Extraction Systems and Analytical Techniques for Food Phenolic Compounds: A Review.

Phenolic compounds are highly valuable food components due to their potential utilisation as natural bioactive and antioxidant molecules for the food, cosmetic, chemical, and pharmaceutical industries. For this purpose, the development and optimisation of efficient extraction methods is crucial to obtain phenolic-rich extracts and, for some applications, free of interfering compounds. It should be accompanied with robust analytical tools that enable the standardisation of phenolic-rich extracts for industrial applications. New methodologies based on both novel extraction and/or analysis are also implemented to characterise and elucidate novel chemical structures and to face safety, pharmacology, and toxicity issues related to phenolic compounds at the molecular level. Moreover, in combination with multivariate analysis, the extraction and analysis of phenolic compounds offer tools for plant chemotyping, food traceability and marker selection in omics studies. Therefore, this study reviews extraction techniques applied to recover phenolic compounds from foods and agri-food by-products, including liquid-liquid extraction, solid-liquid extraction assisted by intensification technologies, solid-phase extraction, and combined methods. It also provides an overview of the characterisation techniques, including UV-Vis, infra-red, nuclear magnetic resonance, mass spectrometry and others used in minor applications such as Raman spectroscopy and ion mobility spectrometry, coupled or not to chromatography. Overall, a wide range of methodologies are now available, which can be applied individually and combined to provide complementary results in the roadmap around the study of phenolic compounds.

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.

Characterization of the lignocellulosic and sugars composition of different olive leaves cultivars.

Olive leaves are an under valorized residue of olive tree pruning and olive fruit harvesting and that are usually removed by either burning or grinding and scattering them on fields. However, as plant material easily available, they may be used as raw material in biorefineries, or for the industrial manufacture of many diverse products, given their lignocellulosic composition. Like other lignocellulosic biomasses, the composition of olive leaves depends on cultivar and to know it is essential for an adequate use. Therefore, this work tackles a characterization analysis of the lignocellulosic fraction of some olive leaf cultivars, both commercial and wild. In general, the cultivars studied did not show large differences in their quantitative composition, except for the content of ethanolic extractives and cellulose of the commercial and wild cultivars. In addition, the high lignin content (around 15%) is remarkable.

Content of phenolic compounds and mannitol in olive leaves extracts from six Spanish cultivars: Extraction with the Soxhlet method and pressurized liquids.

Olive leaves are considered a promising source of bioactives such as phenolic compounds and mannitol. The extraction of high added value products is an issue of great interest and importance from the point of view of their exploitation. However, the content of these compounds can differ between cultivars and extraction methods. In this work, six olive leaves cultivars, including three wild cultivars, and two extraction processes (an innovative and alternative technique, pressurized liquid extraction, and a conventional Soxhlet extraction) were evaluated and compared towards the selective recovery of bioactive compounds. The wild cultivars showed the highest content of phenolic and flavonoid compounds, being oleuropein the compound present in higher amount. Findings also revealed that the highest mannitol content in the extracts was observed with the commercial cultivars, specifically in Arbequina. It is thus possible to decide which cultivars to use in order to obtain the highest yield of each bioproduct.

Valorization of olive mill leaves through ultrasound-assisted extraction.

Olive leaves farmed from trees are valuable for the production of functional extracts. Nonetheless, olive leaves (containing thin branches), which are separated during olives cleaning in the mill, have received little attention. In this context, a multiple response optimization was performed to maximize at once the yield, total phenolic content, oleouropein and antioxidant activity obtained by ultrasound-assisted extraction of this low-cost byproduct. The optimum was achieved using the following operational parameters: solid-to-liquid ratio, 5.9%; ethanol concentration, 47%; extraction time, 50 min. This enabled to obtain an extract with both high level of oleuropein and antioxidant activity. Besides oleuropein, other minor phenolic compounds were characterized in the extract, which could contribute to the antioxidant activity as Pearson correlation suggested. After this extraction step, how the phenolic extraction affects the recovery/profile of other constituents was evaluated, looking for the integral valorization of this resource towards the zero-waste.

Integrated Process for Sequential Extraction of Bioactive Phenolic Compounds and Proteins from Mill and Field Olive Leaves and Effects on the Lignocellulosic Profile.

