Application of Thermo-Malaxation Followed by Three-Phase Centrifugation to Enable the Biorefinery of Alperujo, the Main By-Product of Olive Oil.

The pomace olive oil sector needs to improve its use of the main olive oil by-product, called alperujo, which is currently used mainly for combustion after extraction of pomace oil, with all the problems this process entails due to the high degree of humidity, organic load and phytotoxic substances. In this work, a solution at an industrial level that uses thermo-malaxation at a temperature close to 65 °C for one or two hours followed by centrifugation in three phases is proposed. In this way, over 40% of the pomace oil that is rich in minor compounds, a solid with a lower degree of humidity (55%), and a liquid aqueous fraction that is rich in bioactive compounds such as phenolics and sugars are obtained. This aqueous fraction can be treated through subsequent storage stages to increase its content of the main phenolic, hydroxytyrosol, to up to 1.77 g/L, decreasing its percentage of insoluble solids by up to 1.9%, making it possible to obtain extracts that are rich in hydroxytyrosol using systems that are commonly in place at the industrial level. The aqueous fraction, without phenolics, could be used for energy production. A solid with a slightly higher fat content than the initial alperujo remains, thus the rest of the oil content can be extracted from it using solvent, making it, once defatted, suitable for application in subsequent bioprocesses.

An Acid-Free Alternative to Pectin Production from the Cell Walls of Olive Oil Waste and Different Fruits Using Choline Chloride.

The pectin from the cell walls of olive waste (alperujo) and apple, orange and strawberry fruits was extracted using choline chloride (ChCl) and the yield and chemical and structural compositions were compared to pectin extracted using citric acid (CA) and ammonium oxalate/oxalic acid (AOOA). According to the results, the alperujo pectin extracted using ChCl from alcohol-insoluble residue (AIR) showed a higher yield (2.20-2.88% on the basis of dry weight of AIR) than using CA (0.65-1.22%) but lower than using AOOA (3.92-5.42%). For fruit pectin, the highest yield was obtained using CA (8.81-16%), followed by AOOA (5.4-6.63%), although for apple pectin, ChCl gave a similar yield (5.36%) to AOOA. The uronic acid contents in all ChCl pectins (45.9-70.6% dry basis AIR) were higher or similar to that of the other extracting agents (30.6-65.2%), although a lower level of neutral sugar side chains was detected, with a lower degree of branching and degree of methylation. The NMR and FT-IR spectroscopy of the pectin isolated using ChCl confirmed its slightly different structural composition with respect to CA and AOOA pectin. Therefore, depending on the source material and functionality, pectin isolated using ChCl could be an acid-free alternative to pectin production.

Valorization of strawberry extrudate waste: Recovery of phenolic compounds by direct-hydrothermal treatment and subsequent methane production by mesophilic semi-continuous anaerobic digestion.

Strawberry extrudate (SE) is an underused by-product from strawberry industry. Recovery of the phenolic compounds present in SE would represent a very interesting valorisation option. Two main challenges need to be solved, firstly, the solubilisation and recovery of the phenolic compounds contained in SE, and, after that, the stabilisation of the resulted de-phenolized SE. The present research evaluates the potential of a biorefinery process combining a hydrothermal pre-treatment, followed by a phenolic extraction process and, finally, the anaerobic digestion of the remaining SE for producing energy that will contribute to compensate the energy requirements of the whole system. Following the hydrothermal pre-treatment at 170 °C for 60 min, an extraction of 0.6 ± 0.1 g of gallic acid per kilogram of SE was achieved using an adsorbent resin, representing a recovery rate of 64 %. Long-term semi-continuous anaerobic digestion of de-phenolized SE was evaluated at different organic loading rates to evaluate the stability of the process. The anaerobic digestion of pre-treated SE achieved a stable methane production value of 243 ± 34 mL CH4·g volatile solids-1·d-1 at an organic loading rate (ORL) of 1.25 g volatile solids·L-1·d-1. During the operation at this ORL, the control parameters including pH, alkalinity, soluble chemical organic demand (sCOD), and volatile fatty acid (VFA) remained stable and consistently constant. Specifically, the VFA in the reactor during this stable period achieved a value of 102 ± 128 mg O2/L. Also, an economic balance showed that the minimal price of the generated phenolic extract for having benefited from the proposed biorefinery system was 0.812 €·(g of gallic acid equivalents)-1, a price within the range of phenolic compounds used in the food industry.

