Influence of refining processes on content of bioactive compounds, rheology, and texture of olive pomace oil for use in topical formulations

Resea ch A icle Influence o efining p ocesses on con en o bioac i e compounds, heology, and ex u e o oli e pomace oil o use in opical o mula ions Ca la And ea S anchez-Gu i e ez 1,2 , Ma ia Vic o ia Ruiz-M endez 2  , Ma ia Rosa Jim enez-Cas ellanos 1  and Ma ia Jes us Luce o 1 1 Facul ad de Fa macia, Depa amen o de Fa macia y Tecnolog ıa Fa mac eu ica, Uni e sidad de Se illa, Spain 2 Ins i u o de la G asa, Consejo Supe io de In es igaciones Cien  ıficas, Se illa, Spain Re aining he bioac i e ing edien s o oli e pomace oil is a c ucial s ep in ensu ing hei unc ional o pha maceu ical alue. To help selec he bes me hod o e aining bioac i e compounds in oli e pomace oil, h ee efining p ocesses, namely, chemical, classical physical, o molecula dis illa ion, we e compa ed a di e en empe a u es. Fo each me hod, he con en s o iacylglyce ols, a y acids (oleic acid, linoleic, and linolenic acids), i e penic acids (oleanolic and maslinic acids), and unsaponifiable ma e (squalene, alipha ic, s e ols, and e penic) we e s udied. We obse ed ha physical efining by molecula dis illa ion p o ided oils wi h in e es ing amoun s o bioac i e compounds, especially i e penic acids. O hese oils, he samples submi ed o a empe a u e o 190°C exhibi ed a g ea e albei low acidi y compa ed wi h he o he oils ob ained by chemical and classical physical efining bu exhibi ed highe amoun s o all bioac i e compounds. Con e sely, he molecula dis illa ion efining p ocess had only a sligh e ec on he alues o he consis ency index. The fi mness, cohesi eness, and adhesi eness pa ame e s om ex u e p ofile analysis indica ed ha in gene al, all deodo ized oils had high smoo hness and sp eadabili y bu low adhesi eness. Molecula dis illa ion was concluded o be he bes efining p ocess. Ne e heless, i is necessa y o imp o e he wo king condi ions o his p ocess. P ac ical applica ions: Oli e pomace oil has all unc ional compounds o ex a i gin oli e oil, bu wi h a highe concen a ion o some mino componen s. I is, he e o e, e y in e es ing o use i in skin ca e o mula ions. Howe e , o use in he pha maceu ical field mus be efined. This s udy aimed o p o ide de e mine he influence o di e en efining p ocesses (chemical efining, classical physical efining, o physical efining by molecula dis illa ion a di e en empe a u es) in de e mining he composi ion o bioac i e compounds. The bes efining p ocess was selec ed based on he analy ical de e mina ion o bioac i e compounds by gas ch oma og aphy. Resul s indica ed ha molecula dis illa ion is he bes efining p ocess. Ne e heless, i is necessa y o imp o e he wo king condi ions. Keywo ds: Bioac i e compounds / Oli e pomace oil / Re ining p ocesses / Rheology / Topical o mula ions Recei ed: Sep embe 29, 2016 / Re ised: Ma ch 6, 2017 / Accep ed: Ma ch 13, 2017 DOI: 10.1002/ejl .201600408 1 In oduc ion Oli es a e subjec ed o a mechanical ex ac ion p ocess in o de o sepa a e he di e en pa s ha make up he ui and ob ain he pomace (solid esidue), ex a i gin oli e oil (EVOO juice), and ege able wa e . EVOO has been used since ancien imes as a na u al p oduc o skin-ca e o mula ions and cosme ics [1]. Howe e , oli e pomace oil (OPOC), ob ained om pomace, has always been disca ded o used in o he echnological fields. Ne e heless, in ecen yea s, he e has been g owing in e es in OPOC due o i s Co espondence: P o . Ma ia Jesus Luce o, Facul ad de Fa macia, Dp o. de Fa macia y Tecnolog ıa Fa mac eu ica C/ P o . Ga c ıa Gonz alez n° 2, 41012-Se illa, Spain E-mail: [email p o ec ed] Abb e ia ions: EVOO, ex a i gin oli e oil; OPOC, oli e pomace oil; TPA, ex u e p ofile analysis Addi ional co esponding au ho : P o . Ma ia Rosa Jim enez-Cas ellanos, E-mail: m [email protected] Addi ional co esponding au ho : M. Vic o ia Ruiz-M endez, E-mail: [email p o ec ed] Eu . J. Lipid Sci. Technol. 