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This is the peer reviewed version of the following article: Romero, C. , 1Medina, E. , Mateo,
M. A. and Brenes, M. (2018), New by‐products rich in bioactive substances from the olive oil
mill processing. J. Sci. Food Agric, 98: 225-230, which has been published in final form at
https://doi.org/10.1002/jsfa.8460. This article may be used for non-commercial purposes in
accordance with Wiley Terms and Conditions for Self-Archiving.
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Concepción Romeroa, Eduardo Medinaa, Mª Antonia Mateob, Manuel Brenesa,*
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*Correspondence to: Manuel Brenes, Food Biotechnology Department, Instituto de la
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Grasa (IG$CSIC), Campus University Pablo de Olavide, Ctra. Utrera km 1, 41013$
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Seville, Spain. E$mail brenes@cica.es
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Running title: New olive by$products from olive oil mill processing
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Journal of the Science of Food and Agriculture
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BACKGROUND: Olive oil extraction generates a large amount of residue comprising
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mainly of the pomace and leaves when using the two$phase centrifugation system. The
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aim of this study was to assess the content of phenolic and triterpene compounds in the
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by$products produced in Spanish olive oil mills.
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RESULTS: The olive pomace had lower concentrations than 2 and 3 g kg$1 of phenolic
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and triterpene substances, respectively. The leaves contained a high concentration in
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these substances although those collected from ground$picked olives had lost most of
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their phenolic compounds. Moreover, the sediment from the bottom of the olive oil
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storage tanks did not have a significant amount of these substances. By contrast, the
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new by$product named olive pomace skin has been revealed as a very rich source of
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triterpenic acids because this waste can reach up to 120 g kg$1 of these compounds,
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maslinic acid comprising around 70 % of total triterpenics.
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CONCLUSIONS: Among the by$products generated during extraction of olive oil, the
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olive pomace skin has been discovered as a very rich source of triterpenic acids that can
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reach up to 120 g kg$1 of the waste. These results will contribute to the valorization of
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olive oil by$products.
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: olive; phenolic; triterpene; oleuropein; maslinic; oleanolic
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Olive leaves and olive mill pomace are the two main wastes generated during the
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extraction of olive oil by the two$phase centrifugation system. In Spain, more than 4
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and 0.2 million tons of olive pomace and leaves are annually produced, respectively.
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These by$products are used for composting, combustion, animal feed and soil
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amendment among others.1$4 However, they are also rich in bioactive substances such
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as phenolic compounds and triterpenic acids that could contribute to the revalorization
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of these by$products.5
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There are many methods and patents to extract phenolic compounds and
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triterpenic acids from olive leaves because fresh olive leaves may contain up to 70 g kg$
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and oleanolic acid being the main phenolic and triterpene substances in this material.8
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These bioactive substances have been analyzed in many fresh olive leaf cultivars during
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olive maturation, under different agronomic conditions and taking into account many
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other variables.9$12 However, the olive leaf by$product generated in the Spanish olive
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oil mills comes from two sources, ground$picked or tree$picked olives, which have not
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been characterized.
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and 20 g kg$1 of phenolic compounds and triterpenic acids respectively; 6,7 oleuropein
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Hundreds of articles describe the phenolic composition of olive oil mill
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wastewaters produced during the extraction of olive oil by the three$phase
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centrifugation system.13,14 The characterization of these substances in the two$phase
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olive pomace has also been reported.15$17
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composition of this olive pomace, which is named “Alperujo” in Spain, are scarce.18,19
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However, studies on the triterpene
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Alperujo is a semi$solid waste generated in the Spanish olive oil mills that is
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stored in large open air ponds for several months until the residual oil is extracted by
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physical or chemical methods in the oil extracting plants.20 The de$oiled pomace is used
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for co$generation of electrical power. Currently, large pit fragments are separated from
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fresh Alperujo in the olive oil mills to use them for combustion. However, pieces of
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skin and olive pulp are adhered to these pit fragments and they are separated to obtain
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pits which are free of pulp. Consequently, a new by$product is generated at large scale
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at the olive oil mills that consists mainly of olive skin together with small pieces of
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pulp. This by$product is used for combustion or spreading on the soil but it has attracted
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the attention of enterprises due to its potential content in bioactive substances,
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particularly triterpenic acids. It must be highlighted that triterpenic acids are mainly
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concentrated in the skin of fruits,21,22 although no data are available about the
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composition of this new by$product, rich in the olive skin.
