Enhanced anti-oxidant activity of neoagarooligosaccharides produced by β-agarase derived from Aquimarina agarilytica

The neoagarooligosaccharides have received growing attention owing to their physiological activities. The aim of this study was the isolation of agarase-producing bacteria for production of agar hydrolysates with special emphasis on their anti-oxidant potential. An agarolytic strain NI125 was isolated from Nelson's Island, Alexandria, Egypt. Based on 16S rRNA analysis and extensive phenotypic characterization, it was identified as Aquimarina agarilytica . Maximum enzyme production was achieved after 24 h incubation at 20 ° C, and tryptone was recorded to be the best nitrogen source for agarase production. Extracellular agarase was partially purified by ammonium sulfate precipitation. The substrate specificity assay using p-nitrophenyl-α/β-D-galactopyranoside revealed the cleavage of the β-linkage rather than the α-linkage. Neoagarooligosaccharides produced by the partially purified β-agarase expressed promising anti-oxidant properties, with 23% free radical scavenging potential. Notable enhancement of the anti-oxidant potency of the oligosaccharides was achieved (up to 87% scavenging ability) by sulfation of the agar prior to hydrolysis for 12 h with β-agarase. Results obtained suggest the potential application of the produced neoagarooligosaccharides anti-oxidants as promising additives in food and feed products.

In general, NAOs exhibit various physiological activities hence are used as food additives, pharmaceutical and cosmetics ingredients. In several previous studies, NAOs are reported as anti-tumors (Lee et al., 2017), anti-inflammatory (Enoki et al., 2010;Wang et al., 2017), anti-fatigue (Zhang et al., 2017), tyrosinase inhibitors (Lee et al., 2008), immune modulators (Kang et al., 2017), whitening agents with skin-moisturizing properties (Kim et al., 2017), antiobesity, and anti-diabetic (Hong et al., 2017a). In addition, Hong et al., (2017b) added that the toxicological evaluations indicated that NAOs did not exert any mutational effects, as oral administration to rats and beagle dog models did not express any adverse effects. Moreover, NAOs can potentially serve as prebiotics to stimulate and promote the growth of the bifidobacteria and the lactobacilli, and improve the composition of the gut microbiota (Han et al., 2019a).
Furthermore, the anti-oxidant potential is one of the paramount features of NAOs. Zhu et al., (2016); Xiao et al., (2019b); Zhang et al., (2019), have documented their scavenging hydroxyl free radical, scavenging superoxide anion radical and inhibition of lipid peroxidation activities. Previously, Ji et al., (2010) thought that the antioxidant merits of NAOs may be exerted by direct eliminating of free radicals, inhibiting their generation, or resisting the activation of the oxidation system. Recently, Liang et al., (2018); Wang et al., (2018); Chen and Huang, (2019);  that chemical sulfation of polysaccharides could enhance their water solubility and change their conformation, resulting in the alteration of their biological activities with possible therapeutic uses.
In the current study, agar-degrading Aquimarina agarilytica strain NI125 isolated from marine sediments was employed to produce β-agarase for the hydrolysis of native and sulfated agar, and the antioxidant potential of the produced agar-hydrolysates was evaluated.

Sample collection, isolation, and screening of agarolytic bacteria
Samples of sediments of the Mediterranean Sea were collected from the vicinity of Nelson's Island, (31° 21' 30.996"N 30° 6' 28.858"E), Alexandria, Egypt. Aliquots of appropriate dilutions were spread onto artificial seawater salts (ASW) agar plates composed of; 6.1 g Tris base (pH 7.2), 12.3 g MgSO 4 , 0.74 g KCl, 0.13 g (NH 4 )2HPO 4 , 17.5 g NaCl, 0.14 g CaCl 2 , 0.2 g yeast extract, and 15 g agar\ l (Kim and Hong, 2012). After incubation at 25°C for 2 to 4 d, colonies that formed pits or crater-like depressions were picked and re-streaked on the same medium. Further confirmation of the agarolytic activity was conducted by means of spot inoculation of the purified cultures on the ASW agar plates. Following incubation at 25°C for 24 h, the plates were flooded with Lugol's iodine solution. The appearance of clear zone around the colony indicates its agar-degrading potency (Furusawa et al., 2017). The agarolytic bacteria showing obvious agarase activity were selected for further investigations.

