Effects of Trichoderma Biostimulation on the Phenolic Profile of Extra-Virgin Olive Oil and Olive Oil By-Products.

Olive trees are grown on five continents. Fertilization of fields, pest control management, olive leaves, olive pomaces, and olive mill wastewaters have a substantial environmental impact. It is possible to reduce this problem by using organic products to cultivate and decrease olive oil processing waste by recovering the bioactive molecules. In this work, the effects of biostimulation, with beneficial microbes belonging to the Trichoderma genera, and with Trichoderma secondary metabolites (6PP and the HA) were evaluated on the phenolic profile and the antioxidant potential of extra-virgin olive oil (EVOO) and olive leaf samples to make them more commercially attractive as a source of phytochemicals useful for the pharmaceutical, cosmetic, and food industries. Phenolics were identified and quantified by a spectrometer method using Q Exactive Orbitrap UHPLC-MS/MS (Ultra High Pressure Liquid Chromatography). Antioxidant activity was evaluated spectrophotometrically by the DPPH test. The use of Trichoderma strains, 6PP (6-Pentyl-α-Pyrone) and HA (Harzianic Acid), was demonstrated as an effective strategy to increase the leaves’ economic value as a source of phytochemicals (flavonoids, lignans, and oleuropein) useful for food, pharmaceutical, and cosmetic industries.


Introduction
Olive production is a critical economic sector in many rural regions around the globe. The leading European producers are Spain (2.4 million ha), Italy (1.4 million ha), Greece (1 million ha), Portugal (0.5 million ha), and France (40,000 ha). Other cultivation countries are Tunisia, Turkey, Syria, Morocco, Algeria, Cyprus, Israel, Egypt, Libya, Lebanon, Jordan, Argentina, Mexico, Chile, Peru, Australia, USA, and China [1]. A new eco-friendly way to promote plant development and raise crop productivity is biostimulation with microbes. Biostimulation responds to the market need for organic and more nutritious food [2]. According to the "Global Biostimulants Market 2019-2027", the global market

Plant Material
The impact of bioformulates was tested on Olea europaea var. Ottobratica. Ottobratica is an Olea europaea variety commonly cultivated in the Calabria Region for oil extraction. The 20-year-old trees situated in the area Rombiolo (Vibo Valentia, South Western Calabria, Italy) were selected and labeled. Plant material used for these experiments was provided by Dr. Andrea Sicari (LINFA scarl, Vibo Valentia, Italy).
Field experiments were chosen with plants (15 years old) in excellent nutritional and phytosanitary status with a low ratio of wood and leaves, and with an adequate number of fruiting branches. The experimental field consisted of 9 rows (3 rows per treatment in a randomized experimental design), each containing 20 plants. Treatments were applied after plants sprouted with a monthly cadence starting from February until July for a total number of 6 treatments. Each bioformulate (10 6 ufc/mL of the living microbes TH1 (Trichoderma harzianum) and M10, 10 −6 M of the metabolites harzianic acid and 6-pentyl-α-pyrone) and one water treatment (control sample) were applied to the root system by drenching (around the root system at 10 cm deep) and as foliar spray application (10 L per row of which 5 L was drenching and 5 L was spray). The field test was repeated two times.