2017, 119, 1600408 1600408 (1 o 10) www.ejls .comß2017 WILEY-VCH Ve lag GmbH & Co. KGaA, Weinheim composi ion because i con ains all he unc ional com- pounds o EVOO bu wi h a highe concen a ion o some mino componen s [2]. Thus, in dec easing o de o concen a ion, he bioac i e compounds a e: (i) iacylgly- ce ols as he main componen (>95%); (ii) a y acids, such as oleic acid, used in cosme ic and pha maceu ical o mula- ion, and linoleic and linolenic acids, which influence he me abolic p ocesses o he skin and p omo e ce amide o ma ion [3] and he ac i i y o i amins A and E [4]; (iii) i e penic acids, in pa icula oleanolic and maslinic acids, wi h an i umo , an i-inflamma o y, and bac e icidal ac i i- ies [5]; and (i ) unsaponifiable ma e composed o squalene, used as an emollien o keep skin so and elas ic. Addi ionally, i has an ioxidan p ope ies due o i s isop ene s uc u e [6]. Alipha ic alcohols supp ess he elease o a ious inflamma o y media o s [7]; s e ols and e penic alcohols (e y h odiol and u aol) ha e posi i e e ec s on he inflamma o y p ocess [8]. Chemical and mechanical ex ac ions can be used o ob ain OPOC. In a p e ious pape [9], i was demons a ed ha mechanical ex ac ion p o ides a g ea e pe cen age o he main compounds ( iacylglyce ols). Howe e , due o he high acidi y o he OPOC, i canno be used in pha maceu- icals. This is why he OPOC mus be subjec ed o a efining p ocess, bu wi h minimum losses o bioac i e compounds [10]. The e a e wo efining p ocedu es, chemical (C) o physical (P), wi h di e en s eps. The common me hod o efining oil is by eac ing i wi h an alkali solu ion, which neu alizes he ee a y acids (chemical efining). Ruiz- Mendez e al. [11] demons a ed ha physical p ocedu es esul in minimal losses o bioac i e compounds, such as i e penic acids in he case o OPOC. In physical efining, ege able oils can be neu alized by ee a y acid dis illa ion a high empe a u es, low p essu es, and wi h s eam s eam. Howe e , physical efining can only be applied a e mechanical ex ac ion. This is because he OPOC om chemical ex ac ion has a high con en o chlo ophyll, phospha ides, ca o enes, and o he pollu an s ha canno be emo ed by simple physical means [12]. Gi en he abo e, he aim o his wo k was o s udy he influence o efining p ocesses in de e mining he composi ion o bioac i e compounds o use in opical o mula ions. Fo quan i a i e e alua ion o he compo- si ion, wo ypes o OPOC we e selec ed, ob ained by chemical o mechanical ex ac ion. These oils we e subjec ed o a ious efining p ocesses (chemical efining, classical physical efining, o physical efining by molecula dis illa ion a di e en empe a u es). The bes efining p ocess was selec ed on he basis o he analy ical de e mina ion o bioac i e compounds by gas ch oma og aphy. Finally, heological cha ac e iza ion and ex u e p ofile analysis (TPAs) a e discussed as con enien me hods o mechanical cha ac e iza ion o opical oli e pomace oils (OPOCs). 2 Ma e ials and me hods 2.1 Ma e ials The aw ma e ials used o his pape we e OPOC om he 2012–2013 campaign and we e labeled as ollows: OPOC ob ained by chemical ex ac ion (OPOC C) and, wo OPOCs ob ained by mechanical ex ac ion (OPOC MA and OPOC MB). All OPOCs we e efined a he labo a o y scale. The e o e, OPOC C and OPOC MA we e subjec ed o a chemical efining p ocess, and he ob ained p oduc s we e labeled as OPOD CC and OPOD MAC, espec i ely. On he o he hand, OPOC MA and OPOC MB we e efined by classical physical efining and physical efining by molecula dis illa ion a di e en empe a u es, and he ob ained p oduc s we e labeled as OPOD MAP and OPOD MBP, espec i ely (Fig. 1). Di e en eagen s and sol en s we e used: hexane, die hy l e he , isop opyl e he , e hanol, chlo o o m, dichlo o- me hane phospho ic acid, me hanol, py idine, ace one, ace ic acid, sul u ic acid, po assium hyd oxide, sodium me hyla e, sodium hyd oxide, e hyl ace a e, phenolph ha- lein, and HPLC g ade ace oni ile we e pu chased om Pan eac SA (Ba celona, Spain). T ime hyl chlo osilane, hexame hyl disiloxane, and Sudan I we e pu chased om Sigma–Ald ich (Da ms ad , Ge many). Fil e aids we e supplied by Dicali e (Gen , Belgium). Tonsil sup eme 114 FF was ob ained om S€ ud-Chemie (Toledo, Spain). All eagen s and sol en s used we e o analy ical quali y. As s anda d compounds, 5-a-choles an-3-ol, n-heicosanol, be ulinic acid, lau yl a achida e, and me hyl hep adecanoa e we e pu chased om Sigma–Ald ich SA (S. Louis, USA). Squalene was pu chased om Me ck (Da m- s ad , Ge many). 2.2 Me hods 2.2.1 Chemical efining p ocess The s eps o his me hod a e desc ibed below: Degumming: Fi e hund ed g ams o OPOC C o OPOC MA was weighed in o a essel, and 0.3 w .