# Functional Composition, Nutritional Properties, and Biological Activities of MoroccanSpirulina Microalga

Authors: Rajaa Seghiri; Mourad Kharbach; Azzouz Essamri
Journal: Journal of Food Quality
 (2019)

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## Abstract

The present study aimed to characterize the nutraceutical properties and the antimicrobial effect of MoroccanSpirulina (Arthrospira platensis). The nutritional composition was evaluated, including water content, crude protein, total carbohydrates, lipids, phenolic composition, macro- and micromineral content, fiber content, and energy value. Then, the microbiological analysis and antioxidant activity were measured. The antimicrobial activity was evaluated using the minimum inhibitory concentration method on bacteria and fungi. Moroccan Spirulina contained a large amount of protein (76.65 ± 0.15%), followed by carbohydrates (6.46 ± 0.32%), minerals (20.91 ± 0.88%), crude fiber (4.07 ± 1.42%), lipids (2.45 ± 0.82%), ash (14.56 ± 0.74), and twenty phenolic acids being identified and quantified. Moreover, flavonoid and phenolic contents were present at 15.60 ± 2.74 mg RE/g dw and 4.19 ± 0.21 mg GAE/g dw, respectively. Microbiological risk assessment indicated that this product is safe to be consumed as a human food product. The antioxidant activity was higher in the methanolic fraction (23 mg TE/g dw) (DPPH).


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## Body

## 1. Introduction

The Moroccan coast extends over 3500 km (2900 km for the Atlantic coast and 500 km for the Mediterranean coast). This ecosystem was recognized as vital and fragile but with a considerable ecological value. Despite increasing recognition of different foods and their safety, algae have received small interest in spite of their wide and abundant availability. Algae were one of the first life forms on Earth, and several thousand species existed [1]. On the contrary, microalgae were considered an important part of aquatic biodiversity; they were grown in different environments (sea, freshwater, and desert) and also in different forms (single cells, colonies, or filaments) [2]. Recently, algae have been used as food supplements in order to enhance their nutritional value, as animal feed additives, and even for pharmaceutical uses [3, 4]. However, they have diverse chemical properties and were 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 terrestrial plants [6]. Polyphenolic compounds were known as important natural antioxidants, while numerous classes of flavonoids and other classes of phenolics were found in microalgae. In addition, various polyphenolic molecules were associated with biopharmacological activities including antimicrobial and antioxidant actions [7, 8]. The commercial strains of edible microalgae were mainly dominated by Chlorella, Arthrospira, Dunaliella salina, and Aphanizomenon flos-aquae. Towards a more food-secure future, the Spirulina cyanobacterium (Arthrospira platensis) became trendy health food brands, which explained its culture in several countries [9]. Various companies sell a variety of Arthrospira-based products (tablets and powder) as a food supplement and distribute them to over 20 countries around the world. Therefore, they have been widely introduced (fresh or dried) in the human diet and nutrition.Spirulina is an abundant source of nutritious composition, including protein, vitamins, lipids, fibers, minerals, carbohydrates, and some of natural pigments [10]. Spirulina was recognized as a safe supplement (GRAS) without toxicological effect and was officially approved by the Food and Drug Administration (FDA) and the National Sanitary Surveillance Agency (ANVISA). These main constituents confer a physiological potential and a functional benefit that makes it promising for the food industry to overcome the problem of malnutrition and in health applications against pathogens [11, 12]. Right from the beginning of the applied phycology, the algae’s chemical composition and their main bioactive compounds have received an intense interest [13]. The nutritional and toxicological assessments of commercial algae foodstuff were little studied and often based on reports from field or interlaboratory ecological or physiological studies [14]. According to the best of our knowledge, no studies have been performed on the nutraceutical properties of a strain of Spirulina isolated in Morocco. The present work aims to evaluate the Moroccan Spirulina’s nutritional and pharmacological properties. Firstly, the chemical and nutritional compositions including phenolic profiling were investigated. Secondly, antimicrobial and antioxidant activities were evaluated.


## 2. Materials and Methods

### 2.1. Raw Materials

Spirulina platensis (Arthrospira platensis) used in this study was provided by the Spirulina-Berbère® company, located in the region of Souss-Massa-Drâa, in the south of Morocco. From an artificial pond culture in March 2016, the microalga was grown by company’s process on Zarrouk’s medium implemented under cover in a greenhouse under the following environmental conditions: at pH = 10.25, salinity = 16, and shading = 65% and under the solar light between 23 and 30°C with constant shaking. Growth was monitored by measuring water transparency using the test of Secchi. After harvesting and spinning, microalga cake was dried in a hot air oven at 70°C. Finally, dried samples were crushed and sieved at 1 mm pore diameter to obtain a fine and homogeneous powder using a mill (MF 10 basic, IKA-WERKE). The powder was stored in sealed plastic bags in desiccators at room temperature for further chemical analysis. To facilitate reading, Spirulina was used as a generic name for commercial products from Arthrospira spp.


### 2.2. Chemicals and Reagents

All chemical reagents and solvents used were provided by Sigma-Aldrich (St. Louis, MO). Bacterial strains studied are in the form of batches by the American Type Culture Collection (ATCC), maintained by subculture on nutrient agar favorable to their growth, and obtained from the Fungus Collection Mycology Laboratory of 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 in g/100 g. Analyses were carried out in three replicates.Ash content: it was determined by AACC [15]. Samples were preweighed in a crucible and combusted in a muffle furnace (Nabertherm, 30–3000°C) at 550°C for 4 hours. Then, they were cooled with the door left open and 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 dry weight basis [16].Crude protein (CP): it was quantified using the Kjeldahl method by the AOAC [17].Crude fat (fat): it was determined using the Soxhlet method by the AACC [15]. It was extracted with ethyl ether, and the obtained mixture was concentrated under vacuum. The obtained fat was weighed and expressed as a percentage (%).Total fiber fractions: crude fiber (CF) was assessed according to the AOAC method [17]. The neutral detergent fiber (NDF), acid detergent fiber (ADF), and acid detergent lignin (ADL) were determined according to the method described in [18]. In fact, for each sample, all fiber fractions previously mentioned were sequentially determined. The obtained hemicellulose was calculated according to the method in [19].Mineral composition: macroelements including K+ and Na+ contents were determined using a flame photometer, while Mg2+ and Ca2+ contents were determined using complexometric titration. On the contrary, phosphorus content was measured by using the acidified solution reaction of ammonium molybdate containing ascorbic acid and antimony [20]. Microelements including Cu, Zn, Fe, Mn, and Ni were determined using atomic absorption spectrometry in the air-acetylene flame [21].Carbohydrates (CHO): they were quantified according to the method in [22], by a difference of all other components as weight in grams minus water, fiber, ash, fat, and protein content.Energy value (E): it was calculated according to the method in [22], by calculating the approximate composition data. Energy (kcal) was calculated according to the following equation:(1)Ekcal=CP×4+CHO×4+fat×9.Values were expressed in kcal/100 g.


### 2.4. Microbiological Quality

The microbiological stability of the packaged sample was analyzed by measurement of total aerobic mesophilic flora (NF EN ISO 4833),Staphylococcus (NF EN ISO 6888-2 at 37°C), total coliforms (NFV 08-050 at 30°C), fecal coliforms (NFV 08-060 at 44°C), sulfite-reducing Clostridium bacteria (NFV 08-061), yeasts and molds (NF V08-059 at 28°C), Salmonella (NF EN ISO 6579), and enterobacteria (NF V 08-054).


### 2.5. Physicochemical Analysis and Characterization

#### 2.5.1. Total Polyphenol Assay

It was estimated by the Folin–Ciocalteu method [23]. Gallic acid was used as a standard curve for quantification of PC. The results were expressed as gallic acid equivalent (GAE)/g of dry matter of Spirulina.


#### 2.5.2. Total Flavonoid Assay

It was assessed by applying the aluminum chloride colorimetric method according to the study in [24]. Flavonoid concentrations were deduced from the range of the calibration curve established with quercetin. The results were expressed in milligram equivalents of quercetin per gram of dry matter (mg EQ/g dry matter).HPLC-DAD-QTOF/MS Analysis of Polyphenolic Composition. The Agilent 1100 high-performance liquid chromatography (HPLC) system (Agilent Technologies, Inc., Wilmington, DE, USA) is composed of a diode array detector (DAD) (G1315B), an autosampler (G1330B), and a binary pump (G1312A). The mass spectrometer (MS) detector was connected to an electrospray ionizer (Micromass Quattro Micro; Waters Technologies, Milford, MA, USA). The elution separation was carried out in a reverse phase based on Zorbax column phase C18 (Agilent Technologies; 100 mm × 2.1 mm × 1.7 μm). The following chromatographic conditions were applied: injection volume 10 μl; column temperature 35°C; flow of injection 0.5 ml/min; mobile phase compositions: acetonitrile with 0.1% formic acid (A) and 0.1% formic acid in water (v/v) (B); and gradient elution (v/v): 10% A (0 min), 70% A (18 min), 10% A (2 min), 10% A (3 min), 10% A (2 min), and 10% A (5 min). The negative mode was applied with the following conditions: voltage of capillary 3.0 kV, voltage of cone 20 V, voltage of extractor 2 V, temperature source 100°C, desolvation temperature 350°C, desolvation gas flow 350 l/h, and cone gas flow 30 l/h. The phenolic acid peak identification was carried out by comparison of retention times and MS spectra with those of pure standards and by molecular ion identification (Sigma-Aldrich, France). On the contrary, the quantification of phenolic acids was done based on standard calibration curves. Spirulina samples were ground as powder, dissolved in water in order to obtain 1 mg/mL concentration, and then filtered by a 0.2 μm (PVDF) syringe filter prior to the chromatographic analysis.