Isolation and Characterization of Harzianic Acid and 6-Pentyl-α-Pyrone
Trichoderma metabolites were isolated and characterized, as reported previously by Pascale et al. [10]. Trichoderma strains P1 and M10 were used to arrive at the bioactive compounds. Mycelia were put into 5 L conical flasks containing 1 L of sterile potato dextrose broth (PDB, HiMedia Mumbai, India) for 30 days at 25 • C. A filter paper (Whatman No. 4, Brentford, UK) was used to vacuum-filter the cultures. The filtrate (2 L) was extracted with ethyl acetate (EtOAc). The EtOAc fraction was dried with Na 2 SO 4 and evaporated in a vacuum at 35 • C. A flash chromatography (50 g Si gel 0.2 e 0.5 mm Merck-EMD Darmstadt, Germany) was used to separate metabolites in the yellow oil residue utilizing a gradient of petroleum ether/EtOAc (9:1 v/v to 4:6 v/v) as mobile phase. Then, 80 mg 6PP was obtained. Recovered fractions were chromatographed on thin-layer chromatography (TLC Si gel 60 F 254 Merck-EMD Darmstadt, Germany; mobile phase, petroleum ether/ethyl acetate, (8:2 v/v)) and fractions with similar profiles were combined. Successively, the red residue obtained from M10 was dissolved in CHCl 3 , extracted three times with NaOH 2M, and HA precipitated with HCl 2 M. The pellet was recovered (135 mg), solubilized, and subjected to RP-18 vacuum chromatography (20 g Si gel RP-18, 40-63 µm, Sigma Aldrich, St. Louis, MO, USA), eluting with a gradient of CH 3 OH/H 2 O/CH 3 CN (0.5:9:0.5 v/v/v to 10:0:0 v/v/v) to obtain 45 mg of pure HA. The compounds were detected on TLC using UV radiation (254 or 366 nm) and by immersing the sheets in a 5% (w/v) ethanol solution of 2 M H 2 SO 4 and heating at 110 • C for 10 min. The purified metabolites were characterized by LC/MS QTOF (Quadrupole Time of Flight) analysis recorded with a 6540 UHD Accurate Mass QTOF LC-MS/MS mass spectrometer (Agilent Technologies, Santa Clara, CA, USA) with a dual ESI (Electrospray Ionization) source, coupled to a 1200 series Rapid Resolution HPLC with a DAD (Diode-Array Detection) system (Agilent Technologies). Sample were identical to standards previously presented at Biological Control laboratories of the University of Naples Federico II.

Chemicals
All the chemicals and standards used were from Sigma Aldrich St. Louis, MO, USA, unless specified differently. Hydroxytyrosol was purchased from Indofine (Hillsborough, NJ, USA) and secologanoside was purchased from ChemFaces Biochemical Co., Ltd. (Wuhan, China).

Ultra High-Performance Liquid Chromatograph
Qualitative and quantitative profiles of phenols were obtained using an Ultra-High-Performance Liquid Chromatography (UHPLC, Thermo Fisher Scientific, Waltham, MA, USA) provided with a degassing system (Dionex Ultimate 3000), a quaternary UHPLC pump working at 1250 bar, an autosampler device, and a thermostated column compartment (T = 30 • C) with a Accucore aQ 2.6 µm (100 × 2.1 mm) column (Thermo Scientific, Waltham, MA, USA). The injection volume was 5 µL. The mobile phase was made as follows: Phase A: H 2 O 0.1% of acetic acid, and phase B: Acetonitrile. Phenols were eluted using a 0.4 mL/min flow rate with a gradient programmed: 0 to 5 min 5% phase B, 6 to 25 min 40% phase B, 25.1 to 27 min 100% phase B, 27.1 to 35 min 5% phase B, 35.1 to 45 min 0% phase B. Phenolics' identification occurred by comparing the retention times to the mass spectra of the purified compounds and the standards. The ligstroside quantification was made using the oleuropein in place of the authentic standard not commercially available.

Mass Spectrometry Analysis
Mass experiments were done with a Q Exactive Orbitrap LC-MS/MS (Thermo Fisher Scientific, Waltham, MA, USA) equipped with an ESI source (HESI II, Thermo Fisher Scientific, Waltham, MA, USA) operating in negative ion mode (ESI−). The ion source parameters were spray voltage −3.0 kV, sheath gas (N 2 > 95%) 30, auxiliary gas (N 2 > 95%) 15, capillary temperature 200 • C, S-lens RF level 50, auxiliary gas heater temperature 305 • C. The MS (Mass) detection was conducted in two acquisition modes: Full scan (negative-ion modes) and targeted selected ion monitoring. The parameters of the full scan acquisition mode were: Mass resolving power 35,000 full width at half maximum (at m/z 200), scan range 100-1500 m/z, scan rate 2s −1 , and the automatic gain control target 1 × 10 5 ions for a maximum injection time of 200 ms. The parameters of the targeted selected ion monitoring acquisition mode were: 15s-time window, resolution power 35,000 full width at half maximum (at m/z 200), and quadrupole isolation window 1.2 m/z. The mass list containing exact masses and expected retention times of the target polyphenolic compounds was taken in.