Application of a Cold-Pressing Treatment to Improve Virgin Olive Oil Production and the Antioxidant Phenolic Profile of Its by-Products.

The olive oil sector is continuously evolving in order to improve the quality of olive oil and its by-products. In fact, the trend is to use increasingly greener olives to improve quality by decreasing the extraction yield, thus obtaining a higher content of antioxidant phenolics. The application of a cold-pressing system to the olive before the extraction of oil was tested with three varieties: picual at three different stages of maturity and arbequina and hojiblanca at early stages of maturity. The Abencor system was used for the extraction of virgin olive oil and its by-products. For the quantification of phenols and total sugars for all phases, organic solvent extractions and colorimetric measurements and high-performance liquid chromatography (HPLC) with a UV detector were used. The results show that the new treatment significantly improved the amount of oil extracted by between 1 and 2% and even increased its concentration of total phenols by up to 33%. Regarding the by-products, the concentrations of the main phenols, such as hydroxytyrosol, increased by almost 50%, as did the glycoside. The treatment also facilitated the separation of phases in by-products and improved the phenolic profile, although not in terms of total phenols, but individual phenols with higher antioxidant activity were obtained.

Extraction of polyphenols associated with pectin from olive waste (alperujo) with choline chloride.

The main by-product from olive oil extraction (alperujo) was extracted with hot water, citric acid, natural deep eutectic solvent (choline chloride: citric acid), and only choline chloride. The purified extracts were composed of macromolecular complexes constituting polyphenols associated with pectin. The extracts were structurally characterized by FT-IR and solid-NMR spectroscopy and an in vitro test revealed distinct antioxidant and antiproliferative activity, depending on the extracting agents. The choline chloride-extracted complex contained the highest amount of polyphenols among the examined agents, which exhibited a strong antioxidant activity and significant antiproliferative capacity. However, the complex extracted by hot water showed the highest antiproliferative capacity in vitro against the colon carcinoma Caco-2 cell line. In this finding, choline chloride could be used as a novel, green and promising alternative to the conventional extracting agent for the production of complexes that combine the antioxidant activity of phenolic compounds and the physiological effects of pectic polysaccharides.

Evolution of Hydroxytyrosol, Hydroxytyrosol 4-ß-d-Glucoside, 3,4-Dihydroxyphenylglycol and Tyrosol in Olive Oil Solid Waste or "Alperujo".

The main by-product generated from the olive oil two-phase extraction system, or alperujo, is undoubtedly a rich source of bioactive components, among which phenolics are one of the most important. The evolution of four of its main phenolics: hydroxytyrosol (HT), hydroxytyrosol 4-ß-d-glucoside (Glu-HT), 3,4-dihydroxyphenylglycol (DHPG) and tyrosol (Ty) was studied over two seasons and in ten oil mills under similar climatological and agronomic conditions, for the first time using organic extraction and high-performance liquid chromatography (HPLC-DAD) determination. The results show that HT (200-1600 mg/kg of fresh alperujo) and Ty (10-570 mg/kg) increase, while DHPG (10-370 mg/kg) decreases only in the last month of the season and Glu-HT (1400-0 mg/kg) decreases drastically from the beginning. This evolution is similar between different seasons, with a high correlation between Glu-HT, HT, and Ty. On the other hand, it has been verified that a mixture of alperujos from all the oil mills, which is what the pomace extractor receives, is a viable source of a liquid fraction which is rich in the phenolics studied through organic extractions and especially after the application of a thermal treatment, obtaining values of 4.2 g/L of HT, 0.36 g/L of DHPG, and 0.49 g/L of Ty in the final concentrated liquid fraction.

Date Seed: Rich Source of Antioxidant Phenolics Obtained by Hydrothermal Treatments.

