Antioxidant Nano Phlorotannin Powder from Brown Algae Sargassum serratum: Spray Drying, Antioxidant Activities, Physic-Chemical Characterization

Introduction: Phlorotannins are bioactive-value polymers existing in brown algae and useful for pharmaceutics and functional food. Therefore, the study focused on the spray drying conditions survey, the evaluation of antioxidant activities, and physico-chemical characterization of antioxidant nano phlorotannin powder prepared from brown algae Sargassum serratum. Methods: To survey the effect of spray drying conditions (carriers, carrier-to-solution ratio, compressed air pressure, liquid feed speed, inlet air temperature) on antioxidant activities (total antioxidant, reducing power, DPPH free radical scavenging), and physico-chemical (solubility degree, moisture, particle morphology, and bulk density) of antioxidant phlorotannin powder from brown algae Sargassum serratum commonly growing in the Vietnam sea area. Results: The optimum spray drying condition consisted of the carrier-to-solution ratio of 10%, compressed air pressure of 0.8 bar, the liquid feed speed of 10 ml min -1 , and the inlet temperature of 110°C. At the optimization condition, the antioxidant activity of phlorotannin powder possessed total antioxidant (4.347 ± 0.018 g ascorbic acid equivalent 100 -1 g DP), reducing power activity (9.390 ± 0.024 g FeSO4 equivalent 100 -1 g DP), DPPH free radical scavenging activity ((70.02 ± Original Research Article Cuong; JPRI, 32(9): 71-85, 2020; Article no.JPRI.57412 72 0.26)%), physico-chemical of antioxidant phlorotannin powder consisting of moisture content (5 ± 0.5%), phlorotannin content (2.268 ± 0.010 g phloroglucinol equivalent 100 -1 g DP), solubility degree (100%), and bulk density (<1 g/ml). Phlorotannin content and antioxidant activities were affected by the spray drying condition (p < 0.05) and a strong correlation to each other (R 2 > 0.9). Antioxidant phlorotannin particles possessed nanometer size and the morphology of irregular and microspheres. Conclusion: Antioxidant nano phlorotannin powder could be usefulas functional food and


INTRODUCTION
Phlorotannins are common secondary metabolites compounds that exist in the outer cortical layer of brown algae thalli from 0.4% to 3.0% of the dried algal [1] and non-toxic antioxidant biological polymers [2]. Phlorotannins structures are diverse, because they have four linkage styles from basic units (phloroglucinol), for example, phenyl, ether,and dibenzodioxin [3,4]. Phlorotannin is the largest group of natural pigments in brown algae, evaluated as sources of a functional food [5][6][7] because of their bioactive diversity. For example, antioxidant capacity [6,8,9], anti-inflammatory [6,10], antiallergic [11]; anti-proliferative and anti-diabetic [12], anti-HIV-1 [13], antitumoral [14], antithrombotic and profibrinolytic [15]. Phlorotannin improves against free radical within the human body, the replacement of synthetic antioxidants that cause the diseases in the human [16]. The experiment showed almost phlorotannin dissolve in ethanol and methanol in comparison to another solvent such as aqueous, non-polar solvent, or non-strong polar solvent. The thing caused the limit of application for phlorotannin into functional food, and pharmacy.
Nowadays, there are many methods for solvent movement, such as concentration, spray drying, lyophilized, but spray drying still is the most efficient method for the economy and application, such as longer shelf-life, lower moisture content, and lower shipping cost. Brown algae Sargassum serratum was detected for the first time in Vietnam [17,18] and evaluated to possess high phlorotannin content and good bioactivity, although they grow commonly in the Vietnam sea. The technique control of phlorotannin powder preparation from brown algae will support the efficient use and application of phlorotannins and high-value products increasing from production processing of fucoidan and alginate. Therefore, the impact of different spray drying conditions on phlorotannin content, antioxidant activity (total antioxidant activity, reducing power, and DPPH free radical scavenging), and a correlation between factors were found. Physicochemical such as solubility degree, moisture, particle morphology (surface and size), and bulk density was also analysed for best antioxidant activity powder.