Q Exactive Orbitrap UHPLC-MS/MS Method Validation
The method linearity was evaluated by the regression coefficient of the calibration curve. The calibration curve was determined by using phenolics external standards. Limits of detection (LODs) and limits of quantification (LOQs) were derived from the regression curve (LODs = 3 × standard deviation angular coe f f icient ; LOQs = 10 × standard deviation angular coe f f icient ). Intraday repeatability was evaluated by injecting seven different concentrations of each phenolic standard three times. The interday assay variations were obtained by repeating after seven days the same operating conditions.

Extraction of Phenolics from the Olive Oil
The phenolic compounds were extracted following the method of Vasquez Roncero [11]. In brief: 25 g of oil were dissolved in 25 mL hexane. The polar compounds were extracted three times, with 15 mL of a mixture consisting of methanol:water (3:2 v/v). The three extracts were mixed and treated once with 25 mL hexane. The solvent was evaporated to dryness in a rotary evaporator (Buchi, Switzerland) at 40 • C. The residue was dissolved in 1 mL of methanol and filtered through 0.2 mm nylon filer and immediately frozen and stored at −18 • C until analysis.

Extraction of Phenolics from the Olive Leaves
The sample extraction was performed as described previously by Talhaoui et al. [12]. The dry leaves (0.5 g) were crushed and extracted with MeOH/H 2 O (80:20 v/v) (10 mL). The samples were placed in an ultrasonic bath for 10 min and then centrifuged at 6000 rpm for 10 min. The supernatant was recovered, and the pellet was re-extracted following the procedure applied to the starting sample. The obtained supernatants were dried to rotavapor and dissolved in 4 mL of a methanol/water solution (50:50 v/v). Three replicates of each sample were processed.

Results
The hydrophilic extracts obtained from EVOO and olive leaves were analyzed by Q Exactive Orbitrap UHPLC-MS/MS to identify and quantify the main phenolics.

Validation of the MS Method
Validation parameters for the analysis of the phenolic by UPLC-HRMS-Orbitrap are reported in Table 1.

Identification of the Phenolic Compounds
Nineteen phenolics, including two flavonoids, two phenolic alcohols, six phenolic acids, two lignans, and seven secoiridoids, were identified and quantified. The parameters used to identify phenolics in samples are reported in Table 2.

Quantification of the Phenolic Compound
Different amounts of single phenolics were quantified (Tables 3-6). In vivo experiment for the disease control of the olive plants showed that not only the Trichoderma strains but also their metabolites (6PP and HA) in all samples tested had a positive effect on the luteolin, the pinoresinol, and the acetoxypinoresinol content (Table 3).    The biostimulation decreased the tyrosol concentration in all samples tested and enhanced the hydroxytyrosol concentration in the EVOO samples ( Table 4).
The response of the phenolic acids to the treatments was very different in the EVOO and the leaf samples. All biostimulators had a positive effect in the leaf samples on the 4-hydroxybenzoic acid, the vanillic acid, and the cinnamic acid content, and a positive impact in the EVOO samples on the 3-hydroxybenzoic acid, the vanillic acid, the p-coumaric acid and the ferulic acid content (Table 5).

Antioxidant Activity
The biostimulation of olive trees had a positive effect on the antioxidant activity of the olive leaf samples and of the EVOO samples obtained from the olive tree under M10 treatment (Figure 1). Antioxidants 2020, 9, x FOR PEER REVIEW 8 of 12

Antioxidant Activity
The biostimulation of olive trees had a positive effect on the antioxidant activity of the olive leaf samples and of the EVOO samples obtained from the olive tree under M10 treatment (Figure 1).