### 2.6. Bioactivity Analysis

#### 2.6.1. Preparation of Methanolic Extract

10 g of each powder sample was crushed in a solvent (MeOH) (1 L) for 48 h at 24°C in dark and stirred [25] with slight modifications. The extracts were centrifuged at 5000 rpm for 15 min, 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

The radical-scavenging 2,2-diphenyl-1-picrylhydrazyl (DPPH) test was used. Each test was repeated in triplicate. The procedure was followed as described by Lopes-Lutz [26].The DPPH solution (5.91 mg of DPPH and 50 ml of methanol) was mixed with methanolic extracts at a different concentration (2–600 μg/ml) (1 ml; 0.3 mM) of DPPH and then incubated for 30 min in dark. A solution control (blank) was prepared by dissolving the DPPH solution in pure methanol. The reduction in DPPH free radicals was determined at 517 nm. The positive control was prepared with L-ascorbic acid, and the inhibition ratio (%) was calculated using the following equation:(2)%inhibition=Abs of control–Abs of test sample÷Abs ofcontrol×100%.The antioxidant activity of each sample was determined based on the inhibition curve and was expressed in terms of IC50.


#### 2.6.3. Antimicrobial Assays

The minimum inhibitory concentrations (MICs) of the methanolic extract were assayed according to the procedure described in [27] and modified in [28, 29]. In vitro antibacterial studies were carried out against four bacterial strains: the Gram-positive bacteria S. aureus, B. subtilis, and M. luteus and the Gram-negative bacterium E. coli. Moreover, one mold (Aspergillus niger) and one mushroom wood rot (Coriolus versicolor) were also employed. Quantities of these extracts were added to test tubes containing nutrient agar at 0.2% for bacteria and potato dextrose agar (PDA) for molds to promote germ/compound contact in test tubes. Each contained 13.5 ml tryptic soy agar medium, sterilized by autoclaving (20 min at 121°C), cooled to 45°C, and then poured into Petri dishes. 1.5 ml of each dilution was added in such a way to obtain a final concentration of 1/100 to 1/5000 (v/v). Controls consisting of the medium plus 0.2% agar-agar solution alone were also prepared. The results were considered in the context of calibrated platinum loops in the same volume of inoculum. The inoculum was in the form of culture broth (24 h, 37°C) for bacteria and in the form of suspension in physiological water of spores from a seven-day culture (7 d, 25°C) in the PDA for molds. Each test was done in triplicate to minimize experimental error.


### 2.7. Statistical Analyses

Results were expressed as mean values ± standard deviation of three separate determinations.


## 2.1. Raw Materials

Spirulina platensis (Arthrospira platensis) used in this study was provided by the Spirulina-Berbère® company, located in the region of Souss-Massa-Drâa, in the south of Morocco. From an artificial pond culture in March 2016, the microalga was grown by company’s process on Zarrouk’s medium implemented under cover in a greenhouse under the following environmental conditions: at pH = 10.25, salinity = 16, and shading = 65% and under the solar light between 23 and 30°C with constant shaking. Growth was monitored by measuring water transparency using the test of Secchi. After harvesting and spinning, microalga cake was dried in a hot air oven at 70°C. Finally, dried samples were crushed and sieved at 1 mm pore diameter to obtain a fine and homogeneous powder using a mill (MF 10 basic, IKA-WERKE). The powder was stored in sealed plastic bags in desiccators at room temperature for further chemical analysis. To facilitate reading, Spirulina was used as a generic name for commercial products from Arthrospira spp.


## 2.2. Chemicals and Reagents

All chemical reagents and solvents used were provided by Sigma-Aldrich (St. Louis, MO). Bacterial strains studied are in the form of batches by the American Type Culture Collection (ATCC), maintained by subculture on nutrient agar favorable to their growth, and obtained from the Fungus Collection Mycology Laboratory of 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 in g/100 g. Analyses were carried out in three replicates.Ash content: it was determined by AACC [15]. Samples were preweighed in a crucible and combusted in a muffle furnace (Nabertherm, 30–3000°C) at 550°C for 4 hours. Then, they were cooled with the door left open and 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 dry weight basis [16].Crude protein (CP): it was quantified using the Kjeldahl method by the AOAC [17].Crude fat (fat): it was determined using the Soxhlet method by the AACC [15]. It was extracted with ethyl ether, and the obtained mixture was concentrated under vacuum. The obtained fat was weighed and expressed as a percentage (%).Total fiber fractions: crude fiber (CF) was assessed according to the AOAC method [17]. The neutral detergent fiber (NDF), acid detergent fiber (ADF), and acid detergent lignin (ADL) were determined according to the method described in [18]. In fact, for each sample, all fiber fractions previously mentioned were sequentially determined. The obtained hemicellulose was calculated according to the method in [19].Mineral composition: macroelements including K+ and Na+ contents were determined using a flame photometer, while Mg2+ and Ca2+ contents were determined using complexometric titration. On the contrary, phosphorus content was measured by using the acidified solution reaction of ammonium molybdate containing ascorbic acid and antimony [20]. Microelements including Cu, Zn, Fe, Mn, and Ni were determined using atomic absorption spectrometry in the air-acetylene flame [21].Carbohydrates (CHO): they were quantified according to the method in [22], by a difference of all other components as weight in grams minus water, fiber, ash, fat, and protein content.Energy value (E): it was calculated according to the method in [22], by calculating the approximate composition data. Energy (kcal) was calculated according to the following equation:(1)Ekcal=CP×4+CHO×4+fat×9.Values were expressed in kcal/100 g.


## 2.4. Microbiological Quality

The microbiological stability of the packaged sample was analyzed by measurement of total aerobic mesophilic flora (NF EN ISO 4833),Staphylococcus (NF EN ISO 6888-2 at 37°C), total coliforms (NFV 08-050 at 30°C), fecal coliforms (NFV 08-060 at 44°C), sulfite-reducing Clostridium bacteria (NFV 08-061), yeasts and molds (NF V08-059 at 28°C), Salmonella (NF EN ISO 6579), and enterobacteria (NF V 08-054).


## 2.5. Physicochemical Analysis and Characterization

### 2.5.1. Total Polyphenol Assay

It was estimated by the Folin–Ciocalteu method [23]. Gallic acid was used as a standard curve for quantification of PC. The results were expressed as gallic acid equivalent (GAE)/g of dry matter of Spirulina.


### 2.5.2. Total Flavonoid Assay

It was assessed by applying the aluminum chloride colorimetric method according to the study in [24]. Flavonoid concentrations were deduced from the range of the calibration curve established with quercetin. The results were expressed in milligram equivalents of quercetin per gram of dry matter (mg EQ/g dry matter).HPLC-DAD-QTOF/MS Analysis of Polyphenolic Composition. The Agilent 1100 high-performance liquid chromatography (HPLC) system (Agilent Technologies, Inc., Wilmington, DE, USA) is composed of a diode array detector (DAD) (G1315B), an autosampler (G1330B), and a binary pump (G1312A). The mass spectrometer (MS) detector was connected to an electrospray ionizer (Micromass Quattro Micro; Waters Technologies, Milford, MA, USA). The elution separation was carried out in a reverse phase based on Zorbax column phase C18 (Agilent Technologies; 100 mm × 2.1 mm × 1.7 μm). The following chromatographic conditions were applied: injection volume 10 μl; column temperature 35°C; flow of injection 0.5 ml/min; mobile phase compositions: acetonitrile with 0.1% formic acid (A) and 0.1% formic acid in water (v/v) (B); and gradient elution (v/v): 10% A (0 min), 70% A (18 min), 10% A (2 min), 10% A (3 min), 10% A (2 min), and 10% A (5 min). The negative mode was applied with the following conditions: voltage of capillary 3.0 kV, voltage of cone 20 V, voltage of extractor 2 V, temperature source 100°C, desolvation temperature 350°C, desolvation gas flow 350 l/h, and cone gas flow 30 l/h. The phenolic acid peak identification was carried out by comparison of retention times and MS spectra with those of pure standards and by molecular ion identification (Sigma-Aldrich, France). On the contrary, the quantification of phenolic acids was done based on standard calibration curves. Spirulina samples were ground as powder, dissolved in water in order to obtain 1 mg/mL concentration, and then filtered by a 0.2 μm (PVDF) syringe filter prior to the chromatographic analysis.


## 2.5.1. Total Polyphenol Assay

It was estimated by the Folin–Ciocalteu method [23]. Gallic acid was used as a standard curve for quantification of PC. The results were expressed as gallic acid equivalent (GAE)/g of dry matter of Spirulina.