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Another by$product produced in the olive oil mills is the sediment that settles at
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the bottom of the olive oil tanks during the storage of the oil before commercialization.
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This olive oil lees is composed mainly of fat and water, and it is intended for refining or
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making soap. The phenolic characterization of the solid and aqueous components of this
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by$product has been reported but not the composition of the oily phase, particularly
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triterpenic acids.23,24
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Considering the industrial demand for bioactive substances from olive by$
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products, this study was undertaken to evaluate the content in phenolic and triterpenic
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compounds of the main by$products generated in the olive oil mills such as Alperujo,
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leaves, olive oil lees and olive pomace skins.
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Most samples were obtained from olive oil mill Cooperatives belonging to
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Jaencoop SCA located in the south of Spain. They were taken between December and
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January of the 2013/2014 season.
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Fresh Alperujo was acquired from three olive oil mill Cooperatives, three
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samples from each cooperative on three consecutive days, at the beginning of the
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2013/2014 harvesting season (December) and another nine samples at the end of the
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season (January). All samples were transferred to the laboratory and immediately
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analyzed on the same day without any storage period. The Picual cultivar was processed
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in the three Cooperatives, and the centrifugation equipment was the two$phase system.
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Samples of olive leaves were obtained from one olive oil mill Cooperative at the
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beginning and end of the 2013/2014 season. In accordance with current practice, olives
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of the Picual cultivar were harvested and transported to the Cooperative facilities where
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leaves and small branches were removed. The olive factory has two different olive oil
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extraction lines for fruits that were tree$picked or ground$picked thereby leaves from
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these two different types of harvesting were analyzed.
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Twenty samples of olive pomace skin were obtained from 15 olive oil mill
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Cooperatives located in the south of Spain that had machines to separate the olive
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pomace skin from the pit fragments of the pomace. These samples were taken
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throughout the 2013/2014 harvesting season.
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The oily sediment of 5 storage tanks located in 5 different olive oil mill
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Cooperatives was taken from the bottom of the tanks containing virgin olive oil. They
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were centrifuged at 6000 x g for 5 min (22 ºC), and phenolic and triterpenic compounds
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were analyzed in the oily phase. The virgin olive oil had been preserved for one year in
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the tanks.
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The extraction of phenolic compounds from Alperujo was based on the
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methodology reported elsewhere.25 Around 10 g of fresh Alperujo were mixed in an
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Ultra$Turrax homogenizer (Ika, Breisgau, Germany) with 30 mL of dimethyl sulfoxide
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(DMSO). After 30 min of resting contact, the mixture was centrifuged at 6000 g for 5
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min (22 ºC) and 0.25 mL of the supernatant were diluted with 0.5 mL of DMSO plus
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0.25 mL of 0.2 mM of syringic acid in DMSO (internal standard). The extraction of
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these substances from the leaves was made similarly to the Alperujo as described above,
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but this time the mixing ratio was 2 g of olive leaf and 30 mL of DMSO.
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Samples were filtered through a 0.22 µm pore size nylon filter, and an aliquot
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(20 µL) was injected into the chromatograph. The chromatographic system consisted of
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a Waters 717 plus autosampler, a Waters 600 pump, a Waters column heater module,
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and a Waters 996 photodiode array detector operated with Empower2 software (Waters,
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Mildford, USA.). A 25 cm x 4.6 mm i. d., 5 µm, Spherisob ODS$2 (Waters, Inc.)
