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ThermostablephycocyaninfromtheredmicroalgaCyanidioschyzonmerolae,anewnaturalbluefoodcolorantD.
Y.
Rahman1,2&F.
D.
Sarian1&A.
vanWijk1&M.
Martinez-Garcia1&M.
J.
E.
C.
vanderMaarel1Received:18September2016/Revisedandaccepted:1November2016/Publishedonline:21November2016#TheAuthor(s)2016.
ThisarticleispublishedwithopenaccessatSpringerlink.
comAbstractThedemandfornaturalfoodcolorantsisgrowingasconsumersquestiontheuseofartificialcolorantsmoreandmore.
ThephycobiliproteinC-phycocyaninofArthospiraplatensisisusedasanaturalbluecolorantincertainfoodproducts.
ThethermoacidophilicredmicroalgaCyanidioschyzonmerolaemightprovideanalternativesourceofphycocyanin.
Cyanidioschyzonmerolaebelongstotheor-derCyanidiophyceaeofthephylumRhodophyta.
Itsnaturalhabitataresulfurichotspringsandgeysersfoundnearvolca-nicareasin,e.
g.
,YellowstoneNationalParkintheUSAandinJava,Indonesia.
ItgrowsoptimallyatapHbetween0.
5and3.
0andattemperaturesupto56°C.
ThelowpHatwhichC.
merolaegrowsminimizestheriskofmicrobialcontaminationandcouldlimitproductionloss.
AsC.
merolaelacksacellwall,phycocyaninwithahighpuritynumberof9.
9couldbeextractedbyanosmoticshockusingasimpleultrapurewaterextractionfollowedbycentrifugation.
Thedenaturationmid-pointatpH5was83°C,beingconsiderablyhigherthantheA.
platensisphycocyanin(65°C).
TheC.
merolaephycocyaninwasrelativelystableatpH4and5upto80°C.
ThehighthermostabilityatslightlyacidicpHmakestheC.
merolaephycocyaninaninterestingalternativetoA.
platensisphyco-cyaninasanaturalbluefoodcolorant.
KeywordsFoodcolorant.
Phycocyanin.
Pigments.
Redmicroalga.
CyanidioschyzonmerolaeIntroductionSyntheticdyesareusedtoprovidecolortoallkindsoffoodproducts,confectionary,andbeverages(Antelloetal.
2008).
Consumershavebecomesuspiciousabouttheuseofthesesyntheticcolorantsastheyarelinkedtohavingnegativeeffectonchildren'sbehavior(McCannetal.
2007;Arnoldetal.
2012).
Severallargeretailersarefollowingmajorfoodpro-ducersbybanningfoodproductscontainingartificialcolor-antsfromtheirstores.
Thisleadstoagrowingdemandfornaturalcolorantsderivedfromplantsandalgae.
Formostoftheartificialcolorants,naturalalternativesarerelativelyeasilyavailable(WrolstadandCulter2012).
MorechallengingistofindnaturalalternativestotheartificialbluecolorantssuchasPatentBlueV(E131)orBrilliantBlueFCF(E133).
Bluecolorsarewidespreadinnaturebutitturnsoutthatitisdiffi-culttoreplicatethebluecolor.
AtneutralpH,naturalbluecolorantsarestablebut,especiallyatpHvaluesbelow5,theyaremuchlessstableandfadequickly(Newsomeetal.
2014).
RecentlytheFDAandEFSAhavegivenapprovalforuseofaSpirulina(Arthrospira)platensisextractcontaininghighlevelsofphycocyaninasnaturalbluefoodcolorantforcolor-ingcandyandchewinggum(CodeofFederalRegulation2016).
Besidesthepotentialuseasfoodcolorant,phycocyaninhasalsobeendescribedashavinginterestingpharmaceuticalandnutraceuticalproperties(Eriksen2008).
Phycocyaninisapigment-proteincomplexthatispartofthephycobilisomesfoundincyanobacteriaandmicroalga.
Phycobilisomesarelarge,water-solubleproteincomplexesattachedtothecytoplasmicsurfaceofthethylakoidmembrane(BigginsandBruce1989)andserveasthemajorantenna*M.
