Biorefinery Approach Applied to the Production of Food Colourants and Biostimulants from Oscillatoria sp.

Ci a ion: Mo illas-España, A.; Be mejo, R.; Abdala-Díaz, R.; Ruiz, Á.; La a ga, T.; Acién, G.; Fe nández-Se illa, J.M. Bio e ine y App oach Applied o he P oduc ion o Food Colou an s and Bios imulan s om Oscilla o ia sp.. Biology 2022,11, 1278. h ps://doi.o g/10.3390/ biology11091278 Academic Edi o : John Ko s ad Recei ed: 28 July 2022 Accep ed: 22 Augus 2022 Published: 28 Augus 2022 Publishe ’s No e: MDPI s ays neu al wi h ega d o ju isdic ional claims in published maps and ins i u ional a il- ia ions. Copy igh : © 2022 by he au ho s. Licensee MDPI, Basel, Swi ze land. This a icle is an open access a icle dis ibu ed unde he e ms and condi ions o he C ea i e Commons A ibu ion (CC BY) license (h ps:// c ea i ecommons.o g/licenses/by/ 4.0/). biology A icle Bio e ine y App oach Applied o he P oduc ion o Food Colou an s and Bios imulan s om Oscilla o ia sp. Ainoa Mo illas-España 1,2, Rupe o Be mejo 3, Robe o Abdala-Díaz 4,Ángela Ruiz 1,2 , Tomás La a ga 1,2 , Gab iel Acién1,2,* and JoséMa ía Fe nández-Se illa 1,2 1Depa men o Chemical Enginee ing, Uni e si y o Alme ía, 04120 Alme ia, Spain 2CIESOL Sola Resea ch Cen e, Join Cen e Uni e si y o Alme ía-CIEMAT, C a. Sac amen o s/n, 04120 Alme ía, Spain 3Depa men o Physical and Analy ical Chemis y, Highe Enginee ing Poly echnic School o Lina es, Uni e si y o Jaén, 23700 Lina es, Spain 4Depa amen o de Ecología y Geología, Facul ad de Ciencias, Ins i u o de Bio ecnología y Desa ollo Azul (IBYDA), Uni e sidad de Málaga, 29071 Málaga, Spain *Co espondence: [email p o ec ed] Simple Summa y: In his s udy, a bio e ine y based on Oscilla o ia sp. is de eloped o p oduce high- alue compounds such as C-phycocyanin, used in ood colou an applica ions, and bios imulan s, used in ag icul u e- ela ed applica ions. The esul s con i m ha C-phycocyanin concen a ions anging om 22 o 106 mg/L p oduce colou s simila o comme cial p oduc s; mo eo e , he sa e y o he ex ac ed C-phycocyanin was con i med h ough oxici y es s. The le o e biomass was con i med as a bios imulan , wi h he esul s con i ming a ele an auxin-like posi i e e ec . Finally, an economic analysis was conduc ed o e alua e di e en scena ios, wi h esul s con i ming his as he bes scena io om an economic s andpoin . Abs ac : In his s udy, a bio e ine y based on Oscilla o ia sp. is de eloped o p oduce high- alue compounds such as C-phycocyanin, used in ood colou an applica ions, and bios imulan s, used in ag icul u e- ela ed applica ions. Fi s , he Oscilla o ia biomass p oduc ion was op imized a a pilo scale in an open aceway eac o , wi h biomass p oduc i i ies equi alen o 52 /ha · yea being achie ed using egula e ilize s as he nu ien sou ce. The biomass p oduced con ained 0.5% C-phycocyanins, 95% o which we e ob ained a e eeze– hawing and ex ac ion a pH 6.5 and ionic s eng h (FI) 100 mM, wi h a pu i y a io o 0.71 achie ed in he inal ex ac . This pu i y a io allows o use o he ex ac di ec ly as a ood colou an . Then, he ex ac ’s colou an capaci y on di e en be e ages was e alua ed. The esul s con i m ha C-phycocyanin concen a ions anging om 22 o 106 mg/L p oduce colou s simila o comme cial p oduc s, hus a oiding he need o syn he ic colou an s. The colou emained s able o up o 12 days. Mo eo e , he sa e y o he ex ac ed C-phycocyanin was con i med h ough oxici y es s. The was e biomass was e alua ed o use as a bios imulan , wi h he esul s con i ming a ele an auxin-like posi i e e ec . Finally, an economic analysis was conduc ed o e alua e di e en scena ios. The esul s con i m ha he p oduc ion o bo h C-phycocyanin and bios imulan s is he bes scena io om an economic s andpoin . The e o e, he de eloped biomass p ocessing scheme p o ides an oppo uni y o expand he ange o comme cial applica ions o mic oalgae- ela ed p ocesses. Keywo ds: bio e ine y; phycocyanin; bios imulan s; mic oalgae; oxici y; bioeconomy 1. In oduc ion Mic oalgae, including cyanobac e ia, a e a po en ial sou ce o biop oduc s ha can be used o applica ions in high- alue ma ke s, such as pha maceu icals, cosme ics, nu- aceu icals, and ood; medium- alue ma ke s, such as eed, bioma e ials, bios imulan s, and biopes icides; and low- alue ma ke s, including bio uels and bio e ilise s [ 1 ]. Despi e Biology 2022,11, 1278. h ps://doi.o g/10.3390/biology11091278 h ps://www.mdpi.com/jou nal/biology Biology 2022,11, 1278 2 o 15 hei g ea po en ial, he annual p oduc ion o mic oalgae is ela i