Research ArticleFunctional Composition, Nutritional Properties, and BiologicalActivities of Moroccan Spirulina Microalga

Rajaa Seghiri ,1 Mourad Kharbach,2 and Azzouz Essamri1

1Laboratory of Agro-Resources, Polymers and Process Engineering (LAR2PE), Department of Chemistry, Faculty of Sciences,Ibn Tofaıl University, B.P. 133, 14000 Kenitra, Morocco2Biopharmaceutical and Toxicological Analysis Research Team, Laboratory of Pharmacology and Toxicology,Faculty of Medicine and Pharmacy, University Mohammed V, Rabat, Morocco

Correspondence should be addressed to Rajaa Seghiri; rajaa.seghiri@gmail.com

Received 25 January 2019; Revised 28 April 2019; Accepted 20 May 2019; Published 3 July 2019

Academic Editor: Francisca Hernandez

Copyright © 2019 Rajaa Seghiri et al.  is is an open access article distributed under the Creative Commons Attribution License,which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

 e present study aimed to characterize the nutraceutical properties and the antimicrobial e�ect of Moroccan Spirulina(Arthrospira platensis).  e nutritional composition was evaluated, including water content, crude protein, total carbohydrates,lipids, phenolic composition, macro- and micromineral content, �ber content, and energy value.  en, the microbiologicalanalysis and antioxidant activity were measured.  e antimicrobial activity was evaluated using the minimum inhibitoryconcentration method on bacteria and fungi. Moroccan Spirulina contained a large amount of protein (76.65± 0.15%), followedby carbohydrates (6.46± 0.32%), minerals (20.91± 0.88%), crude �ber (4.07± 1.42%), lipids (2.45± 0.82%), ash (14.56± 0.74), andtwenty phenolic acids being identi�ed and quanti�ed. Moreover, �avonoid and phenolic contents were present at 15.60± 2.74mgRE/g dw and 4.19± 0.21mg GAE/g dw, respectively. Microbiological risk assessment indicated that this product is safe to beconsumed as a human food product.  e antioxidant activity was higher in the methanolic fraction (23mgTE/g dw) (DPPH).

1. Introduction

 e Moroccan coast extends over 3500 km (2900 km for theAtlantic coast and 500 km for the Mediterranean coast).  isecosystem was recognized as vital and fragile but with aconsiderable ecological value. Despite increasing recogni-tion of di�erent foods and their safety, algae have receivedsmall interest in spite of their wide and abundant availability.Algae were one of the �rst life forms on Earth, and severalthousand species existed [1]. On the contrary, microalgaewere considered an important part of aquatic biodiversity;they were grown in di�erent environments (sea, freshwater,and desert) and also in di�erent forms (single cells, colonies,or �laments) [2]. Recently, algae have been used as foodsupplements in order to enhance their nutritional value, asanimal feed additives, and even for pharmaceutical uses[3, 4]. However, they have diverse chemical properties andwere marketed in various forms, mainly powder, tablets,straw, capsules, and liquids, or incorporated to other foods,

such as pasta, gums, and beverages [5]. On the contrary,microalgae are an alternative food of natural antioxidants,which were more varied than those found in other terrestrialplants [6]. Polyphenolic compounds were known as im-portant natural antioxidants, while numerous classes of�avonoids and other classes of phenolics were found inmicroalgae. In addition, various polyphenolic moleculeswere associated with biopharmacological activities includingantimicrobial and antioxidant actions [7, 8]. e commercialstrains of edible microalgae were mainly dominated byChlorella, Arthrospira, Dunaliella salina, and Aphanizome-non �os-aquae. Towards a more food-secure future, theSpirulina cyanobacterium (Arthrospira platensis) becametrendy health food brands, which explained its culture inseveral countries [9]. Various companies sell a variety ofArthrospira-based products (tablets and powder) as a foodsupplement and distribute them to over 20 countries aroundthe world.  erefore, they have been widely introduced(fresh or dried) in the human diet and nutrition.