## 2.5.2. Total Flavonoid Assay

It was assessed by applying the aluminum chloride colorimetric method according to the study in [24]. Flavonoid concentrations were deduced from the range of the calibration curve established with quercetin. The results were expressed in milligram equivalents of quercetin per gram of dry matter (mg EQ/g dry matter).HPLC-DAD-QTOF/MS Analysis of Polyphenolic Composition. The Agilent 1100 high-performance liquid chromatography (HPLC) system (Agilent Technologies, Inc., Wilmington, DE, USA) is composed of a diode array detector (DAD) (G1315B), an autosampler (G1330B), and a binary pump (G1312A). The mass spectrometer (MS) detector was connected to an electrospray ionizer (Micromass Quattro Micro; Waters Technologies, Milford, MA, USA). The elution separation was carried out in a reverse phase based on Zorbax column phase C18 (Agilent Technologies; 100 mm × 2.1 mm × 1.7 μm). The following chromatographic conditions were applied: injection volume 10 μl; column temperature 35°C; flow of injection 0.5 ml/min; mobile phase compositions: acetonitrile with 0.1% formic acid (A) and 0.1% formic acid in water (v/v) (B); and gradient elution (v/v): 10% A (0 min), 70% A (18 min), 10% A (2 min), 10% A (3 min), 10% A (2 min), and 10% A (5 min). The negative mode was applied with the following conditions: voltage of capillary 3.0 kV, voltage of cone 20 V, voltage of extractor 2 V, temperature source 100°C, desolvation temperature 350°C, desolvation gas flow 350 l/h, and cone gas flow 30 l/h. The phenolic acid peak identification was carried out by comparison of retention times and MS spectra with those of pure standards and by molecular ion identification (Sigma-Aldrich, France). On the contrary, the quantification of phenolic acids was done based on standard calibration curves. Spirulina samples were ground as powder, dissolved in water in order to obtain 1 mg/mL concentration, and then filtered by a 0.2 μm (PVDF) syringe filter prior to the chromatographic analysis.


## 2.6. Bioactivity Analysis

### 2.6.1. Preparation of Methanolic Extract

10 g of each powder sample was crushed in a solvent (MeOH) (1 L) for 48 h at 24°C in dark and stirred [25] with slight modifications. The extracts were centrifuged at 5000 rpm for 15 min, 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

The radical-scavenging 2,2-diphenyl-1-picrylhydrazyl (DPPH) test was used. Each test was repeated in triplicate. The procedure was followed as described by Lopes-Lutz [26].The DPPH solution (5.91 mg of DPPH and 50 ml of methanol) was mixed with methanolic extracts at a different concentration (2–600 μg/ml) (1 ml; 0.3 mM) of DPPH and then incubated for 30 min in dark. A solution control (blank) was prepared by dissolving the DPPH solution in pure methanol. The reduction in DPPH free radicals was determined at 517 nm. The positive control was prepared with L-ascorbic acid, and the inhibition ratio (%) was calculated using the following equation:(2)%inhibition=Abs of control–Abs of test sample÷Abs ofcontrol×100%.The antioxidant activity of each sample was determined based on the inhibition curve and was expressed in terms of IC50.


### 2.6.3. Antimicrobial Assays

The minimum inhibitory concentrations (MICs) of the methanolic extract were assayed according to the procedure described in [27] and modified in [28, 29]. In vitro antibacterial studies were carried out against four bacterial strains: the Gram-positive bacteria S. aureus, B. subtilis, and M. luteus and the Gram-negative bacterium E. coli. Moreover, one mold (Aspergillus niger) and one mushroom wood rot (Coriolus versicolor) were also employed. Quantities of these extracts were added to test tubes containing nutrient agar at 0.2% for bacteria and potato dextrose agar (PDA) for molds to promote germ/compound contact in test tubes. Each contained 13.5 ml tryptic soy agar medium, sterilized by autoclaving (20 min at 121°C), cooled to 45°C, and then poured into Petri dishes. 1.5 ml of each dilution was added in such a way to obtain a final concentration of 1/100 to 1/5000 (v/v). Controls consisting of the medium plus 0.2% agar-agar solution alone were also prepared. The results were considered in the context of calibrated platinum loops in the same volume of inoculum. The inoculum was in the form of culture broth (24 h, 37°C) for bacteria and in the form of suspension in physiological water of spores from a seven-day culture (7 d, 25°C) in the PDA for molds. Each test was done in triplicate to minimize experimental error.


## 2.6.1. Preparation of Methanolic Extract

10 g of each powder sample was crushed in a solvent (MeOH) (1 L) for 48 h at 24°C in dark and stirred [25] with slight modifications. The extracts were centrifuged at 5000 rpm for 15 min, 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

The radical-scavenging 2,2-diphenyl-1-picrylhydrazyl (DPPH) test was used. Each test was repeated in triplicate. The procedure was followed as described by Lopes-Lutz [26].The DPPH solution (5.91 mg of DPPH and 50 ml of methanol) was mixed with methanolic extracts at a different concentration (2–600 μg/ml) (1 ml; 0.3 mM) of DPPH and then incubated for 30 min in dark. A solution control (blank) was prepared by dissolving the DPPH solution in pure methanol. The reduction in DPPH free radicals was determined at 517 nm. The positive control was prepared with L-ascorbic acid, and the inhibition ratio (%) was calculated using the following equation:(2)%inhibition=Abs of control–Abs of test sample÷Abs ofcontrol×100%.The antioxidant activity of each sample was determined based on the inhibition curve and was expressed in terms of IC50.


## 2.6.3. Antimicrobial Assays

The minimum inhibitory concentrations (MICs) of the methanolic extract were assayed according to the procedure described in [27] and modified in [28, 29]. In vitro antibacterial studies were carried out against four bacterial strains: the Gram-positive bacteria S. aureus, B. subtilis, and M. luteus and the Gram-negative bacterium E. coli. Moreover, one mold (Aspergillus niger) and one mushroom wood rot (Coriolus versicolor) were also employed. Quantities of these extracts were added to test tubes containing nutrient agar at 0.2% for bacteria and potato dextrose agar (PDA) for molds to promote germ/compound contact in test tubes. Each contained 13.5 ml tryptic soy agar medium, sterilized by autoclaving (20 min at 121°C), cooled to 45°C, and then poured into Petri dishes. 1.5 ml of each dilution was added in such a way to obtain a final concentration of 1/100 to 1/5000 (v/v). Controls consisting of the medium plus 0.2% agar-agar solution alone were also prepared. The results were considered in the context of calibrated platinum loops in the same volume of inoculum. The inoculum was in the form of culture broth (24 h, 37°C) for bacteria and in the form of suspension in physiological water of spores from a seven-day culture (7 d, 25°C) in the PDA for molds. Each test was done in triplicate to minimize experimental error.


## 2.7. Statistical Analyses

Results were expressed as mean values ± standard deviation of three separate determinations.


