Obtaining Phenolic-Enriched Liquid Fractions and Compostable Pomace for Agriculture from Alperujo Using Standard Two-Phase Olive Oil Mill Equipment

Abstract

Olive oil extraction by two-phase systems generates a by-product called “alperujo” which presents several difficulties for its valorization. The present work evaluated an industrial approach, based on the application of thermal treatments to alperujo followed by solid/liquid separation using standard two-phase olive oil mill equipment. Treatments consisted of the thermo-malaxation of alperujo at 70 °C for 45 or 90 min, with or without acid addition, followed by solid/liquid separation in an industrial decanter. The solid was characterized concerning subsequent use for composting, while total and hydrophilic phenolics were analyzed in liquid for their recovery. Additionally, a laboratory-scale trial to compare phenolic purification by ethylic acetate extraction with chromatographic procedures was also included. The static respiration index showed that solid fractions presented higher susceptibility to biodegradation processes than raw alperujo. The phenolic content of treated liquid fractions was higher than in raw alperujo. Total phenolics were maximum at the longest exposure time without acid addition, while hydrophilic phenolics were highest at the shortest exposure time in acidified samples. The use of non-ionic resins seemed attractive for obtaining highly concentrated phenolic fractions. The proposed thermal treatments can be applied in olive oil industries, allowing in situ pomace valorization and the recovery of phenolic-enriched liquid fractions.

1. Introduction

Argentina is the main olive oil producer among South American countries, with over 250.000 t of olive fruits milled per year [1]. Olive oil extraction in Argentina is generally performed by continuous two-phase centrifugation systems. These systems generate a semisolid waste made of olive pulp, olive stone and vegetation water commonly called “alperujo” that represents 80 to 85% of raw fruit weight. Alperujo is composed of water (60–70%), organic matter (cellulose, hemicellulose, lignin, lipids, sugars and phenolic compounds) and a minor proportion of mineral ashes [2]. In Spain, the main olive oil-producing country worldwide, there are many industries that collect and use alperujo as raw material for solvent-mediated oil extraction and energy cogeneration. These industries, called pomace–oil extractors, represent a key stage regarding alperujo valorization and management. In Argentina, as in many other olive oil-producing countries, the scenery is completely different. The lack of pomace–oil extractors favors the implementation of in situ procedures for alperujo management. Among them, its direct application to soil as an organic amendment is the most relevant practice. It represents a simple option for disposing of olive mill by-products, but it still presents technical and environmental limitations and is not a way to take advantage of all alperujo constituents [3]. Composting appears to be an attractive alternative for the in situ valorization of this by-product. However, the chemical characteristics of alperujo such as high water content, high carbon/nitrogen ratio and the presence of biostatic compounds such as phenolics represent obstacles for aerobic biodegradation, considering this by-product as a recalcitrant raw material. In agreement, several reports recommend the addition of bulking agents to decrease the initial water content and dilute the relative content of phenolic compounds to initiate a composting process with alperujo [4,5,6,7]. Among the different phenolic compounds found in alperujo, 3,4-Dihydroxyphenylglycol (DHPG), Hydroxytyrosol (HT) and Tyrosol (Ty) are the most relevant and deeply studied. These hydrophilic compounds exhibit high antioxidant capacity [8] and a wide range of health benefits [9,10,11] as well as technologically promising properties that will help in the formulation of safer and healthier foods [12,13,14,15]. Different reports describe the extraction of hydrophilic phenolic compounds from olive by-products through multistage technologies. The methods usually consist of pretreatments to enhance the solubilization of water-soluble phenolics, followed by a second stage focused on the purification and recovery of individual phenolics of interest. Among other approaches, it is reported that hydrothermal, chemical or ultrasound-mediated pretreatments of alperujo allow the relaxation of the organic matter structure, induce the autohydrolysis of complex molecules and favor the solubilization of HT, Ty and DHPG [16,17,18,19,20,21]. These reactions are triggered by acetic and formic acids generated after the hydrolysis of the hemicellulosic acid groups. The lowering of the pH facilitates the hydrolysis of the sugars in the wall material and consequently the release of phenolics [16]. On the other hand, the treatment changes the lignocellulosic structure, allowing a better separation of the phases, which results in a liquid phase enriched in phenolics and sugars. Regarding pretreatment approaches, steam-based thermal treatments were the first systems that started to be used at the industrial level for recovering phenolic compounds [21]. However, the high pressure and temperature required for its implementation still represent an obstacle to its widespread adoption. In this sense, in recent years, low-temperature hydrothermal treatments (below 90 °C) have been proposed in order to find balance points between the efficiency of phenol recovery and the possible implementation of the technology in standard olive oil industries [17,18]. Concerning phenolic purification systems, the use of organic solvents, chromatographic procedures with ionic and non-ionic resins, supercritical fluids, and deep eutectic solvents have been evaluated to purify and concentrate the phenolic fraction of interest [22,23]. A very comprehensive comparison between phenolic compound extraction systems indicated that liquid–liquid extraction with organic solvents is easy to use, highly efficient and widely applicable; however, poor selectivity and the large amounts of organic solvents required represent obstacles for the subsequent use of resultant extracts [24]. Supercritical fluid extraction is fast and highly selective, but the investment and energy required represent an important disadvantage for scaling up the process. Other recent studies used green solvents or deep eutectic solvents [22], but concluded that at the moment, the costs of the components are very high, and that it is still necessary to find systems that allow the recovery of these solvents for reuse. Taking this into account, the use of chromatographic procedures with adsorption resins has the advantage of being efficient and versatile [8], and selection of the resin and elution conditions is critical to the overall outcome of the extraction process.

