New Strategies in the Cultivation of Olive Trees and Repercussions on the Nutritional Value of the Extra Virgin Olive Oil

The health advantages of extra-virgin olive oil (EVOO) are ascribed mainly to the antioxidant ability of the phenolic compounds. Secoiridoids, hydroxytyrosol, tyrosol, phenolic acid, and flavones, are the main nutraceutical substances of EVOO. Applications of beneficial microbes and/or their metabolites impact the plant metabolome. In this study the effects of application of selected Trichoderma strains or their effectors (secondary metabolites) on the phenolic compounds content and antioxidant potential of the EVOOs have been evaluated. For this purpose, Trichoderma virens (strain GV41) and Trichoderma harzianum (strain T22), well-known biocontrol agents, and two their metabolites harzianic acid (HA) and 6-pentyl-α-pyrone (6PP) were been used to treat plants of Olea europaea var. Leccino and var. Carolea. Then the nutraceutical potential of EVOO was evaluated. Total phenolic content was estimated by Folin–Ciocalteau’s assay, metabolic profile by High-Resolution Mass spectroscopy (HRMS-Orbitrap), and antioxidant activity by DPPH and ABTS assays. Our results showed that in the cultivation of the olive tree, T22 and its metabolites improve the nutraceutical value of the EVOOs modulating the phenolic profile and improving antioxidants activity.


Introduction
The health benefits of the extra-virgin olive oil are ascribed mainly to phenolic compounds, among which the most concentrated are lignans (pinoresinol, acetoxypinoresinol, hydroxypinoresinol, etc.) and secoiridoids (ligstroside, oleuropein, etc.), with the latter located only in the Oleaceae family [1]. Other phenolics in EVOO are flavonoids (luteolin, apigenin, etc.), phenolic alcohols (tyrosol, hydroxytyrosol, etc.), and phenolic acids (hydroxybenzoic acid, ferulic acid, etc.) [2]. These substances modulate aging-associated processes and have antitumor, antiviral, anti-atherogenic, anti-inflammatory, A new chromatographic method was applied for the quantification of individual secondary metabolites, whose validation parameters were reported in Table 2. Limits of detection (LODs) range was from 0.02 to 1.0 mg/L, Limits of quantification (LOQs) 0.033 to 3.0 mg/L, and the linearity range between 88.7 and 1%.
The second group of phenolic compounds by concentration were phenolic alcohols ( Table 4). The third most abundant class of phenolic compounds were lignans (Table 5) followed by flavonoids (Table 6) and phenolic acids (Table 7).
Noteworthy was the content of luteolin in Carolea oil and the ability of Ha biostimulation to improve it.
In Leccino EVOO, the highest concentration of phenols occurred in the oil produced from olives obtained by treating trees with HA (+23%), followed by T22 (+7%). On the contrary, the treatment of the olive trees with GV41 and 6PP decreased their concentration: −4% and −11%, respectively. ( Figure 2).

Phenol Content and Antioxidant Activity
As shown in Figure 1, the EVOO obtained from Leccino variety olives had the highest content of phenols (EVOOLeccino:133.662 mg/kg −1 ; EVOOCarolea:77.871 mg·kg −1 ). The treatment of the Carolea olive trees with the biocontrol agent 6PP, compared to the untreated trees, improved the concentration of phenols in EVOO (+22%), followed by HA (+18%), and T22 (+7%), only the treatment with the living fungus GV41 decreased their concentration (−16%) ( Figure 1). In Leccino EVOO, the highest concentration of phenols occurred in the oil produced from olives obtained by treating trees with HA (+23%), followed by T22 (+7%). On the contrary, the treatment of the olive trees with GV41 and 6PP decreased their concentration: −4% and −11%, respectively. ( Figure  2). DPPH and ABTS assays were used to determine the antioxidant activity of samples. A positive correlation was found between phenolic concentration and antioxidant activity measured by the ABTS test in all oil samples (Leccinooil = 0.970322; Cororeaoil = 0.757275). Concerning the correlation between phenolic concentration and antioxidant activity measured by DPPH test, it was positive (0.91454) in Leccinooil and negative in Coroleaoil (−0.09952).

