Food & Function

PAPER

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aCRIAcq, University of Naples Federico II, PabDepartment of Food Science University of N

80055 Portici, ItalycDepartment of Plant Biology University of N

80055 Portici, Italy

Cite this: Food Funct., 2013, 4, 144

Received 7th August 2012Accepted 10th October 2012

DOI: 10.1039/c2fo30198a

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144 | Food Funct., 2013, 4, 144–152

Microalgae as human food: chemical and nutritionalcharacteristics of the thermo-acidophilic microalgaGaldieria sulphuraria

Giulia Graziani,b Simona Schiavo,c Maria Adalgisa Nicolai,b Silvia Buono,a

Vincenzo Fogliano,*a Gabriele Pintoac and Antonino Pollioac

The use of microalgae as a food source is still poorly developed because of the technical difficulties related

to their cultivation and the limited knowledge about their chemical composition and nutritional value. The

unicellular red microalga Galdieria sulphuraria has a very high daily productivity and its cultivation under

acidic conditions avoided any bacterial contamination. G. sulphuraria can be cultured under autotrophic

and heterotrophic conditions: in this study a screening of 43 strains showed that in the latter case a

duplication of biomass production was obtained. The proximate composition (protein, carbohydrates,

fiber and lipids), the micronutrient content (carotenoids, phycobiliproteins, chlorophylls and vitamins)

together with the antioxidant activity of the biomass produced by a selected strain of G. sulphuraria

under both cultivation conditions were determined. Results showed that the material is rich in proteins

(26–32%) and polysaccharides (63–69%) and poor in lipids. Under heterotrophic cultivation conditions,

the lipid moiety mainly contained monounsaturated fatty acids. Among micronutrients, some B group

vitamins are present, beta-carotene is the main carotenoid and phycobiliproteins are present under both

cultivating conditions. G. sulphuraria proteins are strictly associated with polysaccharide components

and therefore not digestible. In the second part of the work, an extraction protocol using Viscozyme L,

a commercial enzymatic preparation containing a mixture of polysaccharidases, was developed which

made G. sulphuraria proteins a good substrate for human gastrointestinal enzymes. All in all, the data

suggested that G. sulphuraria biomass has a potential use as food ingredients both for protein-rich or

insoluble dietary fibre-rich applications. The low concentration of lipids and the absence of green color

make this microalgae source particularly useful for the addition to many food preparations.

Introduction

Low pH environments derived from the oxidation of sulphurcompounds are diffused worldwide. They are oen associatedwith secondary volcanism and, in this case, their temperaturesrange from 50 to 80 �C.1 In these very selective environments,the dominant photosynthetic eukaryotes belong to the classCyanidiophyceae,2 a class encompassing three genera: Cyani-dium, Galdieria and Cyanidioschyzon.3 Galdieria is the onlygenus of this class able to grow heterotrophically, usingnumerous carbon sources.4 It has been hypothesized that theaptitude of growing under heterotrophic conditions is related tothe ability of Galdieria to thrive also in habitats where light isabsent.5 The trophic exibility ofGaldieriamakes this microalga

rco Gussone Ed 77, I-80055 Portici, Italy

aples Federico II, Parco Gussone Ed 77, I-

aples Federico II, Parco Gussone Ed 77, I-

a promising candidate for the exploitation of heterotrophicmass cultivations.

Large-scale microalgal cultures are presently used in shfeeding and production of ne chemicals, whereas theproduction of biodiesel from microalgae is still in its infancy.6,7

The use of microalgae as a food source for humans and animalshas been increasing since the early 1950’s.8,9

Microalgae can represent a valuable source of vitamins andfatty acids. Because of the high protein content of some speciesit might be useful to use them for manufacturing healthy foodsboth for humans and animals.10

Microalgae are usually photosynthetic organisms, but a fewspecies are able to grow under heterotrophic conditions. Thispossibility opens a promising eld of research, through the useof cheap fermentors instead of expensive photobioreactors,allowing us to obtain high densities of microalgae cells andtherefore a high yield.11

In this respect, Galdieria species are of particular interest asit grows at pH values between 1.5 and 2.0, thus preventingbacterial contamination, one of the major problems of largescale microalgae cultures. Heterotrophic cultures of Galdieria

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sulphuraria contain high levels of phycocianin, a pigment usedas a uorescent marker in diagnostics, and as a dye in food andcosmetics.12 In addition to the production of ne chemicalsGaldieria species also have signicant potential as a source ofprotein and other macronutrients. One of the unique charac-teristics of this alga is the high protein content of the cell wall,as reported in the pioneering work of Bailey and Staehelin,13

who rst suggested that microalgae from Galdieria genera couldhave a nutritional application. The difficulties in introducingmicroalgae based ingredients in many foods are due to thestrong green color and the oxidability of the lipid moiety14 butthe peculiar features of G. sulphuraria have the potential toovercome these bottlenecks.