HindawiJournal of Food QualityVolume 2019, Article ID 3707219, 11 pageshttps://doi.org/10.1155/2019/3707219

Spirulina is an abundant source of nutritious compo-sition, including protein, vitamins, lipids, fibers, minerals,carbohydrates, and some of natural pigments [10]. Spirulinawas recognized as a safe supplement (GRAS) without tox-icological effect and was officially approved by the Food andDrug Administration (FDA) and the National SanitarySurveillance Agency (ANVISA). +ese main constituentsconfer a physiological potential and a functional benefit thatmakes it promising for the food industry to overcome theproblem of malnutrition and in health applications againstpathogens [11, 12]. Right from the beginning of the appliedphycology, the algae’s chemical composition and their mainbioactive compounds have received an intense interest [13].+e nutritional and toxicological assessments of commercialalgae foodstuff were little studied and often based on reportsfrom field or interlaboratory ecological or physiologicalstudies [14]. According to the best of our knowledge, nostudies have been performed on the nutraceutical propertiesof a strain of Spirulina isolated in Morocco. +e presentwork aims to evaluate the Moroccan Spirulina’s nutritionaland pharmacological properties. Firstly, the chemical andnutritional compositions including phenolic profiling wereinvestigated. Secondly, antimicrobial and antioxidant ac-tivities were evaluated.

2. Materials and Methods

2.1. Raw Materials. Spirulina platensis (Arthrospira pla-tensis) used in this study was provided by the Spirulina-Berbere® company, located in the region of Souss-Massa-Draa, in the south of Morocco. From an artificial pondculture in March 2016, the microalga was grown by com-pany’s process on Zarrouk’s medium implemented undercover in a greenhouse under the following environmentalconditions: at pH� 10.25, salinity� 16, and shading� 65%and under the solar light between 23 and 30°C with constantshaking. Growth was monitored by measuring watertransparency using the test of Secchi. After harvesting andspinning, microalga cake was dried in a hot air oven at 70°C.Finally, dried samples were crushed and sieved at 1mm porediameter to obtain a fine and homogeneous powder using amill (MF 10 basic, IKA-WERKE). +e powder was stored insealed plastic bags in desiccators at room temperature forfurther chemical analysis. To facilitate reading, Spirulinawasused as a generic name for commercial products fromArthrospira spp.

2.2. Chemicals and Reagents. All chemical reagents andsolvents used were provided by Sigma-Aldrich (St. Louis,MO). Bacterial strains studied are in the form of batches bythe American Type Culture Collection (ATCC), maintainedby subculture on nutrient agar favorable to their growth, andobtained from the Fungus Collection Mycology Laboratoryof the Forest Research Centre of Rabat, Morocco.

2.3. Nutritional Value. Protein, carbohydrates, lipids, fibers,ash, and micronutrient content were expressed on a

microalga dry weight basis, and the results were presented ing/100 g. Analyses were carried out in three replicates.

Ash content: it was determined by AACC [15]. Sampleswere preweighed in a crucible and combusted in amuffle furnace (Nabertherm, 30–3000°C) at 550°C for4 hours. +en, they were cooled with the door left openand weighed, and the ash content was calculated.Moisture: it was measured by drying at 80°C for 24 h,and the samples and their results were reported on a dryweight basis [16].Crude protein (CP): it was quantified using the Kjeldahlmethod by the AOAC [17].Crude fat (fat): it was determined using the Soxhletmethod by the AACC [15]. It was extracted with ethylether, and the obtained mixture was concentratedunder vacuum. +e obtained fat was weighed andexpressed as a percentage (%).Total fiber fractions: crude fiber (CF) was assessedaccording to the AOAC method [17]. +e neutraldetergent fiber (NDF), acid detergent fiber (ADF),and acid detergent lignin (ADL) were determinedaccording to the method described in [18]. In fact, foreach sample, all fiber fractions previously mentionedwere sequentially determined. +e obtained hemi-cellulose was calculated according to the methodin [19].Mineral composition: macroelements including K+ andNa+ contents were determined using a flame pho-tometer, while Mg2+ and Ca2+ contents were de-termined using complexometric titration. On thecontrary, phosphorus content was measured by usingthe acidified solution reaction of ammonium molyb-date containing ascorbic acid and antimony [20].Microelements including Cu, Zn, Fe, Mn, and Ni weredetermined using atomic absorption spectrometry inthe air-acetylene flame [21].Carbohydrates (CHO): they were quantified accordingto the method in [22], by a difference of all othercomponents as weight in grams minus water, fiber, ash,fat, and protein content.Energy value (E): it was calculated according to themethod in [22], by calculating the approximate com-position data. Energy (kcal) was calculated according tothe following equation:

E(kcal) � CP × 4 + CHO × 4 + fat × 9. (1)

Values were expressed in kcal/100 g.

2.4.Microbiological Quality. +emicrobiological stability ofthe packaged sample was analyzed by measurement of totalaerobic mesophilic flora (NF EN ISO 4833), Staphylococcus(NF EN ISO 6888-2 at 37°C), total coliforms (NFV 08-050 at30°C), fecal coliforms (NFV 08-060 at 44°C), sulfite-reducingClostridium bacteria (NFV 08-061), yeasts and molds

2 Journal of Food Quality

(NF V08-059 at 28°C), Salmonella (NF EN ISO 6579), andenterobacteria (NF V 08-054).

