, which in our case were applied on olives before crushing (Nucciarelli et al., 2022 ). Our results revealed more frequent positive impacts of the hydrocooler treatment on FFA, PV, and spectrophotometric indexes were detected in Frantoio than in Leccino samples, in both fresh and stored oil samples. No significant effects of the treatments on the fatty acid profile were observed in our study. This contrasts with other study results where the internal temperature of the olive fruit was maintained at a lower level (7°C for 16 h) before crushing compared to our hydrocooler experiments, which were conducted at temperatures ranging between 17.8 and 12.5°C applied for a very short time (Guerrini et al., 2021 ). Applying the lower temperatures, the previously cited work highlighted a strong effect on fatty acids (Guerrini et al., 2021 ). Lowering storage temperatures could induce modifications in cell membrane lipid composition, favouring polyunsaturated fatty acids such as linolenic acid (C18:3) to maintain fluidity, as observed in other plant studies and prolonged storage conditions (Xu & Siegenthaler, 1997 ; Lee et al., 2005 ). However, such alterations were not detected in our hydrocooler experiments, possibly due to the short duration of the treatments and the more limited temperature reduction. One of the main goals of our approach at the industrial level was to evaluate the effects of reducing the temperature of the olives in terms of the polyphenol and VOC profiles of the resulting oils. These two parameters are of paramount importance in terms of overall quality of the oils and have been highly affected in lab-scale trials carried out on Leccino olives (Dourou et al., 2020 ). Our results in part confirm these findings in terms of polyphenol content, highlighting that different genotypes react differently to the treatment imposed. In fact, the Leccino oil samples showed, as a general trend, better responses after the hydrocooler treatments in terms of total polyphenol content, with an increase in cinnamic and ferulic acid along with apigenin and methyl luteolin, as well as ODDMLA in 2021. This compositional modification in terms of polyphenols may be related to the more pronounced spiciness reported by the trained panellist for the Hy Leccino oil samples, both fresh and after one year of optimal storage. Considering the VOC profile, previous studies have indicated a reduction in total VOCs in olive oil with elevated temperatures, potentially leading to the development of off-flavours (Boskou, 2006 ; Boselli et al., 2009 ). Temperature control is thus crucial for ensuring a superior olive oil aroma. Cooling olive paste during the extraction process has been proposed as a method for enhancing oil quality, as in the application of a thermal exchanger for rapid olive paste cooling post-crushing (Veneziani et al., 2018 ). The enzyme activities in the LOX pathway, including lipoxygenase (LOX), hydroperoxide lyase (HPL), alcohol dehydrogenase (ADH), and alcohol acyltransferase (AAT) enzymes, are highly sensitive to temperature. 2 The optimal temperature range for these enzymes varies, typically between 15 to 35°C, depending on the specific enzyme (Kiritsakis & Markakis, 1988 ). ADH and AAT exhibit optimal activity around 30–35°C, with lower temperatures resulting in decreased alcohol and ester production (Olias et al., 1993 ). Conversely, HPL, the enzyme responsible for converting 13-hydroperoxide into aldehydes, operates optimally at 15°C, potentially leading to increased aldehyde synthesis and reduced conversion to alcohols and esters (Anthon & Barrett, 2003 ). This may partially explain the changes in the aldehyde/alcohol ratio observed in oils from cooled fruit, with higher levels of this ratio which are considered beneficial in terms of the sensory impact (Cerretani et al., 2008 ). Considering the Leccino cultivar, in oils obtained from olives cooled (at 19°C) before crushing, an increase in VOCs from the LOX pathway, such as 2-hexen-1-ol, 3-hexen-1-ol, and 2-hexenal, has been reported, which also aligns with our findings (Dourou et al., 2020 ). Additionally, an increase in polyphenol content at 15°C in Leccino has also been reported, which is consistent with our results. An increase in C6 VOCs has also been reported in oil produced from olives undergoing cold storage before the extraction process, which suggests a positive correlation between the flavour and lowering olive temperatures (Guerrini et al., 2021 ). However, while in the latter study the polyphenol profile of the oil appeared to be preserved from oxidation under cold conditions, our results in the Frantoio cultivar indicate a significant decrease in these compounds. Still considering VOCs, the hydrocooler treatment led to a reduction in ethanol (off-flavour reduction) in the Frantoio cultivar (Supplementary Fig. 1) and, also in line with previously reported results (Karagoz et al., 2017 ; Dourou et al., 2020 ), a general decreasing trend in C5 compounds such as 1-penten-3-ol and 1-penten-3-one, the latter being a derivative of 1-penten-3-ol metabolism. This highlights the importance of considering the interplay between 1-penten-3-ol and 1-penten-3-one compounds in terms of olive oil quality. This is also important considering that in the literature there is contrasting evidence of their impact, and in general of C5 aroma compounds, on the sensory attributes. Additionally, unlike our findings regarding oil chemical parameters, our VOC results are in line with other previously reported data, with an increase in (E)-2-hexenal across all cultivars after the application of cold treatments, along with a decrease in 1-penten-3-ol (Nucciarelli et al., 2022 ). In addition, in our study a significant increase in alcohols was found, especially 3-hexen-1-ol (green aroma). This highlights the significance of using the postharvest cooling of olives to increase desirable volatile compounds. Studies have highlighted the significant influence of cultivar on the VOC profile of olive oil (Genovese et al., 2015 ; Hbaieb et al., 2016 ; Serrano et al., 2021 ). Additionally, the hydrocooling treatment has the advantage of modifying the volatile profile of the resulting oil. This is corroborated in previous research reporting that high temperatures during malaxation (25–35 °C) altered the composition of metabolites from the LOX pathway, thus reducing pleasant volatile compounds and increasing less attractive ones (Servili et al., 2003 ; Boskou, 2006 ). Considering the sensory profile, our findings reveal a particularly interesting scenario. In several cases, the differences between oils obtained from the control (C) and Hydrocooler-treated (Hy) samples were evident both in the fresh oils and after one year of storage, regardless of the cultivar. Overall, the treatment led to an improvement in both varieties (Leccino and Frantoio), although a clear influence of the harvest year was also observed. While the differences immediately after production were significant, albeit relatively modest, they became more pronounced during storage, ultimately leading to the loss of Extra Virgin Olive Oil (EVOO) classification in the untreated samples. Notably, oils produced with the traditional method showed a decline in quality over time, eventually being rated as unacceptable (HI < 6). This trend highlights the effectiveness of the Hydrocooler treatment in preserving oil quality over time. Regardless of cultivar or harvest year, oils from hydrocooler-treated olives consistently showed greater taste and olfactory intensity, characterized by higher levels of perceived fruitiness, bitterness, and pungency. 5. Conclusions The hydrocooler treatment appeared to be suitable for industrial application in the olive oil extraction process in terms of cooling efficacy and timing and thus could substitute the olive washing step. The results, especially considering the effects of the treatment on the olive VOC profile and sensory attributes, confirmed the positive findings of previous lab-scale research. The observed improvement in specific aroma compounds, and the reduction in off-flavours appeared to be consistent considering the different seasons and genotypes. However, different outcomes in terms of TPC, TTC and other important technological parameters of the oils were noted based on the season and genotype. Overall, pre-processing cooling of olives seems very promising for the improvement of oil quality and sensory profile, especially considering southern Mediterranean countries, which are subjected to very high temperatures during the harvesting period. Declarations Ethics approval and consent to participate Not applicable Consent for publication Not applicable Availability of data and materials The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. Competing interests The authors declare that they have no competing interests. Funding This work was financed by the project grant TIMONE-ID11 (https://www.cerm.unifi.it/timone/timone/), funded by the Italian Ministry of Agricultural, Food and Forestry Policies (MiPAAF) to Claudio Luchinbat and Pietro Tonutti. Gaia Meoni was supported by a research contract co-funded by the European Union - PON Research and Innovation 2014-2020 in accordance with Article 24, paragraph 3a), of Law No. 240 of December 30, 2010, as amended, and Ministerial Decree No. 1062 of August 10, 2021. Ethical statement The research obtained the approval of the Ethics Committee of the University of Pisa (protocol no. 0088081/2019). The research was conducted according to the ethical guidelines, and informed consent was obtained from all participants. Authors' contributions Conceptualization, S.B. and P.T.; methodology, F.V., M.V.C. and G.M.; formal analysis, M.V.C., A.B., I.T. and G.M.; data curation, A.B., I.T., M.V.C. and S.B.; investigation, M.V.C.; resources, P.T. and C.L.; writing-original draft preparation, M.V.C., S.B., A.B., F.V., I.T. and P.T.; writing-review and editing, V.C.M., G.M, L.T., C.L., P.T., A.B., F.V. and S.B.; supervision, S.B. and P.T. All authors have read and agreed to the published version of the manuscript. Acknowledgements We would like to express our sincere gratitude to Fabrizio Rossi and his family for hosting the experiments at their commercial olive mill in Scansano (Frantoio Rossi, Grosseto, Italy). References Angerosa, F., Mostallino, R., Basti, C., & Vito, R. (2001). Influence of malaxation temperature and time on the quality of virgin olive oils. Food Chemistry , 72 (1), 19-28. https://doi.org/10.1016/S0308-8146(00)00194-1 Anthon, G. E., & Barrett, D. M. (2003). 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(2005). Differential impact of low temperature on fatty acid unsaturation and lipoxygenase activity in figleaf gourd and cucumber roots. Biochemical and Biophysical Research Communications , 330 (4), 1194-1198. https://doi.org/10.1016/j.bbrc.2005.03.098 Lozano-Castellón, J., López-Yerena, A., Rinaldi de Alvarenga, J. F., Romero del Castillo-Alba, J., Vallverdú-Queralt, A., Escribano-Ferrer, E., & Lamuela-Raventós, R. M. (2020). Health-promoting properties of oleocanthal and oleacein: Two secoiridoids from extra-virgin olive oil. Critical reviews in food science and nutrition , 60 (15), 2532-2548. https://doi.org/10.1080/10408398.2019.1650715 Macaluso, M., Taglieri, I., Venturi, F., Sanmartin, C., Bianchi, A., De Leo, M., & Zinnai, A. (2021). Influence of the atmosphere composition during malaxation and storage on the shelf life of an unfiltered extra virgin olive oil: Preliminary results. European Journal of Lipid Science and Technology , 123 (2), 2000122. https://doi.org/10.1002/ejlt.202000122 Masella, P., Guerrini, L., Angeloni, G., Spadi, A., Baldi, F., & Parenti, A. (2019). Freezing/storing olives, consequences for extra virgin olive oil quality. International Journal of Refrigeration , 106 , 24-32. https://doi.org/10.1016/j.ijrefrig.2019.06.035 Michalopoulos, G., Kasapi, K. A., Koubouris, G., Psarras, G., Arampatzis, G., Hatzigiannakis, E., & Kokkinos, G. (2020). Adaptation of Mediterranean olive groves to climate change through sustainable cultivation practices. Climate , 8 (4), 54. https://doi.org/10.3390/cli8040054 Modesti, M., Shmuleviz, R., Macaluso, M., Bianchi, A., Venturi, F., Brizzolara, S., Zinnai, A., & Tonutti, P. (2021). Pre-processing cooling of harvested grapes induces changes in berry composition and metabolism, and affects quality and aroma traits of the resulting wine. Frontiers in Nutrition , 8 , 728510. https://doi.org/10.3389/fnut.2021.728510 Modesti, M., Taglieri, I., Bianchi, A., Tonacci, A., Sansone, F., Bellincontro, A., & Sanmartin, C. (2021). E-nose and olfactory assessment: Teamwork or a challenge to the last data? The case of virgin olive oil stability and shelf life. Applied Sciences , 11 (18), 8453. https://doi.org/10.3390/app11188453 Monteleone, E., Caporale, G., Carlucci, A., & Pagliarini, E. (1998). Optimisation of extra virgin olive oil quality. Journal of the Science of Food and Agriculture , 77 (1), 31-37. https://doi.org/10.1002/(SICI)1097-0010(199805)77:1%3C31::AID-JSFA998%3E3.0.CO;2-F Nucciarelli, D., Esposto, S., Veneziani, G., Daidone, L., Urbani, S., Taticchi, A., & Servili, M. (2022). The Use of a Cooling Crusher to Reduce the Temperature of Olive Paste and Improve EVOO Quality of Coratina, Peranzana, and Moresca Cultivars: Impact on Phenolic and Volatile Compounds. Food and Bioprocess Technology , 15 (9), 1988-1996. https://doi.org/10.1007/s11947-022-02862-9 Olias, J. M., Perez, A. G., Rios, J. J., & Sanz, L. C. (1993). Aroma of virgin olive oil: biogenesis of the" green" odor notes. Journal of Agricultural and Food Chemistry , 41 (12), 2368-2373. https://doi.org/10.1021/jf00036a029 Plasquy, E., García Martos, J. M., Florido, M. C., Sola-Guirado, R. R., & García Martín, J. F. (2021). Cold storage and temperature management of olive fruit: The impact on fruit physiology and olive oil quality—A review. Processes , 9 (9), 1543. https://doi.org/10.3390/pr9091543 Sánchez-Quesada, C., Gutiérrez-Santiago, F., Rodríguez-García, C., & Gaforio, J. J. (2022). Synergistic effect of squalene and hydroxytyrosol on highly invasive MDA-MB-231 breast cancer cells. Nutrients , 14 (2), 