An Environmentally Friendly Practice Used in Olive Cultivation Capable of Increasing Commercial Interest in Waste Products from Oil Processing

In the Rural Development Plan (2014–2020), the European Commission encouraged the conversion and supported the maintenance of organic farming. Organic olive oil (bioEVOO) production involves the use of environmentally sustainable fertilizers and the recycling of olive pomace (Pom) and olive vegetation waters (VW) to reduce the environmental impact of these wastes. An ecofriendly way to recycle olive wastes is to reuse them to extract bioactive compounds. In this study, the total phenolic compounds content, their profile and dosage, the antioxidant action in oil, pomace, and vegetation water was evaluated when the Trichoderma harzianum M10 was used as a biostimulant in agriculture. Two spectrophotometric tests (2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2′-azinobis (3-ethylbenzothiazoline-6-sulfonic) acid (ABTS)) evaluated the antioxidant potential of samples, a spectrophotometric method estimated total phenolic content, and an Ultra-High-Performance Liquid Chromatography (UHPLC)–Orbitrap method evaluated the phenolics profile. Our results showed that the biostimulation improved the antioxidant potential and the total concentration of phenolics in the bioEVOO and bio-pomace (bioPom) samples and mainly enhanced, among all classes of phenolic compounds, the production of the flavonoids and the secoiridoids. Moreover, they demonstrated the Trichoderma action in the mevalonate pathway to produce phenols for the first time. The decisive action of the Thricoderma on the production of phenolic compounds increases the economic value of the waste materials as a source of bioactive compounds useful for the pharmaceutical, cosmetic, and food industries.


Plant Material
Bioformulates was tested on Olea europaea var. Leccino. The trees (20-year-old) situated in the South-Western Calabria (Rombiolo, Vibo Valentia, Italy) were selected and marked.
Plant material was offered d by Dr. Andrea Sicari (Linfa Scarl, Vibo Valentia, Italy). Only plants in excellent phytosanitary and nutritional status were used for experimental purposes. Six treatments were applied every month, starting from February until July. One control sample (water treatment), and 10 6 ufc/mL of the living microbes and were applied as spray application on the leaves (10 L per row of which 5 L was sprayed and 5 L was drenching), and around the root system at 10 cm deep. Two times was replicated the field test.

Oil Production
The oil samples were produced in a local three-phase mill. They were conserved in brown bottles without headspace and conserved at a constant temperature (10 ± 2 • C) until analysis.

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

The Phenolics Extraction
The phenolic extraction method proposed by Vasquez Roncero [28] with some modification was carried out. An amount of 25 g of oil was extracted with hexane (25 mL). The organic fraction was treated with MeOH:H 2 O/3:2 (v/v) (15 mL, three times). The extracts (three) were combined and extracted with 25 mL hexane. The hexane was dried at 40 • C in a rotary evaporator (Buchi, Switzerland); the residue was treated with 1 mL of MeOH, filtered through nylon filer (0.2 mm), frozen and stored (−18 • C) until analysis.

Q Exactive Orbitrap LC-MS/MS Method
Ultra-High-Performance Liquid Chromatography (UHPLC, Thermo Fisher Scientific, Waltham, MA, USA) was used to the dosage of the phenolics. The chromatographic instrument was provided with an autosampler device, a Dionex degassing system (Thermo Scientific™ Ultimate 3000, Waltham, MA, USA), a quaternary UHPLC pump (1250 bar), and a column (Accucore aQ 2.6 µm 100 × 2.1 mm Thermo Scientific, Waltham, MA USA, USA) in a thermostat column compartment (T = 30 • C). The mobile phase consisted of two phases: Phase A: acetic acid (0.1%), and phase B: 100% acetonitrile. The following gradient was used for experimental purposes: 5% phase B from 0 to 5 min, 40% phase B from 6 to 25 min, 100% phase B from 25.1 to 27 min, 5% phase B from 27.1 to 35 min, 0% phase B from 35.1 to 45 min. A flow rate of 0.4 mL/min operated.
A Thermo Fisher Scientific Orbitrap LC-MS/MS (Q Exactive, Waltham, MA, USA) was employed to characterize phenolic compounds. The spectrometer was provided with a HESI II (Thermo Fisher Scientific, Waltham, MA, USA). The ion source setting parameters were: spray voltage −3.0 kV, auxiliary gas (N 2 > 95%), sheath gas (N 2 > 95%), auxiliary gas heater temperature 305 • C, capillary temperature 200 • C, radiofrequency that captures and focuses the ions into a tight beam S-lens RF level 50. The MS detection was performed in full scan and targeted selected ion monitoring. Full scan acquisition parameters were scan rate 2 s −1 ; scan range 100-1500 m/z; mass resolving power 35,000 full width at half maximum (at m/z 200); automatic gain control target 1 × 10 5 ions; maximum injection time of 200 ms. The SIM (selected ion monitoring acquisition) parameters were: 35,000 full widths and half maximum (at m/z 200) (resolution power); 15 s (time window); 1.2 m/z (quadrupole isolation window).

