New by-products rich in bioactive substances from the olive oil mill processing

Page 1 of 24 1 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. 2 3 4 5 6 Concepción Romeroa, Eduardo Medinaa, Mª Antonia Mateob, Manuel Brenesa,* 7 8 9 ! 10 12 ! ) # * * # $ % &%'%( + , -(((' + *. ! rR ee 11 " rP Fo ) / 13 *Correspondence to: Manuel Brenes, Food Biotechnology Department, Instituto de la 14 Grasa (IG$CSIC), Campus University Pablo de Olavide, Ctra. Utrera km 1, 41013$ 15 Seville, Spain. E$mail brenes@cica.es 16 ie 17 ev Running title: New olive by$products from olive oil mill processing w 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Journal of the Science of Food and Agriculture JSFA@wiley.com Journal of the Science of Food and Agriculture Page 2 of 24 2 18 19 BACKGROUND: Olive oil extraction generates a large amount of residue comprising 20 mainly of the pomace and leaves when using the two$phase centrifugation system. The 21 aim of this study was to assess the content of phenolic and triterpene compounds in the 22 by$products produced in Spanish olive oil mills. 23 RESULTS: The olive pomace had lower concentrations than 2 and 3 g kg$1 of phenolic 24 and triterpene substances, respectively. The leaves contained a high concentration in 25 these substances although those collected from ground$picked olives had lost most of 26 their phenolic compounds. Moreover, the sediment from the bottom of the olive oil 27 storage tanks did not have a significant amount of these substances. By contrast, the 28 new by$product named olive pomace skin has been revealed as a very rich source of 29 triterpenic acids because this waste can reach up to 120 g kg$1 of these compounds, 30 maslinic acid comprising around 70 % of total triterpenics. 31 CONCLUSIONS: Among the by$products generated during extraction of olive oil, the 32 olive pomace skin has been discovered as a very rich source of triterpenic acids that can 33 reach up to 120 g kg$1 of the waste. These results will contribute to the valorization of 34 olive oil by$products. ev rR ee 36 rP 35 Fo : olive; phenolic; triterpene; oleuropein; maslinic; oleanolic ie 37 w 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 38 39 JSFA@wiley.com Page 3 of 24 Journal of the Science of Food and Agriculture 3 ! 40 41 Olive leaves and olive mill pomace are the two main wastes generated during the 42 extraction of olive oil by the two$phase centrifugation system. In Spain, more than 4 43 and 0.2 million tons of olive pomace and leaves are annually produced, respectively. 44 These by$products are used for composting, combustion, animal feed and soil 45 amendment among others.1$4 However, they are also rich in bioactive substances such 46 as phenolic compounds and triterpenic acids that could contribute to the revalorization 47 of these by$products.5 48 There are many methods and patents to extract phenolic compounds and 49 triterpenic acids from olive leaves because fresh olive leaves may contain up to 70 g kg$ 50 1 51 and oleanolic acid being the main phenolic and triterpene substances in this material.8 52 These bioactive substances have been analyzed in many fresh olive leaf cultivars during 53 olive maturation, under different agronomic conditions and taking into account many 54 other variables.9$12 However, the olive leaf by$product generated in the Spanish olive 55 oil mills comes from two sources, ground$picked or tree$picked olives, which have not 56 been characterized. Fo and 20 g kg$1 of phenolic compounds and triterpenic acids respectively; 6,7 oleuropein rR ee rP 57 Hundreds of articles describe the phenolic composition of olive oil mill 58 wastewaters produced during the extraction of olive oil by the three$phase 59 centrifugation system.13,14 The characterization of these substances in the two$phase 60 olive pomace has also been reported.15$17 61 composition of this olive pomace, which is named “Alperujo” in Spain, are scarce.18,19 ie ev However, studies on the triterpene w 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 62 Alperujo is a semi$solid waste generated in the Spanish olive oil mills that is 63 stored in large open air ponds for several months until the residual oil is extracted by 64 physical or chemical methods in the oil extracting plants.20 The de$oiled pomace is used 65 for co$generation of electrical power. Currently, large pit fragments are separated from 66 fresh Alperujo in the olive oil mills to use them for combustion. However, pieces of 67 skin and olive pulp are adhered to these pit fragments and they are separated to obtain 68 pits which are free of pulp. Consequently, a new by$product is generated at large scale 69 at the olive oil mills that consists mainly of olive skin together with small pieces of 70 pulp. This by$product is used for combustion or spreading on the soil but it has attracted 71 the attention of enterprises due to its potential content in bioactive substances, JSFA@wiley.com Journal of the Science of Food and Agriculture Page 4 of 24 4 72 particularly triterpenic acids. It must be highlighted that triterpenic acids are mainly 73 concentrated in the skin of fruits,21,22 although no data are available about the 74 composition of this new by$product, rich in the olive skin. 75 Another by$product produced in the olive oil mills is the sediment that settles at 76 the bottom of the olive oil tanks during the storage of the oil before commercialization. 