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International Journal of Biological Macromolecules xxx (2014) xxx–xxx

Contents lists available at ScienceDirect

International Journal of Biological Macromolecules

journal homepage: www.elsevier.com/locate/ijbiomac

1 Antioxidant and anticoagulant activity of polyphenol and

2 polysaccharides from fermented Sargassum sp.
3 Q1 P. Shobharani a , V.H. Nanishankar b , P.M. Halami a , N.M. Sachindra c,∗
a
4 Department of Food Microbiology, CSIR-Central Food Technological Research Institute, Mysore 570020, India
b
5 Food Safety and Analytical Quality Control Laboratory, CSIR-Central Food Technological Research Institute, Mysore 570020, India
c
6 Department of Meat and Marine Science, CSIR-Central Food Technological Research Institute, Mysore 570020, India
7

8
23 a r t i c l e i n f o a b s t r a c t
9
10 Article history: The current investigation was carried out with an objective of determining the structural characteristic of
11 Received 8 October 2013 polysaccharides extracted from fermented Sargassum sp. to be used as potent natural heparin substitute
12 Received in revised form 22 January 2014 anticoagulant compound. Sargassum sp. fermented with marine lactic acid bacteria was initially subjected
13 Accepted 3 February 2014
to ethanol precipitation for the recovery of bioactive compounds. Antioxidant activity was maximum in
14 Available online xxx
the soluble fraction whereas anticoagulant activity was observed to be high in the precipitate which corre-
15
lated with the increased polyphenols and total sugars respectively. The precipitate was purified by anion
16 Keywords:
exchange chromatography and the fractions collected were analyzed for total sugars and anticoagulant
17 Fermentation
18 Lactic acid bacteria
activity. There was 2.6–3.9-folds increase in anticoagulant activity in the final purified fractions, with a
19 Alginates maximum activity in case of sample fermented with Enterococcus faecium (6.7 ± 0.22 IU/mg). Structural
20 NMR elucidation of potential anticoagulant polysaccharide by Fourier Transform Infrared Spectroscopy (FT-IR)
21 FTIR and Nuclear Magnetic Resonance (NMR) analysis indicated the presence of alginate rich in mannuronic
22 Polysaccharide acid.
© 2014 Published by Elsevier B.V.

24 1. Introduction as production difficulties, chemical inhomogenicity, variability in 42

physiological activities, bleeding etc. [5]. Hence there is a require- 43

25 Marine organisms are rich source of structurally diverse bioac- ment for safe, natural and easy to use heparin substitute. In this 44

26 tive compound that play a vital role in human health and nutrition. regard, seaweeds that are rich in eminent bioactive molecules have 45

27 Seaweeds are one of the important marine living resources that attracted large number of researchers to investigate anticoagulant 46

28 have been source of food, feed and medicine in western world. activity of seaweed polysaccharide (PS) as an alternative/substitute 47

29 Recently they have been extensively exploited because of their to heparin compound. Owing to the fact that sulfated polysac- 48

30 perceived health benefits including anticoagulant, antioxidant, charides from marine seaweed and heparin share similar ionic 49

31 antimicrobial, anti-inflammatory, anticancer, anti-herpes and anti- structure, research interest toward anticoagulant compound from 50

32 hyperlipidemic activity [1–3]. Thus the potentiality of marine foods marine source is increasing. Sulfated galactan from red seaweed 51

33 and their by-products have been expanded not only in food industry [6], sulfated ␤-arabinan from green seaweed [7] and fucoidan from 52

34 but also in pharmaceutical and biomedical field. brown seaweed [8] have been well demonstrated for their anti- 53

35 According to World Health Organization [4], cardiovascular dis- coagulant activity. But alginates, the major constituent of brown 54

36 eases including heart diseases and stroke related to thrombosis are seaweed cell wall have been less exploited for anticoagulant activ- 55

37 the main cause of death globally and predictions have been made ity. 56

38 that by 2030, almost 3.6 million people will die from these diseases. Further, the other major problem faced in the present day is the 57

39 Heparin, a sulfated polysaccharide has been used as an anticoagu- cytotoxicity and metabolic disorders caused due to free radicals 58

40 lant drug in the area of hematology and transfusion medicine for or reactive oxygen species. Reactive oxygen species or free rad- 59