J.
E.
C.
vanderMaarelm.
j.
e.
c.
van.
der.
maarel@rug.
nl1AquaticBiotechnologyandBioproductEngineeringDepartment,EngineeringandTechnologyInstituteGroningen(ENTEG),UniversityofGroningen,Nijenborgh4,9747AGGroningen,TheNetherlands2ResearchCenterforBiotechnology,IndonesianInstituteofSciences(LIPI),Jl.
RayaBogorKM46,16911Cibinong,Bogor,IndonesiaJApplPhycol(2017)29:1233–1239DOI10.
1007/s10811-016-1007-0complexharvestinglight.
Phycobilisomescanmakeupasmuchas20%ofthecellularproteincontent(Glazer1989).
Phycocyaninisanoligomericproteincomposedofαandβsubunittowhichseveralopenchaintetrapyrolesareattached(Stecetal.
1999;Padyanaetal.
2001;Coyleretal.
2005).
Thetetrapyrolestructuresgivethetypicalbluecolortophycocya-ninwhiletheproteinpartconfersthestabilitywithrespecttopHandtemperature.
Arthrospiraplatensisphycocyaninhasalimitedthermosta-bilityasitdenaturesattemperaturesabove60°C(Jespersenetal.
2005;Martellietal.
2014).
Inthesearchforamorether-mostableand/oracidstablephycocyanin,thethermoacidophilicredmicroalgaCyanidioschyzonmerolaewasexploredasasourceofabluecolorant.
Cyanidioschyzonmerolaeisaunicel-lularmicroalgabelongingtotheorderCyanidiophyceaeofthephylumRhodophyta.
Thisspeciesinhabitshotsulfuricspringsandgeysersinvolcanicareas;itgrowsbestattemperaturesbetween40and56°CandacidicconditionofpH0.
5to3(Cinigliaetal.
2004).
Thephycocyaninofanotherredmicroalga,Galdieriasulphuraria,hasbeeninvestigated(Slothetal.
2006;Srensenetal.
2012)asthisspeciesnotonlygrowsatrelativelyhightemperaturesandlowpH,butalsobecauseitcangrowheterotrophicallyinthedarkonsugaraswellasautotrophicallyinthelight(GrossandSchnarrenberger1995).
Arthrospiraplatensisisgrownoutdoorinopenpondsorracewaysystems(Lee1997;Belay2013),sufferingfrompro-ductivitylossduetoinfectionasitisdifficulttomaintainstricthygienicconditions(RichmondandGrobbelaar1986).
Theex-tremeconditionsappliedtogrowredmicroalgacouldbead-vantageousasinfectionsareunlikelyatthelowpHlevelsap-plied.
Slothetal.
(2006)andSrensenetal.
(2012)investigatedtheheterotrophicgrowthofG.
sulphurariaonglucoseinclosedfermentersandfoundareasonablephycocyaninproductivitybutconcludedthatthepolysacchariderichcellwallofG.
sulphurariamakesitdifficulttodisruptthecellsandextractthephycocyanin.
Inaddition,Srensenetal.
(2012)foundthatthephycocyaninextractedfromG.
sulphurariacultureswasnotpure,asaconsiderableamountofproteinandchlorophyllwasalsoextracted.
Cyanidioschyzonmerolaecouldbeamoreeffectivesourceofphycocyaninasitlacksacellwall(Lee2004)makingtheextractionofthephycocyaninprobablymoreefficient.
ThispaperreportsontheproductionandextractionofphycocyaninfromC.
merolaebyasimpleultrapurewatertreatment.
MaterialsandmethodsGrowthofC.
merolaeandphycocyaninextractionTheredunicellularmicroalgaCyanidioschyzonmerolaewasobtainedfromNationalInstituteforEnvironmentalStudies(NIES,Japan),catalogno1332.
AstockculturewasmaintainedinAllenmediumpH2underconstantlight(100μmolphotonsm2s1)onashakerat150rpmand40°C.
Allenmedium(Allen1959)consistedof1.
32gL1(NH4)2SO4,0.
27gL1KH2PO4,0.
25gL1MgSO4.
7H2O,0.
