Bioprocess and Biosystems Engineering (2020) 43:1231–1240

https://doi.org/10.1007/s00449-020-02318-4

RESEARCH PAPER

Magnetic fields: biomass potential of Spirulina sp. for food supplement

Mayara Copello Veiga1 · Mariana Martins Fontoura1 · Mariana Gonçalves de Oliveira1 · Jorge Alberto Vieira Costa2 ·
Lucielen Oliveira Santos1

Received: 31 January 2020 / Accepted: 23 February 2020 / Published online: 6 March 2020
© Springer-Verlag GmbH Germany, part of Springer Nature 2020

Abstract
This study evaluated the influence of the magnetic field on the chemical composition of Spirulina sp. LEB 18 and its digest-
ibility and protein solubility. The highest protein digestibility of biomass was obtained at 30 °C and with 2.5 g ­L−1 ­NaNO3
(78.4%) in the medium, and the highest solubility was found in the cultivated biomass exposed to 60 mT, 30 °C and 2.5 g
­L−1 ­NaNO3 (89%, pH 6). MF application did not modify the protein concentration of biomass, but reduced the carbohydrate
concentration by 69.1%, showing that the biomass obtained in the culture submitted to MF may be used as an ingredient in
the development of protein supplements.

Keyword  Ferrite magnets · Microalgae · Protein digestibility · Protein solubility

Introduction It is possible to modify its metabolic pathways to obtain

greater biomass production and to induce the synthesis of
Population growth has been stimulated the search for new biomolecules of high commercial interest [4]. However,
sources of food, especially protein-rich foods. The use of for this to happen, new technological approaches should be
microalgae, such as Spirulina for the development of food studied for microalgae cultivations. New technologies may
supplements becomes interesting because of its high protein be tested to increase protein concentration, digestibility and
concentration and the GRAS (Generally Recognized as Safe) solubility of Spirulina biomass, among them, the use of
certification from the Food and Drug Administration [1]. magnetic fields (MF).
Due to its composition, a portion of the market could be sup- MF may stimulate or inhibit growth in biological organ-
plied with foods containing microalgae proteins and lipids, isms and may influence the metabolism of microorganisms
making it a sustainable and cost-effective alternative [2, 3]. by modifying the synthesis of carbohydrates, proteins and
accumulation of essential amino acids [5, 6].
Although this area of study is promising and innovative,
* Lucielen Oliveira Santos it is still an unexplored area. Studies of the effects on the
santoslucielen@gmail.com growth rate, biomass composition and generated products
Mayara Copello Veiga may be found. However, there are no studies on the influence
mayaracopello@hotmail.com of MF on digestibility and protein solubility of the biomass.
Mariana Martins Fontoura Producing more high protein and high digestibility bio-
marianamartins@furg.br mass may be an alternative for human feeding since this
Mariana Gonçalves de Oliveira biomass has a better quantity and quality than conventional
marianaoliveira@furg.br proteins. Food proteins are added to food products due to
Jorge Alberto Vieira Costa their functionality and easy digestion. In the food indus-
dqmjorge@furg.br try, soy and milk proteins are widely used because of their
1
functional properties and nutritional value [3, 7]. Alternative
Laboratory of Biotechnology, School of Chemistry
and Food, Federal University of Rio Grande, Rio Grande,
sources of protein for human consumption need to be devel-
RS 96203‑900, Brazil oped to attend population demand. Microalga proteins stand
2
Laboratory of Biochemical Engineering, School
out favorably with conventional sources in terms of quality
of Chemistry and Food, Federal University of Rio Grande,
Rio Grande, RS 96203‑900, Brazil

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1232 Bioprocess and Biosystems Engineering (2020) 43:1231–1240

and quantity, which makes microalga biomass a promising
source of dietary protein [8].
This study aimed at evaluating the influence of the MF
application on the chemical composition of Spirulina sp.
LEB 18 and its digestibility and protein solubility, so that
it can be used in the food industry as an ingredient or food
supplement.
Assays were performed in triplicate for 16 days in acrylic
vertical tubular photobioreactors (1.8 L working volume).
Culture conditions were 30 °C or 35 °C, 12 h dark/light
photoperiod with a light intensity of 30 µmolphotons ­m−2 s−1.
The initial biomass concentration was 0.2 g L −1. Air fil-
tered (glass wool) was inject constantly (0.3 vvm) by the
compressor.