In this study, the biochemical composition of a selectedG. sulphuraria strain has been evaluated by comparing thechemical constituents of cultures grown in autotrophy orheterotrophy, on a medium supplemented with 3% glycerol,putting particular emphasis on the potential nutritional valueof the alga. The main problem related to the adoption ofmicroalgae proteins as human food is due to their poordigestibility. In this paper, an enzyme-based extraction methodis proposed which made protein a good substrate for humandigestive enzymes.

Materials and methodsAlgal strains and medium

The Galdieria strains investigated belong to the species G. sul-phuraria and are maintained in the ACUF collection of theBiological Science Department of University ‘Federico II’,Naples, Italy (http://www.biologiavegetale.unina.it/acuf.html).

Table 1 Doubling times of G. sulphuraria strains grown in autotrophy or heterotroValues with different letters are significantly different

G. sulphurariaACUF strain number

Autotrophy doublingtime (hours)

Heterotrophy doublingtime (hours)

077/300 70 � 3.5c 41 � 5.5d

075/301 57 � 3.8b 55 � 6.1d

074/302 47 � 7.6b 33 � 4.3c

073/303 48 � 4.0b 41 � 1.8d

072/304 65 � 6.5c 31 � 4.5c

071/305 53 � 6.1b 34 � 2.9c

070/306 50 � 5.1b 17 � 0.3b

068/307 43 � 1.9b 41 � 4.8d

067/308 44 � 4.2b 29 � 3.1c

064/309 39 � 2.3a 16 � 0.5a

022/312 45 � 2.1b 21 � 4.7b

021/313 56 � 8.5b 18 � 2.2b

018/316 119 � 4.3e 20 � 3.0b

017/317 84 � 8.3d 27 � 3.1c

016/318 118 � 8.1e 82 � 6.5e

014/319 65 � 8.5c 43 � 2.8d

013/320 115 � 9.7e 95 � 7.7e

011/322 69 � 7.8c 19 � 0.7b

010/323 48 � 5.5b 30 � 3.9c

009/324 52 � 8.1b 35 � 2.4c

007/325 55 � 3.2b 41 � 4.9d

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The strain numbers are indicated in Table 1. A modied AllenMedium15 with (NH4)2SO4 as a nitrogen source was adopted inall the experiments, and the nitrogen content in the mediumwas xed at 40 mg L�1. Carbon dioxide was supplemented bysparging ltered air into the medium. In the strains grownunder articial light no organic carbon source was supple-mented to the medium. In heterotrophy experiments, organiccarbon was supplemented with 3% glycerol. The pH was set at1.5 and was controlled daily, whereas temperature was main-tained at 36 � 1 �C. The axenic conditions of the cultures werechecked by spreading 100 mL samples of each culture on thesurface of agar plates containing Bacto Yeast Extract (DIFCO212 750) or Malt Extract (DIFCO 211 320). Test plates wereincubated at 37 �C for 3 days and at 25 �C for 1 week.

Selection of the strain for mass culture

Tests to assess strain doubling times in autotrophy andheterotrophy were performed in batch cultures carried out in100 mL Erlenmeyer asks, containing 50 mL of Allen mediuminoculated with samples from agar slants of each strain. Batchculture tests lasted 9 days, and the initial algal concentrationwas 5 � 104 cells per mL.

Typically, the asks were placed on a Plexiglas shakingapparatus under continuous irradiance (150 mE m�2 s�1)provided by daylight uorescent Philips lamps (TLD 30W/55). Inheterotrophy experiments, each ask was wrapped with analuminum foil. Growth experiments were carried out in tripli-cate and repeated two times.

The cultures were sampled daily and the growth of thecultures was followed by measuring their optical density at550 nm with a Secomam250 spectrophotometer. Doubling

phy. Growth experiments were carried out in triplicate and repeated two times.

G. sulphurariaACUF strain number

Autotrophy doublingtime (hours)

Heterotrophy doublingtime (hours)