073gL1CaCl2.
2H2O,11mgL1FeCl3,2.
8mgL1H3BO3,1.
8mgL1MnCl2,0.
218mgL1ZnSO4.
7H2O,0.
05mgL1CuSO4,0.
023mgL1NH4VO3,and0.
024mgL1Na2MoO4.
2H2O.
ThepHofthemediumwasadjustedto2.
0with4MH2SO4andautoclavingat120°Cfor20min.
A1-Lphoto-bioreactor(approximately100μmolphotonsm2s1at40°C)wasusedtogrowC.
merolaeforphycocyaninproduction.
Cultureswereharvestedbycen-trifugationat10,000*gfor10minat4°C.
Thepelletwasresuspendedinultrapurewater,mixedwellfor5min,andkeptforseveralhoursatroomtemperature.
Celldebriswasremovedbycentrifugationat15,000*gat4°Cfor15min;thebluecoloredsupernatantwastransferredtonewtubeforfurtheranalysis.
PurificationofphycocyaninToinvestigatetheinfluenceofthetimeofexposuretoultra-purewaterontheyieldofphycocyanin,200mgwetbiomassweremixedwellwith2-mLultrapurewater(MilliQpurifica-tionsystem)andleftatroomtemperatureforupto300min.
Bluecoloredsupernatantwascollectedbycentrifugationat15,000*gandtransferredintonewtube.
Besidesmixing,twootherextractionmethodswereused,beadbeatingbyshakingthesuspendedcellswithasmallmetalballathighspeedandhigh-pressurehomogenizationbymeansofimplo-sionofthecells.
Thecrudephycocyaninextractobtainedaftermixingwasfurtherpurifiedbyammoniumsulfateprecipitationinthreesteps(20,40,and60%saturation).
Theprecipitatewasrecov-eredbycentrifugationat10,000*gfor30min,thecolorlesssupernatantwasdiscarded,andtheprecipitatewasdissolvedin50mMcitratebufferpH5atroomtemperature.
DeterminationofconcentrationThephycocyaninconcentrationwasestimatedbyusingaspectrophotometer,DR3900(Hach-Lange,TheNetherlands).
Measurementwasconductedat624and652,atwhichphyco-cyaninandallophycocyaninrespectivelyshowmaximumab-sorption(BennettandBogorad1973).
Thepurityofphycocy-aninwasassessedbycalculatingtheratioofA624toA280,whereA280istheabsorbanceoftotalprotein.
Theconcentrationphycocyaninwascalculatedusingthefollowingequation(Silveiraetal.
2007):PhycocyaninmgmL1A624A6520:4745:341234JApplPhycol(2017)29:1233–1239ThermostabilityofphycocyaninTodeterminethedenaturationmidpoint,1mLofphycocyaninsolution(initialODA620=0.
8)wasincubatedatpH5anddifferenttemperaturesinawaterbathfor30min(intervalsof10°Cfrom30to100°C).
Thethermostabilityofthephyco-cyaninwasmeasuredbyincubatingsamplesatpH5and80°Cfollowedbymeasuringtheabsorbanceat624nmatregularintervals(0–150min).
Theremainingconcentrationofphycocyanin(CR,%)relativetotheinitialconcentrationwascalculatedusingthefollowingequation:CR(%)=C/C0*100.
TodeterminethepHstabilityofphycocyanin,thesampleswereincubatedat80°CatdifferentpHvaluesfrom2to5,andtheabsorbanceat624nmwasmeasuredatregularintervals(0to60min).
ResultsanddiscussionGrowthofC.
merolaeandphycocyaninextractionTheextremophilicredmicroalgaC.
merolaewasgrownauto-trophicallyinmineralmediumatpH2and40°Cwithcon-stantillumination(Fig.
1a).
Afterabout3days,itstartedtogrowwithaspecificgrowthrateof0.
15±0.
01day1.
Atday21,thecellshadreachedanOD800ofabout1.
8,whichcorre-spondstoadryweightof1.
051±0.
14gL1.
Aninvivoabsorptionspectrum(300to800nm)wasmadeeveryday,showingthattherewerethreemainabsorptionmaxima,at430,620,and680nm(Fig.