The growing interest in natural compounds is helping to improve the management of agro-industrial by-products such as the date seed as sources of such compounds. In this work, the application of a hydrothermal treatment at 160 and 180 °C for 60 min was studied to achieve the solubilization of its phenolic components and sugars in order to obtain biologically active extracts. The percentage of phenols and total sugars in the final extracts were very similar, at 45 and 25% for the 160 and 180 °C treatments, respectively. The treatment at a higher temperature allowed greater solubilization of other components. The antioxidant activity was measured as free-radical scavenging capacity. For the DPPH• method, expressed as EC50, the results were 0.34 and 0.37 mg/L, the TEAC values for the ABTS• method were 6.61 and 3.28 mg/g dried extract, and the values obtained by the ORAC method were 12.82 and 9.91 mmol Trolox/g dried extract, for 160 and 180 °C, respectively. All these values are higher than those of other plant extracts and extracts obtained using the whole date. Therefore, the date seed is a very important source of phenols, and through thermal and chromatographic processes, it is possible to obtain extracts with high antioxidant activity.

Formation of a bioactive cyclopentenone and its adducts with amino acids in sterilized-fruits and - vegetables baby foods.

The formation of the molecule 4,5-dihydroxy-2-cyclopenten-1-one (DHCP) from the thermal treatment of pectin-containing foods was investigated in small-scale laboratory preparation of sterilized vegetable puree (carrot, zucchini and tomato) and fruit puree (peach and mixture of pear and apple) and in commercial baby foods. DHCP attracts attention due to its cytotoxicity as well as potential antiviral and anti-inflammatory effects. However, its effects and the difficulty of its identification in food are mediated in part by the formation of Michael adducts of DHCP with amino acids. The results revealed that DHCP reacted efficiently with cysteine and glutathione, and to a lesser extent with histidine. Mass spectrometry analysis confirmed the formation of adducts of DHCP with amino acids in a model system, being in a real food system difficult to investigate. However, these formed adducts are of potential interest, although it is not known whether they are safe, bioactive or reversible.

Effect of the Olive Oil Extraction Process on the Formation of Complex Pectin-Polyphenols and Their Antioxidant and Antiproliferative Activities.

The aim of this research was to investigate the interaction of phenols and pectic polysaccharides during the olive oil extraction process. For this, pectin was extracted from fresh olive fruits and compared to the pectin isolated from the paste resulting from the extraction of the olive oil after milling with malaxation at 30 °C/30 min and subsequent centrifugation of the olive paste from the same lot of olive fruits in a system called ABENCOR (AB). The results indicate that these interactions were enhanced during the olive oil extraction process. In addition, the resulting AB extracts exhibited high antioxidant activity (ORAC) and strong antiproliferative activity in vitro against colon carcinoma Caco-2 cell lines compared to olive fruit extracts. The polyphenols associated mainly with the acidic pectin substance, with a higher content in AB extracts, seem to be responsible for these activities, and appear to maintain their activities in part after complexation. However, even in olive fruit extracts with smaller amounts of phenols in their compositions, pectic polysaccharides may also be involved in antioxidant and antiproliferative activities.

Antioxidant Capacity and Phenolic and Sugar Profiles of Date Fruits Extracts from Six Different Algerian Cultivars as Influenced by Ripening Stages and Extraction Systems.

The study investigated the phenols, sugar and the antioxidant capacities of date fruit extracts obtained by organic solvents and by hydrothermal treatment from six different Algerian cultivars at two ripening stages for the first time. The analyzed cultivars exhibited potent antioxidant properties (ferric reducing antioxidant power (FRAP), 1,1-Diphenyl-2-picrylhydrazyl (DPPH) and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS) scavenging capacities) and different phenols regardless of the solvents and the maturity stages. About 18 phenols were identified and quantified, mainly in the hydrothermal extracts. The earlier stages were characterized by high amounts of o-coumaric acid, cinnamic acid and luteolin, with a noticeable absence of quercetin. The tamr stage presented the highest sugar content (78.15-86.85 mg/100 mg dry weight (DW)) with an abundance of glucose. Galactose was present only in some cultivars from the kimri stage (tamjouhert). Uronic acids were mostly detected at the tamr stage (4.02-8.82 mg gallic acid equivalent/100 mg dried weight). The obtained results highlight the potential of using date fruit extracts as natural antioxidants, especially at industrial scales that tend use hydrothermal extraction.