255. https://doi.org/10.3390/nu14020255 Serrano, A., De la Rosa, R., Sánchez-Ortiz, A., Cano, J., Pérez, A. G., Sanz, C., Arias-Calderón, R., Velasco, L., & León, L. (2021). Chemical components influencing oxidative stability and sensorial properties of extra virgin olive oil and effect of genotype and location on their expression. LWT , 136 , 110257. https://doi.org/10.1016/j.lwt.2020.110257 Servili, M., Esposto, S., Lodolini, E., Selvaggini, R., Taticchi, A., Urbani, S., & Gucci, R. (2007). Irrigation effects on quality, phenolic composition, and selected volatiles of virgin olive oils cv. Leccino. Journal of Agricultural and Food Chemistry , 55 (16), 6609-6618. https://doi.org/10.1021/jf070599n Servili, M., Selvaggini, R., Taticchi, A., Esposto, S., & Montedoro, G. (2003). Volatile compounds and phenolic composition of virgin olive oil: optimization of temperature and time of exposure of olive pastes to air contact during the mechanical extraction process. Journal of Agricultural and Food Chemistry , 51 (27), 7980-7988. https://doi.org/10.1021/jf034804k Tsimidou, M. Z. (2006). Olive oil quality. In Olive Oil (pp. 93-111). AOCS press. https://doi.org/10.1016/B978-1-893997-88-2.50010-9 Vendrell Calatayud, M., Brizzolara, S., Meoni, G., Tenori, L., Luchinat, C., & Tonutti, P. (2022, August). Effects of pre-processing cooling treatments of harvested olives on oil volatilome and quality parameters. In XXXI International Horticultural Congress (IHC2022): International Symposium on Postharvest Technologies to Reduce Food Losses 1364 (pp. 127-134). https://doi.org/10.17660/ActaHortic.2023.1364.16 Veneziani, G., Esposto, S., Taticchi, A., Urbani, S., Selvaggini, R., Di Maio, I., & Servili, M. (2017). Cooling treatment of olive paste during the oil processing: Impact on the yield and extra virgin olive oil quality. Food chemistry , 221 , 107-113. https://doi.org/10.1016/j.foodchem.2016.10.067 Veneziani, G., Esposto, S., Taticchi, A., Urbani, S., Selvaggini, R., Sordini, B., & Servili, M. (2018). Characterization of phenolic and volatile composition of extra virgin olive oil extracted from six Italian cultivars using a cooling treatment of olive paste. LWT , 87 , 523-528. https://doi.org/10.1016/j.lwt.2017.09.034 Xu, Y., & Siegenthaler, P. A. (1997). Low temperature treatments induce an increase in the relative content of both linolenic and λ3-hexadecenoic acids in thylakoid membrane phosphatidylglycerol of squash cotyledons. Plant and cell physiology , 38 (5), 611-618. https://doi.org/10.1093/oxfordjournals.pcp.a029211 Additional Declarations No competing interests reported. Supplementary Files Supplementarymaterial.pdf Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6835795","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":495864712,"identity":"8d23cc44-bfe1-405c-bbff-60de470d38cc","order_by":0,"name":"Mario Vendrell Calatayud","email":"data:image/png;base64,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","orcid":"","institution":"Scuola Superiore Sant'Anna","correspondingAuthor":true,"prefix":"","firstName":"Mario","middleName":"Vendrell","lastName":"Calatayud","suffix":""},{"id":495864716,"identity":"b6f7db28-022c-4fec-9b39-5b31338d5b5a","order_by":1,"name":"Gaia Meoni","email":"","orcid":"","institution":"University of Florence","correspondingAuthor":false,"prefix":"","firstName":"Gaia","middleName":"","lastName":"Meoni","suffix":""},{"id":495864718,"identity":"35d642c1-915b-477c-b101-858cfe7f8cd3","order_by":2,"name":"Leonardo Tenori","email":"","orcid":"","institution":"University of Florence","correspondingAuthor":false,"prefix":"","firstName":"Leonardo","middleName":"","lastName":"Tenori","suffix":""},{"id":495864719,"identity":"2e9e8691-14d4-46f9-ba6d-9a5f37c68d79","order_by":3,"name":"Claudio Luchinat","email":"","orcid":"","institution":"University of Florence","correspondingAuthor":false,"prefix":"","firstName":"Claudio","middleName":"","lastName":"Luchinat","suffix":""},{"id":495864721,"identity":"8d47e924-bc1e-4f39-b77f-b5532db38e8e","order_by":4,"name":"Alessandro Bianchi","email":"","orcid":"","institution":"University of Pisa","correspondingAuthor":false,"prefix":"","firstName":"Alessandro","middleName":"","lastName":"Bianchi","suffix":""},{"id":495864722,"identity":"8edc9241-986d-41a4-a12f-478abf1c8af2","order_by":5,"name":"Francesca Venturi","email":"","orcid":"","institution":"University of Pisa","correspondingAuthor":false,"prefix":"","firstName":"Francesca","middleName":"","lastName":"Venturi","suffix":""},{"id":495864723,"identity":"cb1d139c-3be5-4a4b-87bf-a63de3600423","order_by":6,"name":"Isabella Taglieri","email":"","orcid":"","institution":"University of Pisa","correspondingAuthor":false,"prefix":"","firstName":"Isabella","middleName":"","lastName":"Taglieri","suffix":""},{"id":495864724,"identity":"a3362a52-2d79-4163-8a39-842c888e8855","order_by":7,"name":"Pietro Tonutti","email":"","orcid":"","institution":"Scuola Superiore Sant'Anna","correspondingAuthor":false,"prefix":"","firstName":"Pietro","middleName":"","lastName":"Tonutti","suffix":""},{"id":495864725,"identity":"9a1a8276-8f83-4e3e-85ee-204ce08b4c93","order_by":8,"name":"Stefano Brizzolara","email":"","orcid":"","institution":"Scuola Superiore Sant'Anna","correspondingAuthor":false,"prefix":"","firstName":"Stefano","middleName":"","lastName":"Brizzolara","suffix":""}],"badges":[],"createdAt":"2025-06-06 09:38:47","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6835795/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6835795/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":88448236,"identity":"58118529-9f39-4231-a5c2-42a77e57c272","added_by":"auto","created_at":"2025-08-06 13:58:29","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":523139,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003ePresto Fresco hydrocooler equipment supplied by the company Danese Group Srl (Verona, Italy).\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6835795/v1/663f205e8efb3a80b97fb459.jpg"},{"id":88446893,"identity":"5df0ac55-ff72-4d30-b7b3-bbb76712f99e","added_by":"auto","created_at":"2025-08-06 13:50:29","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":194137,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eA: FFA (Free Fatty Acids; mg KOH/g), B: PV (Peroxide Value; meq O\u003c/em\u003e\u003csub\u003e\u003cem\u003e2\u003c/em\u003e\u003c/sub\u003e\u003cem\u003e/100g), and C and D: spectrophotometric indexes (absorbance at 232 and 268 nm) in different years and cultivars of the oil samples analyzed after production. Different letters associated with each column (black bars report the standard deviation) indicate significant differences (within the same cv) between treatments and times (p ≤ 0.05, Tukey post hoc test). Values are the average of the analysis performed in triplicate each year and for each cultivar; Frantoio (2019 and 2020) and Leccino (2020 and 2021). The orange lines represent the UE legal limits of the different parameters below which the olive oil is classified Virgin. F: Frantoio. L: Leccino. C: Control. Hy: Hydrocooler-treated samples. Bar with lines filling: Samples evaluated in 2019. Bar with solid filling: Samples evaluated in 2020. Bar with squared filling: Samples evaluated in 2021.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6835795/v1/5342bc8e62fd274a2e8e4be2.jpg"},{"id":88448235,"identity":"174c2711-6227-4546-a6a5-aabb7586b1b5","added_by":"auto","created_at":"2025-08-06 13:58:29","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":108829,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eTotal Phenol Content (TPC) (A) and Tocopherol (TTC) (B) content of control and hydrocooler treated samples in different years and cultivars in the fresh oil. Different letters associated with each column (black bars report the standard deviation) indicate significant differences between treatments and times (p ≤ 0.05, Tukey post hoc test). Values are the average of the analysis performed in triplicate each year and for each cultivar; Frantoio (2019 and 2020) and Leccino (2020 and 2021). F: Frantoio. L: Leccino. C: Control. Hy: Hydrocooler-treated samples. Bar with lines filling: Samples evaluated in 2019. Bar with solid filling: Samples evaluated in 2020. Bar with squared filling: Samples evaluated in 2021.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6835795/v1/86535c76026d3c06ca31c5f9.jpg"},{"id":88449429,"identity":"9a8f5bf2-768c-45bc-86bc-5f7996b4517b","added_by":"auto","created_at":"2025-08-06 14:14:29","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":116532,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eHeatmap representation of the polyphenols trend after hydrocooler treatment. Panel A: Frantoio (2019 and 2020). Panel B: Leccino (2020 and 2021). Blue color: higher in Hy; red color: lower in Hy. Analysis was performed in triplicate per each year and cultivar. Fold Change (Log2(Treatment/Control)) was used to represent modifications. Asterisks indicate significant differences between Hy and C samples (t-test p≤0.05). AHLA: Aldehydic Hydroxylic Ligstroside Aglycon, AHOA: Aldehydic Hydroxylic Oleuropein Aglycon, DDMOA: Dialdehyde Decarboxy Methyl Oleuropein Aglycon, DDMLA: Dialdehyde Decarboxy Methyl Ligstroside Aglycon, DAL: Dialdehyde Aglicone Ligstroside, DOA: Dialdehyde Oleuropein Aglycon, OAHLA: Oxidized Aldehydic Hydroxylic Ligstroside Aglycon, OAHOA: Oxidized Aldehydic Hydroxylic Oleuropein Aglycon, ODDMOA: Oxidized Dialdehyde Decarboxy Methyl Oleuropein Aglycon, ODDMLA: Oxidized Dialdehyde Decarboxy Methyl Ligstroside Aglycon, Oleuropein: Oleuropein hydrolyzed derivates.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6835795/v1/3569d5c0c197f0950289b3e4.jpg"},{"id":88448456,"identity":"8dee01bf-18fa-483d-b8af-3701790b887a","added_by":"auto","created_at":"2025-08-06 14:06:29","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":341662,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003ePartial Least Square Discriminant Analysis (PLS-DA), Variable importance in projection (VIP), and heatmap representation of the VOCs composition. Frantoio (A and B) and Leccino (C and D). Treatment has been used as response variable, whereas all the quantified VOCs have been used as predictor variables. A and C: Score plots of the models created analysing fresh oil samples. B and D: Score of the variables that contributed the most to the grouping of the samples deriving from the different treatments with the heatmap representation (blue color: higher in hydrocooler; red color: lower in hydrocooler). Analysis was performed in triplicate per year and per cultivar; Frantoio (2019 and 2020) and Leccino (2020 and 2021). The asterisks indicate significant differences between the treated and control samples (t-test p≤0.05).\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6835795/v1/12c81c90731c13442cf6a727.jpg"},{"id":88449908,"identity":"435999ef-c499-40bb-bd4b-6d0611f4acd1","added_by":"auto","created_at":"2025-08-06 14:22:29","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":98935,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003ePartial Least Square Discriminant Analysis (PLS-DA), Variable importance in projection (VIP), and heatmap representation of all analyzed metabolites (polyphenols, VOCs and fatty acids). Treatment has been used as response variable, whereas the analysed metabolites have been used as predictor variables. A: Score plot of the model of oil analysed after production. B: Score of the variables that contribute most to the grouping of the samples deriving from the different treatments with the heatmap representation (blue color: higher in hydrocooler; red color: lower in hydrocooler). F: Frantoio (filled symbols). L: Leccino (hollow symbols). C: Control. Hy: Hydrocooler treated samples. -19: Analysis performed in 2019. -20: Analysis performed in 2020. -21: Analysis performed in 2021. Analysis was performed in triplicate per year and per cultivar; Frantoio (2019 and 2020) and Leccino (2020 and 2021). The asterisks indicate the significant differences between the treatment and control (t-test p≤0.05). DDMLA: Dialdehyde Decarboxy Methyl Ligstroside Aglycon, ODDMLA: Oxidized Dialdehyde Decarboxy Methyl Ligstroside Aglycon, ODDMOA: Oxidized Dialdehyde Decarboxy Methyl Oleuropein Aglycon, DDMOA: Dialdehyde Decarboxy Methyl Oleuropein Aglycon, OAHOA: Oxidized Aldehydic Hydroxylic Oleuropein Aglycon\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Figure6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6835795/v1/a4dd451a98b48fa74c75419c.jpg"},{"id":88446912,"identity":"46bcf8e9-a7c7-4888-8905-cbb7364560cb","added_by":"auto","created_at":"2025-08-06 13:50:30","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":1649462,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eMedians of quantitative descriptors obtained from sensory analysis of Frantoio oils produced in 2019 (\u003c/em\u003e\u003cem\u003e\u003cstrong\u003eA\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e) and 2020 (\u003c/em\u003e\u003cem\u003e\u003cstrong\u003eB\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e) and Leccino oils produced in 2020 (\u003c/em\u003e\u003cem\u003e\u003cstrong\u003eC\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e) and 2021 (\u003c/em\u003e\u003cem\u003e\u003cstrong\u003eD\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e) analysed immediately after the production. Significance level: ***= p \u0026lt; 0.001; **= p \u0026lt; 0.01; *= p \u0026lt; 0.05; without asterisk = not significant (p ≥ 0.05).\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Figure7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6835795/v1/963ee0903237f5829b520f54.jpg"},{"id":88448243,"identity":"81bd62f1-110e-4396-a298-5605c0605e63","added_by":"auto","created_at":"2025-08-06 13:58:30","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":206812,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eA: FFA (Free Fatty Acids; mg (KOH)/g), B: PV (Peroxide Value; meq O2/100g), and spectrophotometric indexes (absorbance at 232 and 268 nm) (C and D respectively) in different years and cultivars of the oil samples analyzed after one year of storage. Different letters associated with each column (black bars report the standard deviation) indicate significant differences (within the same cv) between treatments and times (p ≤ 0.05, Tukey’s test). Values are the average of the analysis performed in triplicate per year and cultivar; Frantoio (2019 and 2020) and Leccino (2020 and 2021). The orange lines represent the UE legal limits of the different parameters below which the olive oil is classified Virgin. F: Frantoio. L: Leccino. C: Control. Hy: Hydrocooler-treated samples. Bar with lines filling: Samples evaluated in 2019. Bar with solid filling: Samples evaluated in 2020. Bar with squared filling: Samples evaluated in 2021.