Preparation of the crude agarase enzyme
For agarase production, the selected bacterial isolates were inoculated into ASW broth (24.6 g NaCl, 1.36 g CaCl 2 .2H 2 O, 0.67 g KCl, 6.29 g MgSO 4 .7H 2 O, 4.66 g MgCl 2 .9H 2 O and 0.18 g NaHCO 3 ), supplemented with 0.2% agar as the sole source of carbon. Cultures were then incubated aerobically at 25 °C with shaking at 180 rpm for 24 h. After incubation, the bacterial cells were removed by centrifugation at 16000 rpm for 15 min., and then the cell-free supernatant was used as the crude enzyme preparation (Gupta et al., 2013).

Quantitative screening for the agarase production
The agarase assay was carried out by estimating the liberated reducing sugars using 3,5-dinitrosalisylic acid (DNS) method in reference to Miller, (1959). Approximately 100 μl of the crude enzyme were added to 3.9 ml of 0.2% agarose solution in Tris-HCl buffer (20 mM, pH 8.0), and then incubated at 40°C for 30 min. After that, about 1 ml of the reaction solution Novel Research in Microbiology Journal, 2019 was mixed with an equal volume of DNS reagent, and then heated in a boiling water bath for 15 min. After cooling to room temperature, the released reducing sugars were estimated by measuring the absorbance at 546 nm against the standard curve of D-galactose. One unit of an enzymatic activity was defined as the amount of enzyme that released 1 μmol of reducing sugar (as D-galactose) from the agar per minute. The protein concentration was measured according to Bradford, (1976), where the bovine serum albumin (BSA) was used for preparing the standard curve.

Phylogenetic analysis of the agarolytic bacterium
The agarolytic strain designated NI125 exhibiting the maximum agarase potential was identified through sequencing of its 16S rRNA gene. In brief, GeneJET™ Genomic DNA Purification Kit (Thermo Scientific, USA) was used for DNA extraction according to the supplier's instructions. The 16S rRNA gene was amplified by the Polymerase Chain Reaction (PCR), using 27F (5'-AGAGTTTGATCMTGGCTCAG-3') and 1492R (5'-TACGGYTACCTTGTTACGACTT-3') primers. PCR was conducted in a total volume of 50 μl containing 2.5 μl 10X DreamTaq buffer, 50 ng genomic DNA template, 0.2 mM of each dNTP, 0.4 μM of each primer, one unit of Dream Taq DNA polymerase (Thermo Scientific, USA), and nuclease-free water up to 50 μl. The reaction conditions were set as follows; initial denaturation at 95°C for 3 min., denaturation at 95°C for 40 s, annealing at 55°C for 40 s, extension at 72°C for 1.5 min. for 32 cycles, and finally an extension step at 72°C for 8 min.
The amplicon was purified using QIAquick PCR purification kit (Qiagen, Germany), while the purified PCR product was sequenced in both directions using the same primers at Macrogen (Seoul, Korea). The forward and reverse DNA sequence reads were assembled using DNA Baser Sequence Assembler software (v.3.5.3). The produced consensus of 16S rRNA sequence was analyzed using BLAST in NCBI and EzTaxon server database according to Yoon et al., (2017). The phylogenetic tree of the strain NI125 was constructed using the neighbor-joining (NJ) method of the MEGA X software, and the bootstrap analysis was based on 1000 replicates.

Phenotypic and biochemical characterization of the agrolytic bacterium
The strain NI125 was subjected to phenotypic characterization according to the standard methods described by Smibert and Krieg, (1994). This strain was incubated on marine agar 2216 (MA, Difco) plates at 25°C for 36 h, unless otherwise stated. The bacterial cell morphology and type of flagellation were observed using the Transmission electron microscopy (TEM) (JEM-1010; JEOL), after negative staining with 1% (w/v) phosphotungstic acid. The temperature range for the bacterial growth was determined on the MA plates incubated at 4-45°C for 7 d.
The salt tolerance was assayed in synthetic ZoBell medium (Difco) containing different concentrations of NaCl ranging from 0 to 15% (w/v), at intervals of 0.5%. The biochemical assays were conducted following the standard protocols of Tindall et al., (2007). The utilization of carbohydrates and metabolism were determined using API 20E strips (bioMérieux, France) according to the manufacturer's instructions, with the exception of the bacterial suspension which was prepared in 2 % (w/v) NaCl.