2.5. Physicochemical Analysis and Characterization

2.5.1. Total Polyphenol Assay. It was estimated by theFolin–Ciocalteu method [23]. Gallic acid was used as astandard curve for quantification of PC. +e results wereexpressed as gallic acid equivalent (GAE)/g of dry matter ofSpirulina.

2.5.2. Total Flavonoid Assay. It was assessed by applying thealuminum chloride colorimetric method according to thestudy in [24]. Flavonoid concentrations were deduced fromthe range of the calibration curve established with quercetin.+e results were expressed in milligram equivalents ofquercetin per gram of dry matter (mg EQ/g dry matter).

HPLC-DAD-QTOF/MS Analysis of Polyphenolic Com-position. +e Agilent 1100 high-performance liquid chro-matography (HPLC) system (Agilent Technologies, Inc.,Wilmington, DE, USA) is composed of a diode array de-tector (DAD) (G1315B), an autosampler (G1330B), and abinary pump (G1312A). +e mass spectrometer (MS) de-tector was connected to an electrospray ionizer (MicromassQuattro Micro; Waters Technologies, Milford, MA, USA).+e elution separation was carried out in a reverse phasebased on Zorbax column phase C18 (Agilent Technologies;100mm× 2.1mm× 1.7 μm).+e following chromatographicconditions were applied: injection volume 10 μl; columntemperature 35°C; flow of injection 0.5ml/min; mobile phasecompositions: acetonitrile with 0.1% formic acid (A) and0.1% formic acid in water (v/v) (B); and gradient elution(v/v): 10% A (0min), 70% A (18min), 10% A (2min), 10% A(3min), 10% A (2min), and 10% A (5min). +e negativemode was applied with the following conditions: voltage ofcapillary 3.0 kV, voltage of cone 20V, voltage of extractor2V, temperature source 100°C, desolvation temperature350°C, desolvation gas flow 350 l/h, and cone gas flow 30 l/h.+e phenolic acid peak identification was carried out bycomparison of retention times and MS spectra with those ofpure standards and by molecular ion identification (Sigma-Aldrich, France). On the contrary, the quantification ofphenolic acids was done based on standard calibrationcurves. Spirulina samples were ground as powder, dissolvedin water in order to obtain 1mg/mL concentration, and thenfiltered by a 0.2 μm (PVDF) syringe filter prior to thechromatographic analysis.

2.6. Bioactivity Analysis

2.6.1. Preparation of Methanolic Extract. 10 g of eachpowder sample was crushed in a solvent (MeOH) (1 L) for48 h at 24°C in dark and stirred [25] with slight modifica-tions. +e extracts were centrifuged at 5000 rpm for 15min,filtered, diluted with 10% of dimethyl sulfoxide (DMSO),and then stored in glass vials in dark at 4°C before use.DMSO was used as a negative control.

2.6.2. Antioxidant Activity. +e radical-scavenging 2,2-diphenyl-1-picrylhydrazyl (DPPH) test was used. Each testwas repeated in triplicate. +e procedure was followed asdescribed by Lopes-Lutz [26].+e DPPH solution (5.91mgof DPPH and 50ml of methanol) was mixed withmethanolicextracts at a different concentration (2–600 μg/ml) (1ml;0.3mM) of DPPH and then incubated for 30min in dark. Asolution control (blank) was prepared by dissolving theDPPH solution in pure methanol. +e reduction in DPPHfree radicals was determined at 517 nm. +e positive controlwas prepared with L-ascorbic acid, and the inhibition ratio(%) was calculated using the following equation:

% inhibition � [(Abs of control –Abs of test sample)

÷Abs of control] × 100%.

(2)

+e antioxidant activity of each sample was determinedbased on the inhibition curve and was expressed in terms ofIC50.

2.6.3. Antimicrobial Assays. +e minimum inhibitoryconcentrations (MICs) of the methanolic extract wereassayed according to the procedure described in [27] andmodified in [28, 29]. In vitro antibacterial studies werecarried out against four bacterial strains: the Gram-positivebacteria S. aureus, B. subtilis, and M. luteus and the Gram-negative bacterium E. coli. Moreover, one mold (Aspergillusniger) and one mushroom wood rot (Coriolus versicolor)were also employed. Quantities of these extracts were addedto test tubes containing nutrient agar at 0.2% for bacteria andpotato dextrose agar (PDA) for molds to promote germ/compound contact in test tubes. Each contained 13.5mltryptic soy agar medium, sterilized by autoclaving (20min at121°C), cooled to 45°C, and then poured into Petri dishes.1.5ml of each dilution was added in such a way to obtain afinal concentration of 1/100 to 1/5000 (v/v). Controlsconsisting of the medium plus 0.2% agar-agar solution alonewere also prepared. +e results were considered in thecontext of calibrated platinum loops in the same volume ofinoculum. +e inoculum was in the form of culture broth(24 h, 37°C) for bacteria and in the form of suspension inphysiological water of spores from a seven-day culture (7 d,25°C) in the PDA for molds. Each test was done in triplicateto minimize experimental error.

