Determination of phenolic compounds in apple and pear pulp by ultra-high performance liquid chromatography coupled with mass spectrometry

João Da id Guima ães Teixei a De e mina ion o phenolic compounds in apple and pea pulp by ul a-high pe o mance liquid ch oma og aphy coupled wi h mass spec ome y ou ub o de 2022 UMinho | 2022 Da id Teixei a De e mina ion o phenolic compounds in apple and pea pulp by ul a-high pe o mance liquid ch oma og aphy coupled wi h mass spec ome y Uni e sidade do Minho Escola de Ciências João Da id Guimã aes Teixei a De e mina ion o phenolic compounds in apple and pea pulp by ul a-high pe o mance liquid ch oma og aphy coupled wi h mass spec ome y Disse ação de Mes ado Mes ado em Química Medicinal T abalho e e uado sob a o ien ação do P o esso Dou o Pie Pa po e da Dou o a Ana Sanches Sil a Uni e sidade do Minho Escola de Ciências ou ub o de 2022 i DIREITOS DE AUTOR E CONDIÇÕES DE UTILIZAÇÃO DO TRABALHO POR TERCEIROS Es e é um abalho académico que pode se u ilizado po e cei os desde que espei adas as eg as e boas p á icas in e nacionalmen e acei es, no que conce ne aos di ei os de au o e di ei os conexos. Assim, o p esen e abalho pode se u ilizado nos e mos p e is os na licença abaixo indicada. Caso o u ilizado necessi e de pe missão pa a pode aze um uso do abalho em condições não p e is as no licenciamen o indicado, de e á con ac a o au o , a a és do Reposi ó io da Uni e sidade do Minho. Licença concedida aos u ilizado es des e abalho A ibuição-NãoCome cial-SemDe i ações CC BY-NC-ND h ps://c ea i ecommons.o g/licenses/by-nc-nd/4.0/ ii Ag adecimen os Du an e a elabo ação des a ese de mes ado, i e a so e de ob e o apoio de á ias pessoas e en idades, às quais desejo deixa uma pala a de ap eço: À Uni e sidade do Minho, e odos os docen es que me acompanha am no pe cu so académico e con ibuí am pa a o meu desen ol imen o pessoal e p o issional. Ao INIAV, I.P., pela opo unidade que me deu e po o na possí el, com as melho es condições, a ealização do abalho des a ese. Aos meus o ien ado es, Dou o a Ana Sanches Sil a, po oda a disponibilidade, conselhos, ajuda e p eocupação que exibiu du an e es e pe íodo e ao P o . Dou o Pie Pa po , meu o ien ado do abalho de ese de mes ado, pela p eocupação, igo e apoio demons ados ao longo des e abalho. À Dou o a Claudia Sanchez e ao Dou o Miguel Leão pela disponibilização das amos as egionais e come ciais de maçã e egionais de pe a, indispensá eis à ealização des e p oje o. Aos meus pais, po odos os es o ços que azem po mim, po odos os conselhos que melho am o meu dia-a-dia, e po se em semp e os meus maio es admi ado es. Ao meu i mão, po es a semp e p esen e e po me o na uma igu a de exemplo pa a si. À minha Dani, po oda a e nu a, ca inho, apoio e amo que me ansmi e. Mui o ob igado po es a es semp e p esen e, p on a a ajuda e mais especialmen e po me aze es ac edi a em mim. A odos os meus amigos, mas em especial à Flá ia, à Cá ia, ao Espe ança, à Inês, ao Ma celo, ao Ma co, ao Miguel, ao Nené, ao Rod igo, ao Tiago e ao Xa i, po du an e es es anos man e em uma amizade p óxima comigo e po se em p o agonis as em belas memó ias. Ag adeço ambém a odos os colegas do INIAV,I.P., pelo excelen e ambien e que p opo ciona am, com especial ên ase à Ana, à Ca men, ao João, à Ma ga ida, à Ma a, ao Rica do e à Ri a. Es e abalho oi ealizado no âmbi o do p oje o clabel+: Alimen os ino ado es “clean label” na u ais, nu i i os e o ien ados pa a o consumido , com a e e ência POCI-01-0247-FEDER- 046080 inanciado pelo P og ama Ope acional Compe i i idade e In e nacionalização (POCI), sob o aco do de pa ce ia COMPETE2020, PORTUGAL2020, a a és do co- inanciamen o do Fundo Eu opeu de Desen ol imen o Regional (FEDER). iii STATEMENT OF INTEGRITY I he eby decla e ha ing conduc ed his academic wo k wi h in eg i y. I con i m ha I ha e no used plagia ism o any o m o undue use o in o ma ion o alsi ica ion o esul s along he p ocess leading o i s elabo a ion. I u he decla e ha I ha e ully acknowledged he Code o E hical Conduc o he Uni e si y o Minho. i De e mina ion o phenolic compounds in apple and pea pulp by ul a-high pe o mance liquid ch oma og aphy coupled wi h mass spec ome y Apples ( Malus domes ica ) and pea s ( Py us L.). a e wo o he mos widely g own and consumed ui s on he plane . They p o ide phenolic compounds, which a e conside ed o ha e posi i e e ec s on human heal h such as an ioxidan and an i-hype ensi e p ope ies. The by- p oduc s o hese ui s a e no cu en ly ully u ilized; hus, i is c i ical o cha ac e ize hem, pa icula ly wi h ega d o hei an ioxidan capabili ies, in o de o iden i y po en ial uses and p e en hei was e. The goal o his s udy was o iden i y he an ioxidan p ope ies o by-p oduc s and edible pa s o six na ional cul i a s o pea s — Bela-Feia, To es No as, Ca apinhei a, Ca apinhei a Roxa, Lambe-os-Dedos, and Amo im — and i e di e en Po uguese apple cul i a s — Pa do Lindo, Pê o de Bo belo, Noi a, Pê o Coimb a, and Repinau — p oduced in he Alcobaça egion (Po ugal) and compa e hem wi h comme cially a ailable cul i a s, also om his egion. The an ioxidan p ope ies we e assessed employing an ioxidan capaci y es s (DPPH adical sca enging and - ca o ene bleaching) as well as o al la onoids and o al phenolic con en assays. In gene al by- p oduc s om apples and pea s, pe o med be e in e ms o an ioxidan capaci y, TFC, and TPC, indica ing hei po en ial as sou ces o bene icial an ioxidan compounds. I is in e es ing o no e ha in some cul i a s such as he Ca apinhei a pea , he maximum an ioxidan p ope ies can be ound in seeds while in o he cul i a s like Noi a apple can be ound in peels. A UHPLC-ToF-MS me hod was also de eloped o he de e mina ion o 20 indi idual phenolics in he mesoca p (pulp) and by-p oduc s o apples and pea s. The analy ical me hod was e alua ed ega ding linea i y, limi o de ec ion, limi o quan i ica ion, accu acy, showing i s sui abili y o he quan i ica ion o phenolic compounds. This me hod was applied o ou samples o he egional cul i a s o ui s, p o iding hei phenolic composi ion. Vanillic acid and ca eic acid we e he phenolics wi h a highe concen a ion in he ui samples. The p incipal componen analysis (PCA) echnique was used o educe he da a dimensionali y while main aining he con ibu ion o he o iginal a iables. F ui s we e success ully di e en ia ed in o g oups based on hei p o enience. Keywo ds: Apples; PCA; Pea s; Phenolic compounds; UHPLC-ToF-MS De e minação de compos os enólicos em polpas de maçãs e pe as po c oma og a ia líquida de ul a esolução acoplada a espec ome ia de massas Maçãs e pe as são duas das u as mais p oduzidas e consumidas em odo o mundo. Es as u as são on es de compos os enólicos, que são conside ados como endo e ei os posi i os pa a a saúde humana, ais como p op iedades an ioxidan es e an i-hipe ensi as. Os sub-p odu os des es u os não es ão a se de idamen e ap o ei ados; po an o é essencial ca ac e izá-los, pa icula men e em elação às suas p op iedades an ioxidan es, de manei a a iden i ica po enciais usos e p e eni o seu despe dício. O obje i o des e abalho oi iden i ica as p op iedades an ioxidan es dos sub-p odu os e pa e edí el de seis cul i a es nacionais de pe as — Bela-Feia, To es No as, Ca apinhei a, Ca apinhei a Roxa, Lambe-os-Dedos, e Amo im — e cinco cul i a es po ugueses de maçãs Pa do Lindo, Pê o de Bo belo, Noi a, Pê o Coimb a, e Repinau — p oduzidos na egião de Alcobaça (Po ugal) e compa á-los com cul i a es come cialmen e disponí eis, ambém des a egião. As capacidades an ioxidan es o am a aliadas aplicando es es de capacidade an ioxidan e (mé odo do adical DPPH e b anqueamen o do -ca o eno) e ambém pela de e minação do eo o al de compos os enólicos e de la onoides. No ge al, os sub-p odu os ob i e am melho es esul ados na capacidade an ioxidan e, TPC e TFC, mos ando o seu po encial como on e de an ioxidan es. É in e essan e e e i que em alguns cul i a es, como na pe a Ca apinhei a, os esul ados são melho es nas semen es e nou os, como na maçã Noi a, nas cascas. Também oi desen ol ido um mé odo UHPLC-ToF-MS pa a a de e minação de 20 compos os enólicos indi iduais no mesoca po (polpa) e sub-p odu os des as ma izes. O mé odo analí ico oi a aliado quan o à linea idade, limi e de de eção, limi e de quan i icação, p ecisão, mos ando se adequado pa a a de e minação de compos os enólicos. O mé odo oi aplicado a 4 amos as dos cul i a es egionais, o necendo a sua composição enólica. Os ácidos anílico e ca eico o am encon ados em maio es concen ações nes as ma izes. Uma écnica de análise de componen es p incipais (PCA) oi u ilizada pa a diminui a dimensão de dados, man endo o con ibu o das a iá eis iniciais. Os u os o am sepa ados com sucesso com base na sua p o eniência. Pala as-cha e: Compos os enólicos, Maçã, PCA, Pe a, UHPLC-ToF-MS i Table o Con en s Ag adecimen os ...................................................................................................................................... ii STATEMENT OF INTEGRITY ................................................................................................................... iii Abs ac ................................................................................................................................................. i Resumo .................................................................................................................................................. Table o Con en s ................................................................................................................................... i Lis o Figu es ........................................................................................................................................ ix Lis o Tables ........................................................................................................................................ xiii Lis o Abb e ia ions .............................................................................................................................. x i 1. In oduc ion ................................................................................................................................... 1 1.1. INIAV, I.P. .............................................................................................................................. 2 1.2. Apple ..................................................................................................................................... 4 1.3. Pea ....................................................................................................................................... 6 1.4. Phenolic compounds .............................................................................................................. 8 1.4.1. Fla onoids ..................................................................................................................... 9 1.4.1.1. Fla onols .............................................................................................................10 1.4.1.2. Fla an-3-ols .........................................................................................................11 1.4.1.3. Fla ones ..............................................................................................................12 1.4.1.4. Iso la ones ..........................................................................................................13 1.4.1.5. Fla anones ..........................................................................................................14 1.4.1.6. An hocyanidins ....................................................................................................14 1.4.2. Phenolic Acids .............................................................................................................15 1.4.2.1. Hyd oxycinnamic acids ........................................................................................16 1.4.2.2. Hyd oxybenzoic acids ..........................................................................................16 1.4.3. S ilbenes ......................................................................................................................17 1.4.4. Lignans........................................................................................................................18 1.5. Me hods o he quan i ica ion o bioac i e compounds .........................................................19 1.5.1. Spec opho ome ic me hods .......................................................................................20 1.5.1.1. DPPH ee adical inhibi ion assay ........................................................................20 1.5.1.2. To al con en o phenolic compounds assay .........................................................21 1.5.2. To al con en o la onoids assay ..................................................................................21 1.5.2.1. β-ca o ene bleaching assay ..................................................................................22 1.5.3. Ch oma og aphic sepa a ion me hods ..........................................................................22 1.5.3.1. High-Pe o mance Liquid Ch oma og aphy (HPLC) ...............................................23 1.5.3.2. Ul a-High-Pe o mance Liquid Ch oma og aphy (UHPLC) .....................................26 1.5.3.3. Gas Ch oma og aphy (GC) ...................................................................................26 xiii Lis o Tables Chap e 1 Table 1.1: Po ugal na ional p oduc ion o apples. Sou ce: GPP 2021. ........................ 5 Table 1.2: In e na ional ade balance ega ding apple comme ce (x 1000€). Sou ce: GPP 2021 ................................................................................................................................ 5 Table 1.3: Po ugal na ional p oduc ion o pea s. Sou ce: GPP 2021........................... 7 Table 1.4: In e na ional ade balance ega ding pea comme ce (x 1000€). Sou ce: GPP 2021 ........................................................................................................................................ 