% oil o phospho ic acid was added. The mix u e was main ained a 25°C o 30 min unde agi a ion, o p ecipi a e phospholipids in he acidic media. Neu aliza ion: Wi hou sepa a ing he gums, he equi ed amoun o NaOH (24° Be) was added, accompa- nied by an excess o 10%. The addi ion was pe o med a 40°C and he empe a u e was hen inc eased o 80°C. The mix u e was s i ed a his empe a u e o 20 min. Then, he soap s ocks we e sepa a ed by cen i uga ion a 2500 pm. Win e iza ion: Wi hou washing, 5% o NaOH (4° Be) was added o he neu alized oil, and he mix u es was kep a 5°C o 18 h. The p ecipi a es we e sepa a ed by cen i uga ion. 1600408 (2 o 10) C. A. S anchez-Gu i e ez e al. Eu . J. Lipid Sci. Technol. 2017, 119, 1600408 ß2017 WILEY-VCH Ve lag GmbH & Co. KGaA, Weinheim www.ejls .com Washes: In o de o emo e aces o dissol ed soap, he win e ized oil was washed h ee imes wi h wa e (10% w/w) o 10 min. The washing wa e was disca ded. Bleaching:The washed oil was d ied unde a acuum o 60 To a 90°C o 15 min wi h an agi a ion o 125 pm. Nex , 1.5% o Tonsil Sup eme 114 and 0.15% o ac i a ed ca bon we e added. The empe a u e was aised o 110°C and held o 10 min. Then, he mix u e was cooled unde a acuum and fil e ed a 50°C. Deodo iza ion: The bleached oils we e deodo ized a 220°C o 3 h unde a acuum o 0.5–1 mba . Once deodo ized, he oils we e fil e ed wi h fil e pape and s o ed a 18°C un il analysis (OPOD CC and OPOD MAC). 2.2.2 Physical efining p ocess 2.2.2.1 Classical physical efining The s eps o his p ocedu e we e as ollows: Degumming: Fi e hund ed g ams o OPOC MA was weighed in o a essel, and 0.5 w .% oil o ci ic acid was added o i . The mix u e was main ained o 10 min a 80°C unde agi a ion, o p ecipi a e phospholipids in he acidic media. Washes: In o de o emo e aces o dissol ed soap, he degummed oil was washed once wi h wa e (10% w/w), o 10 min a 80°C. The washing wa e was disca ded. Bleaching: The washed oil was d ied unde a acuum o 60 To a 90°C o 15 min wi h an agi a ion o 125 pm. Then, 1.5% Tonsil Sup eme 114, 0.1% ac i a ed ca bon, 1% T ysil 300, and 0.1% Dicali e we e added. The empe a u e was aised o 110°C and held o 10 min. Then, he mix u e was fil e ed. Neu alizing deodo iza ion: The bleached oil was deodo ized a 250°C (OPOD MAP) o 3 h unde a acuum o 0.5–1 mba . Wa e apo was used as he ca ie gas a a a e o 6.24 mL/h. Once deodo ized, he oil was fil e ed h ough pape and s o ed a 18°C un il analysis. 2.2.2.2 Molecula dis illa ion efining The s eps o his p ocedu e we e as ollows: Bleaching: The bleaching condi ions we e he same as hose used in he classical physical efining p ocedu e. Molecula dis illa ion: The deodo iza ion was pe - o med in a sho -pa h de ice (mod. KDL5, UIC Gmb, Ge many). The ope a ing condi ions we e fixed as ollows: eed flow o 1.44 l/h, 250 pm as he o a ion speed o he olle s, condense empe a u e o 50°C, and acuum o 0.001 mba . Deodo ized oils we e aken a 140°- C (OPOD140 MBP), 165°C (OPOD165 MBP), 190°- C (OPOD190 MBP), and 215°C (OPOD215 MBP). 2.3 Analy ical s udy Analy ical de e mina ion o bioac i e compounds was ca ied ou o c ude OPOCs (OPOC C, OPOC MA, and OPOC MB) and deodo ized oils ob ained in di e en efining Figu e 1. Ex ac ion and efining p ocesses o oli e pomace oil om pomace. Eu . J. Lipid Sci. Technol. 2017, 119, 1600408 Influence o efining p ocesses o oli e pomace oil 1600408 (3 o 10) ß2017 WILEY-VCH Ve lag GmbH & Co. KGaA, Weinheim www.ejls .com p ocess (OPOD CC, OPOD MAC, OPOD MAP, and di e en OPOD MBP). The acidi y, a y acid composi ion, wax con en , and squalene and alkyl es e s we e de e mined by he me hods ou lined in he Eu opean Union [13]. The unsaponifiable ma e was ex ac ed and quan ified a e saponifica ion o he oils wi h KOH/E OH, ollowing he Spanish s anda d me hod [14]. F om hese ex ac s, he con en o s e ols and alipha ic alcohols was de e mined acco ding he p oposed s anda d me hods [13], by using 5-a- choles an-3-ol and n-heneicosanol as in e nal s anda ds, espec i ely. The i e penic acid con en was de e mined ollowing he me hod p oposed by P e ez-Camino and Ce [2], which consis s o he isola ion o he a y acid ac ion by SPE-NH 2 columns, silyla ion o he ex ac s, and quan ifica ion by gas ch oma og aphy (Mod. 7890A, Agilen Technologies), using be ulinic acid as an in e nal s anda d. 2.4 Rheological cha ac e iza ion Mul i-s ep flow cu e measu emen s we e un using a con olled s ess heome e (AR-2000, TA Ins umen s, New Cas le, USA) and a plane geome y o 60-mm diame e wi h a smoo h su ace. A cons an empe a u e was main ained wi h a Pel ie pla e (25 0.1°C). The expe i- men al p o ocol began wi h a dynamic oscilla o y s udy o de e mine he linea iscoelas ic egion o oils h ough s ess sweeps a a equency o 1 Hz, a pe cen age ole ance o 1%, and maximum poin ime o 120 s. Then, an app op ia e alue o s ess was selec