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Review ArticleCarotenoids Functionality, Sources, and Processing by Supercritical Technology: A Review

Natália Mezzomo and Sandra R. S. Ferreira

EQA-CC/UFSC, Chemical and Food Engineering Department, Federal University o Santa Catarina,CP , - Florianópolis, SC, Brazil

Correspondence should be addressed to Natália Mezzomo; [email protected]

Received November ; Revised January ; Accepted February

Academic Editor: Artur M. S. Silva

Copyright © N. Mezzomo and S. R. S. Ferreira. Tis is an open access article distributed under the Creative CommonsAttribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work isproperly cited.

Carotenoid is a group o pigments naturally present in vegetal raw materials that have biological properties. Tese pigments havebeen used mainly in ood, pharmaceutical, and cosmetic industries. Currently, the industrial production is executed throughchemical synthesis, but natural alternatives o carotenoid production/attainment are in development. Te carotenoid extractionoccurs generallywith vegetal oil and organicsolvents, butsupercriticaltechnology is an alternative techniqueto the recovery o thesecompounds, presenting many advantages when compared to conventional process. Brazil has an ample diversity o vegetal sourcesinadequately investigated and, then, a major development o optimization and validation o carotenoid production/attainment

methods is necessary, so that the benets o these pigments can be delivered to the consumer.

1. Introduction

Carotenoids are a class o compounds that have coloringpower and have been widely used in ood industry, leadingits market to ull development. Carotenoids occur widely innature and, in general, all ruits and vegetables o color aregood sources o these compounds. Currently, the carotenoidsused industrially are synthesized chemically, but a small por-tion is obtained through extraction rom plantsor algae. Con-sidering the act that there is a high demand and consumer

preerence or natural compounds, there is a global trend inincreasing the use o products made with natural ingredients.

Natural alternatives o production/attainment are beingstudied. Te main subjects o these works are the investiga-tion o vegetal sources containing high carotenoids concen-trations and producer microorganisms and their optimumconditions or bioproduction. Many organisms producecarotenoids, but not all are industrially interesting. Yeast isdistinguished or its use as a protein source and growingability on low cost substrates with high sugar content, but theamount and type o carotenoids produced by their microor-ganisms can vary according to the operational conditions o growth.

In order to be able to separate these compounds, easibleextraction methods to obtain them rom natural sources ormicroorganisms are being intensively investigated in orderto determine the optimum conditions to have better peror-mance and stability. Te extraction o pigments rom planttissues and microbial biomass, especially made o at-solublepigments (such as carotenoids), usually uses vegetal oils andorganic solvents. In contrast, the negative impact o mostorganic solvents on the environment and human health isknown. Tus, supercritical technology emerges as an alterna-

tive technique or the carotenoids recovery rom microorgan-isms’ producers or directly rom vegetal sources, and its study is in widespread development.

In this context, the objective o this study was to investi-gate the occurrence, extraction, and production o pigmentsrom natural or bioproduced sources, doing a literaturereview o the technological and nutritional status o thecurrently known major carotenoids.

2. Carotenoids Definition and Structure

“Carotenoids” is a generic term used to designate themajority o pigments naturally ound in animal and plant kingdoms.

Hindawi Publishing CorporationJournal of Chemistry Volume 2016, Article ID 3164312, 16 pageshttp://dx.doi.org/10.1155/2016/3164312

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Mevalonic acid

Geranylgeranyl diphosphate

Initial steps

Phytoene

Lycopene

Phytoene formation

Xanthophylls

Desaturation

Cyclization

Hydroxylation and so forth

-carotene

F : Flowchartsummary o the carotenoidsbiosynthesis stages.Reerence: Britton et al. [].

Tis group o at-soluble pigments comprises more than compounds responsible or the red,orange, and yellow colors.Most carotenoids are hydrocarbons containing carbonatoms and two terminal rings [].

All photosynthetic organisms (including plants algae andcyanobacteria) and some nonphotosynthetic bacteria and

ungi synthesize the carotenoids. wo classes o carotenoidsare ound in nature: (a) the carotenes such as -carotene,which consist o linear hydrocarbons that can be cyclized atone end or both ends o the molecule, and (b) the oxygenatedderivatives o carotenes such as lutein, violaxanthin, neoxan-thin, and zeaxanthin, known as xanthophylls [].

Most carotenoids are tetraterpenoids (C) consistingo units isoprenoids linked so that the molecule is linearand symmetrical, with the order reversed in the center. Tebasic cyclic structure can be modied by hydrogenation,dehydrogenation, cyclization, and oxidation, as shown in Fig-ure . Te system o conjugated double bonds gives these pig-ments high chemical reactivity that can be easily isomerized

and oxidized []. Figure shows the structure o somecarotenoids.

3. Carotenoids Properties and Functions

Due to the coloring properties o carotenoids, they are ofenused in ood, pharmaceutical, cosmetics, and animal eedindustries. In addition to their extensive use as colorants, they are also used in ood ortication because o their possibleactivity as provitamin A and their biological unctions tohealth benet, such as strengthening the immune system,reducing the risk o degenerative diseases [], antioxidantproperties [, ] and antiobesity/hypolipidemic activities [].

From several hundred naturally occurring carotenoids,only have signicant biological activity and they can bedivided into two groups, with and without the provitamin A.Te carotenoids that are precursors o vitamin A should haveat least one ring o -ionone not replaced and side polienicchain with at least carbons [].

Vitamin A is important or growth, development, main-tenance o epithelial tissues, reproduction, immune system,and, in particular, visual cycle acting in the regenerationo photoreceptors []. Its deciency is a serious problem o public health, being the major cause o inant mortality indeveloping countries. A prolonged deciency can producechanges in the skin, night blindness, and corneal ulcers.Besides, it leads to blindness, growth disorders, and learningdifficulties in childhood []. On the other hand, vitamin A inexcess is toxic and can cause congenital malormation duringpregnancy, bone diseasein patients with chronic renal ailure,xerophthalmia, blindness, and death [, ].

Carotenoids are converted to vitamin A as the body needs, with varying degrees o conversion efficiency. Teorms o provitamin A carotene are ound in dark green andyellow-orange leay. Darker colors are associated with higherlevels o this provitamin [].

Carotenoids,alongwith vitamins, arethe substances mostinvestigated as chemopreventive agents, acting as antioxi-dants in biological systems []. Antioxidants can act directly in the neutralization o ree radicals, preventing or reducingdamage caused by these compounds in cells, or indirectly involved in enzyme systems that have antioxidant activity [].

