1(2)37-45 !!!!!!!!!!!!!!!!!!CONTACT&:&INANPI&HIDAYATI&SUMIASIH &!!!!!!!inanpihs@trilogi.ac.id! ©!International!Journal!of!Applied!Biology! 37! Abstract& Citrus!peel!color!is!one!of!the!main!quality!attributes!which!was!caused!by! the!accumulation!of!carotenoids!and!its!derivatives,!especially!β"citraurine.! It!makes!citrus!peel!color!looks!attractive!(orange).!The!orange!color!is!a! mixture! of! β"cryptoxanthin! with! β"citraurin.! The! objectives! of! this! study! were!(1)!to!observe!the!effect!of!precooling!and!duration!of!proper!ethylene! exposure!in!the!formation!of!orange!color!on!citrus!peel,!(2)!to!identify!and! determine!the!β"cryptoxanthin0content!and!total!chlorophyll!on!citrus!peel.! Citrus!was!from!Tuban,!East!Java!while!the!study!was!conducted!at!PKHT!IPB! and!LIPI.!Precooling!and!without!precooling!treatment!prior!to!injection!of! 100!ppm!of!ethylene!exposed!at!15!°C,!duration!of!exposure!control!(0),!24,! and!48!hours.!The!results!show!that!the!best!color!of!the!Citrus!Color!Index! (CCI)!is!the!precooling!treatment!and!the!duration!of!ethylene!exposure!for! 24!hours,!which! can!reduce! total!chlorophyll! content!about!8! times!and! proved! to! increase! β"cryptoxanthin! pigment! content! five! times! in! accelerating!the!formation!of!orange!citrus0reticulata0peel!color!to!bright! orange.!Degreening!has!no!significant!effect!on!total!dissolved!solids!and!the! firmness!level!of!citrus!fruits.! & ISSN!:!2580X2410! eISSN!:!2580X2119! ! ! ! The&Analysis&of&β"cryptoxanthin&and&Zeaxanthin0using&HPLC&in& the&Accumulation&of&Orange&Color&on&Lowland&Citrus&& ! Inanpi!Hidayati!Sumiasih1,2,!Roedhy!Poerwanto1,!Darda!Efendi1,3,!Andria!Agusta4!and! Sri!Yuliani5! ! 1!Agronomy!and!Horticulture!Department,!Faculty!of!Agriculture,!Bogor!Agricultural! University,!Bogor,!Indonesia! 2!Agroecotechnology!Program,!Faculty!of!Bioindustry,!Trilogi!University,!Jakarta,!Indonesia! 3!Center!for!Tropical!Horticulture!Studies!(PKHT)!Baranangsiang!Campus!of!IPB,!Bogor,! Indonesia! 4!Center!for!Biology,!Indonesia!Institute!of!Sciences!(LIPI),!Cibinong,!Indonesia! 5!Center!for!Agricultural!Postharvest!Research!and!Development,!Bogor,!Indonesia! ! ! & & & & & & & & & & & & & & & Introduction& Peel!color!is!one!of!external!quality!attributes!of!citrus!and!plays!important!factor!in! costumer!decision!in!purchasing.!GreenXcolored!citrus!is!usually!considered!sourXtasted!and! unripe,!while!orangeXcolored!citrus!is!considered!otherwise.!Generally,!consumers!choose! orangeXcolored!citrus.!Therefore,!it!is!essential!to!ensure!that!the!citrus!fulfills!the!criteria! when!it!is!ready!to!enter!the!market.! &&&&&&&&OPEN&ACCESS& & & &&&&&&&&&&International&Journal&of&Applied&Biology& Keyword! β"cryptoxhantin0 Citrus0 Chlorophyll0 Degreening0 Ethylene0zeaxanthin! Article&History! Received!23!November!2017! Accepted!21!December!2017!! International Journal of Applied Biology is licensed under a Creative Commons Attribution 4.0 International License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. ! International Journal of Applied Biology, p-ISSN : 2580-2410 e-ISSN : 2580-2119. Journal homepage : http://journal.unhas.ac.id/index.php/ijoab International0Journal0of0Applied0Biology,01(2),020170 ! 