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Figure8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6835795/v1/c1ac76e0d52cb6ce6ca4c7f6.jpg"},{"id":88446925,"identity":"e491b9b2-d66d-474d-90d9-2438b5c944d9","added_by":"auto","created_at":"2025-08-06 13:50:30","extension":"jpg","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":103502,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eTotal Phenol Content (TPC) (A) and Tocopherol Content (TTC) (B) of control and hydrocooler treated samples in different years and cultivars in the stored oil. Different letters associated with each value (± standard deviation of the measurement) indicate significant differences between treatments and times (p ≤ 0.05, Tukey’s test). Values are the average of the analysis performed in triplicate per year and per cultivar; Frantoio (2019 and 2020) and Leccino (2020 and 2021). F: Frantoio. L: Leccino. C: Control. Hy: Hydrocooler-treated samples. Bar with lines filling: Samples evaluated in 2019. Bar with solid filling: Samples evaluated in 2020. Bar with squared filling: Samples evaluated in 2021.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Figure9.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6835795/v1/65b8e4ccacee7a512ccab410.jpg"},{"id":88446923,"identity":"35eda2ea-077e-47cb-92de-02346e6729d2","added_by":"auto","created_at":"2025-08-06 13:50:30","extension":"jpg","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":141820,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003ePartial Least Square Descriptive Analysis (PLS-DA), Variable importance in projection (VIP), and heatmap representation of the fatty acids, polyphenols, and VOCs composition in oil samples 1 year after the extraction. Frantoio (A and B) and Leccino (C and D). Treatment has been used as response variable, whereas fatty acids, polyphenols, and VOCs have been used as predictor variables. A and C: Score plot of the model of analysed oil samples. B and D: Score of the variables that contribute most to the grouping of the samples deriving from the different treatments with the heatmap representation (blue color: higher in hydrocooler; red color: lower in hydrocooler). Analysis was performed in triplicate per year and per cultivar; Frantoio (2019 and 2020) and Leccino (2020 and 2021). The asterisks indicate the significant differences between the treatment and control (t-test p≤0.05). DOA: Dialdehyde Oleuropein Aglycon, DDMLA: Dialdehyde Decarboxy Methyl Ligstroside Aglycon, OAHOA: Oxidized Aldehydic Hydroxylic Oleuropein Aglycon, Oleuropein: Oleuropein hydrolyzed derivate.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Figure10.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6835795/v1/d4820e07d18015627d400b7b.jpg"},{"id":88448460,"identity":"7df37134-33db-4e2b-b70f-afdaedddf978","added_by":"auto","created_at":"2025-08-06 14:06:30","extension":"jpg","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":896920,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eMedians of quantitative descriptors obtained from sensory analysis of Frantoio oils produced in 2020 (\u003c/em\u003e\u003cem\u003e\u003cstrong\u003eA\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e) and Leccino oils produced in 2020 (\u003c/em\u003e\u003cem\u003e\u003cstrong\u003eB\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e) after a year of optimal storage. Significance level: ***= p \u0026lt; 0.001; **= p \u0026lt; 0.01; *= p \u0026lt; 0.05; without asterisk = not significant (p ≥ 0.05). (for technical reasons the organoleptic analysis of the Frantoio samples produced in 2019 and Leccino samples produced in 2021 was not performed).\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Figure11.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6835795/v1/03b5dceec1704a5a08b3b1e5.jpg"},{"id":88448458,"identity":"a4f97bb3-71eb-453c-bbbc-18e8b2fd0e1a","added_by":"auto","created_at":"2025-08-06 14:06:30","extension":"jpg","order_by":12,"title":"Figure 12","display":"","copyAsset":false,"role":"figure","size":996056,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eHedonic index (HI) of all the oil evaluated (Frantoio oils produced in 2019, 2020 and 2020-after 1 year and Leccino oils produced in 2020,2021 and 2020-after 1 year) Different letters indicate significant difference among values (p \u0026lt; 0.05)\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Figure12.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6835795/v1/8b82f9c6e02ddbcd1550824d.jpg"},{"id":88505313,"identity":"6c573291-bdf2-476d-876f-928c77309a3b","added_by":"auto","created_at":"2025-08-07 07:23:39","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":6697244,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6835795/v1/c5599f63-ed4f-4df3-ae5a-2622f5a346c5.pdf"},{"id":88446896,"identity":"811e568d-c4d5-4fbc-a432-912fd0b9ca41","added_by":"auto","created_at":"2025-08-06 13:50:29","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":1024748,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterial.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6835795/v1/9467ea3e850cbfbf6b4ef88d.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Pre-processing cooling of harvested olives: effects on oil composition and quality parameters in two different genotypes","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eThe global olive oil industry faces two interrelated challenges that demand innovative solutions. First, there is a growing demand for olive oil of improved organoleptic quality and enhanced nutraceutical properties. Consumers worldwide seek the delightful taste and aroma of extra virgin olive oil (EVOO) and are increasingly more aware and informed of its health-promoting attributes, mainly related to the monounsaturated fatty acid, polyphenol and tocopherol content (Tsimidou, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Kalua et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Modesti et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). EVOO secoiridoids, such as oleocanthal, oleacein, hydroxytyrosol, and squalene, show therapeutic promise against cancer, arthropathy, cardiovascular diseases, as well as antitumor activity on highly invasive breast tumor cells when acting together (Lozano-Castell\u0026oacute;n et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; S\u0026aacute;nchez-Quesada et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Meeting this demand requires careful cultivation and harvesting practices (anticipated harvest, for example, to avoid collecting over-ripe olives) and innovative processing techniques that preserve and possibly enhance these valuable compounds.\u003c/p\u003e\u003cp\u003eSecond, the specter of climate change looms large, ushering in warmer temperatures, prolonged summer seasons, and, consequently, leading to accelerated ripening and biochemical processes that may negatively affect oil composition and quality (Fraga et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Michalopoulos et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Plasquy et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The delicate balance of fatty acids and phenolic compounds that contribute to the taste and health benefits of olive oil may be jeopardized by this acceleration. Moreover, elevated temperatures can accelerate the degradation of desirable volatiles, or shift their biosynthesis, reducing fruity and floral notes while sometimes increasing less favorable rancid odors (Aparicio et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e1997\u003c/span\u003e; Servili et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). This alteration is particularly important because the aroma profile plays a crucial role in the sensory quality of olives and olive oil, and such changes can diminish their market appeal and sensory acceptability. Similarly to the modifications observed in polyphenol content, the composition of volatile compounds in olives markedly changes during ripening (Beltr\u0026aacute;n et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Boskou, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). Therefore, it is imperative to adapt olive processing methods to address these emerging challenges effectively (Fraga et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eA practice that can aid in addressing these two challenges is temperature control during olive processing. Traditionally, olive paste temperature management during malaxation is aimed to reduce oxidation and retain the oil sensory and nutritional properties. Managing the temperature of olive paste during malaxation is crucial for preserving the sensory and nutritional qualities of olive oil. Optimal temperature ranges from 20\u0026ndash;27\u0026deg;C, where the preservation of volatile compounds and phenolics is maximized, resulting in oils with rich aromas and high antioxidant content. Moderate temperatures (28\u0026ndash;30\u0026deg;C) offer a balance between yield and quality, though with slight reductions in these beneficial compounds. High temperatures (above 30\u0026deg;C) increase oil yield but degrade volatile compounds and phenolics, leading to lower-quality oil with diminished aroma and health benefits (Angerosa et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Kalua et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Veneziani et al., \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). In contrast, little is known about the effects of temperature control applied directly on harvested olives in the immediate pre-processing phase. In the optimal oil production chain, olives should be processed soon after harvest, avoiding any fruit storage. In fact, storing harvested olives has been recognized to decrease oil quality, even if refrigeration is applied for a better management of the mill schedule with the main goal of avoiding keeping olives at high environmental temperatures for long periods (Masella et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eFurthermore, to produce high quality olive oil, nowadays early harvests of still green or partially pigmented olives are commonly performed in many olive-growing areas. This agronomical approach aims at obtaining oils with high levels of polyphenols, and with attractive aroma profiles (green, herbaceous), with hints of spicy and bitter, and, compared to oil obtained from ripe/overripe olives, reduced risks of off-flavors. However, harvesting early in the season could result in handling olives with high internal temperatures. In addition, since the harvested olives are, while waiting to be processed, often kept in bins in open air and under direct sunlight, the fruit temperature and that of the bulk increase, with the risk of off-flavor development and fermentative processes induction (Monteleone et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e1998\u003c/span\u003e; Kyriakidis \u0026amp; Dourou, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2002\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe relationship between the inner temperature of the olives before crushing and its impact on the quality of the resulting olive oil has not been extensively investigated. Two studies previously reported the effect of reducing olive fruit temperature to 18\u0026deg;C and up to 6.5\u0026deg;C before the crushing step of the oil extraction workflow (Dourou et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Guerrini et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). These studies, carried out at lab-scale, highlight the beneficial effects of lowering temperature management of the olives in enhancing the EVOO quality, in particular regarding the phenolic content and the volatile profile, with increases of compounds imparting green aromas. Additionally, a third study focuses on reducing the temperature during the mechanical extraction process, specifically using a cooling crusher (19\u0026deg;C) running experiments in an industrial mill (Nucciarelli et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). This approach led to improvements in the volatile profiles of EVOOs from Coratina, Peranzana, and Moresca cultivars by increasing desirable aldehydes like (\u003cem\u003eE\u003c/em\u003e)-2-hexenal and reducing alcohols such as 1-penten-3-ol and 1-hexanol. Additionally, phenolic compounds were preserved, particularly in Moresca and Peranzana oils, by 17.8% and 12.1%, respectively, due to a reduction in oxidative processes (Nucciarelli et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eReducing inner temperatures of harvested perishable fruits (pre-cooling) is a widely practiced technique in postharvest management, particularly in refrigerated storage facilities. Among the various pre-cooling techniques, hydrocooling is commonly used for summer fruits like cherries, tomatoes, and berries. For example, hydrocooling has been shown to effectively reduce the temperature of cherries by 10\u0026ndash;15\u0026deg;C within 5\u0026ndash;10 minutes, depending on the initial temperature and the water temperature used in the process (Kader, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). Similar results have been observed in tomatoes and berries, where rapid cooling helps to extend shelf life by reducing respiration rates and slowing down the ripening process (Horvitz, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Cherono et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). The technique consists of a rapid cooling down of fruit inner temperature by showering them under cold water (0\u0026deg;C) for few seconds/minutes (less than 1 minute for small fruits) before they enter the cold storage rooms, resulting in a more efficient and immediate effects of the refrigeration.