From the nonenzymatic compounds, on antioxidantdeense, some minerals (copper, manganese, zinc, selenium,and iron), vitamins (ascorbic acid, vitamin E, and vitaminA), carotenoids (-carotene, lycopene, and lutein), bioavon-oids (genistein, quercetin), and tannins (catechins) can bedetached [].

Studies show the relationship between increased con-sumption o oods rich in carotenoids and the risk reductiono various diseases. According to Olson [], carotenoidsquench singlet oxygen, remove peroxy radicals, modulatecarcinogen metabolism, inhibit cell prolieration, stimulatecommunication between cells (gap junctions), and increasethe immune response. ests in vitro and in vivo suggest thatcarotenoids are excellent antioxidants, scavenging and inac-tivating ree radicals [].

Both carotenoids that are precursors o vitamin A and the

nonprecursors,such as lutein, zeaxanthin, and lycopene, haveprotective action against cancer []. Still, rom the importantbiological activities o these compounds, the inhibition o other diseases is detached where ree radicals play role asatherosclerosis, cataracts, macular degeneration, multiplesclerosis, degenerative diseases, and cardiovascular diseases[–].

Due to the high unsaturation rate, actors such as heat,light, and acids cause trans-isomerization o carotenoids,which is the most stable orm in nature, or the cis-orm,promoting a slight loss o color and provitamin activity.Carotenoids are also susceptible to enzymatic or nonenzy-matic oxidation, which depends on the carotenoid structure,

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OH (A)

(B)

(C)

(D)

OH

OH

OH

OH

O

O

OH

OH

(a)

(E)

(F)

(G)

(H)

(b)

F : Carotenoids structure: (a) xanthophylls: (A) zeaxanthin, (B) lutein, (C) beta-cryptoxanthin, and (D) astaxanthin; (b) Carotenes:(E) neurosporene, (F) lycopene, (G) -carotene, and (H) -carotene. Reerence: Britton et al. [].

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the oxygen availability, enzymes, metals, prooxidants andantioxidants, high temperature, and light exposure [].

Carotenoids are hydrophobic compounds, lipophilic,insoluble in water, and soluble in solvents such as acetone,alcohol, and chloroorm []. Because carotenoids are at-soluble pigments widely distributed in nature, they have

broad utility because o their coloring power [].In ood industries, the carotenoids are mainly used as

restoring colorants, used in products submitted to intenseprocessing/storage (which lost part o their natural color),or in order to standardize the ood products color, such asin ruit juices, pasta, beverages, candies, margarines, cheeses,and sausages. Tey are also precursors o many importantchemical compoundsresponsibleor theavor o some oods,such as alkaloids and volatile compounds, and ragrances o some owers and staining o photoprotection []. Indirectly,through their application in eed, these pigments serve tointensiy the color o egg yolk, chicken skin, sh, and milk []. Astaxanthin, the main carotenoid ound in crustaceans,

is an effective agent pigment when incorporated in salmonidsand crustaceans diets [–].

More recently, with the growing interest in maintaininghealth through natural products, carotenoids have also beenadded to oods due to their biological activities listed above,in order to enrich the ood product. Furthermore, in the cos-metic and pharmaceutical industries, they can be used only or the purpose o coloring medicine capsules, supplements,and cosmetics [].

In commercial ormulations, the carotenoids used may be o two types: natural extracts or synthetic colors identicalto natural ones. Te great demand generated by industriesand the growing demand or natural products have resulted

in an increase in research concerning the bioproduction andextraction o carotenoids. In addition to the connotation“natural,” the products are simply obtained by extractionrom natural sources or through microbial production ol-lowed by extraction, which can be achieved in a short periodat any time o year [].

Te pigments can absorb light in particular ultraviolet(UV) and visible spectrum, showing color. Te structureresponsible or light absorption is the chromophore group,which in carotenoids is characterized by conjugated doublebonds. Each carotenoid is characterized by an electronicabsorption spectrum. Tus, absorption spectroscopy is animportant technique in the analysis o carotenoids [].

.. -Carotene. Te -carotene is a thermolabile orangepigment, light, and oxygen sensitive, and it is associated withprotection against heart disease and cancer [, ], dueto its potential protection mechanisms already cited. Still,the oxidation o LDL-cholesterol is a crucial actor or theatherosclerosis development and -carotene acts by inhibit-ing the lipoprotein oxidation [].

A study conducted at the State University o New York showed that the consumption o vegetables rich in-carotenemore than once a week signicantly reduces the lung cancerrisk in relation to individuals who did not consume these

vegetables [].

.. Lycopene. Te pigment lycopene belongs to the subgroupo nonoxygenated carotenoids, being characterized by a sym-metric structure containing conjugated double bonds [].Due to its chemical structure, lycopene stands as one o thebest biological suppressors o ree radicals [–]. Among aseries o measured carotenoids,lycopene wasshownto be one

o the most effective antioxidants andmay donate electronstoneutralize the singlet oxygen molecules and other oxidizingmolecules beore they affect the cells [, ], with twice theantioxidant activity when compared to -carotene [, ].

Clinical and epidemiological studies have conrmed thatdiets rich in lycopene are associated with reduced risk o developing prostate lung and ovary cancers and a lowerincidence o chronic degenerative diseases and cardiovascu-lar diseases [, , ]. It is interesting to note that somemore recent studies indicated that lycopene intake present intomato ruit is more effective in preventing certain types o cancer than the administration o puried lycopene by capsules [].

.. Lutein and Zeaxanthin. Lutein and zeaxanthin are caro-tenoids stored in our body in the retina and lens eyes [].Some studies have shown that high lutein and zeaxanthinintake, particularly rom oods rich in xanthophylls suchas spinach, broccoli, and eggs, are related to the signicantreduction o cataract (over %) and macular degenerationrelated to age (over %) [].

.. Astaxanthin. Astaxanthin is a pigment ound in aquaticanimals, such as lobster, crab, and shrimp. A growing interestin the use o astaxanthin or poultry and sh-arming hasbeen developed [–, ], once this pigment is not synthe-sized by animals and must be added to the diets in order to

obtain a color attractive to consumers.Tis xanthophyll has antioxidant power times greater

than -carotene and times higher than vitamin E []. Inaddition, this pigment has been important in some diseasestreatment and prevention, with antitumor properties andprotection against ree radicals, lipid peroxidation, oxidativedamage to LDL-cholesterol, oxidation o essential polyunsat-urated atty acids, and the UV light effects on cell membranesand tissues [].