38! In!order!to!fulfill!the!consumers’!demand!regarding!the!taste,!the!accumulation!of! orangeXcolored!on!citrus!peel!through!postXharvest!treatment!should!be!done.!The!treatment! is!degreening!with!ethylene.!Degreening!so!far!is!still!not!succeed!to!form!orangeXcolored! citrus.!In!fact,!it!made!the!citrus!to!form!yellowXcolored!peel.!This!result!is!also!unfavorable! because!consumers!consider!yellowXcolored!citrus!is!in!the!verge!of!senescence.!! Degreening!in!tropical!region!is!still!not!success!because!βXcitraurin0pigment!as!the!red! color!formation!is!not!formed.!Only!yellow!color!pigment!β"cryptoxanthin0is!formed.!(Kato!et0 al.,02006;!Fanciullino!et0al.,!2008).!Yellow!on!citrus!peel!is!caused!by!carotenoids!derivative! cryptoxanthin0 (Zhou! et0 al.,! 2010).! Orange! color! can! be! formed! due! to! the! mixture! of! β" cryptoxanthin!and!β"citraurine!(Stewart!&!Wheaton,!1971;!Rodrigo!et0al.,02013).!!While!β" cryptoxanthin!and!zeaxanthin0is!the!pigment!contributed!in!the!formation!of!β"citraurin!(Ma! et0al.,02013).! The!failure!of!degreening!in!Indonesia!is!because!the!citrus!is!not!exposed!to!low! temperature!during!its!growth.!Thus,!β"citraurin0pigment!can!not!be!formed.!It!is!only!formed! at!18X24!oC.!To!substitute!the!existence!of!low!temperature,!the!precooling!treatment!was! introduced.!Precooling!after!harvest!is!expected!to!substitute!low!temperature!treatment! which!could!not!be!fulfilled!when!the!fruit!is!in!the!field.!Ladaniya!(2008)!reported!that!low! temperature!affects!greatly!on!the!color!changes!on!peel.!! According!to!Matsumoto!et0al.,!(2009)!research!about!Satsuma!fruit!storage!at!30!ºC! was!proven!to!increase!zeaxanthin!and!β"carotene0but!no!β"citraurin0pigment!was!found.!The! main!reason!of!failure!was!that!high!temperature!prevents!the!accumulation!of!β"citraurin! and!reduced!orange!color!(Stewart!&!Wheaton,!1971).!High!temperature!on!tropical!regions! limits!the!accumulation!of!important!pigments!on!citrus!and!prevents!its!peel!to!achieve!more! favorable!color.!! Therefore,! degreening! technology! which! could! induce! citrus! peel! orange,! reduce! chlorophyll! content,! and! identify! β"cryptoxanthin0 as! orange! formation! is! needed.! The! research!aims!to!observe!the!effect!of!precooling!and!obtain!the!proper!ethylene!exposure! duration!and!to!identify!and!determine!β"cryptoxanthin0and!Chlorophyll0content!on!citrus! peel!before!and!after!degreening.! & Materials&and&Methods& The!research!was!done!at!PKHT!IPB!and!the!analysis!of!chemical!content!of!peel!was! done!at!LIPI!Cibinong.!It!was!conducted!from!January!2016!until!July!2017.!Materials!used!in! the!research!were!Citrus!reticulate!“Tejakula”,!ethylene!gas,!and!liquid!nitrogen.!Tools!used! were!degreening!box,!cool!storage,!syringe,!refractometer,!color!reader,!spectrophotometer,! and! chromatography,! HPLC! (High! Performance! Liquid! Chromatography).! A! 2! factors! Randomized! Group! Design! was! used! as! research! design.! Treatments! were! harvest! and! exposure! duration.! Harvest! factor! consists! of! 2! levels! that! is! without! precooling! and! precooling.! Exposure! duration! factor! consists! of! 3! levels,! without! ethylene! exposure,! exposure!for!24!hours,!and!sxposure!for!48!hours.!There!were!6!combinations!repeated!3! times!and!18!units!of!experiment.& Citrus0reticulata!“Tejakula”!came!from!low!land!estate!in!Singgahan,!Tuban,!East!Java! (40!m!asl).!After!the!citrus!was!harvested!and!treated,!it!was!brought!to!PKHT!lab,!Bogor.!It! was!sorted!and!put!into!degreening!box!and!sealed!tightly!