41 more than 50 years. However, it has several disadvantages such ical generated by UV light, ionizing radiation, chemical reaction 60

or metabolic processes contribute to metabolic changes that may 61

lead to inflammation, retrolental fibroplasias, arthrosclerosis, lung 62

∗ Corresponding author. Tel.: +91 9449827424. injury, ischemia–reperfusion, cancer and aging [9]. Although free 63

E-mail addresses: sachiprathi@yahoo.com, nmsachindra@cftri.res.in radicals are essential for certain biological processes, their over pro- 64

(N.M. Sachindra). duction or weakened defense system can lead to cellular damage. 65

http://dx.doi.org/10.1016/j.ijbiomac.2014.02.005
0141-8130/© 2014 Published by Elsevier B.V.

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66 Hence exogenous supplementations of antioxidants are essential. inoculated with native marine LAB isolates at a concentration of 125

67 Synthetic antioxidants such as butylated hydroxyonisole, butylated 5 log cfu/ml and incubated at 37 ◦ C for 12 day under static condi- 126

68 hydroxytoluene that are being used in food industries have been tion. After incubation time, fermented samples were centrifuged 127

69 suspected to have cytotoxicity effect [10]. Therefore, there is an and the supernatant seaweed extract was used for further analysis. 128

70 urge for safe and efficient antioxidant from natural sources. Seaweed broth was used as test control and cellulase hydrolyzed 129

71 In Indian context, although seaweeds have been utilized for seaweed without LAB cultures was used as sample control. 130

72 industrial production of agar/alginate and as fertilizers, their appli-

73 cations in other contemporary area are still unexploited. The recent 2.4. Polysaccharide extraction and purification 131

74 finding on seaweeds application in clinical and nutraceutical area

75 has awakened researcher to explore the unexploited domain of Fermented seaweed samples after 12 days of incubation was 132

76 seaweed to develop novel therapeutic compounds. centrifuged at 8000 rpm for 15 min and the supernatant was pre- 133

77 Studies have been carried out to demonstrate antioxidant cipitated with absolute ethanol (99%) at a ratio of 1:3 (v/v) and kept 134

78 potential of seaweeds from Indian water [11]. Recently we have at 4 ◦ C for 24 h. The precipitate and the soluble fraction were sepa- 135

79 demonstrated the enhanced antioxidant and anticoagulant activity rated by centrifugation at 10,000 rpm for 15 min at 4 ◦ C. Precipitate 136

80 by fermentation of brown seaweed Sargassum sp. [12]. The brown was freeze dried and stored at −20 ◦ C until use. Soluble fraction 137

81 algae, Sargassum is a rich source of biologically active compounds. was flash evaporated to remove solvent, freeze dried and stored at 138

82 They have a complex cell wall mainly composed of cellulose −20 ◦ C until use. The precipitate was suspended in 50 mM sodium 139

83 microfibrils embedded in an amorphous matrix of acid polysaccha- acetate buffer (pH 5.0) and the soluble fractions in distilled water 140

84 ride linked to each other by protein. There is an increasing demand for analyzing anticoagulant and antioxidant activity. 141

85 for studying structural characteristics and efficiency of these bioac- The precipitate exhibiting higher anticoagulant activity was 142

86 tive compounds to be used as safe alternatives in biomedical and selected for further purification. The precipitate (60 mg) dissolved 143

87 food industries. In this regard present work has been focused on in 50 mM sodium acetate buffer (pH 5.0) was applied to DEAE cel- 144

88 purification of antioxidant and anticoagulant compound of Sargas- lulose column (18 cm × 3 cm) equilibrated with 250 ml of 50 mM 145

89 sum sp. after fermentation with marine lactic acid bacteria (LAB). sodium acetate buffer (pH 5.0). The column was washed with 146

90 Further data on structural characteristics of the purified polysac- 100 ml of same buffer. Elution was carried out at a flow rate of 147

91 charide (PS) with anticoagulant activity has been presented. 1 ml/min with a linear gradient of 0–1 M NaCl in the same buffer. 148

Fractions of 5 ml was collected and measured for total sugars. Frac- 149

92 2. Materials and methods tions showing higher sugar content were pooled, concentrated by 150

freeze drying and stored at −20 ◦ C until use. The concentrated sam- 151

93 2.1. Materials ple was then checked for anticoagulant activity by activated partial 152

thromboplastin time (APTT) assay. 153

94 Sargassum sp. collected from West coast of India was

95 washed thoroughly with water, dried and milled to form 2.5. Anticoagulant activity 154