ed in o de o comply wi h non- des uc i e condi ions. A e his, a equency sweep was ca ied ou in he ange om 0.01 o 628.3 ad/s. The analysis o esul s is based on he s o age (G0) and loss (G00) moduli, which a e ela ed o he elas ic and iscous componen s, espec i ely. On he o he hand, flow and iscosi y cu es we e de e mined by a o a ional shea assay. Each measu e- men was made in iplica e and was deemed alid when he cu es we e supe imposable. Di e en heo e ical models can be used o analyze he heological esul s o es pe o mance in e ms o shea s ess ( ) and shea a e (g) o iscosi y (h) and shea a e (g). The lowes s anda d e o was applied o he heo e ical heological models. In ou pape , he powe law model (also well-known as Os wald-de Waele model) was used Eq. (1). ¼K:gnð1Þ This model ela es he shea s ess ( ) wi h he shea a e (g), whe e K and n a e he consis ency index and fluid index, espec i ely. Depending on he alue o n, hefluid ypes can be de e mined. Fluids wi h n¼1 a e New onian fluids and hose wi h n6¼1a enon-New onianfluids. The flow beha io can be de e mined depending on he n alue. Thus, n<1 indica es pseudoplas ic o plas ic flow (shea - hinning) and n>1 indica es dila an flow (shea - hickening). 2.5 Tex u e p ofile analysis (TPA) The ex u e is defined by ISO 5492 [15] as: an ins umen al echnique ha s udies he se o heological p ope ies and s uc u e o a pe cei ed p oduc by he mechano- ecep o s, ouch ecep o s, and in some cases, by isuals and audi o y in o ma ion. Ac ually, ex u e is an impo an a ibu e o any p oduc , which a ec s i s p ocessing and handling, and de e mines i s use ul li e as well as accep ance by use s/pa ien s. Sp eadabili y, in p ac ical e ms, is he ease wi h which a p oduc can be sp ead in a hin, e en laye on a su ace. The fi mness o ha dness can be measu ed by he o ce equi ed o ob ain a gi en de o ma ion o he amoun o de o ma ion by a gi en o ce. Al hough ex ensibili y is also a de o ma ion unde an ex e nal load, i is a mo e dynamic p ope y han fi mness o ha dness. Measu emen s o fi mness and sp eadabili y end o be highly co ela ed, bu his ela ionship is usually no pe ec . Tex u al p ope ies o he di e en OPOCs and OPODs we e de e mined by uniaxial comp ession wi h a load cell o 5 kg. A ex u e analyze TA.XT2i (S able Mic o Sys ems L d., Su ey, UK) was used and con olled by so wa e. A conical p obe (HDP/SR, S able Mic o Sys ems) was inse ed in he complemen a y conical sample holde con aining 8 0.1 g o di e en oils. This p obe is an accesso y ha measu es he ease wi h which a p oduc can be applied in a hin, e en laye . Du ing he es , he o ce was inc eased o he poin o maximum dep h as he cone en e ed he essel. The alue o peak o ce (maximum o ce) was aken as he fi mness o he specified dep h. Fi me samples showed a la ge posi i e a ea, ep esen ing he o al amoun o o ce (cohesi eness) equi ed o pe o m he b eaking p ocess. Bo h alues we e used o quan i y he sp eadabili y o samples; howe e , one o he wo measu e- men s o en ga e mo e ele an in o ma ion han he o he . The adhesi e p ope ies o he p oduc (adhesi eness), which a e quan ified by he nega i e zone o he cu e, we e cha ac e ized du ing he e u n mo emen o he p obe [16]. 3 Resul s and discussion 3.1 Chemical cha ac e iza ion o OPOCs and OPODs ob ained by efining p ocesses As men ioned be o e, OPOCs we e efined o emo e unwan ed mino componen s om oils, while causing minimal losses o bioac i e compounds [10]. Table 1 shows he esul s o analy ical de e mina ions o c ude pomace oil 1600408 (4 o 10) C. A. S anchez-Gu i e ez e al. Eu . J. Lipid Sci. Technol. 2017, 119, 1600408 ß2017 WILEY-VCH Ve lag GmbH & Co. KGaA, Weinheim www.ejls .com ob ained by: chemical (OPOC C) and mechanical (OPOC MA) ex ac ions, and deodo ized oil ob ained by he chemical efining p ocess (OPOD CC and OPOD MAC). Table 2 includes he co esponding esul s o oils ob ained by mechanical ex ac ion (OPOC MA and OPOC MB), and deodo ized oils ob ained by physical efining p ocesses (OPOD MAP and OPOD MBP). 