7 Chap e 3 Table 3.1: Reagen s and hei cha ac e is ics used o he de e mina ion o he an ioxidan p ope ies and he quan i ica ion o uc ose in apple and pea samples. .................................. 36 Table 3.2: Ma e ials and hei cha ac e is ics used o he de e mina ion o he an ioxidan p ope ies and he quan i ica ion o uc ose in apple and pea samples. .................................. 38 Table 3.3: Equipmen s used o he de e mina ion o he an ioxidan p ope ies and he quan i ica ion o uc ose in apple and pea samples. .............................................................. 39 Table 3.4: P epa a ion o he olox s anda d solu ions. ............................................. 40 Table 3.5: P epa a ion o he gallic acid s anda d solu ions. ...................................... 41 Table 3.6: P epa a ion o he epica echin s anda d solu ions. .................................... 42 Table 3.7: P epa a ion o he uc ose s anda d solu ions. ......................................... 43 Table 3.8: P epa a ion o he di e en phenolic compounds’ s anda ds. .................... 45 Table 3.9: Mobile phase g adien used o he sepa a ion o phenolic compounds. .... 53 Table 3.10: GC column empe a u e g adien o pes icide sepa a ion. ..................... 54 Chap e 4 Table 4.1: Sol en s udy using he DPPH ee adical inhibi ion sys em. (IP= Inhibi ing pe cen age) ............................................................................................................................ 55 Table 4.2: Sol en s udy using he o al la onoid con en assay. (ECE= Epica echin equi alen s) ............................................................................................................................ 56 Table 4.3: Sol en s udy using he β-ca o ene bleaching assay. (AAC= An ioxidan Ac i i y) .............................................................................................................................................. 56 Table 4.4: Resul s o DPPH ee adical inhibi ion assay o di e en egional apple cul i a s collec ed in 2021 a Alcobaça egion (Po ugal). ........................................................ 57 xi Table 4.5: DPPH ee adical inhibi ion assay esul s o di e en egional pea cul i a s collec ed in 2021 a Alcobaça egion (Po ugal). ...................................................................... 57 Table 4.6: DPPH ee adical inhibi ion assay esul s on he comme cial apple cul i a s. .............................................................................................................................................. 60 Table 4.7: β-ca o ene bleaching assay esul s on he egional apple cul i a s. Ha es ed in 2021 in Alcobaça (Po ugal). ............................................................................................... 64 Table 4.8: β-ca o ene bleaching assay esul s on he pea cul i a s ha es ed in 2021 in Alcobaça (Po ugal). ................................................................................................................ 65 Table 4.9: β-ca o ene bleaching assay esul s on he comme cial apple cul i a s ha es ed in 2021 in Alcobaça (Po ugal). ............................................................................... 67 Table 4.10: To al phenolics con en o he egional apple cul i a s ha es ed in 2021 in Alcobaça (Po ugal). ................................................................................................................ 72 Table 4.11: To al phenolics con en o he egional pea cul i a s ha es ed in 2021 in Alcobaça (Po ugal). ................................................................................................................ 72 Table 4.12: To al phenolics con en o comme cial apple cul i a s ha es ed in 2021 in Alcobaça (Po ugal). ................................................................................................................ 75 Table 4.13: To al la onoids con en o he apple cul i a s ha es ed in 2021 in Alcobaça (Po ugal). .............................................................................................................................. 80 Table 4.14: To al la onoids con en o he pea s cul i a s ha es ed in 2021 in Alcobaça (Po ugal). .............................................................................................................................. 80 Table 4.15: To al la onoids con en o he comme cial apple cul i a s ha es ed in 2021 in Alcobaça ............................................................................................................................. 83 Table 4.16: To al uc ose con en o he apple cul i a s ha es ed in 2021 in Alcobaça (Po ugal). .............................................................................................................................. 88 Table 4.17: To al uc ose con en o he pea cul i a s ha es ed in 2021 in Alcobaça (Po ugal). .............................................................................................................................. 88 Table 4.18: To al uc ose con en assay esul s on he comme cial apple cul i a s ha es ed in 2021 in Alcobaça (Po ugal). ............................................................................... 91 Table 4.19: Linea ange, calib a ion cu es, de e mina ion coe icien s and e en ion ime o he s udied phenolic compounds. .............................................................................. 103 Table 4.20: Limi o quan i ica ion and limi o de ec ion o he s udied phenolic compounds. ......................................................................................................................... 105 x Table 4.21: Reco e y o he analy ical me hod o each o he s udied phenolic compounds a wo o i ica ion le els. .................................................................................... 108 Table 4.22: Phenolic compounds concen a ion in he s udied ui samples. .......... 110 x i Lis o Abb e ia ions AAC An ioxidan ac i i y coe icien DPPH 2,2-diphenyl-1-pic ylhyd azyl ECE Epica echin equi alen s GAE Gallic acid equi alen s GC Gas ch oma og aphy HS-SPME Headspace solid phase mic oex ac ion HPLC High pe o mance liquid ch oma og aphy INIAV Na ional Ins i u e o Ag a ian and Ve e ina y Resea ch IP Inhibi ing pe cen age LC Liquid ch oma og aphy LOD Limi o de ec ion LOQ Limi o quan i ica ion MeOH Me hanol MS Mass spec ome y PCA P incipal componen analysis PDMS Polydime hylsiloxane QuECHERS Quick, Easy, Cheap, E ec i e, Rugged and Sa e Coe icien o co ela ion 2 Coe icien o de e mina ion RSA Radical sca enging ac i i y Re en ion ime SLE Solid-liquid ex ac ion SPME Solid phase mic oex ac ion TE T olox equi alen s TFC To al la onoids con en ToF Time o Fligh TPC To al phenolics con en x ii UHPLC Ul a-high pe o mance liquid ch oma og aphy UV-B Ul a iole B adia ion UV-Vis Ul a iole - isible spec oscopy 1 1. In oduc ion Food loss and was e is de ined as “a dec ease, a all s ages o he ood sys em om p oduc ion o consump ion, in mass and/o quali y, o ood ha was o iginally in ended o human consump ion, ega dless o he cause”.1 The e o e, add essing he sys emic causes (demands and consump ion pa e ns ha a e in luenced by comme ce, a luence, and cul u e), pe mi ing changes in consume beha io , and p omo ing esponsible ood alo iza ion a e he essen ial componen s o p e en ing ood was e. cLabel+ (cLabel+ Inno a i e na u al, nu i ious, and consume -o ien ed clean label ood) is a esea ch and echnological de elopmen p ojec aimed a add essing he issues he ood indus y aces. The cLabel+ p ojec is p omo ed by a conso ium led by Sumol + Compal Ma cas SA and comp ised o 20 en i ies: 8 companies wi h di e se and complemen a y a eas o ac i i y and 12 non-business en i ies om he R&I Sys em wi h a s ong expe ience in de elopmen o p ojec s in he scien i ic a eas o he p ojec . cLabel+ p ojec ocuses on "clean label" concep , which is eme ging as one o he majo cu en ends in he sec o gi en he g owing numbe o cus ome s who a e inc easingly conscious and eady o in o ma ion and who a e looking o al e na i e, mo e anspa en , and na u al ood p oduc s. Focusing he clean label concep , a book chap e 2 dedica ed o he Regula o y amewo k and guidelines o he de elopmen o clean label p oduc s was accep ed o publica ion, whe e I am he i s au ho . In he ame o he wo kpackage 2 o cLabel+ (PPS2), new solu ions o educing and modeling he swee ening powe in oods a e de eloped. In his line, he Na ional Ins i u e o Ag a ian and Ve e ina y Resea ch (INIAV I.P.), as pa ne o he p ojec , planned o s udy he composi ion o egional cul i a s o apples and pea s p oduced a he Alcobaça campus o INIAV, as an al e na i e o educe suga in some ood o mula ions. In his con ex , his disse a ion aimed o cha ac e ize he an ioxidan capaci y and uc ose con en , o egional cul i a s o apples and pea s, o which he e a e s ill sca ce da a on componen s, and compa e hese esul s wi h hose o comme cial cul i a s. Mo eo e , di e en pa s o he ui s (peels, seeds and mesoca p) we e analyzed indi idually in o de o e alua e he po en ial o by-p oduc s o be used a aluable sou ce o na u al an ioxidan s. Mo eo e , an ul a- high esolu ion liquid ch oma og aphy coupled o ime-o - ligh mass spec ome y (UHPLC-ToF- MS) me hod was de eloped and alida ed o de e mine indi idual phenolics and i was applied o ou selec ed samples. 2 Also, a pes icide de ec ion s udy was made o e alua e i he egional cul i a s we e con amina ed wi h hese compounds. P incipal componen analysis was also ca ied ou in his s udy in o de o isualize he ela ionship be ween samples which consis o di e en apple and pea a ie ies and hei chemical p ope ies. 1.1. INIAV, I.P. The p esen ed esea ch s udy was de eloped in he Vai ão campus (Vila do Conde) o he INIAV, I.P, INIAV is he S a e Labo a o y, in he a ea o compe ences o Ag icul u e, Fo es y and Ru al De elopmen , which de elops esea ch ac i i ies in he ag onomic and e e ina y a eas. I is posi ioned a he in e ace be ween he na ional scien i ic and echnological sys em, companies, and e i o ial agen s. INIAV has wo main a eas o ac i i y. The i s is he na ional e e ence labo a o ies, labo a o ies a he o e on o echnology in he Eu opean Union. The e is only one o hese labo a o ies pe coun y, and in he case o Po ugal, INIAV holds he e e ence labo a o ies o animal diseases, plan diseases and pes s, and ood sa e y. The o he majo a ea o ac i i y is esea ch and inno a ion in ag icul u e, ood, and o es y. Figu e 1.1: Dis ibu ion o INIAV´s in as uc u es in Po ugal. 3 INIAV is sp ead h oughou he na ional e i o y (Figu e 1.1), whe e he e a e scien i ic and echnological in as uc u es ha cons i u e an inno a ion ne wo k a he se ice o he ag o- ood sec o . I has a eam o o e 600 collabo a o s, 300 o whom a e highly specialized in hese a eas o knowledge. I wo ks in coope a ion wi h 500 pa ne s in Po ugal, seeking o espond o hei needs and oppo uni ies. Some o INIAV´s mission and assignmen s a e: o De elop he echnological and scien i ic ounda ions o he sec o -speci ic public policies. o P omo e esea ch, expe imen a ion, and demons a ion ac i i ies in he a eas o ag o o es y, c op p o ec ion, ood p oduc ion, animal and plan heal h, ood sa e y, as well as in he a ea o ood echnologies and bio echnology wi h applica ion in hese a eas, in acco dance wi h he public policies de ined o he espec i e sec o s, which ensu e echnical and scien i ic suppo leading o de elopmen and inno a ion and imp o emen o compe i i eness. o Ensu e ha he Na ional Re e ence Labo a o y pe o ms i s du ies, pa icula ly hose ela ed o ood sa e y, animal heal h, and plan heal h. o Coope a e wi h simila scien i ic and echnological ins i u ions, whe he domes ic o o eign, ake pa in science and echnology ac i i ies, such as conso ia, ne wo ks, and o he o ms o collabo a i e wo k, and p omo e knowledge ans e wi h domes ic and in e na ional public and p i a e en i ies, pa icula ly h ough he conclusion o coope a ion ag eemen s and p o ocols, all wi hou comp omising he pu iew o he Minis y o Fo eign A ai s. o Pa icipa e in he c ea ion o o icial con ol plans o ood sa e y, plan heal h, and animal heal h. o Ensu e ha labo a o y analyses a e comple ed wi hin he pa ame e s o o icial con ol plans coo dina ed by he Minis y o Ag icul u e, speci ically h ough he ne wo king o exis ing acc edi ed labo a o ies, in he a eas o i s pu iew. 4 1.2. Apple Apples ( Malus domes ica ), a membe o he osaceae amily, a e ound all o e he wo ld, bu a e pa icula ly common in he no he n hemisphe e, since hey a e na i e om Cen al Asia.3 The e a e o e 7500 di e en cul i a s, each wi h an unique se o cha ac e is ics comp ising size, colo , i mness, shape, ex u e, la o (including swee , sou , and bi e sensa ions), juiciness, a oma, and nu i ional alue.4 Only a small po ion o hese cul i a s a e p oduced and sold wo ldwide, wi h pa icula high sha es o p oduc ion in he Asian con inen (Figu e 1.2).5 In 2020, almos 47% o he 86 million ons o apple p oduced wo ldwide, whe e p oduced in China alone, wi h he Uni ed S a es and Tu key coming in second and hi d wi h a ound 5% o he wo ld p oduc ion each.6 In 2020, 286 housand ons o apples we e p oduced in Po ugal, a om he peak o he 370 housand ons ha we e p oduced in he yea be o e (Table 1.1). Figu e 1.2: P oduc ion sha e o apples by egion, in 2020 6. 5 Table 1.1: Po ugal na ional p oduc ion o apples. Sou ce: GPP 2021. Yea O cha d A ea (ha) To al P oduc ion ( ons) 2012 12 903 220 761 2013 13 661 287 314 2014 13 847 273 721 2015 14 006 324 994 2016 14 159 254 321 2017 13 851 329 371 2018 13 612 263 961 2019 14 311 370 708 2020 14 313 286 075 Apple is he esh ui ha , by a , p esen s a highe p oduc ion in ons, he e o e ep esen s a signi ican sec o in ag icul u al p oduc ion in Po ugal. In he las i e yea s expo s ha e been inc easing, in such a way ha he ade balance u ned posi i e ( ep esen ing a p o i o 8 398 000€). (Table 1.2) Table 1.2: In e na ional ade balance ega ding apple comme ce (x 1000€). Sou ce: GPP 2021 Yea 2012 2013 2014 2015 2016 2017 2018 2019 2020 Expense 31 360 44 029 29 702 32 720 43 049 45 812 40 227 30 204 32 435 Sales 14 091 14 853 21 819 26 765 24 729 30 079 31 789 39 737 40 833 Balance -17 269 29 176 -7 883 -5 955 -18 321 -15 733 -8 438 9 533 8 398 The main supplie s o he domes ic ma ke a e Spain, F ance, Chile, and B azil. The majo i y o expo s go o he Eu opean Union, wi h Spain s anding ou . B azil and he Uni ed Kingdom also accoun o a sizable po ion o expo s (Sou ce: GPP 2021). Acco ding o he Po uguese Food Composi ion da abase om he Na ional Ins i u e o Heal h D Rica do Jo ge, apples a e mos ly composed o wa e (83%, w/w), suga (13.4%, w/w), die a y ibe (2.1%, w/w), and mine als, he mos signi ican o which being po assium, phospho us, sodium, and calcium. The lipid con en is 0.5% (w/w), and he p o ein con en is less han 0.3% (w/w) (Figu e 1.3). (Sou ce: INSA, 2022). Mo eo e , he ene gy alue o an apple is 64 kcal (269 kJ) pe 100g o edible weigh (Sou ce: INSA, 2022). 