(air!tight).!Ethylene!gas!100!ppm! was!injected!to!the!degreening!box!using!5!ml!syringe,!then!the!citrus!was!put!in!cool!storage! at!15!°C.!The!fan!was!activated!during!degreening,!which!is!control,!24!hours,!and!48!hours.!! International0Journal0of0Applied0Biology,01(2),020170 ! 39! ! Hue&Angle&and&Citrus&Color&Indeks0(CCI)& Changes!in!the!color!quality!of!citrus!peel!was!measured!with!Minolta!Color!Reader! type!310.!This!tool!color!notation!system!is!hunter!notation!system!(color!system!L,!a,!and!b).! The! measurement! results! were! expressed! in! the! Citrus! Color! Index! (CCI).! Hunter! color! notation!system!is!characterized!by!three!parameters!of!color,!the!brightness!symbolized!by! L,!the!chromatic!colors!symbolized!by!‘a’!notation!and!color!intensity!with!the!notation!‘b’.! Notation!‘L’!states!the!parameter!of!brightness!(lightness),!with!value!L:!value!0!means!black! and!100!means!white.!While! ‘a’!notation!stating!chromatic!colors!redXgreen!mix!with!+a! (positive)!from!0!to!+100!indicating!red!color!and!value!(negative)!from!0!to!X80!indicating! green!color,!while!the!notation!‘b’!states!chromatic!color,!the!mixture!of!blueXyellow!value!+b! (positive)!from!0!to!+70!indicating!yellow!color!and!the!Xb!(negative)!value!from!0!to!X70! indicating!blue!color!(Andarwulan!et0al.,02011).!! The!value!of!Citrus!Color!Index!(CCI)!is!a!formula!widely!used!to!see!the!quality!of!citrus! peel!color.!According!to!JimenezXCueata!et0al.,! (1981),!Value!Citrus!Color! Index!(CCI)! is!a! formula!that!is!widely!used!to!see!the!quality!of!orange!peel!color.!Based!on!JimenezXCueata! et0al.,!(1981)!that!the!value!of!CCI!is!used!as!followshe!following!equation:!! ! !!"# =#1000.().* 0 0 Citrus!Color!Index!(CCI)!using!following!range:!CCI≤!X5!(dark!green),!X5!!10!(dark!orange).! & Total&Solluble&Solid& The! fruit! flesh! of! several! samples! from! each! treatment! were! taken! and! the! total! soluble!solids!was!measured!using!a!hand!refractometer.!A!drop!of!juice!was!placed!on!hand! refractometer!prism.!! ! Fruit&Firmness& Fruit! firmness! is! determined! by! fruit! resistance! to! puncturing! the! peel! done! by! penetrometer.! ! Total&Chlorophyll&& The! total! content! of! chlorophyll! was! measured! using! spectrophotometry! method.! Citrus!peel!was!weighed!at!0.5!grams!and!then!crushed!(slurry)!and!extracted!with!2!mL! acetris.! The! extract! then! inserted! into! microtube! and! centrifuged! for! 10! seconds.! The! centrifuged!filtrate!was!inserted!in!a!1XmL!reaction!tube,!and!then!3!mL!acetris!was!added! and!placed!in!cuvet!to!be!measured!using!a!spectrophotometer!at!wavelengths!537,!647!and! 663!nm.!According!to!Sims!and!Gamon!(2002),!after!obtaining!an!absorbance!value,!the!total! content!of!chlorophyll!can!be!calculated!by!the!following!equation:! ! !ℎ,- #=#0.01373∗1223# −#0.000897∗1738# −#0.003046∗12;8! !ℎ,< #=#0.02405∗12;8# −#0.004305∗1738#−#0.005507∗12230 ?@A(,#B,@C@DE,# =#!ℎ,- + !ℎ,<0 International0Journal0of0Applied0Biology,01(2),020170 ! 40! 0 Identification&of&β"cryptoxanthin&and&zeaxanthin0Pigments& Material&Preparation& Citrus!peels!are!separated!from!the!edible!portion,!then!immediately!cooled!with! nitrogen!liquid!in!the!dewar!tube!until!next!step.! ! Carotenoid&Extraction& The!identification!of!carotenoids!was!performed!by!the!method!described!by!Kato!et0 al!