3.1.1 Acidi y and i e penic acids In ela ion o he acidi y, i is possible o app ecia e wo impo an aspec s. Fi s , i is no ed ha he oil ob ained by mechanical ex ac ion (OPOC MA and OPOC MB) has less acidi y han ha ob ained by chemical ex ac ion (OPOC C). The e o e, i seems ha he cen i uga ion p ocess is mo e sui able han he sol en ex ac ion p ocess. Mo e nonpola ma e ial was ex ac ed in he fi s s age o he mechanical ex ac ion, mainly iacylglyce ols (TAG), whe eas he la e sol en s ages con ained mo e pola ma e ial as small amoun s o o he lipid componen s including non-es e ified a y acids, pa ial glyce ides, long-chain alcohols, and s e ols. Besides, he chemical ex ac ion p ocess equi es p elimina y d ying unde d as ic condi ions, p oducing oxida ion and hyd olysis eac ions and hus causing he inc ease o ee a y acids. Con e sely, i is obse ed ha he empe a u e o he efining p ocess g ea ly influences he acidi y. Thus, i was obse ed ha using high empe a u es, as in he case o chemical (220°C) and classical physical (250°C) efining, acidi y g ea ly dec eases agains he selec ed empe a u es in molecula dis illa ion. Howe e , hese high empe a u es ha e a significan impac on he concen a ion o bioac i e compounds. In he case o chemical efining, ee a y acids and i e penic acids we e emo ed by saponifica ion. The acidi y was educed below <0.1%; howe e , he oil quali y was deg aded om a heal h poin o iew because i e penic acids had been emo ed. Simila beha io can be obse ed in he case o classical physical efining (Table 2). In con as , he acidi y was less educed by molecula dis illa ion, bu a highe amoun o an i-inflamma o y bioac i e compounds ( i e - penic acids) we e ob ained (Table 2), because hey canno dis ill in he condi ions used in he assays and con ibu e o he highe acidi y alues. This majo acidi y could be a ibu ed o he ac ha a s eam s eam was no in oduced in he molecula dis ille o emo e ee a y acids. 3.1.2 Alkyl es e s As shown in Tables 1 and 2, lowe amoun s o alkyl es e s we e ob ained in all p ocesses o efining. These non-bioac i e Table 1. Analy ical de e mina ions o c ude oli e pomace oil ob ained by chemical (OPOC C) and mechanical (OPOC MA) ex ac ions and deodo ized oil ob ained by chemical efining p ocess (OPOD CC and OPOD MAC) Chemical ex ac ion and chemical e ining Mechanical ex ac ion and chemical e ining OPOC C OPOD CC OPOC MA OPOD MAC Acidi y (%) 20.70 0.14 0.08 0.01 6.89 0.02 0.075 0,01 T i e penic acids (mg/kg) 5121 130 11 171 263 Oleanolic acid 4342 78 0 5027 142 0 Maslinic acid 779 52 0 6114 121 0 Alkyl es e s (%) 3.69 0.02 1.21 0.42 Waxes (mg/kg) 3510 78 5172 173 1798 58 2348 18 Unsaponi iable ma e (%) 2.64 0.18 2.43 0.05 2.14 0.08 1.51 0,08 Squalene (mg/kg) 5048 96 3747 9 6175 121 4567 12 Alipha ic alcohols (mg/kg) 5784 81 4744 7 5263 234 3081 96 S e ols (mg/kg) 5435 158 3065 269 3406 87 2252 58 Choles e ol (%) 0.24 0.01 0.24 0.04 0.24 0.03 0.24 0,03 Campes e ol 3.20 0.02 3.54 0.04 3.20 0.22 2.81 0,16 S igmas e ol 1.23 0.07 1.59 0.18 1.29 0.10 0.86 0.06 Cle os e ol 0.94 0.01 1.34 0.05 1.42 0.23 1.79 0.26 b-Si os e ol 87.71 0.31 84.48 0.03 88.04 0.48 88.11 0.018 Si os enol 1.26 0.13 2.17 0.28 2.36 0.22 2.97 0.21 5-A enas e ol 3.98 0.16 3.60 0.13 0.43 0.06 0.38 0.12 5,24-Es igmas adienol 0.83 0.17 1.94 0.37 2.18 0.02 2.06 0.12 D-7-S igmas enol 0.38 0.04 0.89 0.11 0.46 0.01 0.45 0.15 D-7-A enas e ol 0.23 0.02 0.21 0.04 0.37 0.04 0.33 0.05 T i e penic alcohols (mg/kg) 568 4 511 4 582 48 376 29 Eu . J. Lipid Sci. Technol. 2017, 119, 1600408 Influence o efining p ocesses o oli e pomace oil 1600408 (5 o 10) ß2017 WILEY-VCH Ve lag GmbH & Co. KGaA, Weinheim www.ejls .com compounds appea due o e men a ion o ege able ma e du ing OPOC s o age in ponds [17]. Consis en wi h Yuan e al. [18], an inc ease in empe a u e causes a dec ease o alkyl es e s, as obse ed in he oils a e molecula dis illa ion, because hese compounds a e ola ile a empe a u es >165°C. 3.1.3 Waxes Thesecompoundsa esolidsa oom empe a u e.In gene al, he p esence o long-chain es e s as waxes in ege able oil is used o de e mine he pu i y and classifica ion o he a ious g ades o oli e oil [13]. The wax con en inc eases du ing he chemical and physical efining p ocesses (Tables 1 and 2). This sugges s es e ifica ion eac ions be ween ee a y acids and alipha ic alcohols p esen in he oil a deodo iza ion empe a u es, as epo ed by Tubaileh e al. [19]. Thus, when deodo iza ion empe a u e inc eased, he wax con en inc eased, as obse ed om he oils ob ained by molecula dis illa ion. 3.1.4 Unsaponifiable ma e This is composed o a complex mix u e o compounds o he han glyce ides. The majo bioac i e compounds in OPOC a e squalene, s e ols, and he i e penic alcohols e y h odiol and u aol [8]. As displayed in Tables 1 and 2, he pe cen age o unsaponifiable ma e dec eased wi h efin- ing. This could be a ibu ed mainly o he emo al o squalene in he efining p ocesses. Howe e , in molecula dis illa ion, a significan amoun o squalene was collec