96 fine powder. Ascorbic acid, 3,5-dinitrosalicylic acid (DNS),

97 glucose, NaCl, ferric chloride, gallic acid, calcium chloride Anticoagulant activity of seaweed polysaccharide was evaluated 155

98 were of AR grade from Sisco Research Laboratory, Banga- using human blood collected from healthy individual donors in a 156

99 lore, India. Diethyl aminoethyl (DEAE) cellulose, 2,2-azinobis-3- conical tubes containing 2.5% sodium citrate (9:1, v/v). The plasma 157

100 ethylbenzothiazoline-6-sulphonate (ABTS), peroxidase, synthetic was separated from blood cells by centrifugation at 2000 rpm for 158

101 antioxidant standards, tertiary butyl hydroxyl quinine (TBHQ), ␣- 20 min at 4 ◦ C and stored at −80 ◦ C until use. For APTT assay, cit- 159

102 tocopherol (TP), ethylene diaminetetra acetic acid (EDTA) were rated plasma (50 ␮l) was mixed with 50 ␮l of seaweed extract and 160

103 procured from Sigma–Aldrich Inc, USA. Activated partial throm- incubated at 37 ◦ C for 1 min. APTT reagent (50 ␮l) was added to 161

104 boplastin time (APTT) reagent, prothrombin time (PT) and heparin the mixture and incubated for 1 min at 37 ◦ C. Clotting time was 162

105 were from Tulip diagnostics (P) Ltd., Goa, India. All other chemicals determined after addition of 50 ␮l of 0.05 mM CaCl2 solution. For 163

106 used were of AR grade. PT determination, seaweed extract (50 ␮l) was mixed with 50 ␮l of 164

citrated human plasma and incubated at 37 ◦ C for 2 min. Clotting 165

107 2.2. Bacterial cultures and growth condition time was recorded after addition of 50 ␮l of PT reagent. Similarly 166

assay was carried out with samples from test control (seaweed 167

108 The marine LAB isolates Pediococcus acidilactici (LC), Weisella broth) and sample control (cellulase treated seaweed broth without 168

109 paramesenteroides (Nw-1a), Pediococcus pentosaceus (2Nw-1a) and LAB cultures). Relative clotting factor was determined by divid- 169

110 Enterococcus faecium (P1-2CB-w1) [12] were grown in MRS ing the clotting time of fermented sample by the time obtained 170

111 (deMann Rogosa and Sharpe) broth at 37 ◦ C for 24 h. For crude cellu- under similar condition with blank (distilled water). The activity 171

112 lase enzyme, the native isolate Bacillus megaterium [13] was grown was expressed as international units/mg using parallel standard 172

113 in Luria Bertani (LB) broth supplemented with 0.1% carboxy methyl curve prepared with heparin standard. 173

114 cellulose (CMC) at 37 ◦ C for 24 h under shaking condition (100 rpm).

115 Cell free supernatant of B. megaterium was obtained by centrifuga- 2.6. Antioxidant activity 174

116 tion (8000 rpm for 10 min), filter sterilized and freeze dried. The
117 crude cellulase thus obtained was stored at −20 ◦ C until use. Antioxidant activity of seaweed extract was analyzed by seven 175

different assays such as reducing potential, metal chelation, nitric 176

118 2.3. Preparation of fermented Sargassum sample oxide scavenging, hydrogen peroxide scavenging, ABTS scaveng- 177

ing activity and singlet oxygen quenching activity as described by 178

119 Sargassum sp. was fermented using marine LAB isolates as Sowmya and Sachindra [14]. 179

120 described by Shobharani et al. [12]. Briefly, fine Sargassum powder

121 (1%) was suspended in distilled water supplemented with yeast 2.7. Analytical methods 180

122 extract (0.1%) and glucose (0.5%). This seaweed broth was auto-
123 claved and subjected to initial enzymatic hydrolysis by using crude Total yield (mg) was determined by oven drying of the samples 181

124 cellulase enzyme (4%) at 60 ◦ C for 4 h. Hydrolyzed seaweed was in a preweighed aluminum plate until constant weight is obtained. 182

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183 Total sugars was measured by phenol–sulphuric acid method [15] extracted by hot water extract with a total yield of 21% containing 241