In summary, there are a number of reports that propose different technologies to recover phenolic compounds from alperujo. Evaluation of different pretreatments, comparison of extraction efficiency and analysis of the use of the resulting extracts as natural-based antioxidants are the main aspects investigated. However, the industrial adoption of the proposed technologies is very low and is limited to a few cases implemented in pilot plants and in pomace–oil extractors. Currently, phenolic compound recovery procedures are not widespread in the olive sector. The present work considers the recovery of phenolic compounds from alperujo as the key to allowing or facilitating the use of the remnant solid fraction and enabling the integral management of this by-product. Industrial hydrothermal treatments to obtain phenolic-reduced pomace as a raw material for the composting process and phenolic-enriched liquid fractions as resources for the recovery of hydrophilic phenolics were evaluated. Hydrothermal treatments were applied to raw alperujo using standard two-phase olive mill equipment as an approach for the handling and valorization of this by-product using inactive equipment during the off-season.

2. Materials and Methods

2.1. Origin and Characterization of Raw Material

Fresh alperujo (2400 kg) was obtained from a continuous two-phase centrifugation system (Oliomio 200 Eco, Toscana Enologica Mori, Tavarnelle Val di Pesa, Italy) placed in the olive oil extraction plant of INTA, San Juan, Argentina. Olive fruits (CV Arbequina) were milled at a maturity index (MI) of 3. MI was determined as reported in [25]. Alperujo was collected in 200 L PVC tanks and stored at room temperature (10–25 °C) for 15 days until the end of the olive oil extraction season.

2.2. Chemicals and Analytical Determinations

Raw alperujo and treatment-derived solid fractions were characterized according to the following determinations. Water content was measured gravimetrically at 70 °C [26], pH by potentiometric determination in 1:5 (w/v) water extract [26], and total organic matter and ashes by loss on ignition at 550 °C for 24 h [26].

Total phenolic content was determined using a modified version of the method described by [27] with Folin–Ciocalteu reagent and measured in Shimadzu 1240 UV–Visible spectrophotometer at 725 nm. Results were expressed as mg of caffeic acid per kg of initial sample. Hydrophilic individual phenolics (DHPG, HT and Ty) were measured by a high-performance liquid chromatography (HPLC Hewlett-Packard series 1100), using a Kinetex^® EVO 5 μm C18 (250 × 4.6 mm) Phenomenex^® column. The analysis was performed at room temperature, and the elution was performed at a flow rate of 1.0 mL/ min, with a mobile phase A of acetonitrile and B of ultrapure water, using the following gradient over a total run time of 55 min: 95% B initially, 75% B at 30 min, 50% B at 45 min, 0% B at 47 min, 75% A at 50 min, 95% A at 52 min until the run was completed. The chromatograms were analyzed by integration peaks at 280 nm, utilized with reference calibrations performed with external standards. The results were expressed in mg/L. For both methods, total phenolics and hydrophilic individual phenolics, samples of aqueous fractions were determined directly, while samples from alperujo and treatment-derived solid fractions were previously subjected to methanolic extraction (methanol/water 80:20 v/v using 2 mL per gram of wet sample).

