Algal Research 58 (2021) 102387

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Algal Research
journal homepage: www.elsevier.com/locate/algal

Effect of the addition of Spirulina sp. biomass on the development and

characterization of functional food
Leila Magda Rodrigues Almeida a, *, Larissa Farias da Silva Cruz a,
Bruna Aparecida Souza Machado b, Itaciara Larroza Nunes c, Jorge Alberto Vieira Costa d,
Ederlan de Souza Ferreira a, Paulo Vitor França Lemos a, Janice Izabel Druzian a,
Carolina Oliveira de Souza a
a
Department of Bromatological Analysis, College of Pharmacy, Federal University of Bahia, Av. Adhemar de Barros, s/n◦ - Ondina, Salvador 40170-110, Brazil
b
Department of Food and Biotechnology, University Center SENAI CIMATEC, Avenue Orlando Gomes, 1845 - Piatã, Salvador, BA 41650-010, Brazil
c
Department of Food Science and Technology, Federal University of Santa Catarina, Admar Gonzaga Highway, 1346, Itacorubi, 88034-000 Florianópolis, Santa
Catarina, Brazil
d
Laboratory of Biochemical Engineering, College of Chemistry and Food Engineering, Federal University of Rio Grande, PO Box 474, Rio Grande 96203-900, Brazil

A R T I C L E I N F O A B S T R A C T

Keywords: The Spirulina sp. biomass has been used as an alternative for the production of high added-value functional and
Spirulina sp., nutraceutical foods. This study aimed to develop a ready-made functional sauce enriched with Spirulina sp.
Sauce biomass and evaluate its physicochemical, sensory, antioxidant, and microbiological properties over 45 days of
Enriched
storage. The formulation with 4% Spirulina sp. biomass presented significantly increased protein (34%), fiber
Antioxidant
Functional food
(57%), ash (40%), monounsaturated fatty acid (240%), and mineral (192%) contents than the control. The
storage stability for up to 45 days was noticed in both physicochemical and microbiological parameters. The
phenolic content also doubled, with a 12-fold increase in antioxidant activity, as shown by the IC50. The enriched
sauce presented a high capacity to mask microalgae's characteristic flavor, enhancing the nutritional quality of
the product. Therefore, the sauce enriched with 4% biomass showed the best characteristics among the con­
centrations evaluated. It can be concluded that Spirulina sp. biomass can be used as a natural ingredient to
develop a functional sauce with antioxidant and nutritional potential.

1. Introduction chemical composition, including phenols (caffeic, chlorogenic, salicylic,

Spirulina sp. has various applications in the food and pharmaceutical
industries due to its valuable biomolecules. The development of new
products with high nutritional value based on Spirulina sp. biomass is an
emergent biotechnological approach. This microalga is one of the richest
protein sources of microbial origin (55–70%), containing all essential
amino acids and good digestibility [1,2].
The interest in the production of Spirulina sp. biomass has gained
attention due to its various technological applications. Therefore, some
research has been carried out to investigate the techniques, procedures,
and processes for producing microalgae. The use of wastewater in this
production has also been explored since the content of microalgal
biomass can be used to make bioproducts of industrial interest [3].
The current interest in Spirulina platensis primarily derives from its
synaptic and trans-cinnamic acids), which account for its antioxidant
properties [4] supporting its use as a functional food [5]. In addition,
this microorganism also stands out for its high protein content, with
essential amino acids, vitamins (A, E, C and especially B12), minerals
(iron, calcium, copper, magnesium, manganese, phosphorus, potassium,
sodium and zinc), photosynthetic pigments (carotenoids, phycocyanin
and chlorophyll), and polyunsaturated fatty acids (omega-6 and omega-
3) [6,7].
The presence of alpha-linolenic acid (C18:3 n-3) is very important
from a nutritional standpoint. The Western diet is generally deficient in
this fatty acid, which plays an essential prophylactic role in treating
various chronic diseases [7,8]. The in vitro and in vivo studies have
demonstrated that in addition to nutritional properties, Spirulina sp. has
therapeutic properties that may help to treat diabetes, anemia,

* Corresponding author.
E-mail address: leila.rodrigues@ufba.br (L.M.R. Almeida).