ed in deodo iza ion dis illa es (OPOD140 MBP–OPOD215 MBP). This squalene, as indica ed by Bondioli e al. [20], can be added o ou oil, en iching he concen a ion o his bioac i e compound. Tables 1 and 2 epo he di e en s e ols cha ac e is ic o he OPOC. In ag eemen wi h Ve leyen e al. [21], he pa ial emo al o s e ols du ing he efining p ocess depends la gely on he condi ions applied in he a ious s ages o he p ocess. Thus, wi h chemical and classical physical efining, he amoun o hese bioac i e compounds dec eased (40 and 20%, espec i ely); in con as , he Table 2. Analy ical de e mina ions o c ude pomace oils ob ained by mechanical ex ac ion (OPOC MA and OPOC MB) and, deodo ized oils ob ained by physical efining p ocesses (OPOD MAP and OPOD MBP) Mechanical ex ac ion and classical physical e ining Mechanical ex ac ion and molecula dis illa ion e ining OPOC MA OPOD MAP OPOC MB OPOD140 MBP OPOD165 MBP OPOD190 MBP OPOD215 MBP Acidi y (%) 6.89 0.02 0.12 0.02 11.76 0.01 17.00 0.06 6.30 0.02 2.75 0.01 2.15 0.09 T i e penic acids (mg/ Kg) 11 141 263 49 0.9 38 300 123 8275 257 7619 180 9856 357 8458 567 Oleanolic acid 5027 142 29 0.6 12 400 67 4681 82 4450 141 5450 128 4310 167 Maslinic acid 6114 121 20 0.3 25 900 56 3594 135 3169 39 4406 229 4148 400 Alkyl es e s (%) 1.21 0.00 2.67 1.60 0.18 0.03 0.02 Waxes (mg/kg) 1798 58 2374 126 4633 78 7782 202 8806 106 8827 78 8866 80 Unsaponi iable ma e (%) 2.14 0.08 0.85 0.02 3.57 0,25 3.37 0,05 3.05 0,16 2.80 0,15 2.35 0,14 Squalene (mg/kg) 6175 121 232 12 4516 107 2355 126 2061 81 310 56 85n8 Alipha ic alcohols (mg/ kg) 5263 234 2220 98 6595 567 6546 319 7690 115 5465 282 6954 366 S e ols (mg/kg) 3406 87 2646 29 3979 453 3851 90 4284 54 4131 31 4359 180 Choles e ol (%) 0.24 0.03 0.24 0.01 0.24 0.05 0.24 0.02 0.24 0,02 0.24 0,03 0.20 0,08 Campes e ol 3.20 0.22 3.18 0.12 3.07 0.09 4.03 0.21 4.67 0,05 4.73 0,08 4.67 0,41 S igmas e ol 1.29 0.10 2.01 0.18 1.43 0.18 1.390.06 1.36 0,02 1.26 0,10 1.25 0,52 Cle os e ol 1.42 n0.23 1.83 0.09 1.34 0.11 1.50 0.12 1.41 0.12 1.22 0.41 1.95 0.03 b-Si os e ol 88.04 0.48 87.47 0.14 88.49 0.37 87.71 0.49 87.90 0.02 88.03 0.04 86.62 0.04 Si os enol 2.36 0.22 2.34 0.12 2.05 0.21 2.04 0.03 1.90 0.09 2.01 0.08 2.46 0.22 5- A enas e ol 0.43 0.06 0.33 0.07 0.39 0.04 0.40 0.04 0.19 0.07 0.20 0.08 0.31 0.01 5,24-Es igmas adienol 2.18 0.02 2.02 0.02 2.12 0.02 2.08 0.07 1.75 0.09 1.78 0.09 2.02 0.04 D-7-s igmas enol 0.46 0.01 0.29 0.02 0.38 0.01 0.45 0.03 0.41 0,05 0.39 0,00 0.39 0,00 D-7-A enas e ol 0.37 0.04 0.27 0.01 0.28 0.03 0.17 0.01 0.18 0,02 0.16 0,00 0.15 0,02 T i e penic alcohols (mg/ kg) 582 48 238 38 435 131 838 2 1221 24 1298 53 1393 214 A e age alue n¼3SD. 1600408 (6 o 10) C. A. S anchez-Gu i e ez e al. Eu . J. Lipid Sci. Technol. 2017, 119, 1600408 ß2017 WILEY-VCH Ve lag GmbH & Co. KGaA, Weinheim www.ejls .com amoun o hese s e ols inc eased sligh ly in he molecula dis illa ion. Simila beha io can be obse ed wi h i e - penic alcohols. Conside ing all o he abo e, he physical efining p ocesses, especially molecula dis illa ion, con ibu e o be e pe o mance in e ms o he bioac i e compounds s udied, which can be used in opical o mula ions o hei he apeu ical ac i i ies. Fu he mo e, o all he oils ob ained by molecula dis illa ion, OPOD190 MBP was chosen o be he only one ha simul aneously shows low acidi y and less loss o bioac i e compounds. In summa y, he esul s ob ained o OPOD190 MBP by molecula dis illa ion we e conside ed app op ia e o compa e wi h espec o EVOO, which is a aw ma e ial widely used in opical o mula ions (Table 3). I is no ed ha OPOD190 MBP showed g ea e acidi y han EVOO, al hough s ill low; howe e , we confi med ha ou OPOC exhibi ed highe amoun s o all bioac i e compounds, excep o squalene. 3.2 Rheological cha ac e iza ion Figu e 2 shows he heological beha io in chemical and mechanical ex ac ion (OPOC C and OPOC MA), and chemical efining (OPOD CC and OPOD MAC). Figu e 2A p esen s he oscilla o y equency sweep o he measu emen o iscoelas ici y. In all cases, i was obse ed ha he iscoelas ici y o fluids, G0and G00, is dependen on he applied equency. Mo eo e , he e is a p edominance o he iscous componen in all OPOC as OPOD. G0was only s able a equencies less han 5–10 ad.s 1 , indica ing li le influence o he elas ic componen in he iscoelas ic beha io . A he same ime, an impo an gap was obse ed in he p ofiles be ween G00 and G0, which co obo a es he impac o iscous beha io . Only OPOD CC shows li le di e ence be ween he wo componen