Secoiridoids and Phenolic Alcohols Correlation
Significant correlation indexes correlated secoiridoids and phenolic alcohols (EVOO Leccino − 0.7 and EVOO Carolea + 0.6). These indexes were obtained by correlating the sum of the concentrations of the four most representative secoiridoids (oleuropein-aglycone monoaldehyde+ ligstroside-aglycone monoaldehyde + oleocanthal + oleacein) with the sum of the two phenolic acids (tyrosol + OHTyrosol) (     HA treatment influenced flavonoid production in both monovarietal oils. The antioxidant activity of EVOO Carolea was higher than EVOO Leccino ( Figure 5). HA treatment influenced flavonoid production in both monovarietal oils. The antioxidant activity of EVOOCarolea was higher than EVOO Leccino ( Figure 5).  The total phenolic content was strongly affected by variations of the concentration of oleacein, tyrosol and apigenin (Figure 7).  The 6PP biostimulation interfered with the production of the flavonoids and the lignans in the olives ( Figure 6). HA treatment influenced flavonoid production in both monovarietal oils. The antioxidant activity of EVOOCarolea was higher than EVOO Leccino ( Figure 5).  The total phenolic content was strongly affected by variations of the concentration of oleacein, tyrosol and apigenin (Figure 7).   The total phenolic content was strongly affected by variations of the concentration of oleacein, tyrosol and apigenin (Figure 7).