https://doi.org/10.1016/j.algal.2021.102387
Received 28 February 2021; Received in revised form 6 June 2021; Accepted 11 June 2021
Available online 30 June 2021
2211-9264/© 2021 Elsevier B.V. All rights reserved.
L.M.R. Almeida et al.
malnutrition, obesity, cardiovascular disease, and cancer, among others.
Thus, Spirulina sp. can be used as an alternative for the production of
high added-value functional and nutraceutical foods [1,8–12].
The global food industry has been widely exploiting microalgal as
promising sources for the development of new products. The incorpo­
ration of the biomass of Spirulina sp. in certain foods is considered an
innovation with high technological and commercial potential since it
adds nutritional value to products already known by the population
[13,14]. Compared to Spirulina sp. in commercial powder form, the
foods incorporated with biomass are more accepted by the consumers
due to their better sensorial properties [15].
The novel biomass incorporated food products represent a sustain­
able trend in the food industry. This trend considers the increasing
consumer demand for healthy and practical foods with a high content of
protein and bioactive compounds and low fat and sugar. In this context,
ready-made sauces stand out in the market because of their convenience
and practicality in preparing elaborate dishes and because new products
and formulations are continuously launched [16,4].
Spirulina sp. biomass's main disadvantages are its odor, flavor, and
color. These attributes of biomass are percept by most people as un­
pleasant [1]. However, its incorporation into a sauce can mask these
attributes, with the added benefit of enhancing the nutritional and
functional quality of the product.
Some foods such as yogurts and functional beverages have already
been developed of Spirulina sp. biomass supplementation [14,15,17,18].
Despite this, there is a lack of research on the development and char­
acterization of biomass-based functional sauces. Besides, it is worth
mentioning that enriched sauces are versatile products that could be
used to prepare other foods.
This study aimed to develop a ready-made functional sauce enriched
with Spirulina sp. biomass and evaluate its physicochemical, sensory,
antioxidant, and microbiological properties over 45 days of storage.
2. Materials and methods
2.1. Materials
Spirulina sp. was isolated from Mangueira Lagoon (33◦ 30′ 12′′ S, 53◦
08′ 58′′ W), located between the Atlantic Ocean and Mirim Lake, in the
southern end of the state of Rio Grande do Sul, Brazil [19]. It was grown
under outdoor conditions in Zarrouk medium in a 500-L pilot plant
under the climatic conditions of Salvador, Bahia, Brazil (12◦ 58′ 16′′ S,
38◦ 30′ 30′′ W), according to Jesus et al. [20].
Cultivation was carried out outdoors in raceway tanks which con­
tained 500 L of Spirulina sp. with an initial biomass concentration of 0.5
g L− 1. The cultivation temperature ranged from 27.1 ◦ C to 37.3 ◦ C, and
light intensity varied from 75.85 to 1840.75 μmolphotons m− 2 s− 1. The pH
of the cultivation medium ranged between 9.10 and 10.20 in the
northeast and between 9.97 and 10.60.
The resulting biomass was initially freeze-dried at − 49 ◦ C for 48 h (L
101 Freeze Dryer, Liotop), then macerated in a mortar and pestle and
passed through a 32 mesh (500 μm) for particle size standardization and
to improve solubility. The produced powder was treated with
ultraviolet-C light at 254 nm for 15 min (Class II Biological Safety
Cabinet, ESCO) for decontamination. The other ingredients used to
prepare the sauces were purchased from local shops in the city of
Salvador.
2.2. Methods
2.2.1. Sauce formulations
To define the contents of the sauce components used in the study,
preliminary tests were performed with different proportions of the in­
gredients and Spirulina sp. biomass. The criteria used for the sauce were
parameters such as visual appearance, predominant spice aroma, uni­
formity, homogeneity, and soft texture.
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Algal Research 58 (2021) 102387
Four sauce formulations, containing 0 (control), 2.0, 3.0, and 4.0%
Spirulina sp. biomass (wt%), tomato (69.00 g), onion (11.00 g), soybean
oil (3.60 g), garlic (0.97 g), cumin (0.38 g), sodium chloride (0.31 g),
rosemary (0.24 g), parsley (0.14 g), oregano (0.11 g), and water (14.00
mL) were prepared. The experimental design is given in Fig. 1.
The ingredients were ground, mixed in a blender, transferred to a
metal container, and heated for 10 min at 95 ◦ C (except Spirulina sp.
biomass). After this step, the sauce was left to rest until it reached a
temperature of 60 ◦ C, and then Spirulina sp. biomass was added and
mixed into the sauce.
The freshly prepared sauces were poured into 200 mL glass jars with
metal lids, previously sterilized in boiling water (100 ◦ C) for 30 min. The
sauces were pasteurized by heating the jars for 40 s at 98 ◦ C [21] and
stored in a refrigerator (4–6 ◦ C, 60% of relative humidity).
The formulations were evaluated regarding their parameters pH and
acidity, texture, and sensory acceptance 24 h after preparation. In
addition to the control formulation, the formulation containing Spirulina
sp. biomass that presented the best results for all parameters was
selected for the stability test.
In the stability test, the two formulations (control and a selected
