Microbial Pathogenesis 136 (2019) 103642 Contents lists available at ScienceDisect PREY Ey Microbial Pathogenesis journal homepage: www.clsevier.comilocate/micpath Characterization of the antioxidant and anti-inflammatory properties of a polysaccharide-based bioflocculant from Bacillus subtilis F9 cae Sib Sankar Giri', EunChae Ryu', Se Chang Park” brary of Ac Baiting, als Varnry Main and Rach ss Voy Sit, Sl Natal iy, Sl 886, Sah Kae ARTICLE INFO ABSTRACT ower ‘Microbial foccuants are versatile class of novel blomacromlccues with numerous potential Industrial appl ‘beter ‘ations. This stay sought to investigate the antimicrobial, aniondant, and ant-nlammatory potential of a polysaccharide based biofloculant (PHB) extracted from Bacilus subs F9. To achieve this, the atoniant ‘activity of dterent PBB concentrations( 100 g/m 1000 g/mL) was fst examined in vio using 2.2g for 15 min and the precipitate was dissolved in 0.5M NaCl Cold anhydrous ethanol (Uhre volumes) was again added and the precipitate was gently washed thrice with 75% ethanol, The precipitate was lyophilized to obtain the crude bio- Aoceulants. The partially purified bioflocculant solution (0.1%) was further purified using column chromatography [16] and lyophilized until further use, A working soluton of 4mg/m. in delonized water was prepared for experimental purposes. 23, Detecting antioxidant properties of polysaccharide based bioflocculane ‘The antioxidant activities of purified PBB were evaluated using 2,2 Aliphenyl-1-pierylhydrazyI(DPPH), hydroxyl radicals, and superoxide radical scavenging assays. Accotbic acid (Sigma-Aldrich) was employed a8 a positive control ‘The DPPH-scavenging activities of PBB were determined following the method described by Chen et al. [17]. Briefly, 1 ml, of PBB sample (appropriate dilution) was mixed with 1 mL of DPPH solution (Sigma. Aldrich, USA) (0.2mM) and incubated in the dark for 30 min. Deio- rized water was used as a blank sample. The percentage of scavenged DPPH radicals was calculated as per Equation (1) 11 Asry Gample) / Asry (blank) x 100 @ ‘The hydroxyl radical scavenging properties of PBB were quantified as previously described by Zhai etal. [18]. Briefly, 2mL of PBS (pHi 7.4), Leak of 2.5mM of 1, 10-Phenanthroline (Sigma, USA), 1 mL. of appropriately diluted PBB sample, and 1 mL of 2:5mM FeSO, were combined. We then added 1 mi of 20:M H0,to initiate the reaction. ‘The reaction miature was incubated at 37°C for 90 min, Hydroxyl radical scavenging effect (36) was cleulated according to Equation (2) Scavenging effect (6) [Asan ample) — Assy Clank) / (Asse Ceontra) Asan (lankyy) x 100 @ ‘The superoxide radial scavenging assay was performed according to the method described by Li etal. {9}. Briefly, al of Tis HC buffer (PH 80, 150mM), 1.0mL of 1,2,3>phentriol (.5mM dissolved in 10mM HCD, and 0.5m, of PRB sample was acted sequentially to a tube and combined thoroughly. The reaction tube was incubated at 25°C for 20min, The absorbance wae measured at 32mm and the ‘quantity of superoxide radicals generated by 1,2,3-phentrol auto-oxi- dation was caleuated according to Equation (2). Superoxide scavenging activity (99) ~ [- (Are Ae) / (Aoy- Ago] % 100 @ Where, Aon isthe absorbance ofthe sample in the absence of PBB and 41,2:3-phentsiol, Apis the absorbance of samples containing],2,3- phentriol in the absence of PBB, Als the absorbance of samples‘containing PBB in the absence of 1,2,3.phentrol, and Ass the absor- bance of samples containing PBB and 1,2,3-phentriol. 24, Examining antibacterial activites of polysaccharide based bofteccutant ‘The minimal inhibitory concentration (MIC) of PBB against various, pathogenic bacterial strains was assessed using a broth micro-dilution method in 96-well plates (FALCON, USA) [19]. The bacterial stains actus cereus, Staphylococcus aureus, Listeria monocytogenes, Salmonella ‘ophimurtum, aod Escherichia col) were inoculated in 3m. of Mueller Hinton (ME) broth and incubated overnight ina shaker incubator at 37°C. Broth cultures were diluted two-fold in Sm. of MH broth and incubated for 4 hat 37°C. The fist column on the plate contained only (0.1 ml of MH broth, which acted asa negative grovith control. Columns 21 contained increasing