Application of magnetic fields in cultivation

Material and methods
Magnetic fields (MF) applied to photobioreactors were made
Culture conditions
by ferrite magnets adaptation with a mean intensity of 30
mT (150 × 50 × 10 mm) or 60 mT (50 × 50 × 25 mm). They
The microalga Spirulina sp. LEB 18 belongs to the Culture
were disposed at 180º each other and 150 mm above the base
Collection of the Laboratory of Biochemical Engineering
of the photobioreactor [12]. Thus, the generated MF was
at the Federal University of Rio Grande (FURG), located in
concentrated inside the photobioreactor and MF intensities
Rio Grande, RS, Brazil. This strain was isolated from Man-
were measured by MF meter (Global Mag, TLMP-HALL
gueira Lagoon located in Santa Vitória do Palmar, RS, Bra-
05 k, Brazil). Control cultures (CC) were done without MF
zil (latitude 33° 31′ 08″ S and longitude 53° 22′ 05″ W) [9].
application (Table 1), only 0.005 mT (Earth’s MF). Magnets
Assays were performed on standard Zarrouk medium
were replaced by inert material in CC to maintain the uni-
[10] which contains (g  L−1): ­NaHCO3 (16.8), K ­ 2HPO4
formity luminous received by the microalga. MF was applied
(0.5), ­NaNO3 (2.5), ­K2SO4 (1.0), NaCl (1.0), ­MgSO4.7H2O
throughout the culture period (24 h d−1).
(0.2), ­CaCl2 (0.04), ­FeSO4.7H2O (0.01), EDTA (0.08),
A5 solution (1 mL L−1) and B6 solution (1 mL L−1). The
A5 solution contains (g ­L−1): ­H3BO3 (2.86), ­MnCl2.4H2O Analytical determinations
(1.81), ­ZnSO4.7H2O (0.222), N ­ aMoO4 ­2H2O (0.015) and
­CuSO4.5H2O (0.079) whereas the B6 solution contains Biomass concentration (X, g L−1) was determined daily by
(g  L −1): ­N H 4VO 3 (0.023), KCr(SO 4) 2.12H 2O (0.048), UV–Vis spectrophotometer (QUIMIS Q7980RM, Brazil)
­Na2WO4.2H2O (0.018), ­TiO2 (0.0084) and Co(NO3)2.6H2O using a standard curve of Spirulina sp. LEB 18 (X = 0.5079.
(0.044). A modified Zarrouk medium was also used with the OD670, with R2 = 0.9882). This curve was obtained by relat-
same nutrient composition, but the ­NaNO3 concentration ing the optical density at 670 nm and biomass concentration
was reduced [11] to 1.875 g ­L−1. of Spirulina inoculum, as performed by Costa et al. [13].

Table 1  Culture conditions and Assays T (°C) Concentration MF* Xmax (g ­L−1) Pmax (g ­L−1 ­d−) µmax ­(d−1) Dt (d)
kinetics parameters of assays of ­NaNO3 (g application
with Spirulina sp. LEB 18 ­L−1) (mT)

1 30 2.50 – 1.02 ± 0.05b.c 0.06 ± 0.01a 0.19 ± 0.04a 3.83 ± 0.67a

2 30 2.50 30 1.17 ± 0.07b 0.08 ± 0.02a 0.18 ≤ 0.01a 3.86 ± 0.21a
3 30 2.50 60 1.18 ± 0.02b 0.09 ± 0.02a 0.19 ± 0.01a 3.59 ± 0.11a
4 35 2.50 – 0.98 ± 0.08c 0.07 ± 0.02a 0.20 ± 0.03a 3.44 ± 0.46a
5 35 2.50 30 1.02 ± 0.10b,c 0.06 ± 0.02a 0.17 ± 0.06a 4.33 ± 1.44a
6 35 2.50 60 1.10 ± 0.06b,c 0.08 ± 0.02a 0.16 ± 0.01a 4.26 ± 0.31a
7 30 1.875 – 1.02 ± 0.05b,c 0.06 ± 0.01a 0.18 ± 0.03a 4.00 ± 0.73a
8 30 1.875 30 1.11 ± 0.07b,c 0.08 ± 0.02a 0.17 ± 0.03a 4.15 ± 0.70a
9 30 1.875 60 1.40 ± 0.06a 0.08 ≤ 0.01a 0.21 ± 0.03a 3.41 ± 0.58a
10 35 1.875 – 1.11 ± 0.08b,c 0.08 ± 0.01a 0.18 ± 0.01a 3.91 ± 0.19a
11 35 1.875 30 1.02 ± 0.08b,c 0.05 ± 0.01a 0.14 ± 0.02a 5.18 ± 0.97a
12 35 1.875 60 1.03 ± 0.01b,c 0.09 ± 0.01a 0.18 ± 0.01a 3.93 ± 0.25a