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column at a flow rate of 1 mL min$1 and a temperature of 35 ºC, was used in all
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experiments. Separation was achieved by gradient elution using (A) water (pH 2.5
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adjusted with 0.15% phosphoric acid) and (B) methanol. The initial composition was
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90% A and 10% B. The concentration of B was increased to 30% over 10 min and was
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maintained for 20 min. Subsequently, B was raised to 40% over 10 min, maintained for
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5 min, and then increased to 50%. Finally, B was increased to 60, 70, and 100% in 5$
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min periods. The initial conditions were reached in 10 min. Chromatograms were
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recorded at 280 nm.
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The evaluation of each compound was performed using a regression curve with
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the corresponding standard. Hydroxytyrosol, oleuropein, verbascoside, luteolin, luteolin
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7$glucoside, rutin, and apigenin were purchased from Extrasynthese S. A. (Genay,
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France) and tyrosol, caffeic, vanillicand $coumaric acids from Sigma Chemical Co. (St
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Louis, USA). Hydroxytyrosol$1$glucoside, caffeoyl ester of secologanoside, and
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comselogoside were quantified using the response factors of hydroxytyrosol, caffeic
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acid, and $coumaric acid, respectively. Salidroside and ligustroside were quantified
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using the response factor of tyrosol. Hydroxytyrosol$4$glucoside and the dialdehydic
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form of decarboxymethyl elenolic acid linked to hydroxytyrosol (HyEDA) were
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obtained using a HPLC preparative system.26
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They were extracted from the oil with 0,0$dimethylformamide (DMF).26
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Briefly, 0.6 g of oil were extracted with 3 x 0.6 mL of DMF; the extract was then
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washed with hexane, and N2 was bubbled into the DMF extract to eliminate residual
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hexane. Syringic acid (0.2 mM) was employed as internal standard. Finally, the extract
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was filtered through a 0.22 µm pore size nylon filter and injected into the
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chromatograph.
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The chromatographic system was the same as noted above except that a
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fluorescence detector was put in series with a DAD detector to monitor all the phenolic
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compounds. Hydroxytyrosol glycol and 4$ethylphenol were purchased from Sigma (St.
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Louis, USA), and hydroxytyrosol acetylated, pinoresinol, 1$acetoxypinoresinol,
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ligustrosideaglycon and the dialdehydic form of decarboxymethyl elenolic acid linked
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to tyrosol (TyEDA) were obtained using a preparative HPLC system.26
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The water content of Alperujo, leaves and olive pomace skin was determined by
weighing 10 g of the organ and then oven drying at 105 ºC to constant weight.
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' (
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)
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Ten grams of Alperujo or cut leaves were desiccated at 105 ºC until weight
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stabilization. Subsequently, 1 g of dry and triturated Alperujo or leaf was mixed in a 10
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mL centrifuge tube with 4 mL of methanol/ethanol (1:1, v/v) and vortexed for 1 min,
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centrifuged at 6000 g for 5 min at 20 ºC, and the solvent was separated from the solid
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phase. This step was repeated six times, and the pooled solvent extract was vacuum
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evaporated. The residue was dissolved in 2 mL of methanol, which was filtered through
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a 0.22 µm pore size nylon filter and an aliquot (20 µL) was injected into the liquid
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chromatograph. The chromatographic system and column were the same as those used
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for the phenolic compound analysis. The mobile phase (methanol/acidified water with
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phosphoric acid at pH 3.0, 92:8, v/v) was delivered to the column at a flow rate of 0.8
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mL min$1 and the eluate was monitored at 210 nm. Oleanolic and maslinic acids were
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quantified using external standards (Sigma, USA). The extraction of these substances
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from olive pomace skin was conducted using a procedure similar to that described
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above for Alperujo and leaf but, this time, the mixing ratio was 0.1 g of olive pomace
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skin and 4 mL of methanol/ethanol. The residue was dissolved in 10 mL methanol.
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The extraction of these substances from olive oil was done using a mixture of
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methanol/ethanol (1:1).18 The oil (0.8 g) and the alcoholic solvent (1.6 mL) were
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vortexed for 1 min, centrifuged at 6000 g for 5 min at 20 ºC, and the alcoholic phase
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was separated from the lipid phase. This step was repeated six times. Subsequently, the
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pooled alcoholic extract was vacuum evaporated and the residue dissolved in 2.4 mL
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methanol, which was centrifuged at 6000 g for 5 min at 20 ºC. Finally, the solution was
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filtered through 0.22 µm pore size nylon filter and an aliquot (20 µL) was injected into
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the liquid chromatograph. The chromatographic system and column were the same as
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described above.