Discussion
Two monovarietal EVVOs olives were analyzed to determine the possible impact on their nutraceutical properties when biocontrol strategy was used in the fields. This goal was obtained by treating two monovarietal olive trees (Olea europaea var. Leccino and Olea europaea var Carolea) with two strains of Trichoderma (GV41 and T22), and their metabolites HA and 6PP and evaluating the total content of phenols in the oil, determining the single phenol quality and quantity and comparing these data with the antioxidant activity of the oils. A remarkable variability was found in phenolic composition between the two sets of monovarietal EVOOs analyzed. The phenolic identification was obtained by using an Orbitrap platform in MS and MS/MS levels, and phenolic quantification was performed by using a UPLC-MS technology. The quantification method was validated in terms of linearity, precision, and sensitivity. The correlation factor of the calibration curve 1 established the first one, LODs, and the LOQs confirmed method sensitivity, and the relative standard deviation (RSD) <10% validated the repeatability. Nine secoiridoids, two phenolic alcohols, two lignans, two flavonoids, and six phenolic acids were characterized comparing the mass spectra with standards, except the ligstroside whose identification occurred comparing mass data with literature data, [21] and the hydroxybenzoic acid isomers, for which the retention times and the mass spectra were used. In all olive oils, the secoiridoid derivatives were the most abundant phenols, followed by phenolic alcohols, flavonoids, and phenolic acids. The lignans and the flavonoids were in the aglycon form since they degrade during the malaxation process. A significant correlation index between secoiridoids and phenolic alcohols (Figure 3) confirmed that tyrosol and the OHtyrosol were degradation products of ligstroside and oleuropein [22]. All biostimulant treatments, increased the total polyphenol content in EVOOs, except GV41 and 6PP in the EVOO Leccino. The DPPH test and the ABTS method tested the antioxidant activity. The DPPH detected the ability of an antioxidant to transfer one electron to reduce any compound. The ABTS method determined the aptitude of the antioxidant to quench free radicals by hydrogen donation [23]. In this study, a significative correlation was found between the total phenolic content, and both tests used to determine the antioxidant activity. The DPPH measures were higher than that obtained with the ABTS test in the samples containing higher concentrations of flavonoids, O-diphenols and secoiridoids since DPPH test overestimates slow reacting antioxidants with many phenol groups as lutein, OHTyrosol and secoiridoid derivatives, able to donate hydrogen and improve radical stability by forming an intramolecular hydrogen bond between the free hydrogen of phenoxyl radicals, therefore the ABTS method is the best for the determination of the antioxidant activity in the oil [24]. The T22 biostimulation interferes above all with the production of secoiridoids in the olive. In both oil samples were found an increase of the oleuropein-aglycon mono aldehyde. A decreased concentration of ligstroside decarboxymethyl-aglycone was shown in the EVOO Carolea , probably due to transformation in its degradation product (tyrosol) (Figure 4). The biostimulation with the T22 strain enhanced the concentration of the total phenolic content of 7% in both oil samples (Figures 1 and 2). This increase determines the growth of the antioxidant activity of 20% (DPPH = 22%; ABTS = 20%) in the EVOO Leccino, (Figure 2) and different measures of antioxidant activity in the EVOO Corolea , (Figure 1) according to the method used to determine it (DPPH test = 31% and ABTS test = 4%) (Figure 4), since the higher concentration of secoiridoids and flavonoids in EVOO Carolea than EVOO Leccino , were overestimated in the DPPH method. The biostimulation with HA increased the phenolic content (particularly the flavonoidic fraction) in both monovarietal oils ( Figure 5). The higher concentration of flavonoids in EVOO Carolea than EVOO Leccino (Table 6), determined the overestimation in the DPPH method ( Figure 5). The biostimulation with the 6PP metabolite decreased the total phenol concentration in the EVOO Leccino and enhanced it in the EVOO Corolea (Figures 1 and 2). Consequently, the antioxidant activity decreased in the EVOO Leccino and improved in the EVOO Corolea . more than the control (Figure 6). The different variations of the polyphenol classes concentrations under the microbe or the microbe metabolites biostimulation (Figure 7) suggested that the nutraceutical properties [25] of the EVOO depended on the biostimulant used to grow olive trees (Figure 8). The biostimulation with T22 mainly enhanced the concentration of the secoiridoid fraction of phenols. As known, oleuropein is commercially available as a food supplement used to prevent the oxidation and the inflammatory damage, the cardiovascular and the cancer diseases, and as antiviral and antimicrobial agents [26]. Instead, the HA metabolite increased the flavonoids in the EVOOs. Luteolin has showed antitumorigenic, antimutagenic, antioxidative, immunomodulatory, and anti-inflammatory properties useful in cancer, cardiovascular diseases, and neurodegenerative pathologies prevention [27,28]. Finally, the 6PP metabolite improved lignans concentration in the EVOOs. Pinoresinol and acetoxypinoresinol intake has been related to LDL oxidation prevention, and health properties correlate to estrogen hormonal disfunction such as protection against cancer (prostate and breast) [29]. enhanced the concentration of the secoiridoid fraction of phenols. As known, oleuropein is commercially available as a food supplement used to prevent the oxidation and the inflammatory damage, the cardiovascular and the cancer diseases, and as antiviral and antimicrobial agents. [26] Instead, the HA metabolite increased the flavonoids in the EVOOs. Luteolin has showed antitumorigenic, antimutagenic, antioxidative, immunomodulatory, and anti-inflammatory properties useful in cancer, cardiovascular diseases, and neurodegenerative pathologies prevention [27,28]. Finally, the 6PP metabolite improved lignans concentration in the EVOOs. Pinoresinol and acetoxypinoresinol intake has been related to LDL oxidation prevention, and health properties correlate to estrogen hormonal disfunction such as protection against cancer (prostate and breast) [29].

Study Area
This study was carried out in two experimental sites situated in Calabria, the most southern region of the Italian peninsula (ranges between 38°12′ and 40° latitude North and between 16°30′ and 17°15′ longitude East). The provinces of Calabria are: Catanzaro (CZ, regional capital), Reggio Calabria, Cosenza (CS), Crotone (KR), and Vibo Valentia (VV). The two experimental sites are in the villages of Cariati (CS), and Roccabernarda (KR). The climate of this Region is predominantly Mediterranean, temperatures are very mild, especially in the coastal plains. In the summer the heat is shared by the entire regional territory and only the altitude mitigates the heat or the breezes; peaks of over 35 °C are common. In the case of invasions of very hot African air, the temperatures exceed the 40 °C threshold. In Winter, on the other hand, temperatures remain mild with maxima greater than 10 °C on the coasts and cold in the internal areas and in the mountains, where the snow falls abundantly, and above 1000 m can persist throughout the period from December to March.