concentrations of PBB (100 g/mL. 41000 g/mL). The dilutions of PUB were prepared at concentrations 2x higher than the desired final concentrations followed by adlition of the same volume of inoculum (total volume 100 jL/ Well). The bacterial ‘ell concentrations were adjusted to 10° CFU/ml. Column 12 contained a positive growth control (without EPS), containing 50 ul of MH broth ‘and 50 lof bacterial inoculum. The plates were incubated overnight at 37°Cand MIC was determined. The OD was measured at 600 nm in & microplate reader (Molecular Devices, USA), 2.5. Macrophage ell culture and MIT assay Isolation and culturing of head-kidney (HK) macrophages from ‘ohita was carted out as previously described (20. Cell number was ‘adjusted to 1 % 10® viable cells/ml. Purified HK macrophages were used for the establishment of macrophage cell Hines. Cells were tained in an atmosphere of 5% CO» in Dulbecco's modified Eagle's ‘medium (DMEM) (Gibco-BRL, NY, USA) supplemented with 5% heat: inactivated fetal bovine serum (FBS), 100ug/ml. streptomycin, and 100 g/m peniitn Cell viability asays were performed in 96well microplates using the MIT assay system (abcam, Cambridge, USA). Briefly, cells were seeded into plates at 1 x 10° cells/well and cultured with different ‘concentrations of PBB (100 ug/l, 200 g/mL, 400ug/ml., 600 ng/mL, ‘or 800 g/ml.) for 24hat 37°C, 5% COs. Untreated cells were con sidered to be negative controls. Gells were then stimulated for 4 with 1S 1 yg/mL) and 100 of MT was then added into cach wel for an additonal 4. The formazan product was solubilized by adding 150. ‘of Dimethyl sulfoxide (DMSO) and 100uL of 10% Sodium dodecyl sulfate ($DS) in 0.01 M HCL Viability was estimated by measuring the ‘absorbance at S70nm using an ELISA plate reader. 26, Quantifying nic oxide produetion Production of nitric oxide (NO) was quantified by measuring the nitrite concentration using the Griess assay [21]. A microplate reader Mirai Ramones 196 2019) 109642 was employed to measure the absorbance at 550m. Nitrite con- centrations were calculated from a sodium nitrite standard reference curve [22] 227. Effect ofthe polysaccharide based biloceulation the inflammatory response in HK macrophages HK microphage cells were incubated with 100yg/nl, 200 ug/ml, 400 ig/ml, oF 600 g/m. of PBB for 1 hat 26°C, and stimulated with LS (ug/ml Escherichia coli0L11: Ba) for 24h. The expression levels of TNF-a, 1L-1f, 1L-10, and TGF:p in the culture supernatants were measured by employing a fish specific ELISA kit (MyBioSouree, San Diego, USA) according to manufacturer instructions. ‘The mRNA expression levels of the inflammatory cytokines were measured by Real-time quantitative reverse transription polymerase chain reaction (RT-PCR). Briefly, HK macrophages were stimulated with different concentrations of PBB for 1h and treated with LPS for 24h, Total RNA was extracted from the stimulated HK macrophages ‘sing TRIZo1 reagent (Invitrogen, USA) The extracted RNA was reverse transcribed to eDNA using ® SuperScript® cDNA synthesis kit (Life “Technologies), according to the manufacturee instructions, qRT-qPCR analysis of I-16, 1-10, TNF-a, TGE-B, and factin was performed with (CFX96™ Real-Time PCR (Bio-Rad Laboratories In. Hercules, CA, USA) following the standard protocols. The primer sequences and thermo: cycling conditions are mentioned in Table I, All samples were run in triplicate, Meling curve analysis was performed after amplification to verify the accuracy of each amplicon, Gene expression results were analyzed using the 2~** method after verifying that the primers ef: fectively amplified the target genes with an efficiency of approximately 100% (24). 28, Statistical analyse Data were analyzed using one-way ANOVA. For comparison be "wween two groups Students ests were used. The OriginPro (version 8; Origintab Corporation, Northampton, USA) was used for all statistical analyses. Level of significance was set at P < 0.05, and results were expressed as mean = standard deviation (SD). 