Solution Preparation for Spray Drying
The solution preparation for spray drying contained 550 mL of the sample solution and 257. 5  ). The extract was in turn collected by using filtration through the membrane Whatman No 4. and concentrated until phlorotannin content (3 ± 0.05g phloroglucinol equivalent), total antioxidant activity (6.70 ± 0.02 g ascorbic acid equivalent), reducing power activity (20.80 ± 0.20 g FeSO 4 equivalent), and free radical scavenging activity (87.25 ± 1)% per 500 mL. The concentrated then stored in black bottles at 4°C temperature.
The aids solution (AS): The different spray drying aids (SDA) in other ratio was dissolved into 250 mL of water with 2.75 mL of tween 80 and vortexed for 15 minutes at 1.500 rpm for collecting the solutions of the aid.
The homogenization of the sample solution (550 mL) and the aids solution (257.5 mL) at 2.500 rpm for collecting the drying solution.

Determination of Phlorotannin Content
Phlorotannin content (TPC) was quantified according to the Folin-Ciocalteu's method and using a standard phloroglucinol [20]. One mL of 10% Folin-Ciocalteu was added to 300 μL extract and kept the mixture for 5 minutes and adding 2 mL of 10% Na 2 CO 3 for the vortex. The compound was then incubated for 90 minutes in the dark and measured the absorbance at 750 nm by the UV-Vis Spectrophotometer Jen Way 6400/ 6405.

Total antioxidant activity
Total antioxidant activity (TA) was determined by the reduction of Phosphate-Mo (VI) to Phosphate Mo (V) [21] with the ascorbic acid standard. 100 µL extract diluted by 900 µL of distilled water, then shaken with 3 mL of solution A (0.6M sulfuric acid, 28 mM sodium phosphate, and 4 mM ammonium molybdate), and kept for 90 minutes at 95°C. The absorbance measurement of the compound was at awavelength of 695 nm.

Reducing power
Reducing power was determined based on the reduction of iron (III) to iron (II) with FeSO 4 standard [18]. 0.5 mL phosphate buffer (pH 7.2) and 0.2 mL of 1% K 3 [Fe(CN) 6 ] was added to 500 µL extract, in turn, and kept at 50°C for 20 minutes. 500 µL of 10% CCl 3 COOH, 300 µL of distilled water, and 80 µL of 0.1% FeCl 3 were then added to the mixture, in turn and shaken.
The absorbance measurement wasdetermined at a wavelength of 655 nm.

Free radical scavenging DPPH
Free radical scavenging DPPH was determined based on electron-transfer that produces a violet solution in ethanol [20]. 200 µL, 400 µL, 600 µL, 800 µL, and 1000 µL of extract were poured into each of test-tube, in turn, and added the 3 mL of DPPH (25 mg L -1 ) into each test-tube, called the test sample. The blank sample was similar to the test sample but replaced 3 ml of DPPH by 3 ml of 96% ethanol. The control sample was similarly the blank sample but replaced the extract with DPPH, respectively. All test-tubes were kept in the dark under room temperature for 30 minutes before the absorbance measurement at a wavelength of 550 nm. The percent of free radical scavenging DPPH was calculated the following equation: In here: A sample: the absorbance of the test sample at 550 nm A control: the absorbance of the control sample at 550 nm A blank: the absorbance of the blank sample at 550 nm UV-Vis Spectrophotometer Jen Way 6400/ 6405 machine was used to measure.

The particle morphology
The particle morphology of powder was observed by using scanning electron microscopy (SEM) (FEI, QUANTA 250 FEG).

The bulk density
The powders were poured into a 10 mL graduated cylinder without any pressure and weighed the powders weigh. The bulk density (ρb) of powder was determined based on the sample weight-to-volume ratio [22].

Solubility degree
5 g of antioxidant phlorotannin powder was suspended in 50 mL of distilled water at ambient temperature, stirred for 1 min, and kept for 30 min at 37°C. The mixture was then centrifuged at 1500 rpm at 4°C for 10 min and separated the supernatant that dried at 105°C until constant weight, named A (g). Solubility degree calculation was based on the ratio of A and 5 g.

Fourier-transform infrared spectroscopy
FTIR spectrum analysis was on the machine Tensor 37 (Brucker, Germany) with the spectrum range of 7,500 to 370 cm -1 and beamsplitter of standard KBr. The extended spectral range with a near and middle infrared detector was from 15,000 to 370 cm -1 .

Differential scanning calorimetry
DSC spectrum of antioxidant phlorotannin powder were measured and calculated on the machine (DTA/ DSC/TGA) Labsys Evo S60/58988.