Study Area
This study was carried out in two experimental sites situated in Calabria, the most southern region of the Italian peninsula (ranges between 38 • 12 and 40 • latitude North and between 16 • 30 and 17 • 15 longitude East). The provinces of Calabria are: Catanzaro (CZ, regional capital), Reggio Calabria, Cosenza (CS), Crotone (KR), and Vibo Valentia (VV). The two experimental sites are in the villages of Cariati (CS), and Roccabernarda (KR). The climate of this Region is predominantly Mediterranean, temperatures are very mild, especially in the coastal plains. In the summer the heat is shared by the entire regional territory and only the altitude mitigates the heat or the breezes; peaks of over 35 • C are common. In the case of invasions of very hot African air, the temperatures exceed the 40 • C threshold. In Winter, on the other hand, temperatures remain mild with maxima greater than 10 • C on the coasts and cold in the internal areas and in the mountains, where the snow falls abundantly, and above 1000 m can persist throughout the period from December to March.

Plant Material
The possible impact of bioformulates was tested on two cultivars of Olea europaea: Leccino and Carolea. Plant material were given by Dr. Andrea Sicari (LINFA scarl, Vibo Valentia, Italy).
Plants (15 years old) in excellent nutritional and phytosanitary status with an adequate number of fruiting branches and a low ratio of wood and leaves were used for experimental purposes. The experimental field contained 20 plants split in 12 rows (3 rows per treatment). Six treatments were applied starting from February until July after plants sprouted. Each bioformulate (10 −6 M HA, 10 −6 M 6 PP, 10 6 ufc/mL GV41, and 10 6 ufc/mL T22) and one control sample (water treatment) were applied to the root system (drenching around the root system at 10 cm deep) and the leaves (10 L per row of which 5 L was spray and 5 L was drenching).

Fungal Material
Biological Control laboratories of the University of Naples Federico II provide the Trichoderma strains.

Isolation and Characterization of Harzianic Acid
The Trichoderma strains M10 was used to produce the bioactive molecules. Mycelia were inoculated into 1L of sterile potato dextrose broth (PDB, HiMedia Mumbai, India). Cultures of each strain were grown for 30 days at 25 • C, and then vacuum-filtered through filter paper (Whatman No. 4, Brentford, UK). Ethyl acetate (EtOAc) was used to extract the filtrate (2 L). Organic fractions were dried with Na 2 SO 4 and the solvent evaporated in vacuum at 35 • C. The red residue obtained from M10 was dissolved in CHCl 3

Oil Production
The oils samples were cold produced at a semi-industrial scale in a local two-phase mill. It was kept at a constant temperature (10 ± 2 • C) in dark bottles without headspace until analysis.

Chemicals
All the chemicals used are from Sigma Aldrich St. Louis, MO, USA, unless specified differently.

Extraction of Phenolic Compounds from Olive Oil
The method proposed by Vasquez Roncero [32] was used. In 25 mL hexane were put 25 g oil. 15 mL methanol:water (3:2 v/v) extracted the polar in three times. The extracts combined were treated once with 25 mL hexane. The solvent was evaporated in a rotary evaporator (Büchi, Switzerland) at 40 • C.
The insoluble residue was abundantly washed with CH 3 OH and filtered through 0.2 µm nylon filter and immediately stored at −18 • C until analysis.

