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.

Inhibitory Effect of Olive Phenolic Compounds Isolated from Olive Oil By-Product on Melanosis of Shrimps.

Melanosis is an unsolved problem of the crustacean industry and the cause of great loss of value. This study investigates the effect of two potent, natural antioxidants isolated from olive waste (hydroxytyrosol, HT and 3,4-dihydroxyphenylglycol, DHPG) and three novel HT-derivatives containing selenium and sulfur (dihydroxytyrosyl diselenide, N-hydroxytyrosyl selenourea, and N-hydroxytyrosyl thiourea) on the prevention of melanosis in Atlantic ditch shrimp (Palaemonetes varians) during refrigerated storage. These results clearly demonstrate the positive inhibitory effect of DHPG and dihydroxytyrosyl diselenide on delaying melanosis in vivo, although this effect was not dose dependent. The effect was associated with a concomitant-inhibitory effect on tyrosinase activity in vitro. To our knowledge, so far no studies on the prevention of melanosis have been conducted on this small specie of shrimp which is available in large quantities at any time of the year at low cost. Studies with these promising compounds could then be extended to other more economically important species with a greater guarantee of success.

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.

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.

Selenium and sulphur derivatives of hydroxytyrosol: inhibition of lipid peroxidation in liver microsomes of vitamin E-deficient rats.

PURPOSE: The objective of this study was to evaluate the capacity of modified phenols synthesized from hydroxytyrosol, a natural olive oil phenol, specifically those containing a selenium or sulphur group, to inhibit lipid peroxidation. METHODS: The compounds' abilities to inhibit lipid peroxidation in liver microsomes obtained from vitamin E-deficient rats were compared to hydroxytyrosol. RESULTS: All synthetic compounds had a significant higher ability to inhibit lipid peroxidation than hydroxytyrosol. Selenium derivates displayed a higher antioxidant activity than sulphur derivatives. In addition, the antioxidant activity increased with a higher number of heteroatoms in the hydroxytyrosol molecular structure. CONCLUSION: The study shows, for the first time, the ability of synthetic compounds, derived from the most active phenol present in olives in free form (hydroxytyrosol), and containing one or two atoms of sulphur or selenium, to inhibit the lipid peroxidation of vitamin E-deficient microsomes. The antioxidant activity of five thioureas, a disulfide, a thiol, three selenoureas, a diselenide, and a selenonium were evaluated and the results showed a higher inhibition of lipid peroxidation than the natural phenol. Selenium and sulphur derivatives of hydroxytyrosol are novel antioxidants with the potential to supplement the lack of vitamin E in the diet as natural alternatives for the prevention of diseases related to oxidative damage.

Physical and functional properties of pectin-fish gelatin films containing the olive phenols hydroxytyrosol and 3,4-dihydroxyphenylglycol.

This study describes the development of a composite edible film based on pectin and fish skin protein capable of protecting food from microbial attack and oxidative degradation. The film was prepared with glycerol as plasticizer and the antioxidant and antimicrobial phenolic compounds hydroxytyrosol (HT) and 3,4-dihydroxyphenylglycol (DHPG), extracted from olive fruit, as active agents. The influence of the concentration of plasticizer and active HT/DHPG on the mechanical and functional properties of the films was investigated, with values of water vapor permeability (WVP) between 0.13-0.22gmm/hm2kPa and oxygen permeability (OP) between 9.91-40.76cm3µm/m2dkPa. The release behavior in water at different pH values was also evaluated. The antimicrobial capacity of the novel food coating was tested on strawberries, a fruit with high perishability. The bioactive edible film containing HT/DHPG preserved the strawberries against mold during storage with a significant delay in visible decay.

Complexation of hydroxytyrosol and 3,4-dihydroxyphenylglycol with pectin and their potential use for colon targeting.

Hydroxytyrosol (HT) and 3,4-dihydroxyphenylglycol (DHPG) are two phenolic antioxidants naturally found in olive fruit with anti-inflammatory properties. This study explored the interaction of pectin with HT and DHPG via their encapsulation into pectinate beads. Purification by size exclusion chromatography, changes in the fluorescence spectrum of the HT and pectin, and MALDI TOF-TOF analysis suggested the existence of the phenol-pectin complexes. The entrapment efficiency, swelling properties, and in vitro release of HT and DHPG of the beads were studied. The results show that the beads can entrap the water soluble compounds HT and DHPG in sufficient amounts to reach the colon. The beads consisted of an important amount of pectin-bound HT or DHPG after two hours at gastric pH. This study highlights the potential use of HT-and DHPG-loaded pectinate gel beads for the colon-targeted delivery of these bioactive compounds to help prevent or relieve chronic inflammatory bowel disease.

Influence of pH on the antioxidant phenols solubilised from hydrothermally treated olive oil by-product (alperujo).