Discussion
Olive oil and nonfood olive biomass are promising sources of bioactive molecules, including phenols. The latter, when added to food, cosmetics, and medicines have additive (flavoring, coloring, and texturizing) and functional properties [8,[14][15][16][17]. The plants produce phenolics, mainly in leaves or bark, to attract pollinators, protect against UV radiation, and defend against phytopathogenic bacteria, viruses, and fungi [18]. In this study, the phenolic profile and dosage in extra-virgin olive oil and olive leaves were investigated by using an HPLC-Orbitrap platform in MS and MS/MS levels. The linearity, sensitivity, and precision were studied to validate the MS methods. The correlation factor of the calibration curve = 0.999 and the residues normally distributed confirmed the linearity of the calibration curve, the method sensitivity in the range of the LODs and the LOQs, verified method sensitivity, and the RSD (Relative Standard Deviation) values <15% validated the inter-and intraday repeatability. Two flavonoids, two lignans, seven secoiridoids, six phenolic acids, and two phenolic alcohols were identified and quantified. Identification was made comparing the retention time, mass spectra, accurate mass measurements, and MS2 analyses with standards when they were commercially available or with literature data (ligstroside) [19]. The presence of two hydroxybenzoic acid isomers was confirmed comparing the retention time and mass spectra with standards. The flavonoids and the lignans occurred in the aglycon form in the oil and the leaf samples. It was probably due to degradation during the malaxation process and leaf breakage to allow extraction. The biostimulation treatment increased the concentrations of flavonoids and the lignans in the EVOO and the leaves, although organic agriculture generally affects negatively on polyphenol contents, since the shikimate pathway links the biosynthesis of these compounds and nitrogen metabolism. The protein-phenol competition decreased the action of the phenylalanine ammonia-lyase, an enzyme involved in their biosynthesis [20][21][22]. Instead, Trichoderma strains may improve phenolic concentrations, increasing the plant nutrient uptake mechanism [23] and enhancing plant nitrogen