12 when conside ing only he peels. On he same s udy i was also epo ed ha he le el o ca echin was be ween 0.8 and 3.6 mg pe 100 g o esh ui and 2.0-5.4 mg in he peels.50 Figu e 1.9: S uc u e o he epica echin and ca echin molecules (adap ed om Del Rio e al. 51). 1.4.1.3. Fla ones Fla ones a y om o he la onoids ega ding he la onoid skele on, which in his case i has a double bond be ween C2 and C3, he e is no subs i u ion a he C3 posi ion, and he C4 posi ion is oxidized. A g owing body o e idence sugges s ha la ones including apigenin, lu eolin, and lu eolin-8-C-glucoside (Figu e 1.10) can p o ec skin cells om UVB adia ion.25,51 Fla ones can also p o ec plan s om pa asi es and ungal in ec ions by ac ing as na u al pes icides.52 Repo s on la one consump ion in adul s in Eu ope show esul s ha anges om 0.64 o 9.04 mg/day.53,54 Plan la ones a e usually conjuga ed as 7-O-glycosides. The mos p e alen la one C-glycosides ound a e 6-C and 8-C-glucosides. 13 Figu e 1.10: S uc u e o la one aglycones and some de i a i es (adap ed om Hos e le e al 52). 1.4.1.4. Iso la ones Iso la ones may be ound in a cul i a o plan s, mainly in he oo s and seeds. They' e ound in many di e en species o Fabaceae (Leguminosae) plan s, including soybeans (Genus: Glycine), chickpeas (Genus: Cice ), lupine (Genus: Lupinus), a a beans (Genus: Vicia), Poaceae, ba ley (Genus: Ho deum), and o B assicaceae, like b occoli (Genus: B assica), and cauli lowe (Genus: B assica).55 Iso la ones ha e also been ound in plan s o he han legumes, such as linseed and ed clo e .56 Soybeans and hei p oduc s a e among he iches and mos p e alen sou ces o iso la ones, and hei human heal h bene i s ha e been ex ensi ely esea ched, leading o he disco e y ha hey p omo e bone 57 and p os a e heal h 58 as well as possess an ibac e ial ac i i y.59 Daidzein and genis ein a e wo o he main aglycones. They a e he wo mos ecognized compounds in his subclass, and hey con ibu e signi ican ly o he o e all quan i y o iso la ones 14 ound in a ious ood ma ices, which may each 126 mg/100 g o p oduc in some e men ed oods like miso (p oduc ob ained by e men ing soybeans).56 1.4.1.5. Fla anones In gene al, hese molecules appea as la onoid glycosides.60 In ci us ui s, he loca ion o glycosyla ion is ound a posi ion 7 on he la anone uni – hese a e known as ci us la anones. The mos common la anone-7-O-glycosides a e e ioci in, na ingin, na ingenin, and hespe idin (Figu e 1.11).61 In ecen yea s se e al s udies ha e shown ha ci us la anones may be use ul in he ea men o diabe es 62 and ha hey can enhance he bioa ailabili y o ca o enoids 63 as well as possess an i i al and an i- umo al ac i i ies.64 1.4.1.6. An hocyanidins An hocyanidins a e a p ominen subclass o la onoids ha migh be exploi ed as po en inhibi o s o α-glucosidase. An hocyanidins ha e been ound o be pa icula ly plen i ul in colo ed be ies such as bluebe ies and blackcu an s. As a esul , hese b igh ly colo ed be y ui s may be ich in α-glucosidase inhibi o y an hocyanidins.65 Figu e 1.11: S uc u e o common la anone-7-O-glycosides in ui s. 15 An hocyanidin is gene a ed by he hyd olysis o an hocyanin and has he same la onoid s uc u e as an hocyanin bu lacks he ke one g oups. As a esul , i is an hocyanin in i s aglycone o m.66 The bulk (mo e han 65 pe cen ) o an hocyanins a e glycosidic an hocyanins, which include glycoside ligands and so educe an hocyanin bioac i i ies such as an ioxidan ac i i y.67 Some o he mos common an hocyanidins and an hocyanins a e cyanidin, pela gonidin, delphinidin, pe unidin, peonidin and mal idin (Figu e 1.12). Figu e 1.12: S uc u es o common an hocyanidins and an hocyanins.69 1.4.2. Phenolic Acids Phenolic acids a e phenolic compounds ha ha e a ca boxylic acid g oup in hei s uc u e and a e di ided in wo majo subclasses, hyd oxycinnamic (C6-C3 s uc u e) and hyd oxybenzoic (C6-C1 s uc u e) acids (Figu e 1.13), because o hei wo di e en ca bon s uc u es, as well as he numbe and loca ion o hyd oxyl g oups on he a oma ic ing.68 They a e he mos p ominen class o phenolic compounds, esponsible o gi ing ood hei o ganolep ic p ope ies, including la o , colo , ha shness and as ingency.69 Figu e 1.13: Basic skele on s uc u e o phenolic acids (adap ed om Gu ié ez-G ijal a e al.69) 16 1.4.2.1. Hyd oxycinnamic acids Many ege ables and ui s con ain hyd oxycinnamic acids, a g oup o na u ally occu ing phenylp openoic acid compounds ha a e de i a i es o cinnamic acid. These include ca eic acid, chlo ogenic acid, couma ic acid, sinapic acid, and e ulic acid (Figu e 1.14).70 They ha e a wide a ie y o medicinal e ec s, such as an i-in lamma o y, an ioxidan , neu op o ec i e, and an i- amyloid agg ega ion, which a e ele an o he ea men o condi ions like Alzheime 's disease.71 Compa ed o hyd oxybenzoic acids, hyd oxycinnamic acids a e mo e common in na u e and ypically exis in a cul i a o conjuga ed o ms. Cli o d 72 obse ed ha people ha consume a small amoun o ui s and ege ables will inges 25 mg o hyd oxycinnamic acids pe day, bu people who a e egula co ee consume s will ha e a daily in ake o 500-800 mg (mos ly chlo ogenic and ca eic acid). In he wo k o Pé ez-Jiménez e al. 73 he egis e ed daily in ake o hyd oxycinnamic acids (600 mg/day) was signi ican ly highe han ha o hyd oxybenzoic acids (41 mg/day). 1.4.2.2. Hyd oxybenzoic acids Hyd oxybenzoic acids can be ound in wo di e en o ms: bound o he componen s o cells o conjuga ed (soluble o m) wi h suga s o o ganic acids. Red ui s, onions, and black adish a e a ew excep ions ha ha e sligh ly highe le els o he hyd oxybenzoic acid han o he edible plan s. These ypically accumula e e y low le els o his compound.69 Gallic, ellagic, gen isic, p o oca echuic, sy ingic, salicylic, and anillic acids, as well as 3-hyd oxybenzoic and 4- Figu e 1.14: Chemical s uc u e o e ulic acid.72 17 hyd oxybenzoic acids a e he hyd oxybenzoic acid de i a i es (Figu e 1.15) ha a e p ima ily ound in ui s and ege ables. 74,75 Recen ly, heal h bene i s ha e been ound o be ela ed o de i a i es o hyd oxybenzoic acid, like an i-cance p ope ies in gallic acid 76 and ellagic acid 77 and hepa op o ec i e, ca diop o ec i e, an i-apop o ic, and an ip oli e a i e p ope ies in anillic acid.78 In he s udy epo ed by Pé ez-Jiménez e al. 73 , he in ake o hyd oxybenzoic acids o 4942 pa icipan s was ound o be 41 ± 39 mg/day. 1.4.3. S ilbenes The polyphenolic subs ances known as s ilbenes (Figu e 1.16), which ha e a C6-C2-C6 s uc u e, a e p oduced by he seconda y me abolism o plan s. They a e made up o a ans o cis -e hene double bond ha has had phenyls subs i u ed on bo h o i s ca bon a oms.79 They a e highly ega ded o hei an imic obial quali ies in pa icula ,80 bu also o hei an i- ungal,81,82 an icance , an i-in lamma o y and an ioxidan p ope ies. Figu e 1.15: S uc u e o some common hyd oxybenzoic acid de i a i es. Figu e 1.16: Basic s uc u e o s ilbenes (adap ed om Pa an e al. 85). 18 S ilbenes and hei de i a i es, s ilbenoids - na u al, biologically ac i e subs ances, o m a mul idisciplina y ield ha combines many impo an b anches o chemis y. I is possible o ind his g oup o compounds in na u e (Figu e 1.17). Bo h monome s and p og essi ely mo e complex oligome s o s ilbenoids exis . The ans isome o he monome ic s ilbene aglycone is he mos p e alen con igu a ion, and i s ai ly simple skele on consis s o wo a oma ic ings connec ed by an e hylene b idge. The majo i y o s ilbenes ha a e ound in na u e ha e mul iple phenol g oups.83 F ui s like g apes and bluebe ies a e hei p incipal ood sou ces.79 Figu e 1.17: Gene al skele on o common s ilbenoids (Adap ed om Ri iè e e al.86) 1.4.4. Lignans Lignans a e bioac i e polyphenolic compounds wi h highly complex s uc u es ha a e p oduced when wo coni e yl alcohol esidues combine. They possess neu op o ec i e84, an i i al 85 and an ioxidan 86 p ope ies. They can be b oadly di ided in o plan lignans and mammalian lignans depending on whe e hey o igina ed om.87 While mammalian lignans ha e hyd oxyl g oups in -me a posi ion, plan lignans p ima ily ha e oxygena ed subs i uen s in -pa a posi ions. Some plan lignans ound in many edible plan s a e ans o med in o mammalian lignans like en e odiol and en e olac one (Figu e 1.18) by in es inal mic obio a.88 On he basis o hei s uc u al cha ac e is ics, such as hei ca bon skele ons, he manne in which oxygen is inco po a ed in o he skele ons, and he (E)-S ilbene Z)-S ilbene 2-a ylbenzo u an bis(bibenzyl) ype A phenan h ene 9,10-dihyd ophenan h ene Dihyd os ilbene o bibenzyl 19 pa e n o cycliza ion, lignans a e di ided in o eigh classes, a ylnaph halene, a yl e alin, dibenzocyclooc adiene, dibenzylbu ane, dibenzylbu y olac ol, dibenzylbu y olac one, u an, and u o u an. 89 Figu e 1.18: Me abolism o plan lignans o mammalian lignans.91 The majo i y o plan s ha a e high in ibe ha e lignans in hei composi ion, including sesame seeds, pumpkin seeds, g ains like ye, ba ley, and whea , legumes like len ils and beans, and ege ables like b occoli, aspa agus, ga lic, and ca o s. Foods o en ha e modes lignan le els, usually less han 2 mg/100g. Sesame seeds and laxseed a e he excep ions since hey ha e lignan con en s ha a e se e al imes highe han hose o o he ood sou ces. 90 1.5. Me hods o he quan i ica ion o bioac i e compounds Due o he wide cul i a o bioac i e chemicals ound h oughou plan issues and he cul i a o hei chemical s uc u es, nume ous analy ical p ocedu es o hei iden i ica ion and quan i ica ion ha e had o be de eloped. The ea lies me hods c ea ed we e spec opho ome ic me hods, which a e pa icula ly in e es ing om a quali y con ol pe spec i e since hey o e quick, e icien , and a o dable solu ions. I has become equi ed o adop mo e p ecise echniques, like ch oma og aphic echniques, which allow he indi idual iden i ica ion o each o he bioac i e subs ances o nu i ional ele ance, because spec opho ome ic echniques, despi e ha ing hese ad an ages, ha e some limi a ions. 20 1.5.1. Spec opho ome ic me hods Fo he pu pose o de e mining he amoun o bioac i e chemicals p esen in plan ma ices, nume ous spec opho ome ic echniques ha e been de eloped. These echniques enable he quan i ica ion o a ce ain g oup o bioac i e subs ances , such as he o al con en o phenolic compounds,91 as well as he quan i ica ion o a single bioac i e subs ance's quan i y, such as β- ca o ene 92 o wo a he same ime, like β-ca o ene and lycopene,93 in di e en ood ma ices. Despi e he cul i a o spec opho ome ic echniques cu en ly a ailable, hey ha e some d awbacks. When complex samples a e p esen , ce ain ma ix elemen s may in e e e wi h he analysis and cause he analysis o o e es ima e he amoun o de e mined subs ances. Reduced speci ici y o he me hods is he e o e a signi ican limi a ion.94,95 1.5.1.1. DPPH ee adical inhibi ion assay The DPPH assay is a well-known echnique ha is commonly used since i is s aigh o wa d, inexpensi e, and jus uses a basic spec opho ome e . The e a e a ew s anda ds ha can be used o assess he an ioxidan capaci y in oods, plan ex ac s, and d inks, including asco bic acid, gallic acid, and T olox.96,97 In he DPPH ee adical inhibi ion assay, he addi ion o he ex ac , ha has an ioxidan s, educes an unpai ed alence elec on o he N (ni ogen) a om in DPPH, by gi ing a hyd ogen a om, causing he o ma ion o DPPH-H and a change o colo om pale pu ple o a yellow-colo ed p oduc , in a concen a ion-dependen way (Figu e 1.19).98 An ioxidan s' abili y o educe can hus be assessed by moni o ing he abso bance's decline, on a wa eleng h o 515nm. Figu e 1.19: Reduc ion o DPPH in he DPPH ee adical inhibi ion assay.101 21 1.5.1.2. To al con en o phenolic compounds assay Quan i ica ion o phenolic compounds can be accomplished using a numbe o me hods, such as he Folin-Ciocal eu assay and CuO oxida ion-GC and a high-pe o mance liquid ch oma og aphy (HPLC) me hod.99 In his wo k, we op ed o he Folin-Ciocal eu, because when compa ed o he CuO oxida ion-GC and he HPLC p ocedu es, his es is mo e a o dable and less complica ed. This me hod elies on he Folin-Ciocal eu eagen 's (composed o oxides o ungs en and molybdenum). In he ully oxidized 6+ alence s a e o he me al, he isopolyphospho ungs a es a e colo less while he molybdenum compounds a e yellow.100 They exis in an acidic solu ion and combine o o m mixed he e opolyphospho ungs a es-molybda es. Re e sible one o wo elec on sequen ial educ ions lead o blue species 101 (Figu e 1.20), due o he addi ion o an elec on o a nonbonding o bi al. Figu e 1.20: Reac ion be ween polyphenols and he Folin Ciocal eu eagen .102 1.5.1.3. To al con en o la onoids assay The o al la onoids con en (TFC) in plan s is ypically de e mined calo ime ically. One o he mos common me hods o de e mining TFC in plan and ui ex ac s is he aluminum chlo ide colo ime ic assay, in which Al(III) is used as a complexing agen (Figu e 1.21), o ming a complex wi h he hyd oxyl g oup o la onoids.103 The o al la onoids con en can be accessed wi h he aid 28 The packed columns a e made o signalized glass and packed wi h solid pa icles coa ed wi h he liquid ha makes up he mobile phase.127 The analy e's pola i y, molecula weigh , solubili y, ola ili y, and quan i y in he sample should all be aken in o accoun while selec ing he column. The highe he molecula weigh and pola i y o he subs ance, he lowe i s ola ili y and consequen ly he g ea e he di icul y o analyze.124,128 A de ec o is also pa o a ch oma og aph. This ins umen ampli ies he elec ical signal, iden i ies, and quan i ies he componen s sepa a ed by he column; he analy e o be s udied should be aken in o conside a ion when choosing his ins umen . The e a e a ious de ec o ypes, wi h he ollowing being he mos popula in gas ch oma og aphy: • Flame-ioniza ion de ec o (FID)129 • Elec on cap u e de ec o (ECD)130 • The mal conduc i i y de ec o (TCD)131 • Mass spec ome y de ec o (MS)132 Essen ially, he e a e wo conside a ions in choosing a de ec o . The wo aspec s a e sensi i i y, i.e., he signal he de ec o is capable o c ea ing, and he noise, which desc ibes he de ec o 's ins abili y. The sensi i i y dec eases as noise le el inc eases. 