## 3. Results and Discussion

### 3.1. Gross Biochemical Composition

Microalgae were considered an alternative source of protein, thanks to their high content of proteins [30]. The gross chemical composition of the analyzed Spirulina is summarized in Table 1.Table 1
Global nutritional analysis ofSpirulina (Morocco) (% of dry matter).
Elements
Percentage (%)b
Other micro/macroalgae
Moisturea (%)
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 ± 0.15
60–71 [5, 36, 37],
6–71% [39–45]
3–47% [46, 47]
Lipids (%)
2.45 ± 0.82
6–13% [5, 37]
>60% [39–45]
>5% [46, 47]
CF (%)
4.07 ± 1.42
1.36–7.73% [53]
Carbohydrates (%)c
6.46 ± 0.32
15–25% [5, 36, 37]
10–27% [39–45]
20–68% [46, 47]
Energy (kcal/100 g)
436.18 ± 2.29
ND
Note. aExpressed as percentage of freeze-dried samples. bData are mean values ± SD of three determinations. cCalculated by difference (=100 − crude protein − crude lipid − total dietary fiber (TDF)−ash). Dried Spirulina sp. % at 70°C of cell constituents is calculated after the moisture content was subtracted. ND: not detected.The Moroccan strain studied contains a considerable amount of protein (76.65 ± 0.15%). This value was higher than that of variousSpirulina species harvested from other countries [31, 32], with its amino acid composition of aspartic acid, glutamic acid, serine, glycine, histidine, arginine, threonine, alanine, proline, valine, isoleucine, leucine, phenylalanine, and lysine shown in [33]. It also showed a fairly high protein concentration compared to some of other microalgae (6 to 71%) [34, 35]. It was also higher than that of the most of red and green seaweeds (10 to 47%) of dry matter [36] and brown edible seaweeds (3 to 15% dw) [37]. This result confirms that the protein content in Spirulina was considerably higher than those in some plants [5]. Furthermore, several studies have demonstrated that the protein content of microalgae varied according to species, environmental conditions, and analytical methods for protein determination [38, 39].Lipids represent one of the main sources of energy for human metabolic processes.Spirulina lipid contains polyunsaturated fatty acids (PUFAs), mainly γ-linoleic acid with 30 to 35% of total lipid content, which are functional and structural components of cell membranes, while the main lipid membrane constituents in Spirulina are glycolipids and the essential fatty acids from the ω3 and ω6 families [40]. The Moroccan samples contained 2.45 ± 0.82% dw of fat. This value was much lower than that reported in previous studies on other Spirulina products (6 to 13%) [31, 32]. On the contrary, it was lower than the content of marine and some freshwater microalga species where lipid content was above 60% of dw [34, 35]. Although it was reported that edible lipid seaweed content does not obviously exceed 5% of dry matter, our result is also following the literature range [36, 37]. This result confirms that Spirulina appears very favorable for low-fat diets relatively to vegetables [5, 42]. Furthermore, this difference might relate to the strain, different extraction methods, solvents used, or nutritional and environmental factors [50].Microalga carbohydrates (e.g., starch, glucose, sugars, and other polysaccharides) normally generate energy and cellular structure. The carbohydrate content was about 6.46 ± 0.32%. This value was considerably lower than the carbohydrate concentration average of 15 to 25% reported previously for otherSpirulina strains [31, 32]. Besides, the Spirulina studied showed a carbohydrate content typically below average for many microalgae, overall accounting for 10 to 27% dw [34, 35]. In addition, it was lower than that reported in edible seaweeds which ranged from 20 to 68% [36, 37, 43]. The soluble carbohydrates constitute a major part of the total carbohydrate content, whereas in higher plants, insoluble carbohydrates are a major constituent of total carbohydrates [44]. This result agrees with other reports that showed dried whole microalgae can be used as food due to their higher carbohydrate digestibility. However, like carbohydrate composition, the protein content of algae was linked to the seasonal variation [50].Algae were characterized by a higher ash content. The ash content for Moroccan samples was 14.56 ± 0.74%. This value was higher than that found previously in otherSpirulina species (7.4 to 10.4%) [31, 32, 45]. This value might be high because of inadequate grinding of the samples prior to analysis. Indeed, ash content of our Spirulina was found as 20% which was also compared with freshwater and marine microalgae which range from 4% to 20% [46]. However, it was lower than the ash content shown in edible seaweeds, which in general have a high content of minerals reflected in the ash content [36, 37, 47].Moisture is an important factor for assessing the microalga quality. The dry matter content was 87.70 ± 1.85%. In fact, companies provide standards in their nutritional information and set the moisture standard below 9% [48]. This value could be due to drying methods and drying time [49]. On the contrary, the packaging and the storage conditions might have an indirect influence on the humidity rate. The microalgae should not be dried extremely because it could change the structure of living cells which would degrade their physicochemical properties. Therefore, their overall quality will not be optimal.The dietary fiber content of algae was of high nutritional importance. The present study was focused on the determination of crude fiber (CF, lignocellulose complex) and some polysaccharides of fibrous nature which would be identified further by future studies using highly sophisticated techniques. The cellulose and hemicellulose play an important role as structural components of the cell wall in algae [50]. The crude fiber content was 4.07 ± 1.42% higher than that from seaweeds [51] and than that from terrestrial plants or vegetables [52]. Besides, the value was in the middle of the seaweed content (1.36–7.73%) [53]. Therefore, Moroccan Spirulina might be considered as a good source of dietary fiber like several edible seaweeds.Generally, algae were considered to have a high ash content, essential minerals, and trace elements required for human nutrition. Given the ash content inSpirulina in Table 1, a high mineral content was predicted. Nutrient sufficiency is essential for productivity and longevity. The mineral result analyses are shown in Table 2. The studied microalga contained higher amounts of the macrominerals (32694.32 ± 6175.08 mg/100 g·dw) and trace elements (88.44 ± 3.2 mg/100 g·dw) required for human nutrition [54]. In addition, it contained significant amounts of essential minerals such as P (10088.33 ± 5766.88), Na (14004 ± 397.55), K (2501.66 ± 4.22), Ca (6000 ± 4.66), and Mg (100.33 ± 1.77 mg/100 g·dw), respectively. Trace elements were also detected (Fe, 80.66 ± 1.77; Zn, 5 ± 0.66; Cu, 1.22 ± 0.66; and Mn, 1.56 ± 0.11 mg/100g·dw, respectively). The macroelements found were slightly higher than those found in marine microalgae, which is interesting as Zarrouk’s medium supplies them in excess, except for Mg which displays a lower value compared to them. Otherwise, the microelements Cu, Mn, and Fe were presented in the microalga studied with modest values compared to marine microalgae, excluding the Zn content [55]. These results were relatively higher than those of the most land plants (5–10%) [56]. The mineral content is highly related to physiological and environmental factors, processing, and mineralization methods involving dry mineralization in an oven at 550°C [17, 57]. Consequently, the study confirms that Spirulina’s benefits may be attributed to both mineral and trace element contents.Table 2
Mineral composition ofSpirulina (Morocco) (mg/100g dw).
Elements
Spirulina studied
Other microalgae [55]
Minerals (mg/100 g dw)
P
10088.33 ± 5766.88
1700–3000
Na
14004 ± 397.55
7000–1100
K
2501.66 ± 4.22
600–1200
Ca
6000 ± 4.66
300–2100
Mg
100.33 ± 1.77
100–1100
Trace elements (mg/100 g dw)
Fe
80.66 ± 1.77
100–700
Zn
5 ± 0.66
23.9–370
Cu
1.22 ± 0.66
1.2–65
Mn
1.56 ± 0.11
3.7–59.2


### 3.2. Microbiological Analysis

The edible product was exposed to all contamination vectors. The microbial monitoring of marketedSpirulina powder is necessary since it is possible to affect the product quality by reducing the availability of nutrients. The microbiological quality of edible Spirulina is shown in Table 3. With respect to pathogenic microorganisms, the presence of total coliforms and Staphylococcus (<100 cfu/100 ml) may present a hazard. However, the absence of fecal coliforms indicates hygienic standards of the adopted technology and preparation were respected. Salmonella and sulfito-reducer bacteria, specifically Clostridium perfringens, were also not detected. The presence of enterobacteria, in particular Escherichia coli at 40%, which should be absent according to the EU standard, indicates fecal contamination. Finally, the yeasts and molds present in the ground and feces may have been transmitted via dust by wind, insects, or equipment. In fact, the microbial qualities of the samples from the routinely cultivated biomass by the company were moderately satisfactory according to the European Union and World Health Organization standards. In order to ensure its safety as food, it was necessary to control total coliforms, Staphylococcus, and Escherichia coli which could be responsible for toxicity potential. Otherwise, except the negative findings, these results were in agreement with those shown in [58], which confirmed that the microflora associated with Spirulina crops was generally nonpathogenic. In addition, a high alkalinity of the Spirulina environment is normally an excellent barrier to contamination, whether by bacteria, fungi, or algae. Furthermore, certain substances of both intracellular and extracellular metabolites as antimicrobial agents such as terpenols, sterols, polysaccharides, dibutenolides, peptides, and protein metabolites secreted by or present in Spirulina have a bactericidal or at least bacteriostatic effect on humans [59, 60]. The final microbial load of the product has been reported to depend on how carefully the culture and product are handled at various stages [61]. Therefore, subsequent handling of the product during harvest, drying, and packaging is very important and in this case appears to be generally satisfactory (Figure 1).Table 3
Microbiological quality of edibleSpirulina (cfu/ml).
Pathogens
All aerobic mesophilic flora
Staphylococcus
Total coliforms
Fecal coliforms
Sulfito-reducer bacteria
Yeasts and molds
E. coli
Salmonella
Bacterial count
208
93
26
ND
ND
14
4.15
ND
EU standard
105
Absent
Absent
No data
104
104
Absent
Absent
WHO
No data
No data
No data
No data
103
No data
Absent
EU, European Union [41]; WHO, World Health Organization [42]; ND: not detected.Figure 1
Crosstalk between nutritional and physicochemical properties and bioactivities in the functional foodSpirulina species.


### 3.3. Phytochemical Analysis and Characterization

#### 3.3.1. Total Phenolic Content (TPC) and Total Flavonoid Content (TFC)

Spirulina was considered a good source of nutritional phenolic and flavonoid compounds due to its higher production capacity compared to conventional plant-derived sources. As shown in Figure 2, the TPC and TFC of methanolic extracts from Spirulina were determined in terms of milligrams of gallic acid equivalents per gram of dry weight (mg GAE/g dw) from the calibration curve of gallic acid and milligrams of quercetin equivalents per gram of dry microalga extract (mg QE/g DE) through the calibration curve of quercetin, respectively. The obtained results revealed the presence of polyphenols (0.287 mg GAE/g DW) and total flavonoids (0.166 mg QE/g DE). These results were in agreement with the total phenolic content level (<5 mg GAE/g DW) for different microalgal species and also for different extracts [62, 63]. Phenolic compounds are secondary metabolites commonly found in Spirulina [64], with some of the main forms being salicylic, chlorogenic, synaptic caffeic, and trans-cinnamic acids, which have been known for their chemical protective mechanisms against some agents of biotic and abiotic stresses [65]. It also contains various classes of flavonoids like isoflavones, flavonols, flavanones, and dihydrochalcones. These natural phenolics have probably a broad spectrum of chemical and biological properties including antioxidant and free radical-scavenging activities. Moreover, the total phenolic contents obtained showed significant variations [66] which could be explained by geographical, physiological, and environmental factors and culture conditions [67].Figure 2
Total phenolic content (TPC) and total flavonoid content (TFC) of the methanolic extract ofSpirulina expressed as μg mg GAE/g DW and mg QE/g DE, respectively (n = 3).