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%
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Statistical comparisons of the mean values for each experiment were performed
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by one$way analysis of variance (ANOVA), followed by the Duncan’s multiple range
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test ( < 0.05) using Statistica software version 8.0 (Stat$Soft, Inc., Tulsa, USA).
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#% $ %
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Figure 1 presents the phenolic composition of fresh Alperujo obtained at the
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beginning
and
end
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the
2013/2014
season.
Hydroxytyrosol$4$glucoside,
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hydroxytyrosol and the dialdehydic form of decarboxymethyl elenolic acid linked to
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hydroxytyrosol (HyEDA) were the main phenolic compounds detected in this material,
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followed by verbascoside, tyrosol, salidroside and other substances in minor
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concentration (see Supporting information, Fig. 1S). This chemical characterization is in
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agreement with previous studies,15,16 and a trend was confirmed to lower concentration
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of these substances as the harvesting season progressed, except for hydroxytyrosol and
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tyrosol.16 It must be noted that the major phenolic compound in fresh olives is
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oleuropein, which can reach up to 20 g kg$1 in the olive pulp,8 whereas the total
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phenolic content in the Alperujo analyzed was 1$1.3 g kg$1. Taking into consideration
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that the transfer of phenolic compounds from the olive paste to the oil during the
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malaxation step is low and the differences between the mass molecular weight of
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oleuropein (540 u.m.a.) and hydroxytyrosol (154 u.m.a.),16,27 most of the phenolic
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compounds degraded or transformed during the malaxation step. Klen and Vodopivec
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(2012) estimated that just only half of the initial phenolic content in fresh olives
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remained in the extracted olive paste. In our case, we estimated that around 80% of the
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initial amount of phenolic compounds was lost during the extraction process of olive
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oil. It is well$known that oxidase enzymes act on phenolic compounds, particularly
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hydroxytyrosol and derivatives, to form quinones and polymers. Besides, esterases and
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especially β$glucosidase enhance the rupture of the oleuropein bonds during the milling
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and malaxation steps.
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With regard to the content of triterpenic acids in Alperujo, the concentration of
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these substances was rather similar in all the samples obtained from three different olive
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oil mill Cooperatives regardless of the time of sampling (Figure 2). The mean content
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was around 2.5 g kg$1, with maslinic acid being more abundant than oleanolic) (see
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Supporting information, Fig. 2S). These data are similar to those reported for the
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concentration of triterpenic acids in fresh olive pulp,8 which confirmed the low transfer
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of these substances to virgin olive oil during oil extraction.28 Therefore, Alperujo was
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disclosed as a rather good and reliable source of triterpenic acids, although the
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concentration of these substances in olives is also cultivar dependent.29
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Triterpenic acids are abundant in the plant kingdom and play a role in plant
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defense thereby they are concentrated in the skin of fruits such as olives.8,21,30 Hence,
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the new residue olive pomace skin generated in the olive oil mills must be rich in these
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substances, and it was confirmed on 20 samples analyzed of this new by$product
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(Figure 3). The concentration of triterpenic acids was very much higher than found in
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fresh Alperujo, although a great variability among samples was detected; total
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triterpenic acids ranged from 40 to 140 g kg$1, with maslinic acid representing around
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70% of the total. As it has been commented above, this residue comes from the cleaning
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of the pit fragments present in the olive pomace that contains pieces of olive skin
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together with small pieces of pulp. Because the moisture of these samples ranged from 7
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to 50%, a correlation between moisture and triterpenic acid concentration was studied
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but it failed since a higher concentration in triterpenic acids was not correlated with a
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lower content in moisture. The technology used for the separation of olive skin pomace
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from the pit fragments is new and there are many different variables such as olive
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cultivar and agronomic conditions that can be a reasonable explanation for the great
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variability observed in the samples analyzed. It is also known that the skin of olives is
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rich in phenolic compounds,25 particularly oleuropein, but the level of phenolic
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compounds in this new by$product was insignificant (data not shown). The high loss of
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phenolic compounds in Alperujo occurring during the malaxation step has been
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commented above, but new oxidation and degradative reactions must undergo at the
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further steps needed to obtain the olive skin pomace. Overall, a new by$product which
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is very rich in triterpenic acids has been identified in the olive oil mills.