77 This olive oil lees is composed mainly of fat and water, and it is intended for refining or 78 making soap. The phenolic characterization of the solid and aqueous components of this 79 by$product has been reported but not the composition of the oily phase, particularly 80 triterpenic acids.23,24 Fo 81 Considering the industrial demand for bioactive substances from olive by$ 82 products, this study was undertaken to evaluate the content in phenolic and triterpenic 83 compounds of the main by$products generated in the olive oil mills such as Alperujo, 84 leaves, olive oil lees and olive pomace skins. 85 86 " 88 % # $% "# & % ev 87 rR ee rP 89 Most samples were obtained from olive oil mill Cooperatives belonging to 90 Jaencoop SCA located in the south of Spain. They were taken between December and 91 January of the 2013/2014 season. w ie 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 92 Fresh Alperujo was acquired from three olive oil mill Cooperatives, three 93 samples from each cooperative on three consecutive days, at the beginning of the 94 2013/2014 harvesting season (December) and another nine samples at the end of the 95 season (January). All samples were transferred to the laboratory and immediately 96 analyzed on the same day without any storage period. The Picual cultivar was processed 97 in the three Cooperatives, and the centrifugation equipment was the two$phase system. 98 Samples of olive leaves were obtained from one olive oil mill Cooperative at the 99 beginning and end of the 2013/2014 season. In accordance with current practice, olives 100 of the Picual cultivar were harvested and transported to the Cooperative facilities where JSFA@wiley.com Page 5 of 24 Journal of the Science of Food and Agriculture 5 101 leaves and small branches were removed. The olive factory has two different olive oil 102 extraction lines for fruits that were tree$picked or ground$picked thereby leaves from 103 these two different types of harvesting were analyzed. 104 Twenty samples of olive pomace skin were obtained from 15 olive oil mill 105 Cooperatives located in the south of Spain that had machines to separate the olive 106 pomace skin from the pit fragments of the pomace. These samples were taken 107 throughout the 2013/2014 harvesting season. 108 The oily sediment of 5 storage tanks located in 5 different olive oil mill 109 Cooperatives was taken from the bottom of the tanks containing virgin olive oil. They 110 were centrifuged at 6000 x g for 5 min (22 ºC), and phenolic and triterpenic compounds 111 were analyzed in the oily phase. The virgin olive oil had been preserved for one year in 112 the tanks. ee 114 rP 113 Fo ' 115 The extraction of phenolic compounds from Alperujo was based on the 116 methodology reported elsewhere.25 Around 10 g of fresh Alperujo were mixed in an 117 Ultra$Turrax homogenizer (Ika, Breisgau, Germany) with 30 mL of dimethyl sulfoxide 118 (DMSO). After 30 min of resting contact, the mixture was centrifuged at 6000 g for 5 119 min (22 ºC) and 0.25 mL of the supernatant were diluted with 0.5 mL of DMSO plus 120 0.25 mL of 0.2 mM of syringic acid in DMSO (internal standard). The extraction of 121 these substances from the leaves was made similarly to the Alperujo as described above, 122 but this time the mixing ratio was 2 g of olive leaf and 30 mL of DMSO. w ie ev rR 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 123 Samples were filtered through a 0.22 µm pore size nylon filter, and an aliquot 124 (20 µL) was injected into the chromatograph. The chromatographic system consisted of 125 a Waters 717 plus autosampler, a Waters 600 pump, a Waters column heater module, 126 and a Waters 996 photodiode array detector operated with Empower2 software (Waters, 127 Mildford, USA.). A 25 cm x 4.6 mm i. d., 5 µm, Spherisob ODS$2 (Waters, Inc.) 128 column at a flow rate of 1 mL min$1 and a temperature of 35 ºC, was used in all 129 experiments. Separation was achieved by gradient elution using (A) water (pH 2.5 130 adjusted with 0.15% phosphoric