Highlights glucose monomers followed with traces of


Branched glucan comprised with minor ribose was investigated first time from Enterococcus hirae
Substantially produced Enterococcus hirae EPS was 18.18- 18.57g/L at prolonged condition
FTIR and NMR revealed the 51% of ?-(1-6) and 42% of ?-(1?3) linked branches
Surface microstructure of EPS shows porous and starch like cracked granules
EPS is amorphous and also with good thermostable, water solubility and holding capacity

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Enterococcus hirae EPS was exhibit as potential prebiotic agent in food industry



An appreciable exopolysaccharide producing lactic flora (Enterococcus hirae CBTF3) were isolated from indigenous feces of Irula tribes. The isolate displayed maximum production of 18.18-18.57g/L at prolonged condition about 24 to 72h with MRS broth consisting of 2% sucrose. In TLC and GC-MS analysis confirms predominately glucose monomers followed with traces of ribose and it can be specified as homopolysaccharide of glucan. FTIR and NMR studies confirms the existence of 51% ?-(1-6) and 42% linked branches of ?-(1?3). SEM analysis of glucan-EPS revealed that porous and starch like cracked granules of aggregation. AFM revealed the spherical lumps and densed grainy like network by microstructure of glucan-EPS. The thermal behaviour of glucan-EPS showed degradation temperature of 315.98°C and melting point of 296.67°C. XRD analysis determined the amorphous with CIXrd of 0.48 for glucan-EPS. The Water Solubility Index and Water Holding Capacity of glucan-EPS showed 46.5% and 202.04%. Overall characteristic of glucan-EPS from Enterococcus hirae it can explored as potential prebiotics by its high constitute of ?-(1?3) on resistance to human physiological flow to better stimulation of probiotic bacteria. This is a first study reporting from that branched glucan with minor ribose produced Enterococcus hirae.

Keywords: Exopolysaccharide, Enterococcus hirae, Glucan













In recent studies revealed that human intestinal lactic flora produced multiple functional metabolites i.e. BSH, Bacteriocin, and EPS to strongly influence the success for the base of probiotic attributes (Maria et al., 2006 1;Messaoudi et la., 20132; Ren et al., 2014.,3 ). Amongst EPS play a wide application and also have technological properties to health benefits to consumers (Wang et al., 20084; Ito et al., 20115). General forms of EPS existed as branched or linear in the various origins of plant, algae, fungi and bacteria (Wang et al., 20084). Naturally, EPS microbial had predominately composed of hexose sugar D-glucose, D-mannose, D-galactose, D-xylose and D-arabinose moreover additional rare sugar exists in minor composition as D-ribose, fucose, glucosamine, galactosamine, galactouronic acid and glucouronic acid (Han et al., 20145 ; Li and Shah, 20166; Bramhachari and Dubey, 20067). That EPS develops their biological and functional properties to attractive in industries (Rani et al., 20178). As LAB had EPS from probiotics as commonly attained as GRAS (Generally Recognized As Safe) by its confirming of prolonged colonization and prebiotics in GI-tract (Gardiner et al., 19999; Welman and Maddox, 200810). Additionally, EPS from LAB had been presented to numerous health-promoting effects such as immunomodulating, antitumour, antioxidant and cholesterol-lowering properties (Li et al., 201511).In general, EPS are of two forms during cell growth as excreted and both excreted and cell bound by specific LAB (Li et al., 201511).  EPS are separated into class as homo and heteropolysaccharide by its composition and biosynthetic mechanism.  Homopolysaccharides are consist of one type monomeric units and it can synthesis large amount from the substrate of sucrose by the action of sucrase enzyme whereas heteropolysaccharides composed of multiple sugars synthesized by different glycosyltransferase enzymes (Van Hijum et al., 200612; De Vuyst and Degeest, 199913). In homopolymer of glucan showed viscosifying, stabilizing, emulsifying, sweetening, gelling, water binding and prebiotic agent in the food (Whitfield, 198814; Welman and Maddox, 200810; Das et al.,201415). In glucan produced Enterococcus spp from human gut reported limited and also it possess strong therapeutic with food application such as emulsifying, antimicrobial (Bajaji et al., 201516). To date, Enterococcus hirae have been investigated as probiotics from animal and dosa batter and also beneficial to human of high resistance to colonization against Salmonella enterica ser. Typhimurium than L. ruminis when administrated with in mice model (Gupta1&Tiwari117, 2015; Arokiyaraj et al., 201418; Galvão1 et al., 201719). Also in chemotherapy studies, it high expression immune cells and retain after antibiotic administration (Daille`re et al., 201620). Henceforth there is no report on EPS production and probiotic studies from Enterococcus hirae present in human origin.