2.7. Statistical Analyses. Results were expressed as meanvalues± standard deviation of three separate determinations.

3. Results and Discussion

3.1. Gross Biochemical Composition. Microalgae were con-sidered an alternative source of protein, thanks to their highcontent of proteins [30]. +e gross chemical composition ofthe analyzed Spirulina is summarized in Table 1.

+e Moroccan strain studied contains a considerableamount of protein (76.65± 0.15%). +is value was higherthan that of various Spirulina species harvested from other

Journal of Food Quality 3

countries [31, 32], with its amino acid composition ofaspartic acid, glutamic acid, serine, glycine, histidine, argi-nine, threonine, alanine, proline, valine, isoleucine, leucine,phenylalanine, and lysine shown in [33]. It also showed afairly high protein concentration compared to some of othermicroalgae (6 to 71%) [34, 35]. It was also higher than that ofthe most of red and green seaweeds (10 to 47%) of dry matter[36] and brown edible seaweeds (3 to 15% dw) [37]. +isresult confirms that the protein content in Spirulina wasconsiderably higher than those in some plants [5]. Fur-thermore, several studies have demonstrated that the proteincontent of microalgae varied according to species, envi-ronmental conditions, and analytical methods for proteindetermination [38, 39].

Lipids represent one of the main sources of energy forhuman metabolic processes. Spirulina lipid contains poly-unsaturated fatty acids (PUFAs), mainly c-linoleic acid with30 to 35% of total lipid content, which are functional andstructural components of cell membranes, while the mainlipid membrane constituents in Spirulina are glycolipids andthe essential fatty acids from theω3 andω6 families [40].+eMoroccan samples contained 2.45± 0.82% dw of fat. +isvalue was much lower than that reported in previous studieson other Spirulina products (6 to 13%) [31, 32]. On thecontrary, it was lower than the content of marine and somefreshwater microalga species where lipid content was above60% of dw [34, 35]. Although it was reported that edible lipidseaweed content does not obviously exceed 5% of dry matter,our result is also following the literature range [36, 37]. +isresult confirms that Spirulina appears very favorable for low-fat diets relatively to vegetables [5, 42]. Furthermore, thisdifference might relate to the strain, different extractionmethods, solvents used, or nutritional and environmentalfactors [50].

Microalga carbohydrates (e.g., starch, glucose, sugars,and other polysaccharides) normally generate energy andcellular structure. +e carbohydrate content was about

6.46± 0.32%. +is value was considerably lower than thecarbohydrate concentration average of 15 to 25% reportedpreviously for other Spirulina strains [31, 32]. Besides, theSpirulina studied showed a carbohydrate content typicallybelow average for many microalgae, overall accounting for10 to 27% dw [34, 35]. In addition, it was lower than thatreported in edible seaweeds which ranged from 20 to 68%[36, 37, 43]. +e soluble carbohydrates constitute a majorpart of the total carbohydrate content, whereas in higherplants, insoluble carbohydrates are a major constituent oftotal carbohydrates [44].+is result agrees with other reportsthat showed dried whole microalgae can be used as food dueto their higher carbohydrate digestibility. However, likecarbohydrate composition, the protein content of algae waslinked to the seasonal variation [50].

Algae were characterized by a higher ash content. +eash content for Moroccan samples was 14.56± 0.74%. +isvalue was higher than that found previously in other Spir-ulina species (7.4 to 10.4%) [31, 32, 45]. +is value might behigh because of inadequate grinding of the samples prior toanalysis. Indeed, ash content of our Spirulina was found as20% which was also compared with freshwater and marinemicroalgae which range from 4% to 20% [46]. However, itwas lower than the ash content shown in edible seaweeds,which in general have a high content of minerals reflected inthe ash content [36, 37, 47].

Moisture is an important factor for assessing themicroalga quality.+e drymatter content was 87.70± 1.85%.In fact, companies provide standards in their nutritionalinformation and set the moisture standard below 9% [48].+is value could be due to drying methods and drying time[49]. On the contrary, the packaging and the storage con-ditions might have an indirect influence on the humidityrate. +e microalgae should not be dried extremely becauseit could change the structure of living cells which woulddegrade their physicochemical properties. +erefore, theiroverall quality will not be optimal.