Different lowercase letters in the same column correspond to significant differences (p ≤ 0.10) by the Tukey
test, for the same response
Xmax maximum biomass concentration, Pmax maximum biomass productivity, µmax maximum specific
growth rate, Dt doubling time

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Bioprocess and Biosystems Engineering (2020) 43:1231–1240 1233
The pH was directly measured daily by a digital pHme-
ter (KASVI K39-2014B, China). At the end of the culture,
biomass was separated from the medium by centrifugation
(Hitachi Himac CR-GIII, Japan) at 15,000 × g for 15 min,
resuspended in distilled water and centrifuged again under
the same conditions to remove nutrients from medium cul-
ture. The centrifuged biomass was dried in an air circulation
drying oven for 24 h at 40 °C (ODONTOBRÁS EL-1.2) to
be used for biomass analysis.
Biomass characterization
Protein, carbohydrate and lipid contents were analyzed in
biomass. It was necessary to prepare biomass extracts for
protein and carbohydrate determination. Five milligrams of
dry biomass and 10 mL of distilled water were sonicated in
an ultrasonic probe (COLE PARMER, CPX 130, EUA) for
10 min in 59-s cycles. The samples were kept on ice during
sonication to avoid heating of the sample. This procedure
was performed to release intracellular material from micro-
algae into a liquid medium.
Protein content was determined by the colorimetric
method described by Lowry et al. [14], with a standard
bovine serum albumin curve. Carbohydrate content was
determined by phenol–sulfuric method described by Dubois
et al. [15], with a glucose standard curve.
Lipid determination was performed directly on dry
biomass according to Folch, Lees and Stanley [16].
Extraction was done with chloroform:methanol (2:1) and
methanol:water (2:1) solvents to extract non-polar and polar
lipids (at room temperature), respectively. After biomass
characterization, to continue the study, the culture condi-
tions were used in which the highest protein and the lowest
carbohydrate concentrations were obtained.
It was necessary to determine the protein concentration
by the micro-Kjeldahl method [17] to perform the protein
solubility and digestibility determinations.
This method requires a larger amount of sample, as it is
an official food method, it was used only in the assays that
obtained the best results to perform the complete characteri-
zation of the biomass.
In vitro protein digestibility and protein solubility deter-
minations were performed. Protein digestibility in vitro was
evaluated by the method described by Akeson and Stahmann
[18], where 500 mg sample was suspended in 10 mL solu-
tion containing 1 mg mL−1 pepsin and incubated at 37 °C
for 3 h. After this period, the hydrolysis with pepsin was
stopped by the addition of 10 mL of 0.1 mol L−1 NaOH.
The samples were centrifuged, and the supernatant filtered.
In the precipitate, 10 mL of sodium phosphate buffer solu-
tion (0.2 mol L−1, pH 8.0) were added 2 mg mL−1 of pan-
creatin, and the reaction was maintained at 37 °C for 24 h.
After hydrolysis with pancreatin, 30% trichloroacetic acid
was added to stop the reaction. The sample was centrifuged
again, the supernatant filtered, and the precipitate was dis-
carded. Soluble proteins in pepsin and pancreatin hydrolysis
supernatants were quantified by the method of Lowry et al.
[14] with tyrosine as standard reference. Protein digestibility
of biomass was calculated by Eq. 1, where IVPD is in vitro
protein digestibility, PS the concentration of soluble protein
after the enzymatic digestion determined by the method of
Lowry et al. [14] and PTOTAL the total concentration of pro-
tein in the biomass determined by micro-Kjeldahl described
by AOAC [17].
PS
IVPD (%) = × 100 (1)
PTOTAL
Protein solubility was determined by the method of Morr
et al. [19] at pH 2, 4 and 6. Samples of 500 mg were resus-
pended in 2 mL NaCl (0.1 mol L−1) under stirring. After
complete dispersion of the sample, 40 mL buffer solution
at the corresponding pH was added and the mixture was
stirred for 45 min. Volume was bulked in 50 mL and the
suspension was centrifuged. Soluble protein concentration
was determined according to Lowry et al. [14] and protein
solubility calculated by applying Eq. 2, where PS is protein
solubility, PSS is the concentration of soluble proteins in the
supernatant and PTOTAL the concentration of proteins in the
micro-Kjeldahl determined sample described in AOAC [18].
PSS × 50
PS (%) = × 100 (2)
mSAMPLE ×
PTOTAL
100
Kinetic parameters
Maximum biomass concentration was obtained from bio-
mass concentration ­(Xmax, g L−1). The maximum biomass
productivity ­(Pmax, g L−1 d−1) was obtained in each culture
according to Eq. 3, where X ­ t is the biomass concentration
(g L−1) at time t (d) and ­X0 is the initial biomass concentra-
tion (g L−1).
The maximum specific growth rate (µmax, ­d−1) was calcu-
lated by linear regression of the logarithmic growth phase in
the graph of ln X (g L−1) versus t (d). In this curve, the slope
was the maximum specific growth rate. The doubling time
(Dt) was determined in the exponential growth phase apply-
ing Eq. (4). Efficiency (η) was calculated by applying Eq. (5)
to compare the results obtained from MF-applied cultures in
relation to CC, where CMF is MF-applied responses and CC
responses obtained in the control culture.
(X − X0 )
Pmax = (3)
(t − t0 )
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1234 Bioprocess and Biosystems Engineering (2020) 43:1231–1240

