Multi-response optimization of alginate bleaching technology extracted from brown seaweeds by an eco-friendly agent

Carbohydrate Polymers 251 (2021) 116992

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Carbohydrate Polymers
journal homepage: www.elsevier.com/locate/carbpol

Multi-response optimization of alginate bleaching technology extracted
from brown seaweeds by an eco-friendly agent
Camila Yamashita a, *, Izabel Cristina Freitas Moraes b, Antonio Gilberto Ferreira c,
Ciro Cesar Zanini Branco a, Ivanise Guilherme Branco a
a
b
c

S˜
ao Paulo State University (UNESP), Biological Sciences Department, 19806-900 Assis, S˜
ao Paulo, Brazil
University of S˜
ao Paulo (USP), Food Engineering Department, 13635-900, Pirassununga, S˜
ao Paulo, Brazil
Federal University of Sao Carlos (UFSCAR), Chemistry Department, 13565-905, S˜
ao Carlos, S˜
ao Paulo, Brazil

A R T I C L E I N F O

A B S T R A C T

Keywords:
Ozone

Sodium alginate
Depolymerization
Sargassum
Response surface methodology
Physical properties

Alginate only finds industrial applicability after undergoing a bleaching process to improve its visual appearance.
Box-Behnken Design was used to optimize bleaching parameters (time, oxygen flow and temperature) for sodium
alginate (SA) extracted from seaweeds using ozone as the bleaching agent. The optimal conditions (oxygen flow 2
L/min for 35 min at 25 ◦ C) resulted in an ozone-bleached SA with a mannuronic/guluronic acids ratio of 0.70,
viscosity-average molecular weight of 66.30 kDa and dynamic viscosity of 1.39 mPa.s, aligned to strong and
brittle gels formation, which are potentially suitable for hydrogels and bioink application. Results indicated that
ozonation caused depolymerization of the SA chain. Colorimetric parameters showed that ozone has a great
bleaching efficacy. The bleached sample presented high antioxidant capacity, highlighting that discoloration by
ozone might have minimal effects on the bioactive compounds which are valuable ingredients for food-based
products.

1. Introduction
Seaweeds, also known as marine macroalgae, are becoming an
increasingly attractive resource for human utilization due to their high
growth rates in areas lacking freshwater and arable land (Lorbeer,
Lahnstein, Bulone, Nguyen, & Zhang, 2015). There are several func­
tional marine-derived compounds, for example, the polysaccharides that
are abundant in Phaeophyta (brown algae), an algal phylum which is the
main source of commercially available alginate (Rhein-Knudsen, Ale,
Ajalloueian, & Meyer, 2017). Species of brown seaweed which yield
alginate include Macrocystis pyrifera, Laminaria spp., Ascophyllum nodo­
sum and Sargassum spp. (Gates, 2012).
In the recent decade, alginate isolated from marine algae have shown
a wide range of applications because of their biological activity and

relatively low toxicity (Fleita, El-Sayed, & Rifaat, 2015) in food,
cosmetic and pharmaceutical industries such as biodegradable pack­
aging materials, controlled drug delivery and nanoremediation (Fer­
nando, Lee, Han, & Ahn, 2019; Fernando, Kim, Nah, & Jeon, 2019).
Despite all these applications, Brazil imported alginic acid or alginate
worth approximately $8.33 million, mainly from Chile and China in

2018 (ATLAS OF ECONOMIC COMPLEXITY, 2020).
Alginate is a particular type of polysaccharide present in the cell wall
of brown seaweeds, containing 1,4-linked β-D-mannuronic acid (M) and
α-L-guluronic acid (G) residues arranged in an irregular, blockwise order
along the chain (Davis, Ramirez, Mucci, & Larsen, 2004). These blocks
can be composed of homopolymeric sequences (MM or GG) and heter­
opolymeric sequences (MG) in the same molecule structure (Draget,
Smidsrød, & Skjåk-Bræk, 2005). The M/G ratio and the monosaccharide
distribution can vary in alginates obtained from different species of
brown seaweed, and both these parameters are mainly used to evaluate
the physicochemical and rheological properties of alginate (Andriama­
nantoanina & Rinaudo, 2010). Another property is its antioxidant ca­
pacity (Borazjani, Tabarsa, You, & Rezaei, 2017; Fawzy, Gomaa, Hifney,
& Abdel-Gawad, 2017; Kelishomi et al., 2016; Sellimi et al., 2014; Xiao,
Chen, Li, Huang, & Fu, 2019), that may enhance functional properties
and shelf life (Balboa, Conde, Moure, Falqu´
e, & Domínguez, 2013) of the
alginate-based products, for example, when used as a natural stabilizer,
thickener and gelling additive or in the edible coating production
(Pawar & Edgar, 2012). The development of a feasible and cost-effective
bleaching process of alginate is necessary depending on its final use.

* Corresponding author.