184 and reducing sugars by DNS method [16]. Protein content was esti- 80% sugar and 10% protein. Rodrigues et al. [23] applied papain 242

185 mated according to Lowry’s method using bovine serum albumin digestion followed by precipitation with cetylpyridium chloride 243

186 as standard [17]. and ethanol for a total yield of 3% PS. This indicates that the PS yield 244

vary among different species and extraction procedure applied. 245

187 2.8. Structural elucidation of purified fermented seaweed Antioxidant activities of precipitate and soluble fraction are pre- 246

188 polysaccharide sented in Table 1. All the seven antioxidant assays carried out for 247

different free radicals revealed that the activity was higher in case 248

189 FTIR (Fourier Transform Infrared Spectroscopy) spectra of puri- of soluble fraction as compared to precipitate. The result may be 249

190 fied PS fractions were determined using FT-IR spectrophotometer correlated with higher polyphenol content in soluble fraction than 250

191 model 5700 (M/S Thermo electron Corporation, USA). PS powder in precipitate. Polyphenols with their hydroxyl group are highly 251

192 (2–3 mg) was mixed with KBr and pressed into a disk. The whole IR effective in scavenging free radicals [24]. In comparison with the 252

193 spectrum was analyzed with a scan range of 4000–400 cm−1 . Thirty test control of soluble fraction, enzyme hydrolyzed sample con- 253

194 scans were taken with 4 cm−1 resolution. CO2 and H2 O corrections trol exhibited reduction in reducing activity, metal chelation ability, 254

195 were incorporated. Reproducibility of the normalized spectra was nitric oxide reduction and ABTS activity. Among the soluble frac- 255

196 ±2%. tion of fermented samples, a significant increase (p < 0.05) in the 256

197 NMR (Nuclear Magnetic Resonance) spectra of purified PS frac- antioxidant activity was observed except reducing potential, where 257

198 tion (3 mg dissolved in D2 O) showing highest anticoagulant activity the highest activity (6.94 ± 0.13 ␮g/mg dry weight) was found in 258

199 were recorded on DRX-500 NMR spectrometer equipped with test control. Significantly higher activity (p < 0.05) was observed 259

200 broad band probe of 5 mm diameter (IISC NMR spectra facility, in sample fermented with E. faecium (P1-2CB-w1) compared to 260

201 Bangalore). The spectra were recorded at 65 ◦ C with 8000 scans. those samples fermented with other LAB isolates. E. faecium (P1- 261

2CB-w1) was found to produce high lactic acid with maximum cell 262

202 2.9. Statistical analysis viability as compared to other LAB tested [12], thus facilitating the 263

breakdown of seaweed cell wall and increasing the leaching out of 264

203 Each experiment was carried out in triplicate and significant polyphenols with antioxidant activity. Even in the precipitate the 265

204 differences between samples were analyzed ANOVA and mean sep- activity was higher in the sample obtained by fermentation with E. 266

205 aration was accomplished by Duncan’s Multiple range test [18]. A faecium, except for nitric oxide (NO) scavenging. 267

206 value of p ≤ 0.05 is considered to be statistically significant. In the soluble fraction, with respect to correlation between 268

polyphenol content and antioxidant activity, except for reduc- 269

207 3. Results and discussion ing potential (r = 0.34) and singlet oxygen scavenging (r = 0.69) all 270

the other antioxidant parameter showed correlation co-efficient 271

208 Fermentation is a process where complex macromolecules are above 0.8. In the precipitate only metal chelating activity (r = 0.83), 272

209 broken down to simpler compounds by the action of microor- ABTS scavenging (r = 0.86) and singlet oxygen quenching (r = 0.85) 273

210 ganisms. In our earlier study, the application and advantages of showed good correlation with polyphenol content. On the contrary, 274

211 fermentation using well defined marine LAB isolates for enhanced the precipitate constituting 61.33–86.52% total sugars exhibited 275

212 anticoagulant and antioxidant activity from Sargassum sp. has been lesser antioxidant activity. Hence it can be predicted that polyphe- 276

213 reported [12]. Fermentation by LAB had an additional advantage nol content in the soluble fraction may be responsible for the 277

214 in reducing pH and simultaneous increase of total and reduc- activity. As the demand for antioxidant compounds is increasing, 278

215 ing sugars. In the study, fermentation for a period of 12 days the present investigation of antioxidant compound from fermented 279