\u003c/p\u003e\u003cp\u003eTo scale up the laboratory experiments previously performed by our research group (Dourou et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) into an industrial-scale level, the hydrocooling technique has been applied to olives in a commercial mill in substitution of the olive washing step. The trials were conducted during three harvesting years on two Italian olive cultivars, Frantoio and Leccino, and treatment effects on olive oil quality were investigated by monitoring chemical parameters (free fatty acids, peroxide value, K232, K268, total phenol and tocopherol content) and applying metabolomics to fresh olive oils and to oils stored for one year under optimal conditions.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1. Fruit Material\u003c/h2\u003e\u003cp\u003eOlive fruit belonging to Frantoio and Leccino cvs were harvested between the months of October and November during three harvesting seasons (2019 and 2020 for Frantoio; 2020 and 2021 for Leccino) in a commercial farm located in Scansano (Grosseto, South Tuscany, Italy; 42.64500511746627, 11.360858291024662). Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e reports the Jaen index (ripening index) of the harvested fruit used in the trials from both cultivars in the different harvesting years.\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\u003eRipening index of the harvested Frantoio and Leccino olives subjected to hydrocooler treatment in the trials.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"3\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCULTIVAR\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eYEAR\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eRI (Jaen index)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eFrantoio\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e2019\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e2.55\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e2020\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e2.14\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eLeccino\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e2020\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e1.71\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e2021\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e2.70\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2. Hydrocooler treatment\u003c/h2\u003e\u003cp\u003eThe trials were conducted at a commercial mill facility (Scansano, Grosseto, Italy). After harvest, 150 kg of olives were kept in bins until hydrocooling treatment and/or regular oil extraction procedure. For each cultivar and for each year, two sets of samples were considered: control oil (C), extracted from olives washed with water at ambient temperature; and hydrocooler oil (Hy), produced from olives subjected to hydrocooler treatment.\u003c/p\u003e\u003cp\u003eThe olives were cooled using the hydrocooling technology Presto Fresco (Danese S.r.l., Verona, Italy) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). This hydrocooler boasts compact dimensions (2.5 x 0.8 x 1.5 m, length x width x height).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe equipment, which has been conceptually and technically built for treating other fruit types in plastic boxes, has been equipped with a conveyor belt typically used in commercial olive mill plants. Applying this modification, the equipment was capable of processing about 50 kg/min of olives disposed in a single layer on the conveyor. The hydrocooler trial consisted in the application of a water shower at about 1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u0026deg;C for 30 s. Time and temperature have been selected after a trial performed to optimize hydrocooler treatment protocol in order to achieve specific inner temperatures in the olive fruit. Olive inner temperature was measured with a probe thermometer (Qimei, NY) before entering the equipment and at the exit of hydrocooling to assess treatment efficiency. Additional measurements of temperatures were performed on olive paste after the crushing and malaxation steps, and on the oil after centrifugation process.\u003c/p\u003e\u003cp\u003eOil was extracted from cooled and control olives using a commercial mill produced by Mori-Tem, equipped with a hammer crusher, a malaxator with temperature control, and a centrifuge extractor. This system allows to process relatively small quantities of olives (10 q/h), with a required minimum volume of 150 kg per kneader. For each set of samples, two consecutive extractions were performed for C and Hy treatments by processing 150 kg of olives every time. The malaxation time was 20 minutes at 28\u0026deg;C. To ensure the collection of \u0026ldquo;clean\u0026rdquo; oil from each specific treatment, only the oils from the second extraction were collected and considered for further analysis.\u003c/p\u003e\u003cp\u003eAfter extraction, C and Hy oil samples were stored in dark glass bottles and analyzed in correspondence of: A) 2\u0026ndash;3 weeks after production (fresh oil) and, B) after storage for one year at 15\u0026deg;C in 0.75 L bottles, kept in the dark and with nitrogen on the headspace of the bottle.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3. Chemical parameters\u003c/h2\u003e\u003cp\u003eFree fatty acids (FFA), peroxide value (PV), UV spectrophotometric indices (K232 and K268), total phenol (TPC) and tocopherol content (TTC) were analyzed at Analytical S.r.l. (Florence, Italy) in accordance with official methods described in Regulation EC 2568/91 of the Commission of the European Union\u003csup\u003e25\u003c/sup\u003e.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4. Volatile organic compounds analysis (HS-SPME/GC-MS)\u003c/h2\u003e\u003cp\u003eOlive oil volatile organic compounds (VOCs) were analyzed using the headspace solid-phase microextraction technique (HS-SPME) followed by gas chromatography coupled to mass spectrometry (GC-MS) analysis following a protocol which has been previously published by our research group (Dourou et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Vendrell-Calatayud et al., 2022). Briefly, 2 g of olive oil were placed into a transparent 20 ml crimp vial. The samples were incubated at 60\u0026deg;C for 30 min. VOCs were extracted at the same temperature using a divinylbenzene/carboxen/polydimethylsiloxane fiber (50/30\u0026micro;m, 2 cm, Supelco Ltd., Bellefonte, PA) for 1 h. GC-MS analyses were performed using a Perkin Elmer Clarus 680 gas chromatography coupled to a Perkin Elmer Clarus 600S quadrupole mass selective spectrometer, equipped with a split-splitless PSSI injection port (kept at 250\u0026deg;C). Helium was used as a carrier gas at a constant flow of 1 ml/min.\u003c/p\u003e\u003cp\u003eCompound separation was performed using a Supelcowax-10 capillary column (60 m 0.32 mm i.d., 0.5 \u0026micro;m film thickness, Supelco Ltd, Bellefonte, PA). Column temperature program was the following: 40\u0026deg;C for 6 min, increased to 120\u0026deg;C at a rate of 5\u0026deg;C/min, then increased to 160\u0026deg;C at a rate of 3\u0026deg;C/min and finally to 240\u0026deg;C at 15\u0026deg;C/min, holding this temperature for additional 5 min (46 min of total run time). The volatiles absorbed by the fiber were desorbed in the gas chromatograph injector for 5 min. Volatile compounds present in the headspace of the olive oil under study were identified by matching their mass spectra with the reference mass spectra from the NIST Mass Spectra library, also using the Retention index (Kovats index) and available published literature to improve identification quality. Three technical repetitions per each treatment and sample were performed. The quantification of the different identified VOCs was carried out by calculating the relative intensity, which was obtained through the ratio of the area of each specific compound on the sum of the areas of all the compounds identified in each specific chromatogram. This approach stands for a normalized assessment of the abundance of individual compounds within the chromatographic data, which allows for avoiding variation due to fiber decay.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.5. Fatty acids determination by GC-FID\u003c/h2\u003e\u003cp\u003eFatty acids were analyzed by Analytical s.r.l. (Firenze, Italy) using a methodology in accordance with the EU standard guidelines (Appendix es II and IX of European Community Regulation EEC/2568/91). The fatty acids methyl esters (FAME) were obtained by cold alkaline transesterification using methanolic potassium hydroxide solution and extracted with n-heptane. The analysis of the fatty acid profile was conducted using a Focus GC, Thermo Scientific (Milano, Italy) chromatograph equipped with a split/splitless injector, a FID detector, and a SP-2560 fused silica capillary column (100 m x 0.25 cm i. d. x 0.2 \u0026micro;m film thickness, Supelco, Bellefonte, PA). Helium was used as carrier gas at an internal pressure of 110 kPa. The detector and injector were set at 275\u0026deg;C and 260\u0026deg;C, respectively. The oven temperature was programmed to start at 70\u0026deg;C for the first 4 minutes, increasing to 110\u0026deg;C at a rate of 8\u0026deg;C/min, then increasing to 170\u0026deg;C at a rate of 5\u0026deg;C/min, with a 10-minute hold, and finally increasing to 250\u0026deg;C at a rate of 4\u0026deg;C/min with a 15-minute hold. The split ratio was 1:50, and the injected volume was 1 \u0026micro;L. The results are expressed as the relative percentage of each fatty acid. A control sample of fatty acid methyl ester standard mixture (Supelco 37 FAME Mix, Supelco, Bellefonte, PA) was used to calibrate and identify the FAME by their retention times.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e2.6. Phenolic composition determination by RP-HPLC-DAD\u003c/h2\u003e\u003cp\u003ePhenolic composition was analyzed by Analytical s.r.l. (Firenze, Italy) based on the EC Regulation 432/2012. The polar fractions were extracted by dissolving 2.5 g of olive oil in 5 mL of hexane, followed by the addition of an equal volume of methanol/water (60:40 v/v). The resulting mixture was vigorously vortexed for 2 min and subsequently centrifuged for 10 min at 3500 rpm. The polar phenolic compounds were subjected to reverse-phase high-performance liquid chromatography diode array detector (RP-HPLC-DAD) analysis before and after acidic hydrolysis. An aliquot of 200 \u0026micro;L from the polar fraction was mixed with 200 \u0026micro;L of a 1 M sulfuric acid solution to prepare for RP-HPLC-DAD analysis (hydrolysis). This mixture was then maintained in a water bath at 80\u0026deg;C for 2 h. The hydrolysis was carried out in triplicate for each sample. Subsequently, the dry hydrolysate was resuspended in 200 \u0026micro;L of acetonitrile/water (50:50, v/v) have been added to each hydrolysate. The three replicates were combined to obtain a representative hydrolysate that was filtered through a 0.45 \u0026micro;m pore size regenerated cellulose membrane (Schleicher and Schell, MicroScience GmbH, Dassel, Germany) before injection into the chromatograph. Chromatographic separation of polar phenols was carried out using a Nucleosil C18 column (250 \u0026times; 4.6 mm, 5 \u0026micro;m). The elution system consisted of a gradient of 1% aqueous acetic acid (solvent A) and acetonitrile (solvent B). The gradient program included various steps with specific percentages of solvent B over a 60 min period. The flow rate was 0.5 mL/min, and the injection volume was 20 \u0026micro;L. Detection was performed using both a diode array detector (UV 6000 LP model, cell volume\u0026thinsp;=\u0026thinsp;10 \u0026micro;L, Thermo Separation Products, San Jose, CA, USA) and a fluorescence detector (SSI 502 model, cell volume\u0026thinsp;=\u0026thinsp;8 \u0026micro;L, Scientific Systems Inc., State College, PA, USA) in line with the chromatograph. Different wavelengths have been monitored: 260 nm for vanillic and caffeic acids, 280 nm for pinoresinol, hydroxytyrosol, tyrosol, and secoiridoids derivatives; 310 nm for \u003cem\u003ep\u003c/em\u003e-coumaric acid and o-coumaric acid, 325 nm for ferulic and cinnamic acid, 338 nm for apigenin and 350 nm for luteolin and methyl luteolin. The results of the quantification were expressed as mg/kg performing a calibration curve for each detected compound.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e2.7. Sensory analysis\u003c/h2\u003e\u003cp\u003eEvaluation of the olive oil was performed through quantitative descriptive analysis (QDA) from the expert panel of the University of Pisa. QDA was conducted at room temperature, in a standard sensory laboratory (ISO 8589:2010) located in the Department of Agriculture, Food, and Environment (DAFE) following the method described in the EEC/2568/91 Regulation and later modifications (Cherono et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Macaluso et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The judges (10 assessors: 6 females and 4 males, aged between 23 and 63 years), primarily experts in oil tasting, were selected based on their availability from a larger pool of judges who regularly collaborate with the DAFE at the University of Pisa. All judges underwent standardized training to enhance their ability to recognize, describe, and quantify tastes, odors, and texture properties in accordance with ISO 8586:2023 standards and typically worked collaboratively. A final set of 18 descriptive parameters including both quantitative descriptors olfactory intensity, fruity (olfactory), aromatic richness, taste intensity, fruity, bitter, spicy, sweet, heating, mold, winey, sludge, metallic, rancid, evolutionary state) and hedonic descriptors (olfactory pleasantness, taste pleasantness, and overall pleasantness), were selected. The samples were presented in a different order at each tasting session, and 5 min intervals between each sample were set. Furthermore, a oil sample was randomly replicated to verify the performance of the panel at each tasting session. For evaluation, each assessor was provided with filtered water and asked to cleanse their palate between tastings. Each attribute was evaluated on a 0\u0026ndash;9 scale. All ratings were digitally acquired by the Input Sensory Soft 2.0 (ISS, Centro Studi Assaggiatori, Brescia, Italy). Finally, the overall hedonic index (HI) of the oils, which represents the overall acceptability of the product, was calculated based on the mean of the hedonic parameters, which were converted to a scale from 0 to 10, as previously reported (Bianchi et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). The HI was calculated at time 0 and after 1 year of storage to assess whether the acceptability of the product changed over time. Personal data, including consent forms and identifying information, were securely collected and stored in compliance with national data protection regulations. The sensory evaluations were carried out in accordance with ethical standards regarding human subject involvement, following and health and safety protocols. The study was approved by the Ethics Committee of the University of Pisa (protocol no. 0088081/2019).