ln(2) Results and discussion

Dt = (4)
μmax
Effects of MF on biomass concentration and kinetic
(CMF − CC ) parameters
η(%) = × 100 (5)
CC
Microalga growth was observed under all conditions. How-
ever, MF application at 30 °C stimulated the growth of Spir-
ulina sp. LEB 18 (Fig. 1b). The highest biomass concentra-
Statistical analysis tion (Xmax) was 1.40 ± 0.06 g L−1 (Table 1) with reduction of
­NaNO3 concentration and 60 mT. None of the assays had an
Results were evaluated using variance analysis fol- adaptation phase, showing exponential growth from day one.
lowed by Tukey test, with a confidence interval of 90% In the study of Deamici, Costa and Santos [12] the bio-
(p ≤ 0.10). Student’s t-test was also performed with a sig- mass concentration increased by 89.3% when Spirulina sp.
nificance level of 95%. LEB 18 was exposed to 60 mT for 24 h d−1 with a light
intensity of 60 µmolphotons ­m−2 s−1. In this study, the appli-
cation of 60 mT at 30 °C and 1.875 g L−1 of N ­ aNO3 with a

Fig. 1  Biomass concentration of cultures at 30 °C with a 2.5 g L−1 ­NaNO3 and b 1.875 g ­L−1 ­NaNO3 and 35 °C with c 2.5 g ­L−1 ­NaNO3 and d
1.875 g ­L−1 ­NaNO3