216 after initial hydrolysis by cellulase showed better anticoagulant seaweed could be an interesting subject and hence further studies 280

217 and antioxidant activity as compared to unfermented seaweed. In are required for the characterization of the antioxidant molecule 281

218 the present investigation, polysaccharide from fermented Sargas- from these fermented sample. As the present study was focused 282

219 sum was recovered, purified and characterized. As observed in the further on bioactivities of seaweed PS, work has been continued 283

220 present study, all the four LAB cultures facilitated breakdown of with the ethanol precipitate showing anticoagulant activity. 284

221 seaweed with a total yield (crude sample) ranging from 2.4 to 3.0 g Anticoagulant activity as analyzed by APTT and PT assay 285

222 dry weight which represent 81.3–93.6% of total sugar and 3.1–10.5% revealed that the precipitate showed higher inhibition of coagula- 286

223 proteins. tion cascade as compared to soluble fraction (Table 2). As the blood 287

coagulation mechanism consist of intrinsic and extrinsic pathways 288

224 3.1. Anticoagulant and antioxidant activity of fermented wherein various factors affect the coagulation cascade, both the 289

225 Sargassum APTT and PT assay was carried out respectively for intrinsic and 290

extrinsic pathway inhibition. The ethanol precipitate of fermented 291

226 In the initial step of purification, the fermented samples were seaweed samples exhibited higher APTT activity (4.27–4.71 IU/mg) 292

227 precipitated with ethanol (99%) and the precipitate and soluble in comparison with PT test (0.091–0.110 IU/mg) indicating the 293

228 fractions separated by centrifugation were individually tested for intrinsic pathway inhibition. The APTT value for precipitate was 294

229 both anticoagulant and antioxidant activity. The total yield of pre- significantly (p < 0.05) higher in the precipitate from the fermented 295

230 cipitate was in the range of 75–120 mg (2.5–5.0% of dry weight). sample compared to control, highest (4.71 ± 0.02 IU/mg) being the 296

231 Total sugars of the precipitate also varied in each sample ranging precipitate obtained from the seaweed fermented with E. faecium 297

232 from 61.4–79.6%. Rioux et al. [19] extracted PS from Saccharina (P1-2Cb-w1). As reported by Pomin [25], even a single structural 298

233 longicruris by ethanol precipitation and obtained total yield of 19.4% difference in sugar type can make a great change in anticoag- 299

234 dry weight with 57.8% sugar and 12.4% protein. PS from Sargassum ulant activity, even though other parameters including sulfation 300

235 pallidum extracted through hot water extract followed by ethanol pattern, anomeric configuration, glycosidic linkage and molecu- 301

236 precipitation yielded 1.8 g of dry weight (0.18%) [20] and about lar mass are similar. E. faecium (P1-2CB-w1) fermentation that 302

237 3.96–5.11% from S. latifolium [21]. Camera et al. [22] used proteoly- contributes to higher total sugar and reducing sugar, indicate its 303

238 sis and sequential acetone precipitation to obtain acidic PS from potency in hydrolyzing seaweed cell wall as compared to other 304

239 brown seaweed Canistrocarpus cervicorni with 10.6–23.5% yield LAB cultures. The leached out seaweed PS from E. faecium (P1- 305

240 with total sugar content of 12.2–41.8%. Tichocarpus crinitus PS was 2CB-w1) may have a structural variation that contribute to potent 306

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Table 1
Antioxidant activity of fermented Sargassum sp. on ethanol precipitation.

Sample PP RP MC NO H2 O2 ABTS SO

(A) Antioxidant activity of soluble fraction

Test control 43.32 ± 1.69a 6.94 ± 0.13e 48.27 ± 0.59b 670.97 ± 1.05a 198.11 ± 2.22a 202.17 ± 18.4ab 419.67 ± 0.21a
Sample control 44.32 ± 0.07b 5.36 ± 0.12d 46.79 ± 0.57a 663.14 ± 1.04a 286.05 ± 0.05b 192.67 ± 8.05a 493.92 ± 0.10d
LC 47.39 ± 0.02c 5.00 ± 0.01c 62.54 ± 0.76e 712.91 ± 7.92b 352.74 ± 2.60c 237.64 ± 0.09d 507.98 ± 0.24e
Nw1-a 46.73 ± 0.01c 4.88 ± 0.06b 58.86 ± 0.03c 705.34 ± 1.10b 363.01 ± 1.91d 214.42 ± 0.13c 444.32 ± 0.13b
2Nw-1 48.72 ± 0.01d 4.54 ± 0.04a 60.24 ± 0.06d 874.16 ± 0.69c 427.83 ± 1.67e 207.14 ± 0.28bc 468.16 ± 0.05c
P1-2CB-w1 60.61 ± 0.04e 6.87 ± 0.05e 71.56 ± 0.11f 907.29 ± 1.42d 485.96 ± 4.22f 253.24 ± 0.01e 529.47 ± 0.25f
r 0.34 0.88 0.84 0.84 0.82 0.69