#### 3.3.2. Phenolic Acid and Flavonoid Composition

A chromatographic separation method (HPLC-DAD/MS) was developed for simultaneous identification and quantification of phenolic acids and flavonoids inSpirulina reported to possess vital nutritional and biological activities [68]. A total of twenty phenolic acid and flavonoid compounds were properly identified and quantified compared with phenolic standards (retention times, mass spectra, and their fragmentations) within 30 min. Table 3 shows the quantification results and some information about phenolic acids and flavonoids (molecular formula, molecular weight, [M-H]− values, mass spectra, and retention times) in an aqueous extract from Spirulina. The phenolic and flavonoid amounts in an aqueous extract were determined, and the extract was rich in succinic acid (1122.88 mg/kg), followed by quinic acid (844.17 mg/kg), 3-4-hydroxybenzoic acid (687.07 mg/kg), catechin (584.53 mg/kg), citric acid (64.06 mg/kg), vanillic acid (16.24 mg/kg), gallic acid (2.13 mg/kg), 4-hydroxybenzoic acid (1.07 mg/kg), and trace elements (<1 mg/kg) of rutin, chlorogenic acid, quercetin, rosmarinic acid, salicylic acid, resveratrol, pyrogallol, ferulic acid, 4-hydroxycinnamic acid, 3-hydroxycinnamic acid, and 2-hydroxycinnamic acid. A list of other phenolic acids and flavonoids is also shown in Table 4. The phenolic acid amounts were varied in Spirulina species depending on culturing conditions [67, 69]. Only gallic, caffeic, salicylic, chlorogenic, and trans-cinnamic acids were detected in previous studies with Spirulina algae [69, 70].Table 4
Phenolic and flavonoid compounds determined in theSpirulina-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
191.120
Citric acid
64.06 ± 3.20
C6H8O7
192.123
0.63
191.102
Pyrogallol
0.42 ± 0.02
C6H6O3
126.111
0.64
125.024
Succinic acid
1122.88 ± 56.14
C4H6O4
118.088
0.65
117.018
Gallic acid
2.13 ± 0.11
C7H6O5
170.022
0.66
168.90
Chlorogenic acid
0.86 ± 0.04
C16H18O9
354.311
0.83
353.202
3,4-Hydroxybenzoic acid
687.07 ± 34.35
C7H6O4
154.121
0.95
153.010
4-Hydroxybenzoic acid
1.07 ± 0.05
C7H6O3
138.122
1.30
137.050
Catechin
584.53 ± 29.22
C15H14O6
290.271
1.63
289.064
Vanillic acid
16.24 ± 0.81
C8H8O4
168.148
2.12
167.036
4-Hydroxycinnamic acid
0.12 ± 0.01
C9H8O3
164.160
2.21
163.042
Rutin
0.93 ± 0.05
C27H30O16
610.153
2.63
609.1
3-Hydroxycinnamic acid
0.15 ± 0.01
C9H8O3
164.160
2.98
163.042
Ferulic acid
0.48 ± 0.02
C10H10O4
194.186
3.12
193.050
Quercetin
0.01 ± 0.00
C15H10O7
302.238
3.48
301.000
2-Hydroxycinnamic acid
0.31 ± 0.02
C9H8O3
164.160
3.65
163.042
Salicylic acid
0.08 ± 0.003
C7H6O3
138.122
3.68
137.025
Rosmarinic acid
0.18 ± 0.01
C18H16O8
360.318
4.01
359.054
Resveratrol
0.10 ± 0.005
C14H12O3
228.247
5.83
227.072
Quercitrin
0.04 ± 0.00
C21H20O11
448.38
5.99
447.120
4-Hydroxycoumarin
ND
C9H8O3
164.160
ND
163.042
Aesculin
ND
C15H16O9
340.284
ND
339.072
Epigallocatechin gallate
ND
C22H18O11
458.375
ND
457.078
Esculetin
ND
C9H6O4
178.143
ND
177.018
Kaempferol
ND
C15H10O6
286.239
ND
285.040
Luteolin
ND
C15H10O6
286.239
ND
285.040
Malic acid
ND
C4H6O5
134.087
ND
133.014
Syringic acid
ND
C9H10O5
198.174
ND
197.045
3-Hydroxybenzoic acid
ND
C7H6O3
138.122
ND
137.025
Benzoic acid
ND
C7H6O2
122.123
ND
121.031
Caffeic acid
ND
C9H8O4
180.159
ND
179.035
Epicatechin
ND
C15H14O6
290.271
ND
289.064
Hesperetin
ND
C16H14O6
302.282
ND
301.015
Hesperidin
ND
C28H34O15
610.565
ND
609.172
Naringenin
ND
C15H12O5
272.256
ND
271.061
Naringin
ND
C27H32O14
580.539
ND
579.173
Pyrocatechol
ND
C6H6O2
110.112
ND
109.028
Sinapic acid
ND
C11H12O5
224.212
ND
223.061
Tannic acid
ND
C76H52O46
1701.206
ND
1700.080
ND: not detected.


#### 3.3.3. Antioxidant Bioactivity Analysis

The total antioxidant capacity (TAC) of the methanolic extract was determined using the DPPH radical-scavenging assay. The results are displayed in Figure3.Figure 3
Percentage radical scavenging of the methanolic extract ofSpirulina as per DPPH assay-expressed activity (n = 3) as a function of concentration of the extract.The DPPH radical-scavenging activity was generally quantified in terms of inhibition percentage of the preformed free radical by antioxidants and EC50 (concentration required to obtain a 50% antioxidant effect) [71]. The EC50 was widely used to express the antioxidant capacity and to compare the activity of different compounds. In this study, the EC50 was 23 μg/ml, indicating a very high antioxidant potential [72]. Both the lower EC50 and the higher DPPH activity were related to a high antioxidant activity [73]. The study has been carried out on the DPPH-scavenging activity of the Spirulina extract compared to both vitamins C and E which showed that EC50 was about 40 ppm; i.e., one is better than the other which is comparable, as shown in [71]. Previous reports have revealed that the antioxidant activity of Spirulina may arise from a whole spectrum of natural antioxidant compounds that contribute to the oxidation process inhibition [74]. The phenolic compounds are mostly found in extracts of higher polarity and seem to be related to antioxidant activity or synergistic action, thanks to their redox properties [75]. However, the presence of different antioxidant compounds in the methanol extract was responsible for the free radical-scavenging activity, either individually or synergistically.


#### 3.3.4. Antimicrobial Activity

The growth of pathogenic and contaminant microorganisms in food decreases the nutritional quality and increases food toxicity. The antimicrobial activity of the methanolic extract ofSpirulina against microorganisms that contaminate food products was investigated. The results are presented in Table 5. The methanolic extract had no antimicrobial activity against the bacteria and molds studied. Nevertheless, it had antifungal activity against Coriolus versicolor, with a minimum inhibitory concentration (MIC) between 1/250 and 1/100 v/v of 1 g/10 ml. These results disagree with those of previous studies, in which Spirulina has been declared as bioactive-rich compounds in that microbial growth could be promoted or inhibited [76]. Indeed, it has been reported that the methanolic extract of Spirulina platensis possesses antimicrobial and antifungal potential against many pathogenic plant fungi and against all the bacteria tested [74]. The antimicrobial activity of the methanolic extract of studied Spirulina has been attributed to the presence of functional lipids, mainly γ-linolenic acid, and an antibiotically active fatty acid [77]. In fact, lipids kill microorganisms (bacteria, fungi, and yeasts) by reaching the bacterial membrane and causing their disintegration [78]. Indeed, they are disrupting the extensive meshwork of peptidoglycans in the cell wall without visible changes. γ-Linolenic acid and other fatty acids known for their antimicrobial activity are highly presented in Spirulina. Study [79] showed that the synergistic effect between these fatty acids is involved in their antimicrobial activity [80].Table 5
Inhibitory volumes and minimum inhibitory concentrations of the methanolic extract ofSpirulina (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
0
Bacteria
Bacillus subtilis
+
+
+
+
+
+
+
+
Staphylococcus aureus
+
+
+
+
+
+
+
+
Escherichia coli
+
+
+
+
+
+
+
+
Micrococcus luteus
+
+
+
+
+
+
+
+
Fungi
Aspergillus niger
+
+
+
+
+
+
+
+
Coriolus versicolor
−
+
+
+
+
+
+
+