006/326 45 � 7.3a 26 � 2.5b

005/327 59 � 5.1b 42 � 3.3d

004/328 45 � 5.1a 35 � 4.0b

162/330 40 � 1.3a 58 � 6.0e

139/331 67 � 3.0b 20 � 1.8a

140/332 74 � 3.5c 48 � 4.9d

141/333 60 � 4.2b 18 � 2.0a

142/334 64 � 3.9b 18 � 0.9a

134/338 75 � 4.5c 35 � 3.9c

135/339 42 � 3.5a 20 � 2.1a

136/340 63 � 6.8b 34 � 2.4c

137/341 53 � 3.7b 27 � 3.0b

138/342 56 � 2.9b 22 � 1.2a

214/343 61 � 7.4b 52 � 5.5e

101/345 130 � 1.8f 95 � 8.1f

216/346 78 � 6.2c 47 � 3.9d

192/347 95 � 8.0e 31 � 0.5b

166/348 74 � 4.4c 29 � 1.3b

231/349 56 � 4.9b 38 � 3.7c

080/350 64 � 6.6b 33 � 1.2c

079/351 67 � 3.7b 59 � 6.3e

078/352 67 � 3.0b 47 � 5.5d

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times were calculated according to the formula: K ¼ log2 (N1 �N0)/DT, in which K is doublings per day, and N1 and N0 denotethe population size at the beginning and the end of the timeinterval. At the beginning and end of the tests, the ammoniumconcentration in the culture medium was measured using anammonium selective electrode (Mettler ion meter SG8), and thedissolved oxygen with a HI 9143 Dissolved Oxygen Meter(Hanna Instruments).

Operating conditions for autotrophic and heterotrophic masscultures

For autotrophic and heterotrophic mass cultures, a gradualincrease of the concentration of the selected G. sulphurariastrain 064/309 was performed to reach the required volumes.Dense algal cultures (0.25 g L�1 of dry weight) were transferredfrom 100 mL asks to 500 mL Erlenmeyer asks containing 250mL of Allen Medium. The asks were placed in a ClimaticChamber, under continuous illumination of 100 mE m�2 s�1

and mixing provided by air bubble sparging, until microalgaeconcentration reached a value of about 0.5 g L�1 of dry weight.

Mass cultures were carried out according to the proceduresdescribed elsewhere in detail.16 In brief, glass cylindricalbioreactors having 5 L of internal volume were inoculated withthe selected culture from the 500 mL Erlenmeyer asks. In theheterotrophy experiments, the bioreactors were covered withaluminum foils. The working volume was set at 4 L. Air wassparged at the photobioreactor bottom by means of a porousceramic diffuser, at a volumetric ow rate of 40 NL h�1. Filtersof 0.2 mm were used to sterilize air ow inlet and outlet. Thebioreactors were housed in a thermostated chamber equippedwith the same kind of lamps as previously indicated. In theautotrophic cultures, the light irradiance (IL) was set at 150 mEm�2 s�1. A concentrated Allen medium (2�) was supplementedto the culture two times a week during the test to restore theinitial nitrogen concentration. The nitrogen integration did notdilute the broth since the added liquid volume balanced theperiodic culture sampling. In the heterotrophy experiments, theconcentrated medium added to bioreactors contained also 3%glycerol.

The experiments under the above described fed-batchconditions lasted 30 days. Cell dry weight determination wasmade with duplicate samples of cultures. Cells were centrifugedin an ALC pK121centrifuge at 4000 rpm for 10 minutes, andwashed one time with a 0.5 NaCl solution and two times withdistilled water, to remove culture medium constituents. Then,the algae were collected on pre-weighted ber glass lters(Whatman GF/A), dried at 105 �C for 24 hours, and weighted.

Characterization of lipids

Lipids were extracted from algal biomass using the chlor-oform : methanol mixture. Briey: 20 mL of a mixture chlor-oform : methanol (2 : 1, v/v) were added to 5 g of the freezedried sample followed by 20 min of mixing. This mixing wasrepeated for 10 min both aer adding the second portion of10 mL of chloroform and aer addition of 20 mL of water. Aercentrifugation, the organic phase was evaporated to dryness

146 | Food Funct., 2013, 4, 144–152

and the weight of the residue was determined aer 30 min at105 �C. The lipid extract prepared for total lipid determinationwas suspended in hexane and used for the fatty acid methyla-tion. Fatty acids were converted to their methyl esters beforeanalysis. Hexane extract (1 mL) was added with 200 mL of KOH2 N in methanol for 30 s at room temperature, and 1 mL wasinjected directly in the GC apparatus. The analysis of fatty acidmethyl esters was carried out using a Shimadzu 17A gas chro-matograph equipped with a fused silica capillary column(Phenomenex ZB-WAX, 0.50 mm lm thickness, 60 m � 0.32mm i.d.) and a FID detector. Helium was used as carrier gas at2 mL min�1. The temperature program was 80 �C � 2 min, rate15 �Cmin�1 until 180 �C, constant at 180 �C for 6 min, a secondrate of 5 �C min�1 until 240 �C, constant at 240 �C for 20 min.The injector and FID temperatures were 250 and 270 �C,respectively. Identication of fatty acid was made using refer-ence fatty acids methyl esters (FAME) from Merck (Merck,Darmstadt, Germany).