1.5.3. Ex ac ion echniques Due o he complexi y o he ma ix, i s incompa ibili y wi h ch oma og aphic sys ems, and he ac ha many o he subs ances o be s udied a e in ace amoun s, ch oma og aphic analysis o ood samples equen ly equi es p e ious ea men o he sample 133. The e a e nume ous sample p e ea men me hods a ailable oday o he ex ac ion o bioac i e subs ances om ood ma ices. The mos signi ican me hods ha ha e been equen ly employed o he ex ac ion o phenolic compounds om a ious ood ma ices a e solid phase ex ac ion (SPE)134,135, QuEChERS (Quick, Easy, Cheap, E ec i e, Rugged and Sa e)136,137 and solid-liquid ex ac ion (SLE).138 29 1.5.3.1. Solid-Liquid Ex ac ion (SLE) The mos popula analy ical me hod o p epa ing solid samples is known as solid-liquid ex ac ion (SLE), which in ol es sepa a ing he analy es o in e es om he o he compounds in he ma ix. Th ee key mechanisms con ol he SLE p ocess: he pene a ion o he ex ac an in o he solid ma ix, he di usi i y o he analy es o he ou e space and he solubili y o he analy es in he ex ac an .139 SLE has his o ically been cha ac e ized by a lack o quan i a i e e iciency, and se e al measu es ha e been aken o imp o e i s unc ionali y. The use o high empe a u e wi h high p essu e and he aid wi h auxilia y ene gies, pa icula ly mic owa es and ul asound, s and ou as he mos e ec i e me hods o imp o ing SLE quan i a i ely. These app oaches ha e gi en ise o sample p epa a ion me hods like supe hea ed sol en ex ac ion, mic owa es-assis ed ex ac ion, and ul asound-assis ed ex ac ion. These me hods enable imp o ed pe o mance in addi ion o sho e ex ac ion imes, au oma ion o he p ocedu e, and less consump ion o o ganic sol en s. 1.5.3.2. Solid Phase Mic oEx ac ion (SPME) SPME is a e y s aigh o wa d so p i e ex ac ion-based sample ex ac ion and p econcen a ion echnique.140 Cu en ly, his me hod is equen ly used o examine he ola ile p o ile o a ious meals and d inks, including wine,141,142 and ui s.143,144 Gene ally, analy es p esen in aqueous ma ices a e ex ac ed by being abso bed o adso bed on o a hin used silica ibe co e ed wi h a polyme ic laye ,145 which is housed in a sy inge-shaped appa a us (Figu e 1.25). Figu e 1.25: SPME de ice. 30 SPME can be ca ied ou using di ec imme sion (DI-SPME), headspace (HS-SPME), o wi h he aid o a memb ane (M-SPME) (Figu e 1.26), depending on he ype o analy es being in es iga ed and he complexi y o he sample. HS-SPME exposes he ibe o he apo phase abo e he sample, whe eas DI-SPME places he ibe in di ec con ac wi h he sample. Memb ane ex ac ion is compa able o DI-SPME wi h he sole excep ion ha he ibe is shielded by a semipe meable memb ane. Figu e 1.26: Schema ic ep esen a ion o SPME modes: (A) di ec imme sion, (B) headspace, (C) memb ane assis ed (Adap ed om Pawliszyn151). Since HS-SPME is mo e selec i e han di ec imme sion and, a oiding addi ional in e e ences such as high molecula weigh molecules ha may be p esen in he ma ix, his ex ac ion mode is he mos equen ly employed. 1.6. F uc ose F uc ose is a ke onic suga p esen , in la ge quan i ies, in he juices o plan s and ui s, whe e i equen ly o ms he disaccha ide suc ose when linked o glucose. I is one o he h ee die a y monosaccha ides ha a e di ec ly abso bed in o he blood du ing diges ion, along wi h galac ose and glucose. Howe e , i does no en e he bloods eam in la ge amoun s because i is p ima ily con e ed in o glycogen o iglyce ides once i eaches he li e . 146 Sample headspace Fibe Memb ane A Sample Coa ing Coa ing Sample B C 31 Figu e 1.27: Rela i e swee ness (%) o na u al suga s and swee ene s. Suc ose is e e ence and is se a 100 (Adap ed om Basso e al.153) One o he main asse s o uc ose is i s swee ening powe . In a s udy de eloped by Basso e al. 147 (Figu e 1.27), i was s a ed ha all he swee ene s es ed had a ela i e swee ness lowe han suc ose, and he e o e a e less swee han suc ose. On he o he hand, uc ose p o ed o be almos wice as swee as suc ose. Howe e , i is well es ablished ha his is only ue, when he 6- membe ed ing o m o uc ose is p esen , because when hea ed, his ing o ms a 5-membe ed ing o m and gi es only as much swee ening powe as egula suc ose.148 Some o he bigges na u al sou ces o uc ose a e ui s, pa icula ly, g apes149 and apples,150 and ege ables.151 I is also abundan ly used in he p ocess o making high- uc ose co n sy up (HFCS), a swee ene p oduced wi h co n sy up ha is commonly used in he be e age and in ce eals and p ocessed oods indus ies.152 1.6.1. To al con en o uc ose assay The e a e a a ie y o me hods o de e mine he o al uc ose con en in ood ma ixes, such as he one de eloped by Mo ei a e al., 153 in which a elec osyn hesized molecula ly imp in ed polyme is used, and also he me hod epo ed by Rong ong e al., 154 using nea -in a ed spec oscopy o de e mine di e en cons i uen s o ood p oduc s, like uc ose. A mo e es ablished colo ime ic me hod is he one de eloped by Ashwell, 155 based on he p inciple ha he 0 20 40 60 80 100 120 140 160 180 200 Rela i e Swee ness (%) 32 hyd oxyme hyl u u al o med om uc ose in acid medium156 (Figu e 1.28) eac s wi h eso cinol o gi e a ed colo p oduc . Figu e 1.28: Hyd oxyme hyl u u al o ma ion, om uc ose.156 1.7. Mul i a ia e da a analysis (Chemome ics) Chemome ics is desc ibed by some people as “ he chemical discipline ha uses ma hema ical, s a is ical, and o he me hods employing o mal logic o design o selec op imal measu emen p ocedu es and expe imen s, and o p o ide maximum ele an chemical in o ma ion by analyzing chemical da a”.157 Despi e he b oad de ini ion o chemome ics, i is ob ious ha he use o mul i a ia e da a analysis in da a ob ained om analy ical chemical p ocedu es is i s mos use ul ins umen . The mul i a ia e da a analysis has gained ecogni ion as a po en me hod o s uc u ing and analyzing da a se s in he ields o chemis y and biochemis y.157 In hese ields o esea ch, undamen ally in analy ical chemis y, and ood science, he in o ma ion e ie ed om ins umen al measu emen s is highly complex and equen ly made up o housands o a iables o each sample. Mul i a ia e da a analysis is equi ed o unde s and/sol e he in o ma ion in he da a due o i s complexi y. Bo h quan i a i e and quali a i e analysis can be used o assess he expe imen al da a. The e a e wo ypes o quali a i e analy ic me hods: supe ised lea ning and unsupe ised lea ning.158 P incipal componen analysis (PCA) i s unde he unsupe ised lea ning ca ego y. 33 Figu e 1.29: Mul i a ia e da a analysis me hods used in he e alua ion o Ag o-Food p oduc s. MLR: Mul iple Linea Reg ession, PCR: P inciple Componen Reg ession, PLSR: Pa ial Leas Squa e Reg ession, ANN: A i icial Neu al Ne wo k, SVM: Suppo Vec o Machine, Mul i a ia e da a analysis me hods used in he e alua ion o Ag o-Food p oduc s. MLR: Mul iple Linea Reg ession, PCR: P inciple Componen Reg ession, PLSR: Pa ial Leas Squa e Reg ession, ANN: A i icial Neu al Ne wo k, SVM: Suppo Vec o Machine, LDA: Linea Disc iminan Analysis, PLS- DA: Pa ial Leas Squa es–Disc iminan Analysis, kNN: k- Nea es Neighbo , PCA: P incipal Componen Analysis.159 1.7.1. P incipal componen analysis (PCA) P incipal componen analysis is a s a is ical ool ha allows o educe he dimensionali y o he da a which con ain a se o a iables using linea combina ions. I is used wi h sample esul s o demons a e how and how much he examined a iables a ec he ange o he measu ed alues.160 PCA is a ool ha is used o dec ease edundancy and he numbe o a iables used in he sys em obse a ion, by es ablishing a new da abase whose componen s a e linea ly independen o he p ima y componen s indica ed by he i s se o a iables. These new componen s a e hen o de ed so ha hey ep esen mos o he o iginal a iance.161 1.8. Pes icides The use o pes icides is s ill a eali y and, in ac , is essen ial o p e en ood loss e en while e o s o dec ease o ind al e na i es a e apidly de eloping. The use o pes icides, howe e , also has nega i e impac s on he en i onmen o ood consume s. As a esul , i is c ucial o manage pes icide esidues in ood, and in he Eu opean Union, his is suppo ed by egula ion o sa egua d public sa e y as well as domes ic and in e na ional ade. 34 E ec i e sample p epa a ion and ace-le el de ec ion and iden i ica ion a e i al componen s o analy ical me hods due o he low de ec ion limi s demanded by egula o y bodies and he complex s uc u e o ood ma ices in which he a ge compounds a e con ained. 35 2. Objec i es The main objec i es o his esea ch s udy a e: • De e mina ion o an ioxidan p ope ies o bo h by-p oduc s (peels and seeds) and edible pa (mesoca p) o i e di e en egional apples (Pê o de Bo belo, Pa do Lindo, Repinau, Pê o Coimb a and Noi a) and six egional pea s (Bela-Feia, To es No as, Ca apinhei a, Ca apinhei a Roxa, Lambe-os-Dedos e Amo im), in wo consecu i e ha es yea s (2021 and 2022) in o de o cha ac e ize Po uguese cul i a s o hese ui s conce ning hei bioac i es’ composi ion, o which he e a e sca ce da a, and o con ibu e o conse a ion o ui biodi e si y and consump ion o di e se die s; • De e mina ion o an ioxidan p ope ies o 22 comme cial cul i a s o apples p oduced in he same egion (Alcobaça, Po ugal) in o de o compa e hem wi h egional cul i a s; • E alua e uc ose con en o bo h egional and comme cial cul i a s o apples and pea s o conclude abou hei po en ial o subs i u e suga in ood o mula ions; • Op imiza ion and alida ion o an analy ical me hod o he de e mina ion o 23 indi idual phenolic compounds in ui s by ul a-high pe o mance liquid ch oma og aphy coupled o ime o ligh mass spec ome y (UHPLC-ToF-MS), acco ding o in e na ional guidelines. • Applica ion o he UHPLC-ToF-MS analy ical me hod o selec ed pea and apple samples. • Ca y ou a PCA in o de o e alua e he ela ionship be ween he apple and pea samples and hei chemical p ope ies. • E alua ion o he pes icide esidues con en in apple and pea cul i a s a ge ed o s udy and o conclude abou hei sa e y. • Discussion o he esul s abou he po en ial o he di e en apple and pea cul i a s o be used as sou ce o na u al an ioxidan s o as a sou ce o uc ose. 36 3. Expe imen al Pa This sec ion desc ibes he ma e ials, equipmen and eagen s used, he cha ac e iza ion o he samples, as well as all he expe imen al p ocedu es pe o med h oughou his wo k. The expe imen al p ocedu es we e ca ied ou mos ly in Na ional Ins i u e o Ag a ian and Ve e ina y Resea ch - Polo de Vai ão (Vila do Conde, Po ugal), and in Uni e si y o Minho (B aga, Po ugal) (pes icide analysis). 3.1. Reagen s The eagen s used and hei espec i e cha ac e is ics a e desc ibed in Table 3.1. No e ha all eagen s a e o analy ical g ade and he ul apu e MilliQ® wa e was ob ained om a Milli- Q® Di ec Wa e Pu i ica ion Sys em. Table 3.1: Reagen s and hei cha ac e is ics used o he de e mina ion o he an ioxidan p ope ies and he quan i ica ion o uc ose in apple and pea samples. Reagen s Chemical o mula S a e o ma e Pu i y le el (%) B and E hanol C2H6O Liquid 96.0 Honeywell T olox C14H18O4 Solid 97.0 Sigma-Ald ich DPPH C18H12N5O6 Solid 95.0 Sigma-Ald ich Me hanol CH4O Liquid 99.9 Honeywell Folin-Cioucal eu eagen C6H6O Liquid - Sigma-Ald ich Sodium ca bona e Na2CO3 Solid 99.5 Sigma-Ald ich Epica echin C15H14O6 Solid 90.0 Sigma-Ald ich Sodium ni i e NaNO2 Solid 95.0 Supelco Aluminum chlo ide AlCl3 Solid 98.0 Sigma-Ald ich Sodium hyd oxide NaOH Solid 97.0 Sigma-Ald ich β-Ca o ene C40H56 Solid 93.0 Sigma-Ald ich Chlo o o m CHCl3 Liquid 99.5 Sigma-Ald ich Tween® 40 C62H122O26 Liquid - Sigma-Ald ich Linoleic acid C18H32O2 Liquid 98.0 Sigma-Ald ich F uc ose C6H12O6 Solid 99.0 Sigma-Ald ich Reso cinol C6H6O2 Solid 99.0 Sigma-Ald ich Thiou ea CH4N2S Solid 99.0 Sigma-Ald ich 37 Reagen s Chemical o mula S a e o ma e Pu i y le el (%) B and Ace ic acid C2H4O2 Liquid 99.7 Honeywell Hyd ochlo ic acid HCl Liquid 37.0 Honeywell o-Couma ic acid C9H8O3 Solid 97.0 Sigma-Ald ich Chlo ogenic acid C16H18O9 Solid 95,0 Sigma-Ald ich Hype oside C21H20O12 Solid 99.0 Sigma-Ald ich p-Couma ic acid C9H8O3 Solid 98.0 Sigma-Ald ich Ca eic acid C9H8O4 Solid 98.0 Sigma-Ald ich Ru in Hyd a e C27H30O16 . xH2O Solid 94.0 Sigma-Ald ich T ans- e ulic acid C10H10O4 Solid 99.0 Sigma-Ald ich Lu eolin C15H10O6 Solid 97.0 Supelco Que ce in 3-β-D-glucoside C21H20O12 Solid 90.0 Sigma-Ald ich Que ce in C15H10O7 Solid 95.0 Sigma-Ald ich Gen isic acid C7H6O4 Solid 98.0 Sigma-Ald ich Sinapic acid C11H12O5 Solid 98.0 Sigma-Ald ich Apigenin C15H10O5 Solid 99.0 Supelco 4-Hyd oxybenzoic acid C7H6O3 Solid 99.0 Sigma-Ald ich Sy ingic acid C9H10O5 Solid 95.0 Sigma-Ald ich (−)-Epica echin C15H14O6 Solid 98.0 Sigma-Ald ich Vanillic acid C8H8O4 Solid 97.0 Sigma-Ald ich (+)-Ca echin C15H14O6 Solid 99.0 Supelco Phlo idzin C21H24O10 Solid 98.0 Sigma-Ald ich Que ci in C21H20O11 Solid 99.0 Sigma-Ald ich (±)-Na ingenin C15H12O5 Solid 95.0 Supelco E iodic yol C15H12O6 Solid 95.0 Sigma-Ald ich 3.2. Ma e ials In o de o success ully pe o m he analy ical p ocedu e, ce ain ma e ials we e used ha we e p ope ly calib a ed acco ding o he in e nal labo a o y p o ocols and checked whene e used (Table 3.2). 44 3.4.1.13. Sodium ni i e solu ion (50 mg/mL) The sodium ca bona e solu ion was p epa ed by using a p ecision scale o weigh 0.5 g o sodium ni i e in o a 10 mL olume ic lask and making up he olume wi h ul apu e MilliQ® wa e . 