formulation containing Spirulina sp.) were stored for 45 days under
refrigeration (4–6 ◦ C). Both formulations were periodically evaluated (0,
7, 15, 30, and 45 days) for their acidity, pH, water activity, total pro­
teins, total lipids, carbohydrates, crude fiber, total ash, color, and anti­
oxidant properties - 2, 2-diphenyl-1-picryl-hydrazyl-hydrate (DPPH), 3-
ethylbenzothiazoline-6-sulfonic acid (ABTS), and ferric reducing anti­
oxidant potential (FRAP).
The fatty acid profile and the microbiological analysis were evalu­
ated at the initial time (zero-day) and after 45 days of storage under
refrigeration (4–6 ◦ C). The minerals were evaluated at the final storage
time (45th day). Three replicates of all formulations were prepared and
analyzed in triplicate.
2.2.2. Physicochemical tests
The pH was measured using a digital pH meter (PHS-3D, China). The
titratable acidity (TA) was evaluated by pH titration that consisted of
titrating each sample with 0.1 M sodium hydroxide solution to the
8.1–8.2 pH range (method 945.08 – AOAC [22]). The moisture was
determined using an infrared moisture analyzer (Mx-50) at 105 ◦ C. The
water activity was evaluated by direct determination with an Aqualab
LITE hygrometer (AL1612, Decagon Devices, USA) at 25 ◦ C. The ash
content was determined by the gravimetric method in a muffle furnace
(QUIMIS) at 550 ◦ C (method 923.03 – AOAC [22]).
Total protein content was estimated using the Kjeldahl method and a
protein conversion factor of 6.38 [23]. Total lipids were determined by
the Bligh-Dyer method [24]. The solvent system consisted of chloro­
form, methanol, and water (2:1:1 v v− 1) and a gravimetric quantification
was performed. The total fiber was determined using the nonenzymatic
method based on ethanol precipitation (method 978.10 – AOAC [22]).
Total carbohydrates (TC) was estimated by the difference [25] accord­
ing to Eq. (1).
TC (%) = 100 − (M + TA + TN + TL) (1)
From Eq. (1) M is the sample moisture, TA is the total ash, TN is the
total nitrogen, and TL is the total lipids.
The minerals iron, sodium, calcium, and magnesium were deter­
mined by inductively coupled plasma-optical emission spectrometry
(ICP-OES spectrometer 720 series, Agilent Technologies). The method's
accuracy was confirmed by analyzing certified reference material,
namely, apple leaves (NIST 1515), under the same experimental con­
ditions. The analytical curves for each element were linear throughout
the operating range and covered the sample concentrations.
2.2.3. Texture analysis
The texture of samples stored for at least 24 h at 6 ◦ C was analyzed
using a texture analyzer (Brookfield CT3, USA). The tests were
L.M.R. Almeida et al. Algal Research 58 (2021) 102387

Tomato, onion, soybean oil, garlic, cumin, sodium chloride,

rosemary, parsley, oregano, and water

Mixeds in a blender Heated for 10 min at 95ºC

Incorporation of Spirulina sp. (0, 2, 3 and 4% (m m-1))

Sauce enriched with Spirulina sp. Control sauce

biomass
[2, 3 and 4 % (m m-1))]

Pasteurization by for 40 seconds at 98 ºC

Stored in a refrigerator Analysis

(4-6 °C) for 24 h

The control sauce and the formulation containing

Spirulina sp. biomass that presented the best results for
all parameters were selected for the stability test.

Sauce enriched with 4% Control sauce

Spirulina sp. biomass

Stored for 45 days under refrigeration (4-6 °C)

Periodically evaluated (0, 7, 15, 30, and 45 days)

Analysis

Fig. 1. Experimental design.

performed using a 100-mL beaker with 30 g of sample and a cylinder
probe (diameter, 38.1 mm; height, 20 mm (TA4/1000)). The samples
were compressed to 50% of the original thickness in a double cycle at
0.5 mm⋅s− 1 test speed and 5 g trigger load, over two cycles. The
following texture profile parameters were assessed: firmness (N),
cohesiveness and elasticity (mm) [26]. Data were processed using
Texture Expert for Windows 1.20 software (Stable Micro Systems).
2.2.4. Sensory evaluation
A team of 40 not trained tasters performed the sensory evaluation
[27]. The group consisted of students and staff of the Federal University
of Bahia (UFBA), of both sexes (80% women, 20% men), aged from 19 to
45. The session was conducted by first presenting four sauce samples
corresponding to the formulations with different Spirulina sp. biomass
concentrations and the control formulation.
The samples were served on small, white plates with a disposable
spoon and randomly encoded with three-digit numbers. As a vehicle for
tasting the sauce, 25-g portions of pasta were served in disposable
containers. The tasters evaluated the overall acceptance of the formu­
lations using a nine-point hedonic scale, ranging from 1 (dislike
extremely) to 9 (like extremely), for the following traits: appearance,
aroma, flavor, texture, and overall impression [23,27].
The tasters were also asked about their intention to purchase the
products, on a scale from 1 (definitely would not buy) to 4 (definitely
would buy). The Research Ethics Committee from the Federal University
of Rio Grande approved the study (process number 1.525.184).
2.2.5. Fatty acid identification and quantification
The transesterification of the extracted lipid fraction was performed
to determine the fatty acid composition, according to Nascimento et al.
[28]. The fatty acid methyl esters were separated on a gas chromato­
graph (Clarus 680, Perkin Elmer) equipped with a flame ionization