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Bioprocess and Biosystems Engineering (2020) 43:1231–1240 1235
light intensity of 30 µmolphotons ­m−2 s−1 increased by 37.2%
Xmax, therefore, this is the best condition for biomass yield.
The difference between the results obtained of the previous
study [12] and in this study was the light intensity because
this is an important factor for microalgae growth since
the growth of this microorganism requires a light energy
source is necessary for photosynthesis [4, 20–22].
Lv et al. (2010) [23] cultivated Chlorella vulgaris with
24, 60 and 120 μmol m−2 s−1 and the highest lipid con-
centration and biomass productivity were obtained using
60 μmol m−2 s−1. In addition, the authors observed that
using intensities higher or lower than this, they obtained
the lowest biomass productivity and lipid concentra-
tion. Thus, the light intensity may modify pH, Mg 2+ and
NADPH, indirectly increasing the activity of the main
enzymes of lipid synthesis, while limitation of the light
may reduce enzymatic activity.
In the culture of Chlorella kessleri LEB 113, 30 mT and
60 mT at different exposure times (1 h d−1 and 24 h ­d−1)
were used to verify the effects of microalga growth. The
highest ­Xmax (1.39 g ­L−1) was obtained when 60 mT was
applied for 1 h d­ −1 compared to the control culture (0.76 g
­L−1) [24]. However, when intensities from 0 to 550 mT
were applied to evaluate the growth of Spirulina platensis,
there was an increase in the biomass concentration with
250 mT and the growth inhibition was observed at higher
intensities [25].
Therefore, the highest biomass concentrations found in
this study and previous studies [12, 24, 25], have demon-
strated that the application of higher MF intensities has
a stimulating effect on microalgae growth. This makes
biomass production economically viable, as the magnet
pair value is about $ 6.00 and may be used in large-scale
bioreactors by recirculating the medium in the magnets.
There was inhibition in the ­X max at 35  ºC and with
1.875 g ­L−1 ­NaNO3, applying 30 mT or 60 mT (Fig. 1c,
d). MF application, high temperature and reduced nitrogen
source may have caused stress on microalga cells, inhibit-
ing cell growth. Temperature affects metabolic activities
as well as the availability and absorption of nutrients and
other physical properties of the culture medium. This may
be related to stimulation of the microalga respiration rate
at high temperatures, which causes high cellular energy
expenditure and, consequently, a reduction in biomass con-
centration [26]. Metabolic reactions are based on the dif-
ference in electrical charges and ions in the system, which
may have been altered by MF application. Therefore,
35 °C, with different ­NaNO3 concentrations and MF appli-
cation did not influence the microalga growth (Fig. 1c, d).
However, the highest ­Xmax (Table 1) was reached when
30 °C and 60 mT was used. So, the temperature of 30 °C
should be used to obtain microalga biomass.
The increase in ­Xmax at 30 °C may be related to the ­CO2
partial pressure in the medium being higher at this tempera-
ture than at 35 °C, which leads to an increase in bicarbonate
concentration and consequently increases the photosynthetic
rate [11]. The low partial pressure of ­CO2 in the atmosphere
usually cannot maintain the demand for inorganic carbon
assimilation during the intense growth of microalgae, caus-
ing a growth limitation. However, increased ­CO2 transfer
capacity may increase the availability of inorganic carbon in
the culture, which leads to increased microalga growth [27].
Other authors, such as Hirano, Ohta and Abe [28], Li
et al. [25], Tu et al. [29] reported the same conclusions of
this study under certain experimental conditions there is a
range of MF intensities that inhibition occurs. However,
there is a range in which inhibition does not occur, so that
growth and product uptake are not inhibited. Comparison
of studies showed that MF does not act linearly and fac-
tors, such as exposure time, frequency and intensity, cellular
condition of the strain used, and culture conditions should
be considered.
pH
The pH of cultures ranged from 9.6 to 10.7. Cyanobacteria
must be cultivated at a pH between 8.3 and 11.0, as this
range has a high photosynthetic rate, moreover, at pH val-
ues outside this range, the cultures are subject to contami-
nation [13]. The gradual increase in pH occurs due to the
mechanism of bicarbonate assimilation by cyanobacteria,
the cells incorporate two bicarbonate ions, one is consumed
and internalized as carbon dioxide ­(CO2), while the other is
released as carbonate (­ CO3−2) and leading increased pH in
the medium [30].
The pH of the cultures ranged from 9.6 to 10.1 when
30 °C and 2.5 g L ­ −1 ­NaNO3 without MF application was
used, but with 30 mT the pH range was 9.6 a 10.3 and with
60 mT from 9.6 to 10.7. This behavior shows that depend-
ing on the applied intensity, the MF may change the energy
levels and the spin orientation of the electrons. In addition,
all magnetic forces changes in ion exchange, which may
cause pH change in the medium. However, even with these
changes in pH, the results remained within the range con-
sidered ideal for Spirulina cultivation.
Characterization of biomass
The chemical compositions of Spirulina biomass in the
assays are shown in Table 2. The cultivation of Spirulina
in Zarrouk medium can reach protein concentration from
60 to 70% (dry weight) depending on the culture conditions
[31]. The high concentration of proteins in Spirulina sp. is
an important factor due to the increased nutritional value
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1236 Bioprocess and Biosystems Engineering (2020) 43:1231–1240

Table 2  Chemical composition of Spirulina sp. LEB 18 biomass

Assays T (°C) NaNO3* (g ­L−1) MF** (mT) Protein (%) Carbohydrate (%) Lipid (%) Ashes (%)