s (G00 and G0), which we e e en e e sed a equencies below 0.1 ad/s, indica ing ha G0has g ea e impac on he in e nal s uc u e o his oil. Figu e 2B shows he flow cu es o OPOC C, OPOC MA, OPOD CC, and OPOD MAC. All oils beha e as pseudoplas ic o shea - hinning fluids. Figu e 3 compa es he heological beha io in chemical and classical physical efining (OPOD MAC and OPOD MAP). Fo all oils, he iscous componen (G00) inc eased wi h he applied equency (Fig. 3A). The e o e, OPOD MAC and OPOD MAP we e iscoelas ic fluids wi h a p edominance o he iscous componen , simila o OPOC MA. Fu he mo e, i was obse ed ha OPOD MAP had p og essi ely lowe alues o he elas ic componen (G0), in con as wi h OPOD MAC. The flow beha io can be obse ed in Fig. 3B, which shows ha all oils we e non-New onian fluids wi h pseudo- plas ic beha io . Figu e 4 compa es he heological beha io o he wo physical efining p ocesses, classical physical (OPOD MAP), and molecula dis illa ion (OPOD MBP). Again, all oils we e iscoelas ic fluids wi h a p edominance o he iscous componen , simila o aw OPOCs. None heless, i is obse ed ha he ac ions subjec ed o empe a u es abo e 145°C in he molecula dis illa ion efining p ocess Table 3. Compa a i e s udy o he composi ion o oli e pomace oil ob ained by mechanical ex ac ion and molecula dis illa ion (OPOD190 MBP) espec o ex a i gin oli e oil (EVOO) OPOD190 MBP EVOO Acidi y (%) 2.75 <0.8 T i e penic acids (mg/kg) 9856 90–190 Oleanolic acid 5450 30-80 Maslinic acid 4406 60–110 Alkyl es e s (%) 0.03 – Waxes (mg/kg) 8827 <250 Unsaponi iable ma e (%) 2.80 0.4-2 Squalene (mg/kg) 310 9000 Alipha ic alcohols (mg/kg) 5465 350 S e ols (mg/kg) 4131 1500 T i e penic alcohols (mg/kg) 1298 <100 Figu e 2. Rheological beha io o oli e pomace oils ob ained by chemical and mechanical ex ac ion (OPOC C and OPOC MA), and chemical efining (OPOD CC and OPOD MAC). (A) Oscilla o y equency sweep. (B) Flow cu es. Eu . J. Lipid Sci. Technol. 2017, 119, 1600408 Influence o efining p ocesses o oli e pomace oil 1600408 (7 o 10) ß2017 WILEY-VCH Ve lag GmbH & Co. KGaA, Weinheim www.ejls .com (OPOD165 MBP, OPOD190 MBP, and OPOD215 MBP) had p og essi ely lowe alues o G0, and los hei elas ic p ope y (Fig. 4A). The flow beha io can be obse ed in Fig. 4B. Again, all o he oils s udied exhibi ed pseudoplas ic flow beha io . The flow cu es we e adjus ed o di e en heological heo e ical models. The bes fi was ob ained wi h he powe law model. Table 4 egis e s all he pa ame e s ob ained. The esul s confi m ha all oils s udied beha e as Non- New onian fluids wi h pseudoplas ic o shea - hinning beha io (n<1). Ne e heless, he flow index alues close o 1 deno e ha hey we e almos New onian fluids. The consis ency index (K) alues indica e ha he iscosi y was small in all ins ances. Mo eo e , he chemical efining p ocess did no a ec he flow cha ac e is ics (OPOD CC wi h espec o OPOC C and OPOD MAC wi h espec o OPOC MA). None heless, because OPOD CC and OPOD MAC did no di e in he efining p ocess, he ex ac ion p ocess is esponsible o he esul s ob ained. Thus, mechanical ex ac ion caused a loss o iscosi y, while chemical ex ac ion p omo ed an inc ease o i . This could be ela ed wi h he p ofile o componen ex ac ed by means o sol en , included ee a y acids. I is impo an o men ion ha OPOC MA wi h lowe acidi y esul ed in a dec ease in iscosi y. The opposi e occu ed in OPOC C, he sample wi h highe acidi y (ob ained by sol en ex ac ion), inc easing he iscosi y o he sample [22]. The classical physical efining (OPOD MAP) displayed less iscosi y han OPOC MA, al hough bo h a e almos New onian fluids. The consis ency index alues we e lowe o he physically efined samples han o he c ude OPOC, while he chemical p ocess seemed o no modi y his pa ame e . On he o he hand, he molecula dis illa ion efining p ocess (OPOD MBP) sligh ly a ec ed he alues o he consis ency index. 3.3 Tex u e p ofile analysis (TPA) De e mining he ex u e pa ame e s o he oils can indica e hei mechanical and senso y p ope ies du ing si ua ions o use, o example, sp eadabili y, adhesi eness, and cohesi eness. Figu e 3. Rheological beha io o oli e pomace oils ob ained by mechanical ex ac ion (OPOC MA), and chemical and classical physical efining (OPOD MAC and OPOD MAP). (A) Oscilla o y equency sweep. (B) Flow cu es. Figu e 4. Rheological beha io o deodo ized oli e pomace oils ob ained by classical physical (OPOD MAP) and molecula dis illa ion (OPOD MBP) efining. (A) Oscilla o y equency sweep. (B) Flow cu es. 1600408 (8 o 10) C. A. S anchez-Gu i e ez e al. Eu . J. Lipid Sci. Technol. 