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L.M.R. Almeida et al. Algal Research 58 (2021) 102387

detector (GC-FID) and an Elite-WAX fused silica capillary column (30 m 2.2.9. Statistical analysis
× 0.32 mm × 0.25 μm). The equipment was set to 250 ◦ C injector The analyses were performed in triplicate and expressed as the mean
temperature and 280 ◦ C detector temperature. values ± standard deviation. Data were subjected to analysis of variance
The programmed column temperature was set to 150 ◦ C for 16 min, (ANOVA), followed by the Tukey's test (p < 0.05) to evaluate the dif­
increasing 2 ◦ C per min until 180 ◦ C, remaining at this temperature for ferences between the formulations zero (control), 2, 3 and 4% incor­
25 min, increasing 5 ◦ C until 210 ◦ C, and staying at this temperature for porated with Spirulina sp. biomass 24 h after the preparation.
25 min. Helium was used as a carrier gas at 1.0 mL min− 1. Samples were The formulation that presented the best physicochemical parameters
injected in split mode (1:90) with an injection volume of 1 μL. and sensory attributes was also evaluated during 45 days of storage
Fatty acids were identified by comparing the peak retention times of (zero, 7, 15, 30 and 45 days). The Student's t-test was used to assess the
samples with the retention times of fatty acid methyl ester standards differences between the control and the formulation that presented the
(C4–C24, 18919-AMP, Sigma-Aldrich®, USA). The quantification was best physicochemical parameters and sensory attributes at days zero and
accomplished by adding an internal standard (C23:0 Sigma, USA) to the 45 storage. A linear regression was used to verify the correlation be­
fatty acid methyl esters. The calculations were performed according to tween dependent variables. The tests were performed using the free
Eq. (2). The values were expressed as mg of fatty acids per g lipid (mg software environment for statistical computing and graphics R Com­
g− 1). mander 3.5.1.
( )
mg AFA × WIS × F
Concentration = 1000 × (2) 3. Results and discussion
g AIS × FFA × W
3.1. Preliminary results
AFA = area under the fatty acid methyl ester peak in the sample chro­
matogram;WIS = weight of the internal standard added to sample, in mg;
The control formulation had lower pH and higher acidity. Overall,
F = correction factor for the response of each fatty acid methyl ester in
the four formulations significantly differed in both parameters (p <
the ionization detector relative to C23:0;AIS = area under the internal
0.05). The differences increased with the content of Spirulina sp. biomass
standard peak in the sample chromatogram;W = weight of the sample in
incorporated into the sauce (Table 1). The supplementation with Spir­
milligrams; andFFA = fatty acid methyl ester to the fatty acid conversion
ulina sp. biomass increased the sauces' pH and decreased the product's
factor.
acidity. These differences were attributed to the biomass' pH, which is
neutral or slightly basic, ranging from 7.0 to 8.0.
2.2.6. Phenolic compounds and antioxidant activity
Similarly to pH, total acidity is also an important parameter for
Each extract's total phenolic content was determined using the Folin-
preserving and accepting a food product. Food decomposition, either by
Ciocalteu method described by Singleton and Rossi [29]. The readings
hydrolysis, oxidation, or fermentation, typically alters the hydrogen ion
were performed at 760 nm (Lambda 35 spectrophotometer, Perkin-
concentration and, consequently, the acidity [34]. The total acidity
Elmer). For quantification, a standard curve (50–250 μg⋅mL− 1) was
values differed among the formulations.
prepared with gallic acid, and the results were expressed in milligrams
Considering that the increase in Spirulina sp. biomass concentration
of gallic acid equivalent per gram of sample (mgGAE⋅g− 1). The radical
reduced the acidity, the formulation with 4% biomass had the lowest
scavenging activity of DPPH (2,2-diphenyl-1-picrylhydrazyl, Sigma-
value. This can be explained by the alkaline character of Spirulina sp.
Aldrich Chemical) was determined according to the method proposed
biomass. The Spirulina sp. biomass presented a buffering effect in the
by Vinson et al. [30], reading the samples at 517 nm.
sauce, reducing the concentration of free hydrogen ions. This is due to
Antioxidant activity (half maximal inhibitory concentration IC50)
some components such as proteins, peptides, and amino acids [35,36].
was expressed as the final concentration in mg⋅mL− 1 sample necessary
Interestingly, the addition of biomass in some food products de­
for reducing the concentration of DPPH by 50%. The ferric reducing
creases the pH and increases the total acidity. The biomass addition to
ability of plasma (FRAP) was determined according to the method
fermented products can stimulate some bacteria to produce organic
described by Benzie & Strain [31], reading the samples at 595 nm. A
acids. Thus, explaining the inverse effect in both pH and acidity when
standard curve was constructed with ferrous sulfate (500–2000 μM
compared to those observed in the present study. pH and acidity showed
mL− 1), and the results were expressed as μmol Fe2+g sample− 1.
a significant and inversely proportional correlation (r = − 0.99).
The radical scavenging activity of ABTS•+ (2,2′ -azino-bis (3-ethyl­
Thus, demonstrating that the lower the sauce's pH, the higher the
benzothiazoline-6-sulfonic acid; Sigma-Aldrich Chemical) was assessed
total acidity value will be. The negative correlation between pH and
according to the method proposed by Re et al. [32], reading the samples
acidity is consistent since the titratable acidity is related to the amount
at 734 nm. A standard curve was constructed with Trolox (6-hydroxy-
of organic acids present in the sample. The pH is associated with the
2,5,7,8-tetramethylchroman-2-carboxylic acid; Sigma-Aldrich Chemi­
concentration of H3O+ ions and this concentration is also proportional to
cal) (100–2000 μM mL− 1), and the results were expressed as μM Trolox g
the concentration of acids.
sample− 1.
Thus, both pH and acidity are important parameters for assessing
food conservation during storage since they can undergo changes caused
2.2.7. Color analysis
by microorganisms and enzymatic activity [37,38]. Barkallah et al. [23]
Color analysis was performed on a digital colorimeter (CIE L*a*b*
have also observed an inversely proportional relationship between the
system; CR-5 benchtop colorimeter, Konica Minolta). Calibration was
performed with a standard white plate according to the manufacturer's
instructions. Results were expressed as L*, (luminosity) ranging from Table 1
0 (black) to 100 (white); a*, ranging from − a* (− 60.0 green) to þa* pH and acidity values for the control sauce and the sauces supplemented with
(+60.0 red); and b*, from − b* (− 60.0 blue) to +b* (+60.0 yellow). different Spirulina sp. biomass concentrations after 24 h of storage.
Parameters Control sauce Spirulina sp. biomass content (%)
2.2.8. Microbiological evaluation 2 3 4
The samples were tested for Salmonella spp. (absence or presence in
pH 3.04 ± 0.01a 3.14 ± 0.01b 3.23 ± 0.02c 4.32 ± 0.02d
25 g), total coliforms, coagulase-positive Staphylococcus, mold, and yeast
Total acidity (%) 3.19 ± 0.09d 3.15 ± 0.02c 2.97 ± 0.59b 1.65 ± 0.01a
according to the Compendium of Methods for the Microbiological Ex­
amination of Foods, American Public Health Association (APHA) [33]. Data are expressed as the mean ± standard deviation of triplicates. Different
lowercase letters on the same line indicate a significantly difference between the
sauces analyzed according to the Tukey's test at α = 0.05.