The application of a novel industrial process based on the hydrothermal treatment of olive oil waste (alperujo) led to a final liquid phase that contained a high concentration of simple phenolic compounds. In this study the effect of pH on phenol extraction with ethyl acetate from the aqueous phase of hydrothermally treated alperujo at 160°C/60min (without modification, pH 4.5, and adjusted to pH 2.5) was evaluated, beside the increase of hydroxytyrosol during the storage. The variation of the concentration of phenolic compounds in each extract was analyzed by HPLC. The phenolic extract obtained at pH 4.5 presented a higher proportion of total and individual phenols and better antioxidant capacity in vitro than the extract obtained at pH 2.5. The use of lower pH values enhances the concentration of hydroxytyrosol in the liquid diminishing the storage times.

Antioxidant phenolic extracts obtained from secondary Tunisian date varieties (Phoenix dactylifera L.) by hydrothermal treatments.

Three common non-commercial Tunisian date varieties were treated by two thermal systems, obtaining a liquid fraction which was characterized and its antioxidant capacity determined. The concentration of total phenols in the three varieties (Smeti, Garen Gazel, and Eguwa) was increased by steam explosion treatment up to 5311, 4680, and 3832 mg/kg of fresh dates, and their antioxidant activity up to 62.5, 46.5 and 43.1 mmol Trolox/kg of fresh date, respectively. Both thermal treatments increased the content of phenolic acids. Additionally, a long scale study was carried out in a pilot plant, with steam treatment at 140 °C and 160 °C for 30 min. The liquid phase was extracted and fractionated chromatographically using adsorbent or ionic resins. The phenolic profiles were determined for each fraction, yielding fractions with interesting antioxidant activities with EC50 values of up to 0.08 mg/L or values of TEAC of 0.67 mmol Trolox/g of extract.

Isolation and identification of minor secoiridoids and phenolic components from thermally treated olive oil by-products.

The application of an industrial process based on the hydrothermal treatment of 160 °C/60 min of alperujo, a by-product of olive oil extraction, allows the formation of a liquid phase containing a high concentration of phenolic and secoiridoid compounds. Ethyl acetate was used to extract these phenolic compounds from the aqueous matrix. In this study, the isolation with polyamide and XAD resin allowed detection of the presence of phenolic compounds in minor concentrations. These minor phenols were several oleuropein derivatives that had not been identified in these phenolic extracts previously. The polar compounds, acteosides, secoiridoids, and flavonoids, that remain in the aqueous fraction after extraction with ethyl acetate were identified. We report the presence of known compounds and also detected a novel molecule in alperujo with a molecular weight of 408 whose structure was characterized for first time. This new secoiridoid glucoside was identified as 1-ß-D-glucopyranosyl acyclodihydroelenolic acid.

Isolation and Characterization of a Secoiridoid Derivative from Two-Phase Olive Waste (Alperujo).

A secoiridoid derivative was isolated from the ethyl acetate extract of two-phase olive waste (alperujo). The structure of this compound was fully characterized as s-trans-(E)-3-(1-oxobut-2-en-2-yl)glutaric acid. The spectroscopic data, including one- and two-dimensional nuclear magnetic resonance, mass spectrometry, infrared analysis, and ultraviolet spectrum, were showed. The origin of this compound has not been previously studied, although it most likely results from the breakdown of the oleuropein (or ligstroside) secoiridoid skeleton via oxidation and decarboxylation of the dialdehydic form of elenolic acid, with this transformation being enhanced by extraction of phenolics with ethyl acetate. In addition, the bactericidal activity of (E)-3-(1-oxobut-2-en-2-yl)glutaric acid and extracts containing it was evaluated against two phytopatoghenic microorganisms Pseudomonas syringae and Agrobacterium tumefaciens.

Inhibitory and synergistic effects of natural olive phenols on human platelet aggregation and lipid peroxidation of microsomes from vitamin E-deficient rats.

PURPOSE: This study explored the in vitro antioxidant and anti-platelet activities of hydroxytyrosol, hydroxytyrosol acetate, 3,4-dihydroxyphenylglycol and two phenolic olive extracts. These compounds and extracts were obtained from a new industrial process to hydrothermally treat the alperujo (160 °C/60 min), a by-product of olive oil extraction. METHODS: The extracts and the purified compounds were obtained chromatographically using both ionic and adsorbent resins. The antioxidant activity was determined by measuring inhibition of human platelet aggregation and inhibition of lipid peroxidation in liver microsomes of vitamin E-deficient rats. RESULTS: The positive effect of the extracts on the inhibition of platelet aggregation is showed, being higher in the case of hydroxytyrosol acetate up to 38%, and for the first time, its synergist effect with hydroxytyrosol has been proved, obtaining more than double of inhibition. The phenolic extracts and the isolated phenols showed good results for inhibiting the lipid oxidation, up to 62 and 25%, respectively. A synergistic effect occurred when the hydroxytyrosol acetate and the 3,4-dihydroxyphenylglycol were supplemented by hydroxytyrosol. CONCLUSION: These results suggest the extract and these compounds obtained from a novel industrial process could be natural alternatives for the prevention of diseases related to cardiovascular disorder or oxidative damage.

Properties of lignin, cellulose, and hemicelluloses isolated from olive cake and olive stones: binding of water, oil, bile acids, and glucose.