2).
The430and680nmmaximaaretypicalforchlorophyll(Giltelsonetal.
1999),whilethe620nmmaximumistypicalforphycocyanin(Pateletal.
2005).
Theamountofphycocyaninpercelldidnotseemtovaryasaconstantvalueofabout17.
5to18mgphycocyaninperunitofabsorptionat800nmwasfoundforalmostalltimepoints(Fig.
1b).
Toextractphycocyaninfromthecells,severalcelldisrup-tionmethodsweretested.
Beadbeatingandhigh-pressurehomogenization,bothdisruptivetechniques,werecomparedwithexposureofthecellstoanosmoticshockbymixingthemwithultrapurewater(Fig.
3).
Thelattermethodworkedbestasmostphycocyanin(0.
55mgmL1)wasfoundinsolution.
Cyanidioschyzonmerolaeisknowntolackacellwall(Albertanoetal.
2000),makingitsusceptibletoosmoticshocks.
Whenexposedtoultrapurewater,thecellstakeupwaterandfinallylyse,releasingthecellularcontentsintothewater.
Thewaterphasemainlycontainedphycocyanin(A624,Table1)withapurityindexof9.
92,calculatedastheFig.
2InvivoVISspectrum(300–800nm)ofC.
merolae.
Thestrongabsorptionpeakat680nmischlorophyll(Cp),theabsorptionpeakaround620nmcorrespondstophycocyanin(Pc).
Otherabsorptionpeaksintherangeof400–500nmarefromcarotenoid(Ct)Fig.
3ExtractionyieldofphycocyaninfromC.
merolaewithdifferentextractionmethods,1:beadbeater,2:mixingbyvortex,and3:high-pressurehomogenizerFig.
1GrowthofC.
merolaeonairandlightandtherelatedphycocyaninproductioninvivo.
(blacksquares),biomass,gdryweightL1;(whitecircles)amountofinvivophycocyaninmeasuredat620nm.
Invivophycocyaninamountperamountofbiomassexpressedas1unitofopticaldensityat800nmJApplPhycol(2017)29:1233–12391235ratioofA624toA280(protein).
Smallamountsofchlorophyllwerealsodetectedinthewaterphase(A562andA652,Table1).
C.
merolaephycocyaninextractedbyosmoticshockhasahighpuritynumberThedurationoftheosmoticshockhadacleareffectontheamountofphycocyaninextracted(Fig.
4).
IncubatingC.
merolaecellsinultrapurewaterforupto100mingaverela-tivelylowamountsofphycocyanininthewaterphase(upto0.
28mgmL1).
However,asteepincreaseintheamountofphycocyaninwasobservedbetween100and150minincuba-tion;after150min,almost0.
81±0.
09mgmL1phycocyaninwasreleased.
Longerincubationdidnotresultinasignificantincreaseintheamountofphycocyaninreleased.
Thepurityofthephycocyaninextractobtainedwithultrapurewatertreat-mentisconsiderablyhigherthanthatofextractsobtainedfromSpirulina,withapurityindexrangingfrom0.
46to2.
78(Silveiraetal.
2007;Liaoetal.
2011),fromSynechococcuswithapurityindexof2.
2(GuptaandSainis2010),andfromthecloselyrelatedredmicroalgaG.
sulphurariaofonly1(Srensenetal.
2012).
ThereasonforthemuchhigherpurityindexfoundforthephycocyaninextractedfromC.
merolaecomparedtootherphototrophsisthatthelatterhavea(thick)cellwallrequiringamechanicaltreatmenttodisruptthecellsandreleasethephycocyanin.
AsaresultofthemechanicaltreatmentofG.
sulphuraria,alsochlorophyllaandcaroten-oidsarereleasedintosolution(Slothetal.
2006),resultinginmuchlowerpuritynumberforphycocyanin.
Applyingdiffer-entpurificationtechniquessuchasaqueoustwophaseextrac-tionwithpolyethyleneglycolandwater(Rito-Palomaresetal.
2004),ammoniumsulfateprecipitationofexpendedbedab-sorptionincombinationwithchromatographyresultedinhigherpuritynumbers(ZhangandChen1999;Niuetal.
