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.

Discriminant Analysis and Data Mining CHAID Decision Tree as Tools to Evaluate the Buffering Effect of Hydroxytyrosol on Reactive Oxygen Species in Rooster Sperm Cryopreservation.

Sperm cryopreservation is effective in safeguarding genetic biodiversity in avian species. However, during this process, spermatozoa are very susceptible to plasma membrane peroxidation in the presence of high concentrations of reactive oxygen species (ROS). To mitigate this effect, the addition of exogenous antioxidants, such as hydroxytyrosol (3,4-dihydroxyphenylethanol; HT), an antioxidant derived from olive oil, to the cryopreservation sperm diluent, could be useful. To verify this, a cryopreservation diluent was supplemented with different concentrations (0 µg/mL, 50 µg/mL, 100 µg/mL, and 150 µg/mL) of HT. For this, semen was collected in 10 replicates from 16 roosters of the Utrerana avian breed, and a pool was prepared with the optimum quality ejaculates in each replicate. After cryopreservation, spermatozoa were thawed and different in vitro semen quality parameters were evaluated. A discriminant canonical analysis (DCA) was carried out and revealed that total motility (TM; Lambda = 0.301, F = 26,173), hypo-osmotic swelling test (HOST; Lambda = 0.338, F = 22,065), and amplitude of lateral head displacement (ALH, Lambda = 0.442; F = 14,180) were the variables with the highest discriminant power. Finally, a chi-squared automatic interaction detection (CHAID) decision tree (DT) was performed excluding fresh semen samples and ROS was found to be the most valuable variable to discriminate between the different established freezing groups. Samples in the absence of HT or with low concentrations of this antioxidant showed less desirable ROS values in cryopreserved rooster semen. The present study could lead to the improvement of cryopreservation techniques for the genetic material of local poultry breeds and optimize the conservation programs of endangered native avian breeds.

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.

In vivo evidences of the health-promoting properties of bioactive compounds obtained from olive by-products and their use as food ingredient.

Olea europaea L. is the source of virgin olive oil (VOO). During its extraction, a high amount of by-products (pomace, mill wastewaters, leaves, stones, and seeds) is originated, which possess an environmental problem. If the generation of waste cannot be prevented, its economic value must be recovered and its effects on the environment and climate change must be avoided or minimized. The bioactive compounds (e.g., phenols, pectins, peptides) of these by-product fractions are being investigated as nutraceutical due to the beneficial properties it might have. In this review, the aim is to summarize the in vivo studies carried out in animals and humans with bioactive compounds exclusively obtained from olive by-products, aiming to demonstrate the potential health benefits these products can exert, as well as to describe its use in the food industry as bioactive ingredient. Several food matrices have been fortified with olive by-products fractions, leading to an improvement of properties. Animal and human studies suggest the benefits of ingesting olive-derived products to promote health. However, the investigation until now is scarce and consequently, well-designed human studies are required in order to fully address and confirm the safety and health-promoting properties of olive oil by-products.

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.

Nephroprotective Effect of the Virgin Olive Oil Polyphenol Hydroxytyrosol in Type 1-like Experimental Diabetes Mellitus: Relationships with Its Antioxidant Effect.

The aim of this study was to determine whether hydroxytyrosol administration prevented kidney damage in an experimental model of type 1 diabetes mellitus in rats. Hydroxytyrosol was administered to streptozotocin-diabetic rats: 1 and 5 mg/kg/day p.o. for two months. After hydroxytyrosol administration, proteinuria was significantly reduced (67-73%), calculated creatinine clearance was significantly increased (26-38%), and the glomerular volume and glomerulosclerosis index were decreased (20-30%). Hydroxytyrosol reduced oxidative and nitrosative stress variables and thromboxane metabolite production. Statistical correlations were found between biochemical and kidney function variables. Oral administration of 1 and 5 mg/kg/day of hydroxytyrosol produced an antioxidant and nephroprotective effect in an experimental model of type 1-like diabetes mellitus. The nephroprotective effect was significantly associated with the systemic and renal antioxidant action of hydroxytyrosol, which also influenced eicosanoid production.

Bayesian Analysis of the Effects of Olive Oil-Derived Antioxidants on Cryopreserved Buck Sperm Parameters.

The present study evaluates the effect of olive oil-derived antioxidants, hydroxytyrosol (HT) and 3,4-dihydroxyphenylglycol (DHPG), on cryopreserved caprine sperm using Bayesian inference of ANOVA. For this proposal, sperm was collected, pooled and diluted in freezing media supplemented with different concentrations of HT, DHPG and the mixture (MIX) of both antioxidants. Sperm motility, viability, acrosome integrity, mitochondrial status, and lipid peroxidation (LPO) were assessed in fresh and frozen-thawed sperm samples. The results provided evidence that HT at low concentrations improves sperm motility and viability, and reduces the LPO. Contrastingly, DHPG and MIX exert a positive effect by reducing LPO values as concentration increases. Additionally, mitochondrial potential was reduced when samples were supplemented with HT at low concentrations and mixture of both antioxidants. Conclusively, the addition of olive oil-derived antioxidants (HT at 10 µg/mL and DHPG at 30 µg/mL) implements a protective effect in cryopreserved buck sperm. Bayesian analysis alternatives offer new possibilities to determine the repercussion of antioxidants on sperm, both quantitatively and qualitatively.