Static respiration index (SRI) [28] was measured to assess the susceptibility of alperujo and treatment-derived solid fractions to biodegradation processes. This technique is a static respiration stability assessment method which is performed in mesophilic temperatures (37 °C) with sealed 500 mL flasks. An electrochemical dissolved O₂ electrode is placed in the headspace of the flask and records the O₂ air concentration drops within the flask. Oxygen uptake rate (OUR) is finally expressed in mg O₂/g VS/h and is calculated via the slope of the O₂ concentration drop. The SRI is the maximum averaged OUR calculated during a 24 h period (after the initial lag time).

Insoluble solids in liquid fractions were determined as reported in [18]. Briefly, 10 mL of sample was centrifuged (10,140 g-force, 5 min, 4 °C) in a Sorvall RC-5C refrigerated centrifuge (Du Pont Instruments, Newton, CT, USA). The solid fraction was left in the oven (50 °C until constant weight), the dry weight was determined gravimetrically, and the concentration of insoluble solids was expressed as mg/L of liquid fraction.

Total sugars were determined by the anthrone method [29]. Briefly, 0.2 mL of sample and 0.5 mL of 0.2% anthrone solution were stirred and heated to 100 °C in a thermostatic bath (Vicking, model D1402, Buenos Aires, Argentina) for 5 min. Subsequently, the absorbance was measured at 630 nm in microplate reader (Biotek 800TS, Winooski, VT, USA). The calibration curve was created with glucose aqueous solution in the range of 0.1 g/L and 1 g/L.

2.3. Industrial Pretreatments

Before starting the experimental procedures, the water content of raw alperujo was adjusted to 80% with the addition of water. Pretreatments consisted of heating water-adjusted alperujo at 70 °C for 45 min or 90 min with or without the addition of H₂SO₄ (0.25% v/v). Assay conditions were set based on results obtained by previous reports [16,17], and considering the temperature limitations given by the industrial equipment. Pretreatments were applied by triplicate in 200 kg individual trials using the continuous two-phase centrifugation system (Oliomio 200 Eco, Toscana Enologica Mori, Italy) previously utilized for olive oil extraction. For this purpose, the thermo-malaxer was covered with a ceramic fiber blanket (Luyangwool 1260, Luyang, China) to improve the thermic insulation, connected to a 25 L electrical thermo-tank. Under these conditions, alperujo reached 70 °C in 20 min. After treatment application, solid/liquid separation was performed by the two-phase decanter (RCF: 3500 g). The phases obtained were, on the one hand, the wet solid and, on the other hand, a mixture of oil and vegetation water. Solid fractions obtained were weighted and characterized for use in composting. Liquid fractions obtained were weighed and stored at room temperature (15 to 20 °C) in PVC tanks for 48 h to allow the separation of remnant fats. Finally, the contents of total phenolics, HT, Ty, DHPG and suspended solids were determined in liquid fractions taken by an outlet valve positioned in the middle of each tank.

2.4. Phenolics Purification Trial

Liquid fractions from pretreatment trials with the highest content of total phenolics and HT were selected for phenolic extraction. Three systems were evaluated: (A) adsorption of phenolics with non-ionic resin (Amberlite^® XAD 2, Sigma (Livonia, MI, USA), SKU 10357; n° CAS 9003-70-7) at a 1:3 v/v ratio and elution with 70% ethanol [8]; (B) adsorption of phenolics with ionic resin (WO02064537) at a 1:3 v/v ratio and elution with distilled water [16]; (C) liquid/liquid partition with ethylic acetate (Sigma Aldrich (Burlington, MA, USA) SKU 319902; n° CAS 141-78-6) 2:1 v/v [8].