Anti-Inflammatory and Antioxidant Activity of Hydroxytyrosol and 3,4-Dihydroxyphenyglycol Purified from Table Olive Effluents.

New liquid effluents based on the use of acetic acid in the table olive industry make it easier to extract bioactive compounds to be used for food, cosmetic, and pharmaceutical purposes. The use of water acidified with acetic acid or in brine with or without acetic acid for storing the table olive enhances the extraction of two more active phenolic compounds: hydroxytyrosol (HT) and 3,4-dihydroxyphenylglycol (DHPG). This work has two aims: (1) measure the solubilization of phenolics controlled for two years using more than thirty olive varieties with different ripeness index as a potential source of HT and DHPG, and (2) evaluate the anti-inflammatory activity of the purified phenolics. The effluents with a higher concentration of phenolics were used for the extraction of HT and DHPG in order to evaluate its antioxidant and anti-inflammatory activity in vitro by the determination of pro-inflammatory cytokines such as Human Tumor Necrosis Factor-α (TNF), Interleukin-6 (IL-6), and Interleukin-1ß (Il-1ß). The anti-inflammatory activity of these phenolic extracts was demonstrated by studying the expression of cytokines by qPCR and the levels of these proteins by enzyme-linked immunosorbent assay (ELISA).

Mesophilic Semi-Continuous Anaerobic Digestion of Strawberry Extrudate Pretreated with Steam Explosion.

The production of strawberry concentrate produces a side stream after extrusion that is commonly landfilled. This strawberry extrudate (SE), of lignocellulosic character, contains valuable bioactive compounds such as sugars and phenols. Thermal treatments, such as steam explosion, are currently used for the valorisation of agricultural lignocellulosic wastes due to their ability to impact the structure of the lignocellulose and hemicellulose present in these wastes, favouring the disruption of fibrous material. Steam explosion has already been shown as a promising technology for phenol recovery from SE. Biogas is an additional valuable resource that might be produced from thermally pretreated and de-phenolised SE. This study assessed the influence of a steam-explosion pretreatment and the subsequent recovery of phenolic compounds from the long-term operation of a semi-continuous anaerobic digester of pretreated SE. The anaerobic digestion of SE steam exploded at 220 °C for 5 min and de-phenolised was stable at an OLR of 0.5 g of volatile solids (VS)/(L·d), which permitted a specific production rate of 135 ± 11 mL of CH4/(g of VS·d). The system was not able to operate at an OLR of 1 g of VS/(L·d), which resulted in a failure of the process. Despite the inhibition threshold of phenolic compounds not being achieved, the inhibition of the anaerobic digestion process at an OLR of 1 g of VS/(L·d) was most likely due to the overloading of the system. This was indicated by the accumulation of soluble organic matter and volatile fatty acids. The increase in the propionic acid concentration up to 1300 mg/L when operating at OLRs higher than 0.5 g of VS/(L·d) could be the main factor responsible for the inhibition. An economic evaluation showed that the proposed approach (steam explosion, phenol recovery, and anaerobic digestion) would offer positive benefits, taking into account the high phenolic recovery (0.90 g of gallic acid equivalents/kg of extrudate) and the low sales price of the phenol extract, i.e., EUR 0.610/g of gallic acid equivalents, needed to reach zero net profit.

Biogas Potential of the Side Streams Obtained in a Novel Phenolic Extraction System from Olive Mill Solid Waste.

The olive oil production is an important industrial sector in many Mediterranean areas, but it is currently struggled by the necessity of a proper valorisation of the olive mill solid waste or alperujo. The alperujo is the main by-product generated during the two-phase olive oil extraction, accounting for up to 80% of the initial olive mass. The alperujo is a source of valuable compounds, such as the pomace olive oil or highly interesting phenolic compounds. In the present research, a novel biorefinery approach has been used for phenolic compounds recovery. However, the extraction of these valuables compounds generates different exhausted phases with high organic matter content that are required to be managed. This study consists of the evaluation of the anaerobic biodegradability of the different fractions obtained in a novel biorefinery approach for the integral valorisation of alperujo. The results show that the different phases obtained during the biorefinery of the alperujo can be effectively subjected to anaerobic digestion and no inhibition processes were detected. The highest methane yield coefficients were obtained for the phases obtained after a two-months storages, i.e., suspended solids and liquid phase free of suspended solids, which generated 366 ± 7 mL CH4/g VS and 358 ± 6 mL CH4/g VS, respectively. The phenol extraction process reduced the methane yield coefficient around 25% due to the retention of biodegradable compounds during the extraction process. Regardless of this drop, the anaerobic digestion is a suitable technology for the stabilization of the different generated residual phases, whereas the high market price of the extracted phenols can largely compensate the slight decrease in the methane generation.