acid) and (B) methanol. The initial composition was 131 90% A and 10% B. The concentration of B was increased to 30% over 10 min and was JSFA@wiley.com Journal of the Science of Food and Agriculture Page 6 of 24 6 132 maintained for 20 min. Subsequently, B was raised to 40% over 10 min, maintained for 133 5 min, and then increased to 50%. Finally, B was increased to 60, 70, and 100% in 5$ 134 min periods. The initial conditions were reached in 10 min. Chromatograms were 135 recorded at 280 nm. 136 The evaluation of each compound was performed using a regression curve with 137 the corresponding standard. Hydroxytyrosol, oleuropein, verbascoside, luteolin, luteolin 138 7$glucoside, rutin, and apigenin were purchased from Extrasynthese S. A. (Genay, 139 France) and tyrosol, caffeic, vanillicand $coumaric acids from Sigma Chemical Co. (St 140 Louis, USA). Hydroxytyrosol$1$glucoside, caffeoyl ester of secologanoside, and 141 comselogoside were quantified using the response factors of hydroxytyrosol, caffeic 142 acid, and $coumaric acid, respectively. Salidroside and ligustroside were quantified 143 using the response factor of tyrosol. Hydroxytyrosol$4$glucoside and the dialdehydic 144 form of decarboxymethyl elenolic acid linked to hydroxytyrosol (HyEDA) were 145 obtained using a HPLC preparative system.26 ee rP Fo 146 147 rR 148 They were extracted from the oil with 0,0$dimethylformamide (DMF).26 149 Briefly, 0.6 g of oil were extracted with 3 x 0.6 mL of DMF; the extract was then 150 washed with hexane, and N2 was bubbled into the DMF extract to eliminate residual 151 hexane. Syringic acid (0.2 mM) was employed as internal standard. Finally, the extract 152 was filtered through a 0.22 µm pore size nylon filter and injected into the 153 chromatograph. w ie ev 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 154 The chromatographic system was the same as noted above except that a 155 fluorescence detector was put in series with a DAD detector to monitor all the phenolic 156 compounds. Hydroxytyrosol glycol and 4$ethylphenol were purchased from Sigma (St. 157 Louis, USA), and hydroxytyrosol acetylated, pinoresinol, 1$acetoxypinoresinol, 158 ligustrosideaglycon and the dialdehydic form of decarboxymethyl elenolic acid linked 159 to tyrosol (TyEDA) were obtained using a preparative HPLC system.26 160 161 " JSFA@wiley.com Page 7 of 24 Journal of the Science of Food and Agriculture 7 162 163 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. 164 ' ( 165 ) 166 Ten grams of Alperujo or cut leaves were desiccated at 105 ºC until weight 167 stabilization. Subsequently, 1 g of dry and triturated Alperujo or leaf was mixed in a 10 168 mL centrifuge tube with 4 mL of methanol/ethanol (1:1, v/v) and vortexed for 1 min, 169 centrifuged at 6000 g for 5 min at 20 ºC, and the solvent was separated from the solid 170 phase. This step was repeated six times, and the pooled solvent extract was vacuum 171 evaporated. The residue was dissolved in 2 mL of methanol, which was filtered through 172 a 0.22 µm pore size nylon filter and an aliquot (20 µL) was injected into the liquid 173 chromatograph. The chromatographic system and column were the same as those used 174 for the phenolic compound analysis. The mobile phase (methanol/acidified water with 175 phosphoric acid at pH 3.0, 92:8, v/v) was delivered to the column at a flow rate of 0.8 176 mL min$1 and the eluate was monitored at 210 nm. Oleanolic and maslinic acids were 177 quantified using external standards (Sigma, USA). The extraction of these substances 178 from olive pomace skin was conducted using a procedure similar to that described 179 above for Alperujo and leaf but, this time, the mixing ratio was 0.1 g of olive pomace 180 skin and 4 mL of methanol/ethanol. The residue was dissolved in 10 mL methanol. ev rR ee rP Fo 181 ie 182 w 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 183 The extraction of these substances from olive oil was done using a mixture of 184 methanol/ethanol (1:1).18 The oil (0.8 g) and the alcoholic solvent (1.6 mL) were 185 vortexed for 1 min, centrifuged at 6000 g for 5 min at 20 ºC, and the alcoholic phase 186 was separated from the lipid phase. This step was repeated six times. Subsequently, the 187 pooled alcoholic extract was vacuum evaporated and the residue dissolved in 2.4 mL 188 methanol, which was centrifuged at 6000 g for 5 min at 20 ºC. Finally, the solution was 189 filtered through 0.22 µm pore size nylon filter and an aliquot (20 µL) was injected into 190 the liquid chromatograph. The chromatographic system and column were the same as 191 described above. JSFA@wiley.com Journal of the Science of Food and Agriculture Page 8 of 24 8 192 % 193 Statistical comparisons of the mean values for each experiment were performed 194 by one$way analysis of variance (ANOVA), followed by the Duncan’s multiple range 195 test ( < 0.05) using Statistica software version 8.0 (Stat$Soft, Inc., Tulsa, USA). 