Carotenoid extraction and determination

Carotenoids were extracted from 100 mg of lyophilized micro-algae with 100 mL of dichloromethane (25%) and methanol(75%) according to ref. 17. The extract solution, mixed with thealgal cells, was then separated by centrifugation at 10 000g for 5min, and the supernatant, containing pigments, was collected.Aer the extraction procedure was repeated three times, thepigments were almost completely extracted. The combinedpigment extracts were re-centrifuged at 10 000g for 5 min andkept in the dark at �20 �C for HPLC analysis. Saponication ofcarotenoid esters was carried out according to ref. 18. Six mL of0.107 M NaOH dissolved in methanol, which was freshlyprepared, was added to 30 mL of the pigment extract solutionunder a nitrogen atmosphere. The mixture (36 mL) was evapo-rated and concentrated to 30 mL under nitrogen during theprocess of adding NaOH, and then kept at 5 �C in darknessunder nitrogen for 12 h in a water bath for complete hydrolysisof carotenoid esters. The saponied pigment extract solutionwas directly separated and determined by HPLC as described inref. 19.

Determination of protein, carbohydrates and dietary brecontent

Samples were analyzed for protein concentration by the Kjel-dahl method20 using a nitrogen conversion factor of 6.25.Carbohydrate determination was performed on 2 g of freezedried samples treated with hydrochloric acid (0.2 M) at 85 �C for1 h. Aer neutralization by sodium hydroxide, reducing sugarswere determined using the Fehling test. Total ber content wasdetermined by the AOAC 985.29 gravimetric method.

Determination of vitamin content

The procedure described in ref. 21 was employed for water-soluble vitamins. Two g of freeze dried sample was weighed in a100 mL volumetric amber ask, 40 mL of water and 4 mL ofNaOH 2 M were added and the suspension was vigorouslyshaken, and then 50 mL of phosphate buffer 1 M (pH 5.5) were

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added in order to lower the pH of the nal solution at 7. Thesuspension was sonicated for 10 min in an ultrasonic bath andltered through a 0.22 mmMillipore syringe MillexTM GP lter(Bedford, MA, USA) before the HPLC analysis. HPLC separationwas carried out at a ow rate of 0.8 mL min using an HPLC(Model LC 10, Shimadzu, Osaka, Japan), with a diode arraydetector and a Prodigy ODS3 100 A column (250 � 4.6 mm,particle size 5 mm) (Phenomenex, CA, USA). The mobile phaseswere (A) consisting of an aqueous solution of TFA 0.025% pH2.6, and (B) acetonitrile. Vitamin elution was achieved using thefollowing linear gradient: initial isocratic step 100% A for 5 min,followed by a linear gradient to solvent (B)–solvent (A) (25 : 75,v/v) in 6 min. Then a second linear gradient to solvent (B)–solvent (A) (40 : 60, v/v) in 8 min, this mixture was held for 1min. Finally, the initial conditions were reestablished in 1 minand held for 4 min. Chromatographic detection was performedat 210 nm for vitamins B5, B8 and B12 and at 275 nm for vita-mins B1, C, PP, B6, B9 and B2. Three injections were performedfor each sample.

Vitamin E extraction and analysis was performed as previ-ously described.22

Phycobiliprotein determination

Phycobiliproteins were quantitatively extracted using theprocedure reported in ref. 23. The concentrations of phycocy-anin and phycoerythrin were calculated from the measuredabsorbances using the Kursar and Alberte equation.24

Antioxidant activity

Total antioxidant activity was determined using a directmeasurement of ABTS_+ (2,2-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid) called the ‘QUENCHER’ approach.25 ABTS radicalcation (ABTS_+) was produced by reacting ABTS stock solution(ABTS dissolved in water to a 7 mM concentration) with 2.45mMpotassium persulfate (nal concentration) and allowing themixture to stand in the dark at 4 �C for 12–16 h before use untilthe radical was stable. The ABTS_+ solution was diluted in ahydroalcoholic mixture ethanol–water (50/50) to an absorbanceof 0.70 (�0.02) at 734 nm. The direct measurement of ABTS isdone on a nely ground lyophilized algae sample sieved at500 mM. Samples were diluted with cellulose (1 : 9), and 10 mgof the diluted sample was used for the determination. Aerbeing suspended in 6 mL of ABTS_+ diluted solution, the mixturewas stirred in the dark for 25 min and then centrifuged at 4000rpm for 2 min. The absorbance was read at 734 nm using asblank the same quantity of the ABTS_+ diluted chromophore inwhich had been suspended only cellulose. The antioxidantcapability (AC) was expressed, using Trolox as standard, asmmol of Trolox Equivalent (TE) per kg�1 of dried weight,therefore TEAC notation was used.

Protein extraction by enzymatic treatment

Preliminary extraction of G. sulphuraria fresh and freeze driedmaterial were performed in water, hydroalcoholic solutions,detergents (2% Triton X-100 and 1% SDS) and 9 M urea also inthe presence of a reducing agent (1% b-mercaptoethanol). The

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extraction rate was monitored by colorimetric protein assay(Bio-Rad, Hercules, CA, USA).