Valorisation of Olea europaea L. Olive Leaves through the Evaluation of Their Extracts: Antioxidant and Antimicrobial Activity.

Olea europaea L. leaves constitute a source of bioactive compounds with recognized benefits for both human health and technological purposes. In the present work, different extracts from olive leaves were obtained by the application of two extraction methods, Soxhlet and microwave-assisted extraction (MAE), and six solvents (distilled water, ethanolic and glycerol mixtures solvents). MAE was applied under 40, 60 and 80 °C for 3, 6.5 and 10 min. The effect of the extraction method, solvent and treatment factors (the latter in MAE) on the total phenol content (TPC), the antioxidant activity (AA) and the phenolic profile of the extracts were all evaluated. The extracts showed high values of TPC (up to 76.1 mg GAE/g DW) and AA (up to 78 mg TE/g DW), with oleuropein being the most predominant compound in all extracts. The Soxhlet extraction method exhibited better yields in TPC than in MAE, although both methods presented comparable AA values. The water MAE extract presented the strongest antimicrobial activity against five foodborne pathogens, with minimum inhibitory concentration (MIC) values ranging from 2.5 to 60 mg/mL. MAE water extract is proposed to be exploited in the food and nutraceutical industry in the frame of a sustainable economy.

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).

Strawberry Puree Functionalized with Natural Hydroxytyrosol: Effects on Vitamin C and Antioxidant Activity.

The natural antioxidant hydroxytyrosol (HT) was used to functionalize a strawberry puree. The effect of the antioxidant on the stability of the two bioactive forms of vitamin C (ascorbic acid-AA and dehydroascorbic acid-DHAA) in strawberry puree stored at 4 °C, compared with the effect on a model system of AA in water, was investigated. In the absence of HT, the concentration of vitamin C in strawberry puree decreased but not in the model system. Low concentrations of HT in strawberry puree (0.05 and 0.1 mg HT/g puree) stabilized vitamin C and improved its antioxidant activity. However, at high concentrations of HT (from 0.5 mg HT/g puree), although the antioxidant activity improved, degradation of vitamin C occurred. Therefore, the concentration of HT used to obtain a functionalized strawberry puree it is very important. An adequate concentration increases the antioxidant activity and protects vitamin C from degradation, developing a functional food. However, an inadequate concentration of HT affects the vitamin C content, which is essential for the human diet because it cannot be biosynthetized by the organism.

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.

Effect of olive-derived antioxidants (3,4-dihydroxyphenylethanol and 3,4 dihydroxyphenylglycol) on sperm motility and fertility in liquid ram sperm stored at 15°C or 5°C.

The aim of the present study was to evaluate the effect of the addition of two olive oil-derived antioxidants, hydroxytyrosol (3,4-dihydroxyphenylethanol, HT) and 3,4-dihydroxyphenylglycol (DHPG), on ovine semen during liquid storage at 5°C and 15°C. Semen was collected, pooled, diluted and then divided into aliquots supplemented with different concentrations (5 µg/ml, 10 µg/ml, 50 µg/ml and 100 µg/ml) of HT, DHPG and a mixture (MIX) of both antioxidants. Sperm motility characteristics were assessed in the different samples at 0, 6, 24, 48, 72 and 96 hr after cooling, and a fertility trial was also conducted. The results showed that the antioxidant addition did not significantly improve total and progressive motility in ovine cooled sperm maintained at 15° or 5°C. However, in samples stored at 5°C, LIN (48, 72, 96 hr), STR (0 hr) and WOB (0, 48, 72, 96 hr) values significantly decreased in comparison with control treatment when high antioxidant concentrations were added (MIX100 or HT100). When samples were maintained at 15°C, MIX50 showed significantly higher VCL values than the control treatment after 6 hr cooling, and MIX100 showed significantly lower VCL values at 96 hr after cooling. According to the artificial insemination trial, no significant differences were observed when antioxidants were added. In conclusion, the use of HT and DHPG showed small impact in sperm motility and fertility was not affected (nor detrimentally nor positively) when insemination was carried out using antioxidant-supplemented liquid sperm.

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.

Effect of edible pectin-fish gelatin films containing the olive antioxidants hydroxytyrosol and 3,4-dihydroxyphenylglycol on beef meat during refrigerated storage.

The objective of this research was to evaluate the effect of the addition of two antioxidants naturally present in olives, hydroxytyrosol (HT) and 3,4-dihydroxyphenylglycol (DHPG), to a pectin-fish gelatin edible film on the preservation of raw beef meat during refrigerated storage. A new composite film that included beeswax was also prepared, resulting in a reduction in the film's oxygen permeability. Results showed that the meat samples wrapped with film containing antioxidants reduced the formation of oxidation products in the form of thiobarbituric acid reaction substances (TBARS) compared with control film without antioxidants. HT added at 0.5% to the film with beeswax suppressed the lipid oxidation of beef meat during 7 days of storage at 4 °C, possibly by the combined effect of acting as an oxygen barrier and the specific antioxidant activity. The interference of plasticizer agents (glycerol and sorbitol) incorporated to the film on the TBARS method was showed for the first time.