E-mail address: (C. Yamashita).
/>Received 20 July 2020; Received in revised form 21 August 2020; Accepted 23 August 2020
Available online 1 September 2020
0144-8617/© 2020 Elsevier Ltd. This article is made available under the Elsevier license ( />

C. Yamashita et al.

Carbohydrate Polymers 251 (2021) 116992

Sodium hypochlorite solution is the most commonly used bleaching
agent, however, it is a precursor of organic chloramines which can be
ă
harmful for human health and the environment (Olmez
& Kretzschmar,
2009). Hence the need to explore a more sustainable and suitable agent.
Ozone gas has shown great potential while being utilized for discolor­
ation of wastewaters, dyes and sugarcane juice (Malik, Ghosh, Vaidya, &
Mudliar, 2020; Sartori, Angolini, Eberlin, & de Aguiar, 2017). Addi­
tionally, it is considered as Generally Recognized as Safe in the food
processing industry (O’Donnell, Tiwari, Cullen, & Rice, 2012; Pascual,
Llorca, & Canut, 2007).
Many factors such as gas flow rate, temperature, treatment time and
ozone concentration might affect the effectiveness of color removal by
ozonation (O’Donnell et al., 2012; Tiwari, Muthukumarappan, O’Don­
nell, & Cullen, 2008). Therefore, optimization of discoloration of algi­
nate extracted from brown seaweed is required for maximum efficacy.
Response surface methodology (RSM) is a useful mathematical and
statistical tool that can derive optimal conditions by considering mul­
tiple variables simultaneously from rationally designed experiments
(Bezerra, Santelli, Oliveira, Villar, & Escaleira, 2008).

The seaweeds of the Sargassum genus are abundant along the Bra­
zilian coast and are still poorly studied, which motivates this study. To
the best of our knowledge, there is no report in literature about applying
ozone as a bleaching agent in SA extracted from seaweeds. Therefore,
this study could be a starting point for its industrial utilization as a
bleaching agent in the food industry by applying a white biotechnology.
The present work gives insight into bleaching effects by evaluating the
effect of time, feed gas flow and temperature of exposure to ozone gas on
the chemical composition, rheological properties and colorimetric pa­
rameters of sodium alginate (SA) extracted from brown algae (Sargassum
spp.). A Box–Behnken Design (BBD) and RSM were applied to statisti­
cally optimize the ozonation process conditions, to obtain higher quality
parameters in the minimum time. In addition, the antioxidant capacity
and Fourier transform infrared spectroscopy (FTIR) analysis of SA postbleaching were evaluated.

The solution was placed in a 500 mL glass container which was halffilled by the ozonation reaction. The ozone gas was obtained by the
conversion of oxygen (O2) to ozone (O3) in an ozone generator (Model
GOBSUS, OzonioBras, Araỗatuba, Brazil). The feed gas flow (oxygen)
was controlled by a pressure regulator and the solution temperature was
maintained using a jacket connected to a thermostatic bath. The solution
was kept under magnetic stirring to promote uniform exposure of the
sample to ozone. After ozone treatment, samples were freeze-dried for
further analysis. SA extracted from the seaweed without bleaching
treatment was considered the control sample.
2.3.2. Experimental design and optimization
The BBD with three factors (treatment time (X1), oxygen flow rate
(X2) and temperature (X3)) was used to obtain the maximum postozonation values of M/G ratio, viscosity-average molecular weight
(Mv), dynamic viscosity (μdyn), percentage transmittance (%T) and
lightness (L*) of alginate extracted from brown algae. The experimental
design consisted of 15 experiments, with three replicates at the central

point, which are presented with the independent variables, their levels
and real values in Table 1. Design Expert 11 (Stat-Ease Inc., Minneapolis,
MN, USA) was used for the experimental design, data analysis and
regression modelling. Experimental data from the BBD provided a
maximum adjustment to a second-order polynomial model (Eq. 1),
which relates the dependent and independent variables.
3
∑

Y = β0 +

3
∑

β i Xi +
i=1

i=1

βii Xi2 +

3
∑

βij Xi Xj

(1)

1≤i≤j

where Y is the response, Xi and Xj are the coded independent variables,
β0 is the intercept (regression coefficient of the model) and βi, βii and βij
are the linear, quadratic and interaction coefficients, respectively.
The statistical significance of the coefficients in the regression
equation (p < 0.05) was checked by analysis of variance (ANOVA). The
fitness of the polynomial model equation to the responses was evaluated
with the coefficient of determination (R2), and lack of fit was evaluated
using F-test. Finally, optimization was performed by the desirability
function (0 ≤ d ≤ 1) to determine the most desirable set of bleaching
conditions (Bezerra et al., 2008) for higher M/G, Mv, μdyn and colori­
metric indexes values. In addition, validation of the multivariable model
was performed whereby confirmatory experiments were carried out in
duplicate under optimum conditions. These experimental data were
compared with the predicted results in order to confirm the efficacy of
the model.

2. Material and methods
2.1. Seaweed samples
The brown seaweeds (Sargassum spp.) were collected from Ubatuba,
S˜
ao Paulo State, Brazil, and transported in seawater at low temperature.
The samples were washed with tap water and soaked in chlorinated
water (25 ppm) for 30 min. The algal material was dried in a convection
oven (Model 420-1D, EthikTechnology, Vargem Grande Paulista, Brazil)
at 45 ◦ C for 12 h. The dried samples were ground using a cutting mill and
stored in airtight containers at room temperature.

2.4.

1

H nuclear magnetic resonance (NMR) analysis

The chemical composition of SA was determined by 1H NMR analysis
on a Bruker Avance III spectrometer (Karlsruhe, BW, Germany) oper­
ating at 400 MHz. Spectra were recorded at 80 ◦ C, previously solubilized
in D2O (deuterium oxide), while TSP-d4 (sodium salt of trimethylsilyl­
propionic acid) was used as an internal reference. The data obtained
were analyzed using TopSpin software 3.6.0.