The commercial enzymes used in this study included Vis-cozyme L (a multi-enzyme complex containing a wide range ofcarbohydrases, including arabanase, cellulase, b-glucanase,hemicellulase and xylanase), pepsin and trypsin. Enzymaticdigestion of the freeze dried sample was performed dissolving 3g of powdered sample in 120mL of water and treating with 4mLViscozyme L at pH 4, 37 �C for 24 h. Aer digestion, the tube wascentrifuged at 4000g for 15 min, and Viscozyme digestedmaterial was taken for further digestion with pepsin and trip-sin. Algal biomass was also digested with proteases pepsin andtrypsin with or without a pre treatment with Viscozyme diges-tion. 3 g of powdered sample was treated rst with 2 mL pepsinsolution (800–2500 U mg�1, 100 mg mL�1, pH 2, 37 �C for 2 h)and then with 2 mL tripsin solution (Type II-S; 100 mg mL�1 inphosphate buffer pH 7.6, 37 �C for 2 h). The same enzymaticdigestions were applied also to A. platensis.

The protein prole of freeze-dried algal powder, aer enzy-matic treatments, was determined by SDS-PAGE on 4 % and12.0% acrylamide slab gels. A total of 10 mg of protein sampleswere dissolved in 1mL of sample buffer (0.5M Tris–HCl, pH 6.8,containing 10% (w/v) SDS, 10% (v/v) glycerol, 5% b-mercaptoe-thanol and 0.1% (w/v) bromophenol blue) and heated at 100 �Cfor 5 min. Every sample (10 mL) was applied to each lane of gel.The separationwas performed at 120 V constant voltage for 1.5 h.The polypeptides used as molecular mass markers were: pho-phorylase b (94 kDa), albumin (67 kDa), ovalbumin (43 kDa),carbonic anhydrase (30 kDa), trypsin inhibitor (20.1 kDa) andlactalbumin (14.4 kDa). The separated protein were visualised bystaining the gelwith 0.1% (w/v)CoomassieBrilliant BlueR-250 in10%(v/v) acetic acid: 40% (v/v)methanol anddestainedwith10%(v/v) acetic acid containing 40% (v/v) methanol.

Results and discussionSelection of the G. sulphuraria strain for mass cultures

Forty-three G. sulphuraria strains, coming from either acidic orthermo-acidic environments isolated in different Italian regions,were grown under autotrophic or heterotrophic conditions. Inthe latter case, preliminary experiments indicated that thegrowth-rate of G. sulphuraria is not modied using glycerol as acarbon source instead of glucose or sucrose. Therefore, glycerolwas used in this screening as it is a cheapbyproduct that iswidelyavailable from biodiesel production. The growth performance ofthe G. sulphuraria strains in batch culture experiments issummarized in Table 1, reporting doubling times of each strainsunder the two cultivating conditions: the lower the doublingtime, the higher the daily biomass produced. The culturesmaintained the exponential phase of growthduring the course ofthe experiment (nine days). Ammonium concentration and dis-solved oxygen were measured at the beginning and end of thetests. Also in the case of strain 064/309, which showed the lowestdoubling times, only a 10% reduction of ammonium concen-tration was observed (not shown), whereas the dissolved oxygenremained unchanged, indicating that both nitrogen and oxygenwere not limiting during the course of the experiments.

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As expected, all the G. sulphuraria strains growing experi-ments were characterized by a complete absence of bacterialcontaminations.

It is worth noticing that most strains strongly decreased theirdoubling times when grown in heterotrophy. G. sulphuraria 064/309, isolated in Sicily from a sulfur mine, showed the highestdoubling time under heterotrophic conditions and, based onthese results, it was chosen for mass cultivation. In the 5 Lreactors, under the fed-batch conditions above described, G.sulphuraria 064/309 reached aer 30 days a biomass of 5.7 g L�1

dry weight under autotrophic conditions and 29 g L�1 dryweight in heterotrophy.

Macronutrient composition

Macronutrient composition of G. sulphuraria cultivated underthe two conditions is summarized in Table 2. The proteincontent was higher than that of vegetables, which is a commonfeature for microalgae. The protein content of heterotrophicand autotrophic biomass was 27% and 33%, respectively. Thedifference is mainly due to different amounts of phycobili-proteins, which in autotrophic algae is 8.2%, while in hetero-trophic algae, due to the absence of the photosyntheticprocess, it is only 1.1%. The protein concentration wascomparable to that of other microalgae which have beenproposed as a source of proteins to integrate into the diet indeveloping countries.26

The lipid concentration was very low under both cultureconditions: as shown in Table 2, the percentage of lipids was1.1% in autotrophic and 1.4% under heterotrophic conditions,respectively. Lipid content was much lower than that reportedfor other microalgae.27,28 It is known that the concentration oflipid in microalgae is extremely variable, reaching productivepeaks of 70–80% for those used in oil production.29