## 3.1. Gross Biochemical Composition

Microalgae were considered an alternative source of protein, thanks to their high content of proteins [30]. The gross chemical composition of the analyzed Spirulina is summarized in Table 1.Table 1
Global nutritional analysis ofSpirulina (Morocco) (% of dry matter).
Elements
Percentage (%)b
Other micro/macroalgae
Moisturea (%)
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 ± 0.15
60–71 [5, 36, 37],
6–71% [39–45]
3–47% [46, 47]
Lipids (%)
2.45 ± 0.82
6–13% [5, 37]
>60% [39–45]
>5% [46, 47]
CF (%)
4.07 ± 1.42
1.36–7.73% [53]
Carbohydrates (%)c
6.46 ± 0.32
15–25% [5, 36, 37]
10–27% [39–45]
20–68% [46, 47]
Energy (kcal/100 g)
436.18 ± 2.29
ND
Note. aExpressed as percentage of freeze-dried samples. bData are mean values ± SD of three determinations. cCalculated by difference (=100 − crude protein − crude lipid − total dietary fiber (TDF)−ash). Dried Spirulina sp. % at 70°C of cell constituents is calculated after the moisture content was subtracted. ND: not detected.The Moroccan strain studied contains a considerable amount of protein (76.65 ± 0.15%). This value was higher than that of variousSpirulina species harvested from other countries [31, 32], with its amino acid composition of aspartic acid, glutamic acid, serine, glycine, histidine, arginine, threonine, alanine, proline, valine, isoleucine, leucine, phenylalanine, and lysine shown in [33]. It also showed a fairly high protein concentration compared to some of other microalgae (6 to 71%) [34, 35]. It was also higher than that of the most of red and green seaweeds (10 to 47%) of dry matter [36] and brown edible seaweeds (3 to 15% dw) [37]. This result confirms that the protein content in Spirulina was considerably higher than those in some plants [5]. Furthermore, several studies have demonstrated that the protein content of microalgae varied according to species, environmental conditions, and analytical methods for protein determination [38, 39].Lipids represent one of the main sources of energy for human metabolic processes.Spirulina lipid contains polyunsaturated fatty acids (PUFAs), mainly γ-linoleic acid with 30 to 35% of total lipid content, which are functional and structural components of cell membranes, while the main lipid membrane constituents in Spirulina are glycolipids and the essential fatty acids from the ω3 and ω6 families [40]. The Moroccan samples contained 2.45 ± 0.82% dw of fat. This value was much lower than that reported in previous studies on other Spirulina products (6 to 13%) [31, 32]. On the contrary, it was lower than the content of marine and some freshwater microalga species where lipid content was above 60% of dw [34, 35]. Although it was reported that edible lipid seaweed content does not obviously exceed 5% of dry matter, our result is also following the literature range [36, 37]. This result confirms that Spirulina appears very favorable for low-fat diets relatively to vegetables [5, 42]. Furthermore, this difference might relate to the strain, different extraction methods, solvents used, or nutritional and environmental factors [50].Microalga carbohydrates (e.g., starch, glucose, sugars, and other polysaccharides) normally generate energy and cellular structure. The carbohydrate content was about 6.46 ± 0.32%. This value was considerably lower than the carbohydrate concentration average of 15 to 25% reported previously for otherSpirulina strains [31, 32]. Besides, the Spirulina studied showed a carbohydrate content typically below average for many microalgae, overall accounting for 10 to 27% dw [34, 35]. In addition, it was lower than that reported in edible seaweeds which ranged from 20 to 68% [36, 37, 43]. The soluble carbohydrates constitute a major part of the total carbohydrate content, whereas in higher plants, insoluble carbohydrates are a major constituent of total carbohydrates [44]. This result agrees with other reports that showed dried whole microalgae can be used as food due to their higher carbohydrate digestibility. However, like carbohydrate composition, the protein content of algae was linked to the seasonal variation [50].Algae were characterized by a higher ash content. The ash content for Moroccan samples was 14.56 ± 0.74%. This value was higher than that found previously in otherSpirulina species (7.4 to 10.4%) [31, 32, 45]. This value might be high because of inadequate grinding of the samples prior to analysis. Indeed, ash content of our Spirulina was found as 20% which was also compared with freshwater and marine microalgae which range from 4% to 20% [46]. However, it was lower than the ash content shown in edible seaweeds, which in general have a high content of minerals reflected in the ash content [36, 37, 47].Moisture is an important factor for assessing the microalga quality. The dry matter content was 87.70 ± 1.85%. In fact, companies provide standards in their nutritional information and set the moisture standard below 9% [48]. This value could be due to drying methods and drying time [49]. On the contrary, the packaging and the storage conditions might have an indirect influence on the humidity rate. The microalgae should not be dried extremely because it could change the structure of living cells which would degrade their physicochemical properties. Therefore, their overall quality will not be optimal.The dietary fiber content of algae was of high nutritional importance. The present study was focused on the determination of crude fiber (CF, lignocellulose complex) and some polysaccharides of fibrous nature which would be identified further by future studies using highly sophisticated techniques. The cellulose and hemicellulose play an important role as structural components of the cell wall in algae [50]. The crude fiber content was 4.07 ± 1.42% higher than that from seaweeds [51] and than that from terrestrial plants or vegetables [52]. Besides, the value was in the middle of the seaweed content (1.36–7.73%) [53]. Therefore, Moroccan Spirulina might be considered as a good source of dietary fiber like several edible seaweeds.Generally, algae were considered to have a high ash content, essential minerals, and trace elements required for human nutrition. Given the ash content inSpirulina in Table 1, a high mineral content was predicted. Nutrient sufficiency is essential for productivity and longevity. The mineral result analyses are shown in Table 2. The studied microalga contained higher amounts of the macrominerals (32694.32 ± 6175.08 mg/100 g·dw) and trace elements (88.44 ± 3.2 mg/100 g·dw) required for human nutrition [54]. In addition, it contained significant amounts of essential minerals such as P (10088.33 ± 5766.88), Na (14004 ± 397.55), K (2501.66 ± 4.22), Ca (6000 ± 4.66), and Mg (100.33 ± 1.77 mg/100 g·dw), respectively. Trace elements were also detected (Fe, 80.66 ± 1.77; Zn, 5 ± 0.66; Cu, 1.22 ± 0.66; and Mn, 1.56 ± 0.11 mg/100g·dw, respectively). The macroelements found were slightly higher than those found in marine microalgae, which is interesting as Zarrouk’s medium supplies them in excess, except for Mg which displays a lower value compared to them. Otherwise, the microelements Cu, Mn, and Fe were presented in the microalga studied with modest values compared to marine microalgae, excluding the Zn content [55]. These results were relatively higher than those of the most land plants (5–10%) [56]. The mineral content is highly related to physiological and environmental factors, processing, and mineralization methods involving dry mineralization in an oven at 550°C [17, 57]. Consequently, the study confirms that Spirulina’s benefits may be attributed to both mineral and trace element contents.Table 2
Mineral composition ofSpirulina (Morocco) (mg/100g dw).
Elements
Spirulina studied
Other microalgae [55]
Minerals (mg/100 g dw)
P
10088.33 ± 5766.88
1700–3000
Na
14004 ± 397.55
7000–1100
K
2501.66 ± 4.22
600–1200
Ca
6000 ± 4.66
300–2100
Mg
100.33 ± 1.77
100–1100
Trace elements (mg/100 g dw)
Fe
80.66 ± 1.77
100–700
Zn
5 ± 0.66
23.9–370
Cu
1.22 ± 0.66
1.2–65
Mn
1.56 ± 0.11
3.7–59.2


## 3.2. Microbiological Analysis

The edible product was exposed to all contamination vectors. The microbial monitoring of marketedSpirulina powder is necessary since it is possible to affect the product quality by reducing the availability of nutrients. The microbiological quality of edible Spirulina is shown in Table 3. With respect to pathogenic microorganisms, the presence of total coliforms and Staphylococcus (<100 cfu/100 ml) may present a hazard. However, the absence of fecal coliforms indicates hygienic standards of the adopted technology and preparation were respected. Salmonella and sulfito-reducer bacteria, specifically Clostridium perfringens, were also not detected. The presence of enterobacteria, in particular Escherichia coli at 40%, which should be absent according to the EU standard, indicates fecal contamination. Finally, the yeasts and molds present in the ground and feces may have been transmitted via dust by wind, insects, or equipment. In fact, the microbial qualities of the samples from the routinely cultivated biomass by the company were moderately satisfactory according to the European Union and World Health Organization standards. In order to ensure its safety as food, it was necessary to control total coliforms, Staphylococcus, and Escherichia coli which could be responsible for toxicity potential. Otherwise, except the negative findings, these results were in agreement with those shown in [58], which confirmed that the microflora associated with Spirulina crops was generally nonpathogenic. In addition, a high alkalinity of the Spirulina environment is normally an excellent barrier to contamination, whether by bacteria, fungi, or algae. Furthermore, certain substances of both intracellular and extracellular metabolites as antimicrobial agents such as terpenols, sterols, polysaccharides, dibutenolides, peptides, and protein metabolites secreted by or present in Spirulina have a bactericidal or at least bacteriostatic effect on humans [59, 60]. The final microbial load of the product has been reported to depend on how carefully the culture and product are handled at various stages [61]. Therefore, subsequent handling of the product during harvest, drying, and packaging is very important and in this case appears to be generally satisfactory (Figure 1).Table 3
Microbiological quality of edibleSpirulina (cfu/ml).
Pathogens
All aerobic mesophilic flora
Staphylococcus
Total coliforms
Fecal coliforms
Sulfito-reducer bacteria
Yeasts and molds
E. coli
Salmonella
Bacterial count
208
93
26
ND
ND
14
4.15
ND
EU standard
105
Absent
Absent
No data
104
104
Absent
Absent
WHO
No data
No data
No data
No data
103
No data
Absent
EU, European Union [41]; WHO, World Health Organization [42]; ND: not detected.Figure 1
Crosstalk between nutritional and physicochemical properties and bioactivities in the functional foodSpirulina species.


## 3.3. Phytochemical Analysis and Characterization

### 3.3.1. Total Phenolic Content (TPC) and Total Flavonoid Content (TFC)

Spirulina was considered a good source of nutritional phenolic and flavonoid compounds due to its higher production capacity compared to conventional plant-derived sources. As shown in Figure 2, the TPC and TFC of methanolic extracts from Spirulina were determined in terms of milligrams of gallic acid equivalents per gram of dry weight (mg GAE/g dw) from the calibration curve of gallic acid and milligrams of quercetin equivalents per gram of dry microalga extract (mg QE/g DE) through the calibration curve of quercetin, respectively. The obtained results revealed the presence of polyphenols (0.287 mg GAE/g DW) and total flavonoids (0.166 mg QE/g DE). These results were in agreement with the total phenolic content level (<5 mg GAE/g DW) for different microalgal species and also for different extracts [62, 63]. Phenolic compounds are secondary metabolites commonly found in Spirulina [64], with some of the main forms being salicylic, chlorogenic, synaptic caffeic, and trans-cinnamic acids, which have been known for their chemical protective mechanisms against some agents of biotic and abiotic stresses [65]. It also contains various classes of flavonoids like isoflavones, flavonols, flavanones, and dihydrochalcones. These natural phenolics have probably a broad spectrum of chemical and biological properties including antioxidant and free radical-scavenging activities. Moreover, the total phenolic contents obtained showed significant variations [66] which could be explained by geographical, physiological, and environmental factors and culture conditions [67].Figure 2
Total phenolic content (TPC) and total flavonoid content (TFC) of the methanolic extract ofSpirulina expressed as μg mg GAE/g DW and mg QE/g DE, respectively (n = 3).