3.4.1.14. Aluminum chlo ide solu ion (100 mg/mL) The aluminum chlo ide solu ion was p epa ed by using a p ecision scale o weigh 2.0 g o aluminum chlo ide in o a 20 mL olume ic lask and making up he olume wi h ul apu e MilliQ® wa e . The p epa a ion o his solu ion should be deal ca e ully, because his is an exo he mic eac ion. 3.4.1.15. Sodium hyd oxide solu ion (40 mg/mL) The sodium hyd oxide solu ion was p epa ed by using a p ecision scale o weigh 2.0 g o sodium hyd oxide in o a 50 mL olume ic lask and making up he olume wi h ul apu e MilliQ® wa e . 3.4.1.16. β-ca o ene solu ion (0.2 mg/mL) The β-ca o ene solu ion was p epa ed by using a p ecision scale o weigh 1.0 mg o β- ca o ene in o a 5 mL olume ic lask and making up he olume wi h chlo o o m. This solu ion mus be p epa ed and used on he day o pe o ming he assay. 3.4.1.17. β-ca o ene emulsion The β-ca o ene emulsion was p epa ed by using a p ecision scale o weigh 20 mg o linoleic acid, 200 mg o Tween®40 and adding 2 mL o he β-ca o ene solu ion in o a 250 mL olume ic lask. This mix u e was e apo a ed on a o a y e apo a o a 40 °C. Then, 100 mL o ul apu e MilliQ® wa e , ha was agi a ed o 30 minu es o inco po a e oxygen, we e added, and igo ously agi a ed, un il an emulsion was o med. 45 3.4.1.18. Reso cinol eagen The eso cinol eagen was p epa ed by using a p ecision scale o weigh 1.0 g o eso cinol and 250 mg o hiou ea in o a 100 mL olume ic lask and making up he olume wi h glacial ace ic acid. This solu ion mus be p epa ed and used on he day o pe o ming he assay. 3.4.1.19. Dilu e hyd ochlo ic acid solu ion The dilu ed HCl solu ion was p epa ed in a 250 mL olume ic lask, using a beake o measu e 50 mL o concen a ed HCl and making up he olume wi h ul apu e MilliQ® wa e . 3.4.1.20. Phenolic compounds s anda d solu ions S anda d solu ions o 23 phenolic compounds we e p epa ed by using a p ecision scale o weigh 2.5, 5.0 o 10.0 o he 23 phenolic compounds, in o a 5- o 10-mL olume ic lask and making up he olume wi h e hanol. The di e en concen a ions o he s ock solu ions can be seen in Table 3.8. Table 3.8: P epa a ion o he di e en phenolic compounds’ s anda ds. S anda d S ock solu ion (mg/mL) Wo king solu ion (µg/mL) o-Couma ic acid 1.0 100 Chlo ogenic acid 1.0 100 p-Couma ic acid 1.0 100 Ca eic acid 1.0 100 Ru in Hyd a e 1.0 100 T ans- e ulic acid 1.0 100 Lu eolin 0.5 50 Que ce in 3-β-D-glucoside 0.5 50 Que ce in 1.0 100 46 S anda d S ock solu ion (mg/mL) Wo king solu ion (µg/mL) Gen isic acid 1.0 100 Sinapic acid 1.0 100 Apigenin 0.5 50 4-Hyd oxybenzoic acid 1.0 100 Sy ingic acid 1.0 100 (−)-Epica echin 0.5 50 Vanillic acid 1.0 100 (+)-Ca echin 0.5 50 Phlo idzin 1.0 100 Que ci in 1.0 100 (±)-Na ingenin 1.0 100 E iodic yol 0.5 50 The s anda d solu ions we e s o ed a -24 °C o up o h ee mon hs. 3.4.1.21. Phenolic compounds s anda d mix u e solu ion A e ob aining he s anda d solu ions o he di e en phenolic compounds, a olume o 0.625 o 1.25 mL was ans e ed om each o he solu ions in o a 25 mL olume ic lask and he olume was p e ixed wi h me hanol. This mix u e has a concen a ion o 2.5 µg/mL o each one o he phenolic compounds. This solu ion can be s o ed a -24 °C in da k condi ions o up o h ee mon hs. 3.4.2. P epa a ion o he samples In his s udy 11 samples o egional ui cul i a s we e used, om he Alcobaça campus o INIAV, in which 5 we e apple egional cul i a s (Noi a, Repineau, Pê o Coimb a, Pa do Lindo and Pê o de Bo belo) (Figu e 3.1) and 6 we e pea egional cul i a s (Ca apinhei a, Bela-Feia, To es No as, Ca apinhei a Roxa, Lambe-os-Dedos and Amo im) (Figu e 3.2). 47 Noi a Repinau Pê o Coimb a Pa do Lindo Pê o de Bo belo Ca apinhei a Bela-Feia To es No as Ca apinhei a- Roxa Lambe-os- Dedos Amo i The samples we e collec ed be ween July and No embe 2021 and July and Sep embe 2022. The sampling echnique consis ed o andomly collec ing 10 o 15 ipe ui s o each cul i a o ensu e ha he ha es was ep esen a i e. The samples we e s o ed in he absence o ligh and sen o he Inno a ion Campus o Vai ão, INIAV, I.P. The samples we e sepa a ed in 3 po ions: peels, mesoca p and seeds. The 3 po ions we e homogenized wi h he help o a homogenize and ozen a <-20°C. 3.4.2.1. Fo he de e mina ion o he an ioxidan p ope ies Fo each sample, 2.0 g we e weigh ed be o e eezing in o 50 mL Falcon ubes. Then, on he day o he assays o he de e mina ion o he an ioxidan p ope ies, 20 mL o sol en (e hanol) was added, and he solu ion was mixed on an Ul a-TURRAX homogenize o 3 minu es. The samples we e hen cen i uged a 3600 pm o 10 minu es a oom empe a u e, leading o a good sepa a ion o he wo phases. The supe na an was isola ed and s o ed in he absence o ligh and in a low empe a u e en i onmen (Figu e 3.3). Figu e 3.1: Regional apple cul i a s. Figu e 3.2: Pea egional cul i a s. 48 3.4.2.2. Fo he de e mina ion o he uc ose con en Fi s , he ex ac s ha had been p e iously p epa ed o he s udy o an ioxidan capaci y and o al con en o phenolic compounds and o al la onoids we e used o e alua e he uc ose con en . Howe e , in o de o s udy he in luence o he sol en (e hanol) on he de e mina ion o he uc ose con en , some ex ac s o he ui mesoca p we e also p epa ed in ul apu e wa e . 3.4.3. Expe imen al p ocedu e Compa a i e quan i ica ion o o al phenolic compounds con en and o al la onoids con en was pe o med on he edible pa and he by-p oduc s o he di e en samples. Fu he mo e, he an ioxidan capaci y o he ex ac s was also measu ed by DPPH adical and β- ca o ene bleaching me hods. Also, he o al uc ose con en was analyzed in hese ma ices. 3.4.3.1. DPPH ee adical inhibi ion assay This me hod was ca ied ou as desc ibed by Mou e e al. 162 and modi ied by And ade e al. 163 using T olox as he s anda d. To de e mine he an ioxidan capaci y o he e hanolic ex ac s h ough he DPPH ee adical inhibi ion assay, 50 µL o sample a e mixed wi h 2 mL o he DPPH adical solu ion. Nex , his mix u e is kep in he da k o 30 minu es and ead in a spec opho ome e a 515 nm (Figu e 3.4). Figu e 3.3: Sepa a ion p ocess o he ui s’ po ions/pa s o be analyzed. 49 3.4.3.2. β-ca o ene bleaching assay This me hod was ca ied ou as desc ibed by Mille .164 In he β-ca o ene bleaching me hod, 200 µL o e hanol we e mixed wi h 5 mL o he β-ca o ene emulsion o p epa e he "blank" assay and his was ead in a spec opho ome e a 470 nm. A e his, 200 µL o sample we e also mixed wi h 5 mL o he emulsion and oge he wi h he blank we e kep in a wa e ba h a 55 °C o 2 hou s. A he end o his pe iod, bo h samples and he blank we e ead in a spec opho ome e a 470 nm (Figu e 3.5). The An ioxidan Ac i i y Coe icien (AAC) was calcula ed eso ing o he ollowing equa ion: 𝐴𝐴𝐶 =(𝐴 𝑠𝑎𝑚𝑝𝑙𝑒− 𝐴2 𝑐𝑜𝑛𝑡𝑟𝑜𝑙 𝐴0 𝑐𝑜𝑛𝑡𝑟𝑜𝑙− 𝐴2 𝑐𝑜𝑛𝑡𝑟𝑜𝑙)×1000 Whe e 𝐴0 𝑐𝑜𝑛𝑡𝑟𝑜𝑙 co esponds o he abso bance o he con ol a he ini ial ime and 𝐴2 𝑐𝑜𝑛𝑡𝑟𝑜𝑙 is he abso bance i he con ol a e 2 hou s a 50◦C and 𝐴 𝑠𝑎𝑚𝑝𝑙𝑒 is he abso bance o he sample, also a e being submi ed o 50◦C in a wa e ba h o 2 hou s. The solu ions' abso bance was ead a 470 nm. Figu e 3.4: DPPH ee adical inhibi ion sys em. (Sou ce: Pe sonal collec ion) 50 3.4.3.3. To al phenolics con en (TPC) assay This me hod was ca ied ou as desc ibed by E kan e al. 165 using gallic acid as he s anda d. In his me hod, 1 mL o sample is mixed wi h 7.5 mL o he Folin-Cioucal eu solu ion and a e 5 minu es, 7.5 mL o he sodium ca bona e solu ion a e added. A e 2 hou s, he samples a e ead in a spec opho ome e a 725 nm (Figu e 3.6). 3.4.3.4. To al la onoids con en (TFC) assay This me hod was ca ied ou as desc ibed by Ba bosa e al. 166 using epica echin as he s anda d. In his s udy, 1 mL o sample was mixed wi h 4 mL o ul apu e wa e and 0.3 mL o sodium ni i e. A e 5 minu es, 0.6 mL o aluminum chlo ide was added o he mix u e and a e Figu e 3.5: β -ca o ene bleaching assay. (Sou ce: Pe sonal collec ion) Figu e 3.6: De e mina ion o he o al con en o phenolic compounds (Sou ce: Pe sonal collec ion). 51 ano he 6 minu es, 2 mL o sodium hyd oxide and 2.1 mL o ul apu e MilliQ® wa e we e added. The samples we e hen ead in a spec opho ome e a 510 nm (Figu e 3.7). 3.4.3.5. To al con en o uc ose assay In his s udy o al con en o uc ose was de e mined acco ding o Ashwell,155 using uc ose as he s anda d. B ie ly, 2 mL o sample we e mixed wi h 1 mL o he eso cinol eagen . A e ha , 7 mL o he hyd ochlo ic acid solu ion we e added o he mix u e. The samples mix u es we e kep in a wa e ba h a 80 °C o 10 minu es. A he end o his pe iod, all samples we e cooled unde ap wa e o 5 minu es and ead in a spec opho ome e a 520 nm (Figu e 3.8). Figu e 3.8: De e mina ion o he o al uc ose con en . (Sou ce: Pe sonal collec ion) Figu e 3.7: De e mina ion o he o al con en o la onoids (Sou ce: Pe sonal collec ion). 52 3.5. De e mina ion o Phenolic Compounds by UHPLC-ToF A solid-liquid ex ac ion me hodology was used o ex ac phenolics om samples. Op imiza ion o he ex ac ion p ocedu e led o he inal ex ac ion me hod, desc ibed in Figu e 3.9. Two di e en ex ac ion sol en s we e es ed: me hanol (MeOH) and MeOH:H2O:Fo mic acid (49.95:49.95:0.10 / / ). I should be no ed ha he op imiza ion was ca ied ou using an apple sample (Pa do Lindo mesoca p) ha had been o i ied wi h he s anda d solu ion mix u e o phenolic compounds (1 and 10 g/kg). De ec ion and quan i ica ion we e pe o med wi h a Nexe a X2 Shimadzu UHPLC coupled wi h a 5600+ ToF-MS de ec o (SCIEX, Fos e Ci y, CA, USA) equipped wi h a Tu bo Ion Sp ay elec osp ay ioniza ion sou ce wo king in posi i e mode (ESI+). The phenolic compounds we e sepa a ed using an Acqui y UPLC BEH C18 (2.1 mm × 100 mm, 1.7 μm) analy ical column. The column empe a u e was main ained a 85ºC and he au osample was p og ammed o a empe a u e o 20ºC. The ch oma og aphic sepa a ion ook place in g adien mode using an aqueous solu ion o 0.1% o mic acid (eluen A) and me hanol wi h 0.1% o mic acid (eluen B) as he mobile phase. The injec ion olume o bo h s anda ds and samples was adjus ed o 20 µL. Figu e 3.9: Ex ac ion p ocedu e o he de e mina ion o phenolic compounds by UHPLC-ToF.(Sou ce: Pe sonal collec ion) 53 Table 3.9: Mobile phase g adien used o he sepa a ion o phenolic compounds. Time (min) Mobile Phase A (%) Mobile Phase B (%) 0.01 90 10 0.50 90 10 8.00 20 80 Using he Analys ® TF so wa e (SCIEX, Fos e Ci y, CA, USA) and he ollowing pa ame e s o mass spec ome y, he acquisi ion was ca ied ou in ull scan om 100 o 750 Da: ion sou ce ol age o 5500 V; sou ce empe a u e o 575 C; cu ain gas (CUR) o 30 psi; Gas 1 and Gas 2 o 55 psi; and declus e ing po en ial (DP) o 100 V. To p o ide accu a e mass esolu ion, he ToF-MS de ec o was calib a ed e e y 7 injec ions in he me hod's mass ange. PeakView™ and Mul iQuan ™ so wa e (SCIEX, Fos e Ci y, CA, USA) we e used o phenolic compound iden i ica ion and da a p ocessing. PeakView™ so wa e au oma ically p esen s he iso ope ma ch. Two pa ame e s and hei accompanying equa ions (Equa ions (1) and (2)) we e employed o phenolic compound iden i ica ion: (1) maximum e en ion ime de ia ion (∆RRT) o 0.1 min (Equa ion (1)); and (2) exac mass de ia ion (m) wi h a ole ance o 5 ppm (Equa ion (2)). 𝑅𝑇=(𝑅𝑇spiked samples −𝑅𝑇s anda d 𝑅𝑇s anda d )×100 Equa ion (1) ∆𝑚 (𝑝𝑝𝑚)=(𝐸𝑥𝑎𝑐𝑡 𝑚𝑎𝑠𝑠− 𝐷𝑒𝑡𝑒𝑐𝑡𝑒𝑑 𝑚𝑎𝑠𝑠 𝐸𝑥𝑎𝑐𝑡 𝑚𝑎𝑠𝑠 )×106 Equa ion (2) 3.6. De e mina ion o pes icides by GC-MS Analysis o pes icides in apple and pea pulps was pe o med on a 450-GC gas ch oma og aph om Va ian coupled o a 4000 Pe o mance ion- ap mass spec ome e also om Va ian. 60 Table 4.6: DPPH ee adical inhibi ion assay esul s on he comme cial apple cul i a s. Sample/Cul i a Po ion %RSA Concen a ion (µg TE/g) Gala ab Peels 72.2 892.5 Seeds 16.8 236.8 Mesoca p 10.9 167.6 G anny Smi h Peels 37.0 476.2 Seeds 22.3 301.9 Mesoca p 6.52 115.7 Daline e Peels 42.7 544.0 Seeds 16.9 238.1 Mesoca p 7.76 130.4 Venus Fengal Peels 51.3 645.1 Seeds 19.7 271.3 Mesoca p 9.45 150.3 Rubeli e Peels 46.0 582.6 Seeds 5.51 103.8 Mesoca p 0.78 47.88 Rubin Fuji Peels 29.1 383.1 Seeds 19.7 271.3 Mesoca p 2.02 62.51 S o y Ino ed Peels 62.1 772.8 Seeds 19.2 266.0 Mesoca p 6.97 121.0 E elina Peels 38.6 494.6 Seeds 7.77 130.5 Mesoca p 0.14 15.60 Pixie Peels 50.1 630.7 Seeds 8.70 141.1 Mesoca p 0.30 42.11 Schinga Schnico Peels 54.6 683.7 Seeds 13.3 195.9 Mesoca p 0.00 0.000 Redlum Peels 41.3 526.4 Seeds 13.5 197.6 Mesoca p 0.00 0.000 61 Sample/Cul i a Po ion %RSA Concen a ion (µg TE/g) Fuji Az ec Peels 28.1 370.9 Seeds 8.82 142.9 Mesoca p 0.00 0.000 Bigbucks Peels 68.8 851.6 Seeds 18.7 259.5 Mesoca p 0.00 0.000 Candine Peels 39.8 508.7 Seeds 10.8 165.8 Mesoca p 0.00 0.000 Deca li Peels 42.4 539.7 Seeds 17.8 249.0 Mesoca p 2.69 70.37 Schnico Red Peels 25.5 339.9 Seeds 10.4 161.2 Mesoca p 2.31 65.82 Red eu Peels 55.3 692.6 Seeds 15.4 220.3 Mesoca p 2.69 70.37 B ook ield Peels 39.8 509.4 Seeds 17.4 244.5 Mesoca p 1.54 56.74 Jonap ince Peels 25.0 333.8 Seeds 7.94 132.4 Mesoca p 3.07 74.91 Fuji Spu Peels 53.3 668.4 Seeds 13.3 196.0 Mesoca p 4.87 96.10 Fuji Fub ax Peels 29.2 383.8 Seeds 19.9 273.2 Mesoca p 3.84 83.99 Fengapi Peels 59.3 739.6 Seeds 17.0 239.9 Mesoca p 4.61 93.08 62 0 20 40 60 80 100 120 140 160 180 200 TE (µg TE/g) 0 50 100 150 200 250 300 350 TE (µg TE/g) 0 100 200 300 400 500 600 700 800 900 1000 TE (µg TE/g) Figu e 4.2: G aphical ep esen a ion o he DPPH ee adical inhibi ion assay esul s in comme cial apples. A – Resul s o he peels. B – Resul s o he seeds. C – Resul s o he mesoca ps. 63 I is impo an o highligh ha all he cul i a s, comme cial and egional we e p oduced unde he same condi ions, du ing he same ime pe iod and on INIAV´s o cha d ields a Alcobaça and he e o e minimizing he in luence o edaphoclima ic condi ions. Compa ing he comme cial apple cul i a s wi h hei egional coun e pa s, we can obse e ha , in gene al, he comme cial cul i a s ha e a highe DPPH ee adical inhibi ion pe cen age. The Noi a egional cul i a , howe e , su passes 16 o he comme cial cul i a s, ega ding he peels and 15 ega ding he mesoca p, bu only 9 when accoun ing o he seeds. The highes pe cen age o DPPH ee adical inhibi ion was eached in he peels by he Gala ab cul i a , wi h a 72.2 inhibi ion pe cen age (co esponding o 892.48 µg TE/g o esh ui ) and in he seeds by he G anny Smi h cul i a , wi h 22.3% (301.93 µg TE/g o esh ui ). I is also impo an o men ion ha he DPPH ee adical inhibi ion assay was no able o de ec an ioxidan capaci y in 5 samples co esponding o he edible po ion (mesoca p) o Schinga Schnico, E elina, Bigbucks, Fuji Az ec and Redlum. To u he analyze he DPPH adical inhibi ing p ope ies o he egional cul i a s, a compa ison be ween he 2021 and 2022 ha es s o egional apple and pea cul i a s was made. Only 3 o he apple cul i a s and 5 o he pea cul i a s we e accoun ed due o a ailabili y easons. We can obse e ha ega ding he TE concen a ion (µg TE/g), di e ences lowe han 20% we e ound in he peels and seeds o bo h ui s. The seeds o he Lambe-os-Dedos pea cul i a 0 50 100 150 200 250 300 350 Peels Seeds Mesoca p Peels Seeds Mesoca p Peels Seeds Mesoca p Peels Seeds Mesoca p Peels Seeds Mesoca p Peels Seeds Mesoca p Peels Seeds Mesoca p Peels Seeds Mesoca p Pê o Coimb a Pa do Lindo Repinau Bela-Feia To es No as Ca apinhei a Roxa Lambe-os- Dedos Amo im TE (µg TE/g) 2021 2022 Figu e 4.3: An ioxidan capaci y acco ding o he DPPH adical assay in apples and pea s ha es in 2021 and 2022. 