Table 2 Proximal chemical composition of G. sulphuraria biomass cultivatedunder heterotrophic and autotrophic conditions

NutrientsG. sulphurariaheterotrophic mg g�1

G. sulphurariaautotrophic mg g�1

Proteins 265 � 25 325 � 32Lipids 11.4 � 3.0 17.7 � 3.2Total carbohydrates 691 � 31 629 � 16Dietary bre 541 � 38 540 � 25

Table 3 Fatty acid percentage composition of G. sulphuraria cultivated underheterotrophic and autotrophic conditions

Fatty acidsG. sulphurariaheterotrophic%

G. sulphurariaautotrophic%

C14:0 2.7 � 0.4 0.9 � 0.1C16:0 14.7 � 2.0 39.4 � 2.5C16:1 2.4 � 0.8 n.d.C18:0 n.d. 4.7 � 1.8C18:1 57.5 � 3.7 8.6 � 1.9C18:2 19.5 � 2.6 45.2 � 2.3C18:3 2.7 � 0.0 1.1 � 0.1

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Qualitative prole of the lipid fraction of G. sulphuraria isreported in Table 3. In this case, there were signicant differ-ences between the two growing conditions. Under heterotrophicconditions, the unsaturated/saturated ratio was about ve, whileit decreased nearly to one in the material grown autotrophically;on the other hand, palmitic acid represents, for autotrophicalgae, about 40% of lipid fraction while it was only 20% inheterotrophic algae. Linoleic acid was the major fatty acid inautotrophic algae while oleic acid was the most represented inheterotrophic algae. These results conrmed thatmicroalgae aremetabolically very exible and their total lipid concentration aswell as fatty acid composition can change signicantly depend-ing on the strain and on the growth conditions used.19

The high polyunsaturated fatty percentage with respect tothe total fatty acid content, typical of microalgae, is observed inG. sulphuraria biomass obtained in autotrophic cultivation. Thehigh prevalence of monounsaturated fats is a response toheterotrophic growth reported also in Chlorella zongiensis,30

and probably related to a reduction of thylakoid structurallipids, occurring in the algae grown in the dark.

Regarding the possible use of the whole G. sulphurariabiomass, the low amount of total lipids and the limitedpercentage of polyunsaturated fatty acids can positively inu-ence the storage time limiting the oxidative degradation, whichis usually an important problem when microalgae are incor-porated in foods.31

Carbohydrates are the main macronutrients present in G.sulphuraria. The transition to heterotrophic cultivation did notmodify the carbohydrate content, which was 69.1%, of which54.1% was insoluble dietary ber, whereas in autotrophic algae,carbohydrates represented 62.9%, of which 54.0% was insol-uble dietary ber. The content of insoluble dietary ber washigher than that reported in the literature for other microalgae:the carbohydrate fraction was mainly represented by heteroge-neous soluble polymers, and the cellulosic cell wall representson average about 10% of the algal dry matter.14 In most marinered algae high-molecular-weight, sulfated galactans is the majorcell wall component.32,33 The red unicellular alga Porphyridiumcruentum also have a carbohydrate content ranging from 40 to57% of dry weight, with a prevalence of storage amylopectine-and amilose-polyglucans,27 whereas the Cyanidiophycean Cya-nidium caldarium synthesizes large amounts of storage glycogenpolyglucans, but not amylose.34 The species Galdieria maximagrown heterotrophically on glucose increases its carbohydratescontent by a factor of 4, synthesizing a storage polysaccharidewith a very high branching degree, which is accumulated incytoplasm.35 In G. sulphuraria, the cellulosic-like polymers aremore represented than storage polysaccharides, with an averageamount of about 35% of dried biomass.

The presence of insoluble dietary ber can be regarded as apositive feature of G. sulphuraria biomass. Insoluble dietaryber is able to reach the colon and to stimulate the growth ofpositive microora, thus contributing to the improvement ofthe human intestinal ecosystem. The addition of ber-rich algalproducts to the diet could increase overall dietary ber intake,helping people to reach the recommended dietary allowance(RDA).36

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All in all, G. sulphuraria showed an unusual compositionalprole among microalgae, with a large prevalence of carbohy-drates, a good content of proteins and a very low level of lipids.This pattern is similar to that found in some brown and redmacroalgae, which have been considered as good sources offunctional ingredients.37

Micronutrient and pigment composition

Microalgae are a valuable source of many essential vitamins(e.g., A, B1, B2, B6, B12, C, E, nicotinate, biotin, folic acid andpantothenic acid).14

As shown in Table 4 the concentration of vitamin E was 9 and15 mg kg�1 of dry weight for autotrophic and heterotrophicalgae, respectively. The content of water soluble vitaminschanged under the different culture conditions. In fact,heterotrophic algae contain vitamin B2 (riboavin) and B3

(niacin) respectively at a level of 30 mg kg�1 and 32 mg kg�1,respectively. However, autotrophic algae only contain 20 mgkg�1 vitamin B3 and no vitamin B2.