Three replicates with 250 mL of initial sample were evaluated under each condition. Phenolic-enriched liquid fractions obtained by each system were concentrated in a rotary evaporator (70 °C, 150 rpm) until dried. The total solids obtained were weighed and reconstituted in 10 mL of ethanol/water (1:1 v/v). Reconstituted samples were characterized by the determination of total phenolics, tyrosol, hydroxytyrosol, DHPG and total sugars.

2.5. Experimental Design and Statistical Analysis

Industrial pretreatments were performed under a complete bifactorial design, while phenolic purification trials were performed under a complete factorial design. Analysis of variance (ANOVA) was performed using Infostat Professional Software 2.0 version (UNC). For comparison of means, LSD Fisher (p ≤ 0.05) was employed.

3. Results and Discussion

3.1. Raw Material Characterization

As shown in

Table 1. Chemical composition of alperujo (cv Arbequina, MI: 3). Average values ± standard deviation of three independent determinations are shown.

Analyzed Parameter	n	Mean Value ± SD
pH	3	5.5 ± 0.05
Water content (%)	3	69.0 ± 0.4
Organic matter (%)	3	28.5 ± 2.0
Ash content (%)	3	2.5 ± 0.3
Total soluble phenolics (mg/kg)	3	3121.0 ± 142.2
HT (mg/kg)	3	127.2 ± 54.4
Ty (mg/kg)	3	44.7 ± 9.3
DHPG (mg/kg)	3	5.4 ± 1.2
SRI (mg O2/g VS/h)	3	0.31 ± 0.02

, the chemical composition of alperujo applied as raw material was similar to others in previous reports [2,30]. It was characterized by high water content and slightly acidic pH. Regardless of the water content, it was mainly composed of organic matter and a minor proportion of mineral ashes. Among the organic fraction, this work focused on the content of total phenolics and hydrophilic phenolics as compounds of interest. The total phenolic content (3121 mg/kg) was close to that observed in other reports on alperujo obtained from olives of the same region and cultivar [17]. Regarding hydrophilic phenolics, the summatory of HT, Ty and DHPG in the present work reached less than 6% of the total soluble phenolics. As stated, the amount and profile of olive phenolic compounds are highly variable and dependent mainly on cultivar and maturity stage as well as agronomic and climatic conditions [31,32,33]. During olive ripening, the level of complex phenolics like oleuropein decreases, while the concentration of hydrophilic phenolics like hydroxytyrosol or their glucoside increases [34]. In this sense, the combination of cultivar and the early-stage maturity index of olives in this work could explain the low proportion of hydrophilic phenolics observed in raw alperujo. As observed, raw alperujo presented a static respiration index (SRI) of 0.31 mg O₂/g VS/h. Respiration is directly related to the metabolic activity of a microbial population in a given sample; hence, the SRI is extensively used in composting procedures to analyze the stability/instability of raw materials and compost [35]. Usually, samples with SRI values under 0.5 mg O₂/g VS/h are considered stable, with a low tendency to initiate the biodegradation process itself [28]. In this sense, the SRI value observed in raw alperujo suggests low susceptibility to biodegradation that could be related to the antimicrobial properties given by phenolic compounds [36]. This observation agrees with several reports that recommend the addition of bulking agents to initiate a composting process with alperujo and suggest that high water content, low porosity, and the presence of biostatic compounds such as phenolics are the main factors responsible for this recalcitrant behavior [4,5,6,7].

3.2. Applied Pretreatments and Analysis of Obtained Fractions

Table 2. Main parameters of solid fraction obtained after industrial treatment application. Mean values ± standard deviation of three independent assays are shown.