3. Results 3.1, Polysaccharide based bioflocculantexhibie significant antioxidant acti ‘The free radical scavenging capacity for PBB was measured using DPPH radical scavenging aetivity and was compared with that of the Positive control, ascorbic acid (Table 2). Results revealed that PBB exhibited high capacity for scavenging radicals and this ability in creased steadily with inereasing PBB concentrations with 62.439% ac- Livity observed following treatment with 100 ug/ml. and 81.46% with 800 ig/ml. However, the DPPH radieal scavenging capacity of PAB was ‘able 1 Heatime primer sequences and thermocyclng condos Te Gre Pier 0 9 emeyeig ion cee ap AoncTCTEATCGUAG WC Hew ceo o8 CB COE IOe md TAC IOE = wo stceacoccacracctetcr 95° Ms eet 6705605204 72°C 204 ea se ccacoermeacrreace Cs oydert 8 C55 608°C 20cm 72206 ea rors scrmarreocanceasa 95° Hs eet 8705605204 amd 72°04 ea cin scncenocrreascreeareats 26° m5 4 oye of 5,655 605°C 304 amd 72306 ea‘Table 2 Mirai Ramones 196 2019) 109642 [PPA radical savenging,hydrony radical scavenging, and superoxide radical scavenging ectivities(%) of various concentration (200-1000 9/1) of poly saccharide biofloceuant and positive contol vitamin C (ascorbic acid). Data are presented as mean * SEM. One way ANOVA was used fr data analysis (n ~ 3 P< 000), Data with superscript letter “a” denotes significant iferences with corespond value of ascorbic acid tert ‘onecation G/L) Dre eal eaves peo aca evening ‘peroxide dl evening olocaen 10 cam = 208 se = Lar sua = ae 200 esis is 92 + O88 sean = 127 ‘o 73a La Sem = ae fee an 1090 e372 21" 08 = 188" B74 = 238 combed 10 css 088 ware in found to be lower (p_< 0,05) than the positive contr, except 100 8/ mi PBB. In fact, the DPPH radical scavenging capacity of ascorbie acid ‘ne seon to increase steadily upto 800 ig/ml st which point it began to plateau. Further, the scavenging activity of 1000\g/ml. of PBB (83.72 2 2.119) was similar to that of 600 g/mL of ascorbic acid (43 + 114%, PBB also exhibited concentration-dependent scavenging activities against hydroxyl radicals (Table 2). This specific seavenging activity of PPB was 37.56 = 1.4% atthe concentration 100 ug/ml. and increased to 66.34 = 2.63% when the concentration reached 800 g/mL. Alter natively, ascorbic acid exhibited 43.7 © 2.16% to 72.1 = 2.74% hy- ‘droxyl radical scavenging capacity at concentrations of 100 g/mL and 800 ug/ml, respectively. These values obtained for the positive control ‘were determined tobe significantly higher than corresponding values in PBB, Moreover the hydroxyl seavenging activity exhibited by 800p8/ mL of PBB (66.34 = 2.63%) was slightly ower than that observed for £600 ug/l. of ascorbic acd (68.6 ~ 1.53%). ‘The seavenging activities of PBB and ascorbie acid against super ‘oxide radicals are presented in Table 2. The superoxide racic scavenging capacity of PBB was determined to be $3.29 + 1.8% at 100 ig/ml and increased to 81.74 = 2.26% when the concentration reached 1000 g/t. Altematively, at the same concentrations of as corbie acid, 48.1 = 1.73% and 92.3 = 1.38% superoxide radical scavenging ‘was observed. Interestingly, the superoxide adic scavenging activity observed at 1000 g/mL of PBB (81.74 + 2.26%) was similar to that of 600 ug/ml. of ascorbic acid (80.2 * 2.1990) 32, Antibacterial properties of polysaccharide based biflaculant PBB exhibited antibacterial properties against all of the tested bacterial strains as revealed by MIC values ranging from 3 mg/mL. to (95 mg/ml. (Table 8). Our study found that Ecol, S. aureus, and S. typhimurium were significantly inhibited by PBR concentrations of Smgyml, 45mg/mL, and 7 mg/mL, respectively. However, L. mono: ‘ytogenes was only inhibited following treatment with PBB at a con ‘centration of 9.5 mg/m. 3.3. Cell vibiliy and ni oxide production ‘The effect that PBB elicited on cell viability is shown in Fig 1. PBB ‘concentrations between 100 ug/ml. and 800ug/mL. demonstrated no significant effect on cel viability. Within the contto, eel viability was found to be 99.4%, whereas the viability in cultures exposed to 800 8/ mi of PRB was 98.2%. Results from the MTT assay confirmed that the ‘observed inhibitory effect elicited by PBB was not associated with ey- totoxicty. ‘To determine if PBB exhibited antiinflammatory funetions, we analyzed NO production by LPS-stimulated macrophages. We found that PBB functioned to attenuate the LPS-induced NO production in a concentration dependent manner (Fi. 2)- Ata concentration of 600 15/ ‘i. PBB exhibited maximum inhibition of LPS-induced NO production (approximately 60% inhibition compared to the canta). ‘34, Effect of PBB on inflammatory cytokine production in LPS-stimulated carp head-kdney macrophages To investigate the antiinflammatory effect exhibited by PBB on LS-stimulated macrophages, the production of pro-inflammatory and ant-inflammatory eytokines were evaluated. Cell supematants were analyzed by ELISA co quantify cytokine protein secretion, and qRT-PCR ‘analysis was performed to examine the transcriptional response (Fig. °) (Our results show thatthe levels of TNF and IL-1B increase in LPS: stimulated macrophage cultures; however, pretreatment with PBB acted to significantly prevent this overexpression of TNF-a and Il-1in a eoncentration-dependent manner (Pig, 2A-D). Alternatively, the ex pression level of I-10 and TGF-f were found tobe slightly increased in the LPS-stimulated macrophage eulture media, however, these levels were further augmented by pretreatment with PB (Fi, 3E-HD, Overall, PBB pretreatment inhibited the expression of pro-inflammatory cyto kines while enhancing the expression of anthinflammatory cytokines Gg. 9. 4. Discussion PSs are primarily produced by the metabolic pathways of miero- ‘organisms [19] and are largely polyanione since they are composed of, sulfate, pyruvate, phosphate and uronic acids. The physical properties Of EPS are affected by monosaccharide configuration as well as the aggregation of polymer chains [25]. In the present study, we ‘rable 3 ‘Antimicrobial activity of plyseccharide-bioflocculant (PE) aguas pathogenic becteral tesa ctrl sis IG oem) 0 ac cea MEE 65:29 ° tpl eeu MTCC 727 ts Sapna para ATCCI4028 7 ce ot NTO 443 3 PD exhibived antibacterial setiviis against all the test bacterial strains a re vealed by MIC values ranging from 3 mg/mL to 11 mg/ml (Table 2). Our study found that E col, S.eurus, and S. typhimurium were inhibited With con Eentration of 3 4 and 7 mg/mL, respectively. However, .monoryagees was inhibited with & PB concentration of 11 m/m.120 100 6 4 2 0 1023 4 $ 6 7 Fig. 1. fects of PBB on cel viability in carp macrophages. Cells were pre tected with PBB for 1h, and then stile wih 1 (1g Cell abit was assayed by MTT assy. Data are presente as mean = SEM. ty (%) 100 lth. Fig. 2 fect of PB on LPS-induced NO production in carp macrophages. Data ‘are presented a mean = SEM (n= 3). Nitgte produetion (YD) fnvestigated the antioxidant properties of PBB using three diferent methods. DPPH fre radical is a stable radical containing an unpaired valence electron on one of the nitrogen bridge atoms. These free radi cals must scavenge to donate an electron or an active hydrogen atom, from surrounding antioxidant compounds (26). In the present study, the scavenging activity exhibited by 1000ug/mL. of | PPB (63.72 + 2.11%) was similar to that of 600ug/m. of the postive control, ascorbie acid (84.3 + 1.14%), Furthermore, the DPPH scavenging activity of PBB was found to be higher than that of the EPS isolated from B. subs, which exhibited 61.9% DPPH antioxidant ac Livty ata concentration 800 ig/ml [27]. However, EPS isolated from 1B. amploliguefaciens exhibited a higher DPPH radical scavenging ac tivity (99.39 + 0.63%) at 1000 ug/ml. [25], Hydroxyl radicals function as a strong oxidant that can react with ‘almost all biological molecules, including proteins, lipids, and carbo hydrates and leads to biological damage [29]. In addition, free radical ‘OH can function to accelerate aging by oxidizing most biomacromo- lecules, thereby leading tothe development of rlated oxidative stress disease [90], Thus, eliminating “OH is vital for protection of cells and food systems against oxidative sires. In our study, the purified PBB ‘demonstrated an upward trend for hydroxyl radical scavenging with increasing concentrations. Specifically, at 800 9/mL, 66.34 + 2.63% scavenging was observed, which was similar to that of 600 ug/ml. of ascorbic acid (68,6 + 1,539), This scavenging activity was higher {than that exhibited by EPS from Bifidobacterium bifidum or Lactobacillus plantarum [9]. However, EPS from Bacillus sp. 5-1 elicited higher hy ‘droxyl radical scavenging than PBB (10). In