The vitamin content of microalgae is of fundamental nutri-tional importance for their use as food, however limited andconicting data, with huge variations among the studies, arepresent in the literature. Some of the differences may be due tovariations in culture conditions and harvesting and storageprocedure. Many studies on the vitamin content of differentmicroalgae used in aquacultures were performed;38,39 thevitamin E content of G. sulphuraria, both autotrophic andheterotrophic, was 10 to 20 times lower than the literature data,while water soluble vitamin contents were in accordance withliterature reports. De Roeck-Holtzhauer et al.40 measured thecontent of ten vitamins in ve microalgae; thiamine, riboavin,ascorbic acid, pyridoxyl phosphate and the fat-soluble vitaminsA, D, E and K. The authors concluded that despite variability,microalgae can represent a valuable source of B group vitamins,particularly riboavin.

Microalgae are also a source of pigments like chlorophyll(0.5% to 1% of dry weight), carotenoids (0.1% to 0.2% of dry

Table 4 Pigment and water soluble vitamin contents in dry biomass of G. sul-phuraria cultivated under heterotrophic and autotrophic conditions

G. sulphurariaheterotrophic

G. sulphurariaautotrophic

Vitamins (mg kg�1)B2 30 � 2 n.d.B3 32 � 3 20 � 2E 9 � 1 15 � 3Carotenoids (mg kg�1)b-Carotene n.d. n.d.Cryptoxanthin n.d. n.d.Astaxanthin n.d. 575 � 123Lutein n.d. 387 � 112Chlorophills (mg kg�1)Chlorophyll a n.d. 12.020 � 541Phycobiliproteins (g kg�1)Allophycocyanin 4.5 � 1.2 79.0 � 3.1Phycocyanin 0.5 � 0.0 0.2 � 0.0Phycoerytrin 6.5 � 1.1 3.3 � 0.8

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weight on average and up to 14% of dry weight for b-carotene ofDunaliella) and phycobiliproteins.26

Data reported in Table 4 indicated that carotenoids are onlya minor pigment in G. sulphuraria: they were detected only inthe autotrophic algae, the main ones being astaxanthin andlutein. The absence of carotenoids in heterotrophic algae wasdue to the lack of photosynthesis under these growth cultureconditions. Furthermore, under the autotrophic conditions thepresence of chlorophyll at 1.21%, which is in the standard rangefor microalgae, was detected by chromatographic analysis.

Phycobiliproteins constitute the main pigments of the ana-lysed biomass and they have been proposed for commercial useas a natural dye. In G. sulphuraria, allophycocyanin is thedominant form in the autotrophic algae, while phycoerytrin wasthe main phycobiliprotein in the heterotrophic algae. Theoccurrence of phycobiliproteins in the algal biomass underheterotrophic conditions (phycocyanin and phycoerytrin) was inagreement with literature studies reporting that somesubstances involved in the photosynthetic apparatus continue tobe synthesized in the dark, for example, the light-harvestingpigment phycocyanin from G. sulphuraria.41 In the literature, itwas reported that phycocyanin and allophycocyanin are alwayspresent in Cyanophyceae and Rhodophyceae.42 Moreover, theexpression of genes encoding phycobiliproteins has been shownto vary in response to chemical and physical stimuli includingnutrient availability, temperature, light intensity and in somecases light wavelength. The different growing conditions used inthis study might have stimulated the phycoerythrin productionby G. sulphuraria.43 Data obtained on G. sulphuraria shown inTable 4 are in agreement with those reported in the literature forRhodophyta in which the red color is due to the presence of highamounts of the biliprotein phycoerythrin in addition to the bluephycocyanin and chlorophyll a.44

Antioxidant capacity

Antioxidant capacity (TEAC) of the dried biomass from G. sul-phuraria cultivated under heterotrophic and autotrophicconditions was of 5.6 mmol kg�1 and of 29.6 mmol kg�1 dryweight, respectively. The high value under the autotrophicconditions paralleled the higher concentrations of carotenoidsand pigments well known in the literature for their antioxidantactivity. The absolute value of antioxidant activity is comparableto that reported for Porphyridium cruentum.27 The comparison ofthese data with those reported in the literature for plant foodsindicated that TEAC of G. sulphuraria biomass are comparablewith those of some vegetables.45 These results suggest thatG. sulphuraria (particularly under autotrophic conditions) canbe used as ingredients in food preparations to improve thenutritional value.