ely small, no exceeding 40,000 wo ldwide and lowe han 3000 in Eu ope [ 2 ]. Due o he cu en low p oduc ion capaci y, mos o he biomass p oduced is des ined o he nu aceu ical and ood sec o s, wi h a small sha e being used in cosme ics and aqua eeds. The main eason is ha , in hese applica ions, he biomass alue is su icien o co e he cu en p oduc ion cos s; hese ange om 20–50 €· kg −1 o he biomass p oduced in closed pho obio eac o s and om 5–20 €·kg−1 o hose p oduced in open aceways [1]. To p omo e he u ilisa ion o mic oalgal biomass in o he medium- alue and low- alue biomass sec o s, wo s a egies ha e been sugges ed: (i) o educe he cu en p oduc ion cos s and (ii) o de elop bio e ine y schemes able o p o ide a wide po olio o p oduc s combining high, medium, and low- alue p oduc s. To educe he cu en p oduc ion cos s, new p oduc ion echnologies need o be de eloped and cu en p oduc ion p ocesses need o be scaled up; hese changes could ake place in he medium e m. In con as , he de elopmen o bio e ine y schemes migh be achie ed in he sho e m. The bio e ine y concep in ol es ob aining mul iple p oduc s om he biomass, whe e medium- and low- alue p oduc s a e eco e ed a e he high- alue compounds—such as pigmen s, polyunsa u a ed a y acids, i amins, e c.—ha e been emo ed [ 3 ]. To de elop such bio e ine y schemes, i is necessa y o iden i y he igh p oduc s based on he composi ion o he biomass p oduced and he ma ke p ice/demand o he a ge compounds. Pigmen s such as chlo ophylls, ca o enoids, and phycobilip o eins a e aluable com- pounds ha ha e adi ionally been ob ained om mic oalgae [4]. O hese, phycobilip o- eins a e pa icula ly in e es ing gi en hei a ious applica ions; hey a e cu en ly used as luo escen ma ke s, nu aceu icals, and ood colou an s, among o he hings [ 5 ]. Rega d- ing he ood addi i es ma ke , na u al colou an s a e o g ea in e es . Phycobilip o eins ha e been inco po a ed in o di e en ood ma ices, al hough hei use needs o be u - he suppo ed by oxici y es ing, which is, un il now, an aspec gene ally o e looked. These aluable compounds a e na u ally p oduced by cyanobac e ia, wi h s ains such as Spi ulina and Oscilla o ia being especially well-sui ed o p oducing phycobilip o eins (C-PC) [ 5 ]. Some au ho s ha e claimed ha he use o his p o ein in ood and be e ages is limi ed by hei lack o s abili y o hea , ligh , and pH a ia ions [ 6 ]. The bios imulan s ma ke has also ecei ed inc eased a en ion o e he las decade due o a g ea e demand o sus ainable ag icul u e, as well as he need o boos c op yields and educe he ca bon oo p in om ood p oduc ion. The bios imulan s ma ke was es ima ed o be wo h USD 2.6 billion in 2019 and is p ojec ed o each USD 4.9 billion by 2025, a a CAGR o 11.24% o e he o ecas pe iod [ 7 ]. Mic oalgae a e al eady ecognised as a sou ce o aluable bios imulan s o ag icul u e due o hei amino acid and phy oho mone con en s ac ing as plan g ow h p omo e s [ 8 ]. Se e al mic oalgal s ains ha e been p o en o be na u al p oduce s o phy oho mones, such as indole-3-ace ic acid (IAA), cy okinins, gibbe ellins, abscisic acid (ABA), and jasmonic acid, as well as o he subs ances wi h bios imulan ac i i y [ 9 ]. Cyanobac e ia, such as A h ospi a and Oscilla o ia, can also p oduce IAA, cy okinins, and/o jasmonic acid [ 10 ]; hei use in ag icul u e imp o es he plan s’ ion up ake, an ioxidan con en , cellula me abolism, and lea chlo ophyll con en as well as he soil’s nu ien a ailabili y and humidi y [11,12]. Fo hese easons, Oscilla o ia has been iden i ied as a p omising aw ma e ial o bo h ood colou an s and bios imulan s. This wo k aimed o analyse he possibili y o de eloping bio e ine y schemes using Oscilla o ia o he p oduc ion o ood-g ade C-PC and bios imulan s o use in ag icul u e. The non oxic na u e and he colou ing p ope ies o he C-PC ex ac s we e e alua ed along wi h he posi i e bios imulan e ec s o he le o e s om C-PC ex ac ion. A echnoeconomic analysis was ca ied ou o iden i y he majo bo lenecks in de eloping eal indus ial p ocesses ocused on hese p oduc s. Biology 2022,11, 1278 3 o 15 2. Ma e ials and Me hods 2.1. Mic oalgae P oduc ion The cyanobac e ium Oscilla o ia sp. was p oduced in seawa e using a cul u e medium based on comme cial e ilize s: 0.90 g · L −1 NaNO 3 , 0.25 g · L −1 MgSO 4 , 0.3 g · L −1 K 2 PO 4 , and 0.03 g · L −1 o Ka en ol ® (Kenoga d, Spain). The pho obio eac o used was a 12 m 3 (80 m 2 ) aceway eac o loca ed inside a g eenhouse a he SABANA Demons a ion Plan (IFAPA, Alme ía, Spain). The