Discussion
Olive oil and nonfood olive biomass are promising sources of bioactive molecules, including phenols. The latter, when added to food, cosmetics, and medicines have additive (flavoring, coloring, and texturizing) and functional properties [8,[14][15][16][17]. The plants produce phenolics, mainly in leaves or bark, to attract pollinators, protect against UV radiation, and defend against phytopathogenic bacteria, viruses, and fungi [18]. In this study, the phenolic profile and dosage in extra-virgin olive oil and olive leaves were investigated by using an HPLC-Orbitrap platform in MS and MS/MS levels. The linearity, sensitivity, and precision were studied to validate the MS methods. The correlation factor of the calibration curve = 0.999 and the residues normally distributed confirmed the linearity of the calibration curve, the method sensitivity in the range of the LODs and the LOQs, verified method sensitivity, and the RSD (Relative Standard Deviation) values <15% validated the inter-and intraday repeatability. Two flavonoids, two lignans, seven secoiridoids, six phenolic acids, and two phenolic alcohols were identified and quantified. Identification was made comparing the retention time, mass spectra, accurate mass measurements, and MS2 analyses with standards when they were commercially available or with literature data (ligstroside) [19]. The presence of two hydroxybenzoic acid isomers was confirmed comparing the retention time and mass spectra with standards. The flavonoids and the lignans occurred in the aglycon form in the oil and the leaf samples. It was probably due to degradation during the malaxation process and leaf breakage to allow extraction. The biostimulation treatment increased the concentrations of flavonoids and the lignans in the EVOO and the leaves, although organic agriculture generally affects negatively on polyphenol contents, since the shikimate pathway links the biosynthesis of these compounds and nitrogen metabolism. The protein-phenol competition decreased the action of the phenylalanine ammonia-lyase, an enzyme involved in their biosynthesis [20][21][22]. Instead, Trichoderma strains may improve phenolic concentrations, increasing the plant nutrient uptake mechanism [23] and enhancing plant nitrogen use efficiency [24,25]. Our results support that not only Trichoderma strains have this ability, but, also, their metabolites (6PP and HA) increase the concentration of the polyphenols both in olive leaves and in oil. These findings revealed that the 6PP and the HA are involved in the induction of plant systemic resistance. The different concentrations of the polyphenols under the microbe or the microbe metabolites biostimulation were related to the ability of the living microbe to attach the pathogen by more than one mechanism, including the antibiosis, the competition, the direct mycoparasitism, and the induction of the disease resistance that acts synergistically [24]. The higher concentration of flavonoids in the leaves than in the oil can be explained since the flavonoids are produced in the leaves [26]. The prevalence of the luteolin, among the flavonoids, agreed with the results already detected for the olive species [27]. The high content of luteolin was of great interest, as its intake was associated with antioxidant and health-promoting actions [28,29]. Other phenolics synthesized through the shikimic pathway are phenolic acids [30]. In this study, all biostimulation treatments had a positive effect only on vanillic acid concentration. The concentration of the phenolic acids was affected by the ripening process, except for the vanillic acid content [31]. Therefore, the vanillic acid content was only affected by the effects due to the use of biostimulants in agriculture. Concerning secoiridoid fractions, the biostimulation decreased oleuropein degradation. The higher concentration of oleuropein and the lower concentration of its degradation product (tyrosol) demonstrated it [32] (Figure 2). Antioxidants 2020, 9, x FOR PEER REVIEW 9 of 12 pathogen by more than one mechanism, including the antibiosis, the competition, the direct mycoparasitism, and the induction of the disease resistance that acts synergistically [24]. The higher concentration of flavonoids in the leaves than in the oil can be explained since the flavonoids are produced in the leaves [26]. The prevalence of the luteolin, among the flavonoids, agreed with the results already detected for the olive species [27]. The high content of luteolin was of great interest, as its intake was associated with antioxidant and health-promoting actions [28,29]. Other phenolics synthesized through the shikimic pathway are phenolic acids [30]. In this study, all biostimulation treatments had a positive effect only on vanillic acid concentration. The concentration of the phenolic acids was affected by the ripening process, except for the vanillic acid content [31]. Therefore, the vanillic acid content was only affected by the effects due to the use of biostimulants in agriculture. Concerning secoiridoid fractions, the biostimulation decreased oleuropein degradation. The higher concentration of oleuropein and the lower concentration of its degradation product (tyrosol) demonstrated it [32] (Figure 2). The oleuropein has nutraceutical and pharmaceutical properties. It modulates several oncogenic signaling pathways and treats oxidant and inflammatory-related diseases (obesity, diabetes, neuro deficiency, cardiovascular disease, and hepatic disorder) [34]. Finally, the antioxidant activity was evaluated. In the leaf tests, all biostimulation treatment increased the antioxidant potential of the samples. On the reverse, in EVOO experiments, only the M10 stimulation gave these answers. In this investigation, antioxidant activity was founded by the DPPH test. This method limited the capability of the antioxidant to transfer an electron and to reduce carbonyls, metals, and themes. It provides the highest rankings to the compound with many phenol groups and ortho-substituted with electrondonating alkyl or methoxy groups [35,36]. In EVOO samples, the most representative phenolics The oleuropein has nutraceutical and pharmaceutical properties. It modulates several oncogenic signaling pathways and treats oxidant and inflammatory-related diseases (obesity, diabetes, neuro deficiency, cardiovascular disease, and hepatic disorder) [34]. Finally, the antioxidant activity was evaluated. In the leaf tests, all biostimulation treatment increased the antioxidant potential of the samples. On the reverse, in EVOO experiments, only the M10 stimulation gave these answers.
In this investigation, antioxidant activity was founded by the DPPH test. This method limited the capability of the antioxidant to transfer an electron and to reduce carbonyls, metals, and themes. It provides the highest rankings to the compound with many phenol groups and ortho-substituted with electron-donating alkyl or methoxy groups [35,36]. In EVOO samples, the most representative phenolics (tyrosol, hydroxytyrosol, oleacein, oleuropein-aglycone mono-aldehyde, and ligstroside aglycone-dialdehyde) did not have these characteristics. Thus, the antioxidant potential of the EVOO samples may have been underestimated [37]. On the contrary, in the leaf samples, the B-ring catechol structure and the 2.3-double bond conjugated with a 4-oxo in the structure of the most representative compound (luteolin) may have resulted in an overestimation of the antioxidant potential.

Conclusions
In this study, the effects of olive trees' treatment with beneficial microbes belonging to Trichoderma genera were evaluated in extra-virgin olive oil and olive leaves on phenolic profile and content by using HPLC-Orbitrap platform in MS and MS/MS levels. The recovery, the linearity, the precision, and the accuracy validated the used methods. This study supported the excellent effect of biostimulation on the nutraceutical value of EVOO, and for the first time delineated the effects of the Trichoderma secondary metabolites (6PP and the HA) used in the olive tree cultivation on the phenolics' production and the antioxidant activity in EVOO and leaf samples. Our results suggest the possibility of using HA and 6PP in olive cultivation, as they eliminate some of the limitations related to the application of living biological control agents and the concentration of phenolic compounds in olive leaves, and making these wastes commercially attractive as a source of phytochemicals useful for the pharmaceutical, cosmetic, and food industries.