2.2. Alginate extraction
The SA was extracted according to the protocol reported by McHugh
et al. (2001) with acid and alkaline treatment parameters (temperature,
time and pH) optimized by Lorbeer et al. (2015) and Nogueira (2017),
respectively. The milled algae (6 g) was treated twice with 85 % ethanol
(EtOH 200 mL) with constant stirring for 3 h and dried overnight at 40
◦
C. The dried ground algae was treated with hydrochloric acid (0.1 M,
pH 2.0, 45 ◦ C, 110 min), stirring in a shaking incubator at 250 rpm and a
2% (w/v) solution of sodium carbonate (pH 10, 75.7 ◦ C, 90 min). The
extracted SA was pressure-filtered and precipitated with absolute
ethanol (2v) and the precipitate was dried at 45 ◦ C for 12 h in a con­
vection oven (Model 420-1D, EthikTechnology, Vargem Grande Pau­
lista, Brazil).

2.5. Viscosity-average molecular weight (Mv)
The Mv of sodium alginate was calculated by determining the
intrinsic viscosity on the basis of the Mark–Houwink equation (Eq. 2)
with K = 0.023 dL/g and a = 0.984 as proposed by Clementi, Mancini,
and Moresi (1998)).

[η] = K (Mv)a

2.3. Bleaching treatment of sodium alginate

(2)

where [η] is the intrinsic viscosity, K and a are dependent constants of
the polymer, solvent and temperature, and Mv is the viscosity-average
molecular weight (kDa).
The intrinsic viscosity was measured on a Cannon-Fenske viscometer

2.3.1. Ozone treatment
The SA aqueous solution (1%, w/v) was bleached with ozone gas.
2


C. Yamashita et al.

Carbohydrate Polymers 251 (2021) 116992

Table 1
Box–Behnken design for independent variables and their responses (M/G ratio, Mv (viscosity-average molecular weight), μdyn (dynamic viscosity), %T (transmittance)
and L* (lightness index)).
Treatments

Coded variables

(T)

X1

X2

X3

Actual variables
Time (min)

O2 flow rate (L/min)

Temperature (◦ C)

Observed values
M/G

Mv (kDa)

μdyn (mPa.s)

%T

L*

1
2
3
4
5
6
7

8
9
10
11
12
13
14
15
Control

+1
+1
+1
+1
0
0
0
0
− 1
− 1
− 1
− 1
0
0
0
–

+1
0
0

− 1
+1
+1
− 1
− 1
+1
0
− 1
0
0
0
0
–

0
+1
− 1
0
+1
− 1
+1
− 1
0
+1
0
− 1
0
0
0
–

35
35
35
35
20
20
20
20
5
5
5
5
20
20
20
–

2.0
1.5
1.5
1.0
2.0
2.0
1.0
1.0
2.0
1.5
1.0
1.5

1.5
1.5
1.5
–

25
45
5
25
45
5
45
5
25
45
25
5
25
25
25
–

0.69
0.67
0.66
0.64
0.64
0.68
0.66
0.67

0.74
0.68
0.71
0.75
0.71
0.73
0.71
0.74

63.57
26.11
71.69
70.76
39.00
73.59
27.00
34.17
57.94
27.31
53.79
46.14
24.62
20.29
19.41
139.51

1.68
1.58
1.66
1.53

1.62
1.69
1.67
1.61
3.15
2.45
3.80
3.03
2.07
1.93
1.58
5.62

83.6
57.5
58.7
75.1
56.6
59.1
55.7
57.1
40.8
44.1
37.5
40.8
75.3
81.3
77.6
29.4

76.29
62.24
66.27
71.47
60.14
65.32
64.23
61.25
53.42
54.56
48.36
54.68
70.70
75.97
70.15
48.67

(size 100). Five different concentrations of SA solutions (0.1–0.5 g/dL)
were prepared in 0.1 M NaCl at room temperature and the flow time of
each SA solution was recorded. The intrinsic viscosity (dL/g) was
determined by extrapolating reduced specific viscosity against the
concentration curve to zero (Sellimi et al., 2015).

radical scavenging activity of alginate was measured according to
Rufino et al. (2010). The ABTS+ (Sigma Aldrich, MO, USA) radical was
produced by reaction of the ABTS with potassium persulfate (Sigma
Aldrich, MO, USA). The sample, at four different dilutions (2–8 g/L), and
in triplicate, was mixed with the ABTS radical, and kept in the dark for 6
min. The absorbance was measured (Biospectro Sp-220, Equipar, Curi­
tiba, Brazil) at 734 nm. The standard curve was plotted with Trolox and

the results were expressed as μM Trolox equivalent (TE) per gram of
alginate powder (μM TE/g).

2.6. Flow curves
Steady-shear flow curves for 1% SA solution (w/v) were performed
using an AR2000 rotational rheometer (TA Instruments, New Castle, DE,
USA) with a double concentric cylinder geometry (external radius 17.5
mm, internal radius 16.0 mm; internal radius 15.3 mm, height 56 mm,
gap 2000 (μm)) at 25 ◦ C. Shear stress was determined at shear rates in
the range of 0.01 to 300 s− 1. The experimental data were evaluated and
then adjusted to the Newtonian mathematical equation (Eq. 3).