1 30 2.50 – 70.5 ± 6.5a 22.3 ± 3.6a 14.0 ± 1.5a,b 2.0 ± 0.6e

2 30 2.50 30 66.2 ± 2.0a 8.5 ± 1.5b,c,d 12.4 ± 5.8a,b 3.1 ± 0.8d,e
3 30 2.50 60 73.2 ± 6.1a 6.9 ± 0.4b,c,d 13.4 ± 1.2a,b 11.5 ± 3.7b
4 35 2.50 – 72.8 ± 2.3a 12.5 ± 3.4b,c 16.4 ± 4.7a,b 7.4 ± 0.4b,c,d
5 35 2.50 30 56.8 ± 8.5a 12.2 ± 3.5b 12.9 ± 2.4a,b 5.3 ± 1.7c,d,e
6 35 2.50 60 65.8 ± 2.6a 14.5 ± 1.1c,d 15.0 ± 4.1a,b –***
7 30 1.875 – 58.4 ± 1.4a 12.1 ± 1.8b,c,d 16.9 ± 1.1a 16.1 ± 1.0a
8 30 1.875 30 60.7 ± 4.0a 10.5 ± 1.1b,c,d 11.8 ± 1.5a,b 4.9 ± 0.1c,d,e
9 30 1.875 60 69.5 ± 7.5a 8.7 ± 1.5b,c,d 9.1 ± 1.5b 8.1 ± 0.4b,c
10 35 1.875 – 66.4 ± 0.9a 13.8 ± 2.3d 11.1 ± 2.1a,b 7.3 ± 0.4b,c,d
11 35 1.875 30 67.2 ± 5.9a 8.6 ± 0.3c,d 11.0 ± 2.8a,b 7.2 ± 1.9b,c,d
12 35 1.875 60 69.2 ± 5.2a 11.4 ± 1.6b,c,d 12.6 ± 0.9a,b 4.4 ± 1.0c,d,e

Different lowercase letters in the same column correspond to significant differences (p ≤ 0.10) by the Tukey test, for the same response
*Concentration of sodium nitrate
**Magnetic fields
***Not determined

of biomass, making it an alternative source of protein for
human and animal consumption [32]. Changes in tempera-
ture, ­NaNO3 concentration and MF application did not show
significant differences in protein concentration (p < 0.10).
However, good protein concentrations were observed in dif-
ferent conditions evaluated, remaining in the range from 56
to 73% (m ­m−1).
The high protein content of Spirulina extracts suggests
many possible nutritional applications. Food supplements
with high protein content have high market demand and are
consumed to improve sports performance, hypertrophy and
muscle recovery and overall health [7]. It is also considered
important for the elderly to consume high-quality protein-
rich supplements to compensate for the loss of muscle mass,
providing health benefits [33, 34].
Spirulina has a nutritional profile that makes it an ideal
food supplement because it has a high protein content, as
well as vitamins, minerals and pigments. Carvalho et al.
[35] developed and evaluated six different supplements for
athletes (electrolyte reinforcer, muscle enhancer and recov-
ery supplement), with and without Spirulina. The Spirulina
electrolyte booster, compared to the product without micro-
algae, showed a 0.35% (w/w) increase in mineral content.
The carbohydrate content of the recovery supplement devel-
oped with spirulina was 2% (w/w) higher than recovery sup-
plement without Spirulina. Microalga Spirulina is known
to have active compounds with important functions for the
body. Thus, the composition of the food met the nutritional
needs of athletes.
Due to its satiety efficiency, protein consumption is gen-
erally favored by its role in controlling food intake, favor-
ing weight loss and preventing weight gain in athlete and
non-athlete groups [36]. In addition, combined protein and
carbohydrate intake improves performance and increases
blood glucose levels [37]. Protein added to carbohydrate
may increase insulin; decrease cortisol levels and reduce
muscle damage by an average of 27% and muscle pain by
30% in runners [38].
Lipid concentrations were higher than those reported by
Belay [1] (Table 2), which found 6% (m ­m−1) in Spirulina
sp. biomass. The microalga genus was the same, but not the
same species. Thus, MF acts differently on different strains
of the same microalga, as previously reported, probably due
to differences in the physiological state of the cells. The
higher the culture temperature, the higher lipid concentra-
tion, reaching the highest value between 30 and 35 °C for
most strains of Spirulina [39]. However, there was no signifi-
cant difference (p ≤ 0.10) in lipid concentration at different
temperatures used in cultures. The high ash content in Spir-
ulina makes it important for vegetarians, athletes, elderly,
and children, who need more minerals in their food to stay
healthy if the biomass contains essential minerals in its
constitution [40]. The ash content of the biomass produced
ranged from 2.0 ± 0.6% to 16.1 ± 1.0% (p ≤ 0.10) (Table 2).
When comparing the experiments without MF application,
it was noticed that temperature and N ­ aNO3 concentration
directly influenced the ash concentration of the biomass.
Moreover, in assays at 30 °C with 2.5 g L−1 ­NaNO3, MF
application increased ash concentration, but when applying
the MF with 1.875 g L−1 ­NaNO3, at the same temperature, it
had the opposite effect by decreasing the ash concentration.
The chemical composition of biomass varies according
to the cultivation conditions applied, as well as the strain.
Thus, in studies using Spirulina sp. LEB 18 found ash