Optimization of the conditions for agarase production
The optimal media components and incubation conditions for agarase production were screened using the one-factor-at-a-time (OFAT) method (Jung et al., 2012), keeping the other factors constant.

Effect of incubation period
To study the optimal incubation period for maximum agarase production, ASW broth supplemented with 0.2% agar was inoculated with 2% starter culture of the strain NI125, and then incubated at 25°C. Samples were withdrawn periodically every 6-h intervals over 60 h and assayed for agarase activity.

Effect of incubation temperature
The effect of temperature on optimal agarase production was studied by cultivating the strain NI125 in ASW broth supplemented with 0.2% agar for 24 h, at different temperatures ranging from 12ºC-30ºC, as independent treatments.

Effect of initial pH of the medium
The ASW broth medium supplemented with 0.2% agar was adjusted before sterilization to various levels of pH ranging from 5-10. After 24 h incubation at 20ºC, the cells were harvested by centrifugation and then the cell-free supernatant was analyzed for agarase activity.

Effect of various nitrogen sources
The impact of various nitrogen sources on agarase production was performed using ASW broth containing 0.2% agar and supplemented individually with 1% (w/v) of; casein, tryptone, peptone, beef extract, urea, yeast extract, and NaNO 3 as nitrogen sources. The strain NI125 was inoculated and the cultures were incubated at 20ºC for 24 h, after that the agarase potential was examined.

Partial purification of the agarase enzyme
The strain NI125 was cultivated under optimum conditions for agarase production; consequently, the produced agarase was partially purified using ammonium sulfate fractionation assay as described by Kaur et al., (2017). In brief, the culture was centrifuged at 16,000 xg for 10 min., the supernatant was brought to 20% (w/v) saturation by slow addition of powdered ammonium sulfate, and then left at 4°C overnight. After centrifugation at 21,000 xg for 30 min. at 4°C, the supernatant was brought to 80% (w/v) saturation by slow addition of powdered ammonium sulfate, and then left at 4°C overnight again. Then, the formed precipitate was collected by centrifugation at 21,000 xg for 30 min., re-suspended in 20 mM Tris-HCl, and then dialyzed against the same buffer at 4°C for 24 h. The dialysate (partially purified enzyme) was assayed for agarase potency.

Determination of the substrate specificity
To determine the substrate specificity and cleavage pattern of the partially purified agarase, the enzyme assay was conducted using p-nitrophenyl-α-D-galactopyranoside or p-nitrophenyl-β-Dgalactopyranoside as artificial chromogenic substrates (Chi et al., 2015). The reaction was carried out at 40°C for 2 h and then terminated by the addition of 1 M Na 2 CO 3 . The release of the yellow-colored pnitrophenol was measured by recording the absorbance at 420 nm (A 420 ).

Preparation of the sulfated agar
Sulfation of agar was performed following the chlorosulfonic acid-pyridine (CSA-Pyr) method according to Xie et al., (2016). The sulfation reagent was prepared by dropwise addition of CSA to pyridine at a ratio of 1:4 (v/v) in an ice bath. Subsequently, the mixture was stirred for 30 min. at room temperature. Approximately one gram of agar-agar was suspended in 100 ml of N, N-dimethylformamide (DMF) at room temperature under continuous agitation. After 15 min., the sulfation reagent was added to the agar suspension drop by drop, and then heated in a water bath at 60°C for 4 h with continuous stirring. Afterwards, the mixture was cooled to room temperature and neutralized to pH 7.0 using 4 M NaOH. After that, 95% (v/v) ethanol was added and the mixture was allowed to precipitate at 4°C. After 18 h, the precipitate was re-suspended in dist. water and dialyzed against dist. water; finally the sulfated agar was freeze-dried. The sulfur content of the sulfated polysaccharides was estimated using the benzidine method (Antonopoulos et al., 1962). The degree of substitution (DS) designating the average number of sulfo-groups on each sugar residue was calculated on the basis of the sulfur content in reference to Zhang et al., (2003).
Novel Research in Microbiology Journal, 2019

Preparation of the neoagarooligosaccharides (NAOs)
Partially purified β-agarase (100 U) was added to 100 ml of 50 mM Tris-HCl buffer (pH 8.0) containing 1% (w/v) agar (or sulfated agar), incubated at 40°C for 12 h, and then the reaction was stopped by heating the solution in boiling water for 10 min. About twovolumes of absolute ethanol was added to the reaction mixture to remove the high-molecular-massed polysaccharides. After centrifugation, the soluble fraction was collected and lyophilized. The amount of total sugars was determined using the method of phenol-sulfuric acid according to Dubois et al., (1956). The optical density was measured at 490 nm, and the values for the total sugars were expressed as D-galactose equivalents.