biomass was p oduced in semicon inuous mode a a 0.3 day −1 dilu ion a e. The empe a u e, pH, and dissol ed oxygen concen a ion o he cul u e we e moni o ed online using 5083T and 5120 p obes (C ison Ins umen s, Ba celona, Spain) connec ed o an MM44 con ol- ansmi e uni (C ison Ins umen s, Ba celona, Spain) and Lab iew da a acquisi ion so wa e (Na ional Ins umen s, Aus in, TX, USA). The pH was con olled a 8.0 by on-demand injec ion o CO 2 whe eas he dissol ed oxygen was kep below 250% Sa by on-demand ae a ion. The olume o he eac o was kep cons an by adding eshwa e (e apo a ion) and he pH was con olled by CO2injec ion. 2.2. Ex ac ion and Cha ac e iza ion o Phycocyanin C-phycocyanin was ex ac ed om he eeze-d ied biomass by solubilisa ion in phos- pha e bu e (5 mM, pH 6.5) a a a io o 1:20 (w/ ). The solu ion was s i ed o 5 min a 2000 × gwi h a magne ic s i e (Schwabach, Ge many) and he in acellula ma e ial was eleased by cell osmo ic shock. The sample was hen cen i uged a 12,000 × g o 15 min (Med onic BL-S, P-Selec a, Ba celona, Spain) o eco e he phycobilip o eins solubilised in he supe na an . Subsequen ly, he phycobilip o eins we e p ecipi a ed by adding an ammonium sulpha e solu ion (70% sa u a ion) and le o s and o e nigh in he da k a 4◦C . Following his, he pu i ied bilip o ein pelle s we e esuspended in a small olume o he phospha e bu e (5 mM, pH 6.5) and dialyzed o e nigh a 4 ◦ C agains he same bu e . Then, 1% w/wsodium azide (NaN 3 ) was added o he p o ein solu ion as a p ese a i e and he samples we e s o ed a 4 ◦ C un il u he analysis. The abso bance spec a we e eco ded using a UV– isible spec opho ome e (Lambda 20, Pe kin Elme , UV Da a Man- age so wa e, Wal ham, MA, USA) a 250–750 nm. The concen a ions o phycobilip o eins (PC-Phycocyanin, APC-allophycocyanin, and PE-phycoe y h in) we e es ima ed using he ollowing equa ions: PC mg·mL−1=A615 −0.474 ×(A652) 5.34 (1) APC mg·mL−1=A652 −0.208 ×(A615) 5.09 (2) PE mg·mL−1=A563 −2.41 ×(PC)−0.849 ×(APC) 9.62 (3) 2.3. Colou E alua ion Colou eco dings we e aken using a CM-5 ch oma me e (Konica Minol a, Osaka, Japan) and he CM-S100W Spec aMagic ™ NX so wa e e sion 2.2 (Konica Minol a, Osaka, Japan). The colou ing o be e age samples was s udied (Figu e 1). The s udied be e ages we e iso onic d inks, onic wa e , and wine—all comme cial p oduc s. The selec ed comme cial p oduc s ha e a blue colou , whe eas he s anda d p oduc s we e colou less. The same b and was used o he comme cial and s anda d p oduc s. The samples we e s o ed in a domes ic e ige a o a 5 ±2◦C. Only he onic d ink and wine we e manipula ed be o e he colou ime ic analysis o elimina e hei dissol ed gases. This was pe o med by s i ing o 20 min a 300 × g. The blue p o ein ex ac was added o he be e ages while s i ing cons an ly. The pigmen a ion capaci y o he ex ac was ca ied ou as desc ibed elsewhe e [13]. Biology 2022,11, 1278 4 o 15 Biology 2022, 11, x FOR PEER REVIEW 4 o 16 I-C T-D W-E Figu e 1. Pho og aphs showing a i id p esen a ion o he comme cial be e ages (on he le o each pai ) and he be e ages con aining he C-PC ex ac as a na u al colou an (on he igh o each pai ). Th ee ep esen a i e samples a e shown: I-C ep esen ing iso onic be e ages, T-D ep esen ing onic be e ages, and W-E ep esen ing wine. 2.4. Cy o oxici y The cy o oxici y o he C-PC ex ac s ob ained om he Oscilla o ia sp. biomass was e alua ed h ough a cy o oxici y assay wi h human gingi al ib oblas s. Cells om he HGF-1 cell line (8 × 103 cells well−1) we e incuba ed a C-phycocyanin concen a ions om 3 o 1.95·10−2 mg mL−1 in se ial dilu ions (1:1). These we e placed in a 96-well mic opla e o 72 h a 37 °C wi h 5% CO2 in a humid a mosphe e. Cell p oli e a ion was es ima ed h ough he MTT (3-(4,5-dime hyl hiazol-2-yl)-2,5-diphenyl e azolium b omide) assay, as desc ibed elsewhe e [14]. The ela i e cell iabili y was exp essed as he pe cen age o iable cells compa ed o he con ol. Fou samples om each es ed concen a ion we e included in each expe imen . De e mina ions we e ca ied ou in iplica e in independen expe imen s. 2.5. Bios imulan Capaci y o he Was e Biomass Up o ou di e en bioassays we e conduc ed o e alua e he bios imulan p ope - ies o he Oscilla o ia sp. ex ac s. Fo his, he le o e biomass (a e ex ac ing he C- phycocyanin) was subjec ed o enzyma ic hyd olysis o 4 h in a s i ed- ank eac o a 40 °C and pH 7.5, using Alcalase and Fla ou zyme om No ozyme [15]. The gibbe ellin-like e ec was s udied by measu ing he ge mina ion index o Lepidium sa i um seeds [16]. The auxin-like e ec was de e mined by he ad en i ious oo induc ion o mung beans (Vigna adia a). Comme cial Vigna adia a seeds we e plan ed ollowing p e ious wo k [17]. The cy okinin-like e ec was de e mined h ough he excised cucumbe expansion es [17]. Mo eo e , he an isenescen ac i i y o he na u al cy okinins was de e mined h ough he T i icum (Whea ) chlo ophyll e en ion es [18]. Fo his, seeds om sui able whea cul i a s (T i icum aes i um L.) may be used. The whea seeds we e insed unde unning ap wa e o 4 h. Each seed was plan ed a a 1 cm dep h in mois ened pe li e in glass ays. The ays we e placed in a g ow h chambe and kep a 25 °C, a 60 o 65% ela i e hu- midi y, and illumina ed wi h luo escen lamps o 10 days. The seedling lea es we e col- lec ed and cu (3 cm below hei apical ip) in o 10 mm segmen s. The esh weigh o en cu ings was measu ed wi h an analy ical g ade balance and placed in a 50 mL alcon cen i uge ube ( ou ials pe ea men ) con aining 10 mL o dis illed wa e and 0.5 mg/L BAP (as he con ol), and bios imulan s a 0.5 and 2 g·L−1 (as he ea men ). The ials we e placed back in he da k g ow h chambe o 4 days. A e he incuba ion pe iod, he lea es we e blo ed d y and pu in o es ubes (g adua ed 14 mL alcons) con aining 8 mL o 80% e hanol. The es ubes we e ans e ed o a wa e ba h (wa med o 80 °C). A e 10 min o chlo ophyll ex ac ion, he solu ion was cooled down and supplemen ed wi h 80% e hanol up o 10 mL. To a oid e apo a ion, he es ubes we e co e ed. The cooled chlo- ophyll ex ac was pou ed (wi hou he segmen s) in o spec opho ome e cu e es. The op ical densi y was de e mined a 645 nm and compa ed o 10 mg o esh weigh ; he Figu e 1. Pho og aphs showing a i id p esen a ion o he comme cial be e ages (on he le o each pai ) and he be e ages con aining he C-PC ex ac as a na u al colou an (on he igh o each pai ). Th ee ep esen a i e samples a e shown: I-C ep esen ing iso onic be e ages, T-D ep esen ing onic be e ages, and W-E ep esen ing wine. 2.4. Cy o oxici y The cy o oxici y o he C-PC ex ac s ob ained om he Oscilla o ia sp. biomass was e alua ed h ough a cy o oxici y assay wi h human gingi al ib oblas s. Cells om he HGF-1 cell line (8 × 10 3 cells well −1 ) we e incuba ed a C-phycocyanin concen a ions om 3 o 1.95 · 10 −2 mg mL −1 in se ial dilu ions (1:1). These we e placed in a 96-well mic opla e o 72 h a 37 ◦ C wi h 5% CO 2 in a humid a mosphe e. Cell p oli e a ion was es ima ed h ough he MTT (3-(4,5-dime hyl hiazol-2-yl)-2,5-diphenyl e azolium b omide) assay, as desc ibed elsewhe e [ 14 ]. The ela i e cell iabili y was exp essed as he pe cen age o iable cells compa ed o he con ol. Fou samples om each es ed concen a ion we e included in each expe imen . De e mina ions we e ca ied ou in iplica e in independen expe imen s. 2.5. Bios imulan Capaci y o he Was e Biomass Up o ou di e en bioassays we e conduc ed o e alua e he bios imulan p op- e ies o he Oscilla o ia sp. ex ac s. Fo his, he le o e biomass (a e ex ac ing he C-phycocyanin) was subjec ed o enzyma ic hyd olysis o 4 h in a s i ed- ank eac o a 40 ◦C and pH 7.5, using Alcalase and Fla ou zyme om No ozyme [ 15 ]. The gibbe ellin- like e ec was s udied by measu ing he ge mina ion index o Lepidium sa i um seeds [ 16 ]. The auxin-like e ec was de e mined by he ad en i ious oo induc ion o mung beans (Vigna adia a). Comme cial Vigna adia a seeds we e plan ed ollowing p e ious wo k [ 17 ]. The cy okinin-like e ec was de e mined h ough he excised cucumbe expansion es [ 17 ]. Mo eo e , he an isenescen ac i i y o he na u al cy okinins was de e mined h ough he T i icum (Whea ) chlo ophyll e en ion es [ 18 ]. Fo his, seeds om sui able whea cul i a s (T i icum aes i um L.) may be used. The whea seeds we e insed unde unning ap wa e o 4 h. Each seed was plan ed a a 1 cm dep h in mois ened pe li e in glass ays. The ays we e placed in a g ow h chambe and kep a 25 ◦ C, a 60 o 65% ela i e humidi y, and illumina ed wi h luo escen lamps o 10 days. The seedling lea es we e collec ed and cu (3 cm below hei apical ip) in o 10 mm segmen s. The esh weigh o en cu ings was measu ed wi h an analy ical g ade balance and placed in a 50 mL alcon cen i uge ube ( ou ials pe ea men ) con aining 10 mL o dis illed wa e and 0.5 mg/L BAP (as he con ol), and bios imulan s a 0.5 and 2 g · L −1 (as he ea men ). The ials we e placed back in he da k g ow h chambe o 4 days. A e he incuba ion pe iod, he lea es we e blo ed d y and pu in o es ubes (g adua ed 14 mL alcons) con aining 8 mL o 80% e hanol. The es ubes we e ans e ed o a wa e ba h (wa med o 80 ◦ C). A e 10 min o chlo ophyll ex ac ion, he solu ion was cooled down and supplemen ed wi h 80% e hanol up o 10 mL. To a oid e apo a ion, he es ubes we e co e ed. The cooled chlo ophyll ex ac was pou ed (wi hou he segmen s) in o spec opho ome e cu e es. The op ical densi y was de e mined a 645 nm and compa ed o 10 mg o esh weigh ; he adjus ed Biology 2022,11, 1278 5 o 15 esul s we e compa ed o he con ol and analysed using a s anda d cu e o compa ison made using he loga i hmic concen a ion ange o he speci ic cy okinin (BAP). 