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Bioprocess and Biosystems Engineering (2020) 43:1231–1240 1237
concentrations ranging from 8 [41] to 16% [8], which dem-
onstrates that the content found in this study is in agreement
with the microalga used.
Carbohydrate synthesis was reduced with 30 mT and 60
mT at 30 °C (Table 2) demonstrating that the use of MF
inhibited the carbohydrate synthesis in 61.9% and 69.1%,
respectively. The physiological and biochemical constitu-
tion of microorganisms may be susceptible to the action of
electromagnetic forces [5]. All metabolic reactions are based
on the difference in electrical charges and system ions. Like-
wise, electromagnetic forces cause changes in biological cell
metabolism and the movement of electrons and ions may
cause changes in biomolecules concentration, such as pro-
tein, carbohydrate, and lipid. Therefore, it can modify free
radical activities, cell growth and enzymatic activity [42].
Some of the reasons why electrostimulation is rarely
applied to fermentative processes is the lack of knowledge
of the molecular conditions of most fermentation processes,
particularly those involving the synthesis of extracellular
products. The contradictory results and the lack of reproduc-
ibility are believed to be typical problems of MF research
and differences in researchers results may be due to the
experimental design used, the microorganism strains, the
exposure time, the system used for the generation and meth-
ods of field detection [43]. MF application associated with
different temperatures and ­NaNO3 concentrations reduced
carbohydrate concentration. The smaller concentration
was 6.9 ± 0.4% (Table 2) when 60 mT, 30 ºC and 2.5 g L ­ −1
­NaNO3 were used.
According to ANVISA Resolution no 18 of 2010 [44] in
Brazil to be considered a protein supplement, the ready-to-
eat product must contain at least 50% of its protein derived
energy value. However, as there was no significant differ-
ence in concentration (p ≤ 0.10), it was decided to use the
condition with the lowest carbohydrate concentration in the
biomass for further study, because the low carbohydrate con-
tent and high solubility are required to give excellent nutri-
tional profile to provide an effective functional ingredient for
many nutritional applications [45]. To obtain protein-rich
biomass for the development of food supplements, assays
with 2.5 g L ­ −1 ­NaNO3, 30 °C without MF application or
with 60 mT were done to evaluate the in vitro protein digest-
ibility and the protein solubility of the biomass. The biomass
Table 3  Biomass Assay Proteins (%)
characterization in assays with
Spirulina sp. LEB 18 Control culture 67.5 ± 4.6a
Culture with MF 64.8 ± 6.0a
Control culture: 30 °C, 2.5 g L−1; Culture with magnetic field application: 30 °C, 2.5 g L−1 and 60 mT
*IVPD = In vitro protein digestibility
Different lowercase letters in the same column correspond to significant differences (p ≤ 0.05) by Student’s
t test, for the same response
characterization of these assays was shown in Table 3. Lipid
and protein concentrations in both assays showed no signifi-
cant difference. However, differences in carbohydrate con-
centration were observed. Therefore, both conditions showed
potential for application as an ingredient in the formulation
of protein supplements.
In vitro digestibility
The highest in vitro digestibility was found in control cul-
ture biomass, being 78.4 ± 2.1% and significantly different
(p ≤ 0.05) from the 60 mT assay, where the digestibility was
73.6 ± 1.3% (Table 3).
MF effect on free radical concentration is amplified if the
pair of radicals have opposite charge. Therefore, these radi-
cals are some of the possible sites of action of MF exposure
[46]. MF may also cause oxidative stress in organisms by
altering energy levels and electron spin orientation and con-
centration of free radical, which alters the relative likelihood
of recombination of other interactions with possible bio-
logical consequences [47]. Furthermore, algae proteins are
susceptible to the action of proteolytic enzymes and there are
compounds, such as phenolic compounds and polysaccha-
rides that may limit the digestibility of their proteins [48].
MF affects the production of biomolecules in microorgan-
isms [12, 24, 28, 49]. Therefore, it may have caused changes
that resulted in lower digestibility in MF assays. In the study
of Almeida et al. [50], the authors found 55.2% in vitro pro-
tein digestibility in soybean protein isolate. Thus, Spirulina
biomass had higher digestibility by comparison with soy
protein. In addition, Sinha et al. [51] evaluated in vitro pro-
tein digestibility of whey protein concentrate before and
after hydrolysis, in the former the digestibility was 25% and
in the latter the digestibility increased to 70%. Digestibility
is extremely important when it is desired to apply ingredi-
ents to foods, such as protein supplements. From this, the
protein digestibility of Spirulina biomass without and with
MF application is comparable to whey proteins demonstrat-
ing that this biomass can be used in the food industry. In
relation to digestibility, Spirulina sp. showed 70% [31],
Spirulina sp. LEB 18 showed 70% [8] and 74.1% [52]. The
digestibility of Spirulina pacifica and Spirulina platensis
was 71.4% and 81.9%, respectively [53].
Lipids (%) Carbohydrates (%) Ashes (%) IVPD* (%)
14.0 ± 1.5a 21.2 ± 3.7a 2.0 ± 0.6b 78.4 ± 2.1%
13.4 ± 1.2a 6.1 ± 0.3b 9.4 ± 1.6a 73.6 ± 1.3%
13