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L.M.R. Almeida et al. Algal Research 58 (2021) 102387

pH and the acidity in the yogurts incorporated with Spirulina sp. presence of sulphur volatile compounds present in onions (such as iso-
biomass. allin, methiin, and propiin) [44] and garlic (allicin) [45] Besides, the
Texture, appearance, and flavor are the three organoleptic attributes cumin (o-cymene and cumic alcohol) [46], rosemary (several mono­
that determine consumer acceptability. Texture parameters correlate terpene hydrocarbons) [47], parsley (monoterpenes as myristicin, beta-
with sensory properties and are fundamental food attributes [39]. The phellandrene, and myrcene) [48], and oregano (p-cymene and carva­
textural (firmness, cohesiveness, and elasticity) and sensory parameters crol) [49] have also contributed to masking the biomass' undesirable
for the four formulations can be seen in Table 2. odor.
The firmness, cohesiveness, and elasticity values of the sauces con­ It is worth mention the considerable concentration of spices in the
taining 2%, 3%, and 4% Spirulina sp. biomass significantly differed from sauce and that their odors are very perceptible to the olfactory system.
each other and the control (p < 0.05). Accordingly, supplementation The sauce containing 4% biomass presented the highest flavor score,
with biomass changed the overall texture profile, resulting in increas­ which did not significantly differ from the control score (p = 0.924).
ingly firm and elastic sauces with increasing microalgal concentration. The addition of the microalga substantially changed the color of the
The sauce containing 4% biomass presented the highest firmness value. products from red to dark green. Therefore, the color was perceived as
Like in the present study, Bolanho et al. [40] also reported higher inadequate by the tasters, as shown in the appearance scores (Table 2).
firmness values for cookie formulations with 5% Spirulina sp. biomass This perception could have been caused because the tasters had not been
than for formulations containing zero and 2% of the same biomass. The trained. Thus, the control sauce scored significantly higher (p < 0.05) in
firmness values increase proportionally with biomass concentration. the appearance attribute than the sauces supplemented with 3 and 4%
This trend can be related to the microalgal's high protein content that Spirulina sp. biomass. Bolanho et al. [40] have also reported a similar
increases protein-protein interactions generating a more rigid and result. The cookies enriched with 5% Spirulina sp. biomass scored lower
cohesive gel network [5]. in the appearance attribute than the control.
The incorporation of Spirulina sp. biomass into the sauces also Despite the color difference, purchase intent did not differ signifi­
resulted in significant differences in cohesiveness and elasticity among cantly (p > 0.05) between the control sauce and the sauces supple­
all treatments (p < 0.05). The sauce containing 4% Spirulina sp. biomass mented with Spirulina sp. biomass (Table 2). This attribute score's
presented the lowest cohesiveness and highest elasticity. These results reached values near 3.0, classified in the hedonic scale as “probably
can also be correlated with protein content in biomass. Proteins are would buy”. Although the difference was not significant, the sauce
amphiphilic molecules, and they migrate spontaneously to an air-water containing 4% biomass scored the highest (2.95), with 57.5% of the
interface or an oil-water. Once at the interface, it can interact with the tasters indicating they would buy this sauce. Accordingly, aroma, flavor,
neighboring molecules and form an elastic film withstands thermal and and texture showed no significant change after the addition of 2%, 3%,
mechanical motions. However, for cohesiveness, there may have been a and 4% Spirulina sp. biomass, demonstrating general acceptability by
reduction in the chemical bonding resistance that makes up the product tasters.
[41]. The sauce added with 4% biomass showed the highest scores for the
The sensory analysis showed that the incorporation of Spirulina sp. purchase intent and overall impression. Therefore, indicating that the
biomass did not affect the tasters' acceptance of the product. Incorpo­ appearance did not harm these attributes. Besides, the sauce added with
rating 2, 3, and 4% biomass into the sauces did not significantly affect 4% biomass presented optimal physical characteristics (pH, acidity, and
the aroma, flavor, texture, and overall impression purchase (p > 0.05). texture), ensuring the final product's quality. Higher nutritional values
The undesirable fish flavor and smell from Spirulina sp. biomass have from the formulations with higher content of incorporated biomass are
already been associated with a mixture of several volatile compounds expected due to the proximate composition of Spirulina sp. biomass [20].
[42,43]. The spices' combination aroma used to prepare the sauces in In this study, the sauce containing 4% of Spirulina sp. biomass was
this study was necessary to mask Spirulina sp. biomass's unpleasant chosen as the best final product. This sauce was selected for further
flavor. analyses to evaluate its physicochemical, microbiological, and antioxi­
Thus, the undesirable fish aroma of biomass has been masked by the dant properties over 45 days of storage under refrigeration. Barkallah
et al. [23], have also chosen the formulation for evaluation during
Table 2
storage, based on the product's textural properties and sensory
Textural and sensory parameters for the control sauce and the sauces containing acceptability.
different concentrations of Spirulina sp. biomass.
Parameters Control Spirulina sp. biomass content (%)
3.2. Physicochemical characteristics
sauce
2 3 4
No chemical preservative was added to the sauce formulations. The
Firmness (N) 0.40 ± 0.01a 0.40 ± 0.10a 0.43 ± 0.06b 0.45 ± control of pH, acidity, moisture, and water activity is essential for
0.06b ensuring a product's food safety over time. The pH of both the control
Cohesiveness 1.23 ± 0.18b 1.38 ± 0.12c 1.42 ± 0.00 1.09 ±
d
0.03a
sauce and the sauce supplemented with 4% Spirulina sp. biomass grad­
Elasticity (nm) 3.39 ± 0.47a 4.31 ± 0.20b 4.66 ± 0.01c 5.75 ± ually decreased during the storage period (Table 3). The formulation
0.01d containing microalgal biomass showed a minor decrease (4.9%) than the
Appearance 7.20 ± 0.90b 6.65 ± 6.05 ± 0.70a 5.92 ± control (8.6%) after 45 days. Throughout the storage period, both sauces
0.80ab 1.01a
had pH values lower than 4.5. Similarly, Barkallah et al. [23] reported
Aroma 6.90 ± 0.90a 6.75 ± 0.90a 6.50 ± 0.72a 6.80 ±
0.83a pH values ranging from 4.04 to 4.53 for yogurt fortified with 0.25%
Flavor 6.62 ± 0.71a 6.10 ± 0.93a 6.35 ± 0.75a 6.82 ± Spirulina sp. biomass over 28 days of storage.
1.00a TA values were higher in the control sauce (90–217%), depending on
Texture 7.00 ± 0.65a 6.87 ± 0.84a 6.75 ± 0.80a 7.00 ± storage time (Table 3). The control's TA values significantly (p < 0.05)
0.90a
Overall 6.87 ± 1.12a 6.42 ± 1.30a 6.40 ± 0.95a 6.90 ±
differed from the sauce containing 4% Spirulina sp. biomass. The results
impression 1.04a show that the control sauce was more acidic than the supplemented
Purchase intent 2.82 ± 0.72a 2.65 ± 0.83a 2.75 ± 0.78a 2.95 ± sauce. The analyzed sauces showed a slight increasing trend in acidity
0.75a over time. This increase may indicate the onset of hydrolysis of fatty
Data are expressed as the mean ± standard deviation of triplicates. Different acids present in the products.
lowercase letters on the same line indicate a significant difference between the As a result of the action of enzymes, the hydrolysis of triacylglycerols
sauces analyzed according to Tukey's test at α = 0.05. promotes the release of free fatty acids. These, in turn, are susceptible to