Data Analysis
Remove outlier values by using Dulcan method. Statistic, ANOVA and regression analysis using MS. Excel software. Figures were exhibited by Origin 8.0 software. Experiments were repeated intriplicate (n=3).
One of the reasons causing statistical significance in the drying aids efficiency was the difference in drying aids structure. Maltodextrin has three to 17 of D-glucose units with the primary linkage of α(1→4) glycosidic and a branch structure. Saccharose only consisted of glucose and fructose in their molecules. The molecular weight and the hydrogen groups of glucose are the least. The hydrophobic tail and the hydrophilic head of tween 80 linked to phlorotannin and maltodextrin, respectively, through hydrogen linkage for microparticles formation that depended on many hydroxyl groups and molecular weight of drying aids. Maltodextrin has the most groups of hydrogen and the highest molecular weight. Therefore, the ability of spatial network formation of maltodextrin is better than one of the other drying aids (saccharose, glucose) for wrap and linkage of phlorotannin through tween 80 (polysorbate 80). Maltodextrin was also used in spray drying for the polyphenol-rich extract of apple [23], the bioactive substances-rich extract of cactus, and was the most effective for the protection of pigments and the restriction of antioxidant activities decline of terrestrial plant antioxidants [24], but non-notices on maltodextrin use for phlorotannin powder. The current study showed maltodextrin had a good effect on the drying aid of antioxidant pigment from terrestrial plant and brown algae. Phlorotannin content in powder was higher than polyphenol content of tomatoes powder (12.24 mg gallic acid equivalents 100 -1 g DP) [25] and lower than polyphenol content of tea-green powder (200.37 ± 0.9 mg gallic acid equivalents g -1 DP) [26].

Fig. 2. Changes in phlorotannin content and antioxidant activities of brown algae extract powder according to drying aids ratio
Phlorotannin content and antioxidant activity of the powder was strongly affected by the maltodextrin/solution ratio (p < 0.05) and the highest value as 10% MDS ratio. Phlorotannin content increased by 45% when the MDS ratio increased from 2% to 10% and decreased by 1% as 14% MDS was, compared to 10% MDS. Total antioxidant and reducing power activity of phlorotannin powder were correspondingly increased by 24.66% and 61.02% as using of 10% maltodextrin aids, compared to 2% maltodextrin aids. The total antioxidant activity was not significantly different, and reducing power activity was decreased by 5.1% when the MDS ratio was more than 10%. The highest and lowest value of DPPH free radical scavenging activity was when 10% and 2% of MDS was, respectively (Fig. 2b). A low MDS ratio caused less formation of microparticles. When maltodextrin content increased too high, the viscosity of the solution was increased and difficult dry processing, described on the drying of Gac powder by Kha [27]. Quek et al. (2007) showed that the maltodextrin content affected the dry powder characteristics of watermelon juice and recovery yield that stuck in a drying chamber and cyclone as non-use of maltodextrin [28]. The MDS ratio was strongly related to phlorotannin content and antioxidant activity of powder (R 2 > 0.81) that changed according to the non-linear model of level 2 with the maximum peak at 10% of MDS ratio.
Phlorotannin content, total antioxidant activity, reducing power activity, and DPPH free radical scavenging of powder was affected by compressed air pressure (p < 0.05) and changed as the change of compressed air pressure. The correlation between phlorotannin content and antioxidant activities were (R 2 > 0.8), and the close relation between compressed air pressure and target functions was also recorded (R 2 > 0.75). Phlorotannin content and antioxidant activities of powder were changed according to the non-linear model of level 2, and got the maximum point at a compressed air pressure of 0.8 bar, except for DPPH free radical scavenging activity because this activity had the model of linear change. The difference in each organic material leading compressed air pressure was used differently in spray drying processing. The compressed air pressure was adequate from 0.6 -1.4 bar for spray drying [29,30], except for spray drying of leaves extract Piper betle (4 bar), described by Lee [31], antioxidant powder from Tinospora cordifolia leaf (45 psi (3.103 bar)) [32]. The compressed air pressure, particle weight, and powder weight in the container also affected recovery yield. Moving microparticles in the cyclo were affected by at least two forces (the earth's gravitational and the inertial force) that due to the fogging speed and thrust of the compressed air. Microparticles were blown out of the cycle when the earth's gravitational forces are smaller than the inertial force (thrust of the compressed air), and it found for less powder weight. The larger the weighted particle is, the larger the earth's gravitational force for particle weight. The faster nozzle rotates, the smaller fog particles, and this happens as an increase of pumping speed, and the contact areas between fog particles and hot air increased. When microparticle weight is too small, they cannot be able to overcome the thrust of the compressed air to reach the container, collected powder weight in the condition is less than that in lower compressed air pressure.
Pumping speed affected phlorotannin content, antioxidant activities, and collected powder weight (p < 0.05) that decreased as the increase of feed flow rate. Phlorotannin content decreased to 87% and 62,14% as the feed flow rate of 20 rpm and 50 rpm, respectively, compared to the pumping speed at 10 rpm. Total antioxidant activity corresponded to 94% and 72.36% as the feed flow rate of 20 rpm and 50 rpm, respectively, compared to the feed flow rate at 10 rpm. The reducing power of the powder was decreased by 13% and 37.33% as feed flow rate at 20 rpm and 50 rpm, respectively, compared to the feed flow rate at 10 rpm. The correlation between feed flow rate and phlorotannin content, antioxidant activities, and weight of powder was good (R 2 > 0.92; F > F crit ) and a negative correlation, and the reverse is a positive correlation between phlorotannin content and antioxidant activities. The increase of feed flow rate caused more fog number, higher humidity in cyclo, the longer the drying time of the dewdrops. The difference in extract caused various liquid feed rates, for example, polyphenol (quercetin and vanillin) microencapsulation (10 mL min -1 ) [33] and polyphenol extract of green tea (3 mL min -1 ) [34].