64 p esen ed one o he highes inc eases (83%) o an ioxidan capaci y acco ding o DPPH be ween 2021 and 2022 ha es . On he o he hand, in all o he mesoca p po ions, we could obse e an inc ease o o e 50% o TE (wi h he excep ion o he Pa do Lindo apple and Amo im pea cul i a s, whe e he an ioxidan capaci y o he DPPH adical assay was lowe in 2022). The wo ha es yea s ha e di e en clima ic condi ions ha g ea ly in luence he an ioxidan capaci y o he samples. Howe e , he clima ic condi ions can a ec di e en ly he di e se pea and apple cul i a s analyzed because hey a e ha es ed in di e en pe iods (mon hs o he yea – he Bela-Feia, To es No as, Ca apinhei a Roxa, Lambe-os-Dedos and Amo im pea cul i a s and he Pê o Coimb a apple we e ha es ed in Sep embe and he o he apple cul i a s, in ea ly Oc obe ). 4.1.3. β-ca o ene bleaching assay The An ioxidan ac i i y coe icien s o he egional apples and pea s is shown in Tables 4.7 and 4.8 and Figu e 4.4. Table 4.7: β-ca o ene bleaching assay esul s on he egional apple cul i a s. Ha es ed in 2021 in Alcobaça (Po ugal). Sample/Cul i a Po ion An ioxidan ac i i y coe icien (AAC) Pê o de Bo belo Peels 143.4 Seeds 108.8 Mesoca p 71.70 Pa do Lindo Peels 124.2 Seeds 62.74 Mesoca p 76.82 Repinau Peels 89.63 Seeds 42.25 Mesoca p 81.95 Pê o Coimb a Peels 126.8 Seeds 147.2 Mesoca p 85.79 Noi a Peels 283.0 Seeds 195.9 Mesoca p 76.82 65 Table 4.8: β-ca o ene bleaching assay esul s on he pea cul i a s ha es ed in 2021 in Alcobaça (Po ugal). Sample/Cul i a Po ion An ioxidan ac i i y coe icien (AAC) Bela-Feia Peels 161.3 Seeds 121.6 Mesoca p 83.23 To es No as Peels 88.35 Seeds 85.79 Mesoca p 64.02 Ca apinhei a Peels 162.6 Seeds 165.2 Mesoca p 40.97 Ca apinhei a Roxa Peels 96.03 Seeds 60.18 Mesoca p 52.50 Lambe-os-Dedos Peels 129.3 Seeds 39.69 Mesoca p 60.18 Amo im Peels 216.4 Seeds 152.4 Mesoca p 76.82 In wha conce ns o β-ca o ene bleaching assay, i was ound ha al hough no in all cul i a s, he by-p oduc s ha e a highe AAC han he mesoca p. The Pa do Lindo and Repinau apple cul i a s and he Lambe-os-Dedos pea cul i a ha e AAC alues o he mesoca p highe han he seeds, bu ne e highe han he peels. I was also ound han be ween he wo po ions o by-p oduc s, peels p esen ed highe AAC in mos he apples and pea s cul i a s. The Pê o Coimb a apple and Ca apinhei a pea cul i a s, on he o he hand, ha e highe AAC in he seeds. 66 Compa ing he apple wi h he pea cul i a s, i was ound he peels o apples and pea s p esen ed simila AAC, wi h special a en ion o he Noi a apple, eaching a AAC o 283.0. In he same line, seeds p esen he same pa e n, wi h Noi a apple cul i a eaching a AAC o 195.9. The AAC o mesoca p was ound o be simila among all he s udied samples, howe e Ca apinhei a and Ca apinhei a Roxa pea cul i a s p esen ed much lowe AAC alues han all he o he cul i a s. Among all he ui cul i a s,Noi a apple, as well as Amo im pea , and he Ca apinhei a pea by-p oduc s s and ou ega ding he AAC. 0 50 100 150 200 250 300 Peels Seeds Mesoca p Peels Seeds Mesoca p Peels Seeds Mesoca p Peels Seeds Mesoca p Peels Seeds Mesoca p Peels Seeds Mesoca p Bela-Feia To es No as Ca apinhei a Ca apinhei a Roxa Lambe-os-Dedos Amo im AAC 0 50 100 150 200 250 300 Peels Seeds Mesoca p Peels Seeds Mesoca p Peels Seeds Mesoca p Peels Seeds Mesoca p Peels Seeds Mesoca p Pê o de Bo belo Pa do Lindo Repinau Pê o Coimb a Noi a AAC Figu e 4.4: β-ca o ene bleaching assay esul s in apples and pea s collec ed in 2021 in Alcobaça egion (Po ugal). A - Resul s o apples egional cul i a s. B - Resul s o pea s egional cul i a s 67 Table 4.9: β-ca o ene bleaching assay esul s on he comme cial apple cul i a s ha es ed in 2021 in Alcobaça (Po ugal). Sample/Cul i a Po ion An ioxidan ac i i y coe icien (AAC) Gala ab Peels 241.7 Seeds 208.3 Mesoca p 83.33 G anny Smi h Peels 366.7 Seeds 383.3 Mesoca p 125.0 Daline e Peels 325.0 Seeds 375.0 Mesoca p 266.7 Venus Fengal Peels 225.0 Seeds 225.0 Mesoca p 225.0 Rubeli e Peels 358.3 Seeds 325.0 Mesoca p 308.3 Rubin Fuji Peels 225.0 Seeds 325.0 Mesoca p 200.0 S o y Ino ed Peels 441.7 Seeds 233.3 Mesoca p 116.7 E elina Peels 347.4 Seeds 511.9 Mesoca p 164.5 Pixie Peels 330.9 Seeds 426.0 Mesoca p 96.89 Schinga Schnico Peels 325.4 Seeds 352.8 Mesoca p 82.27 Redlum Peels 309.0 Seeds 135.3 Mesoca p 126.1 68 Sample/Cul i a Po ion An ioxidan ac i i y coe icien (AAC) Fuji Az ec Peels 188.3 Seeds 435.1 Mesoca p 65.81 Bigbucks Peels 376.6 Seeds 343.7 Mesoca p 74.95 Candine Peels 248.6 Seeds 356.5 Mesoca p 111.5 Deca li Peels 323.0 Seeds 180.9 Mesoca p 68.09 Schnico Red Peels 241.3 Seeds 208.2 Mesoca p 71.98 Red eu Peels 324.9 Seeds 371.6 Mesoca p 46.69 B ook ield Peels 365.8 Seeds 289.9 Mesoca p 83.66 Jonap ince Peels 336.6 Seeds 192.6 Mesoca p 101.2 Fuji Spu Peels 356.0 Seeds 389.1 Mesoca p 68.09 Fuji Fub ax Peels 249.0 Seeds 367.7 Mesoca p 40.86 Fengapi Peels 385.2 Seeds 241.3 Mesoca p 87.55 69 The compa ison o apple cul i a s wi h hei egional coun e pa s, allows o conclude ha , in gene al, he comme cial cul i a s ha e a much highe AAC. The Noi a egional cul i a , su passes 9 o he comme cial cul i a s, ega ding he mesoca p and 7 ega ding he peels, and only 3 when compa ing he seeds. The highes an ioxidan ac i i y was eached by he S o y Ino ed cul i a , wi h a AAC o 441.7 in he peels and by he E elina cul i a , wi h a AAC o 511.9 in he seeds. I is also impo an o men ion, ha o he han he Noi a cul i a , he egional apple cul i a s showed conside ably lowe AAC alues, wi h less han 50% o he alues showed by he comme cial cul i a s. 76 Sample/Cul i a Po ion Con en (µg GAE/g) Fuji Az ec Peels 814.8 Seeds 315.7 Mesoca p 106.3 Bigbucks Peels 1864 Seeds 539.0 Mesoca p 131.8 Candine Peels 1027 Seeds 406.1 Mesoca p 134.1 Deca li Peels 1087 Seeds 519.7 Mesoca p 139.6 Schnico Red Peels 729.8 Seeds 303.3 Mesoca p 138.0 Red eu Peels 1417 Seeds 437.0 Mesoca p 130.3 B ook ield Peels 1199 Seeds 553.7 Mesoca p 120.2 Jonap ince Peels 815.6 Seeds 321.1 Mesoca p 143.4 Fuji Spu Peels 1526 Seeds 383.7 Mesoca p 162.0 Fuji Fub ax Peels 857.3 Seeds 504.2 Mesoca p 148.8 Fengapi Peels 1602 Seeds 388.3 Mesoca p 174.3 77 When compa ing he o al phenolics con en o he comme cial apple cul i a s wi h he egional cul i a s, a endency is obse ed. The egional cul i a s, pa icula y Noi a, Pê o de Bo belo and Pê o Coimb a cul i a s, show a highe con en o phenolics han mos o he comme cial cul i a s in all he po ions. O he egional cul i a s su pass he comme cial ones only conce ning he seeds and mesoca ps. All he comme cial cul i a s p esen a phenolics con en in he peels lowe han he Noi a cul i a , bu Gala ab, S o y Ino ed, and Bigbucks comme cial cul i a s showed simila con en s (1852, 1897, and 1864 µg GAE/g o esh ui , espec i ely). While he o al phenolics con en in he seeds anges be ween 278.6 and 566.8 µg GAE/g o esh ui in he comme cial cul i a s, i G anny Smi h cul i a is no aken in o conside a ion, on he o he hand, in he egional cul i a s his con en anged be ween 586.9 – 689.6 µg GAE/g o esh ui , showing ha all o he seeds o egional cul i a s ha e highe phenolics con en han all o he comme cial cul i a s, apa om he G anny Smi h cul i a . The same can be concluded ega ding he mesoca ps, being G anny Smi h cul i a (268.6 µg GAE/g o esh ui ) he only su passing he egional cul i a s wi h lowes TPC, i.e., Noi a, wi h 260.9 µg GAE/g o esh ui . 78 0 50 100 150 200 250 300 GAE (µg GAE/g) 0 100 200 300 400 500 600 700 800 GAE (µg GAE/g) 0 200 400 600 800 1000 1200 1400 1600 1800 2000 GAE (µg GAE/g) Figu e 4.8: To al phenolics con en o comme cial apples. A- Peels; B- Seeds; C-Mesoca p. 79 The di e ences on o al phenolics con en be ween he 2021 and 2022 ha es s o some o he egional cul i a s a e shown in Figu e 4.9. In he o al phenolics con en assay, sligh changes in phenolics con en o peels a e ound in µg GAE/g, wi h he excep ion o he Lambe-os-Dedos pea cul i a , whe e a 46% inc ease was ound. As o he seeds and mesoca ps, he phenolics con en p esen ed highe di e ences, wi h he seeds ob aining alues in 2022 anging om 40-80% o hose ob ained in 2021, in mos , and he mesoca ps p esen ing alues in 2022 anging om 31-61% o hose ob ained in 2021 in he majo i y o he cul i a s. 4.1.5. To al con en o la onoids assay Rega ding he o al la onoids con en (TFC) assay, a calib a ion cu e o epica echin was p epa ed in o de o be able o p esen he esul s as Epica echin Equi alen s (ECE) pe g am. Fo his pu pose, calib a ion s anda ds we e p epa ed om an epica echin s ock solu ion in me hanol wi h a concen a ion o 200 µg/mL. F om his solu ion, he calib a ion s anda d solu ions we e p epa ed by dilu ion, and a e wa ds subjec ed o he p ocedu e o o al phenolics con en assay me hod p ocedu e desc ibed in sec ion h ee. 0 200 400 600 800 1000 1200 1400 1600 Peels Seeds Mesoca p Peels Seeds Mesoca p Peels Seeds Mesoca p Peels Seeds Mesoca p Peels Seeds Mesoca p Peels Seeds Mesoca p Peels Seeds Mesoca p Peels Seeds Mesoca p Pê o Coimb a Pa do Lindo Repinau Bela-Feia To es No as Ca apinhei a Roxa Lambe-os- Dedos Amo im GAE (µg GAE/g) 2021 2022 Figu e 4.9: G aphical compa ison o he o al phenolic con en (µg GAE/g) be ween he 2021 and 2022 ha es s o egional apple and pea cul i a s. 80 The ob ained i s -deg ee equa ion cu e o epica echin, in he ange 5- 150 µg/mL, was Abs = 0.0017 Cepica echin + 0.0165 (Cepica echin - Concen a ion o Epica echin solu ion). The good linea i y ob ained ( 2=0.9997) indica ed sui abili y o he cu e o be used in he quan i ica ion o ECE equi alen s in he samples. The esul s o he samples in µg o epica echin equi alen s pe g am o ui (µg ECE/g) a e p esen ed in Tables 4.13 and 4.14 and Figu e 4.8. Table 4.13: To al la onoids con en o he apple cul i a s ha es ed in 2021 in Alcobaça (Po ugal). Sample/Cul i a Po ion Con en (µg ECE/g) Pê o de Bo belo Peels 778.1 Seeds 228.1 Mesoca p 37.89 Pa do Lindo Peels 295.4 Seeds 210.5 Mesoca p 43.74 Repinau Peels 561.6 Seeds 272.0 Mesoca p 37.89 Pê o Coimb a Peels 748.9 Seeds 359.7 Mesoca p 70.07 Noi a Peels 1284 Seeds 356.8 Mesoca p 64.22 Table 4.14: To al la onoids con en o he pea s cul i a s ha es ed in 2021 in Alcobaça (Po ugal). Sample/Cul i a Po ion Con en (µg ECE/g) Bela-Feia Peels 389.0 Seeds 154.9 Mesoca p 11.55 To es No as Peels 339.3 Seeds 216.4 Mesoca p 0.000 81 Sample/Cul i a Po ion Con en (µg ECE/g) Ca apinhei a Peels 371.4 Seeds 447.5 Mesoca p 61.29 Ca apinhei a Roxa Peels 257.3 Seeds 111.0 Mesoca p 0.000 Lambe-os-Dedos Peels 292.4 Seeds 125.7 Mesoca p 0.000 Amo im Peels 547.0 Seeds 236.8 Mesoca p 23.26 Rega ding he esul s o o al la onoids con en , mesoca p o he analyzed ui s is he pa wi h he lowes con en on hese compounds. In ac , some o he pea cul i a s showed null esul s when subjec ed o his assay. Mo eo e , we can also obse e ha mos o la onoids is loca ed on ui s’ by-p oduc s. Again, i is in he peels ha he con en is highe , when aking he by-p oduc s in conside a ion. Among egional apples, Noi a cul i a s ands ou by ha ing he highes TFC, wi h 1284 µg ECE/g o esh ui . Again, in his assay, he Ca apinhei a pea is he only sample ha has a highe con en in he seeds han in he peels, while all he o he cul i a s ha e a leas wice as much o al la onoids in he peels han in he seeds (wi h he excep ion o he Pa do Lindo apple and he To es No as pea , whe e he la onoid con en is simila be ween he wo po ions). All apple cul i a s, excep Pa do Lindo, ha e a highe o al la onoids con en in he peels han he pea cul i a s. The same can be concluded abou he TFC in he seeds, apa om Pê o de Bo belo apple, ha has a lowe TFC han some pea cul i a s. The TFC on he Ca apinhei a pea seeds s and ou om he o he esul s because, despi e being a pea cul i a , i shows he highes TFC alue o seeds, wi h 447.5 µg ECE/g o esh ui . The TFC ound in he mesoca p o pea cul i a s is o e all null, wi h he excep ion o he Ca apinhei a cul i a , ha showed a TFC o 61.3 µg ECE/g o esh ui , making i he hi d highes among all he samples o bo h ui s, only losing ou o he Pê o Coimb a and Noi a apple cul i a s, wi h 70.1 and 64.2 µg ECE/g o esh ui , espec i ely. All he o he cul i a s ha e a TFC lowe han 50 µg ECE/g o esh ui . 82 To al la onoids con en anging om 7 o 1421 µg ECE/g we e epo ed by Migna d e al. 169, in a compa a i e s udy among 155 apple samples. This shows a e y simila ange o concen a ions in compa ison o ou esul s (38 o 1284 µg ECE/g). Rega ding he s udy ca ied ou by Guan e al. ,171 whe e he compa ison be ween he o al la onoids con en o 22 Asian pea cul i a s is pe o med, he au ho s egis e ed a TFC anging be ween 23 and 104 µg ECE/g o esh ui , while he TPC o egional apple cul i a s anged om 0 µg ECE/g in some mesoca ps, o 547 µg ECE/g ound in he Xuehua cul i a . 