5
L.M.R. Almeida et al. Algal Research 58 (2021) 102387

Table 3 protein, ash, and total fiber than the control (Table 3).
Evaluation of the control sauce's physicochemical characteristics and stability The microalgal's biomass supplementation increased the protein
and the sauce supplemented with 4% Spirulina sp. biomass over 45 days of content by 52% over the control sauce. The high protein increment
storage. noticed can be explained due to the high protein content (around 65%
Parameter/sauce Time (days) wt) present in the Spirulina sp. biomass used in this study [20]. It is
0 7 15 30 45 worth mention that the protein from microalgal biomass is qualitatively
complete because it presents the essential amino acid ratios recom­
pH Control 3.04 ± 2.91 ± 2.87 ± 2.85 ± 2.80 ±
0.01dA 0.02cA 0.01bA 0.02aA 0.01aA
mended by the Food and Agriculture Organization (FAO) [51].
4% 4.32 ± 4.26 ± 4.22 ± 4.14 ± 4.12 ± Incorporating 4% Spirulina sp. biomass into sauce adds nutritional
Sauce 0.01cB 0.03bB 0.02bB 0.01aB 0.01aB value to the product, which may help individuals reach the recom­
Total acidity Control 3.19 ± 3.72 ± 3.76 ± 3.78 ± 3.87 ± mended daily protein intake. A medium portion (60 g) of the sauce
(%) 0.09aB 0.01bB 0.01abB 0.01abB 0.08cB
enriched with Spirulina sp. biomass contributes 25% to 33% of the rec­
4% 1.65 ± 1.70 ± 1.72 ± 1.75 ± 1.78 ±
Sauce 0.01aA 0.02bA 0.02bA 0.07cA 0.01dA ommended protein intake according to the Dietary Reference Intakes
Aw Control 0.99 ± 0.99 ± 0.98 ± 0.98 ± 0.99 ± (DRIs) [52] (15–20%) for a healthy adult.
0,01aB 0,01aB 0,01aB 0.01aB 0.01aB The ash content in the sauce supplemented with Spirulina sp. biomass
4% 0.96 ± 0.95 ± 0.95 ± 0.95 ± 0.94 ± was significantly higher than that in the control sauce. No variations in
Sauce 0,01aA 0,01aA 0,04aA 0.01aA 0.01aA
Moisture (%) Control 86.01 87.63 87.09 86.41 86.13
ashes during storage were observed. The sauce supplemented with 4%
± ± ± ± ± biomass had higher ash content. This can be explained due to the
0.36bB 1.12aB 0.81abB 0.35abB 0.66abB essential minerals such as magnesium, iron, and calcium naturally
4% 83.46 83.55 83.19 83.33 83.40 present in the microalgal biomass. The total fiber content was also
Sauce ± ± ± ± ±
higher in the formulation containing biomass than in the control
0.51aA 0.07aA 0.05aA 0.18aA 0.15aA
Crude protein Control 5.44 ± 5.07 ± 4.93 ± 4.90 ± 4.91 ± formulation.
(%) 0.28aA 0.41aA 0.17aA 0.65ªA 0.45ªA These results corroborate the findings of Bolanho et al. [40], which
4% 8.26 ± 8.03 ± 7.86 ± 7.77 ± 7.81 ± reported higher ash and fiber contents in cookies enriched with 5%
Sauce 0.15aB 0.24aB 0.11aB 0.15aB 0.30aB Spirulina sp. biomass than its control sample. The carbohydrate content
Total lipids Control 2.09 ± 2.08 ± 2.00 ± 2.06 ± 2.04 ±
(%) 0.04aB 0.08aB 0.07aB 0.07ªB 0.03ªB
was higher in the enriched sauce than in the control sauce. Possibly, due
4% 1.95 ± 1.90 ± 2.01 ± 2.01 ± 2.00 ± to the amount of this compound present in the microalgal biomass,
Sauce 0.05aA 0.10aA 0.08aA 0.13aA 0.09aA which is approximately 10% [20]. The minor variations in the carbo­
Carbohydrate Control 4.44 ± 3.24 ± 3.93 ± 4.69 ± 4.90 ± hydrate content during the 45 days were attributed to the indirect
(%) 0.03dA 0.02aA 0.01bA 0.22cA 0.03eA
quantification method used in this study.
4% 5.26 ± 5.47 ± 5.91 ± 5.83 ± 5.73 ±
Sauce 0.02aB 0.03aB 0.04bB 0.03bB 0.05bB
Ash Control 0.43 ± 0.42 ± 0.43 ± 0.42 ± 0.40 ± 3.3. Fatty acids composition
(%) 0.01ªA 0.02ªA 0.01ªA 0.01aA 0.04aA
4% 1.07 ± 1.05 ± 1.03 ± 1.06 ± 1.06 ±
The sauce supplemented with 4% biomass presented 14 fatty acids.
Sauce 0.03aB 0.11aB 0.03aB 0.04aB 0.03aB
Crude fiber Control 0.60 ± 0.57 ± 0.57 ± 0.54 ± 0.54 ±
The C18:2 n-6 was the predominant, followed by C18:1 n-9 (Table 4). In
(%) 0.01aA 0.01aA 0.01aA 0.01ªA 0.01ªA the control sauce, C18:2 n-6 was also the major fatty acid derived from
4% 1.15 ± 1.16 ± 1.14 ± 1.13 ± 1.13 ± the soybean oil added to both formulations. Notably, the main lipid
Sauce 0.02aB 0.01aB 0.02aB 0.01aB 0.01aB source in the control sauce was derived from the lipid fraction of soy­
Aw: water activity. Data are expressed as the mean ± standard deviation of bean oil, which has high levels of polyunsaturated fatty acids [53],
triplicates. Different lowercase letters on the same line indicate a significant whereas the lipids in the sauce supplemented with Spirulina sp. biomass
difference between the averages for the same sauce (control or 4% sauce) at the also included fatty acids derived from microalgal, such as C16:1 n-7,
different times analyzed by the Tukey test (α = 0.05). Different capital letters in C18:1 n-9, and C18:3 n-3.
the same column indicate a significant difference between the control sauce and The sauce supplemented with 4% of biomass presented richer than
the 4% sauce simultaneously according to Student's t-test at α = 0.05. the control because its formulation contained oleic (C18:1 n-9, 136.73
mg⋅g− 1) and α-linolenic (C18:3 n-3, 43.57 mg⋅g− 1) acids. Daily intake of
oxidative processes, resulting in the formation of compounds respon­ food sources of omega-3 fatty acids is important for human nutrition.
sible for rancified taste and odor, changing the quality of the product However, industrialization has led to a gradual increase in the con­
[50]. Çelekli et al. [17], evaluated the increase in the growth and ac­ sumption of refined oils with a high linoleic acid (C18:2 n-6) content and
tivity of probiotic bacteria by adding Spirulina sp. biomass to ayran. The to a decrease in the consumption of foods rich in alpha-linolenic acid
authors found that during storage (21 days; 4 ◦ C), the samples added to (C18:3 n-3), resulting in an inadequate n-6/n-3 ratio [54]. Thus, the
Spirulina sp. also had lower acidity levels than the control. sauce enriched with Spirulina sp. biomass is an alternative practical food
The supplemented sauce's moisture and water activity values were that helps increase n-3 intake, thereby providing health benefits because
smaller than those found for the control sauce. The biomass is a dry the sauce supplemented with microalgal biomass contains C18:3 n-3.
powder (less than 10% of moisture) containing a variable content of In the sauce containing 4% biomass, the sum of monounsaturated
total solids. Beyond the high content of proteins, carbohydrates, and fatty acids (MUFAs) and polyunsaturated fatty acids (PUFAs) prevailed
some lipids, the biomass also has carbonates, bicarbonates, chloride, over the total saturated fatty acids (SFAs). The sauce supplemented with
sulfide, phosphate, and others. The inorganic species comes from the 4% biomass showed an increase of 130.84 mg⋅g− 1 in MUFAs and of
cultivation media and the cellular metabolism of microalgae. approximately 25% in SFAs and PUFAs compared to the control.
The addition of the biomass implies the substitution of a 4% (m m− 1) In contrast to the present study results, Barkallah et al. [23] reported
of high moisture content from the control by a dry content, resulting in a 70.56% increase in SFAs, overshadowing the increase in UFAs
decreased moisture and water activity values observed. Thus, the (29.19%) and MUFAs in the yogurt fortified with Spirulina sp. biomass.
formulation that contains 4% (m m− 1) of this dry powder contains more These differences can be attributed to the Spirulina sp. biomass compo­
macronutrients and total solids than the control. As a result, either the sitions, which strongly depend on the strains and the growth conditions
moisture or the water activity tends to decrease. It can be interpreted as used in the production of this microalga [20].
a substitution of 4% of a mass with high moisture content in the control In general, both sauces' storage for 45 days caused no significant
sample by the dried biomass. The supplemented sauce presented higher change (p > 0.05) in the fatty acid composition. Natural antioxidants