Method Validation of the Phenolics Dosage
The construction of a calibration curve was achieved using three different concentrations of each calibration standard.
The linearity of the method was obtained from the regression coefficient of the calibration curve.
Limits of detection (LODs) = 3 × standard deviation angular coe f f icient Limits of quantification (LOQs) = 10 × standard deviation angular coe f f icient ) Intraday repeatability was performed by injecting each phenolic standard, three times, at seven different concentrations.

Total Polyphenol Content
Total phenol content was obtained by the Folin-Ciocalteu colorimetric method described previously by Gao et al. 2000 [29]. Extracts (0.1 mL) were added to H 2 O (2 mL) and Folin-Ciocalteu reagent (0.2 mL) and were incubated at room temperature (3 min). Sequentially, 1 mL of the sodium carbonate (20%) was added, and the mixture was left (1 h) at room temperature. The total polyphenols were determined in a spectrophotometer (Lambda 25, PerkinElmer, Waltham, MA, USA) (λ = 765 nm). The results were expressed as mg gallic acid equivalents (GAE)/kg of sample. All determinations were performed in triplicate (n = 3).

Statistical Analysis
"Statistica" software version 7.0 (StatSoft, Inc. Tulsa, OK, USA) was used to perform statistical analyses.

Results
A UHPLC-MS/MS method was employed to delineate the phenolic profile in the Extra virgin olive oil (EVOO) and olive pomace. The dosage method was validated according to AOAC instructions (AOAC 2012) [32]. Table 1 showed the parameters used to validate it.

The Phenolics Characterization
Seventeen phenolics, including two flavonoids, two phenolic alcohols, seven secoiridoids, and six phenolic acids, were identified and quantified. Table 2 reports the parameters used to identify phenolics in samples.

The Phenolics Dosage
Tables 3-5 report the dosage of each phenolic compound found in the samples. The Trichoderma biostimulation improved the apigenin concentration in the EVOO, and the olive pomace (Pom), but decreased it together with luteolin in VW. The Pom and the VW samples did not contain lignans (Table 3). The phenolic acid response to the biostimulation was like that seen for the other phenols; there was an increase in the concentration of each compound, but the increase was different from compound to compound (Table 4). Our results confirmed the Trichoderma's ability to increase the concentration of secoiridoids and their degradation products in EVOO, and established a similar activity in Pom, but not in VW (Tables 5  and 6).  Table 6. Variation % of the concentration of each phenolic under biostimulation.

Total Phenolic Concentration and Antioxidant Activity
The biostimulation had a positive effect on the antioxidant activity measured with both methods (ABTS and DPPH). The ABTS method evaluated the antioxidant activity of the Pom and VW more than the DPPH method, and the opposite occurred in the EVOO samples.