### 3.3.2. Phenolic Acid and Flavonoid Composition

A chromatographic separation method (HPLC-DAD/MS) was developed for simultaneous identification and quantification of phenolic acids and flavonoids inSpirulina reported to possess vital nutritional and biological activities [68]. A total of twenty phenolic acid and flavonoid compounds were properly identified and quantified compared with phenolic standards (retention times, mass spectra, and their fragmentations) within 30 min. Table 3 shows the quantification results and some information about phenolic acids and flavonoids (molecular formula, molecular weight, [M-H]− values, mass spectra, and retention times) in an aqueous extract from Spirulina. The phenolic and flavonoid amounts in an aqueous extract were determined, and the extract was rich in succinic acid (1122.88 mg/kg), followed by quinic acid (844.17 mg/kg), 3-4-hydroxybenzoic acid (687.07 mg/kg), catechin (584.53 mg/kg), citric acid (64.06 mg/kg), vanillic acid (16.24 mg/kg), gallic acid (2.13 mg/kg), 4-hydroxybenzoic acid (1.07 mg/kg), and trace elements (<1 mg/kg) of rutin, chlorogenic acid, quercetin, rosmarinic acid, salicylic acid, resveratrol, pyrogallol, ferulic acid, 4-hydroxycinnamic acid, 3-hydroxycinnamic acid, and 2-hydroxycinnamic acid. A list of other phenolic acids and flavonoids is also shown in Table 4. The phenolic acid amounts were varied in Spirulina species depending on culturing conditions [67, 69]. Only gallic, caffeic, salicylic, chlorogenic, and trans-cinnamic acids were detected in previous studies with Spirulina algae [69, 70].Table 4
Phenolic and flavonoid compounds determined in theSpirulina-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
191.120
Citric acid
64.06 ± 3.20
C6H8O7
192.123
0.63
191.102
Pyrogallol
0.42 ± 0.02
C6H6O3
126.111
0.64
125.024
Succinic acid
1122.88 ± 56.14
C4H6O4
118.088
0.65
117.018
Gallic acid
2.13 ± 0.11
C7H6O5
170.022
0.66
168.90
Chlorogenic acid
0.86 ± 0.04
C16H18O9
354.311
0.83
353.202
3,4-Hydroxybenzoic acid
687.07 ± 34.35
C7H6O4
154.121
0.95
153.010
4-Hydroxybenzoic acid
1.07 ± 0.05
C7H6O3
138.122
1.30
137.050
Catechin
584.53 ± 29.22
C15H14O6
290.271
1.63
289.064
Vanillic acid
16.24 ± 0.81
C8H8O4
168.148
2.12
167.036
4-Hydroxycinnamic acid
0.12 ± 0.01
C9H8O3
164.160
2.21
163.042
Rutin
0.93 ± 0.05
C27H30O16
610.153
2.63
609.1
3-Hydroxycinnamic acid
0.15 ± 0.01
C9H8O3
164.160
2.98
163.042
Ferulic acid
0.48 ± 0.02
C10H10O4
194.186
3.12
193.050
Quercetin
0.01 ± 0.00
C15H10O7
302.238
3.48
301.000
2-Hydroxycinnamic acid
0.31 ± 0.02
C9H8O3
164.160
3.65
163.042
Salicylic acid
0.08 ± 0.003
C7H6O3
138.122
3.68
137.025
Rosmarinic acid
0.18 ± 0.01
C18H16O8
360.318
4.01
359.054
Resveratrol
0.10 ± 0.005
C14H12O3
228.247
5.83
227.072
Quercitrin
0.04 ± 0.00
C21H20O11
448.38
5.99
447.120
4-Hydroxycoumarin
ND
C9H8O3
164.160
ND
163.042
Aesculin
ND
C15H16O9
340.284
ND
339.072
Epigallocatechin gallate
ND
C22H18O11
458.375
ND
457.078
Esculetin
ND
C9H6O4
178.143
ND
177.018
Kaempferol
ND
C15H10O6
286.239
ND
285.040
Luteolin
ND
C15H10O6
286.239
ND
285.040
Malic acid
ND
C4H6O5
134.087
ND
133.014
Syringic acid
ND
C9H10O5
198.174
ND
197.045
3-Hydroxybenzoic acid
ND
C7H6O3
138.122
ND
137.025
Benzoic acid
ND
C7H6O2
122.123
ND
121.031
Caffeic acid
ND
C9H8O4
180.159
ND
179.035
Epicatechin
ND
C15H14O6
290.271
ND
289.064
Hesperetin
ND
C16H14O6
302.282
ND
301.015
Hesperidin
ND
C28H34O15
610.565
ND
609.172
Naringenin
ND
C15H12O5
272.256
ND
271.061
Naringin
ND
C27H32O14
580.539
ND
579.173
Pyrocatechol
ND
C6H6O2
110.112
ND
109.028
Sinapic acid
ND
C11H12O5
224.212
ND
223.061
Tannic acid
ND
C76H52O46
1701.206
ND
1700.080
ND: not detected.


### 3.3.3. Antioxidant Bioactivity Analysis

The total antioxidant capacity (TAC) of the methanolic extract was determined using the DPPH radical-scavenging assay. The results are displayed in Figure3.Figure 3
Percentage radical scavenging of the methanolic extract ofSpirulina as per DPPH assay-expressed activity (n = 3) as a function of concentration of the extract.The DPPH radical-scavenging activity was generally quantified in terms of inhibition percentage of the preformed free radical by antioxidants and EC50 (concentration required to obtain a 50% antioxidant effect) [71]. The EC50 was widely used to express the antioxidant capacity and to compare the activity of different compounds. In this study, the EC50 was 23 μg/ml, indicating a very high antioxidant potential [72]. Both the lower EC50 and the higher DPPH activity were related to a high antioxidant activity [73]. The study has been carried out on the DPPH-scavenging activity of the Spirulina extract compared to both vitamins C and E which showed that EC50 was about 40 ppm; i.e., one is better than the other which is comparable, as shown in [71]. Previous reports have revealed that the antioxidant activity of Spirulina may arise from a whole spectrum of natural antioxidant compounds that contribute to the oxidation process inhibition [74]. The phenolic compounds are mostly found in extracts of higher polarity and seem to be related to antioxidant activity or synergistic action, thanks to their redox properties [75]. However, the presence of different antioxidant compounds in the methanol extract was responsible for the free radical-scavenging activity, either individually or synergistically.


### 3.3.4. Antimicrobial Activity

The growth of pathogenic and contaminant microorganisms in food decreases the nutritional quality and increases food toxicity. The antimicrobial activity of the methanolic extract ofSpirulina against microorganisms that contaminate food products was investigated. The results are presented in Table 5. The methanolic extract had no antimicrobial activity against the bacteria and molds studied. Nevertheless, it had antifungal activity against Coriolus versicolor, with a minimum inhibitory concentration (MIC) between 1/250 and 1/100 v/v of 1 g/10 ml. These results disagree with those of previous studies, in which Spirulina has been declared as bioactive-rich compounds in that microbial growth could be promoted or inhibited [76]. Indeed, it has been reported that the methanolic extract of Spirulina platensis possesses antimicrobial and antifungal potential against many pathogenic plant fungi and against all the bacteria tested [74]. The antimicrobial activity of the methanolic extract of studied Spirulina has been attributed to the presence of functional lipids, mainly γ-linolenic acid, and an antibiotically active fatty acid [77]. In fact, lipids kill microorganisms (bacteria, fungi, and yeasts) by reaching the bacterial membrane and causing their disintegration [78]. Indeed, they are disrupting the extensive meshwork of peptidoglycans in the cell wall without visible changes. γ-Linolenic acid and other fatty acids known for their antimicrobial activity are highly presented in Spirulina. Study [79] showed that the synergistic effect between these fatty acids is involved in their antimicrobial activity [80].Table 5
Inhibitory volumes and minimum inhibitory concentrations of the methanolic extract ofSpirulina (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
0
Bacteria
Bacillus subtilis
+
+
+
+
+
+
+
+
Staphylococcus aureus
+
+
+
+
+
+
+
+
Escherichia coli
+
+
+
+
+
+
+
+
Micrococcus luteus
+
+
+
+
+
+
+
+
Fungi
Aspergillus niger
+
+
+
+
+
+
+
+
Coriolus versicolor
−
+
+
+
+
+
+
+


## 3.3.1. Total Phenolic Content (TPC) and Total Flavonoid Content (TFC)

Spirulina was considered a good source of nutritional phenolic and flavonoid compounds due to its higher production capacity compared to conventional plant-derived sources. As shown in Figure 2, the TPC and TFC of methanolic extracts from Spirulina were determined in terms of milligrams of gallic acid equivalents per gram of dry weight (mg GAE/g dw) from the calibration curve of gallic acid and milligrams of quercetin equivalents per gram of dry microalga extract (mg QE/g DE) through the calibration curve of quercetin, respectively. The obtained results revealed the presence of polyphenols (0.287 mg GAE/g DW) and total flavonoids (0.166 mg QE/g DE). These results were in agreement with the total phenolic content level (<5 mg GAE/g DW) for different microalgal species and also for different extracts [62, 63]. Phenolic compounds are secondary metabolites commonly found in Spirulina [64], with some of the main forms being salicylic, chlorogenic, synaptic caffeic, and trans-cinnamic acids, which have been known for their chemical protective mechanisms against some agents of biotic and abiotic stresses [65]. It also contains various classes of flavonoids like isoflavones, flavonols, flavanones, and dihydrochalcones. These natural phenolics have probably a broad spectrum of chemical and biological properties including antioxidant and free radical-scavenging activities. Moreover, the total phenolic contents obtained showed significant variations [66] which could be explained by geographical, physiological, and environmental factors and culture conditions [67].Figure 2
Total phenolic content (TPC) and total flavonoid content (TFC) of the methanolic extract ofSpirulina expressed as μg mg GAE/g DW and mg QE/g DE, respectively (n = 3).