Extraction, bioaccessibility and digestibility of proteins fromG. sulphuraria

The benet derived from the consumption of protein-rich foodsdepends on protein digestibility which can be affected by thelow bioaccessibility of the proteins because of their folding and/or the strict association with other food components.46 In the

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Fig. 1 SDS-PAGE analysis of proteins extracted from freeze-dried biomass of G.sulphuraria and A. platensis panel A: proteins extracted frommicroalgae with 9 Murea and 1% b-mercaptoethanol. Lanes: 1, molecular mass markers; 2, G. sul-phuraria extract (9 M urea and 1% b-mercaptoethanol); A platensis extract (9 Murea and 1% b-mercaptoethanol). Panel B: lanes: 1, molecular mass markers; 2, G.sulphuraria extract (Viscozyme digestion); 3, G. sulphuraria extract (Viscozymedigestion followed by viscozyme, trypsin and pepsin); 4, G. sulphuraria extract(trypsin and pepsin digestion).

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case of G. sulphuraria, most of the proteins are embedded in thepolysaccharide components and the treatment with differentprocedures usually adopted for protein extraction (urea, solventor detergents) do not allow us to achieve a satisfactory proteinsolubilization.

In Fig. 1 panel A, the electrophoretic pattern of proteinsextracted from microalgae G. sulphuraria and A. platensis with 9M urea and 1% b-mercaptoethanol is shown. While the proteinsfrom A. platensis (extracted as control) are well resolved indistinct bands, the extract from G. sulphuraria gave a longsmear, indicating that polysaccharide cell walls are still boundto the proteins which cannot be separated on the SDS gel.

To develop an effective treatment to disrupt the poly-saccharide cell wall, making the protein and other constituentsaccessible for digestive enzymes, a screening of commercialenzymatic preparations was carried out. Viscozyme L, which is amixture of polysaccaridases from Aspergillus sp., proved to bevery effective in disrupting the G. sulphuraria cell wall. In Fig. 1panel B, the SDS prole obtained aer hydrolysis of algal drymatter with Viscozyme L is shown. The electrophoretic patternof G. sulphuraria, aer Viscozyme digestion (lane 2), revealedseveral main bands with apparent molecular weight between97.0 and 45.0 kDa and other less abundant bands with molec-ular masses ranging from approximately 45.0 to 12.0 kDa.

A successive treatment of the Viscozyme-extracted proteinswith a mixture of human digestive enzymes (pepsine and tryp-sine) at physiological conditions resulted in amarkeddecrease ofthe relative intensity ofproteinsathighmolecularweightwith theappearance of new bands at low molecular weight, and particu-larly of an intense band at 28.0 kDa (lane 3). The overall intensityof the bands is decreased, indicating thatmost of the proteins aredegraded intopeptides lower that 10kDa. This evidence indicates

150 | Food Funct., 2013, 4, 144–152

that aer Viscozyme extraction G. sulphuraria proteins became agood substrate for human digestive enzymes. Interestingly, whenG. sulphuraria biomass was incubated directly with digestiveenzymes, skipping the treatment with Viscozyme (lane 4), alimited amount of proteins bands was visible, but an intenseband with molecular mass at about 25.0 kDa could be seen. Thissuggested that digestive enzymes were able to cleave and solu-bilise some parts of the proteins which are not completelywrapped in the cell wall polysaccharides structure.

Despite their high content of protein rich essential aminoacids, dried microalgae have not gained signicant importanceas food or food ingredients thus far. This is due to many tech-nological and sensorial obstacles: the powder-like consistency ofthe dried biomass; its dark green color, easily turning to brown;and its slightly shy odor, which becomes a rancid smell duringthe product’s shelf life. Moreover, the digestibility of microalgaeproteins is limited as well as the amount of high quality (i.e. notbacterial contaminated) microalgae biomass on the market.

Data of this paper suggested that G. sulphuraria biomassobtained under heterotrophic conditions can potentially over-come most of these hurdles. As shown for other microalgaespecies,47,48 the growth in heterotrophy allows reaching high celldensities also using cheap byproducts such as glycerol. G. sul-phuraria biomass was colorless, thus avoiding the appearanceof an unattractive green-brownish color aer incorporation intofoods. It had a low lipid content mainly of monounsaturatedacid, therefore the oxidation during shelf life is negligible.Moreover, the developed enzymatic extraction protocol allowedthe extraction of proteins which proved to be good substrates ofhuman digestive enzymes.

G. sulphuraria can be used to develop new food ingredients,and thanks to its favorable macro and micronutrient proles, itcan be used to design food preparations rich in bioavailableproteins and dietary ber.

Acknowledgements

This work was carried out in the framework of the project“Phytocosmetics: cosmetic formulations based on water solubleextracts from microalgae with replenishing action on skintissue” funded by Institute of Foreign Commerce (ICE) andConference of Italian University Rectors (CRUI).

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