agreement with the results ‘of the present study, EPS extracted from &. amyloliquefaciens GSBs-1 [4] ‘and fom B. anyloliquefacens SMS 2017 [28] exhibited strong hydroxyl radical seavenging capacity. The antioxidant mechanism of PBB may erdaPehgeets 196 (2019 103542 nvolve the electrons or hydrogen atoms from polysaccharide molecules chelating with Fe?" thereby preventing radical chain reactions [31] Superoxide radicals are preeursors for most ROS including singlet ‘oxygen, hydrogen peroxide, and hydroxyl radicals, al of which induce tissue damage or cell death [9,29]. Although the superoxide scavenging activity of PBB (81.74 = 2.26% at 1000 ig/ml.) was found to be sig: nificantly lower than that ofthe same concentration of ascorbic acid (023 + 1.349%), and EPS from B. ayloliquefaciens 3MS_ 2017 ex ibited higher superoxide radical scavenging activity (91.44 + 2.51% at 1000 ug/L) [28] It was higher than that exhibited by EPSs from B. anplotquefaciens GSBa-I (4.86% at $000 ig/ml) [4], EPS from Ba cillus sp. (6843 = 3.21% at 7000 g/ml) [10], EPS from Bifido bacterium bifidum (59.75 = 8.12% at 1000/mL) or Lactobaelis plantarum (65.45 = 8.48% at 1000 g/mL) [9]. The presence of more electron withdrawing groups on polysaccharides results in a weaker dissociation energy of the O-H bond; and the presence of only a few electrophilic groups, such as aldehyde or keto, in PBB may account for its relatively lower seavenging activity compared to that of ascorbic acid [32]. Further, the pH of the eulture media was 7.0 and while, PBB has demonstrated optimal activity at neutral pH [16], {thas been pre viously reported that acidic polysaccharides exhibit stronger superoxide anion scavenging activity compared to neutral polysaccharides {33}. Nevertheless, overall, PBB in our study, exhibited strong antioxidant properties. The presence of recicing gars, monosaccharides, proteins, amino acids, uronic acids and other organic molecules may have co tributed to the observed scavenging activities (271 Im addition to its antioxidant properties, purified PBB also exhibited antimierobial aetvity against various Gram-positive and Gram negative bacteria, which agrees with results from an earlier study that demon- strated inhibitory activity in EPS feom probiotic L. plantarm for the growth of various food-borne pathogenic bacteria [19]. Similarly, EPS Droduced by I. kefranofaciens DNI was seen to inhibit the growth of various food-borne bacterial pathogens [34]. Moreover, EPS from B. bifidum and L. plantarum exhibited antibacterial effects against various bacterial pathogens such as Listeria monocytogens: CMCCS4007, Sta Phylococeus aureus CGMCC26003, Bacilus cereus ATCCI4579, Salmo rela yphimurium ATCCL3311, Shigella sonnei ATCC25931, and E. coll 0157:H7 [9]. Thus, further exploitation of PBB may serve to further elucidate the mechanism responsible for its observed antimicrobial activity against pathogenic bacteria Macrophages play a vital role in acute and chronic inflammatory responses [35]. To determine whether PBB exhibit cytotoxic effects, feolated carp HK macrophage cells were incubated with or without PRE pretreatment (Pig 1. We found that at concentrations between 100 1/ ‘mL-800 ug/ml. PRB did not significantly affect cell viability. Following 24h of treatment, cell viability was determined to be 98.2% in cultures With 800 g/ml. of PBB. This negligible eytotoxie effect confirms the Possible use of this PRE for biological uses. Similarly, in a recent study, biofloceutants isolated from P.jamilae were also described as being non: cytotoxic [13]. In accordance with 1SO-10099%35, 2009, cell viability above 80% is considered to be non-toxie [36] Further, PBB funetioned to attenuate the LPS-induced NO produ tion in a concentration-dependent manner (Vig. 2). NO isa signaling molecule that has important functions in the pathogenesis of flammation. It appears as an antiinflammatory in normal state, how- ever NO functions as a pro-inflammatory mediator thereby promoting inflammation through its overproduction in abnormal physiological states [28]. Therefore, NO inhibitors have been shown to play a vital role in conteolling inflammatory diseases (371. Our results show that 600 ug/ml of PBB resulted