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Leaves are the second by$product in importance generated in the olive oil mills
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without a valuable use. The chemical characterization of olive leaves has been carried
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out extensively on leaves picked from the tree.8,31 In this study, analyses were
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performed on leaves obtained from both tree$picked and ground$picked olives as they
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are currently separated for processing in the Spanish olive oil mills. The concentration
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of triterpenic acids in the olive leaves was very high (Figure 4), in particular that of
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oleanolic acid, which is in line with previous reports.8
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differences were found between ground and tree$picked leaves collected at the
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beginning of the harvesting season. By contrast, leaves from ground$picked olives
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collected at the end of the harvesting season showed a statistically higher content in
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triterpenic acids than those from tree$picked olives. This finding must be related to the
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lower concentration in moisture of the former leaves (20%) than the latter (38%). It
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seems that the loss in humidity of the olive leaves does not give rise to a reduction in
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the triterpenic acid content but enrichment in these substances is originated.
In addition, no statistical
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Moreover, leaves from ground$picked olives had a much lower concentration in
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phenolic compounds that those from tree$picked olives (Figure 5). It seems that
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oleuropein degraded during desiccation of the leaves on the ground. As leaves lose
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moisture, cells die, and a contact between phenolic compounds and degradative
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enzymes occurs giving rise to oxidative and hydrolytic reactions on the oleuropein
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moiety. Therefore, ground$picked leaves are a good source of triterpenic acids but not
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for phenolic compounds.
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Finally, the sediment of the bottom of the tanks where virgin olive oil is
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currently stored for months before commercialization was explored as a potential source
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of bioactive substances. This by$product is mainly composed of oil, followed by water,
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sugars and other substances. The phenolic and triterpene composition of the oil was
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analyzed (Figure 6). The phenolic compounds detected in this oil were the same as
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previously reported in virgin or extra virgin olive oil,26 and the total content ranged
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between 0.3$0.8 g kg$1, which is a relatively low content for considering their recovery.
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On the other hand, this oily phase of the sediment was enriched in triterpenic acids
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during storage of the oil because the concentration of these substances in virgin olive oil
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is currently lower than 0.1 g kg$1.28 However, these results mean that this by$product
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does not seem to be a good source of phenolic or triterpene bioactive substances.
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Olive oil mills generate a high amount of by$products that contain valuable
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bioactive substances whose recovery could contribute to the revalorization of these
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wastes. This study has explored the presence of phenolic and triterpene compounds in
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the main wastes produced in the Spanish olive oil mills. Among them, the olive pomace
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skin has been discovered as a very rich source of triterpenic acids that can reach up to
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120 g kg$1 of the waste, maslinic acid comprising around 70% of the total triterpenics.
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Alperujo has also been disclosed as an abundant and constant source of triterpenic and
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phenolic compounds although most of the latter substances are lost during the extracting
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process. Leaves can be used for the extraction of phenolic compounds, particularly
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oleuropein, but it has been found that those obtained from ground$picked olives lose
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their content in these substances. By contrast, triterpenic acids, mainly oleanolic, are
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very concentrated in this by$product regardless of the place where olives were picked.
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Finally, the sediment formed in the bottom of the storage tanks of olive oils does not
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seem to be a good source of bioactive substances.
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%
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We are grateful to the Cooperatives of the Jaencoop SCA group for the supply of
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olive material. This work was supported by the Spanish Government and European
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Feder funds (project ASOAN, Interconecta ITC$20111073). We thank Alejandra
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Expósito and Juan Antonio Espejo for technical assistance.