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e2.8. Statistical analysis\u003c/h2\u003e\u003cp\u003eMultivariate analyses were performed using R software (R core team, 2023, version 1.1.463-2009-2018 R-studio, Inc.). Differences between olives temperature in the different stages of the process were evaluated by t-test (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u003cb\u003e\u0026le;\u003c/b\u003e\u0026thinsp;0.05). Metabolites and chemical parameter levels (TPC, TTC, FFA, PV, and UV spectrophotometric indices K232 and K268) were evaluated using ANOVA (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u003cb\u003e\u0026le;\u003c/b\u003e\u0026thinsp;0.05) and Tukey HSD post hoc test (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u003cb\u003e\u0026le;\u003c/b\u003e\u0026thinsp;0.05). Fatty acid data were visually represented using histograms to illustrate the relative percentage of different fatty acids in the oil samples. Heatmap representation of the fold change (Formula 1) values were used to better visualize differences between control and treated samples in terms of polyphenols and VOCs level, where the samples were evaluated by t-test (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u003cb\u003e\u0026le;\u003c/b\u003e\u0026thinsp;0.05). Partial Least Square Discriminant Analysis (PLS-DA) has been used for processing metabolomic (polyphenols, fatty acids, and VOCs) data. Treatment has been used as response variables, whereas polyphenols, fatty acids, and VOCs have been used as predictor variables. Variable Importance in Projection (VIP) scores were employed to filter the most important variables for the exploratory data analysis. All the analyses were conducted in triplicate.\u003cdiv id=\"Equ1\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ1\" name=\"EquationSource\"\u003e\n$$\\:{Log}_{2}\\left(\\frac{Treated\\:sample}{Control\\:sample}\\right)\\:$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e1\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eThe data processing of the sensory profile has been carried out by the software Big Sensory Soft 2.0 (Centro Studi Assaggiatori, Brescia, Italy) and the statistical analyses were performed by ANOVA interquartile two ways, choosing samples and panelists as main factors.\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e3.1. Effects of the hydrocooler treatment on the temperature of olives, paste and oil\u003c/h2\u003e\u003cp\u003eDuring three consecutive harvesting seasons (2019, 2020, and 2021), olives from Frantoio and Leccino cultivars were treated using hydrocooler equipment immediately after harvest, just before starting the oil extraction, as described in section \u003cspan refid=\"Sec4\" class=\"InternalRef\"\u003e2.2\u003c/span\u003e. Despite the short treatment duration (approximately 30 s), the olive fruit mesocarp temperature decreased significantly with a Δ temperature ranging between 6.2 and 9.4\u0026deg;C (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). No significant differences were found in the extraction yield between the control and treated olives (Supplementary Table\u0026nbsp;1).\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\u003eTemperatures of the olives (mesocarp) from Frantoio and Leccino cultivars at harvest and following the hydrocooler treatment across the three experimental seasons.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\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=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eYear\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCultivar\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eTemperature\u003c/p\u003e\u003cp\u003eat harvest (\u0026deg;C)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eTemperature\u003c/p\u003e\u003cp\u003epost-hydrocooler (\u0026deg;C)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eΔ TEMPERATURE (\u0026deg;C)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e2019\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eFrantoio\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e21.7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e12.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e9.3\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e2020\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eFrantoio\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e24.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e17.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e7.0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eLeccino\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e23.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e17.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e6.2\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e2021\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eLeccino\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e23.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e14.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e9.4\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\u003eIn addition to the internal temperature of the olives, the paste temperature was monitored immediately after crushing and during malaxation and centrifugation. Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e presents the paste and oil temperatures recorded throughout the various stages of olive processing, for both the paste and the resulting oil in the different trials and for the two cultivars tested.\u003c/p\u003e\u003cp\u003eFor both cultivars and considering all years, significantly lower paste and oil temperatures were recorded for the Hy samples, with paste temperature showing the greatest differences with C samples (of approximately 4\u0026deg;C). Although there were considerable differences in the temperatures recorded at harvest and immediately after the cooling treatment across the different years (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), the variations in paste temperatures after the crushing step were much less pronounced within the same sample groups (C or Hy), and showed more consistent and uniform values.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eTemperature of the paste after crushing and after malaxation, and oil temperature after centrifugation in different trials and for both tested cultivars. Different letters associated with each value (\u0026plusmn;\u0026thinsp;standard deviation) indicate significant differences (within the same cv) between treatments and times (p\u0026thinsp;\u0026le;\u0026thinsp;0.05, Tukey\u0026rsquo;s test). Values represent the mean of analyses conducted in triplicate for each year and cultivar.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"6\"\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\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eYear\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCultivar\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eTreatment\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003ePaste temperature after crushing (\u0026deg;C)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003ePaste temperature after malaxation (\u0026deg;C)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eOil temperature after centrifugation (\u0026deg;C)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e\u003cb\u003e2019\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eFrantoio\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eC\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e26.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e25.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e27.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2a\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eHy\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e22.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4b\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e24.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2b\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e25.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3b\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"3\" rowspan=\"4\"\u003e\u003cp\u003e\u003cb\u003e2020\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eFrantoio\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eC\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e26.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e25.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e26.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2a\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eHy\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e23.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3b\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e25.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3b\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e25.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1b\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eLeccino\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eC\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e25.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e25.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e27.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1a\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eHy\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e21.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1b\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e23.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3b\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e26.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3b\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e\u003cb\u003e2021\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eLeccino\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eC\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e24.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e25.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e27.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3a\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eHy\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e20.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3b\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e23.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2b\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e26.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2b\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"6\"\u003eC: Control; Hy: Hydrocooler-treated\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003e3.2. Evaluation of the quality and composition parameters of the oil samples\u003c/h2\u003e\u003cdiv id=\"Sec14\" class=\"Section3\"\u003e\u003ch2\u003e3.2.1. Effect of the hydrocooler treatment on chemical parameters and fatty acid profile of fresh oil\u003c/h2\u003e\u003cp\u003eFigures \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e show the free fatty acids (FFAs), peroxide values (PVs), spectrophotometric parameters K232 and K268, total phenol content (TPC) and total tocopherol content (TTC) analyzed 2\u0026ndash;3 weeks after oil production in the Hy and C samples.\u003c/p\u003e\u003cp\u003eClear differences between Frantoio and Leccino cultivars in terms of responses to the hydrocooler treatment were present, as well as a strong effect of the harvesting year. This was potentially linked with the differences in the Jean index recorded at harvest (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), and/or the differences registered in the different years in terms of Δ temperature after hydrocooling treatment (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eHy oil samples from the Frantoio cv consistently showed significant reductions in FFAs in both the 2019 and 2020 analyses. In contrast, the Hy Leccino oil samples showed no significant impact on FFA values in 2020 and a slight, but a significant increase in 2021. In terms of oxidative stability through PV, only the Frantoio Hy samples showed a significant decrease in both years. Additionally, K232 and K268 showed different behaviours in the two cultivars in the years studied, with a reduction in these indexes in the Hy samples only in 2019 for Frantoio, and for Leccino oils in 2021 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe Hy Frantoio samples showed a significant and consistent reduction in TPC (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA) in both years. In contrast, the Hy Leccino samples presented a variable response, showing a slight but significant decrease in 2020, and a significant increase in 2021. Hy Frantoio samples showed a significant increase in TTC in 2019, but not in 2020. In comparison with the respective control oil (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB), a reduction in TTC was shown in the Hy Leccino oil samples in 2020, but an increase in 2021.