2017, 119, 1600408 ß2017 WILEY-VCH Ve lag GmbH & Co. KGaA, Weinheim www.ejls .com The fi mness is ela ed wi h he maximum posi i e o ce equi ed o a ain a gi en de o ma ion. Cohesi eness and adhesi eness co espond o he a eas unde he cu e ob ained in he comp ession and wi hd awal o he p obe, espec i ely. Cohesi eness indica es he o ce o b eak he physical/chemical bonds es ablished be ween he subs ances composing each oil. In con as , adhesi eness indica es he o ce equi ed o sepa a e he oil om he walls o he con aining ecipien . The esul s o TPA (Table 5) indica e ha he efining p ocesses influence hese pa ame e s. Thus, he chemical efining p ocess shows ha he deodo ized oils (OPOD CC and OPOD MAC) p esen simila alues. I he chemical and classical physical efining p ocesses a e compa ed, OPOD MAC and MAP OPOD show he lowes alues o all pa ame e s, indica ing g ea e influence o classical physical efining han chemical efining. In he case o molecula dis illa ion and aking in o accoun all empe a u es, changes in he ex u e alues a e less ma ked han in classical physical efining. As obse ed in Tables 1 and 2, waxes and i e penic acids, solid compounds a oom empe a u e, we e highe in sol en ex ac ed oils and p esen ed highe con en s a e physical efining by molecula dis illa ion. n summa y, OPOD CC and OPOD MAC a e cha ac e - ized by low fi mness and cohesi eness, which indica e ha hey a e so and ha e high sp eadabili y. A he same ime, hey ha e li le adhesi eness and will hus o m a hin film on he skin. OPOD MAP p esen s he same cha ac e is ics o ex u e. Molecula dis illa ion p o ides a ba e y o OPOD wi h op imal ex u e cha ac e is ics o so ness and sp eadabil- i y, which allow he o ma ion o a mo e adhesi e film on he skin. The e o e, hese oils will cons i u e he aw ma e ials in he design, al eady in de elopmen , o de ma ological o mula ions. Al hough i will p oceed o imp o e he cha ac e is ics o he oils wi h new echniques o efining. 4 Conclusions Tempe a u e and, in gene al, he wo king condi ions o he efining p ocesses ha e impo an e ec s on he con en o bioac i e compounds. The physical efining p ocesses, especially molecula dis illa ion, con ibu e o be e pe o mance in hese compounds. Fu he mo e, o all o he oils ob ained by molecula dis illa ion, OPOD190 MBP was chosen o be he only one ha simul aneously showed low acidi y and less loss o bioac i e compounds. Howe e , his oil showed highe acidi y han ex a i gin oli e oil bu highe amoun s o all bioac i e compounds, excep squalene. A e he efining p ocess, he OPOCs showed he bes fi o he powe law heological model, possibly due o he emo al o mino componen s in he p ocess. In he OPOC subjec ed o efining, he iscosi y dec eased wi h an inc ease in he deodo iza ion empe a u e and educ ion in he amoun o ee a y acids, hus also a ec ing he na u e o he samples. The fi mness, cohesi eness, and adhesi eness pa ame e s om TPAs showed ha , in gene al, all OPODs ha e high smoo hness and sp eadabili y bu low adhesi eness. In iew o he achie ed esul s, i can be concluded ha molecula dis illa ion is he bes efining p ocess. Ne e he- less, i is necessa y o imp o e he wo king condi ions in o de o educe acidi y and achie e lowe losses, i possible, o bioac i e compounds. The e o e, o he s udies need o be Table 4. Pa ame e s ob ained wi h he Powe Law model o c ude and deodo ized oli e pomace oils s udied K(Pas) nSE Chemical e ining OPOC C 0.151 0.904 1.967 OPOD CC 0.158 0.889 2.414 OPOC MA 0.127 0.933 1.367 OPOD MAC 0.126 0.940 1.295 Physical e ining OPOC MA 0.127 0.933 1.367 OPOD MAP 0.070 0.986 1.238 OPOC MB 0.298 0.849 2.220 OPOD140 MBP 0.211 0.886 2.704 OPOD165 MBP 0.186 0.954 3.357 OPOD190 MBP 0.227 0.899 4.510 OPOD215 MBP 0.257 0.880 4.150 K, consis ency index; n,flow index; SE, s anda d e o . Table 5. Pa ame e s ob ained in Tex u e p ofile analysis o c ude and deodo ized oli e pomace oils s udied Fi mness (N) Cohesi eness (N.s) Adhesi eness (N.s) Chemical e ining OPOC C 0.28 0.22 0.04 OPOD CC 0.29 0.28 0.07 OPOC MA 0.42 0.00 0.21 OPOD MAC 0.30 0.24 0.04 Physical e ining OPOC MA 0.42 0.00 0.21 OPOD MAP 0.17 0.16 0.01 OPOC MB 0.71 0.37 0.16 OPOD140 MBP 0.54 0.34 0.13 OPOD165 MBP 0.36 0.26 0.07 OPOD190 MBP 0.43 0.30 0.09 OPOD215 MBP 0.54 0.32 0.12 Eu . J. Lipid Sci. Technol. 2017, 119, 1600408 Influence o efining p ocesses o oli e pomace oil 1600408 (9 o 10) ß2017 WILEY-VCH Ve lag GmbH & Co. KGaA, Weinheim www.ejls .com