2007;Yanetal.
2011),butthesearestillconsiderablylowerthanthe9.
9foundforC.
merolae.
Phycocyaninsolutionwithapurityindexofatleast0.
7areconsideredtobeafoodgrade,whileapurityindexofatleast4isconsideredtobeanalyticalgrade(CisnerosandRito-Palomares2004).
ThephycocyaninextractedfromC.
merolaegrowncellshasahighpurityindexanyfurtherpurificationisnotrequiredtouseitasanalyticalgradematerial.
Theultrapurewaterextractcontainingphycocyaninwasfurtherpurifiedbyammoniumsulfateprecipitationat20–40%saturationresultinginaconcentratedphycocyaninsolution(54%yield)withapurityindexof18.
07(Table1).
Thepelletfraction20–40%ammoniumsulfatewasdissolvedincitratebuffer(pH5)andwasusedtofurthercharacterizetheC.
merolaephycocyanin.
Theabsorptionspectrumofthisfractionshowedaclearλmaxat624nm(Fig.
5),beingthephycocyanin,andshoulderat562nm,indicativeforphycoer-ythrin(CisnerosandRito-Palomares2004).
TheA.
platensisphycocyaninhasaλmaxof616nmatpH5and620atpH7(Jespersenetal.
2005).
ThethermophileSynechococcuslividusphycocyaninhasλmaxof609nmatpH6(Edwardsetal.
1997),whilePhormidiumluridumphycocyaninhasaλmaxatpH6of622nm(Edwardsetal.
1996).
GlazerandFang(1996)evenreportedaλmaxofabout650nmatpH3forthephycocyaninofSynechococcussp.
Table1AbsorptionatdifferentwavelengthsofC.
merolaecrudeextractandthedifferentfractionofammoniumsulfatetreatedextractCrudeextractSupernatantfractionof20%(NH4)2SO4Supernatantfractionof40%(NH4)2SO4Pelletfractionof40%(NH4)2SO4Supernatantfractionof60%(NH4)2SO4Pelletfractionof60%(NH4)2SO4Volumeofsample(mL)404040104010Dilution1:51:51:51:201:51:20A6520.
0980.
0960.
0320.
0590.
0190.
031A6240.
4960.
4690.
1670.
2710.
0420.
147A5620.
1450.
1350.
0490.
1350.
0170.
048A2800.
050.
0510.
0250.
0150.
0730.
014Purityindex9.
929.
266.
7818.
070.
5810.
78TotalC-PC16.
815.
85.
69.
21.
25Yield10094.
3133.
8154.
067.
3831.
32Fig.
4TheeffectofexposingC.
merolaecellstoultrapurewaterforanincreasingamountoftime(inmin)1236JApplPhycol(2017)29:1233–1239C.
merolaephycocyaninisthermostableThealphaandbetasubunitofthephycocyaninhavingamo-lecularweightof17–18kDawereclearlyvisibleonanSDS-polyacrylamidegel(datanotshown).
Thisisinagreementwiththemolecularweightofotherphycocyaninreportedsofar(GlazerandFang1996;Chaiklahanetal.
2011).
AsC.
merolaecangrowatrelativelyhightemperaturesupto55°C,itistobeexpectedthatthephycocyaninisrelativelythermostable.
Thepurifiedphycocyaninwasincubatedfor30minincitratebuffer(pH5)attemperaturesvaryingfrom20to100°C(Fig.
6a).
Upto75°C,thephycocyaninwasFig.
5VisibleabsorptionspectraofsolubleandprecipitatedphycocyaninfromC.
merolaeatdifferentammoniumsulfateconcentrationsTable2Half-lifeofammoniumsulfate(40%saturation)purifiedphycocyaninofCmerolaeatroomtemperature(22°C)and80°CatpH3,4and5Temperature(°C)pHHalf-life(min)2234005>1200803<5429540JApplPhycol(2017)29:1233–12391237Fig.
6CharacteristicsofphycocyaninfromC.
merolae.
aEffectofincreasingtemperaturesonthesolubility(CR(%))ofphycocyanin(incubationtime:30min).
bpHstabilityoveraperiodof180minat27°Candcat80°C.