Estimation of the anti-oxidant activity of the NAOs
To investigate the anti-oxidant activity of the produced NAOs, DPPH (1,1-diphenyl-2picrylhydrazyl) radical scavenging assay was performed (Yang et al., 2006). The prepared hydrolysate powder containing NAOs was dissolved in dist. water and then subjected to anti-oxidant activity assays. In brief, 1.0 ml of the hydrolysate solution (1mg/ ml) was mixed with 2 ml of 0.2 mM DPPH dissolved in ethanol. After the mixture was shaken and incubated at room temperature for 30 min. in the dark, the absorbance of the resulting solution was measured at 517 nm. The DPPH radical scavenging effect of the sample was calculated as follows: Scavenging ability (%) = (1absorption of sample/absorption of control) × 100

Statistical analysis
The measured data were subjected to the analysis of variance (ANOVA) appropriate to the design. The significant differences between treatments were compared with the critical difference at 5% level of probability by the Duncan's test using PASW 17.0 statistics software (SPSS Inc).

Isolation and screening of the agarolytic bacteria
After incubation for 3 d, colonies that formed pits or holes on the plates were selected, and transferred onto new plates until the colony morphology is unchanged. The plates are stained using Lugol's solution to check for the agarolytic potential. About twelve agarolytic colonies forming deep holes in agar are selected and subjected to quantitative screening. Based on the secondary screening, a promising strain designated NI125 exhibiting superior agarase activity is selected for further investigations.

Phylogenetic analysis
The 16S rRNA gene sequence (1,427 bp) of strain NI125 is deposited in GenBank (Accession No. MK880485). The phylogenetic analysis performed with partial and almost complete sequences of closely related species indicated that strain NI125 is affiliated within the Family Flavobacteriaceae belonging to the Phylum Bacteroidetes. The BLAST analysis of the NI125 16S rRNA gene revealed that it shares 99.72 % similarity with Aquimarina agarilytica ZC1 (Accession No. NR116794.1), 97.32 % with A. agarivorans strain HQM9 (Accession No. NR136817.1), and 96.3 % with A. seongsanensis strain CBA3208 (Accession No. NR152627.1). The NJ tree showing phylogenetic relationships of strain NI125 and the closest bacteria is presented in Fig. 1.

The phenotypic characteristics
The agarolytic strain NI125 is a yellowpigmented, Gram-negative, rod-shaped, and nonmotile bacterium. TEM observations by the negative staining with phosphotungstic acid indicated the absence of flagella (Fig. 2). This strain grows at temperature range of 4-30°C, and the growth occurs in the presence of 0.5-4% NaCl. The strain is positive to oxidase, catalase, alkaline phosphatase, βgalactosidase, and negative to nitrate reduction, indole and H 2 S production. The NI125 strain hydrolyzes agar, starch and casein but not cellulose, gelatin and tween 80. It produces acid from glucose, galactose and maltose but not from sucrose and maltose (Table  1). Based on phylogenetic and phenotypic characteristics, the strain NI125 is identified as Aquimarina agarilytica.

Optimization of the growth conditions for agarase production
In an attempt to determine the optimum conditions for agarase production, the influence of four factors was investigated; namely incubation period, temperature, initial pH, and different nitrogen sources. The time course of agarase production for 60 h indicated that agarase is produced in low level at the first 6 h of incubation, and then the enzyme production is increased gradually. The maximum production level is observed after 24 to 30 h of incubation. A dramatic decrease in agarase production is observed after prolonged cultivation time (Fig.  3a). The optimum temperature for enzyme production is observed at 20°C, where further increase in the temperature resulted in a suppression of enzyme production (Fig. 3b).
A. agarilytica NI125 produced agarase fairly over a wide pH range of 6.0-8.0, and results showed a severe diminishing of agarase production at pH 8.5 (Fig. 3c). Regarding the effect of various nitrogen sources on agarase production, results demonstrated that all the organic nitrogenous compounds except urea served as good nitrogen sources. The maximum production of agarase is achieved when tryptone or casein are used separately. This is followed by beef extract and yeast extract but with lesser proportion. Significant suppression of agarase production is observed when either urea or NaNO 3 are used as nitrogen sources (Fig. 3d).