2.8.2. DPPH assay
The DPPH radical scavenging activity was determined by the method
of Kirby and Schmidt (1997), with slight modifications. Briefly, SA (500
μL), at different concentrations (0.5–1500 μg/mL), was added to 375 μL
of 99 % ethanol and 125 μL of DPPH solution [0.02 % (w/v) in ethanol].
The mixture was incubated for 60 min in the dark at room temperature,
then absorbance was measured at 517 nm. The ability of alginate to
scavenge the radical DPPH was calculated using Eq. 4.
•

(3)

τ = μdyn ∗γ˙

where τ is the shear stress (Pa), μdyn is the dynamic viscosity (Pa.s) and γ˙
is the shear rate (s− 1).

DPPH radical − scavenging activity (%) =

2.7. Determination of colorimetric parameters

Acontrol −

Ablank + Asample
× 100
Acontrol
(4)

where Acontrol is the absorbance of the control (containing all reagents
except the sample), Ablank is the absorbance of the SA solution (con­
taining all reagents except DPPH solution) and Asample is the absorbance
of the SA solution with the DPPH solution.

Ozonized alginate and control samples, both in 1% solution, were
centrifuged and the color of the supernatant was measured in terms of %
transmittance using a spectrophotometer (Biospectro Sp-220, Equipar,
´ndez-Carmona,
Curitiba, Brazil) at 510 nm (McHugh, Herna
Arvizu-Higuera, & Rodríguez-Montesinos, 2001). The color of the
freeze-dried samples was measured using a HunterLab colorimeter
(MiniScan XE, HunterLab, Reston, USA). Before measuring, the color­
imeter was standardized with black and white calibration tiles provided
with the instrument. Results were expressed as lightness index (L*) [0
(black) to 100 (white)]. Hue angle (◦ Hue) was also assessed representing
the both colorimeter readings [a* (green (-) to red (+)) and b* (blue (-)
to yellow (+))] calculated as the arctangent (b*/a*).

2.8.3. β-Carotene/linoleic acid assay

The β-carotene linoleic acid model system was carried out as previ­
ously described by Koleva, Van Beek, Linssen, Groot, and Evstatieva
(2002). A stock solution was prepared with 0.5 mg of β-carotene (Sigma
Aldrich, MO, USA), 25 μL of linoleic acid (Sigma Aldrich, MO, USA) and
200 μL of Tween 80 (Labsynth, SP, Brazil) in 1 mL of chloroform which
was completely evaporated under vacuum in a rotary evaporator.
Distilled water (100 mL) was added and vigorously stirred. Aliquots (2.5
mL) of the β-carotene/linoleic acid emulsion were mixed with SA solu­
tion (0.2 mL) at different concentrations (0.05–1.5 mg/mL), followed by
incubation for 2 h at 50 ◦ C. The absorbance was measured at 470 nm.
The control tube contained no sample. Antioxidant activity was
expressed as percentage inhibition (Eq. 5).
[
(
)]
A0− At
β − carotene − bleaching inhibition (%) = 1 −
× 100
(5)
A0−´A´t

2.8. Antioxidant activity
The antioxidant capacity of SA, obtained under BBD-optimized
conditions, and commercially alginate (Grindsted® Alginate FD 175,
Danisco) was evaluated by ABTS, DPPH and β-carotene-linoleic acid
assays.
2.8.1. ABTS + assay
The ABTS (2,2′ -azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)

where A0 and A’0 are the absorbance of the sample and control,

•

3


C. Yamashita et al.

Carbohydrate Polymers 251 (2021) 116992
1

respectively, measured at time zero, and At and A’t are the absorbance of
the sample and the control, respectively, measured after 2 h.

H NMR spectroscopy establishes its functionality and possible appli­
cations in different industrial sectors. The sample spectra allowed the
identification of individual (M and G) and double (MM, GG and MG =
GM*) fractions of M and G, as well as the M/G ratio. The signal areas of
the anomeric regions between 4.25 and 5.5 ppm signals (ASTM Inter­
national, 2010) confirmed the structure of SA extracted from seaweed.
The limits and adjustments of integration applied to the calculations
(Table 3) (ASTM International, 2010) of the areas denoted A, B, and C in
the anomeric regions are presented in Fig. 2.
The spectra of the different treatments and the control sample of SA
(Fig. 2) showed signals characteristic of the biopolymer which were
similar to those observed by other researchers studying the same
biomaterial (Andriamanantoanina & Rinaudo, 2010; Grasdalen, Larsen,
& Smidsrød, 1979; Penman & Sanderson, 1972). The ozone-bleached SA
presented a prevalence of G content (Table 4), which indicates that
blocks containing mannuronic units (MM or MG) are more sensitive to