A process based on a steam explosion pretreatment and alkali solution post-treatment was applied to fractionate olive stones (whole and fragmented, without seeds) and olive cake into their main constitutive polymers of cellulose (C), hemicelluloses (H), and lignin (L) under optimal conditions for each fraction according to earlier works. The chemical characterization (chromatographic method and UV and IR spectroscopy) and the functional properties (water- and oil-holding capacities, bile acid binding, and glucose retardation index) of each fraction were analyzed. The in vitro studies showed a substantial bile acid binding activity in the fraction containing lignin from olive stones (L) and the alkaline extractable fraction from olive cake (Lp). Lignin bound significantly more bile acid than any other fraction and an amount similar to that bound by cholestyramine (a cholesterol-lowering, bile acid-binding drug), especially when cholic acid (CA) was tested. These results highlight the health-promoting potential of lignin from olive stones and olive cake extracted from olive byproducts.

A study of the precursors of the natural antioxidant phenol 3,4-dihydroxyphenylglycol in olive oil waste.

3,4-Dihydroxyphenylglycol (DHPG) is a potent antioxidant recently found in the free form in olive oil and table olives. DHPG can be recovered from olive oil solid waste by a hydrothermal treatment. It was observed that an increase in the concentration of DHPG occurred when alperujo aqueous extracts were subjected to mild thermal conditions (post-treatment). This fact indicates that certain solubilized compounds or precursors containing DHPG which is released with the post-treatment. In the present study, the precursors of DHPG were identified and characterized after extraction from alperujo using thermal treatment and purification by fractionation on Amberlite® XAD16 polyamide and semi-preparative reverse-phase HPLC columns. Their structures were elucidated using HPLC coupled to diode array detector (DAD) and electrospray ionization mass spectrometry (ESI-MS). The results identified three compounds as precursors, and their structures can be attributed to the diastereoisomeric forms of the two ß-hydroxy derivatives of verbascoside and isoverbascoside (ß-hydroxyacteoside and ß-hydroxyisoacteoside), and 2″-hydroxyoleuropein, all of which contain a DHPG moiety, potentially explaining the increases in the concentration of this phenolic compound in olive oil waste.

Isolation and identification of phenolic glucosides from thermally treated olive oil byproducts.

A liquid phase rich in bioactive compounds, such as phenols and sugars, is obtained from olive oil waste by novel thermal treatment. Two groups of fractions with common characteristics were obtained and studied after thermal treatment, acid hydrolysis, and separation by ultrafiltration, chromatography, and finally Superdex Peptide HR. In the first group, which eluted at the same time as oligosaccharides with a low DP (4-2), an oleosidic secoiridoid structure conjugated to a phenolic compound (hydroxytyrosol) was identified as oleuropeinic acid, and three possible structures were detected. In the second group, glucosyl structures formed by hydroxytyrosol and one, two, or three units of glucose or by tyrosol and glucose have been proposed. Verbascoside, a heterosidic ester of caffeic acid, in which hydroxytyrosol is linked to rhamnose-glucose or one of its isomers was also identified. Neutral oligosaccharides bound to a phenol-containing compound could be antioxidant-soluble fibers with bioactive properties.

New phenolic compounds hydrothermally extracted from the olive oil byproduct alperujo and their antioxidative activities.

The application of a novel process based on the hydrothermal treatment of olive oil waste (alperujo) led to a final liquid phase that contained a high concentration of simple phenolic compounds. This study evaluated the effects of time (15-90 min) on the composition of the phenolic compounds isolated at a fixed temperature of 160 °C. Phenolic compounds were extracted with ethyl acetate. Both qualitative and quantitative HPLC analyses of the extracts showed variation of the concentrations of phenolic compounds with time. In addition, new phenols that were not present in the untreated control have been characterized. The antioxidant activities of different phenolic extracts was measured by various assays conducted in vitro: antiradical capacity (using DPPH and ABTS radicals), ferric reducing power (P(R)), inhibition of primary and secondary oxidation in lipid systems, and other tests, such as inhibition of tyrosinase activity. The results show that the phenolic extracts inhibited oxidation in aqueous and lipid systems to a significantly greater extent than the untreated control, and they performed as well as or better than vitamin E in this capacity.

New hydrothermal treatment of alperujo enhances the content of bioactive minor components in crude pomace olive oil.

The application of a new process based on the hydrothermal treatment of olive oil waste (alperujo) led to a final solid rich in pomace olive oil (POO) enriched in minor components with functional activities. The effects of the time (15-90 min) and the temperature (150, 160, and 170 °C) of the thermal processing of alperujo on the yield, quality, and enrichment of minor components of crude POO were evaluated. The final treated solid had an increase in oil yield up to 97%, with a reduction in solids up to 35.6-47.6% by solubilization. Sterols increased up to 33%, aliphatic alcohols increased up to 92%, triterpenic alcohols increased up to 31%, squalene increased up to 43%, tocopherols increased up to 57%, and oleanolic acid increased up to 16% by the new treatment. The increase maintains a high concentration of functional substances probably even in the refining POO.