High-Value-Added Compound Recovery with High-Temperature Hydrothermal Treatment and Steam Explosion, and Subsequent Biomethanization of Residual Strawberry Extrudate.

This study was on the comparison of hydrothermal treatments at 170 °C (steam injection) and 220 °C (steam explosion) to solubilize the organic matter contained in residual strawberry extrudate, focusing on phenolic compounds that were susceptible to be extracted and on sugars. After the extraction step, the remaining strawberry extrudate phases were subjected to anaerobic digestion to generate biogas that would compensate the energy requirements of the suggested hydrothermal treatments and to stabilize the remaining waste. Hydrothermal treatment at 220 °C allowed the recovery of 2053 mg of gallic acid eq. per kg of residual strawberry extrudate. By contrast, after hydrothermal treatment at 170 °C, only 394 mg of gallic acid eq. per kg of residual strawberry extrudate was recovered. Anaerobic digestion processes were applied to the de-phenolized liquid phase and the solid phase together, which generated similar methane productions, i.e., around 430 mL CH4/g volatile solids, after both 170 °C and 220 °C hydrothermal treatments. Considering the latest observation, hydrothermal treatment at 220 °C is a preferable option for the valorization of residual strawberry extrudate (RSE) due to the high solubilization of valuable phenolic compounds that can be recovered.

New Liquid Source of Antioxidant Phenolic Compounds in the Olive Oil Industry: Alperujo Water.

The current management of alperujo as the main solid by-product from the two-phase olive oil extraction system has led to the appearance of a new liquid effluent that until now was treated together with the alperujo itself. The composition and antioxidant properties of its bioactive components at different depths of the pond were studied using colorimetric and HPLC with UV and MS detectors, DPPH, reducing power and rancimat. The concentration of suspended solids varied between 1.71 and 8.49 g/L, total fat was between 0.74 and 1.47 g/L, and total phenols were found between 3.74 and 4.11 g/L, which included hydroxytyrosol, 3,4-dihydroxyphenylglycol and tyrosol as the main phenols. Two types of extracts were obtained through two industrial systems with ethyl acetate and by chromotography, with an average content in total sugars of 2.1% and 3.16%, total phenols of 17.9% and 28.6% and hydroxytyrosol of 51.5 and 79.0 mg/g of extract, respectively. The activity presented by the chromatographic extract was higher in terms of free radical sequestering capacity, reducing power and the inhibition of lipid oxidation. Obtaining bioactive extracts would improve the formulation of food with natural components and at the same time would be the first step in a biorefinery to improve the management of the new effluent.

Antiproliferative Activity of Olive Extract Rich in Polyphenols and Modified Pectin on Bladder Cancer Cells.

Bladder cancer (BC) is one of the most common human cancers. There is an interest in controlling and treating BC and other types of cancer via the use of natural substances and/or combination chemotherapy. Modified forms of pectin have been reported to possess anticancer bioactivity related to the interaction of galactosyl, a main component of pectin, with galectin-3, a carbohydrate-binding protein that is overexpressed on many types of cancer cells. In this study, the antiproliferative effect on BC of novel modified pectins extracted from olives was evaluated. Pectoliv extracts, with high polyphenol content associated to polysaccharides rich in pectin, exhibited an important antiproliferative capacity in vitro against four human BC cells lines, RT112, T24, J82, and SCaBER. Pectoliv treatment reduced the expression of galectin-1 and galectin-3 and significantly inhibited the agglutination of erythrocytes. Thus, Pectoliv may have the potential for development as a novel galectin-3 inhibitor.

Pectin-rich extracts from olives inhibit proliferation of Caco-2 and THP-1 cells.