## 3.3.2. Phenolic Acid and Flavonoid Composition

A chromatographic separation method (HPLC-DAD/MS) was developed for simultaneous identification and quantification of phenolic acids and flavonoids inSpirulina reported to possess vital nutritional and biological activities [68]. A total of twenty phenolic acid and flavonoid compounds were properly identified and quantified compared with phenolic standards (retention times, mass spectra, and their fragmentations) within 30 min. Table 3 shows the quantification results and some information about phenolic acids and flavonoids (molecular formula, molecular weight, [M-H]− values, mass spectra, and retention times) in an aqueous extract from Spirulina. The phenolic and flavonoid amounts in an aqueous extract were determined, and the extract was rich in succinic acid (1122.88 mg/kg), followed by quinic acid (844.17 mg/kg), 3-4-hydroxybenzoic acid (687.07 mg/kg), catechin (584.53 mg/kg), citric acid (64.06 mg/kg), vanillic acid (16.24 mg/kg), gallic acid (2.13 mg/kg), 4-hydroxybenzoic acid (1.07 mg/kg), and trace elements (<1 mg/kg) of rutin, chlorogenic acid, quercetin, rosmarinic acid, salicylic acid, resveratrol, pyrogallol, ferulic acid, 4-hydroxycinnamic acid, 3-hydroxycinnamic acid, and 2-hydroxycinnamic acid. A list of other phenolic acids and flavonoids is also shown in Table 4. The phenolic acid amounts were varied in Spirulina species depending on culturing conditions [67, 69]. Only gallic, caffeic, salicylic, chlorogenic, and trans-cinnamic acids were detected in previous studies with Spirulina algae [69, 70].Table 4
Phenolic and flavonoid compounds determined in theSpirulina-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
191.120
Citric acid
64.06 ± 3.20
C6H8O7
192.123
0.63
191.102
Pyrogallol
0.42 ± 0.02
C6H6O3
126.111
0.64
125.024
Succinic acid
1122.88 ± 56.14
C4H6O4
118.088
0.65
117.018
Gallic acid
2.13 ± 0.11
C7H6O5
170.022
0.66
168.90
Chlorogenic acid
0.86 ± 0.04
C16H18O9
354.311
0.83
353.202
3,4-Hydroxybenzoic acid
687.07 ± 34.35
C7H6O4
154.121
0.95
153.010
4-Hydroxybenzoic acid
1.07 ± 0.05
C7H6O3
138.122
1.30
137.050
Catechin
584.53 ± 29.22
C15H14O6
290.271
1.63
289.064
Vanillic acid
16.24 ± 0.81
C8H8O4
168.148
2.12
167.036
4-Hydroxycinnamic acid
0.12 ± 0.01
C9H8O3
164.160
2.21
163.042
Rutin
0.93 ± 0.05
C27H30O16
610.153
2.63
609.1
3-Hydroxycinnamic acid
0.15 ± 0.01
C9H8O3
164.160
2.98
163.042
Ferulic acid
0.48 ± 0.02
C10H10O4
194.186
3.12
193.050
Quercetin
0.01 ± 0.00
C15H10O7
302.238
3.48
301.000
2-Hydroxycinnamic acid
0.31 ± 0.02
C9H8O3
164.160
3.65
163.042
Salicylic acid
0.08 ± 0.003
C7H6O3
138.122
3.68
137.025
Rosmarinic acid
0.18 ± 0.01
C18H16O8
360.318
4.01
359.054
Resveratrol
0.10 ± 0.005
C14H12O3
228.247
5.83
227.072
Quercitrin
0.04 ± 0.00
C21H20O11
448.38
5.99
447.120
4-Hydroxycoumarin
ND
C9H8O3
164.160
ND
163.042
Aesculin
ND
C15H16O9
340.284
ND
339.072
Epigallocatechin gallate
ND
C22H18O11
458.375
ND
457.078
Esculetin
ND
C9H6O4
178.143
ND
177.018
Kaempferol
ND
C15H10O6
286.239
ND
285.040
Luteolin
ND
C15H10O6
286.239
ND
285.040
Malic acid
ND
C4H6O5
134.087
ND
133.014
Syringic acid
ND
C9H10O5
198.174
ND
197.045
3-Hydroxybenzoic acid
ND
C7H6O3
138.122
ND
137.025
Benzoic acid
ND
C7H6O2
122.123
ND
121.031
Caffeic acid
ND
C9H8O4
180.159
ND
179.035
Epicatechin
ND
C15H14O6
290.271
ND
289.064
Hesperetin
ND
C16H14O6
302.282
ND
301.015
Hesperidin
ND
C28H34O15
610.565
ND
609.172
Naringenin
ND
C15H12O5
272.256
ND
271.061
Naringin
ND
C27H32O14
580.539
ND
579.173
Pyrocatechol
ND
C6H6O2
110.112
ND
109.028
Sinapic acid
ND
C11H12O5
224.212
ND
223.061
Tannic acid
ND
C76H52O46
1701.206
ND
1700.080
ND: not detected.


## 3.3.3. Antioxidant Bioactivity Analysis

The total antioxidant capacity (TAC) of the methanolic extract was determined using the DPPH radical-scavenging assay. The results are displayed in Figure3.Figure 3
Percentage radical scavenging of the methanolic extract ofSpirulina as per DPPH assay-expressed activity (n = 3) as a function of concentration of the extract.The DPPH radical-scavenging activity was generally quantified in terms of inhibition percentage of the preformed free radical by antioxidants and EC50 (concentration required to obtain a 50% antioxidant effect) [71]. The EC50 was widely used to express the antioxidant capacity and to compare the activity of different compounds. In this study, the EC50 was 23 μg/ml, indicating a very high antioxidant potential [72]. Both the lower EC50 and the higher DPPH activity were related to a high antioxidant activity [73]. The study has been carried out on the DPPH-scavenging activity of the Spirulina extract compared to both vitamins C and E which showed that EC50 was about 40 ppm; i.e., one is better than the other which is comparable, as shown in [71]. Previous reports have revealed that the antioxidant activity of Spirulina may arise from a whole spectrum of natural antioxidant compounds that contribute to the oxidation process inhibition [74]. The phenolic compounds are mostly found in extracts of higher polarity and seem to be related to antioxidant activity or synergistic action, thanks to their redox properties [75]. However, the presence of different antioxidant compounds in the methanol extract was responsible for the free radical-scavenging activity, either individually or synergistically.


## 3.3.4. Antimicrobial Activity

The growth of pathogenic and contaminant microorganisms in food decreases the nutritional quality and increases food toxicity. The antimicrobial activity of the methanolic extract ofSpirulina against microorganisms that contaminate food products was investigated. The results are presented in Table 5. The methanolic extract had no antimicrobial activity against the bacteria and molds studied. Nevertheless, it had antifungal activity against Coriolus versicolor, with a minimum inhibitory concentration (MIC) between 1/250 and 1/100 v/v of 1 g/10 ml. These results disagree with those of previous studies, in which Spirulina has been declared as bioactive-rich compounds in that microbial growth could be promoted or inhibited [76]. Indeed, it has been reported that the methanolic extract of Spirulina platensis possesses antimicrobial and antifungal potential against many pathogenic plant fungi and against all the bacteria tested [74]. The antimicrobial activity of the methanolic extract of studied Spirulina has been attributed to the presence of functional lipids, mainly γ-linolenic acid, and an antibiotically active fatty acid [77]. In fact, lipids kill microorganisms (bacteria, fungi, and yeasts) by reaching the bacterial membrane and causing their disintegration [78]. Indeed, they are disrupting the extensive meshwork of peptidoglycans in the cell wall without visible changes. γ-Linolenic acid and other fatty acids known for their antimicrobial activity are highly presented in Spirulina. Study [79] showed that the synergistic effect between these fatty acids is involved in their antimicrobial activity [80].Table 5
Inhibitory volumes and minimum inhibitory concentrations of the methanolic extract ofSpirulina (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
0
Bacteria
Bacillus subtilis
+
+
+
+
+
+
+
+
Staphylococcus aureus
+
+
+
+
+
+
+
+
Escherichia coli
+
+
+
+
+
+
+
+
Micrococcus luteus
+
+
+
+
+
+
+
+
Fungi
Aspergillus niger
+
+
+
+
+
+
+
+
Coriolus versicolor
−
+
+
+
+
+
+
+


## 4. Conclusions

Given its chemical composition, rich nutritional value, and antimicrobial and antioxidant activities, the MoroccanSpirulina has important nutraceutical potential. Furthermore, pharmacochemical, experimental, and clinical studies on the Moroccan Spirulina are required to identify its mechanism of action as a complement of traditional pharmacopeia.



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*Source: 3707219-2019-07-03.xml*