(2004).!The!pigment!was!extracted!from!the!sample!with!hexane!solution:!aceton:!ethanol! (2:! 1:! 1,! v! /! v)! containing! 0.1%! (w! /! v)! 2.6! ditert! butyl! 4! methylphenol! and! 10%! (w! /! v)! magnesium! carbonate.! After! the! organic! solvent! evaporates! entirely,! the! carotenoidX containing! extract,! which! is! esterified! into! fatty! acid,! is! disacorated! with! 20%! (w! /! v)! methanolic!KOH.!WaterXsoluble!extracts!are!removed!by!saturated!NaCL.!The!pigments!were! partitioned!in!the!dhietyl!ether!phase!and!evaporated.! ! Analysis&of&β"cryptoxanthin,0zeaxanthin&and&β"carotene& The!residue!is!redissolved!in!methyl!tert!butyl!ether!solution:!methanol!(4:!6,!v!/!v).!An! aliquot!(30!μL)!is!separated!by!an!HPLC!reversed!phase!(Shimadzu,!Ascentis)!equipped!with! type!CX18,!at!a!1!mL!minX1!flow!rate.!The!standards!of!β"cryptoxanthin,!zeaxanthin!and!β" carotene!were!prepared!based!on!the!method!described!with!fresh!weight!micrograms!per! gram! (preparing! for! calibration! of! the! standard).! Quantification! of! β"cryptoxanthin,! zeaxanthin0and!β"carotene!was!performed!in!3!replications.!The!data!were!obtained!from! average!HPLC!values!and!calculated!using!the!calibration!equation.!With!standard!pigments! (Figure!1),!and!the!calibration!formula!are!as!follows!(Table!1):! ! & & & & & & & & Figure& 1.& HPLC& chromatogram& for& mixture& of& authentic& sample& of& Zeaxhantin0 β" cryptoxanthin,&and&β"carotene.& Table&1.&The&linier&equation&for&β"carotene,0βQcryptoxanthin,0and0zeaxanthin0pigments& Standard& 0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 min 0 5 10 15 20 25 30 mV Detector+A:450nm+ 6 .8 3 3 /5 9 0 8 0 5 1 0 .7 7 8 /8 9 8 5 7 1 9 .1 5 7 /9 7 0 6 1 Zeaxanthin! βXcryptoxhantin! βXcarotene! International0Journal0of0Applied0Biology,01(2),020170 ! 41! ! Data&Analysis& ! The!results!from!observation!were!tested!by!using!SAS!software!(Statistical!Analysis! System)!The!data!were!analyzed!using!Analysis!of!Variance!at!5%!significance!level.!When!the! result! showed! significant! effect! of! the! treatment,! it! will! be! tested! further! using! Duncan! Multiple!Range!Test!(DMRT)!at!5%!level.! && Results&and&discussion&& The!effect!of!precooling!and!exposure!duration!to!Citrus0reticulata!of!Tuban!origin! showed!the!best!color!change!was!on!the!duration!of!24!hours!exposure!with!precooling!and! degreening! treatment.! Citrus! peel! color! with! precooling! treatment! on! 24Xhour! ethylene! exposure! resulted! in! orange! peel! color,! whereas! without! precooling! showed! a! yellowish! orange!color!(Figure!2,!3).! ! & & & & ! ! ! ! Figure&2.&The&change&of&Citrus0reticulata&‘tejakula’&peel&color&without&precooling&treatment& on&severalðylene&exposure&durations&(9&days&after&treatment)& & & & & & & & & Figure&3.&The&change&of&Citrus0reticulata&‘tejakula’&peel&color&with&precooling&treatment&on& severalðylene&exposure&durations&(9&days&after&treatment)& & Total&soluble&solids&and&and&citrus&firmness& The!measurement!for!measuring!citrus!sweetness!level!is!expressed!in!the!content!of! total!soluble!solids.!Based!on!the!precooling!treatment!and!ethylene!exposure!duration!at!the! final!observation,!it!was!shown!that!the!treatments!gave!no!significant!effects!to!total!soluble! solids!and!citrus!firmness.!! & Table&2.