Te incorporation o astaxanthin in eed or aquacultureis effective or salmonidsand crustaceans pigmentation, so itspresence in shrimp, lobster, crab, and othershellsh exoskele-tons has been investigated [, –]. Wathne et al. []

and Pangantihon-Kühlmann et al. [] observed increase o color intensity in salmon and shrimp, respectively, using dietscontaining astaxanthin in the diet.

4. Carotenoids Occurrence andNatural Production

Carotenoids are natural pigments that occur widely in nature.Plants and some microorganisms produce these pigments;however, animals must obtain them through diet []. Tecarotenoids market is in ull progress and most o these pig-ments used industrially are synthesized chemically, or exam-ple, astaxanthin, canthaxanthin, and -carotene []. Tus,

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natural alternatives are being studied because o growingconcerns about ood saety and the negative aspects o thecoloring production, such as carotenoids, by chemical syn-thesis [].

.. Natural Sources. All color ruits and vegetables are good

carotenoids sources, but because they are at-soluble sub-stances, the absorption largely depends on their preparationwith oils or ats. Te orms o carotene provitamin A areound in dark green and yellow-orange leay. Darker colorsare associated with higher levels o provitamin [].

Among the carotenoids, the -carotene is the mostabundant in oods that has the highest activity provitamin A.It can be ound in large quantities in buriti ( Mauritia vinieraMart.) [], tucumã ( Acrocomia mokay ́ayba Barb. Rodr.),bocaiúva [], acerola, mango [], some varieties o pump-kin [], carrot [], nuts [], camu-camu ( Myrciaria dubia)[], carrot noodles [], rose hip ruits [], and oil palm[], which is an excellent source due to being ree rom any

barrier o vegetal matrix and thus has increased bioavailabil-ity o this pigment “beta-carotene” [].

Lycopene is a red pigment that occurs naturally only in vegetables and algae tissues. Currently, the most cited sourceso lycopene are the tomato and its derivatives, such as juices,soups, sauces, and ketchup [], including the processingwaste [, ] and peel []. However, it can be ound in evenhigher concentrations in cherry, guava, and guava products[], at similar concentrations in watermelon [] and Taipapaya, and in lower quantitiesin Solo and Formosa cultivarso papaya [].

Both lutein and zeaxanthin are present in green and dark greenleay vegetables, like broccoli,Brussels sprouts, spinach,

and parsley []. Some varieties o pumpkin [], acerola[], and leay vegetables in general [] are also good luteinand -carotene sources. Te ropaeolum majus L. edibleower is also a rich lutein source and it still appears in goodquantity in caja (Spondias lutea) [] and camu-camu ( Myr-ciaria dubia) []. Some microalgae also produce lutein, suchas Chlorella vulgaris [], Chlorella sorokiniana MB- [],and the indigenous microalga Scenedesmus obliquus CNW-N []. Zeaxanthin is ound in high concentrations in pequi(Caryocar villosum) [] andalso the native marine microalgaeChlorella saccharophila [].

Astaxanthin is a reddish-pink pigment ound in aquaticanimals, such as lobster, crab, and shrimp [], including

their processing waste [, ]. In addition, the microalgaePhaffia rhodozyma, Chlorella vulgaris, and Haematococcus

pluvialis synthetize large amount o astaxanthin [, ].

.. Bioproduction. According to Silva [], the biotechno-logical production o carotenoids has been increasing due toactors such as theusing possibility o lowcost substrates; des-ignation o natural substances; small area required to biopro-duction; being not subject to environmental conditions suchas climate, season, or soil composition; and control o cultureconditions.

Carotenoids may be biosynthesized by photosyntheticorganisms, as, or example, algae andcyanobacteria(blue and

green), and nonphotosynthetic microorganisms such as bac-teria, ungi, and yeasts [].

Te carotenoids production by biotechnological processhas been widely investigated, dethatching the commercialproduction o -carotene by the ungus Blakeslea trispora[], the marine microalgae Dunaliella [], the astaxanthin

production by the reshwater microalga Haematococcus sp.,and the yeast Phaffia rhodozyma [].Te Dunaliella microalgae are rich in -carotene and

other large application carotenoids. India has the largestmanuacturing industry o these microalgae, where the -carotene is intended or pharmaceutical use. Other majorproducers are located in Australia, United States, China,Mongolia, and Japan; small plants are also ound in Mexico,Chile, Cuba, Iran, and aiwan [].

Te industrial production o astaxanthin by Haematococ-cus also presents great interest due to its high commercial

value o this pigment and the high market growth o aqua-culture.Te world’s major producers are located in the UnitedStates, Japan, and India [].

Te production o natural pigments in industrial scaleand the high value o the products make the biotechnologicalprocess o carotenoids an areao intense study. Te bioprocessproductivity in a given system depends on the nutritional andphysical conditions o the microorganism, affecting not only cell growth as the pigment production. Tereore, microor-ganisms accumulate different carotenoids in response to thestress o environmental conditions [].

Improving the efficiency o carotenoids biosynthesis canincrease production. In addition to growing conditions, thecarotenoids biosynthesis is controlled by the biosyntheticenzymes level and activity, beyond the total carbon ow o the synthesizing system [].

According to Bhosale [], we can achieve a bettercarotenoids production with low cost using stimulants inthe culture medium and adjusting the external conditions o cultivation. Silva [] studied the effects o various chemicals(acid, -ionone, mevalonic acid, diphenylamine, and otheramino acids) in the carotenoids biosynthesis by Rhodotorulayeasts in order to increase and direct the carotenogenesis.Acetic acid had no effect on growth and production o pig-ment, but the -ionone inhibited the growth and caroteno-genesis. Te mevalonic acid stimulated the carotenoids or-mation in % or R. mucilaginosa and % or R. glutinis,without affecting the cells production o these microorgan-isms.

Te cultivation ability o yeast in media with high sugarcontent makes these microorganisms industrially interest-ing. Yeasts such as Xanthophyllomyces dendrorhous [],Rhodotorula glutinis [], Rhodotorula mucilaginosa [],Sporobolomyces [], and Phaffia [] are being studied inorder to maximize the carotenoids bioproduction, aiming atthe industrial use.