Ultra High Pressure Liquid Chromatograph
Polyphenol compounds were isolated and quantified by an Ultra High Pressure Liquid Chromatograph (UHPLC, Thermo Fisher Scientific, Waltham, MA, USA) equipped with a Dionex Ultimate 3000 degassing system, a quaternary UHPLC pump (Thermo Fisher Scientific, Waltham, MA, USA) working at 1250 bar, and a column (Thermo Scientific, Waltham, MA, USA) Accucore aQ 2.6 µm (100 × 2.1 mm) in a thermostated compartment (T = 30 • C). 5 µL of the sample was injected. The eluent phase consists of a gradient programmed as follows: 0 to 5 min −5% of phase B, 25 15 , sheath gas (N 2 > 95%) 30 , auxiliary gas heater temperature 305 • C and S-lens RF level 50]. The mass detection was obtained in two acquisition modes: negative-ion modes (full scan; mass resolving power 35,000 full width at half maximum (at m/z 200), the automatic gain control target 1 × 105 ions for a maximum injection time of 200 ms, and scan range 100-1500 m/z, scan rate 2 s −1 ) and targeted selected ion monitoring [15 s-time window, quadrupole isolation window 1.2 m/z, and resolution power 35,000 full width at half maximum (at m/z 200)].

Validation of the Method Used to Quantify Single Phenols
Method was validated following AOAC instructions (AOAC 2012) [33]. The parameters analyzed were linearity, LOD, LOQ, repeatability, and reproducibility. Three points (in triplicate) were used to build the calibration curves of each compound. Method linearity was obtained from the regression coefficient of the calibration curve LOD (Limits of detection) and LOQ limits of quantification were calculated from the regression curve. Nine different concentrations of each phenolic standard three times gave intraday repeatability.

Total Phenolic Compounds
The total polyphenols amount was evaluated by using the Folin-Ciocalteau's assay as reported by Singleton and Rossi (1965) [34]. In a falcon (15 mL), 2.5 mL dd H 2 O and 625 µL methanolic extract, 625 µL of Folin-Ciocalteau's phenol reagent were shaken. After 6 min, 6.25 mL of 7% Na2CO3 solution was added to the mixture. The solution was diluted with 5 mL dd H 2 O and mixed. The absorbance (Lambda 25, PerkinElmer, Italy) of reagent blank was determined at 760 nm by spectrophotometer after incubation for 90 min at room temperature. All biological replicates of samples were analyzed in triplicate. Total phenolic content was expressed as mg gallic equivalents (GAE)/kg FW.
ABTS method. 2,2 -azinobis (3-Ethylbenzothiazoline-6-sulfonic acid) (ABTS) procedure modified from Re et al. was used (1999) [36]. A concentrate solution of the reagent (stock solution) was prepared dissolving 9.6 mg of ABTS in 2.5 mL of water and adding 44 mL of a solution made by dissolving 37.5 mg of potassium persulphate, K 2 S 2 O 8 , in 1 mL of water. The stock solution was kept in the dark at 4 • C for 8 h before use; the work solution was obtained from the stock solution by dilution using a 1:88 (v/v) ratio. Dilution was adjusted depending on the measured absorbance at λ734 nm (A734) in the work solution, until a value between 0.7 and 0.8. Subsequently, 100 µL of sample and 1 mL of work solution were added, and A734 was measured exactly after 2 min and 30 s. (Lambda 25, PerkinElmer, Italy). Calibration curve was obtained using 6-Hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), a water-soluble analog of αtocopherol, as standard and results were expressed as mmol Trolox equivalent (TE) kg −1 FW. All biological replicates of samples were analyzed in triplicate.

Conclusions
Our results confirmed the ability of the Trichoderma harzianum (strain T22) and its metabolites (6PP and HA) in the defense system of the Olea europaea tree and demonstrate that the ABTS test is the preferred method for determining the antioxidant activity in the EVOO. To the best of our knowledge, the present work is the first report that correlates the nutraceutical properties of the EVOO to a specific biostimulation method used in the field. Our results suggest new possibilities of using Thricoderma and its metabolites to select the nutraceutical properties of the EVOO and recommend the use of Thricoderma metabolites in olive tree cultivation to avoid some of the limitations related to the application of living microbes.