&The&change&of&Citrus0reticulata&“tejakula’&peel&color&with°reening&treatment&on& severalðylene&exposure&durations&(0,&12&days&after&treatment)& Treatment! Total!soluble!solids!(o!brix)! Citrus!firmness!(Kgf)! !! Zeaxantin& Criptoxantin& Caroten& Linier!equation! y!=!86079x!+!44977! y!=!68174x!+!111876! y!=!3915.3x!+!249036! R2! R²!=!0.9938! R²!=!0.9673! R²!=!0.9838! Without! degreening! Degreening, 24!hours! Degreening,! 48!hours! Precooling! Without! precooling!! Without! degreening! ! Degreening,! 24!hours! Degreening,! 48!hours! International0Journal0of0Applied0Biology,01(2),020170 ! 42! 0! 12! 0! 12! Day!after!treatment! Precooling! ! ! ! ! X!Non!precooling! 10.76! 10.76! 0.58! 0.62! X!Precooling! 10.77! 10.72! 0.56! 0.62! Exposure!durations! ! ! ! ! X!0!hour! 10.77! 10.73! 0.57! 0.63! X!24!hours! 10.75! 10.76! 0.57! 0.63! X!48!hours! 10.79! 10.74! 0.57! 0.60! 0 Citrus&Color&Index&Value0(CCI)&& The!use!of!the!Citrus!Color!Index!(CCI)!value!described!colors!such!as!red,!orange!or! green.!The!increase!of!CCI!value!indicates!that!there!is!a!change!of!color!from!green!to!orange.! Precooling!with!degreening!had!an!effect!on!the!CCI!value!on!Citrus0reticulata!to!form!orange! color!faster!than!treatment!without!precooling.!The!result!of!CCI!measurements!of!Citrus0 reticulata!with!precooling!treatment!with!degreening!on!24Xhour!exposure!duration,!it!could! form!orange!color!on!dayX9!after!degreening!with!value!7.94.!While!the!CCI!value!of!Citrus0 reticulata!given!treatment!without!precooling!on!24Xhour!exposure!duration!could!form!an! orange!color!on!dayX12!of!treatment!with!value!7.99!(data!not!shown).& & Total&Chlorophyll& The!color!variation!of!citrus!fruits!is!caused!by!the!main!groups!of!chlorophyll!pigments! and! carotenoids.! The! dominant! pigment! on! unripe! or! ripe! green! fruit! peel! is! chlorophyll! (Rodrigo!et0al.!2013).!The!total!chlorophyll!of!Citrus0reticulata!“tejakula”!was!observed!at!the! time! before! degreening! treatment! (harvest,! 0! days),! without! degreening! (9! days! after! treatment)!and!precooling!treatment!with!degreening!(9!days!after!treatment).!The!total! chlorophyll!in!Citrus0reticulata!“tejakula”!with!precooling!and!degreening!treatment!with!24! hours!exposure!decreased!sharply!with!the!lowest!value!compared!to!other!treatments,!with! a!value!of!9.81!ug!g!FWX1!on!dayX9!after!treatment,!initial!chlorophyll!content!value!before! treatment!113.81!ug!g!FWX1!(Figure!4).!! ! ! ! ! ! ! ! ! ! ! ! ! ! & Figure&4.&The&change&of&total&chlorophyll&on&citrus&peel&& Chlorophyll!degradation!causes!color!changes!during!degreening!and!the!appearance! of!carotenoid!pigment!to!produce!orange!color.!These!results!are!also!similar!to!the!study! conducted! by! Stewart! and! Wheaton! (1972);! Rodrigo! and! Zacarias! (2007)! that! ethylene! 0 20 40 60 80 100 120 140 before!degreening!(0!day! after!harvest) without!degreening!(9!day! after!harvest) Degreening!(9!day!after! harvest) ch lo ro ph yl l!c on te nt (u g! g! FW X1 ) Non!Precooling Precooling International0Journal0of0Applied0Biology,01(2),020170 ! 43! applications! triggered! skin! pigmentation! by! stimulating! chlorophyll! degradation! and! accumulation!of!carotenoids,!including!