196 197 198 #% $ % %! %% Figure 1 presents the phenolic composition of fresh Alperujo obtained at the 199 beginning and end of the 2013/2014 season. Hydroxytyrosol$4$glucoside, 200 hydroxytyrosol and the dialdehydic form of decarboxymethyl elenolic acid linked to 201 hydroxytyrosol (HyEDA) were the main phenolic compounds detected in this material, 202 followed by verbascoside, tyrosol, salidroside and other substances in minor 203 concentration (see Supporting information, Fig. 1S). This chemical characterization is in 204 agreement with previous studies,15,16 and a trend was confirmed to lower concentration 205 of these substances as the harvesting season progressed, except for hydroxytyrosol and 206 tyrosol.16 It must be noted that the major phenolic compound in fresh olives is 207 oleuropein, which can reach up to 20 g kg$1 in the olive pulp,8 whereas the total 208 phenolic content in the Alperujo analyzed was 1$1.3 g kg$1. Taking into consideration 209 that the transfer of phenolic compounds from the olive paste to the oil during the 210 malaxation step is low and the differences between the mass molecular weight of 211 oleuropein (540 u.m.a.) and hydroxytyrosol (154 u.m.a.),16,27 most of the phenolic 212 compounds degraded or transformed during the malaxation step. Klen and Vodopivec 213 (2012) estimated that just only half of the initial phenolic content in fresh olives 214 remained in the extracted olive paste. In our case, we estimated that around 80% of the 215 initial amount of phenolic compounds was lost during the extraction process of olive 216 oil. It is well$known that oxidase enzymes act on phenolic compounds, particularly 217 hydroxytyrosol and derivatives, to form quinones and polymers. Besides, esterases and 218 especially β$glucosidase enhance the rupture of the oleuropein bonds during the milling 219 and malaxation steps. w ie ev rR ee rP Fo 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 220 With regard to the content of triterpenic acids in Alperujo, the concentration of 221 these substances was rather similar in all the samples obtained from three different olive 222 oil mill Cooperatives regardless of the time of sampling (Figure 2). The mean content JSFA@wiley.com Page 9 of 24 Journal of the Science of Food and Agriculture 9 223 was around 2.5 g kg$1, with maslinic acid being more abundant than oleanolic) (see 224 Supporting information, Fig. 2S). These data are similar to those reported for the 225 concentration of triterpenic acids in fresh olive pulp,8 which confirmed the low transfer 226 of these substances to virgin olive oil during oil extraction.28 Therefore, Alperujo was 227 disclosed as a rather good and reliable source of triterpenic acids, although the 228 concentration of these substances in olives is also cultivar dependent.29 229 Triterpenic acids are abundant in the plant kingdom and play a role in plant 230 defense thereby they are concentrated in the skin of fruits such as olives.8,21,30 Hence, 231 the new residue olive pomace skin generated in the olive oil mills must be rich in these 232 substances, and it was confirmed on 20 samples analyzed of this new by$product 233 (Figure 3). The concentration of triterpenic acids was very much higher than found in 234 fresh Alperujo, although a great variability among samples was detected; total 235 triterpenic acids ranged from 40 to 140 g kg$1, with maslinic acid representing around 236 70% of the total. As it has been commented above, this residue comes from the cleaning 237 of the pit fragments present in the olive pomace that contains pieces of olive skin 238 together with small pieces of pulp. Because the moisture of these samples ranged from 7 239 to 50%, a correlation between moisture and triterpenic acid concentration was studied 240 but it failed since a higher concentration in triterpenic acids was not correlated with a 241 lower content in moisture. The technology used for the separation of olive skin pomace 242 from the pit fragments is new and there are many different variables such as olive 243 cultivar and agronomic conditions that can be a reasonable explanation for the great 244 variability observed in the samples analyzed. It is also known that the skin of olives is 245 rich in phenolic compounds,25 particularly oleuropein, but the level of phenolic 246 compounds in this new by$product was insignificant (data not shown). The high loss of 247 phenolic compounds in Alperujo occurring during the malaxation step has been 248 commented above, but