Treatment	Water Content (%)	Organic Matter (%)	Ash (%)	pH	Total Phenolics (mg/kg)	SRI (mg O2/g VS/h)
70 °C, 45 min	64.4 ± 1.1 c	34.2 ± 0.6 b	1.4 ± 0.3 b	5.50 ± 0.15 b	885 ± 63 a	1.17 ± 0.12 b
70 °C, 90 min	60.7 ± 0.5 b	37.6 ± 0.4 b	1.7 ± 0.4 b	5.35 ± 0.14 b	889 ± 71 a	1.17 ± 0.20 b
70 °C, 45 min, 0.25% H2SO4 w/w	62.0 ± 0.7 b	36.4 ± 0.2 b	1.6 ± 0.2 b	4.75 ± 0.18 a	1021 ± 104 b	0.81 ± 0.09 a
70 °C, 90 min, 0.25% H2SO4 w/w	56.7 ± 1.1 a	42.3 ± 0.1 a	1.0 ± 0.2 a	4.80 ± 0.10 a	1073 ± 117 b	0.79 ± 0.30 a

Different letters indicate significant differences among treatments. p < 0.05.

details the conditions of industrial pretreatments applied, and the main parameters of different solid fractions obtained by each treatment. As shown, the water content of solid fractions recovered decreased by the effect of longer exposure time and acid addition. This result agrees with previous reports suggesting that more intensive conditions favor S/L separation [17,18]. A similar pattern was observed in the ash content, suggesting that acid addition and temperature promoted the solubilization of the mineral fraction. It can also be concluded that for the parameters studied, it is not necessary to reach 90 min of treatment. Regarding total phenolics, the values determined in solid fractions were significantly lower (3 to 4 times) than those observed in raw alperujo (Table 1), indicating that all treatments were appropriate for removing phenolics from the solid fraction. The concentration of phenolics in the solid is higher when acid is used because the acid probably improves the accessibility of organic matter from the wall material such as hemicellulose and cellulose, although these phenolics could affect the subsequent application of a bioprocess for their utilization.

Respirometry assays showed that solid fractions obtained by all treatments presented higher SRI values than raw alperujo. As mentioned before, respiration is directly related to the metabolic activity of the microbial population. Samples with SRI values over the stability threshold (0.5 mg O₂/g VS/h) are considered unstable, with suitable properties to initiate the biodegradation process itself [28]. Interestingly, the SRI values of treated solid fractions were not only higher than the SRI of raw alperujo but also considerably higher than the stability threshold. Among the results, solid fractions obtained by non-acidified treatments presented higher SRI values than those obtained by acidified ones. This observation could be explained mainly by the higher phenolic content of acidified treatments as a condition that delays the microbial respiration rate. In agreement, as shown in Figure 1, a strong correlation between SRI values and total phenolics in analyzed samples was observed. Spots from non-acidified treatments (white-filled circles) presented the highest SRI values and the lowest phenolic content. On the opposite side of the correlation curve, spots from raw alperujo samples (black-filled circles) presented the lowest SRI values (under the 0.5 threshold) and the highest phenolic content. Remarkably, spots from acidified samples (white-filled triangles) were under the correlation curve, indicating that the SRI values of these samples were not totally explained by the phenolic content, suggesting that the lower pH could also be affecting metabolic respiration in samples from acidified treatments. Certainly, the higher susceptibility to biodegradation of pomace-derived treatments could make it beneficial for use in composting processes but would not replace the bulking agent addition required to increase porosity and balance the C/N ratio [7]. This result is of great interest for integrated industries, where the olive oil extraction plant is located in the same place as the olive groves. The application of this thermal treatment in olive oil industries would allow the in situ use of solid fractions that represent 60% w/w of the raw alperujo.

Table 3. Main parameters of liquid fraction obtained after industrial treatment application. Mean values ± standard deviations of three independent assays are shown.

Treatment	Liquid Fraction
	Percent of Raw (% w/w)	Insoluble Solids (mg/L)	Phenolics (mg/L)
			Total	HT	Ty	DHPG
70 °C, 45 min	36 ± 3 a	18.0 ± 0.7 b	4230 ± 47 b	475 ± 30 a	137 ± 6 c	43 ± 2 c
70 °C, 90 min	39 ± 2 a	23.8 ± 0.5 c	4334 ± 112 b	615 ± 19 b	127 ± 10 c	37 ± 1 c
70 °C, 45 min, 0.25% H2SO4 w/w	38 ± 1 a	11.7 ± 0.2 a	3472 ± 24 a	849 ± 33 c	61 ± 8 b	27 ± 2 b
70 °C, 90 min, 0.25% H2SO4 w/w	43 ± 3 b	13.0 ± 0.4 a	3551 ± 136 a	519 ± 18 a	47 ± 8 a	16 ± 3 a