6
L.M.R. Almeida et al. Algal Research 58 (2021) 102387

Table 4 Table 5
Fatty acid composition of the control and 4% Spirulina sp. biomass sauces at the Total phenolic content and antioxidant activity of the control sauce and sup­
beginning and end of storage. plemented with 4% biomass after 45 days of storage (4–6 ◦ C).
Class Fatty acid Sauce (day 0) Sauce (day 45) Sauce Time (days)

Control 4% Spirulina Control 4% Spirulina 0 7 15 30 45

sp. biomass sp. biomass
TP Control 164.73 159.84 158.04 157.14 156.96
SFA C11:0 ND 1.66 ± 0.02a ND 1.63 ± 0.03a ± 0.66cA ± ± ± 0.32aA ± 0.67aA
C14:0 0.65 ± 0.95 ± 0.04b 0.69 ± 0.98 ± 0.03b 0.52bcA 1.55abA
0.04a 0.02a 4% 352.84 350.43 349.88 349.40 348.98
C16:0 69.90 ± 86.20 ± 0.3b 69.68 ± 85.25 ± 0.30b Sauce ± 0.08cB ± 0.04bB ± ± ± 0.41Ab
0.30a 0.30a 0.74abB 0.58abB
C17:0 ND 0.72 ± 0.03a ND 0.75 ± 0.04a DPPH Control 127.11 127.48 129.61 130.45 133.92
C18:0 21.44 ± 27.89 ± 0.09b 21.49 ± 27.96 ± 0.06b ± 0.18aB ± 0.54aB ± 0.64bB ± 0.13bB ± 0.03cB
0.50a 0.30a 4% 11.86 ± 11.94 ± 12.05 ± 12.26 ± 12.89 ±
C20:0 2.05 ± 2.44 ± 0.04b 1.96 ± 2.39 ± 0.50b Sauce 0.04aA 0.03abA 0.05bA 0.04bA 0.10bA
0.03a 0.05a FRAP Control 269.90 265.29 263.18 261.60 260.38
C22:0 2.17 ± ND 2.15 ± ND ± 0.05eA ± 0.05dA ± 0.03cA ± 0.73bA ± 0.02aA
0.07a 0.09a 4% 500.96 502.36 498.83 497.27 495.56
Total 96.21 ± 119.86 ± 95.97 ± 118.96 ± Sauce ± 0.05dB ± 0.28eB ± 0.31cB ± 0.07bB ± 0.05aB
(mg⋅g− 1) 0.18a 0.08b 0.12a 0.16b ABTS•+ Control 30.61 ± 30.70 ± 29.60 ± 29.30 ± 29.40 ±
MUFA C16:1c n7 ND 86.20 ± 0.02a ND 86.16 ± 0.01a 0.01bA 0.05bA 0.03aA 0.03aA 0.01Aa
C18:1c n9 92.50 ± 136.73 ± 83.27 ± 136.75 ± 4% 73.50 ± 72.63 ± 73.21 ± 72.51 ± 72.46 ±
0.03a 0.82b 0.01a 0.05b Sauce 0.70bB 0.03abB 0.35bB 0.04aB 0.09aB
C20:1 n9 0.95 ± 1.66 ± 0.01b 0.93 ± 1.64 ± 0.01b
0.02a 0.01a TP: total phenolic compounds (mgGAE⋅g− 1); GAE: gallic acid equivalent; DPPH
Total 93.45 ± 224.29 ± 84.20 ± 224.55 ± (IC50 mg⋅mL− 1); FRAP (mM ferrous sulphate⋅g− 1); ABTS•+ (μM Trolox⋅g− 1). Data
(mg⋅g− 1) 0.02a 0.02b 0.01a 0.02b are expressed as the mean ± standard deviation of triplicates. Different lower­
PUFA C18:2 n6 337.20 ± 367.24 ± 0.5b 336.95 ± 368.17 ± case letters in the same line indicate a significant difference between the aver­
0.6a 0.3a 0.01b ages for the same sauce (control or 4% biomass supplemented sauce) at the
C18:3 n6 0.88 ± 8.51 ± 0.2b 0.88 ± 8.47 ± 0.02b different times analyzed by the Tukey test (α = 0.05). Different capital letters in
0.04a 0.07a the same column indicate a significant difference between the control sauce and
C18:3 n3 ND 43.57 ± 0.7a ND 43.99 ± 0.05a
the 4% biomass supplemented sauce simultaneously by the t-test at α = 0.05.
C20:2 n6 ND 0.31 ± 0.01a ND 0.29 ± 0.05a
Total 338.08 ± 419.93 ± 337.83 ± 420.92 ±
(mg⋅g− 1) 0.32a 0.35b 0.18a 0.03b El Baky et al. [55] reported IC50 values of 55.98 μg⋅g− 1 at time zero
SFA: saturated fatty acid. MUFA: monounsaturated fatty acid. PUFA: poly­ and 100.41 μg⋅g− 1 after 30 days in cookies supplemented with 0.9%
unsaturated fatty acid. Values are expressed as mg of fatty acid per gram of fat. Spirulina sp. biomass, with a decrease in antioxidant activity throughout
Data are expressed as the mean ± standard deviation of triplicates. ND: not storage. Similar results were found in the present study but at different
detected. Different lowercase letters on the same line indicate a significant dif­ magnitudes.
ference between the control sauce and the 4% sauce at the time 0 days, and then The antioxidant potential of foods may be affected by processing and
45 days according to Student's t-test at α = 0.05. storage. The light, pH, temperature, oxygen exposure, and oxidizing
enzymes presence lead to variations in the antioxidant activity, espe­
(phenolic compounds, carotenoids, vitamins, and others) present in the cially by macerating plant tissues or using homogenization methods
Spirulina sp. biomass may have contributed to the sauce's oxidative [57]. Moreover, the compounds with antioxidant properties may not
stability because these compounds prevent lipid peroxidation during respond in the same manner to different radical or oxidant sources in a
storage [55]. Although the oxidative stability of oils is related to the complex food system. Thus, the use of multiple assays to measure the
unsaturation of their fatty acids, commercial vegetable oils are less antioxidant capacities is mandatory for proper evaluations [58].
susceptible to oxidative rancidity, mainly due to natural antioxidants The antioxidant activity was also quantified using the ABTS•+ and
and synthetic additives [56]. Thus, such components present in the FRAP assays. The results showed that similarly to the antioxidant assay
soybean oil may have stabilized the control sauce. using the DPPH radical scavenging method, the sauce supplemented
with 4% biomass had significantly higher (140%) antioxidant capacity
values than did the control sauce. The IC50 values and the total phenolic
3.4. Antioxidant properties
compounds presented directly proportional and high correlated (r =
0.81). The FRAP assay also showed that the sauce supplemented with
Table 5 presents the results from the set of methods used to deter­
4% biomass had a significantly higher (p < 0.05) antioxidant activity
mine the sauces' bioactive compound composition and antioxidant ac­
than the control sauce.
tivity. The sauce containing 4% biomass showed more than twice (p <
The control sauce also showed some antioxidant activity. Possibly,
0.05) of the total phenolic content than the control sauce. The DPPH (p
derived from the bioactive compounds present in the ingredients (spices,
< 0.001) and ABTS•+ (p < 0.05) radical scavenging capacities and ferric
tomato, and oil), which were also used in the sauce containing 4%
reducing ability (FRAP) (p < 0.01) of the supplemented sauce were also
biomass. However, the antioxidant activity values were lower in the
higher than those of the control sauce throughout storage.
control sauce, showing that supplementation with microalgal signifi­
Lower IC50 values were found for the sauce containing 4% biomass,
cantly increased the product's antioxidant capacity. Barkallah et al. [23]
which remained approximately ten times higher than that for the control
also reported that the antioxidant activity was higher in yogurt sup­
sauce during storage (Table 5). The higher antioxidant activity of the
plemented with Spirulina sp. biomass than in the control formulation.
sauce containing the microalgal can be attributed to its phenolic com­
pounds' free radical scavenging activity through hydrogen donation.
Total phenolic content showed a strong negative correlation with 3.5. Mineral contents
antioxidant activity (IC50) both in the control sauce (r = 0.95) and in the
sauce enriched with Spirulina sp. biomass (r = 0.99). At the end of the The iron, magnesium, calcium, sodium, and potassium contents of
storage period, the decreases in total phenolic contents were approxi­ the sauces at baseline are showed in Table 6. Macrominerals (Ca, Mg, K,
mately 1% in the sauce containing biomass and 5% in the control sauce. and Na) and microminerals (Fe, Se) are required in different amounts for

7
L.M.R. Almeida et al. Algal Research 58 (2021) 102387

Table 6 Table 7
Mineral composition of the control sauce and the sauce containing 4% Spirulina Variations in L* (luminosity). a* (red) and b* (yellow) in the control sauce and
sp. biomass with 45 days of storage.a the sauce with 4% Spirulina sp. biomass stored for 45 days at 4–6 ◦ C.
Minerala Control sauce 4% Spirulina sp. sauce Increase (%) Sauce Time (days) Color