(B) Antioxidant activity of precipitate

Test control 0.44 ± 0.01a 1.04 ± 0.001a 4.98 ± 0.03a 141.83 ± 0.11a 30.14 ± 0.17a 25.69 ± 0.31a 105.63 ± 0.07a
Sample control 0.52 ± 0.03b 1.21 ± 0.006b 5.21 ± 0.05b 275.93 ± 3.96d 31.03 ± 0.24b 25.77 ± 1.00a 106.08 ± 0.12c
LC 0.54 ± 0.03b 1.74 ± 0.009c 5.24 ± 0.10b 231.44 ± 1.15c 31.97 ± 0.55c 26.57 ± 0.53b 105.07 ± 0.12b
Nw1-a 0.63 ± 0.07c 1.97 ± 0.001d 5.42 ± 0.08c 159.04 ± 0.39b 51.79 ± 0.07f 26.81 ± 0.18b 105.62 ± 0.02a
2Nw-1 0.76 ± 0.05d 1.49 ± 0.007e 5.15 ± 0.10b 331.73 ± 1.06f 34.56 ± 0.08d 26.82 ± 0.15b 108.52 ± 0.09d
P1-2CB-w1 0.95 ± 0.04e 2.42 ± 0.010f 6.93 ± 0.14d 307.04 ± 7.86e 44.70 ± 0.42e 33.99 ± 0.75c 136.24 ± 0.16e
r 0.79 0.83 0.67 0.54 0.86 0.85

Values are mean ± SD of three trials. Values in columns with same superscripts does not differ significantly (p > 0.05).
LC: Pediococcus acidilactici, Nw-1a: Weisella paramesenteroides, 2Nw-1a: Pediococcus pentosaceus, P1-2CB-w1: Enterococcus faecium. PP: polyphenols (gallic acid equivalent:
␮g/mg dry weight); RP: reducing potential (ascorbic acid equivalent: ␮g/mg dry weight); MC: metal chelation ability (EDTA equivalent: ␮g/mg dry weight); NO: nitric oxide
scavenging (TBHQ equivalent: ␮g/mg dry weight); H2O2: hydrogen peroxide scavenging activity (TP equivalent: ␮g/mg dry weight); ABTS scavenging (TBHQ equivalent:
␮g/mg dry weight); SO: singlet oxygen scavenging (TP equivalent: ␮g/mg dry weight); r: correlation co-efficient between polyphenol and each antioxidant activity.

Table 2
Anticoagulation activity of precipitate and soluble fraction on ethanol precipitation of fermented Sargassum sp.

Sample Activity (heparin IU/mg)

APTT PT

Precipitate Soluble fraction Precipitate Soluble fraction

Test control 3.23 ± 0.04a 1.54 ± 0.05a 0.077 ± 0.002a 0.065 ± 0.003a
Sample control 3.90 ± 0.11b 1.63 ± 0.01b 0.082 ± 0.005a 0.075 ± 0.004b
LC 4.44 ± 0.09d 1.81 ± 0.04c 0.094 ± 0.007b 0.088 ± 0.004d
Nw-1a 4.27 ± 0.08c 1.78 ± 0.07c 0.091 ± 0.001b 0.081 ± 0.002c
2Nw-1 4.50 ± 0.08d 2.54 ± 0.03d 0.095 ± 0.001b 0.089 ± 0.005d
P1-2CB-w1 4.71 ± 0.02e 2.67 ± 0.05e 0.110 ± 0.005c 0.092 ± 0.004d

Values are mean ± SD of three trials. Values in columns with same superscripts does not differ significantly (p > 0.05).
LC: Pediococcus acidilactici, Nw-1a: Weisella paramesenteroides, 2Nw-1a: Pediococcus pentosaceus, P1-2CB-w1: Enterococcus faecium; APTT: activated partial thromboplastin
time; PT: prothrombin time; Blank (distilled water) for APTT assay: clot time – 101.38 s and for PT assay: clot time – 22.2 s; IU: international units with respect to heparin.