3. Resul s and Discussion 3.1. Mic oalgal Biomass P oduc ion P oduc ion o he cyanobac e ia Oscilla o ia sp. in a aceway eac o (loca ed in Alme ia, Spain) was s udied. This s ain was selec ed because o i s g ea po en ial o p oducing bo h C-phycocyanin and bios imulan s. Mo eo e , he s ain ole a es high salini y allowing seawa e o be u ilised in he p oduc ion s ep, hus making he p ocess mo e sus ainable. The use o egula e ilise s as he nu ien sou ce also educes he biomass p oduc ion cos , al hough his could be u he educed i nu ien - ich e luen s such as was ewa e and cen a e ( he liquid ac ion o e luen s om anae obic diges ion o o ganic ma e ) we e used, again con ibu ing o a mo e sus ainable p ocess [19]. Expe imen s we e pe o med in di e en seasons, wi h he en i onmen al condi ions changing h oughou he yea . The sola adia ion anged om 11 o 30 MJ/m 2· day, whe eas he daily a e age empe a u e anged om 11 o 25 ◦ C be ween he win e and summe seasons (Figu e 2). Due o his a ia ion, he pe o mance o he Oscilla o ia cul u es also a ied despi e ope a ing he eac o s a he same dilu ion a e (0.2 day −1 ) and pH (9.0). The biomass concen a ion anged om 0.3 o 0.9 g/L and he biomass p oduc i i y anged om 9.0 o 30.0 g/m 2· day (Figu e 2). The da a we e as expec ed when applying a p e iously alida ed model ha conside ed bo h he biological and enginee ing aspec s o he pho obio eac o [ 20 ]. Fo his, he biological pa ame e alues al eady conside ed we e a maximal g ow h a e o 0.6 day−1 , an i adiance cons an o 120 µ E/m 2· s, and a biomass ex inc ion coe icien o 0.12 m 2 /g. The esul s show ha Oscilla o ia sp. pe o med adequa ely in sp ing and summe , bu no so in he au umn when he biomass concen a ion d opped below 0.5 g/L , making he isk o cul u e washou oo high. The sui able pe iod o p oducing his s ain was om Ma ch o Oc obe when he a e age achie able biomass p oduc i i y was 21 g/m2·day , equi alen o 52 /ha · yea . This biomass p oduc ion capaci y is lowe han he maximum alues al eady epo ed o loca ions wi h empla e clima e (up o 100 /ha·yea ), due bo h o he low pe o mance o his s ain in ou doo eac o s and he ac ha i is no possible o p oduce i all-yea - ound. I has been epo ed ha obus and as -g owing s ains capable o being p oduced h oughou he yea a e equi ed o maximize he pe o mance o mic oalgae- ela ed p ocesses [1]. 3.2. Phycocyanins Ex ac ion Once he Oscilla o ia sp. biomass was p oduced, di e en ex ac ion p ocesses we e assayed o de e mine he bes condi ions o C-PC eco e y om he mic oalgal biomass (Table 1). Fi s ly, a global biomass cha ac e iza ion was de eloped o de e mine he o al amoun o phycocyanins and chlo ophylls con ained in he biomass, wi h alues o 0.5 and 0.82% (w/w) being measu ed, espec i ely. Rega ding he ex ac ion p ocess, he in luence o di e en a iables such as he ionic s eng h (FI), pH, and he s i ing ime in he C-PC ex ac ion we e es ed. The C-PC concen a ion, pu i y, and ex ac ion a io o he phyco- cyanin ex ac s we e de e mined using spec oscopic measu emen s. In addi ion, di e en cell dis up ion s a egies based on di ec osmo ic shock, eeze– hawing, ul asound (US), and hei combina ion ha e been s udied. The esul s show ha he maximal C-PC ex ac ed alue o 0.5% w/wwas ob ained when combining all he cell dis up ion me hods and pe o ming he ex ac ion a pH 6.5 using an FI o 100 mm; in his case, a pu i y a io o 0.173 was measu ed (Table 1). In con as , he highes pu i y a io o 0.190 was achie ed when using eeze– hawing as he cell dis up ion me hod ollowed by ex ac ion a pH 6.5 and FI 100 mm. Taking in o accoun all he ex ac ion pa ame e s e alua ed, i seems he bes p ocedu e would be o use he eeze– hawing me hodology, which gene a es a high p o ein concen a ion in he ex ac as well as high pu i y and ex ac ion a ios. Howe e , in addi ion o good ex ac ion pa ame e s, he economics o he p ocess should also be aken in o accoun . In Biology 2022,11, 1278 6 o 15 his ega