+e dietary fiber content of algae was of high nutritionalimportance. +e present study was focused on the de-termination of crude fiber (CF, lignocellulose complex) andsome polysaccharides of fibrous nature which would beidentified further by future studies using highly sophisti-cated techniques. +e cellulose and hemicellulose play animportant role as structural components of the cell wall inalgae [50]. +e crude fiber content was 4.07± 1.42% higherthan that from seaweeds [51] and than that from terrestrialplants or vegetables [52]. Besides, the value was in themiddleof the seaweed content (1.36–7.73%) [53]. +erefore,Moroccan Spirulinamight be considered as a good source ofdietary fiber like several edible seaweeds.

Generally, algae were considered to have a high ashcontent, essential minerals, and trace elements required forhuman nutrition. Given the ash content in Spirulina inTable 1, a high mineral content was predicted. Nutrientsufficiency is essential for productivity and longevity. +emineral result analyses are shown in Table 2. +e studiedmicroalga contained higher amounts of the macrominerals(32694.32± 6175.08mg/100 g·dw) and trace elements (88.44± 3.2mg/100 g·dw) required for human nutrition [54].

Table 1: Global nutritional analysis of Spirulina (Morocco) (% ofdry matter).

Elements Percentage (%)b Other micro/macroalgaeMoisturea (%) 12.66± 1.7 <9% [48]

Ash (%) 14.56± 0.74 7.4–10.4% [5, 37, 54]4–20% [55, 56]

Protein (%) 76.65± 0.1560–71 [5, 36, 37],6–71% [39–45]3–47% [46, 47]

Lipids (%) 2.45± 0.826–13% [5, 37]>60% [39–45]>5% [46, 47]

CF (%) 4.07± 1.42 1.36–7.73% [53]

Carbohydrates (%)c 6.46± 0.3215–25% [5, 36, 37]10–27% [39–45]20–68% [46, 47]

Energy (kcal/100 g) 436.18± 2.29 NDNote. aExpressed as percentage of freeze-dried samples. bData are meanvalues± SD of three determinations. cCalculated by difference (�100− crudeprotein− crude lipid− total dietary fiber (TDF)−ash). Dried Spirulina sp. %at 70°C of cell constituents is calculated after the moisture content wassubtracted. ND: not detected.

4 Journal of Food Quality

In addition, it contained significant amounts of essentialminerals such as P (10088.33± 5766.88), Na (14004±397.55), K (2501.66± 4.22), Ca (6000± 4.66), and Mg(100.33± 1.77mg/100 g·dw), respectively. Trace elementswere also detected (Fe, 80.66± 1.77; Zn, 5± 0.66; Cu,1.22± 0.66; and Mn, 1.56± 0.11mg/100g·dw, respectively).+e macroelements found were slightly higher than thosefound in marine microalgae, which is interesting as Zar-rouk’s medium supplies them in excess, except for Mg whichdisplays a lower value compared to them. Otherwise, themicroelements Cu, Mn, and Fe were presented in themicroalga studied with modest values compared to marinemicroalgae, excluding the Zn content [55]. +ese resultswere relatively higher than those of the most land plants(5–10%) [56]. +e mineral content is highly related tophysiological and environmental factors, processing, andmineralization methods involving dry mineralization in anoven at 550°C [17, 57]. Consequently, the study confirms thatSpirulina’s benefits may be attributed to both mineral andtrace element contents.

3.2. Microbiological Analysis. +e edible product was ex-posed to all contamination vectors. +e microbial moni-toring of marketed Spirulina powder is necessary since it ispossible to affect the product quality by reducing theavailability of nutrients. +e microbiological quality of ed-ible Spirulina is shown in Table 3.With respect to pathogenicmicroorganisms, the presence of total coliforms andStaphylococcus (<100 cfu/100ml) may present a hazard.However, the absence of fecal coliforms indicates hygienicstandards of the adopted technology and preparation wererespected. Salmonella and sulfito-reducer bacteria, specifi-cally Clostridium perfringens, were also not detected. +epresence of enterobacteria, in particular Escherichia coli at40%, which should be absent according to the EU standard,indicates fecal contamination. Finally, the yeasts and moldspresent in the ground and feces may have been transmittedvia dust by wind, insects, or equipment. In fact, the microbialqualities of the samples from the routinely cultivated bio-mass by the company were moderately satisfactoryaccording to the European Union and World Health Or-ganization standards. In order to ensure its safety as food, itwas necessary to control total coliforms, Staphylococcus, andEscherichia coli which could be responsible for toxicitypotential. Otherwise, except the negative findings, these