β"citraurin.0 ! Identification&of&βQCarotene,0βQCriptoxanthin0and&Zeaxantin0 From! the! result,! we! can! conclude! that! in! order! to! form! orange! color! on! Citrus0 reticulata!"Tejakula"!in!lowland,!precooling!prior!to!degreening!is!needed.!Precooling!and! degreening!treatments!provided!better!and!faster!result!in!orange!color!accumulation.!HPLC! curve!on!zeaxanthin!and!βXcryptoxanthin!pigments!(Figure!5).!The!content!of!βXcryptoxanthin! on!Citrus0reticulata!"Tejakula"!with!precooling!and!degreening!treatments!increased!rapidly! around!22.03!ug!g!FWX1!on!day!9!after!treatment!(Figure!5).! !! ! ! ! ! ! ! ! & & & & & & & & & & Figure&5.&HPLC&chromatogram&of&Citrus0reticulata&"Tejakula"&without&precooling&(a),&and& precooling&(b)&(day&9&after&treatment)&with°reening&& ! The!content!of!zeaxhantin!on!precooling!and!degreening!treatments!and!exposure! duration! for! 24! hours! resulted! in! 37.65! ug! g! FWX1! on! day! 9! after! treatment.! Zeaxhantin! content!without!degreening!shows!lower!value!that!is!12.22!ug!g!FWX1!(data!not!shown).!This! result!is!in!accordance!with!Kato!et0al!(2004);!Rodrigo!et0al!(2013)!who!stated!that!the!change! of!citrus!peel!color!from!green!to!orange!caused!by!nonphotosyntetic!carotenoid!synthesis,! that! is! βXcitraurin! acts! as! the! color! formation! to! redXorange! on! Citrus! nobilis! peel.! The! accumulation!of!this!compound!is!determined!by!precursor!availability!in!carotenoids!which! is! photosynthetic! such! as! zeaxanthin,! βXcryptoxanthin,! karotenoid! nonphotosintetic! (βX citraurin)!which!only!take!place!in!low!temperature.& & Conclusions& The!Citrus!Color!Index!(CCI)!is!the!precooling!treatment!and!the!duration!of!ethylene! exposure!for!24!hours,!which!can!reduce!total!chlorophyll!content!about!8!times!and!proved! to! increase! β"cryptoxanthin! pigment! content! five! times! in! accelerating! the! formation! of! orange!Citrus0reticulata0peel!color!to!bright!orange.!Degreening!has!no!significant!effect!on! total!dissolved!solids!and!the!firmness!level!of!citrus!fruits.! & 0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 min 0 25 50 75 100 125 150 mV Detector+A:450nm+ 9 .0 1 7 /1 0 4 7 4 7 3 1 2 .3 4 3 /7 7 2 2 0 5 1 7 .4 5 7 /8 0 2 0 8 6 (a)! 0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 min 0 25 50 75 100 125 150 175 200 mV Detector+A:450nm+ 5 .8 1 6 /2 1 1 1 2 5 8 9 .9 7 4 /1 3 2 3 3 4 1 1 7 .6 1 6 /3 8 4 5 5 9 (b)! Zeaxanthin0 β"cryptoxanthin0 Zeaxanthin0 β"cryptoxanthin0 International0Journal0of0Applied0Biology,01(2),020170 ! 44! Acknowledgment&& The!author!would!like!to!express!gratitude!to!Center!for!Tropical!Horticulture!Studies! (PKHT)!IPB!and!LIPI!Cibinong!which!have!facilitated!the!research.!The!author!also!would!like! to! express! gratitude! to! Ministry! of! Research,! Technology,! and! Higher! Education! for! the! Doctoral!Dissertation!Research!Grant!with!research!contract!number!0432/K3/KM/2017.!The! author!also!would!like!to!thank!to!Ministry!of!Agriculture!for!the!partnership!grant!which!help! funding!the!research!with!contract!number!714/LB.620/1.1/2/2013.& & References& Andarwulan,!N.,!Kusnandar,!F.!&!Herawati,!D.!2011.!Analysis!of!Food.!Jakarta.!Dian!Rakyat![In! Indonesian]! Gang,! Ma,! Zhang! L.,! Matsuta,! A.,! Matsutani,! K.,! Yamawaki,! K.,! Yahata,! M.,! Wahyudi,! A.,! Motohashi,!R.!&!Kato!M.!2013.!Enzymatic!Formation!of!βXCitraurin!from!bXCryptoxanthin! and! Zeaxanthin! by! Carotenoid! Cleavage! Dioxygenase4! in! the! Flavedo! of! Citrus! Fruit.! 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