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eAnalysis of the fatty acid profile revealed a spectrum of 12 distinct molecules, categorized into monounsaturated, polyunsaturated, and saturated groups. Monounsaturated fatty acids included palmitoleic, margaroleic, oleic, and eicosanoic acids. The polyunsaturated category comprised linoleic and linolenic acids, while the saturated fatty acids comprised palmitic, stearic, arachic, beenic, and lignoceric acids. The isomer trans oleic\u0026thinsp;+\u0026thinsp;linoleic was also identified, showing a comprehensive profile of the different fatty acid composition in the samples examined. However, no significant effects of the hydrocooler treatments were detected in terms of the fatty acids analysed (data not shown). This was the case for both cultivars and in all years, with the only exception of a slight but significant decrease in lignoceric acid recorded for the Hy Leccino samples in 2021.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section3\"\u003e\u003ch2\u003e3.2.2. Effects of the hydrocooler treatment on the polyphenol profile\u003c/h2\u003e\u003cp\u003eThe polyphenol analysis identified a total of 24 molecules, reported in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e and Supplementary Table\u0026nbsp;2. This range of phenolic compounds comprised various classes, including acids, aldehydes, flavones, lignanes, and secoiridoids.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe fold-change analysis reported in the heatmap (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e) varies depending on the treatment and time. Different trends were observed for the Frantoio and Leccino cultivars, also in relation to the different trial years. With the exception of luteolin, the Frantoio oil samples (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA) showed a general decrease in polyphenols in 2019 after the hydrocooler treatment, with significant decreases reported also in 2020, which however, involved different compounds. On the other hand, the Leccino samples (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB) showed a significant increase in specific polyphenols in 2021 such as cinnamic acid, apigenin, methyl luteolin, and oxidized dialdehyde decarboxy methyl lLigstroside aglycon (ODDMLA).\u003c/p\u003e\u003cp\u003eAs reported in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e and supported by the measurements (mg/kg) in Supplementary Table\u0026nbsp;2, the Hy Frantoio oil samples from 2019 showed a reduction in both highly abundant polyphenols, such as pinoresinol, and less abundant compounds, such as apigenin, methyl luteolin, and cinnamic acid. The treatment reduced the abovementioned compounds by the same proportion. The highly abundant secoiridoid derivatives, such as AHOA (aldehydic hydroxylic oleuropein aglycon) and DDMLA (dialdehyde dicarboxymethyl ligstroside aglycon), and compounds such as AHLA (aldehydic hydroxylic ligstroside aglycon), followed the same trend and were greatly affected by the cooling treatment, as well as the less abundant phenolic compound, hydroxytyrosol which showed a decrease after the Hy treatment.\u003c/p\u003e\u003cp\u003eIn contrast, in the analysis of fresh Leccino oil produced in 2021 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB), notable changes in phenolic compounds were identified. Polyphenols such as cinnamic acid and apigenin, as well as methyl luteolin and the secoiridoid derivate ODDMLA, showed a significant increase.\u003c/p\u003e\u003cp\u003eInterestingly, very limited or no changes in the polyphenol profiles were detected in both Leccino and Frantoio oil samples in 2020 (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA and B).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section3\"\u003e\u003ch2\u003e3.2.3. Volatile Organic Compound (VOC) analysis\u003c/h2\u003e\u003cp\u003eVOCs play a crucial role in defining the olive oil aroma and flavour profile, which are key factors in its sensory quality and consumer acceptance. The analysis revealed a total of 31 VOCs, as reported in Supplementary Table\u0026nbsp;3 and Supplementary Figs.\u0026nbsp;1 and 2, with different chemical classes, including organic acids, alcohols, aldehydes, ketones, and other compounds.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eFigure \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e: \u003cem\u003ePartial Least Square Discriminant Analysis (PLS-DA), Variable importance in projection (VIP), and heatmap representation of the VOCs composition. Frantoio (A and B) and Leccino (C and D). Treatment has been used as response variable, whereas all the quantified VOCs have been used as predictor variables. A and C: Score plots of the models created analysing fresh oil samples. B and D: Score of the variables that contributed the most to the grouping of the samples deriving from the different treatments with the heatmap representation (blue color: higher in hydrocooler; red color: lower in hydrocooler). Analysis was performed in triplicate per year and per cultivar; Frantoio (2019 and 2020) and Leccino (2020 and 2021). The asterisks indicate significant differences between the treated and control samples (t-test p\u0026thinsp;\u0026le;\u0026thinsp;0.05).\u003c/em\u003e\u003c/p\u003e\u003cp\u003eConsidering factors1 and 2, for the Frantoio oil samples the model explained a total of 97% of the variability within the dataset (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Again, considering factors1 and 2, for Leccino a total of 95% of the total variability was explained by the model. Both models showed an important effect of the treatment, which induced a clear clustering of Hy (right side of the plot for both models) and C samples (left side of the plot for both models). In addition, another common feature in the two models was that the impact of the year led to a separation of the samples along factor 2. The Frantoio samples from 2019 showed positive values on factor 2, while samples from 2020 were characterized by negative values. Similarly, in Leccino, the influence of the year was evident, with the year 2020 reflecting positive values on factor 2 and the year 2021 being associated with negative values. This distinct positioning indicates a discernible difference between the two years.\u003c/p\u003e\u003cp\u003eFigures \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB and D report the ten variables that contributed the most to the cluster formation based on their different importance in the projection score (VIPs), together with the heatmap representation of the fold change (FC) between the C and Hy samples for these variables, and the results of the univariate statistical analysis. The values corresponding to the relative intensity recorded for each compound can be found in Supplementary Table\u0026nbsp;3.\u003c/p\u003e\u003cp\u003eRegarding the Frantoio oil samples, among the variables that contributed the most to the separation of oil clusters, some VOCs also revealed statistically significant differences between the C and Hy samples. Specifically, 3-hexen-1-ol and 2-hexen-1-ol (which, except for 2-hexen-1-ol in 2019, increased in the Hy oils), and 1-penten-3-one and ethyl alcohol (which, except for 1-penten-3-one in 2020, decreased in the Hy oils) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). The Frantoio VIP list also shows other molecules, despite not being significant after the univariate analysis. Of these, 2-hexenal, which is one of the most important C6 VOC in olive oil, and 1-penten-3-ol, which is considered an off-flavour, appeared to increase and decrease, respectively, in Hy oils (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB).\u003c/p\u003e\u003cp\u003eRegarding the Leccino oil samples, 3-hexen-1-ol and 2-hexen-1-ol were included in the VIP list and showed significant differences between the C and Hy samples, with Hy oils showing higher levels in both years. These C6 alcohols are important contributors to the olive oil aroma and derive from the lipoxygenase (LOX) pathway. In addition, a volatile ester derived from 3-hexen-1-ol, namely 3-hexen-1-ol acetate, was also found to be differentially accumulated in Hy oils, showing higher levels than the C samples in both years (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). The ester 3-hexen-1-ol acetate accumulated in the Hy samples of the Frantoio cultivar, being present in the VIP list (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB), but this result was not supported by statistical significance running univariate analysis.\u003c/p\u003e\u003cp\u003eIn general, most of the modifications observed after the hydrocooler treatment and considering all the years of analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB and D) regarded the class of C6 alcohols, which are associated with a herbal/green flavour.\u003c/p\u003e\u003cp\u003eIn the Hy Frantoio oil, the relative intensity of 3-hexen-ol and 2-hexen-1-ol, which are two highly abundant compounds, increased by approximately ten and two-fold, respectively. In the Frantoio oil samples, the treatment also induced a significant decrease (approximately ten-fold) in ethyl alcohol, which is characterized by an alcoholic note and considered to be an off-flavour. In addition, for the Frantoio cultivar, 1-penten-3-one, which is another highly abundant compound, decreased by about six-fold due to the treatment. Regarding the Leccino oil, 3-hexen-ol acetate, 3-hexen-ol, and 2-hexen-1-ol, which are highly abundant compounds, increased by about twenty, six, and two-fold, respectively. These three compounds contribute to grassy and green aromas.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section3\"\u003e\u003ch2\u003e3.2.4. Multivariate analysis of fatty acids, polyphenols, and VOC data\u003c/h2\u003e\u003cp\u003eTo better understand the effects of the hydrocooler treatment, a multivariate analysis of the datasets composed of fatty acids, polyphenols, and VOCs was performed considering data from both cultivars and both seasons within the same analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). The aim of this analytical approach was to highlight the most influential variables involved in the olive response to hydrocooler treatment regardless of the cultivar and year of application.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe PLS-DA model created (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA) explained approximately 50% of the total variability found in the dataset considering factors 1 and factor 2 together. In the score plot of the model, a clear separation of the oils from the different cultivars can be observed, with the Frantoio samples shown on the left of the graph and the Leccino oils positioned on the right.\u003c/p\u003e\u003cp\u003eThe effect of the year of analysis on the sample positioning, and consequently on the compound concentration, is evident, with important differences observed across different treatments. It is interesting to note that the Frantoio control and treated samples are located a long way from each other in the plot, while samples from Leccino were close together, with a less clear clustering of the different experimental theses. This thus suggests a stronger effect of the treatment on the Frantoio cultivar. Regarding the VIP score analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB), DDMLA (dialdehyde decarboxy methyl ligstroside aglycon) and ODDMLA (oxidated dialdehyde decarboxy methyl ligstroside aglycon) emerged as the most influential polyphenols along with DDMOA (dialdehyde decarboxy methyl oleuropein aglycon) and OAHOA (oxidized aldehydic hydroxylic oleuropein aglycon). At the same time, 2-hexen-1-ol and 3-hexen-1-ol contributed greatly to the differentiation of the cultivars, which was also reflected in the specific VIP score in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB. In contrast, the fatty acids linolenic (C18:3) and oleic (C18:1) showed relatively lower scores, ranking among the least influential variables when considering the top 10 most significant factors, and showing very small changes in the heatmap analysis.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section3\"\u003e\u003ch2\u003e3.2.5. Sensory analysis\u003c/h2\u003e\u003cp\u003eTo better understand how the postharvest hydrocooling treatment affected the olive oil sensory profile, a qualitative descriptive analysis was conducted to compare the sensory attributes of the fresh olive oil of each sample type (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe results indicated that the hydrocooling treatment significantly enhanced the sensory attributes of olive oil from both cultivars. For the Frantoio 2019 oil samples (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA), hydrocooling (FHy) increased olfactory intensity, taste intensity, and spiciness compared to the control (FC), although a decrease in fruity notes was observed. This trend was confirmed in the Frantoio 2020 samples (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB), where the treated oil (FHy) showed significantly higher values for olfactory intensity, olfactory fruity, taste intensity, and bitterness. However, fruity (taste) and spiciness slightly decreased compared to the control (FC).\u003c/p\u003e\u003cp\u003eIn the Leccino cultivar, an increase in bitterness, olfactory intensity, and spiciness was also detected in both the 2020 and 2021 samples. Specifically, in 2020 (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC), the hydrocooling treatment (LHy) resulted in higher levels of spiciness and bitterness compared to the control (LC), but it negatively affected taste and olfactory intensity. In contrast, in 2021 (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD), the treated Leccino samples (LHy) showed the highest levels of taste and olfactory intensity, fruity notes, bitterness, and spiciness.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eOverall, regardless of cultivar and harvest year, hydrocooling generally proved effective in producing oils with enhanced sensory profiles, particularly in terms of fruitiness, bitterness, and spiciness, a trend that aligns with findings from chemical and volatile compound analyses.