d, di ec ex ac ion using osmo ic shock could be used o ob ain e y simila pa ame e s o he maximal ones. The u iliza ion o di e en sol en s and me hods o acili a e he ex ac ion o pigmen s om mic oalgae biomass is a highly ele an opic. In his sense, sol en s such as me hanol, chlo o o m, dime hyl sulphoxide (DMSO) and ace one ha e been p oposed, al hough he use o wa e is p e e ed [ 21 ]. Conce ning me hods, he u iliza ion o mechanical me hods such as mic owa e and ul asound o acili a e he cell b eakage and ex ac ion o molecules has been also epo ed, al hough he mos ecommendable is he u iliza ion o high-p essu e homogeniza ion by i s lowe ene gy consump ion and inc ease in empe a u e ha can dena u e he a ge compounds [22]. Biology 2022, 11, x FOR PEER REVIEW 6 o 16 Figu e 2. Annual a ia ion in (A) en i onmen al condi ions (sola adia ion and cul u e empe a- u e) and (B) biomass p oduc ion (concen a ion and p oduc i i y) o Oscilla o ia sp. cul u es cul i- a ed in an 80 m2 aceway eac o ope a ed in semicon inuous mode a 0.2 day−1. Lines co espond o alues es ima ed using he p e iously de eloped model [20]. 3.2. Phycocyanins Ex ac ion Once he Oscilla o ia sp. biomass was p oduced, di e en ex ac ion p ocesses we e assayed o de e mine he bes condi ions o C-PC eco e y om he mic oalgal biomass (Table 1). Fi s ly, a global biomass cha ac e iza ion was de eloped o de e mine he o al amoun o phycocyanins and chlo ophylls con ained in he biomass, wi h alues o 0.5 and 0.82% (w/w) being measu ed, espec i ely. Rega ding he ex ac ion p ocess, he in- luence o di e en a iables such as he ionic s eng h (FI), pH, and he s i ing ime in he C-PC ex ac ion we e es ed. The C-PC concen a ion, pu i y, and ex ac ion a io o he phycocyanin ex ac s we e de e mined using spec oscopic measu emen s. In addi- ion, di e en cell dis up ion s a egies based on di ec osmo ic shock, eeze– hawing, ul asound (US), and hei combina ion ha e been s udied. 0 5 10 15 20 25 30 35 40 0 5 10 15 20 25 30 35 Dec Feb Ma May Jul Aug Oc Dec Jan Tempe a u e (ºC) Sola adia ion (MJ/m 2 ·day) Sola adia ion Tempe a u e A) 0 10 20 30 40 0.0 0.5 1.0 1.5 2.0 Dec Feb Ma May Jul Aug Oc Dec Jan Biomass p oduc i i y (g/m 2 ·day) Biomass concen a ion (g/L) Biomass concen a ion Biomass p oduc i i y B) Figu e 2. Annual a ia ion in ( A ) en i onmen al condi ions (sola adia ion and cul u e empe a u e) and ( B ) biomass p oduc ion (concen a ion and p oduc i i y) o Oscilla o ia sp. cul u es cul i a ed in an 80 m 2 aceway eac o ope a ed in semicon inuous mode a 0.2 day −1 . Lines co espond o alues es ima ed using he p e iously de eloped model [20]. Biology 2022,11, 1278 7 o 15 Table 1. Ex ac ion p ocesses assayed o C-phycocyanin eco e y (US s ands o ul asound ea men ). Me hod pH FI (mm) C-PC (mg/mL) Pu i y Ra io (Amax/A280)C-PC Ex ac ed (% w/w) Di ec (5 min s i ing) 6.5 5 0.23 0.146 0.33 F eeze– hawing 6.5 5 0.18 0.100 0.26 F eeze– hawing 6.5 50 0.34 0.150 0.37 F eeze– hawing 6.5 100 0.42 0.190 0.45 F eeze– hawing 6.5 175 0.43 0.180 0.46 F eeze– hawing 6.5 250 0.30 0.160 0.49 US + F eeze– hawing 7.0 100 0.34 0.182 0.49 US + F eeze– hawing 6.5 100 0.34 0.187 0.48 US + F eeze– hawing 5.5 100 0.33 0.180 0.47 US + F eeze– hawing 5.0 100 0.33 0.170 0.46 US + S i ing 45 min + F eeze– hawing 6.5 100 0.32 0.173 0.50 Biomass cha ac e iza ion: C-PC = 0.5% d.w . biomass; Chlo ophylls = 0.82% d.w . biomass (0.67%chlA; 0.15% chlC). The C-PC ex ac om Oscilla o ia sp. was mainly composed o C-phycocyanin ( C-PC ). A p elimina y analysis o he ex ac ’s phycobilip o ein con en e ealed ha he ex ac con ained 2.08 mg C-PC · mL −1 as he p incipal componen (91% w/ ). O he p o eins we e p esen in lesse ye signi ican p opo ions, such as APC (9% w/ ). The C-phycocyanin pu- i y g ade is de ined as he a io be ween i s peak abso bance a 616 nm and he abso bance o p o eins a 280 nm (A616/A280). When his a io is highe han 0.7, i is conside ed o be o ood-g ade pu i y [ 23 ]. In his wo k, he C-PC pu i y a io was 0.71, demons a ing ha i could be used in es s as a ood colou ing agen wi hou u he pu i ica ion. In his case, only a small p opo ion o APC was p esen , which did no signi ican ly a ec he inal blue colou o he C-PC ex ac (APC is also a blue p o ein, i s shade being ligh e han ha o C-PC). Thus, as he main ex ac cons i uen , C-phycocyanin con e s an a ac i e, deep-da k-blue colou . The pH is a ele an ac o in luencing phycobilip o ein s abili y. The be e ages e alua ed in his wo k had a pH o 3.0 (iso onic d ink and onic wa e ) and a