Discussion
Liquid and solid olive processing waste contain high amounts of organic materials that are not easily degradable. When these wastes are put into the environment, they create odor nuisance, an oily shine, enhance the oxygen demand, and are toxic to plant life. Therefore, the direct release of olive processing waste is forbidden, and some actions must be required before discarding into the environment. Some studies showed that olive processing waste might also be considered as an economic resource. Some practices are proposed to recycle and reuse them; using them as starting material to extract beneficial products for human health such as antioxidants is interesting. Olive pomace and olive vegetation water are sources of phenols. The industry requires phenolic compounds to produce functional foods, supplements, food additives, and the formulation of cosmetics and drugs [5,33,34]. Trichoderma species promote the production of phytochemicals, including phenolic compounds [23,26], whose production varies according to the strain used [35]. Previous studies have discussed that the Trichoderma can enhance phenols in EVOO and olive leaves [1,35]. In this work, we tested the ability of the Trichoderma to increase the concentration of phenolic compounds in the olive pomace and the olive vegetation water. Moreover, we determined the concentration of each compound, considering that the interest in phenolic compounds from industry depends on their chemical structure to which biological action is linked. Phenolic profile and dosage were investigated by an HPLC-Orbitrap method validated in terms of linearity, precision, and sensitivity, as recommended by the AOAC (2012) guidelines [36]. The linearity of the method was confirmed by the coefficient of regression (r 1) of the calibration curve. The sensitivity was verified by the inclusion of the concentration detected in the LODs and the LOQs range. The repeatability was confirmed by Relative Standard Deviation (RSD) values <6%. Phenolics identification was performed by comparing their mass spectra with those obtained by the standards analyses. The identification of the two hydroxybenzoic acid isomers was obtained through comparing the retention time and mass spectra with standards. The ligstroside identification, as it was not commercially available, was confirmed by comparing the chromatographic evidence and the spectroscopic data with those reported in the literature [37]. Biostimulation improved the total phenolic content in Pom and EVOO samples in accordance to our previous results [1]. On the other hand, biostimulation decreased the total phenolic concentration in VW. The main phenols found in the Pom and VW were the same as those in the EVOO, but their total concentrations, expressed as mmol Trolox/kg, were higher in the olive vegetation water control, followed by bioPom, Pom, bioVW, EVOO and bioEVOO. Among these, secoiridoids and their degradation products were the most concentrated compounds in the bioEVOO, the EVOO, and the VW samples. In contrast, the flavonoids were the most representative compounds in the bioPom, the Pom samples, and the bioVW. The lignans were found only in the bioEVOO and the EVOO. The resveratrol was in the bioVW and the VW samples. Therefore, the most variable phenolics were secologanoside, resveratrol, and lignans. Concerning secoiridoids fraction, secoiridoids biosynthesis in the plant occurs through two biosynthetic pathways: the shikimic pathway and the mevalonate pathway ( Figure 1). Biostimulation with Thricoderma M10 enhanced the production of the oleuropein and ligstroside. It preserved them from the degradation during the malaxation process, as shown by their higher concentration and the lower concentrations of their degradation products (oleuropein-aglycone di-aldehyde, ligstroside-aglycone mono-aldehyde, tyrosol, and hydroxytyrosol) in the bioEVOO vs. the EVOO. Moreover, the higher concentration of ligstroside and secologanoside in the bioEVOO respect of the EVOO sample indicated the biostimulation's ability to enhance the secoiridoid biosynthesis mainly through the mevalonate pathway ( Figure 2). Finally, the negative variation of the percentage content of oleuropein in the pomace and strongly positive increase of its precursor, the secologanoside, in the pomace were further confirmation (Table 6). Moreover, biocontrol agriculture enhanced the production of resveratrol, as shown by higher concentrations of the resveratrol and lower concentrations of its precursors (the cinnamic acid, and the p-coumaric acid) in the bioVW vs. VW sample (Figure 2). This datum is noteworthy since resveratrol has some nutraceutical properties, such as anti-inflammatory and anti-oxidative effects, and disturbs the start and progression of many illnesses such as some cancer types, and neurological and cardiovascular disorders through several mechanisms. In vitro and in vivo evidence confirmed the resveratrol's ability as a therapeutic agent [38]. The lignans were absent in the Pom and the VW samples, as they have high solubility in fats (logP 3.1) [39]. The improvement of total phenolics concentrations, determined using the Trichoderma in culture, is followed by an increase in the antioxidant activity in these samples. The values of the antioxidant activity, measured with the DPPH test, are overestimated in samples where the flavonoid concentration is high (Figure 3 and Table 3) [40]. It is clear that the use of biostimulants is useful to increase the concentration of phenolic compounds with antioxidant activity, not only in the EVOO, but also in the Pom, transforming the latter from an environmental problem into a source of bioactive molecules of nutraceutical, food, pharmaceutics, and cosmetic interest. Moreover, the Thricoderma improves the nutraceutical value of the bioEVOO, and decreases the losses of phenolic compounds in vegetation waters, favoring the transformation of phenolic alcohols into secoiridoids, lignans, and flavonoids which have higher properties for human health.

Conclusions
For the first time, this study delineates the effects of the Trichoderma used in olive tree cultivation on the antioxidant activity and the phenol production in olive pomace and olive vegetation water. Our results confirmed the Trichoderma's ability to improve the concentration of phenolics and antioxidant activity in EVOO, establishing the same ability in the olive pomace. Finally, they demonstrate that biostimulation principally determines, among the phenolic compounds, the biosynthesis of flavonoids and secoiridoids, two classes of phenolic compounds with well-known health properties, making olive pomace and olive vegetation water commercially appealing as a source of botanicals convenient for the food, cosmetic, and pharmaceutical industries. Finally, the Trichoderma action in the mevalonate pathway, producing phenols, was highlighted for the first time.

Conflicts of Interest:
The authors declare no conflicts of interest.