Different letters indicate significant differences among treatments. p < 0.05.

details conditions of industrial pretreatments applied, and the main parameters of different liquid fractions obtained by each treatment. As shown, the percentage of liquid fractions recovered presented slight variations between treatments, being increased by the effect of longer exposure time and acid addition. This result agrees with previous reports, suggesting that more intensive conditions promote higher rupture of cellular structures and favor S/L separation [17,18]. A different pattern was observed when the total insoluble solids were evaluated. This parameter increased with exposure time in non-acidified treatments and presented significantly lower values in both samples compared to acidified ones. This result could indicate that longer exposure time promotes the rupture of cellular structures, thus increasing the suspended solids; on the other hand, the combination of temperature and acid addition probably favored the hydrolysis and subsequent solubilization of suspended solids. This fact was previously observed in other reports at a laboratory scale and in more intensive conditions [16,37].

Regarding phenolic compounds, total soluble phenolics in all liquid fractions recovered were 10% to 40% higher than those observed in raw alperujo. This result agrees with published data [17] and supports the importance of hydrothermal treatments to recover phenolic-enriched liquid fractions. Among treatments, total phenolic content was higher in liquid fractions obtained by non-acidified treatments than by acidified ones, without presenting significant differences within exposure time. This result suggests that acid addition could favor hydrolysis and partial reduction of these compounds. The content of total soluble phenolics observed in both non-acidified treatments was slightly higher than those previously reported under similar conditions (3214 mg/L at 65 °C, 60 min) [18] and could be explained by differences in the cultivar and maturity index of the raw material.

Interestingly, individual hydrophilic phenolics like HT, Ty and DHPG presented dissimilar patterns among them. HT values were maximum at the shortest exposure time in acidified treatments, while Ty and DHPG presented maximum levels in non-acidified treatments without significant differences among exposure times. These results suggest different susceptibilities to time and acid addition and agree with previous reports [17,37]. Remarkably, the summatory of HT, Ty and DHPG reached 16% to 27% of total soluble phenolics in liquid fractions obtained by all applied treatments. These percentages are considerably higher than the 6% observed in raw alperujo (Table 1), agree with the hypothesis indicating that applied treatments induced hydrolysis of complex polyphenolics enhancing the amount of simpler hydrophilic ones, and support the importance of pretreatment application.

Table 4. Theoretical amounts of total phenolics, HT, Ty and DHPG in liquid fractions obtained from 200 kg of raw alperujo sample by each treatment. Mean values ± standard deviations are shown.

Treatment	Phenolics in Liquid Fractions (g)
	Total	HT	Ty	DHPG
70 °C, 45 min	304 ± 15.3 b	34.8 ± 3.5 a	9.5 ± 0.4 c	3.0 ± 0.3 c
70 °C, 90 min	338 ± 8.7 c	47.5 ± 0.6 c	9.5 ± 0.6 c	3.0 ± 0.1 c
70 °C, 45 min, 0.25% H2SO4 w/w	264 ± 6.7 a	66.1 ± 1.3 d	4.2 ± 0.5 a	2.2 ± 0.1 b
70 °C, 90 min, 0.25% H2SO4 w/w	307 ± 4.2 b	43.7 ± 0.5 b	4.0 ± 0.5 b	1.6 ± 0.2 a

Different letters indicate significant differences among treatments. p < 0.05.

indicates the theoretical amounts of phenolics in liquid fractions, considering the initial weight of raw alperujo (200 kg), percent of liquid fraction obtained and phenolic concentration in each treatment. As shown, the amounts of total soluble phenolics as well as Ty and DHPG were maximum at a longer exposure time without acid addition (70 °C, 90 min). On the other hand, the amount of HT presented the highest values in acidified treatments at a shorter exposure time (70 °C, 45 min, 0.25% H₂SO₄ w/w).

This estimation made with the analyzed data shows the amount of each of the main phenolics that could be extracted from the liquid fraction, although current industry systems allow about 50% of the available phenolics to be obtained. This source can be considered a good source of these bioactive compounds.