0 200 400 600 800 1000 1200 Peels Seeds Mesoca p Peels Seeds Mesoca p Peels Seeds Mesoca p Peels Seeds Mesoca p Peels Seeds Mesoca p Pê o de Bo belo Pa do Lindo Repinau Pê o Coimb a Noi a ECE (µg ECE/g) 0 200 400 600 800 1000 1200 Peels Seeds Mesoca p Peels Seeds Mesoca p Peels Seeds Mesoca p Peels Seeds Mesoca p Peels Seeds Mesoca p Peels Seeds Mesoca p Bela-Feia To es No as Ca apinhei a Ca apinhei a Roxa Lambe-os-Dedos Amo im ECE (µg ECE/g) Figu e 4.10: To al la onoids con en o egional apples and pea s collec ed in 2021 a Alcobaça egion (Po ugal). A- Resul s o apples egional cul i a s. B- Resul s o pea s egional cul i a s. 83 Table 4.15: To al la onoids con en o he comme cial apple cul i a s ha es ed in 2021 in Alcobaça Sample/Cul i a Po ion Con en (µg ECE/g) Gala ab Peels 1472 Seeds 327.6 Mesoca p 61.29 G anny Smi h Peels 933.2 Seeds 409.5 Mesoca p 61.29 Daline e Peels 909.8 Seeds 245.6 Mesoca p 23.26 Venus Fengal Peels 1115 Seeds 233.9 Mesoca p 34.96 Rubeli e Peels 1039 Seeds 181.3 Mesoca p 40.81 Rubin Fuji Peels 833.7 Seeds 327.6 Mesoca p 0.000 S o y Ino ed Peels 1603 Seeds 301.2 Mesoca p 37.89 E elina Peels 988.8 Seeds 277.8 Mesoca p 55.44 Pixie Peels 1311 Seeds 263.2 Mesoca p 61.29 Schinga Schnico Peels 1161 Seeds 231.0 Mesoca p 37.89 Redlum Peels 865.9 Seeds 184.2 Mesoca p 43.74 84 Sample/Cul i a Po ion Con en (µg ECE/g) Fuji Az ec Peels 775.2 Seeds 207.6 Mesoca p 23.26 Bigbucks Peels 1416 Seeds 365.6 Mesoca p 64.22 Candine Peels 1041 Seeds 368.5 Mesoca p 46.66 Deca li Peels 532.4 Seeds 272.0 Mesoca p 20.33 Schnico Red Peels 333.4 Seeds 175.4 Mesoca p 17.41 Red eu Peels 880.5 Seeds 307.1 Mesoca p 8.628 B ook ield Peels 687.4 Seeds 304.1 Mesoca p 2.776 Jonap ince Peels 564.5 Seeds 143.2 Mesoca p 11.55 Fuji Spu Peels 795.7 Seeds 219.3 Mesoca p 49.59 Fuji Fub ax Peels 462.1 Seeds 286.6 Mesoca p 58.37 Peels 892.2 Fengapi Seeds 263.2 Mesoca p 61.29 85 The compa ison o he o al la onoids con en be ween comme cial and egional apple cul i a s allows o conclude he e isn’ a conside able di e ence be ween he egional and he comme cial cul i a s ega ding he seeds and he mesoca p po ions. Rega ding he seeds, Pê o Coimb a and Noi a egional cul i a s show a highe o al la onoids con en (359.7 and 356.8 µg ECE/g o esh ui , espec i ely) han all he comme cial cul i a s, wi h excep ion o he G anny Smi h, Bigbucks and Candine cul i a s (409.5, 365.6, and 368.5 µg ECE/g o esh ui , espec i ely). I is also possible o obse e ha he TFC in he peels’ anges be ween 561.6 and 1284 µg GAE/g o esh ui in he egional cul i a s and ha hese alues a e in gene al lowe han he TFC alues o he comme cial cul i a s. The Noi a egional cul i a s ands ou as he egional cul i a wi h he highes TFC, being only su passed by 4 o he comme cial cul i a s (Gala ab, S o y Ino ed, Pixie and Bigbucks – eaching TFC alues be ween 1311 and 1603 µg ECE/g o esh ui ). 92 Sample/Cul i a Po ion Con en (mg/g) Fuji Az ec Peels 79.83 Seeds 100.0 Mesoca p 43.82 Bigbucks Peels 78.74 Seeds 99.85 Mesoca p 49.56 Candine Peels 61.82 Seeds 91.94 Mesoca p 47.54 Deca li Peels 78.59 Seeds 94.73 Mesoca p 56.39 Schnico Red Peels 99.85 Seeds 56.08 Mesoca p 78.59 Red eu Peels 56.39 Seeds 91.32 Mesoca p 69.27 B ook ield Peels 56.70 Seeds 92.87 Mesoca p 50.96 Peels 50.96 Joanap ince Seeds 88.06 Mesoca p 43.35 Fuji Spu Peels 63.06 Seeds 108.6 Mesoca p 60.74 Fuji Fub ax Peels 86.50 Seeds 105.6 Mesoca p 58.25 Fengapi Peels 81.23 Seeds 108.4 Mesoca p 48.63 93 0 10 20 30 40 50 60 70 80 mg F uc ose/g F ui 0 20 40 60 80 100 120 140 160 mg F uc ose/g F ui 0 20 40 60 80 100 120 140 mg F uc ose/g F ui Figu e 4.14: To al uc ose con en o comme cial apples ha es ed in 2021 in Alcobaça (Po ugal). 94 Compa ing uc ose con en in he egional apple cul i a s wi h i s con en in he comme cial cul i a s allows o conclude ha he comme cial cul i a s, in gene al, ha e a highe uc ose con en han he Noi a apple cul i a (73.6 mg uc ose/g ui ). Howe e , 17 o he comme cial apple cul i a s ha e a lowe con en han he o he 4 egional cul i a s. I was possible o conclude ha he uc ose con en in he peels was always lowe in he comme cial cul i a s, excep o he Pixie and Schnico Red cul i a s, in which he con en is 67.6 and 56.1 mg uc ose/g ui , espec i ely. I is also e y in e es ing o conclude ha all he egional cul i a s possess a highe uc ose con en , anging om 85.6 o 107 mg uc ose/g ui in he mesoca p han all o he comme cial cul i a s, whe eas he uc ose con en a ies be ween 37.6 – 78.6 mg uc ose/g ui . The di e ences be ween he uc ose con en o he egional cul i a s in 2021 and 2022 ha es s a e shown in Figu e 4.15. The uc ose con en in he pa ame e whe e he g ea es di e ences we e ound in all he po ions o bo h ui s be ween he 2021 and 2022 ha es s. I is possible o obse e ha mos o he peels o he pea cul i a s, su e ed a conside able dec ease o hei uc ose con en , wi h he Bela-Feia and Amo im cul i a s being he mos a ec ed by his dec ease, (238 and 187%, espec i ely). In a gene al way, i is possible o conclude ha he o he cul i a s ha e also had a 0 50 100 150 200 250 Peels Seeds Mesoca p Peels Seeds Mesoca p Peels Seeds Mesoca p Peels Seeds Mesoca p Peels Seeds Mesoca p Peels Seeds Mesoca p Peels Seeds Mesoca p Peels Seeds Mesoca p Pê o Coimb a Pa do Lindo Repinau Bela-Feia To es No as Ca apinhei a Roxa Lambe-os- Dedos Amo im mg F uc ose/g ui 2021 2022 Figu e 4.15: Compa ison o he o al uc ose con en (mg F uc ose/g ui ) o apples and pea s o egional cul i a s ha es ed in 2021 and 2022. 95 signi ican dec ease on hei uc ose con en , bu ha in some cases, like he Pa do Lindo and Repinau seeds, he uc ose con en has ac ually inc eased by 32 and 49%, espec i ely. 4.3. P incipal componen analysis The p incipal componen analysis (PCA) is a ma hema ical p ocedu e ha uses an o hogonal ans o ma ion o con e a se o obse a ions o possibly co ela ed a iables in o a se o alues o linea ly unco ela ed a iables called p incipal componen s. The numbe o p incipal componen s is always less han o equal o he numbe o o iginal a iables. The main objec i e is he dimensionali y educ ion keeping he con ibu ion o all ini ial a iables in o de o p o ide a isual pa e n ecogni ion. In his con ex he biplo g aph which p ojec he samples (sco es) and he a iables (loadings) in o a wo o h ee dimensional g aph, shows i s o all how he samples a e ela ed o each o he and in a second place how each a iable a ec each sample. To ensu e he cons uc ion o ep esen a i e desc ip i e indices wi h he highes possible capaci y o disc imina e be ween uses, he ui s we e cha ac e ized as desc ibed abo e by means o 6 chemical pa ame e s. These pa ame e s a e an ioxidan ac i i y (DPPH), an ioxidan ac i i y coe icien (Bca o en), o al phenolics con en (Fenol), o al la onoids con en (Fla on), o al uc ose con en (F u ose) and DPPH ee adical inhibi ing pe cen age (PI). Figu e 4.16: Dend og am wi h hie a chical g ouping o he samples. 96 The dend og am shows ha he Noi a apple peel (MNC) sample is e y di e en chemically om he es o he analyzed samples. In gene al, he e a e sepa a e clus e s o apples and pea s wi h he excep ion o he Amo im pea seeds, To es No as pea seeds and Ca apinhei a Roxa pea seeds, ha al hough belonging o he pea cul i a s, s ill ha e signi ican simila i ies wi h some apple species. Figu e 4.17: Co ela ion cha among he s udied a iables. The co ela ion cha shows ha he uc ose con en is independen om all o he pa ame e s. All o he pa ame e s a e co ela ed wi h pa icula emphasis on he DPPH-PI pai , which is o be expec ed because bo h o hese pa ame e s' esul s come om he same assay. PCA ans o ms he o iginal a iable in o new ones called p incipal componen s using eigen ec o s, which a e calcula ed a ibu ing a coe icien o each o iginal a iables p opo ional o hei con ibu ion in o his ans o ma ion ( o a ion) in o de o maximize he a iances o he i s ew componen s. The main objec i e is he dimensionali y educ ion keeping a he same ime he con ibu ion o all ini ial a iables in o de o p o ide a isual pa e n ecogni ion. In his con ex 97 he biplo g aph which p ojec he samples (sco es) and he a iables (loadings) in o a wo- o h ee- dimensional g aph, shows i s o all how he samples a e ela ed o each o he and in a second place how each a iable a ec each sample. The samples si ua ed close o each o he a e simila while he ones wi h highe dis ance be ween hem a e di e en conside ing he s udied p ope ies. The g oups we e o med using k-means clus e ing. Rega ding o he loadings ec o s an angle (θ) close o 0º p o ides a co ela ion coe icien o 1, since his las one is calcula ed om cos(θ) and means ha he e a e highly co ela ed. An angle equal o 90º gi es a co ela ion coe icien o 0 indica ing ha no co ela ion exis s be ween hem, meaning in o he wo ds ha he e is independen . Finally, an angle o 180 º which gi es a co ela ion coe icien o -1 means ha he e a e in e sely p opo ional, indica ing ha an inc ease o he i s one co esponds o a dec ease o he second one. The loading si ua ed close o a gi en sample has g ea e e ec on he de ini ion o he chemical/biological beha io o his sample. These wo PCs explain 95 % o he obse ed a ia ion in he o iginal da a. In his case he a iable PI was no included in he s udy, since i is highly co ela ed wi h he DPPH pa ame e , and i has he same e ec on he dis ibu ion o he samples. i was ound a signi ican co ela ion among he DPPH pa ame e and he con en on o al la onoids, o al phenolics compounds, and he an ioxidan ac i i y coe icien . These ha e a highe in luence in he samples o clus e s 1 and 2. The uc ose a iable allows he di e en ia ion be ween apple and pea species. Figu e 4.18: Biplo cha PC2 s PC1. 98 Since he Noi a apple peel sample has a e y dis inc se o pa ame e s, we decided o emo e i om he plo ing and e-do he biplo cha . Fou clus e s we e ound in he samples whe e each clus e con ains mo e chemically simila samples. The a iable " o al uc ose con en " allows o di e en ia e apples om pea s. Addi ionally, wi h he comme cial apple cul i a s and he 2022 ha es s o some egional cul i a s, he ui s we e again cha ac e ized as desc ibed abo e, his ime by means o 5 chemical pa ame e s. These pa ame e s a e an ioxidan ac i i y (DPPH), an ioxidan ac i i y coe icien (Bca ), o al phenolic con en (Phen), o al la onoid con en (Fla ), and o al uc ose con en (F uc). Figu e 4.19: Biplo cha PC2 s PC1. (A e emo ing he Noi a apple peel sample) 99 This hea map hea map be ween samples and a iables shows he impac o each a iable on a gi en sample (Figu e 4.20) and shows ha , as s a ed p e iously, he uc ose con en in comple ely independen om he o he pa ame e s. Also, he an ioxidan ac i i y coe icien has a lowe co ela ion wi h he o he 3 pa ame e s, han hey ha e wi h each o he . This can be con i med by he co ela ion plo (Figu e 4.21) below. Figu e 4. 21: Co ela ion plo be ween he s udied pa ame e s. Figu e 4.20: Hie a chical clus e ing hea map o he samples and he s udied pa ame e s. 100 The co ela ion ma ix is he a iance/co a iance ma ix o he s anda dized da a and shows he coe icien s o he co ela ion be ween a iables. The co ela ion be ween he phenolics con en and he la onoids con en and be ween he la onoids con en and an ioxidan capaci y (0.93) a e he highes among all he co ela ions obse ed. The e is also a e y high co ela ion be ween he o al phenolics con en and he an ioxidan capaci y (0.91). Howe e , he co ela ion obse ed be ween he an ioxidan ac i i y coe icien and hese h ee pa ame e s (0.6, 0.54, 0.49) is no as high, as ound be o e. The uc ose con en shows almos no co ela ion wi h he o he pa ame e s (<0.21). Figu e 4.22: biplo cha PC2 s PC1. These wo PCs (Figu e 4.22) explain 88 % o he obse ed a ia ion in he o iginal da a. We can obse e ha 3 clus e s eme ge, wi h he le one (in ed) consis ing o only comme cial apple cul i a s. This esul shows ha he comme cial apples can be sepa a ed om he o he egional species and PCA can be used o ce i y he o igin and he cul i a o he ui samples. 4.4. Classi ica ion wi h machine lea ning algo i hms Due o he a ailabili y o mul i a ia e da a ma ices, i is possible o e icien ly ex ac he mos ele an in o ma ion om da a using s a is ical and ma hema ical me hods, and g oup indi iduals in classes. In o de o place new, unknown samples in one o he ecognized classes based on hei measu emen pa e n, supe ised pa e n ecogni ion echniques use in o ma ion 101 abou he samples' membe ship in a gi en g oup (class o ca ego y). Fo ou case, he samples we e di ided in o 3 classes (1.00 – comme cial apples; 2.0 – egional pea s; 3.0 – egional apples). Fo he pu pose o de eloping a model o he classi ica ion o he samples, supe ised pa e n ecogni ion needs a aining se o samples which belong o ecognized ca ego ies. Since we had a small amoun o da a, he decision ee (DT) algo i hm was applied, and he da a we e spli in o a aining g oup (60% o he samples) and a es g oup (40% o he samples). The sco es we ob ained we e 0.9660 o he aining g oup, meaning ha he model was able o s a e, wi h 97% con idence, ha he samples belong o he s a ed g oup. Rega ding he es g oup, as expec ed, he esul s we e a bi lowe , 0.8056, bu s ill p esen ing a e y accep able con idence alue o 80%. These esul s can be seen in Figu e 4.23. The con usion ma ix shows how he da a we e dis ibu ed among h ee classes, also showing he alse posi i es. 4.5. Quan i a i e Analysis o Phenolic Compounds by UHPLC-ToF 4.5.1. Selec ion o he sol en o ex ac ion The selec ion o he ex ac ion sol en is a c ucial s ep in o de o ob ain accu a e analy ical esul s. The ex ac ion o polyphenols om plan ma e ial equen ly in ol es he use o sol en s such me hanol o e hanol,174,175 some imes wi h some amoun o wa e . Figu e 4.23: Con usion ma ix o he aining g oup (a he le ) and he es g oup (a he igh ). 108 4.5.2.3. Accu acy o he me hod Due o he ac ha mos ex ac ion echniques esul in some analy e loss o can p esen some in e e ence, he de e mina ion o he ex ac ion e iciency o he analy ical p ocedu e is a equi ed s ep o any analy ical me hod. In he absence o e e ence ma e ials, he accu acy o he me hod can be de e mined h ough eco e y assays. The eco e y s udy was pe o med by analyzing a sample be o e and a e addi ion o a known concen a ion o he mix u e o phenolic compounds. The eco e ies we e measu ed a e spiking samples a wo di e en o i ica ion le els (0.1 and 1 mg/100 g). The esul s a e shown on Table 4.21. Table 4.21: Reco e y o he analy ical me hod o each o he s udied phenolic compounds a wo o i ica ion le els. Phenolic Compound [M+H]+ (m/z) Fo i ica ion le el (mg/100g) Reco e y Pe cen age (%) 4-Hyd oxybenzoic Acid 139.03 0.1 91.75 1 97.29 Gen isic Acid 155.03 0.1 80.15 1 99.51 Ca eic Acid 181.04 0.1 95.75 1 87.93 Chlo ogenic Acid 355.10 0.1 n.d. 1 78.27 o-Couma ic Acid 165.05 0.1 85.55 1 88.24 p-Couma ic Acid 165.05 0.1 81.48 1 87.54 Sinapic Acid 225.07 0.1 82.22 1 86.53 Sy ingic Acid 199.05 0.1 70.24 1 88.45 ans-Fe ulic Acid 195.06 0.1 n.d. 1 82.20 Vanillic Acid 169.04 0.1 75.99 1 88.92 109 Phenolic Compound [M+H]+ (m/z) Fo i ica ion le el (mg/100g) Reco e y Pe cen age (%) Phlo idzin 437.14 0.1 75.35 1 85.83 Que ce in 303.04 0.1 84.36 1 86.68 Que ce in-3-B-D-Glucoside 465.10 0.1 93.97 1 88.05 Que ci in 449.10 0.1 77.51 1 85.39 Ru in 611.15 0.1 80.46 1 87.11 (+-)-Na ingenin 273.07 0.1 80.58 1 87.03 (+)-Ca echin 291.08 0.1 75.20 1 76.86 E iodyc iol 289.06 0.1 84.30 1 87.81 Apigenin 271.05 0.1 85.11 1 67.05 (-)-Epica echin 291.08 0.1 72.36 1 83.28 Lu eolin 287.05 0.1 87.00 1 75.15 n.d.