307 anticoagulant activity. The APTT values were comparatively lower fulvellum by natural fermentation followed by DEAE cellulose frac- 332

308 in soluble fraction (1.54–2.67 IU/mg), but still highest in seaweed tionation and obtained a yield of 0.594 mg which represents 1.6% 333

309 fermented with E. faecium (P1-2CB-w1). PT values were also higher recovery. Cho et al. [27] isolated sulfated PS from Enteromorpha pro- 334

310 in precipitate (0.077–0.11 IU/mg) compared to soluble fraction lifera by hot water extraction followed by ethanol precipitation and 335

311 (0.065–0.092 IU/mg). Similar observation was made by Camara fractionation with ion exchange chromatography to obtain three 336

312 et al. [22] wherein PS from Canistrocarpus cervicornis effectively fractions with total yield of 25.1% containing total sugar ranging 337

313 acted on intrinsic system and lacked the effect on extrinsic path- from 50.8 to 62.5% and protein 1.0 to 13.9%. 338

314 way. Albuquerque et al. [5] extracted sulfated PS by proteolytic A comparative data for anticoagulant activity, total sugars, 339

315 digestion followed by acetone precipitation that prolonged coag- reducing sugar, proteins and purification fold of crude, ethanol 340

316 ulation time by 4.88-folds higher than Clexane (4.1 ␮g/ml). As the precipitate and active fractions from DEAE Cellulose column is 341

317 ethanol precipitate rich in sugars exhibited higher anticoagulant presented in Table 4. The activity increased by 2.6–3.9-folds on 342

318 activity, it was taken up for further purification and structural elu- purification by DEAE cellulose column with a recovery of 0.5–1.04%. 343

319 cidation.

Table 3
320 3.2. Purification of seaweed PS Anticoagulation activity of DEAE fractions of Sargassum polysaccharides.

Sample Activity (IU/mg)

321 Purification of fermented seaweed PS was carried out by anion
Fraction 1 Fraction 2 Fraction 3
322 exchange chromatography using DEAE cellulose. Total sugar con-
323 tent in different fraction collected during fractionation indicated LC 0.32 ± 0.012b 4.59 ± 0.063a 2.74 ± 0.148b
324 that each samples fermented with different LAB showed elution Nw-1a 0.27 ± 0.045a 4.98 ± 0.027b 0.26 ± 0.011a
2Nw-1a 0.26 ± 0.033a 6.52 ± 0.539c 0.29 ± 0.027a
325 at different NaCl concentration. Accordingly 3 fractions from each P1-2CB-w1 0.33 ± 0.009b 6.70 ± 0.224c 0.25 ± 0.013a
326 samples with higher sugar content were separately pooled, freeze
Values are mean ± SD of three trials. Values in columns with same superscripts does
327 dried and analyzed for anticoagulant activity. A higher anticoag-
not differ significantly (p > 0.05).
328 ulant activity (4.59–6.70 IU/mg) was observed in fraction 2 of all LC: Pediococcus acidilactici, Nw-1a: Weisella paramesenteroides, 2Nw-1a: Pediococ-
329 the samples (Table 3). Total yield of this active fractions were cus pentosaceus, P1-2CB-w1: Enterococcus faecium, activity (IU/mg): anticoagulation
330 in the range of 0.5–1.04% constituting 24.2–50.8% of total sugar activity as analyzed by activated partial thromboplastin time (APTT) assay; IU: inter-
national units with respect to heparin. Blank (distilled water): clot time – 101.38 s.
331 and 3.6–14.7% protein. De Zoysa et al. [26] extracted PS from S.

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Table 4
Purification table.