pH o 3.2 (wine). To ob ain a s able medium o he C-PC, he pH o he be e ages was co ec ed o 5.0, a le el a which he ex ac ’s blue colou is s able. 3.3. U iliza ion o he C-PC Ex ac as a Colou an To de e mine he easibili y o using he al eady ob ained C-PC ex ac as a colou ing agen , a se o expe imen s we e pe o med using di e en so d inks. The colou exhibi ed by he blue C-PC ex ac ob ained in his wo k was compa ed wi h he colou o he e e ence comme cial p oduc s ha we e colou ed using syn he ic molecules. The mos common syn he ic blue ood colou s a e an h aquinone blue (E-130), blue pa en V (E-131), indigo ine (E-132), and b illian blue (E-133) while he mos equen na u al molecule is an hocyanin (E-163) [ 24 ]. The s anda d comme cial p oduc was pigmen ed using he blue C-PC ex ac o achie e a colou ha was as close as possible o ha o he e e ence p oduc s. Fo his, i was necessa y o add a speci ic olume o C-PC ex ac o he uncolou ed comme cial be e ages (Table 2). I should be no ed ha he e ec o he ex ac addi ion on ex u e and iscosi y was negligible gi en ha he added quan i ies needed o ep oduce he colou s o he co esponding ma ke p oduc s we e lowe han 106 mg · L −1 in all he es s. Simila esul s we e epo ed in p e ious wo ks whe e na u al pigmen s did no a ec he heological p ope ies o dai y p oduc s [ 25 ]. In his wo k, onic be e ages equi ed a smalle amoun o C-PC o ma ch he colou o he comme cial p oduc s. Samples D and E had s aining ac o s o 22 and 47.9 mg · L −1 , espec i ely. In con as , Sample B was he p oduc equi ing he mos p o ein, ha ing a s aining ac o o 105.8 mg · L −1 . These esul s show he C-PC ex ac ’s po en ial as a na u al colou an while he low s aining ac o alues con i m i s economic iabili y. Figu e 1shows he samples colou ed wi h he C-PC ex ac compa ed wi h he e e ence p oduc s ound on he ma ke . These images demons a e ha he colou o each sample was simila o he colou o he Biology 2022,11, 1278 8 o 15 comme cial e e ence p oduc . In addi ion, Figu e 3shows he colou ing cu es o he di e en be e ages. When he ∆ E*ab alue is in he 0.0–0.5 ange, he colou di e ence is impe cep ible o he human eye, whe eas alues in he 0.5–1.5 ange a e sligh ly di e en and highe alues a e easily de ec ed by an un ained consume [26]. Table 2. S aining ac o s o he assayed be e ages: da a a e ob ained om he colou ing cu es o he p oduc s ([C-PC] ex ac = 2.08 mg/mL−1). P oduc Type Comme cial B and Ex ac Volume Added (µL) ∆E*ab Reached S aining Fac o (mg·L−1) Iso onic D inks A 610 7.92 86.4 B 750 8.06 105.8 C 650 7.94 92.3 Tonic D ink D 150 1.41 22.0 Wine E 130 5.62 47.9 Biology 2022, 11, x FOR PEER REVIEW 8 o 16 3.3. U iliza ion o he C-PC Ex ac as a Colou an To de e mine he easibili y o using he al eady ob ained C-PC ex ac as a colou ing agen , a se o expe imen s we e pe o med using di e en so d inks. The colou exhib- i ed by he blue C-PC ex ac ob ained in his wo k was compa ed wi h he colou o he e e ence comme cial p oduc s ha we e colou ed using syn he ic molecules. The mos common syn he ic blue ood colou s a e an h aquinone blue (E-130), blue pa en V (E- 131), indigo ine (E-132), and b illian blue (E-133) while he mos equen na u al mole- cule is an hocyanin (E-163) [24]. The s anda d comme cial p oduc was pigmen ed using he blue C-PC ex ac o achie e a colou ha was as close as possible o ha o he e e - ence p oduc s. Fo his, i was necessa y o add a speci ic olume o C-PC ex ac o he uncolou ed comme cial be e ages (Table 2). I should be no ed ha he e ec o he ex ac addi ion on ex u e and iscosi y was negligible gi en ha he added quan i ies needed o ep oduce he colou s o he co esponding ma ke p oduc s we e lowe han 106 mg·L−1 in all he es s. Simila esul s we e epo ed in p e ious wo ks whe e na u al pig- men s did no a ec he heological p ope ies o dai y p oduc s [25]. In his wo k, onic be e ages equi ed a smalle amoun o C-PC o ma ch he colou o he comme cial p od- uc s. Samples D and E had s aining ac o s o 22 and 47.9 mg·L−1, espec i ely. In con as , Sample B was he p oduc equi ing he mos p o ein, ha ing a s aining ac o o 105.8 mg·L−1. These esul s show he C-PC ex ac ’s po en ial as a na u al colou an while he low s aining ac o alues con i m i s economic iabili y. Figu e 1 shows he samples col- ou ed wi h he C-PC ex ac compa ed wi h he e e ence p oduc s ound on he ma ke . These images demons a e ha he colou o each sample was simila o he colou o he comme cial e e ence p oduc . In addi ion, Figu e 3 shows he colou ing cu es o he di e en be e ages. When he ΔE*ab alue is in he 0.0–0.5 ange, he colou di e ence is impe cep ible o he human eye, whe eas alues in he 0.5–1.5 ange a e sligh ly di e en and highe alues a e easily de ec ed by an un ained consume [26]. Figu e 3. Colou ing cu es o be e ages ob ained om colou ing assays using he C-PC ex ac om Oscilla o ia sp. ([C-PC] ex ac = 2.08 mg mL−1). 