BlacksquarespH2,TrianglespH3,whitecirclespH4,WhitesquarespH5solubleandremainedclearlyblue;attemperaturesabove75°C,itstartedtoprecipitateandat90°C,ithadcompletelyprecipitated.
Thedenaturationmidpoint,whichcanbedefinedasthattemperature(Tm)atwhich50%ofthephycocyaninisstillinsolution(CR=50%),ofC.
merolaephycocyaninisat83°C(Fig.
6a).
AtroomtemperatureandpH5,the40%ammoniumsulfatepurifiedphycocyaninfractionwasstable;morethan85%stayedinsolutionfor180min(Fig.
6b).
AtpH4,thisfractionwasalreadylessstable;about50%ofthephy-cocyaninwaslostafter180minofincubation(Fig.
6b).
At80°CandpHof4or5,thecolorfadedmuchmorerapidly(Fig.
6c).
AtpHof2and3,thebluecolordisappearedwithinseveralminutesbothatroomtemperatureandat80°C(Fig.
6b,c).
Thehalf-lifeofthe40%ammoniumsulfatepuri-fiedphycocyaninatroomtemperaturewasseveralhundredminutesatpH4and5whileatpH2and3,thephycocyaninfadedtocolorlessinlessthan5min(Table2).
Athightem-peratureof80°C,thephycocyaninhadahalf-lifeof29to40minatpH4and5,respectively.
AtpH2and3and80°C,thephycocyaninlostitscolorwithinseveralminutes(Table2).
PhycocyaninpredominantlyexistsasahexameratpH5,anditisbelievedthatthehexamericformgivessomeprotec-tionagainstdenaturation(Edwardsetal.
1996).
AtpH7,itispredominantlyinamonomericortrimericform,resultinginlowerthermostability(Edwardsetal.
1997;Jespersenetal.
2005).
Verylikely,theC.
merolaephycocyaninisalsopresentinmonomericortrimericformatlowpH,asitloosesitsthermostabilityrapidlyattheselowpHvalues.
ThestabilityoftheC.
merolaephycocyaninathighertemperaturesandapHof4or5ismuchbetterthanthatoftheS.
platensisphy-cocyanin.
TheTmoftheA.
platensisphycocyaninisbetween55and62°C(Jespersenetal.
2005;Martellietal.
2014).
TheC.
merolaephycocyaninproteinsequencecontainsfourcys-teineresidues,whereastheA.
platensisphycocyaninse-quence,being75%identicaltotheC.
merolaesequence,hastwocysteineresidues(UniProtKB-P72509).
Cysteinecanformcovalentdisulfidebondsthatcontributetothethermo-stabilityofaprotein(Fass2012).
Addinghighamountsofsugars(40to55%)suchasfructoseorglucoseimprovedthethermostabilityofA.
platensisphycocyanin,indicatingthatitcouldbeusedinhighsugarfoodproductssuchasconfection-aryandpastries(Martellietal.
2014).
AsC.
merolaephyco-cyaninalreadyhasahigherthermostabilityofitsown,itcouldbeusedinlowsugarproductsthatareexposedtohighertem-peraturesduringproduction.
ConclusionsPhycocyanincaneasilybeextractedfromautotrophicallygrownC.
merolaecellsbyanosmoticshockprocedurewithultrapurewater.
Thephycocyaninobtainedinthiswayhasahighpuritynumber(9.
9)andisthermostableupto83°CatneutralandslightacidicpH.
ThesepropertiesmaketheC.
merolaephycocyaninaninterestingalternativetoA.
platensisphycocyaninasanaturalbluefoodcolorant.
AcknowledgementsDYRwassupportedbytheUbboEmmiusPhDscholarshipprogramoftheUniversityofGroningen.
OpenAccessThisarticleisdistributedunderthetermsoftheCreativeCommonsAttribution4.
0InternationalLicense(http://creativecommons.
org/licenses/by/4.
0/),whichpermitsunrestricteduse,distribution,andreproductioninanymedium,providedyougiveappro-priatecredittotheoriginalauthor(s)andthesource,providealinktotheCreativeCommonslicense,andindicateifchangesweremade.