in maximum inhibition of LPS-induced NO production, which was in agreement with a previous study, that re Ported on biofloceulant isolated from Pacrbacils jamilachaving the ability to effectively reduce the level of LPS-induced NO production in peripheral blood mononicear cells [13] Inflammatory cytokines, particularly TNF-a, IL-1, and IL-6 play key roles in the induetion and perpetuation of inflammation caused byssone ict ato 196 019) 1862 A w : : 3 a te . j iis : i aii z Gee : i ad i BG : 0 é rosqont) 0 0 mom amore ae ee ee c a: 3 Z i: z si = au : at i BE t i i = i 1 = o a Peneenly 00 1 aD MO rEsuenty 00mm E ‘ a2 z° ae i sis re aey ; ry gf i» m2 § 1 o 0 ao ronegnl 0 0 am man an © resggniy 00mm an an eee ee z Bass Hip BL: Bao Bis Z4 Fig. 3 feet of on the production of INF, IL-1, 10, and TF 8 n LPS-stimulated earp macrophage cells Levels cytokines were measured by ELISA. The mRNA levels of cytokines were measured by RT-PCR. The gure show the representative results of three independent experimen and data are presented 3s ‘macrophages. Elevated levels of TNF-< has the potential to cause tise ‘damage or even sepsis and death [38]. High doses of LPS (g/mL) have been shown to Furthermore, LPS is a strong stimulator of macrophages and activates several signal transduction pathways producing vatious proin- flammatory cytokines in humans and animals, including fish [20,3 induce strong inflammatory responses in fish‘Our results demonstrated that treatment with PBB served 10 sig: nificantly reduce the expression of TNF-a and IL-1 at the mRNA level, which was confirmed by quantifying the corresponding protein levels Via ELISA. Conversely, PBB was found to significantly augment the production of specific antiinflammatory cytokines, namely, TGF-B and UA10 compared to the control group (Pig. 2). I-10 elicits ant flammatory effets, which contributes to its role in inhibiting mono- e/macrophages to secrete inflammatory mediators such as TNF-a, AAP, and 1-6 [40]. These findings suggest that PBB may suppress the production of pro-inflammatory cytokines, however the detailed me ‘chanisms need to be further elucidated. EPS from B. amyloliquefacins 3MS2017 exhibited ant-inflammatory activity (28). Previously, bio Floceulant from Paenibacilus jamilae was shown to exhibit anti: flammatory activity through the inhibition of LPS-induced TNF-a and 11-1 production in PBMCS, and through enhanced production of 110 and TGF (13) ‘5. Conclusions “The PDB extracted from 1 subills F9 was characterized for anti ‘oxidant and antvinflammatory properties. PBB exhibited potent scavenging capacity against DPPH, hydroxyl, and superoxide radicals, which suggests that it has the potential to be exploited further as & natural source of antioxidants for nutraceuticals or functional food development. I¢ also elicited strong antibacterial properties against ‘multiple pathogenic bacteria; and acted to inhibit the pro-inflammatory response while inteasing the antiinflammatory response of LPS-st- mulated macrophages. Although, PBB exhibited potential beneficial biological activities, further studies are required to determine its structure function relationship and better understand the antioxidant, antiinflammatory, and antibacterial nature of B,subilis FO PBB. Also, scale up of bioflocculant ranted for Punding statement ‘This research was supported by the Korea Research Fellowship, Program’ of the National Research Foundation of Korea, Ministry of Science and ICT (KRF: 2016H1D3A1909005), and Cooperative Research Program for Agriculture Science and ‘Technology Development (Supportive managing project of Center for Companion Animals Research) by Rural Development Administration (©3013985032018) Acknowledgements ‘This research was supported by the “Korea Research Fellowship Program’ of the National Rescarch Foundation of Korea, Ministry of Science and ICT (KRF: 2016H1D3A1909005), and Cooperative Research Program for Agriculture Seience and ‘Technology Development (Supportive managing project of Center for Companion ‘Animals Research) by Rural Development Administration (©3013985032018. Conflicts of interest “There is no confit of interest to declare, References 1D]. 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