Effect of inlet air temperature
Phlorotannin content, total antioxidant activity, reducing power, DPPH free radical scavenging of the powder got the highest value, corresponding to 2.268 ± 0.010 g phloroglucinol equivalent, 4.347 ± 0.018 g ascorbic acid .390 ± 0.024 g FeSO 4 equivalent, and (70.02 ± 0.26)% per 100 g DP, respectively, at the inlet air temperature of 110°C. Total antioxidant activity and reducing power activity of powder were decreased by 46.84% (2.311 ± 0.020 g ascorbic acid equivalent 100 -1 g DP) and to 62.59% (3.513 ± 0.024g FeSO 4 equivalent 100 -1 g DP) in comparison to the inlet air temperature of 110°C. DPPH free radical scavenging activity of powder got the lowest value at the inlet air temperature of 160°C and was 37.28% in comparison to the inlet air temperature of 110°C (Fig. 5b). At the inlet air temperature of 160°C, phlorotannin content, total antioxidant activity, and reducing power also got the lowest value.
The inlet air temperature affected phlorotannin content and antioxidant activities of powder (p < 0.05). Phlorotannin content of powder at various inlet air temperature was statistically significant, and the difference also happened for antioxidant activities. The inlet air temperature increased to 160°C, phlorotannin content of powder was decreased by 59.26%, compared to the inlet air temperature of 110°C. The changes in phlorotannin content and antioxidant activities complied to thelinear equation when the inlet air temperature was altered from 110°C to 160°C and had a close correlation with each other (R 2 > 0.87; F > F crit ). The moisture diffusion rate at the particle surface and inside particles were relatively balanced. Microparticles formation happened rapidly, phlorotannin content and antioxidant activity of microparticles were good, found at the inlet air temperature of 110°C. When the inlet air temperature was above 110°C, phlorotannin content and antioxidant activities decreased, the surface of microparticles was drier, even burnt, compared to the inlet air (a) (b)

Fig. 5. Effect of inlet drying temperatureon phlorotannin content and total antioxidant activity of powder
temperature of 110°C. Inlet air temperature in the current study was lower than for bayberry polyphenols extract (150°C) [35] and polyphenolrich grape marc extract (140°C) [36]. The difference in extract characteristics and polyphenol characteristics caused various input air temperature. Bayberry polyphenols and polyphenol-rich grape marc extract contained mainly water [35,36], phlorotannin extract consisted of ethanol and water. The evaporation temperature of ethanol is lower than that of water, caused by rapid particle formation. In the current study, lower drying temperature led to less electricity cost for drying temperature.

