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

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.

Carbohydrate Polymers 251 (2021) 116992 Contents lists available at ScienceDirect 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 L/min for 35 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 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: ca_yamashita@msn.com (C Yamashita) https://doi.org/10.1016/j.carbpol.2020.116992 Received 20 July 2020; Received in revised form 21 August 2020; Accepted 23 August 2020 Available online September 2020 0144-8617/© 2020 Elsevier Ltd This article is made available under the Elsevier license (http://www.elsevier.com/open-access/userlicense/1.0/) 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 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 ∑ Y = β0 + ∑ β i Xi + i=1 i=1 βii Xi2 + ∑ 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 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 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 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 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 C Yamashita et al Carbohydrate Polymers 251 (2021) 116992 Table 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* 10 11 12 13 14 15 Control +1 +1 +1 +1 0 0 − − − − 0 – +1 0 − +1 +1 − − +1 − 0 0 – +1 − +1 − +1 − +1 − 0 – 35 35 35 35 20 20 20 20 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 25 45 45 25 45 25 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 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− 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 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 • (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 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 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 (%) = − × 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, • C Yamashita et al Carbohydrate Polymers 251 (2021) 116992 respectively, measured at time zero, and At and A’t are the absorbance of the sample and the control, respectively, measured after 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 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 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− by acquiring 32 scans with cm− resolution 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 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 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 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 − 0.03 ∗ X3 0.92 0.283 [Mv] 22.45 + 5.87 ∗ X1 + 6.05 ∗ X2 − 13.27 ∗ X3 + 19.60 ∗ X1 + 20.22 ∗ X2 0.92 0.051 [μdyn] 1.70 − 0.7 ∗ X1 + 0.6 ∗ X1 0.93 0.359 [%T] 74.38 + 13.96 ∗ X1 − 0.22 ∗ X3 − 12.37 ∗ X1 − 14.49 ∗ X3 0.90 0.081 [L*] 63.31 + 8.16 ∗ X1 0.62 0.002* * Significant (p < 0.05) C Yamashita et al Carbohydrate Polymers 251 (2021) 116992 Fig 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 C Yamashita et al 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 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 shows the color of freeze-dried SA after different bleaching treatments Table 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 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 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 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 C Yamashita et al Carbohydrate Polymers 251 (2021) 116992 Fig 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 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 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 (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 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 L/min for 35 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 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 Ozone bleaching treatments (Ti (i = 1, 2, 3…, 15)) according to Table of sodium alginate solutions after freeze drying C Yamashita et al 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− attributed to OH– bending vibration, whereas a weak band near 2925 cm− was attributed to CH– stretching vibration (Khajouei et al., 2018; Yu, Zhang, & Graham, 2017) The absorption peaks around 1635 and 1410 cm− 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− 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− 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− 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 Antioxidant activity of control, ozonized, and commercial sodium alginate samples: (a) DPPH radical scavenging activity and (b) β-carotene/linoleic acid method C Yamashita et al 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 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environmental 10 ... 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 L/min... H., Gu´ egan, J P., Jefti´c, J., & Benvegnu, T (2016) Extracted and depolymerized alginates from brown algae Sargassum vulgare of Lebanese origin: Chemical, rheological, and antioxidant properties... (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