hydrolysis of glycosidic linkages (Andriamanantoanina & Rinaudo,
2010). Considering that gel formation depends mainly on the presence
of zones rich in GG blocks (Haug & Larsen, 1962), the results of the
present study suggest that ozone-bleached SA may favor stronger gel
formation. Therefore, it might be advantageous in applications where
high mechanical resistance is needed such as cell entrapment (Draget
et al., 2005).
The M/G ratios from the different treatments ranged from 0.64 to
0.75 (Tables 2 and 4), within the range (0.51–1.56) found for Sargassum
algae (Bertagnolli, Espindola, Kleinübing, Tasic, & da Silva, 2014; Bor­
azjani et al., 2017; Davis et al., 2004; Fenoradosoa et al., 2010; Khajouei
et al., 2018; Larsen, Salem, Sallam, Mishrikey, & Beltagy, 2003;
Mohammed et al., 2018; Sari-Chmayssem et al., 2016; Torres et al.,
2007), which classifies them as having an intermediate M/G ratio since
the amount of G is greater than that of M (Borazjani et al., 2017). Re­
ported M/G ratios vary widely because structural composition is influ­
enced by the different species, season and location of algae collection, as
well as the SA extraction procedure (Bertagnolli et al., 2014; Fernando,
Kim et al., 2019; Gomez, Lambrecht, Lozano, Rinaudo, & Villar, 2009)
and SA bleaching treatment (Mohammed, Rivers, Stuckey, & Ward,
2020). Oxygen flow (X2) and temperature (X3) had a similar influence on
the M/G ratio (Fig. 1a), better results being obtained with central level
treatments (X1 = 0, X2 = 0, X3 = 0). However, treatment time (X1) had
the opposite effect on the response.

2.8.4. Statistical analysis
Statistical analysis was performed using Microcal™ Origin® 6.0
software (Microcal, Northampton, MA, USA). Significant differences
were identified by one-way ANOVA followed by a post-hoc Tukey test. A
probability value of p < 0.05 was considered to be statistically

significant.
2.9. FTIR analysis
Commercially available alginate (Grindsted® Alginate FD 175,
Danisco) and ozone-bleached alginates obtained under the optimum
conditions were evaluated using FTIR analysis. The Fourier transform
infrared spectroscopy (FTIR) was performed on a Spectrum One spec­
trometer (Perkin Elmer, Waltham, MA, USA) with universal attenuated
total reflectance (UATR) accessory at room temperature. Spectra were
analyzed with Origin® 6.0 software (Microcal, Northampton, MA, USA).
Spectra were recorded in the range of 4000–600 cm− 1 by acquiring 32
scans with 4 cm− 1 resolution.
3. Results and discussion
3.1. Model fitting and statistical analysis
The experimental results of four dependent variables for each run in
the experimental design are shown in Table 1. Multiple regression
analysis was performed for each predicted response, generating a
polynomial model exploring the relationship between independent and
dependent variables. The empirical second-order model fitted well to
the experimental data, except for L* which fitted best to the first-order
model. The suitability of the model for describing the experimental
data was assessed by the statistical parameter R2 and p-values of lack of
fit (Table 2). The coefficients of determination (R2) were greater than
0.90, except for L*, for which a value close to the minimum limit was
observed (R2 = 0.7), hence the fit of the model to the experimental data
was considered satisfactory (Moore, 2010). Regarding p-values, the
models obtained were adequate for factor–response descriptions, with
95 % confidence, except for L* response, where a lack of fit to the model
was observed. The response surface plots for all responses are shown in
Fig. 1 and will be subsequently discussed individually.

3.3. Effect of ozonation on Mv of SA
The response surface plots for Mv (Fig. 1b) and its respective equa­
tion (Table 2) show that when the maximum and minimum bleaching
time (X1) and oxygen flow (X2) are used, there is an increase in the
intrinsic viscosity and Mv. In contrast, an increase in temperature (X3)
leads to a reduction in the response values.
The intrinsic viscosity and Mv of SA exposed to different ozonation
treatments ranged from 0.42 to 1.58 dL/g (data not shown) and
19.41–73.59 kDa, respectively (Table 1). Intrinsic viscosity values were
much lower when compared to commercial alginate (6.6 dL/g) (Gomez
et al., 2009) and those extracted from Sargassum (3.42–15.20 dL/g)
(Fenoradosoa et al., 2010; Khajouei et al., 2018; Larsen et al., 2003;
Sari-Chmayssem et al., 2016; Torres et al., 2007). Mv values were within
the range for commercial alginate (32–400 kDa) (Fernando, Kim et al.,
2019), although higher values (103–734 kDa) were reported for algi­
nates obtained from Sargassum algae (Borazjani et al., 2017; Fenor­
adosoa et al., 2010; Khajouei et al., 2018; Larsen et al., 2003;
Sari-Chmayssem et al., 2016; Torres et al., 2007). The intrinsic viscosity
and Mv of the control sample were within the range in the literature and
they were much higher than those for ozonized sodium alginate, indi­
cating the ozonation reduces these parameters. This may be due to the
cleavage of glycosidic linkages in alginate by ozone (Yin, Wen, Li, Li, &
Long, 2019) causing its depolymerization (Watthanaphanit & Saito,
2013).

3.2. Effect of ozonation on SA M/G ratio
Determination of the structural and sequential composition of SA by
Table 2
Models and statistical parameters obtained from Box–Behnken experimental
design. Effect of time (X1), oxygen flow (X2) and temperature (X3) of sodium

alginate solution bleaching on M/G, viscosity-average molecular weight (Mv),
dynamic viscosity (μdyn), transmittance (%T) and lightness (L*) index.
Response

Coded predictive model

R2

p
value
(lack
of fit)

[M/G]

0.72 − 0.03 ∗ X1 + 0.01 ∗ X2 − 0.02 ∗ X3 −
0.02 ∗ X2 2 − 0.03 ∗ X3 2

0.92

0.283

[Mv]

22.45 + 5.87 ∗ X1 + 6.05 ∗ X2 − 13.27 ∗ X3 + 19.60 ∗
X1 2 + 20.22 ∗ X2 2

0.92

0.051

[μdyn]