The extraction of bioactive compounds in a biorefinery context could be a way to valorize agri-food byproducts, but there is a remaining part that also requires attention. Therefore, in this work the integrated extraction of phenolic compounds, including the bioactive oleuropein, and proteins from olive mill leaves was addressed following three schemes, including the use of ultrasound. This affected the total phenolic content (4475.5-6166.9 mg gallic acid equivalents/100 g), oleuropein content (675.3-1790.0 mg/100 g), and antioxidant activity (18,234.3-25,459.0 µmol trolox equivalents/100 g). No effect was observed on either the protein recovery or the content of sugars and lignin in the extraction residues. Concerning the recovery of proteins, three operational parameters were evaluated by response surface methodology. The optimum (63.1%) was achieved using NaOH 0.7 M at 100 °C for 240 min. Then, the selected scheme was applied to olive leaves from the field, observing differences in the content of some of the studied components. It also changed the lignocellulosic profile of the extraction residues of both leaf types, which were enriched in cellulose. Overall, these results could be useful to diversify the valorization chain in the olive sector.

Extraction of oleuropein and luteolin-7-O-glucoside from olive leaves: Optimization of technique and operating conditions.

Olive leaves have become a promising source of phenolic compounds and flavonoids with high added value. Phenolic compounds and flavonoids are important sources of antioxidants and bioactives, and one of the processes used to effectively produce them is extraction via solvents, using aqueous ethanol solutions. To obtain the highest extraction yield per kg of biomass, olive leaves were extracted using a conventional technique (dynamic maceration) and an emerging technology, such as pressurized liquid extraction. Studies of the factors that influence these processes were performed: temperature, leaf moisture content, solvent/solid, and aqueous ethanol concentration were optimized using the central composite and Box-Behnken experiment designs. Pressurized liquid extraction resulted in more efficient oleuropein and luteolin-7-O-glucoside extraction than dynamic maceration. The operational conditions for maximizing the recovery of phenolic compounds and flavonoids and antioxidant capacity were determined to be 190 °C, leaf moisture content of 5%, and aqueous ethanol concentration of 80%.

Protein extraction from agri-food residues for integration in biorefinery: Potential techniques and current status.

The biorefinery concept is attracting scientific and policy attention as a promising option for enhancing the benefits of agri-food biomass along with a reduction of the environmental impact. Obtaining bioproducts based on proteins from agri-food residues could help to diversify the revenue stream in a biorefinery. In fact, the extracted proteins can be applied as such or in the form of hydrolyzates due to their nutritional, bioactive and techno-functional properties. In this context, the present review summarizes, exemplifies and discusses conventional extraction methods and current trends to extract proteins from residues of the harvesting, post-harvesting and/or processing of important crops worldwide. Moreover, those extraction methods just integrated in a biorefinery scheme are also described. In conclusion, a plethora of methods exits but only some of them have been applied in biorefinery designs, mostly at laboratory scale. Their economic and technical feasibility at large scale requires further study.

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.

Obtaining sugars and natural antioxidants from olive leaves by steam-explosion.

In this work, steam-explosion treatment was evaluated as a procedure to recover sugars and natural antioxidants from olive tree leaves. The treatment was carried out following a Box-Behnken experimental design, with three factors, temperature (180-220°C), process time (2-10min) and milling time (0-15s). Response surface methodology showed that temperature was the most influential factor, followed by process time, while the best results were achieved with whole leaves. The operational conditions for simultaneously maximizing the sugars and natural antioxidants recoveries resulted to be 180°C, 8.3min and whole leaf; under these conditions 18.39g and 1950mg were obtained from 100g dry olive leaves, respectively. This is equivalent to 70% recovery of the initial sugars present in olive leaves, with a very low formation of inhibitory compounds and an important amount of natural products with antioxidant capacity such as oleuropein, hydroxytyrosol and flavonoids.

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.

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.

Effect of a new thermal treatment in combination with saprobic fungal incubation on the phytotoxicity level of alperujo.

Byproducts generated from food industries, such as olive oil mills, have been studied to decrease harmful pollution and their environmental consequences. In this work, a new thermal pretreatment and saprobic fungal incubation to detoxify alperujo (two-phase olive mill waste) have been evaluated in view of its use as fertilizer in agriculture. The sequential use of both methods simplifies the thermal conditions and incubation times of the fungal treatment. Optimization of the thermal treatment from 150 to 170 °C for 45 and 15 min, respectively, reduced the incubation time with Coriolpsis rigida from 20 to 10 weeks needed to reduce phytotoxic effects on tomato plants. Therefore, the combination of thermal and biological treatments will allow the development of the potential benefits of alperujo to improve nutrients in agricultural soil.

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.