Partial purification of the agarase enzyme
The extracellular agarase produced under the optimum conditions was partially purified through ammonium sulfate fractionation of the culture supernatant. The specific enzyme activity is increased from 4.91 U/mg protein to 14.73 U/mg protein after ammonium sulfate precipitation and dialysis. Three-fold purification of the agarase with an overall yield of 52.4% is achieved.

Determination of the substrate specificity
The partially purified agarase exhibited a strong hydrolytic activity towards p-nitrophenylβ-D-galactopyranoside (OD 540 = 0.974), however  it showed negligible activity towards p-nitrophenyl-α-D-galactopyranoside (OD 540 =0.004) indicating the specific cleavage of the β-linkage rather than the αlinkage.

Preparation of the sulfated agar
In the present study, a chemical modification of the agar was performed to increase the sulfur content. Agar was sulfated by the CSA-Pyr method; results revealed that the sulfur content of the sulfated agar derivative is increased by 8.7% (± 0.6), while the DS is increased by 6.09 (± 0.03), compared with the untreated agar.

Estimation of the anti-oxidant activity of the NAOs
For investigation of the anti-oxidant activity of the NAOs, agar and the sulfated agar were hydrolyzed by the partially purified β-agarase for 12 h, whereas the free radical scavenging activity of the produced Novel Research in Microbiology Journal, 2019 agar-hydrolysates was estimated using the stable free radical DPPH. Results revealed the promising antioxidative properties of oligosaccharides obtained by the enzymatic hydrolysis of agar by the β-agarase enzyme derived from strain NI125. Products of the enzymatic hydrolysis showed 23% scavenging ability. On the other hand, the sulfated NAOs exhibited a significant improvement of the radical scavenging activity. More than 87% inhibition of the DPPH is achieved by the NAOs produced by hydrolysis of the sulfated agar through the action of the β-agarase enzyme.