results were in agreement with those shown in [58], whichconfirmed that the microflora associated with Spirulinacrops was generally nonpathogenic. In addition, a high al-kalinity of the Spirulina environment is normally an ex-cellent barrier to contamination, whether by bacteria, fungi,or algae. Furthermore, certain substances of both intracel-lular and extracellular metabolites as antimicrobial agentssuch as terpenols, sterols, polysaccharides, dibutenolides,peptides, and protein metabolites secreted by or present inSpirulina have a bactericidal or at least bacteriostatic effecton humans [59, 60]. +e final microbial load of the producthas been reported to depend on how carefully the cultureand product are handled at various stages [61]. +erefore,subsequent handling of the product during harvest, drying,and packaging is very important and in this case appears tobe generally satisfactory (Figure 1).

3.3. Phytochemical Analysis and Characterization

3.3.1. Total Phenolic Content (TPC) and Total FlavonoidContent (TFC). Spirulina was considered a good source ofnutritional phenolic and flavonoid compounds due to itshigher production capacity compared to conventional plant-derived sources. As shown in Figure 2, the TPC and TFC ofmethanolic extracts from Spirulina were determined interms of milligrams of gallic acid equivalents per gram of dryweight (mg GAE/g dw) from the calibration curve of gallicacid and milligrams of quercetin equivalents per gram of drymicroalga extract (mg QE/g DE) through the calibrationcurve of quercetin, respectively. +e obtained resultsrevealed the presence of polyphenols (0.287mg GAE/g DW)and total flavonoids (0.166mg QE/g DE). +ese results werein agreement with the total phenolic content level (<5mgGAE/g DW) for different microalgal species and also fordifferent extracts [62, 63]. Phenolic compounds are sec-ondary metabolites commonly found in Spirulina [64], withsome of the main forms being salicylic, chlorogenic, synapticcaffeic, and trans-cinnamic acids, which have been knownfor their chemical protective mechanisms against someagents of biotic and abiotic stresses [65]. It also containsvarious classes of flavonoids like isoflavones, flavonols,flavanones, and dihydrochalcones. +ese natural phenolicshave probably a broad spectrum of chemical and biologicalproperties including antioxidant and free radical-scavengingactivities. Moreover, the total phenolic contents obtained

Table 2: Mineral composition of Spirulina (Morocco) (mg/100g dw).

Elements Spirulina studied Other microalgae [55]

Minerals (mg/100 g dw)

P 10088.33± 5766.88 1700–3000Na 14004± 397.55 7000–1100K 2501.66± 4.22 600–1200Ca 6000± 4.66 300–2100Mg 100.33± 1.77 100–1100

Trace elements (mg/100 g dw)

Fe 80.66± 1.77 100–700Zn 5± 0.66 23.9–370Cu 1.22± 0.66 1.2–65Mn 1.56± 0.11 3.7–59.2

Journal of Food Quality 5

showed signi�cant variations [66] which could be explainedby geographical, physiological, and environmental factorsand culture conditions [67].

3.3.2. Phenolic Acid and Flavonoid Composition. A chro-matographic separation method (HPLC-DAD/MS) wasdeveloped for simultaneous identi�cation and quanti�cationof phenolic acids and �avonoids in Spirulina reported topossess vital nutritional and biological activities [68]. A totalof twenty phenolic acid and �avonoid compounds wereproperly identi�ed and quanti�ed compared with phenolicstandards (retention times, mass spectra, and their frag-mentations) within 30min. Table 3 shows the quanti�cationresults and some information about phenolic acids and�avonoids (molecular formula, molecular weight, [M-H]−values, mass spectra, and retention times) in an aqueousextract from Spirulina.  e phenolic and �avonoid amountsin an aqueous extract were determined, and the extract wasrich in succinic acid (1122.88mg/kg), followed by quinicacid (844.17mg/kg), 3-4-hydroxybenzoic acid (687.07mg/kg), catechin (584.53mg/kg), citric acid (64.06mg/kg),vanillic acid (16.24mg/kg), gallic acid (2.13mg/kg), 4-hydroxybenzoic acid (1.07mg/kg), and trace elements(<1mg/kg) of rutin, chlorogenic acid, quercetin, rosmarinicacid, salicylic acid, resveratrol, pyrogallol, ferulic acid, 4-hydroxycinnamic acid, 3-hydroxycinnamic acid, and 2-hydroxycinnamic acid. A list of other phenolic acids and

�avonoids is also shown in Table 4.  e phenolic acidamounts were varied in Spirulina species depending onculturing conditions [67, 69]. Only gallic, ca�eic, salicylic,chlorogenic, and trans-cinnamic acids were detected inprevious studies with Spirulina algae [69, 70].