1.70 − 0.7 ∗ X1 + 0.6 ∗ X1 2

0.93

0.359

[%T]

74.38 + 13.96 ∗ X1 − 0.22 ∗ X3 − 12.37 ∗ X1 2 −
14.49 ∗ X3 2

0.90

0.081

[L*]

63.31 + 8.16 ∗ X1

0.62

0.002*

*

Significant (p < 0.05).
4

C. Yamashita et al.

Carbohydrate Polymers 251 (2021) 116992

Fig. 1. Response surface plot of time (X1), oxygen flow (X2) and temperature (X3) of sodium alginate solution bleaching for (a) M/G ratio, (b) viscosity-average
molecular weight (Mv), (c) dynamic viscosity (μdyn), (d) transmittance (%T) and (e) lightness index (L*). Fixed variables were kept constant at the central levels.

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Carbohydrate Polymers 251 (2021) 116992

significant reduction of dynamic viscosity, but also led to a reduction of
molecular weight, as also reported previously. The decrease in molec­
ular weight and rheological performance may be a consequence of the
hydrolysis of some polysaccharide linkages, as also verified using acidic
treatment for SA extraction–purification (Gomez et al., 2009). In addi­
tion, bleaching treatment can also lead to a decrease in molecular weight
and rheological properties due to degradation of the linkage in the MG
and MM blocks than the GG blocks (Mohammed et al., 2020), as
mentioned previously. The obtained response surface (Fig. 1c) shows
that only the bleaching time had an influence on dynamic viscosity.

Table 3
Relationships used for 1H NMR quantitative analysis of alginates.
Individual fraction

Double fraction

G = 0.5 x (IA + IC + 0.5x (IB1 +IB2 +IB3))
M = IB4 +0.5 x (IB1 +IB2 +IB3)
M/G ratio = M/G
GG = 0.5 x (IA + IC - 0.5x (IB1 +IB2 +IB3))
MG = GM* = 0.5 x (IB1 +IB2 +IB3)
MM = IB4

*
For long chains (polymerization degree > 20), corrections for reducing end
residues are neglected, so FGM= FMG.

Intrinsic viscosity and Mv values of ozone-treated SA solutions
decreased by between 47 % and 86 % (Table 1) compared to the control
sample. SA extracted from two Sargassum algae species exposed to so­
dium hypochlorite bleaching showed intrinsic viscosity of 1.92 and 2.06
dL/g with a reduction of 90 % and 50 %, respectively, compared to their
control samples (Andriamanantoanina & Rinaudo, 2010). Thus,
considering a diluted polymer solution system, it is widely accepted that
intrinsic viscosity appears to be a continuous function of its Mv, ac­
cording to the Mark–Houwink equation (Eq. 2). The results of the pre­
sent study show that ozone application in alginate solutions has a strong
effect on reduction of intrinsic viscosity and Mv, in similar proportions
to sodium hypochlorite bleaching treatment, which is probably by
shortening the polymer chain because of glycosidic bond breakage
(Kelishomi et al., 2016). These properties might be suitable for
alginate-based hydrogels and 3D printing applications due to its
biodegradability and printability, respectively (Reakasame & Boccac­
cini, 2018).

3.5. Effect of ozonation on sodium alginate color
Food color is an important sensory attribute which plays a significant
role in food acceptance. Native SA extracted from seaweed bears the
characteristic brown color of the algae. The use of this polysaccharide
without any bleaching treatment can adversely affect the visual quality
of the final product, leading to reduced consumer acceptance. Fig. 4
shows the color of freeze-dried SA after different bleaching treatments.

Table 4
Alginate composition and structural parameters.

3.4. Effect of ozonation on SA dynamic viscosity (μdyn)
Steady-shear flow curves of 1% SA solutions are represented in Fig. 3.
All different ozonation treatments and the control sample exhibited
rheological behavior close to that of Newtonian fluids (flow behavior
index around 1) with a correlation coefficient (R2) above 0.99 (Fig. 3).
Similar rheological behavior was observed in SA extracted from algae of
the same genus Sargassum (Khajouei et al., 2018; Torres et al., 2007) and
bleached with sodium hypochlorite (Andriamanantoanina & Rinaudo,
2010). Ozone application to the control sample not only caused a

Treatments

FG

FM

FGG

FMM

FGM

M/G

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
Control

0.59
0.60
0.60
0.61
0.61
0.59
0.60

0.60
0.58
0.59
0.59
0.57
0.59
0.58
0.59
0.57

0.41
0.40
0.40
0.39
0.39
0.41
0.40
0.40
0.42
0.41
0.41
0.43
0.41
0.42
0.41
0.43

0.47
0.49
0.48

0.50
0.50
0.47
0.49
0.48
0.44
0.48
0.44
0.43
0.47
0.45
0.46
0.41

0.29
0.29
0.28
0.28
0.28
0.29
0.28
0.29
0.29
0.29
0.27
0.29
0.29
0.30
0.29
0.26

0.12
0.11
0.12
0.11
0.11
0.12
0.12
0.11
0.14
0.12
0.15
0.14
0.12
0.12
0.13
0.16

0.69
0.67
0.66
0.64
0.64
0.68
0.66
0.67
0.74
0.68
0.71
0.75

0.71
0.73
0.71
0.74

Fig. 2. 1H NMR spectra of control sodium alginate (untreated) and alginate exposed to different ozonation treatments; peaks A and C are integrated while peaks B1,
B2, B3 and B4 are obtained by deconvolution.
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Carbohydrate Polymers 251 (2021) 116992

Fig. 3. Rheograms of shear stress as a function of shear rate of 1% sodium alginate solutions submitted to different treatments.