Discussion
Marine environments are natural sources of massive products with admirable biological activities. Nowadays, algae-derived oligosaccharides have received eminent attention in numerous applications of the industrial biotechnology. Many algal polysaccharides have commercial interests in various industrial applications. Furthermore, the chemical or enzymatic modification of the marine polysaccharides to produce oligosaccharides with new biophysical and biochemical features is a topic of progressing interest (Jutur et al., 2016;Han et al., 2019a;Chen et al., 2019a;Li et al., 2019a). Recent studies of Xu et al., (2018); Yu et al., (2019) reported the significant bioactivities of the agar oligosaccharides produced by various agarases enzymes derived from agardegrading bacteria.
The optimization studies revealed the significant impact of incubation period and temperature on the production of the extracellular agarase by A. agarilytica NI125. The current results showed that maximum production of agarase is achieved at low temperature (20°C); however, the optimum temperature for production of the same enzyme by the Pseudoalteromonas strain JYBCL was recorded to be 25°C (Jung et al., 2012). Regarding the nitrogen source, tryptone and casein stimulate maximum production of agarase by A. agarilytica NI125. The optimum nitrogen source for production of agarase is varied; organic nitrogen sources are preferred by some bacteria while inorganic nitrogen is favorable for others. Yeast extract, tryptone, and peptone were recorded to be the best nitrogen source for agarase production by Agarivorans albus YKW-34, Pseudoalteromonas sp. JYBCL 1, and Rhodococcus sp. Q5, respectively (Fu et al., 2009;Feng et al., 2012;Jung et al., 2012). It is assumed that the organic nitrogen sources can easily replenish the existing internal pool of amino acids within the microbial cell, thus facilitating the availability of these amino acids for protein synthesis. On the other hand, Lakshmikanth et al., (2006); Roseline and Sachindra, (2016) revealed that ammonium nitrate or sodium nitrate induced significant production of agarase by Pseudomonas aeruginosa AG LSL-11, and Acinetobacter junii PS12B. By employing the OFAT optimization method, more than 4-fold increment in agarase production by A. agarilytica NI125 is achieved. However, several studies of Abd El Aty In the present investigation, the substrate specificity assessment suggests that partially purified agarase belongs to β-agarase that produces neoagarooligosaccharides from agar. These findings are consistent with the results of several recent studies dealing with agarases from various sources, describing the cleavage of β-bonds in agar and agarose (Han et al., 2019b;Li et al., 2019b;Choi et al., 2019;Chen et al., 2019b). Agarases are characterized as α-agarases and β-agarases according to the cleavage pattern. Li et al., (2018);Hafizah et al., (2019);Liu et al., (2019);Lee et al., (2019) revealed that the basic products of the α-agarases and β-agarases are agarobiose and neoagarobiose, respectively. Results of this study revealed the significant anti-oxidant potential of the NAOs produced by hydrolysis of agar using βagarases derived from A. agarilytica NI125. It was reported that agar-derived oligosaccharides have high economic values, due to their physiological and biological activities. In agreement with these findings, Zhu et al., (2016) demonstrated that oligosaccharides produced by the enzymatic treatment of agar with agarase enzyme derived from Stenotrophomonas sp. NTA had inhibitory effects on hydroxyl, DPPH, and ABTS radicals, with potent anti-oxidative potency. Similarly, oligosaccharides derived from agar by Vibrio natriegens β-agarase exhibited excellent antioxidative activity (Zhang et al., 2019).
It is worth mention that agar sulfation before hydrolysis resulted in the production of NAOs with notable enhanced anti-oxidant potential. Upon hydrolysis of the sulfated agar derivatives with DS value of 6.09 which is higher than the un-sulfated agar, the produced oligosaccharides demonstrated 87% scavenging ability; however, the oligosaccharides produced from the un-sulfated agar expressed 28% scavenging ability only. The previous study of Wang et al., (2004) suggested that oligosaccharides with the sulfate group or with higher molecular masses showed stronger anti-oxidative activities than those without the sulfate group or with smaller molecular masses. The current findings agree with the recent investigations suggesting that sulfate modification is an effective method to improve the antioxidant activities of various polysaccharides (Xiao et al., 2019a;Olasehinde et al., 2019;Huang et al., 2019). It was thought that sulfation of the polysaccharides promotes their scavenging ability to free radicals by activation of hydrogen atoms on the anomeric carbons, thus providing stronger hydrogen supply capacity and reduce the aggressiveness of the free radicals. Thus, these sulfated polysaccharides possess a greater capacity to donate hydrogen to the superoxide anion (Chen et al., 2015). Beside the antioxidative activities, agar-derived oligosaccharides may inhibit the growth of bacteria, slow down the degradation of starch, and used as low-calorie additives to improve food qualities (Giordano et al., 2006). Moreover, Li et al., (2014); Zhang et al., (2019) revealed that NAOs obtained from the enzymatic hydrolysis of agarose stimulated the growth of Bifidobacteria sp., Lactobacillus delbrueckii subsp. bulgaricus and Sterptococcus thermophilus in vitro and in vivo without side effects. In this study, A. agarilytica strain NI125 is found to produce an extracellular β-agarase enzyme that digests agar producing biologically active oligosaccharides with promising anti-oxidative properties. Furthermore, the present work shed light on the crucial role of agar sulfation prior to hydrolysis with respect to enhancing the anti-oxidant power of the produced oligosaccharides.

Conclusion
The present investigation explores the potential of a newly isolated psychrophilic marine bacterium, A. agarilytica strain NI125, for production of β-agarase. More than 4-folds enhancement in the productivity is achieved by cultivation of this strain in ASW broth supplemented with 1% tryptone for 24 h at 20°C. The partially purified enzyme exhibited a specific activity of 14.73 U/mg protein, with apparent potential towards the beta linkage, so is classified as β-agarase. The anti-oxidant potential of agar hydrolysates is significantly improved by sulfation of agar before the enzymatic hydrolysis. The produced oligosaccharides possessing up to 87% scavenging ability; thus could be used as promising additives in food and feed products. Nevertheless, further investigations are required to study the β-agarase at the molecular level, and to characterize the sulfated NAOs with special emphasis on their physiological activities. Novel