Microorganisms that are technologically interesting andwith high potential to be used in carotenoids bioproduc-tion are the cyanobacteria Anabaena variabilis, Aphani-zomenon os-aquae, and Nostoc commune in the productiono canthaxanthin []; algae Chlorella pyrenoidosa, Dicty-coccus cinnabarinus, Dunaliella salina, Dunaliella tertiolecta,

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Haematococcus pluvialis, and Spongiococcum excetricum inthe production o lutein, canthaxanthin [], -carotene[], -carotene [], astaxanthin [], and lutein [],respectively; ungi and Blakeslea trispora yeasts in the pro-duction o -carotene and lycopene []; Dacrymyces deli-quescens in the production o lutein []; Phaffia rhodozyma

in the production o astaxanthin and -carotene [];Rhodosporidium sp., Rhodotorula glutinis, and Rhodotorula graminis to produce torulene and -carotene []; Sporid-iobolus salmonicolor in production o -carotene []; andthe Mycobacterium brevicaie, Mycobacterium lacticola, Strep-tomyces chrestomyceticus, and Rhodococcus maris bacteria inthe production o canthaxanthin, astaxanthin, canthaxan-thin, and xanthophylls [], respectively.

5. Carotenoids Extraction and Recovery

Te industrial bioproduction o carotenoids is well estab-lished and has been expanding commercially. However, theoperations o product extraction and recovery and directextraction rom marine or vegetal sources are still underdevelopment in the literature. Moreover, the extraction pro-cesses contribute to increasing productioncosts, emphasizingthe need or detailed research in this area.

Te main techniques o extraction used in studies o carotenoids recovery are the conventional technique usingorganic solvents or vegetal oils and supercritical uid extrac-tion. Each o these techniques is based on different unda-mentals, according to the main mechanism responsible orthe extraction, as it will be explained below.

.. Raw Material Pretreatment beore Extraction. Kaiser et al.[] evaluateda method on a small scale combining hydroly-sis technique, in order to acilitate the carotenoids extractionbioproduced by Micrococcus luteus and Rhodotorula glutinis,with minimum degradation. Te methodology was evalu-ated by combining two enzymes (lysozyme and lipase orlyticase and lipase), ollowed by mechanical treatment withreezing and ultrasonic cycles ollowed by chemical treat-ment with dimethylsuloxide. For the extraction, they useda methanol/chloroorm mixture, stabilized with butylatedhydroxytoluene (BH) and -tocopherol. For the recovery and reproducibility evaluation o the extraction method,carotenoids internal standard was used. Te extractionmethod proved to be a sensitive tool in the carotenoidsdetermination derived rom microorganisms.

Perdigão et al. [] studied the pigments extraction romlobster, shrimp, and crab shells. Te raw materials were orwere not subjected to cooking by immersion in boiling water,drying in an oven with air circulation at ∘C during hours,and crushing to a particle size o mm. Ten, the sampleswere added to rened soybean oil on a : (w/v) ratio andheated at ∘C in a water bath during min. Te carotenoidenriched oil was separated rom the matrix by centriugationand the carotenoids levels were measured. Pretreated samplesusing cooking presented the highest pigment content, whichmeans that this pretreatment can cause a break on thecarotenoid-protein complex, acilitating extraction. Te redlobster (Panulirus argus) showed the highest concentration

o astaxanthin (. mg/ g) when compared to the greenlobster (Panulirus laevicauda) (. mg/g); the pink (Penaeus (Farantepenaeus) subtilis) and white (P. (Litope-naeus) schmitti) shrimp showed carotenoids content o .and . mg astaxanthin/ g o pigmented oil, respectively;and the aratu crab (Goniopsis cruentata) showed a con-

siderable astaxanthin content (. mg/g), ollowed by the uçá crab (Ucides cordatus cordatus) (.mg/ g) andguaiamum crab (Cardisoma guanhumi) (. mg/ g).

Mezzomo et al. [] evaluated the use o isolated orcombined pretreatments in order to break the carotenoid-protein complex rom pink shrimp (P. brasiliensis and P.

paulensis) processing residue, simpliying the carotenoidextraction. Te pretreatment processes evaluated were quick cooking during min, drying at∘C during h, and millingin domestic blender. Te results indicated that the best raw material pretreatment, in terms o extraction yield and totalcarotenoid content, is the combination o the three proce-dures tested.

Gu et al. [] compared three previous treatments to theextraction (ultrasound, grinding, and addition o HCl), andin order to optimize the extraction process, they evaluatedthe temperature ( to ∘C), solute/solvent ratio ( to v/m), and time ( to min) effects on extraction yieldo carotenoids rom R. sphaeroides. Te results proved thatthe pretreatment using HCl wasthe most effective method orthe carotenoids extraction. In the optimization stage, the bestresults o carotenoids extraction rom R. sphaeroides wereound when they used ∘C o temperature and optimumsolute/solvent ratio o , and maximum carotenoids extrac-tion was obtained at the time o min.

Guillou et al. [] affirmed that, according to the lit-erature, total extraction o the carotenoids rom crab shellcan be only achieved using acetone or methanol, afer shelldecalcication with acetic acid. Omara-Alwala et al. []reported that theuse o propionic acid increasedby %in theastaxanthin recovery rom lobsters waste. Guillou et al. []obtained a % higher extraction o astaxanthin in shrimp(Pandalus borealis) waste subjected to silage when comparedwith the “in natura” (crude) extraction residue.

Enzymatic hydrolysis has been considered a viablemethod as pretreatment or the astaxanthin recovery [,]. Holanda [] perormed organic solvent extraction o carotenoids rom shrimp waste and obtained an extractiontwo times more efficient than using oil, in both untreatedwaste andsoluble andinsoluble ractions obtained afer enzy-

matic hydrolysis. In the solvent extraction, the recovery was to % higher afer enzymatic hydrolysis. Te best recovery was . mg astaxanthin/ g dry residue, when hydrolysisis done with alcalase. Most o astaxanthin (approximately %) was rom the insoluble raction and the authors oundthat the orange color o the pigment still stood in the matrix,indicating that both the oil and the solvents used were notable to extract the total astaxanthin present in the insolubleraction.

Chen and Meyers [] observed a slight increase (rom to %) in the astaxanthin extraction with an increase o thehydrolysis time or shrimp (Solenocera melantho) waste withseveral commercial proteases. Still, extraction differences

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were observed when distinct proteolytic enzymes were used.According to the authors, the increase in extraction, observedwith increasing time o hydrolysis and the enzyme used, isprobably due to a break in carotenoid-protein link that wouldallow the recovery o oil or solvent.

Due to the strong association o carotenoids with the

cells o the producers’ microorganisms and in order tomaximize the extraction o the pigments, Valduga et al. []tested different methods o cell disruption and solventextraction. It was ound that when using a liquid nitrogenand dimethylsuloxide combination or cell disruption andextraction with acetone and methanol ( : , v/v) mixture,they obtained the highest carotenoids recovery rom the S.salmonicolor yeast grown on agar YM (Yeast Malt ExtractAgar).