new oxidation and degradative reactions must undergo at the 249 further steps needed to obtain the olive skin pomace. Overall, a new by$product which 250 is very rich in triterpenic acids has been identified in the olive oil mills. w ie ev rR ee rP Fo 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 251 Leaves are the second by$product in importance generated in the olive oil mills 252 without a valuable use. The chemical characterization of olive leaves has been carried 253 out extensively on leaves picked from the tree.8,31 In this study, analyses were 254 performed on leaves obtained from both tree$picked and ground$picked olives as they 255 are currently separated for processing in the Spanish olive oil mills. The concentration JSFA@wiley.com Journal of the Science of Food and Agriculture Page 10 of 24 10 256 of triterpenic acids in the olive leaves was very high (Figure 4), in particular that of 257 oleanolic acid, which is in line with previous reports.8 258 differences were found between ground and tree$picked leaves collected at the 259 beginning of the harvesting season. By contrast, leaves from ground$picked olives 260 collected at the end of the harvesting season showed a statistically higher content in 261 triterpenic acids than those from tree$picked olives. This finding must be related to the 262 lower concentration in moisture of the former leaves (20%) than the latter (38%). It 263 seems that the loss in humidity of the olive leaves does not give rise to a reduction in 264 the triterpenic acid content but enrichment in these substances is originated. In addition, no statistical 265 Moreover, leaves from ground$picked olives had a much lower concentration in 266 phenolic compounds that those from tree$picked olives (Figure 5). It seems that 267 oleuropein degraded during desiccation of the leaves on the ground. As leaves lose 268 moisture, cells die, and a contact between phenolic compounds and degradative 269 enzymes occurs giving rise to oxidative and hydrolytic reactions on the oleuropein 270 moiety. Therefore, ground$picked leaves are a good source of triterpenic acids but not 271 for phenolic compounds. rR ee rP Fo 272 Finally, the sediment of the bottom of the tanks where virgin olive oil is 273 currently stored for months before commercialization was explored as a potential source 274 of bioactive substances. This by$product is mainly composed of oil, followed by water, 275 sugars and other substances. The phenolic and triterpene composition of the oil was 276 analyzed (Figure 6). The phenolic compounds detected in this oil were the same as 277 previously reported in virgin or extra virgin olive oil,26 and the total content ranged 278 between 0.3$0.8 g kg$1, which is a relatively low content for considering their recovery. 279 On the other hand, this oily phase of the sediment was enriched in triterpenic acids 280 during storage of the oil because the concentration of these substances in virgin olive oil 281 is currently lower than 0.1 g kg$1.28 However, these results mean that this by$product 282 does not seem to be a good source of phenolic or triterpene bioactive substances. 283 ! w ie ev 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 !$ % % 284 Olive oil mills generate a high amount of by$products that contain valuable 285 bioactive substances whose recovery could contribute to the revalorization of these 286 wastes. This study has explored the presence of phenolic and triterpene compounds in 287 the main wastes produced in the Spanish olive oil mills. Among them, the olive pomace JSFA@wiley.com Page 11 of 24 Journal of the Science of Food and Agriculture 11 288 skin has been discovered as a very rich source of triterpenic acids that can reach up to 289 120 g kg$1 of the waste, maslinic acid comprising around 70% of the total triterpenics. 290 Alperujo has also been disclosed as an abundant and constant source of triterpenic and 291 phenolic compounds although most of the latter substances are lost during the extracting 292 process. Leaves can be used for the extraction of phenolic compounds, particularly 293 oleuropein, but it has been found that those obtained from ground$picked olives lose 294 their content in these substances. By contrast, triterpenic acids, mainly oleanolic, are 295 very concentrated in this by$product regardless of the place where olives were picked. 296 Finally, the sediment formed in the bottom of the storage tanks of olive oils does not 297 seem to be a good source of bioactive substances. Fo 298 299 ! rP *$# +#"# % 300 We are grateful to the Cooperatives of the Jaencoop SCA group for the supply of 301 olive material. This work was supported by the Spanish Government and European 302 Feder funds (project ASOAN, Interconecta ITC$20111073). We thank Alejandra 303 Expósito and Juan Antonio Espejo for technical assistance. 