3.3.3. Antioxidant Bioactivity Analysis.  e total antioxi-dant capacity (TAC) of the methanolic extract was de-termined using the DPPH radical-scavenging assay.  eresults are displayed in Figure 3.

 e DPPH radical-scavenging activity was generallyquanti�ed in terms of inhibition percentage of the pre-formed free radical by antioxidants and EC50 (concen-tration required to obtain a 50% antioxidant e�ect) [71]. e EC50 was widely used to express the antioxidantcapacity and to compare the activity of di�erent com-pounds. In this study, the EC50 was 23 μg/ml, indicating avery high antioxidant potential [72]. Both the lower EC50and the higher DPPH activity were related to a high an-tioxidant activity [73].  e study has been carried out onthe DPPH-scavenging activity of the Spirulina extractcompared to both vitamins C and E which showed thatEC50 was about 40 ppm; i.e., one is better than the otherwhich is comparable, as shown in [71]. Previous reportshave revealed that the antioxidant activity of Spirulinamayarise from a whole spectrum of natural antioxidantcompounds that contribute to the oxidation process in-hibition [74].  e phenolic compounds are mostly foundin extracts of higher polarity and seem to be related toantioxidant activity or synergistic action, thanks to theirredox properties [75]. However, the presence of di�erent

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

TFC TPC

Figure 2: Total phenolic content (TPC) and total �avonoid content(TFC) of the methanolic extract of Spirulina expressed as μgmgGAE/g DW and mg QE/g DE, respectively (n� 3).

Nutritionalcomposition

Mineral assay

Physicochemicalanalysis

Biologicalactivities

Nutraceutical value

Functional food:Spirulina sp.

Food safetycontrol

Figure 1: Crosstalk between nutritional and physicochemicalproperties and bioactivities in the functional food Spirulina species.

Table 3: Microbiological quality of edible Spirulina (cfu/ml).

Pathogens All aerobic mesophilic�ora Staphylococcus Total

coliformsFecal

coliformsSul�to-reducer

bacteriaYeasts andmolds E. coli Salmonella

Bacterialcount 208 93 26 ND ND 14 4.15 ND

EUstandard 105 Absent Absent No data 104 104 Absent Absent

WHO No data No data No data No data 103 Nodata Absent

EU, European Union [41]; WHO, World Health Organization [42]; ND: not detected.

6 Journal of Food Quality

antioxidant compounds in the methanol extract was re-sponsible for the free radical-scavenging activity, eitherindividually or synergistically.

3.3.4. Antimicrobial Activity. +e growth of pathogenic andcontaminant microorganisms in food decreases the nu-tritional quality and increases food toxicity. +e anti-microbial activity of the methanolic extract of Spirulinaagainst microorganisms that contaminate food productswas investigated. +e results are presented in Table 5. +emethanolic extract had no antimicrobial activity againstthe bacteria and molds studied. Nevertheless, it had an-tifungal activity against Coriolus versicolor, with a min-imum inhibitory concentration (MIC) between 1/250 and1/100 v/v of 1 g/10ml. +ese results disagree with those ofprevious studies, in which Spirulina has been declared asbioactive-rich compounds in that microbial growth could

be promoted or inhibited [76]. Indeed, it has been re-ported that the methanolic extract of Spirulina platensispossesses antimicrobial and antifungal potential againstmany pathogenic plant fungi and against all the bacteriatested [74]. +e antimicrobial activity of the methanolicextract of studied Spirulina has been attributed to thepresence of functional lipids, mainly c-linolenic acid, andan antibiotically active fatty acid [77]. In fact, lipids killmicroorganisms (bacteria, fungi, and yeasts) by reach-ing the bacterial membrane and causing their disinte-gration [78]. Indeed, they are disrupting the extensivemeshwork of peptidoglycans in the cell wall withoutvisible changes. c-Linolenic acid and other fatty acidsknown for their antimicrobial activity are highly pre-sented in Spirulina. Study [79] showed that the synergisticeffect between these fatty acids is involved in their anti-microbial activity [80].

Table 4: Phenolic and flavonoid compounds determined in the Spirulina-evaluated samples (mg/kg).