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#,# # !#%
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composting: enhancement of the composting rate and compost quality by grape
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2. Rodríguez$Lucena P, Hernández D, Hernández$Apaolaza L and Lucena, J J,
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and Outor$Monteiro D, Effects of the dietary incorporation of olive leaves on
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Journal of the Science of Food and Agriculture
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Acidification of Alperujo paste prevents off$odors during their storage in open
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Triterpenic acids in table
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400
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401
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32. Talhaoui, N, Gómez$Caravaca A, León L, de la Rosa R, Fernández$Gutiérrez, A
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404
olive fruits from six different cultivars. J Agric Food Chem 43:10466$10467
405
(2015).
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,
,
.. Phenolic compounds in Alperujo obtained from three different olive oil mill
Cooperatives at the beginning and the end of the harvesting season 2013/2014. Standard
deviation of nine samples is drawn on the bars. For each compound, vertical bars with
different letters indicate significant differences according to Duncan’s multiple$range
test ( < 0.05). Hy, hydroxytyrosol; HyEDA, dialdehydic form of decarboxymethyl
elenolic acid linked to hydroxytyrosol; Hy$4$glucoside, hydroxytyrosol$4$glucoside;
others is the sum of vanillic, caffeic and $coumaric acids, luteolin$7$glucoside, rutin,
ester of caffeic acid linked to secologanoside and comselogoside.
-8 Triterpenic acids in Alperujo obtained from three different olive oil mill
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,
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Cooperatives (A, B and C) at the beginning and the end of the harvesting season
2013/2014. Standard deviation of triplicates is drawn on the bars. For each season,
ee
vertical bars with different letters indicate significant differences according to Duncan’s
multiple$range test ( < 0.05).
,
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3. Concentration of triterpenic acids in 20 samples of olive pomace skin
ev
obtained from 15 olive oil mill factories. Standard deviation of duplicates is drawn on
the bars.
,
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28 Triterpenic acids content in leaves obtained from ground$picked and tree$
picked olives. Samples were obtained from a local olive oil mill Cooperative, and taken
at the beginning and the end of the harvesting season 2013/2014. For each season,
vertical bars with different letters indicate significant differences according to Duncan’s
multiple$range test ( < 0.05).
,
/8 Phenolic compound content in leaves obtained from tree$picked and ground$
picked olives. Samples were obtained from a local olive oil mill Cooperative at the end
of the harvesting season 2013/2014. Others are hydroxytyrosol$1$glucoside,
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Journal of the Science of Food and Agriculture
Page 16 of 24
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hydroxytyrosol$4glucoside, verbascoside, luteolin$7$glucoside, salidroside, rutin, ester
of caffeic acid linked to secologanoside and comselogoside.
,
48 Concentration of triterpenic acids and total phenolic compounds in several
samples of olive oils obtained from the bottom of the storage tanks after one year of
preservation. Samples were from 5 different olive oil Cooperatives (A$E) located in the
Jaen province. Standard deviation of triplicates is drawn on the bars. Phenolic
compounds quantified were hydroxytyrosol, hydroxytyrosol glycol, hydroxtyrosol
acetylated, tyrosol, pinoresinol, 1$acetoxy pinoresinol, 4$ethylphenol, luteolin, apigenin,
Fo
oleuropein and ligustroside aglycons, and the dialdehydic form of decarboxymethyl
elenolic acid linked to hydroxytyrosol and tyrosol.
.%8 HPLC chromatograms of phenolic compounds in alperujo and olive leaf. 1,
ee
,
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hydroxytyrosol, 2, hydroxytyrosol 1$glucoside; 3, hydroxytyrosol 4$glucoside; 4,
salidroside; 5, tyrosol; 6, vanillic acid; 7, caffeic acid; IS, internal standard; 8,
$
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coumaric acid; 9, verbascoside; 10, HyEDA; 11, luteolin 6$glucoside; 12, oleuropein;
13, rutin; 14, ester of caffeic acid linked to secologanoside; 15, comselogoside.
-%8 HPLC chromatograms of triterpenic acids in olive leaf (A), alperujo (B) and
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,
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olive pomace skin (C). 1, maslinic acid; 2, oleanolic acid.
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Figure 1
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Figure 2
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