Rawson et al. [] studied the effect o ultrasound andblanching pretreatments on carotenoid compounds o hotair- and reeze-dried carrot discs. Ultrasound pretreatmentollowed by hot-air-drying at the highest amplitude and

treatment time investigated resulted in higher retention o carotenoids in dried carrot discs than blanching ollowed by hot-air-drying. In addition, the reeze-dried samples had ahigher retention o carotenoid compounds compared to hotair dried samples. Tis study showed that ultrasound pre-treatment is a potential alternative to conventional blanchingtreatment in the carrots drying.

Te effect o enzymatic pretreatment, using pectinaseon the carotenoids content rom Gac ruit ( Momordicacochinchinensis Spreng) aril, was evaluated by Kha et al. [].Te results indicated the highest oil extraction and enhance-ment o the carotenoids content, specially the -caroteneconcentration, when using the enzyme concentration at

.% (w/w) or pretreatment beore air-drying.Strati et al. [] also studied the use o pectinase and

cellulase enzymes to assist the high-pressure extraction o carotenoids rom tomato waste. As a result, authors provedthat the total carotenoid and lycopene extraction yields wereincreased by the use o the enzymes beore the extraction,when compared to the nonenzyme process.

.. Conventional echnique o Extraction. Te extractionand recovery o carotenoids, either rom bioproduction ordirectly rom plant and animal sources, can conduce using

vegetable oils or organic solvents.

Te extraction with organic solvents is based on using

high operating temperatures and demonstrates solvent-components interactions, which is a unction o chemicalaffinity between the species in the system []. Carotenoidsare compounds o low polarity and thereore soluble in sol-

vents o low polarity such as hexane. However, these organicmolecules have also a polar part, soluble in polar sol-

vents, increasing the range o organic solvents useul in thecarotenoids extraction.

When an organic solvent is used in its boiling tempera-ture, the solvent surace tension and viscosity are very smallwhen compared to a lower temperature. Tus, the solvent canreach easily the solute on certain matrix spaces, solubilizinga greater amount and variety o solutes [].

Te solvent recovery is a crucial period in the extractionprocess with organic solvents, mainly due to environmen-tal and economic problems. Tus, extraction with organicsolvents has other major disadvantages, that is, the possiblethermaldegradation o the extract andthe incomplete solventremoval; it is a lazy process and has low selectivity [].

Nevertheless, the extraction with organic solvents iseffective in the extraction o carotenoids [, ]. However,as previously reported, the hightemperatures required ortheremoval o the solvents can result in degradation o pigment,and the nal product may contain trace amounts o solventand, consequently, reduce their potential or use in oodproducts [].

Vegetable oils have been used as a solvent orextraction o carotenoids. Te advantage o using vegetable oils is that they are considered a good barrier against oxygen, slowing oxi-dation processes, in addition to being used as energy sourcein the subsequent application in oods [, , , ]. Tesolvent removing step is not used when using vegetable oil,and the resultant product consists in a mixture o oil/extractrich in carotenoids, and the process may not present thedrawbacks o extracts thermal degradation.

Gildberg and Stenberg [] used extraction with organicsolvent (HCl and water) o shrimp (Pandalus borealis) wasteand ound concentration o . mg astaxanthin/ g o residue, and this value was times higher than the valuesound in literature or oil extraction. Chen and Meyers []ound . mg astaxanthin/ g o craysh waste, usingsolvent, against . mg astaxanthin/ g with oil extraction.Meyers and Bligh [] achieved concentration o . mgastaxanthin/ g o residue in craysh waste with extractionusing organic solvent. Tese differences are due to distinctavailable quantities o carotenoids in eed, environmentalconditions and species [], and the method used or extrac-tion and quantication o astaxanthin. Variables such as theparticle size o the waste, temperature, and residue/solventratio can also lead to differences in extraction.

Shahidi and Synowiecki [] and Saito and Regier []used shrimp (Pandalus borealis) waste and crab(Chionoecetesopilio) shell or astaxanthin and chitin extraction. In theextraction o carotenoids, they used cod liver oil ( amountso oil : part o residue, v/m) at ∘C during min. Ten,the mixture was ltered and dried under vacuum at ∘C,separating the water rom the carotenoids extract. Te resultsindicated % o astaxanthin extraction rom shrimp waste(. mg/ g dry residue) and % o the crab shell

(. mg/g dry weight).Sachindraand Mahendrakar [] determined the extrac-

tion yield o carotenoids rom shrimp waste, extracted usingdifferent vegetable oils (sunower oil, groundnut oil, gingerly oil, mustard oil, soy oil, coconut oil, and rice bran oil). Temethod applied consists in the homogenization and mixingo shrimp waste with the selected oil, ollowed by heating,ltration, centriugation, and separation o pigmented oil.Te results o the carotenoid yield (spectrophotometrically determined) indicated superiority o the sunower oil andwere signicantly inuenced by level o oil to waste, time, andtemperature o heating beore centriugation to separate pig-mentedoil. Te best conditions orextraction o shrimpwaste

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carotenoids in sunower oil were determined to be oil level towaste o , temperature o ∘C, and heating time o min.

aungbodhitham et al. [] evaluated a carotenoidsextracting method in order to be used by a wide variety o

vegetal sources. As representative sample was used tomato juice and to the extraction method were selected low envi-

ronmental impact solvents (ethanol and hexane). Tey usedmixtures o these solvents, with a : (v/v) ratio, respectively,o ethanol and hexane, resulting in good conversions o carotenoids in juice (% o lycopene, %-carotene, and% o -carotene). In order to evaluate the method appli-cability to other kinds o ruits and vegetables, the authorsused carrots, spinach, and tomato juice added to carotenoids.Te added carotenoids recovery was, respectively, or tomato

juice, carrot, and spinach as ollows: %, %, and .%or -carotene and .%, .%, and .% or the -carotene. Tese results indicated that the extraction methodor different vegetal matrixes was established.

.. Supercritical Fluid Extraction (SFE). Te supercriticaltechnology using carbon dioxide (CO

2) near the critical

point as a solvent in the extraction o compounds such ascarotenoids has been considered as an alternative to employ-ment in ood and pharmaceutical industries []. Te useo supercritical uid extraction (SFE) in industrial processeshas been increasing, mainly due to the environmental andquality actors involved: it is a process ree o toxic waste, doesnot require extracts postprocessing to solvent removal anddoes not cause thermal extracts degradation, allows the low temperatures use, and prevents oxidation reactions, due tothe light and oxygen absence on extraction column. Further-more, it is a exible process due to the continuous adjustmentpossibility o the solvent solvation power and selectivity [–].