Those days south Indian tribe are well practitioner in natural medicine to cure numerous diseases and highly nutritious unexploited dietary fibre based food, whether cooked or fermented as food source in human diet (Sandhya et al., 2006; Ayyanar et al., 2005; Ignacimuthu et al., 1998; Sarkar et al., 2015; Rajyalakshmi & Geervani., 1993). By the survey of EFSA from Europeans dietary fibre dietician to frame a guideline of dietary intake of 25 g/day or above of the traditional diet of humans, it improves bowel movement, reduce CHD and type-II diabetics (Shen Q et al., 2011). Additionally, earlier researchers also supported in different traditional dietary fibre dietician had reduced disease condition or without any ill effect by increase in intestinal lactic flora, other beneficial microbes, SCFA production and saccharolytic enzymes than normal dieticians (Mai V et al., 2009; Burkitt DP., 1971; Pan Y et al., 2016; Shen Q et al., 2011). In south Indian origin, particularly Tamil Nadu, one of the unique groups of traditional dietary fibre consuming tribes is Irulas and named as Irula kizhangu from Dioscorea spp. They tubers were collected day by day and prepare koji as a staple side dish in all matched with daily foods. They lived in thick jungle folk and also an immense knowledge of the healing properties of herbal medicine that can cure various ailments (Ravishankar et al., 2007). To the best of our knowledge, no reports of EPS produced probiotic LAB from Irula tribes Intestinal origin in Tamil Nadu.  Therefore, the present studies were evaluated the intestinal lactic flora obtained from healthy volunteers of Irula tribes living in the village of Dharmapuri district in Tamil Nadu. To check the probiotic bacterial battery of acid and bile tolerance. Then subjected to identify EPS production probiotic LAB and to characterize the EPS by high producing strain








2. Materials and Methods

2.1 Fecal LAB

A high probiotic base of acid, bile and EPS production LAB was isolated from indigenous feces of Irula tribal.  Then the bacterial gene was sequenced and identified as Enterococcus hirae. Further the sequence was submitted and got the ref.no: KX577639 and it can be used for throughout the examination for characterization.

2.2 Production, Extraction and purification of CBTF3-EPS

The Eh-EPS was produced, extracted and purified by the method of Garcia- Garibay and Marshall 1999 with slight modifications. Briefly, overnight culture was inoculated in MRS broth supplement with 2% sucrose and incubated at 30°C in a rotatory shaker at 125 rpm. The aliquots were collected at three different intervals of time (24, 48, 72 h), centrifuged at 12,000 x g- rpm for 15 minutes. The supernatant was collected and treated with 2% TCA to remove proteins. EPS was precipitated with ice cold ethanol (thrice the volume) and left overnight. The precipitate was collected after centrifugation at 19,200xg for 15min and dissolved in Milli-Q water for 48 h. The dialyzed contents were then freeze dried by lyophilisation for 48 h and the EPS content was quantified by the phenol-sulfuric acid method ().

2.2. FTIR analysis of CBTF3-EPS

The major functional group of Eh-EPS were detected using FTIR spectrophotometer. Ten mg of the Eh-EPS powder were mixed with KBr followed by compressing the combination into pellet form. Then spectrum was scanned between 400-4000 cm?1 using (Bruker Vector22 instruments, Germany).