0 5 10 15 20 25 30 35 0 100 200 300 400 500 600 700 800 900 1000 ∆E*ab Ex ac olume (μL) Iso onic Upg ade Iso onic Powe ade Iso onic Isod ink Tonic No dic Wine Don Luciano Figu e 3. Colou ing cu es o be e ages ob ained om colou ing assays using he C-PC ex ac om Oscilla o ia sp. ([C-PC] ex ac = 2.08 mg mL−1). Th ee di e en comme cial iso onic be e ages we e s udied o es he po en ial colou ing capaci y o he C-PC ex ac (Table 2). The inal colou s p esen no able di e ences, al hough hese a e p obably no enough o consume s o ind hem una ac i e. Fu he s udies will assess consume accep ance o he p oduc s. The p oduc s de eloped in his wo k had he addi ional ad an age o being p oduced using a na u al colou an . Recen wo k demons a ed ha , i consume s a e awa e o he heal h and en i onmen al bene i s o inco po a ing na u al mic oalgae-de i ed p oduc s in o oods, no only hei pu chase in en ion bu also he amoun o money hey a e willing o pay o he p oduc is g ea e [ 27 ]. Sample B egis e ed he highes s aining ac o (Table 2) and i s colou ing cu e sugges s ha a la ge olume o he B-PE ex ac (750 µ L) was needed o achie e he inal sample colou (Figu e 3). Howe e , in all he s udied samples, he s aining ac o s we e low, p o ing ha a ac i e colou s can be achie ed wi h a e y low quan i y o he C-PC. Biology 2022,11, 1278 9 o 15 Cy o oxici y es s we e pe o med o e alua e he sa e y o he p oduced ex ac s o be used as colou an s. The esul s show ha he e was C-PC ex ac cy o oxici y up o a concen a ion o 196.87 µ g mL −1 , wi h a su i al a e o 38%. F om his concen a ion, he highes su i al a e was 80% a a concen a ion o 98.43 µ g mL −1 (Figu e 4A). The IC 50 ob ained was 169.86 µ g mL −1 . Addi ionally, a cy o oxici y analysis was pe o med on Sigma’s pu e phycocyanin (99.9%) o see i he cy o oxici y was due o he C-PC i sel o o o he ex ac p o eins o componen s. The analysis showed ha high-pu i y C-PC is no oxic o he HFG-1 cell line (Figu e 4B). Based on hese igu es, i can be concluded ha some compounds al eady con ained in he ex ac om algae biomass can be sligh ly oxic. As he concen a ion ange a which cy o oxici y was obse ed was much highe han he dose needed o p o ide adequa e colou , i was concluded ha he colou ing ex ac s could be used as colou an s. Howe e , mo e esea ch is necessa y o iden i y and emo e he o he compounds accompanying he C-PC in he ex ac s, which may be esponsible o oxici y in he samples a high concen a ions. Biology 2022, 11, x FOR PEER REVIEW 10 o 16 Figu e 4. Su i al o he HGF-1 cell line as a unc ion o he C-phycocyanin concen a ion o he ob ained ex ac (A) and Sigma’s 99.9% pu i y o phycocyanin (B). 3.4. S abili y o Colou ed Be e ages The s abili y o ood p oduc s is a pi o al aspec o conside ; so, colou s abili y de- e mina ions we e de eloped o es he be e ages o which he C-PC ex ac was added. Figu e shows he colou changes o he p oduc s du ing an 11-day s o age pe iod. Re- ga ding he iso onic and onic samples, e y sligh a ia ions we e obse ed du ing he s o age pe iod. In his case, he onic sample showed he highes a* alues while he iso- onic sample egis e ed he lowes . Simila ly, he L* (Figu e 5B) and b* (Figu e 5D) alues o he iso onic and onic samples emained cons an and s able om day 0 o day 11. Despi e a ia ions and upwa d ends, he ΔE*ab did no exceed a alue o 0.75 o e he en i e s udy pe iod o he onic and iso onic samples. A di e en end was obse ed o he wine sample, which s a ed wi h a alue o ΔE*ab = 0.8 on day 1 and concluded wi h ΔE*ab = 2.6 on day 11. I is well-known ha ΔE*ab alues below 3.0 canno be easily de- ec ed by he naked human eye and a e in e p e ed as he same colou by consume s [28]. Acco ding o hese da a, i could be concluded ha he colou was s able in all he samples 0 20 40 60 80 100 120 140 0 500 1000 1500 2000 2500 3000 3500 Su i al (%) cell line HGF-1 Phycocyaninc oncen a ion (µg·mL -1 ) A) 0 20 40 60 80 100 120 140 0 500 1000 1500 2000 2500 3000 3500 Su i al (%) cell line HGF-1 Phycocyanin concen a ion (µg·mL -1 ) B) Figu e 4. Su i al o he HGF-1 cell line as a unc ion o he C-phycocyanin concen a ion o he ob ained ex ac (A) and Sigma’s 99.9% pu i y o phycocyanin (B).