Te supercritical CO2 is an essentialnonpolarsolvent and

its solvation power varies with its density. It permits extract-ing a large variety o lipophilic compounds depending on thepressure applied [],suchas carotenoids. Te solute solubil-ity in the supercritical uid increases with the operating pres-sure at a constant temperature by increasing the density o thesolvent. When increasing the pressure, the solute solubility also increases, but solvent selectivity is reduced [].

Te supercritical extraction can be an alternative to conventional methods or carotenoids extraction [] and canbe used with the pure supercritical solvent, usually CO

2, or

with addition o a cosolvent.

... SFE with Pure CO. De França et al. [] perormed theextraction o -carotene rom buriti ( Mauritia exuosa) pulpwith supercritical CO

2 to obtain high-concentrated oil rac-

tions in this carotenoid. Te results indicated that the super-critical CO

2 at MPa o pressure and temperature o ∘C is

capable o removing approximately % o the beta-carotenecontent in relation to the total amount extracted with hexane.

Mendes et al. [] perormed the SFE o lipids andcarotenoids o microalgae Chlorella vulgaris in temperaturesoand∘C and pressures rom to MPa. Both the lipidcontents such as carotenoids increased with elevating pres-sure. A ew years later, the authors [] expanded the research

or microalgae (Botryococcus braunii, Chlorella vulgaris,Dunaliella salina,and Arthrospira maxima) with the objectiveo maximizing the extraction o carotenoids and other lipidsby SFE with CO

2. SFE experiments were conducted at

temperatures o and ∘C and pressures rom to MPa.As a result, the authors reported that at ∘C and MPa

the microalgae Botryococcus braunii produced a supercrit-ical gold and clear extract when compared to the extractobtained by conventional extraction. For the microalgaeChlorella vulgaris, the SFE produced extracts composed by canthaxanthin and astaxanthin, and the carotenoids yield washigher when increasing pressure. As or the algae Dunaliellasalina, which produces -carotene in high yields, the resultsindicate that the cis-isomer o this carotenoid is much moresoluble in supercritical CO

2 at MPa and ∘C. Finally, or

the cyanobacteria Arthrospira maxima, the main lipid wasidentied as -linolenic acid.

Maćıas-Sánchez et al. [] conducted the SFE o caroten-oids and chlorophyll rom marine microalgae Nannochlorop-sis gaditana. For this, theyevaluated the inuence o tempera-ture( to∘C) and pressure (rom to MPa), obtainingempirical relationships between carotenoids and chlorophyllyields, according to the operational parameters. Te resultsshowed that it is necessary to work in pressure o MPa at∘C to obtain a signicant yield o the pigments evaluated.

Maćıas-Sánchez et al. [] carried out the extraction withsupercritical CO

2 rom dry biomass o the cyanobacterium

Synechococcus sp., in order to concentrate carotenoids andchlorophyll. Te experiments conduced rom to MPao pressure and rom to ∘C o temperature. Te highestconcentration o carotenoids was obtained at MPa and∘C, while the highest carotenoids/chlorophyll ratio wasobtained at MPa and ∘C, due to the highest selectivity achieved by acilitating the separation and purication o twopigments groups. According to the authors, the supercriticalCO

2 is an adequate solvent or carotenoids extraction due to

the low polarity o these components and, then, the processbecomes more selective in the presence o more polarpigments such as chlorophyll.

Machmudah et al. [] perormed the SFE o the rose ruit(Rosa canina) at pressures rom to MPa, temperaturesrom to ∘C, and CO

2 ow rate rom to mL/min,

in order to optimize the carotenoids extraction. Te totalcarotenoids concentration rom the rose ruit was rom .to . mg/g o ruit, and the maximum concentrationwas obtained at ∘C, MPa, and mL/min. Te main

carotenoids present in the extracts were lycopene (.–. mg/g), the -carotene (.–. mg/g), and lutein(.–. mg/g). Optimization results showed signicanteffects: the temperature in thetotal carotenoids yield;the tem-perature, pressure, and CO

2 ow rate in the lycopene yield;

and pressure and CO2

ow rate in the -carotene and luteinyields.

Filho et al. [] perormed the carotenoids extractionusing supercritical CO

2 rom pitanga (Eugenia uniora L.).

Te conditions evaluated were two levels o temperature (and ∘C) and seven levels o pressure (, , , , ,, and MPa). Te major carotenoids identied in thesupercritical extracts were lycopene, the rubixanthin, and

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-cryptoxanthin, and the maximum carotenoids recovery (%) was obtained at ∘C and MPa, ocusing mainly onlycopene (%) and rubixanthin (%). Te different oper-ating conditions established different extracts compositionaccording to the SFE yield and carotenoids concentration,indicating that supercritical CO

2 is selective depending on

the temperature and pressure.de la Fuente et al. [] studied the lycopene and

astaxanthin solubility in supercritical CO2

as a unction o temperature ( to ∘C) and pressure ( to MPa). Teresults indicated that, in general, both the lycopene andastaxanthin solubilities in supercritical CO

2 were higher

when pressure and temperature were elevated, varying rom

. to . × −6 or lycopene and rom , to . × −6 orastaxanthin.

Mezzomo et al. [] studied thetechnical andthe econom-ical viability to concentrate the carotenoid components by means o SFE rom pink shrimp (P. paulensis and P. paulensis)processing waste. Te variation o raw material moisture

content (. and .%), the solvent ow rate (. and. g/min), and the conditions o temperature ( and ∘C)and pressure (– MPa) were evaluated. Te results, repre-sented by total carotenoid content, carotenoid prole, astax-anthin yield, and UV-Vis scanning spectrometry o extractsindicated that the SFE was avorable at. g/min o CO

2 ow

rate and at .% o raw material moisture content. Te high-est astaxanthin yield was obtained by pure CO

2 at bar/

. K, and the cost analysis suggested a more lucrativeprocess.

Kha et al. [] evaluated the particle size o the raw mate-rial and SFE time, afer an enzymatic pretreatment, on the oilyield and carotenoids content in the resultant oil using super-

critical CO2 extraction rom Gac ruit ( Momordica cochinchi-nensis Spreng) aril. Te authors conclude that the carotenoidscontent enhanced by suitable particle size o . mm andextraction time o min, as well as the Gac oil, containshigh amount o carotenoids, specially the beta-caroteneand lycopene.