2.3. NMR studies of CBTF3-EPS

                NMR spectra of 1D (C13 and H1) and 2D 1H–1H correlated spectroscopy (COSY), 1H–1H total correlated spectroscopy (TOCSY), 1H–13C heteronuclear single quantum coherence spectroscopy (HSQC) and 1H–13C heteronuclear multiple bond correlation spectroscopy (HMBC) experiments were held out by Bruker AV III 500 MHz FT NMR Spectrometer. Twenty five milligrams of purified EPS were dissolved in 1ml of 99.66% D2O and analysed. For 1D NMR (C13 and H1), the sample solution was submitted to a delay (D1) and acquisition time (AQ) of (1.00, 2.00 sec) and (0.55, 1.58 sec). Subsequently, 2D NMR (COSY, TOCSY, HSQC and HMBC) was done with a delay (D1) and acquisition time (AQ) of (1.45, 1.43, and 1.48 sec) and (0.18, 0.37 and 0.09 sec). The chemical shift of NMR data was expressed in parts per million (ppm). 

2.4. Analysis of monomeric composition

2.4.1. TLC and GC-MS analysis of CBTF3-EPS

For GC-MS and TLC analysis was similarly followed by Khatua and Acharya, 2016 on GC-model (Thermo GC- Trace Ultra Version: 5.0) and the chromatogram was matched and commercial carbohydrates present in the NIST database respectively.

2.5.4. SEM and AFM analysis of CBTF3-EPS

Two milligram EPS was dissolved in 1ml of Milli-Q water at the concentration of 1mg/ml. Then the sample was constantly stirred at 1 h at 50?C in a sealed bottle under N2 stream to get a uniform dispersion. After that the samples were cooled, the suspension was diluted to a concentration of 0.01 mg/mL.  Then 5?l of suspension was equally spread on mica disc and dried at room temperature after ethanol fixation. AFM images were captured by scanning probe microscope (Multiple View 2000, Nanonics Isarel) in tapping mode. The cantilever oscillation was set at the appropriate frequency of 158 kHz with driving amplitude of 0.430 V. EPS was fixed on the aluminium stub and gold sputtered and examined through SEM (JEOL, Model: 6390) at an accelerating voltage of 10 Kv (Ahmed et al., 2013).

2.5.5. TG, DSC and XRD pattern of CBTF3-EPS

Ten milligram of dried EPS was passed through the thermal system (TG-DTA/DSC Model: Q600 SDT). Then TG–DSC thermograms were directed to the temperature of 0–400?C underneath the flow of nitrogen air at the rate of 10?C/min. Afterward that the individual graph were plotted proportion between weight loss in percentage and heat flow against temperature (Wang et al., 2010). X- ray diffraction studies were carried out to study the amorphous and crystalline nature of EPS. The diffraction scan was performed at diverse ranges of two-theta angles (10-80°C) was done. XRD was accomplished on X-ray powder diffractometer (Shimadzu XRD 7000) with a Cu K- tube. The X- ray produces ? radiation at the speed of 2°/min at a diffraction angle 2? of 4° and 50° at 40 kV and 30 mA with PW3011/20 proportional detector. Crystallinity index (CIxrd) was calculated from the area under crystalline peaks stabilized with corresponding to the total scattering area. Equipped

2.6. Water Solubility Index (WSI)

                The water solubility index of EPS was recorded using the method of Anderson et al., (1982). EPS quantity of 0.5 g was mixed with 6mL of distilled water into a centrifuge tube and continuously stirred for 30 min in a water bath at 30° C to produce a uniform suspension. Then the suspension was centrifuged at 3000×g for 10min. The supernatant was placed in a petri dish and dried at 105°C for 4 hours to get the dry solid weight. Next the centrifuged wet residue was weighed by Reddy et al., 2013. Then the WSI was calculated according to the following equation,

            WSI = (weight of dry solids in supernatant × weight of dry sample) × 100                  (2)

2.7. Water Holding Capacity (WHC)

                WHC of the EPS was determined by dissolving 0.2g specimen in 10 mL of Milli-Q water and kept at 40° C for 10 minutes. Then the sample was centrifuged at 15,000 rpm for 30 minutes and the supernatant was discarded. Then pellet was dropped on the pre-weighed filter paper to allow for complete drainage of water.  The filter paper was weight and the results were documented (Ahmed et al., 2013).  Then percentage of Water-Holding Capacity was calculated using below equation

                                   WHC % =   Total sample weight after water absorption × 100 ((3)

                                                               Total dry sample weight