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003e3.3. Influence of temperature treatment on olive oil quality one year after production\u003c/h2\u003e\u003cp\u003eTo assess the impact of the hydrocooler treatment on the oil quality and composition during storage, C and Hy oil samples from both cultivars were kept in 0.75 l dark-glass bottles at 15\u0026deg;C in the dark, also adding nitrogen gas in the headspace of the bottle for one year, as outlined in section \u003cspan refid=\"Sec4\" class=\"InternalRef\"\u003e2.2\u003c/span\u003e.\u003c/p\u003e\u003cdiv id=\"Sec20\" class=\"Section3\"\u003e\u003ch2\u003e3.3.1. Chemical parameters\u003c/h2\u003e\u003cp\u003eFigure \u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e presents the FFA (acidity), PV, and spectrophotometric parameters K232 and K268, while Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e shows the TPC and TTC analyzed in oil samples after one year of storage. To identify a general trend and possible significant differences induced by the treatment on oil quality, data were grouped by cultivar and separated per year of analysis (2019 and 2020 for Frantoio, and 2020 and 2021 for Leccino).\u003c/p\u003e\u003cp\u003eThe analysis of chemical quality parameters revealed clearly different responses of the Frantoio and Leccino oil samples. Notably, Hy Frantoio samples showed a lower FFA value than that of the control in 2019 (as observed in the fresh oil), while no difference was detected in 2020. In contrast, Leccino exhibited no change in FFA in 2020 but a reduction in 2021. The peroxide value decreased consistently in the Hy Frantoio samples for both years, while Leccino remained stable in 2020 and showed a decrease in PV only in 2021, when C samples exceeded the VOO limit. Considering the behaviour of the two spectrophotometric indexes in oils from Leccino, no clear trend induced by the cooling treatment was recorded, despite a significant decrease recorded for k232 and k268 in 2021 and 2020, respectively. Vice versa, In the Frantoio oil samples, a reduction in K232 and K268 after the cooling treatment was consistently detected in both years.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe TPC in both Frantoio and Leccino samples was reduced by the hydrocooler treatment in the 2019/2020 season but showed an increase in 2020/2021 after the cooling, thus demonstrating a season-specific behaviour (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e). The TTC in Frantoio exhibited an increase in 2019 but a decrease in 2020 in response to the cooling treatment, while in Leccino the opposite trend was observed, with a decrease in 2020 and an increase in 2021.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec21\" class=\"Section3\"\u003e\u003ch2\u003e3.3.2. Multivariate analysis of fatty acid, polyphenol, and VOC data\u003c/h2\u003e\u003cp\u003ePLS-DA analyses of the fatty acid, polyphenol, and VOC data of the oil one year after production were performed separately for the two cultivars (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003e). The model in Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003eA refers to the Frantoio cultivar and explained a total of 89% of the variability considering factors 1 and 2 together. On the other hand, the model in Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003eC refers to the Leccino oil and explains a total of 92% of the variability considering the first two factors. The PLS-DA score plot shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003eA illustrates the substantial and persistent differences between C and Hy olive oil samples from the Frantoio cultivar. A clear distinction between Hy and C samples is shown even after one year of storage under optimal conditions, and also the differences between the harvest seasons is still visible. A similar result was also obtained for the Leccino cultivar.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eConsidering the Frantoio oil, after one year of storage of the C samples, which were initially homogeneous in the fresh oil, now exhibited a notable difference between the two years, thus indicating a variable long-term modification depending on the specific season. VIP analysis of the Frantoio samples (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003eB) revealed beenic acid (C22:0), methyl luteolin, 2-hexenal, and ferulic acid as primary variables contributing to the distinction between C and Hy samples. Variations between years introduced inconsistency, with lower polyphenols such as methyl luteolin and hydroxytyrosol only in 2019, while ferulic acid was higher only in 2020 (phenolic concentration values and relative intensity of VOCs are shown in Supplementary Tables\u0026nbsp;4 and 5, respectively). On the other hand, for the Leccino samples, VIP analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003eD) revealed important variables influencing cluster separation, with lignoceric acid (C24:0), DOA (dialdehyde oleuropein aglycon), ferulic acid, and hydroxytyrosol acetate emerging as primary contributors to the differences between the C and Hy oil samples.\u003c/p\u003e\u003cp\u003eInterestingly, also after one year of storage some common variables in the top 10 VIP list were identified as being affected by the hydrocooler treatment in both cultivars, namely 2-hexenal, ferulic acid, and DOA.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec22\" class=\"Section3\"\u003e\u003ch2\u003e3.3.4. Sensory analysis\u003c/h2\u003e\u003cp\u003eTo verify the influence of hydrocooler treatment on sample shelf life, a descriptive analysis was performed after one year of storage only for the 2020 samples (Frantoio e Leccino (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e11\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eAfter one year of storage, the control oils (FC and LC) developed sensory defects, regardless of the cultivar (Frantoio or Leccino). Specifically, the presence of rancid and winey notes negatively impacted the overall sensory evaluation, resulting in the oils losing their eligibility for the Extra Virgin Olive Oil (EVOO) classification. In contrast, the hydrocooling-treated samples (Hy) maintained higher sensory quality throughout the observation period, regardless of cultivar. More specifically, Frantoio oil treated with hydrocooling (FHy) showed significant improvements in olfactory and taste intensity, bitterness, fruitiness, and spiciness compared to the control oils (FC) (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e11\u003c/span\u003eA). A similar trend was observed in the Leccino 2020 oils (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e11\u003c/span\u003eB).\u003c/p\u003e\u003cp\u003eAs previously noted, the hedonic quality of a product is a key factor in determining consumer acceptance. Figure\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e12\u003c/span\u003e presents the Hedonic Index (HI) calculated for all analyzed oils, with a threshold value of six set for acceptability. All oils -except LC-21, FC-20 (after 1 year), and LC-20 (after 1 year)-scored above this threshold. In general, oils produced with the hydrocooling treatment (Hy) were consistently more appreciated than those without treatment (C). This trend was maintained after one year of storage: both LHy-20 and FHy-20 samples scored above the acceptability threshold, while the control oils (LC-20 and FC-20) were rated below it, consistent with the presence of sensory defects such as rancid and winey notes.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eThe olive oil industry has increasingly recognized the significant impact on oil quality of temperature control during the malaxation process. Studies have shown it can enhance the oil's nutraceutical properties by preserving key volatile compounds and antioxidants (Angerosa et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Kalua et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Veneziani et al., \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Veneziani et al., \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Temperature reduction has been successfully applied in other sectors of the fruit industry, such as in viticulture where cooling harvested grapes (\u003cem\u003eVitis vinifera\u003c/em\u003e) for 24 h before vinification resulted in changes in the aroma of the resulting wines. This thus indicated that the management of fruit temperature before processing could have an impact on the composition and aroma of the final produce (Xu \u0026amp; Siegenthaler, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e1997\u003c/span\u003e). The previously published research at the lab-scale has led to the application of these principles to olive oil production, and thus the mill trials discussed here (Dourou et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Guerrini et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). These trials showed the strict relation between lowering the temperature of the olive and the increase in the green aromas of the olive oil, thus highlighting the potential interest in this treatment for the olive oil industry. This work demonstrates that hydrocooler technology can be directly used in the mill plant without delaying the olive oil processing, since the treatment could replace the washing step used in traditional mills.\u003c/p\u003e\u003cp\u003eBased on the preliminary results obtained during the initial mill experiments (2019), the objective of the treatment, in terms of the optimal quality of the final oil, was to cool the olives until the fruit reached an internal temperature of approximately 17\u0026ndash;18\u0026deg;C. The internal temperature of the mesocarp varies over the years, probably due to the climate and weather at harvest time, as well as cultivar-related differences. The analysis of temperature data demonstrated a consistent and significant reduction in temperature induced by the treatment of both cultivars in each year of analysis. This significance was observed at each step evaluated, highlighting the treatment efficiency in decreasing the temperatures of the olive, paste and oil throughout the extraction process (Tables\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e and \u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). At the same time, the differences in terms of temperature at harvest and the different cooling treatments of the internal olive tissue appeared to change in the different years (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). However, these differences were greatly reduced after crushing, when the temperature of the samples from different years and the cultivars appeared more homogeneous within each treatment type (C and Hy, Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). This helped in homogenizing the effects of the cooling in different seasons.\u003c/p\u003e\u003cp\u003eConsidering the different olive genotypes, the effects of the treatment appeared to be strongly influenced by the different cultivars used (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). While Frantoio exhibited several improvements in chemical parameters, as indicated by decreased FFA levels in both years, Leccino showed some negative trends. Similarly, Frantoio demonstrated an improved oxidative stability with consistent decreases in PV across both years, whereas Leccino showed a worsening trend with increased values. Regarding K232, no consistent pattern was observed across cultivars or years. However, an improvement was noted in K268 levels for Frantoio in 2019 and Leccino in 2021, as both showed decreases (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), thus suggesting positive changes in these parameters. Additionally, the use of the hydrocooler and olive cooling had an impact on the TPC and TTC. Two studies performed on several cultivars (Frantoio, Leccino, Gentile, Ogliarola Garganica, Moraiolo, San Felice, Coratina, Peranzana, and Ottobratica) by cooling the paste after crushing showed no impact on the peroxide, free fatty acid, and spectrophotometric values (Veneziani et al., \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Veneziani et al., \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). However, another study regarding the cooled storage of Frantoio and Moraiolo olives using refrigerated cells before oil extraction showed higher spectrophotometric values, but no differences in FFA or PV were detected (Guerrini et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). This aligns with the findings of our study and suggests a strong effect of the genotype in response to the hydrocooler treatment. Another study on the effects of applying a cooling crusher on the quality of the final olive oil quality, showed no significant impact on chemical parameters. On the other hand, our results demonstrated several notable modifications, as outlined in the results section (Nucciarelli et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). This discrepancy may be attributed to the different responses of the olive genotypes used in each study and/or to the different cooling strategies, which in our case were applied on olives before crushing (Nucciarelli et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Our results revealed more frequent positive impacts of the hydrocooler treatment on FFA, PV, and spectrophotometric indexes were detected in Frantoio than in Leccino samples, in both fresh and stored oil samples.\u003c/p\u003e\u003cp\u003eNo significant effects of the treatments on the fatty acid profile were observed in our study. This contrasts with other study results where the internal temperature of the olive fruit was maintained at a lower level (7\u0026deg;C for 16 h) before crushing compared to our hydrocooler experiments, which were conducted at temperatures ranging between 17.8 and 12.5\u0026deg;C applied for a very short time (Guerrini et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Applying the lower temperatures, the previously cited work highlighted a strong effect on fatty acids (Guerrini et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Lowering storage temperatures could induce modifications in cell membrane lipid composition, favouring polyunsaturated fatty acids such as linolenic acid (C18:3) to maintain fluidity, as observed in other plant studies and prolonged storage conditions (Xu \u0026amp; Siegenthaler, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e1997\u003c/span\u003e; Lee et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). However, such alterations were not detected in our hydrocooler experiments, possibly due to the short duration of the treatments and the more limited temperature reduction.