Espinosa-Pardo et al. [] evaluated extracts obtainedrom peach palm ruit (Bactris gasipaes) using SFE, interms o yield, total phenolic content, total avonoids, totalcarotenoids, and antioxidant activity by -carotene bleachingmethod. Te authors concluded that supercritical CO

2 allows

or obtaining rich extracts in carotenoids and although itpresents lower yield than conventional extraction, the SFE

represents a technique with greater advantages. Te bestoperation condition or SFE was bar/∘C, given that thehighest concentration o carotenoids was obtained.

Goto et al. [] developed a wet extraction process usingliqueed (subcritical) dimethyl ether (DME) as solvent ataround MPa. Tey applied liqueed DME or the extrac-tion o lipids andunctional compounds, such as carotenoids,rom various kinds o algae. Te results indicated that sincethe water content o biomaterials was very high, dryingprocess was necessary. Moreover, the subcritical (liqueed)DME is unique and suitable or extraction o water and oily substances rom biomaterials with high water content. Inaddition, it can eliminate the process or cell disruption and

solvent evaporation and, then, has potential to cut energy consumption o extraction procedures.

Algae contain lipids and unctional compounds such ascarotenoids. Particularly, microalgae are recently ocused asa source o biouel. o extract these components, organicsolvent or supercritical carbon dioxide has been used.

... SFE with CO + Cosolvent. One o the main character-istics o supercritical CO

2 is that it presents a limitedsolvation

power when used to polar solutes. Te addition o an organicsolvent as a modier can enhance the CO

2 efficiency by

increasing the compound’s solubility, reducing its interac-tions with the matrix, or changing it to simpliy the extraction[]. Hawthorne and Miller [] stated that an effectivecosolvent must be more polar than the supercritical solventand a good solvent in the liquid state or the solute ocused.

Lim et al. [] studied the separation o astaxanthin romPhaffia rhodozyma by SFE with CO

2 supercial ow rate

varying rom . to . cm/min, pressure rom . to MPa, temperature rom to ∘C, and use o ethanolas cosolvent at concentrations rom to %. Te bestcarotenoids recovery (%) and astaxanthin (%)using pureCO

2 at . cm/min were obtained at ∘C and MPa.

Te % ethanol showed an increase o astaxanthin yield o and % at and ∘C, respectively, when operated at MPa. When using two subsequent steps with pressuresranging rom to MPa, the astaxanthin concentration onhigh pressure increased by times at ∘C and times at∘C, and the astaxanthin concentrations were . to timesgreater than those obtained in conventional extraction usingacetone.

Katherine et al. [] conducted a study o supercriticalCO

2 as solvent and ethanol as cosolvent aiming to establish

conditions to obtain maximum extraction o lycopene romrozen watermelon by varying the operating temperature(–∘C), pressure (.–. MPa), and the ethanol con-centration ( to %). Te results had the highest lycopeneconcentration obtained at lower temperature (∘C) andpressure (. MPa) and using the highest concentration o cosolvent (%). However, this extraction yield was . timeslower than that obtained with resh ruit, indicating that thestorage o the watermelon in the studied conditions causes asignicant loss o lycopene. Te temperature was the param-eter that showed the greatest effect on lycopene yield and,considering the storage losses, the authors ollowed the study evaluating the lycopene yield on the SFE temperature (rom

to ∘C), pressure o . MPa, and the ethanol additiono %, obtaining a lycopene yield at ∘C o % higher than∘C.

López et al. [] made a comparison between the conventional extraction with acetone and supercritical uidextraction, with pure CO

2 or adding ethanol (–%), o

astaxanthin rom crustaceans, at conditions o to MPaandto∘C in a dynamic extraction during to min. Ina subsequent step, the column was depressurized and ushedwith . mL o acetone. Te highest astaxanthin yield wasobtained at MPa and ∘C, and the SFE time o minwas considered ideal to remove this pigment. Te range o ethanol content studied was chosen according to the polarity

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o the component and determined by the SFE data rom theliterature. According to the authors, the ethanol addition wasnecessary to ensure a signicantastaxanthinextraction,beingthat the optimum concentration was %. Still, according tothe authors, the SFE is simpler and aster than the conven-tional techniques o extraction, as well as being more efficient

and accurate, and does not need to use large amounts o toxicsolvents. In addition, the SFE extract can be obtained at low temperatures and, then, it is not exposed to thermal degrada-tion.

Liau et al. [] investigated cosolvent modied supercrit-ical carbon dioxide extraction o lipids and carotenoids romthe microalgae species o Nannochloropsis oculata. Continu-ous modication by ethanol o supercritical carbon dioxideextractions showed that the addition ratio was important orextraction efficiency o lipids and carotenoids. Te extractionconducted at MPa, ∘C, and .% o ethanol additionyielded . mg/g o carotenoids.

As observed, ethanol, a polar solvent, has been used toincrease the polarity o supercritical CO

2 on the extraction o

a wide variety o compounds. However, when the compoundsto be extracted are nonpolar, as most carotenoids, thiscosolvent is not the best option or increasing the solubility o CO

2. In addition, the ethanol must be removed rom the nal

product, requiring the use o heat and, thus, having the samedisadvantages mentioned in the conventional extraction:postprocessing need and possible thermal degradation o theextract [].

Otherwise, Bamberger et al. [] reported that the solu-bility o less volatile lipids in supercritical CO

2 is signicantly

increased by the presence o more triglycerides species on thesystem. According to Mendes et al.[], to increase theextrac-tion yield and protect the carotenoids rom degradation, theaddition o vegetable oil as cosolvent is an interesting alterna-tive. Besidesall the above advantages,Krichnavaruk et al. []cited that using vegetableoil as a modiermakes a subsequentseparation o the oil product unnecessary. According tothese considerations, the vegetable oil seems to be a goodcosolvent in the SFE o carotenoids.

Vasapollo et al. [] perormed the lycopene extractionrom tomato with supercritical CO

2 added or notby vegetable

oil (almond, peanut, hazelnut, and sunower seed). Teexperiments were conducted evaluating the temperature(rom to ∘C), pressure (rom . to MPa), and CO

2

ow rate (rom to kg/h). Te hazelnut oil presentedhigher yields when compared to other oils and pure CO

2;

beyond that, it is cheaper and has less acidity, preventingthe lycopene degradation during extraction. Te highestlycopene concentration (%) o tomato was obtained atMPa and ∘C with the addition o hazelnut oil at aconcentration o % and CO

2 ow rate o kg/h.