ReferencesAlbertanoP,CinigliaC,PintoG,PollioA(2000)ThetaxonomicpositionofCyanidium,CyanidioschyzonandGaldieria:anupdate.
Hydrobiologia433:137–143AllenMB(1959)StudieswithCyanidiumcaldarium,ananomalouslypigmentedchlorophyte.
ArchMikrobiol32:270–277AntelloFS,CostaJAV,KalSJ(2008)ThermaldegradationkineticsofthephycocyaninfromSpirulinaplatensis.
BiochemEng41:43–47ArnoldLE,LofthouseN,HurtE(2012)Artificialfoodcolorsandatten-tion-deficit/hyperactivitysymptoms:conclusiontodyefor.
Neurotherapeutics9:599–609BelayA(2013)BiologyandindustrialproductionofArthrospira(Spirulina).
In:RichmondA,HuQ(eds)Handbookofmicroalgalculture:appliedphycologyandbiotechnology.
Blackwell,Oxford,pp.
339–358BennettA,BogoradL(1973)Complementarychromaticadaptationinafilamentousblue-greenalga.
JCellBiol58:419–435BigginsJ,BruceD(1989)Regulationofexcitationenergytransferinorganismcontainingphycobilins.
PhotosynthRes20:1–34ChaiklahanR,ChirasuwanN,LohaV,TiaS,BunnagB(2011)SeparationandpurificationofphycocyaninfromSpirulinasp.
usingamembraneprocess.
BioresourceTechnol102:7159–7164CinigliaC,YoonHS,PollioA,PintoG,BhayyacharyaD(2004)HiddenbiodiversityoftheextremophilicCyanidialesredalgae.
MolEcol13:1827–1838CisnerosM,Rito-PalomaresM(2004)Asimplifiedstrategyforthere-leaseandpimaryrecoveryofC-phycocyaninproducedbySpirulinaplatensis.
ChemBiochemEngQ18:385–390CodeofFederalRegulation(2016)Spirulinaextract.
https://www.
govregs.
com/regulations/title21_chapterI_part73_subpartA_section73.
530,accessed13July2016CoylerCL,KinkadeCS,ViskariPJ,LandersJP(2005)Analysisofcyanobacterialpigmentandproteinsbyelectrophoreticandchro-matographicmethod.
AnalBiochemChem382:559–569EdwardsMR,MacCollR,EiseleLE(1996)SomephysicalpropertiesofanusualC-phycocyaninisolatedfromaphotosynthethicthermopile.
BiochemBiophysActa1276:64–70EdwardsMR,HouerC,StackRF,EiseleLE,MacCollR(1997)ThermophilicC-phycocyanin:effectoftemperature,monomersta-bility,andstructure.
BiochemBiophysActa1321:157–164EriksenNT(2008)Productionofphycocyanin—apigmentwithapplica-tioninbiology,biotechnology,foods,andmedicine.
ApplMicrobialBiotechnol80:1–14JespersenL,StrmdahlLD,OlsenK,SkibstedLH(2005)Heatandlightstabilityofnaturalbluecolorantsforuseinconfectioneryandbev-erages.
EurFoodResTechnol220:261–2661238JApplPhycol(2017)29:1233–1239FassD(2012)Disulfidebondinginproteinbiophysics.
AnnuRevBiophys41:63–79GiltelsonAA,BuschmannC,LichtenthalerHK(1999)ThechlorophyllfluorescenratioF735/F700asanaccuratemeasureofthechlorophyllcontentinplants.
RemoteSensEnviron69:296–302GlazerAN(1989)Thelightguide:directionalenergytransferinapho-tosyntheticantenna.
J.
BiolChem264:1–4GlazerAN,FangS(1996)Chromophorecontentofblue-greenalgalphycobiliproteins.
JBiolChem248:659–662GrossW,SchnarrenbergerC(1995)HeterotrophicgrowthoftwostrainsoftheacidophilicredalgaeGaldieriasulphuraria.
PlantCellPhysiol36:633–638GuptaA,SainisJK(2010)IsolationofC-phycocyaninfromSynechococcussp.