Three olive modified pectin extracts have been produced by heat and acid treatment of the major by-product of olive oil production. Their effect on proliferation of the colon carcinoma Caco-2 and the leukemia monocytic THP-1 cell lines has been studied in order to determine possible anti-tumor properties. All extracts inhibited proliferation at some of the concentrations ranging from 1 to 10 mg ml-1. Interestingly none of the extracts inhibited the growth of confluent Caco-2 cells, showing the specificity of the antiproliferative effect for the transformed Caco-2 phenotype. All the extracts inhibited agglutination of red blood cells by galectin-3, a lectin involved in tumor growth, metastasis, and immune cell regulation that has been proposed as a mediator of the anti-tumor effects of modified pectins. In addition, activation of caspase-3 in THP-1 cells indicates that treatment with the pectin-rich extracts triggers apoptosis. These results point to a possible use as health-promoting food ingredients or supplements.

Performance evaluation of mesophilic semi-continuous anaerobic digestion of high-temperature thermally pre-treated olive mill solid waste.

The aim of the present work was to evaluate the effects of a thermal pre-treatment of olive mill solid waste (OMSW) and phenol extraction process on the semi-continuous anaerobic digestion of this pre-treated waste during a prolonged operational period (275 days) in order to assess the organic loading rates (OLR) of 1 ad 2 g Volatile Solids (VS)/(L·d). The anaerobic digestion of thermally pre-treated and de-phenolized OMSW was stable at an OLR of 1 g VS/(L·d), which permitted a specific production rate of 172 ±â€¯60 mL CH4/(g VS·d). However, the system was not able to operate at an OLR of 2 g VS/(L·d), which resulted in the total failure of the process. Regardless of the applied OLR, the phenolic compounds were effectively degraded and the inhibition thresholds were not reached. The inhibition of the anaerobic digestion process at an OLR of 2 g VS/(L·d) was probably due to the overloading of the system, indicated by the accumulation of organic matter and volatile fatty acids. The operation of the anaerobic digester under stable conditions allowed for high profitability for the proposed bio-refinery concept, which would still be profitable at a phenol extract price above 51.8 €/kg, which is 90% lower than the current price of 520 €/kg.

The accumulation of volatile fatty acids and phenols through a pH-controlled fermentation of olive mill solid waste.

This work aims to compare the use of olive mill solid waste as substrate in pH-controlled fermentation at acid (pH = 5), neutral (uncontrolled, pH ≈ 7) and alkaline (pH = 9) operating pH levels. The results obtained in this study indicate that operating pH strongly affected the anaerobic microorganisms and, hence, different target compounds could be obtained by adjusting the operating pH. Fermentation at neutral pH resulted in the conversion of 93.5% of the fed chemical oxygen demand to methane. However, fermentations at pH 5 and 9 resulted in the inhibition of the methanogenic activity. At pH 9, volatile fatty acids reached a maximum concentration of 3.69 g O2/L, where acetic acid represented up to 79.3% of the total volatile fatty acids. Unlike volatile fatty acid production, an optimal operation of fermentation at pH 5 could allow the recovery of phenols such as vanillin.

Strawberry dietary fiber functionalized with phenolic antioxidants from olives. Interactions between polysaccharides and phenolic compounds.

The interaction of strawberry cell wall with hydroxytyrosol (HT) and 3,4-dihydroxyphenylglycol (DHPG), two potent phenolic antioxidants naturally found in olive fruit with important biological properties, was investigated. The interaction occurred with drying and seemed to be more complex, strong and irreversible than a simple association. MALDI TOF-TOF analysis suggested covalent (ester bond) and non-covalent (strong hydrogen-bonding, mostly) interactions. The oxygen radical absorbance capacity (ORAC) assay confirmed that the phenols maintained partially their antioxidant activity after binding to the soluble dietary fraction. This soluble dietary fiber was obtained following digestion simulated in vitro with gastric and intestinal fluids. Although the antioxidant activity of HT and DHPG was affected by the dietary fiber interaction, this activity was restored when polysaccharide size was reduced by enzymatic treatment, suggesting that a similar process could occur in the colon. Thus, the use of this novel antioxidant-enriched soluble dietary fiber as a functional food ingredient could potentially promote intestinal health.