3.3. Phenolics Purification Trial

Based on the results shown in Table 4, liquid fractions derived from hydrothermal treatments performed at 70 °C for 90 min without acid addition and at 70 °C for 45 min with acid addition were selected for subsequent purification assays.

Table 5. Total solids, total sugars and ash content of dry samples obtained by three different purification systems (non-ionic resins, ionic resins and ethyl acetate partition). Mean values ± standard deviations of three independent assays are shown.

Treatment-Derived Liquid Fraction	Liquid Fraction (mL)	Purification System	Total Solids (g)	Total Sugars (mg/g)	Ash Content (mg/g)
70 °C, 90 min	250	Non-ionic resin	3.05 ± 0.12 b	122.8 ± 11.6 c	1.5 ± 0.1 b
	250	Ionic resin	0.91 ± 0.07 a	40.2 ± 3.8 b	1.5 ± 0.3 b
	250	Ethyl acetate	0.83 ± 0.05 a	29.8 ± 1.4 a	0.3 ± 0.1 a
70 °C, 45 min,0.25% H2SO4 w/w	250	Non-ionic resin	2.49 ± 0.17 b	121.3 ± 13.1 c	2.5 ± 0.3 c
	250	Ionic resin	0.55 ±0.09 a	23.3 ± 4.4 a	1.7 ± 0.3 b
	250	Ethyl acetate	0.78 ±0.15 a	14.0 ± 1.9 a	0.6 ± 0.2 a

Different letters indicate significant differences among treatments. p < 0.05.

shows the chemical parameters of dry samples obtained by the three different purification systems performed with 250 mL of the liquid fractions. As observed, total solids and total sugars were significantly higher in samples obtained by non-ionic resins than in both other purification systems. Noticeably, total solids were maximum when liquid fractions derived from non-acidified treatments were processed. Regarding ash content, samples obtained by ionic and non-ionic resins presented higher values than samples obtained by ethyl acetate, indicating that minerals in liquid fractions were retained in these resins and posteriorly eluted. This observation was more evident in liquid fractions derived from acidified treatments, probably due to higher levels of mineral ashes in acidified liquid fractions.

The analysis of phenolic compounds in ethanol-reconstituted fractions obtained by different purification systems indicated that total phenolics as well as HT, Ty and DHPG were highest when non-ionic resins were applied as the purification system (

Table 6. Total phenolics (Total), hydroxytyrosol (HT), tyrosol (Ty), dihydroxyphenyl glycol (DHPG) and HP/Total ratio in ethanol-reconstituted fractions obtained by three different purification systems.

Treatment-Derived Liquid Fraction	Purification System		Phenolics in Ethanol-Reconstituted Fractions (mg/L)
		Total	HT	Ty	DHPG	HP/Total
70 °C, 90 min	Non-ionic resin	45,780 ± 938 d	13,558 ± 677 e	1696 ± 84 d	662 ± 33 e	0.35
	Ionic resin	19,047 ± 276 b	4763 ± 238 b	856 ± 42 b	90 ± 5 b	0.30
	Ethyl acetate	16,828 ± 712 b	6968 ± 348 d	1020 ± 51 c	234 ± 22 c	0.49
70 °C, 45 min, 0.25% H2SO4 w/w	Non-ionic resin	38,844 ± 546 c	7287 ± 364 d	1100 ± 55 c	394 ± 22 d	0.22
	Ionic resin	9773 ± 307 a	1714 ± 86 a	446 ± 22 a	41 ± 2 a	0.23
	Ethyl acetate	14,232 ± 644 b	5941 ± 297 c	1001 ± 53 c	208 ± 9 c	0.49

HP/Total: (HT + Ty + DHPG)/Total phenolics. Different letters indicate significant differences among treatments. p < 0.05.

). Remarkably, all parameters were maximum when liquid fractions derived from non-acidified treatments were processed, reaching values of HT up to 100-fold higher than those observed in raw alperujo.