\u003c/p\u003e\u003cp\u003eOne of the main goals of our approach at the industrial level was to evaluate the effects of reducing the temperature of the olives in terms of the polyphenol and VOC profiles of the resulting oils. These two parameters are of paramount importance in terms of overall quality of the oils and have been highly affected in lab-scale trials carried out on Leccino olives (Dourou et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Our results in part confirm these findings in terms of polyphenol content, highlighting that different genotypes react differently to the treatment imposed. In fact, the Leccino oil samples showed, as a general trend, better responses after the hydrocooler treatments in terms of total polyphenol content, with an increase in cinnamic and ferulic acid along with apigenin and methyl luteolin, as well as ODDMLA in 2021. This compositional modification in terms of polyphenols may be related to the more pronounced spiciness reported by the trained panellist for the Hy Leccino oil samples, both fresh and after one year of optimal storage.\u003c/p\u003e\u003cp\u003eConsidering the VOC profile, previous studies have indicated a reduction in total VOCs in olive oil with elevated temperatures, potentially leading to the development of off-flavours (Boskou, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Boselli et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Temperature control is thus crucial for ensuring a superior olive oil aroma. Cooling olive paste during the extraction process has been proposed as a method for enhancing oil quality, as in the application of a thermal exchanger for rapid olive paste cooling post-crushing (Veneziani et al., \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). The enzyme activities in the LOX pathway, including lipoxygenase (LOX), hydroperoxide lyase (HPL), alcohol dehydrogenase (ADH), and alcohol acyltransferase (AAT) enzymes, are highly sensitive to temperature.\u003csup\u003e2\u003c/sup\u003e The optimal temperature range for these enzymes varies, typically between 15 to 35\u0026deg;C, depending on the specific enzyme (Kiritsakis \u0026amp; Markakis, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e1988\u003c/span\u003e). ADH and AAT exhibit optimal activity around 30\u0026ndash;35\u0026deg;C, with lower temperatures resulting in decreased alcohol and ester production (Olias et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e1993\u003c/span\u003e). Conversely, HPL, the enzyme responsible for converting 13-hydroperoxide into aldehydes, operates optimally at 15\u0026deg;C, potentially leading to increased aldehyde synthesis and reduced conversion to alcohols and esters (Anthon \u0026amp; Barrett, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). This may partially explain the changes in the aldehyde/alcohol ratio observed in oils from cooled fruit, with higher levels of this ratio which are considered beneficial in terms of the sensory impact (Cerretani et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2008\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eConsidering the Leccino cultivar, in oils obtained from olives cooled (at 19\u0026deg;C) before crushing, an increase in VOCs from the LOX pathway, such as 2-hexen-1-ol, 3-hexen-1-ol, and 2-hexenal, has been reported, which also aligns with our findings (Dourou et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Additionally, an increase in polyphenol content at 15\u0026deg;C in Leccino has also been reported, which is consistent with our results. An increase in C6 VOCs has also been reported in oil produced from olives undergoing cold storage before the extraction process, which suggests a positive correlation between the flavour and lowering olive temperatures (Guerrini et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). However, while in the latter study the polyphenol profile of the oil appeared to be preserved from oxidation under cold conditions, our results in the Frantoio cultivar indicate a significant decrease in these compounds.\u003c/p\u003e\u003cp\u003eStill considering VOCs, the hydrocooler treatment led to a reduction in ethanol (off-flavour reduction) in the Frantoio cultivar (Supplementary Fig.\u0026nbsp;1) and, also in line with previously reported results (Karagoz et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Dourou et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), a general decreasing trend in C5 compounds such as 1-penten-3-ol and 1-penten-3-one, the latter being a derivative of 1-penten-3-ol metabolism. This highlights the importance of considering the interplay between 1-penten-3-ol and 1-penten-3-one compounds in terms of olive oil quality. This is also important considering that in the literature there is contrasting evidence of their impact, and in general of C5 aroma compounds, on the sensory attributes. Additionally, unlike our findings regarding oil chemical parameters, our VOC results are in line with other previously reported data, with an increase in (E)-2-hexenal across all cultivars after the application of cold treatments, along with a decrease in 1-penten-3-ol (Nucciarelli et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). In addition, in our study a significant increase in alcohols was found, especially 3-hexen-1-ol (green aroma). This highlights the significance of using the postharvest cooling of olives to increase desirable volatile compounds. Studies have highlighted the significant influence of cultivar on the VOC profile of olive oil (Genovese et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Hbaieb et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Serrano et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Additionally, the hydrocooling treatment has the advantage of modifying the volatile profile of the resulting oil. This is corroborated in previous research reporting that high temperatures during malaxation (25\u0026ndash;35 \u0026deg;C) altered the composition of metabolites from the LOX pathway, thus reducing pleasant volatile compounds and increasing less attractive ones (Servili et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Boskou, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2006\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eConsidering the sensory profile, our findings reveal a particularly interesting scenario. In several cases, the differences between oils obtained from the control (C) and Hydrocooler-treated (Hy) samples were evident both in the fresh oils and after one year of storage, regardless of the cultivar. Overall, the treatment led to an improvement in both varieties (Leccino and Frantoio), although a clear influence of the harvest year was also observed. While the differences immediately after production were significant, albeit relatively modest, they became more pronounced during storage, ultimately leading to the loss of Extra Virgin Olive Oil (EVOO) classification in the untreated samples. Notably, oils produced with the traditional method showed a decline in quality over time, eventually being rated as unacceptable (HI\u0026thinsp;\u0026lt;\u0026thinsp;6). This trend highlights the effectiveness of the Hydrocooler treatment in preserving oil quality over time. Regardless of cultivar or harvest year, oils from hydrocooler-treated olives consistently showed greater taste and olfactory intensity, characterized by higher levels of perceived fruitiness, bitterness, and pungency.\u003c/p\u003e"},{"header":"5. Conclusions","content":"\u003cp\u003eThe hydrocooler treatment appeared to be suitable for industrial application in the olive oil extraction process in terms of cooling efficacy and timing and thus could substitute the olive washing step. The results, especially considering the effects of the treatment on the olive VOC profile and sensory attributes, confirmed the positive findings of previous lab-scale research. The observed improvement in specific aroma compounds, and the reduction in off-flavours appeared to be consistent considering the different seasons and genotypes. However, different outcomes in terms of TPC, TTC and other important technological parameters of the oils were noted based on the season and genotype. Overall, pre-processing cooling of olives seems very promising for the improvement of oil quality and sensory profile, especially considering southern Mediterranean countries, which are subjected to very high temperatures during the harvesting period.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003eEthics approval and consent to participate\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eConsent for publication\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAvailability of data and materials\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eCompeting interests\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eFunding\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was financed by the project grant TIMONE-ID11 (https://www.cerm.unifi.it/timone/timone/), funded by the Italian Ministry of Agricultural, Food and Forestry Policies (MiPAAF) to Claudio Luchinbat and Pietro Tonutti. Gaia Meoni was supported by a research contract co-funded by the European Union - PON Research and Innovation 2014-2020 in accordance with Article 24, paragraph 3a), of Law No. 240 of December 30, 2010, as amended, and Ministerial Decree No. 1062 of August 10, 2021.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eEthical statement\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe research obtained the approval of the Ethics Committee\u0026nbsp;of the University of Pisa (protocol no. 0088081/2019). The research was conducted according to the ethical guidelines, and informed consent was obtained from all participants.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAuthors\u0026apos; contributions\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization, S.B. and P.T.; methodology, F.V., M.V.C. and G.M.; formal analysis, M.V.C., A.B., I.T. and G.M.; data curation, A.B., I.T., M.V.C. and S.B.; investigation, M.V.C.; resources, P.T. and C.L.; writing-original draft preparation, M.V.C., S.B., A.B., F.V., I.T. and P.T.; writing-review and editing, V.C.M., G.M, L.T., C.L., P.T., A.B., F.V. and S.B.; supervision, S.B. and P.T. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAcknowledgements\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe would like to express our sincere gratitude to Fabrizio Rossi and his family for hosting the experiments at their commercial olive mill in Scansano (Frantoio Rossi, Grosseto, Italy).\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAngerosa, F., Mostallino, R., Basti, C., \u0026amp; Vito, R. (2001). Influence of malaxation temperature and time on the quality of virgin olive oils. \u003cem\u003eFood Chemistry\u003c/em\u003e, \u003cem\u003e72\u003c/em\u003e(1), 19-28. https://doi.org/10.1016/S0308-8146(00)00194-1\u003c/li\u003e\n\u003cli\u003eAnthon, G. E., \u0026amp; Barrett, D. M. (2003). 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Low temperature treatments induce an increase in the relative content of both linolenic and \u0026lambda;3-hexadecenoic acids in thylakoid membrane phosphatidylglycerol of squash cotyledons. \u003cem\u003ePlant and cell physiology\u003c/em\u003e, \u003cem\u003e38\u003c/em\u003e(5), 611-618. https://doi.org/10.1093/oxfordjournals.pcp.a029211\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":true,"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":"Olea europaea, hydrocooler, temperature, olive oil, VOCs, off-flavors","lastPublishedDoi":"10.21203/rs.3.rs-6835795/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6835795/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe quality of olive oil is influenced by various factors including the olive environmental growth conditions. High temperatures in the later stages of fruit development can hasten ripening, with a detrimental impact on the fatty acid and polyphenol profiles. Harvesting and processing olives at high internal temperatures further degrades the oil quality, and especially affects the aromatic traits. To mitigate this problem, trials were conducted with the pre-processing rapid cooling of harvested olives from Frantoio and Leccino cultivars using hydrocooling technology. The aim was to assess the effects of quick cooling treatments of olives on oil quality and composition. The results indicated that cooled olive samples consistently produced virgin olive oil, and exhibited improved chemical quality parameters, particularly in Frantoio cultivars. The cooling treatment showed genotype-dependent effects, improving specific aroma compounds, and reducing off-flavours, although different outcomes were reported based on the season and genotype. Overall, pre-processing cooling seems to be a promising method for improving positive aroma compounds and minimizing off-flavors.\u003c/p\u003e","manuscriptTitle":"Pre-processing cooling of harvested olives: effects on oil composition and quality parameters in two different genotypes","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-06 13:50:24","doi":"10.21203/rs.3.rs-6835795/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":"6c34b4e4-1803-4933-839d-4450c3ea088f","owner":[],"postedDate":"August 6th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-03-16T12:42:46+00:00","versionOfRecord":[],"versionCreatedAt":"2025-08-06 13:50:24","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6835795","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6835795","identity":"rs-6835795","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}