304 #,# # !#% ev 305 rR ee 306 1. Cayuela M L, Sánchez$Monedero M A and Roig A, Two$phase olive mil waste 307 composting: enhancement of the composting rate and compost quality by grape 308 stalks addition. 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García A, Brenes M, Romero C, García P and Garrido A, Use of 1$ 387 acetoxypinoresinol to authenticate Picual olive oils. Int J Food Sci Technol 388 36:615$625 (2002). 389 27. Klen T J and Vodopivec B M, The fate of olive fruit phenols during commercial 390 olive oil processing: traditional press 391 centrifuge. LWT$Food Sci Technol 217267$274 (2012). 392 28. Pérez$Camino M Fo C and continuous two$and three$phase Cert A, Quantitative determination of 393 hydroxypentacyclic triterpene acids in vegetable oils. J Agric Food Chem 394 26:1558$1562 (1999). 395 396 rP 29. Romero C, García A, Medina E, Ruíz$Méndez M V, de Castro and Brenes M, Triterpenic acids in table 397 30. olives. Food Chem ..0:670$674 (2010). 398 31. Olmo$García L, Bajoub A, Fernández$Gutiérrez A and Carrasco$Pancorbo A, 399 Evaluating the potential of LC coupled to three alternative detection systems 400 (ESI$IT, APCI$TOF and DAD) for the targeted determination of triterpenic 401 acids and dialcohols in olive tissues. Talanta ./57355$366 (2016). rR ee 402 32. Talhaoui, N, Gómez$Caravaca A, León L, de la Rosa R, Fernández$Gutiérrez, A 403 and Segura$Carretero A, Pattern of variation of fruit traits and phenol content in 404 olive fruits from six different cultivars. J Agric Food Chem 43:10466$10467 405 (2015). w ie ev 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 406 407 408 409 JSFA@wiley.com Page 15 of 24 Journal of the Science of Food and Agriculture 15 , , .. Phenolic compounds in Alperujo obtained from three different olive oil mill Cooperatives at the beginning and the end of the harvesting season 2013/2014. Standard deviation of nine samples is drawn on the bars. For each compound, vertical bars with different letters indicate significant differences according to Duncan’s multiple$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 rP , Fo 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$range test ( < 0.05). , rR 3. Concentration of triterpenic acids in 20 samples of olive pomace skin ev obtained from 15 olive oil mill factories. Standard deviation of duplicates is drawn on the bars. , w ie 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 28 Triterpenic acids content in leaves obtained from ground$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, JSFA@wiley.com Journal of the Science of Food and Agriculture Page 16 of 24 16 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$ethylphenol, luteolin, apigenin, Fo oleuropein and ligustroside aglycons, and the dialdehydic form of decarboxymethyl elenolic acid linked to hydroxytyrosol and tyrosol. .%8 HPLC chromatograms of phenolic compounds in alperujo and olive leaf. 1, ee , rP hydroxytyrosol, 2, hydroxytyrosol 1$glucoside; 3, hydroxytyrosol 4$glucoside; 4, salidroside; 5, tyrosol; 6, vanillic acid; 7, caffeic acid; IS, internal standard; 8, $ rR 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 ie , ev olive pomace skin (C). 1, maslinic acid; 2, oleanolic acid. w 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 JSFA@wiley.com Page 17 of 24 Figure 1 w ie ev rR ee rP Fo 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Journal of the Science of Food and Agriculture JSFA@wiley.com Journal of the Science of Food and Agriculture Figure 2 w ie ev rR ee rP Fo 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 JSFA@wiley.com Page 18 of 24 Page 19 of 24 Figure 3 rP Fo 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 w ie ev rR ee 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Journal of the Science of Food and Agriculture JSFA@wiley.com Journal of the Science of Food and Agriculture Figure 4 w ie ev rR ee rP Fo 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 JSFA@wiley.com Page 20 of 24 Page 21 of 24 Figure 5 w ie ev rR ee rP Fo 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Journal of the Science of Food and Agriculture JSFA@wiley.com Journal of the Science of Food and Agriculture Figure 6 w ie ev rR ee rP Fo 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 JSFA@wiley.com Page 22 of 24 Page 23 of 24 Figure 1S w ie ev rR ee rP Fo 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Journal of the Science of Food and Agriculture JSFA@wiley.com Journal of the Science of Food and Agriculture Figure 2S w ie ev rR ee rP Fo 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 JSFA@wiley.com Page 24 of 24