Interestingly, when non-acidified liquid fractions were processed, the ratio of hydrophilic phenolics/total phenolics (HP/Total) obtained by the three purification systems was substantially higher than that observed in initial liquid fractions (0.18). These results suggest that the three systems allowed the selective purification and concentration of these compounds. As shown in Table 6, the highest HP/Total ratios were obtained by ethyl acetate, followed by non-ionic resins and ionic resins. On the other hand, when acidified liquid fractions were processed, only the HP/Total ratio obtained by ethyl acetate was higher than that observed in the initial liquid fraction (0.27), suggesting that the lower pH of this fraction could be altering the adsorption/desorption properties of these resins. This observation attains interest since it is reported that acid addition can be a simple practice to increase the HT content and improve the storage conditions of alperujo liquid fractions [37].

The phenolic concentration and profile in reconstituted samples of non-ionic resins presented values comparable to the results obtained by other authors using similar resins [38,39]. On the other hand, the HT concentration in reconstituted samples of ionic resins was significantly lower than reported previously [16]. Other reports evaluated the combination of conventional purification systems (liquid–liquid extraction with ethyl acetate), followed by purification with non-ionic resins, obtaining better results than the use of each system separately and higher than those observed in the present study [8]; however, the use of organic solvents during the process limits the subsequent use of the obtained natural antioxidant. Regarding non-conventional methods, ultrasound-, microwave-, supercritical fluid- and deep eutectic solvent-assisted phenolic extraction have shown good results at the laboratory scale; however, as mentioned before, there are technological difficulties that prevent their industrial scaling up [23,24].

Considering the obtained results, the use of non-ionic resins to obtain highly concentrated phenolic fractions seems attractive. By this system, the level of total phenolics, as well as hydrophilic phenolics of interest, was maximum. Besides the phenolic content, the use of ethanol solution to desorb and elute phenolics from non-ionic resins presented several technological advantages. Compared to the system based on ethyl acetate partition, it does not require an organic solvent, reducing the risk associated with organic solvent handling and allowing the obtainment of natural products. Additionally, compared to the systems with ionic resins and eluted with distilled water, the energy required for concentration is significantly lower and the ethanol is recovered and available for further cycles. Furthermore, at an industrial scale, the system can be operated with bioethanol that presents low price and high availability, and there is modular chromatographic equipment on the market that facilitates the process scale-up, and a wide range of commercial non-ionic resins that are commonly used in industrial processes. On the other hand, there are other characteristics that must be studied in depth towards the obtainment of phenolic-based natural antioxidants. Specifically, the considerably higher value of total solids observed in samples obtained by non-ionic resins (Table 5) could be an obstacle to increasing dry-based phenolic concentration. Moreover, the total sugar levels observed in dry samples derived from non-ionic resins (Table 5) could impair or interfere with phenolic compounds, altering their scavenging activity and limiting their use as natural antioxidants [40]. In this sense, further analysis of antioxidant activity is required to elucidate and advance in the development of phenolic-based natural antioxidants. However, it should also be considered that a large part of these sugars can be bound to phenolics, so although their antioxidant capacity is slightly diminished, it has been shown to improve their bioavailability, while at the same time allowing the production of oligosaccharides or soluble fiber with antioxidant and anti-inflammatory activity [41].

4. Conclusions

Taken together, the results obtained in this article show that by means of standard two-phase olive oil mill equipment, it is possible to apply hydrothermal treatments to alperujo and obtain two main useful fractions: phenolic-reduced solid fractions, with increased susceptibility to aerobic bioprocesses such as composting, and phenolic-enriched liquid fractions that can be potentially used as raw materials for phenolic-based natural antioxidant purification. Regarding the analysis of phenolic purification systems, the use of non-ionic resins to obtain highly concentrated phenolic fractions seemed attractive and presented several technological advantages. Certainly, further analysis including scaling up and a deep analysis of antioxidant activity is required to advance the development of phenolic-based natural antioxidants from alperujo. The use of a two-phase olive oil mill system, which is normally used for the extraction of virgin olive oil, out of season and in countries where olive pomace is not centralized in the pomace oil extraction industry, could be a good tool to improve the use of this by-product. Applying this thermal treatment in olive oil industries would allow the in situ utilization of solid fractions (that represent 60% w/w of raw alperujo) and obtain phenolic-enriched liquid fractions that could be derived from purification processes.