(AnacystisnidulansBD1).
JApplPhycol22:231–233LeeYK(1997)CommercialproductionofmicroalgainAsia-Pacificrim.
JApplPhycol9:403–411LeeRE(2004)Phycology,4thedn.
CambridgeUniversityPress,Cambridge,pp.
108–109LiaoX,ZhangB,WangX,YanH,ZhangX(2011)PurificationofC-phycocyaninfromSpirulinaplatensisbysingle-stepion-exchangechromatography.
Chromatographia73:291–296MartelliG,FolliC,VistaL,DagliaM,FerrariD(2014)ThermalstabilityimprovementofbluecolorantC-phycocyaninfromSpirulinaplatensisforfoodindustryapplication.
ProcessBiochem49:154–159McCannD,BarretA,CooperA,CrumplerD,DalneL,GrimshawK,KitchinE,LokK,PorteousL,PrinceE,Sanuga-BarkeE,WarnetJO,StevensonJ(2007)Foodadditivesandhyperactivein3-year-oldand8/9-year-oldchildreninthecommunity:arandomized,double-blinded,placebo-controlledtrial.
Lancet370:1560–1567NewsomeAG,CulterCA,vanBreemenRB(2014)Nature'spalette:thesearchfornaturalbluecolorants.
JAgricFoodChem62:6498–6511NiuJF,WangGC,LinX,ZhouBC(2007)Large-scalerecoveryofphycocyaninfromSpirulinaplatensisusingexpandedbedadsorp-tionchromatography.
JChromatogrB850:267–276PadyanaAK,BhatVB,MadyasthaKM,RajashankarKB,RamakumarS(2001)Crystalstructureoflight-harvestingproteinC-phycocyaninfromSpirulinaplatensis.
BiochemBiophysResCommun282:893–898PatelA,MishraS,PawarR,GhoshPK(2005)Purificationandcharac-terizationofphycocyaninfromcyanobacterialspeciesofmarineandfreshwaterhabitat.
ProtExprPur40:248–255RichmondA,GrobbelaarJU(1986)FactoraffectingtheoutputrateofSpirulinaplatensiswithreferencetomasscultivation.
Biomass10:253–264Rito-PalomaresM,NunezL,AmadrD(2004)Practicalapplicationofaqueoustwo-phasesystemfordevelopmentofaprototypeprocessforc-phycocyaninrecoveryfromSpirulinamaxima.
JChemTechnolBiotechnol76:1273–1280SilveiraS,BurkertJFM,CostaJAV,BurkertCAV,KalilSJ(2007)OptimizationofphycocyaninextractionfromSpirulinaplatensisusingfactorialdesign.
BioresourceTechnol98:1629–1634SlothJK,WiebeMK,EriksenNT(2006)Accumulationofphycocyanininheterotrophicandmixotrophicculturesoftheacidophilicredal-gaeGaldieriasulphuraria.
EnzMicrobTechnol38:168–175SrensenL,HantkeA,EriksenNT(2012)Purificationofthephotosyn-theticpigmentC-phycocyaninfromheterotrophicGaldieriasulphuraria.
JSciFoodAgric93:2933–2938StecB,TroxlerRF,TeeterMM(1999)CrystalstructureofC-phycocyaninfromCyanidiumcaldariumprovidesanewperspec-tiveonphycobilisomesassembly.
BiophysJ76:2912–2921WrolstadRE,CulterCA(2012)AlternativestothoseartificialFDandCfoodcolorant.
AnnuRevFoodSciTechnol3:59–77YanS,ZhuL,SuH,ZhangX,ChenX,ZhouB,ZhangY(2011)SinglestepchromatographyforsimultaneouspurificationofC-phycocyaninandallophycocyaninwithhighpurityandrecoveryfromSpirulina(Arthrospira)platensis.
JApplPhycol3:1–6ZhangY,ChenF(1999)AsimplemethodforefficientseparationandpurificationofC-phycocyaninandallophycocyaninfromSpirulinaplatensis.
BiotechnolTechnol13:601–603JApplPhycol(2017)29:1233–12391239