3.5.1. Effect of ozonation on SA transmittance (%T)
The control sample exhibited the lowest transmittance value and,
consequently, showed the darkest color. Transmittance values of ozon­
ized samples ranged from 37.5% to 83.6% (Table 1), where treatment 1
was the lightest solution (highest transmittance value) followed by the
center point treatments. These results are close to those verified in a 1%
solution of commercial SA (90.4 %). Discoloration of colored alginate
after ozone treatment could be due to the breakage of double bonds of
alginate followed by reformation of single bonds (Nagasawa, Mitomo,
Yoshii, & Kume, 2000).
A three-dimensional response surface of the quadratic model
describing the transmittance response (Fig. 2d) shows that the O2 flow
(X2) has no significant influence on %T, while the temperature (X3) has a

negative influence on the response at 5 and 45 ◦ C. However, treatment
time (X1) has the greatest influence. These results indicate that in order
to obtain a higher %T, i.e., a lighter solution, ozonation must be per­
formed for the longest exposure time, independent of the oxygen flow
used.
3.5.2. Effect of ozonation on SA lightness index (L*)
The control sample showed the lowest L* value, while the highest
values were verified for treatments 14 and 1 (Table 1). These values
correlate closely to that of Sargassum from Caribbean region bleached
with sodium hypochlorite where a value of 79.08 was observed
(Mohammed et al., 2020). Although the linear model fits the experi­
mental data better than the quadratic one, it was not able to satisfac­
torily predict L*, since the lack of adjustment was significant in relation
to the pure error (Table 2), which is not desirable. Despite the lack of
adjustment of the model to the L* experimental data, treatment time
(X1) showed a positive effect on the response: as bleaching time in­
creases, the L* value increases (Fig. 1e). The color results obtained were
satisfactory: treatments 14 and 1 presented higher transmittance and L*
index values, exhibiting lighter alginate powders (Fig. 4). These color­
imetric results are in accordance with the obtained Hue angle (◦ H)
values since treatments with high L* values presented the highest ◦ H
(above 89) while the control exhibited the lowest value (76.58).
3.6. Optimization and validation of bleaching of SA extracted from brown
algae
The multi-response optimization of alginate bleaching parameters
(oxygen flow 2 L/min for 35 min at 25 ◦ C) to obtain maximum response
values was performed using the desirability function (0 ≤ d ≤ 1); the
higher this value, the more accurate the independent variables in the
optimization (Bezerra et al., 2008). Applying the methodology of the
desired function, the predicted responses (M/G ratio = 0.68; Mv =74.19

kDa; μdyn = 1.6 mPa.s; %T = 75.97; L* = 71.47) was obtained with
desirability value of 0.912.
The bleaching of SA was validated experimentally under the optimal
parameters, presenting M/G = 0.70, Mv =66.30 kDa, μdyn = 1.39 mPa.s,
%T = 87.8 and L* = 58.35. In general, these values are in accordance
with the treatment 1 results in the BBD, since the optimized conditions
were the same, except for the lightness index value that was lower, as the
predictive model of this parameter was not significant. Therefore, the
model is satisfactory for predicting the effect of each ozonation

Fig. 4. Ozone bleaching treatments (Ti (i = 1, 2, 3…, 15)) according to Table 1
of sodium alginate solutions after freeze drying.
7


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Carbohydrate Polymers 251 (2021) 116992

parameter (time, temperature and oxygen flow) on SA quality as eval­
uated by chemical composition, rheological properties and colorimetric
parameters.

to prevent β-carotene bleaching.
At 1.5 mg/mL, the control and ozone-treated samples showed 26 %
and 10 % antioxidant activity, respectively. A higher value was verified
by Sellimi et al. (2015) who observed 60 % antioxidant activity at 1.5
mg/mL in SA extracted from Cystoseira barbata algae. However, studies
evaluating β-carotene/linoleic acid antioxidant capacity for algae of the
genus Sargassum have not been found for comparison, since antioxidant

activity may vary between algae of the same species as well as in
different genera (Luo, Wang, Yu, & Su, 2010).
Molecular weight and M/G ratio are influential factors on the anti­
oxidant activity of alginate, as alginate with a low Mv and high M/G
ratio exhibits good antioxidant properties (Fawzy et al., 2017). As
ozone-treated alginate presented lower Mv values than the control
samples, the β-carotene results are in agreement with this correlation.
Falkeborg et al. (2014) depolymerized alginate by enzymatic activity
and suggested a mechanism involving radical addition for antioxidant
activity due to the presence of double bonds between C-4 and C-5
(Falkeborg et al., 2014).