1238
Fig. 2  Protein solubility of Spirulina sp. LEB 18. Different lower-
case letters in the same column correspond to significant differences
(p ≤ 0.05) by Student’s t test, for the same response
Protein digestibility of commercial protein concentrates
varies from 55.2% for soybean protein [50] to 70% in whey
concentrate [51]. Thus, the protein digestibility in the assays
without and with MF application had higher results than
the commercialized concentrates. However, MF application
stimulates the biomass production, making this an alterna-
tive to obtaining biomass for use as an ingredient in food
supplements.
Protein solubility
Figure 2 shows the solubility of Spirulina sp. LEB 18 with
and without MF application at different pH. The highest pro-
tein solubility of biomass was found at pH 6 in culture with
MF application (89.1%). Biomass solubility in MF-applied
cultures was higher than the control culture at all pH tested.
Breakdown of protein molecules into smaller peptides may
justify an increase in protein solubility [8]. Thus, MF appli-
cation may cause changes in cell structure and morphology,
which may have resulted in protein breakdown into smaller
peptides.
Protein solubility is considered a critical property by
food manufacturers because the high solubility allows for
rapid and complete dispersion of protein molecules. So, it
is important to know the solubility when determining the
degree of protein extraction and purification. Due to this,
the maintenance of protein solubility has been the focus of
researchers and food scientists [7]. The solubility of pro-
teins depends on the pH. Thus, solubility increases at pH
above the isoelectric point (IP) and decreases at pH below
the IP. The increase in solubility is due to the increase in the
13
Bioprocess and Biosystems Engineering (2020) 43:1231–1240
electrostatic repulsion between the protein molecules and
the hydration of the charged residues [54].
Due to their functional properties and nutritional value,
whey and soybean proteins in their concentrated and isolated
forms are widely used in the food industry [7]. The solubility
of commercial whey protein concentrates at pH 4 and 7 was
about 59.1% and 73.9%, respectively [55]. In commercial
soybean protein concentrates at the same pH, the solubility
of 30% at pH 4 and 50% at pH 7 was found [56].
Results of protein solubility in the biomass of Spirulina
sp. LEB 18 without MF application were like those previ-
ously reported [8], which found solubility of 30%, 10% and
50% at pH 3, 4 and 7, respectively. Therefore, it can be said
that the protein solubility of Spirulina biomass is compara-
ble with commercial whey and soybean protein concentrates,
so this biomass is best suited for use in the food industry.
Conclusion
Magnetic field application stimulated 37.2% (1.40 ± 0.06 g
­L−1) the growth of Spirulina sp. LEB 18 when 60 mT and
30 °C was used and reduced 69.1% carbohydrate concentra-
tion. Therefore, this condition is suitable to obtain biomass
for use as an ingredient in food supplements. The protein
digestibility of biomass in the control culture and in the cul-
ture with MF application at 30 °C and nitrate concentration
of 2.5 g L­ −1 was higher than those found in commercial
protein concentrates. The biomass with MF application had
high protein solubility at all pH tested (89% at pH 6). Bio-
mass obtained with MF application is promising for use in
food and have the potential to be used as an ingredient in the
manufacture of protein supplements.
Acknowledgements  This study was financed in part by the Coorde-
nação de Aperfeiçoamento de Pessoal de Nível Superior—Brasil
(CAPES)—Finance Code 001.
Compliance with ethical standards 
Conflict of interest  The authors declare that they have no conflict of
interest.
Ethical approval  This article does not contain any studies with human
participants or animals performed by any of the authors.
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