Iron 1.18 ± 0.58a 4.20 ± 0.44b 356 L* a* b*

Magnesium 15.71 ± 2.43a 35.82 ± 3.09b 228
Control 0 23.62 ± 0.02Bd 34.38 ± 0.52Ba 21.44 ± 0.12Ba
Calcium 29.75 ± 1.33a 51.80 ± 2.21b 174
7 23.50 ± 0.01Bc 34.72 ± 0.02Ba 21.52 ± 0.02Ba
Sodium 169.74 ± 2.14a 354.67 ± 2.56b 209
15 23.43 ± 0.01Bb 34.64 ± 0.04Ba 21.50 ± 0.01Ba
Potassium 243.20 ± 2.51a 437.94 ± 3.14b 180
30 23.38 ± 0.03Bab 34.60 ± 0.06Ba 21.44 ± 0.05Ba
a
Data are expressed as mg 100 g− 1 on a wet basis. Data are expressed as the 45 23.37 ± 0.02Ba 34.53 ± 0.1Ba 21.42 ± 0.07Ba
mean ± standard deviation of triplicates. Different lowercase letters on the same 4% Sauce 0 5.79 ± 0.04Ab 1.45 ± 0.01Ac − 1.36 ± 0.01Aa
line indicate a significant difference between the control sauce and the 4% sauce 7 5.62 ± 0.09Aa 1.38 ± 0.01Ab − 1.34 ± 0.01Aac
15 5.66 ± 0.01Aa 1.36 ± 0.01Ab − 1.35 ± 0.01Aab
according to Student's t-test at α = 0.05.
30 5.67 ± 0.03Aab 1.32 ± 0.02Aa − 1.34 ± 0.01Abc
45 5.66 ± 0.04Aab 1.31 ± 0.01Aa − 1.33 ± 0.01Ac
human development because their proper intake is important to prevent
Data are expressed as the mean ± standard deviation of triplicates. Different
health problems related to nutritional deficiencies [59]. capital letters in the same column indicate a significant difference between the
The supplemented sauce with 4% biomass had significantly (p < control sauce and the 4% biomass sauce simultaneously by the t-test (α = 0.05).
0.05) higher mineral values than the control sauce at 45 days of storage. Different lower case letters in the same column indicate a significant difference
The increased values ranged from 174 to 356%, depending on the between the averages for the same sauce (control or 4% sauce) at the different
mineral (Table 6). Potassium was the major macromineral in the sauce times analyzed by the Tukey test at α = 0.05.
containing 4% biomass, followed by iron and magnesium. According to
the DRIs [60], 4.7 g of potassium per day is recommended for healthy blue-green color [64]. Due to this characteristic, the sauce supplemented
adults. The intake of two portions (120 g) of the sauce containing the with biomass had a strong dark green color.
microalgal would meet 11% of the potassium daily intake
recommendation.
Calcium availability is lower in fruits and vegetables than in milk and 3.7. Microbiological quality
its derivatives. In some population groups' diets as lactose intolerants,
vegans, elderlies, and celiacs, calcium is one of the most limiting mineral Microbiological analyses at baseline zero and after 45 days of storage
elements [61,62]. Thus, consumption of the sauce containing 4% showed an absence of Salmonella sp., mold, yeast, coliforms (≤3.0 most
biomass could increase 174% in this mineral, therefore, becoming an probable number (MPN)g− 1 product), and coagulase-positive Staphylo­
alternative for increasing the diversification of sources and dietary coccus (102 colony forming units (CFU)g− 1 product). Preservatives, such
intake of this element. as sorbic and benzoic acids, are commonly used to prolong the foods'
High iron contents were found in the sauce supplemented with 4% shelf life [65]. However, current consumer patterns show a trend toward
biomass. According to the Recommended Dietary Allowances [63], the a preference for products without chemical preservatives.
recommended iron intake per day is 8 mg for men aged from 19 to 50 The novel sauce developed meets the demands of consumers of meat,
years and 18 mg for non-pregnant women in the same age group. pasta and, salads, among other foods. Even without any addition of
Therefore, consuming two portions (120 g) of the supplemented chemical preservatives, the main microbiological and chemical issues of
sauce would suffice to meet more than half the recommended daily the formulation could be controlled during storage. Besides, the unde­
intake of this micronutrient for male adults. Despite this, women would sirable characteristic microalgal flavor has been successfully masked.
require a higher intake. Supplementing the sauce with 4% biomass The addition of 4% Spirulina sp. results in a milder sour taste due to
resulted in a satisfactory increase in mineral content, increasing the reduced acidity and increased pH (from 3.0 to 4.3). Moreover, the sauce
nutritional value of the product, providing an alternative for increasing is microbiologically stable for 45 days at 4–6 ◦ C. The growth of mold,
the inclusion of this compound in the population's diet. yeast, coliforms, Staphylococcus, and Salmonella is inhibited if measures
are taken to ensure low initial contamination with these microorgan­
3.6. Color measurement of sauce samples isms. Other parameters are enhanced, such as protein (34%), fiber
(48%), ash (57%), and mineral contents.
Table 7 relates the color parameters (L*, a*, and b*) of the control The total lipid content of the sauce with Spirulina sp. was similar to
and 4% biomass sauce formulations during storage. For the control that of the control, despite the increase in SFAs and PUFAs and the high
sauce, the parameters a* and b* indicate that the color remained stable MUFA content. The phenolic content also doubled, with a 12-fold in­
during the 45 days of storage. In the sauce with 4% biomass, it was crease in antioxidant activity, as shown by the IC50. This suggests that
observed that the parameters L, a*, and b* showed reductions over the the sauce may be protected from the various factors associated with lipid
45 days, achieving greater chromaticity directed to green intensity (b*) oxidation.
at the end of the storage period. This protection apparently results from the elimination of oxygen
When comparing the control sauce with the 4% biomass, significant due to the presence of antioxidant compounds. The sauce supplemented
differences (p < 0.05) were observed for all parameters (L, a* and b*) of with Spirulina sp. biomass represents a new approach to the food area,
the study. The differences were due to the microalgal's natural charac­ with a composition that can mask the unpleasant flavor of the micro­
teristic color (green-blue), which predominated in relation to the other algae, with the advantage of enhancing the nutritional and functional
ingredients. The results from L, a*, b* parameters corroborate with those quality of the product for dietary supplementation. The results obtained
obtained in the sensorial analysis. in this study demonstrated that the biomass-based-ready-made func­
The control sauce had a higher L* (luminosity) value than the sauce tional sauce has potential nutritional and sensory properties advantages.
supplemented with 4% biomass. Thus, indicating that the supplemen­
tation decreased luminosity causing the product's darkening. The a* and 4. Conclusion
b* values were lower in the sauce containing biomass (p < 0.001) than
the control sauce throughout the storage period, indicating that green The sauces prepared with 2%, 3%, and 4% Spirulina sp. biomass
prevails over yellow. The Spirulina sp. biomass contains many pigments, resulted in innovative products with good acceptance due to the sensory
including chlorophyll and phycocyanin, responsible for the microalgal's properties. The formulations developed in this study masked the strong

8
L.M.R. Almeida et al.
characteristic flavor of the microalgal and had properties that meet the
current consumer demand for healthy and practical foods.
Moreover, the data reported in this study indicate that the sauce
enriched with 4% Spirulina sp. biomass could provide health benefits
due to its high protein, fiber, mineral, monounsaturated, and poly­
unsaturated fatty acid content, and antioxidant potential. The 4%
biomass supplemented presented better attributes than the control
formulation. The sauce enriched with 4% Spirulina sp. biomass may be a
feasible option to improve the population's dietary intake that routinely
consumes processed and easily prepared foods.
CRediT authorship contribution statement
Leila Magda Rodrigues Almeida: Conceptualization, Methodology,
Writing - Original Draft, Writing - Review & Editing. Investigation,
Formal analysis, Visualization. Larissa Farias da Silva Cruz: Method­
ology, Investigation. Bruna Aparecida Souza Machado: Writing -
Original Draft. Itaciara Larroza Nunes: Resources, Project adminis­
tration, Funding acquisition. Jorge Alberto Vieira Costa: Resources,
Project administration, Funding acquisition. Ederlan de Souza Fer­
reira: Supervision, Resources. Paulo Vitor França Lemos: Writing -
Original Draft, Language. Janice Izabel Druzian: Conceptualization,
Supervision, Resources, Project administration, Funding acquisition.
Carolina Oliveira de Souza: Conceptualization, Writing - Review &
Editing, Supervision, Resources, Project administration, Funding
acquisition.
Statement of informed consent
No conflicts. An Informed consent was obtained from all individual
participants included in the study. All sensory research procedures fol­
lowed were in accordance with the ethical standards of the local com­
mittee responsible for human experimentation and with The Code of
Ethics of the World Medical Association (Declaration of Helsinki of
1975, as revised in 2013).
Funding
This work was supported by the Ministry of Science, Technology,
Innovations and Communications (n◦ 01200.005005/2014-49).
Declaration of competing interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgments
The authors are grateful to FAPESB (Foundation for Research Sup­
port of the State of Bahia) for scholarship.
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