Phenolic acids Spirulina Molecular formula Molecular weight (M)HPLC-ESI/MS (m/z)

RT (min) [M-H]−

Quinic acid 844.17± 42.21 C7H12O6 192.167 0.51 191.120Citric acid 64.06± 3.20 C6H8O7 192.123 0.63 191.102Pyrogallol 0.42± 0.02 C6H6O3 126.111 0.64 125.024Succinic acid 1122.88± 56.14 C4H6O4 118.088 0.65 117.018Gallic acid 2.13± 0.11 C7H6O5 170.022 0.66 168.90Chlorogenic acid 0.86± 0.04 C16H18O9 354.311 0.83 353.2023,4-Hydroxybenzoic acid 687.07± 34.35 C7H6O4 154.121 0.95 153.0104-Hydroxybenzoic acid 1.07± 0.05 C7H6O3 138.122 1.30 137.050Catechin 584.53± 29.22 C15H14O6 290.271 1.63 289.064Vanillic acid 16.24± 0.81 C8H8O4 168.148 2.12 167.0364-Hydroxycinnamic acid 0.12± 0.01 C9H8O3 164.160 2.21 163.042Rutin 0.93± 0.05 C27H30O16 610.153 2.63 609.13-Hydroxycinnamic acid 0.15± 0.01 C9H8O3 164.160 2.98 163.042Ferulic acid 0.48± 0.02 C10H10O4 194.186 3.12 193.050Quercetin 0.01± 0.00 C15H10O7 302.238 3.48 301.0002-Hydroxycinnamic acid 0.31± 0.02 C9H8O3 164.160 3.65 163.042Salicylic acid 0.08± 0.003 C7H6O3 138.122 3.68 137.025Rosmarinic acid 0.18± 0.01 C18H16O8 360.318 4.01 359.054Resveratrol 0.10± 0.005 C14H12O3 228.247 5.83 227.072Quercitrin 0.04± 0.00 C21H20O11 448.38 5.99 447.1204-Hydroxycoumarin ND C9H8O3 164.160 ND 163.042Aesculin ND C15H16O9 340.284 ND 339.072Epigallocatechin gallate ND C22H18O11 458.375 ND 457.078Esculetin ND C9H6O4 178.143 ND 177.018Kaempferol ND C15H10O6 286.239 ND 285.040Luteolin ND C15H10O6 286.239 ND 285.040Malic acid ND C4H6O5 134.087 ND 133.014Syringic acid ND C9H10O5 198.174 ND 197.0453-Hydroxybenzoic acid ND C7H6O3 138.122 ND 137.025Benzoic acid ND C7H6O2 122.123 ND 121.031Caffeic acid ND C9H8O4 180.159 ND 179.035Epicatechin ND C15H14O6 290.271 ND 289.064Hesperetin ND C16H14O6 302.282 ND 301.015Hesperidin ND C28H34O15 610.565 ND 609.172Naringenin ND C15H12O5 272.256 ND 271.061Naringin ND C27H32O14 580.539 ND 579.173Pyrocatechol ND C6H6O2 110.112 ND 109.028Sinapic acid ND C11H12O5 224.212 ND 223.061Tannic acid ND C76H52O46 1701.206 ND 1700.080ND: not detected.

Journal of Food Quality 7

4. Conclusions

Given its chemical composition, rich nutritional value, andantimicrobial and antioxidant activities, the MoroccanSpirulina has important nutraceutical potential. Further-more, pharmacochemical, experimental, and clinical studieson the Moroccan Spirulina are required to identify itsmechanism of action as a complement of traditionalpharmacopeia.

Data Availability

 e data used to support the �ndings of this study are in-cluded within the article.

Conflicts of Interest

 e authors declare that they have no con�icts of interest.

Acknowledgments

 e authors are grateful to the National Center for Agri-cultural Research, a Research Unit on Agri-Food Technologyand Quality (Rabat); University Center for Analysis, Ex-pertise, Transfer of Technology and Incubation, University

Ibn Tofaıl, Kenitra; Research Laboratory of Biotechnologyand Biomolecule Engineering (ERBGB), Faculty of Scienceand Technology, Abdelmalek Essaadi University, Tangier;and Center of Forest Research, at Research Unit of Medi-cines and Microbiology, Rabat, Morocco, for the researchfacilities.  e algal supplier “Spirulina-Berbere” (Morocco)is thanked for providing samples.

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Table 5: Inhibitory volumes and minimum inhibitory concentrations of the methanolic extract of Spirulina (Morocco).

Concentration 1/100 1/250 1/500 1/1000 1/2000 1/3000 1/5000 Control(mg/ml) 0.001 0.0004 0.002 0.001 0.00005 0.000033 0.00002 0Bacteria

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y = 27.785x – 21.67R2 = 0.9242

0

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8 Journal of Food Quality

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