Fermenting Sample Total yield Activity Total Total sugars Reducing sugars Total protein Purification
culture (IU/mg) activity (IU) fold

mg % (mg) % (mg) % (mg) %

LC Crude 3000 100 1.51 4530 2437.50 81.3 731.25 24.4 92.61 3.1 1
Ethanol precipitate 90 3.0 4.44 399.6 61.33 68.1 25.31 28.1 19.00 21.1 2.9
DEAE active fraction 22 0.73 4.59 100.98 5.14 24.2 1.03 4.7 0.83 3.6 3.0

Nw-1a Crude 3000 100 1.85 5550 2484.38 82.8 795.31 26.5 192.72 6.4 1
Ethanol precipitate 75 2.5 4.27 320.25 59.52 79.6 24.61 32.8 14.46 19.3 2.3
DEAE active fraction 15 0.5 4.98 74.7 3.63 25.7 2.81 18.7 1.27 8.5 2.7

2Nw-1a Crude 2400 100 1.69 4056 2203.13 91.8 660.94 27.5 251.63 10.5 1
Ethanol precipitate 105 4.37 4.49 471.45 64.44 61.4 16.41 15.6 24.13 22.9 2.7
DEAE active fraction 20 0.83 6.52 130.4 6.93 34.6 3.437 17.2 1.56 7.8 3.9

P1-2CB-w1 Crude 2400 100 2.58 6192 2246.88 93.6 674.06 28.1 151.30 6.3 1
Ethanol precipitate 120 5.0 4.71 565.3 86.52 72.1 39.37 32.8 32.03 26.7 1.8
DEAE active fraction 25 1.04 6.70 167.5 12.70 50.8 5.078 20.3 3.68 14.7 2.6

LC: Pediococcus acidilactici, Nw-1a: Weisella paramesenteroides, 2Nw-1a: Pediococcus pentosaceus, P1-2CB-w1: Enterococcus faecium, activity (IU/mg): anticoagulation activity
as analyzed by activated partial thromboplastin time (APTT) assay; IU: international units with respect to heparin. Blank (distilled water): clot time – 101.38 s.
Purification factor = [activity (IU/mg) of fraction/activity (IU/mg) of crude sample].

344 The total and reducing sugar varied among the active fraction of structural characteristic of PS extracted from fermented seaweed 360

345 each sample. Highest total sugar (50.8%) and reducing sugar (20.3%) with higher anticoagulant activity. 361

346 were detected in the active fraction of precipitate obtained from FT-IR analysis was carried out to determine the spectral fea- 362

347 seaweed fermented with E. faecium (P1-2CB-w1). Maximum recov- tures of polysaccharides extracted from Sargassum sp. fermented 363

348 ery (1.04%) of polysaccharide was obtained from the same sample with E. faecium (P1-2CB-w1) which exhibited higher anticoag- 364

349 and it showed significantly (p < 0.05) higher anticoagulant activity ulant activity (Fig. 1). IR spectrum showed specific absorption 365

350 (6.7 ± 0.22 IU/mg). bands at around 3407 cm−1 readily assignable to OH stretching 366

vibration mode. Two more sharp absorption bands at 1602 and 367

351 3.3. Structural elucidation of seaweed polysaccharide 1411 cm−1 assigned to carboxylate O C O asymmetric stretch- 368

ing and to C OH deformation vibration with contribution to 369

352 Specific structural characterization of PS is very essential for O C O symmetric vibration COO stretching was observed. In addi- 370

353 any therapeutic application. Biological activity of PS is related to tion, a weak band at 2930 cm−1 attributed to C H stretching 371

354 molecular size, type and amount of sugar and sulfate content [28]. was observed which are characteristic bands of alginate (Fig. 1). 372

355 Although their structure varies according to season, specific and The absorption bands at 1092 cm−1 and 1036 cm−1 confirmed 373

356 geographical location, several investigations have been carried out the C O stretching vibration and C O and C C stretching vibra- 374

357 on structure and functions of PS isolated from different Sargassum tions of pyranose ring. Apart from these absorption bands, the 375

358 sp. [29,30]. As the structure of PS is the major factor affecting presence of mannuronic and guluronic units was confirmed by 376

359 their biological activity, an attempt was made to understand the the observed absorptions bands at 820 cm−1 [31,32]. The distinct 377

Fig. 1. FT-IR spectra of purified polysaccharide from Sargassum fermented by P1-2CB-w1 (Enterococcus faecium).

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Fig. 2. (A) 13 C NMR spectra of purified polysaccharide from Sargassum fermented by P1-2CB-w1 (Enterococcus faecium); (B) a proposed structure of mannuronate rich alginate
from Sargassum sp. fermented by P1-2CB-w1 (Enterococcus faecium).

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