Sun and emelli [] evaluated the SFE efficiency oncarotenoids extraction rom carrot using canola oil as cosol-

vent, compared with conventional extraction using hexaneand acetone. SFE experiments were conducted evaluatingthe effect o raw material moisture (.–.%), particlesize (.– mm), temperature (–∘C), pressure (–bar), concentration o canola oil (–%), and CO

2 ow rate

(.–L/min). When canola oil was added, the extraction

yields o - and -carotene increased more than twice thoseo lutein, and more than our times compared to the yieldobtained with pure CO

2. Te increase o both temperature

and pressure had a positive effect on carotenes yield, whilelarger particle sizes had a negative impact on carotenes yield.-Carotene and -carotene yields decreased with increasing

raw material moisture, while the lutein yield increased. Tehighest carotenoids yield was obtained in superior CO2 ow rate, but a greater variety o carotenoids was solubilized whenthe supercritical CO

2 was used in the lower ow rate. Tus,

the highest carotenoids yield was obtained at ∘C, bar,% canola oil, particle diameter o .–. mm, .% raw material moisture, and L/min CO

2 ow rate.

Te effects o different modiers on the compositions,yield, and antioxidant activity o carotenoid by SFE o pump-kin (Cucurbita maxima) were studied by Shi et al. []. Tedifferent concentrations o single, mixed binary or ternary modiers were designed as ollows: () single modiers:ethanol (%, % or %), water (%, % or %), or oil (%,

% or %) and () mixed binary modiers: ethanol (% or%) + water (% or %), water (% or %) + oil (% or%), or ethanol (% or %) + oil (% or %). Te combina-tion o ethanol and water, or ethanol and oil, or water and oilin SFE showed that the yields o carotenoids were higher thanthe single modier with equal amount. Finally, authors con-cluded that the modier and operating temperature o SFEnotably inuenced the selectivity o carotenoids, especially the ratio o cis-b-carotene/total carotenoids.

Krichnavaruk et al. [] investigated the use o ethanol,soybean oil, or olive oil as cosolvents to the supercritical CO

2

extraction o astaxanthin rom Haematococcus pluvialis, incomparison with SFE using pure CO

2. Without the addition

o cosolvents, only % o astaxanthin was extracted underthe conditions o ∘C and MPa, whereas with the useo soybean oil and olive oil as cosolvent at a concentrationo % achieved an increase o % and %, respectively,o the efficiency o astaxanthin extraction. As the authorsconclude, the results o their study clearly demonstrated thesuperiority power o extraction when using cosolvents, suchas soybean oil and olive oil, in the high-pressure extraction o astaxanthin rom H. pluvialis.

able summarized the inormation o mentioned worksthat applied the SFE or carotenoid extraction.

6. Final Comments

Brazil has an underused agricultural potential with a variety o ruits and vegetables species low or inadequately investi-gated. Tis could potentially demonstrate an increase in therange o possible sources o carotenoids with broad utility inood, cosmetics, and medicines.

Different microorganisms and bioproduction techniquesare being developed, as well as extraction methods o thesecompounds, aimingto improve the process by the determina-tion o ideal operating conditions to reach an optimum yieldo carotenoids depending on the matrix.

More optimization studies and also validation methodsare required to pursue the perormance o technical studies to

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: Inormation about recent studies ocusing on the supercritical uid extraction o carotenoids rom natural materials.

Raw material Solvent applied Pressure and temperature applied Carotenoid o interest Reerence

Buriti ( M. exuosa) pulp Carbon dioxide and MPa, and ∘C -Carotene []

C. vulgaris Carbon dioxide and MPa, and ∘C

Canthaxanthin andastaxanthin

[]

C. vulgaris, D. salina, and A. maxima Carbon dioxide – MPa, and ∘C otal carotenoid content,-carotene []

Nannochloropsis gaditana Carbon dioxide – MPa, –∘C otal carotenoid content []

Synechococcus sp. Carbon dioxide – MPa, –∘C otal carotenoid content []

Rose ruit (R. canina) Carbon dioxide – MPa, –∘C

otal carotenoid content,lycopene, -carotene, and

lutein

[]

Pitanga ruit (E. uniora L.) Carbon dioxide – MPa, and ∘C

otal carotenoid content,lycopene, rubixanthin and

-cryptoxanthin

[]

Gac ruit( Momordica cochinchinensisSpreng) aril

Carbon dioxide MPa, ∘C -carotene, lycopene []

Peach palm pulp(Bactrisgasipaes)

Carbon dioxide – MPa, –∘C otal carotenoid content []

Japanese algae(Chlorella vulgaris,Undaria pinnatida)

Dimethyl ether – MPa, –∘C Fucoxanthin []

Phaffia rhodozyma Carbon dioxide + ethanol .– MPa, –∘C

otal carotenoid content,astaxanthin

[]

Frozen watermelon Carbon dioxide + ethanol .–. MPa, –∘C Lycopene []

Crustaceans Carbon dioxide + ethanol –MPa, –∘C Astaxanthin []

Nannochloropsis oculata Carbon dioxide + ethanol MPa, ∘C otal carotenoid content []

omato Carbon dioxide + several

vegetal oils .– MPa, –∘C Lycopene []

Carrot Carbon dioxide + canola oil .–. MPa, –∘

C otal carotenoid content, -

and -carotene, and lutein []

Haematococcus pluvialis

Carbon dioxide, carbondioxide + ethanol, and

carbon dioxide + vegetaloils

MPa, ∘C Astaxanthin []

Pink shrimp (P. paulensisand P. paulensis) processingwaste

Carbon dioxide, carbondioxide +

hexane/isopropanolmixture, and carbon

dioxide + sunower oil

– MPa, and ∘Cotal carotenoid content,

carotenoid prole, andastaxanthin

[]

Pumpkin(Cucurbita maxima)

Carbon dioxide + ethanol,water and/or olive oil

MPa, and ∘C otal carotenoid content,

carotenoid prole []

scale up the processes and the determination o their eco-nomical viability, which are essential to reach industrialproduction and, thus, the consumer.

Tus, the unctional and nutritional benets o these nat-ural pigments (such as precursors o vitamin A in the treat-ment o diseases like cancer through its antioxidant activity,among others) and their coloring properties must be effec-tively achieved and applied.

Competing Interests

Te authors declare that they have no competing interests.

Acknowledgments

Te authors wish to thank CNPq, Project no. /-, and CAPES, Project no. ./- (AUXPE:/), Brazilian unding agencies, or the nancial sup-port and scholarships that sustain this work.

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