3.7. Structure and antioxidant activity of SA obtained under optimized
bleaching conditions
3.7.1. ABTS radical scavenging activity
The ABTS antioxidant activity of alginate was determined in the
control, ozonized and commercial samples (Grindsted® Alginate FD
175, Danisco) as 49.86 ± 0.45, 24.62 ± 0.01 and 12.36 ± 0.09 μM TE/g,
respectively. The control sample had the greatest effect on free radical
elimination, followed by ozone-treated and commercial samples. The
ozone-bleached alginate had twice the antioxidant activity of the com­
mercial sample, which is treated with sodium hypochlorite, showing
that ozone gas could be a more viable option as a bleaching agent for
preserving bioactive compounds.
3.7.2. DPPH radical scavenging activity
Antioxidant activity assessed with DPPH radicals was dependent on
the concentration of the alginate solution (Fig. 5a); there was a linear
increase in antioxidant activity with the concentration until reaching the
maximum value, remaining constant at the highest alginate concentra­
tions. Above 0.15 mg/mL, control and ozonized samples showed similar

results at all concentrations, with free radical scavenging activity values
of 54.48 % and 50.84 %, respectively.
Previous studies have reported high antioxidant activity (approxi­
mately 70 %) in alginate extracted from Cystoseira barbata algae (Sellimi
et al., 2015) and in SA of the genus Sargassum extracted with acid
treatment, as performed in this study (Borazjani et al., 2017), at a
concentration of 0.5 mg/mL, in both studies. Comparing these results
with the present study, the antioxidant activity values were relatively
high, since they were obtained at a much lower concentration.
The ozone bleaching process did not cause a significant decrease in
alginate DPPH antioxidant activity. The commercial SA presented the
lowest value (42.57 %) of antioxidant activity. Therefore, ozonation
may be less harmful to antioxidant compounds as compared to com­
mercial sodium hypochlorite treatment. Studies using ozone as a disin­
fectant indicate that this compound does not impair the antioxidant
activity of fruits and vegetables (Alothman, Kaur, Fazilah, Bhat, &
´n, Chaves, & Vicente, 2009;
Karim, 2010; Rodoni, Casadei, Concello
Yeoh, Ali, & Forney, 2014).

3.7.4. FTIR analysis
The main functional groups and chemical bonds of ozone-bleached,
commercial and control alginates can be revealed by representative
FTIR absorbance spectra. As shown in Fig. 6, commercial, control and
ozonized samples showed a similar and wide absorption peak near 3260
cm− 1 attributed to OH– bending vibration, whereas a weak band near
2925 cm− 1 was attributed to CH– stretching vibration (Khajouei et al.,
2018; Yu, Zhang, & Graham, 2017). The absorption peaks around 1635
and 1410 cm− 1 indicate the presence of asymmetric and symmetric
stretching vibration of carboxylate (COO) groups, respectively (Bor­

azjani et al., 2017), whose presence is also verified in alginate salts
extracted at alkaline pH (Daemi & Barikani, 2012). The presence of COO
groups is in accordance with the alginates being extracted at alkaline pH
and thus extracted as alginate salts (Daemi & Barikani, 2012). Ozonized
and commercial alginates presented a similar band at ~1300 cm− 1
which can be assigned to C–CH and OCH––– deformations caused by
alginate depolymerization through different bleaching treatments (Yu
et al., 2017). Two peaks at 1080 and 1027 cm− 1 were related to (CO–)
stretching vibrations of M and C–O (and CC–) stretching vibrations of
the pyranose rings of G, respectively (Khajouei et al., 2018). A strong
absorption band at 1050 cm− 1 indicates the elongation of CO– groups
´rtolo, 2011). The anomeric or
(Pereira, Tojeira, Vaz, Mendes, & Ba
fingerprint region of SA (750–950 cm− 1) is related to the vibration of
uronic acid residues (Khajouei et al., 2018; Sellimi et al., 2015). The
FTIR spectrum of commercial alginate showed a band at 880 cm− 1,
weaker in the spectra of the two SA (ozone-bleached and control), which
can be interpreted as being indicative of CH– deformation vibration of
β-D-mannuronic acid (Khajouei et al., 2018). This indicates that com­
mercial alginate might have more M than the extracted and bleached

3.7.3. β-Carotene/linoleic acid bleaching assay
In this system, the presence of antioxidants prevents the destruction
of β-carotene and hence the orange color is maintained. As can be seen
(Fig. 5b), all samples displayed concentration- and time-dependent
radical scavenging activity but the samples demonstrated a low ability

Fig. 5. Antioxidant activity of control, ozonized, and commercial sodium alginate samples: (a) DPPH radical scavenging activity and (b) β-carotene/linoleic
acid method.
8

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Carbohydrate Polymers 251 (2021) 116992

CRediT authorship contribution statement
Camila Yamashita: Conceptualization, Data curation, Formal
analysis, Investigation, Methodology, Writing - original draft, Writing review & editing. Izabel Cristina Freitas Moraes: Formal analysis,
Writing - review & editing. Antonio Gilberto Ferreira: Formal analysis,
Writing - review & editing. Ciro Cesar Zanini Branco: Funding acqui­
sition, Resources, Writing - review & editing. Ivanise Guilherme
Branco: Conceptualization, Writing - review & editing, Supervision.
Acknowledgments
This work was supported by the Coordination of Improvement of
Higher Education Personnel (CAPES – to CY), S˜
ao Paulo Research
Foundation (FAPESP – Grant 2014/22952-6 to CCZB) and National
Council of Scientific and Technological Development (CNPq – Grants
306567/2014-8, 432172/2016-5, and 302993/2017-7 to CCZB).
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4. Conclusion
The results reported in this study showed that ozone is a promising
clarifying agent for bleaching SA. Colorimetric results demonstrated the
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