- no de e mined The esul s showed ha he eco e y o he analy es anged om 70.24 o 95.75% o he lowes concen a ion le el, and om 67.05 o 99.51% o he highes concen a ion anges. Reco e y be ween 80 and 120% is conside ed accep able, howe e , i he eco e y pe cen age is o e 70%, esul s can be conside ed sa is ac o y. In his pe spec i e, he eco e y o he di e en phenolics is accep able, and he ex ac ion p ocess is sui able o he ex ac ion o mos o he phenolic compounds om apple and pea ui ma ices. 110 4.5.3. Applica ion o he me hod o apple and pea samples A e con i ming ha he me hod was app op ia e o de e mine phenolic compounds in ui ma ices, i was applied o ou selec ed egional ui samples: peels o he Amo im pea , peels o he Noi a apple, mesoca p o he Pa do Lindo apple and mesoca p o Lambe-os-Dedos pea cul i a s. These ex ac s we e chosen among all he egional cul i a s because hey p esen ed he bes o e all esul s, among he by-p oduc s and mesoca p, espec i ely, on he assays pe o med ela ed o an ioxidan p ope ies. Table 4.22 shows he phenolic compounds concen a ions e alua ed in he ou selec ed, wo egional apples and wo egional pea s cul i a s. Values ha e been co ec ed wi h eco e y alue (mean eco e y o bo h spiking le els). Table 4.22: Phenolic compounds concen a ion in he s udied ui samples. (H.B.A – Hyd oxybenzoic Acid. H.C.A. – Hyd ocycinnamic Acid. DHC - Dihyd ochalcone) Type o phenol S anda d Concen a ion (µg/g esh ui ) Noi a Peel Pa do Lindo Mesoca p Amo im Peel Lambe-os- Dedos Mesoca p H.B.A. 4-Hyd oxybenzoic Acid n.d. n.d. n.d. n.d. H.B.A. Gen isic Acid 2.58 ± 0.41 < LOQ 2.06 ± 0.40 < LOQ H.C.A. Ca eic Acid n.d. n.d. 6.73 ± 0.55 1.98 ± 0.05 H.C.A. Chlo ogenic Acid 0.08 ± 0.01 0.83 ± 0.01 0.41 ± 0.01 < LOQ H.C.A. o-Couma ic Acid < LOQ < LOQ n.d. n.d. H.C.A. p-Couma ic Acid 2.58 ± 0.04 n.d. 2.12 ± 0.02 n.d. H.C.A. Sinapic Acid 0.58 ± 0.06 n.d. 0.35 ± 0.01 0.56 ± 0.04 H.B.A. Sy ingic Acid < LOQ n.d. 0.93 ± 0.04 1.91 ± 0.01 H.C.A. ans-Fe ulic Acid < LOQ n.d. < LOQ < LOQ H.B.A. Vanillic Acid 0.99 ± 0.03 < LOQ 9.32 ± 0.59 9.77 ± 0.41 DHC Phlo idzin 0.69 ± 0.07 n.d. n.d. n.d. Fla onol Que ce in 0.12 ± 0.01 n.d. < LOQ n.d. Iso la one Que ce in-3-B-D- Glucoside 0.10 ± 0.00 < LOQ 0.11 ± 0.01 < LOQ Fla one Que ci in 0.30 ± 0.01 0.09 ± 0.00 0.09 ± 0.00 < LOQ Fla one Ru in n.d. n.d. < LOQ n.d. Fla anone (+-)-Na ingenin 0.06 ± 0.01 n.d. < LOQ n.d. Fla an-3-ol (+)-Ca echin < LOQ < LOQ < LOQ 0.15 ± 0.01 111 Type o phenol S anda d Concen a ion (µg/g esh ui ) Noi a Peel Pa do Lindo Mesoca p Amo im Peel Lambe-os- Dedos Mesoca p Fla one Apigenin n.d. n.d. n.d. n.d. Fla an-3-ol (-)-Epica echin 0.08 ± 0.01 < LOQ 0.10 ± 0.01 0.09 ± 0.01 Fla one Lu eolin < LOQ n.d. < LOQ n.d. To al 8.16 ± 0.69 0.92 ± 0.12 22.22 ± 1.66 14.46 ± 0.65 In gene al, he esul s ob ained o almos all o he po ions o he s udied ui s a e sa is ac o y, wi h he excep ion o he Pa do Lindo apple mesoca p, whe e mos o he phenolic compounds unde s udy could no be de ec ed. These compounds migh no ha e been p esen in he ma ix o a cul i a o easons, such as he ui cul i a no ha ing he phenolic compounds unde s udy o he me hod no being sensi i e enough o ind he ace le els o hese compounds in he ma ix. Bo h he po ions o he pea cul i a s showed a highe o al phenolic composi ion among he s udied phenolic compounds, han he di e en po ions o apple cul i a s. This is pa icula ly due o hei high le el o anillic acid (9.32 ± 0.59 and 9.77 ± 0.41 µg/g o esh ui ) and ca eic acid (6.73 ± 0.55 and 1.98 ± 0.05 µg/g o esh ui ) – Figu e 4.26 shows a UHPLC-To -MS ch oma og am o he Amo im pea cul i a ega ding his compound. Looking a he phenolic p o ile o he apple cul i a s, gen isic acid was he one wi h he highes concen a ion ound (2.58 µg/g o esh ui in he Noi a peels), and p-couma ic acid (2.58 µg/g o esh ui ). Rega ding he pea cul i a s, anillic acid was ound o be he mos p e alen Figu e 4.26: Ch oma og am o he Amo im pea sample ega ding Ca eic Acid. 112 polyphenol in he s udied ma ices, ollowed by ca eic acid. I is impo an o poin ou ha his high con en o anillic acid was no expec ed, since o he s udies, like he one epo ed by Es- Sba a e al., 180 p esen ed lowe esul s (<1.09 mg/L) han he esul s ob ained in he p esen s udy. Vanillic acid was also ound o be p esen on he peels o he Noi a cul i a apple, bu in much lowe quan i ies. Vanillic Acid is a hyd oxybenzoic phenolic acid, widely dis ibu ed in a wide cul i a o ui s, such as ap ico s181 and apples.182 Phenolic acids we e p esen in highe concen a ions han all he o he phenolic compounds subclasses. The e is no da a ega ding egional cul i a s in he scien i ic li e a u e, so he compa ison was made wi h comme cial cul i a s o apples and pea s. Howe e , he comme cial cul i a s ound in he li e a u e showed a highe concen a ion o indi idual phenolics, ha may be due o di e se ci cums ances, including no also he di e ences in he cul i a s, edaphoclima ic condi ions, cul i a ion p ac ices, ui ipeness, and ex ac ion me hod, as well as he ac ha ou ui samples, due o he delayed a i al o he analy ical s anda ds and due o UHPLC sys em a ailabili y easons, had o be s o ed o almos one yea , a -20 oC. This may ha e led o he deg ada ion o he phenolic compounds o he ozen samples. A ecen s udy by Radenko s e al. 183, p esen ed apple by-p oduc s as a sou ce o phenolic compounds o ood applica ions. In his s udy, apples om he cul i a Gi a we e ai d ied and he e o e he con en o majo polyphenols is exp essed in µg/g (d y weigh ). The au ho s epo ed alues o 137, 44 and 21 µg/g (d y weigh ) o chlo ogenic acid, (−)-epica echin and (+)-ca echin, espec i ely, when using 96% e hanol (also di e en om he 90% e hanol selec ed in ou s udy) o he ex ac ion p ocedu e. Ma e al. 184, also epo ed he phenolic composi ion o apple cul i a s (Fuji) exp essed in µg/g (d y weigh ), using h ee di e en d ying me hods - ho ai d ying, hea pump d ying and eeze d ying, some o which wi h di e en d ying empe a u es. The bes esul s we e ob ained wi h he hea pump d ying me hod o all o he analyzed phenolics, apa om chlo ogenic acid, ha was de ec ed in a highe le el, when he sample was eeze-d ied. When he apple peel was d ied by hea pump a 65 °C, i showed ela i ely high d ying a e, exhibi ed high e en ion o phenolics, and p esen ed high an ioxidan capaci y. Bílko á e al. 185 , when ecen ly s udying he bene i s o ul a-low oxygen condi ions in long- e m s o age, epo ed a much highe phenolics con en in apple cul i a s (Angold, Gala, Nicole a, Rucla, Golden Delicious, among o he s) pa icula ly in ega d o he chlo ogenic acid con en , ha consis ed in o e 50% o he o al phenolic composi ion o he s udied cul i a s, eaching a 113 concen a ion o 99.6 µg/g o esh weigh in he Angold cul i a . In ou s udy hough, he chlo ogenic acid con en was only esidual, wi h 0.83 µg/g ( esh weigh ) in he mesoca p o he Pa do Lindo cul i a . The au ho s epo ed ha he analysis was made igh a e ha es ing he ui s, while ou analysis occu ed se e al mon hs la e due o he easons s a ed ea lie . This may explain he majo di e ence seen in his phenolic compound concen a ion. Some o he cul i a s (Rubin) p esen ed simila o al phenolics con en o he cul i a s e alua ed in he p esen s udy. A ecen s udy ca ied ou by Salaza -O bea e al .186, e alua ed he e ec o di e en p ocessing echniques, in indus ial se up, in he s abili y o phenolic compounds in Golden Delicious apples. The au ho s epo ed he esul s in mg/100g o esh weigh . Howe e , he au ho s did no speci y he indi idual con en o each phenolic compound. Ins ead, hey p esen ed he sum o phenolic compounds. I is possible o obse e ha he o al phenolics con en de e mined by hese au ho s was 86 mg/100g o esh weigh , showing ela i ely highe con en han ou egional samples. In a s udy conduc ed by Sal a e al., 187 he phenolic composi ion, as well as he an ioxidan ac i i y, o pea cul i a s (Rocha, Comice, Passe C assane, Gene al Lecle c and Aba e) was e alua ed. The esul s indica e ha he con en o chlo ogenic acid was also he highes among he s udied phenolics. In he Rocha pea , he con en was 62.4 mg/100 g ( esh weigh ) o chlo ogenic acid. In he o he cul i a s, ca eic acid was he highes among he s udied phenolics, wi h he Aba e cul i a showing a concen a ion o ca eic acid o 12.9 mg/100 g ( esh weigh ), while he Rocha pea p esen ed 11.1 mg/100 g ( esh weigh ). 4.6. Pes icide de ec ion analysis The use o pes icides is s ill a eali y and, in ac , is essen ial o p e en ood loss e en while e o s o dec ease o ind al e na i es a e apidly de eloping. The use o pes icides, howe e , also has nega i e impac s on he en i onmen o ood consume s. As a esul , i is c ucial o manage pes icide esidues in ood, and in he Eu opean Union, his is suppo ed by egula ion o sa egua d public sa e y as well as domes ic and in e na ional ade. E ec i e sample p epa a ion and ace-le el de ec ion and iden i ica ion a e i al componen s o analy ical me hods due o he low de ec ion limi s demanded by egula o y bodies and he complex s uc u e o ood ma ices in which he a ge compounds a e con ained. 114 In his s udy, we s a ed by analyzing he indi idual samples o apple and pea ma ices, ecu ing o he me hod desc ibed be o e. Figu e 4.27: Ch oma og am o he mesoca p o he Noi a egional apple. As we can see om Figu e 4.27, he ch oma og am shows ha he e isn´ a de ec able amoun o any pes icide in his sample o he mesoca p o he Noi a egional apple cul i a . The e o e, we hough abou he possibili y o epea ing he s udy in a andomly bough comme cial apple, and again, he ch oma og am shows ha no quan i iable amoun o any o he pes icides is p esen in he mesoca p - Figu e 4.28 no in he peels – Figu e 4.29. Figu e 4.28: Ch oma og am o he mesoca p o he comme cial apple. 115 Figu e 4.29: Ch oma og am o he peels o he comme cial apple. This led us o doub i he ex ac ion p ocess was being e ec i e. Many s udies ha e p o en ha in some ood ma ices, me hod op imiza ion is necessa y so ha he analy es a e p esen in he ex ac . We decided o spike he sample wi h an alkaline ungicide widely used o con ol plan diseases, me alaxyl (me hyl N-(me hoxyace yl)-N-(2,6-xylyl)-DL-alanina e). Resul s show ha a single peak (Figu e 4.30), wi h = 21.165 was obse ed and ha ha peak e ec i ely co esponds o me alaxyl (Figu e 4.31), so we can say ha he ex ac ion me hod is app op ia e and ha he egional apple and pea samples a e no con amina ed wi h aceable amoun s o pes icides. Figu e 4.30: Ch oma og am o he peels o he egional apple spiked wi h me alaxyl. 116 Figu e 4.31: Lib a y sea ch o he iden i ied peak (co esponds o me alaxyl - me hyl N-(me hoxyace yl)-N-(2,6- xylyl)-DL-alanina e) and mass spec um o he same compound. We also used a comme cially a ailable pes icide mix u e solu ion, con aining 155 pes icides (A achmen 1), o ensu e ha he me hod was capable o de ec ing hese compounds (Figu e 4.32). The ch oma og am shows se e al peaks ha co espond o pes icides known in ou lib a y. Figu e 4.32: Ch oma og am o he 155-pes icide mix u e. 117 5. Conclusions Ea ing ui is e y impo an o ha e a balanced die and heal hy habi s and also o p e en diseases. In ac , The Wo ld Heal h O ganiza ion (WHO) ecommends consuming a leas 400 g each day o ob ain hei heal h and nu i ion bene i s.188 This is a majo con ibu ing ac o o he high demand and he e o e high p oduc ion alues o some ui s, including apples and pea s. Howe e , only abou 70% o he ui is edible, and his o igina es a high olume o by-p oduc s o hese ui s. Mo eo e , ui s by-p oduc s ha e a high con en o phenolic compounds, he e o e he e is bo h a need and an asse o euse hese by-p oduc s. In his wo k, an analy ical me hod o he de e mina ion o phenolic compounds in ui pulps and by-p oduc s by ul a-high pe o mance liquid ch oma og aphy coupled o ime-o - ligh mass spec ome y, was de eloped. The ollowing analy ical pa ame e s we e cha ac e ized o each phenolic compound: wo king ange, linea i y, limi o de ec ion, limi o quan i ica ion, and accu acy. The me hod was applied o ou samples o egional ui s ( wo mesoca ps and wo peels, one o each ui ). The linea anges we e be ween 0.25 and 1250 µg/mL. The limi s o quan i ica ion ob ained we e all lowe han 100 µg/mL in he case o phenolic acids and 5 µg/mL in he case o o he phenolics. Reco e y es s showed alues be ween 70 and 96% o he lowe o i ica ion le el (0.1 mg/100g) and 67 and 99% o he highe o i ica ion le el (1.0 mg/100g). The highes con en o a phenolic compound ound in he ui samples was anillic acid, wi h 9.77 µg/g o esh Lambe-os-Dedos pea , ollowed by ca eic acid wi h 6.73 µg/g o esh Amo im peel. The an ioxidan capaci y o he egional cul i a s o apples and pea s, as well as some comme cial cul i a s o apples, was compa ed. Be ween he egional cul i a s o apples and pea s, he apple cul i a s p esen a highe con en o o al phenolics and o al la onoids, as well as a highe an ioxidan capaci y, in gene al. The same hing can be said ega ding he o al uc ose con en because he apple cul i a s p esen highe alues o uc ose han he egional pea cul i a s. The egional apple cul i a s, howe e , do no ha e as high alues o hese pa ame e s as he comme cial cul i a s do. Rega dless o ha , some egional cul i a s, pa icula ly he Noi a apple and he Amo im pea , al hough ha ing low p oduc ion olume, should be conside ed o be used e.g., in he ood o cosme ic indus ies, due o no only o he cha ac e is ics e idenced by his wo k, bu also due o i s o ganolep ic p ope ies. 124 Spec opho ome ic De ec ion. Food Chemis y 2016, 212, 43–47. h ps://doi.o g/10.1016/j. oodchem.2016.05.082. (92) Biswas, A. 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