Differential scanning calorimetry
The temperature zone of sub-T relaxation, glass transition, crystallization, melting and decomposition exhibited very clear based on the notice of Qiuju et al. [37]. The sub-T relaxation of phlorotannin powder occurred before T g point. The glass transitions (T g ) determination of antioxidant phlorotannin powder was to base the heating curve change from onset to the endpoint of them that occurred located 67.12°C and the width of the clear transition. The midpoint T g and the endpoint T g of antioxidant phlorotannin powder were 100.21 and 117.54°C. According to Agata et al. T g values of carbohydrate-protein systems were higher than single disaccharides consisting of lactose and trehalose [38], and the current study showed Tg temperature values of the phlorotannin-maltodextrin system were lower than β-lactoglobulin-vitamin A-trehalose and βlactoglobulin-cholecalciferol-lactose powder [38]. The difference in Tg could be from the different solvent using before the spray drying, described the Tg temperatures of foods depending on their moisture content and chemical compositions [39][40][41]. The temperature zone of crystallization, melting and destroying appeared in turn to the peak of 1, 2 and 3 from left to right. The onset, midpoint, and endpoint of crystallization temperature (T c ) corresponded to 153.08°C, 150.84°C, and 155.83°C, respectively. The temperature zone belongs to the exotherm zone (give up heat) (Fig. 6a) , and 673 cm -1 . The peak 3286 cm -1 related to the hydroxyl stretching vibration and H-bonding [42]. The peak 2925 cm -1 exhibited the CH and C-H stretching vibration belonging to the group of methoxy compounds (2928 cm -1 ), described by Rengasamy et al. [43] and John [42] and C-H asymmetry stretching (2937 cm -1 ) [44]. The peak 1641 cm -1 exhibited the stretching of carbonyl groups (C=O), noticed by Rengasamy et al. [43] and Senthilkumar et al. [45]. According to Mayra et al., C-C stretch (in-ring) of phenolic groups from 1500-1400 cm -1 [46] and in the current study, the peak 1414 cm -1 occurred. The peak 1359 cm -1 belonged to the stretching of C-O groups (1383 cm -1 ), found in a previous study [47]. Basing on the peak 1243 cm -1 showed the COO-(C-O) stretching [48]. The sulfonate radicals appeared in antioxidant phlorotannin powder, exhibited via the peak 1148 cm -1 [42] (Fig. 6b). The peak 1078 related to the vibration of hydroxyl groups [47]. The C-F stretch found in the spectrum FTIR (the peak 1014 cm -1 ) exhibiting aliphatic fluoro compounds existence in phlorotannin structure. The stretching of =C-H and =CH 2 presented in the spectrum FTIR via the peak 931 cm ) [49]. The C-H groups at the sitespara of aromatic ring exhibited in the peak 848 cm -1 [42]. The peak 761 cm -1 showed occurring the C-H groups of aromatic phenyl. The C-H bend (rocking) of -C≡C-H was from 700-610 cm -1 and in the current study, the peak 700 cm -1 and the peak 673 cm 1 presented [50].

The particle morphology, the bulk density, the moisture and the solubility degree of powder
The solubility degree of antioxidant phlorotannin powder was 100%, observed on all powder sample that dried at different conditions. Drying temperature, aids kind, and aids ratio did not affect the solubility degree of powder (p > 0.05). Drying temperature and aids kind correlated negatively to the bulk density of powder (p < 0.05) that was in the range of 0.53 to 0.67 g/ml, reported in a previous study [51,52]. The antioxidant phlorotannin particles possessed different morphologies (irregular microcapsule and microspheres) (Fig. 7a). The moisture content of the antioxidant nano phlorotannin powder got 5 ± 0.5%.Maltodextrin formed a hard wall outside the microcapsule. 99.2% and 0.8% of particles got an average size of 68.23 and 479.3 d.nm, respectively (Fig. 7b). The average size of antioxidant phlorotannin powder was 211.0 d.nm that got the average level in the size of nano polyphenol particles as reported in the previous studies (30 -1300 d.nm) [53].The nanosize of antioxidant phlorotannin powder in the current study was lower than the notice of Marco et al. [52] and did not find in the previous studies on phlorotannin powder.

CONCLUSION
Antioxidant nano phlorotannin powder from brown algae Sargassum seratum got the highest phlorotannin content and antioxidant activity with moisture content (5 ± 0.5%) at the optimum condition (10% carrier-to-solution ratio, 0.8 bar compressed air pressure, 10 ml min -1 liquid feed speed, and 110 o C inlet temperature), compared to other conditions. Nano phlorotannin correlated strongly and possessed antioxidant activities. Antioxidant nano phlorotannin powder dissolved fully into the water with their bulk density under 1 g/ml. 100% of phlorotannin powder (99.2% of powder size under 70 d.nm) possessed the nanoparticles and the irregular and microspheres morphology. The current study showed the characterization of the transition temperature (DSC) and the functional group (FTIR) of nanopowder but did not present NMR, DTA, TGA, and TEM. Different bioactivities, flow properties, and other bioactive substances attachment of antioxidant nano phlorotannin powder should be studied and evaluated in further studies. Antioxidant nano phlorotannin powder preparing from brown algae Sargassum serratum has the high potency of application into the field of the functional food and the pharmaceuticals as a bioactive material.

CONSENT
It is not applicable.

ETHICAL APPROVAL
It is not applicable.

COMPETING INTERESTS
Author has declared that no competing interests exist.